Sample records for acanthamoeba castellanii encystment

  1. DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation.

    PubMed

    Moon, Eun-Kyung; Hong, Yeonchul; Lee, Hae-Ahm; Quan, Fu-Shi; Kong, Hyun-Hee

    2017-04-01

    Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba . To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1-3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba . In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.

  2. Francisella tularensis type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks post Infection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    El-Etr, S H; Margolis, J; Monack, D

    2009-07-28

    Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown, and the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype (REP)more » is caused by factor(s) secreted by amoebae and/or F. tularensis into the co-culture media. Further, our results indicate that in contrast to LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks post infection and that induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that the interactions between F. tularensis strains and amoeba may play a role in the environmental persistence of F. tularensis.« less

  3. Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii Trophozoites and Cysts

    PubMed Central

    Matin, Abdul

    2011-01-01

    The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission. PMID:22355201

  4. Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts.

    PubMed

    Matin, Abdul; Jung, Suk-Yul

    2011-12-01

    The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.

  5. Acanthamoeba castellanii interactions with Streptococcus pneumoniae and Streptococcus pyogenes.

    PubMed

    Siddiqui, Ruqaiyyah; Yee Ong, Timothy Yu; Jung, Suk Yul; Khan, Naveed Ahmed

    2017-12-01

    Among the genus Streptococcus, S. pyogenes and S. pneumoniae are the major causes of pharyngitis, impetigo, pneumonia and meningitis in humans. Streptococcus spp. are facultative anaerobes that are nutritionally fastidious, yet survive in the environment and target the predisposed population. Antibacterial disinfectants have been partially effective only, indicating the need for novel preventative measures and to understand mechanisms of bacterial resistance. Acanthamoeba is a free-living protist that is known to harbour microbial pathogens, provide shelter, and assist in their transmission to susceptible population. The overall aim of this study was to determine whether S. pyogenes and S. pneumoniae can interact with A. castellanii by associating, invading, and surviving inside trophozoites and cysts. It was observed that both S. pyogenes and S. pneumoniae were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. Notably, S. pyogenes and S. pneumoniae survived the encystation process, avoided phagocytosis, multiplied, and exhibited higher recovery from the mature cysts, compared with the trophozoite stage (approximately 2 bacteria per amoebae ratio for cyst stage versus 0.02 bacteria per amoeba ration for trophozoite stage). As Acanthamoeba cysts are resilient and can disperse through the air, A. castellanii can act as a vector in providing shelter, facilitating growth and possibly genetic exchanges. In addition, these interactions may contribute to S. pyogenes and S. pneumoniae survival in harsh environments, and transmission to susceptible population and possibly affecting their virulence. Future studies will determine the molecular mechanisms associated with Acanthamoeba interactions with Streptococcus and the evolution of pathogenic bacteria and in turn expedite the discovery of novel therapeutic and/or preventative measures. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Acanthamoeba castellanii STAT protein.

    PubMed

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

  7. Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila

    PubMed Central

    2013-01-01

    Background Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts. Methods Here, we used a keratitis isolate of A. castellanii of the T4 genotype and studied its interactions with two bacterial genera which have not been tested before, Enterobacter aerogenes, and Aeromonas hydrophila, as well as E. coli. Assays were performed to determine bacterial association with and invasion of A. castellanii. Additionally, bacterial survival intracellular of A. castellanii trophozoites as well as cysts was determined. Results All three bacterial isolates tested, associated, invaded, and survived inside A. castellanii trophozoites as well as A. castellanii cysts. However, E. aerogenes and E. coli exhibited significantly reduced association with and invasion of A. castellanii as compared with A. hydrophila (P < 0.01 using paired T-test, one tail distribution). In the long term survival assays, all three bacterial isolates tested remained viable inside A. castellanii trophozoites, while amoeba remained intact; however A. hydrophila exhibited higher survival inside amoebae (14.54 ± 3.3 bacteria:amoeba ratio) compared with E. aerogenes (3.96 ± 0.7 bacteria:amoeba ratio) and E. coli (5.85 ± 1.1 bacteria:amoeba ratio). A. hydrophila, E. coli, and E. aerogenes remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (14.13 ± 0.89 A. hydrophila:amoeba ratio, 10.13 ± 1.17 E. aerogenes:amoeba ratio, and 11.95 ± 0.7 E. coli:amoeba ratio). Conclusions A. hydrophila and E. aerogenes also joined the ranks of other bacteria that could benefit from A. castellanii

  8. Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila.

    PubMed

    Yousuf, Farzana Abubakar; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2013-06-07

    Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts. Here, we used a keratitis isolate of A. castellanii of the T4 genotype and studied its interactions with two bacterial genera which have not been tested before, Enterobacter aerogenes, and Aeromonas hydrophila, as well as E. coli. Assays were performed to determine bacterial association with and invasion of A. castellanii. Additionally, bacterial survival intracellular of A. castellanii trophozoites as well as cysts was determined. All three bacterial isolates tested, associated, invaded, and survived inside A. castellanii trophozoites as well as A. castellanii cysts. However, E. aerogenes and E. coli exhibited significantly reduced association with and invasion of A. castellanii as compared with A. hydrophila (P < 0.01 using paired T-test, one tail distribution). In the long term survival assays, all three bacterial isolates tested remained viable inside A. castellanii trophozoites, while amoeba remained intact; however A. hydrophila exhibited higher survival inside amoebae (14.54 ± 3.3 bacteria:amoeba ratio) compared with E. aerogenes (3.96 ± 0.7 bacteria:amoeba ratio) and E. coli (5.85 ± 1.1 bacteria:amoeba ratio). A. hydrophila, E. coli, and E. aerogenes remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (14.13 ± 0.89 A. hydrophila:amoeba ratio, 10.13 ± 1.17 E. aerogenes:amoeba ratio, and 11.95 ± 0.7 E. coli:amoeba ratio). A. hydrophila and E. aerogenes also joined the ranks of other bacteria that could benefit from A. castellanii. Because cysts can be airborne, these

  9. In vitro adhesion of Acanthamoeba castellanii to soft contact lenses depends on water content and disinfection procedure.

    PubMed

    Reverey, Julia F; Fromme, Roland; Leippe, Matthias; Selhuber-Unkel, Christine

    2014-08-01

    To compare the potential of different soft contact lenses to be contaminated with Acanthamoeba castellanii as a function of material parameters and cleaning procedures. Different unworn soft hydrogel and silicone hydrogel contact lenses were incubated with human pathogenic A. castellanii. The adhesion of the acanthamoebae was investigated on the contact lenses and put into relation to their material parameters. The efficacy of a recommended contact lens cleaning procedure in reducing A. castellanii adhesion was investigated. We found that material parameters such as elastic modulus, silicone content, ionic properties and swelling do not influence the adhesion of acanthamoebae to soft contact lenses. A material parameter that influenced adhesion significantly was the water content of the lens. With increasing water content, the adhesion of acanthamoebae increased. By following the cleaning instructions of the manufacturer the contamination of the lenses with A. castellanii could be reduced to a minimum, as shown both on contact lenses and in control experiments. With this study we show that for the tested lenses, the adhesion of A. castellanii to contact lenses is independent of the silicone content of the lens, but depends nonlinearly on the water content of the lens. Furthermore, we demonstrate that applying proper lens cleaning procedures minimizes the risk of acanthamoebae adhesion to contact lenses. Copyright © 2013 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  10. The role of Src kinase in the biology and pathogenesis of Acanthamoeba castellanii

    PubMed Central

    2012-01-01

    Background Acanthamoeba species are the causative agents of fatal granulomatous encephalitis in humans. Haematogenous spread is thought to be a primary step, followed by blood–brain barrier penetration, in the transmission of Acanthmaoeba into the central nervous system, but the associated molecular mechanisms remain unclear. Here, we evaluated the role of Src, a non-receptor protein tyrosine kinase in the biology and pathogenesis of Acanthamoeba. Methods Amoebistatic and amoebicidal assays were performed by incubating amoeba in the presence of Src kinase-selective inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyrimidine). Using this inhibitor, the role of Src kinase in A. castellanii interactions with Escherichia coli was determined. Zymographic assays were performed to study effects of Src kinase on extracellular proteolytic activities of A. castellanii. The human brain microvascular endothelial cells were used to determine the effects of Src kinase on A. castellanii adhesion to and cytotoxicity of host cells. Results Inhibition of Src kinase using a specific inhibitor, PP2 (4-amino-5-(4 chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d] pyrimidine) but not its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d] pyrimidine), had detrimental effects on the growth of A. castellanii (keratitis isolate, belonging to the T4 genotype). Interestingly, inhibition of Src kinase hampered the phagocytic ability of A. castellanii, as measured by the uptake of non-invasive bacteria, but, on the contrary, invasion by pathogenic bacteria was enhanced. Zymographic assays revealed that inhibition of Src kinases reduced extracellular protease activities of A. castellanii. Src kinase inhibition had no significant effect on A. castellanii binding to and cytotoxicity of primary human brain microvascular endothelial cells, which constitute the blood–brain barrier. Conclusions For the first

  11. Superdiffusion dominates intracellular particle motion in the supercrowded cytoplasm of pathogenic Acanthamoeba castellanii

    NASA Astrophysics Data System (ADS)

    Reverey, Julia F.; Jeon, Jae-Hyung; Bao, Han; Leippe, Matthias; Metzler, Ralf; Selhuber-Unkel, Christine

    2015-06-01

    Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements, and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin, and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved by actively driven motion, while purely thermally driven diffusion is negligible.

  12. Insights into the prominent effect of mahanimbine on Acanthamoeba castellanii: Cell profiling analysis based on microscopy techniques

    NASA Astrophysics Data System (ADS)

    Hashim, Fatimah; Amin, Nakisah Mat

    2017-02-01

    Mahanimbine (MH), has been shown to have antiamoeba properties. Therefore, the aim of this study was to assess the growth inhibitory mechanisms of MH on Acanthamoeba castellanii, a causative agents for Acanthamoeba keratitis. The IC50 value obtained for MH against A. castellanii was 1.18 µg/ml. Light and scanning electron microscopy observation showed that most cells were in cystic appearance. While transmission electron microscopy observation revealed changes at the ultrastructural level and fluorescence microscopy observation indicated the induction of apoptosis and autophagic activity in the amoeba cytoplasms. In conclusion, MH has very potent anti-amoebic properties on A. castellanii as is shown by cytotoxicity analyses based on microscopy techniques.

  13. Bowman's layer encystment in cases of persistent Acanthamoeba keratitis.

    PubMed

    Yokogawa, Hideaki; Kobayashi, Akira; Yamazaki, Natsuko; Ishibashi, Yasuhisa; Oikawa, Yosaburo; Tokoro, Masaharu; Sugiyama, Kazuhisa

    2012-01-01

    The purpose of this study was to report Acanthamoeba encystment in Bowman's layer in Japanese cases of persistent Acanthamoeba keratitis (AK). Laser confocal microscopic images of the cornea were obtained in vivo from 18 consecutive eyes from 17 confirmed AK patients. Retrospectively, 14 cases treated over 4 months were categorized as a nonpersistent group and three cases that required prolonged therapy for more than 6 months were categorized as a persistent group. Clinical outcomes based on final best-corrected visual acuity were retrospectively analyzed, and selected confocal images were evaluated qualitatively for abnormal findings. The final best-corrected visual acuity was significantly lower (P < 0.01) for patients in the persistent group compared with that in the nonpersistent group. At the initial visit, in vivo confocal microscopy demonstrated Acanthamoeba cysts exclusively in the epithelial layer in both the nonpersistent group (80%) and the persistent group (100%). At a subsequent follow-up visit, numerous Acanthamoeba cysts were observed in the epithelial cell layer and in Bowman's layer in all patients with persistent AK, but Acanthamoeba cysts were undetectable in all cases with nonpersistent AK tested. Invasion of cysts into Bowman's layer was characteristically observed in patients with persistence of AK. This finding suggests that invasion of Acanthamoeba cysts into Bowman's layer may be a useful predictor for a persistent clinical course.

  14. Legionella pneumophila prevents proliferation of its natural host Acanthamoeba castellanii

    PubMed Central

    Mengue, Luce; Régnacq, Matthieu; Aucher, Willy; Portier, Emilie; Héchard, Yann; Samba-Louaka, Ascel

    2016-01-01

    Legionella pneumophila is a ubiquitous, pathogenic, Gram-negative bacterium responsible for legionellosis. Like many other amoeba-resistant microorganisms, L. pneumophila resists host clearance and multiplies inside the cell. Through its Dot/Icm type IV secretion system, the bacterium injects more than three hundred effectors that modulate host cell physiology in order to promote its own intracellular replication. Here we report that L. pneumophila prevents proliferation of its natural host Acanthamoeba castellanii. Infected amoebae could not undergo DNA replication and no cell division was observed. The Dot/Icm secretion system was necessary for L. pneumophila to prevent the eukaryotic proliferation. The absence of proliferation was associated with altered amoebal morphology and with a decrease of mRNA transcript levels of CDC2b, a putative regulator of the A. castellanii cell cycle. Complementation of CDC28-deficient Saccharomyces cerevisiae by the CDC2b cDNA was sufficient to restore proliferation of CDC28-deficient S. cerevisiae and suggests for the first time that CDC2b from A. castellanii could be functional and a bona fide cyclin-dependent kinase. Hence, our results reveal that L. pneumophila impairs proliferation of A. castellanii and this effect could involve the cell cycle protein CDC2b. PMID:27805070

  15. Glycogen phosphorylase in Acanthamoeba spp.: determining the role of the enzyme during the encystment process using RNA interference.

    PubMed

    Lorenzo-Morales, Jacob; Kliescikova, Jarmila; Martinez-Carretero, Enrique; De Pablos, Luis Miguel; Profotova, Bronislava; Nohynkova, Eva; Osuna, Antonio; Valladares, Basilio

    2008-03-01

    Acanthamoeba infections are difficult to treat due to often late diagnosis and the lack of effective and specific therapeutic agents. The most important reason for unsuccessful therapy seems to be the existence of a double-wall cyst stage that is highly resistant to the available treatments, causing reinfections. The major components of the Acanthamoeba cyst wall are acid-resistant proteins and cellulose. The latter has been reported to be the major component of the inner cyst wall. It has been demonstrated previously that glycogen is the main source of free glucose for the synthesis of cellulose in Acanthamoeba, partly as glycogen levels fall during the encystment process. In other lower eukaryotes (e.g., Dictyostelium discoideum), glycogen phosphorylase has been reported to be the main tool used for glycogen breakdown in order to maintain the free glucose levels during the encystment process. Therefore, it was hypothesized that the regulation of the key processes involved in the Acanthamoeba encystment may be similar to the previously reported regulation mechanisms in other lower eukaryotes. The catalytic domain of the glycogen phosphorylase was silenced using RNA interference methods, and the effect of this phenomenon was assessed by light and electron microscopy analyses, calcofluor staining, expression zymogram assays, and Northern and Western blot analyses of both small interfering RNA-treated and control cells. The present report establishes the role of glycogen phosphorylase during the encystment process of Acanthamoeba. Moreover, the obtained results demonstrate that the enzyme is required for cyst wall assembly, mainly for the formation of the cell wall inner layer.

  16. Glycogen Phosphorylase in Acanthamoeba spp.: Determining the Role of the Enzyme during the Encystment Process Using RNA Interference▿

    PubMed Central

    Lorenzo-Morales, Jacob; Kliescikova, Jarmila; Martinez-Carretero, Enrique; De Pablos, Luis Miguel; Profotova, Bronislava; Nohynkova, Eva; Osuna, Antonio; Valladares, Basilio

    2008-01-01

    Acanthamoeba infections are difficult to treat due to often late diagnosis and the lack of effective and specific therapeutic agents. The most important reason for unsuccessful therapy seems to be the existence of a double-wall cyst stage that is highly resistant to the available treatments, causing reinfections. The major components of the Acanthamoeba cyst wall are acid-resistant proteins and cellulose. The latter has been reported to be the major component of the inner cyst wall. It has been demonstrated previously that glycogen is the main source of free glucose for the synthesis of cellulose in Acanthamoeba, partly as glycogen levels fall during the encystment process. In other lower eukaryotes (e.g., Dictyostelium discoideum), glycogen phosphorylase has been reported to be the main tool used for glycogen breakdown in order to maintain the free glucose levels during the encystment process. Therefore, it was hypothesized that the regulation of the key processes involved in the Acanthamoeba encystment may be similar to the previously reported regulation mechanisms in other lower eukaryotes. The catalytic domain of the glycogen phosphorylase was silenced using RNA interference methods, and the effect of this phenomenon was assessed by light and electron microscopy analyses, calcofluor staining, expression zymogram assays, and Northern and Western blot analyses of both small interfering RNA-treated and control cells. The present report establishes the role of glycogen phosphorylase during the encystment process of Acanthamoeba. Moreover, the obtained results demonstrate that the enzyme is required for cyst wall assembly, mainly for the formation of the cell wall inner layer. PMID:18223117

  17. Acanthamoeba castellanii : growth on human cell layers reactivates attenuated properties after prolonged axenic culture

    PubMed Central

    Koehsler, Martina; Leitsch, David; Duchêne, Michael; Nagl, Markus; Walochnik, Julia

    2009-01-01

    The free-living, but potentially pathogenic, bacteriovorous amoebae of the genus Acanthamoeba can be easily grown axenically in a laboratory culture. This, however, often leads to considerable losses in virulence, and encystment capacity, and to changes in drug susceptibility. We evaluated potential options for a reactivation of a number of physiological properties, attenuated by prolonged axenic laboratory culture, including encystment potential, protease activity, heat resistance, growth rates and drug susceptibility against N-chlorotaurine (NCT). Toward this end, a strain that had been grown axenically for 10 years was repeatedly passaged on human HEp-2 cell monolayers or treated with 5′-azacytidine (AzaC), a methyltransferase inhibitor, and trichostatin A (TSA), a histone deacetylase inhibitor, in order to uplift epigenetic gene regulation. Culture on human cell monolayers resulted in significantly enhanced encystment potentials and protease activities, and higher susceptibility against NCT, whereas the resistance against heat shock was not altered. Treatment with AzaC/TSA resulted in increased encystment rates and protease activities, indicating the participation of epigenetic mechanisms. However, lowered resistances against heat shock indicate that possible stress responses to AzaC/TSA have to be taken into account. Repeated growth on human cell monolayers appears to be a potential method to reactivate attenuated characteristics in Acanthamoeba. PMID:19732153

  18. Bowman’s layer encystment in cases of persistent Acanthamoeba keratitis

    PubMed Central

    Yokogawa, Hideaki; Kobayashi, Akira; Yamazaki, Natsuko; Ishibashi, Yasuhisa; Oikawa, Yosaburo; Tokoro, Masaharu; Sugiyama, Kazuhisa

    2012-01-01

    Background The purpose of this study was to report Acanthamoeba encystment in Bowman’s layer in Japanese cases of persistent Acanthamoeba keratitis (AK). Methods Laser confocal microscopic images of the cornea were obtained in vivo from 18 consecutive eyes from 17 confirmed AK patients. Retrospectively, 14 cases treated over 4 months were categorized as a nonpersistent group and three cases that required prolonged therapy for more than 6 months were categorized as a persistent group. Clinical outcomes based on final best-corrected visual acuity were retrospectively analyzed, and selected confocal images were evaluated qualitatively for abnormal findings. Results The final best-corrected visual acuity was significantly lower (P < 0.01) for patients in the persistent group compared with that in the nonpersistent group. At the initial visit, in vivo confocal microscopy demonstrated Acanthamoeba cysts exclusively in the epithelial layer in both the nonpersistent group (80%) and the persistent group (100%). At a subsequent follow-up visit, numerous Acanthamoeba cysts were observed in the epithelial cell layer and in Bowman’s layer in all patients with persistent AK, but Acanthamoeba cysts were undetectable in all cases with nonpersistent AK tested. Conclusion Invasion of cysts into Bowman’s layer was characteristically observed in patients with persistence of AK. This finding suggests that invasion of Acanthamoeba cysts into Bowman’s layer may be a useful predictor for a persistent clinical course. PMID:22927735

  19. The effect of environmental and physiological conditions on excystation of Acanthamoeba castellanii belonging to the T4 genotype.

    PubMed

    Lakhundi, Sahreena; Khan, Naveed Ahmed; Siddiqui, Ruqaiyyah

    2014-08-01

    Excystation in Acanthamoeba is an important property for the onset of infection as well as infection recurrence, post-treatment. The overall aim of this study was to determine the effects of several environmental and physiological parameters on excystation in Acanthamoeba castellanii belonging to the T4 genotype. Cysts were prepared by inoculating A. castellanii trophozoites on non-nutrient agar plates for up to 2 weeks. To determine the effects of various conditions on excystation, A. castellanii cysts were inoculated in growth medium i.e. PYG and incubated at varying temperatures (4-40 °C), various pHs (4-9), artificial light/dark cycles and 5% of CO2. Optimum excystation was observed when cysts were incubated at 30 °C in growth medium at neutral pH. Extremes of temperature and pH reduced excystation, while light/dark cycles had no effect on excystation of A. castellanii. On the other hand, 5% of CO2 enhanced excystation and growth of excysting amoebae. To determine the effect of serum on A. castellanii excystation, assays were performed in the presence of varying concentrations of heat-inactivated foetal bovine serum (FBS) (5-100%). The results revealed that FBS promoted excystation. The involvement of G proteins in excystation was also determined. Using propranolol hydrochloride, a G protein inhibitor, the results revealed that G proteins play a role in A. castellanii differentiation. Furthermore, organic solvents (methanol/ethanol) completely blocked excystation. None of the aforementioned conditions had any effect on the viability of A. castellanii. A complete understanding of excystation in A. castellanii will be of value to counter infection recurrence.

  20. Effects of pesticides, polychlorinated biphenyls and metals on the growth and reproduction of Acanthamoeba castellanii

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Prescott, L.M.; Kubovec, M.K.; Tryggestad, D.

    1977-07-01

    The effects of pollutants (pesticides, PCB and metals) were studied in the free-living amoeba, Acanthamoeba castellanii. Eight pesticides were used--the insecticides dieldrin, aldrin and sevin, and the herbicides linuron, stam F-34, IPC, atrazine and simazine. It was shown that the sensitivity of A. castellanii to pesticides varied greatly. The population growth was inhibited by linuron, stam F-34, IPC, sevin and atrazine at a level of 10 mg/l. The polychlorinated biphenyl, Arochor 1254, had no significant effect at a concentration of 0.01 mg/l (10 ppb). The studies with metal ions showed that A. castellanii was unaffected by moderately high levels ofmore » Cu and Zn, but was sensitive to the presence of Pb and mercuric ions.« less

  1. Survival of taylorellae in the environmental amoeba Acanthamoeba castellanii

    PubMed Central

    2014-01-01

    Background Taylorella equigenitalis is the causative agent of contagious equine metritis, a sexually-transmitted infection of Equidae characterised in infected mares by abundant mucopurulent vaginal discharge and a variable degree of vaginitis, cervicitis or endometritis, usually resulting in temporary infertility. The second species of the Taylorella genus, Taylorella asinigenitalis, is considered non-pathogenic, although mares experimentally infected with this bacterium can develop clinical signs of endometritis. To date, little is understood about the basic molecular virulence and persistence mechanisms employed by the Taylorella species. To clarify these points, we investigated whether the host-pathogen interaction model Acanthamoeba castellanii was a suitable model for studying taylorellae. Results We herein demonstrate that both species of the Taylorella genus are internalised by a mechanism involving the phagocytic capacity of the amoeba and are able to survive for at least one week inside the amoeba. During this one-week incubation period, taylorellae concentrations remain strikingly constant and no overt toxicity to amoeba cells was observed. Conclusions This study provides the first evidence of the capacity of taylorellae to survive in a natural environment other than the mammalian genital tract, and shows that the alternative infection model, A. castellanii, constitutes a relevant alternative system to assess host-pathogen interactions of taylorellae. The survival of taylorellae inside the potential environmental reservoir A. castellanii brings new insight, fostering a broader understanding of taylorellae biology and its potential natural ecological niche. PMID:24641089

  2. Acanthamoeba castellanii is not be an adequate model to study human adenovirus interactions with macrophagic cells

    PubMed Central

    Cateau, Estelle; Leveque, Nicolas; Kaaki, Sihem; Beby-Defaux, Agnès; Rodier, Marie-Hélène

    2017-01-01

    Free living amoebae (FLA) including Acanthamoeba castellanii, are protozoa that feed on different microorganisms including viruses. These microorganisms show remarkable similarities with macrophages in cellular structures, physiology or ability to phagocyte preys, and some authors have therefore wondered whether Acanthamoeba and macrophages are evolutionary related. It has been considered that this amoeba may be an in vitro model to investigate relationships between pathogens and macrophagic cells. So, we intended in this study to compare the interactions between a human adenovirus strain and A. castellanii or THP-1 macrophagic cells. The results of molecular and microscopy techniques following co-cultures experiments have shown that the presence of the adenovirus decreased the viability of macrophages, while it has no effect on amoebic viability. On another hand, the viral replication occurred only in macrophages. These results showed that this amoebal model is not relevant to explore the relationships between adenoviruses and macrophages in in vitro experiments. PMID:28591183

  3. The type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba.

    PubMed

    Siddiqui, Ruqaiyyah; Malik, Huma; Sagheer, Mehwish; Jung, Suk-Yul; Khan, Naveed Ahmed

    2011-08-01

    The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P<0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. The association of TIF-IA and polymerase I mediates promoter recruitment and regulation of ribosomal RNA transcription in Acanthamoeba castellanii.

    PubMed

    Gogain, Joseph C; Paule, Marvin R

    2005-01-01

    Large amounts of energy are expended for the construction of the ribosome during both transcription and processing, so it is of utmost importance for the cell to efficiently regulate ribosome production. Understanding how this regulation occurs will provide important insights into cellular growth control and into the coordination of gene expression mediated by all three transcription systems. Ribosomal RNA (rRNA) transcription rates closely parallel the need for protein synthesis; as a cell approaches stationary phase or encounters conditions that negatively affect either growth rate or protein synthesis, rRNA transcription is decreased. In eukaryotes, the interaction of RNA polymerase I (pol I) with the essential transcription initiation factor IA (TIF-IA) has been implicated in this downregulation of transcription. In agreement with the first observation that rRNA transcription is regulated by altering recruitment of pol I to the promoter in Acanthamoeba castellanii, we show here that pol I and an 80-kDa homologue of TIF-IA are found tightly associated in pol I fractions competent for specific transcription. Disruption of the pol I-TIF-IA complex is mediated by a specific dephosphorylation of either pol I or TIF-IA. Phosphatase treatment of TIF-IA-containing A. castellanii pol I fractions results in a downregulation of both transcriptional activity and promoter binding, reminiscent of the inactive pol I fractions purified from encysted cells. The fraction of pol I competent for promoter recruitment is enriched in TIF-IA relative to that not bound by immobilized promoter DNA. This downregulation coincides with an altered electrophoretic mobility of TIF-IA, suggesting at least it is phosphorylated.

  5. siRNA-loaded liposomes: Inhibition of encystment of Acanthamoeba and toxicity on the eye surface.

    PubMed

    Faber, Kathrin; Zorzi, Giovanni K; Brazil, Nathalya T; Rott, Marilise B; Teixeira, Helder F

    2017-09-01

    Current treatments for Acanthamoeba keratitis are unspecific. Because of the presence of the resilient cyst form of the parasite, the infection is persistent. Silencing the key protein of cyst formation, glycogen phosphorylase, has shown potential for reducing encystment processes of the Acanthamoeba trophozoite. However, a suitable carrier to protect and deliver siRNA sequences is still needed. DOPE:DSPE-PEG liposomes were prepared by three different techniques and used to associate a therapeutic siRNA sequence. Liposomes prepared by film hydration followed by membrane extrusion were considered the most adequate ones with average size of 250 nm and zeta potential of +45 mV, being able to associate siRNA for at least 24 hr in culture medium. siRNA-liposomes could inhibit up to 66% of the encystment process. Cell viability studies demonstrated MTT reduction capacity higher than 80% after 3 hr incubation with this formulation. After 24 hr of incubation, LDH activity ranged for both the formulations from around 4% to 40%. In vivo tolerance studies in mice showed no macroscopic alteration in the eye structures up to 24 hr after eight administrations during 1 day. Histological studies showed regular tissue architecture without any morphological alteration. Overall, these results suggest that the formulations developed are a promising new strategy for the treatment of ocular keratitis caused by Acanthamoeba spp. © 2017 John Wiley & Sons A/S.

  6. Cellular Response of the Amoeba Acanthamoeba castellanii to Chlorine, Chlorine Dioxide, and Monochloramine Treatments ▿

    PubMed Central

    Mogoa, Emerancienne; Bodet, Charles; Morel, Franck; Rodier, Marie-Hélène; Legube, Bernard; Héchard, Yann

    2011-01-01

    Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii. PMID:21602398

  7. Hydroxynonenal-stimulated activity of the uncoupling protein in Acanthamoeba castellanii mitochondria under phosphorylating conditions.

    PubMed

    Woyda-Ploszczyca, Andrzej; Jarmuszkiewicz, Wieslawa

    2013-05-01

    The influence of 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the amoeba Acanthamoeba castellanii uncoupling protein (AcUCP) in isolated phosphorylating mitochondria was studied. Under phosphorylating conditions, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. The HNE-induced proton leak decreased the yield of oxidative phosphorylation in an HNE concentration-dependent manner. The present study describes how the contributions of ATP synthase and HNE-induced AcUCP in phosphorylating respiration vary when the rate of succinate oxidation is decreased by limiting succinate uptake or inhibiting complex III activity within the range of a constant membrane potential. In phosphorylating mitochondria, at a given HNE concentration (100 μM), the efficiency of AcUCP in mitochondrial uncoupling increased as the respiratory rate decreased because the AcUCP contribution remained constant while the ATP synthase contribution decreased with the respiratory rate. HNE-induced uncoupling can be inhibited by GTP only when ubiquinone is sufficiently oxidized, indicating that in phosphorylating A. castellanii mitochondria, the sensitivity of AcUCP activity to GTP depends on the redox state of the membranous ubiquinone.

  8. Effects of harmful cyanobacteria on the freshwater pathogenic free-living amoeba Acanthamoeba castellanii.

    PubMed

    Urrutia-Cordero, Pablo; Agha, Ramsy; Cirés, Samuel; Lezcano, María Ángeles; Sánchez-Contreras, María; Waara, Karl-Otto; Utkilen, Hans; Quesada, Antonio

    2013-04-15

    Grazing is a major regulating factor in cyanobacterial population dynamics and, subsequently, considerable effort has been spent on investigating the effects of cyanotoxins on major metazoan grazers. However, protozoan grazers such as free-living amoebae can also feed efficiently on cyanobacteria, while simultaneously posing a major threat for public health as parasites of humans and potential reservoirs of opportunistic pathogens. In this study, we conducted several experiments in which the freshwater amoeba Acanthamoeba castellanii was exposed to pure microcystin-LR (MC-LR) and six cyanobacterial strains, three MC-producing strains (MC-LR, MC-RR, MC-YR, MC-WR, [Dha7] MC-RR) and three strains containing other oligopeptides such as anabaenopeptins and cyanopeptolins. Although the exposure to high concentrations of pure MC-LR yielded no effects on amoeba, all MC-producing strains inflicted high mortality rates on amoeba populations, suggesting that toxic effects must be mediated through the ingestion of toxic cells. Interestingly, an anabaenopeptin-producing strain caused the greatest inhibition of amoeba growth, indicating that toxic bioactive compounds other than MCs are of great importance for amoebae grazers. Confocal scanning microscopy revealed different alterations in amoeba cytoskeleton integrity and as such, the observed declines in amoeba densities could have indeed been caused via a cascade of cellular events primarily triggered by oligopeptides with protein-phosphatase inhibition capabilities such as MCs or anabaenopeptins. Moreover, inducible-defense mechanisms such as the egestion of toxic, MC-producing cyanobacterial cells and the increase of resting stages (encystation) in amoebae co-cultivated with all cyanobacterial strains were observed in our experiments. Consequently, cyanobacterial strains showed different susceptibilities to amoeba grazing which were possibly influenced by the potentiality of their toxic secondary metabolites. Hence, this

  9. Silicone hydrogel contact lenses surface promote Acanthamoeba castellanii trophozoites adherence: qualitative and quantitative analysis.

    PubMed

    Omaña-Molina, Maritza A; González-Robles, Arturo; Salazar-Villatoro, Lizbeth; Bernal-Escobar, Alexander; Durán-Díaz, Angel; Méndez-Cruz, Adolfo René; Martínez-Palomo, Adolfo

    2014-05-01

    To describe the adhesion properties of Acanthamoeba castellanii trophozoites to silicone hydrogel contact lenses of first generation (lotrafilcon A), second generation (galyfilcon A), and third generation (comfilcon A) and correlate the results with their specific surface characteristics, time of interaction, and suspension media. Qualitative and quantitative assessments of the adhesion of 200 trophozoites of A. castellanii on contact lenses in culture medium (Bacto Casitone) and isotonic saline (IS) at different time points (15 minutes and 6 hours) were determined. By scanning electron microscopy, A. castellanii trophozoites were observed firmly adhered to the surface of hydrogel lenses after 15 minutes of interaction. The surface of lotrafilcon A lenses on which amoebae adhere better (16.4±10.2 amoebae/lens section) is rough and folded, which increases the contact surface with trophozoites, allowing acanthopodia to attach firmly. Contrarily, galyfilcon A lenses have a smoother surface, and lower numbers of amoebae were observed adhered to these lenses (4.7±2.9 amoebae/lens section). Even fewer amoebae adhered to the smoother surface of the comfilcon A lens (2.2±1.7 amoebae/lens section). Trophozoites showed similar behavior in both Bacto Casitone medium and IS. A rough surface may contribute to better adhesion of amoebae to silicone hydrogel lenses. Although a reduced numbers of trophozoites adhered to smooth lenses, trophozoites are a risk factor for amoebic keratitis. Isotonic saline facilitated trophozoite survival, suggesting that homemade saline solutions may contribute to the persistence of trophozoites, especially when there is no proper hygiene regimen used with the contact lens cases.

  10. Occurrence of Acanthamoeba genotypes in Central West Malaysian environments.

    PubMed

    Basher, Mohamad Hafiz Abdul; Ithoi, Init; Mahmud, Rohela; Abdulsalam, Awatif Mohamed; Foead, Agus Iwan; Dawaki, Salwa; Atroosh, Wahib Mohammed Mohsen; Nissapatorn, Veeranoot; Abdullah, Wan Omar

    2018-02-01

    Acanthamoeba species are ubiquitous free-living protozoa that can be found worldwide. Occasionally, it can become parasitic and the causative agent of acanthamoebic keratitis (AK) and Granulomatous Amoebic Encephalitis (GAE) in man. A total of 160 environmental samples and 225 naturally-infected animal corneal swabs were collected for Acanthamoeba cultivation. Acanthamoeba was found to be high in samples collected from environments (85%, 136/160) compared to infected animal corneas (24.89%, 56/225) by microscopic examination. Analysis of nucleotide sequence of 18S rRNA gene of all the 192 cultivable Acanthamoeba isolates revealed 4 genotypes (T3, T4. T5 and T15) with T4 as the most prevalent (69.27%, 133/192) followed by T5 (20.31%), T15 (9.90%) and T3 (0.52%). Genotype T4 was from the strain of A. castellanii U07401 (44.27%), A. castellanii U07409 (20.83%) and A. polyphagaAY026243 (4.17%), but interestingly, only A. castellanii U07401 was detected in naturally infected corneal samples. In environmental samples, T4 was commonly detected in all samples including dry soil, dust, wet debris, wet soil and water. Among the T4, A. castellanii (U07409) strains were detected high occurrence in dry (45%) followed by aquatic (32.50%) and moist (22.50%) samples but however A. castellanii (U07401) strains were dominant in dry samples of soil and dust (93.10%). Subsequently, genotype T5 of A. lenticulata (U94741) strains were dominant in samples collected from aquatic environments (58.97%). In summary, A. castellanii (U07401) strains were found dominant in both environmental and corneal swab samples. Therefore, these strains are possibly the most virulent and dry soil or dusts are the most possible source of Acanthamoeba infection in cats and dogs corneas. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

    PubMed

    Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

    1995-11-11

    The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF.

  12. Acanthamoeba migration in an electric field.

    PubMed

    Rudell, Jolene Chang; Gao, Jing; Sun, Yuxin; Sun, Yaohui; Chodosh, James; Schwab, Ivan; Zhao, Min

    2013-06-21

    We investigated the in vitro response of Acanthamoeba trophozoites to electric fields (EFs). Acanthamoeba castellanii were exposed to varying strengths of an EF. During EF exposure, cell migration was monitored using an inverted microscope equipped with a CCD camera and the SimplePCI 5.3 imaging system to capture time-lapse images. The migration of A. castellanii trophozoites was analyzed and quantified with ImageJ software. For analysis of cell migration in a three-dimensional culture system, Acanthamoeba trophozoites were cultured in agar, exposed to an EF, digitally video recorded, and analyzed at various Z focal planes. Acanthamoeba trophozoites move at random in the absence of an EF, but move directionally in response to an EF. Directedness in the absence of an EF is 0.08 ± 0.01, while in 1200 mV/mm EF, directedness is significantly higher at -0.65 ± 0.01 (P < 0.001). We find that the trophozoite migration response is voltage-dependent, with higher directionality with higher voltage application. Acanthamoeba move directionally in a three-dimensional (3D) agar system as well when exposed to an EF. Acanthamoeba trophozoites move directionally in response to an EF in a two-dimensional and 3D culture system. Acanthamoeba trophozoite migration is also voltage-dependent, with increased directionality with increasing voltage. This may provide new treatment modalities for Acanthamoeba keratitis.

  13. Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

    PubMed

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Ateeq, Muhammad; Raza Shah, Muhammad; Kulsoom, Huma; Khan, Naveed Ahmed

    2015-01-01

    Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

  14. Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

    PubMed Central

    Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

    1995-01-01

    The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF. Images PMID:7501455

  15. Resistance of Acanthamoeba cysts to disinfection in multiple contact lens solutions.

    PubMed

    Johnston, Stephanie P; Sriram, Rama; Qvarnstrom, Yvonne; Roy, Sharon; Verani, Jennifer; Yoder, Jonathan; Lorick, Suchita; Roberts, Jacquelin; Beach, Michael J; Visvesvara, Govinda

    2009-07-01

    Acanthamoebae are free-living amoebae found in the environment, including soil, freshwater, brackish water, seawater, hot tubs, and Jacuzzis. Acanthamoeba species can cause keratitis, a painful vision-threatening infection of the cornea, and fatal granulomatous encephalitis in humans. More than 20 species of Acanthamoeba belonging to morphological groups I, II, and III distributed in 15 genotypes have been described. Among these, Acanthamoeba castellanii, A. polyphaga, and A. hatchetti are frequently identified as causing Acanthamoeba keratitis (AK). Improper contact lens care and contact with nonsterile water while wearing contact lenses are known risk factors for AK. During a recent multistate outbreak, AK was found to be associated with the use of Advanced Medical Optics Complete MoisturePlus multipurpose contact lens solution, which was hypothesized to have had insufficient anti-Acanthamoeba activity. As part of the investigation of that outbreak, we compared the efficacies of 11 different contact lens solutions against cysts of A. castellanii, A. polyphaga, and A. hatchetti (the isolates of all species were genotype T4), which were isolated in 2007 from specimens obtained during the outbreak investigation. The data, generated with A. castellanii, A. polyphaga, and A. hatchetti cysts, suggest that the two contact lens solutions containing hydrogen peroxide were the only solutions that showed any disinfection ability, with 0% and 66% growth, respectively, being detected with A. castellanii and 0% and 33% growth, respectively, being detected with A. polyphaga. There was no statistically significant difference in disinfection efficacy between the 11 solutions for A. hatchetti.

  16. The effect of viroid infection of citrus trees on the amoebicidal activity of 'Maltese half-blood' (Citrus sinensis) against trophozoite stage of Acanthamoeba castellanii Neff.

    PubMed

    Zouaghi, Ghaya; Najar, Asma; Chiboub, Olfa; Sifaoui, Ines; Abderrabba, Manef; Lorenzo Morales, Jacob

    2017-12-01

    In order to promote a local Tunisian product, this study was designed to examine, for the first time, the anti-Acanthamoeba activity (Acanthamoeba castellanii Neff) of the essential oils of Tunisian Citrus sinensis peels (Maltese half-blood) and the effect of viroid plant infection on this activity. To do so, three samples of peels' essential oils were studied: from a healthy plant (Control), a plant inoculated with Citrus exocortis viroid (CEVd) and one inoculated with hot stunt cachexia viroid (HSVd). The samples were extracted by hydrodistillation from dried peels and characterized by GC-MS. Limonene was the major component with a percentage ranging from 90.76 to 93.34% for (CEVd) sample and (Control), respectively. Anti-Acanthamoeba activity of the tested oils was determined by the Alamar Blue ® assay. Primary results showed a strong potential anti-Acanthamoeba activity with an IC 50 ranging from 36.6 to 54.58 μg/ml for (HSVd) and (CEVd) samples, respectively. In terms of the effect of viroid infection, a strong positive correlation was observed between different chemical classes and anti-Acanthamoeba activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Compositional complexity of the mitochondrial proteome of a unicellular eukaryote (Acanthamoeba castellanii, supergroup Amoebozoa) rivals that of animals, fungi, and plants.

    PubMed

    Gawryluk, Ryan M R; Chisholm, Kenneth A; Pinto, Devanand M; Gray, Michael W

    2014-09-23

    We present a combined proteomic and bioinformatic investigation of mitochondrial proteins from the amoeboid protist Acanthamoeba castellanii, the first such comprehensive investigation in a free-living member of the supergroup Amoebozoa. This protist was chosen both for its phylogenetic position (as a sister to animals and fungi) and its ecological ubiquity and physiological flexibility. We report 1033 A. castellanii mitochondrial protein sequences, 709 supported by mass spectrometry data (676 nucleus-encoded and 33 mitochondrion-encoded), including two previously unannotated mtDNA-encoded proteins, which we identify as highly divergent mitochondrial ribosomal proteins. Other notable findings include duplicate proteins for all of the enzymes of the tricarboxylic acid (TCA) cycle-which, along with the identification of a mitochondrial malate synthase-isocitrate lyase fusion protein, suggests the interesting possibility that the glyoxylate cycle operates in A. castellanii mitochondria. Additionally, the A. castellanii genome encodes an unusually high number (at least 29) of mitochondrion-targeted pentatricopeptide repeat (PPR) proteins, organellar RNA metabolism factors in other organisms. We discuss several key mitochondrial pathways, including DNA replication, transcription and translation, protein degradation, protein import and Fe-S cluster biosynthesis, highlighting similarities and differences in these pathways in other eukaryotes. In compositional and functional complexity, the mitochondrial proteome of A. castellanii rivals that of multicellular eukaryotes. Comprehensive proteomic surveys of mitochondria have been undertaken in a limited number of predominantly multicellular eukaryotes. This phylogenetically narrow perspective constrains and biases our insights into mitochondrial function and evolution, as it neglects protists, which account for most of the evolutionary and functional diversity within eukaryotes. We report here the first comprehensive

  18. Exposure to Synthetic Gray Water Inhibits Amoeba Encystation and Alters Expression of Legionella pneumophila Virulence Genes

    PubMed Central

    Lu, Jingrang; Ashbolt, Nicholas J.

    2014-01-01

    Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems. PMID:25381242

  19. Exposure to synthetic gray water inhibits amoeba encystation and alters expression of Legionella pneumophila virulence genes.

    PubMed

    Buse, Helen Y; Lu, Jingrang; Ashbolt, Nicholas J

    2015-01-01

    Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Interactions between Human Norovirus Surrogates and Acanthamoeba spp.

    PubMed Central

    Hsueh, Tun-Yun

    2015-01-01

    Human noroviruses (HuNoVs) are the most common cause of food-borne disease outbreaks, as well as virus-related waterborne disease outbreaks in the United States. Here, we hypothesize that common free-living amoebae (FLA)—ubiquitous in the environment, known to interact with pathogens, and frequently isolated from water and fresh produce—could potentially act as reservoirs of HuNoV and facilitate the environmental transmission of HuNoVs. To investigate FLA as reservoirs for HuNoV, the interactions between two Acanthamoeba species, A. castellanii and A. polyphaga, as well as two HuNoV surrogates, murine norovirus type 1 (MNV-1) and feline calicivirus (FCV), were evaluated. The results showed that after 1 h of amoeba-virus incubation at 25°C, 490 and 337 PFU of MNV-1/ml were recovered from A. castellanii and A. polyphaga, respectively, while only few or no FCVs were detected. In addition, prolonged interaction of MNV-1 with amoebae was investigated for a period of 8 days, and MNV-1 was demonstrated to remain stable at around 200 PFU/ml from day 2 to day 8 after virus inoculation in A. castellanii. Moreover, after a complete amoeba life cycle (i.e., encystment and excystment), infectious viruses could still be detected. To determine the location of virus associated with amoebae, immunofluorescence experiments were performed and showed MNV-1 transitioning from the amoeba surface to inside the amoeba over a 24-h period. These results are significant to the understanding of how HuNoVs may interact with other microorganisms in the environment in order to aid in its persistence and survival, as well as potential transmission in water and to vulnerable food products such as fresh produce. PMID:25841006

  1. Evidence for a Hydrogenosomal-Type Anaerobic ATP Generation Pathway in Acanthamoeba castellanii

    PubMed Central

    Gray, Michael W.; Roger, Andrew J.

    2013-01-01

    Diverse, distantly-related eukaryotic lineages have adapted to low-oxygen environments, and possess mitochondrion-related organelles that have lost the capacity to generate adenosine triphosphate (ATP) through oxidative phosphorylation. A subset of these organelles, hydrogenosomes, has acquired a set of characteristic ATP generation enzymes commonly found in anaerobic bacteria. The recipient of these enzymes could not have survived prior to their acquisition had it not still possessed the electron transport chain present in the ancestral mitochondrion. In the divergence of modern hydrogenosomes from mitochondria, a transitional organelle must therefore have existed that possessed both an electron transport chain and an anaerobic ATP generation pathway. Here, we report a modern analog of this organelle in the habitually aerobic opportunistic pathogen, Acanthamoeba castellanii. This organism possesses a complete set of enzymes comprising a hydrogenosome-like ATP generation pathway, each of which is predicted to be targeted to mitochondria. We have experimentally confirmed the mitochondrial localizations of key components of this pathway using tandem mass spectrometry. This evidence is the first supported by localization and proteome data of a mitochondrion possessing both an electron transport chain and hydrogenosome-like energy metabolism enzymes. Our work provides insight into the first steps that might have occurred in the course of the emergence of modern hydrogenosomes. PMID:24086244

  2. The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum.

    PubMed

    Wojtkowska, Małgorzata; Buczek, Dorota; Stobienia, Olgierd; Karachitos, Andonis; Antoniewicz, Monika; Slocinska, Małgorzata; Makałowski, Wojciech; Kmita, Hanna

    2015-07-01

    Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

  3. Statins and Voriconazole Induce Programmed Cell Death in Acanthamoeba castellanii

    PubMed Central

    López-Arencibia, Atteneri; Sifaoui, Ines; Reyes-Batlle, María; Valladares, Basilio; Martínez-Carretero, Enrique; Piñero, José E.; Maciver, Sutherland K.; Lorenzo-Morales, Jacob

    2015-01-01

    Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a life-threatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba. Here, we study the processes of programmed cell death in Acanthamoeba, induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC50s) and IC90s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with quantitative and qualitative techniques. Also, the changes related to phosphatidylserine exposure on the external cell membrane and cell permeability were studied. Finally, because caspases are key to PCD pathways, caspase activity was evaluated in Acanthamoeba. All the drugs assayed in this study induced PCD in Acanthamoeba. To the best of our knowledge, this is the first study where PCD induced by drugs is described quantitatively and qualitatively in Acanthamoeba. PMID:25733513

  4. Quantitation by flow microfluorometry of total cellular DNA in Acanthamoeba

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Coulson, P.B.; Tyndall, R.

    1978-01-01

    The DNA content of five speciea of Acanthamoeba was determined by flow microfluorometry. Acanthamoeba castellanii (AC-30), acanthamoeba polyphaga (APG and P-23), acanthamoeba rhysodes, acanthamoeba culbertsoni (A-1), and acanthamoeba royreba were grown in a casitone based medium 24 to 48 hr. The trophozoites were harvested, fixed in 70% ethanol (acidified), pretreated with RNase, stained with propidium diiodide, and evaluated for DNA-bound fluorescence. All species tested had DNA values between 2.0 to 5.0 pg/cell. These results placed DNA/cell values of Acanthamoeba slightly lower than DNA/cell values of other eucaryotic cells and much lower than Amoeba proteus values. These results indicate that FMFmore » may be a useful adjunct in distinguishing Acanthamoeba cells from either eucaryotic cells or some other amoeba. However, differences in DNA/cell between species of Acanthamoeba are small and would not be useful in identification of species.« less

  5. Photochemotherapeutic Strategy against Acanthamoeba Infections

    PubMed Central

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Anwar, Ayaz; Shah, Muhammad Raza; Khoja, Shahrukh

    2015-01-01

    Acanthamoeba is a protist pathogen that can cause serious human infections, including blinding keratitis and a granulomatous amoebic encephalitis that almost always results in death. The current treatment for these infections includes a mixture of drugs, and even then, a recurrence can occur. Photochemotherapy has shown promise in the treatment of Acanthamoeba infections; however, the selective targeting of pathogenic Acanthamoeba has remained a major concern. The mannose-binding protein is an important adhesin expressed on the surface membranes of pathogenic Acanthamoeba organisms. To specifically target Acanthamoeba, the overall aim of this study was to synthesize a photosensitizing compound (porphyrin) conjugated with mannose and test its efficacy in vitro. The synthesis of mannose-conjugated porphyrin was achieved by mixing benzaldehyde and pyrrole, yielding tetraphenylporphyrin. Tetraphenylporphyrin was then converted into mono-nitrophenylporphyrin by selectively nitrating the para position of the phenyl rings, as confirmed by nuclear magnetic resonance (NMR) spectroscopy. The mono-nitrophenylporphyrin was reduced to mono-aminophenylporphyrin in the presence of tin dichloride and confirmed by a peak at m/z 629. Finally, mono-aminoporphyrin was conjugated with mannose, resulting in the formation of an imine bond. Mannose-conjugated porphyrin was confirmed through spectroscopic analysis and showed that it absorbed light of wavelengths ranging from 425 to 475 nm. To determine the antiacanthamoebic effects of the derived product, amoebae were incubated with mannose-conjugated porphyrin for 1 h and washed 3 times to remove extracellular compound. Next, the amoebae were exposed to light of the appropriate wavelength for 1 h. The results revealed that mannose-conjugated porphyrin produced potent trophicidal effects and blocked excystation. In contrast, Acanthamoeba castellanii incubated with mannose alone and porphyrin alone did not exhibit an antiamoebic effect

  6. Chemiluminescence of Acanthamoeba castellanii.

    PubMed Central

    Lloyd, D; Boveris, A; Reiter, R; Filipkowski, M; Chance, B

    1979-01-01

    1. Chemiluminescence of Acanthomoeba castellanii in the presence of O2 was of similar intensity in organisms harvested early or late during exponential growth [when cyanide (1 mM) stimulates or inhibits respiration respectively]. 2. Cyanide (up to 1.5 mM) stimulated photoemission in both types of organism by 250--300 photons/s per 10(7) cells above the value observed under aerobic conditions. 3. 'Dibromothymoquinone' (2,5-dibromo-6-isopropyl-3-methyl-p-benzoquinone) (up to 80 microM) further increased chemiluminescence. 4. Similar responses were also demonstrated in whole homogenates and in subcellular fractions; 36% of the chemiluminescence was provided by a fraction sedimenting at 100000g-min, and 20% in that fraction that was non-sedimentable at 200000g-min. 5. Mitochondrial substrates (succinate, 2-oxoglutarate, NADH) in the presence or absence of ADP and Pi or peroxisomal substrates (glycollate, urate or ethanol) gave no increases in light emission by whole homogenates or in any of the fractions. 6. It is suggested that reactions responsible for production of chemiluminescence are those primarily producing superoxide anions and leading to lipid peroxidation and singlet-oxygen formation. Photoemission enhancement and superoxide dismutase inhibition showed similar cyanide concentration-dependencies. PMID:534514

  7. Sampling gene diversity across the supergroup Amoebozoa: large EST data sets from Acanthamoeba castellanii, Hartmannella vermiformis, Physarum polycephalum, Hyperamoeba dachnaya and Hyperamoeba sp.

    PubMed

    Watkins, Russell F; Gray, Michael W

    2008-04-01

    From comparative analysis of EST data for five taxa within the eukaryotic supergroup Amoebozoa, including two free-living amoebae (Acanthamoeba castellanii, Hartmannella vermiformis) and three slime molds (Physarum polycephalum, Hyperamoeba dachnaya and Hyperamoeba sp.), we obtained new broad-range perspectives on the evolution and biosynthetic capacity of this assemblage. Together with genome sequences for the amoebozoans Dictyostelium discoideum and Entamoeba histolytica, and including partial genome sequence available for A. castellanii, we used the EST data to identify genes that appear to be exclusive to the supergroup, and to specific clades therein. Many of these genes are likely involved in cell-cell communication or differentiation. In examining on a broad scale a number of characters that previously have been considered in simpler cross-species comparisons, typically between Dictyostelium and Entamoeba, we find that Amoebozoa as a whole exhibits striking variation in the number and distribution of biosynthetic pathways, for example, ones for certain critical stress-response molecules, including trehalose and mannitol. Finally, we report additional compelling cases of lateral gene transfer within Amoebozoa, further emphasizing that although this process has influenced genome evolution in all examined amoebozoan taxa, it has done so to a variable extent.

  8. Detection of glycoproteins in the Acanthamoeba plasma membrane

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Paatero, G.I.L.; Gahmberg, C.G.

    1988-11-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presencemore » of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.« less

  9. The capsule plays an important role in Escherichia coli K1 interactions with Acanthamoeba.

    PubMed

    Jung, Suk-Yul; Matin, Abdul; Kim, Kwang Sik; Khan, Naveed Ahmed

    2007-03-01

    Escherichia coli K1 is shown to bind to, associate with, invade and survive inside Acanthamoeba, but the precise mechanisms associated with these events are unclear. We have previously shown that outer membrane protein A and lipopolysaccharide are critical bacterial determinants involved in E. coli K1 interactions with Acanthamoeba. Using an isogenic K1 capsule-deletion mutant (lacking the neuDB genes cluster that is necessary for the production of cytoplasmic precursors to the exopolysaccharide capsule), we observed that the capsule modulates and enhances E. coli K1 association and survival inside Acanthamoeba. The capsule-deletion mutant exhibited significantly reduced association compared with the wild type strain, E44. Similarly, the K1 capsule-deletion mutant exhibited limited ability for invasion/uptake by and survival inside Acanthamoeba. Next, we determined whether E. coli K1 survive inside Acanthamoeba during the encystment process and that viable bacteria can be isolated from the mature cysts. Using encystment assays, our findings revealed that E. coli K1, but not its capsule-deletion mutant, exhibit survival inside Acanthamoeba cysts. We believe this is the first demonstration that the K1 capsule plays an important role in E. coli K1 interactions with Acanthamoeba.

  10. Composition of the mitochondrial electron transport chain in acanthamoeba castellanii: structural and evolutionary insights.

    PubMed

    Gawryluk, Ryan M R; Chisholm, Kenneth A; Pinto, Devanand M; Gray, Michael W

    2012-11-01

    The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of 'supernumerary' subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits. © 2012 Elsevier B.V. All rights reserved.

  11. The use of dimethyl sulfoxide in contact lens disinfectants is a potential preventative strategy against contracting Acanthamoeba keratitis.

    PubMed

    Siddiqui, Ruqaiyyah; Aqeel, Yousuf; Khan, Naveed Ahmed

    2016-10-01

    Acanthamoeba castellanii is the causative agent of blinding keratitis. Though reported in non-contact lens wearers, it is most frequently associated with improper use of contact lens. For contact lens wearers, amoebae attachment to the lens is a critical first step, followed by amoebae binding to the corneal epithelial cells during extended lens wear. Acanthamoeba attachment to surfaces (biological or inert) and migration is an active process and occurs during the trophozoite stage. Thus retaining amoebae in the cyst stage (dormant form) offers an added preventative measure in impeding parasite traversal from the contact lens onto the cornea. Here, we showed that as low as 3% DMSO, abolished A. castellanii excystation. Based on the findings, it is proposed that DMSO should be included in the contact lens disinfectants as an added preventative strategy against contracting Acanthamoeba keratitis. Copyright © 2016 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  12. Failure of chemotherapy in the first reported cases of Acanthamoeba keratitis in Pakistan

    PubMed Central

    Siddiqui, Ruqaiyyah; Chaudhry, Tanveer; Lakhundi, Sahreena; Ahmad, Khabir; Khan, Naveed Ahmed

    2014-01-01

    Acanthamoeba keratitis is a painful and progressive infection of the cornea that can result in loss of vision. Here, for the first time in Pakistan, we report two cases of Acanthamoeba keratitis. The first patient was a 37-year-old female who presented with severe itching, redness, pain, along with loss of vision. The patient was a regular soft contact lens wearer. The second patient was a 25-year-old female who had been using soft contact lenses for the past two years. She presented with a burning sensation and extreme pain, along with loss of vision. Both patients were treated for a possible microbial keratitis with topical moxifloxacin hydrochloride drops, vancomycin drops, propamidine isethionate ointment, amphotericin B drops, and amikacin drops. However, the response was inadequate and both patients were referred for corneal transplant. Acanthamoeba castellanii was isolated by placing contact lenses and contact lens cases on non-nutrient agar plates containing a lawn of non-invasive Escherichia coli K-12 HB101 bacteria. The polymerase chain reaction (PCR) using genus-specific probes confirmed the identity of Acanthamoeba spp., whereas the morphological characteristics of trophozoites and cysts were suggestive of A. castellanii in both cases. With growing use of contact lenses for vision correction/cosmetic use coupled with sub-standard lens care in this region and the possibility of non-contact lens-associated Acanthamoeba keratitis, a need for increased awareness of this sight-threatening infection is discussed further. PMID:24548160

  13. Differential expression of virulence genes in Legionella pneumophila growing in Acanthamoeba and human monocytes.

    PubMed

    Mou, Qianqian; Leung, Polly H M

    2018-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, is widely distributed throughout natural and artificial water systems and can replicate in macrophages and amoebae. Amoebae are the natural hosts of L. pneumophila, whereas macrophages are incidentally infected. The life cycle of L. pneumophila comprises a replicative phase within the Legionella-containing vacuole (LCV) and a transmissive phase during which bacterial cells become motile and are released via killing of the host. Although the host death mechanisms induced by L. pneumophila have been studied, the expression patterns of related L. pneumophila genes have not been reported. The present study compared the expression patterns of host cell death-associated genes in L. pneumophila grown in the human monocytic cell line THP-1 and Acanthamoeba castellanii. Notably, when L. pneumophila was grown in THP-1, expression of the gene flaA, which is involved in the induction of pyroptosis, was downregulated during the course of infection. In contrast, sdhA associated indirectly with host death, was upregulated. Expression of the genes vipD and sidF, which are involved in the induction and suppression of apoptosis, changed by less than 2-fold. Notably, a lower percentage of pyroptotic cells was observed among infected THP-1 cells relative to uninfected cells, and the latter exhibited stronger expression of caspase-1. A different pattern was observed when L. pneumophila was grown in A. castellanii: flaA and vipD were activated, whereas sdhA and sidF were downregulated during the later stage of replication. The percentage of non-viable (annexin-V + PI + or annexin-V + PI - ) A. castellanii organisms increased with Legionella infection, and the expression of metacaspase-1, which is involved in encystation was up-regulated at late infection time. In summary, L. pneumophila can multiply intracellularly in both amoebae and macrophages to induce cell death and secondary infection, and this characteristic is

  14. A novel transcription initiation factor (TIF), TIF-IE, is required for homogeneous Acanthamoeba castellanii TIF-IB (SL1) to form a committed complex.

    PubMed

    Radebaugh, C A; Kubaska, W M; Hoffman, L H; Stiffler, K; Paule, M R

    1998-10-16

    The fundamental transcription initiation factor (TIF) for ribosomal RNA expression by eukaryotic RNA polymerase I, TIF-IB, has been purified to near homogeneity from Acanthamoeba castellanii using standard techniques. The purified factor consists of the TATA-binding protein and four TATA-binding protein-associated factors with relative molecular weights of 145,000, 99,000, 96,000, and 91,000. This yields a calculated native molecular weight of 460, 000, which compares well with its mass determined by scanning transmission electron microscopy (493,000) and its sedimentation rate, which is close to RNA polymerase I (515,000). Both impure and nearly homogeneous TIF-IB exhibit an apparent equilibrium dissociation constant of 56 +/- 3 pM. However, although impure TIF-IB can form a promoter-DNA complex resistant to challenge by other promoter-containing DNAs, near homogeneous TIF-IB cannot do so. An additional transcription factor, dubbed TIF-IE, restores the ability of near homogeneous TIF-IB to sequester DNA into a committed complex.

  15. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anderson, Iain J.; Watkins, Russell F.; Samuelson, John

    Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit adversity of environments.more » Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptorserine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.« less

  16. Acanthamoeba and bacteria produce antimicrobials to target their counterpart

    PubMed Central

    2014-01-01

    Background In the microbial ecosystem, microbes compete for space and nutrients. Consequently, some have developed the ability to kill or inhibit the growth of other competing microbes by producing antimicrobial substances. As the ‘producer’ species are generally immune to these substances, their compounds act on the competing microbial species and give the producer more space and access to nutrients for growth. Many currently used antibiotics were developed by exploiting this potential of certain microbes. Findings Here, the free-living amoeba, Acanthamoeba castellanii, was investigated for its antibacterial activity against representative Gram positive and Gram negative bacteria, while bacterial isolates were tested for their anti-amoebic properties. Conditioned medium from A. castellanii showed remarkable bactericidal properties against methicillin-resistant Staphylococcus aureus (MRSA) exhibiting almost 100% kill rate, but had limited effect against Acinetobacter sp., Pseudomonas aeruginosa and vancomycin-resistant Enterococcus faecalis (VRE). Similarly, the conditioned medium of E. coli K1 and Enterobacter sp., exhibited potent anti-Acanthamoebic effects in a concentration-dependent manner. Conditioned media of Acanthamoeba, E. coli K1 and Enterobacter sp. showed no cytotoxicity in vitro when tested against human brain microvascular endothelial cells. Active molecule/s in aforementioned amoebic and two bacterial conditioned media were 5 – 10 kDa, and <5 kDa respectively. Conclusions A. castellanii conditioned medium showed potent bactericidal properties against MRSA. The active molecule(s) are heat- and pronase-resistant, and in the 5 to 10 kDa molecular mass range. Contrary to this, E. coli K1 and Enterobacter sp., conditioned medium showed anti-amoebic effects that are <5 kDa in molecular mass, suggestive of active metabolites. PMID:24479709

  17. Chemical composition and anti-Acanthamoeba activity of Melaleuca styphelioides essential oil.

    PubMed

    Albouchi, Ferdaous; Sifaoui, Ines; Reyes-Batlle, Maria; López-Arencibia, Atteneri; Piñero, José E; Lorenzo-Morales, Jacob; Abderrabba, Manef

    2017-12-01

    Acanthamoeba infections cause serious humans diseases, such as amoebic keratitis and granulomatous amoebic encephalitis. Melaleuca essential oil has been reported to be effective in treating bacterial and fungal infections. However, the anti-parasitic effects of this essential oil are not well studied. In the present study, we first characterized the composition of the essential oil, extracted from the Tunisian Melaleuca styphelioides leaves, and then tested its effect on the Acanthamoeba castellanii Neff. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the major common compounds were Caryophyllene oxide (23.42%), Spathulenol (20.5%), Isoaromadendrene epoxide (7.45%), Ledol (5.98%), α-Pinene (3.82%), Isopinocarveol (2.18%). Our data also showed that M. styphelioides essential oil inhibited the growth of Acanthamoeba with an IC 50 value of 69.03 ± 9.17 μg/ml. This work suggests M. styphelioides essential oil as a potential anti amoeba drug. Nevertheless, further studies are still needed to further verify the cytotoxicity of the studied oil on human macrophages and check its applicability to treat Acanthamoeba infections in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Inactivation of Acanthamoeba spp. and Other Ocular Pathogens by Application of Cold Atmospheric Gas Plasma

    PubMed Central

    Shama, Gilbert; Andrew, Peter W.

    2016-01-01

    Currently there are estimated to be approximately 3.7 million contact lens wearers in the United Kingdom and 39.2 million in North America. Contact lens wear is a major risk factor for developing an infection of the cornea known as keratitis due to poor lens hygiene practices. While there is an international standard for testing disinfection methods against bacteria and fungi (ISO 14729), no such guidelines exist for the protozoan Acanthamoeba, which causes a potentially blinding keratitis most commonly seen in contact lens wearers, and as a result, many commercially available disinfecting solutions show incomplete disinfection after 6 and 24 h of exposure. Challenge test assays based on international standard ISO 14729 were used to determine the antimicrobial activity of cold atmospheric gas plasma (CAP) against Pseudomonas aeruginosa, Candida albicans, and trophozoites and cysts of Acanthamoeba polyphaga and Acanthamoeba castellanii. P. aeruginosa and C. albicans were completely inactivated in 0.5 min and 2 min, respectively, and trophozoites of A. polyphaga and A. castellanii were completely inactivated in 1 min and 2 min, respectively. Furthermore, for the highly resistant cyst stage of both species, complete inactivation was achieved after 4 min of exposure to CAP. This study demonstrates that the CAP technology is highly effective against bacterial, fungal, and protozoan pathogens. The further development of this technology has enormous potential, as this approach is able to deliver the complete inactivation of ocular pathogens in minutes, in contrast to commercial multipurpose disinfecting solutions that require a minimum of 6 h. PMID:26994079

  19. Amoebicidal Activity of Caffeine and Maslinic Acid by the Induction of Programmed Cell Death in Acanthamoeba

    PubMed Central

    Martín-Navarro, Carmen M.; López-Arencibia, Atteneri; Sifaoui, Ines; Reyes-Batlle, María; Fouque, Emilie; Osuna, Antonio; Valladares, Basilio; Piñero, José E.; Héchard, Yann; Maciver, Sutherland K.

    2017-01-01

    ABSTRACT Free-living amoebae of the genus Acanthamoeba are the causal agents of a sight-threatening ulceration of the cornea called Acanthamoeba keratitis, as well as the rare but usually fatal disease granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba infections, they are generally lengthy and/or have limited efficacy. For the best clinical outcome, treatments should target both the trophozoite and the cyst stages, as cysts are known to confer resistance to treatment. In this study, we document the activities of caffeine and maslinic acid against both the trophozoite and the cyst stages of three clinical strains of Acanthamoeba. These drugs were chosen because they are reported to inhibit glycogen phosphorylase, which is required for encystation. Maslinic acid is also reported to be an inhibitor of extracellular proteases, which may be relevant since the protease activities of Acanthamoeba species are correlated with their pathogenicity. We also provide evidence for the first time that both drugs exert their anti-amoebal effects through programmed cell death. PMID:28320723

  20. Role of phospholipase A2 (PLA2) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis

    PubMed Central

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2013-01-01

    The aim of this study is to determine if the mannose-induced protein (MIP-133) from Acanthamoeba castellanii trophozoites induces apoptosis of corneal epithelial cells through a cytosolic phospholipase A2α (cPLA2α)-mediated pathway. The efficacy of cPLA2α inhibitors to provide protection against Acanthamoeba keratitis was examined in vivo. Chinese hamster corneal epithelial (HCORN) cells were incubated with or without MIP-133. MIP-133 induces significant increase in cPLA2α and macrophage inflammatory protein-2 (MIP-2/CXCL2) levels from corneal cells. Moreover, cPLA2α inhibitors, MAFP (Methyl-arachidonyl fluorophosphonate) and AACOCF3 (Arachidonyl trifluoromethyl ketone), significantly reduce cPLA2α and CXCL2 from these cells (P< 0.05). Additionally, cPLA2α inhibitors significantly inhibit MIP-133-induced apoptosis in HCORN cells (P< 0.05). Subconjunctival injection of purified MIP-133 in Chinese hamster eyes induced cytopathic effects resulting in corneal ulceration. Animals infected with A. castellanii-laden contact lenses and treated with AACOCF3 and CAY10650, showed significantly less severe keratitis as compared with control animals. Collectively, the results indicate that cPLA2α is involved in MIP-133 induced apoptosis of corneal epithelial cells, polymorphonuclear neutrophil infiltration, and production of CXCL2. Moreover, cPLA2α inhibitors can be used as a therapeutic target in Acanthamoeba keratitis. PMID:23792108

  1. Subinhibitory Concentrations of Antimicrobial Agents Reduce the Uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 Cells by Altering the Expression of Virulence-Associated Antigens

    PubMed Central

    Lück, P. Christian; Schmitt, Jürgen W.; Hengerer, Arne; Helbig, Jürgen H.

    1998-01-01

    We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reduced Legionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionella antigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected. PMID:9797218

  2. Inefficacy of marketed contact lens disinfection solutions against keratitis-causing Acanthamoeba castellanii belonging to the T4 genotype.

    PubMed

    Lakhundi, Sahreena; Khan, Naveed Ahmed; Siddiqui, Ruqaiyyah

    2014-06-01

    The aim of this study was to assess the anti-amoebic effects of marketed contact lens disinfecting solutions. Using amoebistatic, amoebicidal, and cysticidal assays, nine different contact lens solutions were tested including: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Replenish, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+, Kontex Soak. In vitro growth inhibition (amoebistatic) assays were performed by incubating Acanthamoeba castellanii with aforementioned contact lens disinfection solutions as per manufacturer's instructions in the growth medium for up to 48h at 30°C. To determine amoebicidal and cysticidal effects, amoebae were incubated with contact lens solutions in phosphate buffered saline for 24h and viability was determined by haemocytometer counting as well as re-inoculating them in the growth medium. For controls, solutions were tested against bacterial corneal pathogen, Pseudomonas aeruginosa, as well as amoebae were incubated with the solvent alone. Of the nine contact lens solutions tested, none of them showed potent amoebicidal effects. Only DuraPlus and OptiFree Replenish exhibited trophozoite lysis of 85.3% and 73.7% respectively. In contrast, all contact lens disinfection solutions except Kontex Clean, Kontex Normal, Kontex Multisol extra+, tested showed amoebistatic effects. Importantly, none of the contact lens disinfection solutions exhibited cysticidal effects using qualitative assays, i.e., cysts treated with aforementioned solutions re-emerged as viable amoebae upon inoculation in the growth medium. However, more than 3-log reduction was observed when ReNu MultiPlus, DuraPlus and OptiFree Express were tested against P. aeruginosa which is in accordance with the ISO Stand-Alone Primary acceptance criteria. These findings are of great concern for contact lens users. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Number of Bacteria and Time of Coincubation With Bacteria Required for the Development of Acanthamoeba Keratitis.

    PubMed

    Nakagawa, Hayate; Hattori, Takaaki; Koike, Naohito; Ehara, Tomoko; Narimatsu, Akitomo; Kumakura, Shigeto; Matsumoto, Tetsuya; Goto, Hiroshi

    2017-03-01

    We hypothesized that bacteria may be a factor contributing to the development of Acanthamoeba keratitis (AK). We investigated interactions between Acanthamoeba and Pseudomonas aeruginosa for the development of keratitis in rabbit corneas. Acanthamoeba castellanii (ATCC50492) and P. aeruginosa (PAO-1) were used. Two densities of P. aeruginosa (high, 1 × 10/mL; low, 3 × 10/mL) and 2 durations of coincubation (long, 6 h; short, 2 h) of Acanthamoeba with 1 × 10/mL of P. aeruginosa were tested. Acanthamoeba alone or Acanthamoeba coincubated with P. aeruginosa was inoculated into rabbit corneas. After inoculation, levofloxacin (LVFX) eye drops were administered. The clinical score of the cornea was evaluated after inoculation. Acanthamoeba alone did not produce keratitis during a 5-day observation period. Rabbit corneas inoculated with Acanthamoeba coincubated with low-density P. aeruginosa followed by topical LVFX were clear with few infiltrates. Corneas inoculated with Acanthamoeba coincubated with high-density P. aeruginosa followed by LVFX treatment developed severe keratitis, and clinical scores were significantly higher compared with high-density P. aeruginosa alone followed by LVFX treatment (scores 7, 9.6, 8.5 vs. 3, 3.5, 3.25 on days 1-3, all P < 0.01). The long (6 h) coincubation time of Acanthamoeba with high-density P. aeruginosa resulted in more severe keratitis compared with short (2 h) coincubation (scores, 9.7, 12.7, 12.1, 9.8, 8.7 vs. 7, 9.6, 8.5, 6.9, 5.6 on days 1-5, all P < 0.01). These results suggest that the presence of bacteria is essential and a critical number of bacteria is required for the development of AK. The time of coexistence with bacteria may be an important determinant of the severity of AK.

  4. Evaluation of Acanthamoeba Myosin-IC as a Potential Therapeutic Target

    PubMed Central

    Lorenzo-Morales, Jacob; López-Arencibia, Atteneri; Reyes-Batlle, María; Piñero, José E.; Valladares, Basilio; Maciver, Sutherland K.

    2014-01-01

    Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a fatal encephalitis. We have targeted myosin-IC by using small interfering RNA (siRNA) silencing as a therapeutic approach, since it is known that the function of this protein is vital for the amoeba. In this work, specific siRNAs against the Acanthamoeba myosin-IC gene were developed. Treated and control amoebae were cultured in growth and encystment media to evaluate the induced effects after myosin-IC gene knockdown, as we have anticipated that cyst formation may be impaired. The effects of myosin-IC gene silencing were inhibition of cyst formation, inhibition of completion of cytokinesis, inhibition of osmoregulation under osmotic stress conditions, and death of the amoebae. The finding that myosin-IC silencing caused incompletion of cytokinesis is in agreement with earlier suggestions that the protein plays a role in cell locomotion, which is necessary to pull daughter cells apart after mitosis in a process known as “traction-mediated cytokinesis”. We conclude that myosin-IC is a very promising potential drug target for the development of much-needed antiamoebal drugs and that it should be further exploited for Acanthamoeba therapy. PMID:24468784

  5. Killing of diverse eye pathogens (Acanthamoeba spp., Fusarium solani, and Chlamydia trachomatis) with alcohols.

    PubMed

    Aqeel, Yousuf; Rodriguez, Raquel; Chatterjee, Aparajita; Ingalls, Robin R; Samuelson, John

    2017-02-01

    Blindness is caused by eye pathogens that include a free-living protist (Acanthamoeba castellanii, A. byersi, and/or other Acanthamoeba spp.), a fungus (Fusarium solani), and a bacterium (Chlamydia trachomatis). Hand-eye contact is likely a contributor to the spread of these pathogens, and so hand washing with soap and water or alcohol-based hand sanitizers (when water is not available) might reduce their transmission. Recently we showed that ethanol and isopropanol in concentrations present in hand sanitizers kill walled cysts of Giardia and Entamoeba, causes of diarrhea and dysentery, respectively. The goal here was to determine whether these alcohols might kill infectious forms of representative eye pathogens (trophozoites and cysts of Acanthamoeba, conidia of F. solani, or elementary bodies of C. trachomatis). We found that treatment with 63% ethanol or 63% isopropanol kills >99% of Acanthamoeba trophozoites after 30 sec exposure, as shown by labeling with propidium iodide (PI) and failure to grow in culture. In contrast, Acanthamoeba cysts, which contain cellulose fibers in their wall, are relatively more resistant to these alcohols, particularly isopropanol. Depending upon the strain tested, 80 to 99% of Acanthamoeba cysts were killed by 63% ethanol after 2 min and 95 to 99% were killed by 80% ethanol after 30 sec, as shown by PI labeling and reduced rates of excystation in vitro. Both ethanol and isopropanol (63% for 30 sec) kill >99% of F. solani conidia, which have a wall of chitin and glucan fibrils, as demonstrated by PI labeling and colony counts on nutrient agar plates. Both ethanol and isopropanol (63% for 60 sec) inactivate 96 to 99% of elementary bodies of C. trachomatis, which have a wall of lipopolysaccharide but lack peptidoglycan, as measured by quantitative cultures to calculate inclusion forming units. In summary, alcohols kill infectious forms of Acanthamoeba, F. solani, and C. trachomatis, although longer times and higher ethanol

  6. Killing of diverse eye pathogens (Acanthamoeba spp., Fusarium solani, and Chlamydia trachomatis) with alcohols

    PubMed Central

    2017-01-01

    Background Blindness is caused by eye pathogens that include a free-living protist (Acanthamoeba castellanii, A. byersi, and/or other Acanthamoeba spp.), a fungus (Fusarium solani), and a bacterium (Chlamydia trachomatis). Hand-eye contact is likely a contributor to the spread of these pathogens, and so hand washing with soap and water or alcohol–based hand sanitizers (when water is not available) might reduce their transmission. Recently we showed that ethanol and isopropanol in concentrations present in hand sanitizers kill walled cysts of Giardia and Entamoeba, causes of diarrhea and dysentery, respectively. The goal here was to determine whether these alcohols might kill infectious forms of representative eye pathogens (trophozoites and cysts of Acanthamoeba, conidia of F. solani, or elementary bodies of C. trachomatis). Methodology/Principal findings We found that treatment with 63% ethanol or 63% isopropanol kills >99% of Acanthamoeba trophozoites after 30 sec exposure, as shown by labeling with propidium iodide (PI) and failure to grow in culture. In contrast, Acanthamoeba cysts, which contain cellulose fibers in their wall, are relatively more resistant to these alcohols, particularly isopropanol. Depending upon the strain tested, 80 to 99% of Acanthamoeba cysts were killed by 63% ethanol after 2 min and 95 to 99% were killed by 80% ethanol after 30 sec, as shown by PI labeling and reduced rates of excystation in vitro. Both ethanol and isopropanol (63% for 30 sec) kill >99% of F. solani conidia, which have a wall of chitin and glucan fibrils, as demonstrated by PI labeling and colony counts on nutrient agar plates. Both ethanol and isopropanol (63% for 60 sec) inactivate 96 to 99% of elementary bodies of C. trachomatis, which have a wall of lipopolysaccharide but lack peptidoglycan, as measured by quantitative cultures to calculate inclusion forming units. Conclusions/Significance In summary, alcohols kill infectious forms of Acanthamoeba, F. solani, and

  7. Characterization of actin filament severing by actophorin from Acanthamoeba castellanii

    PubMed Central

    1991-01-01

    Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem. 261:477-485). Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1:1 covalent complex of actin and actophorin. Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steady-state actin polymer concentration gave a Kd of 0.2 microM for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration. The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate. PMID:1757465

  8. [Investigation of the in vitro effects of Melissa officinalis L., Mentha x piperita L. and Ocimum basilicum L. (Lamiaceae) essential oils on the cysts and trophozoites of Acanthamoeba castellani].

    PubMed

    Ergüden, Ceren; Özkoç, Soykan; Öztürk, Bintuğ; Bayram Delibaş, Songül

    2016-10-01

    Acanthamoeba species are free living amoeba found widely all over the world. They are responsible for Acanthamoeba keratitis (AK), an infection which is especially seen in contact lens users and after minor corneal traumas, that may lead blindness. At present, antifungals and antiseptics are used for the treatment of AK cases, however, some problems such as long treatment periods and the occurrence of side effects, resistance of cyst forms against drugs, emphasize the need for new drugs. There are some published studies that pointed out the effectiveness of plant extracts and essential oils on Acanthamoeba spp. The aim of this study was to investigate the in vitro effects of essential oils of Mentha x piperita L. (peppermint), Melissa officinalis L. (lemon balm) and Ocimum basilicum L. (basil) belonging to Lamiaceae family, on the cysts and trophozoites of Acanthamoeba castellanii. The strain used in our study, namely A. castellanii T4 genotype, is the most frequently isolated amoeba from environment and also the causative agent of AK and granulomatous amebic encephalitis. For the determination of amebicidal activity, essential oils obtained from Mentha x priperita L., Melissa officinalis L. and Ocimum basilicum L. by Neo-Clevenger type of distillation apparatus have been used. In vitro experiments were performed by using 96-well microplates. Cyst and trophozoite solutions were added on the essential oil dilutions to obtain the last concentrations of 40, 20, 10, 5, 2.5 and 1.25 µg/ml for the cysts, and 10, 5, 2.5, 1.25, 0.625 and 0.313 µg/ml for the trophozoites. After the incubation of microplates at 30oC for 1, 6, 24, 48 and 72 hours, the viability of parasitic forms were evaluated under the light microscope followed by staining trypan blue. It was found that, each essential oil showed amebicidal effect on A.castellani cysts and trophozoites dependent on dosage and time, when compared with the control group, The maximum lethal effect occured with Melissa

  9. Cellulose degradation: a therapeutic strategy in the improved treatment of Acanthamoeba infections.

    PubMed

    Lakhundi, Sahreena; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2015-01-14

    Acanthamoeba is an opportunistic free-living amoeba that can cause blinding keratitis and fatal brain infection. Early diagnosis, followed by aggressive treatment is a pre-requisite in the successful treatment but even then the prognosis remains poor. A major drawback during the course of treatment is the ability of the amoeba to enclose itself within a shell (a process known as encystment), making it resistant to chemotherapeutic agents. As the cyst wall is partly made of cellulose, thus cellulose degradation offers a potential therapeutic strategy in the effective targeting of trophozoite encased within the cyst walls. Here, we present a comprehensive report on the structure of cellulose and cellulases, as well as known cellulose degradation mechanisms with an eye to target the Acanthamoeba cyst wall. The disruption of the cyst wall will make amoeba (concealed within) susceptible to chemotherapeutic agents, and at the very least inhibition of the excystment process will impede infection recurrence, as we bring these promising drug targets into focus so that they can be explored to their fullest.

  10. Long-term Survival and Virulence of Mycobacterium leprae in Amoebal Cysts

    PubMed Central

    Wheat, William H.; Casali, Amy L.; Thomas, Vincent; Spencer, John S.; Lahiri, Ramanuj; Williams, Diana L.; McDonnell, Gerald E.; Gonzalez-Juarrero, Mercedes; Brennan, Patrick J.; Jackson, Mary

    2014-01-01

    Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs

  11. Long-term survival and virulence of Mycobacterium leprae in amoebal cysts.

    PubMed

    Wheat, William H; Casali, Amy L; Thomas, Vincent; Spencer, John S; Lahiri, Ramanuj; Williams, Diana L; McDonnell, Gerald E; Gonzalez-Juarrero, Mercedes; Brennan, Patrick J; Jackson, Mary

    2014-12-01

    Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs

  12. Detachment of trophozoites of Acanthamoeba species from soft contact lenses with BEN22 detergent, BioSoak, and Renu multi-purpose solutions.

    PubMed

    Raali, E; Vaahtoranta-Lehtonen, H H; Lehtonen, O P

    2001-07-01

    BEN22 detergent was studied for its ability to detach Acanthamoeba from soft contact lenses without mechanical cleaning or separate cleaning agents. Trophozoites of Acanthamoeba castellanii and A. polyphaga were adhered onto nonionic, high water content soft contact lenses. The lenses were immersed for 2 hours in contact lens care solutions and the remaining trophozoites were counted microscopically. The counts were compared to the counts on the same lens before treatment. BEN22 (50:50 mixture of L-alpha-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate and 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate) (Kassell Industries, Inc., Wisconsin Dells, WI) in a concentration of 0.05% detached the trophozoites to a statistically significant greater extent than saline, but commercial ReNu Multi-Purpose Solution (Bausch & Lomb, Italy) and BioSoak (Finnsusp Ltd., Finland) did so as well. ReNu Multi-Purpose Solution was more effective than 0.005% BEN22 in detaching the trophozoites of both of the Acanthamoeba strains. After the 2 hour immersion period, a maximum of 97% of the initial trophozoites were detached. The variation between individual lenses was significantly greater than that within the different areas of one lens. BEN22 had no reliable detaching effect on Acanthamoeba. The variation between lenses was great, and the rate of detachment was low with all the agents tested indicating that immersion and rinsing in the solutions tested cannot be considered as a safe substitute for proper disinfection against Acanthamoeba in contact lens care.

  13. Effects of multipurpose solutions on the adhesion of Acanthamoeba to rigid gas permeable contact lenses.

    PubMed

    Lee, Ga-Hyun; Yu, Hak-Sun; Lee, Ji-Eun

    2016-03-01

    To evaluate the effect of multipurpose contact lens care solutions (MPSs) on the adhesion of Acanthamoeba to rigid gas permeable (RGP) contact lenses. Acanthamoeba castellanii (AC) trophozoites were inoculated onto untreated RGP contact lenses (FP, Extra, or Menicon Z), and numbers of trophozoites adhering to lenses were counted under a phase contrast microscope at 18 h post-inoculation (controls). Similarly, adhering trophozoites were counted at 6 h post-inoculation on each of the three RGP lens types with one of three MPSs (Boston Simplus, Menicare Plus, and O2 Care). Scanning electron microscopic examinations were performed to compare lens surfaces. Adhesion of AC trophozoites to untreated FP was greater than to untreated Extra or Menicon Z. Surfaces of Extra and Menicon Z lenses were waxier, smoother, and more homogeneous than those of FP lenses. After treatment with Boston Simplus, adhesion of AC trophozoites was significantly reduced for all lens types as compared with controls (p < 0.0001). Treatments with Menicare Plus or O2 Care reduced the number of adherent AC trophozoites significantly on FP lenses only as compared with controls (p < 0.0001). The adhesion rates of AC trophozoites to RGP lenses depended on lens surfaces. Boston Simplus reduced the adhesion rate of AC trophozoites more than Menicare Plus or O2 Care. Appropriate RGP lens and MPS selection could decrease the prevalence of Acanthamoeba keratitis. © 2016 The Authors Ophthalmic & Physiological Optics © 2016 The College of Optometrists.

  14. Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

    PubMed Central

    Mullins, R. Dyche; Kelleher, Joseph F.; Xu, James; Pollard, Thomas D.

    1998-01-01

    The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells. PMID:9529382

  15. Interactions of neuropathogenic Escherichia coli K1 (RS218) and its derivatives lacking genomic islands with phagocytic Acanthamoeba castellanii and nonphagocytic brain endothelial cells.

    PubMed

    Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

    2014-01-01

    Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α -hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity.

  16. Interactions of Neuropathogenic Escherichia coli K1 (RS218) and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    PubMed Central

    Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

    2014-01-01

    Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity. PMID:24818136

  17. Analysis of Giardin expression during encystation of Giardia lamblia

    USDA-ARS?s Scientific Manuscript database

    The present study analyzed giardin transcription in trophozoites and cysts during encystation of Giardia lamblia. Encystment was induced using standard methods, and the number of trophozoites and cysts were counted at various time-points during encystation. At all time points, RNA from both stages...

  18. Acanthamoeba: An Overview of the Challenges to the Development of a Consensus Methodology of Disinfection Efficacy Testing for Contact Lens Care Products.

    PubMed

    Brocious, Jeffrey; Tarver, Michelle E; Hampton, Denise; Eydelman, Malvina

    2018-04-24

    With the increasing incidence of more pathogens that can cause microbial keratitis (MK), it is necessary to periodically reassess disinfection multipurpose solutions testing requirements to ensure that relevant organisms to challenge them are being used. Current testing protocols have included common pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens, Candida albicans, and Fusarium solani but have omitted less common pathogens such as Acanthamoeba. Specifically, Acanthamoeba sp. has recently been identified as a prevalent cause of MK in certain countries. Developing an appropriate protocol for this unique organism presents a challenge, given its two distinct life stages, methods to grow the organism, encystment techniques, and many other parameters that can affect testing outcomes. Therefore, the appropriate combination of these parameters is crucial to developing a protocol that ensures consistent, accurate results. The FDA has recognized the importance of establishing a standardized testing protocol for this pathogen and embarked on research efforts to provide a recommended testing protocol for testing contact lens care products.

  19. Compositions and methods for pathogen transport

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    El-Etr, Sahar; Farquar, George R.

    This disclosure provides a method for transporting a pathogen under ambient conditions, by culturing the pathogen with an amoeba under conditions that favor the incorporation of the pathogen into a trophozoite, starving the amoeba until it encysts, then culturing under conditions that favor conversion of the amoeba back to a trophozoite. In one aspect, the conditions that favor incorporation of the pathogen into the cyst of the amoeba comprises contacting the pathogen with the amoeba in an iron rich environment. Virus and/or bacteria are pathogens that can be transported by the disclosed method. Amoeba that are useful in the disclosedmore » methods include, without limitation Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria gruberi. The disclosed methods have utility in: transporting pathogens from military field hospitals and clinics to the laboratory; transporting pathogens from global satellite laboratories to clinical laboratories; long term storage of pathogens; enriching contaminated patient samples for pathogens of interest; biosurveillance and detection efforts.« less

  20. Membrane Trafficking Modulation during Entamoeba Encystation.

    PubMed

    Herman, Emily; Siegesmund, Maria A; Bottery, Michael J; van Aerle, Ronny; Shather, Maulood Mohammed; Caler, Elisabet; Dacks, Joel B; van der Giezen, Mark

    2017-10-09

    Entamoeba histolytica is an intestinal parasite that infects 50-100 million people and causes up to 55,000 deaths annually. The transmissive form of E. histolytica is the cyst, with a single infected individual passing up to 45 million cysts per day, making cyst production an attractive target for infection control. Lectins and chitin are secreted to form the cyst wall, although little is known about the underlying membrane trafficking processes supporting encystation. As E. histolytica does not readily form cysts in vitro, we assessed membrane trafficking gene expression during encystation in the closely related model Entamoeba invadens. Genes involved in secretion are up-regulated during cyst formation, as are some trans-Golgi network-to-endosome trafficking genes. Furthermore, endocytic and general trafficking genes are up-regulated in the mature cyst, potentially preserved as mRNA in preparation for excystation. Two divergent dynamin-related proteins found in Entamoeba are predominantly expressed during cyst formation. Phylogenetic analyses indicate that they are paralogous to, but quite distinct from, classical dynamins found in human, suggesting that they may be potential drug targets to block encystation. The membrane-trafficking machinery is clearly regulated during encystation, providing an additional facet to understanding this crucial parasitic process.

  1. Co-isolation of Vahlkampfia and acanthamoeba in acanthamoeba-like keratitis in a Spanish population.

    PubMed

    Arnalich-Montiel, Francisco; Lorenzo-Morales, Jacob; Irigoyen, Cristina; Morcillo-Laiz, Rafael; López-Vélez, Rogelio; Muñoz-Negrete, Francisco; Piñero, Jose E; Valladares, Basilio

    2013-05-01

    To report the co-isolation incidence of Acanthamoeba and Vahlkampfia in amoebic keratitis from a tertiary care institution in Madrid, Spain. In this retrospective case series, 7 eyes of 7 consecutive patients with culture-proven or polymerase chain reaction-positive Acanthamoeba keratitis were seen at a tertiary care institution from January 2010 to April 2011, and their charts were reviewed. Two of 7 patients showed mixed Acanthamoeba and Vahlkampfia keratitis. Good clinical response to the treatment was strongly correlated with early diagnosis, whereas delayed diagnosis resulted in poor response to the treatment in single or mixed infection. Co-isolation of Vahlkampfia and Acanthamoeba in Acanthamoeba-like keratitis has recently been detected in our population. This finding should raise awareness of the existence of other amoeba different from Acanthamoeba causing keratitis. There are not enough cases yet to determine the impact of mixed amoebic keratitis in the prognosis of this disease.

  2. Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection

    PubMed Central

    Swart, A. Leoni; Harrison, Christopher F.; Eichinger, Ludwig; Steinert, Michael; Hilbi, Hubert

    2018-01-01

    Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of “effector” proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors. PMID:29552544

  3. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Das, S.; Gillin, F.D.

    1987-05-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of /sup 3/H GlcNAc from UDP-GlcNAcmore » into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei.« less

  4. Killing the dead: chemotherapeutic strategies against free-living cyst-forming protists (Acanthamoeba sp. and Balamuthia mandrillaris).

    PubMed

    Siddiqui, Ruqaiyyah; Aqeel, Yousuf; Khan, Naveed Ahmed

    2013-01-01

    The opportunist free-living protists such as Acanthamoeba spp. and Balamuthia mandrillaris have become a serious threat to human life. As most available drugs target functional aspects of pathogens, the ability of free-living protists to transform into metabolically inactive cyst forms presents a challenge in treatment. It is hoped, that the development of broad spectrum antiprotist agents acting against multiple cyst-forming protists to provide target-directed inhibition will offer a viable drug strategy in the treatment of these rare infections. Here, we present a comprehensive report on upcoming drug targets, with emphasis on cyst wall biosynthesis along with the related biochemistry of encystment pathways, as we strive to bring ourselves a step closer to being able to combat these deadly diseases. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

  5. Environmental mimics and the Lvh type IVA secretion system contribute to virulence-related phenotypes of Legionella pneumophila.

    PubMed

    Bandyopadhyay, Purnima; Liu, Shuqing; Gabbai, Carolina B; Venitelli, Zeah; Steinman, Howard M

    2007-02-01

    Legionella pneumophila, the causative organism of Legionnaires' disease, is a fresh-water bacterium and intracellular parasite of amoebae. This study examined the effects of incubation in water and amoeba encystment on L. pneumophila strain JR32 and null mutants in dot/icm genes encoding a type IVB secretion system required for entry, delayed acidification of L. pneumophila-containing phagosomes, and intracellular multiplication when stationary-phase bacteria infect amoebae and macrophages. Following incubation of stationary-phase cultures in water, mutants in dotA and dotB, essential for function of the type IVB secretion system, exhibited entry and delay of phagosome acidification comparable to that of strain JR32. Following encystment in Acanthamoeba castellanii and reversion of cysts to amoeba trophozoites, dotA and dotB mutants exhibited intracellular multiplication in amoebae. The L. pneumophila Lvh locus, encoding a type IVA secretion system homologous to that in Agrobacterium tumefaciens, was required for restoration of entry and intracellular multiplication in dot/icm mutants following incubation in water and amoeba encystment and was required for delay of phagosome acidification in strain JR32. These data support a model in which the Dot/Icm type IVB secretion system is conditionally rather than absolutely required for L. pneumophila virulence-related phenotypes. The data suggest that the Lvh type IVA secretion system, previously thought to be dispensable, is involved in virulence-related phenotypes under conditions mimicking the spread of Legionnaires' disease from environmental niches. Since environmental amoebae are implicated as reservoirs for an increasing number of environmental pathogens and for drug-resistant bacteria, the environmental mimics developed here may be useful in virulence studies of other pathogens.

  6. Adhesion of Acanthamoeba on Cosmetic Contact Lenses

    PubMed Central

    2017-01-01

    Background This study aimed to evaluate the adhesion of Acanthamoeba trophozoites on cosmetic contact lenses (CLs) with and without CL care multipurpose solution (MPS) treatment. Methods Acanthamoeba lugdunensis L3a trophozoites were inoculated onto disks trimmed from CLs: 1-day Acuvue moist, 1-day Acuvue define, Acuvue 2, and Acuvue 2 define. After 18-hour inoculation, the number of adherent trophozoites was counted under phase contrast microscopy. The effects of MPS, Opti-Free Express, soaking CLs for 6 hours, on Acanthamoeba adhesion were analyzed. Scanning electron microscopic examination was performed for assessment of Acanthamoeba attached on the lens surface. Results Acanthamoeba trophozoites showed greater adhesion to cosmetic CL (P = 0.017 for 1-day CL and P = 0.009 for 2-week CL) although there was no significant difference between the types of cosmetic CL. On all lenses, the number of adherent Acanthamoeba was significantly reduced after treatment with MPS (P < 0.001 for 1-day Acuvue moist, P = 0.046 for 1-day Acuvue define, P < 0.001 for Acuvue 2, and P = 0.015 for Acuvue 2 define), but there was still significant difference between conventional and cosmetic CLs (P = 0.003 for 1-day CL and P < 0.001 for 2-week CL, respectively). More attachment of Acanthamoeba was observed on colored area and the acanthopodia of Acanthamoeba was placed on the rough surface of colored area. Conclusion Acanthamoeba showed a greater affinity for cosmetic CL and mostly attached on colored area. Although MPS that contained myristamidopropyl dimethylamine reduced the adhesion rate, there was a significant difference between conventional and cosmetic CLs. PMID:29318793

  7. Diapause in tardigrades: a study of factors involved in encystment.

    PubMed

    Guidetti, Roberto; Boschini, Deborah; Altiero, Tiziana; Bertolani, Roberto; Rebecchi, Lorena

    2008-07-01

    Stressful environmental conditions limit survival, growth and reproduction, or these conditions induce resting stages indicated as dormancy. Tardigrades represent one of the few animal phyla able to perform both forms of dormancy: quiescence and diapause. Different forms of cryptobiosis (quiescence) are widespread and well studied, while little attention has been devoted to the adaptive meaning of encystment (diapause). Our goal was to determine the environmental factors and token stimuli involved in the encystment process of tardigrades. The eutardigrade Amphibolus volubilis, a species able to produce two types of cyst (type 1 and type 2), was considered. Laboratory experiments and long-term studies on cyst dynamics of a natural population were conducted. Laboratory experiments demonstrated that active tardigrades collected in April produced mainly type 2 cysts, whereas animals collected in November produced mainly type 1 cysts, indicating that the different responses are functions of the physiological state at the time they were collected. The dynamics of the two types of cyst show opposite seasonal trends: type 2 cysts are present only during the warm season and type 1 cysts are present during the cold season. Temperature represents the environmental factor involved in induction, maintenance and termination of the cyst. We also obtained evidence that A. volubilis is able to perform both diapause and cryptobiosis, even overlapping the two phenomena. The induction phase of tardigrade encystment can be compared to the induction phase of insect diapause, also indicating an involvement of endogenous factors in tardigrade encystment. As in insect diapause, tardigrade encystment can be considered a diapausing state controlled by exogenous and endogenous stimuli.

  8. Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation.

    PubMed

    Robles-Martinez, Leobarda; Mendez, Tavis L; Apodaca, Jennifer; Das, Siddhartha

    2017-01-01

    The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2-3 fold higher than of GlcCer. Although both activities follow Michaelis-Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Label-free imaging of acanthamoeba using multimodal nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Kobayashi, Tsubasa; Cha, Yu-Rok; Kaji, Yuichi; Oshika, Tetsuro; Leproux, Philippe; Couderc, Vincent; Kano, Hideaki

    2018-02-01

    Acanthamoeba keratitis is a disease in which amoebae named Acanthamoeba invade the cornea of an eye. To diagnose this disease before it becomes serious, it is important to detect the cyst state of Acanthamoeba in the early stage of infection. In the present study, we explored spectroscopic signitures of the cyst state of Acanthamoeba using multimodal nonlinear optical microscopy with the channels of multiplex coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and third harmonic generation (THG). A sharp band at around 1603 cm-1 in the CARS (Im[χ(3)]) spectrum was found at the cyst state of Acanthamoeba, which possibly originates from ergosterol and/or 7-dehydrostigmasterol. It can be used as a maker band of Acanthamoeba for medical treatment. Keyword: Acanthamoeba keratitis, coherent anti-Stokes Raman scattering, CARS, second harmonic generation, SHG, microspectroscopy, multiphoton microscopy

  10. A case of trauma related Acanthamoeba keratitis.

    PubMed

    Kamel, A G M; Faridah, H; Yusof, S; Norazah, A; Nakisah, M A

    2004-12-01

    Acanthamoeba is an uncommon cause of keratitis but one of the most severe because of the prolonged and painful course of the disease and poor visual outcome. Although contact lens use is the principal risk factor, about 10% of cases occur following trauma and exposure to contaminated soil or water. Two cases of Acanthamoeba keratitis involving women contact lens wearers have previously been reported in Malaysia but this is the first time, a non contact lens related Acanthamoeba keratitis is reported. The case involved a 28 year old Indonesian male construction worker who had a trauma of the right eye during work. His eye was struck by sand and dust particles after which he quickly washed with water from an open tank at the construction site. He experienced pain, redness, glaring and blurring of vision of the right eye three days later. The diagnosis was missed at initial presentation but culture of the corneal scraping had proven Acanthamoeba as the aetiological agent. The history and clinical findings of this trauma related Acanthamoeba keratitis are briefly discussed.

  11. Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens.

    PubMed

    Abjani, Farhat; Khan, Naveed Ahmed; Jung, Suk Yul; Siddiqui, Ruqaiyyah

    2017-12-01

    The aim of this study was (i) to assess the antimicrobial effects of contact lens disinfecting solutions marketed in Malaysia against common bacterial eye pathogens and as well as eye parasite, Acanthamoeba castellanii, and (ii) to determine whether targeting cyst wall would improve the efficacy of contact lens disinfectants. Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal and amoebicidal assays of six different contact lens solutions including Oxysept ® , AO SEPT PLUS, OPTI-FREE ® pure moist ® , Renu ® fresh™, FreshKon ® CLEAR and COMPLETE RevitaLens™ were performed using Manufacturers Minimum recommended disinfection time (MRDT). The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, and Acanthamoeba castellanii. In addition, using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, we determined whether combination of both agents can enhance efficacy of marketed contact lens disinfectants against A. castellanii trophozoites and cysts, in vitro. The results revealed that all contact lens disinfectants tested showed potent bactericidal effects exhibiting 100% kill against all bacterial species tested. In contrast, none of the contact lens disinfectants had potent effects against Acanthamoeba cysts viability. When tested against trophozoites, two disinfectants, Oxysept Multipurpose and AO-sept Multipurpose showed partial amoebicidal effects. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, the findings revealed that combination of both agents in contact lens disinfectants abolished viability of A. castellanii cysts and trophozoites. Given the inefficacy of contact lens disinfectants tested in this study, these findings present a significant concern to public health. These

  12. Isolation of Acanthamoeba from the rhizosphere of maize and lucerne plants

    NASA Astrophysics Data System (ADS)

    Orosz, Erika; Farkas, Ágnes; Ködöböcz, László; Becsak, Péter; Danka, József; Kucsera, István; Füleky, György

    2013-04-01

    Acanthamoeba species are free-living amoebae that can be found in almost every range of environments. Within this genus, a number of species are recognized as human pathogens, potentially causing Acanthamoeba keratitis, granulomatous amoebic encephalitis, and chronic granulomatous lesions. Soil and water samples were taken from experimental station at Julianna Major of Plant Protection Institute of Centre for Agricultural Research, Hungarian Academy of Sciences. We detected living Acanthamoeba spp. based on culture- confirmed detection combined with the molecular taxonomic identification method. Living Acanthamoeba spp. were detected in thirteen (65%) samples. The presence of Acanthamoeba spp. in the samples depends significantly on the rhizosphere plants. The most frequently identified living Acanthamoeba genotype was T4 followed by T11, T2/T6 and T17. Genotypes T4 and T11 of Acanthamoeba, are responsible for Acanthamoeba keratitis as well as granulomatous amoebic encephalitis, and should therefore be considered as a potential health risk associated with human activities in the environment.

  13. Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion.

    PubMed

    Krtková, Jana; Thomas, Elizabeth B; Alas, Germain C M; Schraner, Elisabeth M; Behjatnia, Habib R; Hehl, Adrian B; Paredez, Alexander R

    2016-08-23

    Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia's sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-Rac(CA)) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-Rac(CA)-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis. The encystation process is crucial for the transmission of giardiasis and the life cycle of many protists. Encystation for Giardia lamblia involves the assembly of a protective cyst wall

  14. Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

    PubMed

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2014-05-29

    Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P < 0.05). Protease-activated receptor 2 antagonist significantly inhibited aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P < 0.05). Protease-activated receptor 1 agonists, but not aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen

  15. Giardia Colonizes and Encysts in High-Density Foci in the Murine Small Intestine

    PubMed Central

    Barash, N. R.; Nosala, C.; Pham, J. K.; McInally, S. G.; Gourguechon, S.; McCarthy-Sinclair, B.

    2017-01-01

    ABSTRACT Giardia lamblia is a highly prevalent yet understudied protistan parasite causing significant diarrheal disease worldwide. Hosts ingest Giardia cysts from contaminated sources. In the gastrointestinal tract, cysts excyst to become motile trophozoites, colonizing and attaching to the gut epithelium. Trophozoites later differentiate into infectious cysts that are excreted and contaminate the environment. Due to the limited accessibility of the gut, the temporospatial dynamics of giardiasis in the host are largely inferred from laboratory culture and thus may not mirror Giardia physiology in the host. Here, we have developed bioluminescent imaging (BLI) to directly interrogate and quantify the in vivo temporospatial dynamics of Giardia infection, thereby providing an improved murine model to evaluate anti-Giardia drugs. Using BLI, we determined that parasites primarily colonize the proximal small intestine nonuniformly in high-density foci. By imaging encystation-specific bioreporters, we show that encystation initiates shortly after inoculation and continues throughout the duration of infection. Encystation also initiates in high-density foci in the proximal small intestine, and high density contributes to the initiation of encystation in laboratory culture. We suggest that these high-density in vivo foci of colonizing and encysting Giardia likely result in localized disruption to the epithelium. This more accurate visualization of giardiasis redefines the dynamics of the in vivo Giardia life cycle, paving the way for future mechanistic studies of density-dependent parasitic processes in the host. IMPORTANCE Giardia is a single-celled parasite causing significant diarrheal disease in several hundred million people worldwide. Due to limited access to the site of infection in the gastrointestinal tract, our understanding of the dynamics of Giardia infections in the host has remained limited and largely inferred from laboratory culture. To better understand

  16. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment

    PubMed Central

    Lorenzo-Morales, Jacob; Khan, Naveed A.; Walochnik, Julia

    2015-01-01

    Free-living amoebae of the genus Acanthamoeba are causal agents of a severe sight-threatening infection of the cornea known as Acanthamoeba keratitis. Moreover, the number of reported cases worldwide is increasing year after year, mostly in contact lens wearers, although cases have also been reported in non-contact lens wearers. Interestingly, Acanthamoeba keratitis has remained significant, despite our advances in antimicrobial chemotherapy and supportive care. In part, this is due to an incomplete understanding of the pathogenesis and pathophysiology of the disease, diagnostic delays and problems associated with chemotherapeutic interventions. In view of the devastating nature of this disease, here we present our current understanding of Acanthamoeba keratitis and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be potential targets for improved diagnosis, therapeutic interventions and/or for the development of preventative measures. Novel molecular approaches such as proteomics, RNAi and a consensus in the diagnostic approaches for a suspected case of Acanthamoeba keratitis are proposed and reviewed based on data which have been compiled after years of working on this amoebic organism using many different techniques and listening to many experts in this field at conferences, workshops and international meetings. Altogether, this review may serve as the milestone for developing an effective solution for the prevention, control and treatment of Acanthamoeba infections. PMID:25687209

  17. Acanthamoeba keratitis challenges a case report.

    PubMed

    Cristina, Stan; Cristina, Vlăduţiu; Mihaela, Popovici

    2016-01-01

    Acanthamoeba keratitis is a rare, chronic, mainly contact lens-related infection caused by a free-living amoeba found ubiquitously in water and soil. A case of a 9-year-old child, who presented to our clinic with painful, red left eye, associated with photophobia, and decreased visual acuity, wais reported. The clinical examination revealed a discoid opacity inferiorly bounded by a dense, gray infiltrate. The progressive nature of the corneal infiltrate, the epithelial defect, and the lack of response to treatment was highly suggestive for Acanthamoeba keratitis. The distinctiveness of this case was the presence of Acanthamoeba keratitis in a child without a history of trauma or contact lens usage, the lack of an appropriate diagnosis and management of this vision-threatening infection.

  18. Failure of molecular diagnostics of a keratitis-inducing Acanthamoeba strain.

    PubMed

    Scheid, Patrick L; Balczun, Carsten

    2017-12-01

    An otherwise healthy 49-year-old female patient presented at the local hospital with severe keratitis in both inflamed eyes. She was a contact lens wearer and had no history of a corneal trauma. In our laboratory for medical parasitology Acanthamoebae were detected microscopically from the cornea scraping and from the fluid of the contact lens storage case after xenical culture and showed the typical cyst morphology of Acanthamoebae group II. The diagnosis of "Acanthamoeba keratitis" was established and successful therapy was provided. While the morphological microscopic method led to the correct diagnosis in this case, an in-house multiplex qPCR and a commercial qPCR showed false negative results regarding Acanthamoeba sp. The subsequent sequencing revealed the Acanthamoeba genotype T4. In the present case report, the inability to detect Acanthamoebae using qPCR only is presented. Therefore, we recommend the utilization of combined different assays for optimal diagnostic purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. A comparison of regimen methods for the removal and inactivation of bacteria, fungi and Acanthamoeba from two types of silicone hydrogel lenses.

    PubMed

    Kilvington, Simon; Lonnen, James

    2009-04-01

    To compare the antimicrobial efficacy of commercial contact lens solutions when used according to the manufacturers' recommended regimens with two types of silicone hydrogel lenses. Four multipurpose contact lens care solutions were examined, representing manufacturer recommended regimens of "rub & rinse", "no rub, rinse" or "no rub, no rinse". Test organisms were Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Fusarium solani, Candida albicans and Acanthamoeba castellanii (trophozoites and cysts). Organisms, in the presence of organic soil, were inoculated on to Acuvue Oasys or Air Optix lenses and subjected to the solution manufacturer's recommended regimen. The number of surviving organisms on the lenses and in the soak solution was enumerated in accordance with ISO 14729. ISO 14729 dictates that for a given organism the combined average number of surviving microbes from the lenses and disinfectant soaking solution must be Acanthamoeba with both lens types. Solutions employing "no rub, rinse" were less satisfactory but significantly better than "no rub, no rinse". Significant differences were found in organism survival on the lenses with greater numbers remaining on the Air Optix compared to Oasys (p<0.01-0.0001). The findings of this study demonstrate that the use of a manual rubbing step is more effective than rinsing or soaking alone in removing pathogenic microbes from silicone hydrogel lenses. Accordingly, it would seem prudent to recommend that contact lens care systems include a rub step as part of the hygiene regimen.

  20. First time identification of Acanthamoeba genotypes in the cornea samples of wild birds; Is Acanthamoeba keratitis making the predatory birds a target?

    PubMed

    Karakavuk, Muhammet; Aykur, Mehmet; Şahar, Esra Atalay; Karakuş, Mehmet; Aldemir, Duygu; Döndüren, Ömer; Özdemir, Hüseyin Gökhan; Can, Hüseyin; Gürüz, Adnan Yüksel; Dağcı, Hande; Döşkaya, Mert

    2017-12-01

    Acanthamoeba is a free-living amoeba which can be isolated from environment and among others well known as an opportunist protozoan parasite causing infections in humans and animals. Eyes are extremely important for the wild birds and losing sight ability due to Acanthamoeba can be dangerous. The studies on Acanthamoeba infection in wild birds is very few in world and Turkey therefore we aimed to screen deceased wild birds found in İzmir and Manisa provinces located in western Turkey using PCR and non-nutrition agar (NNA) plate method. Cornea samples were obtained from 18 deceased wild birds. During the external examination, signs of keratitis were observed in two Eurasian sparrowhawks (Accipiter nisus). All of the corneal samples were analyzed by two PCR methods and NNA plate. According to results, the Acanthamoeba positivity in corneal samples was 16.6% and 5.5% by PCR and plate method, respectively. According to sequencing data, two of isolates belonged to genotype T5 and one was genotype T4. In conclusion, Acanthamoeba infection was detected in wild bird cornea samples with/without keratitis for the first time in the world. The result of this study also show that Acanthamoeba can be a cause of keratitis in wild birds of Turkey and thus these predator birds can be a target of other wild animals due to loss of sight ability. In terms of public health, these results show the importance of wild birds as a source of Acanthamoeba infection in nature. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Severe Acanthamoeba sclerokeratitis in a non-contact lens wearer.

    PubMed

    Hirano, K; Sai, S

    1999-06-01

    To report a case of severe Acanthamoeba sclerokeratitis. A 70-year-old male non-contact lens wearer was examined for severe pain in the left eye which began about 40 days after cataract surgery. In spite of a careful search, it required 6 weeks to detect Acanthamoeba. Systemic and topical fluconazol and miconazol did not help and the keratitis progressed into necrotic sclerokeratitis with protrusion of uveal tissue through the thin sclera. Those findings slowly got worse before the Acanthamoeba sclerokeratitis resolved 6 months later with scar formation. We describe the terminal and cicatricial stages of Acanthamoeba keratitis, and report that the healing process can follow the terminal stage and the eye does not need to be enucleated.

  2. Genotyping of clinical isolates of Acanthamoeba genus in Venezuela.

    PubMed

    Wagner, Carolina; Reyes-Batlle, María; Ysea, María Alejandra Vethencourt; Pérez, Mónica V Galindo; de Rondón, Carmen Guzmán; Paduani, Anaibeth J Nessi; Pérez, Angelyseb Dorta; López-Arencibia, Atteneri; Sifaoui, Ines; de Galindo, María Virginia Pérez; de Suárez, Eva Pérez; Martínez-Carretero, Enrique; Valladares, Basilio; Piñero, José E; Lorenzo-Morales, Jacob

    2016-12-01

    Free-living amoebae of Acanthamoeba genus are opportunistic pathogens distributed worldwide. Strains included in this genus are causative agents of a fatal encephalitis and a sight-threating keratitis in humans and other animals. In this study, 550 clinical samples which were collected between 1984 and 2014 from different patients with suspected infections due to Acanthamoeba were initially screened for the presence of this amoebic genus at the Laboratorio de Amibiasis-Escuela de Bioanálisis at the Universidad Central de Venezuela. Samples were cultured in 2% Non-Nutrient agar plates seeded with a layer of heat killed Escherichia coli. From the 550 clinical samples included in this study, 18 of them were positive for Acanthamoeba genus after culture identification. Moreover, positive samples were confirmed after amplification of the Diagnostic Fragment 3 (DF3) of the Acanthamoeba18S rDNA genus and sequencing was carried out in order to genotype the isolated strains of Acanthamoeba. Furthermore, the pathogenic potential of the strains was checked by performing thermotolerance and osmotolerance assays. Sequencing of the DF3 region resulted in the identification of genotype T4 in all the isolated strains. Moreover, most isolates were thermotolerant or both thermotolerant and osmotolerant and thus were classified as potentially pathogenic strains. To the best of our knowledge, this is the first report on the molecular characterization at the genotype level of Acanthamoeba strains in Venezuela.

  3. Area 51: How do Acanthamoeba invade the central nervous system?

    PubMed

    Siddiqui, Ruqaiyyah; Emes, Richard; Elsheikha, Hany; Khan, Naveed Ahmed

    2011-05-01

    Acanthamoeba granulomatous encephalitis generally develops as a result of haematogenous spread, but it is unclear how circulating amoebae enter the central nervous system (CNS) and cause inflammation. At present, the mechanisms which Acanthamoeba use to invade this incredibly well-protected area of the CNS and produce infection are not well understood. In this paper, we propose two key virulence factors: mannose-binding protein and extracellular serine proteases as key players in Acanthamoeba traversal of the blood-brain barrier leading to neuronal injury. Both molecules should provide excellent opportunities as potential targets in the rational development of therapeutic interventions against Acanthamoeba encephalitis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Seasonal distribution of potentially pathogenic Acanthamoeba species from drinking water reservoirs in Taiwan.

    PubMed

    Kao, Po-Min; Hsu, Bing-Mu; Hsu, Tsui-Kang; Liu, Jorn-Hon; Chang, Hsiang-Yu; Ji, Wen-Tsai; Tzeng, Kai-Jiun; Huang, Shih-Wei; Huang, Yu-Li

    2015-03-01

    In order to detect the presence/absence of Acanthamoeba along with geographical variations, water quality variations and seasonal change of Acanthamoeba in Taiwan was investigated by 18S ribosomal RNA (rRNA) gene TaqMan quantitative real-time PCR. Samples were collected quarterly at 19 drinking water reservoir sites from November 2012 to August 2013. Acanthamoeba was detected in 39.5 % (30/76) of the water sample, and the detection rate was 63.2 % (12/19) from samples collected in autumn. The average concentration of Acanthamoeba was 3.59 × 10(4) copies/L. For geographic distribution, the detection rate for Acanthamoeba at the northern region was higher than the central and southern regions in all seasons. Results of Spearman rank test revealed that heterotrophic plate count (HPC) had a negative correlation (R = -0.502), while dissolved oxygen (DO) had a positive correlation (R = 0.463) in summer. Significant differences were found only between the presence/absence of Acanthamoeba and HPC in summer (Mann-Whitney U test, P < 0.05). T2 and T4 genotypes of Acanthamoeba were identified, and T4 was the most commonly identified Acanthamoeba genotypes. The presence of Acanthamoeba in reservoirs presented a potential public health threat and should be further examined.

  5. Acanthamoeba spp. in Contact Lenses from Healthy Individuals from Madrid, Spain.

    PubMed

    Gomes, Thiago Dos Santos; Magnet, Angela; Izquierdo, Fernando; Vaccaro, Lucianna; Redondo, Fernando; Bueno, Sara; Sánchez, Maria Luisa; Angulo, Santiago; Fenoy, Soledad; Hurtado, Carolina; Del Aguila, Carmen

    2016-01-01

    Acanthamoeba keratitis (AK) is a painful and potentially blinding corneal infection caused by Acanthamoeba spp. In Madrid, environmental studies have demonstrated a high presence of these free-living amoebae in tap water. Since most of AK cases occur in contact lenses (CL) wearers with inadequate hygiene habits, the presence of Acanthamoeba in discarded CL has been studied and compared with other common etiological agents of keratitis, such as Pseudomonas aeruginosa and Staphylococcus aureus. One hundred and seventy-seven healthy individuals from Madrid contributed their discarded CL and answered a questionnaire on hygiene habits. DNA was extracted from the CL solution and analyzed by real-time PCR for Acanthamoeba, Pseudomonas aeruginosa and Staphylococcus aureus. These CL and their solutions were also cultured on non-nutrient agar to isolate Acanthamoeba. Among the 177 samples, Acanthamoeba DNA was detected in 87 (49.2%), P. aeruginosa DNA in 14 (7.9%) and S. aureus DNA in 19 (10.7%). Cultivable amoebae, however, were observed in only one sample (0.6%). This isolate was genotyped as T4. The habits reported by this CL owner included some recognized risk factors for AK, but in this study only the practice of "not cleaning the CL case" presented some statistical significant association with Acanthamoeba DNA presence. Detection of the investigated bacterial DNA did not demonstrate statistical significant association with the studied practices, but the presence of P. aeruginosa revealed a possible inhibition of Acanthamoeba in these samples. The PCR results suggest a high presence of Acanthamoeba spp. in healthy CL wearers from Madrid, but we can assume that CL solutions are properly disinfecting the CL since only 1.1% of the positive PCR samples correspond to viable amoebae and, after four years, only one participant reported stronger ocular problems. Nevertheless, more studies are necessary to corroborate this hypothesis.

  6. ACANTHAMOEBA SP.S-11 PHAGOCYTOTIC ACTIVITY ON MYCOBACTERIUM LEPRAE IN DIFFERENT NUTRIENT CONDITIONS.

    PubMed

    Paling, Sepling; Wahyuni, Ratna; Ni'matuzahroh; Winarni, Dwi; Iswahyudi; Astari, Linda; Adriaty, Dinar; Agusni, Indropo; Izumi, Shinzo

    2018-01-01

    Mycobacterium leprae ( M. leprae ) is a pathogenic bacterium that causes leprosy. The presence of M. leprae in the environment is supported by microorganisms that act as the new host for M. leprae . Acanthamoeba 's potential to be a host of M. leprae in the environment. Acanthamoeba sp. is Free Living Amoeba (FLA) that classified as holozoic, saprophytic, and saprozoic. The existence of nutrients in the environment influence Acanthamoeba ability to phagocytosis or pinocytosis. This study is aimed to determine Acanthamoeba sp.S-11 phagocytic activity to Mycobacterium leprae ( M. leprae ) which cultured in non-nutrient media and riched-nutrient media. This research conducted by culturing Acanthamoeba sp.S-11 and M. leprae on different nutrient media conditions. M. leprae intracellular DNA were isolated and amplified by M. leprae specific primers through Real Time PCR (Q-PCR). The results showed that Acanthamoeba co-cultured on non-nutrient media were more active to phagocyte M. leprae than on rich-nutrient media. The use of non-nutrient media is recommended to optimize Acanthamoeba sp. phagocytic activity to M. leprae .

  7. The assembly of GM1 glycolipid- and cholesterol-enriched raft-like membrane microdomains is important for giardial encystation.

    PubMed

    De Chatterjee, Atasi; Mendez, Tavis L; Roychowdhury, Sukla; Das, Siddhartha

    2015-05-01

    Although encystation (or cyst formation) is an important step of the life cycle of Giardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1- and cholesterol-enriched LRs were isolated from Giardia by density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest that Giardia is able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Acanthamoeba keratitis: an emerging disease gathering importance worldwide?

    PubMed

    Lorenzo-Morales, Jacob; Martín-Navarro, Carmen María; López-Arencibia, Atteneri; Arnalich-Montiel, Francisco; Piñero, José E; Valladares, Basilio

    2013-04-01

    Acanthamoeba keratitis (AK) is increasingly being recognized as a severe sight-threatening ocular infection worldwide. Although contact lens wear is the leading risk factor for AK, Acanthamoeba parasites are also an important cause of keratitis in non-contact lens wearers. Diagnosis of AK is challenging, and the available treatments are lengthy and not fully effective against all strains. The pathogenesis of Acanthamoeba is still under study, and the identification of the key factors involved in this process should be useful for the development of fully effective therapies. This review focuses on recent developments on AK pathogenesis and diagnosis as well as novel strategies for the evaluation of anti-amoebic agents that could be applied in the near future against these pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

    PubMed Central

    Merino, María C.; Zamponi, Nahuel; Vranych, Cecilia V.; Touz, María C.; Rópolo, Andrea S.

    2014-01-01

    Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation. PMID:25058047

  10. Vectorial role of Acanthamoeba in Legionella propagation in water for human use.

    PubMed

    Magnet, A; Peralta, R H S; Gomes, T S; Izquierdo, F; Fernandez-Vadillo, C; Galvan, A L; Pozuelo, M J; Pelaz, C; Fenoy, S; Del Águila, C

    2015-02-01

    Legionella spp. is the causative agent of Legionnaires' disease and is transmitted through aerosols emanating from man-made water systems. Legionella resistance to water treatments has been related to its association with environmental amoebae such as Acanthamoeba. Due to the high presence of this protozoon in Spain and the high rate of notification of Legionnaires' disease of this country, the aims of this work were to study the coexistence of these bacteria and protozoa in water as well as their interaction. The usefulness of Acanthamoeba co-culture for the isolation of environmental Legionella was also studied. For this purpose, 70 water samples were collected in 2011 from three Drinking Water Treatment Plants, three Wastewater Treatment Plants and five Natural Pools in Spain. Acanthamoeba was found by PCR in 87.1% (61/70) samples and, by culture in 85.7% (60/70) samples. Legionella was detected by PCR in 58.6% (41/70) of water samples, in 5.7% (4/70) by agar culture and 75.7% (53/70) by Acanthamoeba co-culture. From the 54 Acanthamoeba water isolates, Legionella was detected in 43 of them independently of Acanthamoeba's genotype (T3, T4 and T11). Legionella feeleii, Legionella birminghamiensis, Legionella gresilensis/berliardensis, Legionella fairfieldensis, Legionella drozanski and Legionella falloni were identified. In conclusion, our results showed that environmental Acanthamoeba is infected by Legionella to a high percentage, and due to its ubiquity, high resistance and its pathogenic potential per se, new methods for its elimination should be studied. Also, the high effectivity of Acanthamoeba co-culture for Legionella detection has been shown. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Pathobiology and Immunobiology of Acanthamoeba Keratitis: Insights from Animal Models
.

    PubMed

    Neelam, Sudha; Niederkorn, Jerry Y

    2017-06-01

    Acanthamoeba keratitis (AK) is a rare but sight-threatening disease caused by pathogenic species of Acanthamoeba . Despite its ubiquitous nature, the incidence of AK is relatively low compared to other forms of infectious keratitis. Although contact lens wear is a major risk factor, exposure to contaminated water and ocular trauma are also associated with AK. Once a patient develops AK the prognosis is very poor unless an aggressive treatment regimen is initiated early. Some of the intriguing features of AK are the lack of immunological memory, resistance of the dormant cyst form to treatment, differences between the pathogenic strains and soil isolates of Acanthamoeba and the unique role of the innate immune system in controlling this disease. Understanding the series of steps involved in the pathogenesis of the disease and the host immune response against Acanthamoeba antigens is crucial for developing effective therapeutic strategies targeting the disease.

  12. Encystment of Azotobacter nigricans grown diazotrophically on kerosene as sole carbon source.

    PubMed

    García-Esquivel, Gabriela; Calva-Calva, Graciano; Ferrera-Cerrato, Ronald; Fernández-Linares, Luis Carlos; Vázquez, Refugio Rodríguez; Esparza-García, Fernando José

    2009-03-01

    Encystment of Azotobacter nigricans was induced by its diazotrophic cultivation on kerosene. Its growth and nitrogenase activity were affected by kerosene in comparison to cultures grown on sucrose. Electron microscopy of vegetative cells showed that when nitrogenase activity was higher and the poly-beta-hydroxybutyrate granules were not present to a significant extent, peripheral bodies were abundant. After 8 days of culture on kerosene, the presence of cysts with intracellular bunches of poly-beta-hydroxybutyrate granules was observed. Germination of cysts bears germinating multicelled yet unbroken capsule cysts with up to three cells inside. This is the first report of encystment induction of Azotobacter species grown on kerosene.

  13. Acanthamoeba keratitis in Scotland: risk factors for contact lens wearers.

    PubMed

    Seal, D V; Kirkness, C M; Bennett, H G; Peterson, M

    1999-01-01

    To investigate risk factors for Acanthamoeba keratitis amongst contact lens wearers in Scotland. Patients with Acanthamoeba keratitis in the Scottish study, all of whom wore contact lenses, were compared with 46 healthy asymptomatic contact lens-wearing controls. They were all visited at home for contact lens and environmental microbiological sampling. In addition, all 288 optical practices in the West of Scotland were polled for contact lens types and disinfecting solutions sold in 1995, and a sample, each of whom fitted more than 500 contact lenses per year, were polled for a second time. Independently, a poll was commissioned by the Eyecare Information Service in July/August 1995 to estimate the numbers of contact lens wearers in Scotland and the UK. Industry was polled for numbers of each contact lens disinfecting regimen sold in Scotland in 1995. West of Scotland, UK. All contact lens wearers among the 3 million population of the West of Scotland Health Board Areas. Risk factors for Acanthamoeba infection and recommendations for its prevention. When Acanthamoeba infection occurred, patients' home water systems were frequently (54%) found to be colonised by this amoeba. Patients more frequently washed their storage cases in tap water than controls (P<0.05) with resulting contamination, kept storage cases wet rather than air drying them (P<0.05), and had coliform bacteria cultured from the storage case (P<0.05) and had viable Acanthamoeba within the storage case (P<0.0001). Overall, patients were found to have significantly more risk factors than controls (P<0.0001). The noncompliant use of chlorine tablet disinfection, or failure to disinfect contact lenses at all, was associated with increased risk (P<0.05). Ionic high water content contact lenses (FDA group 4 material), when used without disinfection or with non-compliant use of low chlorine (Soflab) tablet-based disinfection, were associated with increased risk of Acanthamoeba infection (P<0.05). In log

  14. The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation

    PubMed Central

    2013-01-01

    Background Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion. Results We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains. Conclusions Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens. PMID:23889909

  15. Detection and molecular characterization of Acanthamoeba spp. in stray cats from Madrid, Spain.

    PubMed

    Montoya, Ana; Miró, Guadalupe; Saugar, José María; Fernández, Beatriz; Checa, Rocío; Gálvez, Rosa; Bailo, Begoña; Marino, Valentina; Piñero, José E; Lorenzo-Morales, Jacob; Fuentes, Isabel

    2018-05-01

    Acanthamoeba spp. is a widespread protozoan that has been isolated from air, dust, soil, water and biological samples. An opportunistic pathogen of humans and animals, it may cause ocular keratitis, encephalitis, and even multisystem disease. The frequency of Acanthamoeba in animals is unknown. The aim of present study was determine the presence of Acanthamoeba spp. in immunocompromised stray cats - animals possibly more likely to harbour the infection given their immunocompromised status and frequenting of contaminated environments. Of 307 cats examined, 55 were positive for feline immunodeficiency virus and/or feline leukaemia virus and therefore included in the study. Corneal scrapings were obtained to isolate Acanthamoeba spp. by culture and molecular detection by conventional and real time PCR. None of the samples examined directly by molecular methods were positive for Acanthamoeba spp. However, two (3.6%) cases of the cultured samples provided positive results, which were confirmed by subsequent molecular analysis. Sequencing assigned one isolate to genotype T4 and the other to T2. Since Acanthamoeba spp. may also infect animals and humans, the present findings may raise some public health and veterinary concerns. Copyright © 2018. Published by Elsevier Inc.

  16. The association of contact lens solution use and Acanthamoeba keratitis

    PubMed Central

    Joslin, Charlotte E.; Tu, Elmer Y.; Shoff, Megan E.; Booton, Gregory C.; Fuerst, Paul A.; McMahon, Timothy T.; Anderson, Robert J.; Dworkin, Mark S.; Sugar, Joel; Davis, Faith G.; Stayner, Leslie T.

    2009-01-01

    Purpose Diagnosis of Acanthamoeba keratitis, a rare but serious corneal infection, has recently increased significantly at the University of Illinois at Chicago (UIC) Cornea Service. The purpose is to investigate Acanthamoeba keratitis risk factors. Design Retrospective case-control study. Methods Setting University, tertiary care hospital. Patients Fifty-five Acanthamoeba keratitis cases with contact lens use were diagnosed between May 1, 2003 and September 15, 2006. Clinic-matched controls with contact lens use were recruited. Subjects completed surveys targeting lens hygiene, contact lens solution use, and water exposure. Main Outcome Measure Acanthamoeba keratitis. Results Thirty-nine (73.6%) cases and 113 (65.3%) controls participated; 38 cases had complete contact lens data. Thirty-five of 38 cases (92.1%) and 47 of 100 controls (47.0%) used soft lenses. Analysis was performed on 30 cases and 39 controls with matched pairs with soft lens use. Exclusive use of AMO Complete MoisturePlus Multi-Purpose Solution was independently associated with Acanthamoeba keratitis in multivariable analysis (55.2% vs. 10.5%; OR, 16.67; 95% CI, 2.11–162.63; p = 0.008). However, 38.8% of cases reported no use of AMO Complete MoisturePlus Multi-Purpose Solution or used it in combination with other solutions. Although not statistically significant, additional hygiene-related variables (solution ‘reuse’, lack of ‘rubbing’, and showering with lenses) suggest a pattern of risk,. Conclusions AMO Complete MoisturePlus Multi-Purpose Solution use is independently associated with Acanthamoeba keratitis among soft contact lens users. However, it does not explain all cases, suggesting additional factors. Further research into environmental risk factors and hygiene practices is warranted, especially considering this is the second outbreak of an atypical, contact lens-related infection. PMID:17588524

  17. A multisystemic Acanthamoeba infection in a dog in Tenerife, Canary Islands, Spain.

    PubMed

    Valladares, María; Reyes-Batlle, María; Mora-Peces, Inmaculada; Martín-Navarro, Carmen M; López-Arencibia, Atteneri; Dorta-Gorrín, Alexis; Comyn-Afonso, Estefanía; Martínez-Carretero, Enrique; Maciver, Sutherland K; Piñero, José E; Valladares, Basilio; Lorenzo-Morales, Jacob

    2014-10-15

    A 22-month-old male Spanish water dog was hospitalized after its physical examination revealed fever and movement difficulty. After 24h, the dog was found to have a high fever (39.5 °C) and was treated empirically with doxycycline/ciprofloxacin. At 48 h, after submission the fever rose to 41 °C and the animal presented with a stiff neck and dehydration. Peripheral blood and cerebrospinal fluid (CSF) were sampled and trophozoites with an Acanthamoeba-like morphology were observed in the CSF. PCR specific for Acanthamoeba, Naegleria fowleri and Balamuthia mandrillaris were performed and the CSF sample found positive for Acanthamoeba. Lungs, kidney, liver and spleen samples were collected post mortem. All collected organ samples were positive for Acanthamoeba by PCR, thus confirming a multisystemic infection. Water samples taken at a suspected site of infection yielded an almost identical PCR fragment to those of the clinical samples, indicating that this was probably where the infection originated. This is the first report of a fatal case of Acanthamoeba disseminated infection in a dog in Spain. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Genotypic, physiological, and biochemical characterization of potentially pathogenic Acanthamoeba isolated from the environment in Cairo, Egypt.

    PubMed

    Tawfeek, Gihan Mostafa; Bishara, Sawsan Abdel-Hamid; Sarhan, Rania Mohammad; ElShabrawi Taher, Eman; ElSaady Khayyal, Amira

    2016-05-01

    Acanthamoebae are the most common opportunistic amphizoic protozoa that cause life-threatening granulomatous amoebic encephalitis in immunocompromised individuals and sight-threatening amoebic keratitis (AK) in contact lens wearers. The present work aimed to determine the presence of Acanthamoeba isolates in different environmental sources: water, soil, and dust in Cairo, Egypt and to characterize the pathogenic potential of the isolated Acanthamoeba using physiological and biochemical assays as well as determination of the genotypes in an attempt to correlate pathogenicity with certain genotypes. The study included the collection of 22 corneal scrapings from patients complaining of symptoms and signs indicative of acanthamoeba keratitis (AK) and 75 environmental samples followed by cultivation on non-nutrient agar plates preseeded with E. coli. Positive samples for Acanthamoeba were subjected to osmo- and thermo-tolerance assays and zymography analysis. Potentially pathogenic isolates were subjected to PCR amplification using genus-specific primer pair. Isolates were classified at the genotype level based on the sequence analysis of Acanthamoeba 18S rRNA gene (diagnostic fragment 3). The total detection rate for Acanthamoeba in environmental samples was 33.3 %, 31.4 % in water, 40 % in soil, and 20 % in dust samples. Three and two Acanthamoeba isolates from water and soil sources, respectively, had the potential for pathogenicity as they exhibited full range of pathogenic traits. Other 12 isolates were designated as weak potential pathogens. Only ten of the environmental isolates were positive in PCR and were classified by genotype analysis into T4 genotype (70 %), T3 (10 %) and T5 (20 %). Potential pathogens belonged to genotypes T4 (from water) and T5 (from soil) while weak potential pathogens belonged to genotypes T3 (from water) and T4 (from water and soil). Additionally, T7 genotype was isolated from keratitis patients. There is a considerable

  19. Acanthamoeba keratitis: current status and urgent research priorities.

    PubMed

    Khan, Naveed Ahmed; Anwar, Ayaz; Siddiqui, Ruqaiyyah

    2018-05-10

    First discovered in the early 1970s, Acanthamoeba keratitis has remained a major eye infection and presents a significant threat to the public health, especially in developing countries. The aim is to present a timely review of our current understanding of the advances made in this field in a comprehensible manner and includes novel concepts and provides clear directions for immediate research priorities. We undertook a search of bibliographic databases for peer-reviewed research literature and also summarized our published results in this field. The present review focuses on novel diagnostic and therapeutic strategies in details which can provide access to management and treatment of Acanthamoeba keratitis. This coupled with the recently available genome sequence information together with high throughput genomics technology and innovative approaches should stimulate interest in the rational design of preventative and therapeutic measures. Current treatment of Acanthamoeba keratitis is problematic and often leads to infection recurrence. Better understanding of diagnosis, pathogenesis, pathophysiology and therapeutic regimens, would lead to novel strategies in treatment and prophylaxis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Viability and morphological changes of Acanthamoeba spp. cysts after treatment with Effective microorganisms (EM).

    PubMed

    Sampaotong, Tanitta; Lek-Uthai, Usa; Roongruangchai, Jantima; Roongruangchai, Kosol

    2016-06-01

    Acanthamoeba is a free-living opportunistic protozoan parasite that is found in diverse environments. It can cause keratitis, mostly related to inappropriate use of contact lenses, as well as life threatening diseases including encephalitis, disseminated sinusitis, and skin ulcers. This study investigated morphological changes and fine structures of the cyst form of Acanthamoeba spp. after treatment with effective microorganisms (EM™) using light and scanning electron microscopies. Acanthamoeba cysts treated with 1:2, 1:4, 1:6, and undiluted EM™ showed higher percentages of non-viable cysts than those treated with 1:8, 1:10, 1:100, 1:200, and 1:400 EM™ and at 5 days post-treatment developed from cystic stage to trophozoite stage. Acanthamoeba cysts treated at concentrations of 1:2, 1:4, 1:6, and undiluted EM™ exhibited cytoplasmic clumping and shrinkage of amoeba cells away from cyst walls. The effective EM™ concentration lethal to Acanthamoeba spp. cyst could provide information to monitor the environmental control system.

  1. Microscopic diffusion in hydrated encysted eggs of brine shrimp

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mamontov, Eugene

    We have studied microscopic diffusion of water in fully hydrated encysted eggs of brine shrimp (Artemia). We utilized quasielastic neutron scattering. Dry eggs of brine shrimp were rehydrated using (1) water without additives, (2) eutectic mixture of water and dimethyl sulfoxide, and (3) a concentrated aqueous solution of lithium chloride. Despite the complexity of the hydrated multicellular organism, measurable microscopic diffusivity of water is rather well defined. Pure hydration water in eggs exhibits freezing temperature depression, whereas hydration water in eggs mixed with dimethyl sulfoxide or lithium chloride does not crystallize at all. The characteristic size of the voids occupiedmore » by water or aqueous solvents in hydrated brine shrimp eggs is between 2 and 10 nm. Those voids are accessible to co-solvents such as dimethyl sulfoxide and lithium chloride. There is no evidence of intracellular water in the hydrated eggs. The lack of intracellular water in the fully hydrated (but still under arrested development) state must be linked to the unique resilience against adverse environmental factors documented not only for the anhydrous, but also hydrated encysted eggs of brine shrimp.« less

  2. Microscopic diffusion in hydrated encysted eggs of brine shrimp

    DOE PAGES

    Mamontov, Eugene

    2017-05-24

    We have studied microscopic diffusion of water in fully hydrated encysted eggs of brine shrimp (Artemia). We utilized quasielastic neutron scattering. Dry eggs of brine shrimp were rehydrated using (1) water without additives, (2) eutectic mixture of water and dimethyl sulfoxide, and (3) a concentrated aqueous solution of lithium chloride. Despite the complexity of the hydrated multicellular organism, measurable microscopic diffusivity of water is rather well defined. Pure hydration water in eggs exhibits freezing temperature depression, whereas hydration water in eggs mixed with dimethyl sulfoxide or lithium chloride does not crystallize at all. The characteristic size of the voids occupiedmore » by water or aqueous solvents in hydrated brine shrimp eggs is between 2 and 10 nm. Those voids are accessible to co-solvents such as dimethyl sulfoxide and lithium chloride. There is no evidence of intracellular water in the hydrated eggs. The lack of intracellular water in the fully hydrated (but still under arrested development) state must be linked to the unique resilience against adverse environmental factors documented not only for the anhydrous, but also hydrated encysted eggs of brine shrimp.« less

  3. [Development of a liquid fermentation system and encystment for a nitrogen-fixing bacterium strain having biofertilizer potential].

    PubMed

    Camelo-Rusinque, Mauricio; Moreno-Galván, Andrés; Romero-Perdomo, Felipe; Bonilla-Buitrago, Ruth

    The indiscriminate use of chemical fertilizers has contributed to the deterioration of the biological, physical and chemical properties of the soil, resulting in the loss of its productive capacity. For this reason, the use of biofertilizers has emerged as a technological alternative. The objective of this research was to develop a suitable liquid fermentation system and encystment for the multiplication of Azotobacter chroococcum AC1 strain, a bacterium employed in a biofertilizer formulation produced at present by CARPOICA, Colombia. Sequential statistical designs were used to determine the conditions in the fermentation system. The interaction between agitation, aeration and pH was evaluated on the viable biomass (CFU/ml) of AC1. In addition, the encystment ability of the strain was evaluated using two encystment agents and the potential plant growth-promoting rhizobacteria (PGPR) activity was assessed by different techniques, such as nitrogen fixation by ARA, phosphate solubilization by the phospho-molybdenum-blue reaction and indolic compound production by colorimetric reaction using the Salkowski reagent. Results showed significant effects (p<0.05) on the viable biomass in the three conditions (pH, aeration and agitation) tested individually, in one dual interaction and one tripartite interaction, were demonstrated to have a positive effect on the response variable aeration and agitation. The addition of the two encystment agents evaluated, AE01 and AE02, demonstrated the ability of AC1 to form cysts under stress conditions. Likewise, fermentation and encystment conditions did not affect the biological activities tested. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  4. Taken to the limit--Is desiccation stress causing precocious encystment of trematode parasites in snails?

    PubMed

    O'Dwyer, Katie; Poulin, Robert

    2015-12-01

    When hosts experience environmental stress, the quantity and quality of resources they provide for parasites may be diminished, and host longevity may be decreased. Under stress, parasites may adopt alternative strategies to avoid fitness reductions. Trematode parasites typically have complex life cycles, involving asexual reproduction in a gastropod first intermediate host. A rare phenomenon, briefly mentioned in the literature, and termed 'precocious encystment' involves the next stage in the parasites' life cycle (metacercarial cyst) forming within the preceding stage (redia), while still inside the snail. In the trematode Parorchis sp. NZ using rocky shore snails exposed to long periods outside water, we hypothesised that this might be an adaptive strategy against desiccation, preventing parasite emergence from the snail. To test this, we first investigated the effect of prolonged desiccation on the survival of two species of high intertidal snails. Secondly, we measured the reproductive output (cercarial production) of the parasite under wet and dry conditions. Finally, we quantified the influence of desiccation stress on the occurrence of precocious encystment. Snail mortality was higher under dry conditions, indicating stress, and it was somewhat exacerbated for infected snails. Parasite reproductive output differed between wet and dry conditions, with parasites of snails kept in dry conditions producing more cercariae when placed in water. Little variation was observed in the occurrence of precocious encystment, although some subtle patterns emerged. Given the stresses associated with living in high intertidal environments, we discuss precocious encystment as a possible stress response in this trematode parasite. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Identification of 18S ribosomal DNA genotype of Acanthamoeba from hot spring recreation areas in the central range, Taiwan

    NASA Astrophysics Data System (ADS)

    Hsu, Bing-Mu; Ma, Po-Hua; Liou, Tai-Sheng; Chen, Jung-Sheng; Shih, Feng-Cheng

    2009-04-01

    SummaryAcanthamoeba is a free-living amoebae ubiquitous to aquatic environments. Within the genus a few species are recognized as opportunistic potential human pathogens, which cause granulomatous amoebic encephalitis (GAE) and keratitis. Infections of keratitis are frequently reported through wearing lens while swimming in the non-disinfected aquatic environment. Contaminations in hot tubs, spas and public baths are also possible. As a result, in this study, we identified Acanthamoeba based on the PCR amplification with a genus-specific primer pair and investigated the distribution of Acanthamoeba at five hot spring recreation areas in central range, Taiwan. We gathered data on factors potentially associated with the pathogen's distribution, including various sampling sites, aquatic environment, physical and microbiological water quality parameters. Spring water was collected from 55 sites and Acanthamoeba was detected in 9 (16.4%). The most frequently detected was Acanthamoeba griffini, followed by Acanthamoeba jacobsi. Legionella were detected in 18 (32.7%) of the sites sampled in this study. The species of Legionella identified included Legionella pneumophila serotype 6, serotype 1, and Legionella erythra. Overall, 9.1% of the samples contained both Acanthamoeba and Legionella. The prevalence of Acanthamoeba was contrary to the levels of microbiological indicators recommended by Taiwan CDC, and no significant differences (Mann-Whitney U test, P < 0.05) were observed between the presence/absence of Acanthamoeba and water quality parameters. Results of this survey confirm the existence of Acanthamoeba in Taiwan spring recreation areas. Acanthamoeba, the organism responsible for the majority of Acanthamoeba keratitis and can serve as vehicles for facultative pathogens, should be considered a potential threat for health associated with human activities in spring recreation areas of Taiwan.

  6. Surveillance and Molecular Identification of Acanthamoeba and Naegleria Species in Two Swimming Pools in Alexandria University, Egypt

    PubMed Central

    AL-HERRAWY, Ahmad Z.; KHALIL, Mahmoud I.; EL-SHERIF, Soheir S.; OMAR, Fatima A. E.; LOTFY, Wael M.

    2017-01-01

    Background: Swimming in contaminated water was reported to be associated with Acanthamoeba and N. fowleri human infections. The present study was carried out with the aim of isolation and identification of the different species of Acanthamoeba and Naegleria from two swimming pools in Alexandria University. Methods: Samples were collected from the swimming pools of Alexandria University Stadium and Faculty of Agriculture-Alexandria University during the period from May 2012 to April 2013. Results: Free-living amoebae were prevalent in the collected samples. Molecular characterization confirmed the identity of ten Acanthamoeba isolates and seven Naegleria isolates. Acanthamoeba T3, T4, T5, T11 and T15 genotypes were identified. Acanthamoeba T4 was the most prevalent genotype. Conclusion: The relatively high prevalence of Acanthamoeba, especially genotype T4, indicates the presence of a health hazard to swimmers particularly those wearing contact lenses. Naegleria fowleri was not found during the present study. PMID:28761479

  7. Transcriptomic Profiling of High-Density Giardia Foci Encysting in the Murine Proximal Intestine

    PubMed Central

    Pham, Jonathan K.; Nosala, Christopher; Scott, Erica Y.; Nguyen, Kristofer F.; Hagen, Kari D.; Starcevich, Hannah N.; Dawson, Scott C.

    2017-01-01

    Giardia is a highly prevalent, understudied protistan parasite causing significant diarrheal disease worldwide. Its life cycle consists of two stages: infectious cysts ingested from contaminated food or water sources, and motile trophozoites that colonize and attach to the gut epithelium, later encysting to form new cysts that are excreted into the environment. Current understanding of parasite physiology in the host is largely inferred from transcriptomic studies using Giardia grown axenically or in co-culture with mammalian cell lines. The dearth of information about the diversity of host-parasite interactions occurring within distinct regions of the gastrointestinal tract has been exacerbated by a lack of methods to directly and non-invasively interrogate disease progression and parasite physiology in live animal hosts. By visualizing Giardia infections in the mouse gastrointestinal tract using bioluminescent imaging (BLI) of tagged parasites, we recently showed that parasites colonize the gut in high-density foci. Encystation is initiated in these foci throughout the entire course of infection, yet how the physiology of parasites within high-density foci in the host gut differs from that of cells in laboratory culture is unclear. Here we use BLI to precisely select parasite samples from high-density foci in the proximal intestine to interrogate in vivo Giardia gene expression in the host. Relative to axenic culture, we noted significantly higher expression (>10-fold) of oxidative stress, membrane transporter, and metabolic and structural genes associated with encystation in the high-density foci. These differences in gene expression within parasite foci in the host may reflect physiological changes associated with high-density growth in localized regions of the gut. We also identified and verified six novel cyst-specific proteins, including new components of the cyst wall that were highly expressed in these foci. Our in vivo transcriptome data support an

  8. Transcriptomic Profiling of High-Density Giardia Foci Encysting in the Murine Proximal Intestine.

    PubMed

    Pham, Jonathan K; Nosala, Christopher; Scott, Erica Y; Nguyen, Kristofer F; Hagen, Kari D; Starcevich, Hannah N; Dawson, Scott C

    2017-01-01

    Giardia is a highly prevalent, understudied protistan parasite causing significant diarrheal disease worldwide. Its life cycle consists of two stages: infectious cysts ingested from contaminated food or water sources, and motile trophozoites that colonize and attach to the gut epithelium, later encysting to form new cysts that are excreted into the environment. Current understanding of parasite physiology in the host is largely inferred from transcriptomic studies using Giardia grown axenically or in co-culture with mammalian cell lines. The dearth of information about the diversity of host-parasite interactions occurring within distinct regions of the gastrointestinal tract has been exacerbated by a lack of methods to directly and non-invasively interrogate disease progression and parasite physiology in live animal hosts. By visualizing Giardia infections in the mouse gastrointestinal tract using bioluminescent imaging (BLI) of tagged parasites, we recently showed that parasites colonize the gut in high-density foci. Encystation is initiated in these foci throughout the entire course of infection, yet how the physiology of parasites within high-density foci in the host gut differs from that of cells in laboratory culture is unclear. Here we use BLI to precisely select parasite samples from high-density foci in the proximal intestine to interrogate in vivo Giardia gene expression in the host. Relative to axenic culture, we noted significantly higher expression (>10-fold) of oxidative stress, membrane transporter, and metabolic and structural genes associated with encystation in the high-density foci. These differences in gene expression within parasite foci in the host may reflect physiological changes associated with high-density growth in localized regions of the gut. We also identified and verified six novel cyst-specific proteins, including new components of the cyst wall that were highly expressed in these foci. Our in vivo transcriptome data support an

  9. Acanthamoeba keratitis: the role of domestic tap water contamination in the United Kingdom.

    PubMed

    Kilvington, Simon; Gray, Trevor; Dart, John; Morlet, Nigel; Beeching, John R; Frazer, David G; Matheson, Melville

    2004-01-01

    The incidence of acanthamoeba keratitis (AK) in the UK is some 15 times that in the United States and seven times that in Holland. To investigate reasons for this higher frequency, a study of the role of domestic tap water as a potential source of AK was undertaken. Tap outlets from the homes of 27 patients with culture-proven AK were sampled and cultured for free-living amoebae (FLA). For all Acanthamoeba isolates, mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) and cytochrome oxidase (cox 1/2) sequence typing was performed to determine the similarity between corneal and tap water isolates. FLA, including Acanthamoeba, were isolated from 24 (89%) of 27 homes, and the presence within the homes varied significantly with tap water temperature and location: 19 (76%) of 25 bathroom sink cold taps sampled compared with 6 (24%) of 25 hot and 9 (47%) of 19 kitchen cold taps compared with 3 (16%) of 19 of hot kitchen taps. Acanthamoeba were isolated from 8 (30%) of 27 homes (five bathroom sink cold taps, one cloakroom cold tap, one bath, and one bedroom sink mixer [hot/cold] taps). In six cases, identical Acanthamoeba mtDNA profiles were found for the clinical and home tap water isolates. In keeping with UK plumbing practice, 24 of 27 homes had internal roof water storage tanks to supply domestic taps, but the mains fed the kitchen cold tap. Water storage tanks promote colonization of domestic water with FLA, including Acanthamoeba, and hence increase the risk of AK. This accounts for the significantly greater incidence of AK in the UK and supports advice to avoid using tap water in contact lens care routines.

  10. Broad spectrum antimicrobial activity of melimine covalently bound to contact lenses.

    PubMed

    Dutta, Debarun; Cole, Nerida; Kumar, Naresh; Willcox, Mark D P

    2013-01-07

    To develop a stable antimicrobial contact lens, which is effective against the International Organization for Standardization (ISO) panel microorganisms, Acanthamoeba castellanii and drug resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus. Melimine was covalently incorporated into etafilcon A lenses. The amount of peptide present on the lens surface was quantified using amino acid analysis. After coating, the heat stability (121°C), lens surface hydrophobicity (by captive bubble), and in vitro cytotoxicity to mouse L929 cells of the lenses were investigated. Antimicrobial activity against the micro-organisms was evaluated by viable plate count and fluorescence microscopy, measuring the proportion of cell death compared with control lenses with no melimine. The most effective concentration was determined to be 152 ± 44 μg lens(-1) melimine on the lens surface. After coating, lenses were relatively hydrophilic and were nontoxic to mammalian cells. The activity remained high after autoclaving (e.g., 3.1, 3.9, 1.2, and 1.0 log inhibition against P. aeruginosa, S. aureus, A. castellanii, and Fusarium solani, respectively). Fluorescence microscopy confirmed significantly reduced (P < 0.001) adhesion of viable bacteria to melimine contact lenses. Viable count confirmed that lenses were active against all the bacteria and fungi from the ISO panel, Acanthamoeba and gave at least 2 log inhibition against all the multidrug resistant S. aureus and P. aeruginosa strains. Melimine may offer excellent potential for development as a broad spectrum antimicrobial coating for contact lenses, showing activity against all the bacterial and fungal ISO panel microorganisms, Acanthamoeba, and antibiotic resistant strains of P. aeruginosa and S. aureus.

  11. [A new strategy for enhancing acanthamoebicidal activity with synthesis of nanoflower of Laurocerausus officinalis Roemer (cherry laurel) fruit extracts].

    PubMed

    Baldemir, Ayşe; Karaman, Ülkü; Yusufbeyoğlu, Sadi; Eken, Ayşe; Ildız, Nilay; İlgün, Selen; Çolak, Cemil; Kaçmaz, Gamze; Öçsoy, İsmail; Çankaya, Soner

    2018-01-01

    Pathogenic Acanthamoeba species often cause infection known as Acanthamoeba keratitis among people who use contact lenses. It is a type of infection that can result in corneal ulceration, visual loss or even blindness, if not treated. There are various therapeutic options available in the treatment of Acanthamoeba infections but they are usually tough treatments with limited efficacy. For instance, hydrogen peroxide (H 2 O 2 ) is a commonly used contact lens disinfectant which is effective against Acanthamoeba but it is toxic to the cornea. For these reasons, new and more efficacious treatment options are required for Acanthamoeba infections. In this context, plants are considered natural resources for the discovery of new drugs. Laurocerasus officinalis Roem. (cherry laurel) (Rosaceae) grows in Black Sea region; and it is known as "Taflan", "Laz kirazı" or "Karayemis". Local people are using the seeds against diabetes, while the fruits are consuming as food, and used fordiuretic and passing kidney stones. It has also been reported that the seeds of the cherry laurel are used as an antiparasitic agent in this area. The aim of the study was to confirm the traditionally use of antiparasitic activity of this fruit and to increase the potential effect by means of organic-inorganic hybrid synthesis. Total phenol contents of methanol extracts prepared from endocarp, mesocarp and seeds of the fruit were calculated. The effects of methanol extracts and nano flower (NFs) plants synthesized from these extracts on the proliferation of Acanthamoeba castellanii were investigated. Thus, for the first time, novel organic-inorganic nanobio-antiparasitic agents called NFs were produced from cherry laurel and the increase in the amoebicidal activity of the NFs was elucidated. The characterization of NFs were determined with Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectrometer (FT-IR) and Energy-Dispersive X-ray (EDX) techniques. In addition, the catalytic

  12. High occurrence of Acanthamoeba genotype T4 in soil sources from Bolívar State, Venezuela.

    PubMed

    Wagner, Carolina; Reyes-Batlle, María; Hernán, Aurora; Rojas, Elsy; Pérez, Gladymar; López-Arencibia, Atteneri; Sifaoui, Ines; Martínez-Carretero, Enrique; Piñero, José E; Valladares, Basilio; Lorenzo-Morales, Jacob

    2016-09-01

    Pathogenic strains of Acanthamoeba are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, twenty-seven soil samples were collected in the Bolivar State in Venezuela and checked for the presence of Acanthamoeba. Samples were cultivated onto 2% non-nutrient agar plates seeded with a layer of heat killed E. coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 51.8% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotype T4 in all samples. To the best of our knowledge, this is the first report of genotype T4 in soil sources from Venezuela. Further studies should be carried out in this State and in the country in order to determine the current occurrence of Acanthamoeba in Venezuelan environments.

  13. Isolation and genotyping of free-living environmental isolates of Acanthamoeba spp. from bromeliads in Southern Brazil.

    PubMed

    Landell, Melissa Fontes; Salton, Juliana; Caumo, Karin; Broetto, Leonardo; Rott, Marilise B

    2013-07-01

    Species of Acanthamoeba are frequently isolated from distinct environmental sources such as water, soil, dust and air. They are responsible to cause infections and disease in humans and animals. In addition, Acanthamoeba sp. are considered an important reservoir of bacteria, virus and fungi, which act as "Trojan horses" to protect these microorganisms of harsh environmental conditions. In this study, nine Acanthamoeba isolates from bromeliads phylloplane were identified based on the morphology of cyst and trophozoite forms. The genotype level was accessed by the sequence analysis of Acanthamoeba small-subunit rRNA gene. Genotypic characterization grouped five isolates in the genotype T2/T6, three in the T4 genotype and one in the genotype T16. The results obtained indicate that the genotype T2/T6 is common on phylloplane. To predict the pathogenic potential of the Acanthamoeba isolates, thermo and osmotolerance assays were employed, although all isolates were capable of surviving at temperatures of 37°C, other tests will be conducted in the future to determine the potential pathogenic of the isolates. Altogether, our results revealed the importance of the presence of Acanthamoeba associated with bromeliads in Rio Grande do Sul, Brazil, and the necessity for further studies to determine the environmental distribution and the role of these species. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. The Social Amoeba Polysphondylium pallidum Loses Encystation and Sporulation, but Can Still Erect Fruiting Bodies in the Absence of Cellulose

    PubMed Central

    Du, Qingyou; Schaap, Pauline

    2014-01-01

    Amoebas and other freely moving protists differentiate into walled cysts when exposed to stress. As cysts, amoeba pathogens are resistant to biocides, preventing treatment and eradication. Lack of gene modification procedures has left the mechanisms of encystation largely unexplored. Genetically tractable Dictyostelium discoideum amoebas require cellulose synthase for formation of multicellular fructifications with cellulose-rich stalk and spore cells. Amoebas of its distant relative Polysphondylium pallidum (Ppal), can additionally encyst individually in response to stress. Ppal has two cellulose synthase genes, DcsA and DcsB, which we deleted individually and in combination. Dcsa- mutants formed fruiting bodies with normal stalks, but their spore and cyst walls lacked cellulose, which obliterated stress-resistance of spores and rendered cysts entirely non-viable. A dcsa-/dcsb- mutant made no walled spores, stalk cells or cysts, although simple fruiting structures were formed with a droplet of amoeboid cells resting on an sheathed column of decaying cells. DcsB is expressed in prestalk and stalk cells, while DcsA is additionally expressed in spores and cysts. We conclude that cellulose is essential for encystation and that cellulose synthase may be a suitable target for drugs to prevent encystation and render amoeba pathogens susceptible to conventional antibiotics. PMID:25113829

  15. Encystment of parasitic freshwater pearl mussel (Margaritifera margaritifera) larvae coincides with increased metabolic rate and haematocrit in juvenile brown trout (Salmo trutta).

    PubMed

    Filipsson, Karl; Brijs, Jeroen; Näslund, Joacim; Wengström, Niklas; Adamsson, Marie; Závorka, Libor; Österling, E Martin; Höjesjö, Johan

    2017-04-01

    Gill parasites on fish are likely to negatively influence their host by inhibiting respiration, oxygen transport capacity and overall fitness. The glochidia larvae of the endangered freshwater pearl mussel (FPM, Margaritifera margaritifera (Linnaeus, 1758)) are obligate parasites on the gills of juvenile salmonid fish. We investigated the effects of FPM glochidia encystment on the metabolism and haematology of brown trout (Salmo trutta Linnaeus, 1758). Specifically, we measured whole-animal oxygen uptake rates at rest and following an exhaustive exercise protocol using intermittent flow-through respirometry, as well as haematocrit, in infested and uninfested trout. Glochidia encystment significantly affected whole-animal metabolic rate, as infested trout exhibited higher standard and maximum metabolic rates. Furthermore, glochidia-infested trout also had elevated levels of haematocrit. The combination of an increased metabolism and haematocrit in infested fish indicates that glochidia encystment has a physiological effect on the trout, perhaps as a compensatory response to the potential respiratory stress caused by the glochidia. When relating glochidia load to metabolism and haematocrit, fish with low numbers of encysted glochidia were the ones with particularly elevated metabolism and haematocrit. Standard metabolic rate decreased with substantial glochidia loads towards levels similar to those of uninfested fish. This suggests that initial effects visible at low levels of encystment may be countered by additional physiological effects at high loads, e.g. potential changes in energy utilization, and also that high numbers of glochidia may restrict oxygen uptake by the gills.

  16. Isolation and Genotyping of Acanthamoeba spp. as Neglected Parasites in North of Iran.

    PubMed

    Shokri, Azar; Sarvi, Shahabeddin; Daryani, Ahmad; Sharif, Mehdi

    2016-08-01

    Acanthamoeba, a free-living amoeba, is widely distributed in the environment, water sources, soil, dust, and air. It can cause keratitis in contact lens wearers with poor hygiene and also fatal granulomatous amebic encephalitis (GAE) in immunocompromised hosts. The aim of this study was to gain some insights into the distribution and genotypes of the potentially pathogenic species of Acanthamoeba present in water sources in north of Iran. Total 43 Acanthamoeba species were isolated from 77 water samples taken from different water sources within the Mazandaran province in Northern Iran (Sari city and suburbs). Isolates were identified based on cyst and trophozoite morphological characteristics as well genetics. PCR fragments corresponding to the small-subunit 18S rRNA gene were sequenced for 20 of 43 positive isolates. The results revealed that 83.3% of sequenced isolates belonged to the T4 genotype and the rest belonged to the T2 genotype. Our results indicated that Acanthamoeba is widely distributed in Sari city. As the incidence in Iran of amoebic keratitis has increased in recent years, the exact estimation of the prevalence of this amoeba and its predominant genotype may play a crucial role in prevention of the disease. Sari city has several rivers, seashores, and natural recreational amenities, which attract visitors during the year. This is the first report of Acanthamoeba genotypes from water sources in Sari city, Mazandaran province of Iran, and the results suggest that more attention is needed to protect the visiting population and immunocompromised individuals.

  17. Inhibition of 3-Hydroxy-3-Methylglutaryl–Coenzyme A Reductase and Application of Statins as a Novel Effective Therapeutic Approach against Acanthamoeba Infections

    PubMed Central

    Lorenzo-Morales, Jacob; Machin, Rubén P.; López-Arencibia, Atteneri; García-Castellano, José Manuel; de Fuentes, Isabel; Loftus, Brendan; Maciver, Sutherland K.; Valladares, Basilio; Piñero, José E.

    2013-01-01

    Acanthamoeba is an opportunistic pathogen in humans, whose infections most commonly manifest as Acanthamoeba keratitis or, more rarely, granulomatous amoebic encephalitis. Although there are many therapeutic options for the treatment of Acanthamoeba, they are generally lengthy and/or have limited efficacy. Therefore, there is a requirement for the identification, validation, and development of novel therapeutic targets against these pathogens. Recently, RNA interference (RNAi) has been widely used for these validation purposes and has proven to be a powerful tool for Acanthamoeba therapeutics. Ergosterol is one of the major sterols in the membrane of Acanthamoeba. 3-Hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase is an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, one of the precursors for the production of cholesterol in humans and ergosterol in plants, fungi, and protozoa. Statins are compounds which inhibit this enzyme and so are promising as chemotherapeutics. In order to validate whether this enzyme could be an interesting therapeutic target in Acanthamoeba, small interfering RNAs (siRNAs) against HMG-CoA were developed and used to evaluate the effects induced by the inhibition of Acanthamoeba HMG-CoA. It was found that HMG-CoA is a potential drug target in these pathogenic free-living amoebae, and various statins were evaluated in vitro against three clinical strains of Acanthamoeba by using a colorimetric assay, showing important activities against the tested strains. We conclude that the targeting of HMG-CoA and Acanthamoeba treatment using statins is a novel powerful treatment option against Acanthamoeba species in human disease. PMID:23114753

  18. Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pan, Hong; The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012; Wu, Xinyi, E-mail: xywu8868@163.com

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-{beta}. Black-Right-Pointing-Pointer Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. Black-Right-Pointing-Pointer Hypoxia inhibits Acanthamoeba-induced the activation of NF-{kappa}B and ERK1/2 in HCECs. Black-Right-Pointing-Pointer Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. Black-Right-Pointing-Pointer LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cellsmore » has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-{beta} (IFN-{beta}) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-{beta}. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-{kappa}B and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response

  19. Study of Gene Trafficking between Acanthamoeba and Giant Viruses Suggests an Undiscovered Family of Amoeba-Infecting Viruses.

    PubMed

    Maumus, Florian; Blanc, Guillaume

    2016-12-14

    The nucleocytoplasmic large DNA viruses (NCLDV) are a group of extremely complex double-stranded DNA viruses, which are major parasites of a variety of eukaryotes. Recent studies showed that certain unicellular eukaryotes contain fragments of NCLDV DNA integrated in their genome, when surprisingly many of these organisms were not previously shown to be infected by NCLDVs. These findings prompted us to search the genome of Acanthamoeba castellanii strain Neff (Neff), one of the most prolific hosts in the discovery of giant NCLDVs, for possible DNA inserts of viral origin. We report the identification of 267 markers of lateral gene transfer with viruses, approximately half of which are clustered in Neff genome regions of viral origins, transcriptionally inactive or exhibit nucleotide-composition signatures suggestive of a foreign origin. The integrated viral genes had diverse origin among relatives of viruses that infect Neff, including Mollivirus, Pandoravirus, Marseillevirus, Pithovirus, and Mimivirus However, phylogenetic analysis suggests the existence of a yet-undiscovered family of amoeba-infecting NCLDV in addition to the five already characterized. The active transcription of some apparently anciently integrated virus-like genes suggests that some viral genes might have been domesticated during the amoeba evolution. These insights confirm that genomic insertion of NCLDV DNA is a common theme in eukaryotes. This gene flow contributed fertilizing the eukaryotic gene repertoire and participated in the occurrence of orphan genes, a long standing issue in genomics. Search for viral inserts in eukaryotic genomes followed by environmental screening of the original viruses should be used to isolate radically new NCLDVs. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  20. Can artificial tears prevent Acanthamoeba keratitis? An in vitro approach.

    PubMed

    Magnet, Angela; Gomes, Thiago Santos; Pardinas, Carmen; Garcia de Blas, Natalia; Sadaba, Cruz; Carrillo, Eugenia; Izquierdo, Fernando; Del Castillo, José Manuel Benítez; Hurtado, Carolina; Del Aguila, Carmen; Fenoy, Soledad

    2018-01-22

    The use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation. Viability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry. Trypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane. The present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out.

  1. Relationship of tadpole stage to location of echinostome cercaria encystment and the consequences for tadpole survival

    USGS Publications Warehouse

    Schotthoefer, A.M.; Cole, Rebecca A.; Beasley, V.R.

    2003-01-01

    The effect of echinostome infections on the survival of Rana pipiens tadpoles was examined in relation to developmental stage of tadpoles. Individual tadpoles of Gosner stages 25, 27, 32–33, and 37–39 were exposed to 1 of 4 levels of cercariae (0, 20, 50, or 100). Only tadpoles at stage 25, the earliest stage infected, died within a 5-day experimental period. This stage-specific mortality rate could be explained, in part, by the stage-specific location of encystment of cercariae, which was documented in a separate experiment. In accordance with kidney development, cercariae predominately encysted in the pronephroi during early stages of tadpole development (stages 25 through 31–32) and only in the mesonephroi and associated ducts at later stages (stages 37 through 46). As the mesonephros develops, renal capacity presumably increases. Thus, tadpoles died only when metacercariae concentrated in the functional portion of the kidney with the most limited renal capacity. As tadpoles aged, they also became less susceptible to infections. On average, 69.5% of cercariae that were exposed to stage 25–26 tadpoles successfully encysted, compared with only 8.4% of cercariae exposed to stage 37–38 tadpoles. Exposures of metamorphic frogs (poststage 46) to cercariae revealed that these individuals can become infected with echinostomes. Collectively, our data highlight the host stage–dependent dynamics of tadpole–echinostome interactions.

  2. [Regulation of cortical cytoskeleton dynamics during migration of free-living amoebae].

    PubMed

    Kłopocka, Wanda; Redowicz, Maria Jolanta; Wasik, Anna

    2009-01-01

    Amoeba proteus and smaller by an order of magnitude (and evolutionary younger) Acanthamoeba castellanii have been for many years model cells for studies of amoeboidal (crawling) type of movement, characteristic also for some of metazoan cells such as fibroblasts, granulocytes and macrophages. Amoeboidal migration is indispensable of organization and dynamics of actin-based cytoskeleton. While there is a number of data on molecular mechanisms of motility of A. castellanii, there is very little known about bases of migration of A. proteus. Noteworthy, a large A. proteus (length approximately 600 microm) have been from over a century an object for studies on biology and physiology of cellular migration. This review describes the current knowledge on molecular aspects of force generation required for migration of these two amoebae and attempts to compare the functioning and regulation of actin cytoskeleton in these free-living unicellular species.

  3. Isolation of Acanthamoeba Spp. from Drinking Waters in Several Hospitals of Iran

    PubMed Central

    Bagheri, HR; Shafiei, R; Shafiei, F; Sajjadi, SA

    2010-01-01

    Background Acanthamoeba is an opportunistic amphizoic protozoan found in different water sources including swimming pool as well as in sewage. The aim of this study was to investigate the prevalence of Acanthamoeba in tap-water samples in Iran. Method In this descriptive cross-sectional study, 94 samples of cold and warm tap-water were collected from different wards of hospitals in 13 cities of Iran in 2007–2008. Free residual chlorine, pH, and temperature of samples were measured. After filtration through multipore nylon membrane, samples were cultured on non-nutrient agar. Then we investigated existence of Acanthamoeba by reverse contrast phase microscope. Results Acanthamoeba was found in 45 samples (48%). Thirty-four and 11 positive samples were collected from cold and warm tap water, respectively. The samples belonged to the category of 20–30°C temperature with 0–2 ppm free residual chlorine and pH 6–7.4 showed the most coincidence to the positive cases. The greatest proportion of positive samples was obtained from Mashhad hospitals, while all samples collected from Arak and Semnan hospitals were negative. Conclusion considering the results of this study and the pathogenic role of this protozoan on patients with immunodeficiency, as well as capability of this microorganism in carrying other pathogens such as Legionella, further studies are needed. What is more important, potable water in hospitals should follow the procedure of treatment and sanitation, in order to prevent the relevant nosocomial infections. PMID:22347240

  4. Legionella pneumophila: Virulent and Avirulent Interaction with Acanthamoeba castellanii.

    DTIC Science & Technology

    1993-08-01

    HCl 0.40 g Ferric Pryophsphate, Soluble 0.25 g ACES Buffer 10.0 g Activated Charcoal 2.0 g Alpha - Ketoglutaric Acid 1.0 g Agar 15.0 g Polyndixin B...Growth enhancement also occurs with the addition of trace metals such as calcium , cobalt, copper, magnesium, manganese, nickel, vandium and zinc (125...by the addition of ACES buffer (BCYE), (116). This formulation with the addition of a- ketoglutarate facilitates good recovery of legionellae from

  5. Acanthamoeba genotypes T3 and T4 as causative agents of amoebic keratitis in Mexico.

    PubMed

    Omaña-Molina, Maritza; Vanzzini-Zago, Virginia; Hernandez-Martinez, Dolores; Gonzalez-Robles, Arturo; Salazar-Villatoro, Lizbeth; Ramirez-Flores, Elizabeth; Oregon-Miranda, Eric; Lorenzo-Morales, Jacob; Martinez-Palomo, Adolfo

    2016-02-01

    Free-living amoebae (FLA) are widely distributed worldwide. Some genera included in this group act as opportunistic pathogens causing fatal encephalitis and Acanthamoeba keratitis (AK), a sight-threatening infection of the cornea associated with the use of soft contact lenses that could even end in blindness if an early diagnosis and treatment are not achieved. Furthermore, the numbers of AK cases keep rising worldwide mainly due to an increase of contact lens wearers and lack of hygiene in the maintenance of lenses and their cases. In Mexico, no cases of AK have been described so far although the isolation of other pathogenic FLA such as Naegleria fowleri and Balamuthia mandrillaris from both clinical and environmental sources has been reported. The present study reports two cases of Acanthamoeba keratitis diagnosed in two patients admitted to the Hospital "Luis Sánchez Bulnes" for Blindness Prevention in Mexico City, Mexico. Corneal scrapes and contact lenses were checked for the presence of Acanthamoeba strains in both patients. Strains were axenized after initial isolation to classify at the genotype level. After sequencing the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA (rDNA) gene of Acanthamoeba, genotype T3 and genotype T4 were identified in clinical case 1 and 2, respectively. To our knowledge, these are the first reported cases of AK in Mexico in the literature and the first description of Acanthamoeba genotypes T3 and T4 as causative agents of amoebic infection.

  6. An apparent Acanthamoeba genotype is the product of a chimeric 18S rDNA artifact.

    PubMed

    Corsaro, Daniele; Venditti, Danielle

    2018-02-01

    Free-living amoebae of the genus Acanthamoeba are potentially pathogenic protozoa widespread in the environment. The detection/diagnosis as well as environmental survey strategies is mainly based on the identification of the 18S rDNA sequences of the strains that allow the recovery of various distinct genotypes/subgenotypes. The accurate recording of such data is important to better know the environmental distribution of distinct genotypes and how they may be preferentially associated with disease. Recently, a putative new acanthamoebal genotype T99 was introduced, which comprises only environmental clones apparently with some anomalous features. Here, we analyze these sequences through partial treeing and BLAST analyses and find that they are actually chimeras. Our results show that the putative T99 genotype is very likely formed by chimeric sequences including a middle fragment from acanthamoebae of genotype T13, while the 5'- and 3'-end fragments came from a nematode and a cercozoan, respectively. Molecular phylogenies of Acanthamoeba including T99 are consequently erroneous as genotype T99 does not exist in nature. Careful identification of Acanthamoeba genotypes is therefore critical for both phylogenetic and diagnostic applications.

  7. Cell wall formation in zoospores of Allomyces arbuscula. II. Development of surface structure of encysted haploid zoospores, rhizoids, and hyphae.

    PubMed

    Kroh, M; Hendriks, H; Kirby, E G; Sassen, M M

    1976-08-01

    Development of haploid meiospores of Allomyces arbuscula into germling cells with rhizoids and hyphae was followed during incubation in complete growth medium. The surface structure of encysted meiospores, rhizoids and hyphae before and after extraction of amorphous materials with ethanolic KOH was studied by means of carbon-platinum replicas. After 2--3 min incubation in complete medium 10% of the meiospores were surrounded by a cell wall containing microfibrils embedded in a matrix. Structure of cell walls of encysted meiospores, rhizoids, and hyphae differ from one another by the location of amorphous materials and by the arrangement of chitin microfibrils.

  8. The Trojan Horse of the microbiological arms race: phage-encoded toxins as a defence against eukaryotic predators.

    PubMed

    Arnold, Jason W; Koudelka, Gerald B

    2014-02-01

    Phage-encoded Shiga toxin (Stx) acts as a bacterial defence against the eukaryotic predator Tetrahymena. To function as an effective bacterial anti-predator defence, Stx must kill a broad spectrum of predators. Consistent with that assertion, we show here that bacterially encoded Stx efficiently kills the bacteriovore Acanthamoeba castellanii in co-culture. We also show that, in addition to Stx, the phage-encoded exotoxin, diphtheria toxin (Dtx) expressed by Corynebacterium diphtheriae also can function as part of an anti-predator strategy; it kills Acanthamoeba in co-culture. Interestingly, only exotoxins produced by bacteria internalized by the Acanthamoeba predator are cytolethal; the presence of purified Dtx or Stx in culture medium has no effect on predator viability. This finding is consistent with our results indicating that intoxication of Acanthamoeba by these exotoxins does not require a receptor. Thus bacteria, in the disguise of a food source, function as a 'Trojan Horse', carrying genes encoding an exotoxin into target organisms. This 'Trojan Horse' mechanism of exotoxin delivery into predator cells allows intoxication of predators that lack a cell surface receptor for the particular toxin, allowing bacteria-bearing exotoxins to kill a broader spectrum of predators, increasing the fitness of the otherwise 'defenceless' prey bacteria. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  9. [Early clinical diagnosis of acanthamoeba keratitis. A study of 70 eyes].

    PubMed

    Bernauer, W; Duguid, G I; Dart, J K

    1996-05-01

    Acanthamoeba keratitis is an uncommon condition which is usually associated with contact lens wear. The use of home made saline and poor hygiene are important risk factors. Early diagnosis is crucial since these cases respond well to medical therapy. The purpose of this paper is to describe and demonstrate early clinical signs. Between September 1992 and October 1994, 70 cases of acanthamoeba keratitis, one of them bilateral, were prospectively monitored at Moorfields Eye Hospital in London. A database of all patients was set up and the clinical findings, diagnostic methods, therapeutic interventions and the outcome were recorded. 66 patients (96%) were contact lens wearers, 64 of them (97%) wore soft lenses. The mean interval between first symptoms and correct diagnosis was 42%. The most frequent initial diagnoses were "unclear keratoconjunctivitis" and "herpetic keratitis". Early corneal findings included punctate keratopathy (n = 14; 20%), pseudodendrites (n = 4; 6%), epithelial infiltrates (n = 17; 24%), diffuse or focal sub-epithelial infiltrates (n = 36; 51%) and radial keratoneuritis (n = 5; 7%). Ring infiltrates (n = 13; 18%) and corneal ulceration (n = 13) were late signs. When the above corneal findings are observed, particularly in contact lens wearers, the diagnosis of acanthamoeba keratitis should be considered. The diagnosis of "herpetic keratitis" in association with contact lens wear should be encountered with scepticism.

  10. [The effect of active immunization with Acanthamoeba culbertsoni in mice born to immune mother].

    PubMed

    Kong, H H; Seo, S A; Shin, C O; Im, K I

    1993-06-01

    Acanthamoeba culbertsoni is a pathogenic free-living amoeba causing primary amoebic meningoencephalitis (PAME) in human and mouse. Several reports on the immune responses in mice with this amoebic infection have been published, but the effects of transferred passive immunity on the active immunization in offspring mice have not been demonstrated. This experiment was done to observe the effect of active immunization with Acanthamoeba culbertsoni in mice born to immune mothers. Acanthamoeba culbertsoni was cultured in the CGV medium axenically. Female BALB/c mice weighing about 20g were immunized through the intraperitoneal injection of Acanthamoeba culbertsoni trophozoites 1 x 10(6) each three times at the interval of one week. Offspring mice were immunized two times. The mice were inoculated intranasally with 1 x 10(4) trophozoites under secobarbital anesthesia. There was a statistical difference in mortality between the transferred immunity group and the active immunization group. Statistical differences were not demonstrated in antibody titer between both groups. But L3T4+ T cell/Ly2+ T cell ratio was increased in the transferred immunity group more than active immunization group of the offspring mice at the age of 5 weeks. There was no differences statistically in mortality between both groups. It was recognized that active immunization in offspring mice born to immune mother could modulate the immune status according to the time of immunization.

  11. Development and optimization of new culture media for Acanthamoeba spp. (Protozoa: Amoebozoa).

    PubMed

    Martín-Pérez, Tania; Criado-Fornelio, Angel; Ávila-Blanco, Manuel; Pérez-Serrano, Jorge

    2018-06-01

    The isolation and growth in axenic liquid media of Acanthamoeba strains is necessary in order to carry out primary in vitro drug screening. Amoebic isolates which are hard to grow in the current liquid media have been reported. Such circumstances hampers the ability of conducting drug sensitivity tests. Therefore, finding suitable universal growth media for Acanthamoeba species is required. The present study was aimed at the development of liquid medium suitable for growing a fastidious (F) genotype T3 Acanthamoeba isolate, and eventually for other genotypes of this genus as well. Trophozoite growth was indirectly monitored by respiration analysis with oxygen-sensitive microplates (OSM) and further confirmed by manual counting. Media were empirically designed and tested first in a non-fastidious (NF) T3 isolate and then tested with 14 different strains, including the fastidious one. Combinations of nutritive components such as meat/vegetable broth, LB medium, malt and skimmed milk led to the design of new media suitable for culturing all the isolates tested, in conditions similar to those obtained in standard culture media such as PYG or CERVA. Copyright © 2018. Published by Elsevier GmbH.

  12. Scleral and intraocular amoebic dissemination in Acanthamoeba keratitis.

    PubMed

    Arnalich-Montiel, Francisco; Jaumandreu, Laia; Leal, Marina; Valladares, Basilio; Lorenzo-Morales, Jacob

    2013-12-01

    To review an Acanthamoeba keratitis case series for the documented extracorneal spread of the amoeba. A retrospective review of an observational case series from a single institution. Three patients with 4 instances of microbiologically confirmed extracorneal amoebic spread were identified. Patient 1 had nodular scleritis after undergoing penetrating keratoplasty and was treated successfully with double freeze-thaw cryotherapy; patient 2 had intraocular dissemination of the amoeba detected in a retrocorneal membrane; and patient 3 had, after undergoing tectonic keratoplasty, intraocular dissemination of the amoeba that was treated successfully with intraocular and systemic voriconazole and, afterwards, a nodular scleritis treated with double freeze-thaw cryotherapy and a large-diameter corneal graft to treat corneal recurrence. Acanthamoeba can migrate to the sclera or to the intraocular tissues in some instances, such as in long-standing disease or in penetrating keratoplasty. A prompt biopsy for microbiological analysis and early treatment are required, if this is suspected. Voriconazole can be effective for intraocular invasion when used orally and intraocularly. Scleral involvement might require a surgical approach with double freeze-thaw cryotherapy to treat the localized disease.

  13. Genotyping, physiological features and proteolytic activities of a potentially pathogenic Acanthamoeba sp. isolated from tap water in Brazil.

    PubMed

    Magliano, Ana C M; da Silva, Flávia Maia; Teixeira, Marta M G; Alfieri, Silvia C

    2009-11-01

    Acanthamoeba spp., known to cause keratitis and granulomatous encephalitis in humans, are frequently isolated from a variety of water sources. Here we report for the first time the characterization of an Acanthamoeba sp. (ACC01) isolated from tap water in Brazil. This organism is currently being maintained in an axenic growth medium. Phylogenetic analysis based on SSU rRNA gene sequences positioned the new isolate in genotype T4, closest to the keratitis-causing isolate, A. polyphaga ATCC 30461 ( approximately 99% similarity). Acanthamoeba ACC01 and A. polyphaga 30461 both grew at 37 degrees C and were osmotically resistant, multiplying in hyperosmolar medium. Both isolates secreted comparable amounts of proteolytic enzymes, including serine peptidases that were optimally active at a near neutral/alkaline pH and resolved identically in gelatin gels. Incubation of gels at pH 4.0 with 2mM DTT also indicated the secretion of similar cysteine peptidases. Altogether, the results point to the pathogenic potential of Acanthamoeba ACC01.

  14. Isolation and molecular characterization of potentially pathogenic Acanthamoeba genotypes from diverse water resources including household drinking water from Khyber Pakhtunkhwa, Pakistan.

    PubMed

    Tanveer, Tania; Hameed, Abdul; Muazzam, Ambreen Gul; Jung, Suk-Yul; Gul, Asma; Matin, Abdul

    2013-08-01

    Acanthamoeba, an opportunistic protozoan pathogen, is ubiquitous in nature, and therefore plays a predatory role and helps control microbial communities in the ecosystem. These Acanthamoeba species are recognized as opportunistic human pathogens that may cause blinding keratitis and rare but fatal granulomatous encephalitis. To date, there is not a single report demonstrating Acanthamoeba isolation and identification from environmental sources in Pakistan, and that is the aim of this study. Acanthamoeba were identified by morphological characteristics of their cysts on non-nutrient agar plates seeded with Escherichia coli. Additionally, the polymerase chain reaction (PCR) was performed with genus-specific primers followed by direct sequencing of the PCR product for molecular identification. Furthermore, our PCR and sequencing results confirmed seven different pathogenic and nonpathogenic genotypes, including T2-T10, T4, T5, T7, T15, T16, and T17. To the best of our knowledge, we have identified and isolated Acanthamoeba sp., for the first time, from water resources of Khyber Pakhtunkhwa, Pakistan. There is an urgent need to address (1) the pathogenic potential of the identified genotypes and (2) explore other environmental sources from the country to examine the water quality and the current status of Acanthamoeba species in Pakistan, which may be a potential threat for public health across the country.

  15. Eukaryotic cell encystation and cancer cell dormancy: is a greater devil veiled in the details of a lesser evil?

    PubMed

    Baig, Abdul Mannan; Khan, Naveed Ahmed; Abbas, Farhat

    2015-03-01

    Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy as dormant cells evade not only the anticancer drugs but also the host immune system. These dormant cells veil themselves from detection by imaging and/or using biomarkers, which imposes an additional problem in targeting such cells. A similar form of hibernation process known as encystation is studied in detail for pathogenic unicellular eukaryotic microorganisms. By examination using microarray gene expression profiles, immunocytochemistry tools, and siRNAs during the process of encystation, understanding the covert features of cancer cell dormancy as proposed could be possible. This knowledge can be extended to dormant cancer cells to uncover the mechanisms that underlie this ghost, yet dangerous state of human cancers. We propose a strategy to induce dormancy and exit this state by application of knowledge gained from the encystation induction and retrieval processes in pathogenic eukaryotic microorganisms. Given that early detection and characterization of dormant malignant tumor cells is important as a general strategy to monitor and prevent the development of overt metastatic disease, this homology may enable the design of therapies that could either awake the dormant cell from dormancy to make it available for therapies or prolong such a phase to make cancer appear as a chronic disease.

  16. Three-Dimensional Ultrastructural Study of Oil and Astaxanthin Accumulation during Encystment in the Green Alga Haematococcus pluvialis

    PubMed Central

    Matsuura, Hazuki; Nango, Nobuhito; Hirata, Aiko; Kawano, Shigeyuki

    2013-01-01

    Haematococcus pluvialis is a freshwater species of green algae and is well known for its accumulation of the strong antioxidant astaxanthin, which is used in aquaculture, various pharmaceuticals, and cosmetics. High levels of astaxanthin are present in cysts, which rapidly accumulate when the environmental conditions become unfavorable for normal cell growth. It is not understood, however, how accumulation of high levels of astaxanthin, which is soluble in oil, becomes possible during encystment. Here, we performed ultrastructural 3D reconstruction based on over 350 serial sections per cell to visualize the dynamics of astaxanthin accumulation and subcellular changes during the encystment of H. pluvialis. This study showcases the marked changes in subcellular elements, such as chloroplast degeneration, in the transition from green coccoid cells to red cyst cells during encystment. In green coccoid cells, chloroplasts accounted for 41.7% of the total cell volume, whereas the relative volume of astaxanthin was very low (0.2%). In contrast, oil droplets containing astaxanthin predominated in cyst cells (52.2%), in which the total chloroplast volume was markedly decreased (9.7%). Volumetric observations also demonstrated that the relative volumes of the cell wall, starch grains, pyrenoids, mitochondria, the Golgi apparatus, and the nucleus in a cyst cell are smaller than those in green coccid cells. Our data indicated that chloroplasts are degraded, resulting in a net-like morphology, but do not completely disappear, even at the red cyst stage. PMID:23326471

  17. Azasterols impair Giardia lamblia proliferation and induces encystation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Maia, Claudia; Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro, Rio de Janeiro; Attias, Marcia

    2007-11-16

    The effects of sterol biosynthesis inhibitors on growth and fine structure of Giardia lamblia P1 strain cultures were analyzed. Azasterols demonstrated high efficacy in killing cells. The IC{sub 50} values for 22,26-azasterol and 24(R,S),25-epiminolanosterol were 7 {mu}M and 170 nM, respectively. Morphological analysis showed that azasterols induced changes in G. lamblia ultrastructure. The most significant alterations were: (a) considerable increase of the size of the peripheral vesicles, which are part of the parasite endosomal-lysosomal system; (b) appearance of autophagosomal structures; and (c) induction of differentiation, followed by an abnormal enlargement of encystation secretory vesicles. We propose that azasterols are effectivemore » chemotherapeutic drugs against Giardia lamblia in vitro and may have another target in cells besides sterol biosynthesis.« less

  18. Infections caused by pathogenic free-living amebas (Balamuthia mandrillaris and Acanthamoeba sp.) in horses.

    PubMed

    Kinde, Hailu; Read, Deryck H; Daft, Barbara M; Manzer, Michael; Nordhausen, Robert W; Kelly, Daryl J; Fuerst, Paul A; Booton, Gregory; Visvesvara, Govinda S

    2007-05-01

    This article describes amebic infections in 4 horses: granulomatous amebic encephalitis caused by Balamuthia mandrillaris and Acanthamoeba culbertsoni and systemic infections caused by Acanthamoeba sp. The former infection occurred in 1 of 4 horses spontaneously without any underlying conditions; the latter amebic infection was perhaps "opportunistic" considering the visceral involvement by this protozoan in association with Aspergillus sp. and/or Escherichia coli and Pseudomonas sp. The clinicopathologic findings and demonstration of the amebic organisms using immunohistochemical techniques, culture, polymerase chain reactions, and electron microscopy are presented.

  19. Characterisation of the Rac/PAK pathway in Amoeba proteus.

    PubMed

    Kłopocka, W; Moraczewska, J; Redowicz, M J

    2005-04-01

    Molecular mechanisms underlying the unique locomotion of the highly motile Amoeba proteus still remain poorly understood. Recently, we have shown that blocking the endogenous amoebal Rac-like protein(s) leads to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. To elucidate the mechanism of the Rac pathway in Amoeba proteus, we tested the effects of blocking the endogenous myosin I heavy chain kinase (MIHCK), one of the Rac effectors in Acanthamoeba castellanii and Dictyostelium discoideum, with anti-MIHCK antibodies in migrating amoebae, as well as the effect of inhibiting Rac and MIHCK on the actin polymerisation process. Antibodies against A. castellanii MIHCK detected an A. proteus protein with a molecular mass (ca. 95 kDa) similar to the A. castellanii kinase. The cellular distribution of MIHCK in A. proteus was very similar to those of Rac-like protein in amoebae and MIHCK in A. castellanii. Amoebae microinjected with anti-MIHCK antibodies moved slower and protruded fewer wide pseudopodia (5-6) than the control cells (9-10), resembling to some extent the phenotype of cells microinjected with anti-Rac antibodies. The in vitro studies indicate that the A. proteus Rac-like protein, but not the MIHCK isoform, is engaged in the regulation of the nucleation step of the actin polymerisation process. These observations suggest that MIHCK may be one of the effectors for Rac in these extremely large cells.

  20. Molecular characterization of Acanthamoeba strains isolated from the oral cavity of hemodialysis patients in Iran.

    PubMed

    Niyyati, Maryam; Arab-Mazar, Zahra; Lasjerdi, Zohreh; Lorenzo-Morales, Jacob; Espotin, Adel; Yadegarynia, Davood; Gachkar, Latif; Rahmati Roodsari, Sara

    2017-11-01

    Free-living amoebae (FLA) of the genus Acanthamoeba are opportunistic pathogenic agents able to cause life-threatening infections in immunosuppressed patients. Chronic kidney disease impairs adaptive and innate immunity. Thus, patients with chronic kidney disease are prone to opportunistic infections by potentially pathogenic FLA. Therefore, in the present study, the investigation of Acanthamoeba genotypes isolated from the oral cavity of hemodialysis patients of reference hospitals in Iran was aimed, using both morphology and molecular (sequence-based analysis) tools. Furthermore, classification of the strains at the genotype level was performed on the basis of differences in the diagnostic fraction 3 (DF3) region of the 18S rRNA gene. The pathogenic potential of the isolated amoebae was also determined using thermotolerance and osmotolerance assays. Out of the 187 oral cavity samples investigated, nine (4.8%) were positive for FLA. DNA sequencing of the ASA.A1 region of the 18S rRNA gene revealed that the isolated strains belonged to the Acanthamoeba T1 and T4 genotypes. Genotype T1 was isolated for the first time from a patient in Iran. Interestingly, the T1 strain (AN2 strain) exhibits a high pathogenic potential in tolerance assays. The pathogenicity assay revealed that five strains were able to grow at high temperatures (37-40 °C) and high osmolarity (0.5 and 1 M D-mannitol) conditions; thus, they were considered as potentially pathogenic strains. Moreover, two of the patients were positive for Vermamoeba genus. The present study is the first report of genotype T1 isolation in Iran and the first to identify the occurrence of Acanthamoeba and Vermamoeba genera in patients undergoing hemodialysis worldwide. Monitoring hemodialysis and renal failure patients should be a priority for possible control of Acanthamoeba and other FLA-related diseases.

  1. Disinfection capacity of PuriLens contact lens cleaning unit against Acanthamoeba.

    PubMed

    Hwang, Thomas S; Hyon, Joon Young; Song, Jae Kyung; Reviglio, Victor E; Spahr, Harry T; O'Brien, Terrence P

    2004-01-01

    The PuriLens contact lens system is indicated for cleaning and disinfection of soft (hydrophilic) contact lenses by means of subsonic agitation to remove lens deposits and microorganisms, and ultraviolet irradiation of the storage solution for disinfection. The capacity of the PuriLens system to disinfect storage solutions contaminated with known concentrations of Staphylococcus aureus, Pseudomonas aeruginosa, and Acanthamoeba species was evaluated. An in vitro assessment of the antibacterial and antiparasitic efficacy of the PuriLens system was performed. Separated batches of the storage solution for the cleansing system were contaminated with stock strains of S. aureus and P. aeruginosa. A comparison of the microbiologic content was made between the solution before and after the cycle. The PuriLens system effectively eradicated S. aureus and P. aeruginosa organisms after a 15-minute cycle. However, viable cysts of acanthamoeba were recovered in the solution after the 15-minute cycle. The PuriLens system is highly efficient in protecting against contamination with common bacterial ocular pathogens. Acanthamoeba cysts, however, can survive in the solution or contact lens bath undergoing integrated subsonic debridement and indirect ultraviolet light disinfection. Use of chemical disinfecting solutions that contain agents such as chlorhexidine or other cationic antiseptics may be advisable in conjunction with use of the PuriLens device, especially in high-risk settings.

  2. Rapid diagnosis of acanthamoeba keratitis using non-nutrient agar with a lawn of E. coli.

    PubMed

    Borin, Samuel; Feldman, Ilan; Ken-Dror, Shifra; Briscoe, Daniel

    2013-02-27

    A patient presented with a corneal foreign body in his only eye. He was treated with prophylactic antibiotics and sent home, but deteriorated. He returned to the hospital 5 days later, and on slit-lamp examination, there was ciliary injection, corneal oedema and a 1 mm × 1 mm corneal abscess with mild anterior uveitis. Corneal scrapings were taken for culture on a non-nutrient agar with a lawn of Escherichia coli, on chocolate agar and on blood agar. He was treated with fortified gentamicin and cefazolin drops. He improved and was discharged 4 days after admission. On day 5, the culture results showed acanthamoeba. He was brought back to the hospital and treated with hourly chlorhexidine drops, ofloxacin six times daily and neomycin/dexamethasone drops once daily. On day 7, he was discharged to continue treatment at home, at which time his visual acuity in that eye was 6/9, and slit-lamp examination showed punctate keratitis and a stromal opacity with mild peripheral infiltration. Culture on non-nutrient agar with a lawn of E. coli is a rapid, reliable and less invasive alternative to corneal biopsy for the diagnosis of acanthamoeba infection. We suggest using this method where acanthamoeba is suspected. Owing to the risk of corneal abscess, orthokeratology should be avoided in an amblyopic patient or an only eye. Acanthamoeba infection may be masked by other eye diseases.

  3. Sterol biosynthesis de nova via cycloartenol by the soil amoeba Acanthamoeba polyphaga.

    PubMed Central

    Raederstorff, D; Rohmer, M

    1985-01-01

    The soil amoeba Acanthamoeba polyphaga is capable of synthesizing its sterols de novo from acetate. The major sterols are ergosterol and poriferasta-5,7,22-trienol. Furthermore C28 and C29 sterols of still unknown structure with an aromatic B-ring are also synthesized by the amoeba. The first cyclic sterol precursor is cycloartenol, which is the sterol precursor in all photosynthetic phyla. No trace of lanosterol, which is the sterol precursor in animals and fungi, could be detected. These results show that at least some of the biochemical processes of Acanthamoeba polyphaga might be phylogenetically related to those of unicellular algae. Addition of exogenous sterols to the culture medium does not influence the sterol biosynthesis and the sterol composition of the cells. PMID:4074326

  4. Photodynamic inactivation of Acanthamoeba polyphaga with curcuminoids: an in vitro study

    NASA Astrophysics Data System (ADS)

    Corrêa, Thaila Q.; Geralde, Mariana C.; Carvalho, Mariana T.; Bagnato, Vanderlei S.; Kurachi, Cristina; de Souza, Clovis W. O.

    2016-03-01

    Acanthamoeba polyphaga are free-living amoebae that can be considered potentially pathogenic organisms by cause serious human infections, including keratitis and granulomatous amoebic encephalitis that usually results in death. Photodynamic inactivation (PDI) has been used for the biological control of microorganisms and can be promise in the control of Acanthamoeba infections. This study evaluated the in vitro effectiveness of PDI in A. polyphaga using curcuminoids salt as photosensitizer (PS) besides observing morphological changes caused by this PS in this organism, in confocal microscopy. A. polyphaga trophozoites were grown at 37°C in PYG medium for 48 to 72 hours. After, the trophozoites were incubated with PS solution during one hour and the samples were irradiated using light-emitting diodes at 460 nm at light doses 30 and 50 J/cm2. The results revealed reduction of 27.7%, 61.4% and 82.5% at 30 J/cm2 and 75.2%, 85.0% and 95.9% at 50 J/cm2, respectively, at curcuminoid salt concentrations of 500, 1000 and 1500 μg/mL. Through fluorescence images, it was possible to visualize the curcuminoid salt's uptake by the trophozoites. The PS showed toxicity to amoebae, in the dark, but the irradiation in PDI contributed to amoebae death effect. These data suggest that PDI may be an application of therapeutic intervention against Acanthamoeba infections, since it was effective in the inactivation of these amoebae.

  5. In vivo laser confocal microscopy findings of radial keratoneuritis in patients with early stage Acanthamoeba keratitis.

    PubMed

    Kobayashi, Akira; Yokogawa, Hideaki; Yamazaki, Natsuko; Ishibashi, Yasuhisa; Oikawa, Yosaburo; Tokoro, Masaharu; Sugiyama, Kazuhisa

    2013-07-01

    To investigate in vivo corneal changes of keratoneuritis in early stage Acanthamoeba keratitis (AK) using in vivo laser confocal microscopy. Single-center, prospective, clinical study. Thirteen eyes (12 patients; 5 men and 7 women; mean age ± standard deviation, 22.3 ± 4.2 years) with keratoneuritis resulting from early stage AK were included in this study. In vivo laser confocal microscopy was performed, paying special attention to keratoneuritis. Selected confocal images of corneal layers were evaluated qualitatively for shape and degree of light reflection of abnormal cells and deposits. In all patients, Acanthamoeba cysts were observed clearly in the basal epithelial cell layer as highly reflective round particles with a diameter of 10 to 20 μm. Bowman's layer infiltration of Acanthamoeba cysts was observed in only 1 case, and no cases showed stromal or nerve infiltration of Acanthamoeba cysts. In the stroma, all cases showed highly reflective activated keratocytes forming a honeycomb pattern; these changes were significant around the keratoneuritis. Infiltration of inflammatory cells, possibly polymorphonuclear cells, was observed along with keratocyte bodies in all cases. Numerous highly reflective spindle-shaped materials were observed around the keratoneuritis. Most notably, highly reflective patchy lesions were observed around the keratoneuritis in 11 cases (84.6%). Inflammatory cells also were observed in the endothelial cell layer in 4 cases (30.8%). In vivo laser confocal microscopy identified consistent corneal abnormalities around keratoneuritis in early stage AK patients, of which highly reflective patchy lesions may be characteristic of keratoneuritis. Further morphologic studies of corneas with early stage AK in a larger number of patients may elucidate the clinical significance of radial keratoneuritis and may help us to understand the interaction between Acanthamoeba organisms and host corneal cells or nerves. Copyright © 2013 American

  6. Acanthamoeba in the eye, can the parasite hide even more? Latest developments on the disease.

    PubMed

    Juárez, M M; Tártara, L I; Cid, A G; Real, J P; Bermúdez, J M; Rajal, V B; Palma, S D

    2018-06-01

    Acanthamoeba spp. is a free living protozoan in the environment, but can cause serious diseases. Acanthamoeba keratitis (AK), a severe and painful eye infection, must be treated as soon as possible to prevent ulceration of the cornea, loss of visual acuity, and eventually blindness or enucleation. Although the disease affects principally contact lens (CLs) wearers, it is recognized nowadays as a cause of keratitis also in non-CLs wearers. Although the number of infections caused by these amoebae is low, AK is an emerging disease presenting an extended number of cases each year worldwide mostly due to the increasing use of CLs, but also to better diagnostic methods and awareness. There are two principal causes that lead to severe outcomes: misdiagnosis or late diagnosis of the causal agent, and lack of a fully effective therapy due to the existence of a highly resistant cyst stage of Acanthamoeba. Recent studies have reported different genotypes that have not been previously associated with this disease. In addition, Acanthamoeba can act as a reservoir for phylogenetically diverse microorganisms. In this regard, recently giant viruses called Pandoravirus have been found within genotypes producing keratitis. What potential risk this poses is not yet known. This review focuses on an overview of the present status and future prospects of this re-emerging pathology, including features of the parasite, epidemiology, clinical aspects, diagnosis, and treatment. Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  7. Essential oil composition and anti Acanthamoeba studies of Teucrium ramosissimum.

    PubMed

    Ghazouani, Nessrine; Sifaoui, Ines; Bachrouch, Olfa; Abderrabba, Manef; E Pinero, José; Lorenzo-Morales, Jacob

    2017-12-01

    The aim of the present study was to evaluate the chemical composition of the essential oil obtained from the aerial parts of T. ramosissimum by hydrodistillation and to investigate their anti-Acanthamoeba activity. Identification and quantification were realized by gas chromatography-mass spectrometry (GC-MS) and gas chromatography with flame ionization detection by (GC-FID). Sixty-eight compounds representing 97.78% of the essential oil were identified, of which δ-cadinene (18.63%), δ-cadinol (18.70%), β-eudesmol (12.13%), γ-gurjunene (4.34%) and 8-cedrene (3.99%) were the main compounds. This essential oil contained a complex mixture consisting mainly on sesquiterpenes (80.62%) and monoterpene fractions (14.34%). The findings of the anti-Acanthamoeba assay indicate that T. ramosissimum essential oil have a good activity with an IC 50  = 25.73 ± 0.75 μg/mL. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. In vitro anti-Acanthamoeba synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst forms.

    PubMed

    Heredero-Bermejo, I; Sánchez-Nieves, J; Soliveri, J; Gómez, R; de la Mata, F J; Copa-Patiño, J L; Pérez-Serrano, J

    2016-07-25

    Acanthamoeba sp. are the causative agents of severe illnesses in humans such as Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Medical therapy is not yet well established. Treatments of AK last for several months and generate toxicity, resistances appear due to the cysts stage and recurrences can occur. In this study has been demonstrated that the combination of chlorhexidine digluconate (CLX) and carbosilane dendrimers containing ammonium or guanidine moieties has in vitro synergistic effect against Acanthamoeba polyphaga. This synergy provokes an important reduction in the minimal trophozoite amoebicidal concentration (MTAC) of CLX, which means a reduction of their toxic effects on human cells. Moreover, some CLX/dendrimer combinations show important activity against the cyst resistance stage. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

    PubMed Central

    Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

    2007-01-01

    Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment. PMID:17875991

  10. Donor CD8+ T Cells Prevent Toxoplasma gondii De-Encystation but Fail To Rescue the Exhausted Endogenous CD8+ T Cell Population

    PubMed Central

    Bhadra, Rajarshi; Cobb, Dustin A.

    2013-01-01

    Functional exhaustion of CD8+ T cells due to increased expression of inhibitory molecule PD-1 (Programmed Death-1) causes reactivation of latent disease during later phases of chronic toxoplasmosis. Onset of disease recrudescence results in decreased parasite cyst burden concomitant with parasites undergoing stage conversion from a primarily encysted, quiescent bradyzoite to a fast-replicating, highly motile tachyzoite. Thus, reduced cyst burden is one of the early hallmarks of disease recrudescence. This was further validated by depleting gamma interferon (IFN-γ), a cytokine known to control latent toxoplasmosis, in chronically infected prerecrudescent mice. Since CD8+ T cells (an important source of IFN-γ) lose their functionality during the later phases of chronic toxoplasmosis, we next examined if adoptive transfer of functional CD8+ T cells from acutely infected donors to the chronically infected prerecrudescent hosts could impede parasite de-encystation and rescue exhausted CD8+ T cells. While the transfer of immune CD8+ T cells temporarily restricted the breakdown of cysts, the exhausted endogenous CD8+ T cell population was not rescued. Over time, the donor population got deleted, resulting in parasite de-encystation and host mortality. Considering that donor CD8+ T cells fail to become long-lived, one of the cardinal features of memory CD8+ T cells, it bears the implication that memory CD8 differentiation is impaired during chronic toxoplasmosis. Moreover, our data strongly suggest that while adoptive immunotherapy can prevent parasite de-encystation transiently, reduced antigen burden in the chronic phase by itself is insufficient for rescue of exhausted CD8+ T cells. The conclusions of this study have profound ramifications in designing immunotherapeutics against chronic toxoplasmosis. PMID:23817617

  11. Acanthamoeba belonging to T3, T4, and T11: genotypes isolated from air-conditioning units in Santiago, Chile.

    PubMed

    Astorga, Berbeli; Lorenzo-Morales, Jacob; Martín-Navarro, Carmen M; Alarcón, Verónica; Moreno, Johanna; González, Ana C; Navarrete, Elizabeth; Piñero, José E; Valladares, Basilio

    2011-01-01

    Free-living amoebae (FLA) of the genus Acanthamoeba are widely distributed in the environment, in the air, soil, and water, and have also been isolated from air-conditioning units. The objective of this work was to investigate the presence of this genus of FLA in the air-conditioning equipment at the Institute of Public Health of Chile in Santiago, Chile. Water and air samples were collected from air-conditioning systems and were checked for the presence of Acanthamoeba spp. Positive samples were further classified at the genotype level after sequencing the highly variable diagnostic fragment 3 (DF3) region of the 18S rRNA gene. This is the first report of the T3, T4, and T11 genotypes of Acanthamoeba in air-conditioning units from Chile. Overall, the widespread distribution of potentially pathogenic Acanthamoeba strains in the studied source demands more awareness within the public and health professionals in Chile as this pathogen is emerging as a risk for human health worldwide. © 2011 The Author(s) Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

  12. Isolation and molecular identification of Acanthamoeba spp from oasis water in Tunisia.

    PubMed

    Dendana, F; Trabelsi, H; Neiji, S; Sellami, H; Kammoun, S; Makni, F; Feki, J; Cheikhrouhou, F; Ayadi, A

    2018-04-01

    In the southern Tunisia Oasis, we conducted 211 water with drawals from various water traffic sites. This water is used for agriculture, swimming or various other human activities. Acanthamoeba genus was detected in 82% of collected samples. Sequencing of the amplification products with primers P892C/P892 has allowed us to detect genotypic variation with predominance of T4 genotype (51%) and presence of the genotypes T14, T5, T3, T16, T15, T10, T11, T9 and T7. They T4, T3, T5, T15, T11 and T10 genotypes have a high potential for pathogenicity and a very high degree of virulence due to their production of serine proteases and extracellular cysteine enzymes involved in tissue degradation of the host. T4 genotype was the most abundant in the environment as well as in infections caused by Acanthamoeba spp. T5 genotype was ranked second and T3 genotype was less abundant in the environment and its pathogenicity is discussed. Acanthamoeba strains with the genotypes T16, T9 and T7 were considered non pathogenic. In fact, they have been isolated only from the environment. However, for these strains, their role as a reservoir can be a real risk to human health. Copyright © 2018. Published by Elsevier Inc.

  13. Acanthamoeba, an opportunistic microorganism: a review.

    PubMed

    Martinez, A J; Janitschke, K

    1985-01-01

    Granulomatous amebic encephalitis due to Acanthamoeba spp. usually occurs in chronically ill and debilitated individuals. Some of these patients may have received immunosuppressive therapy. Another infection due to Acanthamoeba spp. has been corneal ulcerations which usually occur after minimal trauma to the corneal epithelium (1). In contrast, primary amebic meningoencephalitis due to Naegleria fowleri usually occurs in healthy, young individuals with a history of swimming in heated swimming pools, in manmade lakes or with recent contact with contaminated water and practising water-related sports. Subclinical infections due to free-living amebas are probably common in healthy individuals with the protozoa living as "normal flora" in the nose and throat. It is possible that in humans, antibodies and cell-mediated immunity protect the host in such ordinary circumstances against invasive infection. In debilitated and chronically ill individuals, depressed cellmediated immunity may allow these protozoa to proliferate, allowing a fulminant "opportunistic" infection to develop. In the case of acanthamoebic keratitis, it is important to keep in mind that the temperature and moist environment of the eye serve as a good medium for the growth and proliferation of the amebas and is not necessarily associated with immunosuppression but rather with trauma. This review confirms that opportunistic free-living amebic infections occur with increased frequency in patients treated with steroids, radiotherapy, chemotherapeutic drugs or with broad-spectrum antibiotics and suggest that the mechanism of such infection may be depressed cell-mediated immunity or some other alteration of the immune system, like acquired immunodeficiency syndrome (AIDS).

  14. Bringing forward the new generation of alkoxy-thiourea as potential treatment for Acanthamoeba keratitis

    NASA Astrophysics Data System (ADS)

    Khairul, Wan M.; Goh, Yit-Peng; Daud, Adibah Izzati; Nakisah, M. A.

    2017-02-01

    Alkoxy substituted thiourea derivatives with general formula of A-ArC(O)NHC(S)NHAr-D which A represents the methoxy group and D denotes -OCnH2n+1 have been successfully synthesised and characterized. In turn, all the synthesised molecules were assayed for anti-amoebic activities towards Acanthamoeba sp to examine the cytotoxicity effect at their IC50 and membrane permeability. As predicted, the findings showed that the synthesised molecules owing promising anti-amoebic activity towards Acanthamoeba sp. To support, the Acridine-orange/Propidium iodide (AOPI) staining result under fluorescence microscopy revealed the treated amoeba cells by these alkoxy thiourea derivatives exhibited loss in their membrane permeability.

  15. Presence of Acanthamoeba in the ocular surface in a Spanish population of contact lens wearers.

    PubMed

    Rodríguez-Martín, Javier; Rocha-Cabrera, Pedro; Reyes-Batlle, María; López-Arencibia, Atteneri; Sifaoui, Ines; Rizo-Liendo, Aitor; Bethencourt-Estrella, Carlos J; Piñero, José E; Lorenzo-Morales, Jacob

    2018-06-26

    Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK) which mainly affects contact lens wearers and it is commonly related to poor hygiene of contact lenses and their cases. Moreover, treatment of AK is complex due to the existence of a highly resistant cyst stage and if not diagnosed early has poor prognosis, leading to blindness and/or keratoplasty. Even though AK is increasing worldwide as well as awareness among patients and clinicians, it is still a poorly studied pathogen. Additionally, a remaining question to be answered is whether these opportunistic pathogens are present in the ocular surface of healthy contact lens wearers since they are the main group at risk.In order to carry out this study, sterile Schirmer strip tests were collected from a group of individuals all of them contact lens wearers who were attending a local ophthalmology clinic in Tenerife, Canary Islands, Spain. The collected samples (100 eyes of 50 patients) were cultured in 2% Non-Nutrient Agar (NNA) plates and positive plates (16) were then cultured in axenic conditions for further analyses. Molecular analysis classified all isolated strains belonged to Acanthamoeba genotype T4 and osmotolerance and thermotolerance assays revealed that all strains were potentially pathogenic. In conclusion, the ocular surface of contact lens wearers included in this study was colonized by potentially pathogenic strains of Acanthamoeba and should be considered as a risk for AK infection in this region and worldwide.

  16. The correlation of Acanthamoeba from the ventilation system with other environmental parameters in commercial buildings as possible indicator for indoor air quality.

    PubMed

    Ooi, Soo Shen; Mak, Joon Wah; Chen, Donald K F; Ambu, Stephen

    2017-02-07

    The free-living protozoan Acanthamoeba is an opportunistic pathogen that is ubiquitous in our environment. However, its role in affecting indoor air quality and ill-health of indoor occupants is relatively unknown. The present study investigated the presence of Acanthamoeba from the ventilation system and its correlation with other indoor air quality parameters, used in the industry code of practice and its potential as an indicator for indoor air quality. Indoor air quality assessments were carried out in nine commercial buildings with approval from the building management, and the parameters assessed were as recommended by the Department of Occupational Safety and Health. The presence of Acanthamoeba was determined through dust swabs from the ventilation system and indoor furniture. Logistic regression was performed to study the correlation between assessed parameters and occupants' complaints. A total of 107 sampling points were assessed and 40.2% of the supplying air diffuser and blowing fan and 15% of the furniture were positive for cysts. There was a significant correlation between Acanthamoeba detected from the ventilation system with ambient total fungus count (r=0.327; p=0.01) and respirable particulates (r=0.276; p=0.01). Occupants' sick building syndrome experience also correlated with the presence of Acanthamoeba in the ventilation system (r=0.361; p=0.01) and those detected on the furniture (r=0.290; p=0.01). Logistic regression showed that there was a five-fold probability of sick building syndrome among occupants when Acanthamoeba was detected in the ventilation system.

  17. The correlation of Acanthamoeba from the ventilation system with other environmental parameters in commercial buildings as possible indicator for indoor air quality

    PubMed Central

    OOI, Soo Shen; MAK, Joon Wah; CHEN, Donald K.F.; AMBU, Stephen

    2016-01-01

    The free-living protozoan Acanthamoeba is an opportunistic pathogen that is ubiquitous in our environment. However, its role in affecting indoor air quality and ill-health of indoor occupants is relatively unknown. The present study investigated the presence of Acanthamoeba from the ventilation system and its correlation with other indoor air quality parameters, used in the industry code of practice and its potential as an indicator for indoor air quality. Indoor air quality assessments were carried out in nine commercial buildings with approval from the building management, and the parameters assessed were as recommended by the Department of Occupational Safety and Health. The presence of Acanthamoeba was determined through dust swabs from the ventilation system and indoor furniture. Logistic regression was performed to study the correlation between assessed parameters and occupants’ complaints. A total of 107 sampling points were assessed and 40.2% of the supplying air diffuser and blowing fan and 15% of the furniture were positive for cysts. There was a significant correlation between Acanthamoeba detected from the ventilation system with ambient total fungus count (r=0.327; p=0.01) and respirable particulates (r=0.276; p=0.01). Occupants’ sick building syndrome experience also correlated with the presence of Acanthamoeba in the ventilation system (r=0.361; p=0.01) and those detected on the furniture (r=0.290; p=0.01). Logistic regression showed that there was a five-fold probability of sick building syndrome among occupants when Acanthamoeba was detected in the ventilation system. PMID:27476379

  18. Fibrous Catalyst-Enhanced Acanthamoeba Disinfection by Hydrogen Peroxide.

    PubMed

    Kilvington, Simon; Winterton, Lynn

    2017-11-01

    Hydrogen peroxide (H2O2) disinfection systems are contact-lens-patient problem solvers. The current one-step, criterion-standard version has been widely used since the mid-1980s, without any significant improvement. This work identifies a potential next-generation, one-step H2O2, not based on the solution formulation but rather on a case-based peroxide catalyst. One-step H2O2 systems are widely used for contact lens disinfection. However, antimicrobial efficacy can be limited because of the rapid neutralization of the peroxide from the catalytic component of the systems. We studied whether the addition of an iron-containing catalyst bound to a nonfunctional propylene:polyacryonitrile fabric matrix could enhance the antimicrobial efficacy of these one-step H2O2 systems. Bausch + Lomb PeroxiClear and AOSept Plus (both based on 3% H2O2 with a platinum-neutralizing disc) were the test systems. These were tested with and without the presence of the catalyst fabric using Acanthamoeba cysts as the challenge organism. After 6 hours' disinfection, the number of viable cysts was determined. In other studies, the experiments were also conducted with biofilm formed by Stenotrophomonas maltophilia and Elizabethkingia meningoseptica bacteria. Both control systems gave approximately 1-log10 kill of Acanthamoeba cysts compared with 3.0-log10 kill in the presence of the catalyst (P < .001). In the biofilm studies, no viable bacteria were recovered following disinfection in the presence of the catalyst compared with ≥3.0-log10 kill when it was omitted. In 30 rounds' recurrent usage, the experiments, in which the AOSept Plus system was subjected to 30 rounds of H2O2 neutralization with or without the presence of catalytic fabric, showed no loss in enhanced biocidal efficacy of the material. The catalytic fabric was also shown to not retard or increase the rate of H2O2 neutralization. We have demonstrated the catalyst significantly increases the efficacy of one-step H2O2

  19. The potential pathogenicity of chlorhexidine-sensitive Acanthamoeba strains isolated from contact lens cases from asymptomatic individuals in Tenerife, Canary Islands, Spain.

    PubMed

    Martín-Navarro, Carmen M; Lorenzo-Morales, Jacob; Cabrera-Serra, M Gabriela; Rancel, Fernando; Coronado-Alvarez, Nieves M; Piñero, José E; Valladares, Basilio

    2008-11-01

    Pathogenic strains of the genus Acanthamoeba are causative agents of a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. The prevalence of this infection has risen in the past 20 years, mainly due to the increase in number of contact lens wearers. In this study, the prevalence of Acanthamoeba in a risk group constituted by asymptomatic contact lens wearers from Tenerife, Canary Islands, Spain, was evaluated. Contact lenses and contact lens cases were analysed for the presence of Acanthamoeba isolates. The isolates' genotypes were also determined after rDNA sequencing. The pathogenic potential of the isolated strains was subsequently established using previously described molecular and biochemical assays, which allowed the selection of three strains with high pathogenic potential. Furthermore, the sensitivity of these isolates against two standard drugs, ciprofloxacin and chlorhexidine, was analysed. As the three selected strains were sensitive to chlorhexidine, its activity and IC(50) were evaluated. Chlorhexidine was found to be active against these strains and the obtained IC(50) values were compared to the concentrations of this drug present in contact lens maintenance solutions. It was observed that the measured IC(50) was higher than the concentration found in these maintenance solutions. Therefore, the ineffectiveness of chlorhexidine-containing contact lens maintenance solutions against potentially pathogenic strains of Acanthamoeba is demonstrated in this study.

  20. Phylogeography, genetic variability and structure of Acanthamoeba metapopulations in Iran inferred by 18S ribosomal RNA sequences: A systematic review and meta-analysis.

    PubMed

    Spotin, Adel; Moslemzadeh, Hamid Reza; Mahami-Oskouei, Mahmoud; Ahmadpour, Ehsan; Niyyati, Maryam; Hejazi, Seyed Hossein; Memari, Fatemeh; Noori, Jafar

    2017-09-01

    To verify phylogeography and genetic structure of Acanthamoeba populations among the Iranian clinical isolates and natural/artificial environments distributed in various regions of the country. We searched electronic databases including Medline, PubMed, Science Direct, Scopus and Google Scholar from 2005 to 2016. To explore the genetic variability of Acanthamoeba sp, 205 sequences were retrieved from keratitis patients, immunosuppressed cases and environmental sources as of various geographies of Iran. T4 genotype was the predominant strain in Iran, and the rare genotypes belonged to T2, T3, T5 (Acanthamoeba lenticulata), T6, T9, T11, T13 and T15 (Acanthamoeba jacobsi). A total of 47 unique haplotypes of T4 were identified. A parsimonious network of the sequence haplotypes demonstrated star-like feature containing haplogroups IR6 (34.1%) and IR7 (31.2%) as the most common haplotypes. In accordance with the analysis of molecular variance, the high value of haplotype diversity (0.612-0.848) of Acanthamoeba T4 represented genetic variability within populations. Neutrality indices of the 18S ribosomal RNA demonstrated negative values in all populations which represented a considerable divergence from neutrality. The majority of genetic diversity belonged to the infected contact lens and dust samples in immunodeficiency and ophthalmology wards, which indicated potential routes for exposure to a pathogenic Acanthamoeba sp. in at-risk individuals. A pairwise fixation index (F ST ) was from low to high values (0.02433-0.41892). The statistically F ST points out that T4 is genetically differentiated between north-west, north-south and central-south metapopulations, but not differentiated between west-central, west-south, central-south, and north-central isolates. An occurrence of IR6 and IR7 displays that possibly a gene flow of Acanthamoeba T4 occurred after the founder effect or bottleneck experience through ecological changes or host mobility. This is the first

  1. Virulent T4 Acanthamoeba causing keratitis in a patient after swimming while wearing contact lenses in Southern Brazil.

    PubMed

    Fabres, Laura Fuhrich; Maschio, Vinicius José; Santos, Denise Leal Dos; Kwitko, Sergio; Marinho, Diane Ruschel; Araújo, Bruno Schneider de; Locatelli, Claudete Inês; Rott, Marilise Brittes

    2018-06-26

    Several strains of free-living amoebae belonging to the genus Acanthamoeba can cause a painful sight-threatening disease of the cornea known as Acanthamoeba keratitis (AK). The numbers of AK cases keep rising worldwide mainly due to an increase in contact lens wearers and lack of hygiene in the maintenance of contact lenses and their cases. We report a case of AK in a healthy young woman admitted to the Hospital de Clinicas in Porto Alegre, southern Brazil. Corneal scrapings were examined for the presence of Acanthamoeba strains. The initial isolate was characterized by morphological and genotypic properties. The isolate belonged to group III according to Pussard and Pons' cyst morphology. Analysis of its 18S rDNA sequence identified the isolate as genotype T4. The T4 genotype is the most commonly reported among keratitis isolates and the most common in environmental samples.

  2. Black cobra (Naja naja karachiensis) lysates exhibit broad-spectrum antimicrobial activities

    PubMed Central

    Sagheer, Mehwish; Siddiqui, Ruqaiyyah; Iqbal, Junaid; Khan, Naveed Ahmed

    2014-01-01

    It is hypothesized that animals living in polluted environments possess antimicrobials to counter pathogenic microbes. The fact that snakes feed on germ-infested rodents suggests that they encounter pathogenic microbes and likely possess antimicrobials. The venom is used only to paralyze the rodent, but the ability of snakes to counter potential infections in the gut due to disease-ridden rodents requires robust action of the immune system against a broad range of pathogens. To test this hypothesis, crude lysates of different organs of Naja naja karachiensis (black cobra) were tested for antimicrobial properties. The antimicrobial activities of extracts were tested against selected bacterial pathogens (neuropathogenic Escherichia coli K1, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Streptococcus pneumonia), protist (Acanthamoeba castellanii), and filamentous fungus (Fusarium solani). The findings revealed that plasma and various organ extracts of N. n. karachiensis exhibited antimicrobial activity against E. coli K1, MRSA, P. aeruginosa, S. pneumoniae, A. castellanii, and F. solani in a concentration-dependent manner. The results of this study are promising for the development of new antimicrobials. PMID:24625321

  3. Acanthamoeba keratitis: study of the 5-year incidence in Israel.

    PubMed

    Graffi, Shmuel; Peretz, Avi; Jabaly, Haneen; Koiefman, Anna; Naftali, Modi

    2013-11-01

    Acanthamoeba keratitis (AK) is not a notifiable disease in Israel, so there are no accurate incidence rates for this condition in Israel. The aim of this study was to estimate the incidence of AK in Israel for the years 2008-2012. We distributed a survey questionnaire to laboratory managers in Israel. The laboratories were affiliated to medical institutes that either provided ophthalmology services or served community ophthalmology clinics. Our questionnaire requested survey respondents to provide information regarding the methods used to diagnose AK, and the number of positive and negative cultures for Acanthamoebae species performed for each of the years from 2008 to 2012. Six laboratories used non-nutrient agar with Escherichia coli as the culture medium, one used calcofluor-white staining with fluorescent microscopy, and two used PCR for diagnosing AK. Twenty-three AK cases were identified, to give an estimated incidence of 1/1 668 552. AK is mostly attributable to the use of contact lenses. As contact lenses are popular in Israel, we expected a higher incidence rate. A lower than expected incidence rate may indicate insufficient awareness of AK in Israel.

  4. Deciphering the genomes of 16 Acanthamoeba species does not provide evidence of integration of known giant virus-associated mobile genetic elements.

    PubMed

    Chelkha, Nisrine; Colson, Philippe; Levasseur, Anthony; La Scola, Bernard

    2018-06-02

    Giant viruses infect protozoa, especially amoebae of the genus Acanthamoeba. These viruses possess genetic elements named Mobilome. So far, this mobilome comprises provirophages which are integrated into the genome of their hosts, transpovirons, and Maverick/Polintons. Virophages replicate inside virus factories within Acanthamoeba and can decrease the infectivity of giant viruses. The virophage infecting CroV was found to be integrated in the host of CroV, Cafeteria roenbergensis, thus protecting C. roenbergensis by reduction of CroV multiplication. Because of this unique property, assessment of the mechanisms of replication of virophages and their relationship with giant viruses is a key element of this investigation. This work aimed at evaluating the presence and the dynamic of these mobile elements in sixteen Acanthamoeba genomes. No significant traces of the integration of genomes or sequences from known virophages were identified in all the available Acanthamoeba genomes. These results brought us to hypothesize that the interactions between mimiviruses and their virophages might occur through different mechanisms, or at low frequency. An additional explanation could be that our knowledge of the diversity of virophages is still very limited. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Acanthamoeba keratitis in patients wearing scleral contact lenses.

    PubMed

    Sticca, Matheus Porto; Carrijo-Carvalho, Linda C; Silva, Isa M B; Vieira, Luiz A; Souza, Luciene B; Junior, Rubens Belfort; Carvalho, Fábio Ramos S; Freitas, Denise

    2018-06-01

    To report a series of cases of Acanthamoeba keratitis (AK) in scleral lens wearers with keratoconus to determine whether this type of contact lens presents a greater risk for development of infection. This study reports three patients who wore scleral contact lenses to correct keratoconus and developed AK. The diagnoses of AK were established based on cultures of the cornea, scleral contact lenses, and contact lens paraphernalia. This study investigated the risk factors for infections. The possible risks for AK in scleral contact lens wearers are hypoxic changes in the corneal epithelium because of the large diameter and minimal tear exchange, use of large amounts of saline solution necessary for scleral lens fitting, storing the scleral lens overnight in saline solution rather than contact lens multipurpose solutions, not rubbing the contact lens during cleaning, and the space between the cornea and the back surface of the scleral lens that might serve as a fluid reservoir and environment for Acanthamoeba multiplication. Two patients responded well to medical treatment of AK; one is still being treated. The recommendations for use and care of scleral contact lenses should be emphasized, especially regarding use of sterile saline (preferably single use), attention to rubbing the lens during cleaning, cleaning of the plunger, and overnight storage in fresh contact lens multipurpose solutions without topping off the lens solution in the case. Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  6. Detection of Acanthamoeba spp. in water samples collected from natural water reservoirs, sewages, and pharmaceutical factory drains using LAMP and PCR in China.

    PubMed

    Lass, Anna; Guerrero, Milena; Li, Xiuping; Karanis, Gabriele; Ma, Liqing; Karanis, Panagiotis

    2017-04-15

    Various species of amoebas belonging to the genus Acanthamoeba are widely distributed in many parts of the world. Some strains of these protozoans may exist as parasites and pose risks to human health as causative agents of serious human diseases. Currently in China there is a lack of information about the distribution of Acanthamoeba strains in the environment. Accordingly, 261 environmental water samples taken from rivers, sewage, and pharmaceutical factory drains were collected in Qinghai Province, China. The material was filtered and then analysed with both LAMP and PCR assays. Of the samples examined, Acanthamoeba DNA was found in 32 (14.68%) samples with the use of LAMP; in 13 of these samples, DNA from this amoeba was also detected using PCR. Sequencing of selected positive samples confirmed that the PCR products were fragments of the Acanthamoeba 18S rRNA gene and that isolates represent the T4 genotype, known as the most common strain related to AK cases. The results indicate that surface water, as well as water taken from sewage and pharmaceutical drains, may be a source of acanthamoebic strains potentially pathogenic for humans in China. It has been also demonstrated that LAMP assays is more sensitive than PCR and can be regarded as useful tool for screening the environment for Acanthamoeba spp. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Acanthamoeba of three morphological groups and distinct genotypes exhibit variable and weakly inter-related physiological properties.

    PubMed

    Possamai, Cynara Oliveira; Loss, Ana Carolina; Costa, Adriana Oliveira; Falqueto, Aloisio; Furst, Cinthia

    2018-05-01

    Free-living amoeba of the genus Acanthamoeba can eventually act as parasites, causing infections in humans. Some physiological characteristics of Acanthamoeba have been related to the grade of pathogenicity, allowing inferences about the pathogenic potential. The main goal of this study was to characterize isolates of Acanthamoeba obtained in Brazil and evaluate properties associated with their pathogenicity. A total of 39 isolates obtained from keratitis cases (n = 16) and environmental sources (n = 23) were classified into morphological groups and genotyped by sequencing the 18S rDNA fragments ASA.S1 and GTSA.B1. Samples were also tested regarding their thermo-tolerance, osmo-tolerance, and cytopathogenicity in MDCK cells. Isolates were identified and classified as follows: group I (T17, T18); group II (T1, T3, T4, T11); and group III (T5, T15), with the predominance of genotype T4 (22/39). Clinical isolates were genotyped as T3 (1/16), T4 (14/16) and T5 (1/16). The majority of isolates (38/39) were able to grow at 37 °C, but tolerance to 40 °C was more frequent among environmental samples. The tolerance to 1 M mannitol was infrequent (4/39), with three of these corresponding to clinical samples. The variable ability to cause cytopathic effects was observed among isolates of distinct genotypes and origins. This study identified, for the first time, T1, T15, and T18 in Brazil. It also indicated a weak association between the clinical origin of the isolates and tolerance to high temperatures, high osmolarity, and cytopathogenicity, demonstrating that some in vitro parameters do not necessarily reflect a higher propensity of Acanthamoeba to cause a disease.

  8. mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus.

    PubMed

    Legendre, Matthieu; Audic, Stéphane; Poirot, Olivier; Hingamp, Pascal; Seltzer, Virginie; Byrne, Deborah; Lartigue, Audrey; Lescot, Magali; Bernadac, Alain; Poulain, Julie; Abergel, Chantal; Claverie, Jean-Michel

    2010-05-01

    Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system.

  9. A Review of the Current Research Trends in the Application of Medicinal Plants as a Source for Novel Therapeutic Agents Against Acanthamoeba Infections

    PubMed Central

    Niyyati, Maryam; Dodangeh, Samira; Lorenzo-Morales, Jacob

    2016-01-01

    Acanthamoeba keratitis (AK) is a sight-threating infection of the cornea that mostly affects contact lens wearers. Until now, AK treatment remains very difficult due to the existence of a highly resistant cyst stage in the life cycle of Acanthamoeba which is extremely resistant to most of the available anti-amoebic compounds. Moreover, current treatment of AK is usually based in the combination of various therapeutic agents such as polyhexamethylene biguanide or chlorhexidine and propamidine isethionate. However, all the mentioned compounds have also showed toxic side effects on human keratocytes and presented poor cysticidal effect at the concentrations currently used in the established AK treatments. Nowadays, the elucidation of novel compounds with antimicrobial and anticancer properties from plant and herbs with medicinal properties have encouraged researchers to evaluate plants as a source of new molecules with anti-trophozoite and cysticidal effects. Thus, in recent years, many natural products have been reported to present potent anti-Acanthamoeba properties with good selectivity and minimal toxic effects. Therefore, the chemical drugs currently used for AK treatment, their drawbacks as well as the current research in medicinal plants as a source of potent anti-Acanthamoeba compounds are described in this review. PMID:28243287

  10. Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila.

    PubMed

    Köhler, R; Bubert, A; Goebel, W; Steinert, M; Hacker, J; Bubert, B

    2000-01-01

    The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila. To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (Macrophage Infectivity Potentiator) promoter (P(mip)) and the sod (SuperOxide Dismutase) promoter (P(sod)) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1ten-fold higher intensity. No fluorescence was observed in L. pneumophila transformed with the promoterless gfp gene. Comparison of fluorescence yields between various L. pneumophila strains that differ in their virulence characteristics and were transformed with the P(mip)-gfp carrying plasmid revealed no differences in GFP expression. Infection studies using Acanthamoeba castellanii as host and recombinant L. pneumophila strains carrying the P(mip)-gfp and P(sod)-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by. L. pneumophila strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent L. pneumophila strain Corby was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin D, cycloheximide, and methylamine on the uptake of Legionella by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process.

  11. Comparison of Fluorescence Microscopy and Different Growth Media Culture Methods for Acanthamoeba Keratitis Diagnosis.

    PubMed

    Peretz, Avi; Geffen, Yuval; Socea, Soergiu D; Pastukh, Nina; Graffi, Shmuel

    2015-08-01

    Acanthamoeba keratitis (AK), a potentially blinding infection of the cornea, is caused by a free-living protozoan. Culture and microscopic examination of corneal scraping tissue material is the conventional method for identifying Acanthamoeba. In this article, we compared several methods for AK diagnosis of 32 patients: microscopic examination using fluorescent dye, specific culture on growth media-non-nutrient agar (NNA), culture on liquid growth media-peptone yeast glucose (PYG), and TYI-S-33. AK was found in 14 patients. Thirteen of the specimens were found AK positive by fluorescence microscopic examination, 11 specimens were found AK positive on PYG growth media, and 9 specimens were found AK positive on TYI-S-33 growth media. Only five specimens were found AK positive on NNA growth media. Therefore, we recommend using fluorescence microscopy technique and culture method, especially PYG liquid media. © The American Society of Tropical Medicine and Hygiene.

  12. [The Investigation of Acanthamoeba and Other Free Living Amoeba in Swab Samples Obtained from Conjunctiva and Eye Lid].

    PubMed

    Yünlü, Önder; Özçelik, Semra; Arıcı, Mustafa Kemal

    2015-09-01

    In the study, it is aimed to determine the prevalence of Acanthamoeba and other free-living amoeba (FLA) species in the swab samples obtained from conjunctiva and lower eye lid. For this purpose, swab samples from the 500 patients'eye lid and conjunctiva were obtained who admitted to Cumhuriyet University, Research and Application Hospital, Department of Ophthalmology with variety of reasons. Swab samples were carried out using sterile cotton swab in steril tubes. The swab samples were inoculated onto non-nutrient agar (NNA). Live Escherichia coli was used as food source for the growth of the FLA. The NNA plates were incubated at 300C and examined daily using ligth microscope for two weeks. For morphotyping of the trophozoites and cysts of the FLA were used taxonomic keys. Two of the 500 swab samples (0.4%) were positive for FLA. One of them (0.2%) were identified as Acanthamoeba spp. and other was identified as Hartmannella spp. However, these patients did not reveal any complaints yet. FLA both themselves and bacteria carrying in their body as reservoirs are potential pathogen. The rapid spread of Acanthamoeba keratitis in recent years reveal that these microorganisms are in contact with the eyes.

  13. Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

    PubMed Central

    Khan, Naveed Ahmed; Iqbal, Junaid

    2015-01-01

    In the face of harsh conditions and given a choice, a cell may (i) undergo programmed cell death, (ii) transform into a cancer cell, or (iii) enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes. PMID:25648302

  14. Assessment of the efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and Tetrahymena spp.

    PubMed

    Vaerewijck, M J M; Sabbe, K; Baré, J; Spengler, H-P; Favoreel, H W; Houf, K

    2012-03-01

    The efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and two Tetrahymena spp. was determined based on the European Standard EN 1276:2009 suspension test. Trophozoite viability was assessed by determination of the membrane integrity using flow cytometry as a fast screening technique. Bovine serum albumin was added to simulate clean (0.3 g/liter) and dirty (3 g/liter) conditions. Benzalkonium chloride caused cell lysis at concentrations above 50 mg/liter under clean and dirty conditions. A concentration of 50 mg of free chlorine per liter had a strong biocidal effect on acanthamoebae and tetrahymenae after 15 min under clean and dirty conditions. Our results suggest that benzalkonium chloride and sodium hypochlorite were effective against the three microorganisms at concentrations commonly applied in the food industry.

  15. Inside the lifestyle of the virophage.

    PubMed

    Desnues, C; Raoult, D

    2010-01-01

    We sought to better characterize Sputnik, the first isolated virophage, and to analyze its parasitic lifestyle during co-infection with Marseillevirus (a new giant virus) in Acanthamoeba castellanii. A combination of electron microscopy, immunofluorescence microscopy, and real-time PCR was used to characterize the kinetics of the viral replication cycle. RT-PCR was performed to detect RNAs inside the Sputnik virions. Sputnik is a new viral entity carrying an almost complete ready-to-use set of viral RNAs (20 out of 21). Sputnik does not replicate with Marseillevirus but delays its replication cycle. While Marseillevirus is successfully internalized by A. castellanii following co-infections with Mamavirus and Sputnik, it does not initiate a replication cycle. In contrast, both Marseillevirus and Mamavirus can replicate in the amoeba in case of co-infection, but the development of one is exclusive from the other inside a single amoeba cell. This work provides new insight into the Sputnik replication cycle with another giant virus and confirms that Sputnik is a virophage. It shows new dimensions of the interactions existing among giant viruses. Copyright 2010 S. Karger AG, Basel.

  16. Legionella norrlandica sp. nov., isolated from the biopurification systems of wood processing plants.

    PubMed

    Rizzardi, Kristina; Winiecka-Krusnell, Jadwiga; Ramliden, Miriam; Alm, Erik; Andersson, Sabina; Byfors, Sara

    2015-02-01

    Fourteen isolates of an unknown species identified as belonging to the genus Legionella by selective growth on BCYE agar were isolated from the biopurification systems of three different wood processing plants. The mip gene sequence of all 14 isolates was identical and a close match alignment revealed 86 % sequence similarity with Legionella pneumophila serogroup 8. The whole genome of isolate LEGN(T) was sequenced, and a phylogenetic tree based on the alignment of 16S rRNA, mip, rpoB, rnpB and the 23S-5S intergenic region clustered LEGN(T) with L. pneumophila ATCC 33152(T). Analysis of virulence factors showed that strain LEGN(T) carries the majority of known L. pneumophila virulence factors. An amoeba infection assay performed to assess the pathogenicity of strain LEGN(T) towards Acanthamoeba castellanii showed that it can establish a replication vacuole in A. castellanii but does not significantly affect replication of amoebae. Taken together, the results confirm that strain LEGN(T) represents a novel species of the genus Legionella, for which the name Legionella norrlandica sp. nov. is proposed. The type strain is LEGN(T) ( = ATCC BAA-2678(T) = CCUG 65936(T)). © 2015 IUMS.

  17. How Could Contact Lens Wearers Be at Risk of Acanthamoeba Infection? A Review

    PubMed Central

    Ibrahim, Youhanna W.; Boase, David L.; Cree, Ian A.

    2010-01-01

    Contact lens wear is highly influential on the incidence of ulcerative keratitis worldwide, particularly in developed countries. The association between Acanthamoeba keratitis and contact lens wear is firmly established; it may account for up to 95% of the reported cases. Before the popularisation of soft contact lens wear, Acanthamoeba keratitis was extremely rare. In 2000 it was estimated that the number of contact lens wearers worldwide was about 80 million, out of whom 33 million were in the United States and 90% of them wore hydrogel soft lenses. Contact lens-related problems depend on many factors, such as lens material, wearing modality, lens hygiene, type of lens-caring solution, the degree of compliance of the lens user with lens wear and care procedures, lens overwear, sleeping in lenses, rate of changing lenses, and lens case hygiene. This paper is a thorough review of the literature aiming to highlight the role of one of the main risk factors of infectious keratitis, contact lens wear, and also to show the responsibility of lens users in aggravating this risk.

  18. TATA box-binding protein (TBP) is a constituent of the polymerase I-specific transcription initiation factor TIF-IB (SL1) bound to the rRNA promoter and shows differential sensitivity to TBP-directed reagents in polymerase I, II, and III transcription factors.

    PubMed

    Radebaugh, C A; Matthews, J L; Geiss, G K; Liu, F; Wong, J M; Bateman, E; Camier, S; Sentenac, A; Paule, M R

    1994-01-01

    The role of the Acanthamoeba castellanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.

  19. Acanthamoeba on Sabouraud's agar from a patient with keratitis

    PubMed Central

    Baradkar, Vasant; Samal, Badhuli; Mali, Swapna A; Kulkarni, Ketaki; Shastri, Jayanthi

    2011-01-01

    A 25-year-old transgender patient came with complaints of watery discharge, red eye and photophobia in the left eye since 2 days. The patient had a history of wearing colored contact lenses since 4 years and cleaning the lens with tap water. Culture of lenses on Mac Conkey and blood agar yielded Klebsiella pneumoniae and Pseudomonas aeruginosa. Sabouroud's agar showed yeast cells and double-walled cysts of Acanthamoeba species. On further incubation of Sabouroud's agar, the cysts transformed to trophozoites. Parallel results were obtained on tap water agar. The previous therapy of moxifloxacin was changed to local Neosporin application. PMID:23508061

  20. Detection of serum antibodies in children and adolescents against Balamuthia mandrillaris, Naegleria fowleri and Acanthamoeba T4.

    PubMed

    Lares-Jiménez, Luis Fernando; Borquez-Román, Manuel Alejandro; Alfaro-Sifuentes, Rosalía; Meza-Montenegro, María Mercedes; Casillas-Hernández, Ramón; Lares-Villa, Fernando

    2018-06-01

    The presence of free-living amoebae of the genera Naegleria, Acanthamoeba and Balamuthia, which contain pathogenic species for humans and animals, has been demonstrated several times and in different natural aquatic environments in the northwest of Mexico. With the aim of continuing the addition of knowledge about immunology of pathogenic free-living amoebae, 118 sera from children and adolescents, living in three villages, were studied. Humoral IgG response against B. mandrillaris, N. fowleri and Acanthamoeba sp. genotype T4, was analyzed in duplicate to titers 1: 100 and 1: 500 by enzyme-linked immunosorbent assay (ELISA). Children and adolescents ages ranged between 5 and 16 years old, with a mean of 9 years old, 55% males. All tested sera were positive for the 1: 100 dilution, and in the results obtained with the 1: 500 dilution, 116 of 118 (98.3%) were seropositive for N. fowleri, 101 of 118 (85.6%) were seropositive for Acanthamoeba sp. genotype T4, and 43 of 118 (36.4%) were seropositive for B. mandrillaris. The statistical analysis showed different distributions among the three communities and for the three species of pathogenic free-living amoebae, including age. Lysed and complete cells used as Balamuthia antigens gave differences in seropositivity. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus

    PubMed Central

    Legendre, Matthieu; Audic, Stéphane; Poirot, Olivier; Hingamp, Pascal; Seltzer, Virginie; Byrne, Deborah; Lartigue, Audrey; Lescot, Magali; Bernadac, Alain; Poulain, Julie; Abergel, Chantal; Claverie, Jean-Michel

    2010-01-01

    Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic “virion factory,” we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5′ and 3′ extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus “virophage.” These results—validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)—will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system. PMID:20360389

  2. Germination and amplification of anthrax spores by soil-dwelling amoebas.

    PubMed

    Dey, Rafik; Hoffman, Paul S; Glomski, Ian J

    2012-11-01

    While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.

  3. Distribution of Acanthamoeba Genotypes Isolated from Recreational and Therapeutic Geothermal Water Sources in Southwestern Iran

    PubMed Central

    Niyyati, Maryam; Saberi, Reza; Latifi, Alireza; Lasjerdi, Zohreh

    2016-01-01

    A comprehensive survey was conducted along 10 km of geothermal rivers in southwestern Iran. A total of 40 water samples were tested for the presence of Acanthamoeba spp., and genotypes were determined by targeting the diagnostic fragment 3 region of the 18S rRNA gene. The pathogenic potential of all positive isolates was also identified using tolerance ability test. High occurrences of Acanthamoeba (50%) were detected in the sampling areas. Based on sequencing analysis, isolates belonging to T4 (93.7%) and T2 (6.25%) genotypes were reported. Thermo- and osmotolerance tests revealed that five strains are highly pathogenic. Since every collection site of this study was associated with high human activity, posting of warning signs, monitoring of recreational water sources, and awareness of high-risk people are of utmost importance. To the best of our knowledge, the present research is the first to report T2 genotype from geothermal water sources in Iran. PMID:27127409

  4. Distribution of Acanthamoeba Genotypes Isolated from Recreational and Therapeutic Geothermal Water Sources in Southwestern Iran.

    PubMed

    Niyyati, Maryam; Saberi, Reza; Latifi, Alireza; Lasjerdi, Zohreh

    2016-01-01

    A comprehensive survey was conducted along 10 km of geothermal rivers in southwestern Iran. A total of 40 water samples were tested for the presence of Acanthamoeba spp., and genotypes were determined by targeting the diagnostic fragment 3 region of the 18S rRNA gene. The pathogenic potential of all positive isolates was also identified using tolerance ability test. High occurrences of Acanthamoeba (50%) were detected in the sampling areas. Based on sequencing analysis, isolates belonging to T4 (93.7%) and T2 (6.25%) genotypes were reported. Thermo- and osmotolerance tests revealed that five strains are highly pathogenic. Since every collection site of this study was associated with high human activity, posting of warning signs, monitoring of recreational water sources, and awareness of high-risk people are of utmost importance. To the best of our knowledge, the present research is the first to report T2 genotype from geothermal water sources in Iran.

  5. Enhanced killing of Acanthamoeba cysts with a plant peroxidase-hydrogen peroxide-halide antimicrobial system.

    PubMed

    Hughes, Reanne; Andrew, Peter W; Kilvington, Simon

    2003-05-01

    The activity of H(2)O(2) against the resistant cyst stage of the pathogenic free-living amoeba Acanthamoeba was enhanced by the addition of KI and either horseradish peroxidase or soybean peroxidase or, to a lesser degree, lactoperoxidase. This resulted in an increase in the cysticidal activity of 3% (wt/vol) H(2)O(2), and there was >3-log killing in 2 h, compared with the 6 h required for comparable results with the peroxide solution alone (P < 0.05). With 2% H(2)O(2), enhancement was observed at all time points (P < 0.05), and total killing of the cyst inoculum occurred at 4 h, compared with 6 h for the peroxide alone. The activity of sublethal 1% H(2)O(2) was enhanced to give 3-log killing after 8 h of exposure (P < 0.05). No enhancement was obtained when KCl or catalase was used as a substitute in the reaction mixtures. The H(2)O(2) was not neutralized in the enhanced system during the experiments. However, in the presence of a platinum disk used to neutralize H(2)O(2) in contact lens care systems, the enhanced 2% H(2)O(2) system gave 2.8-log killing after 6 h or total cyst killing by 8 h, and total neutralization of the H(2)O(2) occurred by 4 h. In contrast, 2% H(2)O(2) alone resulted in <0.8-log killing of cysts in the presence of the platinum disk due to rapid (<1 h) neutralization of the peroxide. Our observations could result in significant improvement in the efficacy of H(2)O(2) contact lens disinfection systems against Acanthamoeba cysts and prevention of acanthamoeba keratitis.

  6. Divalent Metal Cations Potentiate the Predatory Capacity of Amoeba for Cryptococcus neoformans.

    PubMed

    Fu, Man Shun; Casadevall, Arturo

    2018-02-01

    Among the best-studied interactions between soil phagocytic predators and a human-pathogenic fungus is that of Acanthamoeba castellanii and Cryptococcus neoformans The experimental conditions used in amoeba-fungus confrontation assays can have major effects on whether the fungus or the protozoan is ascendant in the interaction. In the presence of Mg 2+ and Ca 2+ in phosphate-buffered saline (PBS), C. neoformans was consistently killed when incubated with A. castellanii A. castellanii survived better in the presence of Mg 2+ and Ca 2+ , even when incubated with C. neoformans In the absence of Mg 2+ and Ca 2+ , C. neoformans survived when incubated with A. castellanii , and the percentage of dead amoebae was higher than when incubated without yeast cells. These results show that the presence of Mg 2+ and Ca 2+ can make a decisive contribution toward tilting the outcome of the interaction in favor of the amoeba. Of the two metals, Mg 2+ had a stronger effect than Ca 2+ The cations enhanced A. castellanii activity against C. neoformans via enhanced phagocytosis, which is the major mechanism by which amoebae kill fungal cells. We found no evidence that amoebae use extracellular killing mechanisms in their interactions with C. neoformans In summary, the presence of Mg 2+ and Ca 2+ enhanced the cell adhesion on the surfaces and the motility of the amoeba, thus increasing the chance for contact with C. neoformans and the frequency of phagocytosis. Our findings imply that the divalent cation concentration in soils could be an important variable for whether amoebae can control C. neoformans in the environment. IMPORTANCE The grazing of soil organisms by phagocytic predators such as amoebae is thought to select for traits that enable some of them to acquire the capacity for virulence in animals. Consequently, knowledge about the interactions between amoebae and soil microbes, such as pathogenic fungi, is important for understanding how virulence can emerge. We show that the

  7. Molecular and morphological characterization of Acanthamoeba isolated from corneal scrapes and contact lens wearers in Argentina.

    PubMed

    Casero, Rodolfo D; Mongi, Florencia; Laconte, Laura; Rivero, Fernando; Sastre, Dario; Teherán, Aníbal; Herrera, Giovanny; Ramírez, Juan David

    2017-10-01

    In this study, we describe the frequency of Acanthamoeba keratitis (AK) in patients that assisted in the Ophthalmology Department and determine the species/genotypes of free living amoebas (FLA) isolates. FLA from Corneal scrapes (CS) and contact lens (CL) wearers were studied by morphological and molecular characterization. A database was constructed with sociodemographic, clinical findings and history of use of CL variables. During January 2000 and September 2016 patients with corneal pathology admitted to the Ophthalmology Service of the University Hospital in Córdoba city, Argentina were included in the study. FLA were detected in 1.5% (11/739) and in 17% (11/65) of CS and CL analyzed respectively. FLA isolates from CL users evidenced an 80.9% of inappropriate lens maintenance, 4.8% (1/21) were not CL users that have been in contact with waters in outdoor environment and 14,3% (3/21) with no data about CL users. Acanthamoeba was confirmed in 100% and 82% of CS and LC respectively. The most frequent symptom associated with AK was red eye and photophobia. FLA from CS belonged to group II but 82% (9/11) and 18% (2/11) from CL belonged to group II and III respectively. T4 genotype and A. polyphaga species were detected in 100% of Acanthamoeba isolates. Poor CL hygiene practices, highlights the need for improved education about the severity of AK and consequences of improper CL hygiene. Genotype T4 detected in 100% of both CS and CL samples, consistently with previous findings indicating that this genotype is by far the most prevalent isolated from ocular infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Ultradian metronome: timekeeper for orchestration of cellular coherence.

    PubMed

    Lloyd, David; Murray, Douglas B

    2005-07-01

    Dynamic intracellular spatial and temporal organization emerges from spontaneous synchronization of a massive array of weakly coupled oscillators; the majority of subcellular processes are implicated in this integrated expression of cellular physiology. Evidence for this view comes mainly from studies of Saccharomyces cerevisiae growing in self-synchronized continuous cultures, in which a temperature-compensated ultradian clock (period of approximately 40 min) couples fermentation with redox state in addition to the transcriptome and cell-division-cycle progression. Functions for ultradian clocks have also been determined in other yeasts (e.g. Schizosaccharomyces pombe and Candida utilis), seven protists (e.g. Acanthamoeba castellanii and Paramecium tetraurelia), as well as cultured mammalian cells. We suggest that ultradian timekeeping is a basic universal necessity for coordinated intracellular coherence.

  9. srRNA evolution and phylogenetic relationships of the genus Naegleria (Protista: Rhizopoda).

    PubMed

    Baverstock, P R; Illana, S; Christy, P E; Robinson, B S; Johnson, A M

    1989-05-01

    A rapid RNA sequencing technique was used to partially sequence the small-subunit ribosomal RNA (srRNA) of four species of the amoeboid genus Naegleria. The extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. However, the pattern of variation among the Naegleria species was quite different from that found for those species of tetrapods characterized to date. A phylogenetic analysis of the consensus Naegleria sequence showed that Naegleria was not monophyletic with either Acanthamoeba castellanii or Dictyostelium discoideum, two other amoebas for which sequences were available. It was shown that the semiconserved regions of the srRNA molecule evolve in a clocklike fashion and that the clock is time dependent rather than generation dependent.

  10. Intracellular Localization and Trafficking of Serine Proteinase AhSub and Cysteine Proteinase AhCP of Acanthamoeba healyi

    PubMed Central

    Moon, E.-K.; Lee, S.-T.; Chung, D.-I.; Kong, H.-H.

    2006-01-01

    Proteinases have been proposed to play important roles in pathogenesis and various biologic actions in Acanthamoeba. Although genetic characteristics of several proteases of Acanthamoeba have been reported, the intracellular localization and trafficking of these enzymes has yet to be studied. In the present study, we analyzed the intracellular localization and trafficking of two proteinases, AhSub and AhCP, of Acanthamoeba healyi by transient transfection. Full-length AhSub-enhanced green fluorescent protein (EGFP) fusion protein was found in intracellular vesicle-like structures of transfected amoebae. Time-lapse photographs confirmed the secretion of the fluorescent material of the vesicle toward the extracellular space. The mutated AhSub, of which the pre or prepro region was deleted, was found to localize diffusely throughout the cytoplasm of the amoeba rather than concentrated in the secretory vesicle. Transfection of the construct containing the pre region only showed the same localization and trafficking of the full-length AhSub. A cysteine proteinase AhCP-EGFP fusion protein showed similar localization in the vesicle-like structure in the amoeba. However, using Lyso Tracker analysis, these vesicular structures of AhCP were confirmed to be lysosomes rather than secretory vesicles. The AhCP construct with a deletion of the prepro region showed a dispersed distribution of fluorescence in the cytoplasm of the cells. These results indicated that AhSub and AhCP would play different roles in Acanthameoba biology and that the pre region of AhSub and pro region of AhCP are important for proper intracellular localization and trafficking of each proteinase. PMID:16400174

  11. Acanthamoeba culbertsoni isolated from a clinical case with intraocular dissemination: Structure and in vitro analysis of the interaction with hamster cornea and MDCK epithelial cell monolayers.

    PubMed

    González-Robles, Arturo; Omaña-Molina, Maritza; Salazar-Villatoro, Lizbeth; Flores-Maldonado, Catalina; Lorenzo-Morales, Jacob; Reyes-Batlle, María; Arnalich-Montiel, Francisco; Martínez-Palomo, Adolfo

    2017-12-01

    Acanthamoeba culbertsoni trophozoites, previously isolated from a human keratitis case with severe intraocular damage, were maintained in axenic culture. Co-incubation of amoebae with MDCK cell monolayers demonstrated an apparent preference of the amoebae to introduce themselves between the cells. The trophozoites appeared to cross the cell monolayer through the tight junctions, which resulted in decreased trans-epithelial resistance (TER) measurements. Unexpectedly, after co-incubation of amoebae with hamster corneas, we observed that the trophozoites were able to cross the different cell layers and reach the corneal stroma after only 12 h of interaction, in contrast to other Acanthamoeba species. These observations suggest that this A. culbertsoni isolate is particularly pathogenic. Further research with diverse methodologies needs to be performed to explain the unique behavior of this Acanthamoeba strain. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. The association of contact lens solution use and Acanthamoeba keratitis.

    PubMed

    Joslin, Charlotte E; Tu, Elmer Y; Shoff, Megan E; Booton, Gregory C; Fuerst, Paul A; McMahon, Timothy T; Anderson, Robert J; Dworkin, Mark S; Sugar, Joel; Davis, Faith G; Stayner, Leslie T

    2007-08-01

    To investigate Acanthamoeba keratitis (AK) risk factors. Diagnosis of AK, a rare but serious corneal infection, has recently increased significantly at the University of Illinois at Chicago (UIC) Cornea Service. Retrospective case-control study. settings: University, tertiary care hospital. patients: Fifty-five AK cases with contact lens use were diagnosed between May 1, 2003 and September 15, 2006. Clinic-matched controls with contact lens use were recruited. Subjects completed surveys targeting lens hygiene, contact lens solution use, and water exposure. main outcome measure: Acanthamoeba keratitis. Thirty-nine (73.6%) cases and 113 (65.3%) controls participated; 38 cases had complete contact lens data. Thirty-five of 38 cases (92.1%) and 47 of 100 controls (47.0%) used soft lenses. Analysis was performed on 30 cases and 39 controls with matched pairs with soft lens use. Exclusive use of Advance Medical Optics (AMO) Complete MoisturePlus Multi-Purpose Solution was independently associated with AK in multivariable analysis (55.2% vs 10.5%; odds ratio [OR], 16.67; 95% confidence interval [CI] 2.11 to 162.63; P = .008). However, 38.8% of cases reported no use of AMO Complete MoisturePlus Multi-Purpose Solution either alone or in combination with other solutions. Although not statistically significant, additional hygiene-related variables (solution "reuse," lack of "rubbing," and showering with lenses) suggest a pattern of risk. AMO Complete MoisturePlus Multi-Purpose Solution use is independently associated with AK among soft contact lens users. However, it does not explain all cases, suggesting additional factors. Further research into environmental risk factors and hygiene practices is warranted, especially considering this is the second outbreak of an atypical, contact lens-related infection.

  13. The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna.

    PubMed

    Tyson, Jessica Y; Vargas, Paloma; Cianciotto, Nicholas P

    2014-12-01

    The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires' disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts. © 2014 The Authors.

  14. Molecular characterization of Acanthamoeba strains isolated from domestic dogs in Tenerife, Canary Islands, Spain.

    PubMed

    Valladares, María; Reyes-Batlle, María; Martín-Navarro, Carmen M; López-Arencibia, Atteneri; Dorta-Gorrín, Alexis; Wagner, Carolina; Martínez-Carretero, Enrique; Piñero, José E; Valladares, Basilio; Lorenzo-Morales, Jacob

    2015-06-01

    The present study describes two cases of Acanthamoeba infections (keratitis and ascites/peritonitis) in small breed domestic dogs in Tenerife, Canary Islands, Spain. In both cases, amoebic trophozoites were observed under the inverted microscope and isolated from the infected tissues and/or fluids, without detecting the presence of other viral, fungal or bacterial pathogens. Amoebae were isolated using 2 % non-nutrient agar plates and axenified for further biochemical and molecular analyses. Osmotolerance and thermotolerance assays revealed that both isolates were able to grow up to 37 °C and 1 M of mannitol and were thus considered as potentially pathogenic. Moreover, the strains were classified as highly cytotoxic as they cause more than 75 % of toxicity when incubated with two eukaryotic cell lines. In order to classify the strains at the molecular level, the diagnostic fragment 3 (DF3) region of the 18S rDNA of Acanthamoeba was amplified and sequenced, revealing that both isolates belonged to genotype T4. In both cases, owners of the animals did not allow any further studies or follow-up and therefore the current status of these animals is unknown. Furthermore, the isolation of these pathogenic amoebae should raise awareness with the veterinary community locally and worldwide.

  15. Revisiting the Acanthamoeba species that form star-shaped cysts (genotypes T7, T8, T9, and T17): characterization of seven new Brazilian environmental isolates and phylogenetic inferences.

    PubMed

    Magliano, Ana C M; Teixeira, Marta M G; Alfieri, Silvia C

    2012-01-01

    Free-living amoebae of the genus Acanthamoeba are the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1-T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmental Acanthamoeba isolates, all assigned to group I. Phylogenetic analyses based on partial (~1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related to A. comandoni (genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2·0% sequence divergence) that likely represents a new Acanthamoeba species. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40°C, and resist under hyperosmotic conditions. Additionally, media conditioned by each of the new Acanthamoeba isolates induced the disruption of SIRC and HeLa cell monolayers.

  16. The fundamental ribosomal RNA transcription initiation factor-IB (TIF-IB, SL1, factor D) binds to the rRNA core promoter primarily by minor groove contacts.

    PubMed

    Geiss, G K; Radebaugh, C A; Paule, M R

    1997-11-14

    Acanthamoeba castellanii transcription initiation factor-IB (TIF-IB) is the TATA-binding protein-containing transcription factor that binds the rRNA promoter to form the committed complex. Minor groove-specific drugs inhibit TIF-IB binding, with higher concentrations needed to disrupt preformed complexes because of drug exclusion by bound TIF-IB. TIF-IB/DNA interactions were mapped by hydroxyl radical and uranyl nitrate footprinting. TIF-IB contacts four minor grooves in its binding site. TIF-IB and DNA wrap around each other in a right-handed superhelix of high pitch, so the upstream and downstream contacts are on opposite faces of the helix. Dimethyl sulfate protection assays revealed limited contact with a few guanines in the major groove. This detailed analysis suggests significant DNA conformation dependence of the interaction.

  17. Resistance of Acanthamoeba Cysts to Disinfection Treatments Used in Health Care Settings▿

    PubMed Central

    Coulon, Céline; Collignon, Anne; McDonnell, Gerald; Thomas, Vincent

    2010-01-01

    Free-living amoebae that belong to the genus Acanthamoeba are widespread in the environment, including water. They are responsible for human infections and can host pathogenic microorganisms. Under unfavorable conditions, they form cysts with high levels of resistance to disinfection methods, thus potentially representing a threat to public health. In the present study we evaluated the efficacies of various biocides against trophozoites and cysts of several Acanthamoeba strains. We demonstrated that disinfectant efficacy varied depending on the strains tested, with environmental strains demonstrating greater resistance than collection strains. Trophozoites were inactivated by all treatments except those using glutaraldehyde as an active compound: for these treatments, we observed resistance even after 30 min exposure. Cysts resisted many treatments, including certain conditions with glutaraldehyde and other biocides. Moist heat at 55°C was not efficient against cysts, whereas exposure at 65°C was. Several chemical formulations containing peracetic acid, hydrogen peroxide, or ortho-phthalaldehyde presented greater efficacy than glutaraldehyde, as did ethanol and sodium hypochlorite; however, some of these treatments required relatively long incubation times to achieve cyst inactivation. Amoebal cysts can be highly resistant to some high-level disinfectants, which has implications for clinical practice. These results highlight the need to consider the effective disinfection of protozoa in their vegetative and resistant forms due to their intrinsic resistance. This is important not only to prevent the transmission of protozoa themselves but also due to the risks associated with a range of microbial pathogens that are found to be associated intracellularly with these microorganisms. PMID:20519477

  18. The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA.

    PubMed

    Tiaden, André; Spirig, Thomas; Weber, Stefan S; Brüggemann, Holger; Bosshard, Rachel; Buchrieser, Carmen; Hilbi, Hubert

    2007-12-01

    Legionella pneumophila is an opportunistic human pathogen that replicates within environmental amoebae including Acanthamoeba castellanii and Dictyostelium discoideum. The Icm/Dot type IV secretion system promotes phagocytosis and intracellular replication of L. pneumophila in an endoplasmic reticulum-derived 'Legionella-containing vacuole' (LCV). L. pneumophila adopts a biphasic life cycle consisting of a replicative growth phase and a transmissive (stationary) phase, the latter of which is characterized by the preferential expression of genes required for motility and virulence. A bioinformatic analysis of the L. pneumophila genome revealed a gene cluster homologous to the Vibrio cholerae cqsAS genes, encoding a putative quorum sensing autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a novel response regulator (lqsR). We report here that an L. pneumophila lqsR deletion mutant grew in broth with the same rate as wild-type bacteria, but entered the replicative growth phase earlier. Overexpression of lqsR led to an elongated morphology of the bacteria. The lqsR mutant strain was found to be more salt-resistant and impaired for intracellular growth in A. castellanii, D. discoideum and macrophages, formation of the ER-derived LCV and toxicity. Moreover, L. pneumophila lacking LqsR, as well as strains lacking the stationary sigma factor RpoS or the two-component response regulator LetA, were phagocytosed less efficiently by A. castellanii, D. discoideum or macrophages. The expression of lqsR was dependent on RpoS and, to a lesser extent, also on LetA. DNA microarray experiments revealed that lqsR regulates the expression of genes involved in virulence, motility and cell division, consistent with a role for LqsR in the transition from the replicative to the transmissive (virulent) phase. Our findings indicate that LqsR is a novel pleiotropic regulator involved in RpoS- and LetA-controlled interactions of L. pneumophila with phagocytes.

  19. A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

    PubMed Central

    Al-Khouri, Anna Maria; Paule, Marvin R.

    2002-01-01

    In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852

  20. A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding.

    PubMed

    Al-Khouri, Anna Maria; Paule, Marvin R

    2002-02-01

    In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.

  1. Safety of fish therapeutants to glochidia of the plain pocketbook mussel during encystment on largemouth bass

    USGS Publications Warehouse

    Rach, J.J.; Brady, T.; Schreier, Theresa M.; Aloisi, D.

    2006-01-01

    Mussel biologists and fisheries managers have developed propagation techniques to duplicate the natural glochidia infestation on host fish. However, in intensive culture situations, fish diseases may threaten the survival of both fish and their attached glochidia and chemical treatments may be required to control a disease epizootic. Five therapeutants were evaluated for their safety to largemouth bass Micropterus salmoides encysted with mussel glochidia by comparing the number of sloughed glochidia in the chemical treatment groups with that of an untreated control group. Largemouth bass were infested with glochidia from the plain pocketbook mussel Lampsilis cardium and treated with 20 mg chloramine-T/L, 2 mg Cutrine/L, or 200 mg formalin/L (trial 1) and 200 mg formalin/L, 100 mg hydrogen peroxide/L, or 20,000 mg sodium chloride/L (trial 2). Chemicals were applied for 60 min (15 min in the case of sodium chloride in trial 2) once every other day, for a total of three treatments (six in the case of formalin in trial 2). After the first treatment, aquaria were siphoned each weekday to determine the number of sloughed glochidia or transformed juveniles. In trial 1, the initial mean number of glochidia per fish ranged from 257 to 294, and approximately 94% of the glochidia transformed to juveniles. In trial 2, the initial mean number of glochidia per fish ranged from 97 to 115, and approximately 91% of the glochidia transformed to juveniles. The mean percent of sloughed glochidia varied by less than 2% among all test groups in each trial. There were no significant differences (P < 0.05) in the number of sloughed glochidia or transformed juveniles among control or treatment groups in either trial. Therapeutic treatment of diseased fish with chloramine-T, Cutrine, formalin, hydrogen peroxide, or sodium chloride at the treatment regimens evaluated are viable options for enhancing the survival of fish encysted with glochidia.

  2. Morphological characteristics of developmental stages of Acanthamoeba and Naegleria species before and after staining by various techniques.

    PubMed

    Ithoi, Init; Ahmad, Arine-Fadzlun; Mak, J W; Nissapatorn, Veeranoot; Lau, Yee-Ling; Mahmud, Rohela

    2011-11-01

    Seven stains were studied to determine the best color and contrast for staining the developmental stages of free living pathogenic Acanthamoeba and Naegleria species. The acid-fast bacilli stain (AFB) produced a blue color without contrast; trichrome-eosin and modified Field's showed various color contrasts; Giemsa, iron-hematoxylin, modified AFB and Gram produced only one color which distinguished the nucleus, nucleolus, cytoplasm, food- and water-vacuoles. The motile organs (acanthopodia, pseudopodia, lobopodia and flagella) were also clearly differentiated but produced a similar color as the cytoplasm. These motile organelles were first induced by incubating at 37 degrees C for at least 15 minutes and then fixing with methanol in order to preserve the protruding morphology prior to staining. The trichrome-eosin and iron-hematoxylin stains showed good color contrast for detecting all three stages, the trophozoite, cyst and flagellate; Giemsa and Gram stained the trophozoite and flagellate stages; the modified Field's and modified AFB stains stained only the trophozoite stage. Depending on the purpose, all these stains (except the AFB stain) can be used to identify the developmental stages of Acanthamoeba and Naegleria for clinical, epidemiological or public health use.

  3. The fate of Helicobacter pylori phagocytized by Acanthamoeba polyphaga demonstrated by fluorescent in situ hybridization and quantitative polymerization chain reaction tests

    EPA Science Inventory

    Helicobacter pylori able to express green fluorescent protein, as well as an ATCC strain, and a clinical isolate of this pathogen were evaluated for their ability to survive predation by Acanthamoeba polyphaga. Ingestion was evaluated by microscopic observation of the GFP-H. pyl...

  4. Phosphorylation of Eukaryotic Initiation Factor-2α during Stress and Encystation in Entamoeba Species

    PubMed Central

    Hendrick, Holland M.; Welter, Brenda H.; Sykes, Steven E.; Sullivan, William J.; Temesvari, Lesly A.

    2016-01-01

    encystation in E. invadens, a model encystation system. Together, these data demonstrate that the eIF2α-dependent stress response system is operational in Entamoeba species. PMID:27930733

  5. Acanthamoeba polyphaga mimivirus and other giant viruses: an open field to outstanding discoveries

    PubMed Central

    2014-01-01

    In 2003, Acanthamoeba polyphaga mimivirus (APMV) was first described and began to impact researchers around the world, due to its structural and genetic complexity. This virus founded the family Mimiviridae. In recent years, several new giant viruses have been isolated from different environments and specimens. Giant virus research is in its initial phase and information that may arise in the coming years may change current conceptions of life, diversity and evolution. Thus, this review aims to condense the studies conducted so far about the features and peculiarities of APMV, from its discovery to its clinical relevance. PMID:24976356

  6. UV inactivation of pathogenic and indicator microorganisms

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chang, J.C.; Ossoff, S.F.; Lobe, D.C.

    1985-06-01

    Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4more » times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts.« less

  7. Acanthamoeba Keratitis among Rigid Gas Permeable Contact Lens Wearers in the United States, 2005 through 2011.

    PubMed

    Cope, Jennifer R; Collier, Sarah A; Schein, Oliver D; Brown, Allison C; Verani, Jennifer R; Gallen, Rachel; Beach, Michael J; Yoder, Jonathan S

    2016-07-01

    To describe the clinical presentation and outcomes of Acanthamoeba keratitis (AK) in rigid gas permeable (RGP) contact lens wearers and to identify modifiable risk factors. Case-control investigation. Patients were RGP contact lens-wearing United States residents with a diagnosis of AK from 2005 through 2011. Controls were RGP contact lens wearers with no history of AK who were at least 12 years of age. Patients were identified during 2 multistate AK outbreak investigations. Controls from the first investigation in 2007 were identified using a reverse address directory. In the second investigation, controls were recruited from participating ophthalmology and optometry practices. Patients and controls were interviewed by phone using a standardized questionnaire. Odds ratios (ORs) and Fisher exact P values were calculated to assess risk factors associated with infection. Acanthamoeba keratitis, a rare eye disease primarily affecting contact lens wearers, is caused by free-living amebae, Acanthamoeba species. We identified 37 patients in the 2 investigations, 10 (27%) from the 2007 investigation and 27 (73%) from 2011. There were 17 healthy controls, 9 (53%) from 2007 and 8 (47%) from 2011. Among patients, 9 (24%) wore RGP lenses for orthokeratology or therapeutic indication; no controls wore RGP lenses for these indications. Significant risk factors for AK were wearing lenses for orthokeratology (OR, undefined; P = 0.02), sleeping while wearing lenses (OR, 8.00; P = 0.04), storing lenses in tap water (OR, 16.00; P = 0.001), and topping off contact lens solution in the case (OR, 4.80; P = 0.01). After stratifying by use of RGP lenses for orthokeratology, storing lenses in tap water and topping off remained significant exposures. Nearly one quarter of patients were orthokeratology wearers. Using tap water to store RGP lenses and topping off solution in the lens case were modifiable risk behaviors identified in RGP wearers who wore lenses for both orthokeratology

  8. Resistance of Legionella and Acanthamoeba mauritaniensis to heat treatment as determined by relative and quantitative polymerase chain reactions.

    PubMed

    Dobrowsky, Penelope H; Khan, Sehaam; Khan, Wesaal

    2017-10-01

    Legionella and Acanthamoeba spp. persist in harvested rainwater pasteurized at high temperatures (> 72°C) and the interaction mechanisms exhibited between these organisms need to be elucidated. The resistance of two Legionella reference strains (Legionella pneumophila ATCC 33152 and Legionella longbeachae ATCC 33462), three environmental strains [Legionella longbeachae (env.), Legionella norrlandica (env.) and Legionella rowbothamii (env.)] and Acanthamoeba mauritaniensis ATCC 50676 to heat treatment (50-90°C) was determined by monitoring culturability and viability [ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR)]. The expression of metabolic and virulence genes of L. pneumophila ATCC 33152 (lolA, sidF, csrA) and L. longbeachae (env.) (lolA) in co-culture with A. mauritaniensis ATCC 50676 during heat treatment (50-90°C) was monitored using relative qPCR. While the culturability (CFU/mL) and viability (gene copies/mL) of the Legionella strains reduced significantly (p < 0.05) following heat treatment (60-90°C), L. longbeachae (env.) and L. pneumophila ATCC 33152 were culturable following heat treatment at 50-60°C. Metabolically active trophozoites and dormant cysts of A. mauritaniensis ATCC 50676 were detected at 50°C and 60-90°C, respectively. For L. pneumophila ATCC 33152, lolA expression remained constant, sidF expression increased and the expression of csrA decreased during co-culture with A. mauritaniensis ATCC 50676. For L. longbeachae (env.), while lolA was up-regulated at 50-70°C, expression was not detected at 80-90°C and in co-culture. In conclusion, while heat treatment may reduce the number of viable Legionella spp. in monoculture, results indicate that the presence of A. mauritaniensis increases the virulence of L. pneumophila during heat treatment. The virulence of Legionella spp. in co-culture with Acanthamoeba spp. should thus be monitored in water distribution systems where temperature (heat) is utilized for treatment

  9. Amebicidal activity of the essential oils of Lippia spp. (Verbenaceae) against Acanthamoeba polyphaga trophozoites.

    PubMed

    Santos, Israel Gomes de Amorim; Scher, Ricardo; Rott, Marilise Brittes; Menezes, Leociley Rocha; Costa, Emmanoel Vilaça; Cavalcanti, Sócrates Cabral de Holanda; Blank, Arie Fitzgerald; Aguiar, Jaciana dos Santos; da Silva, Teresinha Gonçalves; Dolabella, Silvio Santana

    2016-02-01

    Amoebic keratitis and granulomatous amoebic encephalitis are caused by some strains of free-living amoebae of the genus Acanthamoeba. In the case of keratitis, one of the greatest problems is the disease recurrence due to the resistance of parasites, especially the cystic forms, to the drugs that are currently used. Some essential oils of plants have been used as potential active agents against this protist. Thus, the aim of this study was to determine the amebicidal activity of essential oils from plants of the genus Lippia against Acanthamoeba polyphaga trophozoites. To that end, 8 × 10(4) trophozoites were exposed for 24 h to increasing concentrations of essential oils from Lippia sidoides, Lippia gracilis, Lippia alba, and Lippia pedunculosa and to their major compounds rotundifolone, carvone, and carvacrol. Nearly all concentrations of oils and compounds showed amebicidal activity. The IC50 values for L. sidoides, L. gracilis L. alba, and L. pedunculosa were found to be 18.19, 10.08, 31.79, and 71.47 μg/mL, respectively. Rotundifolone, carvacrol, and carvone were determined as the major compounds showing IC50 of 18.98, 24.74, and 43.62 μg/mL, respectively. With the exception of oil from L. alba, the other oils evaluated showed low cytotoxicity in the NCI-H292 cell line. Given these results, the oils investigated here are promising sources of compounds for the development of complementary therapy against amoebic keratitis and granulomatous amoebic encephalitis and can also be incorporated into cleaning solutions to increase their amebicidal efficiency.

  10. Effects of topical anaesthetics and fluorescein on the real-time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis.

    PubMed

    Goldschmidt, P; Rostane, H; Saint-Jean, C; Batellier, L; Alouch, C; Zito, E; Bourcier, T; Laroche, L; Chaumeil, C

    2006-11-01

    The early microbiological diagnosis of corneal infections may prevent the condition from worsening. To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real-time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real-time PCR. The capacities of the real-time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis.

  11. Molecular detection of Acanthamoeba spp., Naegleria fowleri and Vermamoeba (Hartmannella) vermiformis as vectors for Legionella spp. in untreated and solar pasteurized harvested rainwater.

    PubMed

    Dobrowsky, Penelope H; Khan, Sehaam; Cloete, Thomas E; Khan, Wesaal

    2016-10-10

    Legionella spp. employ multiple strategies to adapt to stressful environments including the proliferation in protective biofilms and the ability to form associations with free-living amoeba (FLA). The aim of the current study was to identify Legionella spp., Acanthamoeba spp., Vermamoeba (Hartmannella) vermiformis and Naegleria fowleri that persist in a harvested rainwater and solar pasteurization treatment system. Pasteurized (45 °C, 65 °C, 68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples were screened for Legionella spp. and the heterotrophic plate count was enumerated. Additionally, ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) was utilized for the quantification of viable Legionella spp., Acanthamoeba spp., V. vermiformis and N. fowleri in pasteurized (68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples, respectively. Of the 82 Legionella spp. isolated from unpasteurized tank water samples, Legionella longbeachae (35 %) was the most frequently isolated, followed by Legionella norrlandica (27 %) and Legionella rowbothamii (4 %). Additionally, a positive correlation was recorded between the heterotrophic plate count vs. the number of Legionella spp. detected (ρ = 0.710, P = 0.048) and the heterotrophic plate count vs. the number of Legionella spp. isolated (ρ = 0.779, P = 0.0028) from the tank water samples collected. Solar pasteurization was effective in reducing the gene copies of viable V. vermiformis (3-log) and N. fowleri (5-log) to below the lower limit of detection at temperatures of 68-93 °C and 74-93 °C, respectively. Conversely, while the gene copies of viable Legionella and Acanthamoeba were significantly reduced by 2-logs (P = 0.0024) and 1-log (P = 0.0015) overall, respectively, both organisms were still detected after pasteurization at 93 °C. Results from this study indicate that Acanthamoeba spp. primarily acts as the vector and aids in

  12. [Survey of the number of Acanthamoeba keratitis cases in Japan].

    PubMed

    Toriyama, Koji; Suzuki, Takashi; Ohashi, Yuichi

    2014-01-01

    To investigate the trend in the number of Acanthamoeba keratitis (AK) cases in Japan. A survey was conducted in 48 university hospitals. Patients who were diagnosed with AK from January 2007 to December 2011 were enrolled. The trend in the number of cases and the type of contact lenses (CLs) that patients used were studied. A total of 524 patients was studied. The numbers of AK cases in each year, from 2007 to 2011, were 105, 152, 155, 65, and 47. The number dropped markedly after 2009. The percentage of conventional soft CLs and frequent replacement soft CL users that needed daily care such as rubbing-washing also dropped after 2008. The number of AK cases in Japan has been decreasing in recent years. The cause is uncertain, but one possibility is that information about proper CL care promulgated by ophthalmic societies in recent years is producing results.

  13. Effects of topical anaesthetics and fluorescein on the real‐time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis

    PubMed Central

    Goldschmidt, P; Rostane, H; Saint‐Jean, C; Batellier, L; Alouch, C; Zito, E; Bourcier, T; Laroche, L; Chaumeil, C

    2006-01-01

    Background The early microbiological diagnosis of corneal infections may prevent the condition from worsening. Aim To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real‐time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. Methods Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real‐time PCR. Results The capacities of the real‐time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). Conclusions The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis. PMID:16899529

  14. Prognostic factors in Acanthamoeba keratitis.

    PubMed

    Kaiserman, Igor; Bahar, Irit; McAllum, Penny; Srinivasan, Sathish; Elbaz, Uri; Slomovic, Allan R; Rootman, David S

    2012-06-01

    To assess the prognostic factors influencing visual prognosis and length of treatment after acanthamoeba keratitis (AK). Forty-two AK eyes of 41 patients treated between 1999 and 2006 were included. A diagnosis of AK was made on the basis of culture results with a corresponding clinical presentation. We calculated the prognostic effect of the various factors on final visual acuity and the length of treatment. Multivariate regression analysis was used to adjust for the simultaneous effects of the various prognostic factors. Mean follow-up was 19.7 ± 21.0 months. Sixty-four percent of cases had > 1 identified risk factor for AK, the most common risk factor being contact lens wear (92.9% of eyes). At presentation, median best spectacle corrected visual acuity (BCVA) was 20/200 (20/30 to Hand Motion [HM]) that improved after treatment to 20/50 (20/20 to Counting Fingers [CF]). Infection acquired by swimming or related to contact lenses had significantly better final BCVA (p = 0.03 and p = 0.007, respectively). Neuritis and pseudodendrites were also associated with better final BCVA (p = 0.04 and p = 0.05, respectively). Having had an epithelial defect on presentation and having been treated with topical steroid were associated with worse final best spectacle corrected visual acuity (BSCVA) (p = 0.0006 and p = 0.04). Multivariate regression analysis found a good initial visual acuity (p = 0.002), infections related to swimming (p = 0.01), the absence of an epithelial defect (p = 0.03), having been treated with chlorhexidine (p = 0.05), and not having receive steroids (p = 0.003) to significantly forecast a good final BCVA. We identified several prognostic factors that can help clinicians evaluate the expected visual damage of the AK infection and thus tailor treatment accordingly. Copyright © 2012 Canadian Ophthalmological Society. All rights reserved.

  15. [Fundamental studies on legionellosis--the growth with in Acanthamoeba sp. and antibiotics susceptibility of Legionella spp. isolated from soil samples in Japan].

    PubMed

    Furuhata, Katsunori; Miyamoto, Hiroshi; Hara, Motonobu; Fukuyama, Masafumi

    2003-02-01

    As part of an epidemiological study of legionellosis, we investigated the growth within Acanthamoeba sp. and antibiotic susceptibility of 62 strains of Legionella spp. isolated from surface soils nationwide in 2001. 1) All strains tested grew in Acanthamoeba sp., suggesting that the strains were pathogenic. The minimum bacterial number required for the growth in the amoeba was 10(3)-10(8) CFU/ml and there were differences between the strains. 2) Susceptibility to 10 drugs was investigated using the Etest. The MIC90 values of imipenem, as a beta-lactam, and rifampicin, as an antitubercular agent, were 0.047 microgram/ml and 0.064 microgram/ml, respectively, showing high sensitivity. In contrast, sensitivity to minocycline, as a tetracycline, and piperacillin, as a beta-lactam, was low and the MIC90 values were 12 micrograms/ml and 16 micrograms/ml, respectively. Sensitivity to minocycline was particularly low, with a MIC value of 32 micrograms/ml, in two strains. The above findings suggested that all soil-derived strains were pathogenic, and susceptibility of the strains tended to be slightly lower than that of clinical isolates.

  16. Macronuclear chromatin structure dynamics in Colpoda inflata (Protista, Ciliophora) resting encystment.

    PubMed

    Tiano, L; Chessa, M G; Carrara, S; Tagliafierro, G; Delmonte Corrado, M U

    1999-01-01

    The chromatin structure dynamics of the Colpoda inflata macronucleus have been investigated in relation to its functional condition, concerning chromatin body extrusion regulating activity. Samples of 2- and 25-day-old resting cysts derived from a standard culture, and of 1-year-old resting cysts derived from a senescent culture, were examined by means of histogram analysis performed on acquired optical microscopy images. Three groups of histograms were detected in each sample. Histogram classification, clustering and matching were assessed in order to obtain the mean histogram of each group. Comparative analysis of the mean histogram showed a similarity in the grey level range of 25-day- and 1-year-old cysts, unlike the wider grey level range found in 2-day-old cysts. Moreover, the respective mean histograms of the three cyst samples appeared rather similar in shape. All this implies that macronuclear chromatin structural features of 1-year-old cysts are common to both cyst standard cultures. The evaluation of the acquired images and their respective histograms evidenced a dynamic state of the macronuclear chromatin, appearing differently condensed in relation to the chromatin body extrusion regulating activity of the macronucleus. The coexistence of a chromatin-decondensed macronucleus with a pycnotic extrusion body suggests that chromatin unable to decondense, thus inactive, is extruded. This finding, along with the presence of chromatin structural features common to standard and senescent cyst populations, supports the occurrence of 'rejuvenated' cell lines from 1-year-old encysted senescent cells, a phenomenon which could be a result of accomplished macronuclear renewal.

  17. Assessment of the antiprotozoal activity of Pulicaria inuloides extracts, an Algerian medicinal plant: leishmanicidal bioguided fractionation.

    PubMed

    Fadel, Hamza; Sifaoui, Ines; López-Arencibia, Atteneri; Reyes-Batlle, María; Hajaji, Soumaya; Chiboub, Olfa; Jiménez, Ignacio A; Bazzocchi, Isabel L; Lorenzo-Morales, Jacob; Benayache, Samir; Piñero, José E

    2018-02-01

    The lack of an effective chemotherapy for treatment of protozoan disease urges a wide investigation for active compounds, and plant-derived compounds continue to provide key leads for therapeutic agents. The current study reports the in vitro antiprotozoal evaluation of the Algerian medicinal plant Pulicaria inuloides against Leishmania amazonensis, Trypanosoma cruzi, and Acanthamoeba castellanii str. Neff. All the extracts from the aerial part showed to be present a higher leishmanicidal activity than anti-Acanthamoeba or Trypanosoma. Therefore, bioguided fractionation of the active CHCl 3 extract led to the isolation and characterization of the flavonol, quercetagetin-3,5,7,3'-tetramethyl ether (1) as the main component. The structure of compound 1 was established by extensive 1D and 2D NMR spectroscopic analysis (COSY, HSQC, HMBC, and ROESY experiments), chemical transformation (derivatives 2 and 3), and comparison with data in the literature. Compound 1 and derivatives 2 and 3 were further evaluated against the promastigote and amastigote stage of L. amazonensis. Compounds 1-3 exhibited moderate leishmanicidal activity with IC 50 values ranging from 0.234 to 0.484 mM and from 0.006 to 0.017 mM for the promastigote and amastigote forms, respectively, as well as low toxicity levels on macrophages (CC 50 ranging from 0.365 to 0.664 mM). This study represents the first report of the antiprotozoal evaluation of Pulicaria inuloides, and the results highlight this species as a promising source of leishmanicidal agents.

  18. Evaluation of the activity of new cationic carbosilane dendrimers on trophozoites and cysts of Acanthamoeba polyphaga.

    PubMed

    Heredero-Bermejo, Irene; Copa-Patiño, Jose Luis; Soliveri, Juan; Fuentes-Paniagua, Elena; de la Mata, Francisco Javier; Gomez, Rafael; Perez-Serrano, Jorge

    2015-02-01

    Dendrimers are repetitively branched molecules with a broad spectrum of applications, mainly for their antimicrobial properties and as nanocarriers for other molecules. Recently, our research group have synthesized and studied their activity against Acanthamoeba sp., causative agent of a severe ocular disease in humans: Acanthamoeba keratitis. New cationic carbosilane dendrimers were tested against the protozoa forms at different concentrations and for different incubation times. Trophozoite viability was determined by manual counting and cyst viability by observing excystment in microplates with fresh culture medium. Cytotoxicity was checked on HeLa cells using the microculture tetrazolium assay. Alterations were observed by optical microscopy and by flow cytometry staining with propidium iodide. Six out of the 18 dendrimers tested were non-cytotoxic and effective against the trophozoite form, having one of them (dendrimer 14 with an IC50 of 2.4 + 0.1 mg/L) a similar activity to chlorhexidine digluconate (IC50 1.7 + 0.1 mg/L). This dendrimer has a polyphenoxo core and a sulphur atom close to the six -NH3+ terminal groups. On the other hand, only two dendrimers showed some effect against cysts (dendrimers 14 and 17). However, their minimum cysticidal concentrations were cytotoxic and less effective than the control drug. The alterations on the amoeba morphology produced by the treatment with dendrimers were size reduction, increased complexity, loss of acanthopodia and cell membrane disruption. In conclusion, these results suggest that some dendrimers may be studied in animal models to test their effect and that new dendrimers with similar features should be synthesized.

  19. From amoeba to macrophages: exploring the molecular mechanisms of Legionella pneumophila infection in both hosts.

    PubMed

    Escoll, Pedro; Rolando, Monica; Gomez-Valero, Laura; Buchrieser, Carmen

    2013-01-01

    Legionella pneumophila is a Gram-negative bacterium and the causative agent of Legionnaires' disease. It replicates within amoeba and infects accidentally human macrophages. Several similarities are seen in the L. pneumophila-infection cycle in both hosts, suggesting that the tools necessary for macrophage infection may have evolved during co-evolution of L. pneumophila and amoeba. The establishment of the Legionella-containing vacuole (LCV) within the host cytoplasm requires the remodeling of the LCV surface and the hijacking of vesicles and organelles. Then L. pneumophila replicates in a safe intracellular niche in amoeba and macrophages. In this review we will summarize the existing knowledge of the L. pneumophila infection cycle in both hosts at the molecular level and compare the factors involved within amoeba and macrophages. This knowledge will be discussed in the light of recent findings from the Acanthamoeba castellanii genome analyses suggesting the existence of a primitive immune-like system in amoeba.

  20. Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities.

    PubMed

    Müller, Maren Stella; Scheu, Stefan; Jousset, Alexandre

    2013-01-01

    Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil. Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of agricultural systems.

  1. Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities

    PubMed Central

    Müller, Maren Stella; Scheu, Stefan; Jousset, Alexandre

    2013-01-01

    Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil. Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of agricultural systems. PMID:23840423

  2. Antimicrobial efficacy of a novel povidone iodine contact lens disinfection system.

    PubMed

    Yamasaki, Katsuhide; Saito, Fumio; Ota, Ritsue; Kilvington, Simon

    2018-06-01

    Contact lens (CL) wear is a risk factor for the acquisition of microbial keratitis. Accordingly, compliance to manufacturers' recommended hygiene and disinfection procedures are vital to safe (CL) use. In this study we evaluated a novel povidone-iodine (PI) (CL) disinfection system (cleadew, Ophtecs Corporation, Japan) against a range of bacterial, fungal and Acanthamoeba. Antimicrobial assays were conducted according to ISO 14729 using the recommended strains of bacteria and fungi, with and without the presence of organic soil. Regrowth of bacteria and fungi in the disinfection system was also examined. The activity on biofilms formed from Stenotrophomonas maltophilia and Achromobacter sp. was evaluated. Efficacy against A. castellanii trophozoites and cysts was also investigated. The PI system gave >4 log 10 kill of all bacteria and fungi following the manufacturer's recommended disinfection and cleaning time of 4h, with or without the presence of organic soil. No regrowth of organisms was found after 14days in the neutralized solution. In the biofilm studies the system resulted in at least a 7 log 10 reduction in viability of bacteria. For Acanthamoeba, >3 log 10 kill of trophozoites and 1.1-2.8 log 10 kill for the cyst stage was obtained. The PI system effective against a variety of pathogenic microorganisms under a range of test conditions. Strict compliance to recommended CL hygiene procedures is essential for safe CL wear. The use of care systems such as PI, with broad spectrum antimicrobial activity, may aid in the prevention of potentially sight threatening microbial keratitis. Copyright © 2017. Published by Elsevier Ltd.

  3. [A comparative study on hydrolase activities in Acanthamoeba culbertsoni and A. royreba

    PubMed

    Kim, Yong Kyu; Kim, Tae Ue; Joung, In Sil; Im, Kyung Il

    1988-06-01

    Specific or non-specific cytolytic processes of free-living amoebae causing meningoencephalitis have been emphasized and the cytolytic ability related to hydrolases in Entamoeba sp. and Naegleria sp. has also been reported since the latter half of 1970's. However, no information on hydrolase activities in Acanthamoeba sp. is available. Hydrolases in Acanthamoeba culbertsoni, a pathogenic species of free-living amoebae, were assayed and compared with those in a non-pathogenic species, A. royreba. Pathogenicity of these two species was confirmed through experimental infection to BALB/c mice. Hydrolase activities and cytotoxic effects between pathogenic and non-pathogenic species were compared in the trophozoites cultured in CGV media and in CHO cell line, respectively. The results are summarized as follows: The mice infected with A. culbertsoni were all dead 15 days after nasal inoculation, and the mean survival time was 8.5 days. Also the mice infected with this pathogenic species mani fested typical meningoencephalitis, whereas the mice infected with A. royreba did not. Hydrolases detected both in the cell extracts and culture media were acid phosphatase, beta-N-acetyl galactosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase, neutral proteinase and acid proteinase, all of which were detected with remarkably higher rate in A.culbertsoni than in A. royreba. A. culbertsoni revealed strong cytotoxicity for the target CHO cells, whereas A. royreba did not show any specific cytotoxicity. About 80% of the target cells mixed with A. culbertsoni were dead 48 hours after cultivation, and more than 95% of the target cells were dead 72 hours after cultivation. Hydrolase activities in A. culbertsoni cultured with the target cell line were assayed according to the culture time. The activities of acid phosphatase, beta-N-acetyl glucosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase and acid proteinase in this pathogenic amoeba were detected higher in amoeba

  4. Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

    PubMed Central

    Mullins, R. Dyche; Stafford, Walter F.; Pollard, Thomas D.

    1997-01-01

    The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actinrelated proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of ∼13 × 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a K d of 2.3 μM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actinrich regions of Acanthamoeba. PMID:9015304

  5. Visual outcome in Japanese patients with Acanthamoeba keratitis.

    PubMed

    Yamazoe, K; Yamamoto, Y; Shimazaki-Den, S; Shimazaki, J

    2012-04-01

    To identify prognostic factors affecting visual outcome in Acanthamoeba keratitis (AK) treated with topical chlorhexidine gluconate (CHG). A total of 35 eyes in 34 patients with AK were treated with 0.02% topical CHG. Patients were divided into two groups according to the final visual outcome: Group 1, final visual acuity (VA) of 20/25 or greater (22 eyes); Group 2, less than 20/25 (13 eyes). We compared these groups and evaluated the effectiveness of topical CHG compared with outcomes in previous reports. Ring infiltrate was observed more often in Group 2 (4.5% vs 61.5%, OR 33.6, 95% confidence interval (CI) 3.4-333.9, P<0.01). The duration between onset and diagnosis of AK was significantly longer (24.9 days vs 48.4 days, OR 1.03, 95% CI 1.00-1.06, P = 0.04) and VA at initial examination (log MAR) significantly lower (0.47 vs 1.59, OR 25.5, 95% CI 3.4-186.7, P<0.01) in Group 2 (visual outcome <20/25). Multivariate analysis revealed that only VA at initial examination was independently associated with worse visual outcome (adjusted OR 24.5, 95% CI 1.9-312.6, P=0.01). Seventeen (85.0%) of the 20 eyes diagnosed within 1 month and 24 (82.8%) of 29 eyes diagnosed within 2 months achieved a VA of 20/40 or greater. VA at initial examination was the most predictive factors for final visual outcome in AK. Topical CHG was comparably effective to other treatments, including polyhexamethyl biguanide and propamidine isethionate.

  6. Nucleotide sequence of an exceptionally long 5.8S ribosomal RNA from Crithidia fasciculata.

    PubMed Central

    Schnare, M N; Gray, M W

    1982-01-01

    In Crithidia fasciculata, a trypanosomatid protozoan, the large ribosomal subunit contains five small RNA species (e, f, g, i, j) in addition to 5S rRNA [Gray, M.W. (1981) Mol. Cell. Biol. 1, 347-357]. The complete primary sequence of species i is shown here to be pAACGUGUmCGCGAUGGAUGACUUGGCUUCCUAUCUCGUUGA ... AGAmACGCAGUAAAGUGCGAUAAGUGGUApsiCAAUUGmCAGAAUCAUUCAAUUACCGAAUCUUUGAACGAAACGG ... CGCAUGGGAGAAGCUCUUUUGAGUCAUCCCCGUGCAUGCCAUAUUCUCCAmGUGUCGAA(C)OH. This sequence establishes that species i is a 5.8S rRNA, despite its exceptional length (171-172 nucleotides). The extra nucleotides in C. fasciculata 5.8S rRNA are located in a region whose primary sequence and length are highly variable among 5.8S rRNAs, but which is capable of forming a stable hairpin loop structure (the "G+C-rich hairpin"). The sequence of C. fasciculata 5.8S rRNA is no more closely related to that of another protozoan, Acanthamoeba castellanii, than it is to representative 5.8S rRNA sequences from the other eukaryotic kingdoms, emphasizing the deep phylogenetic divisions that seem to exist within the Kingdom Protista. Images PMID:7079176

  7. Raman spectroscopic study on the excystation process in a single unicellular organism amoeba (Acanthamoeba polyphaga)

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Chung; Perevedentseva, Elena; Cheng, Chia-Liang

    2015-05-01

    An in vivo Raman spectroscopic study of amoeba (Acanthamoeba polyphaga) is presented. The changes of the spectra during the amoeba cyst activation and excystation are analyzed. The spectra show the changes of the relative intensities of bands corresponding to protein, lipid, and carotenoid components during cyst activation. The presence of carotenoids in the amoeba is observed via characteristic Raman bands. These signals in the Raman spectra are intense in cysts but decrease in intensity with cyst activation and exhibit a correlation with the life cycle of amoeba. This work demonstrates the feasibility of using Raman spectroscopy for the detection of single amoeba microorganisms in vivo and for the analysis of the amoeba life activity. The information obtained may have implications for the estimation of epidemiological situations and for the diagnostics and prognosis of the development of amoebic inflammations.

  8. Raman spectroscopic study on the excystation process in a single unicellular organism amoeba (Acanthamoeba polyphaga).

    PubMed

    Lin, Yu-Chung; Perevedentseva, Elena; Cheng, Chia-Liang

    2015-05-01

    An in vivo Raman spectroscopic study of amoeba (Acanthamoeba polyphaga) is presented. The changes of the spectra during the amoeba cyst activation and excystation are analyzed. The spectra show the changes of the relative intensities of bands corresponding to protein, lipid, and carotenoid components during cyst activation. The presence of carotenoids in the amoeba is observed via characteristic Raman bands. These signals in the Raman spectra are intense in cysts but decrease in intensity with cyst activation and exhibit a correlation with the life cycle of amoeba. This work demonstrates the feasibility of using Raman spectroscopy for the detection of single amoeba microorganisms in vivo and for the analysis of the amoeba life activity. The information obtained may have implications for the estimation of epidemiological situations and for the diagnostics and prognosis of the development of amoebic inflammations.

  9. Balamuthia mandrillaris, Free-Living Ameba and Opportunistic Agent of Encephalitis, Is a Potential Host for Legionella pneumophila Bacteria

    PubMed Central

    Shadrach, Winlet Sheba; Rydzewski, Kerstin; Laube, Ulrike; Holland, Gudrun; Özel, Muhsin; Kiderlen, Albrecht F.; Flieger, Antje

    2005-01-01

    Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment. PMID:15870307

  10. The virophage as a unique parasite of the giant mimivirus.

    PubMed

    La Scola, Bernard; Desnues, Christelle; Pagnier, Isabelle; Robert, Catherine; Barrassi, Lina; Fournous, Ghislain; Merchat, Michèle; Suzan-Monti, Marie; Forterre, Patrick; Koonin, Eugene; Raoult, Didier

    2008-09-04

    Viruses are obligate parasites of Eukarya, Archaea and Bacteria. Acanthamoeba polyphaga mimivirus (APMV) is the largest known virus; it grows only in amoeba and is visible under the optical microscope. Mimivirus possesses a 1,185-kilobase double-stranded linear chromosome whose coding capacity is greater than that of numerous bacteria and archaea1, 2, 3. Here we describe an icosahedral small virus, Sputnik, 50 nm in size, found associated with a new strain of APMV. Sputnik cannot multiply in Acanthamoeba castellanii but grows rapidly, after an eclipse phase, in the giant virus factory found in amoebae co-infected with APMV4. Sputnik growth is deleterious to APMV and results in the production of abortive forms and abnormal capsid assembly of the host virus. The Sputnik genome is an 18.343-kilobase circular double-stranded DNA and contains genes that are linked to viruses infecting each of the three domains of life Eukarya, Archaea and Bacteria. Of the 21 predicted protein-coding genes, eight encode proteins with detectable homologues, including three proteins apparently derived from APMV, a homologue of an archaeal virus integrase, a predicted primase-helicase, a packaging ATPase with homologues in bacteriophages and eukaryotic viruses, a distant homologue of bacterial insertion sequence transposase DNA-binding subunit, and a Zn-ribbon protein. The closest homologues of the last four of these proteins were detected in the Global Ocean Survey environmental data set5, suggesting that Sputnik represents a currently unknown family of viruses. Considering its functional analogy with bacteriophages, we classify this virus as a virophage. The virophage could be a vehicle mediating lateral gene transfer between giant viruses.

  11. Status of the effectiveness of contact lens solutions against keratitis-causing pathogens.

    PubMed

    Siddiqui, Ruqaiyyah; Lakhundi, Sahreena; Khan, Naveed Ahmed

    2015-02-01

    The aim of this study was to assess the antimicrobial effects of marketed contact lens disinfecting solutions. Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal, fungicidal and amoebicidal assays of eight different contact lens solutions including: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra(+), Kontex Soak were performed. The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Fusarium solani and Acanthamoeba castellanii. The results revealed that ReNu MultiPlus, DuraPlus and OptiFree Express were effective in killing bacterial and fungal pathogens as per manufacturer's minimum recommended disinfection time. Ultimate Plus was effective against F. solani and MRSA but ineffective against P. aeruginosa, S. marcescens and S. aureus. Of concern however, is that none of the locally formulated contact lens disinfecting solutions from Pakistan, i.e., Kontex Clean, Kontex Normal, Kontex Multisol extra(+) and Kontex Soak were effective against any of the keratitis-causing organisms tested. All eight contact lens disinfecting solutions were unable to destroy Acanthamoeba cysts. Because such ineffective contact lens disinfection solutions present a major risk to public health, these findings are of great concern to the health officials and to the manufacturers of the contact lens disinfection solutions and effective solutions are needed, along with emphasis on proper hygiene for contact lens care and special guidelines for developing countries regarding the manufacture and storage of contact lens disinfecting solutions. Copyright © 2014 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  12. Survey on medicinal plants traditionally used in Senegal for the treatment of tuberculosis (TB) and assessment of their antimycobacterial activity.

    PubMed

    Diop, ElHadji Assane; Queiroz, Emerson Ferreira; Kicka, Sébastien; Rudaz, Serge; Diop, Tahir; Soldati, Thierry; Wolfender, Jean-Luc

    2018-04-24

    In West Africa, populations are used to taking traditional medicine as a first aid against common health problems. In this aspect, many plants are claimed to be effective in the treatment of Tuberculosis (TB), which according to the World Health Organization (WHO) remains one of the world's deadliest communicable diseases. The main aim of this study was to identify plants used to treat TB-symptoms by the population of Senegal and to evaluate their possible concomitant use with clinically approved TB-drugs. This approach allowed the selection of plants effectively used in traditional medicine. In order to verify if the usage of some of these plants can be rationalized, the activity of their traditional preparations was assessed with both an intracellular and extracellular antimycobacterial host-pathogen assays. An ethnopharmacological survey conducted on 117 TB-patients and 30 healers in Senegal from March to May 2014. The questionnaires were focused on the use of medicinal plants to treat common TB -symptoms (cough longer than 2 weeks, fever, night sweats, weight loss and bloody sputum). Local plant names, utilized organs (herbal drugs) and traditional formulations of the plants were recorded. Extracts were prepared by mimicking the traditional decoction in boiling water and screened for their antimycobacterial activity using Mycobacterium marinum, as a validated TB surrogate, and an Acanthamoeba castellanii - M. marinum whole-cell based host-pathogen assay, to detect anti-infective activities. By the end of the survey, nearly 30 plants were cited and the 12 most cited herbal drugs were collected and their usage documented by extensive literature search. Extracts of the chosen herbs were screened with the described assays; with a main focus on traditional formulas (mainly herbal decoctions). Two of the water extracts from Combretum aculeatum and Guiera senegalensis showed significant antimycobacterial activities when compared to the positive control drug (rifampin

  13. Acanthamoeba and other free-living amoebae in bat guano, an extreme habitat.

    PubMed

    Mulec, Janez; Dietersdorfer, Elisabeth; Üstüntürk-Onan, Miray; Walochnik, Julia

    2016-04-01

    Several representatives of the so-called free-living amoebae (FLA) are of medical relevance, not only as facultative pathogens but also as vehicles for pathogenic bacteria. Some FLA can survive and even grow under extreme environmental conditions. Bat guano is an exceptional habitat, the conditions becoming gradually more extreme with aging. In the current study, samples of bat guano of different ages from five caves in Slovenia were screened for the presence of FLA. FLA were isolated from almost all guano samples, including guano with a pH of 3.5. Only the two samples that had been drawn from >20-year-old guano were negative for FLA. Generally, FLA diversity correlated to high concentrations of cultivable bacteria (∼10(8) CFU/g) and fungi (∼10(5) CFU/g). Interestingly, the absence of FLA in seasoned guanos was mirrored by the presence of dictyostelid slime moulds. The isolated amoebae were identified as belonging to the genera Acanthamoeba, Copromyxa, Naegleria, Sappinia, Tetramitus, Thecamoeba, Vahlkampfia, Vannella and Vermamoeba. To the best of our knowledge, this is the first study on the diversity of FLA in guano.

  14. Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus.

    PubMed

    Zauberman, Nathan; Mutsafi, Yael; Halevy, Daniel Ben; Shimoni, Eyal; Klein, Eugenia; Xiao, Chuan; Sun, Siyang; Minsky, Abraham

    2008-05-13

    Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the "stargate", allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane-containing viruses.

  15. Distinct DNA Exit and Packaging Portals in the Virus Acanthamoeba polyphaga mimivirus

    PubMed Central

    Zauberman, Nathan; Mutsafi, Yael; Halevy, Daniel Ben; Shimoni, Eyal; Klein, Eugenia; Xiao, Chuan; Sun, Siyang; Minsky, Abraham

    2008-01-01

    Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the “stargate”, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses. PMID:18479185

  16. The rising tide of Acanthamoeba keratitis in Auckland, New Zealand: a 7-year review of presentation, diagnosis and outcomes (2009-2016).

    PubMed

    McKelvie, James; Alshiakhi, Moaz; Ziaei, Mohammed; Patel, Dipika V; McGhee, Charles Nj

    2018-02-07

    Acanthamoeba is an increasingly prevalent cause of vision-threatening microbial keratitis. To assess the incidence, clinical presentation, diagnosis and outcomes of patients with Acanthamoeba keratitis (AK) in Auckland, New Zealand over a 7-year period. Retrospective observational consecutive case series. Fifty-eight eyes of 52 patients diagnosed with AK. All cases of AK were identified using a cross-referenced search of clinical, laboratory and pharmacy records from March 2009 to May 2016. Demographic and clinical data were collected including age, gender, risk factors, clinical manifestations, initial diagnosis, diagnostic investigations, treatment, presenting and final visual acuity and surgical interventions. Contact lens (CL) use was noted in 96% of unilateral and 100% of bilateral cases. The mean duration of symptoms at presentation was 21 days and the mean duration from presentation to definitive diagnosis was 14 days. Initial diagnosis was recorded as CL-related keratitis in 70.6%, viral keratitis in 15.5% and AK in 12.0%. The diagnosis was confirmed with In vivo confocal microscopy (IVCM) in 67.2%, corneal scrape in 22.4%, corneal biopsy in 1.7% and clinically in 8.6%. IVCM sensitivity was 83.0%. Surgical intervention was required in four patients, all with delayed diagnosis (range 63-125 days). The incidence of AK has more than doubled when compared with the preceding 7-year period. AK is a rare vision-threatening protozoal infection with rapidly-increasing incidence in New Zealand, predominantly affecting CL users. Diagnosis is often challenging and when delayed is associated with worse outcomes. IVCM offers rapid diagnosis with high sensitivity. © 2018 Royal Australian and New Zealand College of Ophthalmologists.

  17. Mycobacterium gilvum Illustrates Size-Correlated Relationships between Mycobacteria and Acanthamoeba polyphaga

    PubMed Central

    Lamrabet, Otmane

    2013-01-01

    Mycobacteria are isolated from soil and water environments, where free-living amoebae live. Free-living amoebae are bactericidal, yet some rapidly growing mycobacteria are amoeba-resistant organisms that survive in the amoebal trophozoites and cysts. Such a capacity has not been studied for the environmental rapidly growing organism Mycobacterium gilvum. We investigated the ability of M. gilvum to survive in the trophozoites of Acanthamoeba polyphaga strain Linc-AP1 by using optical and electron microscopy and culture-based microbial enumerations in the presence of negative controls. We observed that 29% of A. polyphaga cells were infected by M. gilvum mycobacteria by 6 h postinfection. Surviving M. gilvum mycobacteria did not multiply and did not kill the amoebal trophozoites during a 5-day coculture. Extensive electron microscopy observations indicated that M. gilvum measured 1.4 ± 0.5 μm and failed to find M. gilvum organisms in the amoebal cysts. Further experimental study of two other rapidly growing mycobacteria, Mycobacterium rhodesiae and Mycobacterium thermoresistibile, indicated that both measured <2 μm and exhibited the same amoeba-mycobacterium relationships as M. gilvum. In general, we observed that mycobacteria measuring <2 μm do not significantly grow within and do not kill amoebal trophozoites, in contrast to mycobacteria measuring >2 μm (P < 0.05). The mechanisms underlying such an observation remain to be determined. PMID:23275502

  18. A Rabbit Model of Acanthamoeba Keratitis: Use of Infected Soft Contact Lenses After Corneal Epithelium Debridement With a Diamond Burr.

    PubMed

    Ortillés, Ángel; Goñi, Pilar; Rubio, Encarnación; Sierra, Marta; Gámez, Ekaterina; Fernández, María T; Benito, María; Cristóbal, José Á; Calvo, Begoña

    2017-02-01

    To develop a rabbit model of Acanthamoeba keratitis (AK) as the best method to reproduce the natural course of this disease. To induce AK, infected contact lenses (1000 amoebae/mm2, 90% trophozoites) were placed over the previously debrided corneal surface, in combination with a temporary tarsorrhaphy. Environmental and clinical strains of Acanthamoeba spp. (genotype T4) were used. Three groups (1L, n = 32; 2L-21d, n = 5; 2L-3d, n = 23) were established according to the number of contact lenses used (1L, 1 lens; 2L-21d and 2L-3d, 2 lenses) and the placement day of these (1L, day 1; 2L-21d, days 1 and 21; 2L-3d, days 1 and 3). The infection was quantified by a clinical score system and confirmed using corneal cytology and culture, polymerase chain reaction and histopathologic analysis. The infection rate obtained was high (1L, 87.5%; 2L-21d, 100%; 2L-3d, 82.6%), although no clinical signs were observed in the 50% of the infected animals in group 1L. Among groups, group 2L-3d showed more cases of moderate and severe infection. Among strains, no statistically significant differences were found in the infection rate. In the control eyes, cross infection was confirmed when a sterile contact lens was placed in the previously debrided corneas but not if the eye remained intact. The combination of two infected contact lenses after corneal debridement seems to be an alternative model, clinically and histopathologically similar to its human counterpart, to induce the different AK stages and reproduce the course of the disease in rabbits.

  19. Multiple identification of most important waterborne protozoa in surface water used for irrigation purposes by 18S rRNA amplicon-based metagenomics.

    PubMed

    Moreno, Y; Moreno-Mesonero, L; Amorós, I; Pérez, R; Morillo, J A; Alonso, J L

    2018-01-01

    Understanding waterborne protozoan parasites (WPPs) diversity has important implications in public health. In this study, we evaluated a NGS-based method as a detection approach to identify simultaneously most important WPPs using 18S rRNA high-throughput sequencing. A set of primers to target the V4 18S rRNA region of WPPs such as Cryptosporidium spp., Giardia sp., Blastocystis sp., Entamoeba spp, Toxoplasma sp. and free-living amoebae (FLA) was designed. In order to optimize PCR conditions before sequencing, both a mock community with a defined composition of representative WPPs and a real water sample inoculated with specific WPPs DNA were prepared. Using the method proposed in this study, we have detected the presence of Giardia intestinalis, Acanthamoeba castellanii, Toxoplasma gondii, Entamoeba histolytica and Blastocystis sp. at species level in real irrigation water samples. Our results showed that untreated surface irrigation water in open fields can provide an important source of WPPs. Therefore, the methodology proposed in this study can establish a basis for an accurate and effective diagnostic of WPPs to provide a better understanding of the risk associated to irrigation water. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  20. Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

    PubMed Central

    1975-01-01

    Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles. PMID:1174130

  1. Short-Term and Long-Term Survival and Virulence of Legionella pneumophila in the Defined Freshwater Medium Fraquil.

    PubMed

    Mendis, Nilmini; McBride, Peter; Faucher, Sébastien P

    2015-01-01

    Legionella pneumophila (Lp) is the etiological agent responsible for Legionnaires' disease, a potentially fatal pulmonary infection. Lp lives and multiplies inside protozoa in a variety of natural and man-made water systems prior to human infection. Fraquil, a defined freshwater medium, was used as a highly reproducible medium to study the behaviour of Lp in water. Adopting a reductionist approach, Fraquil was used to study the impact of temperature, pH and trace metal levels on the survival and subsequent intracellular multiplication of Lp in Acanthamoeba castellanii, a freshwater protozoan and a natural host of Legionella. We show that temperature has a significant impact on the short- and long-term survival of Lp, but that the bacterium retains intracellular multiplication potential for over six months in Fraquil. Moreover, incubation in Fraquil at pH 4.0 resulted in a rapid decline in colony forming units, but was not detrimental to intracellular multiplication. In contrast, variations in trace metal concentrations had no impact on either survival or intracellular multiplication in amoeba. Our data show that Lp is a resilient bacterium in the water environment, remaining infectious to host cells after six months under the nutrient-deprived conditions of Fraquil.

  2. An investigation of virulence factors of Legionella pneumophila environmental isolates.

    PubMed

    Arslan-Aydoğdu, Elif Özlem; Kimiran, Ayten

    Nine Legionella pneumophila strains isolated from cooling towers and a standard strain (L. pneumophila serogroup 1, ATCC 33152, Philadelphia 1) were analyzed and compared in terms of motility, flagella structure, ability to form biofilms, enzymatic activities (hemolysin, nucleases, protease, phospholipase A, phospholipase C, acid phosphatase, alkaline phosphatase and lipase), hemagglutination capabilities, and pathogenicity in various host cells (Acanthamoeba castellanii ATCC 30234, mouse peritoneal macrophages and human peripheral monocytes). All the isolates of bacteria appeared to be motile and polar-flagellated and possessed the type-IV fimbria. Upon the evaluation of virulence factors, isolate 4 was found to be the most pathogenic strain, while 6 out of the 9 isolates (the isolates 1, 2, 3, 4, 5, and 7) were more virulent than the ATCC 33152 strain. The different bacterial strains exhibited differences in properties such as adhesion, penetration and reproduction in the hosts, and preferred host type. To our knowledge, this is the first study to compare the virulence of environmental L. pneumophila strains isolated in Turkey, and it provides important information relevant for understanding the epidemiology of L. pneumophila. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  3. VBNC Legionella pneumophila cells are still able to produce virulence proteins.

    PubMed

    Alleron, Laëtitia; Khemiri, Arbia; Koubar, Mohamad; Lacombe, Christian; Coquet, Laurent; Cosette, Pascal; Jouenne, Thierry; Frere, Jacques

    2013-11-01

    Legionella pneumophila is the agent responsible for legionellosis. Numerous bacteria, including L. pneumophila, can enter into a viable but not culturable (VBNC) state under unfavorable environmental conditions. In this state, cells are unable to form colonies on standard medium but are still alive. Here we show that VBNC L. pneumophila cells, obtained by monochloramine treatment, were still able to synthesize proteins, some of which are involved in virulence. Protein synthesis was measured using (35)S-labeling and the proteomes of VBNC and culturable cells then compared. This analysis allowed the identification of nine proteins that were accumulated in the VBNC state. Among them, four were involved in virulence, i.e., the macrophage infectivity potentiator protein, the hypothetical protein lpl2247, the ClpP protease proteolytic subunit and the 27 kDa outer membrane protein. Others, i.e., the enoyl reductase, the electron transfer flavoprotein (alpha and beta subunits), the 50S ribosomal proteins (L1 and L25) are involved in metabolic and energy production pathways. However, resuscitation experiments performed with Acanthamoeba castellanii failed, suggesting that the accumulation of virulence factors by VBNC cells is not sufficient to maintain their virulence. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Coincidental loss of bacterial virulence in multi-enemy microbial communities.

    PubMed

    Zhang, Ji; Ketola, Tarmo; Örmälä-Odegrip, Anni-Maria; Mappes, Johanna; Laakso, Jouni

    2014-01-01

    The coincidental virulence evolution hypothesis suggests that outside-host selection, such as predation, parasitism and resource competition can indirectly affect the virulence of environmentally-growing bacterial pathogens. While there are some examples of coincidental environmental selection for virulence, it is also possible that the resource acquisition and enemy defence is selecting against it. To test these ideas we conducted an evolutionary experiment by exposing the opportunistic pathogen bacterium Serratia marcescens to the particle-feeding ciliate Tetrahymena thermophila, the surface-feeding amoeba Acanthamoeba castellanii, and the lytic bacteriophage Semad11, in all possible combinations in a simulated pond water environment. After 8 weeks the virulence of the 384 evolved clones were quantified with fruit fly Drosophila melanogaster oral infection model, and several other life-history traits were measured. We found that in comparison to ancestor bacteria, evolutionary treatments reduced the virulence in most of the treatments, but this reduction was not clearly related to any changes in other life-history traits. This suggests that virulence traits do not evolve in close relation with these life-history traits, or that different traits might link to virulence in different selective environments, for example via resource allocation trade-offs.

  5. Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa.

    PubMed

    Gonzales-Gustavson, Eloy; Cárdenas-Youngs, Yexenia; Calvo, Miquel; da Silva, Marcelle Figueira Marques; Hundesa, Ayalkibet; Amorós, Inmaculada; Moreno, Yolanda; Moreno-Mesonero, Laura; Rosell, Rosa; Ganges, Llilianne; Araujo, Rosa; Girones, Rosina

    2017-03-01

    In this study, the use of skimmed milk flocculation (SMF) to simultaneously concentrate viruses, bacteria and protozoa was evaluated. We selected strains of faecal indicator bacteria and pathogens, such as Escherichia coli and Helicobacter pylori. The viruses selected were adenovirus (HAdV 35), rotavirus (RoV SA-11), the bacteriophage MS2 and bovine viral diarrhoea virus (BVDV). The protozoa tested were Acanthamoeba, Giardia and Cryptosporidium. The mean recoveries with q(RT)PCR were 66% (HAdV 35), 24% (MS2), 28% (RoV SA-11), 15% (BVDV), 60% (E. coli), 30% (H. pylori) and 21% (Acanthamoeba castellanii). When testing the infectivity, the mean recoveries were 59% (HAdV 35), 12% (MS2), 26% (RoV SA-11) and 0.7% (BVDV). The protozoa Giardia lamblia and Cryptosporidium parvum were studied by immunofluorescence with recoveries of 18% and 13%, respectively. Although q(RT)PCR consistently showed higher quantification values (as expected), q(RT)PCR and the infectivity assays showed similar recoveries for HAdV 35 and RoV SA-11. Additionally, we investigated modelling the variability and uncertainty of the recovery with this method to extrapolate the quantification obtained by q(RT)PCR and estimate the real concentration. The 95% prediction intervals of the real concentration of the microorganisms inoculated were calculated using a general non-parametric bootstrap procedure adapted in our context to estimate the technical error of the measurements. SMF shows recoveries with a low variability that permits the use of a mathematical approximation to predict the concentration of the pathogen and indicator with acceptable low intervals. The values of uncertainty may be used for a quantitative microbial risk analysis or diagnostic purposes. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  6. Detection limits of Legionella pneumophila in environmental samples after co-culture with Acanthamoeba polyphaga

    PubMed Central

    2013-01-01

    Background The efficiency of recovery and the detection limit of Legionella after co-culture with Acanthamoeba polyphaga are not known and so far no investigations have been carried out to determine the efficiency of the recovery of Legionella spp. by co-culture and compare it with that of conventional culturing methods. This study aimed to assess the detection limits of co-culture compared to culture for Legionella pneumophila in compost and air samples. Compost and air samples were spiked with known concentrations of L. pneumophila. Direct culturing and co-culture with amoebae were used in parallel to isolate L. pneumophila and recovery standard curves for both methods were produced for each sample. Results The co-culture proved to be more sensitive than the reference method, detecting 102-103 L. pneumophila cells in 1 g of spiked compost or 1 m3 of spiked air, as compared to 105-106 cells in 1 g of spiked compost and 1 m3 of spiked air. Conclusions Co-culture with amoebae is a useful, sensitive and reliable technique to enrich L. pneumophila in environmental samples that contain only low amounts of bacterial cells. PMID:23442526

  7. Effectiveness of sampling methods employed for Acanthamoeba keratitis diagnosis by culture.

    PubMed

    Muiño, Laura; Rodrigo, Donoso; Villegas, Rodrigo; Romero, Pablo; Peredo, Daniel E; Vargas, Rafael A; Liempi, Daniela; Osuna, Antonio; Jercic, María Isabel

    2018-06-18

    This retrospective, observational study was designed to evaluate the effectiveness of the sampling methods commonly used for the collection of corneal scrapes for the diagnosis of Acanthamoeba keratitis (AK) by culture, in terms of their ability to provide a positive result. A total of 553 samples from 380 patients with suspected AK received at the Parasitology Section of the Public Health Institute of Chile, between January 2005 and December 2015, were evaluated. A logistic regression model was used to determine the correlation between the culture outcome (positive or negative) and the method for sample collection. The year of sample collection was also included in the analysis as a confounding variable. Three hundred and sixty-five samples (27%) from 122 patients (32.1%) were positive by culture. The distribution of sample types was as follows: 142 corneal scrapes collected using a modified bezel needle (a novel method developed by a team of Chilean corneologists), 176 corneal scrapes obtained using a scalpel, 50 corneal biopsies, 30 corneal swabs, and 155 non-biological materials including contact lens and its paraphernalia. Biopsy provided the highest likelihood ratio for a positive result by culture (1.89), followed by non-biological materials (1.10) and corneal scrapes obtained using a modified needle (1.00). The lowest likelihood ratio was estimated for corneal scrapes obtained using a scalpel (0.88) and cotton swabs (0.78). Apart from biopsy, optimum corneal samples for the improved diagnosis of AK can be obtained using a modified bezel needle instead of a scalpel, while cotton swabs are not recommended.

  8. Related Giant Viruses in Distant Locations and Different Habitats: Acanthamoeba polyphaga moumouvirus Represents a Third Lineage of the Mimiviridae That Is Close to the Megavirus Lineage

    PubMed Central

    Yoosuf, Niyaz; Yutin, Natalya; Colson, Philippe; Shabalina, Svetlana A.; Pagnier, Isabelle; Robert, Catherine; Azza, Said; Klose, Thomas; Wong, Jimson; Rossmann, Michael G.; La Scola, Bernard; Raoult, Didier; Koonin, Eugene V.

    2012-01-01

    The 1,021,348 base pair genome sequence of the Acanthamoeba polyphaga moumouvirus, a new member of the Mimiviridae family infecting Acanthamoeba polyphaga, is reported. The moumouvirus represents a third lineage beside mimivirus and megavirus. Thereby, it is a new member of the recently proposed Megavirales order. This giant virus was isolated from a cooling tower water in southeastern France but is most closely related to Megavirus chiliensis, which was isolated from ocean water off the coast of Chile. The moumouvirus is predicted to encode 930 proteins, of which 879 have detectable homologs. Among these predicted proteins, for 702 the closest homolog was detected in Megavirus chiliensis, with the median amino acid sequence identity of 62%. The evolutionary affinity of moumouvirus and megavirus was further supported by phylogenetic tree analysis of conserved genes. The moumouvirus and megavirus genomes share near perfect orthologous gene collinearity in the central part of the genome, with the variations concentrated in the terminal regions. In addition, genomic comparisons of the Mimiviridae reveal substantial gene loss in the moumouvirus lineage. The majority of the remaining moumouvirus proteins are most similar to homologs from other Mimiviridae members, and for 27 genes the closest homolog was found in bacteria. Phylogenetic analysis of these genes supported gene acquisition from diverse bacteria after the separation of the moumouvirus and megavirus lineages. Comparative genome analysis of the three lineages of the Mimiviridae revealed significant mobility of Group I self-splicing introns, with the highest intron content observed in the moumouvirus genome. PMID:23221609

  9. Relationship between Legionella pneumophila and Acanthamoeba polyphaga: Physiological status and susceptibility to chemical inactivation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Barker, J.; Farrell, I.; Brown, M.R.W.

    1992-08-01

    Survival studies were conducted on Legionella pneumophila cells that had been grown intracellulary in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 [times] MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h andmore » 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure of PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.« less

  10. Protozoan Cysts Act as a Survival Niche and Protective Shelter for Foodborne Pathogenic Bacteria

    PubMed Central

    Lambrecht, Ellen; Baré, Julie; Chavatte, Natascha; Bert, Wim; Sabbe, Koen

    2015-01-01

    The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 μg/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain- and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed. PMID:26070667

  11. Intermediary metabolism in protists: a sequence-based view of facultative anaerobic metabolism in evolutionarily diverse eukaryotes.

    PubMed

    Ginger, Michael L; Fritz-Laylin, Lillian K; Fulton, Chandler; Cande, W Zacheus; Dawson, Scott C

    2010-12-01

    Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks. In the last 2-3 years, genome sequencing and transcript profiling has unveiled several examples of heterotrophic and phototrophic protists that are unexpectedly well-equipped for ATP production using a facultative anaerobic metabolism, including some protists that can (Chlamydomonas reinhardtii) or are predicted (Naegleria gruberi, Acanthamoeba castellanii, Amoebidium parasiticum) to produce H(2) in their metabolism. It is possible that some enzymes of anaerobic metabolism were acquired and distributed among eukaryotes by lateral transfer, but it is also likely that the common ancestor of eukaryotes already had far more metabolic versatility than was widely thought a few years ago. The discussion of core energy metabolism in unicellular eukaryotes is the subject of this review. Since genomic sequencing has so far only touched the surface of protist diversity, it is anticipated that sequences of additional protists may reveal an even wider range of metabolic capabilities, while simultaneously enriching our understanding of the early evolution of eukaryotes. Copyright © 2010 Elsevier GmbH. All rights reserved.

  12. IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system.

    PubMed

    Bardill, J Patrick; Miller, Jennifer L; Vogel, Joseph P

    2005-04-01

    Legionella pneumophila replicates inside alveolar macrophages and causes an acute, potentially fatal pneumonia called Legionnaires' disease. The ability of this bacterium to grow inside of macrophages is dependent on the presence of a functional dot/icm type IV secretion system (T4SS). Proteins secreted by the Dot/Icm T4SS are presumed to alter the host endocytic pathway, allowing L. pneumophila to establish a replicative niche within the host cell. Here we show that a member of the SidE family of proteins interacts with IcmS and is required for full virulence in the protozoan host Acanthamoeba castellanii. Using immunofluorescence microscopy and adenylate cyclase fusions, we show that SdeA is secreted into host cells by L. pneumophila in an IcmS-dependent manner. The SidE-like proteins are secreted very early during macrophage infection, suggesting that they are important in the initial formation of the replicative phagosome. Secreted SidE family members show a similar localization to other Dot/Icm substrates, specifically, to the poles of the replicative phagosome. This common localization of secreted substrates of the Dot/Icm system may indicate the formation of a multiprotein complex on the cytoplasmic face of the replicative phagosome.

  13. Exploring Virulence Determinants of Filamentous Fungal Pathogens through Interactions with Soil Amoebae.

    PubMed

    Novohradská, Silvia; Ferling, Iuliia; Hillmann, Falk

    2017-01-01

    Infections with filamentous fungi are common to all animals, but attention is rising especially due to the increasing incidence and high mortality rates observed in immunocompromised human individuals. Here, Aspergillus fumigatus and other members of its genus are the leading causative agents. Attributes like their saprophytic life-style in various ecological niches coupled with nutritional flexibility and a broad host range have fostered the hypothesis that environmental predators could have been the actual target for some of their virulence determinants. In this mini review, we have merged the recent findings focused on the potential dual-use of fungal defense strategies against innate immune cells and soil amoebae as natural phagocytes. Well-established virulence attributes like the melanized surface of fungal conidia or their capacity to produce toxic secondary metabolites have also been found to be protective against the model amoeba Dictyostelium discoideum . Some of the recent advances during interaction studies with human cells have further promoted the adaptation of other amoeba infection models, including the wide-spread generalist Acanthamoeba castellanii , or less prominent representatives like Vermamoeba vermiformis . We further highlight prospects and limits of these natural phagocyte models with regard to the infection biology of filamentous fungi and in comparison to the phagocytes of the innate immune system.

  14. Isolation of new Brazilian giant viruses from environmental samples using a panel of protozoa.

    PubMed

    Dornas, Fábio P; Khalil, Jacques Y B; Pagnier, Isabelle; Raoult, Didier; Abrahão, Jônatas; La Scola, Bernard

    2015-01-01

    The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini, and Vermamoeba vermiformis (VV) as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in A. polyphaga (46.38%) and the lowest in VV (0%). Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation.

  15. The Legionella pneumophila Collagen-Like Protein Mediates Sedimentation, Autoaggregation, and Pathogen-Phagocyte Interactions

    PubMed Central

    Abdel-Nour, Mena; Duncan, Carla; Prashar, Akriti; Rao, Chitong; Ginevra, Christophe; Jarraud, Sophie; Low, Donald E.; Ensminger, Alexander W.; Terebiznik, Mauricio R.

    2014-01-01

    Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae. PMID:24334670

  16. A conserved OmpA-like protein in Legionella pneumophila required for efficient intracellular replication.

    PubMed

    Goodwin, Ian P; Kumova, Ogan K; Ninio, Shira

    2016-08-01

    The OmpA-like protein domain has been associated with peptidoglycan-binding proteins, and is often found in virulence factors of bacterial pathogens. The intracellular pathogen Legionella pneumophila encodes for six proteins that contain the OmpA-like domain, among them the highly conserved uncharacterized protein we named CmpA. Here we set out to characterize the CmpA protein and determine its contribution to intracellular survival of L. pneumophila Secondary structure analysis suggests that CmpA is an inner membrane protein with a peptidoglycan-binding domain at the C-teminus. A cmpA mutant was able to replicate normally in broth, but failed to compete with an isogenic wild-type strain in an intracellular growth competition assay. The cmpA mutant also displayed significant intracellular growth defects in both the protozoan host Acanthamoeba castellanii and in primary bone marrow-derived macrophages, where uptake into the cells was also impaired. The cmpA phenotypes were completely restored upon expression of CmpA in trans The data presented here establish CmpA as a novel virulence factor of L. pneumophila that is required for efficient intracellular replication in both mammalian and protozoan hosts. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Intermediary Metabolism in Protists: a Sequence-based View of Facultative Anaerobic Metabolism in Evolutionarily Diverse Eukaryotes

    PubMed Central

    Ginger, Michael L.; Fritz-Laylin, Lillian K.; Fulton, Chandler; Cande, W. Zacheus; Dawson, Scott C.

    2011-01-01

    Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks. In the last 2–3 years, genome sequencing and transcript profiling has unveiled several examples of heterotrophic and phototrophic protists that are unexpectedly well-equipped for ATP production using a facultative anaerobic metabolism, including some protists that can (Chlamydomonas reinhardtii) or are predicted (Naegleria gruberi, Acanthamoeba castellanii, Amoebidium parasiticum) to produce H2 in their metabolism. It is possible that some enzymes of anaerobic metabolism were acquired and distributed among eukaryotes by lateral transfer, but it is also likely that the common ancestor of eukaryotes already had far more metabolic versatility than was widely thought a few years ago. The discussion of core energy metabolism in unicellular eukaryotes is the subject of this review. Since genomic sequencing has so far only touched the surface of protist diversity, it is anticipated that sequences of additional protists may reveal an even wider range of metabolic capabilities, while simultaneously enriching our understanding of the early evolution of eukaryotes. PMID:21036663

  18. Experimental demonstration of the possible role of Acanthamoeba polyphaga in the infection and disease progression in Buruli Ulcer (BU) using ICR mice

    PubMed Central

    Azumah, Bright K.; Addo, Phyllis G.; Dodoo, Alfred; Awandare, Gordon; Mosi, Lydia; Boakye, Daniel A.

    2017-01-01

    The transmission of Buruli ulcer (BU), caused by Mycobacterium ulcerans (MU), remains puzzling although a number of hypothesis including through bites of infected aquatic insects have been proposed. We report the results of experiments using ICR mice that give credence to our hypothesis that Acanthamoeba species may play a role in BU transmission. We cocultured MU N2 and MU 1615 which expresses red fluorescent protein (RFP) and Acanthamoeba polyphaga (AP), and confirmed infected AP by Ziehl-Neelsen (ZN) staining. We tested for viability of MU inside AP and observed strong RFP signals inside both trophozoites and cysts after 3 and 42 days of coculturing respectively. ICR mice were topically treated, either on shaved intact or shaved pinpricked rumps, with one of the following; MU N2 only (2.25 x 106 colony forming units [CFU] / ml), MU N2:AP coculture (2.96 x 104 CFU: 1.6 x 106 cells/ml), AP only (1.6 x 106 cells/ml), PYG medium and sterile distilled water. Both MU N2 only and MU N2:AP elicited reddening on day (D) 31; edema on D 45 and D 44 respectively, and ulcers on D 49 at pinpricked sites only. To ascertain infectivity and pathogenicity of MU N2 only and MU N2:AP, and compare their virulence, the standard mouse footpad inoculation method was used. MU N2:AP elicited reddening in footpads by D 3 compared to D 14 with MU N2 only of the same dose of MU N2 (2.96 x 104 CFU). ZN-stained MU were observed in both thin sectioned and homogenized lesions, and aspirates from infected sites. Viable MU N2 were recovered from cultures of the homogenates and aspirates. This study demonstrates in ICR mice MU transmission via passive infection, and shows that punctures in the skin are prerequisite for infection, and that coculturing of MU with AP enhances pathogenesis. PMID:28329001

  19. Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: Predominance of heterotypic encystment and implications for mating interactions and biogeography

    NASA Astrophysics Data System (ADS)

    Brosnahan, Michael L.; Kulis, David M.; Solow, Andrew R.; Erdner, Deana L.; Percy, Linda; Lewis, Jane; Anderson, Donald M.

    2010-02-01

    We report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic Group III isolates. However, hypnozygotes produced in Group I/III co-cultures complete no more than three divisions after germinating. Previous reports have suggested a mate recognition mechanism whereby hypnozygotes produced in co-cultures could arise from either homotypic (inbred) or heterotypic (outbred) gamete pairs. To determine the extent to which each occurs, a nested PCR assay was developed to determine parentage of individual hypnozygotes. The vast majority of hypnozygotes from pairwise Group I/III co-cultures were outbred, so that inviability was a result of hybridization, not inbreeding. These findings support the assertion that complete speciation underlies the phylogenetic structure of the Alexandrium tamarense species complex. Additionally, the ribosomal DNA (rDNA) copy numbers of both hybrid and single ribotype hypnozygotes were reduced substantially from those of haploid motile cells. The destruction of rDNA loci may be crucial for the successful mating of genetically distant conjugants and appears integral to the process of encystment. The inviability of Group I/III hybrids is important for public health because the presence of hybrid cysts may indicate ongoing displacement of a nontoxic population by a toxic one (or vice versa). Hybrid inviability also suggests a bloom control strategy whereby persistent, toxic Group I blooms could be mitigated by introduction of nontoxic Group III cells. The potential for hybridization in nature was investigated by applying the nested PCR assay to hypnozygotes from Belfast Lough, Northern Ireland, a region where Group I and III populations co-occur. Two hybrid cysts were identified in 14 successful assays, demonstrating that Group I and III populations do interbreed in that region. However, an analysis of mating data

  20. Molecular identification of t4 and t5 genotypes in isolates from acanthamoeba keratitis patients.

    PubMed

    Ledee, D R; Iovieno, A; Miller, D; Mandal, N; Diaz, M; Fell, J; Fini, M E; Alfonso, E C

    2009-05-01

    Acanthamoeba keratitis (AK) is a rare but sight-threatening ocular infection. Outbreaks have been associated with contaminated water and contact lens wear. The epidemiology and pathology may be associated with unique genotypes. We determined the Rns genotype for 37 clinical isolates from 23 patients presenting at the University of Miami Bascom Palmer Eye Institute with confirmed AK infections in 2006 to 2008. The genus-specific ASA.S1 amplicon allowed for rapid genotyping of the nonaxenic cultures. Of the 37 isolates, 36 were of the T4 genotype. Within this group, 13 unique diagnostic fragment 3 sequences were identified, 3 of which were not in GenBank. The 37th isolate was a T5, the first in the United States and second worldwide to be found in AK. For five patients with isolates from the cornea and contact lens/case, identical sequences within each patient cluster were observed, confirming the link between contact lens contamination and AK infection. Genotyping is an important tool in the epidemiological study of AK. In this study, it allowed for the detection of new strains and provided an etiological link between source and infection. Additionally, it can allow for accurate categorizing of physiological differences, such as strain virulence, between isolates and clades.

  1. Acanthamoeba-Cytopathic Protein Induces Apoptosis and Proinflammatory Cytokines in Human Corneal Epithelial Cells by cPLA2α Activation

    PubMed Central

    Tripathi, Trivendra; Smith, Ashley Dawn; Abdi, Mahshid; Alizadeh, Hassan

    2012-01-01

    Purpose. We have shown that Acanthamoeba interacts with a mannosylated protein on corneal epithelial cells and stimulates trophozoites to secrete a mannose-induced 133 kDa protease (MIP-133), which facilitates corneal invasion and induces apoptosis. The mechanism of MIP-133–induced apoptosis is unknown. The aim of this study was to determine if MIP-133 induces apoptosis and proinflammatory cytokines/chemokines in human corneal epithelial (HCE) cells via the cytosolic phospholipase A2α (cPLA2α) pathway. Methods. HCE cells were incubated with or without MIP-133 at doses of 7.5, 15, and 50 μg/mL for 6, 12, and 24 hours. The effects of cPLA2α inhibitors on cPLA2α, arachidonic acid (AA) release, and apoptosis were tested in vitro. Inhibition of cPLA2α involved preincubating HCE cells for 1 hour with cPLA2α inhibitors (10 μM methyl-arachidonyl fluorophosphonate [MAFP] or 20 μM arachidonyl trifluoromethyl ketone [AACOCF3]) with or without MIP-133 for 24 hours. Expression of cPLA2α mRNA and enzyme was examined by RT-PCR and cPLA2 activity assays, respectively. Apoptosis of corneal epithelial cells was determined by caspase-3 and DNA fragmentation assays. Expression of IL-8, IL-6, IL-1β, and IFN-γ was examined by RT-PCR and ELISA. Results. MIP-133 induced significant cPLA2α (approximately two to four times) and AA release (approximately six times) from corneal cells while cPLA2α inhibitors significantly reduced cPLA2α (approximately two to four times) and AA release (approximately three times) (P < 0.05). cPLA2α inhibitors significantly inhibited MIP-133–induced DNA fragmentation approximately 7 to 12 times in HCE cells (P < 0.05). MIP-133 specifically activates cPLA2α enzyme activity in HCE cells, which is blocked by preincubation with anti–MIP-133 antibody. In addition, MIP-133 induced significant IL-8, IL-6, IL-1β, and IFN-γ production, approximately two to three times (P < 0.05). Conclusions. MIP-133 interacts with phospholipids on plasma

  2. Reduced expression of the global regulator protein CsrA in Legionella pneumophila affects virulence-associated regulators and growth in Acanthamoeba castellanii.

    PubMed

    Forsbach-Birk, Vera; McNealy, Tamara; Shi, Chunwei; Lynch, Damien; Marre, Reinhard

    2004-07-01

    Legionella bacteria have a developmental cycle in which they go from existing in the aquatic environment to replicating inside eukaryotic host cells. The adaptation to the new environment requires an efficient regulatory system. Overexpression of CsrA, a global regulatory protein found in a variety of gram-negative bacteria has been shown to suppress virulence-associated traits in Legionella pneumophila. Since evidence resulting only from overproduction may not be sufficient to validate the role of a regulatory protein, a csrA mutant strain, CsrA(-), with a drastically reduced production of CsrA, was created. Using RNA slot blots and Western blotting it was shown that fliA and flaA, genes which contribute to flagellation, were expressed early in the mutant. Additionally, in CsrA(-) the levels of the stationary-phase sigma factor, RpoS, and a recently described regulator of virulence traits, LetE, were increased. Growth curves of CsrA(-) bacteria were delayed with pigment production occurring at the same OD578 but at reduced levels in the mutant. Replication ability of the CsrA(-) mutant in amoebae was also affected. Based on these results, we could show that CsrA is involved in the regulation of the bacterial switch from the replicative to the transmissible form.

  3. [Cell-mediated immunity in mice infected with Acanthamoeba culbertsoni].

    PubMed

    Kim, M J; Shin, C O; Im, K I

    1990-09-01

    Observations were made on the differences of cell-mediated responses in mice of three infection groups differently scheduled in their severity with pathogenic Acanthamoeba culbertsoni. Infections were done by dropping 5 microliters saline suspension containing 3 x 10(3), 1 x 10(4), or 1 x 10(5) trophozoites, respectively. Amoebae were cultured axenically in CGV medium and inoculated into the right nasal cavity of C3H/HeJ mice aging around 6-8 weeks, under the anesthesia by intraperitoneal injection of secobarbital. Delayed type hypersensitivity (DTH) responses in footpad and blastogenic responses of mouse spleen cells using (3H)-thymidine and the serum antibody titer were measured up to day 14 after infection, and natural killer cell activities were measured up to day 5 after infection. The results obtained in this study were as follows: 1. The mice infected with 3 x 10(3) trophozoites showed mortality rate of 17%, and 34% in the mice infected with 1 x 10(4) trophozoites and 65% with 1 x 10(5) trophozoites. 2. In regard to DTH responses in all experimental groups, the level increased on day 7 and declined on day 14 after infection, but their differences could not be noted between infected and control groups. 3. The blastogenic responses of splenocytes treated with amoeba lysates and lipopolysaccharides (LPS) showed no difference from the control group. The blastogenic responses of splenocytes treated with concanavalin A were declined significantly in the experimental group as compared with the control group, but the blastogenic responses of splenocytes treated with polyinosinic acid were not different from the control group. There was also no difference among three infected groups. 4. The cytotoxic activity of the natural killer cells was activated on day 1 after infection and declined to the level of control group on day 2 in all experimental groups. On day 5 after infection, the natural killer cell cytotoxicity was significantly suppressed as compared with the

  4. Pathogenic free-living amoebae in Korea

    PubMed Central

    Shin, Ho-Joon

    2004-01-01

    Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis. PMID:15381859

  5. The Legionella pneumophila IcmS-LvgA protein complex is important for Dot/Icm-dependent intracellular growth.

    PubMed

    Vincent, Carr D; Vogel, Joseph P

    2006-08-01

    Many bacterial pathogens require a functional type IV secretion system (T4SS) for virulence. Legionella pneumophila, the causative agent of Legionnaires' disease, employs the Dot/Icm T4SS to inject a large number of protein substrates into its host, thereby altering phagosome trafficking. The L. pneumophila T4SS substrate SdeA has been shown to require the accessory factor IcmS for its export. IcmS, defined as a type IV adaptor, exists as a heterodimer with IcmW and this complex functions in a manner similar to a type III secretion chaperone. Here we report an interaction between IcmS and the previously identified virulence factor LvgA. Similar to the icmS mutant, the lvgA mutant appears to assemble a fully functional Dot/Icm complex. Both LvgA and IcmS are small, acidic proteins localized to the cytoplasm and are not exported by the Dot/Icm system, suggesting they form a novel type IV adaptor complex. Inactivation of lvgA causes a minimal defect in growth in the human monocytic cell line U937 and the environmental host Acanthamoeba castellanii. However, the lvgA mutant was severely attenuated for intracellular growth of L. pneumophila in mouse macrophages, suggesting LvgA may be a critical factor that confers host specificity.

  6. Amoebozoa Possess Lineage-Specific Globin Gene Repertoires Gained by Individual Horizontal Gene Transfers

    PubMed Central

    Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

    2014-01-01

    The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

  7. Surfactin from Bacillus subtilis displays an unexpected anti-Legionella activity.

    PubMed

    Loiseau, Clémence; Schlusselhuber, Margot; Bigot, Renaud; Bertaux, Joanne; Berjeaud, Jean-Marc; Verdon, Julien

    2015-06-01

    A contaminant bacterial strain was found to exhibit an antagonistic activity against Legionella pneumophila, the causative agent of Legionnaires' disease. The bacterial strain was identified as a Bacillus subtilis and named B. subtilis AM1. PCR analysis revealed the presence of the sfp gene, involved in the biosynthesis of surfactin, a lipopeptide with versatile bioactive properties. The bioactive substances were extracted from AM1 cell-free supernatant with ethyl acetate and purified using reversed phase HPLC (RP-HPLC). Subsequent ESI-MS analyses indicated the presence of two active substances with protonated molecular ions at m/z 1008 and 1036 Da, corresponding to surfactin isoforms. Structures of lipopeptides were further determined by tandem mass spectrometry and compared to the spectra of a commercially available surfactin mixture. Surfactin displays an antibacterial spectrum almost restricted to the Legionella genus (MICs range 1-4 μg/mL) and also exhibits a weak activity toward the amoeba Acanthamoeba castellanii, known to be the natural reservoir of L. pneumophila. Anti-biofilm assays demonstrated that 66 μg/mL of surfactin successfully eliminated 90 % of a 6-day-old biofilm. In conclusion, this study reveals for the first time the potent activity of surfactin against Legionella sp. and preformed biofilms thus providing new directions toward the use and the development of lipopeptides for the control of Legionella spread in the environment.

  8. IroT/mavN, a new iron-regulated gene involved in Legionella pneumophila virulence against amoebae and macrophages.

    PubMed

    Portier, Emilie; Zheng, Huaixin; Sahr, Tobias; Burnside, Denise M; Mallama, Celeste; Buchrieser, Carmen; Cianciotto, Nicholas P; Héchard, Yann

    2015-04-01

    Legionella pneumophila is a pathogenic bacterium commonly found in water. Eventually, it could be transmitted to humans via inhalation of contaminated aerosols. Iron is known as a key requirement for the growth of L. pneumophila in the environment and within its hosts. Many studies were performed to understand iron utilization by L. pneumophila but no global approaches were conducted. In this study, transcriptomic analyses were performed, comparing gene expression in L. pneumophila in standard versus iron restricted conditions. Among the regulated genes, a newly described one, lpp_2867, was highly induced in iron-restricted conditions. Mutants lacking this gene in L. pneumophila were not affected in siderophore synthesis or utilization. On the contrary, they were defective for growth on iron-depleted solid media and for ferrous iron uptake. A sequence analysis predicts that Lpp_2867 is a membrane protein, suggesting that it is involved in ferrous iron transport. We thus named it IroT, for iron transporter. Infection assays showed that the mutants are highly impaired in intracellular growth within their environmental host Acanthamoeba castellanii and human macrophages. Taken together, our results show that IroT is involved, directly or indirectly, in ferrous iron transport and is a key virulence factor for L. pneumophila. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  9. The CpxRA two-component system contributes to Legionella pneumophila virulence.

    PubMed

    Tanner, Jennifer R; Li, Laam; Faucher, Sébastien P; Brassinga, Ann Karen C

    2016-06-01

    The bacterium Legionella pneumophila is capable of intracellular replication within freshwater protozoa as well as human macrophages, the latter of which results in the serious pneumonia Legionnaires' disease. A primary factor involved in these host cell interactions is the Dot/Icm Type IV secretion system responsible for translocating effector proteins needed to establish and maintain the bacterial replicative niche. Several regulatory factors have been identified to control the expression of the Dot/Icm system and effectors, one of which is the CpxRA two-component system, suggesting essentiality for virulence. In this study, we generated cpxR, cpxA and cpxRA in-frame null mutant strains to further delineate the role of the CpxRA system in bacterial survival and virulence. We found that cpxR is essential for intracellular replication within Acanthamoeba castellanii, but not in U937-derived macrophages. Transcriptome analysis revealed that CpxRA regulates a large number of virulence-associated proteins including Dot/Icm effectors as well as Type II secreted substrates. Furthermore, the cpxR and cpxRA mutant strains were more sodium resistant than the parental strain Lp02, and cpxRA expression reaches maximal levels during postexponential phase. Taken together, our findings suggest the CpxRA system is a key contributor to L. pneumophila virulence in protozoa via virulence factor regulation. © 2016 John Wiley & Sons Ltd.

  10. Drug effects on drug targets: inhibition of enzymes by neuroleptics, antimycotics, antibiotics and other drugs on human pathogenic amoebas and their anti-proliferative effects.

    PubMed

    Ondarza, Raúl N

    2007-11-01

    This paper reviews the inhibition of various enzymes by neuroleptics, anti-mycotics, antibiotics and other drugs on three species of human pathogenic amoebas, mainly Entamoeba histolytica, Acanthamoeba polyphaga and Naegleria fowleri, and their antiproliferative effects. A recent patent registered by Philip relates to the combination of an antibacterial formulation and antifungal agent for producing a therapeutically effective quantity of an antimicrobial that is suitable for suppressing or treating fungal growth. The rationale behind this patent focused on essential and valid targets with a description of the main pathogenic characteristics of these amoebas. The study of new targets, such as trypanothione and trypanothione reductase, and the drug effects of selected agents were arranged into six main groups: A) Inhibition of disulfide reducing enzymes by neuroleptics, antimycotics and antibiotics; B) Comparative evaluation of the efficacies of several drugs with antiproliferative activities; C) Inhibition of the enzymes for the synthesis of trypanothione, such as ornithine decarboxylase, spermidine synthase and trypanothione synthetase; D) Inhibition of the glycolytic enzyme PPi-dependent phosphofructokinase (PFK) from Entamoeba and Naegleria by pyrophosphate analogues, different from the host enzyme; E) Inhibition of enzymes secreted by these parasites to invade the human host, for example cysteine proteinases; and F) Inhibition of encystment pathways and cyst-wall assembly proteins.

  11. The Relation of Ocular Surface Irregularity and Visual Disturbance in Early Stage Acanthamoeba Keratitis.

    PubMed

    Matsumoto, Yukihiro; Kodama, Asako; Goto, Eiki; Kawakita, Tetsuya; Dogru, Murat; Tsubota, Kazuo

    2017-01-01

    To evaluate the relation between ocular surface irregularity and visual disturbance in early stage Acanthamoeba keratitis (AK). Fifteen patients with culture-proven AK underwent routine ophthalmic examinations, including best-corrected visual acuity (BCVA) measurement, slitlamp biomicroscope examination, and corneal fluorescein dye staining test, in both the eyes. We also evaluated the corneal sensitivity with Cochet-Bonnet esthesiometer, tear functions by Schirmer's test, and ocular surface irregularity by corneal topography and compared the results with the contralateral healthy eyes in this study. The mean logarithm of the minimum angle of resolution BCVA (0.71±0.77) was significantly lower in the eyes with AK (P=0.002). Epithelial disorders were present in all eyes, and radial keratoneuritis in 14 eyes (93.3%). The mean corneal sensitivity (39.3±24.1 mm) was significantly lower in eyes with AK compared with the healthy eyes (P=0.005). The mean Schirmer's test value (22.5±12.0 mm) in eyes with AK was significantly higher compared with the healthy eyes (P=0.01). The ocular surface irregularity indices (the surface regularity index, 2.47±0.42; the surface asymmetry index, 3.24±1.31) were significantly higher in eyes with AK compared with contralateral healthy eyes (P<0.0001 and P<0.0001, respectively). The ocular surface disease in AK is associated with decrease in corneal sensitivity and increase in Schirmer's test value and ocular surface irregularity indices. The visual disturbance in AK may owe not only to corneal haze but also to ocular surface irregularity.

  12. Gold Nanoparticle Conjugation Enhances the Antiacanthamoebic Effects of Chlorhexidine

    PubMed Central

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Anwar, Ayaz; Shah, Muhammad Raza

    2015-01-01

    Acanthamoeba keratitis is a serious infection with blinding consequences and often associated with contact lens wear. Early diagnosis, followed by aggressive topical application of drugs, is a prerequisite in successful treatment, but even then prognosis remains poor. Several drugs have shown promise, including chlorhexidine gluconate; however, host cell toxicity at physiologically relevant concentrations remains a challenge. Nanoparticles, subcolloidal structures ranging in size from 10 to 100 nm, are effective drug carriers for enhancing drug potency. The overall aim of the present study was to determine whether conjugation with gold nanoparticles enhances the antiacanthamoebic potential of chlorhexidine. Gold-conjugated chlorhexidine nanoparticles were synthesized. Briefly, gold solution was mixed with chlorhexidine and reduced by adding sodium borohydride, resulting in an intense deep red color, indicative of colloidal gold-conjugated chlorhexidine nanoparticles. The synthesis was confirmed using UV-visible spectrophotometry that shows a plasmon resonance peak of 500 to 550 nm, indicative of gold nanoparticles. Further characterization using matrix-assisted laser desorption ionization-mass spectrometry showed a gold-conjugated chlorhexidine complex at m/z 699 ranging in size from 20 to 100 nm, as determined using atomic force microscopy. To determine the amoebicidal and amoebistatic effects, amoebae were incubated with gold-conjugated chlorhexidine nanoparticles. For controls, amoebae also were incubated with gold and silver nanoparticles alone, chlorhexidine alone, neomycin-conjugated nanoparticles, and neomycin alone. The findings showed that gold-conjugated chlorhexidine nanoparticles exhibited significant amoebicidal and amoebistatic effects at 5 μM. Amoebicidal effects were observed by parasite viability testing using a Trypan blue exclusion assay and flow-cytometric analysis using propidium iodide, while amoebistatic effects were observed using growth

  13. In vitro encystment of Himasthla elongata cercariae (Digenea, Echinostomatidae) in the haemolymph of blue mussels Mytilus edulis as a tool for assessing cercarial infectivity and molluscan susceptibility.

    PubMed

    Levakin, I A; Losev, E A; Nikolaev, K E; Galaktionov, K V

    2013-06-01

    Infectivity of Himasthla elongata cercariae to mussels, their second intermediate hosts, and resistance by these hosts to infection were assessed on the basis of the cercariae's ability to encyst in mussel haemolymph in vitro. A series of experimental in vivo infections of mussels with batches of cercariae, each batch released from a different single infected mollusc and referred to as a clone (due to their shared genotype), demonstrated that the results of the in vitro tests corresponded to the actual indices of infectivity/susceptibility of the parasites and their hosts. Most cercarial clones had high infectivity, with a few clones having very high or, at the other extreme, very low infectivity. A similar pattern was revealed in mussel resistance to cercarial infection. Most of the molluscs tested were moderately susceptible to cercarial infection, but at each extreme a small fraction (less than 10%) displayed very high or very low susceptibility. It was shown that there were no totally compatible or totally incompatible 'cercaria clone/mussel' combinations. Results obtained are compared with the data on intra-population variability using the characters parasite infectivity/host compatibility for trematode/mollusc-first intermediate host associations. Results are made relevant to actual infection levels in mussel settlements at the White Sea.

  14. Acanthamoeba keratitis in 194 patients: risk factors for bad outcomes and severe inflammatory complications.

    PubMed

    Carnt, Nicole; Robaei, Dana; Minassian, Darwin C; Dart, John K G

    2018-01-03

    To determine demographic and clinical features of patients with Acanthamoeba keratitis (AK) that are independent risk factors both for bad outcomes and for severe inflammatory complications (SIC). A retrospective audit of medical records of AK cases at Moorfields Eye Hospital from July 2000 to April 2012, including 12 earlier surgical cases. Cases with a bad outcome were defined as those having one or more of the following: corneal perforation, keratoplasty, other surgery (except biopsy), duration of antiamoebic therapy (AAT) ≥10.5 months (the 75th percentile of the whole cohort) and final visual acuity ≤20/80. SICs were defined as having scleritis and/or a stromal ring infiltrate. Multivariable analysis was used to identify independent risk factors for both bad outcomes and SICs. Records of 194 eyes (194 patients) were included, having bad outcomes in 93 (48%). Bad outcomes were associated with the presence of SIC, aged >34 years, corticosteroids used before giving AAT and symptom duration >37 days before AAT. The development of SIC was independently associated with aged >34 years, corticosteroids used before giving AAT and herpes simplex virus (HSV) keratitis treatment before AAT. The prompt diagnosis of AK, avoidance of a misdiagnosis of HSV keratitis and corticosteroid use before the exclusion of AK as a potential cause of keratitis are essential to the provision of a good outcome for patients and for the avoidance of SIC. Older age is an unmodifiable risk factor that may reflect differences in the immune response to AK in this patient subset. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  15. Resurgence of Acanthamoeba keratitis in Auckland, New Zealand: a 7-year review of presentation and outcomes.

    PubMed

    Patel, Dipika V; Rayner, Sandra; McGhee, Charles N J

    2010-01-01

    To investigate the presentation, clinical characteristics and outcomes of Acanthamoeba keratitis (AK) in Auckland, New Zealand over a 7-year period. Retrospective analysis of all cases of AK treated by the tertiary corneal service at Auckland City Hospital/ University of Auckland Department of Ophthalmology (August 2001 to May 2008). Data were collected regarding age, gender, contact lens history, presenting signs and symptoms, diagnosis at first presentation, time to final diagnosis, identifiable risk factors, presenting and final visual acuity, results of microbiological testing, medical treatment, surgical interventions, recurrence of disease and length of follow up. All photographs and in vivo confocal microscopy images were reviewed. Twenty-five eyes of 25 patients were identified with a diagnosis of AK (mean age 40 +/- 13 years). Ninety-six per cent were contact lens wearers. Mean time to diagnosis was 41 +/- 49 days (range 0-181 days, median 21 days). Fourteen patients (56%) had been treated with topical corticosteroids prior to the diagnosis. Early diagnosis of AK (<21 days) was associated with significantly better final visual acuity and did not require any surgical intervention compared with those diagnosed at a later stage. Six patients, all in the late diagnosis group, required surgical intervention. AK has become significantly more common in New Zealand in the current decade. This study highlights the fundamental importance of early diagnosis and appropriate management in ensuring favourable outcomes. Practitioners should maintain a clinical suspicion of AK, especially as 96% of the subjects in this study were contact lens wearers.

  16. Exploring Anti-Bacterial Compounds against Intracellular Legionella

    PubMed Central

    Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

    2013-01-01

    Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an ‘accidental’ human pathogen and cause a severe pneumonia known as Legionnaires’ disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoeba castellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

  17. Salicylate inhibition of acanthamoebal attachment to contact lenses.

    PubMed

    Beattie, Tara K; Tomlinson, Alan; Seal, David V; McFadyen, Angus K

    2011-12-01

    Sodium salicylate has shown potential as a component of contact lens care solutions designed to reduce Acanthamoebal attachment to contact lenses. This study determined the minimum effective concentration required to significantly reduce amoebal attachment. Different concentrations of sodium salicylate (10, 15, and 20 mM) were applied during exposure of unworn or bacterial biofilm-coated hydrogel contact lenses to Acanthamoeba castellanii trophozoites. Salicylate was applied at stage 1 intervention during biofilm formation on lenses, at stage 2 intervention during amoebal exposure, or at both stages. A significant reduction in amoebal attachment was achieved when 10 mM salicylate was included during stage 1 alone; however, 15 mM was required for stage 2 intervention to significantly reduce attachment to clean or biofilm-coated lenses. For stages 1 and 2 combined intervention, 10 mM sodium salicylate produced a significant reduction in amoebal attachment. In situ, within a contact lens case, biofilm formation and amoebal attachment would occur concurrently; therefore, stages 1 and 2 intervention would be closest to the real-life situation, thus indicating that 10 mM of salicylate would be an effective minimum concentration for reducing amoebal attachment to hydrogel contact lenses. Inclusion of components in contact lens care solution, such as sodium salicylate, which reduce Acanthamoebal attachment, has the potential to enhance effectiveness, particularly where amoebicidal efficacy may be limited, thus reducing the risk of contact lens-associated Acanthamoebal infection.

  18. Reciprocal expression of integration host factor and HU in the developmental cycle and infectivity of Legionella pneumophila.

    PubMed

    Morash, Michael G; Brassinga, Ann Karen C; Warthan, Michelle; Gourabathini, Poornima; Garduño, Rafael A; Goodman, Steven D; Hoffman, Paul S

    2009-04-01

    Legionella pneumophila is an intracellular parasite of protozoa that differentiates late in infection into metabolically dormant cysts that are highly infectious. Regulation of this process is poorly understood. Here we report that the small DNA binding regulatory proteins integration host factor (IHF) and HU are reciprocally expressed over the developmental cycle, with HU expressed during exponential phase and IHF expressed postexponentially. To assess the role of these regulatory proteins in development, chromosomal deletions were constructed. Single (ihfA or ihfB) and double deletion (Deltaihf) IHF mutants failed to grow in Acanthamoeba castellanii unless complemented in trans when expressed temporally from the ihfA promoter but not under P(tac) (isopropyl-beta-d-thiogalactopyranoside). In contrast, IHF mutants were infectious for HeLa cells, though electron microscopic examination revealed defects in late-stage cyst morphogenesis (thickened cell wall, intracytoplasmic membranes, and inclusions of poly-beta-hydroxybutyrate), and were depressed for the developmental marker MagA. Green fluorescent protein promoter fusion assays indicated that IHF and the stationary-phase sigma factor RpoS were required for full postexponential expression of magA. Finally, defects in cyst morphogenesis noted for Deltaihf mutants in HeLa cells correlated with a loss of both detergent resistance and hyperinfectivity compared with results for wild-type cysts. These studies establish IHF and HU as markers of developmental stages and show that IHF function is required for both differentiation and full virulence of L. pneumophila in natural amoebic hosts.

  19. An atypical proprotein convertase in Giardia lamblia differentiation.

    PubMed

    Davids, B J; Gilbert, M A; Liu, Q; Reiner, D S; Smith, A J; Lauwaet, T; Lee, C; McArthur, A G; Gillin, F D

    2011-02-01

    Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologs distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation. Copyright © 2010 Elsevier B

  20. An Atypical Proprotein Convertase in Giardia lamblia Differentiation

    PubMed Central

    Davids, B. J.; Gilbert, M. A.; Liu, Q.; Reiner, D. S.; Smith, A. J.; Lauwaet, T.; Lee, C.; McArthur, A. G.; Gillin, F. D.

    2010-01-01

    Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologues distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1 mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation. PMID:21075147

  1. Genome segregation and packaging machinery in Acanthamoeba polyphaga mimivirus is reminiscent of bacterial apparatus.

    PubMed

    Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi; Kondabagil, Kiran

    2014-06-01

    Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized

  2. Genome Segregation and Packaging Machinery in Acanthamoeba polyphaga Mimivirus Is Reminiscent of Bacterial Apparatus

    PubMed Central

    Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi

    2014-01-01

    ABSTRACT Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. IMPORTANCE Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside

  3. Detection and identification of free-living amoeba from aquatic environment in Taiwan

    NASA Astrophysics Data System (ADS)

    Jiun Tzeng, Kai; Che Tung, Min; Hsu, Bing Mu; Tsai, Hsiu Feng; Huang, Po Hsiang; Hao Huang, Kuan; Kao, Po Min; Shen, Shu Min; Chen, Jung Sheng

    2013-04-01

    Free-living amoebae including Acanthamoeba, Naegleria, Balamuthia and Hartmannella are widely distributed in water, soil, and air. They can infect humans and can lead to serious illness even death. The aim of this study is to investigate the presence of free-living amoebae from aquatic environment in Taiwan, and to compare the differences between Acanthamoeba and Naegleria in different cultivation methods and conditions. In this study, we used molecular method with specific primers by Polymerase Chain Reaction (PCR) to amplify and to analyze the occurrence of free-living amoebae in aquatic environment. We collected 92 samples from environmental water in Taiwan. The results show that 33 water samples (35.9%) and 11 water samples (12.0%) were detected positive for Acanthamoeba and Naegleria, respectively. Furthermore, both Acanthamoeba and Naegleria can be cultured by PYG in 30° C, but not all free-living amoebae can be enriched and isolated by using storage-cultivation method. Due to the presence of Acanthamoeba and Naegleria in aquatic environment, the water quality monitoring should be more conscious. Keywords: free-living amoebae; Acanthamoeba; Naegleria; Balamuthia; Hartmannella; PCR

  4. Disseminated Acanthamoeba Infection Presenting With Cutaneous Lesions in an Immunocompromised Patient: A Case Report, Review of Histomorphologic Findings, and Potential Diagnostic Pitfalls.

    PubMed

    Morrison, Annie O; Morris, Robert; Shannon, Amie; Lauer, Scott R; Guarner, Jeannette; Kraft, Colleen S

    2016-02-01

    Free-living amoebas are exceedingly rare causes of cutaneous infections and present unique diagnostic and therapeutic challenges. We describe a case of disseminated acanthamoebiasis with cutaneous manifestations and summarize additional diagnostic, prognostic, and therapeutic highlights. A 58-year-old man with relapsed chronic lymphocytic leukemia had several weeks of progressive, painful ulcerations on the forehead, arms, abdomen, and thighs. A biopsy was performed for histopathologic evaluation. The biopsy specimen showed inflammatory infiltrate with abscess formation involving the epidermis, dermis, and subcutis. Scattered cells showed nuclei with a prominent central karyosome, dispersed chromatin, and either abundant foamy basophilic cytoplasm or two well-demarcated cytoplasmic walls. Acanthamoeba species was confirmed by polymerase chain reaction from the formalin-fixed, paraffin-embedded tissue. Cutaneous lesions from acanthamoebiasis are exceptionally rare but should be included in the differential diagnosis of necrotic cutaneous lesions in immunocompromised patients. Although infrequently encountered, pathologists need to be aware of the morphologic features of free-living amoebas. Immunohistochemical and molecular studies can confirm the diagnosis. Multiagent treatment regimens, when initiated empirically, have been more successful than single-agent regimens, but infections involving the central nervous system are almost universally fatal. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Phospholipids Trigger Cryptococcus neoformans Capsular Enlargement during Interactions with Amoebae and Macrophages

    PubMed Central

    Chrisman, Cara J.; Albuquerque, Patricia; Guimaraes, Allan J.; Nieves, Edward; Casadevall, Arturo

    2011-01-01

    A remarkable aspect of the interaction of Cryptococcus neoformans with mammalian hosts is a consistent increase in capsule volume. Given that many aspects of the interaction of C. neoformans with macrophages are also observed with amoebae, we hypothesized that the capsule enlargement phenomenon also had a protozoan parallel. Incubation of C. neoformans with Acanthamoeba castellanii resulted in C. neoformans capsular enlargement. The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability. Analysis of amoebae extracts showed that the likely stimuli for capsule enlargement were protozoan polar lipids. Extracts from macrophages and mammalian serum also triggered cryptococcal capsular enlargement. C. neoformans capsule enlargement required expression of fungal phospholipase B, but not phospholipase C. Purified phospholipids, in particular, phosphatidylcholine, and derived molecules triggered capsular enlargement with the subsequent formation of giant cells. These results implicate phospholipids as a trigger for both C. neoformans capsule enlargement in vivo and exopolysaccharide production. The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro. Protozoan- or mammalian-derived polar lipids could represent a danger signal for C. neoformans that triggers capsular enlargement as a non-specific defense mechanism against potential predatory cells. Hence, phospholipids are the first host-derived molecules identified to trigger capsular enlargement. The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists. PMID:21637814

  6. Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila

    PubMed Central

    Sahr, Tobias; Brüggemann, Holger; Jules, Matthieu; Lomma, Mariella; Albert-Weissenberger, Christiane; Cazalet, Christel; Buchrieser, Carmen

    2009-01-01

    Summary To transit from intra- to extracellular environments, L. pneumophila differentiates from a replicative/non-virulent to a transmissive/virulent form using the two-component system LetA/LetS and the global repressor protein CsrA. While investigating how both regulators act coordinately we characterized two ncRNAs, RsmY and RsmZ that link the LetA/LetS and CsrA regulatory networks. We demonstrate that LetA directly regulates their expression and show that RsmY and RsmZ are functional in E. coli and are able to bind CsrA in vitro. Single mutants have no (ΔrsmY) or a little (ΔrsmZ) impact on virulence, but the ΔrsmYZ strain shows a drastic defect in intracellular growth in Acanthamoeba castellanii and THP-1 monocyte-derived macrophages. Analysis of the transcriptional programs of the ΔletA, ΔletS and ΔrsmYZ strains revealed that the switch to the transmissive phase is partially blocked. One major difference between the ΔletA, ΔletS and ΔrsmYZ strains was that the latter synthesizes flagella. Taken together, LetA activates transcription of RsmY and RsmZ, which sequester CsrA and abolish its post-transcriptional repressive activity. However, the RsmYZ-CsrA pathway appears not to be the main or only regulatory circuit governing flagella synthesis. We suggest that rather RpoS and LetA, by influencing LetE and probably cyclic-di-GMP levels, regulate motility in L. pneumophila. PMID:19400772

  7. Evolution and function of eukaryotic-like proteins from sponge symbionts.

    PubMed

    Reynolds, David; Thomas, Torsten

    2016-10-01

    Sponges (Porifera) are ancient metazoans that harbour diverse microorganisms, whose symbiotic interactions are essential for the host's health and function. Although symbiosis between bacteria and sponges are ubiquitous, the molecular mechanisms that control these associations are largely unknown. Recent (meta-) genomic analyses discovered an abundance of genes encoding for eukaryotic-like proteins (ELPs) in bacterial symbionts from different sponge species. ELPs belonging to the ankyrin repeat (AR) class from a bacterial symbiont of the sponge Cymbastela concentrica were subsequently found to modulate amoebal phagocytosis. This might be a molecular mechanism, by which symbionts can control their interaction with the sponge. In this study, we investigated the evolution and function of ELPs from other classes and from symbionts found in other sponges to better understand the importance of ELPs for bacteria-eukaryote interactions. Phylogenetic analyses showed that all of the nine ELPs investigated were most closely related to proteins found either in eukaryotes or in bacteria that can live in association with eukaryotes. ELPs were then recombinantly expressed in Escherichia coli and exposed to the amoeba Acanthamoeba castellanii, which is functionally analogous to phagocytic cells in sponges. Phagocytosis assays with E. coli containing three ELP classes (AR, TPR-SEL1 and NHL) showed a significantly higher percentage of amoeba containing bacteria and average number of intracellular bacteria per amoeba when compared to negative controls. The result that various classes of ELPs found in symbionts of different sponges can modulate phagocytosis indicates that they have a broader function in mediating bacteria-sponge interactions. © 2016 John Wiley & Sons Ltd.

  8. Phylogenetic Analysis of Mitochondrial Outer Membrane β-Barrel Channels

    PubMed Central

    Wojtkowska, Małgorzata; Jąkalski, Marcin; Pieńkowska, Joanna R.; Stobienia, Olgierd; Karachitos, Andonis; Przytycka, Teresa M.; Weiner, January; Kmita, Hanna; Makałowski, Wojciech

    2012-01-01

    Transport of molecules across mitochondrial outer membrane is pivotal for a proper function of mitochondria. The transport pathways across the membrane are formed by ion channels that participate in metabolite exchange between mitochondria and cytoplasm (voltage-dependent anion-selective channel, VDAC) as well as in import of proteins encoded by nuclear genes (Tom40 and Sam50/Tob55). VDAC, Tom40, and Sam50/Tob55 are present in all eukaryotic organisms, encoded in the nuclear genome, and have β-barrel topology. We have compiled data sets of these protein sequences and studied their phylogenetic relationships with a special focus on the position of Amoebozoa. Additionally, we identified these protein-coding genes in Acanthamoeba castellanii and Dictyostelium discoideum to complement our data set and verify the phylogenetic position of these model organisms. Our analysis show that mitochondrial β-barrel channels from Archaeplastida (plants) and Opisthokonta (animals and fungi) experienced many duplication events that resulted in multiple paralogous isoforms and form well-defined monophyletic clades that match the current model of eukaryotic evolution. However, in representatives of Amoebozoa, Chromalveolata, and Excavata (former Protista), they do not form clearly distinguishable clades, although they locate basally to the plant and algae branches. In most cases, they do not posses paralogs and their sequences appear to have evolved quickly or degenerated. Consequently, the obtained phylogenies of mitochondrial outer membrane β-channels do not entirely reflect the recent eukaryotic classification system involving the six supergroups: Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta. PMID:22155732

  9. The Megavirus Chilensis Cu,Zn-Superoxide Dismutase: the First Viral Structure of a Typical Cellular Copper Chaperone-Independent Hyperstable Dimeric Enzyme

    PubMed Central

    Lartigue, Audrey; Burlat, Bénédicte; Coutard, Bruno; Chaspoul, Florence; Claverie, Jean-Michel

    2014-01-01

    ABSTRACT Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme's activation is independent of a chaperone both for disulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts. IMPORTANCE Mimiviridae are giant viruses encoding more than 1,000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole's oxidative stress. The megavirus chilensis virion contains a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some megavirus

  10. Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments.

    PubMed

    Hsu, Bing-Mu; Huang, Chin-Chun; Chen, Jung-Sheng; Chen, Nai-Hsiung; Huang, Jen-Te

    2011-10-15

    This study compares five genera of free-living amoebae (FLA) hosts by Legionella spp. in the fixed and floating biofilm samples from spring environments. Detection rate of Legionella spp. was 26.9% for the floating biofilms and 3.1% for the fixed biofilms. Acanthamoeba spp., Hartmanella vermiformis, and Naegleria spp. were more frequently detected in floating biofilm than in fixed biofilm samples. The percentage of pathogenic Acanthamoeba spp. among all the genus Acanthamoeba detected positive samples was 19.6%. The potential pathogenic Naegleria spp. (for example, Naegleria australiensis, Naegleria philippinensis, and Naegleria italica) was 54.2% to all the Naegleria detected positive samples. In the study, 12 serotypes of possible pneumonia causing Legionella spp. were detected, and their percentage in all the Legionella containing samples was 42.4%. The FLA parasitized by Legionella included unnamed Acanthamoeba genotype, Acanthamoeba griffini, Acanthamoeba jacobsi, H. vermiformis, and N. australiensis. Significant differences were also observed between the presence/absence of H. vermiformis and Legionella parasitism in FLA. Comparisons between the culture-confirmed method and the PCR-based detection method for detecting FLA and Legionella in biofilms showed great variation. Therefore, using these analysis methods together to detect FLA and Legionella is recommended. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Legionella pneumophila OxyR Is a Redundant Transcriptional Regulator That Contributes to Expression Control of the Two-Component CpxRA System.

    PubMed

    Tanner, Jennifer R; Patel, Palak G; Hellinga, Jacqueline R; Donald, Lynda J; Jimenez, Celine; LeBlanc, Jason J; Brassinga, Ann Karen C

    2017-03-01

    Nominally an environmental organism, Legionella pneumophila is an intracellular parasite of protozoa but is also the causative agent of the pneumonia termed Legionnaires' disease, which results from inhalation of aerosolized bacteria by susceptible humans. Coordination of gene expression by a number of identified regulatory factors, including OxyR, assists L. pneumophila in adapting to the stresses of changing environments. L. pneumophila OxyR (OxyR Lp ) is an ortholog of Escherichia coli OxyR; however, OxyR Lp was shown elsewhere to be functionally divergent, such that it acts as a transcription regulator independently of the oxidative stress response. In this study, the use of improved gene deletion methods has enabled us to generate an unmarked in-frame deletion of oxyR in L. pneumophila Lack of OxyR Lp did not affect in vitro growth or intracellular growth in Acanthamoeba castellanii protozoa and U937-derived macrophages. The expression of OxyR Lp does not appear to be regulated by CpxR, even though purified recombinant CpxR bound a DNA sequence similar to that reported for CpxR elsewhere. Surprisingly, a lack of OxyR Lp resulted in elevated activity of the promoters located upstream of icmR and the lpg1441-cpxA operon, and OxyR Lp directly bound to these promoter regions, suggesting that OxyR Lp is a direct repressor. Interestingly, a strain overexpressing OxyR Lp demonstrated reduced intracellular growth in A. castellanii but not in U937-derived macrophages, suggesting that balanced expression control of the two-component CpxRA system is necessary for survival in protozoa. Taken together, this study suggests that OxyR Lp is a functionally redundant transcriptional regulator in L. pneumophila under the conditions evaluated herein. IMPORTANCE Legionella pneumophila is an environmental pathogen, with its transmission to the human host dependent upon its ability to replicate in protozoa and survive within its aquatic niche. Understanding the genetic factors that

  12. A year long study of the presence of free living amoeba in Spain.

    PubMed

    Magnet, A; Fenoy, S; Galván, A L; Izquierdo, F; Rueda, C; Fernandez Vadillo, C; Del Aguila, C

    2013-12-01

    Free-living amoeba such as Acanthamoeba and Balamuthia mandrillaris can act as opportunistic parasites on a wide range of vertebrates and they are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices and their ubiquitous distribution. This work was carried out in order to study the presence of these free-living amoebae (FLA) and their possible seasonality in a continental-Mediterranean climate in different types of water. For this purpose, a total of 223 water samples were collected during one year from four drinking water treatment plants (DWTP), seven wastewater treatment plants (WWTP) and six locations of influence (LI) on four river basins from Spain. Water samples were concentrated using the IDEXX Filta-Max(®) system and analyzed by a triplex real time PCR that detects Acanthamoeba, B. mandrillaris and Naegleria fowleri. Agar plates were also seeded for Acanthamoeba culture. From the three FLA studied, N. fowleri was not detected in any sample while B. mandrillaris was found at the entrance of a DWTP; this being, to our knowledge, the first report of these protozoa in water worldwide. On the other hand, the presence of Acanthamoeba observed was higher, 94.6% of the studied points were positive by real time PCR and 85.2% by culture, resulting in 99.1% positive for Acanthamoeba with both methods. All genetically analyzed Acanthamoeba were genotype T4 but nine different T4/DF3 sequences were observed, three of them being described for the first time, assigning new codes. No seasonal distribution of Acanthamoeba was found. These facts should serve as a warning to contact lens wearers of the risk of a poor hygiene when handling their contact lenses. It should also serve as a signal to physicians to consider FLA as a possible causative agent of nervous system infections as well as Acanthamoeba keratitis due to their high environmental presence shown in this

  13. A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate.

    PubMed

    Weber, Stephen; Stirnimann, Christian U; Wieser, Mara; Frey, Daniel; Meier, Roger; Engelhardt, Sabrina; Li, Xiaodan; Capitani, Guido; Kammerer, Richard A; Hilbi, Hubert

    2014-12-05

    The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. bdhA-patD operon as a virulence determinant, revealed by a novel large-scale approach for identification of Legionella pneumophila mutants defective for amoeba infection.

    PubMed

    Aurass, P; Pless, B; Rydzewski, K; Holland, G; Bannert, N; Flieger, A

    2009-07-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular parasite of eukaryotic cells. In the environment, it colonizes amoebae. After being inhaled into the human lung, the bacteria infect and damage alveolar cells in a way that is mechanistically similar to the amoeba infection. Several L. pneumophila traits, among those the Dot/Icm type IVB protein secretion machinery, are essential for exploiting host cells. In our search for novel Legionella virulence factors, we developed an agar plate assay, designated the scatter screen, which allowed screening for mutants deficient in infecting Acanthamoeba castellanii amoebae. Likewise, an L. pneumophila clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established Legionella virulence genes, for example, dot/icm genes, were identified. Importantly, 70 novel virulence-associated genes were found. One of those is L. pneumophila bdhA, coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that bdhA is cotranscribed with patD, encoding a patatin-like protein of L. pneumophila showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, the L. pneumophila bdhA-patD mutant showed a severe replication defect in amoebae and U937 macrophages. Our data suggest that the operon is involved in poly-3-hydroxybutyrate utilization and phospholipolysis and show that the bdhA-patD operon is a virulence determinant of L. pneumophila. In summary, the screen for amoeba-sensitive Legionella clones efficiently isolated mutants that do not grow in amoebae and, in the case of the bdhA-patD mutant, also human cells.

  15. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila.

    PubMed

    Brüggemann, Holger; Hagman, Arne; Jules, Matthieu; Sismeiro, Odile; Dillies, Marie-Agnès; Gouyette, Catherine; Kunst, Frank; Steinert, Michael; Heuner, Klaus; Coppée, Jean-Yves; Buchrieser, Carmen

    2006-08-01

    Adaptation to the host environment and exploitation of host cell functions are critical to the success of intracellular pathogens. Here, insight to these virulence mechanisms was obtained for the first time from the transcriptional program of the human pathogen Legionella pneumophila during infection of its natural host, Acanthamoeba castellanii. The biphasic life cycle of L. pneumophila was reflected by a major shift in gene expression from replicative to transmissive phase, concerning nearly half of the genes predicted in the genome. However, three different L. pneumophila strains showed similar in vivo gene expression patterns, indicating that common regulatory mechanisms govern the Legionella life cycle, despite the plasticity of its genome. During the replicative phase, in addition to components of aerobic metabolism and amino acid catabolism, the Entner-Doudoroff pathway, a NADPH producing mechanism used for sugar and/or gluconate assimilation, was expressed, suggesting for the first time that intracellular L. pneumophila may also scavenge host carbohydrates as nutrients and not only proteins. Identification of genes only upregulated in vivo but not in vitro, may explain higher virulence of in vivo grown L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes predicted to promote transmission and manipulation of a new host cell, therewith priming it for the next attack. These including substrates of the Dot/Icm secretion system, other factors associated previously with invasion and virulence, the motility and the type IV pilus machineries, and > 90 proteins not characterized so far. Analysis of a fliA (sigma28) deletion mutant identified genes coregulated with the flagellar regulon, including GGDEF/EAL regulators and factors that promote host cell entry and survival.

  16. Virulence Phenotypes of Legionella pneumophila Associated with Noncoding RNA lpr0035

    PubMed Central

    Jayakumar, Deepak; Early, Julie V.

    2012-01-01

    The Philadelphia-1 strain of Legionella pneumophila, the causative organism of Legionnaires' disease, contains a recently discovered noncoding RNA, lpr0035. lpr0035 straddles the 5′ chromosomal junction of a 45-kbp mobile genetic element, pLP45, which can exist as an episome or integrated in the bacterial chromosome. A 121-bp deletion was introduced in strain JR32, a Philadelphia-1 derivative. The deletion inactivated lpr0035, removed the 49-bp direct repeat at the 5′ junction of pLP45, and locked pLP45 in the chromosome. Intracellular multiplication of the deletion mutant was decreased by nearly 3 orders of magnitude in Acanthamoeba castellanii amoebae and nearly 2 orders of magnitude in J774 mouse macrophages. Entry of the deletion mutant into amoebae and macrophages was decreased by >70%. The level of entry in both hosts was restored to that in strain JR32 by plasmid copies of two open reading frames immediately downstream of the 5′ junction and plasmid lpr0035 driven by its endogenous promoter. When induced from a tac promoter, plasmid lpr0035 completely reversed the intracellular multiplication defect in macrophages but was without effect in amoebae. These data are the first evidence of a role for noncoding RNA lpr0035, which has homologs in six other Legionella genomes, in entry of L. pneumophila into amoebae and macrophages and in host-specific intracellular multiplication. The data also demonstrate that deletion of a direct-repeat sequence restricts the mobility of pLP45 and is a means of studying the role of pLP45 mobility in Legionella virulence phenotypes. PMID:22966048

  17. Innovative Methodology in the Discovery of Novel Drug Targets in the Free-Living Amoebae

    PubMed

    Baig, Abdul Mannan

    2018-04-25

    Despite advances in drug discovery and modifications in the chemotherapeutic regimens, human infections caused by free-living amoebae (FLA) have high mortality rates (~95%). The FLA that cause fatal human cerebral infections include Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba spp. Novel drug-target discovery remains the only viable option to tackle these central nervous system (CNS) infection in order to lower the mortality rates caused by the FLA. Of these FLA, N. fowleri causes primary amoebic meningoencephalitis (PAM), while the A. castellanii and B. Mandrillaris are known to cause granulomatous amoebic encephalitis (GAE). The infections caused by the FLA have been treated with drugs like Rifampin, Fluconazole, Amphotericin-B and Miltefosine. Miltefosine is an anti-leishmanial agent and an experimental anti-cancer drug. With only rare incidences of success, these drugs have remained unsuccessful to lower the mortality rates of the cerebral infection caused by FLA. Recently, with the help of bioinformatic computational tools and the discovered genomic data of the FLA, discovery of newer drug targets has become possible. These cellular targets are proteins that are either unique to the FLA or shared between the humans and these unicellular eukaryotes. The latter group of proteins has shown to be targets of some FDA approved drugs prescribed in non-infectious diseases. This review out-lines the bioinformatic methodologies that can be used in the discovery of such novel drug-targets, their chronicle by in-vitro assays done in the past and the translational value of such target discoveries in human diseases caused by FLA. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Detection and Identification of Free-living Amoeba from Environmental Water in Taiwan by PCR Method

    NASA Astrophysics Data System (ADS)

    Tsai, H. F.; Hsu, B. M.; Huang, K. H.; She, C. Y.; Kao, P. M.; Shen, S. M.; Tseng, S. F.; Chen, J. S.

    2012-04-01

    Acanthamoeba, Naegleria, Balamuthia and Hartmannella all belong to free-living amoebae that are present ubiquitously in the environment including water, soil, and air. Free-living amoebae are parasites which can infect humans and can lead to serious illness and even death. The aim of this study is to investigate the presence of free-living amoebae in aquatic environment in Taiwan, and to compare the differences between Acanthamoeba and Naegleria in diverse cultivation methods and conditions. In this study, we used molecular method by PCR amplification with specific primers to analyze the occurrence of free-living amoebae. We collected 176 samples from environmental water including drinking water treatment plants, stream water, and hot spring recreational areas in Taiwan. Based on the results of PCR, 43 water samples (24.4%) were detected positive for free-living amoebae. The most common Acanthamoeba genotype isolated from samples including T2, T4, T5, T12, and T15. N. australiensis and N. lovaniensis were also identified by molecular biology techniques. Furthermore, we found that both Acanthamoeba and Naegleria can be cultured by PYG in 30° C, but not all free-living amoebae can be isolated and enriched by using storage-cultivation method. Because of the widespread presence of Acanthamoeba and Naegleria in aquatic environments, the water quality and safety of aquatic environments should be more conscious in Taiwan and worldwide. Keywords: free-living amoebae; Acanthamoeba; Naegleria; Balamuthia; Hartmannella; PCR

  19. The role of an organic matrix during the formation of siliceous scales in the heliozoon Actinophrys sol (actinophryida, protista).

    PubMed

    Newman, P J; Patterson, D J

    1993-07-25

    Actinophrys sol is a freshwater heliozoon which has trophic and encysted body forms. During encystment, siliceous scales are laid down in silica deposition vesicles. The scales form one layer of a multi-layered cyst wall. Scale production is described using light microscopy, transmission electron microscopy, scanning electron microscopy, and X-ray microanalysis. Silica is laid down on an organic matrix which is visible prior to silicification and after removal of silica with hydrofluoric acid. Actinophrys sol can be cultured under silica impoverished conditions, with the result that the siliceous plates are absent. The cysts continue to form but are fragile. Silica is not a prerequisite for the processes of encystment and cyst formation. Copyright © 1993 Gustav Fischer Verlag · Stuttgart · Jena · New York. Published by Elsevier GmbH.. All rights reserved.

  20. Bilateral Acanthamoeba ulcer in a user of disposable soft contact lenses: a tragic incident or a consequence of the aggressive policy of soft contact lens trading?

    PubMed

    Sousa, Sidney Júlio de Faria E; Dias, Vanderson Glerian; Marcomini, Luís Antonio Gorla

    2008-01-01

    This is the report of a case of bilateral Acanthamoeba keratitis in a 19-year-old woman who bought a pair of disposable soft contact lenses in a boutique. She wore this same pair of lenses for 3 months daily without the appropriate care. This led to bilateral corneal transplantation with cataract extraction and also trabeculectomy in the right eye. When last seen, both grafts were crystal clear but the visual acuities were far from satisfactory. She also had bilateral secondary glaucoma, barely controlled by topical medication. Actually, the physical features and the wearing time characteristics of the disposable soft contact lenses created unprecedented difficulties to the medical surveillance of their wearers. Without the right assistance they tend to become careless regarding routine cleaning. They also feel free to buy less expensive lenses, to use saline instead of lens solutions, to violate the limits of wearing time and to extend the use over the sleeping period. Additionally, the aggressive marketing and the wide distribution of these lenses increase the chances that economically or educationally unprepared people will acquire them. The question that remains is: Is the present case an accidental event or an example of what is likely to happen in the future if the indiscriminate selling of disposable soft contact lenses continues to evolve?

  1. Acanthamoebicidal activity of periglaucine A and betulinic acid from Pericampylus glaucus (Lam.) Merr. in vitro.

    PubMed

    Mahboob, Tooba; Azlan, Abdul-Majid; Shipton, Fiona Natalia; Boonroumkaew, Patcharaporn; Nor Azman, Nadiah Syafiqah; Sekaran, Shamala Devi; Ithoi, Init; Tan, Tian-Chye; Samudi, Chandramathi; Wiart, Christophe; Nissapatorn, Veeranoot

    2017-12-01

    Acanthamoeba species are pathogenic protozoa which account for amoebic keratitis, conjunctivitis and granulomatous amoebic encephalitis. These amoebae form cysts which resist drugs and more effective acanthamoebicidal agents are needed. Medicinal plants could be useful in improving the current treatment strategies for Acanthamoeba infections. In the present study, we examined the amoebicidal effects of Pericampylus glaucus (Lam.) Merr., a medicinal plant used for the treatment of conjunctivitis in Malaysia. Pathogenic Acanthamoeba triangularis were isolated from environmental water samples and treated with different concentrations of fractions obtained from Pericampylus glaucus (Lam.) Merr. as well as main constituents for 24-72 h. Chlorhexidine was used as a reference drug. Ethanol fraction of stem showed significant (p < 0.05) inhibition of trophozoites survival. Betulinic acid and periglaucine A from this plant at 100 μg/mL inhibited more than 70% survival of both cysts and trophozoites. The calculated therapeutic index for betulinic acid and periglaucine A was 170 and 1.5 for trophozoites stage and 3.75 and 8.5 for cysts stage. The observed amoebicidal efficacies indicate the beneficial aspects of this plant in the treatment of Acanthamoeba infection. Periglaucine A could also be of value for the treatment of Acanthamoeba infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Transcriptome analyses of the Giardia lamblia life cycle

    PubMed Central

    Birkeland, Shanda R.; Preheim, Sarah P.; Davids, Barbara J.; Cipriano, Michael J.; Palm, Daniel; Reiner, David S.; Svärd, Staffan G.; Gillin, Frances D.; McArthur, Andrew G.

    2010-01-01

    We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of Giardia lamblia. PMID:20570699

  3. Raman-spectroscopy-based biosensing for applications in ophthalmology

    NASA Astrophysics Data System (ADS)

    Rusciano, Giulia; Capriglione, Paola; Pesce, Giuseppe; Zito, Gianluigi; Del Prete, Antonio; Cennamo, Giovanni; Sasso, Antonio

    2013-05-01

    Cell-based biosensors rely on the detection and identification of single cells as well as monitoring of changes induced by interaction with drugs and/or toxic agents. Raman spectroscopy is a powerful tool to reach this goal, being non-destructive analytical technique, allowing also measurements of samples in aqueous environment. In addition, micro-Raman measurements do not require preliminary sample preparation (as in fluorescence spectroscopy), show a finger-print spectral response, allow a spatial resolution below typical cell sizes, and are relatively fast (few s or even less). All these properties make micro-Raman technique particularly promising for high-throughput on-line analysis integrated in lab-on-a-chip devices. Herein, we demonstrate some applications of Raman analysis in ophthalmology. In particular, we demonstrate that Raman analysis can provide useful information for the therapeutic treatment of keratitis caused by Acanthamoeba Castellanii (A.), an opportunistic protozoan that is widely distributed in the environment and is known to produce blinding keratitis and fatal encephalitis. In particular, by combining Raman analysis with Principal Component Analysis (PCA), we have demonstrated that is possible to distinguish between live and dead cells, enabling, therefore to establish the effectiveness of therapeutic strategies to vanquish the protozoa. As final step, we have analyzed the presence of biochemical differences in the conjunctival epithelial tissues of patients affected by keratitis with respect to healthy people. As a matter of facts, it is possible to speculate some biochemical alterations of the epithelial tissues, rendering more favorable the binding of the protozoan. The epithelial cells were obtained by impression cytology from eyes of both healthy and keratitis-affected individuals. All the samples were analyzed by Raman spectroscopy within a few hours from cells removal from eyes. The results of this analysis are discussed.

  4. The Rising Dominance of Shigella sonnei: An Intercontinental Shift in the Etiology of Bacillary Dysentery.

    PubMed

    Thompson, Corinne N; Duy, Pham Thanh; Baker, Stephen

    2015-01-01

    Shigellosis is the major global cause of dysentery. Shigella sonnei, which has historically been more commonly isolated in developed countries, is undergoing an unprecedented expansion across industrializing regions in Asia, Latin America, and the Middle East. The precise reasons underpinning the epidemiological distribution of the various Shigella species and this global surge in S. sonnei are unclear but may be due to three major environmental pressures. First, natural passive immunization with the bacterium Plesiomonas shigelloides is hypothesized to protect populations with poor water supplies against S. sonnei. Improving the quality of drinking water supplies would, therefore, result in a reduction in P. shigelloides exposure and a subsequent reduction in environmental immunization against S. sonnei. Secondly, the ubiquitous amoeba species Acanthamoeba castellanii has been shown to phagocytize S. sonnei efficiently and symbiotically, thus allowing the bacteria access to a protected niche in which to withstand chlorination and other harsh environmental conditions in temperate countries. Finally, S. sonnei has emerged from Europe and begun to spread globally only relatively recently. A strong selective pressure from localized antimicrobial use additionally appears to have had a dramatic impact on the evolution of the S. sonnei population. We hypothesize that S. sonnei, which exhibits an exceptional ability to acquire antimicrobial resistance genes from commensal and pathogenic bacteria, has a competitive advantage over S. flexneri, particularly in areas with poorly regulated antimicrobial use. Continuing improvement in the quality of global drinking water supplies alongside the rapid development of antimicrobial resistance predicts the burden and international distribution of S. sonnei will only continue to grow. An effective vaccine against S. sonnei is overdue and may become one of our only weapons against this increasingly dominant and problematic

  5. Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

    PubMed

    Seong, Gi-Sang; Sohn, Hae-Jin; Kang, Heekyoung; Seo, Ga-Eun; Kim, Jong-Hyun; Shin, Ho-Joon

    2017-12-01

    Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Analyses of RNA Polymerase II genes from free-living protists: phylogeny, long branch attraction, and the eukaryotic big bang.

    PubMed

    Dacks, Joel B; Marinets, Alexandra; Ford Doolittle, W; Cavalier-Smith, Thomas; Logsdon, John M

    2002-06-01

    The phylogenetic relationships among major eukaryotic protist lineages are largely uncertain. Two significant obstacles in reconstructing eukaryotic phylogeny are long-branch attraction (LBA) effects and poor taxon sampling of free-living protists. We have obtained and analyzed gene sequences encoding the largest subunit of RNA Polymerase II (RPB1) from Naegleria gruberi (a heterolobosean), Cercomonas ATCC 50319 (a cercozoan), and Ochromonas danica (a heterokont); we have also analyzed the RPB1 gene from the nucleomorph (nm) genome of Guillardia theta (a cryptomonad). Using a variety of phylogenetic methods our analysis shows that RPB1s from Giardia intestinalis and Trichomonas vaginalis are probably subject to intense LBA effects. Thus, the deep branching of these taxa on RPB1 trees is questionable and should not be interpreted as evidence favoring their early divergence. Similar effects are discernable, to a lesser extent, with the Mastigamoeba invertens RPB1 sequence. Upon removal of the outgroup and these problematic sequences, analyses of the remaining RPB1s indicate some resolution among major eukaryotic groups. The most robustly supported higher-level clades are the opisthokonts (animals plus fungi) and the red algae plus the cryptomonad nm-the latter result gives added support to the red algal origin of cryptomonad chloroplasts. Clades comprising Dictyostelium discoideum plus Acanthamoeba castellanii (Amoebozoa) and Ochromonas plus Plasmodium falciparum (chromalveolates) are consistently observed and moderately supported. The clades supported by our RPB1 analyses are congruent with other data, suggesting that bona fide phylogenetic relationships are being resolved. Thus, the RPB1 gene has apparently retained some phylogenetically meaningful signal, making it worthwhile to obtain sequences from more diverse protist taxa. Additional RPB1 data, especially in combination with other genes, should provide further resolution of branching orders among protist

  7. Analysis of the Legionella longbeachae Genome and Transcriptome Uncovers Unique Strategies to Cause Legionnaires' Disease

    PubMed Central

    Rusniok, Christophe; Lomma, Mariella; Dervins-Ravault, Delphine; Newton, Hayley J.; Sansom, Fiona M.; Jarraud, Sophie; Zidane, Nora; Ma, Laurence; Bouchier, Christiane; Etienne, Jerôme; Hartland, Elizabeth L.; Buchrieser, Carmen

    2010-01-01

    Legionella pneumophila and L. longbeachae are two species of a large genus of bacteria that are ubiquitous in nature. L. pneumophila is mainly found in natural and artificial water circuits while L. longbeachae is mainly present in soil. Under the appropriate conditions both species are human pathogens, capable of causing a severe form of pneumonia termed Legionnaires' disease. Here we report the sequencing and analysis of four L. longbeachae genomes, one complete genome sequence of L. longbeachae strain NSW150 serogroup (Sg) 1, and three draft genome sequences another belonging to Sg1 and two to Sg2. The genome organization and gene content of the four L. longbeachae genomes are highly conserved, indicating strong pressure for niche adaptation. Analysis and comparison of L. longbeachae strain NSW150 with L. pneumophila revealed common but also unexpected features specific to this pathogen. The interaction with host cells shows distinct features from L. pneumophila, as L. longbeachae possesses a unique repertoire of putative Dot/Icm type IV secretion system substrates, eukaryotic-like and eukaryotic domain proteins, and encodes additional secretion systems. However, analysis of the ability of a dotA mutant of L. longbeachae NSW150 to replicate in the Acanthamoeba castellanii and in a mouse lung infection model showed that the Dot/Icm type IV secretion system is also essential for the virulence of L. longbeachae. In contrast to L. pneumophila, L. longbeachae does not encode flagella, thereby providing a possible explanation for differences in mouse susceptibility to infection between the two pathogens. Furthermore, transcriptome analysis revealed that L. longbeachae has a less pronounced biphasic life cycle as compared to L. pneumophila, and genome analysis and electron microscopy suggested that L. longbeachae is encapsulated. These species-specific differences may account for the different environmental niches and disease epidemiology of these two Legionella

  8. Protist predation can select for bacteria with lowered susceptibility to infection by lytic phages.

    PubMed

    Örmälä-Odegrip, Anni-Maria; Ojala, Ville; Hiltunen, Teppo; Zhang, Ji; Bamford, Jaana K H; Laakso, Jouni

    2015-05-07

    Consumer-resource interactions constitute one of the most common types of interspecific antagonistic interaction. In natural communities, complex species interactions are likely to affect the outcomes of reciprocal co-evolution between consumers and their resource species. Individuals face multiple enemies simultaneously, and consequently they need to adapt to several different types of enemy pressures. In this study, we assessed how protist predation affects the susceptibility of bacterial populations to infection by viral parasites, and whether there is an associated cost of defence on the competitive ability of the bacteria. As a study system we used Serratia marcescens and its lytic bacteriophage, along with two bacteriovorous protists with distinct feeding modes: Tetrahymena thermophila (particle feeder) and Acanthamoeba castellanii (surface feeder). The results were further confirmed with another study system with Pseudomonas and Tetrahymena thermophila. We found that selection by protist predators lowered the susceptibility to infections by lytic phages in Serratia and Pseudomonas. In Serratia, concurrent selection by phages and protists led to lowered susceptibility to phage infections and this effect was independent from whether the bacteria shared a co-evolutionary history with the phage population or not. Bacteria that had evolved with phages were overall more susceptible to phage infection (compared to bacteria with history with multiple enemies) but they were less vulnerable to the phages they had co-evolved with than ancestral phages. Selection by bacterial enemies was costly in general and was seen as a lowered fitness in absence of phages, measured as a biomass yield. Our results show the significance of multiple species interactions on pairwise consumer-resource interaction, and suggest potential overlap in defending against predatory and parasitic enemies in microbial consumer-resource communities. Ultimately, our results could have larger scale

  9. Growth curves and morphology of three Treponema subtypes isolated from digital dermatitis in cattle.

    PubMed

    Döpfer, D; Anklam, K; Mikheil, D; Ladell, P

    2012-09-01

    Digital dermatitis (DD) is an infectious claw disease of cattle that causes painful lesions, principally along the coronary band of the claws. In the US alone, the estimated economic impact of DD is estimated to be $190 million. The etiology of DD remains unclear and there is no reliable laboratory test, so DD is most often diagnosed clinically. Spirochetal bacteria of the genera Treponema have been implicated in DD infections following their isolation using culture techniques, serological detection of bovine antibodies against treponemes, and amplification of treponemal 16s DNA sequences by PCR. During in vitro growth of spirochetes and treponemes isolated from DD, morphological changes have been observed indicating the presence of a spiral form and an encysted form. It is not known why encysted forms appear or what role they have in the progression of DD. The current study established growth curves for three subtypes of treponemes, Treponema denticola-like, Treponema phagedenis-like, and Treponema medium-like, while photographically monitoring changes in morphology. In addition to observing spiral and encysted forms, two intermediate forms were also observed. These appeared as either spiral forms with spherical bodies or as enveloped clusters of granules. The observation of encysted forms adds further support to the theory that treponemes causing recurrent infections deep in bovine skin have mechanisms to facilitate persistence and the chronic character of DD. Published by Elsevier Ltd.

  10. Hartmannella vermiformis inhibition of Legionella pneumophila cultivability.

    PubMed

    Buse, Helen Y; Donohue, Maura J; Ashbolt, Nicholas J

    2013-10-01

    Hartmannella vermiformis and Acanthamoeba polyphaga are frequently isolated from drinking water and permissive to Legionella pneumophila parasitization. In this study, extracellular factor(s) produced by H. vermiformis and A. polyphaga were assessed for their effects on cultivability of L. pneumophila. Page's amoeba saline (PAS) was used as an encystment medium for H. vermiformis and A. polyphaga monolayers, and the culture supernatants (HvS and ApS, respectively) were assessed against L. pneumophila growth. Compared to PAS and ApS, HvS significantly inhibited L. pneumophila strain Philadelphia-1 (Ph-1) cultivability by 3 log(10) colony forming unit (CFU) mL(-1) after 3 days of exposure compared to <0.5 log(10) CFU mL(-1) reduction of strain Lp02 (P < 0.001). Flow cytometric analysis revealed changes in the percentage and cultivability of three bacterial subpopulations: intact/slightly damaged membrane (ISM), undefined membrane status (UD), and mixed type (MT). After 3 days of HvS exposure, the MT subpopulation decreased significantly (31.6 vs 67.2 %, respectively, P < 0.001), while the ISM and UD subpopulations increased (+26.7 and +6.9 %, respectively) with the ISM subpopulation appearing as viable but nonculturable (VBNC) cells. HvS was separated into two fractions based on molecular weight, with more than 99 % of the L. pneumophila inhibition arising from the <5 kDa fraction (P < 0.001). Liquid chromatography indicated the inhibitory molecule(s) are likely polar and elute from a Novapak C18 column between 6 and 15 min. These results demonstrate that H. vermiformis is capable of extracellular modulation of L. pneumophila cultivability and probably promote the VBNC state for this bacterium.

  11. A New Family of Giardial Cysteine-Rich Non-VSP Protein Genes and a Novel Cyst Protein

    PubMed Central

    Birkeland, Shanda R.; Preheim, Sarah P.; Cipriano, Michael J.; McArthur, Andrew G.; Gillin, Frances D.

    2006-01-01

    Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, “HCNCp” (High Cysteine Non-variant Cyst protein), that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs) that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many “CxxC” tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple “CxC” motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp C-terminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp) containing ≥20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents, vaccine

  12. Migration and site selection of Ornithodiplostomum ptychocheilus (Trematoda: Digenea) metacercariae in the brain of fathead minnows ( Pimephales promelas).

    PubMed

    Matisz, Chelsea E; Goater, Cameron P; Bray, Douglas

    2010-04-01

    The migration of subadult parasites to preferred sites within final hosts is well characterized. In contrast, the migration of larval stages of trematodes to specific sites within their second intermediate hosts is poorly understood. We used a serial necropsy approach to characterize the migration of Ornithodiplostomum ptychocheilus diplostomules from the point of cercarial penetration, to encystment within the outermost tissues of the brain of fathead minnows. Diplostomules utilized peripheral nerves to access the central nerve cord, or they used specific cranial nerves to directly access the brain. Within 3 h of exposure to cercariae, 46% of all diplostomules were observed within the medulla of the brain. Diplostomules subsequently utilized specific neural tracts to reach lateral regions of the outermost tissue layer of the optic lobes, the stratum marginale. Diplostomules remained in this layer during their 4-week growth phase, then shifted site to the adjacent meninges for encystment. Characterization of a habitat shift for developing versus encysted metacercariae helps explain the results of previous ecological studies that document transient changes in the effects of metacercariae on the surivival, behaviour, and anti-parasite defences of infected fish.

  13. Expansion of the molecular and morphological diversity of Acanthamoebidae (Centramoebida, Amoebozoa) and identification of a novel life cycle type within the group.

    PubMed

    Tice, Alexander K; Shadwick, Lora L; Fiore-Donno, Anna Maria; Geisen, Stefan; Kang, Seungho; Schuler, Gabriel A; Spiegel, Frederick W; Wilkinson, Katherine A; Bonkowski, Michael; Dumack, Kenneth; Lahr, Daniel J G; Voelcker, Eckhard; Clauß, Steffen; Zhang, Junling; Brown, Matthew W

    2016-12-28

    Acanthamoebidae is a "family" level amoebozoan group composed of the genera Acanthamoeba, Protacanthamoeba, and very recently Luapeleamoeba. This clade of amoebozoans has received considerable attention from the broader scientific community as Acanthamoeba spp. represent both model organisms and human pathogens. While the classical composition of the group (Acanthamoeba + Protacanthamoeba) has been well accepted due to the morphological and ultrastructural similarities of its members, the Acanthamoebidae has never been highly statistically supported in single gene phylogenetic reconstructions of Amoebozoa either by maximum likelihood (ML) or Bayesian analyses. Here we show using a phylogenomic approach that the Acanthamoebidae is a fully supported monophyletic group within Amoebozoa with both ML and Bayesian analyses. We also expand the known range of morphological and life cycle diversity found in the Acanthamoebidae by demonstrating that the amoebozoans "Protostelium" arachisporum, Dracoamoeba jormungandri n. g. n. sp., and Vacuolamoeba acanthoformis n.g. n.sp., belong within the group. We also found that "Protostelium" pyriformis is clearly a species of Acanthamoeba making it the first reported sporocarpic member of the genus, that is, an amoeba that individually forms a walled, dormant propagule elevated by a non-cellular stalk. Our phylogenetic analyses recover a fully supported Acanthamoebidae composed of five genera. Two of these genera (Acanthamoeba and Luapeleameoba) have members that are sporocarpic. Our results provide high statistical support for an Acanthamoebidae that is composed of five distinct genera. This study increases the known morphological diversity of this group and shows that species of Acanthamoeba can include spore-bearing stages. This further illustrates the widespread nature of spore-bearing stages across the tree of Amoebozoa. This article was reviewed by Drs. Eugene Koonin, Purificacion Lopez-Garcia and Sandra Baldauf. Sandra

  14. Effect of low concentrations of benzalkonium chloride on acanthamoebal survival and its potential impact on empirical therapy of infectious keratitis.

    PubMed

    Tu, Elmer Y; Shoff, Megan E; Gao, Weihua; Joslin, Charlotte E

    2013-05-01

    The significant antiacanthamoebal effect of benzalkonium chloride, at or below concentrations used for preservation of common ophthalmic preparations, should be understood both when choosing empiric antibiotic therapy for infectious keratitis and when assessing the persistent rise in Acanthamoeba cases in the United States since 2003. To characterize the antiacanthamoebal efficacy of low concentrations of benzalkonium chloride (BAK) for drug preservation and therapeutic effect against Acanthamoeba. Experimental study with a review of the literature. Laboratory. A concentration of 10(4) trophozoites of 3 well-characterized clinical strains of Acanthamoeba were exposed at 0.5, 2.0, 3.5, 5.0, and 6.5 hours to BAK (0.001%, 0.002%, and 0.003%), moxifloxacin hydrochloride (0.5%), and moxifloxacin (0.5%) + BAK (0.001% and 0.003%) with hydrogen peroxide (3%) and amoeba saline controls. Amoeba survival was calculated using the most probable number method recorded as log kill values. The relationship of BAK concentration and exposure time as well as the relative effect of BAK and moxifloxacin on acanthamoebal survival were analyzed. Amoebicidal activity of BAK is both time dependent and concentration dependent in pooled and strain-stratified analyses (P < .001). Moxifloxacin demonstrated no significant independent inhibitory effect or additive effect to BAK efficacy on acanthamoebal survival. The profound antiacanthamoebal effect of BAK, 0.003%, was similar to that of hydrogen peroxide for certain strains. Low concentrations of BAK, previously demonstrated to concentrate and persist in ocular surface epithelium, exhibit significant antiacanthamoebal activity in vitro at or below concentrations found in commercially available ophthalmic anti-infectives. The unexplained persistence of the Acanthamoeba keratitis outbreak in the United States, clusters abroad, and clinical studies reporting resolution or modification of Acanthamoeba keratitis without specific antiacanthamoebal

  15. Morphologic and Genomic Analyses of New Isolates Reveal a Second Lineage of Cedratviruses.

    PubMed

    Rodrigues, Rodrigo Araújo Lima; Andreani, Julien; Andrade, Ana Cláudia Dos Santos Pereira; Machado, Talita Bastos; Abdi, Souhila; Levasseur, Anthony; Abrahão, Jônatas Santos; La Scola, Bernard

    2018-07-01

    Giant viruses have been isolated and characterized in different environments, expanding our knowledge about the biology of these unique microorganisms. In the last 2 years, a new group was discovered, the cedratviruses, currently composed of only two isolates and members of a putative new family, "Pithoviridae," along with previously known pithoviruses. Here we report the isolation and biological and genomic characterization of two novel cedratviruses isolated from samples collected in France and Brazil. Both viruses were isolated using Acanthamoeba castellanii as a host cell and exhibit ovoid particles with corks at either extremity of the particle. Curiously, the Brazilian cedratvirus is ∼20% smaller and presents a shorter genome of 460,038 bp, coding for fewer proteins than other cedratviruses. In addition, it has a completely asyntenic genome and presents a lower amino acid identity of orthologous genes (∼73%). Pangenome analysis comprising the four cedratviruses revealed an increase in the pangenome concomitant with a decrease in the core genome with the addition of the two novel viruses. Finally, phylogenetic analyses clustered the Brazilian virus in a separate branch within the group of cedratviruses, while the French isolate is closer to the previously reported Cedratvirus lausannensis Taking all together, we propose the existence of a second lineage of this emerging viral genus and provide new insights into the biodiversity and ubiquity of these giant viruses. IMPORTANCE Various giant viruses have been described in recent years, revealing a unique part of the virosphere. A new group among the giant viruses has recently been described, the cedratviruses, which is currently composed of only two isolates. In this paper, we describe two novel cedratviruses isolated from French and Brazilian samples. Biological and genomic analyses showed viruses with different particle sizes, genome lengths, and architecture, revealing the existence of a second lineage of

  16. DNA transposons have colonized the genome of the giant virus Pandoravirus salinus.

    PubMed

    Sun, Cheng; Feschotte, Cédric; Wu, Zhiqiang; Mueller, Rachel Lockridge

    2015-06-12

    Transposable elements are mobile DNA sequences that are widely distributed in prokaryotic and eukaryotic genomes, where they represent a major force in genome evolution. However, transposable elements have rarely been documented in viruses, and their contribution to viral genome evolution remains largely unexplored. Pandoraviruses are recently described DNA viruses with genome sizes that exceed those of some prokaryotes, rivaling parasitic eukaryotes. These large genomes appear to include substantial noncoding intergenic spaces, which provide potential locations for transposable element insertions. However, no mobile genetic elements have yet been reported in pandoravirus genomes. Here, we report a family of miniature inverted-repeat transposable elements (MITEs) in the Pandoravirus salinus genome, representing the first description of a virus populated with a canonical transposable element family that proliferated by transposition within the viral genome. The MITE family, which we name Submariner, includes 30 copies with all the hallmarks of MITEs: short length, terminal inverted repeats, TA target site duplication, and no coding capacity. Submariner elements show signs of transposition and are undetectable in the genome of Pandoravirus dulcis, the closest known relative Pandoravirus salinus. We identified a DNA transposon related to Submariner in the genome of Acanthamoeba castellanii, a species thought to host pandoraviruses, which contains remnants of coding sequence for a Tc1/mariner transposase. These observations suggest that the Submariner MITEs of P. salinus belong to the widespread Tc1/mariner superfamily and may have been mobilized by an amoebozoan host. Ten of the 30 MITEs in the P. salinus genome are located within coding regions of predicted genes, while others are close to genes, suggesting that these transposons may have contributed to viral genetic novelty. Our discovery highlights the remarkable ability of DNA transposons to colonize and shape

  17. Identification of CpxR as a positive regulator of icm and dot virulence genes of Legionella pneumophila.

    PubMed

    Gal-Mor, Ohad; Segal, Gil

    2003-08-01

    To date, 24 Legionella pneumophila genes (icm and dot genes) have been shown to be required for intercellular growth and host cell killing. A previous report indicated that the regulation of these genes is complicated and probably involves several regulatory proteins. In this study, a genetic screen performed in Escherichia coli identified the CpxR response regulator as an activator of the L. pneumophila icmR gene. Construction of an L. pneumophila cpxR insertion mutant showed that the expression of icmR is regulated by CpxR. In addition, a conserved CpxR binding site (GTAAA) was identified in the icmR regulatory region and L. pneumophila His-tagged CpxR protein was shown to bind to the icmR regulatory region using a mobility shift assay. Besides its dramatic effect on the icmR level of expression, the CpxR regulator was also found to affect the expression of the icmV-dotA and icmW-icmX operons, but to a lesser extent. The role of CpxA, the cognate sensor kinase of CpxR, was also examined and its effect on the icmR level of expression was found to be less pronounced than the effect of CpxR. The RpoE sigma factor, which was shown to coregulate genes together with CpxR, was examined as well, but it did not influence icm and dot gene expression. In addition, when the cpxR mutant strain, in which the expression of the icmR gene was dramatically reduced, and the cpxA and rpoE mutant strains were examined for their ability to grow inside Acanthamoeba castellanii and HL-60-derived human macrophages, no intracellular growth defect was observed. This study presents the first evidence for a direct regulator (CpxR) of an icm-dot virulence gene (icmR). The CpxR regulator together with other regulatory factors probably concerts with the expression of icm and dot genes to result in successful infection.

  18. Sel1 repeat protein LpnE is a Legionella pneumophila virulence determinant that influences vacuolar trafficking.

    PubMed

    Newton, Hayley J; Sansom, Fiona M; Dao, Jenny; McAlister, Adrian D; Sloan, Joan; Cianciotto, Nicholas P; Hartland, Elizabeth L

    2007-12-01

    The environmental pathogen Legionella pneumophila possesses five proteins with Sel1 repeats (SLRs) from the tetratricopeptide repeat protein family. Three of these proteins, LpnE, EnhC, and LidL, have been implicated in the ability of L. pneumophila to efficiently establish infection and/or manipulate host cell trafficking events. Previously, we showed that LpnE is important for L. pneumophila entry into macrophages and epithelial cells. In further virulence studies here, we show that LpnE is also required for efficient infection of Acanthamoeba castellanii by L. pneumophila and for replication of L. pneumophila in the lungs of A/J mice. In addition, we found that the role of LpnE in host cell invasion is dependent on the eight SLR regions of the protein. A truncated form of LpnE lacking the two C-terminal SLR domains was unable to complement the invasion defect of an lpnE mutant of L. pneumophila 130b in both the A549 and THP-1 cell lines. The lpnE mutant displayed impaired avoidance of LAMP-1 association, suggesting that LpnE influenced trafficking of the L. pneumophila vacuole, similar to the case for EnhC and LidL. We also found that LpnE was present in L. pneumophila culture supernatants and that its export was independent of both the Lsp type II secretion system and the Dot/Icm type IV secretion system. The fact that LpnE was exported suggested that the protein may interact with a eukaryotic protein. Using LpnE as bait, we screened a HeLa cell cDNA library for interacting partners, using the yeast two-hybrid system. Examination of the protein-protein interaction between LpnE and a eukaryotic protein, obscurin-like protein 1, suggested that LpnE can interact with eukaryotic proteins containing immunoglobulin-like folds via the SLR regions. This investigation has further characterized the contribution of LpnE to L. pneumophila virulence and, more specifically, the importance of the SLR regions to LpnE function.

  19. The Hidden Sexuality of Alexandrium Minutum: An Example of Overlooked Sex in Dinoflagellates.

    PubMed

    Figueroa, Rosa I; Dapena, Carlos; Bravo, Isabel; Cuadrado, Angeles

    2015-01-01

    Dinoflagellates are haploid eukaryotic microalgae in which rapid proliferation causes dense blooms, with harmful health and economic effects to humans. The proliferation mode is mainly asexual, as the sexual cycle is believed to be rare and restricted to stressful environmental conditions. However, sexuality is key to explaining the recurrence of many dinoflagellate blooms because in many species the fate of the planktonic zygotes (planozygotes) is the formation of resistant cysts in the seabed (encystment). Nevertheless, recent research has shown that individually isolated planozygotes in the lab can enter other routes besides encystment, a behavior of which the relevance has not been explored at the population level. In this study, using imaging flow cytometry, cell sorting, and Fluorescence In Situ Hybridization (FISH), we followed DNA content and nuclear changes in a population of the toxic dinoflagellate Alexandrium minutum that was induced to encystment. Our results first show that planozygotes behave like a population with an "encystment-independent" division cycle, which is light-controlled and follows the same Light:Dark (L:D) pattern as the cycle governing the haploid mitosis. Resting cyst formation was the fate of just a small fraction of the planozygotes formed and was restricted to a period of strongly limited nutrient conditions. The diploid-haploid turnover between L:D cycles was consistent with two-step meiosis. However, the diel and morphological division pattern of the planozygote division also suggests mitosis, which would imply that this species is not haplontic, as previously considered, but biphasic, because individuals could undergo mitotic divisions in both the sexual (diploid) and the asexual (haploid) phases. We also report incomplete genome duplication processes. Our work calls for a reconsideration of the dogma of rare sex in dinoflagellates.

  20. The Hidden Sexuality of Alexandrium Minutum: An Example of Overlooked Sex in Dinoflagellates

    PubMed Central

    Figueroa, Rosa I.; Dapena, Carlos; Bravo, Isabel; Cuadrado, Angeles

    2015-01-01

    Dinoflagellates are haploid eukaryotic microalgae in which rapid proliferation causes dense blooms, with harmful health and economic effects to humans. The proliferation mode is mainly asexual, as the sexual cycle is believed to be rare and restricted to stressful environmental conditions. However, sexuality is key to explaining the recurrence of many dinoflagellate blooms because in many species the fate of the planktonic zygotes (planozygotes) is the formation of resistant cysts in the seabed (encystment). Nevertheless, recent research has shown that individually isolated planozygotes in the lab can enter other routes besides encystment, a behavior of which the relevance has not been explored at the population level. In this study, using imaging flow cytometry, cell sorting, and Fluorescence In Situ Hybridization (FISH), we followed DNA content and nuclear changes in a population of the toxic dinoflagellate Alexandrium minutum that was induced to encystment. Our results first show that planozygotes behave like a population with an “encystment-independent” division cycle, which is light-controlled and follows the same Light:Dark (L:D) pattern as the cycle governing the haploid mitosis. Resting cyst formation was the fate of just a small fraction of the planozygotes formed and was restricted to a period of strongly limited nutrient conditions. The diploid-haploid turnover between L:D cycles was consistent with two-step meiosis. However, the diel and morphological division pattern of the planozygote division also suggests mitosis, which would imply that this species is not haplontic, as previously considered, but biphasic, because individuals could undergo mitotic divisions in both the sexual (diploid) and the asexual (haploid) phases. We also report incomplete genome duplication processes. Our work calls for a reconsideration of the dogma of rare sex in dinoflagellates. PMID:26599692

  1. Primary amebic meningoencephalitis due to Naegleria fowleri in a South American tapir.

    PubMed

    Lozano-Alarcón, F; Bradley, G A; Houser, B S; Visvesvara, G S

    1997-05-01

    Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are known to cause fatal central nervous system (CNS) disease in human beings. N. fowleri causes acute, fulminating primary amebic meningoencephalitis (PAM), which generally leads to death within 10 days. Acanthamoeba spp. and B. mandrillaris cause chronic granulomatous amebic encephalitis, which may last for 8 weeks. Acanthamoeba spp. and B. mandrillaris also cause CNS disease in animals. N. fowleri, however, has been described only in human beings. This report is the first of PAM in an animal, a South American tapir. Dry cough, lethargy, and coma developed in the animal, and its condition progressed to death. At necropsy, lesions were seen in the cerebrum, cerebellum, and lungs. The CNS had severe, suppurative meningoencephalitis with many neutrophils, fibrin, plasma cells, and amebas. Amebas were 6.5 microns to 9 microns in diameter and had a nucleus containing a large nucleolus. Amebas in the sections reacted with a monoclonal antibody specific for N. fowleri in the immunofluorescent assay and appeared bright green.

  2. Acanthamoeba Keratitis FAQs

    MedlinePlus

    ... improperly (such as using tap water or homemade solutions to clean the lenses) Swimming, using a hot ... manufacturer’s guidelines. Never reuse or top off old solution. Use fresh cleaning or disinfecting solution each time ...

  3. Intraocular cysts of toxoplasma gondii in patients with necrotizing retinitis following periocular/intraocular triamcinolone injection.

    PubMed

    Nijhawan, Raje; Bansal, Reema; Gupta, Nalini; Beke, Nikhil; Kulkarni, Pandurang; Gupta, Amod

    2013-10-01

    To report the detection of Toxoplasma gondii cysts in intraocular aspirates of patients with necrotizing retinitis following periocular/intraocular corticosteroid injection. Case report. Two patients (2 eyes) with widespread necrotizing retinitis in a steroid-exposed eye posed a diagnostic challenge and underwent pars plana vitrectomy (PPV). Intraocular samples (vitreous fluid, retinal tissue, and subretinal aspirate in case 1, and vitreous fluid in case 2) were subjected to cytological examination. The subretinal aspirate (case 1) revealed encysted bradyzoites of Toxoplasma gondii. Vitreous fluid (case 2) tested positive for anti-toxoplasma antibodies and the smear showed encysted forms of Toxoplasma gondii on cytology. CONCLUSION. Toxoplasma gondii cysts were detected in eyes with necrotizing retinitis that developed secondary to injudicious use of corticosteroids.

  4. Toxicity of peracetic acid (PAA) to tomonts of IIchthyophthirius multifiliis.

    PubMed

    Meinelt, T; Matzke, S; Stübert, A; Pietrock, M; Wienke, A; Mitchell, A J; Strauss, D L

    2009-09-07

    The free-living infective theront of Ichthyophthirius multifiliis historically has been thought to be the only stage susceptible to treatment. Here we introduce a technique to determine the toxicity of compounds to the newly released tomont, the encysted tomont and the developing tomites within the tomont that emerge as theronts. The toxicity of Wofasteril E400 (40% peracetic acid, PAA) to free-living forms of I. multifiliis was determined shortly after tomonts were physically removed from the surface of the fish and at 2.5 and 24 h after removal. Results indicate that 0.6 to 0.9 mg l(-1) PAA killed 39 to 82% of the newly released tomonts within 48 h when treated immediately. In a second experiment, tomonts were allowed to settle for 2.5 h after sampling from the skin and then treated for 12 h; concentrations > or =0.5 mg l(-1) PAA produced significantly fewer theronts than the controls. In a third experiment, encysted tomonts that were exposed to PAA 24 h after sampling from the skin and treated for 2 or 4 h produced a variable amount of theronts, but the concentrations tested (0.5 to 3.0 mg l(-1)) did not halt theront production. This research demonstrates that encysted I. multifiliis are less susceptible to chemical treatments.

  5. Cloning and expression of chitinases of Entamoebae.

    PubMed

    de la Vega, H; Specht, C A; Semino, C E; Robbins, P W; Eichinger, D; Caplivski, D; Ghosh, S; Samuelson, J

    1997-04-01

    Entamoeba histolytica (Eh) and Entamoeba dispar (Ed) are protozoan parasites that infect hundreds of millions of persons. In the colonic lumen, amebae form chitin-walled cysts, the infectious stage of the parasite. Entamoeba invadens (Ei), which infects reptiles and is a model for amebic encystation, produces chitin synthase and chitinase during encystation. Ei cysts formation is blocked by the chitinase-inhibitor allosamidin. Here molecular cloning techniques were used to identify homologous genes of Eh, Ed, and Ei that encode chitinases (EC 3.2.1.14). The Eh gene (Eh cht1) predicts a 507-amino acid (aa) enzyme, which has 93 and 74% positional identities with Ed and Ei chitinases, respectively. The Entamoeba chitinases have signal sequences, followed by acidic and hydrophilic sequences composed of multiple tandemly arranged 7-aa repeats (Eh and Ed) or repeats varying in length (Ei). The aa compositions of the chitinase repeats are similar to those of the repeats of the Eh and Ed Ser-rich proteins. The COOH-terminus of each chitinase has a catalytic domain, which resembles those of Brugia malayi (33% positional identity) and Manduca sexta (29%). Recombinant entamoeba chitinases are precipitated by chitin and show chitinase activity with chitooligosacharide substrates. Consistent with previous biochemical data, chitinase mRNAs are absent in Ei trophozoites and accumulate to maximal levels in Ei encysting for 48 h.

  6. Echinostoma trivolvis (Digenea: Echinostomatidae) second intermediate host preference matches host suitability.

    PubMed

    Wojdak, Jeremy M; Clay, Letitia; Moore, Sadé; Williams, Taylore; Belden, Lisa K

    2013-02-01

    Many trematodes infect a single mollusk species as their first intermediate host, and then infect a variety of second intermediate host species. Determining the factors that shape host specificity is an important step towards understanding trematode infection dynamics. Toward this end, we studied two pond snails (Physa gyrina and Helisoma trivolvis) that can be infected as second intermediate hosts by the trematode Echinostoma trivolvis lineage a (ETa). We performed laboratory preference trials with ETa cercariae in the presence of both snail species and also characterized host suitability by quantifying encystment and excystment success for each host species alone. We tested the prediction that trematodes might preferentially infect species other than their obligate first intermediate host (in this case, H. trivolvis) as second intermediate hosts to avoid potentially greater host mortality associated with residing in first intermediate hosts. In our experiments, ETa had roughly equivalent encystment success in Helisoma and Physa snails, but greater excystment success in Physa, when offered each species in isolation. Also, the presence of the symbiotic oligochaete Chaetogaster limnaei in a subset of Helisoma snails reduced encystment success in those individuals. When both hosts were present, we found dramatically reduced infection prevalence and intensity in Helisoma-ETa cercariae strongly preferred Physa. Thus, the presence of either an alternative host, or a predator of free-living parasites, offered protection for Helisoma snails from E. trivolvis lineage a infection.

  7. Isolation and identification of pathogenic free-living amoeba from surface and tap water of Shiraz City using morphological and molecular methods.

    PubMed

    Armand, B; Motazedian, M H; Asgari, Q

    2016-01-01

    Free-living amoebae (FLA) are the most abundant and widely distributed protozoa in the environment. An investigation was conducted to determine the presence of free-living amoebae (FLA), Acanthamoeba and Vermamoeba in waterfronts of parks and squares and tap water of Shiraz City, Iran. FLA are considered pathogenic for human. These ubiquitous organisms have been isolated from different environments such as water, soil, and air. Eighty-two water samples were collected from different places of Shiraz City during the summer of 2013. All samples were processed in Dept. of Parasitology and Mycology, Shiraz University of Medical Sciences, Fars, Iran. Samples were screened for FLA and identified by morphological characters in the cultures, PCR amplification targeting specific genes for each genus and sequencing determined frequent species and genotypes base on NCBI database. Overall, 48 samples were positive for Acanthamoeba and Vermamoeba in non-nutrient agar culture based on morphological characteristics. The PCR examination was done successfully. Sequencing results were revealed T4 (62.96 %) genotypes as the most common genotype of Acanthamoeba in the Shiraz water sources. In addition, T5 (33.33 %) and T15 (3.71 %) were isolated from water supplies. Vermamoeba vermiformis was known the dominant species from this genus. The high frequency of Acanthamoeba spp. and Vermamoeba in different environmental water sources of Shiraz is an alert for the public health related to water sources. The result highlights a need for taking more attention to water supplies in order to prevent illnesses related to free-living amoebae.

  8. Morphological and molecular identification of free living amoeba isolated from hospital water in Tunisia.

    PubMed

    Trabelsi, Houaida; Dendana, F; Neji, S; Sellami, H; Cheikhrouhou, F; Makni, F; Ayadi, A

    2016-01-01

    Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections. The aim of our study was to detect FLA and Acanthamoeba spp. in hospital water circuits. Eighty-four water samples were collected over a period of 4 months (September-December 2011) from different wards of the Sfax University Hospital (surgical services, intensive care unit, operating theater, and water storage tanks). FLA were detected in 53.5 % of samples as follows: surgical services (80 %), operating theater and surgical intensive care unit (13.3 %), medical intensive care unit (0 %), water storage tanks (6.6 %). The predominant morphotype was the acanthopodial (89 %). The others morphotypes were as follows: monopodial (40 %), dactylopodial (22 %), rugosa (62 %), eruptive (24 %), fan shaped (18 %), and polypodial (18 %). Acanthamoeba was found in 40 samples (47.6 %). 64.2 % of isolates were identified as Acanthamoeba spp. by PCR, using primers to amplify a region of 18S rDNA which showed variation in the product length. Sequence analysis of five PCR products identified Acanthamoeba sp. These isolates belong to T4, T10, and T11 genotypes, and to our knowledge this is the first report of the T10 and T11 genotype in Tunisia.The occurrence of potentially pathogenic FLA in the hospital environment may represent a health risk for patients, since these organisms can cause severe opportunistic illness and also can harbor pathogenic agents. Thus, increased awareness regarding these parasites and recognition of their importance, particularly in immunocompromised patients is crucial.

  9. Parasitic amoebae found in water bodies of Ukraine.

    PubMed

    Patsyuk, Marina

    2017-12-01

    Two parasitic amoebian species are found in mollusks of the water bodies of Ukraine. Vahlkampfia sp. is found in hepatopankreas of Unio conus Spengler, 1793, and Acanthamoeba sp. is observed in mantle cavity of Viviparus viviparus Linnaeus, 1758. For these protist species, the mollusks are shown to be intermediate hosts where amoebae feed and reproduce. An experimental infection with Vahlkampfia sp. and Acanthamoeba sp. was not successful, no pathological changes in mollusks were observed. These amoebae are successfully cultured in fresh water and agar medium, hence we can safely consider them free-living. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Differential exposure, duration, and sensitivity of unionoidean bivalve life stages to environmental contaminants

    USGS Publications Warehouse

    Cope, W.G.; Bringolf, R.B.; Buchwalter, D.B.; Newton, T.J.; Ingersoll, C.G.; Wang, N.; Augspurger, T.; Dwyer, F.J.; Barnhart, M.C.; Neves, R.J.; Hammer, E.

    2008-01-01

    Freshwater mussels (superfamily Unionoidea) are in serious global decline and in urgent need of protection and conservation. The declines have been attributed to a wide array of human activities resulting in pollution and water-quality degradation, and habitat destruction and alteration. Linkages among poor water quality, pollutant sources, and mussel decline in rivers and streams have been associated with results of laboratory-based tests of specific pollutants. However, uncertainties remain about the relationship of laboratory data to actual contaminant exposure routes for various mussel species, life stages, and in the habitats occupied during these exposures. We evaluated the pathways of exposure to environmental pollutants for all 4 life stages (free glochidia, encysted glochidia, juveniles, adults) of unionoidean mussels and found that each life stage has both common and unique characteristics that contribute to observed differences in exposure and sensitivity. Free glochidia typically are exposed only briefly (e.g., seconds to days) through surface water, whereas adults sustain exposure over years to decades through surface water, pore water, sediment, and diet. Juveniles live largely burrowed in the sediment for the first 0 to 4 y of life. Thus, sediment, pore water, and diet are the predominant exposure routes for this life stage, but surface water also might contribute to exposure during certain periods and environmental conditions. The obligate parasitic stage (encysted glochidia stage) on a host fish might be exposed from surface water while partially encysted or from toxicants in host-fish tissue while fully encysted. Laboratory methods for testing for acute and chronic exposures in water have advanced, and toxicant-specific information has increased in recent years. However, additional research is needed to understand interactions of life history, habitat, and long-term exposure to contaminants through water, pore water, sediment, and diet so that the

  11. Dendritiform Keratopathy Associated with Exposure to Polyquarternium-1, a Common Ophthalmic Preservative.

    PubMed

    Matoba, Alice Y; Peterson, Jeff R; Wilhelmus, Kirk R

    2016-03-01

    To describe dendritiform keratopathy associated with exposure to polyquaternium-1, a common preservative found in contact lens solutions and tear replacement products. Case series. Sixteen patients who demonstrated dendritiform keratopathy during topical ophthalmic exposure to polyquaternium-1. Records were reviewed of all patients diagnosed with dendritiform keratopathy between 1999 and 2014 who had documented exposure to contact lens care disinfecting solutions or artificial tear solutions containing polyquaternium-1. Patients were excluded who had coexisting potential causes for dendritiform keratopathy, such as prior herpes simplex keratitis, varicella-zoster viral keratitis, the linear form of Thygeson's superficial keratitis, epithelial regeneration line, Acanthamoeba keratitis, mucus plaque keratopathy, medication-related keratopathy, or limbal stem cell deficiency characterized by conjunctivalization of the corneal epithelium. Effect of discontinuation of exposure to polyquaternium-1 on the dendritiform keratopathy. Sixteen patients demonstrated dendritiform keratopathy after exposure to the preservative polyquaternium-1. Thirteen patients had a history of recent exposure to contact lens disinfecting solutions (Opti-Free, Equate) containing polyquaternium-1. Three patients used a tear replacement product (Systane) containing a polyquaternium-1 preservative. Four patients were treated with antiviral medications for presumed herpes simplex keratitis; 4 patients underwent diagnostic testing for Acanthamoeba keratitis. Two additional patients were diagnosed sequentially with herpes simplex keratitis, then Acanthamoeba keratitis before referral. All dendritiform lesions resolved within 2 to 6 weeks after elimination of exposure to polyquaternium-1. Ophthalmic products containing polyquaternium-1 may cause dendritiform keratopathy that may be confused with infections of the superficial cornea, such as herpes simplex virus keratitis or Acanthamoeba keratitis

  12. [Evaluation of chemokines in tears of patients with infectious keratitis].

    PubMed

    Hori, Shinsuke; Shoji, Jun; Inada, Noriko; Sawa, Mitsuru

    2013-02-01

    To investigate the chemokine profile in tears of patients with infectious keratitis. Subjects were 32 eyes of 16 patients with infectious keratitis and 5 eyes of 5 healthy volunteers as a control. The patients with infectious keratitis were classified into two groups of eyes: 10 with bacterial keratitis and 6 with Acanthamoeba keratitis. Tear fluid was obtained from both eyes of the patients with infectious keratitis and from the right eyes of the control subjects using filter paper. Chemokine concentration (unit: Odu/mm2) and its profile in tears was analyzed using an antibody-array. In terms of chemokine profile in the bacterial keratitis group, the expression volume of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in the diseased eyes was significantly higher than in the healthy eyes (p < 0.05). The expression volume of mucosae-associated epithelial chemokines (MECs) in the diseased eyes of the bacterial keratitis group was significantly lower than in the healthy eyes of that group (p < 0.05). In the Acanthamoeba keratitis group, chemokines were not significantly increased in the diseased eyes compared with those in the healthy eyes. However, MCP-1 was increased in tears of the Acanthamoeba keratitis group. Regarding the chemokine ratio, the IL-8/MEC ratio in the diseased eyes of the Pseudomonas keratitis group and the MCP-1/IL-8 in the diseased eyes of the Acanthamoeba keratitis group showed a significantly high level (p < 0.05). We concluded that the analyses of the chemokine profile and chemokine ratio in the tears of infectious keratitis patients is useful as a clinical tear laboratory test to interpret the pathologic condition of infectious keratitis

  13. Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga.

    PubMed

    Bekliz, Meriem; Azza, Said; Seligmann, Hervé; Decloquement, Philippe; Raoult, Didier; La Scola, Bernard

    2018-05-15

    The Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis. IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of

  14. Presence of rotavirus and free-living amoebae in the water supplies of Karachi, Pakistan.

    PubMed

    Yousuf, Farzana Abubakar; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2017-06-01

    Rotavirus and pathogenic free-living amoebae are causative agents of important health problems, especially for developing countries like Pakistan where the population has limited access to clean water supplies. Here, we evaluated the prevalence of rotavirus and free-living amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) in drinking water supplies of Karachi, Pakistan. Six water filtration plants that supply drinking water to the population of Karachi were investigated. Additionally, drinking water samples from households were analyzed for the presence of rotavirus and free-living amoebae. Rotavirus was present in 35% of the water samples collected from water filtration plants; however, domestic tap water samples had a prevalence of only 5%. Out of 20 water samples from filtration plants, 13 (65%) were positive for Acanthamoeba spp., and one (5%) was positive for B. mandrillaris. Out of 20 drinking water samples collected from different areas of Karachi, 35% were positive for Acanthamoeba spp. Rotavirus was detected in 5% of the drinking water samples tested. Overall, these findings showed for the first time the presence of rotavirus, in addition to pathogenic free-living amoebae in drinking water supplies of Karachi that could be an important public health risk for the affected population.

  15. Free-living amoebae isolated in the Central African Republic: epidemiological and molecular aspects.

    PubMed

    Farra, Alain; Bekondi, Claudine; Tricou, Vianney; Mbecko, Jean Robert; Talarmin, Antoine

    2017-01-01

    Among the many species of free-living amoebae infecting humans, only Naegleria fowleri , a few species of Acanthamoeba, Balamuthia mandrillaris recently Sappinia diploïdea and Paravahlkampfia Francina are responsible for human diseases especially deadly encephalitis outside of Acanthamoeba keratitis related. In the Central African Republic (CAR), no studies have previously been conducted about free amoebae and no suspicious cases of encephalitis or amoebic keratitis was reported even though the ecosystem supported the proliferation of these microorganisms. The objective of this study was to identify free-living amoebae present in CAR and to define the molecular characteristic. Bathing sites and cerebrospinal fluid from patients died of bacterial meningitis untagged were explored by culture and PCR and the amplicons were sequenced which allowed to characterize the species found. Only species of the genus Tetramitus, namely T. Entericus, T. waccamawensis and T.sp similar to those already described in the world and not pathogenic for humans were found in bathing sites, the cerebrospinal fluid meanwhile remained negative. Although no pathogen species such as Naegleria fowleri or species of Acanthamoeba have been isolated, this study worth pursuing because this investigation was very limited in space because of the insecurity in the country.

  16. Dinoflagellates: Fossil motile-stage tests from the upper cretaceous of the Northern New Jersey coastal plain

    USGS Publications Warehouse

    May, F.E.

    1976-01-01

    Fossil dinoflagellate tests have been considered to represent encysted, nonmotile stages. The discovery of flagellar porelike structures and probable trichocyst pores in the Upper Cretaceous genus Dinogymnium suggests that motile stage tests are also preserved as acid-resistant, organic-walled microfossils.

  17. The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit.

    PubMed

    Kessler, Aline; Schell, Ursula; Sahr, Tobias; Tiaden, André; Harrison, Christopher; Buchrieser, Carmen; Hilbi, Hubert

    2013-02-01

    Legionella pneumophila is an amoeba-resistant opportunistic pathogen that performs cell-cell communication through the signalling molecule 3-hydroxypentadecane-4-one (LAI-1, Legionella autoinducer-1). The lqs (Legionella quorum sensing) gene cluster encodes the LAI-1 autoinducer synthase LqsA, the cognate sensor kinase LqsS and the response regulator LqsR. Here we show that the Lqs system includes an 'orphan' homologue of LqsS termed LqsT. Compared with wild-type L. pneumophila, strains lacking lqsT or both lqsS and lqsT show increased salt resistance, greatly enhanced natural competence for DNA acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using Acanthamoeba castellanii revealed that ΔlqsT and ΔlqsS-ΔlqsT, as well as ΔlqsA and other lqs mutant strains are impaired for intracellular growth and cannot compete against wild-type bacteria upon co-infection. In contrast to the ΔlqsS strain, ΔlqsT does not produce extracellular filaments. The phenotypes of the ΔlqsS-ΔlqsT strain are partially complemented by either lqsT or lqsS, but are not reversed by overexpression of lqsA, suggesting that LqsT and LqsS are the sole LAI-1-responsive sensor kinases in L. pneumophila. In agreement with the different phenotypes of the ΔlqsT and ΔlqsS strains, lqsT and lqsS are differentially expressed in the post-exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of lqsT, are upregulated in absence of lqsS. Reciprocally regulated genes encode components of a 133 kb genomic 'fitness island' or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the L. pneumophila Lqs system comprising two partially antagonistic LAI-1-responsive sensor kinases, LqsT and LqsS, which regulate distinct pools of genes implicated in pathogen-host cell interactions, competence, expression of a

  18. Filling Knowledge Gaps for Mimivirus Entry, Uncoating, and Morphogenesis

    PubMed Central

    Andrade, Ana Cláudia dos Santos Pereira; Rodrigues, Rodrigo Araújo Lima; Oliveira, Graziele Pereira; Andrade, Kétyllen Reis; Bonjardim, Cláudio Antônio; La Scola, Bernard; Kroon, Erna Geessien

    2017-01-01

    ABSTRACT Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii, causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus. IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we

  19. Filling Knowledge Gaps for Mimivirus Entry, Uncoating, and Morphogenesis.

    PubMed

    Andrade, Ana Cláudia Dos Santos Pereira; Rodrigues, Rodrigo Araújo Lima; Oliveira, Graziele Pereira; Andrade, Kétyllen Reis; Bonjardim, Cláudio Antônio; La Scola, Bernard; Kroon, Erna Geessien; Abrahão, Jônatas Santos

    2017-11-15

    Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii , causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus. IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we conducted a

  20. The neurotropic parasite Toxoplasma gondii increases dopamine metabolism

    USDA-ARS?s Scientific Manuscript database

    The common parasite Toxoplasma gondii induces behavioral alterations in its hosts including phenotypes increasing the likelihood of its transmission in rodents and reports of psychobehavioral alterations in humans. We have found that elevated levels of dopamine are associated with the encysted stage...

  1. Characterization of IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii

    USDA-ARS?s Scientific Manuscript database

    Toxoplasma gondii infects approximately one third of the human population and animals habiting terrestrial and aquatic environments. Its environmentally resistant oocysts are excreted by felids, and the stage encysted in tissues (tissue cysts), are important in the horizontal transmission of T. gon...

  2. Biofumigation for control of pale potato cyst nematodes: activity of brassica leaf extracts and green manures on Globodera pallida in vitro and in soil.

    PubMed

    Lord, James S; Lazzeri, Luca; Atkinson, Howard J; Urwin, Peter E

    2011-07-27

    The effects of brassica green manures on Globodera pallida were assessed in vitro and in soil microcosms. Twelve of 22 brassica accessions significantly inhibited the motility of G. pallida infective juveniles in vitro. Green manures of selected brassicas were then incorporated into soil containing encysted eggs of G. pallida. Their effect on egg viability was estimated by quantifying nematode actin 1 mRNA by RT-qPCR. The leaf glucosinolate profiles of the plants were determined by high-performance liquid chromatography. Three Brassica juncea lines (Nemfix, Fumus, and ISCI99) containing high concentrations of 2-propenyl glucosinolate were the most effective, causing over 95% mortality of encysted eggs of G. pallida in polyethylene-covered soil. The toxic effects of green manures were greater in polyethylene-covered than in open soil. Toxicity in soil correlated with the concentration of isothiocyanate-producing glucosinolate but not total glucosinolate in green manures.

  3. Presence of rotavirus and free-living amoebae in the water supplies of Karachi, Pakistan

    PubMed Central

    Yousuf, Farzana Abubakar; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2017-01-01

    ABSTRACT Rotavirus and pathogenic free-living amoebae are causative agents of important health problems, especially for developing countries like Pakistan where the population has limited access to clean water supplies. Here, we evaluated the prevalence of rotavirus and free-living amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) in drinking water supplies of Karachi, Pakistan. Six water filtration plants that supply drinking water to the population of Karachi were investigated. Additionally, drinking water samples from households were analyzed for the presence of rotavirus and free-living amoebae. Rotavirus was present in 35% of the water samples collected from water filtration plants; however, domestic tap water samples had a prevalence of only 5%. Out of 20 water samples from filtration plants, 13 (65%) were positive for Acanthamoeba spp., and one (5%) was positive for B. mandrillaris. Out of 20 drinking water samples collected from different areas of Karachi, 35% were positive for Acanthamoeba spp. Rotavirus was detected in 5% of the drinking water samples tested. Overall, these findings showed for the first time the presence of rotavirus, in addition to pathogenic free-living amoebae in drinking water supplies of Karachi that could be an important public health risk for the affected population. PMID:28591260

  4. Practical Biology.

    ERIC Educational Resources Information Center

    Journal of Biological Education, 1983

    1983-01-01

    Describes equipment for recording human breathing (stethographs, tambors, spirometers), construction of pooters (pipettes), field exercise on examining epiphyte distribution on tree bark, and vertebrate skeleton models for teaching locomotion. Includes abstract of student research project examining pH on Colpoda encystment and reviews of…

  5. 78 FR 59555 - Endangered and Threatened Wildlife and Plants; Designation of Critical Habitat for the Fluted...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-26

    ... physical and chemical water quality parameters (such as temperature, dissolved oxygen, pH, and conductivity... unknown. High temperatures can reduce dissolved oxygen concentrations in the water, which slows growth... encystment, increase oxygen consumption, reduce the speed in which they orient themselves in the substrate...

  6. The Environmental Evaluation Work Group: FY 1979 Studies of the Winter Navigation Demonstration Program, St. Lawrence River Fisheries Study

    DTIC Science & Technology

    1979-07-31

    examined were infected with black spot, the externally encysted stage of the tremetode ( Uvulifer ambloplitis ). At Chimney Bay and Tibbits Creek, 7...and liver surface from 124 perch revealed that 112 (90.3%) were infected with cestodes, believed to be bass tapeworm (Proteocephalus ambloplitis ) or

  7. Scanning electron microscope observations of brine shrimp larvae from space shuttle experiments

    NASA Technical Reports Server (NTRS)

    DeBell, L.; Paulsen, A.; Spooner, B.

    1992-01-01

    Brine shrimp are encysted as gastrula stage embryos, and may remain dehydrated and encysted for years without compromising their viability. This aspect of brine shrimp biology is desirable for studying development of animals during space shuttle flight, as cysts placed aboard a spacecraft may be rehydrated at the convenience of an astronaut, guaranteeing that subsequent brine shrimp development occurs only on orbit and not on the pad during launch delays. Brine shrimp cysts placed in 5 ml syringes were rehydrated with salt water and hatched during a 9 day space shuttle mission. Subsequent larvae developed to the 8th larval stage in the sealed syringes. We studied the morphogenesis of the brine shrimp larvae and found the larvae from the space shuttle experiments similar in rate of growth and extent of development, to larvae grown in sealed syringes on the ground. Extensive differentiation and development of embryos and larvae can occur in a microgravity environment.

  8. Developmental changes in the adhesive disk during Giardia differentiation.

    PubMed

    Palm, Daniel; Weiland, Malin; McArthur, Andrew G; Winiecka-Krusnell, Jadwiga; Cipriano, Michael J; Birkeland, Shanda R; Pacocha, Sarah E; Davids, Barbara; Gillin, Frances; Linder, Ewert; Svärd, Staffan

    2005-06-01

    Giardia lamblia is a protozoan parasite infecting the upper mammalian small intestine. Infection relies upon the ability of the parasite to attach to the intestine via a unique cytoskeletal organelle, the ventral disk. We determined the composition and structure of the disk throughout the life cycle of the parasite and identified a new disk protein, SALP-1. SALP-1 is an immunodominant protein related to striated fiber-assemblin (SFA). The disk is disassembled during encystation and stored as four fragments in the immobile cyst. Serial Analysis of Gene Expression (SAGE) showed that the mRNA levels of the disk proteins decreased in encystation but two-dimensional protein gels showed that the protein levels were more constant. The parasite emerges without a functional disk but the four disk fragments are quickly reassembled into two new disks on the dividing, early excysting form. Thus, disk proteins are stored within the cyst, ready to be used in the rapid steps of excystation.

  9. Monitoring of Waterborne Parasites in Two Drinking Water Treatment Plants: A Study in Sarawak, Malaysia.

    PubMed

    Richard, Reena Leeba; Ithoi, Init; Abd Majid, Mohamad Azlan; Wan Sulaiman, Wan Yusoff; Tan, Tian Chye; Nissapatorn, Veeranoot; Lim, Yvonne Ai Lian

    2016-06-28

    The occurrence of waterborne parasites coupled with water parameters at various processing sites of two drinking water treatment plants (A and B) and seven distribution system (DS) sites in Sarawak, Malaysia were studied. Ten liters of water underwent immunomagnetic separation (IMS) technique to detect the presence of Giardia and Cryptosporidium (oo)cysts. The remaining supernatant was used to detect other parasites whilst 50 mL of water sample was each used in the detection of free-living amoebae and fecal coliforms. Sampled water was positive for Giardia (32.9%; 28/85), Cryptosporidium (18.8%; 16/85) followed by Spirometra ova-like (25.9%; 22/85), Blastocystis-like (25.9%; 22/85), nematode larvae-like (8.2%; 7/85) and Taenia ova-like (1.2%; 1/85). Meanwhile, 90.2% (55/61) samples were positive for Acanthamoeba and Naegleria via cultivation and of these, 11 isolates were confirmed as Acanthamoeba genotype T3 (5/7) and T4 (2/7) followed by Naegleria sp. (4/11), Naegleria italica (2/11), Naegleria australiensis (1/11), Naegleria angularis (1/11) and Vahlkampfia sp. (3/11). Cryptosporidium, Acanthamoeba and Naegleria were also detected in one of the seven tested DS sites. Only Giardia and Cryptosporidium showed significant correlations with fluoride and fecal coliforms. These results describe the occurrence of waterborne parasites that will assist key stakeholders in mitigating contamination at the specific sites.

  10. Monitoring of Waterborne Parasites in Two Drinking Water Treatment Plants: A Study in Sarawak, Malaysia

    PubMed Central

    Richard, Reena Leeba; Ithoi, Init; Abd Majid, Mohamad Azlan; Wan Sulaiman, Wan Yusoff; Tan, Tian Chye; Nissapatorn, Veeranoot; Lim, Yvonne Ai Lian

    2016-01-01

    The occurrence of waterborne parasites coupled with water parameters at various processing sites of two drinking water treatment plants (A and B) and seven distribution system (DS) sites in Sarawak, Malaysia were studied. Ten liters of water underwent immunomagnetic separation (IMS) technique to detect the presence of Giardia and Cryptosporidium (oo)cysts. The remaining supernatant was used to detect other parasites whilst 50 mL of water sample was each used in the detection of free-living amoebae and fecal coliforms. Sampled water was positive for Giardia (32.9%; 28/85), Cryptosporidium (18.8%; 16/85) followed by Spirometra ova-like (25.9%; 22/85), Blastocystis-like (25.9%; 22/85), nematode larvae-like (8.2%; 7/85) and Taenia ova-like (1.2%; 1/85). Meanwhile, 90.2% (55/61) samples were positive for Acanthamoeba and Naegleria via cultivation and of these, 11 isolates were confirmed as Acanthamoeba genotype T3 (5/7) and T4 (2/7) followed by Naegleria sp. (4/11), Naegleria italica (2/11), Naegleria australiensis (1/11), Naegleria angularis (1/11) and Vahlkampfia sp. (3/11). Cryptosporidium, Acanthamoeba and Naegleria were also detected in one of the seven tested DS sites. Only Giardia and Cryptosporidium showed significant correlations with fluoride and fecal coliforms. These results describe the occurrence of waterborne parasites that will assist key stakeholders in mitigating contamination at the specific sites. PMID:27367710

  11. Chapter 42. Waterborne and Foodborne Parasites

    USDA-ARS?s Scientific Manuscript database

    This chapter identifies the most prominent parasites in North America that are acquired through contaminated food and water including protozoa (Acanthamoeba, Naegleria, Entamoeba, Giardia, Cryptosporidium, Cystoisospora, Cyclospora, Toxoplasma, and Balantidium), nematodes (Trichinella, Angiostrongyl...

  12. Physiologic and systemic acute phase inflammatory responses in young horses repeatedly infected with cyathostomins and Strongylus vulgaris.

    PubMed

    Andersen, U V; Reinemeyer, C R; Toft, N; Olsen, S N; Jacobsen, S; Nielsen, M K

    2014-03-17

    Migrating Strongylus vulgaris and encysted cyathostomin larvae cause a localized inflammatory response in horses. It is unknown whether these larvae elicit a systemic acute phase response (APR), evidenced by changes in serum amyloid A (SAA), haptoglobin (Hp), iron (Fe), albumin, or albumin/globulin (A/G) ratio. In this study, 28 horses were randomly allocated to receive either pyrantel tartrate or a pelleted placebo formulation in their daily feed. Concurrent with treatment, all the horses were administered 5000 pyrantel-susceptible cyathostomin infective larvae once daily, 5 days a week, for 24 weeks. Beginning in the fifth week, the horses also received 25 S. vulgaris larvae once weekly for the remainder of the study. At regular biweekly intervals, fecal samples were collected for quantitative egg counts, and whole blood and serum samples were collected for measurement of packed cell volume, total protein, albumin, globulin, A/G ratio, SAA, Hp, and Fe. On days 161-164, all the horses were euthanatized and necropsied. Samples were collected for enumeration of total luminal worm burdens, encysted cyathostomin larval populations, and migrating S. vulgaris larvae. Concentrations of Hp, Fe, and A/G ratio were associated significantly with strongyle burdens. Only treated male horses had significant increases in serum albumin. Larval S. vulgaris did not associate with Fe, whereas Fe was associated negatively with both total cyathostomin burdens and encysted L4s. The A/G ratios differed significantly between the two treatment groups. Significant differences between groups and individual time points were also observed for Hp and Fe, whereas SAA concentrations remained low throughout the study. In general, this study illustrated that experimental inoculations with S. vulgaris and cyathostomins may be associated with changes in Hp, Fe, and serum proteins, but not with SAA. Overall, these changes suggest that mixed strongyle infections elicit a mild acute phase reaction

  13. Self-mating in the definitive host potentiates clonal outbreaks of the apicomplexan parasites Sarcocystis neurona and Toxoplasma gondii.

    PubMed

    Wendte, Jered M; Miller, Melissa A; Lambourn, Dyanna M; Magargal, Spencer L; Jessup, David A; Grigg, Michael E

    2010-12-23

    Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause devastating disease

  14. Self-Mating in the Definitive Host Potentiates Clonal Outbreaks of the Apicomplexan Parasites Sarcocystis neurona and Toxoplasma gondii

    PubMed Central

    Wendte, Jered M.; Miller, Melissa A.; Lambourn, Dyanna M.; Magargal, Spencer L.; Jessup, David A.; Grigg, Michael E.

    2010-01-01

    Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause devastating disease

  15. Effects of X-ray on the metacestodes of Echinococcus granulosus in vitro.

    PubMed

    Mao, Rui; Wu, Ge; Wang, Hui; Lu, Pengfei; Li, Jun; Li, Haitao; Ainiwaer, Aimudula; Bai, Yiwei; Shu, Mingyang; Bao, Yongxing; Zhang, Wenbao

    2017-09-21

    Radiotherapy may represent an alternative treatment modality for cystic echinococcosis (CE), but there is no adequate evidence for it up to now. In this study, we aim to investigate the parasiticidal effects of X-ray on the metacestodes of Echinococcus granulosus in vitro. Protoscoleces obtained from sheep naturally infected with CE were cultivated in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37 °C in 5% CO 2 . Upon encystation on day 14, the metacestodes were subjected to various intensities of X-ray. Metacestode structures were observed using light microscope and transmission electron microscopy (TEM), and Real-Time PCR was carried out to determine the expression of EgTPX, EgHSP70, EgEPC1 and Caspase-3. On day 14, encystation was noticed in the majority of protoscoleces in the control group. In the X-ray groups, the encystation rate showed significant decrease compared with that of the control group (P < 0.05), especially the groups subjected to a dose of ≥40 Gy (P < 0.01). Light microscope findings indicated the hooklets on the rostellum were deranged in the irradiation group, and malformation was noticed in the suckers in a dose dependent manner. For the TEM findings, the cellular structure of the germinal layer of the cysts was completely interrupted by X-ray on day 7. The expression of EgTPX, EgHSP70, EgEPC1 and Caspase-3 was up-regulated after irradiation, especially at a dose of ≥45Gy (P < 0.05). X-ray showed parasiticidal effects on the metacestodes of E. granulosus. Irradiation triggered increased expression of EgTPX, EgHSP70, EgEPC1 and Caspase-3.

  16. Effect of Common Drinking Water Disinfectants, Chlorine and Heat, on Free Legionella and Amoebae-Associated Legionella.

    PubMed

    Cervero-Aragó, Sílvia; Rodríguez-Martínez, Sarah; Puertas-Bennasar, Antoni; Araujo, Rosa M

    2015-01-01

    Chlorine and thermal treatments are the most commonly used procedures to control and prevent Legionella proliferation in drinking water systems of large buildings. However, cases of legionellosis still occur in facilities with treated water. The purpose of this work was to model the effect of temperature and free chlorine applied in similar exposure conditions as in drinking water systems on five Legionella spp. strains and two amoebal strains of the genera Acanthamoeba. Inactivation models obtained were used to determine the effectiveness of the treatments applied which resulted more effective against Legionella than Acanthamoeba, especially those in cystic stages. Furthermore, to determine the influence of the relationship between L. pneumophila and Acanthamoeba spp. on the treatment effectiveness, inactivation models of the bacteria-associated amoeba were also constructed and compared to the models obtained for the free living bacteria state. The Legionella-amoeba association did not change the inactivation models, but it reduced the effectiveness of the treatments applied. Remarkably, at the lowest free chlorine concentration, 0.5 mg L-1, as well as at the lowest temperatures, 50°C and 55°C, the influence of the Legionella-amoeba associate state was the strongest in reducing the effectiveness of the treatments compared to the free Legionella state. Therefore, the association established between L. pneumophila and amoebae in the water systems indicate an increased health risk in proximal areas of the system (close to the tap) where lower free chlorine concentrations and lower temperatures are commonly observed.

  17. Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries

    PubMed Central

    Abdul Majid, Mohamad Azlan; Mahboob, Tooba; Mong, Brandon G. J.; Jaturas, Narong; Richard, Reena Leeba; Tian-Chye, Tan; Phimphila, Anusorn; Mahaphonh, Panomphanh; Aye, Kyaw Nyein; Aung, Wai Lynn; Chuah, Joon; Ziegler, Alan D.; Yasiri, Atipat; Sawangjaroen, Nongyao; Lim, Yvonne A. L.; Nissapatorn, Veeranoot

    2017-01-01

    Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public. PMID:28212409

  18. Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries.

    PubMed

    Abdul Majid, Mohamad Azlan; Mahboob, Tooba; Mong, Brandon G J; Jaturas, Narong; Richard, Reena Leeba; Tian-Chye, Tan; Phimphila, Anusorn; Mahaphonh, Panomphanh; Aye, Kyaw Nyein; Aung, Wai Lynn; Chuah, Joon; Ziegler, Alan D; Yasiri, Atipat; Sawangjaroen, Nongyao; Lim, Yvonne A L; Nissapatorn, Veeranoot

    2017-01-01

    Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public.

  19. The Danger of Using Tap Water with Contact Lenses

    EPA Pesticide Factsheets

    Acanthamoeba is a microbe that is very common in tap water. It has two forms: the trophozoite and the cyst. These trophozoites and cysts can stick to the surface of your contact lenses and then infect your eye.

  20. First record and description of metacercariae of Curtuteria arguinae n. sp. (Digenea: Echinostomatidae), parasite of cockles Cerastoderma edule (Mollusca: Bivalvia) in Arcachon Bay, France.

    PubMed

    Desclaux, Céline; Russell-Pinto, Fernanda; de Montaudouin, Xavier; Bachelet, Guy

    2006-06-01

    A new Himasthlinae species, Curtuteria arguinae, is described as metacercariae from the cockle Cerastoderma edule (L.), collected at Banc d'Arguin (southwestern France). These metacercariae encysted preferentially in the mantle and also in the foot of cockles. Encysted and chemically excysted metacercariae were studied by light microscopy and scanning electron microscopy, respectively. Excysted metacercariae were elongated and curved ventrally. They bore a 33-spine circumoral collar. Sensory papillae were arranged around the oral sucker and also symmetrically along the ventral surface body, from the collar to the acetabulum. The dorsal and ventral tegument surfaces were densely packed with similar pointed spines. The posterior end of the body was without any spines. Among the Curtuteria species described previously, only Curtuteria haematopodis Smogorjewskaja and Iskova, 1966 had the same number of circumoral collar spines. A 6-yr field survey showed that the cockle population at Banc d'Arguin was subjected to a summer infection of C. arguinae. Curtuteria arguinae phenology of infection is characterized by interannual variability and seasonality (beginning in July-August and maximum in autumn). The first intermediate and final hosts remain unknown.

  1. Population dynamics in echinococcosis and cysticercosis: regulation of Taenia hydatigena and T. ovis in lambs through passively transferred immunity.

    PubMed

    Gemmell, M A; Lawson, J R; Roberts, M G; Griffin, J F

    1990-08-01

    A comparison has been made of the interactions between passively transferred and actively acquired immunity in regulating populations of Taenia hydatigena and T. ovis. When ewes were grazed prior to parturition under a high infection pressure, immunity was transferred to their offspring for up to 8 weeks. A qualitative difference between the species was the destruction of larval T. ovis prior to their establishment ('pre-encystment immunity') and that of T. hydatigena after they had become established ('post-encystment immunity') in the challenged lambs. The major difference in terms of population regulation between the two parasites was that infection occurred with T. hydatigena but not with T. ovis in those lambs reared from birth for 16 weeks under high infection pressure. Passive, like active immunity, is a density-dependent constraint. It plays an important role in the population regulation of T. ovis, but not of T. hydatigena. This is discussed in terms of transmission in the natural environment, an hypothesis on humoral protection and the need to elucidate pathways of protection when immunization schedules are being evaluated for controlling the taeniid zoonoses.

  2. Updating strategies for isolating and discovering giant viruses.

    PubMed

    Khalil, Jacques Yaacoub Bou; Andreani, Julien; La Scola, Bernard

    2016-06-01

    Almost fifteen years ago, the discovery of Acanthamoeba polyphaga mimivirus, the first giant virus, changed how we define a virus. It was discovered incidentally in a process of isolating Legionella sp. from environmental samples in the context of pneumonia epidemics using a co-culture system with Acanthamoeba. Since then, much effort and improvement has been put into the original technique. In addition to the known families of Mimiviridae and Marseilleviridae, four new proposed families of giant viruses have been isolated: Pandoravirus, Pithovirus, Faustovirus and Mollivirus. Major improvements were based on enrichment systems, targeted use of antibiotics and high-throughput methods. The most recent development, using flow cytometry for isolation and presumptive identification systems, opens a path to large environmental surveys that may discover new giant virus families in new protozoa supports used for culture support. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Drug Target Discovery Methods In Targeting Neurotropic Parasitic Amoebae.

    PubMed

    Baig, Abdul Mannan; Waliani, Nuzair; Karim, Saiqa

    2018-02-21

    Neurotropic parasitic amoebal infections have imposed an enormous challenge to chemotherapy in patients who fall victims to the infections caused by them. Conventional antibiotics that are given to treat these infections have a low patient compliance because of the serious adverse effects that are associated with their use. Additionally, the growing incidence of the development of drug resistance by the neurotropic parasites like Naegleria fowleri, Balamuthia mandrillaris, and Acanthamoeba spp has made the drug therapy more challenging. Recent studies have reported some cellular targets in the neurotropic parasitic Acanthamoeba that are used as receptors by human neurotransmitters like acetylcholine. This Viewpoint attempts to highlight the novel methodologies that use drug assays and structural modeling to uncover cellular targets of diverse groups of drugs and the safety issues of the drugs proposed for their use in brain infections caused by the neurotropic parasitic amoebae.

  4. Microbial diversities (16S and 18S rDNA gene pyrosequencing) and environmental pathogens within drinking water biofilms grown on the common premise plumbing materials unplasticized polyvinylchloride and copper

    EPA Science Inventory

    Drinking water (DW) biofilm communities influence the survival of opportunistic pathogens, e.g. Legionella pneumophila, via parasitization of free-living amoebae such as Acanthamoebae. Yet knowledge about the microbial composition of DW biofilms developed on common in-premise pl...

  5. Antigenic switching of TSA 417, a trophozoite variable surface protein, following completion of the life cycle of Giardia lamblia.

    PubMed Central

    Meng, T C; Hetsko, M L; Gillin, F D

    1993-01-01

    Expression of TSA 417, the predominant cysteine-rich variable surface protein of Giardia lamblia WB clone C6 trophozoites, did not change during encystation in vitro. However, in vitro excystation of cysts derived in vitro or in vivo consistently produced TSA 417 nonexpressing trophozoite populations, suggesting that completion of the life cycle leads to antigenic switching. Images PMID:8225614

  6. Identification of Antigens of Pathogenic Free-Living Amoebae by Protein Immunoblotting with Rabbit Immune and Human Sera

    DTIC Science & Technology

    1994-09-01

    development of immunoreactive bands was restricted to molecular masses of greater than 18.5 kDa for Naegleria , Hartmannella, and Vahlkampfia antigens. Two...detected between representative species of the three subgroups of Acanthamoeba. Naegleria antigen was likewise serologically distinct, as were

  7. Counting Legionella cells within single amoeba host cells

    EPA Science Inventory

    Here we present the first attempt to quantify L. pneumophila cell numbers within individual amoebae hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 134...

  8. Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing

    EPA Science Inventory

    The potential effect of in-premise plumbing temperatures (24, 32, 37 and 41 °C) on the growth of five different L. pneumophila strains within free-living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. Compared to controls only fed E...

  9. Inactivation and Removal of Free-Living Amoebae.

    EPA Science Inventory

    Free-living amoebae (FLA) are ubiquitous protozoan that are predominantly harmless to humans. There are a few genera that cause disease in humans, Balamuthia, Naegleria, and Acanthamoeba. These organisms are not easily removed by physical means or inactivated by chemic...

  10. Fimbrolide-coated antimicrobial lenses: their in vitro and in vivo effects.

    PubMed

    Zhu, Hua; Kumar, Ajay; Ozkan, Jerome; Bandara, Rani; Ding, Aidong; Perera, Indrani; Steinberg, Peter; Kumar, Naresh; Lao, William; Griesser, Stefani S; Britcher, Leanne; Griesser, Hans J; Willcox, Mark D P

    2008-05-01

    To examine the ability of contact lenses coated with fimbrolides, inhibitors of bacterial quorum sensing, to prevent microbial adhesion and their safety during short-term clinical assessment. A fimbrolide was covalently attached to commercially available high Dk contact lenses. Subsequently Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens, or Acanthamoeba sp. were added to the lenses and control uncoated contact lenses. Lenses plus microbes were incubated for 24 h, then washed thoroughly to remove non-adherent microbes. Lenses were macerated and resulting slurry plated onto agar plates. After appropriate incubation, the numbers of colony forming units of bacteria (or numbers of Acanthamoeba trophozoites measured using a hemocytometer) from fimbrolide-coated and uncoated lenses were examined. A Guinea Pig model of lens wear was used to assess the safety of lenses worn on a continuous basis for 1 month. In a separate study, 10 subjects wore fimbrolide-coated lenses for 24 h. The responses of the Guinea Pigs and human volunteers to the lenses were assessed by slit lamp examination. The fimbrolides-coated lenses reduced the adhesion of all bacterial strains tested, with reductions occurring of between 67 and 92%. For Acanthamoeba a reduction of 70% was seen. There were no significant differences in ocular responses to fimbrolide-coated lenses compared with controls in either the 1 month animal model or overnight human trial. Fimbrolide-coated lenses show promise as an antibacterial and anti-acanthamoebal coating on contact lenses and appear to be safe when worn on the eye in an animal model.

  11. Effect of Common Drinking Water Disinfectants, Chlorine and Heat, on Free Legionella and Amoebae-Associated Legionella

    PubMed Central

    Cervero-Aragó, Sílvia; Rodríguez-Martínez, Sarah; Puertas-Bennasar, Antoni; Araujo, Rosa M.

    2015-01-01

    Chlorine and thermal treatments are the most commonly used procedures to control and prevent Legionella proliferation in drinking water systems of large buildings. However, cases of legionellosis still occur in facilities with treated water. The purpose of this work was to model the effect of temperature and free chlorine applied in similar exposure conditions as in drinking water systems on five Legionella spp. strains and two amoebal strains of the genera Acanthamoeba. Inactivation models obtained were used to determine the effectiveness of the treatments applied which resulted more effective against Legionella than Acanthamoeba, especially those in cystic stages. Furthermore, to determine the influence of the relationship between L. pneumophila and Acanthamoeba spp. on the treatment effectiveness, inactivation models of the bacteria-associated amoeba were also constructed and compared to the models obtained for the free living bacteria state. The Legionella-amoeba association did not change the inactivation models, but it reduced the effectiveness of the treatments applied. Remarkably, at the lowest free chlorine concentration, 0.5 mg L-1, as well as at the lowest temperatures, 50°C and 55°C, the influence of the Legionella-amoeba associate state was the strongest in reducing the effectiveness of the treatments compared to the free Legionella state. Therefore, the association established between L. pneumophila and amoebae in the water systems indicate an increased health risk in proximal areas of the system (close to the tap) where lower free chlorine concentrations and lower temperatures are commonly observed. PMID:26241039

  12. Histopathology of fish. II. The salmon-poisoning fluk

    USGS Publications Warehouse

    1956-01-01

    THE SALMON-POISONING FLUKE is misnamed as far as the fish culturist is concerned, for the disease affects dogs, not fish. There is considerable evidence, however, that fish may also suffer from the complex chain of events leading from snail to dying dog. Histological studies indicate that young salmon and trout may be severely damaged by the encysted stage of the fluke.

  13. Naegleria fowleri: enolase is expressed during cyst differentiation.

    PubMed

    Chávez-Munguía, Bibiana; Segovia-Gamboa, Norma; Salazar-Villatoro, Lizbeth; Omaña-Molina, Maritza; Espinosa-Cantellano, Martha; Martínez-Palomo, Adolfo

    2011-01-01

    Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri. © 2011 The Author(s). Journal of Eukaryotic Microbiology © 2011 International Society of Protistologists.

  14. POTENTIALLY PATHOGENIC FREE-LIVING AMOEBAE IN SOME FLOOD-AFFECTED AREAS DURING 2011 CHIANG MAI FLOOD

    PubMed Central

    Wannasan, Anchalee; Uparanukraw, Pichart; Songsangchun, Apichart; Morakote, Nimit

    2013-01-01

    SUMMARY The survey was carried out to investigate the presence of potentially pathogenic free-living amoebae (FLA) during flood in Chiang Mai, Thailand in 2011. From different crisis flood areas, seven water samples were collected and tested for the presence of amoebae using culture and molecular methods. By monoxenic culture, FLA were detected from all samples at 37 °C incubation. The FLA growing at 37 °C were morphologically identified as Acanthamoeba spp., Naegleria spp. and some unidentified amoebae. Only three samples (42.8%), defined as thermotolerant FLA, continued to grow at 42 °C. By molecular methods, two non-thermotolerant FlA were shown to have 99% identity to Acanthamoeba sp. and 98% identity to Hartmannella vermiformis while the two thermotolerant FLA were identified as Echinamoeba exundans (100% identity) and Hartmannella sp. (99% identity). This first report of the occurrence of FLA in water during the flood disaster will provide information to the public to be aware of potentially pathogenic FLA. PMID:24213194

  15. Potentially pathogenic free-living amoebae in some flood-affected areas during 2011 Chiang Mai flood.

    PubMed

    Wannasan, Anchalee; Uparanukraw, Pichart; Songsangchun, Apichart; Morakote, Nimit

    2013-01-01

    The survey was carried out to investigate the presence of potentially pathogenic free-living amoebae (FLA) during flood in Chiang Mai, Thailand in 2011. From different crisis flood areas, seven water samples were collected and tested for the presence of amoebae using culture and molecular methods. By monoxenic culture, FLA were detected from all samples at 37 °C incubation. The FLA growing at 37 °C were morphologically identified as Acanthamoeba spp., Naegleria spp. and some unidentified amoebae. Only three samples (42.8%), defined as thermotolerant FLA, continued to grow at 42 °C. By molecular methods, two non-thermotolerant FlA were shown to have 99% identity to Acanthamoeba sp. and 98% identity to Hartmannella vermiformis while the two thermotolerant FLA were identified as Echinamoeba exundans (100% identity) and Hartmannella sp. (99% identity). This first report of the occurrence of FLA in water during the flood disaster will provide information to the public to be aware of potentially pathogenic FLA.

  16. Effect of UV irradiation (253.7 nm) on free Legionella and Legionella associated with its amoebae hosts.

    PubMed

    Cervero-Aragó, Sílvia; Sommer, Regina; Araujo, Rosa M

    2014-12-15

    Water systems are the primary reservoir for Legionella spp., where the bacteria live in association with other microorganisms, such as free-living amoebae. A wide range of disinfection treatments have been studied to control and prevent Legionella colonization but few of them were performed considering its relation with protozoa. In this study, the effectiveness of UV irradiation (253.7 nm) using low-pressure lamps was investigated as a disinfection method for Legionella and amoebae under controlled laboratory conditions. UV treatments were applied to 5 strains of Legionella spp., 4 strains of free-living amoeba of the genera Acanthamoeba and Vermamoeba, treating separately trophozoites and cysts, and to two different co-cultures of Legionella pneumophila with the Acanthamoeba strains. No significant differences in the UV inactivation behavior were observed among Legionella strains tested which were 3 logs reduced for fluences around 45 J/m(2). UV irradiation was less effective against free-living amoebae; which in some cases required up to 990 J/m(2) to obtain the same population reduction. UV treatment was more effective against trophozoites compared to cysts; moreover, inactivation patterns were clearly different between the genus Acanthamoeba and Vermamoeba. For the first time data about Vermamoeba vermiformis UV inactivation has been reported in a study. Finally, the results showed that the association of L. pneumophila with free-living amoebae decreases the effectiveness of UV irradiation against the bacteria in a range of 1.5-2 fold. That fact demonstrates that the relations established between different microorganisms in the water systems can modify the effectiveness of the UV treatments applied. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Characterization of giardin protein expression during encystation of Giardia duodenalis

    USDA-ARS?s Scientific Manuscript database

    Giardia duodenalis trophozoites attach to the gut surface by means of a ventral disk that contains various giardin proteins that appear to be important to VD structural integrity. One approach to preventing giardiasis is to stimulate giardin-specific antibodies and thereby block trophozoite attachme...

  18. Chronology of parasite-induced alteration of fish behaviour: effects of parasite maturation and host experience.

    PubMed

    Shirakashi, S; Goater, C P

    2005-02-01

    We monitored temporal changes in the magnitude of altered host behaviour in minnows (Pimephales promelas) experimentally infected with metacercariae of a brain-encysting trematode (Ornithodiplostomum ptychocheilus). This parasite develops and then encysts in a region of the brain that mediates the optomotor response (OMR), an innate behaviour that links visual stimuli with motor performance. The OMR of infected and uninfected minnows was evaluated between 0 and 10 weeks post-infection (p.i.), an interval spanning the development period of metacercariae to infectivity in birds. Trials involved monitoring the time an individual minnow spent following a spinning drum that had been painted with alternating black and white stripes. At 2 and 4 weeks p.i., infected minnows followed the drum 40% less often than controls. Differences between controls and infected fish declined thereafter, and were undetectable by 10 weeks p.i. Both control and infected fish habituated equally rapidly to the spinning drum. However, the difference in performance between controls and infected fish was 29% for experienced fish and 48% for fish that had never experienced the drum. Because maximum parasite-induced reduction in OMR coincided with the period of maximum parasite development, the behavioural effects are most likely due to unavoidable pathology in the brain associated with developing larvae.

  19. The life cycle of Ascocotyle (Phagicola) longa (Digenea: Heterophyidae), a causative agent of fish-borne trematodosis.

    PubMed

    Simões, Susana Balmant Emerique; Barbosa, Helene Santos; Santos, Cláudia Portes

    2010-03-01

    The complete life cycle of the trematode Ascocotyle (Phagicola) longa (Digenea: Heterophyidae) is elucidated by natural observation validated by experimental infections. The natural first intermediate host of A. (P.) longa, an agent of human heterophyiasis in Brazil, is the cochliopid snail Heleobia australis (new first intermediate host). Metacercariae were found encysted in the body musculature, heart, stomach, liver, kidney, spleen, gonads and mesentery of mullets Mugil liza. Hamsters Mesocricetus auratus were experimentally infected with metacercariae of A. (P.) longa obtained from the mullets, and the adults recovered were used to infect the snails H. australis. Rediae and cercariae of A. (P.) longa are described for the first time. The ultrastructure of the tegument of A. (P.) longa shows a change in spination pattern from the cercaria with single-pointed spines to the metacercaria and adult with multipointed, brush-shaped spines. The life cycle of A. (P.) longa is related to estuaries and coastal lagoons where the recruitment of mugilid juveniles occurs. The high prevalence (100%) of A. (P.) longa encysted in the mullets examined within the urban area of Rio de Janeiro indicates the potentially great public health impact of the consumption of raw mullets. Copyright 2009 Elsevier B.V. All rights reserved.

  20. The role of host sex in parasite dynamics: individual based model simulations of host-parasite interactions in a semi-enclosed embayment.

    NASA Astrophysics Data System (ADS)

    Velo Suarez, L.; Arancio, M.; Sourisseau, M.

    2016-02-01

    Parasitic dinoflagellates of the genus Amoebophrya infect free-living dinoflagellates, some of which can cause harmful algal blooms (HABs). During a field study in Salt Pond (MA, USA), we found a significant influence of Amoebophrya spp. on populations of Alexandrium fundyense. Parasitism appeared to exhibit a significant top down influence on A. fundyense populations and a dramatic life-cycle transition from vegetative division to sexual fusion was recorded. Despite our intensive sampling in Salt Pond, host-parasite interactions were undersampled owing to the very short time scales relevant to host-Amoebophrya spp. dynamics. In the present work, we explored the role of sexual reproduction and excystment/encystment processes using an Individual Based Model (IBM). The model was parameterized using published data and laboratory experiments carried out to analyze Amoebophrya spp. functional response. Observed-simulated differences in host-parasite dynamics support the hypothesis of parasite-host simultaneous dormancy, and further excystment months later to propagate both species. Results suggest that coexistence of A. fundyense and Amoebophrya spp. and their annual persistence in Salt Pond might rely on a sexual response/encystment. Understanding host-parasite interactions and coexistence strategies will improve our knowledge of Alexandrium spp. blooms and assess the impact of parasites on natural plankton assemblages in coastal systems.

  1. 3D virtual histology at the host/parasite interface: visualisation of the master manipulator, Dicrocoelium dendriticum, in the brain of its ant host.

    PubMed

    Martín-Vega, Daniel; Garbout, Amin; Ahmed, Farah; Wicklein, Martina; Goater, Cameron P; Colwell, Douglas D; Hall, Martin J R

    2018-06-05

    Some parasites are able to manipulate the behaviour of their hosts to their own advantage. One of the most well-established textbook examples of host manipulation is that of the trematode Dicrocoelium dendriticum on ants, its second intermediate host. Infected ants harbour encysted metacercariae in the gaster and a non-encysted metacercaria in the suboesophageal ganglion (SOG); however, the mechanisms that D. dendriticum uses to manipulate the ant behaviour remain unknown, partly because of a lack of a proper and direct visualisation of the physical interface between the parasite and the ant brain tissue. Here we provide new insights into the potential mechanisms that this iconic manipulator uses to alter its host's behaviour by characterising the interface between D. dendriticum and the ant tissues with the use of non-invasive micro-CT scanning. For the first time, we show that there is a physical contact between the parasite and the ant brain tissue at the anteriormost part of the SOG, including in a case of multiple brain infection where only the parasite lodged in the most anterior part of the SOG was in contact with the ant brain tissue. We demonstrate the potential of micro-CT to further understand other parasite/host systems in parasitological research.

  2. Small-sized euryhaline fish as intermediate hosts of the digenetic trematode Cryptocotyle concavum

    NASA Astrophysics Data System (ADS)

    Zander, C. D.; Kollra, H.-G.; Antholz, B.; Meyer, W.; Westphal, D.

    1984-03-01

    Cercariae of the trematode Cryptocotyle concavum, which encyst in skin and/or kidney of sticklebacks and gobies, were studied in the Schlei Fjord (western Baltic Sea). Mean incidence of dermal cysts was 48 % in Gasterosteus aculeatus and 37 % in Pungitius pungitius. No cysts were found in the kidneys of sticklebacks. While 97 % of Pomatoschistus microps had encysted metacercariae in the kidneys, only 2 % had cysts in the skin. Pomatoschistus minutus, however, showed hardly any cyst infestation of either skin or kidney. In P. microps the intensity of infestation by metacercariae was frequently more than 50 cysts; in contrast, sticklebacks rarely exhibited more than 5 dermal cysts. Infested fish were larger than 10 mm in total length, the incidence rate increasing with growth. Parasitic infestation depends on ambient salinity: C. concavum was not found at salinities below 4 ‰. In contrast to the high incidence in fish, the first hosts — the snails Hydrobia stagnalis and H. neglecta — showed remarkably low infection rates (3 to 5 %). The findings reported are related to the distribution of C. concavum, the mode of life of infested fish, the feeding habits of the final hosts and the infestation of P. microps by other parasites. Evidently, P. microps represents an optimal second host for C. concavum.

  3. Identification and significance of Naegleria fowleri isolated from the hot spring which related to the first primary amebic meningoencephalitis (PAM) patient in Taiwan.

    PubMed

    Tung, Min-Che; Hsu, Bing-Mu; Tao, Chi-Wei; Lin, Wei-Chen; Tsai, Hsiu-Feng; Ji, Dar-Der; Shen, Shu-Min; Chen, Jung-Sheng; Shih, Feng-Cheng; Huang, Yu-Li

    2013-08-01

    Naegleria fowleri can cause primary amoebic meningoencephalitis, a rapidly developing and highly lethal infectious disease. The first confirmed case of primary amoebic meningoencephalitis in Taiwan was reported in November 2011, in which the patient visited a thermal spring recreational area 1 week prior to hospitalisation. Water sampling was performed to verify the presence of Naegleria at the facility. According to our results, 32% and 20% of recreational water samples were contaminated with Naegleria spp. and Acanthamoeba spp., respectively. The genotypes of Naegleria identified at the hot spring included N. fowleri, Naegleria australiensis and Naegleria lovaniensis. Using PCR, it was determined that the strain of N. fowleri in one sample possessed the same genotype 2 as the clinical isolate. Thus, the thermal spring was suggested to be the likely source of infection. This is the first known instance of simultaneously isolating N. fowleri from both a patient as well as from a hot spring in Taiwan. Following this initial study, the pools at the thermal spring recreational area were drained, scrubbed and disinfected, and a follow-up study was performed 1 month later. Naegleria fowleri was not detected in follow-up testing; however, other Naegleria spp. were identified. We postulate that the biofilm in the waterlines may have provided a reservoir for free-living amoebae. The presence/absence of Acanthamoeba and Naegleria spp. did not differ significantly with any measured parameters related to water quality; however, a high percentage of the thermal water pool samples were contaminated with Naegleria or Acanthamoeba. Thus, amoebic contamination may present a serious threat to the health of humans who engage in leisure activities at thermal springs. Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  4. Formulation and stability of an extemporaneous 0.02% chlorhexidine digluconate ophthalmic solution.

    PubMed

    Lin, Shu-Chiao; Huang, Chih-Fen; Shen, Li-Jiuan; Wang, Hsueh-Ju; Lin, Chia-Yu; Wu, Fe-Lin Lin

    2015-12-01

    Acanthamoeba keratitis is difficult to treat because Acanthamoeba cysts are resistant to the majority of antimicrobial agents. Despite the efficacy of 0.02% chlorhexidine in treating Acanthamoeba keratitis, a lack of data in the literature regarding the formulation's stability limits its clinical use. The objective of this study was to develop an optimal extemporaneous 0.02% chlorhexidine digluconate ophthalmic formulation for patients in need. With available active pharmaceutical ingredients, 0.02% chlorhexidine digluconate sample solutions were prepared by diluting with BSS Plus Solution or acetate buffer. Influences of the buffer, type of container, and temperature under daily-open condition were assessed based on the changes of pH values and chlorhexidine concentrations of the test samples weekly. To determine the beyond-use date, the optimal samples were stored at 2-8°C or room temperature, and analyzed at time 0 and at Week 1, Week 2, Week 3, Week 4, Week 5, Week 8, Week 12, and Week 24. Despite chlorhexidine exhibiting better stability in acetate buffer than in BSS solution, its shelf-life was < 14 days when stored in a light-resistant low-density polyethylene container. The acetate-buffered 0.02% chlorhexidine digluconate solution stored in light-resistant high-density polyethylene eyedroppers did not exhibit significant changes in pH or strength at any time interval. The acetate-buffered 0.02% chlorhexidine digluconate ophthalmic solution stored in light-resistant high-density polyethylene eyedroppers demonstrated excellent stability at 2-25°C for 6 months after being sealed and for 1 month after opening. This finding will enable us to prepare 0.02% chlorhexidine digluconate ophthalmic solutions based on a doctor's prescription. Copyright © 2014. Published by Elsevier B.V.

  5. Amoebae from antarctic soil and water.

    PubMed Central

    Brown, T J; Cursons, R T; Keys, E A

    1982-01-01

    Samples of soil and water were taken from the McMurdo Sound-Dry Valley region of Antarctica. Of the 70 samples cultured, 22 yielded amoebae capable of clonal growth at 30 degrees C. None of the isolates was pathogenic for mice. Acanthamoeba isolates appeared to show better survival potential than Naegleria isolates. PMID:7125658

  6. Balamuthia mandrillaris meningoencephalitis: the first case in southeast Asia.

    PubMed

    Intalapaporn, Poj; Suankratay, Chusana; Shuangshoti, Shanop; Phantumchinda, Kammant; Keelawat, Somboon; Wilde, Henry

    2004-06-01

    We present a case of 23-year-old man with acute meningoencephalitis, accompanied by inflammation of a nasal ulcer. He had been healthy until six months prior to admission to the hospital when he had a motorcycle accident. A star-shaped wound at his nose was incurred after falling into a swamp. A computed tomogram of the brain showed two nonenhancing hypodense lesions at the left caudate nucleus and the right parietal lobe, ependymitis and leptomeningeal enhancement. A skin biopsy showed chronic noncaseous granulomatous inflammation without demonstrated microorganisms. The patient did not respond to the empirical treatment with cloxacillin, ceftriaxone, and amphotericin B, and eventually died on the thirteenth day of hospitalization. At autopsy, hematoxylin and eosin-stained brain sections showed a chronic necrotizing inflammation with numerous amebic trophozoites and rare cysts. Definitive identification of Balamuthia mandrillaris was made by fluorescent immunohistochemical analysis. There were 10 Naegleria fowleri primary amebic meningoencephalitis, eight Acanthamoeba granulomatous amebic encephalitis, and three Acanthamoeba meningitis in Thailand. To our knowledge, this case is the first reported case of B. mandrillaris in Southeast Asia.

  7. [Developmental cycle of Stomylotrema vicarium].

    PubMed

    Ostrowski de Nuñez, M

    1978-11-01

    With metacercariae encysted naturally in the visceral cavity of Megadytes glauca-larvae (Dityscidae), day old unfed chicken and a Belonopterus cayennensis were infected and adults of Stomylotrema vicarium Braun, 1901 obtained. Metacercariae and adults have an excretory bladder with lateral ramifications and 4 groups of 10, exceptionally 12 flame cells on each body side. Very young metacercariae without cysts presented a big stylet. A corresponding virgula-cercaria in Ampullaria canaliculata is supposed.

  8. A Unique cis-Encoded Small Noncoding RNA Is Regulating Legionella pneumophila Hfq Expression in a Life Cycle-Dependent Manner.

    PubMed

    Oliva, Giulia; Sahr, Tobias; Rolando, Monica; Knoth, Maike; Buchrieser, Carmen

    2017-01-10

    Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires' disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis-encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5' untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti-hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and

  9. Genomic analysis of a Raoultella ornithinolytica strain causing prosthetic joint infection in an immunocompetent patient.

    PubMed

    Beye, Mamadou; Hasni, Issam; Seng, Piseth; Michelle, Caroline; La Scola, Bernard; Raoult, Didier; Fournier, Pierre-Edouard

    2018-06-21

    We sequenced the genome of Raoultella ornithinolytica strain Marseille-P1025 that caused a rare case of prosthetic joint infection in a 67-year-old immunocompetent male. The 6.7-Mb genome exhibited a genomic island (RoGI) that was unique among R. ornithinolytica strains. RoGI was likely acquired by lateral gene transfer from a member of the Pectobacterium genus and coded for a type IVa secretion system found in other pathogenic bacteria and that may have conferred strain Marseille-P1025 an increased virulence. Strain Marseille-P1025 was also able to infect, multiply within, and kill Acanthamoaeba castellanii amoebae.

  10. Increased surfacing behavior in longnose killifish infected by brain-encysting trematode.

    PubMed

    Fredensborg, B L; Longoria, A N

    2012-10-01

    Some parasites modify the behavior of intermediate hosts to increase the probability of transmission to the next host in their life cycle. In habitats where this is common, parasites play an important role in predator-prey links and food web dynamics. In this study we used laboratory observations to investigate the behavior of longnose killifish, Fundulus similis, that were naturally infected with metacercariae of the trematode, Euhaplorchis sp. A, from Laguna Madre, south Texas. In particular, we examined whether there was a relationship between the number of metacercariae lodged on the brain of the infected fish and behaviors that made the fish more conspicuous to avian final hosts. We also quantified the abundance and cercariae production of this parasite in its first intermediate snail host, Cerithidea pliculosa , and examined the seasonal variation of Euhaplorchis sp. A in F. similis . Our data demonstrated that Euhaplorchis sp. A affected the surfacing behavior of F. similis in an intensity-dependent manner. Fish with many infections spent longer time at the surface of the water than fish with few infections. Our data also show that Euhaplorchis sp. A is a common parasite in the first intermediate host and produces close to 4,000 cercariae m(-2) day(-1). Consequently 97% of all fish collected and necropsied were infected, with little seasonal variation in the mean abundance of the parasite. Based on our data, Euhaplorchis sp. A is likely important to predator-prey links in Gulf of Mexico estuary food webs, similar to the closely related Euhaplorchis californiensis in southern California. We expect that other closely related species elsewhere may have similar effects on other fish hosts, emphasizing the need for incorporating trophically transmitted parasites in estuarine food web studies.

  11. Alternative mounting media for preservation of some protozoa.

    PubMed

    Criado-Fornelio, A; Heredero-Bermejo, I; Pérez-Serrano, J

    2014-10-01

    Protozoa resistant stages are disintegrated when mounted in toluene-based media. To overcome such problem, three toluene-free mountants were tested on preserve Acanthamoeba spp and gregarines. Two commercial glues based on cyanoacrylate or trimethoxysilane were suitable for preserving both cysts and trophozoites. Hoyer's medium showed good results for mounting gregarine oocysts. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. First record of cysts in the tidal tardigrade Echiniscoides sigismundi

    NASA Astrophysics Data System (ADS)

    Clausen, Lykke K. B.; Andersen, Kasper N.; Hygum, Thomas L.; Jørgensen, Aslak; Møbjerg, Nadja

    2014-12-01

    Tardigrades are microscopic metazoans that withstand environmental extremes by entering dormant states, such as cryptobiosis (latent life). In addition, they may also form cysts. Here, we present the first report of cyst formation in a marine heterotardigrade, i.e., Echiniscoides sigismundi, which constitutes a cryptic species complex present worldwide in tidal zones. The cysts were initially discovered during experimental series constructed to investigate osmotic stress tolerance. The animals, which eventually formed cysts, showed signs of an imminent molt at the beginning of experimentation. We use the term "cyst" for stages, where a total of three or more cuticles have been synthesized. Our observations show that encystment in E. sigismundi involves synthesizing of at least two new cuticle layers. Legs with discharged claws are present in connection with the first outer cuticle, as well as the second cuticular layer. In the most developed cyst, a third cuticle lacking claws seems to surround the animal, which is delineated by a fourth cuticle. Many features are shared with the well-studied cysts of eutardigrades. The cysts of E. sigismundi, however, lack pigmentation and have an extra set of claws, and the animal inside retains buccopharyngeal sclerified parts, until discharging the third cuticle. The finding of cysts in a marine heterotardigrade is novel and confirms that encystment also occurs within this major evolutionary lineage.

  13. The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

    PubMed Central

    Frisardi, Marta; Ghosh, Sudip K.; Field, Jessica; Van Dellen, Katrina; Rogers, Rick; Robbins, Phillips; Samuelson, John

    2000-01-01

    The infectious stage of amebae is the chitin-walled cyst, which is resistant to stomach acids. In this study an extraordinarily abundant, encystation-specific glycoprotein (Jacob) was identified on two-dimensional protein gels of cyst walls purified from Entamoeba invadens. Jacob, which was acidic and had an apparent molecular mass of ∼100 kDa, contained sugars that bound to concanavalin A and ricin. The jacob gene encoded a 45-kDa protein with a ladder-like series of five Cys-rich domains. These Cys-rich domains were reminiscent of but not homologous to the Cys-rich chitin-binding domains of insect chitinases and peritrophic matrix proteins that surround the food bolus in the insect gut. Jacob bound purified chitin and chitin remaining in sodium dodecyl sulfate-treated cyst walls. Conversely, the E. histolytica plasma membrane Gal/GalNAc lectin bound sugars of intact cyst walls and purified Jacob. In the presence of galactose, E. invadens formed wall-less cysts, which were quadranucleate and contained Jacob and chitinase (another encystation-specific protein) in secretory vesicles. A galactose lectin was found to be present on the surface of wall-less cysts, which phagocytosed bacteria and mucin-coated beads. These results suggest that the E. invadens cyst wall forms when the plasma membrane galactose lectin binds sugars on Jacob, which in turn binds chitin via its five chitin-binding domains. PMID:10858239

  14. Fine needle aspiration cytology of subcutaneous cysticercosis of the breast. Case report and pathogenic discussion.

    PubMed

    Vuong, P N

    1989-01-01

    A case of subcutaneous cysticercosis of the breast diagnosed by fine needle aspiration cytology is reported. The cytologic preparations showed the presence of many inflammatory cells mixed with spiked spherules resembling the rambutan tropical fruit. These spherules arose from the degenerated integument of the parasite found in the histologically excised cyst. They are suggestive of the presence of an encysted flat worm, but are not specific for cysticercosis, whose identification is based on the study of the cephalic extremity of the parasite.

  15. Amoebae as battlefields for bacteria, giant viruses, and virophages.

    PubMed

    Slimani, Meriem; Pagnier, Isabelle; Raoult, Didier; La Scola, Bernard

    2013-04-01

    When amoebae are simultaneously infected with Acanthamoeba polyphaga Mimivirus (APM) and the strictly intracellular BABL1 bacterium, the latter is always lost after serial subculturing. We showed that the virophage Sputnik 1, by reducing APM fitness, preserved BABL1 growth in acute and chronic models. This capability of a virophage to modulate the virulence of mimiviruses highlights the competition that occurs between them during natural host infection.

  16. [Contribution to the study of Microphallidae Travassos 1920 (trematoda). XXXII. Microphallus breviatus n. sp., a species with an abbreviated evolutive cycle from a Mediterranean pond in the Languedoc].

    PubMed

    Deblock, S; Maillard, C

    1975-01-01

    Contribution to the study of Microphallidae Travassos, 1920 (Trematoda). XXXII. - Microphallus breviatus n. sp., a short life-cycle species of a mediterranean pond of Languedoc. The whole larval life-cycle of M. breviatus takes place in one host, Hydrobia ventrosa (Montagu), Mollusc Hydrobiidae. Hepato-pancreatic sporocysts produce morphologically altered xiphidio-cercariae which become encysted metacercariae in the sporocyts themselves. This species is defined in the genus by the anatomic characteristics of its cercariae and metacercariae, allied with its uncommon biology including two hosts only.

  17. Amoebae as Battlefields for Bacteria, Giant Viruses, and Virophages

    PubMed Central

    Slimani, Meriem; Pagnier, Isabelle; Raoult, Didier

    2013-01-01

    When amoebae are simultaneously infected with Acanthamoeba polyphaga Mimivirus (APM) and the strictly intracellular BABL1 bacterium, the latter is always lost after serial subculturing. We showed that the virophage Sputnik 1, by reducing APM fitness, preserved BABL1 growth in acute and chronic models. This capability of a virophage to modulate the virulence of mimiviruses highlights the competition that occurs between them during natural host infection. PMID:23388714

  18. Local and systemic inflammatory and immunologic reactions to cyathostomin larvicidal therapy in horses.

    PubMed

    Nielsen, M K; Loynachan, A T; Jacobsen, S; Stewart, J C; Reinemeyer, C R; Horohov, D W

    2015-12-15

    Encysted cyathostomin larvae are ubiquitous in grazing horses. Arrested development occurs in this population and can lead to an accumulation of encysted larvae. Large numbers of tissue larvae place the horse at risk for developing larval cyathostominosis. This disease complex is caused by mass emergence of these larvae and is characterized by a generalized acute typhlocolitis and manifests itself as a profuse protein-losing watery diarrhea with a reported case-fatality rate of about 50%. Two anthelmintic formulations have a label claim for larvicidal therapy of these encysted stages; moxidectin and a five-day regimen of fenbendazole. There is limited knowledge about inflammatory and immunologic reactions to larvicidal therapy. This study was designed to evaluate blood acute phase reactants as well as gene expression of pro-inflammatory cytokines, both locally in the large intestinal walls and systemically. Further, mucosal tissue samples were evaluated histopathologically as well as analyzed for gene expression of pro- and anti-inflammatory cytokines, cluster of differentiation (CD) cell surface proteins, and select transcription factors. Eighteen juvenile horses with naturally acquired cyathostomin infections were randomly assigned to three treatment groups; one group served as untreated controls (Group 1), one received a five-day regimen of fenbendazole (10mg/kg) (Group 2), and one group received moxidectin (0.4mg/kg) (Group 3). Horses were treated on day 0 and euthanatized on days 18-20. Serum and whole blood samples were collected on days 0, 5, and 18. All horses underwent necropsy with collection of tissue samples from the ventral colon and cecum. Acute phase reactants measured included serum amyloid A, iron and fibrinogen, and the cytokines evaluated included interferon γ, tumor necrosis factor α, transforming growth factor (TGF)-β, and interleukins 1β, 4, 5, 6, and 10. Transcription factors evaluated were FoxP3, GATA3 and tBet, and CD markers included

  19. Evaluation of the mucosal inflammatory responses to equine cyathostomins in response to anthelmintic treatment.

    PubMed

    Steuer, A E; Loynachan, A T; Nielsen, M K

    2018-05-01

    Members of Cyathostominae are pervasive parasites of equids that can cause larval cyathostominosis, a potentially life-threatening disease that occurs when a multitude of encysted larvae synchronously excyst from the wall of the large intestine. Moxidectin and fenbendazole are the two current labeled drugs that target the encysted larval stages; however, there is limited knowledge of the local inflammatory response to the larvae and to the two treatments in clinically healthy horses. This study is the first to evaluate the local inflammatory response to cyathostomin larvae and to larvicidal treatment at 2 and 5 weeks post treatment. Thirty-six ponies with naturally acquired cyathostomin infections were randomly allocated into 3 groups: Group 1, fenbendazole at 10 mg/kg for 5 days, Group 2, a single dose of moxidectin at 0.4 mg/kg, and Group 3, untreated controls. Tissue samples from the cecum and dorsal and ventral colons were used for histopathological and immunohistochemical evaluation. Tissues were stained with routine hematoxylin and eosin (H&E) for light microscopy and immunohistochemically for MAC387, CD20, and CD3 for differentiation of activated macrophages, B cells, and T cells, respectively. Semiquantitative scores were assigned for all inflammatory cell types and fibrous connective tissue. Larvae observed by light microscopy were enumerated and classified by stage. Mucosal ulcerations and submucosal granulomas were also enumerated. Mean macrophage scores were higher in the moxidectin group than the fenbendazole group (p = 0.0185) and the control group had a higher activated macrophage score than both treatment groups (p = 0.0104, p = 0.0004). T lymphocyte scores were higher in the moxidectin group when compared to the control group (p = 0.0069). Goblet cell hyperplasia scores were elevated at 5 weeks post treatment compared to 2 weeks post treatment (p = 0.0047) and were elevated in the ventral colon compared to the dorsal colon

  20. Comparison of the larvicidal efficacies of moxidectin or a five-day regimen of fenbendazole in horses harboring cyathostomin populations resistant to the adulticidal dosage of fenbendazole.

    PubMed

    Reinemeyer, C R; Prado, J C; Nielsen, M K

    2015-11-30

    Despite widespread acknowledgement of cyathostomin resistance to adult icidal dosages of benzimidazole (BZD) anthelmintics, many strongyle control programs continue to feature regularly scheduled larvicidal treatment with fenbendazole (FBZ). However, no studies have been conducted to evaluate the efficacy of larvicidal regimens against encysted cyathostomins in a BZD-resistant (BZD-R) population. A masked, randomized, controlled clinical study was conducted with 18 juvenile horses harboring populations of cyathostomins that were considered BZD-R on the basis of fecal egg count reduction (FECR). Horses were blocked by prior history, ranked by egg counts, and allocated randomly to one of three treatment groups: 1--control, 2--FBZ >10mg/kg once daily for five consecutive days, or 3--moxidectin (MOX) >0.4 mg/kg once. Fecal samples were collected prior to treatment and seven and 14 days after the final dose of anthelmintic. On Days 18-20, complete replicates of horses were euthanatized and necropsied, and 1% aliquots of large intestinal contents were recovered for determination of complete worm counts. The cecum and ventral colon were weighed, and measured proportions of the respective organ walls were processed for quantitation and characterization of encysted cyathostomin populations. The five-day regimen of FBZ achieved 44.6% fecal egg count reduction, had 56.4% activity against luminal adults and larvae, and was 38.6% and 71.2% effective against encysted early third stage (EL3) and late third stage/ fourth stage (LL3/L4) cyathostomin larvae, respectively. In contrast, MOX provided 99.9% FECR, removed 99.8% of luminal stages, and exhibited 63.6% and 85.2% efficacy against EL3 and LL3/L4 mucosal cyathostomins, respectively. Although BZD-R was the most feasible explanation for the lower larvicidal efficacies of FBZ, mean larval counts of moxidectin-treated horses were not significantly different from controls or those treated with FBZ. The lack of significant

  1. Oral immunization of BALB/c mice with Giardia duodenalis recombinant cyst wall protein inhibits shedding of cysts.

    PubMed

    Larocque, R; Nakagaki, K; Lee, P; Abdul-Wahid, A; Faubert, G M

    2003-10-01

    The process of encystation is a key step in the Giardia duodenalis life cycle that allows this intestinal protozoan to survive between hosts during person-to-person, animal-to-person, waterborne, or food-borne transmission. The release of cysts from infected persons and animals is the main contributing factor to contamination of the environment. Genes coding for cyst wall proteins (CWPs), which could be used for developing a transmission-blocking vaccine, have been cloned. Since the immunogenicity of recombinant Giardia CWP is unknown, we have investigated the immunogenicity of recombinant CWP2 (rCWP2) and its efficacy in interfering with the phenomenon of encystation taking place in the small bowels of BALB/c mice vaccinated with the recombinant protein. Here we report that the immunization of BALB/c mice with rCWP2 stimulated the immune system in a manner comparable to that for a live infection with Giardia muris cysts. Fecal and serum anti-rCWP2 immunoglobulin A (IgA) antibodies were detected in the immunized mice. In addition, anti-rCWP2 IgG1 and IgG2a antibodies were detected in the serum. mRNAs coding for Th1 and Th2 types of cytokines were detected in spleen and Peyer's patch cells from immunized mice. When the vaccinated mice were challenged with live cysts, the animals shed fewer cysts. We conclude that rCWP2 is a possible candidate antigen for the development of a transmission-blocking vaccine.

  2. Study 8: Prevalence and load of Nanophyetus salmincola infection in outmigrating steelhead trout from five Puget Sound rivers

    USGS Publications Warehouse

    Chen, M.F.; Stewart, B.A.; Senkvik, Kevin; Hershberger, Paul

    2015-01-01

    Nanophyetus salmincola is a parasitic trematode, or flatworm, that infects salmonid fishes in the Pacific Northwest, including Washington, Oregon, and portions of California. The adult worm lives in the intestine of fish-eating birds and mammals. Eggs shed into the water hatch into miracidia which penetrate the first intermediate host, one of two species of snail Juga plicifera or J. silicula. Asexual reproduction occurs within the snail. Free-swimming cercaria are released from the snail and penetrate the secondary intermediate host, often a salmonid fish, in fresh and brackish water. The cercaria encyst as metacercaria in various organs of the fish, including gills, muscle and heart, but favor the posterior kidney. Penetration and migration by the cercaria through the fish causes damage to nearly every organ system. Once encysted, metacercaria survive the ocean phase of salmonid life cycle. N. salmincola is a likely contributor to mortality of juvenile coho salmon (Oncorhynchus kisutch) during the early ocean rearing phase, and it is the most prevalent pathogen of outmigrating steelhead in the estuaries of the Pacific Northwest.A field survey was implemented from March-June 2014 to compare the prevalence and parasite load of N. salmincola infections in outmigrating steelhead from five Puget Sound watersheds and to assess changes in infection levels that occurred during the smolt out-migration through each watershed. N. salmincola infection prevalence and parasite loads were determined by counting metacercaria in posterior kidney samples. Tissue samples were collected and examined by standard histological methods.

  3. Radiation Resistance of Soil Azotobacter

    PubMed Central

    Vela, Gerard R.; Wyss, Orville

    1965-01-01

    Vela, Gerard R. (School of Aerospace Medicine, Brooks Air Force Base, Tex.), and Orville Wyss. Radiation resistance of soil Azotobacter. J. Bacteriol. 89:1280–1285. 1965.—Quantitative recovery of Azotobacter from soils subjected to γ-radiation from a cobalt-60 source showed the soil populations to be much more highly resistant than isolates from such cultures grown on laboratory media. Even in the encysted state, the laboratory populations were reduced 10,000-fold by exposure to 200 kr, whereas the soil populations were not measurably reduced by that dose. PMID:14292998

  4. A case of ectopic intraabdominal fascioliasis presented with acute abdomen.

    PubMed

    Tanir, Gönül; Karaman, Ayşe; Tüfekçı, Sehra Birgül; Erdoğan, Duygu; Tuygun, Nilden; Ozkan, Ayşegül Taylan

    2011-06-01

    Human fascioliasis with Fasciola species occurs worldwide and is most common among rural people who tend sheep and eat uncooked water vegetables, particularly watercress. The natural history of the acute phase begins with ingestion of metacercariae encysted on various kinds of aquatic vegetation such as watercress. Fascioliasis primarily involves the liver, bile ducts, gallbladder, and occasionally ectopic sites. We describe herein a case of ectopic fascioliasis. This uncommon form of disease was peritonitis; both visceral and parietal peritoneal layers were affected with the formation of multiple nodules and ascites.

  5. Surveillance and evaluation of the infection risk of free-living amoebae and Legionella in different aquatic environments.

    PubMed

    Ji, Wen-Tsai; Hsu, Bing-Mu; Chang, Tien-Yu; Hsu, Tsui-Kang; Kao, Po-Min; Huang, Kuan-Hao; Tsai, Shiou-Feng; Huang, Yu-Li; Fan, Cheng-Wei

    2014-11-15

    Free-living amoebae (FLA) are ubiquitous in various aquatic environments. Several amoebae species are pathogenic and host other pathogens such as Legionella, but the presence of FLA and its parasites as well as the related infection risk are not well known. In this study, the presence of pathogenic FLA and Legionella in various water bodies was investigated. Water samples were collected from a river, intake areas of drinking water treatment plants, and recreational hot spring complexes in central and southern Taiwan. A total of 140 water samples were tested for the presence of Acanthamoeba spp., Naegleria spp., Vermamoeba vermiformis, and Legionella. In addition, phylogenetic characteristics and water quality parameters were also assessed. The pathogenic genotypes of FLA included Acanthamoeba T4 and Naegleria australiensis, and both were abundant in the hot spring water. In contrast, Legionella pneumophila was detected in different aquatic environments. Among the FLA assessed, V. vermiformis was most likely to coexist with Legionella spp. The total bacteria level was associated with the presence of FLA and Legionella especially in hot spring water. Taken together, FLA contamination in recreational hot springs and drinking water source warrants more attention on potential legionellosis and amoebae infections. Copyright © 2014. Published by Elsevier B.V.

  6. Real-time PCR systems targeting giant viruses of amoebae and their virophages.

    PubMed

    Ngounga, Tatsiana; Pagnier, Isabelle; Reteno, Dorine-Gaelle Ikanga; Raoult, Didier; La Scola, Bernard; Colson, Philippe

    2013-01-01

    Giant viruses that infect amoebae, including mimiviruses and marseilleviruses, were first described in 2003. Virophages were subsequently described that infect mimiviruses. Culture isolation with Acanthamoeba spp. and metagenomic studies have shown that these giant viruses are common inhabitants of our biosphere and have enabled the recent detection of these viruses in human samples. However, the genomes of these viruses display substantial genetic diversity, making it a challenge to examine their presence in environmental and clinical samples using conventional and real-time PCR. We designed and evaluated the performance of PCR systems capable of detecting all currently isolated mimiviruses, marseilleviruses and virophages to assess their prevalence in various samples. Our real-time PCR assays accurately detected all or most of the members of the currently delineated lineages of giant viruses infecting acanthamoebae as well as the mimivirus virophages, and enabled accurate classification of the mimiviruses of amoebae in lineages A, B or C. We were able to detect four new mimiviruses directly from environmental samples and correctly classified these viruses within mimivirus lineage C. This was subsequently confirmed by culture on amoebae followed by partial Sanger sequencing. PCR systems such as those implemented here may contribute to an improved understanding of the prevalence of mimiviruses, their virophages and marseilleviruses in humans.

  7. Broad Spectrum of Mimiviridae Virophage Allows Its Isolation Using a Mimivirus Reporter

    PubMed Central

    Gaia, Morgan; Pagnier, Isabelle; Campocasso, Angélique; Fournous, Ghislain; Raoult, Didier; La Scola, Bernard

    2013-01-01

    The giant virus Mimiviridae family includes 3 groups of viruses: group A (includes Acanthamoeba polyphaga Mimivirus), group B (includes Moumouvirus) and group C (includes Megavirus chilensis). Virophages have been isolated with both group A Mimiviridae (the Mamavirus strain) and the related Cafeteria roenbergensis virus, and they have also been described by bioinformatic analysis of the Phycodnavirus. Here, we found that the first two strains of virophages isolated with group A Mimiviridae can multiply easily in groups B and C and play a role in gene transfer among these virus subgroups. To isolate new virophages and their Mimiviridae host in the environment, we used PCR to identify a sample with a virophage and a group C Mimiviridae that failed to grow on amoeba. Moreover, we showed that virophages reduce the pathogenic effect of Mimivirus (plaque formation), establishing its parasitic role on Mimivirus. We therefore developed a co-culture procedure using Acanthamoeba polyphaga and Mimivirus to recover the detected virophage and then sequenced the virophage's genome. We present this technique as a novel approach to isolating virophages. We demonstrated that the newly identified virophages replicate in the viral factories of all three groups of Mimiviridae, suggesting that the spectrum of virophages is not limited to their initial host. PMID:23596530

  8. Oxygen and the spatial structure of microbial communities.

    PubMed

    Fenchel, Tom; Finlay, Bland

    2008-11-01

    Oxygen has two faces. On one side it is the terminal electron acceptor of aerobic respiration - the most efficient engine of energy metabolism. On the other hand, oxygen is toxic because the reduction of molecular O2 creates reactive oxygen species such as the superoxide anion, peroxide, and the hydroxyl radical. Probably most prokaryotes, and virtually all eukaryotes, depend on oxygen respiration, and we show that the ambiguous relation to oxygen is both an evolutionary force and a dominating factor driving functional interactions and the spatial structure of microbial communities.We focus on microbial communities that are specialised for life in concentration gradients of oxygen, where they acquire the full panoply of specific requirements from limited ranges of PO2, which also support the spatial organisation of microbial communities. Marine and lake sediments provide examples of steep O2 gradients, which arise because consumption or production of oxygen exceeds transport rates of molecular diffusion. Deep lakes undergo thermal stratification in warm waters, resulting in seasonal anaerobiosis below the thermocline, and lakes with a permanent pycnocline often have permanent anoxic deep water. The oxycline is here biologically similar to sediments, and it harbours similar microbial biota, the main difference being the spatial scale. In sediments, transport is dominated by molecular diffusion, and in the water column, turbulent mixing dominates vertical transport. Cell size determines the minimum requirement of aerobic organisms. For bacteria (and mitochondria), the half-saturation constant for oxygen uptake ranges within 0.05-0.1% atmospheric saturation; for the amoeba Acanthamoeba castellanii it is 0.2%, and for two ciliate species measuring around 150 microm, it is 1-2 % atmospheric saturation. Protection against O2 toxicity has an energetic cost that increases with increasing ambient O2 tension. Oxygen sensing seems universal in aquatic organisms. Many aspects

  9. Genotypic characterization and assessment of infectivity of human waterborne pathogens recovered from oysters and estuarine waters in Brazil.

    PubMed

    Leal, Diego Averaldo Guiguet; Souza, Doris Sobral Marques; Caumo, Karin Silva; Fongaro, Gislaine; Panatieri, Lua Ferreira; Durigan, Maurício; Rott, Marilise Brittes; Barardi, Célia Regina Monte; Franco, Regina Maura Bueno

    2018-06-15

    Waterborne, food-borne and sewage-borne pathogens are a major global concern, with the annual recurrence, most notably during the summer, of outbreaks of gastroenteritis of unconfirmed etiology associated with recreational activities in marine environments. The consumption of contaminated water-based foodstuffs is also related to outbreaks of human illness. The main goals of the present study were: i) to identify the genetic assemblages of Giardia duodenalis cysts in growing and depurated oysters destined for human consumption on the southern coast of São Paulo, Brazil; ii) to verify the main circulating G. duodenalis assemblages and their subtypes in different brackish waters used for the production of mollusks and for recreational purposes; iii) to track the contamination of growing and depurated oysters by the human adenovirus and identify the infectivity of adenoviral particles recovered from oysters before and after depuration; iv) to evaluate the occurrence and genotype of the free-living amoebae of the genus Acanthamoeba in brackish water and oysters from all the sites described above. Four sampling sites in the Cananeia estuary were selected to search for pathogenic and amphizoic protozoa (Giardia and Acanthamoeba respectively): site 1: oyster growth, site 2: catchment water (before UV depuration procedure), site 3: filter backwash (filtration stage of water treatment) and site 4: oyster depuration tank. Oysters at sites 1 and 4 were evaluated for the presence of adenovirus (HAdV). Analysis consisted of conventional microbiological as well as molecular methods. Giardia duodenalis were detected in all the water sites analyzed and the molecular analysis revealed that sub-assemblage AII was the most frequently distributed throughout the estuarine environment, although one sample was identified as belonging to the assemblage C. Acanthamoeba were also isolated from different locations of the estuarine area, and were detected at all the analyzed sites. The

  10. Prevalence of pathogenic free-living amoeba and other protozoa in natural and communal piped tap water from Queen Elizabeth protected area, Uganda.

    PubMed

    Sente, Celsus; Erume, Joseph; Naigaga, Irene; Mulindwa, Julius; Ochwo, Sylvester; Magambo, Phillip Kimuda; Namara, Benigna Gabriela; Kato, Charles Drago; Sebyatika, George; Muwonge, Kevin; Ocaido, Michael

    2016-08-03

    Pathogenic water dwelling protozoa such as Acanthamoeba spp., Hartmannella spp., Naegleria spp., Cryptosporidium spp. and Giardia spp. are often responsible for devastating illnesses especially in children and immunocompromised individuals, yet their presence and prevalence in certain environment in sub-Saharan Africa is still unknown to most researchers, public health officials and medical practitioners. The objective of this study was to establish the presence and prevalence of pathogenic free-living amoeba (FLA), Cryptosporidium and Giardia in Queen Elizabeth Protected Area (QEPA). Samples were collected from communal taps and natural water sites in QEPA. Physical water parameters were measured in situ. The samples were processed to detect the presence of FLA trophozoites by xenic cultivation, Cryptosporidium oocysts by Ziehl-Neelsen stain and Giardia cysts by Zinc Sulphate floatation technique. Parasites were observed microscopically, identified, counted and recorded. For FLA, genomic DNA was extracted for amplification and sequencing. Both natural and tap water sources were contaminated with FLA, Cryptosporidium spp. and Giardia spp. All protozoan parasites were more abundant in the colder rainy season except for Harmannella spp. and Naegleria spp. which occurred more in the warmer months. The prevalence of all parasites was higher in tap water than in natural water samples. There was a strong negative correlation between the presence of Acanthamoeba spp., Hartmannella spp., Cryptosporidium spp. and Giardia spp. with Dissolved Oxygen (DO) (P < 0.05). The presence of Cryptosporidium spp. showed a significant positive correlation (P < 0.05) with conductivity, pH and Total Dissolved Solids (TDS); whereas the presence of Giardia spp. had only a strong positive correlation with TDS. Molecular genotyping of FLA produced 7 Acanthamoeba, 5 Echinamoeba, 2 Hartmannella, 1 Bodomorpha, 1 Nuclearia and 1 Cercomonas partial sequences. All water collection sites were

  11. MSCs: The Sentinel and Safe-Guards of Injury.

    PubMed

    Caplan, Arnold I

    2016-07-01

    Mesenchymal stem cells (MSCs) were originally named because they could differentiate in a variety of mesenchymal phenotypes in culture. Evidence indicates that MSCs arise from perivascular cells, pericytes, when the blood vessels are broken or inflamed. These pericyte/MSCs are situated on every blood vessel in the body. The MSCs sense the micro-environment of the injury site and secrete site-specific factors that serve several important reparative functions: first, a curtain of molecules from the front of the MSCs provide a barrier from the interrogation of the over-aggressive immune system. Second, from the back of the MSCs, a different set of bioactive agents inhibit scar formation and establish a regenerative micro-environment. Third, if bacteria are sensed by the MSCs, they produce powerful protein antibiotics that kill the bacteria on contact. Last, the MSCs surround and encyst intruding solid objects like a piece of wood (a "splinter") or other foreign objects. The MSCs act as a combination paramedic and emergency room (ER) staff to survey the damage, isolate foreign components, stabilize the injured tissues, provide antibiotics and encysting protection before a slower, medicinal sequence can be initiated to regenerate the damaged tissue. The MSCs, thus, act as sentinels to safeguard the individual from intrusion and chronic injury. A societal treatment system has evolved, paramedics and ER procedures, which mirror in a macro-sense what MSCs orchestrate in a micro-sense. Key to this new understanding is that MSCs are not "stem cells," but rather as Medicinal Signaling Cells as the therapeutic agents. © 2015 Wiley Periodicals, Inc.

  12. Isolated Myocysticercosis.

    PubMed

    Vignesh, S; Bhole, Chetan; Bafna, Chander; Nirala, Neeraj; Prajapati, Mehul; Samar, Neera; Meena, R L

    2016-03-01

    Cysticercosis, a parasitic disease caused by larval form of Taenia solium, is a major health concern in the developing world. The encysted larval stage can affect any part of the body, but are most frequently detected in brain, eye, skeletal muscle and subcutaneous tissue. Most muscular cysticercosis are almost always associated with central nervous system involvement or with multiple intramuscular cysts or both. Here we report an unusual case of cysticercosis in right rectus muscle which was an isolated muscle involvement without any other systemic manifestation. © Journal of the Association of Physicians of India 2011.

  13. Taeniasis: A possible cause of ileal bleeding.

    PubMed

    Settesoldi, Alessia; Tozzi, Alessandro; Tarantino, Ottaviano

    2017-12-16

    Taenia spp. are flatworms of the class Cestoda, whose definitive hosts are humans and primates. Human infestation (taeniasis) results from the ingestion of raw meat contaminated with encysted larval tapeworms and is considered relatively harmless and mostly asymptomatic. Anemia is not recognized as a possible sign of taeniasis and taeniasis-induced hemorrhage is not described in medical books. Its therapy is based on anthelmintics such praziquantel, niclosamide or albendazole. Here we describe a case of acute ileal bleeding in an Italian man affected with both Taenia spp. infestation resistant to albendazole and Helicobacter pylori -associated duodenal ulcers.

  14. Detection of viable Helicobacter pylori inside free-living amoebae in wastewater and drinking water samples from Eastern Spain.

    PubMed

    Moreno-Mesonero, Laura; Moreno, Yolanda; Alonso, José Luis; Ferrús, M Antonia

    2017-10-01

    Helicobacter pylori is one of the most concerning emerging waterborne pathogens. It has been suggested that it could survive in water inside free-living amoebae (FLA), but nobody has studied this relationship in the environment yet. Thus, we aimed to detect viable H. pylori cells from inside FLA in water samples. Sixty-nine wastewater and 31 drinking water samples were collected. FLA were purified and identified by PCR and sequencing. For exclusively detecting H. pylori inside FLA, samples were exposed to sodium hypochlorite and assayed by specific PMA-qPCR, DVC-FISH and culture. FLA were detected in 38.7% of drinking water and 79.7% of wastewater samples, even after disinfection. In wastewater, Acanthamoeba spp. and members of the family Vahlkampfiidae were identified. In drinking water, Acanthamoeba spp. and Echinamoeba and/or Vermamoeba were present. In 39 (58.2%) FLA-positive samples, H. pylori was detected by PMA-qPCR. After DVC-FISH, 21 (31.3%) samples harboured viable H. pylori internalized cells. H. pylori was cultured from 10 wastewater samples. To our knowledge, this is the first report that demonstrates that H. pylori can survive inside FLA in drinking water and wastewater, strongly supporting the hypothesis that FLA could play an important role in the transmission of H. pylori to humans. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. Recognition of Naegleriae ameba surface protein epitopes by anti-human CD45 antibodies.

    PubMed

    Ravine, Terrence J; Polski, Jacek M; Jenkins, James

    2010-04-01

    Phagocytosis is a highly conserved mechanism exhibited by both free-living amebas and mammalian blood cells. Similarities demonstrated by either cell type during engulfment of the same bacterial species may imply analogous surface proteins involved in receptor-mediated endocytosis. The increased availability of anti-human leukocyte antibodies or clusters of differentiation (CD) markers used in conjunction with flow cytometric (FCM) and/or immunohistochemical (IHC) analysis provides investigators with a relatively easy method to screen different cell populations for comparable plasma membrane proteins. In this study, we incubated Naegleria and Acanthamoeba amebas with several directly conjugated anti-human leukocyte monoclonal antibodies (mAb) for similarly recognized amebic epitopes. CD marker selection was based upon a recognized role of each mAb in phagocyte activation and/or uptake of bacteria. These included CD14, CD45, and CD206. In FCM, only one CD45 antibody demonstrated strong reactivity with both Naegleria fowleri and Naegleria gruberi that was not expressed in similarly tested Acanthamoeba species. Additional testing of N. gruberi by IHC demonstrated reactivity to a different CD45 antibody. Our results suggest a possible utility of using anti-human leukocyte antibodies to screen amebic cells for similarly expressed protein epitopes. In doing so, several important items must be considered when selecting potential mAbs for testing to increase the probability of a positive result.

  16. Identification of free-living amoebae and amoeba-associated bacteria from reservoirs and water treatment plants by molecular techniques.

    PubMed

    Garcia, Alicia; Goñi, Pilar; Cieloszyk, Joanna; Fernandez, Maria Teresa; Calvo-Beguería, Laura; Rubio, Encarnacion; Fillat, Maria Francisca; Peleato, Maria Luisa; Clavel, Antonio

    2013-04-02

    The occurrence of free-living amoebae (FLA) was investigated in 83 water samples from reservoirs and water treatment plants, with culture positive in 64 of them (77.1%). Polymerase chain reaction (PCR) of partial 18S rRNA gene and ITS region was performed in order to identify amoeba isolates, and the presence of Legionella pneumophila , Mycobacterium spp., Pseudomonas spp., and Microcystis aeruginosa was investigated in 43 isolates of amoebae by multiplex PCR. Of the isolated amoebae, 31 were Acanthamoeba spp., 21 were Hartmannella vermiformis, 13 were Naegleria spp., and one was Vanella spp. T2, T4, and T5 genotypes of Acanthamoeba have been identified, and T4 isolates were grouped into five subgenotypes and graphically represented with a Weblog application. Inside amoebae, L. pneumophila was detected in 13.9% (6/43) of the isolates, and Pseudomonas spp. and Mycobacterium spp. were detected in 32.6% (14/43) and 41.9% (18/43), respectively. No statistical correlation was demonstrated between FLA isolation and seasonality, but the presence of intracellular bacteria was associated with warm water temperatures, and also the intracellular presence of Mycobacterium spp. and Pseudomonas spp. were associated. These results highlight the importance of amoebae in natural waters as reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

  17. Artyfechinostomum malayanum: Metacercariae Encysted in Pila sp. Snails Purchased from Phnom Penh, Cambodia

    PubMed Central

    Sohn, Woon-Mok; Yong, Tai-Soon; Eom, Keeseon S.; Sinuon, Muth; Jeoung, Hoo-Gn; Chai, Jong-Yil

    2017-01-01

    The metacercariae of Artyfechinostomum malayanum (Leiper, 1911) Mendheim, 1943 were discovered in Pila sp. snails purchased from a market in Phnom Penh, Cambodia. They were isolated from the snails using the artificial digestion technique and were orally fed to 2 hamsters, 1 rat, and 2 mice to obtain the adult flukes. The metacercariae were round, 145–165 μm in diameter, having a cyst wall of 6–10 μm in thickness, a head collar and collar spines, and characteristic features of excretory granules. Adult flukes were recovered in the small intestines of the animals at days 14 and 32 post infection and were morphologically observed using a light microscope and a scanning electron microscope. They were plump or elongated, ventrally curved, 6.0–8.1×1.6–2.0 mm in size, and characterized by the head collar bearing 43 collar spines, including 5 end group ones on each side, a long cirrus sac extending beyond the posterior margin of the ventral sucker, a submedian ovary, and 2 deeply lobed testes. Eggs in uteri were operculate, ovoid to ellipsoid, and 120–135×68–75 μm in size. In scanning electron microscopy, the head collar was prominent with collar spines looking like horns. Scale-like tegumental spines were densely distributed on the ventral surface between the head collar and ventral sucker. Sensory papillae were distributed mainly on the tegument around suckers. By this study, it has been first confirmed that the life cycle of A. malayanum exists in Cambodia. PMID:28719961

  18. Molecular Detection of Legionella spp. and their associations with Mycobacterium spp., Pseudomonas aeruginosa and amoeba hosts in a drinking water distribution system

    EPA Pesticide Factsheets

    Quantity of Legionella spp., Mycobacterium spp., Acanthamoeba,Vermamoeba vermiformis and Pseudomonas aeruginosa were estimated using qPCR methods.This dataset is associated with the following publication:Lu , J., I. Struewing, E. Vereen, A.E. Kirby, K. Levy, C. Moe, and N. Ashbolt. Molecular detection of Legionella spp. and their associations with Mycobacterium spp., Pseudomonas aeruginosa and amoeba hosts in a drinking water distribution system (Journal Article). JOURNAL OF APPLIED MICROBIOLOGY. Blackwell Publishing, Malden, MA, USA, 120(2): 509-521, (2016).

  19. Control of Cryptococcus neoformans in nature by biotic factors.

    PubMed

    Ruiz, A; Neilson, J B; Bulmer, G S

    1982-03-01

    Two bacterial species (Pseudomonas aeruginosa and Bacillus subtilis) isolated from pigeon droppings, displayed anti Cryptococcus neoformans activity on 4 of 6 media and sterilized pigeon droppings. Acanthamoeba palestinensis trophozoites isolated from pigeon droppings ingested and killed 99.9% of C neoformans cells after 7 days of incubation. Mites and sow bugs (Metoponorthus pruinosus) isolated from pigeon droppings appear to be fungivorous. These findings suggest that many organisms that occur in pigeon droppings influence C. neoformans persistence, reproduction, morphology and distribution in nature.

  20. Experimental life history and biological characteristics of Fasciola gigantica (Digenea: Fasciolidae).

    PubMed

    Phalee, Anawat; Wongsawad, Chalobol; Rojanapaibul, Amnat; Chai, Jong-Yil

    2015-02-01

    This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for maturation study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cercariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the average recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that experimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study reports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained information can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts.

  1. Experimental Life History and Biological Characteristics of Fasciola gigantica (Digenea: Fasciolidae)

    PubMed Central

    Phalee, Anawat; Wongsawad, Chalobol; Rojanapaibul, Amnat; Chai, Jong-Yil

    2015-01-01

    This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for maturation study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cercariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the average recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that experimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study reports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained information can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts. PMID:25748710

  2. Location on chitin in the cyst wall of Entamoeba invadens with colloidal gold tracers.

    PubMed

    Arroyo-Begovich, A; Cárabez-Trejo, A

    1982-04-01

    Chitin was located in the cyst wall of Entamoeba invadens with colloidal gold-linked wheat germ agglutinin. Cysts stained differentially from trophozoites when encysting cultures were treated with the gold tracer; cysts acquired a wine-red coloration while, in general trophozoites remained unstained. Observation of cells with the electron microscope revealed that the tracer particles were bound specifically to the walls of the surface of the cyst when cells were exposed in suspension, and to the cyst wall cross-section, when cells were exposed to the tracer in thin section, indicating that chitin fibers were distributed on the surface as well as throughout the matrix of the cyst wall.

  3. The First Record for the Americas of Loxodes rex, a Flagship Ciliate with an Alleged Restricted Biogeography.

    PubMed

    Hines, Hunter N; McCarthy, Peter J; Esteban, Genoveva F

    2016-01-01

    As the foundations of food webs, protozoa are essential to the success of an ecological system. These organisms are often overlooked, and research in the Americas is sparse. Recent samplings conducted in freshwater canals and ponds in Florida, USA, have revealed Loxodes rex, an alleged endemic ciliate species. Originally described as endemic to tropical Africa, L. rex has been considered a prime candidate for proof of microbial endemism. Our studies have shown this giant, non-encysting ciliate to be thriving in subtropical Florida. Our observations are novel and include both the first record of occurrence for the Americas and the first high-quality in vivo images for this charismatic species.

  4. Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants.

    PubMed

    Corsaro, Daniele; Pages, Gemma Saucedo; Catalan, Vicente; Loret, Jean-François; Greub, Gilbert

    2010-06-01

    In this study, we enlarged our previous investigation focusing on the biodiversity of chlamydiae and amoebae in a drinking water treatment plant, by the inclusion of two additional plants and by searching also for the presence of legionellae and mycobacteria. Autochthonous amoebae were recovered onto non-nutritive agar, identified by 18S rRNA gene sequencing, and screened for the presence of bacterial endosymbionts. Bacteria were also searched for by Acanthamoeba co-culture. From a total of 125 samples, we recovered 38 amoebae, among which six harboured endosymbionts (three chlamydiae and three legionellae). In addition, we recovered by amoebal co-culture 11 chlamydiae, 36 legionellae (no L. pneumophila), and 24 mycobacteria (all rapid-growers). Two plants presented a similar percentage of samples positive for chlamydiae (11%), mycobacteria (20%) and amoebae (27%), whereas in the third plant the number of recovered bacteria was almost twice higher. Each plant exhibited a relatively high specific microbiota. Amoebae were mainly represented by various Naegleria species, Acanthamoeba species and Hartmannella vermiformis. Parachlamydiaceae were the most abundant chlamydiae (8 strains in total), and in this study we recovered a new genus-level strain, along with new chlamydiae previously reported. Similarly, about 66% of the recovered legionellae and 47% of the isolated mycobacteria could represent new species. Our work highlighted a high species diversity among legionellae and mycobacteria, dominated by putative new species, and it confirmed the presence of chlamydiae in these artificial water systems. Copyright 2010 Elsevier GmbH. All rights reserved.

  5. Some secrets are revealed: parasitic keratitis amoebae as vectors of the scarcely described pandoraviruses to humans.

    PubMed

    Scheid, Patrick; Balczun, Carsten; Schaub, Günter A

    2014-10-01

    In this article, the results of a long effort to derive valuable phylogenetic data about an extraordinary spore-like infectious particle (endocytobiont) within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens and the inflamed eye of a patient with keratitis are presented. The development of these endocytobionts has already been demonstrated with electron microscopic photo sequences, leading to a relevant model of its development presented here. The molecular biological investigation following the discovery of two other Pandoravirus species within aquatic sediments in 2013 led to the taxonomic affiliation of our endocytobiont with the genus Pandoravirus. A range of endocytobionts (intracellular biofilms) have been found in recent years, among which are several viruses which obligatorily proliferate within free-living amoebae. In human medicine, foreign objects which are placed in or on humans cause problems with microorganisms in biofilms. Contact lenses are especially important, because they are known as a source of a rapid formation of biofilm. These were the first Pandoraviruses described, and because this is additionally the first documented association with humans, we have clearly demonstrated how easily such (viral) endocytobionts can be transferred to humans. This case counts as an example of parasites acting as vectors of phylogenetically different microorganisms especially when living sympatric within their biocoenosis of biofilms. As the third part of the "Pandoravirus trilogy", it finally reveals the phylogenetic nature of these "extraordinary endocytobionts" within Acanthamoebae.

  6. Effect of disinfectant, water age, and pipe material on occurrence and persistence of Legionella, mycobacteria, Pseudomonas aeruginosa, and two amoebas.

    PubMed

    Wang, Hong; Masters, Sheldon; Hong, Yanjuan; Stallings, Jonathan; Falkinham, Joseph O; Edwards, Marc A; Pruden, Amy

    2012-11-06

    Opportunistic pathogens represent a unique challenge because they establish and grow within drinking water systems, yet the factors stimulating their proliferation are largely unknown. The purpose of this study was to examine the influence of pipe materials, disinfectant type, and water age on occurrence and persistence of three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa), broader genera (Legionella and mycobacteria), and two amoeba hosts (Acanthamoeba spp. and Hartmanella vermiformis). Triplicate simulated distribution systems (SDSs) compared iron, cement, and PVC pipe materials fed either chlorinated or chloraminated tap water and were sampled at water ages ranging from 1 day to 5.7 days. Quantitative polymerase chain reaction quantified gene copies of target microorganisms in both biofilm and bulk water. Legionella, mycobacteria, P. aeruginosa, and both amoebas naturally colonized the six SDSs, but L. pneumophila and M. avium were not detected. Disinfectant type and dose was observed to have the strongest influence on the microbiota. Disinfectant decay was noted with water age, particularly in chloraminated SDSs (due to nitrification), generally resulting in increased microbial detection frequencies and densities with water age. The influence of pipe material became apparent at water ages corresponding to low disinfectant residual. Each target microbe appeared to display a distinct response to disinfectant type, pipe materials, water age, and their interactions. Differences between the first and the second samplings (e.g., appearance of Legionella, reduction in P. aeruginosa and Acanthamoeba) suggest a temporally dynamic drinking water microbial community.

  7. Free-living protozoa in the gastrointestinal tract and feces of pigs: Exploration of an unknown world and towards a protocol for the recovery of free-living protozoa.

    PubMed

    Chavatte, N; Lambrecht, E; Van Damme, I; Sabbe, K; Houf, K

    2016-07-30

    Associations with free-living protozoa (FLP) have been implicated in the persistence of foodborne pathogenic bacteria in food-related environments. To date however no information is available on the presence of FLP in the gastrointestinal tract (GIT) of pigs, which represents an important reservoir for zoonotic foodborne bacteria and hence a potential location for associations with FLP. This is at least partly due to the lack of adequate protocols to recover FLP from intestinal content and feces. In the present study different protocols to recover FLP from the porcine GIT and feces were tested. The most effective protocols were then applied to explore the presence of live FLP in the pig GIT and feces. A filtration based protocol was identified as the most suitable method to recover viable FLP from the porcine GIT and feces. Cultivable FLP were recovered from different parts of the GIT, suggesting at least a transient presence of FLP in this habitat. Free-living amoebae species (Acanthamoeba spp., Hyperamoeba sp., Vannella sp., Vermamoeba vermiformis, hartmannellids and vahlkampfiids) but also ciliates (Colpoda sp. and Tetrahymena/Glaucoma lookalike) and flagellates (cercomonads, bodonids and glissomonads) were recovered and cultured from pig intestinal content. Acanthamoeba hatchetti and Filamoeba sinensis were isolated for the first time from pig intestinal content. Despite high gastric acidity, non-cyst forming amoeba species were also detected which suggests survival of their trophozoites in the animal GIT. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Brain-Eating Amoebae: Predilection Sites in the Brain and Disease Outcome

    PubMed Central

    Ong, Timothy Yu Yee; Siddiqui, Ruqaiyyah

    2017-01-01

    ABSTRACT Acanthamoeba spp. and Balamuthia mandrillaris are causative agents of granulomatous amoebic encephalitis (GAE), while Naegleria fowleri causes primary amoebic meningoencephalitis (PAM). PAM is an acute infection that lasts a few days, while GAE is a chronic to subacute infection that can last up to several months. Here, we present a literature review of 86 case reports from 1968 to 2016, in order to explore the affinity of these amoebae for particular sites of the brain, diagnostic modalities, treatment options, and disease outcomes in a comparative manner. PMID:28404683

  9. Entamoeba mitosomes play an important role in encystation by association with cholesteryl sulfate synthesis

    PubMed Central

    Mi-ichi, Fumika; Miyamoto, Tomofumi; Takao, Shouko; Jeelani, Ghulam; Hashimoto, Tetsuo; Hara, Hiromitsu; Nozaki, Tomoyoshi; Yoshida, Hiroki

    2015-01-01

    Hydrogenosomes and mitosomes are mitochondrion-related organelles (MROs) that have highly reduced and divergent functions in anaerobic/microaerophilic eukaryotes. Entamoeba histolytica, a microaerophilic, parasitic amoebozoan species, which causes intestinal and extraintestinal amoebiasis in humans, possesses mitosomes, the existence and biological functions of which have been a longstanding enigma in the evolution of mitochondria. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. However, because the final metabolites of sulfate activation remain unknown, the overall scheme of this metabolism and the role of mitosomes in Entamoeba have not been elucidated. In this study we purified and identified cholesteryl sulfate (CS) as a final metabolite of sulfate activation. We then identified the gene encoding the cholesteryl sulfotransferase responsible for synthesizing CS. Addition of CS to culture media increased the number of cysts, the dormant form that differentiates from proliferative trophozoites. Conversely, chlorate, a selective inhibitor of the first enzyme in the sulfate-activation pathway, inhibited cyst formation in a dose-dependent manner. These results indicate that CS plays an important role in differentiation, an essential process for the transmission of Entamoeba between hosts. Furthermore, we show that Mastigamoeba balamuthi, an anaerobic, free-living amoebozoan species, which is a close relative of E. histolytica, also has the sulfate-activation pathway in MROs but does not possess the capacity for CS production. Hence, we propose that a unique function of MROs in Entamoeba contributes to its adaptation to its parasitic life cycle. PMID:25986376

  10. Optimized DNA extraction methods for encysted embryos of the endangered fairy shrimp, Branchinecta sandiegonensis

    USGS Publications Warehouse

    Steele, A.N.; Simovich, M.A.; Pepino, D.; Schroeder, K.M.; Vandergast, A.G.; Bohonak, A.J.

    2009-01-01

    The San Diego fairy shrimp Branchinecta sandiegonensis is a federally endangered species endemic to vernal pools in southern California, USA. Filling events in these habitats are highly variable, with some pools failing to hold water long enough for reproduction over many successive years. Studies of this species are thus hindered by the relatively rare appearance of aquatically active life history phases. Because diapausing cysts are abundant and present at all times, they provide an underutilized opportunity for both species identification and genetic studies. However, methods for extracting DNA from cysts are technically challenging because of their structure and size. Here we present a protocol for extracting DNA from B. sandiegonensis cysts in sufficient quantities for "DNA Barcoding", microsatellite analysis and other genotyping and sequencing applications. The technique will aid in population genetic studies and species identification (since taxonomic keys only distinguish among adults), and will be applicable to other crustaceans with similar diapausing cysts. ?? Springer Science+Business Media B.V. 2008.

  11. Putative SF2 helicases of the early-branching eukaryote Giardia lamblia are involved in antigenic variation and parasite differentiation into cysts

    PubMed Central

    2012-01-01

    Background Regulation of surface antigenic variation in Giardia lamblia is controlled post-transcriptionally by an RNA-interference (RNAi) pathway that includes a Dicer-like bidentate RNase III (gDicer). This enzyme, however, lacks the RNA helicase domain present in Dicer enzymes from higher eukaryotes. The participation of several RNA helicases in practically all organisms in which RNAi was studied suggests that RNA helicases are potentially involved in antigenic variation, as well as during Giardia differentiation into cysts. Results An extensive in silico analysis of the Giardia genome identified 32 putative Super Family 2 RNA helicases that contain almost all the conserved RNA helicase motifs. Phylogenetic studies and sequence analysis separated them into 22 DEAD-box, 6 DEAH-box and 4 Ski2p-box RNA helicases, some of which are homologs of well-characterized helicases from higher organisms. No Giardia putative helicase was found to have significant homology to the RNA helicase domain of Dicer enzymes. Additionally a series of up- and down-regulated putative RNA helicases were found during encystation and antigenic variation by qPCR experiments. Finally, we were able to recognize 14 additional putative helicases from three different families (RecQ family, Swi2/Snf2 and Rad3 family) that could be considered DNA helicases. Conclusions This is the first comprehensive analysis of the Super Family 2 helicases from the human intestinal parasite G. lamblia. The relative and variable expression of particular RNA helicases during both antigenic variation and encystation agrees with the proposed participation of these enzymes during both adaptive processes. The putatives RNA and DNA helicases identified in this early-branching eukaryote provide initial information regarding the biological role of these enzymes in cell adaptation and differentiation. PMID:23190735

  12. Characterization of a Rab11-like GTPase, EhRab11, of Entamoeba histolytica.

    PubMed

    McGugan, Glen C; Temesvari, Lesly A

    2003-07-01

    The Entamoeba histolytica Rab11 family of small molecular weight GTPases consists of three members, EhRab11, EhRab11B, and EhRab11C. The functions of these Rabs in Entamoeba have not been determined. Therefore, as an approach to elucidate the role of the Rab11 family of GTPases in Entamoeba, immunofluorescence microscopy was undertaken to define the subcellular localization of one member of this family, EhRab11. Under conditions of growth, EhRab11 displayed a punctate pattern in the cytoplasm of trophozoites. EhRab11 did not colocalize with markers for the Golgi apparatus, endoplasmic reticulum, pinosomes, phagosomes, or compartments formed by receptor-mediated endocytosis, suggesting that this Rab may not play a role in vesicle trafficking between these organelles. Under conditions of iron and serum starvation, EhRab11 was translocated to the periphery of the cell. The altered cellular localization was accompanied by multinucleation of the cells as well as the acquisition of detergent resistance by the cells, features that are characteristic of Entamoeba cysts. The translocation of EhRab11 to the periphery of the cell during iron and serum starvation was specific as the subcellular localizations of two other Rab GTPases, EhRab7 and EhRabA, were not altered under the same conditions. In addition, the formation of multinucleated cells by inhibition of cytokinesis was not sufficient to induce the translocation of EhRab11 to the cell periphery. Taken together, the data suggest that iron and serum starvation may induce encystation in E. histolytica and that EhRab11 may play a role in this process. Moreover, these studies are the first to describe a putative role for a Rab GTPase in encystation in Entamoeba sp.

  13. Pseudocollinia brintoni gen. nov., sp. nov. (Apostomatida: Colliniidae), a parasitoid ciliate infecting the euphausiid Nyctiphanes simplex.

    PubMed

    Gómez-Gutiérrez, J; Strüder-Kypke, M C; Lynn, D H; Shaw, T C; Aguilar-Méndez, M J; López-Cortés, A; Martínez-Gómez, S; Robinson, C J

    2012-05-15

    A novel parasitoid ciliate, Pseudocollinia brintoni gen. nov., sp. nov. was discovered infecting the subtropical sac-spawning euphausiid Nyctiphanes simplex off both coasts of the Baja California peninsula, Mexico. We used microscopic, and genetic information to describe this species throughout most of its life cycle. Pseudocollinia is distinguished from other Colliniidae genera because it exclusively infects euphausiids, has a polymorphic life cycle, and has a small cone-shaped oral cavity whose left wall has a field of ciliated kinetosomes and whose opening is surrounded on the left and right by 2 'oral' kineties (or ciliary rows) that terminate at its anterior border. Two related species that infect different euphausiid species from higher latitudes in the northeastern Pacific Ocean, Collinia beringensis Capriulo and Small, 1986, briefly redescribed herein, and Collinia oregonensis Gómez-Gutiérrez, Peterson, and Morado, 2006, are transferred to the genus Pseudocollinia. P. brintoni has between 12 and 18 somatic kineties, and its oral cavity has only 2 oral kineties, while P. beringensis comb. nov. has more somatic kineties, including 3 oral kineties. P. oregonensis comb. nov. has an intermediate number of somatic kineties. P. beringensis comb. nov. also infects Thysanoessa raschi (a new host species). SSU rRNA and cox1 gene sequences demonstrated that Pseudocollinia ciliates are apostome ciliates and that P. brintoni is different from P. beringensis comb. nov. High densities of rod-shaped bacteria (1.7 µm length, 0.2 to 0.5 µm diameter) were associated with P. brintoni. After euphausiid rupture, high concentrations of P. brintoni and bacteria cluster to form 3 to 6 cm long filaments where tomites encyst and transform to the phoront stage; this is a novel place for encystation. P. brintoni may complete its life cycle when the euphausiids feed on these filaments.

  14. Alternative life-history and transmission strategies in a parasite: first come, first served?

    PubMed

    Poulin, R; Lefebvre, F

    2006-01-01

    Alternative transmission strategies are common in many parasitic organisms, often representing discrete phenotypes adopted in response to external cues. The facultative truncation of the normal 3-host life-cycle to a 2-host cycle in many trematodes provides an example: some individuals mature precociously, via progenesis, in their intermediate host and produce eggs without the need to reach a definitive host. The factors that determine how many and which individuals adopt the truncated life-cycle within a parasite population remain unknown. We investigated the occurrence of progenesis in the trematode Stegodexamene anguillae within its fish intermediate host. Location within the host was a key determinant of progenesis. Although the size and egg output of progenetic metacercariae encysted in host gonads did not differ from those of the few progenetic metacercariae in other host tissues, the likelihood of metacercariae becoming progenetic was much higher for those in the gonads than those elsewhere in the host. Progenetic parasites can only evacuate their eggs along with host eggs or sperm, providing a link between the parasite's transmission strategy and its location in the host. Host size and sex, and the presence of other parasite species in the host, did not affect the occurrence of progenesis in S. anguillae. However, the proportion of metacercariae in host gonads and the proportion of progenetic metacercariae both decreased with increasing numbers of S. anguillae per host. These results suggest that progenesis is adopted mostly by the parasites that successfully establish in host gonads. These are generally the first to infect a fish; subsequent arrivals settle in other tissues as the gonads quickly become saturated with parasites. In this system, the site of encystment within the fish host both promotes and constrains the adoption of a facultative, truncated life-cycle by the parasite.

  15. Ectopic fascioliasis mimicking a colon tumor

    PubMed Central

    Makay, Ozer; Gurcu, Baris; Caliskan, Cemil; Nart, Deniz; Tuncyurek, Muge; Korkut, Mustafa

    2007-01-01

    Fasciola hepatica, a leaf shaped trematode that is common in cattle, sheep and goats, is acquired by eating raw water plants like watercress or drinking water infected with the encysted form of the parasite. The varied clinical presentations of fascioliasis still make a high index of suspicion mandatory. Besides having a wide spectrum of hepatobiliary symptoms like obstructive jaundice, cholangitis and liver cirrhosis, the parasitic infection also has extrabiliary manifestations. Until recently, extrahepatic fascioliasis has been reported in the subcutaneous tissue, brain, lungs, epididymis, inguinal lymph nodes, stomach and the cecum. In this report, a strange manifestation of the fasciola infection in a site other than the liver, a colonic fascioliasis, is presented. PMID:17552017

  16. Waterborne parasites and physico-chemical assessment of selected lakes in Malaysia.

    PubMed

    Onichandran, Subashini; Kumar, Thulasi; Lim, Yvonne A L; Sawangjaroen, Nongyao; Andiappan, Hemah; Salibay, Cristina C; Chye, Tan Tian; Ithoi, Init; Dungca, Julieta Z; Sulaiman, Wan Y W; Ling, Lau Yee; Nissapatorn, Veeranoot

    2013-12-01

    The objective of this study was to assess the physico-chemical parameters and waterborne parasites in selected recreational lakes from Malaysia. Samples were collected from seven stations of Recreational Lake A (RL-A) and six stations of Recreational Lake B (RL-B). The samples were processed to detect the presence of Giardia spp. and Cryptosporidium spp. using immunomagnetic separation kit, helminth eggs or ova by bright field microscopy and Acanthamoeba spp. by cultivation in non-nutrient agar. Chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite and physical parameters such as dissolved oxygen, electrical conductivity, pH, salinity, temperature and total dissolved solid were also measured. Both lakes were freshwater with salinity ranging from 0.05 to 0.09 ppt. Most stations of these lakes were contaminated with Cryptosporidium spp., Giardia spp., Ascaris spp. and hookworm. Schistosoma spp. was found in RL-B only, while Acanthamoeba spp. was found in all stations. Of all sampling sites, station 5 of RL-B is the most contaminated. Linear regression and correlation analysis revealed that Giardia spp. and Schistosoma spp. showed a significant negative correlation with turbidity (p < 0.01). Based on the preliminary data obtained, it is clearly shown that there is a necessity to implement the detection of waterborne parasites and physico-chemical analysis in Malaysia. Future work on heavy metals (chromium, copper, mercury and zinc) is recommended to enhance the overall water quality monitoring and to take appropriate safety measures to ensure maintenance of good water standards.

  17. Microbial keratitis in overnight orthokeratology: review of the first 50 cases.

    PubMed

    Watt, Kathleen; Swarbrick, Helen A

    2005-09-01

    Despite growing evidence for clinical efficacy of orthokeratology (OK) for the temporary reduction of myopic refractive error, there has been an increasing number of reports of microbial keratitis (MK) in association with overnight wear of OK lenses. This article analyzes the first 50 cases of MK reported in overnight OK, in order to define the spectrum of the disease and to identify possible risk factors. All reported cases of presumed MK in overnight OK from 2001 onwards were included in the analysis. Demographic data of patients affected and lenses worn, and details of the disease process and possible risk factors were extracted from these reports. Most cases of MK in OK were reported from East Asia (80%) and most affected patients were Asian (88%). The peak age range was from 9 to 15 years (61%). Although Pseudomonas aeruginosa was the predominant organism implicated in this series of cases (52%), an alarmingly high frequency of Acanthamoeba infection (30%) was found. Inappropriate lens care procedures, patient noncompliance with practitioner instructions, and persisting in lens wear despite discomfort emerged as potential risk factors. The high frequency of MK in overnight OK in young Asian patients is likely to reflect the demographics of the OK lens-wearing population. The high frequency of Acanthamoeba infection strongly suggests that tap water rinsing should be eliminated from the lens care regimen for overnight OK. This study does not reveal the absolute incidence or relative risk of MK in overnight OK, and it is therefore premature to ascribe increased risk to this lens-wearing modality compared with other contact lens modalities.

  18. Amoebae, Giant Viruses, and Virophages Make Up a Complex, Multilayered Threesome

    PubMed Central

    Diesend, Jan; Kruse, Janis; Hagedorn, Monica; Hammann, Christian

    2018-01-01

    Viral infection had not been observed for amoebae, until the Acanthamoeba polyphaga mimivirus (APMV) was discovered in 2003. APMV belongs to the nucleocytoplasmatic large DNA virus (NCLDV) family and infects not only A. polyphaga, but also other professional phagocytes. Here, we review the Megavirales to give an overview of the current members of the Mimi- and Marseilleviridae families and their structural features during amoebal infection. We summarize the different steps of their infection cycle in A. polyphaga and Acanthamoeba castellani. Furthermore, we dive into the emerging field of virophages, which parasitize upon viral factories of the Megavirales family. The discovery of virophages in 2008 and research in recent years revealed an increasingly complex network of interactions between cell, giant virus, and virophage. Virophages seem to be highly abundant in the environment and occupy the same niches as the Mimiviridae and their hosts. Establishment of metagenomic and co-culture approaches rapidly increased the number of detected virophages over the recent years. Genetic interaction of cell and virophage might constitute a potent defense machinery against giant viruses and seems to be important for survival of the infected cell during mimivirus infections. Nonetheless, the molecular events during co-infection and the interactions of cell, giant virus, and virophage have not been elucidated, yet. However, the genetic interactions of these three, suggest an intricate, multilayered network during amoebal (co-)infections. Understanding these interactions could elucidate molecular events essential for proper viral factory activity and could implicate new ways of treating viruses that form viral factories. PMID:29376032

  19. [IMPACT OF CASPIAN SEA LEVEL FLUCTUATIONS ON THE EPIZOOTIC ACTIVITY OF THE CASPIAN SANDY NATURAL PLAGUE FOCUS].

    PubMed

    Popov, N V; Udovikov, A I; Eroshenko, G A; Karavaeva, T B; Yakovlev, S A; Porshakov, A M; Zenkevich, E S; Kutyrev, V V

    2016-01-01

    There is evidence that in 1923-2014 the sharp aggravations of the epizootic situation of plague in the area of its Caspian sandy natural focus after long interepizootic periods are in time with the ups of the Caspian Sea in the extrema of 11-year solar cycles. There were cases of multiple manifestations of plague in the same areas in the epizootic cycles of 1946-1954, 1979-1996, 2001, and 2013-2014. The paper considers the possible role of amebae of the genus Acanthamoeba and nematodes, the representatives of the orders Rhabditida and Tylenchida in the microfocal pattern of plague manifestations.

  20. Microbial keratitis in South India: influence of risk factors, climate, and geographical variation.

    PubMed

    Bharathi, M Jayahar; Ramakrishnan, R; Meenakshi, R; Padmavathy, S; Shivakumar, C; Srinivasan, M

    2007-01-01

    To determine the influence of risk factors, climate, and geographical variation on the microbial keratitis in South India. A retrospective analysis of all clinically diagnosed infective keratitis presenting between September 1999 and August 2002 was performed. A standardised form was filled out for each patient, documenting sociodemographic features and information pertaining to risk factors. Corneal scrapes were collected and subjected to culture and microscopy. A total of 3,183 consecutive patients with infective keratitis were evaluated, of which 1,043 (32.77%) were found to be of bacterial aetiology, 1,095 (34.4%) were fungal, 33 (1.04%) were Acanthamoeba, 76 (2.39%) were both fungal and bacterial, and the remaining 936 (29.41%) were found to be culture negative. The predominant bacterial and fungal pathogens isolated were Streptococcus pneumoniae (35.95%) and Fusarium spp. (41.92%), respectively. Most of the patients (66.84%) with fungal keratitis were between 21 and 50 years old, and 60.21% of the patients with bacterial keratitis were older than 50 (p < 0.0001) (95% CI: 5.19-7.19). A majority of patients (64.75%) with fungal keratitis were agricultural workers (p < 0.0001) [odds ratio (OR): 1.4; 95% CI: 1.19-1.61], whereas bacterial keratitis occurred more commonly (57.62%) in nonagricultural workers (p < 0.0001) (OR: 2.88; 95% CI: 2.47-3.36). Corneal injury was identified in 2,256 (70.88%) patients, and it accounted for 92.15% in fungal keratitis (p < 0.0001) (OR: 7.7; 95% CI: 6.12-9.85) and 100% in Acanthamoeba keratitis. Injuries due to vegetative matter (61.28%) were identified as a significant cause for fungal keratitis (p < 0.0001) (OR: 23.6; 95% CI: 19.07-29.3) and due to mud (84.85%) for Acanthamoeba keratitis (p < 0.0001) (OR: 26.01; 95% CI: 3.3-6.7). Coexisting ocular diseases predisposing to bacterial keratitis accounted for 68.17% (p < 0.0001) (OR: 33.99; 95% CI: 27.37-42.21). The incidence of fungal keratitis was higher between June and

  1. Exposure to synthetic greywater inhibits amoebae encystation and alters expression of Legionella pneumophila virulence genes

    EPA Science Inventory

    Water conservation efforts have focused on greywater (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoebae (FLA) hosts within GW. Using synthetic gre...

  2. Third-stage larvae of the enoplid nematode Dioctophyme renale (Goeze, 1782) in the freshwater turtle Trachemys dorbigni from southern Brazil.

    PubMed

    Mascarenhas, C S; Müller, G

    2015-09-01

    The giant kidney worm Dioctophyme renale is normally found in wild carnivores and domestic dogs, with aquatic oligochaetes acting as intermediate hosts. In the present study a prevalence of 50% of third-stage larvae of D. renale was recorded in 60 specimens of the freshwater turtle Trachemys dorbigni from southern Brazil. Larvae were encysted in muscles, the coelomic cavity and mesentery, the serous lining of the stomach and on the surfaces of the lung, heart, liver, pancreas, spleen and intestines. There are no previous records of reptiles being part of the life cycle of D. renale, although fish and amphibians normally act as paratenic hosts. This is the first report of third-stage D. renale larvae in the freshwater turtle, T. dorbigni.

  3. Morphological and molecular identification of the fish-borne metacercaria of Ascocotyle (Phagicola) longa Ransom, 1920 in Mugil liza from Argentina.

    PubMed

    Martorelli, S R; Lino, A; Marcotegui, P; Montes, M M; Alda, P; Panei, C J

    2012-12-21

    This is the first report of Ascocotyle (Phagicola) longa Ransom, 1920 (Digenea: Heterophyidae) in Argentina confirmed by morphological and molecular studies. The metacercaria was found encysted in myotomal musculature, heart and mesentery of the mullet Mugil liza (Pisces: Mugilidae) from Samborombon bay. We provide a morphological description of the metacercaria which we identified using species-specific primers for A. (Phagicola) longa and nucleotid sequence. This worldwide parasite has been reported as one of the causative agents of heterophyiosis, an emerging fish-borne disease of humans, contracted by the consumption of raw mullet. The discovery of A. (Phagicola) longa in Argentina represents a warning of the potentially great impact of this parasite on public health. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Twelve-year analysis of microbial keratitis trends at a UK tertiary hospital.

    PubMed

    Tan, S Z; Walkden, A; Au, L; Fullwood, C; Hamilton, A; Qamruddin, A; Armstrong, M; Brahma, A K; Carley, F

    2017-08-01

    PurposeTo investigate the frequencies, trends, and in vitro drug susceptibilities of the causative pathogens in microbial keratitis in Manchester Royal Eye Hospital.Patients and methodsCorneal scrape results recorded by the microbiology service between 2004 and 2015 were extracted from an established database. A total of 4229 corneal scrape specimens were identified from an established database. First-line antibiotic treatment in our centre during the study period was ofloxacin and second line was cefuroxime and gentamicin.ResultsMean age was 45.9±21.0. A total of 1379 samples (32.6%) were culture positive. One hundred forty-eight (10.7%) specimens cultured multiple organisms. Of the 1539 organisms identified, 63.3% were Gram-positive bacteria, 27.3% Gram-negative bacteria, 7.1% fungi, and 2.3% Acanthamoebae. A decreasing trend in Gram-positive isolates was found together with a stable trend in Gram negatives and an increasing trend in Acanthamoeba and fungi. There appeared to be a significant increasing trend of Moraxella infection (P=0.001). In all, 83.1 and 90.8% of Gram-positive and -negative isolates tested were susceptible to ofloxacin, respectively. Cefuroxime covered 86.6% of Gram-positive and 61.4% of Gram-negative isolates, whereas gentamicin covered 88.8 and 96.5% of Gram-positive and -negative isolates, respectively.ConclusionWe found a change in the type of Gram-negative organisms isolated over time, with the Moraxella species on the rise. Reassuringly, no significant increase in resistance was observed in vitro for any of the commonly used antibiotics. Ofloxacin remains a good first-line antibiotic treatment but duo-therapy does have broader coverage and should be considered in non-responsive cases.

  5. Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae

    PubMed Central

    Arslan, Defne; Legendre, Matthieu; Seltzer, Virginie; Abergel, Chantal; Claverie, Jean-Michel

    2011-01-01

    Mimivirus, a DNA virus infecting acanthamoeba, was for a long time the largest known virus both in terms of particle size and gene content. Its genome encodes 979 proteins, including the first four aminoacyl tRNA synthetases (ArgRS, CysRS, MetRS, and TyrRS) ever found outside of cellular organisms. The discovery that Mimivirus encoded trademark cellular functions prompted a wealth of theoretical studies revisiting the concept of virus and associated large DNA viruses with the emergence of early eukaryotes. However, the evolutionary significance of these unique features remained impossible to assess in absence of a Mimivirus relative exhibiting a suitable evolutionary divergence. Here, we present Megavirus chilensis, a giant virus isolated off the coast of Chile, but capable of replicating in fresh water acanthamoeba. Its 1,259,197-bp genome is the largest viral genome fully sequenced so far. It encodes 1,120 putative proteins, of which 258 (23%) have no Mimivirus homologs. The 594 Megavirus/Mimivirus orthologs share an average of 50% of identical residues. Despite this divergence, Megavirus retained all of the genomic features characteristic of Mimivirus, including its cellular-like genes. Moreover, Megavirus exhibits three additional aminoacyl-tRNA synthetase genes (IleRS, TrpRS, and AsnRS) adding strong support to the previous suggestion that the Mimivirus/Megavirus lineage evolved from an ancestral cellular genome by reductive evolution. The main differences in gene content between Mimivirus and Megavirus genomes are due to (i) lineages specific gains or losses of genes, (ii) lineage specific gene family expansion or deletion, and (iii) the insertion/migration of mobile elements (intron, intein). PMID:21987820

  6. High intensity and prevalence of two species of trematode metacercariae in the fathead minnow (Pimephales promelas) with no compromise of minnow anti-predator competence.

    PubMed

    Wisenden, Brian D; Martinez-Marquez, Jorge Y; Gracia, Emilia S; McEwen, Daniel C

    2012-08-01

    Opportunity for parasites to manipulate host behavioral phenotype may be influenced by several factors, including the host ecology and the presence of cohabiting parasites in the same host. Metacercariae of Ornithodiplostomum ptychocheilus and "black spot" Crassiphiala bulboglossa have similar life cycles. Each parasite uses a littoral snail as a first intermediate host, fathead minnows as a second intermediate host, and a piscivorous bird as a final host. Metacercariae of black spot encyst in the dermal and epidermal tissues, while metacercariae of O. ptychocheilus encyst on the brain over a region that coordinates optomotor responses. Because of site differences within the host, we predicted that O. ptychocheilus metacercariae might manipulate the behavioral phenotype of minnows to facilitate transmission to the final host, but metacercariae of black spot would not. In our study population, prevalence was 100% for O. ptychocheilus , with an overall median intensity of 105 metacercariae per minnow. Prevalence of black spot was 60%, with a median abundance and intensity of 12 and 20 metacercariae per minnow for the overall sample and for infected fish, respectively. Minnows accumulated both parasites over time, producing significant correlations between intensity and minnow body length and between intensities of the 2 parasites. Minnows infected with black spot had on average twice as many O. ptychocheilus metacercariae as similar-sized minnows without any black spot cercariae. We found no correlation between body condition of minnows and intensity for either parasite. We measured 2 aspects of anti-predator competence to test for effects linked to parasite intensity. We found no correlation between intensity of either species of parasite and latency to behavioral response to attack from a mechanical model heron, nor was there any effect of parasite intensity on a measure of shoaling affinity. The absence of any detectable effect of metacercariae on anti

  7. Pathogen filtration to control plant disease outbreak in greenhouse production

    NASA Astrophysics Data System (ADS)

    Jeon, Sangho; Krasnow, Charles; Bhalsod, Gemini; Granke, Leah; Harlan, Blair; Hausbeck, Mary; Zhang, Wei

    2016-04-01

    Previous research has been extensively focused on understanding the fate and transport of human microbial pathogens in soil and water environments. However, little is known about the transport of plant pathogens, although these pathogens are often found in irrigation waters and could cause severe crop damage and economical loss. Water mold pathogens including Phytophthora spp. and Pythium spp. are infective to a wide range of vegetable and floriculture crops, and they are primarily harbored in soils and disseminated through water flow. It is challenging to control these pathogens because they often quickly develop resistance to many fungicides. Therefore, this multi-scale study aimed to investigate physical removal of plant pathogens from water by filtration, thus reducing the pathogen exposure risks to crops. In column-scale experiments, we studied controlling factors on the transport and retention of Phytophthora capsici zoospores in saturated columns packed with iron oxide coated-sand and uncoated-sand under varying solution chemistry. Biflagellate zoospores were less retained than encysted zoospores, and lower solution pH and greater iron oxide content increased the retention of encysted zoospores. These results provided insights on environmental dispersal of Phytophthora zoospores in natural soils as well as on developing cost-effective engineered filtration systems for pathogen removal. Using small-scale greenhouse filtration systems, we further investigated the performance of varying filter media (i.e., granular sand, iron oxide coated ceramic porous media, and activated carbon) in mitigating disease outbreaks of Phytophthora and Pythium for greenhouse-grown squash and poinsettia, respectively, in comparison with fungicide treatment. For squash, filtration by iron oxide coated media was more effective in reducing the Phytophthora infection, comparing to sand filtration and fungicide application. For poinsettia, sand filtration performed better in controlling

  8. On dormancy strategies in tardigrades.

    PubMed

    Guidetti, Roberto; Altiero, Tiziana; Rebecchi, Lorena

    2011-05-01

    In this review we analyze the dormancy strategies of metazoans inhabiting "hostile to life" habitats, which have a strong impact on their ecology and in particular on the traits of their life history. Tardigrades are here considered a model animal, being aquatic organisms colonizing terrestrial habitats. Tardigrades evolved a large variety of dormant stages that can be ascribed to diapause (encystment, cyclomorphosis, resting eggs) and cryptobiosis (anhydrobiosis, cryobiosis, anoxibiosis). In tardigrades, diapause and cryptobiosis can occur separately or simultaneously, consequently the adoption of one adaptive strategy is not necessarily an alternative to the adoption of the other. Encystment and cyclomorphosis are characterized by seasonal cyclic changes in morphology and physiology of the animals. They share several common features and their evolution is strictly linked to the molting process. A bet-hedging strategy with different patterns of egg hatching time has been observed in a tardigrade species. Four categories of eggs have been identified: subitaneous, delayed-hatching, abortive and diapause resting eggs, which needs a stimulus to hatch (rehydration after a period of desiccation). Cryptobiotic tardigrades are able to withstand desiccation (anhydrobiosis) and freezing (cryobiosis) at any stage of their life-cycle. This ability involves a complex array of factors working at molecular (bioprotectans), physiological and structural levels. Animal survival and the accumulation of molecular damage are related to the time spent in the cryptobiotic state, to the abiotic parameters during the cryptobiotic state, and to the conditions during initial and final phases of the process. Cryptobiosis evolved independently at least two times in tardigrades, in eutardigrades and in echiniscoids. Within each evolutionary line, the absence of cryptobiotic abilities is more related to selective pressures to local habitat adaptation than to phylogenetic relationships. The

  9. High prevalence of Angiostrongylus cantonensis (rat lungworm) on eastern Hawai'i Island: A closer look at life cycle traits and patterns of infection in wild rats (Rattus spp.).

    PubMed

    Jarvi, Susan I; Quarta, Stefano; Jacquier, Steven; Howe, Kathleen; Bicakci, Deniz; Dasalla, Crystal; Lovesy, Noelle; Snook, Kirsten; McHugh, Robert; Niebuhr, Chris N

    2017-01-01

    The nematode Angiostrongylus cantonensis is a zoonotic pathogen and the etiological agent of human angiostrongyliasis or rat lungworm disease. Hawai'i, particularly east Hawai'i Island, is the epicenter for angiostrongyliasis in the USA. Rats (Rattus spp.) are the definitive hosts while gastropods are intermediate hosts. The main objective of this study was to collect adult A. cantonensis from wild rats to isolate protein for the development of a blood-based diagnostic, in the process we evaluated the prevalence of infection in wild rats. A total of 545 wild rats were sampled from multiple sites in the South Hilo District of east Hawai'i Island. Adult male and female A. cantonensis (3,148) were collected from the hearts and lungs of humanely euthanized Rattus rattus, and R. exulans. Photomicrography and documentation of multiple stages of this parasitic nematode in situ were recorded. A total of 45.5% (197/433) of rats inspected had lung lobe(s) (mostly upper right) which appeared granular indicating this lobe may serve as a filter for worm passage to the rest of the lung. Across Rattus spp., 72.7% (396/545) were infected with adult worms, but 93.9% (512/545) of the rats were positive for A. cantonensis infection based on presence of live adult worms, encysted adult worms, L3 larvae and/or by PCR analysis of brain tissue. In R. rattus we observed an inverse correlation with increased body mass and infection level of adult worms, and a direct correlation between body mass and encysted adult worms in the lung tissue, indicating that larger (older) rats may have developed a means of clearing infections or regulating the worm burden upon reinfection. The exceptionally high prevalence of A. cantonensis infection in Rattus spp. in east Hawai'i Island is cause for concern and indicates the potential for human infection with this emerging zoonosis is greater than previously thought.

  10. High prevalence of Angiostrongylus cantonensis (rat lungworm) on eastern Hawai‘i Island: A closer look at life cycle traits and patterns of infection in wild rats (Rattus spp.)

    PubMed Central

    Quarta, Stefano; Jacquier, Steven; Howe, Kathleen; Bicakci, Deniz; Dasalla, Crystal; Lovesy, Noelle; Snook, Kirsten; McHugh, Robert; Niebuhr, Chris N.

    2017-01-01

    The nematode Angiostrongylus cantonensis is a zoonotic pathogen and the etiological agent of human angiostrongyliasis or rat lungworm disease. Hawai‘i, particularly east Hawai‘i Island, is the epicenter for angiostrongyliasis in the USA. Rats (Rattus spp.) are the definitive hosts while gastropods are intermediate hosts. The main objective of this study was to collect adult A. cantonensis from wild rats to isolate protein for the development of a blood-based diagnostic, in the process we evaluated the prevalence of infection in wild rats. A total of 545 wild rats were sampled from multiple sites in the South Hilo District of east Hawai‘i Island. Adult male and female A. cantonensis (3,148) were collected from the hearts and lungs of humanely euthanized Rattus rattus, and R. exulans. Photomicrography and documentation of multiple stages of this parasitic nematode in situ were recorded. A total of 45.5% (197/433) of rats inspected had lung lobe(s) (mostly upper right) which appeared granular indicating this lobe may serve as a filter for worm passage to the rest of the lung. Across Rattus spp., 72.7% (396/545) were infected with adult worms, but 93.9% (512/545) of the rats were positive for A. cantonensis infection based on presence of live adult worms, encysted adult worms, L3 larvae and/or by PCR analysis of brain tissue. In R. rattus we observed an inverse correlation with increased body mass and infection level of adult worms, and a direct correlation between body mass and encysted adult worms in the lung tissue, indicating that larger (older) rats may have developed a means of clearing infections or regulating the worm burden upon reinfection. The exceptionally high prevalence of A. cantonensis infection in Rattus spp. in east Hawai‘i Island is cause for concern and indicates the potential for human infection with this emerging zoonosis is greater than previously thought. PMID:29252992

  11. Whole-genome sequencing of a pandoravirus isolated from keratitis-inducing acanthamoeba.

    PubMed

    Antwerpen, M H; Georgi, E; Zoeller, L; Woelfel, R; Stoecker, K; Scheid, P

    2015-03-26

    Following the recent discovery of two Pandoravirus species in 2013, a previously described endocytobiont isolated from the inflamed eye of a patient with keratitis was subjected to whole-genome sequencing (WGS). Here, we present the complete genome sequence of a new Pandoravirus isolate. Copyright © 2015 Antwerpen et al.

  12. A standardized method of propagating the marine fish parasite, Amyloodinium ocellatum.

    PubMed

    Bower, C E; Turner, D T; Biever, R C

    1987-02-01

    The peridinian dinoflagellate Amyloodinium ocellatum was propagated by serial passage in clownfish (Amphiprion ocellaris) and hybrid striped bass (Morone chrysops X Morone saxatilis). Each 25-50-mm fish was exposed to 4,000-6,000 dinospores in 400 ml of artificial seawater for 30 min. Two days after exposure, trophonts were harvested by immersing the fishes in fresh water. After encystment, tomonts were axenized by multiple washes with sterile distilled water and sterile artificial seawater containing penicillin and streptomycin, and then incubated in the antibiotic solution. High yields of both tomonts and dinospores of the same sizes and ages were obtained, and host mortalities were eliminated. Microbial growth in incubating cultures was inhibited until after dinospores had emerged from tomonts, and dinospores remained infective for at least 4 days at 26 C.

  13. Molecular survey of occurrence and quantity of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa and amoeba hosts in municipal drinking water storage tank sediments.

    PubMed

    Lu, J; Struewing, I; Yelton, S; Ashbolt, N

    2015-07-01

    To examine the occurrence and quantity of potential pathogens and an indicator of microbial contamination in the sediments of municipal drinking water storage tanks (MDWSTs), given the absence of such data across the United States. Sediment samples (87 MDWST) from eighteen locations across ten states of the United States were collected and assayed by qPCR for a range of potential enteric and opportunistic microbial pathogens and a sewage-associated Bacteroides marker. Potential opportunistic pathogens dominated, with the highest detection of occurrence (per cent positive detection; average cell equivalence (CE)) being Mycobacterium spp. (88·9%; 6·7 ± 8·5 × 10(4) CE g(-1) ), followed by Legionella spp. (66·7%; 5·2 ± 5·9 × 10(3) CE g(-1) ), Pseudomonas aeruginosa (22·2%; 250 ± 880 CE g(-1) ) and Acanthamoeba spp. (38·9%; 53 ± 70 CE g(-1) ), with no detected Naegleria fowleri. Most enteric pathogens (Campylobacter jejuni, Escherichia coli 0157:H7, Salmonella enterica, Cryptosporidium parvum and Giardia duodenalis) were not detected, except for a trace signal for Campylobacter spp. There was significant correlation between the qPCR signals of Legionella spp. and Acanthamoeba spp. (R(2) = 0·61, n = 87, P = 0·0001). Diverse Legionella spp. including Leg. pneumophila, Leg. pneumophila sg1 and Leg. anisa were identified, each of which might cause legionellosis. These results imply that potential opportunistic pathogens are common within MDWST sediments and could act as a source of microbial contamination, but need downstream growth to be of potential concern. The results imply that opportunistic pathogen risks may need to be managed by regular tank cleaning or other management practices. 2015 The Society for Applied Microbiology.

  14. Opportunistic Pathogens and Microbial Communities and Their Associations with Sediment Physical Parameters in Drinking Water Storage Tank Sediments.

    PubMed

    Qin, Ke; Struewing, Ian; Domingo, Jorge Santo; Lytle, Darren; Lu, Jingrang

    2017-10-26

    The occurrence and densities of opportunistic pathogens (OPs), the microbial community structure, and their associations with sediment elements from eight water storage tanks in Ohio, West Virginia, and Texas were investigated. The elemental composition of sediments was measured through X-ray fluorescence (XRF) spectra. The occurrence and densities of OPs and amoeba hosts (i.e., Legionella spp. and L . pneumophila , Mycobacterium spp., P. aeruginosa , V. vermiformis, Acanthamoeba spp.) were determined using genus- or species-specific qPCR assays. Microbial community analysis was performed using next generation sequencing on the Illumina Miseq platform. Mycobacterium spp. were most frequently detected in the sediments and water samples (88% and 88%), followed by Legionella spp. (50% and 50%), Acanthamoeba spp. (63% and 13%), V. vermiformis (50% and 25%), and P. aeruginosa (0 and 50%) by qPCR method. Comamonadaceae (22.8%), Sphingomonadaceae (10.3%), and Oxalobacteraceae (10.1%) were the most dominant families by sequencing method. Microbial communities in water samples were mostly separated with those in sediment samples, suggesting differences of communities between two matrices even in the same location. There were associations of OPs with microbial communities. Both OPs and microbial community structures were positively associated with some elements (Al and K) in sediments mainly from pipe material corrosions. Opportunistic pathogens presented in both water and sediments, and the latter could act as a reservoir of microbial contamination. There appears to be an association between potential opportunistic pathogens and microbial community structures. These microbial communities may be influenced by constituents within storage tank sediments. The results imply that compositions of microbial community and elements may influence and indicate microbial water quality and pipeline corrosion, and that these constituents may be important for optimal storage tank

  15. Molecular Detection of Legionella spp. and their associations with Mycobacterium spp., Pseudomonas aeruginosa and amoeba hosts in a drinking water distribution system.

    PubMed

    Lu, J; Struewing, I; Vereen, E; Kirby, A E; Levy, K; Moe, C; Ashbolt, N

    2016-02-01

    This study investigated waterborne opportunistic pathogens (OPs) including potential hosts, and evaluated the use of Legionella spp. for indicating microbial water quality for OPs within a full-scale operating drinking water distribution system (DWDS). To investigate the occurrence of specific microbial pathogens within a major city DWDS we examined large volume (90 l drinking water) ultrafiltration (UF) concentrates collected from six sites between February, 2012 and June, 2013. The detection frequency and concentration estimates by qPCR were: Legionella spp. (57%/85 cell equivalent, CE l(-1) ), Mycobacterium spp. (88%/324 CE l(-1) ), Pseudomonas aeruginosa (24%/2 CE l(-1) ), Vermamoeba vermiformis (24%/2 CE l(-1) ) and Acanthamoeba spp. (42%/5 cyst equivalent, CE l(-1) ). There was no detection of the following microorganisms: human faecal indicator Bacteroides (HF183), Salmonella enterica, Campylobacter spp., Escherichia coli O157:H7, Giardia intestinalis, Cryptosporidium spp. or Naegleria fowleri. There were significant correlations between the qPCR signals of Legionella spp. and Mycobacterium spp., and their potential hosts V. vermiformis and Acanthamoeba spp. Sequencing of Legionella spp. demonstrated limited diversity, with most sequences coming from two dominant groups, of which the larger dominant group was an unidentified species. Other known species including Legionella pneumophila were detected, but at low frequency. The densities of Legionella spp. and Mycobacterium spp. were generally higher (17 and 324 folds, respectively) for distal sites relative to the entry point to the DWDS. Legionella spp. occurred, had significant growth and were strongly associated with free-living amoebae (FLA) and Mycobacterium spp., suggesting that Legionella spp. could provide a useful DWDS monitoring role to indicate potential conditions for non-faecal OPs. The results provide insight into microbial pathogen detection that may aid in the monitoring of microbial water

  16. A New Zamilon-like Virophage Partial Genome Assembled from a Bioreactor Metagenome

    PubMed Central

    Bekliz, Meriem; Verneau, Jonathan; Benamar, Samia; Raoult, Didier; La Scola, Bernard; Colson, Philippe

    2015-01-01

    Virophages replicate within viral factories inside the Acanthamoeba cytoplasm, and decrease the infectivity and replication of their associated giant viruses. Culture isolation and metagenome analyses have suggested that they are common in our environment. By screening metagenomic databases in search of amoebal viruses, we detected virophage-related sequences among sequences generated from the same non-aerated bioreactor metagenome as recently screened by another team for virophage capsid-encoding genes. We describe here the assembled partial genome of a virophage closely related to Zamilon, which infects Acanthamoeba with mimiviruses of lineages B and C but not A. Searches for sequences related to amoebal giant viruses, other Megavirales representatives and virophages were conducted using BLAST against this bioreactor metagenome (PRJNA73603). Comparative genomic and phylogenetic analyses were performed using sequences from previously identified virophages. A total of 72 metagenome contigs generated from the bioreactor were identified as best matching with sequences from Megavirales representatives, mostly Pithovirus sibericum, pandoraviruses and amoebal mimiviruses from three lineages A–C, as well as from virophages. In addition, a partial genome from a Zamilon-like virophage, we named Zamilon 2, was assembled. This genome has a size of 6716 base pairs, corresponding to 39% of the Zamilon genome, and comprises partial or full-length homologs for 15 Zamilon predicted open reading frames (ORFs). Mean nucleotide and amino acid identities for these 15 Zamilon 2 ORFs with their Zamilon counterparts were 89% (range, 81–96%) and 91% (range, 78–99%), respectively. Notably, these ORFs included two encoding a capsid protein and a packaging ATPase. Comparative genomics and phylogenetic analyses indicated that the partial genome was that of a new Zamilon-like virophage. Further studies are needed to gain better knowledge of the tropism and prevalence of virophages in

  17. Opportunistic Pathogens and Microbial Communities and Their Associations with Sediment Physical Parameters in Drinking Water Storage Tank Sediments

    PubMed Central

    Qin, Ke; Struewing, Ian; Domingo, Jorge Santo; Lytle, Darren

    2017-01-01

    The occurrence and densities of opportunistic pathogens (OPs), the microbial community structure, and their associations with sediment elements from eight water storage tanks in Ohio, West Virginia, and Texas were investigated. The elemental composition of sediments was measured through X-ray fluorescence (XRF) spectra. The occurrence and densities of OPs and amoeba hosts (i.e., Legionella spp. and L. pneumophila, Mycobacterium spp., P. aeruginosa, V. vermiformis, Acanthamoeba spp.) were determined using genus- or species-specific qPCR assays. Microbial community analysis was performed using next generation sequencing on the Illumina Miseq platform. Mycobacterium spp. were most frequently detected in the sediments and water samples (88% and 88%), followed by Legionella spp. (50% and 50%), Acanthamoeba spp. (63% and 13%), V. vermiformis (50% and 25%), and P. aeruginosa (0 and 50%) by qPCR method. Comamonadaceae (22.8%), Sphingomonadaceae (10.3%), and Oxalobacteraceae (10.1%) were the most dominant families by sequencing method. Microbial communities in water samples were mostly separated with those in sediment samples, suggesting differences of communities between two matrices even in the same location. There were associations of OPs with microbial communities. Both OPs and microbial community structures were positively associated with some elements (Al and K) in sediments mainly from pipe material corrosions. Opportunistic pathogens presented in both water and sediments, and the latter could act as a reservoir of microbial contamination. There appears to be an association between potential opportunistic pathogens and microbial community structures. These microbial communities may be influenced by constituents within storage tank sediments. The results imply that compositions of microbial community and elements may influence and indicate microbial water quality and pipeline corrosion, and that these constituents may be important for optimal storage tank management

  18. Occurrence and Control of Legionella in Recycled Water Systems

    PubMed Central

    Jjemba, Patrick K.; Johnson, William; Bukhari, Zia; LeChevallier, Mark W.

    2015-01-01

    Legionella pneumophila is on the United States Environmental Protection Agency (USEPA) Candidate Contaminant list (CCL) as an important pathogen. It is commonly encountered in recycled water and is typically associated with amoeba, notably Naegleria fowleri (also on the CCL) and Acanthamoeba sp. No legionellosis outbreak has been linked to recycled water and it is important for the industry to proactively keep things that way. A review was conducted examine the occurrence of Legionella and its protozoa symbionts in recycled water with the aim of developing a risk management strategy. The review considered the intricate ecological relationships between Legionella and protozoa, methods for detecting both symbionts, and the efficacy of various disinfectants. PMID:26140674

  19. Coprology of Panthera tigris altaica and Felis bengalensis euptilurus from the Russian Far East.

    PubMed

    González, P; Carbonell, E; Urios, V; Rozhnov, V V

    2007-08-01

    Fecal samples from the Siberian tiger (Panthera tigris altaica) and the Amur cat (Felis bengalensis euptilurus) from Far Eastern Russia, were examined for parasites. A natural sedimentation methodology was used and a complete examination of all the sediment was performed. This fecal investigation allowed us to isolate and identify several developmental stages of gastrointestinal, hepatic, and respiratory parasites. Five parasites were found from P. t. altaica: 11 trematodes (Platynosomum fastosum) and 4 nematodes (Strongyloides sp., Ancylostomatidae, Toxascaris leonina, and Toxocara cati). Five parasites were found from F. b. euptilurus: 1 cestode (Diplopylidium sp.) and 4 nematodes (Trichuris sp., Ancylostomatidae, Toxascaris leonina, and Aelurostrongylus abstrusus). In addition, trophozoites of the amoeba Acanthamoeba sp. were detected in tiger feces.

  20. cDNA-AFLP analysis of differential gene expression related to cell chemotactic and encystment of Azospirillum brasilense.

    PubMed

    Li, Huamin; Cui, Yanhua; Wu, Lixian; Tu, Ran; Chen, Sanfeng

    2011-12-20

    Our previous study indicated org35 was involved in chemotaxis and interacted with nitrogen fixation transcriptional activator NifA via PAS domain. In order to reveal the role of org35 in nitrogen regulation, the downstream target genes of org35 were identified. We here report differentially expressed genes in org35 mutants comparing with wild type Sp7 by means of cDNA-AFLP. Four up-regulated transcript-derived fragments (TDFs) homologues of chemotaxis transduction proteins were found, including CheW, methyl-accepting chemotaxis protein and response regulator CheY-like receiver. Three distinct TDFs (AB46, AB58 and AB63) were similar to PHB de-polymerase C-terminus, cell shape-determining protein and flagellin domain protein. And 11 TDFs showed similarities with signal transduction proteins, including homologous protein of the nitrogen regulation protein NtrY and nitrate/nitrite response regulator protein NarL. These data suggested that the Azospirillum brasilense org35 was a multi-effecter and involved in chemotaxis, cyst development and regulation of nitrogen fixation. Copyright © 2010 Elsevier GmbH. All rights reserved.

  1. Toxicity of anti-fouling biocides to encysted metacercariae of Echinoparyphium recurvatum (Digenea: Echinostomatidae) and their snail hosts.

    PubMed

    Morley, N J; Leung, K M Y; Morritt, D; Crane, M

    2004-07-01

    The toxicity of the anti-fouling biocides tributyltin (TBTO), copper, and Irgarol 1051 (irgarol) at a nominal concentration of 10 microg/l over a 30 day period were investigated against the viability of metacercarial cysts of the digenean parasite Echinoparyphium recurvatum resident within the body of two common freshwater snails, Lymnaea peregra and Physa fontinalis. Reduced parasite viability was found under most exposures in both snail species. However a greater effect of toxicant exposure was found in cysts within P. fontinalis compared to those in L. peregra. This was associated with an increased mortality of the host snail. Among all tested biocides, TBTO exposures induced the highest mortality to both the parasite and their hosts. These results suggest that parasite viability is interlinked with survival of the host snail. The mechanisms of differing toxicity between host species and its relevance to successful parasite transmission to the next host are discussed.

  2. Presence of pathogenic microorganisms in power-plant cooling waters. Report for October 1, 1979-September 30, 1981

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tyndall, R.L.

    1982-10-01

    Cooling waters from eleven geographically disparate power plants were tested for the presence of Naegleria fowleri and Legionella pneumophila (LDB). Control source waters for each plant were also tested for these pathogens. Water from two of the eleven plants contained pathogenic Naegleria, and infectious Legionella were found in seven of the test sites. Pathogenic Naegleria were not found in control waters, but infectious Legionella were found in five of the eleven control source water sites. Concentrations of nitrite, sulfate, and total organic carbon correlated with the concentrations of LDB. A new species of Legionella was isolated from one of themore » test sites. In laboratory tests, both Acanthamoeba and Naegleria were capable of supporting the growth of Legionella pneumophila.« less

  3. Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry.

    PubMed

    Mustapha, Pascale; Epalle, Thibaut; Allegra, Séverine; Girardot, Françoise; Garraud, Olivier; Riffard, Serge

    2015-04-01

    The viability of three Legionella pneumophila strains was monitored after chlorine dioxide (ClO2) treatment using a flow cytometric assay. Suspensions of L. pneumophila cells were submitted to increasing concentrations of ClO2. Culturable cells were still detected when using 4 mg/L, but could no longer be detected after exposure to 6 mg/L of ClO2, although viable but not culturable (VBNC) cells were found after exposure to 4-5 mg/L of ClO2. When testing whether these VBNC were infective, two of the strains were resuscitated after co-culture with Acanthamoeba polyphaga, but neither of them could infect macrophage-like cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  4. Free-living pathogenic and nonpathogenic amoebae in Maryland soils.

    PubMed Central

    Sawyer, T K

    1989-01-01

    Tests for potentially pathogenic amoebae were carried out on soil samples from the following sites: (i) farmlands fertilized with municipal sewage wastes, (ii) a stream receiving sewage effluent from a sludge lagoon, (iii) a ravine receiving storm runoff from a cattle farm, (iv) farmlands not fertilized with sewage wastes, and (v) a vegetated shoreline of a waterfront estate not used for farming or livestock production. Study sites were located on the eastern shore of Maryland, bordered to the north by Delaware and to the south by Virginia. Twenty-four species of soil amoebae, including five potentially pathogenic Acanthamoeba species (members of the family Acanthamoebidae), were identified. All of the sites yielded two or more of the potential pathogens. PMID:2757373

  5. An optimised multi-host trematode life cycle: fishery discards enhance trophic parasite transmission to scavenging birds.

    PubMed

    Born-Torrijos, Ana; Poulin, Robert; Pérez-Del-Olmo, Ana; Culurgioni, Jacopo; Raga, Juan Antonio; Holzer, Astrid Sibylle

    2016-10-01

    Overlapping distributions of hosts and parasites are critical for successful completion of multi-host parasite life cycles and even small environmental changes can impact on the parasite's presence in a host or habitat. The generalist Cardiocephaloides longicollis was used as a model for multi-host trematode life cycles in marine habitats. This parasite was studied to quantify parasite dispersion and transmission dynamics, effects of biological changes and anthropogenic impacts on life cycle completion. We compiled the largest host dataset to date, by analysing 3351 molluscs (24 species), 2108 fish (25 species) and 154 birds (17 species) and analysed the resultant data based on a number of statistical models. We uncovered extremely low host specificity at the second intermediate host level and a preference of the free-swimming larvae for predominantly demersal but also benthic fish. The accumulation of encysted larvae in the brain with increasing fish size demonstrates that parasite numbers level off in fish larger than 140mm, consistent with parasite-induced mortality at these levels. The highest infection rates were detected in host species and sizes representing the largest fraction of Mediterranean fishery discards (up to 67% of the total catch), which are frequently consumed by seabirds. Significantly higher parasite densities were found in areas with extensive fishing activity than in those with medium and low activity, and in fish from shallow lagoons than in fish from other coastal areas. For the first time, C. longicollis was also detected in farmed fish in netpens. Fishing generally drives declines in parasite abundance, however, our study suggests an enhanced transmission of generalist parasites such as C. longicollis, an effect that is further amplified by the parasite's efficient host-finding mechanisms and its alteration of fish host behaviour by larvae encysted in the brain. The anthropogenic impact on the distribution of trophically

  6. Effects of aging on resistance to Trichinella spiralis infection in rodents exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

    PubMed

    Luebke, R W; Copeland, C B; Andrews, D L

    1999-08-13

    Immune function, including resistance to infection, decreases as humans and rodents age. We have shown that preinfection exposure of young (9-11 weeks) mice or rats to TCDD decreased resistance to Trichinella spiralis (Ts) infection, expressed as delayed onset or completion of parasite elimination and as increased muscle burdens of larvae. It has also been shown that aged mice express lower constitutive levels of resistance to Ts infection, compared to young adult animals. This study tested the hypothesis that the age-related decrease in constitutive levels of resistance to Ts infection exacerbates the decreased resistance to infection that follows TCDD exposure. This hypothesis addresses the concern that TCDD may pose a greater threat to the elderly than to the population at large. Animals were given a single oral dose of 1, 10, or 30 microg TCDD/kg, 7 days before infection. Eleven days later, young (approximately 10 weeks) control rodents had eliminated a greater proportion of the original parasite burden from the intestine than aged control animals. Nevertheless, parasite elimination was decreased by TCDD exposure only in young rodents. The effect of TCDD exposure on numbers of encysted larvae was evaluated only in rats. Increased larvae burdens occurred in young rats at 30 microg TCDD/kg and at 10 or 30 microg TCDD/kg in aged rats. Parasite-specific splenocyte and lymph node cell proliferation was suppressed following dioxin exposure in young mice; cells from aged mice were markedly less responsive to stimulation, yet less sensitive to TCDD exposure. The response to parasite antigens was not affected in aged rats exposed to TCDD, although elevated mitogen-driven B-cell proliferation was observed. These results indicate that age-related constitutive immunosuppression did not exacerbate TCDD-induced suppression of T-cell mediated adult parasite expulsion; rather, advanced age provided some degree of protection. On the other hand, a lower dose of TCDD was required

  7. Nematode eel parasite found inside acanthocephalan cysts--a "Trojan horse" strategy?

    PubMed

    Emde, Sebastian; Rueckert, Sonja; Kochmann, Judith; Knopf, Klaus; Sures, Bernd; Klimpel, Sven

    2014-11-18

    The invasive eel parasite Anguillicoloides crassus (syn. Anguillicola crassus) is considered one of the major causes for the decline of the European eel (Anguilla anguilla) panmictic population. It impairs the swim bladder function and reduces swimming performance of its host. The life cycle of this parasite involves different intermediate and paratenic hosts. Despite an efficient immune system of the paratenic fish hosts acting against infections with A. crassus, levels of parasitized eels remain high in European river systems. Recently, the round goby Neogobius melanostomus (Gobiidae) has become dominant in many rivers in Europe and is still spreading at a rapid pace. This highly invasive species might potentially act as an important, so far neglected paratenic fish host for A. crassus. Based on own observations and earlier single sightings of A. crassus in N. melanostomus, 60 fresh individuals of N. melanostomus were caught in the Rhine River and examined to assess the infection levels with metazoan parasites, especially A. crassus. Glycerin preparations were used for parasite identification. The parasite most frequently found in N. melanostomus was the acanthocephalan Pomphorhynchus sp. (subadult stage) which occurred mainly encysted in the mesenteries and liver. Every third gobiid (P = 31.7%) was infected by A. crassus larvae (L3) which exclusively occurred inside the acanthocephalan cysts. No intact or degenerated larvae of A. crassus were detected elsewhere in the goby, neither in the body cavity and mesenteries nor in other organs. Affected cysts contained the acanthocephalan larvae and 1-12 (mI =3) living A. crassus larvae. Additionally, encysted larvae of the nematode Raphidascaris acus were detected in the gobies, but only in the body cavity and not inside the acanthocephalan cysts. Based on our observations, we suggest that A. crassus might actively bypass the immune response of N. melanostomus by invading the cysts of acanthocephalan parasites of the

  8. Proteomic Study of Entamoeba histolytica Trophozoites, Cysts, and Cyst-Like Structures

    PubMed Central

    Luna-Nácar, Milka; Navarrete-Perea, José; Moguel, Bárbara; Bobes, Raúl J.; Laclette, Juan P.; Carrero, Julio C.

    2016-01-01

    The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition

  9. Molecular cloning and characterization of an SRCAP chromatin remodeling homologue in Toxoplasma gondii.

    PubMed

    Sullivan, William J; Monroy, M Alexandra; Bohne, Wolfgang; Nallani, Karuna C; Chrivia, John; Yaciuk, Peter; Smith, Charles K; Queener, Sherry F

    2003-05-01

    We have identified and mapped a gene in Toxoplasma gondii that encodes a homologue of SRCAP (Snf2-related CBP activator protein), a member of the SNF/SWI family of chromatin remodeling factors. The genomic locus (TgSRCAP) is present as a single copy and contains 16 introns. The predicted cDNA contains an open reading frame of 8,775 bp and encodes a protein of 2,924 amino acids. We have identified additional SRCAP-like sequences in Apicomplexa for comparison by screening genomic databases. An analysis of SRCAP homologues between species reveals signature features that may be indicative of SRCAP members. Expression of mRNA encoding TgSRCAP is upregulated when tachyzoite (invasive form) parasites are induced to differentiate into bradyzoites (encysted form) in vitro. Recombinant TgSRCAP protein is functionally equivalent to the human homologue, being capable of increasing transcription mediated by CREB.

  10. Nematode larvae infecting Priacanthus arenatus Cuvier, 1829 (Pisces: Teleostei) in Brazil.

    PubMed

    Kuraiem, Bianca P; Knoff, Marcelo; Felizardo, Nilza N; Gomes, Delir C; Clemente, Sérgio C São

    2016-05-31

    From July to December, 2013, thirty Priacanthus arenatus specimens commercialized in the cities of Niterói and Rio de Janeiro, State of Rio de Janeiro, were acquired. The fish were necropsied and filleted to investigate the presence of nematode larvae. Twenty fish (66.7%) out of the total were parasitized by nematode larvae. A total of 2024 larvae were collected; among them, 30 third-instar larvae of Anisakis sp. showed prevalence (P) = 20%, mean abundance (MA) = 1, and the mean intensity (MI) = 5, and infection sites (IS) = caecum, stomach, liver, and mesentery; and 1,994 third-instar larvae (1,757 encysted and 237 free) of Hysterothylacium deardorffoverstreetorum with P = 66.7%, MA = 66.5, and MI = 99.7, and IS = spleen, caecum, stomach, liver, mesentery, and abdominal muscle. This is the first study to report H. deardorffoverstreetorum and Anisakis sp. larvae parasitizing P. arenatus.

  11. Host fish suitability for glochidia of Ligumia recta

    USGS Publications Warehouse

    Khym, J.R.; Layzer, J.B.

    2000-01-01

    In the early 1900s several hosts were identified for the black sandshell Ligumia recta. Recent attempts to propagate juvenile L. recta with two of the reported hosts (bluegill Lepomis macrochirus and largemouth bass Micropterus salmoides) have produced inconsistent results and few juveniles. We conducted this study to determine which of the reported hosts or other fish hosts were the most suitable for glochidial metamorphosis. The duration of glochidial metamorphosis varied among seasons. Despite similar water temperatures, juveniles metamorphosed sooner and over a shorter period of time in the spring than early fall; the modal day of metamorphosis differed by 78 d. Relatively few juveniles were recovered from bluegill and largemouth bass in three trials. White crappie Pomoxis annularis and black crappie P. nigromaculatus were marginally suitable hosts. Although glochidia encysted on all hosts, >10x more juveniles metamorphosed on sauger Stizostedion canadense compared to other hosts tested.

  12. Enrichment of free-living amoebae in biofilms developed at upper water levels in drinking water storage towers: An inter- and intra-seasonal study.

    PubMed

    Taravaud, Alexandre; Ali, Myriam; Lafosse, Bernard; Nicolas, Valérie; Féliers, Cédric; Thibert, Sylvie; Lévi, Yves; Loiseau, Philippe M; Pomel, Sébastien

    2018-08-15

    Free-living amoebae (FLA) are ubiquitous organisms present in various natural and artificial environments, such as drinking water storage towers (DWST). Some FLA, such as Acanthamoeba sp., Naegleria fowleri, and Balamuthia mandrillaris, can cause severe infections at ocular or cerebral level in addition to being potential reservoirs of other pathogens. In this work, the abundance and diversity of FLA was evaluated in two sampling campaigns: one performed over five seasons in three DWST at three different levels (surface, middle and bottom) in water and biofilm using microscopy and PCR, and one based on the kinetics analysis in phase contrast and confocal microscopy of biofilm samples collected every two weeks during a 3-month period at the surface and at the bottom of a DWST. In the seasonal study, the FLA were detected in each DWST water in densities of ~20 to 25amoebaeL -1 . A seasonal variation of amoeba distribution was observed in water samples, with maximal densities in summer at ~30amoebaeL -1 and minimal densities in winter at ~16amoebaeL -1 . The FLA belonging to the genus Acanthamoeba were detected in two spring sampling campaigns, suggesting a possible seasonal appearance of this potentially pathogenic amoeba. Interestingly, a 1 log increase of amoebae density was observed in biofilm samples collected at the surface of all DWST compared to the middle and the bottom where FLA were at 0.1-0.2amoebae/cm 2 . In the kinetics study, an increase of amoebae density, total cell density, and biofilm thickness was observed as a function of time at the surface of the DWST, but not at the bottom. To our knowledge, this study describes for the first time a marked higher FLA density in biofilms collected at upper water levels in DWST, constituting a potential source of pathogenic micro-organisms. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Waterborne parasites: a current status from the Philippines.

    PubMed

    Onichandran, Subashini; Kumar, Thulasi; Salibay, Cristina C; Dungca, Julieta Z; Tabo, Hazel Al; Tabo, Norbel; Tan, Tian-Chye; Lim, Yvonne Al; Sawangjaroen, Nongyao; Phiriyasamith, Sucheep; Andiappan, Hemah; Ithoi, Init; Lau, Yee-Ling; Nissapatorn, Veeranoot

    2014-05-28

    Despite the amount of awareness created, waterborne disease still poses threat, especially in developing countries. Due to the scarcity of reported data on waterborne parasites, the consumption of unsafe water prolongs. Thus, the occurrences of waterborne parasites from various samples were investigated from one of the Southeast Asian country, the Philippines. A total of thirty three samples, each consisting of twelve liters, were collected and processed to obtain the sediment. Ten liters of sample each was processed to detect Cryptosporidium spp. and Giardia spp. using an immunomagnetic separation method prior to enumeration via fluorescence microscope. Meanwhile, the remaining two liters were cultured to detect Acanthamoeba and Naegleria through microscopy examination and polymerase chain reaction (PCR) analysis. Twelve samples (36.4%) from river (5), swimming pool (1), pond (3), rain tank (1), and natural lake (2) were positive for Cryptosporidium spp., 17 (45.5%) samples from river (9), pond (2), swimming pool (1), rain tank (1), and natural lake (4) were positive for Giardia spp. while, 13 (33.3%) samples from river (3), swimming pool (2), pond (2), dispenser (1), well (1), tap (2) and natural lake (2) were positive for Acanthamoeba spp. and 5 (18.2%) samples from river (1), natural lake (1), tap (1), dispenser (1) and mineral (1) were Naegleria spp. positive. Physical parameters such as temperature, conductivity, total dissolved solid (TDS), salinity, dissolved oxygen (DO), pH, and turbidity and chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite were also measured. The highest chemical contamination was observed at pond 2. A good correlation was observed between Giardia and nitrite (r = 0.736, p < 0.01) and Giardia and nitrate (r = 0.502, p < 0.01). This study was aimed to create greater awareness of parasitic contamination in the water environment in the Philippines and also to act as a platform of the current

  14. Waterborne parasites: a current status from the Philippines

    PubMed Central

    2014-01-01

    Background Despite the amount of awareness created, waterborne disease still poses threat, especially in developing countries. Due to the scarcity of reported data on waterborne parasites, the consumption of unsafe water prolongs. Thus, the occurrences of waterborne parasites from various samples were investigated from one of the Southeast Asian country, the Philippines. Methods A total of thirty three samples, each consisting of twelve liters, were collected and processed to obtain the sediment. Ten liters of sample each was processed to detect Cryptosporidium spp. and Giardia spp. using an immunomagnetic separation method prior to enumeration via fluorescence microscope. Meanwhile, the remaining two liters were cultured to detect Acanthamoeba and Naegleria through microscopy examination and polymerase chain reaction (PCR) analysis. Results Twelve samples (36.4%) from river (5), swimming pool (1), pond (3), rain tank (1), and natural lake (2) were positive for Cryptosporidium spp., 17 (45.5%) samples from river (9), pond (2), swimming pool (1), rain tank (1), and natural lake (4) were positive for Giardia spp. while, 13 (33.3%) samples from river (3), swimming pool (2), pond (2), dispenser (1), well (1), tap (2) and natural lake (2) were positive for Acanthamoeba spp. and 5 (18.2%) samples from river (1), natural lake (1), tap (1), dispenser (1) and mineral (1) were Naegleria spp. positive. Physical parameters such as temperature, conductivity, total dissolved solid (TDS), salinity, dissolved oxygen (DO), pH, and turbidity and chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite were also measured. The highest chemical contamination was observed at pond 2. A good correlation was observed between Giardia and nitrite (r = 0.736, p < 0.01) and Giardia and nitrate (r = 0.502, p < 0.01). Conclusion This study was aimed to create greater awareness of parasitic contamination in the water environment in the Philippines and also to

  15. Activation of an AMP-activated protein kinase is involved in post-diapause development of Artemia franciscana encysted embryos.

    PubMed

    Zhu, Xiao-Jing; Dai, Jie-Qiong; Tan, Xin; Zhao, Yang; Yang, Wei-Jun

    2009-03-16

    Cysts of Artemia can remain in a dormant state for long periods with a very low metabolic rate, and only resume their development with the approach of favorable conditions. The post-diapause development is a very complicated process involving a variety of metabolic and biochemical events. However, the intrinsic mechanisms that regulate this process are unclear. Herein we report the specific activation of an AMP-activated protein kinase (AMPK) in the post-diapause developmental process of Artemia. Using a phospho-AMPKalpha antibody, AMPK was shown to be phosphorylated in the post-diapause developmental process. Results of kinase assay analysis showed that this phosphorylation is essential for AMPK activation. Using whole-mount immunohistochemistry, phosphorylated AMPK was shown to be predominantly located in the ectoderm of the early developed embryos in a ring shape; however, the location and shape of the activation region changed as development proceeded. Additionally, Western blotting analysis on different portions of the cyst extracts showed that phosphorylated AMPKalpha localized to the nuclei and this location was not affected by intracellular pH. Confocal microscopy analysis of immunofluorescent stained cyst nuclei further showed that AMPKalpha localized to the nuclei when activated. Moreover, cellular AMP, ADP, and ATP levels in developing cysts were determined by HPLC, and the results showed that the activation of Artemia AMPK may not be associated with cellular AMP:ATP ratios, suggesting other pathways for regulation of Artemia AMPK activity. Together, we report evidence demonstrating the activation of AMPK in Artemia developing cysts and present an argument for its role in the development-related gene expression and energy control in certain cells during post-diapause development of Artemia.

  16. A Student's Guide to Giant Viruses Infecting Small Eukaryotes: From Acanthamoeba to Zooxanthellae.

    PubMed

    Wilhelm, Steven W; Bird, Jordan T; Bonifer, Kyle S; Calfee, Benjamin C; Chen, Tian; Coy, Samantha R; Gainer, P Jackson; Gann, Eric R; Heatherly, Huston T; Lee, Jasper; Liang, Xiaolong; Liu, Jiang; Armes, April C; Moniruzzaman, Mohammad; Rice, J Hunter; Stough, Joshua M A; Tams, Robert N; Williams, Evan P; LeCleir, Gary R

    2017-03-17

    The discovery of infectious particles that challenge conventional thoughts concerning "what is a virus" has led to the evolution a new field of study in the past decade. Here, we review knowledge and information concerning "giant viruses", with a focus not only on some of the best studied systems, but also provide an effort to illuminate systems yet to be better resolved. We conclude by demonstrating that there is an abundance of new host-virus systems that fall into this "giant" category, demonstrating that this field of inquiry presents great opportunities for future research.

  17. Pathology Associated with White Spot Virus (WSV) Infection in Wild Broodstock of Tiger Prawns (Penaeus monodon)

    PubMed Central

    Kua, Beng Chu; Rashid, Noraziah Mat

    2012-01-01

    A total of six wild broodstocks of tiger prawns, Penaeus monodon, were found positive for White Spot Virus (WSV) with an IQ2000 detection kit. Using histopathology, the intranuclear inclusion of haemocyte due to WSV infection was observed in the epithelium cells of the antennal gland, stomach and gills. This result confirmed that the wild broodstocks were positive with WSV without showing any white spot. Additionally, histopathological examination also revealed an accumulation of haemocytes around the hepatopancreatic tubules resulting from bacterial infection. Encapsulation and nodule formation, as well as related necrosis, were also observed around the hepatopancreatic tubules infected with a metazoan parasite. Encysted tylocephalum larval cestodes were observed in the hepatopancreas, with haemocytic aggregation being observed around the infected tubules. These findings showed some bacterial and parasitic infections which, in addition to the viral infection itself, could contribute to the 80% mortality rate in wild broodstocks infected with WSV. PMID:24575228

  18. Exceptionally well-preserved Cretaceous microfossils reveal new biomineralization styles.

    PubMed

    Wendler, Jens E; Bown, Paul

    2013-01-01

    Calcareous microplankton shells form the dominant components of ancient and modern pelagic sea-floor carbonates and are widely used in palaeoenvironmental reconstructions. The efficacy of these applications, however, is dependent upon minimal geochemical alteration during diagenesis, but these modifying processes are poorly understood. Here we report on new biomineralization architectures of previously unsuspected complexity in calcareous cell-wall coverings of extinct dinoflagellates (pithonellids) from a Tanzanian microfossil-lagerstätte. These Cretaceous 'calcispheres' have previously been considered biomineralogically unremarkable but our new observations show that the true nature of these tests has been masked by recrystallization. The pristine Tanzanian fossils are formed from fibre-like crystallites and show archeopyles and exquisitely constructed opercula, demonstrating the dinoflagellate affinity of pithonellids, which has long been uncertain. The interwoven fibre-like structures provide strength and flexibility enhancing the protective function of these tests. The low-density wall fabrics may represent specific adaptation for oceanic encystment life cycles, preventing the cells from rapid sinking.

  19. Molecular and chemical dialogues in bacteria-protozoa interactions.

    PubMed

    Song, Chunxu; Mazzola, Mark; Cheng, Xu; Oetjen, Janina; Alexandrov, Theodore; Dorrestein, Pieter; Watrous, Jeramie; van der Voort, Menno; Raaijmakers, Jos M

    2015-08-06

    Protozoan predation of bacteria can significantly affect soil microbial community composition and ecosystem functioning. Bacteria possess diverse defense strategies to resist or evade protozoan predation. For soil-dwelling Pseudomonas species, several secondary metabolites were proposed to provide protection against different protozoan genera. By combining whole-genome transcriptome analyses with (live) imaging mass spectrometry (IMS), we observed multiple changes in the molecular and chemical dialogues between Pseudomonas fluorescens and the protist Naegleria americana. Lipopeptide (LP) biosynthesis was induced in Pseudomonas upon protozoan grazing and LP accumulation transitioned from homogeneous distributions across bacterial colonies to site-specific accumulation at the bacteria-protist interface. Also putrescine biosynthesis was upregulated in P. fluorescens upon predation. We demonstrated that putrescine induces protozoan trophozoite encystment and adversely affects cyst viability. This multifaceted study provides new insights in common and strain-specific responses in bacteria-protozoa interactions, including responses that contribute to bacterial survival in highly competitive soil and rhizosphere environments.

  20. Life cycle of Stephanoprora uruguayense (Digenea: Echinostomatidae) in Argentina.

    PubMed

    Ostrowski de Núñez, Margarita

    2007-10-01

    The life cycle of Stephanoprora uruguayense Holcman et Olagüe, 1989, was experimentally resolved. In an artificial pond in the Zoological Garden in Buenos Aires City, Argentina, Heleobia parchappei (Hydrobiidae) was found to be releasing large-tailed cercariae with a prepharyngeal body, but lacking collar spines and corpuscles in the excretory system. Metacercariae, which encysted on the gills of naturally and experimentally infected Cnesterodon decemmaculatus (Poecilidae), developed collar spines and corpuscles in the excretory system in 7 days. Sexually mature adults were recovered from chicks and immature adults from mice fed metacercariae from C. decemmaculatus. Eggs shed in chick feces developed to miracidia within 10 days; sporocysts were found on the gills of snails. Stephanoprora uruguayense and S. denticulata from Europe are similar in adult morphology, but can be distinguished by morphological and behavioral features of larvae. Likewise, although S. denticulata and S. paradenticulata from Venezuela are similar to S. uruguayense in adult morphology, they differ considerably in larval morphology and intermediate hosts.