Science.gov

Sample records for por cryptosporidium parvum

  1. The Cryptosporidium parvum Kinome

    PubMed Central

    2011-01-01

    Background Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase. Results The C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC50 values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation. Conclusions Identification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search

  2. INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH OZONE

    EPA Science Inventory

    Ozone inactivation rates for Cryptosporidium parvum (C. parvum) oocysts were determined with an in-vitro excystation method based on excysted sporozoite counts. Results were consistent with published animal infectivity data for the same C. parvum strain. The inactivation kinetics...

  3. INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH OZONE

    EPA Science Inventory

    Ozone inactivation rates for Cryptosporidium parvum (C. parvum) oocysts were determined with an in-vitro excystation method based on excysted sporozoite counts. Results were consistent with published animal infectivity data for the same C. parvum strain. The inactivation kinetics...

  4. AN EVALUATION OF CRYPTOSPORIDIUM PARVUM GENOTYPING

    EPA Science Inventory

    We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Crytosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, a...

  5. AN EVALUATION OF CRYPTOSPORIDIUM PARVUM GENOTYPING

    EPA Science Inventory

    We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Crytosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, a...

  6. Cryptosporidium Parvum Transport Through Natural Porous Media

    NASA Astrophysics Data System (ADS)

    Araujo, J. B.; Santamaria, J.; Blandford, W. P.; Gerba, C. P.; Brusseau, M. L.

    2005-12-01

    The objective of this study was to quantify the transport of Cryptosporidium parvum through saturated natural porous media. A series of miscible-displacement experiments were conducted, varying the properties of the porous media and electrolyte solution to help elucidate retention mechanisms. Significant removal (~99%) of oocysts was observed for transport in a sandy soil. Similar removals were also observed for experiments conducted with deionized water in place of the 0.01M NaCl electrolyte solution and experiments with a sub sample of the sandy soil that was treated with nitric acid. Effluent recoveries were greater for experiments conducted using coarser porous media. These results indicate straining contributed to the retention of Cryptosporidium parvum in our system.

  7. Long-Term Transport of Cryptosporidium Parvum

    NASA Astrophysics Data System (ADS)

    Andrea, C.; Harter, T.; Hou, L.; Atwill, E. R.; Packman, A.; Woodrow-Mumford, K.; Maldonado, S.

    2005-12-01

    The protozoan pathogen Cryptosporidium parvum is a leading cause of waterborne disease. Subsurface transport and filtration in natural and artificial porous media are important components of the environmental pathway of this pathogen. It has been shown that the oocysts of C. parvum show distinct colloidal properties. We conducted a series of laboratory studies on sand columns (column length: 10 cm - 60 cm, flow rates: 0.7 m/d - 30 m/d, ionic strength: 0.01 - 100 mM, filter grain size: 0.2 - 2 mm, various solution chemistry). Breakthrough curves were measured over relatively long time-periods (hundreds to thousands of pore volumes). We show that classic colloid filtration theory is a reasonable tool for predicting the initial breakthrough, but it is inadequate to explain the significant tailing observed in the breakthrough of C. parvum oocyst through sand columns. We discuss the application of the Continuous Time Random Walk approach to account for the strong tailing that was observed in our experiments. The CTRW is generalized transport modeling framework, which includes the classic advection-dispersion equation (ADE), the fractional ADE, and the multi-rate mass transfer model as special cases. Within this conceptual framework, it is possible to distinguish between the contributions of pore-scale geometrical (physical) disorder and of pore-scale physico-chemical heterogeneities (e.g., of the filtration, sorption, desorption processes) to the transport of C. parvum oocysts.

  8. Rotifers ingest oocysts of Cryptosporidium parvum

    USGS Publications Warehouse

    Fayer, R.; Trout, J.M.; Walsh, E.; Cole, R.A.

    2000-01-01

    Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein-conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase-contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Euchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable.

  9. Crystal Structure of Cryptosporidium parvum Pyruvate Kinase

    PubMed Central

    Cook, William J.; Senkovich, Olga; Aleem, Khadijah; Chattopadhyay, Debasish

    2012-01-01

    Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time. PMID:23056503

  10. Treating Cryptosporidium parvum infection in calves.

    PubMed

    Nasir, A; Avais, M; Khan, M S; Khan, J A; Hameed, S; Reichel, M P

    2013-08-01

    The present study evaluated the therapeutic efficacy of azithromycin, co-trimoxazole and kalvangi (Nigella sativa, also known as Black Cumin) against Cryptosporidium parvum infection in calves under field conditions. The experimental calves were treated with azithromycin (group A) at 1500 mg/calf/day, co-trimoxazole (group B) at 30 mg Kg-1 and kalvangi seeds powder (group C) at 750 mg Kg-1 BW orally for 7 days. Calves in the group D were naturally infected with C. parvum , untreated animals (positive control) while the calves in the group E were uninfected negative control animals. A significant decrease (p < 0.05) in oocyst counts for calves in groups A, B and C was observed compared to group D. When the oocyst counts amongst the treatment groups A, B and C were compared, a significant decrease (p < 0.05) was observed in group A. On day 21 post-treatment, the efficacy of azithromycin, co-trimoxazole and kalvangi in calves was 88.2% (95% C.I. ± 15.4), 45% (95% C.I. ± 21.8) and 27.8% (95% C.I. ± 20.7), respectively. This study confirmed previous reports of azithromycin efficacy against C. parvum infection, but found co-trimoxazole and kalvangi to be ineffective for this infection under these treatment regimens.

  11. Infectious Cryptosporidium parvum oocysts in final reclaimed effluent

    USGS Publications Warehouse

    Gennaccaro, A.L.; McLaughlin, M.R.; Quintero-Betancourt, W.; Huffman, D.E.; Rose, J.B.

    2003-01-01

    Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.

  12. Molecular epidemiological analyses of Cryptosporidium parvum virus 1 (CSpV1), a symbiotic virus of Cryptosporidium parvum, in Japan.

    PubMed

    Murakoshi, Fumi; Ichikawa-Seki, Madoka; Aita, Junya; Yaita, Seiko; Kinami, Aiko; Fujimoto, Katsuhisa; Nishikawa, Yoshifumi; Murakami, Shin; Horimoto, Taisuke; Kato, Kentaro

    2016-01-04

    We show that Cryptosporidium parvum virus 1 (CSpV1), a member of the family Partitiviridae, genus Cryspovirus that can infect Cryptosporidium parvum, is a new candidate for high-resolution tool for tracing C. parvum. CSpV1 was detected in all C. parvum-positive samples tested. Phylogenetic analysis of dsRNA1 sequence from CSpV1 can distinguish infected areas of C. parvum on the national level. Sequences detected in samples from Iwate prefecture and other islands (Tanegashima, and Okinawa) belonged to a single clade. This system can differentiate the samples from Hokkaido and south part of Japan as well as from other countries. Samples from Iwate, Tanegashima, and Okinawa belonged to a single subclade, respectively. Therefore, the CSpV1 dsRNA sequences reflect the regional distribution of their host and have potential as a high-resolution tool to trace C. parvum IIaA15G2R1 subtype.

  13. Cryptosporidium parvum Infection Following Contact with Livestock

    PubMed Central

    Suler, Denis; Mullins, David; Rudge, Travis; Ashurst, John

    2016-01-01

    Context: Scours, or calf diarrhea, is an infectious gastrointestinal disease commonly found in the calves of dairy farms. It primarily presents with diarrhea that can be life threatening to the animal and is also contagious and threatening to the other livestock. Cryptosporidium is one of the major causes of scours and can be transmitted to humans via fecal-oral route, resulting in diarrheal illnesses. Cryptosporidiosis infection usually occurs as a waterborne outbreak with the potential to affect many people at once. Case Report: We report a case of a 24-year-old female farmer who presented to the emergency department with diarrhea after taking care of ill cattle with similar symptoms. Fecal cultures were positive for Cryptosporidium parvum. Given the patient was immunocompetent, no further treatment was warranted. Conclusion: Confirmed cases should be reported, however, treatment is only recommended in children and immunocompromised adults. Clinicians should educate patients on the importance of proper hygiene and handling techniques in order to decrease transmission and recurrence of the protozoan infection. PMID:27583243

  14. Cryptosporidium parvum is not transmissible to fish, amphibians, or reptiles.

    PubMed

    Graczyk, T K; Fayer, R; Cranfield, M R

    1996-10-01

    A recent report suggested that an isolate of Cryptosporidium parvum had established infections in fish, amphibians, and reptiles and raises concern that animals other than mammals might be a potential source of waterborne Cryptosporidium oocysts. To test this possibility, viable C. parvum oocysts, infectious for neonatal BALB/c mice, were delivered by gastric intubation to bluegill sunfish, poison-dart frogs, African clawed frogs, bearded dragon lizards, and corn snakes. Histological sections of the stomach, jejunum, ileum, and cloaca prepared from tissues collected on days 7 and 14 postinoculation (PI) were negative for Cryptosporidium developmental stages. However, inoculum-derived oocysts were detectable by fluorescein-labeled monoclonal antibody in feces of inoculated animals from day 1 to day 12 PI in fish and frogs, and up to day 14 PI in lizards. Snakes did not defecate for 14 days PI. Impression smears taken at necropsy on days 7 and 14 PI revealed C. parvum oocysts in the lumen of the cloaca of 2 fish and 1 lizard on day 7 PI only. Because tissue stages of the pathogen were not found, it appears that C. parvum was not heterologously transmitted to lower vertebrates. Under certain circumstances, however, such as after the ingestion of C. parvum-infected prey, lower vertebrates may disseminate C. parvum oocysts in the environment.

  15. Cryptosporidium parvum studies with dairy products.

    PubMed

    Deng, M Q; Cliver, D O

    1999-02-02

    Cryptosporidium parvum is a protozoan parasite capable of causing massive waterborne outbreaks. This study was conducted to model the transfer of C. parvum oocysts from contaminated water via food contact surfaces into yogurt and ice-cream, as well as to examine oocyst survival. Propidium iodide staining, combined with a direct immunofluorescence assay, was used for oocyst viability determination. Oocysts were recovered from milk products by a sucrose flotation-based procedure, with average recoveries of 82.3, 60.7, and 62.5% from low (1%) fat milk, 9% fat ice-cream, and 98% fat-free yogurt, respectively. Oocysts were also recovered, by rinsing with tap water, from stainless steel surfaces inoculated with oocyst suspension, with average recoveries of 93.1% when the surface was still wet and 69.0% after the surface had air-dried at room temperature. Viability of oocysts on the surface was significantly affected by desiccation; 5% of the oocysts remained viable after 4 h of air-drying at room temperature, while the proportion of viable oocysts was 81, 69, and 45% after air-drying for 10 min, 1 h, and 2 h, respectively. In contrast, oocyst viability only dropped from 82 to 75% after 30 min contact at room temperature with 5% bleach solution (equivalent to 0.26% NaOCl). Transfer of oocysts from milk and stainless steel surfaces into yogurt, and oocyst survival during the process were analyzed. Yogurt was made from pasteurized low fat milk and live yogurt starter by incubating at 37 degrees C for 48 h and then stored at 4 degrees C. Oocyst viability decreased from 83% (80%) to approximately 60% after 48 h at 37 degrees C and to approximately 58% following 8 days of storage, similar to oocyst survival in the controls using pasteurized milk without the addition of live yogurt. Oocyst survival in ice-cream was investigated by inoculating oocysts into ice-cream mix, and mixing and freezing in an ice-cream freezer, and hardening at -20 degrees C. Although approximately 20

  16. Cryptosporidium parvum DNA replication in cell-free culture.

    PubMed

    Zhang, L; Sheoran, A S; Widmer, G

    2009-10-01

    The lack of robust methods for culturing Cryptosporidium parasites remains a major challenge and is hampering efforts to screen for anti-cryptosporidial drugs. In existing culture methods, monolayers of mammalian epithelial cells are inoculated with oocysts. The system supports an initial phase of asexual proliferation of the parasite. For reasons that are not clear, development rapidly declines within 2-3 days. The unexpected report of Cryptosporidium parvum culture in the absence of host cells, and the failure of others to reproduce the method, prompted us to apply quantitative PCR to measure changes in C. parvum DNA levels in cell-free cultures, and parasite-specific antibodies to identify different life cycle stages. Based on this approach, which has not been applied previously to analyze C. parvum growth in cell-free culture, we found that the concentration of C. parvum DNA increased by about 5-fold over 5 days of culture. Immuno-labeling of cultured organisms revealed morphologically distinct stages, only some of which reacted with Cryptosporidium-specific monoclonal antibodies. These observations are indicative of a modest proliferation of C. parvum in cell-free culture.

  17. A sensitive method for detecting and genotyping Cryptosporidium parvum oocysts

    USDA-ARS?s Scientific Manuscript database

    Cryptosporidium parvum oocysts represent a considerable health risk to humans and animals because the parasite has a low infectious dose and usually exists in low numbers in environmental samples, which makes detection problematic. The purpose of this study was to evaluate Cryspovirus as a target f...

  18. DIFFERENTIATING HUMAN FROM ANIMAL ISOLATES OF CRYPTOSPORIDIUM PARVUM

    EPA Science Inventory

    We analyzed 9s Cryptosporidium parvum isolates from humans and animals by a polymerase chain reaction/restriction fragment length polymorphism method based on the thrombospondin-related anonymous protein 2 gene sequence. Used as a molecular marker, this method can differentiate ...

  19. A COMPARISON OF ENUMERATION TECHNIQUES FOR CRYPTOSPORIDIUM PARVUM OOCYSTS

    EPA Science Inventory

    A variety of methods have been used to enumerate Cryptosporidium parvum oocysts from source or drinking waters. The reliability of these counting methods varies, in part, with suspension density, sample purity, and other factors. Frequently, the method of determination of suspens...

  20. A COMPARISON OF ENUMERATION TECHNIQUES FOR CRYPTOSPORIDIUM PARVUM OOCYSTS

    EPA Science Inventory

    A variety of methods have been used to enumerate Cryptosporidium parvum oocysts from source or drinking waters. The reliability of these counting methods varies, in part, with suspension density, sample purity, and other factors. Frequently, the method of determination of suspens...

  1. Chlorine disinfection of recreational water for Cryptosporidium parvum.

    PubMed Central

    Carpenter, C.; Fayer, R.; Trout, J.; Beach, M. J.

    1999-01-01

    We examined the effects of chlorine on oocyst viability, under the conditions of controlled pH and elevated calcium concentrations required for most community swimming pools. We found that fecal material may alter the Ct values (chlorine concentration in mg/L, multiplied by time in minutes) needed to disinfect swimming pools or other recreational water for Cryptosporidium parvum. PMID:10458969

  2. The Effect of pH on Stability and Sorption of Cryptosporidium parvum Oocysts by Nanoparticles

    EPA Science Inventory

    Cryptosporidium parvum (C. parvum) are waterborne pathogens, which are released into the environment through infected human or animal feces. Their ability to survive outside their host organisms in harsh environmental conditions presents one of the most challenging tasks in resea...

  3. The Effect of pH on Stability and Sorption of Cryptosporidium parvum Oocysts by Nanoparticles

    EPA Science Inventory

    Cryptosporidium parvum (C. parvum) are waterborne pathogens, which are released into the environment through infected human or animal feces. Their ability to survive outside their host organisms in harsh environmental conditions presents one of the most challenging tasks in resea...

  4. Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

    PubMed

    Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

    2015-03-01

    The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes.

  5. Quantitative-PCR assessment of Cryptosporidium parvum cell culture infection.

    PubMed

    Di Giovanni, George D; LeChevallier, Mark W

    2005-03-01

    A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.

  6. Identification of genetic heterogeneity in the Cryptosporidium parvum ribosomal repeat.

    PubMed Central

    Carraway, M; Tzipori, S; Widmer, G

    1996-01-01

    Oocysts of the protozoan parasite Cryptosporidium parvum are found in most surface waters and can contaminate municipal water supplies, as demonstrated by recent outbreaks of cryptosporidiosis. A method capable of fingerprinting C. parvum isolates from the environment would facilitate the study of epidemiology and transmission cycles and aid in the implementation of preventive measures to reduce water contamination by oocytes. In this study, we report polymorphism in C. parvum isolates on the basis of analysis of random amplified polymorphic DNA and nucleotide sequences in a region of the 18S rRNA and the internal transcribed spacer 1. Isolate-specific primers for these two regions were designed, and PCR tests capable of discriminating between isolates were developed. In both PCR assays, the five C. parvum isolates analyzed segregated into two subgroups. One group consisted of isolates that originated directly from human patients, and the other group had various host origins and had been propagated in laboratory animals. These results demonstrate the feasibility of distinguishing C. parvum isolates by sequence-specific PCR tests. PMID:8593074

  7. Cryptosporidium parvum in oysters from commercial harvesting sites in the Chesapeake Bay.

    PubMed

    Fayer, R; Lewis, E J; Trout, J M; Graczyk, T K; Jenkins, M C; Higgins, J; Xiao, L; Lal, A A

    1999-01-01

    Oocysts of Cryptosporidium parvum, a zoonotic waterborne pathogen, can be removed by bivalve molluscs from contaminated water and retained on gills and in hemolymph. We identified oocysts of C. parvum in oysters from seven sites in the Chesapeake Bay area. These findings document the presence of C. parvum infectious for humans in oysters intended for human consumption.

  8. Gamma irradiation of Cryptosporidium parvum oocysts affects intracelluar levels of the viral symbiont CPV

    USDA-ARS?s Scientific Manuscript database

    Previous studies have shown a dose-dependent effect of gamma irradiation on Cryptosporidium parvum development in neonatal mice and newborn calves. In mice, C. parvum oocysts exposed to 200 Gy showed nearly complete inability to develop as measured by C. parvum-specific quantitative PCR of ileal ti...

  9. Cryptosporidium parvum has an active hypusine biosynthesis pathway

    PubMed Central

    Mittal, Nimisha; Morada, Marie; Tripathi, Pankaj; Gowri, V.S.; Mandal, Swati; Quirch, Alison; Park, Myung Hee; Yarlett, Nigel; Madhubala, Rentala

    2014-01-01

    The protozoan parasite Cryptosporidium parvum causes severe enteric infection and diarrheal disease with substantial morbidity and mortality in untreated AIDS patients and children in developing or resource-limited countries. No fully effective treatment is available. Hypusination of eIF5A is an important post-translational modification essential for cell proliferation. This modification occurs in a two step process catalyzed by deoxyhypusine synthase (DHS) followed by deoxyhypusine hydroxylase. An ORF of 1086 bp was identified in the C. parvum (Cp) genome which encodes for a putative polypeptide of 362 amino acids. The recombinant CpDHS protein was purified to homogeneity and used to probe the enzyme’s mechanism, structure, and inhibition profile in a series of kinetic experiments. Sequence analysis and structural modeling of CpDHS were performed to probe differences with respect to the DHS of other species. Unlike Leishmania, Trypanosomes and Entamoeba, Cryptosporidium contains only a single gene for DHS. Phylogenetic analysis shows that CpDHS is more closely related to apicomplexan DHS than kinetoplastid DHS. Important residues that are essential for the functioning of the enzyme including NAD+ binding residues, spermidine binding residues and the active site lysine are conserved between CpDHS and human DHS. N1-guanyl-1.7-diaminoheptane (GC7), a potent inhibitor of DHS caused an effective inhibition of infection and growth of C. parvum in HCT-8 cells. PMID:24893338

  10. Zoonotic Cryptosporidium parvum in Romanian newborn lambs (Ovis aries).

    PubMed

    Imre, Kálmán; Luca, Cătălina; Costache, Marieta; Sala, Claudia; Morar, Adriana; Morariu, Sorin; Ilie, Marius S; Imre, Mirela; Dărăbuş, Gheorghe

    2013-01-16

    This study was undertaken to investigate the occurrence and public health significance of Cryptosporidium species/genotypes and subtypes in a newborn lambs. A total of 175 diarrheic fecal samples from lambs (younger than 21 days) were collected in seven sheep flocks located in western Romania, and were microscopically examined for the presence of Cryptosporidium oocysts after staining with modified Ziehl-Neelsen technique. Twenty-four (13.7%) fecal samples were tested Cryptosporidium positive by microscopy and were subjected for molecular characterization. All positive samples were successfully amplified through a nested polymerase chain reaction (PCR) of the small subunit (SSU) rRNA gene (18S). Cryptosporidium species were determined by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using the conventional SspI and VspI restriction enzymes. The identified species were: Cryptosporidium parvum (20/24), C. ubiquitum (2/24) and C. xiaoi (2/24), respectively. PCR-RFLP results for C. ubiquitum and C. xiaoi isolates were confirmed by DNA sequencing. Subsequently, subtyping of seven randomly selected C. parvum isolates, based on sequence analysis of the GP60 gene, revealed the presence of five different subtypes (IIaA17G1R1, IIaA16G1R1, IIdA20G1, IIdA24G1 and IIdA22G2R1) belonging in two zoonotic subtype families (IIa and IId). These findings may suggest the potential role of the newborn lambs as a source for human cryptosporidiosis. This is the first published report about the presence of C. ubiquitum and C. xiaoi in lambs from Romania. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Analysis of Cryptosporidium parvum oocyst transport in porous media

    NASA Astrophysics Data System (ADS)

    Kim, Song-Bae; Yavuz Corapcioglu, M.

    2004-08-01

    Cryptosporidium parvum is a protozoan parasite, transmitted through aqueous environments in the form of an oocyst. In this study, a transport model into which sorption, filtration and inactivation mechanisms are incorporated is applied to simulate laboratory column data, and the suitability of a kinetic model to describe the C. parvum oocyst transport and removal in porous media is compared with an equilibrium model. The kinetic model is applied to simulate previous column experimental data and successfully simulates the concentration peak; the late time tailing effect appeared in the breakthrough curves, indicating that the kinetic model is more suitable than the equilibrium one at simulating the fate and transport of the oocysts in porous media. Simulation illustrates that sorption causes retardation along with a tailing in the breakthrough curve. Additionally, filtration acts as a major mechanism of removing the oocysts from the aqueous phase, whereas the role of inactivation in reducing the viable oocyst concentration is minimal.

  12. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.

    PubMed

    Vinayak, Sumiti; Pawlowic, Mattie C; Sateriale, Adam; Brooks, Carrie F; Studstill, Caleb J; Bar-Peled, Yael; Cipriano, Michael J; Striepen, Boris

    2015-07-23

    Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.

  13. First report of Cryptosporidium parvum 'ferret' genotype in American mink (Mustela vison Shreber 1777).

    PubMed

    Gómez-Couso, H; Méndez-Hermida, F; Ares-Mazás, E

    2007-03-01

    A total of 51 faecal samples from wild and farmed mink were analysed by a direct immunofluorescence antibody test. Cryptosporidium oocysts were identified in eight, apparently healthy, farmed American mink (Mustela vison). The isolates were identified as Cryptosporidium parvum 'ferret' genotype by PCR-RFLP and sequencing analysis of a 341-base-pair fragment of the Cryptosporidium oocyst wall protein (COWP) gene. This is the first report of Cryptosporidium in American mink.

  14. Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis.

    PubMed

    Feng, X; Rich, S M; Akiyoshi, D; Tumwine, J K; Kekitiinwa, A; Nabukeera, N; Tzipori, S; Widmer, G

    2000-08-01

    Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.

  15. Removal of Cryptosporidium parvum in bank filtration systems

    NASA Astrophysics Data System (ADS)

    Harter, T.; Atwill, E. R.; Hou, L. L.

    2003-04-01

    The protozoan pathogen Cryptosporidium parvum is a leading cause of waterborne disease. Many surface water systems therefore depend on filtration systems, including bank filtration systems, for the removal of the pathogenic oocysts. To better understand the effectiveness, e.g., of bank filtration systems, we have implemented a series of columns studies under various environmental conditions (column length: 10 cm - 60 cm, flow rates: 0.7 m/d - 30 m/d, ionic strength: 0.01 - 100 mM, filter grain size: 0.2 - 2 mm, various solution chemistry). We show that classic colloid filtration theory is a reasonable tool for predicting the initial breakthrough of C. parvum in pulsed injections of the oocyst through sand columns, although the model does not account for the significant tailing that occurs in C. parvum transport. Application of colloid filtration theory to bank filtration system is further limited by the intrinsic heterogeneity of the geologic systems used for bank filtration. We couple filtration theory with a stochastic subsurface transport approach and with percolation theory to account for the effects of intrinsic heterogeneity. We find that a 1-log removal can be achieved even under relatively adverse conditions (low collision efficiency, high velocity) if 85% - 90% of the sedimentary hydrofacies located within the bank filtration system or of the coarsest known hydrofacies connecting the riverbed with the extraction system has a grain-size distribution with a 10% passing diameter equal to 1 mm. One millimeter is a standard sieve size in sediment analysis.

  16. Viability of Cryptosporidium parvum during ensilage of perennial ryegrass.

    PubMed

    Merry, R J; Mawdsley, J L; Brooks, A E; Davies, D R

    1997-01-01

    The survival of Cryptosporidium parvum during ensilage of perennial ryegrass was examined in laboratory silos with herbage prepared in one of three different ways; either untreated, inoculated with a strain of Lactobacillus plantarum or by direct acidification with formic acid. The pH values of all silages initially fell below 4.5, but only formic acid-treated silage remained stable at less than pH 4 after 106 d, with the pH of the untreated and inoculant-treated silages rising to above 6. The formic acid-treated silage had a high lactic acid concentration (109 g kg-1 dry matter (DM)) and low concentrations of propionic and butyric acids after 106 d. However, the untreated and inoculant-treated silages showed an inverse relationship, with low lactic acid concentrations and high concentrations of acetic, propionic and butyric acids. These silages also contained ammonia-N concentrations in excess of 9 g kg-1 DM. In terms of the viability of Cryptosporidium parvum oocysts very few differences were seen after 14 d of ensilage with ca 50% remaining viable, irrespective of treatment and total numbers had declined from the initial level of 5.9 x 10(4) to 1 x 10(4) g(-1) fresh matter. Total oocyst numbers remained approximately the same until the end of the ensiling period, with the percentage of viable oocysts declining to 46, 41 and 32% respectively for formic acid, inoculant and untreated silages. The results are discussed in terms of changes occurring during the silage fermentation, in particular the products which may influence the survival of Cryptosporidium and implications for agricultural practice and the health of silage fed livestock.

  17. Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst.

    PubMed

    Carey, C M; Lee, H; Trevors, J T

    2004-02-01

    Cryptosporidium parvum and Cryptosporidium hominis are obligate enteric protozoan parasites which infect the gastrointestinal tract of animals and humans. The mechanism(s) by which these parasites cause gastrointestinal distress in their hosts is not well understood. The risk of waterborne transmission of Cryptosporidium is a serious global issue in drinking water safety. Oocysts from these organisms are extremely robust, prevalent in source water supplies and capable of surviving in the environment for extended periods of time. Resistance to conventional water treatment by chlorination, lack of correlation with biological indicator microorganisms and the absence of adequate methods to detect the presence of infectious oocysts necessitates the development of consistent and effective means of parasite removal from the water supply. Additional research into improving water treatment and sewage treatment practices is needed, particularly in testing the efficiency of ozone in oocyst inactivation. Timely and efficient detection of infectious C. parvum and C. hominis oocysts in environmental samples requires the development of rapid and sensitive techniques for the concentration, purification and detection of these parasites. A major factor confounding proper detection remains the inability to adequately and efficiently concentrate oocysts from environmental samples, while limiting the presence of extraneous materials. Molecular-based techniques are the most promising methods for the sensitive and accurate detection of C. parvum and C. hominis. With the availability of numerous target sequences, RT-PCR will likely emerge as an important method to assess oocyst viability. In addition, a multiplex PCR for the simultaneous detection of C. parvum, C. hominis and other waterborne pathogens such as Giardia lamblia would greatly benefit the water industry and protect human health.

  18. Efficacy of vegetated buffer strips for retaining Cryptosporidium parvum.

    PubMed

    Tate, Kenneth W; Pereira, Maria Das Gracas C; Atwill, Edward R

    2004-01-01

    Overland and shallow subsurface hydrologic transport of pathogenic Cryptosporidium parvum oocysts from cattle feces into surface drinking water supplies is a major concern on annual grasslands in California's central and southern Sierra Nevada foothills. Soil boxes (0.5 m wide x 1.1 m long x 0.3 m deep) were used to evaluate the ability of grass vegetated buffer strips to retain 2 x 10(8) spiked C. parvum oocysts in 200-g fecal deposits during simulated rainfall intensities of 30 to 47.5 mm/h over 2 h. Buffers were comprised of Ahwahnee sandy loam (coarse-loamy, mixed, active, thermic Mollic Haploxeralfs; 78:18:4 sand to silt to clay ratio; dry bulk density = 1.4 g/cm(3)) set at 5 to 20% land slope, and >/=95% grass cover (grass stubble height = 10 cm; biomass = 900 kg/ha dry weight). Total number of oocysts discharged from each soil box (combined overland and subsurface flow) during the 120-min simulation ranged from 1.5 x 10(6) to 23.9 x 10(6) oocysts. Observed overall mean log(10) reduction of total C. parvum flux per meter of vegetated buffer was 1.44, 1.19, and 1.18 for buffers at 5, 12, and 20% land slope, respectively. Rainfall application rate (mm/h) was strongly associated with oocyst flux from these vegetated buffers, resulting in a decrease of 2 to 4% in the log(10) reduction per meter buffer for every additional mm/h applied to the soil box. These results support the use of strategically placed vegetated buffers as one of several management strategies that can reduce the risk of waterborne C. parvum attributable to extensive cattle grazing on annual grassland watersheds.

  19. Hydrophobic and electrostatic cell surface properties of Cryptosporidium parvum.

    PubMed

    Drozd, C; Schwartzbrod, J

    1996-04-01

    Microbial adhesion to hydrocarbons and microelectrophoresis were investigated in order to characterize the surface properties of Cryptosporidium parvum. Oocysts exhibited low removal rates by octane (only 20% on average), suggesting that the Cryptosporidium sp. does not demonstrate marked hydrophobic properties. A zeta potential close to -25 mV at pH 6 to 6.5 in deionized water was observed for the parasite. Measurements of hydrophobicity and zeta potential were performed as a function of pH and ionic strength or conductivity. Hydrophobicity maxima were observed at extreme pH values, with 40% of adhesion of oocysts to octane. It also appeared that ionic strength (estimated by conductivity) could influence the hydrophobic properties of oocysts. Cryptosporidium oocysts showed a pH-dependent surface charge, with zeta potentials becoming less negative as pH was reduced, starting at -35 mV for alkaline pH and reaching 0 at isoelectric points for pH 2.5. On the other hand, variation of surface charge with respect to conductivity of the suspension tested in this work was quite small. The knowledge of hydrophobic properties and surface charge of the parasite provides information useful in, for example, the choice of various flocculation treatments, membrane filters, and cleaning agents in connection with oocyst recovery.

  20. MOLECULAR CLONING AND ANALYSIS OF THE CRYPTOSPORIDIUM PARVUM AMINOPEPTIDASE N GENE. (R829180)

    EPA Science Inventory

    Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules fo...

  1. MOLECULAR CLONING AND ANALYSIS OF THE CRYPTOSPORIDIUM PARVUM AMINOPEPTIDASE N GENE. (R828035)

    EPA Science Inventory

    Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules fo...

  2. CHARACTERIZATION OF CRYPTOSPORIDIUM PARVUM BY MATRIX-ASSISTED LASER DESORPTION -- IONIZATION TIME OF FLIGHT MASS SPECTROMETRY

    EPA Science Inventory

    Matrix assisted laser desorption/ionization (MALDI) mass spectrometry was used to investigate whole and freeze thawed Cryptosporidium parvum oocysts. Whole oocysts revealed some mass spectral features. Reproducible patterns of spectral markers and increased sensitivity were obtai...

  3. Sensitive quantitative detection/identification of infectious Cryptosporidium parvum oocysts by signature lipid biomarker analysis

    SciTech Connect

    White, D.C. |; Alugupalli, S.; Schrum, D.P.

    1997-08-01

    Unique signature lipid biomarkers were found in the acid-fast oocytes of Cryptosporidium parvum. This makes possible the rapid detection/identification and potential infectivity directly from drinking water membrane filtrates.

  4. AGING OF CRYPTOSPORIDIUM PARVUM OOCYSTS STUDIED BY MALDI-TF MS

    EPA Science Inventory

    Cryptosporidium parvum is an obligate protozoan parasite found in surface waters. It is the etiological agent for cryptosporidiosis, a parasitic infection that causes severe gastrointestinal illness which is potentially fatal among immuno-compromised individuals. This water borne...

  5. Attachment, persistence and infectivity of Cryptosporidium parvum oocysts in experimentally contaminated fruits and vegetables

    USDA-ARS?s Scientific Manuscript database

    Cryptosporidium parvum is an environmentally resistant, abundant, and ubiquitous protozoan parasite that causes severe diarrheal disease in humans and livestock. Consumer dietary preference towards fresh and organically grown produce correlates with a heightened occurrence of foodborne outbreaks of ...

  6. EVALUATING IN VITRO INFECTIVITY FOR MEASURING UV DISINFECTION OF CRYPTOSPORIDIUM PARVUM OOCYSTS IN FINISHED WATER

    EPA Science Inventory

    UV technology to inactivate Cryptosporidium parvum oocysts has become well established in the US. The challenge now is to effectively demonstrate UV reactor performance and disinfection capacity with various finished water matrices and under different operational conditions. In s...

  7. AGING OF CRYPTOSPORIDIUM PARVUM OOCYSTS STUDIED BY MALDI-TF MS

    EPA Science Inventory

    Cryptosporidium parvum is an obligate protozoan parasite found in surface waters. It is the etiological agent for cryptosporidiosis, a parasitic infection that causes severe gastrointestinal illness which is potentially fatal among immuno-compromised individuals. This water borne...

  8. EVALUATING IN VITRO INFECTIVITY FOR MEASURING UV DISINFECTION OF CRYPTOSPORIDIUM PARVUM OOCYSTS IN FINISHED WATER

    EPA Science Inventory

    UV technology to inactivate Cryptosporidium parvum oocysts has become well established in the US. The challenge now is to effectively demonstrate UV reactor performance and disinfection capacity with various finished water matrices and under different operational conditions. In s...

  9. MOLECULAR CLONING AND ANALYSIS OF THE CRYPTOSPORIDIUM PARVUM AMINOPEPTIDASE N GENE. (R829180)

    EPA Science Inventory

    Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules fo...

  10. MOLECULAR CLONING AND ANALYSIS OF THE CRYPTOSPORIDIUM PARVUM AMINOPEPTIDASE N GENE. (R828035)

    EPA Science Inventory

    Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules fo...

  11. Giardia sp. Cysts and Infectious Cryptosporidium parvum Oocysts in the Feces of Migratory Canada Geese (Branta canadensis)

    PubMed Central

    Graczyk, Thaddeus K.; Fayer, Ronald; Trout, James M.; Lewis, Earl J.; Farley, C. Austin; Sulaiman, Irshad; Lal, Altaf A.

    1998-01-01

    Fecal droppings of migratory Canada geese, Branta canadensis, collected from nine sites near the Chesapeake Bay (Maryland), were examined for the presence of Cryptosporidium parvum and Giardia spp. Cryptosporidium sp. oocysts were found in feces at seven of nine sites, and Giardia cysts were found at all nine sites. The oocysts from three sites were infectious for mice and molecularly identified as the zoonotic genotype of Cryptosporidium parvum. Waterfowl can disseminate infectious C. parvum oocysts in the environment. PMID:9647860

  12. Fecundity of Cryptosporidium parvum is Correlated with Intracellular Levels of the Viral Symbiont CPV

    USDA-ARS?s Scientific Manuscript database

    Differences in the virulence and fecundity of Cryptosporidium parvum isolates have been observed by several researchers studying cryptosporidiosis. The purpose of the present study was to determine if there was a correlation between intracellular levels of the viral symbiont CPV in C. parvum and fe...

  13. Development of a Real-Time Quantitative PCR Assay to Detect Cryptosporidium parvum Oocysts in Soil

    EPA Science Inventory

    The risk of Cryptosporidium parvum (C. parvum) contamination is a serious issue with respect to drinking water, as evidenced by the cryptosporidiosis outbreak in Milwaukee WI, in 1993, which involved over 400,000 infections and at least 54 deaths. Ground-water contamination by C...

  14. Development of a Real-Time Quantitative PCR Assay to Detect Cryptosporidium parvum Oocysts in Soil

    EPA Science Inventory

    The risk of Cryptosporidium parvum (C. parvum) contamination is a serious issue with respect to drinking water, as evidenced by the cryptosporidiosis outbreak in Milwaukee WI, in 1993, which involved over 400,000 infections and at least 54 deaths. Ground-water contamination by C...

  15. AGING OF CRYPTOSPORIDIUM PARVUM OOCYSTS STUDIED BY MALDI-TOF MS

    EPA Science Inventory

    Cryptosporidium parvum is a protozoan parasite, and it causes a potentially fatal gastrointestinal illness. This water borne pathogen has been the subject of several high profile disease outbreaks in the US and abroad. C. parvum presents challenges for both compliance monitoring ...

  16. AGING OF CRYPTOSPORIDIUM PARVUM OOCYSTS STUDIED BY MALDI-TOF MS

    EPA Science Inventory

    Cryptosporidium parvum is a protozoan parasite, and it causes a potentially fatal gastrointestinal illness. This water borne pathogen has been the subject of several high profile disease outbreaks in the US and abroad. C. parvum presents challenges for both compliance monitoring ...

  17. Photocatalytic inactivation of Cryptosporidium parvum on nanostructured titanium dioxide films.

    PubMed

    Sunnotel, O; Verdoold, R; Dunlop, P S M; Snelling, W J; Lowery, C J; Dooley, J S G; Moore, J E; Byrne, J A

    2010-03-01

    Control of waterborne gastrointestinal parasites represents a major concern to water industries worldwide. In developed countries, pathogens in drinking water supplies are normally removed by sand filtration followed by chemical disinfection. Cryptosporidium spp. are generally resistant to common disinfection techniques and alternative control strategies are being sought. In the current study, the photocatalytic inactivation of C. parvum oocysts was shown to occur in buffer solution (78.4% after 180 min) and surface water (73.7% after 180 min). Viability was assessed by dye exclusion, excystation, direct examination of oocysts and a novel gene expression assay based on lactate dehydrogenase 1 (LDH1) expression levels. Collectively, this confirmed the inactivation of oocysts and scanning electron microscopy (SEM) confirmed cleavage at the suture line of oocyst cell walls, revealing large numbers of empty (ghost) cells after exposure to photocatalytic treatment.

  18. Adaptation and immunogenicity of Cryptosporidium parvum to immunocompetent mice.

    PubMed

    Matsuo, Tomohide; Tsuge, Yasuko; Umemiya-Shirafuji, Rika; Fujino, Takashi; Matsui, Toshihiro

    2014-03-01

    The adaptation and immunogenisity of Cryptosporidium parvum isolated from Siberian chipmunks (SC1 strain) in immunocompetent (ICR) mice were examined. The oocysts were received to the severe combined immunodeficiency (SCID) mice by repeated passage. The oocysts collected from the 18th SCID mice were inoculated to 5 ICR mice. The mice began to shed oocysts from 6 days after inoculation, the patency was 5 days, and the maximum oocysts per gram of feces (OPG) value was 10(4). The maximum of OPG value was gradually increased by successive passage, and finally that in the 22nd mice reached 10(6) (patency: 11 days). It is considered that these results indicate completion of their adaptation to ICR mice. To examine the immunogenicity of C. parvum to ICR mice, 8 groups of 5 mice each were inoculated with 1.3 × 10(6) oocysts of SC1 strain, which were collected after adaptation to SCID mice. All groups shed oocysts from 6th day, and their patency was from 8 to 12 days. On the 21st day after the primary infection, these mice were challenged with 1.3 × 10(6) oocysts. No oocysts shed from any groups, although 2 control groups shed oocysts from the 6th day, and their OPG values were more than 10(6). These results suggest that this strain has strong immunogenicity against ICR mice. Therefore, the immunological healthy mice were considered a useful experimental model to investigate immunological and drug treatments in the strain of C. parvum.

  19. The use of an atmospheric cold plasma jet to inactivate Cryptosporidium parvum oocysts on cilantro

    USDA-ARS?s Scientific Manuscript database

    Introduction: In 2015, the CDC reported a rise in outbreaks linked to parasites like Cryptosporidium. Outbreaks of Cryptosporidium parvum have been associated with contaminated drinking or recreational water; however, there is growing concern that oocysts may become a more common contaminant in food...

  20. Tracking Cryptosporidium parvum by sequence analysis of small double-stranded RNA.

    PubMed Central

    Xiao, L.; Limor, J.; Bern, C.; Lal, A. A.

    2001-01-01

    We sequenced a 173-nucleotide fragment of the small double-stranded viruslike RNA of Cryptosporidium parvum isolates from 23 calves and 38 humans. Sequence diversity was detected at 17 sites. Isolates from the same outbreak had identical double-stranded RNA sequences, suggesting that this technique may be useful for tracking Cryptosporidium infection sources. PMID:11266306

  1. Development of a Novel, Rapid Integrated Cryptosporidium parvum Detection Assay

    PubMed Central

    Kozwich, Diane; Johansen, Kristine A.; Landau, Keli; Roehl, Christopher A.; Woronoff, Sam; Roehl, Patrick A.

    2000-01-01

    The aim of this study was to develop a reverse transcription-PCR assay and lateral flow detection protocol for specific identification of Cryptosporidium parvum. The method which we developed is sensitive and specific and has a low limit of detection. In our protocol a solid phase material, the Xtra Bind Capture System, was used for extraction and purification of double-stranded RNA (dsRNA) specific for C. parvum. The Xtra Bind Capture System interfaced with pellets concentrated from water samples collected with previously developed filtration devices. The pellets were resuspended in reagent water (final volume, 0.5 ml), and an equal amount of rupture buffer and the Xtra Bind Capture System was added to the resuspended pellet mixture. The dsRNA target sequences in a 0.5-ml portion were captured by the solid phase material via hybridization. The debris and potential inhibitors were removed by washing the Xtra Bind material several times with buffer. The Xtra Bind material with its bound dsRNA was added directly to an amplification reaction mixture, and the target was amplified without elution from the Xtra Bind material. A PCR was performed in the presence of the Xtra Bind Capture System, which resulted in robust amplification of the target. The detection system which we used was adapted from lateral flow chromatography methods typically used for antigen-antibody reactions. The result was a colored line that was visible if the organism was present. When this method was used, we were able to reproducibly and correctly identify 10 oocysts added to 0.5 ml of reagent water. When the protocol was evaluated with a small set of environmental samples, the level of detection was as low as 1 oocyst/liter. The total time from resuspension of the pellet to detection was about 3 h, which is considerably less than the 5 h required for immunomagnetic separation followed by an indirect immunofluorescence assay and microscopy. PMID:10877759

  2. Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein.

    PubMed

    Jenkins, Mark C; O'Brien, Celia N; Trout, James M

    2008-02-01

    Monoclonal antibodies (MAb) were prepared against the 40-kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. In immunoblotting analysis, MAbCPV40-1 bound to a 40-kDa protein in extracts of C. parvum oocysts. This 40-kDa protein was localized in the sporozoite cytoplasm by immunofluorescence (IFA) staining with MAbCPV40-1. In a dot-blot assay, MAbCPV40-1 was capable of detecting 10(2) non-bleach-treated and 10(2)-10(3) bleach-treated C. parvum oocysts. MAbCPV40-1 was capable of detecting CPV40 antigen in both soluble and total C. parvum oocyst protein extracts, indicating a potential use for detecting this parasite in environmental samples.

  3. Quantification of hsp70 mRNA from the Cryptosporidium parvum in soil by reverse transcription real-time PCR

    EPA Science Inventory

    As one of the leading causes of waterborne enteric disease, Cryptosporidium parvum poses significant threat to public health. Besides water, soil can also become an important environmental source of C. parvum once polluted. Detection of viable C. parvum in soil is a key issue whe...

  4. Quantification of hsp70 mRNA from the Cryptosporidium parvum in soil by reverse transcription real-time PCR

    EPA Science Inventory

    As one of the leading causes of waterborne enteric disease, Cryptosporidium parvum poses significant threat to public health. Besides water, soil can also become an important environmental source of C. parvum once polluted. Detection of viable C. parvum in soil is a key issue whe...

  5. Transport of Cryptosporidium parvum Oocysts in a Silicon Micromodel

    SciTech Connect

    Liu, Yuanyuan; Zhang, Changyong; Hilpert, Markus; Kuhlenschmidt, Mark S.; Kuhlenschmidt, Theresa B.; Nguyen, Thanh H.

    2012-02-01

    Effective removal of Cryptosporidium parvum oocysts by granular filtration requires the knowledge of oocyst transport and deposition mechanisms, which can be obtained based on real time microscopic observation of oocyst transport in porous media. Attachment of oocysts to silica surface in a radial stagnation point flow (RSPF) cell and in a micromodel, which has 2-dimensional (2-D) microscopic pore structures consisting of an array of cylindrical collectors, was studied and compared. Real time transport of oocysts in the micromodel was recorded to determine the attached oocyst distributions in transversal and longitudinal directions. In the micromodel, oocysts attached to the forward portion of clean collectors, where the flow velocity was lowest. After initial attachment, oocysts attached onto already attached oocysts. As a result, the collectors ripened and the region available for flow was reduced. Results of attachment and detachment experiments suggest that surface charge heterogeneity allowed for oocyst attachment. In addition to experiments, Lattice-Boltzmann simulations helped understanding the slightly non-uniform flow field and explained differences in the removal efficiency in the transversal direction. However, the hydrodynamic modeling could not explain differences in attachment in the longitudinal direction.

  6. Survival of Cryptosporidium parvum oocysts under various environmental pressures.

    PubMed Central

    Robertson, L J; Campbell, A T; Smith, H V

    1992-01-01

    The survival of various isolates of Cryptosporidium parvum oocysts under a range of environmental pressures including freezing, desiccation, and water treatment processes and in physical environments commonly associated with oocysts such as feces and various water types was monitored. Oocyst viability was assessed by in vitro excystation and by a viability assay based on the exclusion or inclusion of two fluorogenic vital dyes. Although desiccation was found to be lethal, a small proportion of oocysts were able to withstand exposure to temperatures as low as -22 degrees C. The water treatment processes investigated did not affect the survival of oocysts when pH was corrected. However, contact with lime, ferric sulfate, or alum had a significant impact on oocyst survival if the pH was not corrected. Oocysts demonstrated longevity in all water types investigated, including seawater, and when in contact with feces were considered to develop an enhanced impermeability to small molecules which might increase the robustness of the oocysts when exposed to environmental pressures. PMID:1482175

  7. Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts

    PubMed Central

    Gaiser, Birgit K.; Bhandari, Bidha; Bartley, Paul M.; Katzer, Frank; Bridle, Helen

    2015-01-01

    Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 μg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 μg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 μg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall. PMID:26497464

  8. Cryptosporidium species and Cryptosporidium parvum subtypes in dairy calves and goat kids reared under traditional farming systems in Turkey.

    PubMed

    Taylan-Ozkan, Aysegul; Yasa-Duru, Sibel; Usluca, Selma; Lysen, Colleen; Ye, Jianbin; Roellig, Dawn M; Feng, Yaoyu; Xiao, Lihua

    2016-11-01

    Molecular characterizations of Cryptosporidium spp. in ruminants reared under traditional animal management systems are scarce and studies conducted thus far have revealed largely an absence of the pathogenic and zoonotic species Cryptosporidium parvum in pre-weaned animals. In this study, we examined Cryptosporidium species and subtype distribution in free-range pre-weaned dairy calves and goat kids with diarrhea. Cryptosporidium-positive specimens from pre-weaned calves on 10 farms and goat kids on 4 farms in Ankara, Balikesir, Corum, Kirikkale, and Kirsehir Provinces, Turkey were genotyped by PCR-restriction length polymorphism analysis of the small subunit rRNA gene, which identified C. parvum in 27 calves and 9 goat kids and Cryptosporidium ryanae in 1 calf. Among the C. parvum isolates successfully subtyped by DNA sequence analysis of the 60 kDa glycoprotein gene, three subtypes were detected in calves, including IIaA13G2R1 (20/23), IIdA18G1 (2/23), and IIdA20G1b (1/23), and four subtypes were detected in goat kids, including IIaA13G2R1 (3/8), IIaA15G1R1 (2/8), IIdA22G1 (2/8), and IIdA18G1 (1/8). Data of the study suggest that dairy calves reared in a traditional cow-calf system in Turkey are mainly infected with a C. parvum subtype rarely seen elsewhere, whereas goat kids are infected with diverse subtypes. As all five C. parvum subtypes found in this study are known human pathogens, pre-weaned farm animals could play a potential role in the transmission of human cryptosporidiosis.

  9. Role of Tumor Necrosis Factor Alpha in Development of Immunity against Cryptosporidium parvum Infection

    PubMed Central

    Lean, I-Sarah; Lacroix-Lamandé, Sonia; Laurent, Fabrice; McDonald, Vincent

    2006-01-01

    Tumor necrosis factor (TNF-α) significantly reduced Cryptosporidium parvum development in a murine enterocyte cell line, and a key mechanism of action appeared to be inhibition of parasite invasion. However, TNF-α-deficient mice controlled infection as effectively as wild-type mice. This suggests that TNF-α might have only a redundant role for establishing immunity against C. parvum. PMID:16790816

  10. Structure-activity relationship study of selective benzimidazole-based inhibitors of Cryptosporidium parvum IMPDH

    PubMed Central

    Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Sharling, Lisa; Zhang, Minjia; Liu, Xiaoping; Ray, Soumya S.; MacPherson, Iain S.; Striepen, Boris; Hedstrom, Lizbeth; Cuny, Gregory D.

    2012-01-01

    Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The C. parvum and C. hominis genomes indicate that the only route to guanine nucleotides is via inosine 5'-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazole-based inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure-activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro. PMID:22310229

  11. Effect of sunlight on the infectivity of Cryptosporidium parvum in seawater.

    PubMed

    Nasser, Abid M; Telser, Lital; Nitzan, Yeshayahu

    2007-09-01

    The prevalence of pathogenic microorganisms in seawater can result in waterborne and food borne outbreaks. This study was performed to determine the effect of sunlight and salinity on the die-off of Cryptosporidium parvum. Cryptosporidium parvum oocysts, Escherichia coli, and MS2 coliphage were seeded into tap water and seawater samples and then exposed to sunlight. The die-off of C. parvum in seawater, as measured by infectivity, was greater under sunlight (-3.08 log10) than under dark conditions (-1.31 log10). While, no significant difference was recorded in the die-off of C. parvum, under dark conditions, in tap water as compared to seawater (P < 0.05), indicating that the synergistic effect of salinity and sunlight was responsible for the enhanced die-off in seawater. The die-off of MS2 coliphage and E. coli was greater than that observed for C. parvum under all tested conditions. This indicates that these microorganisms cannot serve as indicators for the presence of C. parvum oocysts in seawaters. The results of the study suggest that C. parvum can persist as infectious oocysts for a long time in seawater and can thus pose a serious hazard by direct and indirect contact with humans.

  12. Molecular characterization of Cryptosporidium parvum from two different Japanese prefectures, Okinawa and Hokkaido.

    PubMed

    Ichikawa-Seki, Madoka; Aita, Junya; Masatani, Tatsunori; Suzuki, Moemi; Nitta, Yoshiki; Tamayose, Genta; Iso, Takehiro; Suganuma, Keisuke; Fujiwara, Takashi; Matsuyama, Keita; Niikura, Tadamasa; Yokoyama, Naoaki; Suzuki, Hiroshi; Yamakawa, Kazuhiro; Inokuma, Hisashi; Itagaki, Tadashi; Zakimi, Satoshi; Nishikawa, Yoshifumi

    2015-04-01

    Infectious diarrhea is the most frequent cause of morbidity and mortality in neonatal calves. Cryptosporidium parvum is one of the main pathogens associated with calf diarrhea. Although diarrhea is a symptom of infection with various pathogens, investigations to detect the types of pathogens have never been performed in Japan. This study investigated the prevalence of four major diarrhea-causing pathogens in calves: C. parvum, rotavirus, coronavirus, and enterotoxigenic Escherichia coli (E. coli K99). Commercial immunochromatography testing of all four pathogens and molecular analysis of C. parvum with diarrhea in calves from southernmost Okinawa and northernmost Hokkaido, Japan, were conducted. The frequencies of C. parvum, rotavirus, coronavirus, and E. coli (K99) in Okinawa were 50%, 28%, 2.3%, and 4.7%, respectively. Watery fecal stools were significantly correlated with C. parvum (p<0.05). In oocyst calculations for C. parvum, no significant difference was observed between the single-infection cases and the mixed-infection cases with rotavirus. Interestingly, molecular analyses targeting small subunit ribosomal RNA as well as glycoprotein 60 (GP60) genes revealed that the C. parvum nucleotide sequences from the two prefectures were identical, indicating that C. parvum with a uniform characteristic is distributed throughout Japan. GP60 subtyping analysis identified C. parvum from Okinawa and Hokkaido as belonging to the IIaA15G2R1 subtype, a known zoonotic subtype. Hence, control of cryptosporidiosis is important not only for pre-weaned calves, but also for human health.

  13. PCR-restriction fragment length polymorphism analysis of a diagnostic 452-base-pair DNA fragment discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum isolates of human and animal origin.

    PubMed

    Guyot, K; Follet-Dumoulin, A; Recourt, C; Lelièvre, E; Cailliez, J C; Dei-Cas, E

    2002-04-01

    Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes.

  14. Cryptosporidium parvum GP60 subtypes in dairy cattle from Buenos Aires, Argentina

    USDA-ARS?s Scientific Manuscript database

    Cryptosporidium parvum from 73 dairy calves less than two months old from Buenos Aires province (Argentina) were molecularly characterized using sequence analysis of the GP60 gene. Seventy five sequences were obtained, and seven different subtypes were identified, all belonging to the IIa subtype f...

  15. Improved Cryptosporidium parvum oocysts propagation using dexamethasone suppressed CF-1 mice

    EPA Science Inventory

    This study evaluates Cryptosporidium parvum oocyst production in dexamethasone suppressed CF-1 and C57BL/6 mice. Both models can yield 1 x 109 total oocysts over a 20 day production period; however, only 20 CF-1 mice are required to reliably achieve this goal compared...

  16. Effect of Lot Variability on Ultraviolet Radiation Inactivation Kinetics of Cryptosporidium parvum Oocysts

    EPA Science Inventory

    Numerous studies have demonstrated the efficiency of ultraviolet (UV) radiation for the inactivation of oocysts of Cryptosporidium parvum. In these studies inactivation is measured as reduction in oocysts. A primary goal is to estimate the UV radiation required to achiev...

  17. Short-Term Exposure to Membrane-Active Antibiotics Inhibits Cryptosporidium parvum Infection in Cell Culture

    PubMed Central

    Giacometti, Andrea; Cirioni, Oscar; Del Prete, Maria Simona; Barchiesi, Francesco; Scalise, Giorgio

    2000-01-01

    A cell culture system and double fluorogenic staining were used to study the susceptibility of Cryptosporidium parvum to membrane-active antibiotics. Buforin II and magainin II exerted a cytotoxic effect on sporozoites but did not consistently affect oocyst viability. Lasalocid and nigericin demonstrated less activity against sporozoites but reduced the infectivity of oocysts. PMID:11083662

  18. PREVALENCE AND CONCENTRATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS IN BEEF CATTLE PADDOCK SOILS AND FORAGE

    USDA-ARS?s Scientific Manuscript database

    Cryptosporidium parvum is an enteric coccidian protozoan that receives a great amount of interest because of its widespread occurrence in surface waters, its high degree of infectivity, and the difficulty of risk management associated with its presence and control. Information about environmental l...

  19. DOSE RESPONSE OF CRYPTOSPORIDIUM PARVUM IN OUTBRED NEONATAL CD-1 MICE

    EPA Science Inventory

    Cryptosporidium parvum infectivity in a neonatal CD-1 mouse model was used to determine the dose needed to infect 50% of the population. The 50% infective dose was estimated to be 79 oocysts. It was observed that a mean oral inoculum of 23 oocysts produced infection in 2 of 25 ne...

  20. Coupled Factors Influencing the Transport and Retention of Cryptosporidium Parvum Oocysts in Saturated Porous Media

    USDA-ARS?s Scientific Manuscript database

    The coupled role of solution ionic strength (IS), system hydrodynamics and pore structure on the transport and retention of viable Cryptosporidium parvum oocyst was investigated via batch, packed-bed column, and micromodel systems. The experiments were conducted over a wide range of IS (0.1-100 mM)...

  1. A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum

    EPA Science Inventory

    To better understand and study the infection of the protozoan parasite Cryptosporidium parvum, a more sensitive in vitro assay is required. In vivo, this parasite infects the epithelial cells of the microvilli layer in the small intestine. While cell infection models using colon,...

  2. Effect of Lot Variability on Ultraviolet Radiation Inactivation Kinetics of Cryptosporidium parvum Oocysts

    EPA Science Inventory

    Numerous studies have demonstrated the efficiency of ultraviolet (UV) radiation for the inactivation of oocysts of Cryptosporidium parvum. In these studies inactivation is measured as reduction in oocysts. A primary goal is to estimate the UV radiation required to achiev...

  3. INTESTINAL AND PULMONARY INFECTION BY Cryptosporidium parvum IN TWO PATIENTS WITH HIV/AIDS

    PubMed Central

    REINA, Fábio Tadeu Rodrigues; RIBEIRO, Camila Aparecida; de ARAÚJO, Ronalda Silva; MATTÉ, Maria Helena; CASTANHO, Roberto Esteves Pires; TANAKA, Ioshie Ibara; VIGGIANI, Ana Maria Ferreira Sornas; MARTINS, Luciamáre Perinetti Alves

    2016-01-01

    We describe two patients with HIV/AIDS who presented pulmonary and intestinal infection caused by Cryptosporidium parvum, with a fatal outcome. The lack of available description of changes in clinical signs and radiographic characteristics of this disease when it is located in the extra-intestinal region causes low prevalence of early diagnosis and a subsequent lack of treatment. PMID:27007564

  4. Improved Cryptosporidium parvum oocysts propagation using dexamethasone suppressed CF-1 mice

    EPA Science Inventory

    This study evaluates Cryptosporidium parvum oocyst production in dexamethasone suppressed CF-1 and C57BL/6 mice. Both models can yield 1 x 109 total oocysts over a 20 day production period; however, only 20 CF-1 mice are required to reliably achieve this goal compared...

  5. A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum

    EPA Science Inventory

    To better understand and study the infection of the protozoan parasite Cryptosporidium parvum, a more sensitive in vitro assay is required. In vivo, this parasite infects the epithelial cells of the microvilli layer in the small intestine. While cell infection models using colon,...

  6. Changes in the Levels of Cryspovirus During In Vitro Development of Cryptosporidium parvum

    USDA-ARS?s Scientific Manuscript database

    The purpose of this study was to develop and utilize semi-quantitative RT-PCR and PCR assays for measuring the level of cryspovirus, the viral symbiont of Cryptosporidium parvum, during in vitro development of the protozoan. Cultures of human carcinoma cells (HCT-8) were inoculated with excysting C...

  7. EFFECT OF LOT VARIABILITY ON ULTRAVIOLET RADIATION INACTIVATION KINETICS OF CRYPTOSPORIDIUM PARVUM OOCYSTS

    EPA Science Inventory

    The primary goal of this paper is to account for the effect of lot variability in determining the required ultraviolet (UV) radiation to inactivate Cryptosporidium parvum oocysts in mouse infectivity studies. The number of infectious oocysts (infective dose) per mouse is estima...

  8. Heparin interacts with elongation factor 1α of Cryptosporidium parvum and inhibits invasion

    PubMed Central

    Inomata, Atsuko; Murakoshi, Fumi; Ishiwa, Akiko; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Cagayat Recuenco, Frances; Horimoto, Taisuke; Kato, Kentaro

    2015-01-01

    Cryptosporidium parvum is an apicomplexan parasite that can cause serious watery diarrhea, cryptosporidiosis, in human and other mammals. C. parvum invades gastrointestinal epithelial cells, which have abundant glycosaminoglycans on their cell surface. However, little is known about the interaction between C. parvum and glycosaminoglycans. In this study, we assessed the inhibitory effect of sulfated polysaccharides on C. parvum invasion of host cells and identified the parasite ligands that interact with sulfated polysaccharides. Among five sulfated polysaccharides tested, heparin had the highest, dose-dependent inhibitory effect on parasite invasion. Heparan sulfate-deficient cells were less susceptible to C. parvum infection. We further identified 31 parasite proteins that potentially interact with heparin. Of these, we confirmed that C. parvum elongation factor 1α (CpEF1α), which plays a role in C. parvum invasion, binds to heparin and to the surface of HCT-8 cells. Our results further our understanding of the molecular basis of C. parvum infection and will facilitate the development of anti-cryptosporidial agents. PMID:26129968

  9. Natural infection with zoonotic subtype of Cryptosporidium parvum in Capybara (Hydrochoerus hydrochaeris) from Brazil.

    PubMed

    Meireles, Marcelo Vasconcelos; Soares, Rodrigo Martins; Bonello, Fábio; Gennari, Solange Maria

    2007-06-20

    A total of 145 capybara (Hydrochoerus hydrochaeris) fecal samples from the state of São Paulo, Brazil, were screened for Cryptosporidium spp. oocysts using the malachite green method. Eight samples (5.52%) showed positive results and were further submitted to nested PCR reaction for amplification of fragments of 18S rRNA gene and 60-kDa glycoprotein gene for determination of species, alleles and subtypes of Cryptosporidium. Sequencing of the PCR products of the 18S rRNA gene fragments and 60-kDa glycoprotein gene fragments showed that for both genes all Cryptosporidium isolates from capybara were respectively 100% genetically similar to a bovine isolate of C. parvum and to C. parvum subtype IIaA15G2R1. To the best of our knowledge this is the first report of Cryptosporidium infection in this rodent. The finding of zoonotic C. parvum infection in a semi-aquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies may be contaminated with zoonotic Cryptosporidium oocysts from wildlife.

  10. Multiplication of the waterborne pathogen Cryptosporidium parvum in an aquatic biofilm system

    PubMed Central

    2013-01-01

    Background In natural aquatic environments biofilms are known to act as environmental reservoirs for Cryptosporidium parvum oocysts. However, the fate of these oocysts within biofilms has yet to be determined. Methods This study aimed to identify if biofilms have the ability to support the multiplication of Cryptosporidium by measuring the change in parasite number over time using quantitative polymerase chain reaction (qPCR) and detecting the possible extracellular developmental stages using a combination of confocal microscopy and immunolabelling techniques. Pseudomonas aeruginosa biofilm flow cell systems were established and C. parvum oocysts were constantly supplied over a six day period. Results A significant (P < 0.001) increase in Cryptosporidium was detected as the biofilm matured, with the total number of C. parvum multiplying 2–3 fold during this period. With this, various Cryptosporidium developmental stages (sporozoites, trophozoites, type I and II meronts) were identified from the biofilm. Conclusion This is the first study demonstrating that biofilms not only serve as an environmental reservoir for oocysts, but are also capable of supporting the multiplication of Cryptosporidium over time in an aquatic environment. PMID:24330483

  11. An immunomagnetic separation-real-time PCR method for quantification of Cryptosporidium parvum in water samples.

    PubMed

    Fontaine, Melanie; Guillot, Emmanuelle

    2003-07-01

    The protozoan parasite Cryptosporidium parvum is known to occur widely in both raw and drinking water and is the cause of waterborne outbreaks of gastroenteritis throughout the world. The routinely used method for the detection of Cryptosporidium oocysts in water is based on an immunofluorescence assay (IFA). It is both time-consuming and nonspecific for the human pathogenic species C. parvum. We have developed a TaqMan polymerase chain reaction (PCR) test that accurately quantifies C. parvum oocysts in treated and untreated water samples. The protocol consisted of the following successive steps: Envirochek capsule filtration, immunomagnetic separation (IMS), thermal lysis followed by DNA purification using Nanosep centrifugal devices and, finally, real-time PCR using fluorescent TaqMan technology. Quantification was accomplished by comparing the fluorescence signals obtained from test samples with those from standard dilutions of C. parvum oocysts. This IMS-real-time PCR assay permits rapid and reliable quantification over six orders of magnitude, with a detection limit of five oocysts for purified oocyst solutions and eight oocysts for spiked water samples. Replicate samples of spiked tap water and Seine River water samples (with approximately 78 and 775 oocysts) were tested. C. parvum oocyst recoveries, which ranged from 47.4% to 99% and from 39.1% to 68.3%, respectively, were significantly higher and less variable than those reported using the traditional US Environmental Protection Agency (USEPA) method 1622. This new molecular method offers a rapid, sensitive and specific alternative for C. parvum oocyst quantification in water.

  12. Effects of Surfactants on Cryptosporidium parvum Mobility in Agricultural Soils from Illinois and Utah

    NASA Astrophysics Data System (ADS)

    Darnault, C. J.; Koken, E.; Jacobson, A. R.; Powelson, D.

    2011-12-01

    The occurence of the parasitic protozoan Cryptosporidium parvum in rural and agricultural watersheds due to agricultural activities and wildlife is inevitable. Understanding the behavior of C. parvum oocysts in the environment is critical for the protection of public health and the environment. To better understand the mechanisms by which the pathogen moves through soils and contaminates water resources, we study their mobility under conditions representative of real-world scenarios, where both C. parvum and chemicals that affect their fate are present in soils. Surfactants occur widely in soils due to agricultural practices such as wastewater irrigation and the application of pesticides or soil wetting agents. They affect water tension and, consequently, soil infiltration processes and the air-water interfaces in soil pores where C. parvum may be retained. We investigate the effects of surfactants on the mobility of C. parvum oocysts in agricultural soils from Illinois and Utah under unsaturated flow conditions. As it is critical to examine C. parvum in natural settings, we also developed a quantification method using RT-PCR for monitoring C. parvum oocysts in environmental soil and water samples. We optimized physico-chemical parameters to disrupt C. parvum oocysts and extract their DNA, and developed isolation methods to separate C. parvum oocysts from colloids in natural soil samples. The results of this research will lead to the development of an accurate and sensitive molecular method for the monitoring of C. parvum oocysts in environmental soil and water samples, and will further our understanding of the mechanisms controlling the behavior of C. parvum oocysts in soils, in particular the role of vadose zone processes, sorption to soil and surfactants.

  13. Removal effect of the water purifier for home use against Cryptosporidium parvum oocysts.

    PubMed

    Matsui, Toshihiro; Kajima, Junko; Fujino, Takashi

    2004-08-01

    The removal effects of the faucet mounted type water purifier for home use were examined against Cryptosporidium parvum oocysts. The water purifier is composed of a layer of granular activated carbon and the hollow fiber membrane filter. The cartridges were unused, 25%, 50% and 75% flow down by Arizona-dust of U. S. A. Two respective cartridges were used of the examination. The faucet and the water purifier were connected by anti-pressure tube, and 3.0 x 10(7) oocysts of Cryptosporidium parvum were injected into anti-pressure tube while water was running. Twenty liter of collected purified water was examined under the fluorescent microscope. Any oocysts in the purified water collected from all cartridges were not found. Therefore, we considered this purifier as an effective one in removing Cryptosporidium oocysts from drinking water.

  14. Spinacia oleracea L. leaf stomata harboring Cryptosporidium parvum oocysts: a potential threat to food safety.

    PubMed

    Macarisin, Dumitru; Bauchan, Gary; Fayer, Ronald

    2010-01-01

    Cryptosporidium parvum is a cosmopolitan microscopic protozoan parasite that causes severe diarrheal disease (cryptosporidiosis) in mammals, including humans and livestock. There is growing evidence of Cryptosporidium persistence in fresh produce that may result in food-borne infection, including sporadic cases as well as outbreaks. However, drinking and recreational waters are still considered the major sources of Cryptosporidium infection in humans, which has resulted in prioritization of studies of parasite etiology in aquatic environments, while the mechanisms of transmission and parasite persistence on edible plants remain poorly understood. Using laser scanning confocal microscopy together with fluorescein-labeled monoclonal antibodies, C. parvum oocysts were found to strongly adhere to spinach plants after contact with contaminated water, to infiltrate through the stomatal openings in spinach leaves, and to persist at the mesophyll level. These findings and the fact that this pathogenic parasite resists washing and disinfection raise concerns regarding food safety.

  15. Fabrication and characterization of a microporous polymeric micro-filter for isolation of Cryptosporidium parvum oocysts

    NASA Astrophysics Data System (ADS)

    Ebrahimi Warkiani, Majid; Lou, Chao-Ping; Gong, Hai-Qing

    2011-03-01

    A rapid and effective method to concentrate Cryptosporidium parvum oocysts present in large volumes of drinking water into smaller volumes is critical for accurate detection and quantification of C. parvum oocysts from drinking water. Filtration-based concentration techniques have been widely used to recover C. parvum oocysts into a small volume for downstream analysis. We present a rapid method for fabrication of a polymeric micro-filter with ordered pores and a smooth surface using UV lithography and MEMS technology. To support the filter membrane, we also developed a technique for integrated fabrication of a support mesh. We demonstrated that the filter is able to isolate the oocysts which can be further detected using fluorescent techniques. Sample loading and back-flushing using the micro-filter resulted in 95-99% recovery with a concentration ratio above 2000 of the spiked C. parvum oocysts, which showed significantly improved performance compared with current commercial filters.

  16. CHANGES IN MOUSE CIRULATING LEUKOCYTE NUMBERS IN C57BL/6 MICE IMMUNOSUPPRESSED FOR CRYPTOSPORIDIUM PARVUM OOCYST PRODUCTION

    EPA Science Inventory

    The Iowa strain of Cryptosporidium parvum will not propagate in immunocompetent mice, but will successfully infect genetically immunocompromised Nude or SCID mice as well as immunocompetent mice which have been immunosuppressed with glucocorticoids. Using dexamethasone - tetracy...

  17. Evaluation of water treatment plant UV reactor efficiency against Cryptosporidium parvum oocyst infectivity in immunocompetent suckling mice.

    PubMed

    Le Goff, L; Khaldi, S; Favennec, L; Nauleau, F; Meneceur, P; Perot, J; Ballet, J-J; Gargala, G

    2010-03-01

    To assess the efficiency of a medium-pressure UV reactor under full-scale water treatment plant (WTP) conditions on the infectivity of Cryptosporidium parvum oocysts in an Naval Medical Research Institute (NMRI) suckling mice infectivity model. Six/seven-day-old mice were administered orally 2-10x10(4)Cryptosporidium parvum oocysts. Compared with nonirradiated oocysts, 40 mJ cm(-2) UV irradiation of ingested oocysts resulted 7 days later in a 3.4-4.0 log10 reduction in the counts of small intestine oocysts, using a fluorescent flow cytometry assay. Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development. Present results suggest that in WTP conditions, a medium-pressure UV reactor is efficient in reducing the infectivity of Cryptosporidium parvum oocysts, one of the most resistant micro-organisms present in environmental waters.

  18. A COMPARISON OF FOUR FLUORESCENT ANTIBODY BASED METHODS FOR PURIFYING, DETECTING AND CONFIRMING CRYPTOSPORIDIUM PARVUM IN SURFACE WATERS

    EPA Science Inventory

    Cryptosporidiosis has been traced to drinking contaminated surface water, either not treated, or ineffectively treated. Testing to detect Cryptosporidium parvum in surface water has been suggested to help prevent future outbreaks. This study compared purifications and detection...

  19. CHANGES IN MOUSE CIRULATING LEUKOCYTE NUMBERS IN C57BL/6 MICE IMMUNOSUPPRESSED FOR CRYPTOSPORIDIUM PARVUM OOCYST PRODUCTION

    EPA Science Inventory

    The Iowa strain of Cryptosporidium parvum will not propagate in immunocompetent mice, but will successfully infect genetically immunocompromised Nude or SCID mice as well as immunocompetent mice which have been immunosuppressed with glucocorticoids. Using dexamethasone - tetracy...

  20. A COMPARISON OF FOUR FLUORESCENT ANTIBODY BASED METHODS FOR PURIFYING, DETECTING AND CONFIRMING CRYPTOSPORIDIUM PARVUM IN SURFACE WATERS

    EPA Science Inventory

    Cryptosporidiosis has been traced to drinking contaminated surface water, either not treated, or ineffectively treated. Testing to detect Cryptosporidium parvum in surface water has been suggested to help prevent future outbreaks. This study compared purifications and detection...

  1. Detection and molecular characterisation of Cryptosporidium parvum in British European hedgehogs (Erinaceus europaeus).

    PubMed

    Sangster, Lucy; Blake, Damer P; Robinson, Guy; Hopkins, Timothy C; Sa, Ricardo C C; Cunningham, Andrew A; Chalmers, Rachel M; Lawson, Becki

    2016-02-15

    Surveillance was conducted for the occurrence of protozoan parasites of the genus Cryptosporidium in European hedgehogs (Erinaceus europaeus) in Great Britain. In total, 108 voided faecal samples were collected from hedgehogs newly admitted to eight wildlife casualty treatment and rehabilitation centres. Terminal large intestinal (LI) contents from three hedgehog carcasses were also analysed. Information on host and location variables, including faecal appearance, body weight, and apparent health status, was compiled. Polymerase Chain Reaction (PCR) targeting the 18S ribosomal RNA gene, confirmed by sequencing, revealed an 8% (9/111) occurrence of Cryptosporidium parvum in faeces or LI contents, with no significant association between the host or location variables and infection. Archived small intestinal (SI) tissue from a hedgehog with histological evidence of cryptosporidiosis was also positive for C. parvum by PCR and sequence analysis of the 18S rRNA gene. No other Cryptosporidium species were detected. PCR and sequencing of the glycoprotein 60 gene identified three known zoonotic C. parvum subtypes not previously found in hedgehogs: IIdA17G1 (n=4), IIdA19G1 (n=1) and IIdA24G1 (n=1). These subtypes are also known to infect livestock. Another faecal sample contained C. parvum IIcA5G3j which has been found previously in hedgehogs, and for which there is one published report in a human, but is not known to affect livestock. The presence of zoonotic subtypes of C. parvum in British hedgehogs highlights a potential public health concern. Further research is needed to better understand the epidemiology and potential impacts of Cryptosporidium infection in hedgehogs.

  2. Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to CPV40 capsid protein

    USDA-ARS?s Scientific Manuscript database

    Monoclonal antibodies (MAb) were prepared against the 40 kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. By immunoblotting analysis, one MAb, designated MAbCPV40-1, bound to a 40 kDa protein in extracts of C. parvum oocysts, which...

  3. In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

    PubMed Central

    Elliot, B C; Wisnewski, A V; Johnson, J; Fenwick-Smith, D; Wiest, P; Hamer, D; Kresina, T; Flanigan, T P

    1997-01-01

    Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection. PMID:9284173

  4. Cryptosporidium parvum scavenges LDL-derived cholesterol and micellar cholesterol internalized into enterocytes

    PubMed Central

    Ehrenman, Karen; Wanyiri, Jane W.; Bhat, Najma; Ward, Honorine D.; Coppens, Isabelle

    2013-01-01

    Cryptosporidium spp. are responsible for devastating diarrhea in immunodeficient individuals. In the intestinal tract, the developmental stages of the parasite are confined to the apical surfaces of epithelial cells. Upon invasion, Cryptosporidium incorporates the microvillous membrane of the enterocyte to form the parasitophorous vacuole (PV) and sequesters itself from the host cytoplasm by rearranging the host cytoskeleton. Cryptosporidium parvum has minimal anabolic capabilities and relies on transporters and salvage pathways to meet its basic metabolic requirements. The cholesterol salvage pathway is crucial for the development of protozoan parasites. In this study, we have examined the sources of cholesterol from C. parvum infecting enterocytes. We illustrated that the intracellular stages of Cryptosporidium as well as the oocysts shed by the host, contain cholesterol. Incubation of infected enterocytes in lipoprotein-free medium impairs parasite development and results in substantial decrease in cholesterol content associated with the PV. Among lipoproteins, LDL constitutes an important source of cholesterol for Cryptosporidium. Dietary cholesterol incorporated into micelles is internalized into enterocytes by the NPC1L1 transporter. We showed that C. parvum also obtains cholesterol from micelles in enterocytes. Pharmacological blockade of NPC1L1 function by ezetimibe or moderate down-regulation of NPC1L1 expression decreases parasite infectivity. These observations indicate that, despite its dual sequestration from the intestinal lumen and the host cytoplasm, C. parvum can, in fact, obtain cholesterol both from the gut’s lumen and the host cell. This study highlights the evolutionary advantages for epicellular pathogens to access to nutrients from the outside and inside of the host cell. PMID:23311949

  5. Preliminary Characterization of MEDLE-2, a Protein Potentially Involved in the Invasion of Cryptosporidium parvum

    PubMed Central

    Li, Baoling; Wu, Haizhen; Li, Na; Su, Jiayuan; Jia, Ruilian; Jiang, Jianlin; Feng, Yaoyu; Xiao, Lihua

    2017-01-01

    Cryptosporidium spp. are important causes of diarrhea in humans, ruminants, and other mammals. Comparative genomic analysis indicated that genetically related and host-adapted Cryptosporidium species have different numbers of subtelomeric genes encoding the Cryptosporidium-specific MEDLE family of secreted proteins, which could contribute to differences in host specificity. In this study, a Cryptosporidium parvum-specific member of the protein family MEDLE-2 encoded by cgd5_4590 was cloned and expressed in Escherichia coli. Immunofluorescent staining with antibodies generated from the recombinant protein showed the expression of the protein in sporozoites and development stages. In vitro neutralization assay with the antibodies partially blocked the invasion of sporozoites. These results support the potential involvement of MEDLE-2 in the invasion of host cells. PMID:28912761

  6. Efficacy of Common Laboratory Disinfectants on the Infectivity of Cryptosporidium parvum Oocysts in Cell Culture

    PubMed Central

    Weir, Susan C.; Pokorny, Nicholas J.; Carreno, Ramon A.; Trevors, Jack T.; Lee, Hung

    2002-01-01

    Nine liquid disinfectants were tested for their ability to reduce infectivity of Cryptosporidium parvum oocysts in cell culture. A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxide-amended windshield washer fluid reduced infectivity 1,000-fold. Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure. The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against C. parvum oocysts. PMID:11976138

  7. Multicentric Evaluation of a New Real-Time PCR Assay for Quantification of Cryptosporidium spp. and Identification of Cryptosporidium parvum and Cryptosporidium hominis

    PubMed Central

    Chapey, E.; Dutoit, E.; Guyot, K.; Hasseine, L.; Jeddi, F.; Menotti, J.; Paraud, C.; Pomares, C.; Rabodonirina, M.; Rieux, A.; Derouin, F.

    2013-01-01

    Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed. PMID:23720792

  8. Cryptosporidium parvum infections in Bergen, Norway, during an extensive outbreak of waterborne giardiasis in autumn and winter 2004.

    PubMed

    Robertson, L J; Forberg, T; Hermansen, L; Gjerde, B K; Alvsvåg, J O; Langeland, N

    2006-03-01

    During a large waterborne giardiasis outbreak in Norway, many diarrheic patients were found to have Cryptosporidium infections. Gene sequencing identified these infections as Cryptosporidium parvum infections, although they were not identical. Whether these infections were due to a simultaneous outbreak of waterborne cryptosporidiosis or reflected background levels not normally detected is discussed.

  9. Cryptosporidium erinacei and C. parvum in a group of overwintering hedgehogs.

    PubMed

    Hofmannová, Lada; Hauptman, Karel; Huclová, Kristýna; Květoňová, Dana; Sak, Bohumil; Kváč, Martin

    2016-10-01

    This study describes cryptosporidiosis in an overwintering group of 15 European hedgehogs (Erinaceus europaeus), comprising 3 adults and 12 juveniles. Four juvenile hedgehogs were hospitalised with anorexia, malodorous diarrhoea and dehydration. Immediate parasitological examinations revealed the presence of Cryptosporidium sp. in these animals and also in 5 other juveniles. All hedgehogs were coproscopically monitored for 4 months over the winter season. Shedding of Cryptosporidium oocysts persisted from 6 to 70 days. Repeated shedding of Cryptosporidium oocysts occurred in 3 animals after 4 months subsequent to the first outbreak. Clinical signs were observed only at the beginning of the outbreak (apathy, anorexia, general weakness, mild dehydration, and malodorous faeces with changed consistence - soft/diarrhoea) in the 4 hospitalised juveniles. Overall 11 hedgehogs were Cryptosporidium-positive, both microscopically and by PCR methods. Sequence analyses of SSU rRNA and gp60 genes revealed the presence of C. parvum IIdA18G1 subtype in all positive hedgehogs. Moreover, 3 hedgehogs had a mixed infection of the zoonotic C. parvum and C. erinacei XIIIaA19R13 subtype. Cryptosporidium infections can be rapidly spread among debilitated animals and the positive hedgehogs released back into the wild can be a source of the infection for individuals weakened after hibernation.

  10. Effect of chlorine, blanching, freezing, and microwave heating on Cryptosporidium parvum viability inoculated on green peppers.

    PubMed

    Duhain, G L M C; Minnaar, A; Buys, E M

    2012-05-01

    Cryptosporidium parvum oocysts have been found on the surface of vegetables in both developed and developing countries. C. parvum can contaminate vegetables via various routes, including irrigation water. This study investigated the effect of individual treatments of chlorine, blanching, blast freezing, and microwave heating, as well as combined treatments of chlorine and freezing, and chlorine and microwave heating on the viability of C. parvum oocysts inoculated on green peppers. The viability of the oocysts after the treatments was assessed using propidium iodide and a flow cytometer. Based on the propidium iodide staining, the chlorine treatments did not affect the viability of the oocysts. Blast freezing significantly inactivated 20% of the oocysts. Microwave heating and blanching significantly inactivated 93% of oocysts. Treatment with chlorine followed by blast freezing did not affect the viability of the oocysts significantly. Treatment with chlorine and microwave heating was significantly more effective than microwave heating alone and inactivated 98% of the oocysts. The study indicates that C. parvum oocysts are sensitive to heat and, to some extent, to blast freezing, but are resistant to chlorine. Therefore, the use of chlorine during vegetable processing is not a critical control point for C. parvum oocysts, and the consumption of raw or minimally processed vegetables may constitute a health risk as C. parvum oocysts can still be found viable on ready-to-eat, minimally processed vegetables.

  11. Effect of high-rate algal ponds on viability of Cryptosporidium parvum oocysts.

    PubMed

    Araki, S; Martín-Gomez, S; Bécares, E; De Luis-Calabuig, E; Rojo-Vazquez, F

    2001-07-01

    The physicochemical conditions of high-rate algal ponds were responsible for a more than 97% reduction in the infectivity of Cryptosporidium parvum oocysts in neonatal mice. The use of semipermeable bags of cellulose showed that pH, ammonia, and/or light seems to be a major factor for the inactivation of oocysts in wastewater, supporting the importance of alga-based systems for safer reuse of treated wastewater.

  12. Susceptibility of germfree or antibiotic-treated adult mice to Cryptosporidium parvum.

    PubMed

    Harp, J A; Wannemuehler, M W; Woodmansee, D B; Moon, H W

    1988-08-01

    Adult mice are more resistant than neonatal mice to intestinal colonization with the protozoan parasite Cryptosporidium parvum. Development of a mature intestinal flora may play a role in this resistance. We compared susceptibilities to colonization with C. parvum in adult conventional mice, adult germfree mice, and adult conventional mice treated with oral antibiotics to deplete the intestinal flora. Germfree mice of both CD1 and BALB/c strains were colonized at day 7 following inoculation with C. parvum oocysts isolated from the feces of an infected, diarrheic calf. Age-matched conventional mice of the same strains were comparatively resistant to colonization. Conventional mice treated with antibiotics remained resistant to colonization. These results suggest that the microflora in the intestine was not the sole determinant of resistance or susceptibility to colonization. The germfree adult mouse as an experimental model of cryptosporidiosis is discussed.

  13. Inactivation of Cryptosporidium parvum Oocysts in Fresh Apple Cider by UV Irradiation

    PubMed Central

    Hanes, D. E.; Worobo, R. W.; Orlandi, P. A.; Burr, D. H.; Miliotis, M. D.; Robl, M. G.; Bier, J. W.; Arrowood, M. J.; Churey, J. J.; Jackson, G. J.

    2002-01-01

    This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm2. Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider. PMID:12147528

  14. Detection of Infectious Cryptosporidium parvum Oocysts in Mussels (Mytilus galloprovincialis) and Cockles (Cerastoderma edule)

    PubMed Central

    Gomez-Bautista, M.; Ortega-Mora, L. M.; Tabares, E.; Lopez-Rodas, V.; Costas, E.

    2000-01-01

    Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 103 oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination. PMID:10788352

  15. A rapid IL-17 response to Cryptosporidium parvum in the bovine intestine.

    PubMed

    Drinkall, Emma; Wass, Matthew J; Coffey, Tracey J; Flynn, Robin J

    2017-09-01

    Cryptosporidium parvum causes diarrhoea, due to villi damage, in livestock and humans globally. Immunity develops after repeated infections but initial infections can be severe, highlighting the importance of early infection dynamics. We have modelled early C. parvum infection in bovine jejunum biopsies. IL-17A accumulated over time peaking at 9h post-infection, with no effect of infection on IL-1β; antibiotics positively influenced IL-17A as higher levels were found in cultures with antibiotics. Infection of primary fibroblasts resulted in lower plaque formation when fibroblasts were primed with IL-17A. Our results indicate a role for IL-17A in reducing C. parvum-dependent host cell damage. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability.

    PubMed Central

    Korich, D G; Mead, J R; Madore, M S; Sinclair, N A; Sterling, C R

    1990-01-01

    Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water. PMID:2339894

  17. Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability

    SciTech Connect

    Korich, D.G.; Mead, J.R.; Madore, M.S.; Sinclair, N.A.; Sterling, C.R. )

    1990-05-01

    Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.

  18. Resistance of calves to Cryptosporidium parvum: effects of age and previous exposure.

    PubMed Central

    Harp, J A; Woodmansee, D B; Moon, H W

    1990-01-01

    Cryptosporidium parvum is a coccidian parasite that causes diarrheal disease in many vertebrate species, including young (less than or equal to 1 month old) calves. Older calves and adult cattle are resistant to infection. In this study, newborn calves were raised in isolation from C. parvum for 1 week to 3 months before experimental challenge with the parasite. Calves orally challenged with C. parvum at 1 week of age shed oocysts in their feces and had diarrhea after challenge exposure. When these calves were rechallenged at 1 and 3 months of age, they neither shed oocysts nor had diarrhea. There was no significant increase in the mean anticryptosporidium enzyme-linked immunosorbent assay serum antibody titer in these calves following any of the challenge exposures. Calves orally inoculated with C. parvum for the first time at 1 month of age shed oocysts, had diarrhea after challenge exposure, and were resistant to rechallenge at 3 months of age. These calves had a twofold increase in serum antibody titer after the first challenge and no increase after the second challenge. Calves orally inoculated with C. parvum for the first time at 3 months of age shed oocysts, and two of seven animals had diarrhea. These calves had a 10-fold increase in serum antibody to C. parvum after exposure. This study demonstrates that calves raised in isolation from C. parvum remain susceptible to challenge until at least 3 months of age. Furthermore, within this time period, initial exposure and recovery renders calves resistant to further challenge with the parasite. The data also suggest that exposure of young calves to C. parvum may inhibit the development of a serum antibody response to the parasite. PMID:2365460

  19. Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water.

    PubMed

    McGuigan, K G; Méndez-Hermida, F; Castro-Hermida, J A; Ares-Mazás, E; Kehoe, S C; Boyle, M; Sichel, C; Fernández-Ibáñez, P; Meyer, B P; Ramalingham, S; Meyer, E A

    2006-08-01

    To determine whether batch solar disinfection (SODIS) can be used to inactivate oocysts of Cryptosporidium parvum and cysts of Giardia muris in experimentally contaminated water. Suspensions of oocysts and cysts were exposed to simulated global solar irradiation of 830 W m(-2) for different exposure times at a constant temperature of 40 degrees C. Infectivity tests were carried out using CD-1 suckling mice in the Cryptosporidium experiments and newly weaned CD-1 mice in the Giardia experiments. Exposure times of > or =10 h (total optical dose c. 30 kJ) rendered C. parvum oocysts noninfective. Giardia muris cysts were rendered completely noninfective within 4 h (total optical dose >12 kJ). Scanning electron microscopy and viability (4',6-diamidino-2-phenylindole/propidium iodide fluorogenic dyes and excystation) studies on oocysts of C. parvum suggest that inactivation is caused by damage to the oocyst wall. Results show that cysts of G. muris and oocysts of C. parvum are rendered completely noninfective after batch SODIS exposures of 4 and 10 h (respectively) and is also likely to be effective against waterborne cysts of Giardia lamblia. These results demonstrate that SODIS is an appropriate household water treatment technology for use as an emergency intervention in aftermath of natural or man-made disasters against not only bacterial but also protozoan pathogens.

  20. CP2 gene as a useful viability marker for Cryptosporidium parvum.

    PubMed

    Lee, Soo-Ung; Joung, Migyo; Ahn, Myoung-Hee; Huh, Sun; Song, Hyunje; Park, Woo-Yoon; Yu, Jae-Ran

    2008-02-01

    The validity of the CP2 gene of Cryptosporidium parvum as a viability marker was evaluated using absolute quantitative real-time polymerase chain reaction (qPCR) assays. Total ribonucleic acid (RNA) was isolated from live and heat-killed C. parvum oocysts, and complementary deoxyribonucleic acid was synthesized and used as a template. The most accurate number of viable C. parvum oocysts was predicted when the CP2 gene was used as a target gene. The lower detection limit of the CP2 gene was ten oocysts, which was the most sensitive among examined target genes. With heat shock induction, only hsp70 messenger RNA (mRNA) was induced, and the predicted viable oocyst number was increased by heat shock for this marker. The CP2, hsp70, Cryptosporidium oocyst wall protein, and beta-tubulin mRNAs were not detected in heat-killed oocysts, but the 18S ribosomal ribonucleic acid (rRNA) showed heat stability until 48 h after heat killing. Although the 18S rRNA demonstrated the fastest response in crossing point (CP) value among the examined primer sets in qPCR, overestimation of viable oocysts was noted in the analysis with this gene. In conclusion, the CP2 gene was identified as the most sensitive, reliable, and accurate candidate of a viability marker of C. parvum by qPCR evaluation.

  1. Cryptosporidium parvum-induced ileo-caecal adenocarcinoma and Wnt signaling in a mouse model.

    PubMed

    Benamrouz, Sadia; Conseil, Valerie; Chabé, Magali; Praet, Marleen; Audebert, Christophe; Blervaque, Renaud; Guyot, Karine; Gazzola, Sophie; Mouray, Anthony; Chassat, Thierry; Delaire, Baptiste; Goetinck, Nathalie; Gantois, Nausicaa; Osman, Marwan; Slomianny, Christian; Dehennaut, Vanessa; Lefebvre, Tony; Viscogliosi, Eric; Cuvelier, Claude; Dei-Cas, Eduardo; Creusy, Colette; Certad, Gabriela

    2014-06-01

    Cryptosporidium species are apicomplexan protozoans that are found worldwide. These parasites constitute a large risk to human and animal health. They cause self-limited diarrhea in immunocompetent hosts and a life-threatening disease in immunocompromised hosts. Interestingly, Cryptosporidium parvum has been related to digestive carcinogenesis in humans. Consistent with a potential tumorigenic role of this parasite, in an original reproducible animal model of chronic cryptosporidiosis based on dexamethasone-treated or untreated adult SCID mice, we formerly reported that C. parvum (strains of animal and human origin) is able to induce digestive adenocarcinoma even in infections induced with very low inoculum. The aim of this study was to further characterize this animal model and to explore metabolic pathways potentially involved in the development of C. parvum-induced ileo-caecal oncogenesis. We searched for alterations in genes or proteins commonly involved in cell cycle, differentiation or cell migration, such as β-catenin, Apc, E-cadherin, Kras and p53. After infection of animals with C. parvum we demonstrated immunohistochemical abnormal localization of Wnt signaling pathway components and p53. Mutations in the selected loci of studied genes were not found after high-throughput sequencing. Furthermore, alterations in the ultrastructure of adherens junctions of the ileo-caecal neoplastic epithelia of C. parvum-infected mice were recorded using transmission electron microscopy. In conclusion, we found for the first time that the Wnt signaling pathway, and particularly the cytoskeleton network, seems to be pivotal for the development of the C. parvum-induced neoplastic process and cell migration of transformed cells. Furthermore, this model is a valuable tool in understanding the host-pathogen interactions associated with the intricate infection process of this parasite, which is able to modulate host cytoskeleton activities and several host-cell biological

  2. Chlorine Dioxide Inactivation of Cryptosporidium parvum Oocysts and Bacterial Spore Indicators

    PubMed Central

    Chauret, Christian P.; Radziminski, Chris Z.; Lepuil, Michael; Creason, Robin; Andrews, Robert C.

    2001-01-01

    Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg · min/liter were needed to inactivate approximately 0.5 log10 and 2.0 log10 units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg · min/liter were required to achieve approximately 2.0 log10 units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity. PMID:11425712

  3. ENHANCED PRODUCTION OF CRYPTOSPORIDIUM PARVUM OOCYSTS IN IMMUNOSUPPRESSED MICE

    EPA Science Inventory

    Recently there has been an increase in the need for fresh C. parvum oocysts for engineering and biomedical research applications. In our laboratory the emphsis has shifted from the use of dairy calves to inbred C57BL/67n mice, primarily for reasons of ease of collection and proce...

  4. ENHANCED PRODUCTION OF CRYPTOSPORIDIUM PARVUM OOCYSTS IN IMMUNOSUPPRESSED MICE

    EPA Science Inventory

    Recently there has been an increase in the need for fresh C. parvum oocysts for engineering and biomedical research applications. In our laboratory the emphsis has shifted from the use of dairy calves to inbred C57BL/67n mice, primarily for reasons of ease of collection and proce...

  5. Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum.

    PubMed

    Guo, Yaqiong; Tang, Kevin; Rowe, Lori A; Li, Na; Roellig, Dawn M; Knipe, Kristine; Frace, Michael; Yang, Chunfu; Feng, Yaoyu; Xiao, Lihua

    2015-04-18

    Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis-associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome. Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45-767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5' and 3' ends of chromosome 6 and the gp60 region, largely the result of genetic recombination. The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to

  6. [Cryptosporidium parvum infection in a pregnant immunocompetent woman with occupational risk].

    PubMed

    Neira, Patricia; Muñoz, Nelson; Rosales, José

    2010-08-01

    Cryptosporidioses is a parasitic zoonoses generated by diverse Cryptosporidium species. This coccidiosis affects multiple vertebrate species, including human beings. In Chile, as it happens in other countries, cryptosporidioses is a low frequency infection in immunocompetent individuals, acquiring a big relevance in immunocompromised ones. We present the following case: a recently graduated student from Veterinary medical school, with a 20 week pregnancy, living in "Laguna Verde" area in the Region of Valparaiso and who was infected with Cryptosporidium sp. Etiologic diagnosis was made by Ziehl Neelsen, and nested PCR followed by PCR product sequencing. During the same period, the infection was detected in her cats which were asymptomatic. In all of them, her and the cats, the species identified was Cryptosporidium parvum. Her husband and her other pets were all asymptomatic and non infected. This is the first report of a possible cryptosporidioses transmission between humans and cat.

  7. Low-Pressure UV Inactivation and DNA Repair Potential of Cryptosporidium parvum Oocysts

    PubMed Central

    Shin, Gwy-Am; Linden, Karl G.; Arrowood, Michael J.; Sobsey, Mark D.

    2001-01-01

    Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25°C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm2 (=30 J/m2), the reduction reached the cell culture assay detection limit of ∼3 log10. At UV doses of 1.2 and 3 mJ/cm2, the log10 reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage. PMID:11425717

  8. Transport of Cryptosporidium parvum in Surface Waters: Interplay of Hydrodynamic Processes, Sediments, and Biofilms

    NASA Astrophysics Data System (ADS)

    Searcy, K. E.; Packman, A. I.; Atwill, E. R.; Harter, T.

    2005-05-01

    Understanding the movement of pathogens in the environment is necessary to ensure the safety and protection of municipal water supply systems. Cryptosporidium parvum is a human pathogen of particular concern as it is common in surface waters of the United States, it can survive for long periods of time in the environment, and it is difficult to disinfect in water treatment plants. The transport of oocysts through watersheds can be mediated by interactions with the stream channel and suspended particles in the water column. For example, the association of C. parvum oocysts with suspended particles can alter the effective physical properties of the oocysts and increase their settling velocity. The hydrodynamic coupling of the overlying water with the pore water of the sediment bed can carry oocysts from the surface water into the sediment bed. Surface-attached communities of microorganisms, called biofilms, are ubiquitous in surface water systems and can capture C. parvum oocysts. Laboratory experiments were conducted at multiple scales (flowcell, batch, and flume) to determine the association of oocysts with sediments and biofilm communities and to assess the impact of this association on C. parvum transport. The effects of flow conditions, water chemistry, sediment composition, biofilm composition, and biofilm structure on these associations were all evaluated. The experimental results demonstrate that oocyst-sediment-biofilm interactions have significant implications for the propagation of C. parvum oocysts through watersheds and should generally be considered when predicting the fate of pathogens in the environment.

  9. Serological detection and epidemiology of Neospora caninum and Cryptosporidium parvum antibodies in cattle in southern Egypt.

    PubMed

    Fereig, Ragab M; AbouLaila, Mahmoud Rezk; Mohamed, Samy G A; Mahmoud, Hassan Y A H; Ali, Alsagher O; Ali, Asmaa F; Hilali, Mosaad; Zaid, Anis; Mohamed, Adel Elsayed Ahmed; Nishikawa, Yoshifumi

    2016-10-01

    Neospora caninum and Cryptosporidium parvum are intracellular protozoan parasites that are distributed worldwide and of major economical concern in cattle industry. N. caninum can cause abortion storms and high culling rates, whereas C. parvum has zoonotic implications and can cause diarrhea in calves. There are currently no data on the prevalence of neosporosis and cryptosporidiosis in humans or animals in southern Egypt. Prevalence of these two infections was determined in a sample of cattle from two different areas in southern Egypt, Sohag and Qena, using enzyme-linked immunosorbent assay. A total 301 cattle were sampled, of which 18.9% were positive for N. caninum, 35.9% were positive for C. parvum and 10.0% were positive for both. Geographical location and breeding system were considered as potential risk factors for C. parvum infection. A higher prevalence of infection was identified on small scale farms, compared with larger, intensive systems, with a prevalence of 50.2% compared with 37.8%, respectively. Animals in Sohag had a significantly higher prevalence compared with Qena, with a seroprevalence of 46.1% compared with 31.6%, respectively. In brief, marked seroprevalence recorded in this study indicates a high incidence of N. caninum and C. parvum infections in cattle, and this necessitates the application of more effective strategies for combating these types of infections on farms in Egypt. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Comparative efficacy of conventional primer sets in detection of Cryptosporidium parvum for diagnostic use.

    PubMed

    Kar, Sirri; Daugschies, Arwid; Bangoura, Berit

    2010-02-01

    In this study, the sensitivity and specificity of different previously described primer sets for Cryptosporidium parvum detection by polymerase chain reaction (PCR) was evaluated. For this purpose, the primer sets defined by Cacciò et al. (FEMS Microbiol Lett 170(1):173-179, 1999) (tub), Widmer et al. (Appl Environ Microbiol 64(11):4477-4481, 1998) (btub) and Rochelle et al. (Appl Environ Microbiol 63:2029-2037, 1997) (cphsp), respectively, were used. Deoxyribonucleic acid (DNA) was isolated from three different sample materials: (1) from the faeces of an experimentally C. parvum-infected calf, (2) from purified C. parvum oocysts, and (3) from C. parvum-infected HCT-8 cell cultures. The DNA samples were subjected to PCR reactions with each of the three given primer sets to investigate sensitivity and suitability for routine use. The primers described by Cacciò et al. (FEMS Microbiol Lett 170(1):173-179, 1999) (TUB) were superior regarding sensitivity and specificity in terms of detection of C. parvum in faeces, in purified oocysts and also in cell culture, and may thus be applied for routine diagnostic use in common sample materials.

  11. Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

    PubMed Central

    Di Giovanni, George D.; Rochelle, Paul A.

    2012-01-01

    This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water. PMID:22038611

  12. Biofilms Reduce Solar Disinfection of Cryptosporidium parvum Oocysts

    PubMed Central

    Hargreaves, B. R.; Jellison, K. L.

    2012-01-01

    Solar radiation reduces Cryptosporidium infectivity. Biofilms grown from stream microbial assemblages inoculated with oocysts were exposed to solar radiation. The infectivity of oocysts attached at the biofilm surface and oocysts suspended in water was about half that of oocysts attached at the base of a 32-μm biofilm. PMID:22467508

  13. Detection of Giardia lamblia and Cryptosporidium parvum by direct immunofluorescence assay in stool specimen.

    PubMed

    Rahman, M M; Hossain, M A; Paul, S K; Ahmed, S; Islam, A; Ehsan, M A; Alam, M M; Kabir, M R; Sarkar, S R

    2014-07-01

    Giardia and Cryptosporidium are the pathogens which transmitted through contaminated soil and contaminated water are significant causes of diarrhea and nutritional disorders in institutional and community peoples. Children and immune compromise persons are more vulnerable for these infections. Both Giardiasis and Cryptosporidiosis were included in 2004 as WHO Neglected Disease. So this is a major public health problem in developing countries. The present study was carried out to detect the Giardia and Cryptosporidium from diarrheic or patient having loose stool by Direct Immunofluorescence assay. The study was conducted during July 20012 to February 2013 and the work was done in Mymensingh Medical College in the department of Microbiology and in Bangladesh Agricultural University in the department of Veterinary Medicine. A total of 100 loose stools were collected from school children of different area and hospital under sadar upazilla, Mymensingh. The detection of Giardia lamblia and Cryptosporidium parvum showed the individual prevalence 8% and 4% respectively. The highest cyst/oocyst count was 85,000 and 1,000/gm of stool and the lowest being 100 and 50/gm of stool for Giardiasis and Cryptosporidiosis respectively. The detection rate of Giardia and Cryptosporidium by Immunofluorescence assay was relatively higher than the previous study done in Bangladesh and this was the first report from Bangladesh over human stool specimen using Immunofluorescence assay. So, Immunofluorescence assay could be adapted for rapid and accurate detection of Giardia and Cryptosporidium.

  14. Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler's Diarrhea.

    PubMed

    Shin, Ji-Hun; Lee, Sang-Eun; Kim, Tong Soo; Ma, Da-Won; Chai, Jong-Yil; Shin, Eun-Hee

    2016-10-01

    This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler's diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10(3) oocysts for C. parvum, >1×10(4) cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.

  15. Cryptosporidium parvum Caused a Large Outbreak Linked to Frisée Salad in Finland, 2012.

    PubMed

    Åberg, R; Sjöman, M; Hemminki, K; Pirnes, A; Räsänen, S; Kalanti, A; Pohjanvirta, T; Caccio, S M; Pihlajasaari, A; Toikkanen, S; Huusko, S; Rimhanen-Finne, R

    2015-12-01

    Over 250 individuals fell ill in five outbreaks caused by Cryptosporidium parvum in Finland, October-November 2012. The cases were connected by lunch meals at restaurants in four different cities. In two outbreaks, the same C. parvumIIdA17G1 subtype was found in patients' stool samples which supports a single source of infection. Frisée salad was the only common food item served at the restaurants, and consumption of lunch salad containing the frisée salad was associated with the illness. Lunch customers who responded that they had eaten lunch salad were three times more likely to have become ill than those who had not answered whether they had eaten the salad or not (RR 2.66; 95% Cl 1.02-6.9, P-value <0.01). Cryptosporidiosis should be considered as a causal agent in long-lasting watery diarrhoea combined with abdominal cramps, and clinical samples should be tested for Cryptosporidium at the same time bacteria and viruses are tested. Measures to prevent contamination of 'ready-to-eat vegetables' with Cryptosporidium oocysts and methods to test frozen food samples should be developed. © 2015 Blackwell Verlag GmbH.

  16. The Ultra-Structural Similarities between Cryptosporidium parvum and the Gregarines.

    PubMed

    Aldeyarbi, Hebatalla M; Karanis, Panagiotis

    2016-01-01

    Using a transmission electron microscopy-based approach, this study details the striking similarities between Cryptosporidium parvum and the gregarines during in vitro axenic development at high ultra-structural resolution. C. parvum zoites displayed three unusual regions within uninucleated parasites: epimerite-like, protomerite-like, and the cell body; these regions exhibited a high degree of morphological similarity to gregarine-like trophozoites. The presence of a mucron-like bulging structure at the side of the free ovoid gregarine-like zoites was observed after 2 h of cultivation. An irregular pattern of epicytic-like folds were found to cover the surface of the parasites 24 h postcultivation. Some extracellular stages were paired in laterocaudal or side-side syzygy, with the presence of a fusion zone between some of these zoites. The present findings are in agreement with phylogenetic studies that have proposed a sister relationship with gregarines. Cryptosporidium appears to exhibit tremendous variety in cell structure depending on the surrounding environment, thereby mimicking the "primitive" gregarines in terms of the co-evolution strategy between the parasites and their environments. Given this degree of similarity, different aspects of the evolutionary biology of Cryptosporidium need to be examined, considering the knowledge gained from the study of gregarines.

  17. Die-off of Cryptosporidium parvum in soil and wastewater effluents.

    PubMed

    Nasser, A M; Tweto, E; Nitzan, Y

    2007-01-01

    To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. The die-off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1-log reduction in the oocysts infectivity was observed at 30 degrees C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 degrees C resulted in a 3-log(10) reduction in their infectivity while no change of oocysts viability was recorded. Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die-off rate of C. parvum. Previous die-off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die-off data have significant implications on the management of wastewater reuse in warm environments.

  18. Detection system of Cryptosporidium parvum oocysts by brackish water benthic shellfish (Corbicula japonica) as a biological indicator in river water.

    PubMed

    Izumi, T; Yagita, K; Endo, T; Ohyama, T

    2006-11-01

    The brackish water benthic shellfish, Corbicula japonica, was experimentally exposed to Cryptosporidium parvum oocysts at 1.51x10(4)oocysts/clam/day for 7 or 14 days. Oocysts were predominantly eliminated through the feces of Corbicula japonica in both cases by microscopic and PCR methods. The fecal excretion rates of oocysts within 4 days after the last exposure to Corbicula japonica were 87.6% for the 7-day exposure group and 86.0% for the 14-day exposure group. The tissue residue level of oocysts in the gastrointestinal tract 3 days after the last exposure was 2.7% of total exposed oocysts and that of 7 days was 1.1% for the 7-day exposure case and 1.6 and 0.5% for the 14-day exposure case, respectively, maintaining infectivity to cultured cells (HCT-8) in vitro. At the same time, field tests of Corbicula japonica for collecting oocysts showed that this clam could certainly collect Cryptosporidium parvum oocysts in the natural river and, furthermore, the gene type of C. parvum could be also identified proving its effectiveness as a biological indicator. The present study showed that the brackish water benthic shellfish Corbicula japonica may be capable of gathering and preserving Cryptosporidium parvum oocysts to a considerable extent under the natural ecological conditions, and further suggests the effectiveness of Corbicula japonica as a practical and general bioindicator for estimates of river water contamination by oocysts of Cryptosporidium parvum.

  19. Development of electrochemical based sandwich enzyme linked immunosensor for Cryptosporidium parvum detection in drinking water.

    PubMed

    Thiruppathiraja, Chinnasamy; Saroja, Veerappan; Kamatchiammal, Senthilkumar; Adaikkappan, Periyakaruppan; Alagar, Muthukaruppan

    2011-10-01

    Cryptosporidium parvum is one of the most important biological contaminants in drinking water and generates significant risks to public health. Due to low infectious dose of C. parvum, remarkably sensitive detection methods are required for water and food industry analysis. This present study describes a simple, sensitive, enzyme amplified sandwich form of an electrochemical immunosensor using dual labeled gold nanoparticles (alkaline phosphatase and anti-oocysts monoclonal antibody) in indium tin oxide (ITO) as an electrode to detect C. parvum. The biosensor was fabricated by immobilizing the anti-oocysts McAb on a gold nanoparticle functionalized ITO electrode, followed by the corresponding capture of analytes and dual labeled gold nanoparticle probe to detect the C. parvum target. The outcome shows the sensitivity of electrochemical immune sensor enhanced by gold nanoparticles with a limit of detection of 3 oocysts/mL in a minimal processing period. Our results demonstrated the sensitivity of the new approach compared to the customary method and the immunosensors showed acceptable precision, reproducibility, stability, and could be readily applied to multi analyte determination for environmental monitoring.

  20. Prevalence of Cryptosporidium parvum in private drinking water cisterns in Bani-Kenanah district, northern Jordan.

    PubMed

    Abo-Shehada, Mahmoud; Hindyia, Mona; Saiah, Abbass

    2004-10-01

    Due to water scarcity in Jordan, the water authority only pump the water once or twice a week to the population. Thus people in rural areas, including the Bani-Kenanah district, make the most of their water resources by storing rainwater in private reservoirs for use during periods of water shortage. These reservoirs include; underground cisterns and concrete or metal tanks. The water collected in these reservoirs is at risk of contamination. During the period March-July 2002, the three types of reservoirs from 368 households were surveyed for presence of Escherichia coli and Cryptosporidium parvum, indicators of contamination. The cistern was the most contaminated reservoir with 17% (95% CI: 13,22) for E. coli (significant, P<0.05), and 2% (95% CI: 1,4) for C. parvum. Only 1% (95% CI: 1,6) of the metal reservoirs had E. coli, while concrete reservoirs were free. No C. parvum oocysts were detected in either the concrete or metal reservoirs. Reservoirs opening at floor level and the bucket kept outside the reservoir were significant (P<0.05) enhancing risk factors for contamination with C. parvum.

  1. Efficacy of using harmless Bacillus endospores to estimate the inactivation of Cryptosporidium parvum oocysts in water.

    PubMed

    Garvey, Mary; Clifford, Eoghan; O'Reilly, Edmond; Rowan, Neil J

    2013-06-01

    The need to use complex in vitro cell culture, expensive equipment, and highly-trained technicians that are available only to specialist laboratories has significantly limited studies assessing the potential of pulsed UV light (PUV) to inactivate the waterborne parasite Cryptosporidium parvum in drinking water. This constitutes the first study to report on the use of different non-pathogenic Bacillus endospores as potential surrogate organisms to indicate the PUV inactivation performance of a C. parvum oocyst suspended in water. Findings showed that PUV effectively inactivated approximately 5 log10 CFU/ml Bacillus megaterium and Bacillus pumilus endospores suspended in water at a UV dose of 9.72 μJ/cm(2) that also inactivated statistically similar levels of C. parvum oocysts (P < 0.05), as determined by combined in vitro HCT-8 cell culture and quantitative PCR. Specifically, this study demonstrated that B. megaterium exhibited greater or similar PUV-inactivation kinetic data compared to that of similarly treated C. parvum over the UV dose range 6.4 to 12.9 μJ/cm(2). Therefore, the former may be used as an indicator organism for safely investigating the PUV-inactivation performance of this chlorine-resistant, waterborne parasite at the waste-water treatment plant level. Findings presented will impact positively on future water quality studies and on public health.

  2. ANTIPARASITIC ACTIVITY OF SILVER AND COPPER OXIDE NANOPARTICLES AGAINST ENTAMOEBA HISTOLYTICA AND CRYPTOSPORIDIUM PARVUM CYSTS.

    PubMed

    Saad, Halim A; Soliman, Mohamed I; Azzam, Ahmed M; Mostafa, B

    2015-12-01

    Nanoparticles (NPs) have received more attention as antiparasitic agents. In the present study, silver and copper nanoparticles were synthesized and characterized using scanning electron microscopy (SEM), transmission electron microscope (TEM) and X-ray fluorescence (XRF). The antiparasitic activity of Ag and CuO nanoparticles were tested against two of the most environmentally spread parasites in Egypt (Entamoeba histolytica and Cryptosporidium parvum). The average sizes of synthesized Ag NPs and CuO NPs were 9 & 29 nm respectively and a significant reduction for cysts viability (p > 0.05) was observed for CuO NPs against E. histolytica cysts and Ag NPs against C. parvum oocysts. Moreover, LC50-3h of CuO NPs for E. histolytica and C. parvum were 0.13 and 0.72 mg/l, while Ag NPs recorded 0.34 and 0.54 mg/l respectively. Accordingly, these NPs could be suggested as a new nanoform agent for safe and effective treatment of E. histolytica and C. parvum parasites.

  3. Immunogold labeling of stages of Cryptosporidium parvum recognized by immunoglobulins in hyperimmune bovine colostrum.

    PubMed

    Fayer, R; Barta, J R; Guidry, A J; Blagburn, B L

    1991-06-01

    Ultrathin sections of mouse ileum infected with Cryptosporidium parvum were stained by immunogold techniques. Sections first were stained with polyvalent antibodies in whey from hyperimmune bovine colostrum (HBC), then stained by secondary antibodies in rabbit antibovine IgA, IgM, IgG1, and IgG2, and lastly labeled by goat anti-rabbit gold conjugate. Examination of the immunostained specimens by electron microscopy revealed that each bovine immunoglobulin isotype in the whey recognized antigens in meronts, merozoites, microgametocytes, microgametes, and macrogamonts. Based on these findings it is hypothesized that antigens in all stages of C. parvum provide targets of opportunity for the antiparasitic activity of HBC whey antibodies thereby accounting for its efficacy as an immunotherapeutic agent.

  4. Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) oocyst and sporozoite antigens recognized by bovine colostral antibodies.

    PubMed

    Tilley, M; Fayer, R; Guidry, A; Upton, S J; Blagburn, B L

    1990-09-01

    Colostral whey from seven hyperimmunized and two control cows (hyperimmune bovine colostrum) was examined by Western immunoblotting for the presence of antibody against oocysts and sporozoites of Cryptosporidium parvum, using rabbit anti-bovine immunoglobulin A (IgA), IgG1, IgG2, and IgM antibodies, followed by a horseradish peroxidase goat anti-rabbit polyvalent antibody. Although considerable variation was found in binding activity between cows on different immunization protocols, IgA and IgG1 in whey recognized a greater variety of C. parvum antigens than did IgG2 and IgM. A band at 9 to 10 kilodaltons appeared unique in that it was recognized only by IgA.

  5. Application of an ELISA-type screen printed electrode-based potentiometric assay to the detection of Cryptosporidium parvum oocysts.

    PubMed

    Laczka, Olivier; Skillman, Lucy; Ditcham, William G; Hamdorf, Brenton; Wong, Danny K Y; Bergquist, Peter; Sunna, Anwar

    2013-11-01

    We report a novel electrochemical method for the rapid detection of the parasitic protozoan, Cryptosporidium parvum. An antibody-based capture format was transferred onto screen-printed electrodes and the presence of horseradish peroxidase-labelled antibodies binding to the oocysts was potentiometrically detected. This method allowed the detection of 5 × 10(2)Cryptosporidium oocysts per mL in 60 min.

  6. Effect of batch-process solar disinfection on survival of Cryptosporidium parvum oocysts in drinking water.

    PubMed

    Méndez-Hermida, F; Castro-Hermida, J A; Ares-Mazás, E; Kehoe, S C; McGuigan, K G

    2005-03-01

    The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m(-2)) at 40 degrees C. Viability assays (4',6'-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively.

  7. Effect of Batch-Process Solar Disinfection on Survival of Cryptosporidium parvum Oocysts in Drinking Water

    PubMed Central

    Méndez-Hermida, F.; Castro-Hermida, J. A.; Ares-Mazás, E.; Kehoe, S. C.; McGuigan, K. G.

    2005-01-01

    The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m−2) at 40°C. Viability assays (4′,6′-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively. PMID:15746372

  8. Modeling the U.S. national distribution of waterborne pathogen concentrations with application to Cryptosporidium parvum

    NASA Astrophysics Data System (ADS)

    Crainiceanu, Ciprian M.; Stedinger, Jery R.; Ruppert, David; Behr, Christopher T.

    2003-09-01

    This paper provides a general statistical methodology for modeling environmental pathogen concentrations in natural waters. A hierarchical model of pathogen concentrations captures site and regional random effects as well as random laboratory recovery rates. Recovery rates were modeled by a generalized linear mixed model. Two classes of pathogen concentration models are differentiated according to their ultimate purpose: water quality prediction or health risk analysis. A fully Bayesian analysis using Markov chain Monte Carlo (MCMC) simulation is used for statistical inference. The applicability of this methodology is illustrated by the analysis of a national survey of Cryptosporidium parvum concentrations, in which 93% of the observations were zero counts.

  9. Effect of a commercial disinfectant ('Virkon') on mouse experimental infection by Cryptosporidium parvum.

    PubMed

    Ares-Mazás, E; Lorenzo, M J; Casal, J A; Fernández da Ponte, B; Castro, J A; Freire, F

    1997-06-01

    Cryptosporidium parvum oocysts obtained from naturally-infected calves were exposed to 1-10% 'Virkon' for 10-360 min, then inoculated intragastrically into coccidium-free neonatal mice. Prevalence and intensity of infection were determined seven days later by examination of intestinal homogenates. Although we were unable to abolish infectivity for the mice, the intensity of infection was considerably reduced after long periods of exposure (up to > 90%, depending on disinfectant concentration), indicating that this product may have some value for disinfection when extended exposure is possible (e.g., soaking laboratory glassware).

  10. Coupled Effects of Vadose Zone Hydrodynamics and Anionic Surfactant Aerosol-22 on the Transport of Cryptosporidium parvum in Soil

    NASA Astrophysics Data System (ADS)

    Darnault, C. J.; Jacobson, A. R.; Powelson, D.; Baveye, P.; Peng, Z.; Yu, C.

    2013-12-01

    Cryptosporidium parvum is a microbial pathogen that may be found in soil, surface and groundwater resources. We studied their transport behavior under conditions where both C. parvum oocysts and chemicals that may affect their mobility are present in soils. Surfactants occur widely in soils due to agricultural practices such as wastewater irrigation and application of agrichemicals. Surfactants decrease the surface tension of the soil solution, which may reduce the ability of C. parvum oocysts to be retained at gas-water interfaces. Understanding the fate and transport of C. parvum oocysts following land application of manure and use of surfactants in rural and agricultural watersheds is critical to assess the threat to water resources. We investigated the coupled effects of vadose zone hydrodynamics and an anionic surfactant Aerosol-22 on the transport of C. parvum oocysts in natural structured and non-structured agricultural or range soils from Illinois and Utah. Column transport experiments consisted of unsaturated flow subject to macropore and fingered flows resulting from simulated rainfall with and without surfactant. To assess the behavior of C. parvum oocysts in soils, the breakthrough and distribution of C. parvum oocysts in soil profiles were obtained using qPCR. We observed that surfactant enhanced the transport of C. parvum oocysts when preferential flow paths are present. However, when the interconnection between macropores is not established in the soils, surfactant limited the transport of C. parvum oocysts through the soil matrix by forming oocyst-surfactant-Ca flocs.

  11. Investigating Attachment Behaviors of Cryptosporidium Parvum Oocysts Using Collision Efficiency in Laboratory Column Experiments

    NASA Astrophysics Data System (ADS)

    Park, Y.; Hou, L.; Atwill, R.; Packman, A. I.; Harter, T.

    2009-12-01

    Cryptosporidium is one of the most common enteric parasites of humans and domestic animals, and a number of outbreaks of Cryprosporidiosis, a diarrheal disease caused by Cryptosporidium have been reported worldwide. Natural porous media has been demonstrated to be an effective filter for removing Cryptosporidium parvum from contaminated water and the amount of Cryptosporidium filtered is known to be highly dependent on physical and chemical conditions of the porous media and the water. Cryptosporidium deposition in saturated porous media involves two main steps: approach and attachment. In contrast to the approach mechanisms, attachment processes have not been systematically described to predict a priori because theories that represent attachment behavior (colloid stability) such as DLVO are insufficient to explain experimental data. For this reason, attachment efficiency is calculated based on empirical data, typically experimental breakthrough curves in laboratory columns or field experiments. In this study, collision (attachment) efficiencies (α) of C. parvum oocyst were calculated to test the effect of chemical property changes on the association of oocysts with sand grains. The breakthrough curve data obtained from twelve column experiments and three models were employed to calculate single collector efficiency (η) and α. The first ten experiments were conducted by changing ionic strength and pH, and mixing with natural sediments under the same physical properties (same η). Our experiment results show that iron coating or clay/suspended solids mixture drastically enhanced oocyst deposition. The experiments also showed that increase in ionic strength and decrease in pH enhanced the attachment efficiency. However, the experiment with 100mM NaCl resulted in low attachment efficiency and the experiment with pH 8.5 showed similar attachment efficiency to the one at pH 7. Based on the results from two additional experiments with different flow velocities, it

  12. Low-level detection of Cryptosporidium parvum in field water using optical microfluidic biosensors

    NASA Astrophysics Data System (ADS)

    Angus, Scott V.; Kwon, Hyuck-Jin; Yoon, Jeong-Yeol

    2012-03-01

    Cryptosporidium parvum is a difficult-to-detect protozoan that causes diarrhea in the healthy adults and death in immunocompromised individuals. While it is easy to understand the transmission routes of Cryptosporidium, it is currently difficult to identify low concentrations of Cryptosporidium, especially when following EPA method 1623, which can easily require tens of liters of water to get a positive signal. The current detection method is unacceptable and severely inefficient when taking into account the time that goes into concentrating a sample, actual assays, and training associated with the assays. Using our method, it is possible to use only 15 μL of sample, which is an immunoagglutination assay that uses Mie scatter intensity changes to detect different Cryptosporidium concentrations. In addition to creating a standard curve using a clean sample matrix (i.e., phosphate buffered saline), field samples were collected from a chlorine treated swimming pool, a sump located on a farm, and a turtle pond. Each sample had different intensity changes but the trend represented within the data was the same. This assay has a detection limit of 100-101 oocysts/mL and can be done in as little as 10 minutes.

  13. Molecular Cloning and Expression of a Gene Encoding Cryptosporidium parvum Glycoproteins gp40 and gp15

    PubMed Central

    Cevallos, Ana Maria; Zhang, Xiaoping; Waldor, Matthew K.; Jaison, Smitha; Zhou, Xiaoyin; Tzipori, Saul; Neutra, Marian R.; Ward, Honorine D.

    2000-01-01

    Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host cell interactions and the molecular mechanisms involved in the pathogenesis of cryptosporidiosis are unknown. In this study we have shown that gp40, a mucin-like glycoprotein, is localized to the surface and apical region of invasive stages of the parasite and is shed from its surface. gp40-specific antibodies neutralize infection in vitro, and native gp40 binds specifically to host cells, implicating this glycoprotein in C. parvum attachment to and invasion of host cells. We have cloned and sequenced a gene designated Cpgp40/15 that encodes gp40 as well as gp15, an antigenically distinct, surface glycoprotein also implicated in C. parvum-host cell interactions. Analysis of the deduced amino acid sequence of the 981-bp Cpgp40/15 revealed the presence of an N-terminal signal peptide, a polyserine domain, multiple predicted O-glycosylation sites, a single potential N-glycosylation site, and a hydrophobic region at the C terminus, a finding consistent with what is required for the addition of a GPI anchor. There is a single copy of Cpgp40/15 in the C. parvum genome, and this gene does not contain introns. Our data indicate that the two Cpgp40/15-encoded proteins, gp40 and gp15, are products of proteolytic cleavage of a 49-kDa precursor protein which is expressed in intracellular stages of the parasite. The surface localization of gp40 and gp15 and their involvement in the host-parasite interaction suggest that either or both of these glycoproteins may serve as effective targets for specific preventive or therapeutic measures for cryptosporidiosis. PMID:10858228

  14. Giardia duodenalis and Cryptosporidium parvum infections in adult goats and their implications for neonatal kids.

    PubMed

    Castro-Hermida, J A; Delafosse, A; Pors, I; Ares-Mazás, E; Chartier, C

    2005-11-12

    During the kidding season between January and April 2003, 10 farms were selected and divided into two groups of five. The farms in group A had had serious diarrhoeal illness and losses in neonatal kids the previous year, and there were Cryptosporidium parvum infections in kids associated with diarrhoea during the survey. On the farms in group B, there was no history of diarrhoeal disease the previous year and neither C parvum oocysts nor diarrhoea were detected in neonatal kids during the survey. Faecal samples were collected once from 10 adult goats aged between one and seven years on each farm. To assess more accurately the pattern of output of oocysts of C parvum and cysts of Giardia duodenalis by periparturient adult goats, one farm was selected from each group, faecal samples were collected weekly before and after kidding from 12 goats on the farm in group A and from 10 goats on the farm in group B. There was no significant difference in the prevalence of G duodenalis cysts between the group A farms (14 per cent) and the group B farms (12 per cent), and the numbers of cysts excreted ranged from 143 to 400 cysts per gram of faeces (cpg) on the group A farms and 72 to 334 cpg on the group B farms. There was a significant difference (P=0.03) in the prevalence of C parvum oocysts at the group level between the group A farms (20 per cent) and the group B farms (6 per cent). All the adult goats excreted cysts and oocysts at some date around the kidding period; the number of animals excreting cysts of G duodenalis or oocysts of C parvum increased when they gave birth, and seven to 10 times more cysts and oocysts were shed in the three weeks around kidding than in the period more than three weeks from kidding (P<0.001).

  15. The Association of Cryptosporidium parvum With Suspended Sediments: Implications for Transport in Surface Waters

    NASA Astrophysics Data System (ADS)

    Searcy, K. E.; Packman, A. I.; Atwill, E. R.; Harter, T.

    2003-12-01

    Understanding the transport and fate of microorganisms in surface waters is of vital concern in protecting the integrity and safety of municipal water supply systems. The human pathogen Cryptosporidium parvum is a particular public health interest, as it is ubiquitous in the surface waters of the United States, it can persist for long periods in the environment, and it is difficult to disinfect in water treatment plants. Due to its small size (5 um), low specific gravity (1.05 g/cm3), and negative surface charge, C. parvum oocysts are generally considered to move through watersheds from their source to drinking water reservoirs with little attenuation. However, the transport of the oocysts in surface waters may be mediated by interactions with suspended sediments. Batch experiments were conducted to determine the extent of C. parvum oocyst attachment to several inorganic and organic sediments under varying water chemical conditions, and settling column experiments were performed to demonstrate how these associations influence the effective settling velocity of C. parvum oocysts. Results from these experiments showed that C. parvum oocysts do associate with inorganic and organic sediments and often settle at the rate of the suspended sediment. The size and surface charge of the host suspended sediment influenced the extent of oocyst attachment as oocysts preferentially associated with particles greater than 3 um, and fewer oocysts associated with particles having a highly negative surface charge. Background water chemical conditions including ionic strength, ion composition, and pH did not have a significant effect on oocyst attachment to suspended sediments.

  16. Occurrence of Cryptosporidium and Giardia in recycled waters used for irrigation and first description of Cryptosporidium parvum and C. muris in Greece.

    PubMed

    Spanakos, Gregory; Biba, Anastasia; Mavridou, Athena; Karanis, Panagiotis

    2015-05-01

    Here, we present the first time findings regarding the occurrence of Cryptosporidium and Giardia in sewage waters and the first molecular characterization of Cryptosporidium species in Greece. Biological treatment plants from three regions in Greece have been investigated. The detection of Cryptosporidium oocysts was by modified Ziehl-Neelsen acid fast (MZN-AF) and by immunofluorescence microscopy (IFT) for Cryptosporidium and Giardia (oo)cysts, whereas nested PCR based on the SSU rDNA assay was used for molecular detection of Cryptosporidium followed by sequencing for the genetic characterization of the species. In total, 73 samples (37 raw sewage samples and 38 of treated water samples) were collected and analyzed. Of the 73 water samples, 4 samples were Cryptosporidium-positive by IFT and staining, 12 samples were Cryptosporidium-positive by nested PCR; 9 samples were Giardia-positive by IFT. We showed that Cryptosporidium cysts are found both in the input and the discharge of the biological treatment plants. Molecular characterization of Cryptosporidium based on the small subunit ribosomal DNA gene resulted in the determination of Cryptosporidium parvum and Cryptosporidium muris Greek isolates. This is the first report of Cryptosporidium and Giardia occurrence in wastewaters and the first molecular identification of Cryptosporidium species in Greek environments. As the treated water is used for irrigation, or it is discharged into the sea, our findings indicate that biological treatment facilities constitute a possible risk for public health because the related species are prevalent in humans; the results invite for further epidemiological investigations to evaluate the real public health risk in Greece.

  17. A unique hexokinase in Cryptosporidium parvum, an apicomplexan pathogen lacking the Krebs cycle and oxidative phosphorylation.

    PubMed

    Yu, Yonglan; Zhang, Haili; Guo, Fengguang; Sun, Mingfei; Zhu, Guan

    2014-09-01

    Cryptosporidium parvum may cause virtually untreatable infections in AIDS patients, and is recently identified as one of the top four diarrheal pathogens in children in developing countries. Cryptosporidium differs from other apicomplexans (e.g., Plasmodium and Toxoplasma) by lacking many metabolic pathways including the Krebs cycle and cytochrome-based respiratory chain, thus relying mainly on glycolysis for ATP production. Here we report the molecular and biochemical characterizations of a hexokinase in C. parvum (CpHK). Our phylogenetic reconstructions indicated that apicomplexan hexokinases including CpHK were highly divergent from those of humans and animals (i.e., at the base of the eukaryotic clade). CpHK displays unique kinetic features that differ from those in mammals and Toxoplasma gondii (TgHK) in the preference towards various hexoses and its capacity to use ATP and other NTPs. CpHK also displays substrate inhibition by ATP. Moreover, 2-deoxy-D-glucose (2DG) could not only inhibit the CpHK activity, but also the parasite growth in vitro at concentrations nontoxic to host cells (IC(50) = 0.54 mM). While the exact action of 2-deoxy-D-glucose on the parasite is subject to further verification, our data suggest that CpHK and the glycolytic pathway may be explored for developing anti-cryptosporidial therapeutics. Copyright © 2014 Elsevier GmbH. All rights reserved.

  18. Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter.

    PubMed

    Taguchi, Tomoyuki; Shinozaki, Youhei; Takeyama, Haruko; Haraguchi, Satoshi; Yoshino, Masato; Kaneko, Masao; Ishimori, Yoshio; Matsunaga, Tadashi

    2006-11-01

    This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.

  19. Cell Culture-Taqman PCR Assay for Evaluation of Cryptosporidium parvum Disinfection

    PubMed Central

    Keegan, Alexandra R.; Fanok, Stella; Monis, Paul T.; Saint, Christopher P.

    2003-01-01

    Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log10 units) with LP-UV (20 mJ/cm2) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with <0.1 log10 unit being inactivated. These results demonstrate the inability of MIOX to inactivate Cryptosporidium. The assay is a valuable tool for the evaluation of disinfection systems for drinking water and recycled water. PMID:12732515

  20. Quantitative risk assessment for zoonotic transmission of Cryptosporidium parvum infection attributable to recreational use of farmland.

    PubMed

    Hill, A; Nally, P; Chalmers, R M; Pritchard, G C; Giles, M

    2011-08-01

    Cryptosporidiosis caused by Cryptosporidium parvum infection is a major cause of enteric illness in man and there is a significant reservoir in animals, particularly young ruminant species. To preliminary assess the magnitude of the risk posed by contact with faeces produced by infected livestock, two microbiological risk assessments have been developed: one for the risk of human infection with C. parvum while camping on contaminated land recently grazed by infected suckler cattle and a comparable risk assessment for camping on land recently spread with contaminated cattle slurry. Using a worst-case scenario approach, the upper level of risk was estimated to be one infection in every 6211 person-visits for a camping event on land recently grazed by infected cattle. Translated into camping events of 100 persons, this risk estimate would most likely lead to zero (98% likelihood) or one infection (1% likelihood). The results for cattle slurry model are similar despite different pathways. Sensitivity analysis was conducted for the grazing cattle model only. This suggested that the time between grazing and camping was the most important control strategy, but increasing hand-washing frequency and the removal of cattle faeces before camping would also be beneficial. If the upper level of risk were to be judged unacceptable then further data would be required to more accurately estimate the risk of infection through these scenarios. Further research would also be required to assess the fraction of cases attributable to camping and/or environmental contact with Cryptosporidium oocysts.

  1. Report of fatal mixed infection with Cryptosporidium parvum and Giardia intestinalis in neonatal calves.

    PubMed

    Matsuura, Yuu; Matsubayashi, Makoto; Nukata, Satoko; Shibahara, Tomoyuki; Ayukawa, Osamu; Kondo, Yasuko; Matsuo, Tomohide; Uni, Shigehiko; Furuya, Masaru; Tani, Hiroyuki; Tsuji, Naotoshi; Sasai, Kazumi

    2017-03-01

    In the production and management of beef and dairy cattle, controlling diarrhea is one of the important concerns. Pathogenic agents of the disease, protozoan parasites including Cryptosporidium spp., are difficult to control, making prevention, diagnoses, and treatment of diarrhea. In the present study, we investigated a farm with a history of calf deaths over a period of 10 years in order to determine the cause of disease and to clarify the detailed distribution of the pathogens. In four examined calves that were reared in calf pens, all were positive with Cryptosporidium and/or Giardia, while the other breeding stock and adult cattle were negative. Molecular analyses revealed that the isolates from calves were C. parvum subtype IIaA15G2R1 as a zoonotic and G. intestinalis assemblage E. Other pathogenic bacteria and diarrhea-causing viruses were not detected. After treating the calf pens with boiling water and milk of lime (Ca[OH]2), oocysts of C. parvum and cysts of G. intestinalis were not found and no additional calves died. This is the first report to describe the mixed infection of both parasites in Japan.

  2. Identification of Cryptosporidium parvum Oocysts by an Artificial Neural Network Approach

    PubMed Central

    Widmer, Kenneth W.; Oshima, Kevin H.; Pillai, Suresh D.

    2002-01-01

    Microscopic detection of Cryptosporidium parvum oocysts is time-consuming, requires trained analysts, and is frequently subject to significant human errors. Artificial neural networks (ANN) were developed to help identify immunofluorescently labeled C. parvum oocysts. A total of 525 digitized images of immunofluorescently labeled oocysts, fluorescent microspheres, and other miscellaneous nonoocyst images were employed in the training of the ANN. The images were cropped to a 36- by 36-pixel image, and the cropped images were placed into two categories, oocyst and nonoocyst images. The images were converted to grayscale and processed into a histogram of gray color pixel intensity. Commercially available software was used to develop and train the ANN. The networks were optimized by varying the number of training images, number of hidden neurons, and a combination of these two parameters. The network performance was then evaluated using a set of 362 unique testing images which the network had never “seen” before. Under optimized conditions, the correct identification of authentic oocyst images ranged from 81 to 97%, and the correct identification of nonoocyst images ranged from 78 to 82%, depending on the type of fluorescent antibody that was employed. The results indicate that the ANN developed were able to generalize the training images and subsequently discern previously unseen oocyst images efficiently and reproducibly. Thus, ANN can be used to reduce human errors associated with the microscopic detection of Cryptosporidium oocysts. PMID:11872458

  3. Selective and potent urea inhibitors of Cryptosporidium parvum inosine 5′ monophosphate dehydrogenase

    PubMed Central

    Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Liu, Xiaoping; Sharling, Lisa; Gollapalli, Deviprasad R.; Striepen, Boris; Hedstrom, Lizbeth; Cuny, Gregory D.

    2012-01-01

    Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5′-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (CpIMPDH) identified by high throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC50 < 2 nM) against CpIMPDH, excellent selectivity > 1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model. PMID:22950983

  4. A BAYESIAN METHOD OF ESTIMATING KINETIC PARAMETERS FOR THE INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH CHLORINE DIOXIDE AND OZONE

    EPA Science Inventory

    The main objective of this paper is to use Bayesian methods to estimate the kinetic parameters for the inactivation kinetics of Cryptosporidium parvum oocysts with chlorine dioxide or ozone which are characterized by the delayed Chick-Watson model, i.e., a lag phase or shoulder f...

  5. Experimental infection with Cryptosporidium parvum IIaA21G1R1 subtype in immunosuppressed mice

    USDA-ARS?s Scientific Manuscript database

    Cryptosporidium parvum subtype IIaA21G1R1 oocysts were used to infect dexamethasone immunosuppressed N: NIH Swiss mice. Histology showed developmental stages in the duodenum, proximal and distal jejunum, ileum, cecum and colon, with the small intestine remaining infected until day 35 post infection....

  6. MODELING CRYPTOSPORIDIUM PARVUM OOCYST INACTIVATION AND BROMATE IN A FLOW-THROUGH OZONE CONTACTOR TREATING NATURAL WATER

    EPA Science Inventory

    A reactive transport model was developed to simultaneously predict Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of natural water. A mechanistic model previously established to predict bromate formation in organic-free synthetic waters w...

  7. A COMPARISON OF FOUR FLUORESCENT ANTIBODY-BASED METHODS FOR PURIFYING, DETECTING, AND CONFIRMING CRYPTOSPORIDIUM PARVUM IN SURFACE WATERS

    EPA Science Inventory

    Cryptosporidiosis has been traced to drinking contaminated surface water, which was either not treated or was ineffectively treated. Testing to detect Cryptosporidium parvum in surface water has been suggested to help prevent future outbreaks. In the present study, the same sampl...

  8. MODELING CRYPTOSPORIDIUM PARVUM OOCYST INACTIVATION AND BROMATE IN A FLOW-THROUGH OZONE CONTACTOR TREATING NATURAL WATER

    EPA Science Inventory

    A reactive transport model was developed to simultaneously predict Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of natural water. A mechanistic model previously established to predict bromate formation in organic-free synthetic waters w...

  9. A COMPARISON OF FOUR FLUORESCENT ANTIBODY-BASED METHODS FOR PURIFYING, DETECTING, AND CONFIRMING CRYPTOSPORIDIUM PARVUM IN SURFACE WATERS

    EPA Science Inventory

    Cryptosporidiosis has been traced to drinking contaminated surface water, which was either not treated or was ineffectively treated. Testing to detect Cryptosporidium parvum in surface water has been suggested to help prevent future outbreaks. In the present study, the same sampl...

  10. Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum Oocysts

    USDA-ARS?s Scientific Manuscript database

    The structure and composition of the oocyst wall are primary factors determining the survival of Cryptosporidium parvum oocysts outside the host. An external polymer matrix (glycocalyx) may mediate interactions with environmental surfaces and, thus, affect the transport of oocysts in water, soil, an...

  11. A BAYESIAN METHOD OF ESTIMATING KINETIC PARAMETERS FOR THE INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH CHLORINE DIOXIDE AND OZONE

    EPA Science Inventory

    The main objective of this paper is to use Bayesian methods to estimate the kinetic parameters for the inactivation kinetics of Cryptosporidium parvum oocysts with chlorine dioxide or ozone which are characterized by the delayed Chick-Watson model, i.e., a lag phase or shoulder f...

  12. Sequential inactivation of Cryptosporidium parvum using ozone and chlorine.

    PubMed

    Li, H; Finch, G R; Smith, D W; Belosevic, M

    2001-12-01

    Inactivation of bovine-derived C. parvum oocysts was studied at bench-scale in oxidant demand free 0.05 M phosphate buffer using free chlorine alone or ozone followed by free chlorine at temperatures of 1 degrees C, 10 degrees C and 22 degrees C at pH 6. Animal infectivity using neonatal CD-1 mice was used for evaluation of oocyst viability after treatment. Kinetic models based on the linear Chick-Watson model were developed for free chlorine inactivation and ozone/free chlorine sequential inactivation for 0.4 or 1.6 log-units of ozone primary kill. At 22 degrees C. ozone pre-treatment increased the efficacy of free chlorine for about 4-6 times depending on the level of ozone primary kills. Gross kills of the ozone/free chlorine sequential inactivation were a function of ozone primary kills and increased linearly with the free chlorine C(avg)t (arithmetic average of the initial and final residual x contact time) product. Temperature was critical for both single and sequential inactivation, and the efficacy of free chlorine after 1.6 log-units of ozone primary inactivation decreased by a factor of 1.8 for every 10 degrees C temperature decrease. Given an ozone primary kill of 1.6 log-units, the free chlorine C(avg)t products required for a gross kill of 3.0 log-units were 1000, 2000 and 3,300 mgmin/L for 22 degrees C. 10 degrees C and 1degrees C, respectively.

  13. Cryptosporidium parvum Long-Chain Fatty Acid Elongase▿ †

    PubMed Central

    Fritzler, Jason M.; Millership, Jason J.; Zhu, Guan

    2007-01-01

    We report the presence of a new fatty acyl coenzyme A (acyl-CoA) elongation system in Cryptosporidium and the functional characterization of the key enzyme, a single long-chain fatty acid elongase (LCE), in this parasite. This enzyme contains conserved motifs and predicted transmembrane domains characteristic to the elongase family and is placed within the ELO6 family specific for saturated substrates. CpLCE1 gene transcripts are present at all life cycle stages, but the levels are highest in free sporozoites and in stages at 36 h and 60 h postinfection that typically contain free merozoites. Immunostaining revealed localization to the outer surface of sporozoites and to the parasitophorous vacuolar membrane. Recombinant CpLCE1 displayed allosteric kinetics towards malonyl-CoA and palmitoyl-CoA and Michaelis-Menten kinetics towards NADPH. Myristoyl-CoA (C14:0) and palmitoyl-CoA (C16:0) display the highest activity when used as substrates, and only one round of elongation occurs. CpLCE1 is fairly resistant to cerulenin, an inhibitor for both type I and II fatty acid synthases (i.e., maximum inhibitions of 20.5% and 32.7% were observed when C16:0 and C14:0 were used as substrates, respectively). These observations ultimately validate the function of CpLCE1. PMID:17827345

  14. Biofilm roughness determines Cryptosporidium parvum retention in environmental biofilms.

    PubMed

    DiCesare, E A Wolyniak; Hargreaves, B R; Jellison, K L

    2012-06-01

    The genus Cryptosporidium is a group of waterborne protozoan parasites that have been implicated in significant outbreaks of gastrointestinal infections throughout the world. Biofilms trap these pathogens and can contaminate water supplies through subsequent release. Biofilm microbial assemblages were collected seasonally from three streams in eastern Pennsylvania and used to grow biofilms in laboratory microcosms. Daily oocyst counts in the influx and efflux flow allowed the calculation of daily oocyst retention in the biofilm. Following the removal of oocysts from the influx water, oocyst attachment to the biofilm declined to an equilibrium state within 5 days that was sustained for at least 25 days. Varying the oocyst loading rate for the system showed that biofilm retention could be saturated, suggesting that discrete binding sites determined the maximum number of oocysts retained. Oocyst retention varied seasonally but was consistent across all three sites; however, seasonal oocyst retention was not consistent across years at the same site. No correlation between oocyst attachment and any measured water quality parameter was found. However, oocyst retention was strongly correlated with biofilm surface roughness and roughness varied among seasons and across years. We hypothesize that biofilm roughness and oocyst retention are dependent on environmentally driven changes in the biofilm community rather than directly on water quality conditions. It is important to understand oocyst transport dynamics to reduce risks of human infection. Better understanding of factors controlling biofilm retention of oocysts should improve our understanding of oocyst transport at different scales.

  15. Characterization of Cryptosporidium parvum by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

    PubMed Central

    Magnuson, Matthew L.; Owens, James H.; Kelty, Catherine A.

    2000-01-01

    Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used to investigate whole and freeze-thawed Cryptosporidium parvum oocysts. Whole oocysts revealed some mass spectral features. Reproducible patterns of spectral markers and increased sensitivity were obtained after the oocysts were lysed with a freeze-thaw procedure. Spectral-marker patterns for C. parvum were distinguishable from those obtained for Cryptosporidium muris. One spectral marker appears specific for the genus, while others appear specific at the species level. Three different C. parvum lots were investigated, and similar spectral markers were observed in each. Disinfection of the oocysts reduced and/or eliminated the patterns of spectral markers. PMID:11055915

  16. MicroRNA-221 controls expression of intercellular adhesion molecule-1 in epithelial cells in response to Cryptosporidium parvum infection

    PubMed Central

    Gong, Ai-Yu; Hu, Guoku; Zhou, Rui; Liu, Jun; Feng, Yaoyu; Soukup, Garrett A.; Chen, Xian-Ming

    2011-01-01

    Cryptosporidium parvum is a protozoan parasite that infects gastrointestinal epithelial cells and causes diarrheal disease in humans and animals globally. Pathological changes following C. parvum infection include crypt hyperplasia, a modest inflammatory reaction with increased infiltration of lymphocytes into intestinal mucosa. Expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), on infected epithelial cell surfaces may facilitate adhesion and recognition of lymphocytes at infection sites. MicroRNAs (miRNAs) are small RNA molecules of 23 nucleotides that negatively regulate protein-coding gene expression via translational suppression or mRNA degradation. We recently reported that microRNA-221 (miR-221) regulates ICAM-1 translation through targeting the ICAM-1 3′-untranslated region (UTR). In this study, we tested the role of miR-221 in regulating ICAM-1 expression in epithelial cells in response to C. parvum infection using an in vitro model of human biliary cryptosporidiosis. Up-regulation of ICAM-1 at both message and protein levels was detected in epithelial cells following C. parvum infection. Inhibition of ICAM-1 transcription with actinomycin D could only partially block C. parvum-induced ICAM-1 expression at the protein level. Cryptosporidium parvum infection decreased miR-221 expression in infected epithelial cells. When cells were transfected with a luciferase reporter construct covering the miR-221 binding site in the ICAM-1 3′-UTR and then exposed to C. parvum, an enhanced luciferase activity was detected. Transfection of miR-221 precursor abolished C. parvum-stimulated ICAM-1 protein expression. In addition, expression of ICAM-1 on infected epithelial cells facilitated epithelial adherence of co-cultured Jurkat cells. These results indicate that miR-221-mediated translational suppression controls ICAM-1 expression in epithelial cells in response to C. parvum infection. PMID:21236259

  17. Comparison of microscopy, rapid immunoassay, and molecular techniques for the detection of Giardia lamblia and Cryptosporidium parvum.

    PubMed

    Elsafi, Salah H; Al-Maqati, Thekra N; Hussein, Mohi I; Adam, Ahmed A; Hassan, Mohamed M Abu; Al Zahrani, Eidan M

    2013-04-01

    Giardia lamblia and Cryptosporidium parvum are recognized as the most common protozoan infections in Saudi Arabia. Microscopic examination of stool samples, either direct or concentrated, for the recovery of G. lamblia cysts and trophozoites and C. parvum oocysts is still the most commonly used for the diagnosis of both parasites. We compared the conventional parasitological techniques of iodine-stained wet mount for G. lamblia and Kinyoun's acid-fast for C. parvum against ImmunoCard STAT® Cryptosporidium/Giardia and real-time polymerase chain reaction (PCR) detecting the 18S rRNA gene of G. lamblia and conventional PCR detecting the same gene of C. parvum at a tertiary hospital in Dhahran, Saudi Arabia. Out of 148 stool samples, 19 and 12 true positives were identified for G. lamblia and C. parvum, respectively, using a composite reference standard. In this case, true positives and negatives were considered as those with at least two positive or negative results out of the three tests. Both ImmunoCard STAT! and PCR methods were more sensitive than the microscopic tests of a single stool specimen of 85.7% (CI=62.6-96.2%) and 85.7% (CI=56.2-97.5%) for G. lamblia and C. parvum, respectively. However, specificity of microscopic tests was higher than other techniques for both parasites. Although PCR seems to be most sensitive for both G. lamblia and C. parvum, its low specificity may render its superiority over other techniques. When a single stool sample is used for detection of G. lamblia and C. parvum, better results can be obtained when coupled with serological testing. Although PCR is the most sensitive method for the detection of both G. lamblia and C. parvum, its use requires attention in relation to the increased possible false positives.

  18. Modeling Fate and Transport of Cryptosporidium Parvum Oocysts in Overland and Near- surface Flow

    NASA Astrophysics Data System (ADS)

    Bhattarai, R.; Kalita, P.; Kuhlenschmidt, M. S.

    2008-12-01

    Cryptosporidium parvum is a manure-borne protozoan parasite which is common in the environment. It has been recognized as an important microbial contaminant of water and can cause infection and diarrhea in many mammalian hosts, including humans. The laboratory experiments carried out have demonstrated that recovery of C. parvum oocysts was significantly affected by climatic and surface conditions like slope, rainfall and surface cover. The objective of this study is to develop a model for simulating transport of C. parvum oocysts in overland and near-surface flow. Modeling can help understanding oocysts transport pathways. Accordingly, best management practices (BMP) can be developed. Transport of oocysts in overland flow can be simulated mathematically by including terms for the concentration of the oocysts in the liquid phase (in suspension or free-floating) and the solid phase (adsorbed to the fine solid particles like clay). Oocysts adsorption, advection and decay processes are considered. These processes are solved using numerical technique to predict spatial and temporal changes in oocyst concentrations in solid and liquid phases. The model results are compared with experimental data to validate the model outcome. The model output reproduced observed recovery kinetics for 1.5% slope but not for higher slopes (3.0% and 4.5%).

  19. Triazole inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

    PubMed Central

    Maurya, Sushil K.; Gollapalli, Deviprasad R.; Kirubakaran, Sivapriya; Zhang, Minjia; Johnson, Corey R.; Benjamin, Nicole N.; Hedstrom, Lizbeth; Cuny, Gregory D.

    2010-01-01

    Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. This protozoan parasite cannot salvage guanine or guanosine and therefore relies on inosine 5′-monophosphate dehydrogenase (IMPDH) for biosynthesis of guanine nucleotides and hence for survival. Since C. parvum IMPDH is highly divergent from the host counterpart, selective inhibitors could potentially be used to treat cryptosporidiosis with minimal effects on its mammalian host. A series of 1,2,3-triazole containing ether CpIMPDH inhibitors are described. A structure-activity relationship study revealed that a small alkyl group on the alpha-position of the ether was required with the (R)-enantiomer significantly more active than the (S)-enantiomer. Electron-withdrawing groups in the 3- and/or 4-positions of the pendent phenyl ring were best and conversion of the quinoline containing inhibitors to quinoline-N-oxides retained inhibitory activity both in the presence and absence of bovine serum albumin. The 1,2,3-triazole CpIMPDH inhibitors provide new tools for elucidating the role of IMPDH in C. parvum and may serve as potential therapeutics for treating cryptosporidiosis. PMID:19624136

  20. In vitro inhibitory effects of plant-derived by-products against Cryptosporidium parvum

    PubMed Central

    Teichmann, Klaus; Kuliberda, Maxime; Schatzmayr, Gerd; Pacher, Thomas; Zitterl-Eglseer, Karin; Joachim, Anja; Hadacek, Franz

    2016-01-01

    Disposal of organic plant wastes and by-products from the food or pharmaceutical industries usually involves high costs. In the present study, 42 samples derived from such by-products were screened in vitro against Cryptosporidium parvum, a protozoan parasite that may contaminate drinking water and cause diarrhoea. The novel bioassay was previously established in the microtitre plate format. Human ileocaecal adenocarcinoma (HCT-8) cell cultures were seeded with C. parvum oocysts and parasite development was monitored by an indirect fluorescent antibody technique (IFAT) and microscopic assessment for clusters of secondary infection (CSI). Minimum inhibitory concentrations (MICs) and potential detrimental effects on the host cells were determined. An ethanolic extract from olive (Olea europaea) pomace, after oil pressing and phenol recovery, reproducibly inhibited C. parvum development (MIC = 250–500 μg mL−1, IC50 = 361 (279–438) μg mL−1, IC90 = 467 (398–615) μg mL−1). Accordingly, tyrosol, hydroxytyrosol, trans-coniferyl alcohol and oleuropein were selected as reference test compounds, but their contributions to the observed activity of the olive pomace extract were insignificant. The established test system proved to be a fast and efficient assay for identifying anti-cryptosporidial activities in biological waste material and comparison with selected reference compounds. PMID:27627637

  1. In vitro inhibitory effects of plant-derived by-products against Cryptosporidium parvum.

    PubMed

    Teichmann, Klaus; Kuliberda, Maxime; Schatzmayr, Gerd; Pacher, Thomas; Zitterl-Eglseer, Karin; Joachim, Anja; Hadacek, Franz

    2016-01-01

    Disposal of organic plant wastes and by-products from the food or pharmaceutical industries usually involves high costs. In the present study, 42 samples derived from such by-products were screened in vitro against Cryptosporidium parvum, a protozoan parasite that may contaminate drinking water and cause diarrhoea. The novel bioassay was previously established in the microtitre plate format. Human ileocaecal adenocarcinoma (HCT-8) cell cultures were seeded with C. parvum oocysts and parasite development was monitored by an indirect fluorescent antibody technique (IFAT) and microscopic assessment for clusters of secondary infection (CSI). Minimum inhibitory concentrations (MICs) and potential detrimental effects on the host cells were determined. An ethanolic extract from olive (Olea europaea) pomace, after oil pressing and phenol recovery, reproducibly inhibited C. parvum development (MIC = 250-500 μg mL(-1), IC50 = 361 (279-438) μg mL(-1), IC90 = 467 (398-615) μg mL(-1)). Accordingly, tyrosol, hydroxytyrosol, trans-coniferyl alcohol and oleuropein were selected as reference test compounds, but their contributions to the observed activity of the olive pomace extract were insignificant. The established test system proved to be a fast and efficient assay for identifying anti-cryptosporidial activities in biological waste material and comparison with selected reference compounds. © K. Teichmann et al., published by EDP Sciences, 2016.

  2. Pseudo-Second-Order Calcium-Mediated Cryptosporidium parvum Oocyst Attachment to Environmental Biofilms.

    PubMed

    Luo, Xia; Jedlicka, Sabrina; Jellison, Kristen

    2017-01-01

    Cryptosporidium parvum oocysts are able to infect a wide range of mammals, including humans, via fecal-oral transmission. The remobilization of biofilm-associated C. parvum oocysts back into the water column by biofilm sloughing or bulk erosion poses a threat to public health and may be responsible for waterborne outbreaks; thus, the investigation of C. parvum attachment mechanisms to biofilms, particularly the physical and chemical factors controlling oocyst attachment to biofilms, is essential to predict the behavior of oocysts in the environment. In our study, biofilms were grown in rotating annular bioreactors using prefiltered stream water (0.2-μm retention) and rock biofilms (6-μm retention) until the mean biofilm thickness reached steady state. Oocyst deposition followed a calcium-mediated pseudo-second-order kinetic model. Kinetic parameters (i.e., initial oocyst deposition rate constant and total number of oocysts adhered to biofilms at equilibrium) from the model were then used to evaluate the impact of water conductivity on the attachment of oocysts to biofilms. Oocyst deposition was independent of solution ionic strength; instead, the presence of calcium enhanced oocyst attachment, as demonstrated by deposition tests. Calcium was identified as the predominant factor that bridges the carboxylic functional groups on biofilm and oocyst surfaces to cause attachment. The pseudo-second-order kinetic profile fit all experimental conditions, regardless of water chemistry and/or lighting conditions.

  3. Immunodetection of the microvillous cytoskeleton molecules villin and ezrin in the parasitophorous vacuole wall of Cryptosporidium parvum (Protozoa: Apicomplexa).

    PubMed

    Bonnin, A; Lapillonne, A; Petrella, T; Lopez, J; Chaponnier, C; Gabbiani, G; Robine, S; Dubremetz, J F

    1999-11-01

    Microvilli - actin - villin - ezrin - Cryptosporidium parvum The sporozoites and merozoites of the Apicomplexan protozoan Cryptosporidium parvum (C. parvum) invade the apical side of enterocytes and induce the formation of a parasitophorous vacuole which stays in the brush border area and disturbs the distribution of microvilli. The vacuole is separated from the apical cytoplasm of the cell by an electron-dense layer of undetermined composition. In order to characterize the enterocyte cytoskeleton changes that occur during C. parvum invasion and development, we used both confocal immunofluorescence and immunoelectron microscopy to examine at the C.parvum-enterocyte interface the distribution of three components of the microvillous skeleton, actin, villin and ezrin. In infected cells, rhodamine-phalloidin and anti-villin and anti-ezrin antibodies recognized ring-like structures surrounding the developing parasites. By immunoelectron microscopy, both villin and ezrin were detected in the parasitophorous vacuole wall surrounding the luminal and lateral sides of the intracellular parasite. In contrast, anti-beta and anti-gamma actin antibodies showed no significant labelling of the vacuolar wall. These observations indicate that the parasitophorous vacuole wall contains at least two microvillus-derived components, villin and ezrin, as well as a low amount of F-actin. These data suggest that C.parvum infection induces a rearrangement of cytoskeleton molecules at the apical pole of the host cell that are used to build the parasitophorous vacuole.

  4. Real-time nucleic acid sequence-based amplification (NASBA) assay targeting MIC1 for detection of Cryptosporidium parvum and Cryptosporidium hominis oocysts.

    PubMed

    Hønsvall, Birgitte K; Robertson, Lucy J

    2017-01-01

    Both Cryptosporidium parvum and Cryptosporidium hominis are often associated with cryptosporidiosis in humans, but whereas humans are the main host for C. hominis, C. parvum is zoonotic and able to infect a variety of species. The oocyst transmission stages of both species of parasites are morphologically identical and molecular techniques, usually polymerase chain reaction (PCR), are required to distinguish between oocysts detected by standard methods in environmental samples, such as water. In this study, we developed two primer sets for real-time nucleic acid sequence-based amplification (NASBA), targeting the MIC1 transcript in C. parvum (CpMIC1) and C. hominis (ChMIC1). Using these primer sets, we were not only able to detect low numbers of C. parvum and C. hominis oocysts (down to 5 oocysts in 10 μl, and down to 1 oocyst using diluted RNA samples), but also distinguish between them. One of the primer sets targeted an exon only occurring in CpMIC1, thereby providing a tool for distinguishing C. parvum from other Cryptosporidium species. Although mRNA has been suggested as a tool for assessing viability of Cryptosporidium oocysts, as it is short-lived and may have high transcription, this NASBA assay detected MIC1 mRNA in inactivated oocysts. RNA within the oocysts seems to be protected from degradation, even when the oocysts have been killed by heating or freeze-thawing. Thus, our approach detects both viable and non-viable oocysts, and RNA does not seem to be a suitable marker for assessing oocyst viability.

  5. A cysteine protease inhibitor rescues mice from a lethal Cryptosporidium parvum infection.

    PubMed

    Ndao, Momar; Nath-Chowdhury, Milli; Sajid, Mohammed; Marcus, Victoria; Mashiyama, Susan T; Sakanari, Judy; Chow, Eric; Mackey, Zachary; Land, Kirkwood M; Jacobson, Matthew P; Kalyanaraman, Chakrapani; McKerrow, James H; Arrowood, Michael J; Caffrey, Conor R

    2013-12-01

    Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.

  6. Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of Cryptosporidium parvum Oocysts

    PubMed Central

    Quilez, Joaquin; Sanchez-Acedo, Caridad; Avendaño, Catalina; del Cacho, Emilio; Lopez-Bernad, Fernando

    2005-01-01

    Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4′,6′-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection. PMID:15870337

  7. Efficacy of two peroxygen-based disinfectants for inactivation of Cryptosporidium parvum oocysts.

    PubMed

    Quilez, Joaquin; Sanchez-Acedo, Caridad; Avendaño, Catalina; del Cacho, Emilio; Lopez-Bernad, Fernando

    2005-05-01

    Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4',6'-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.

  8. A Cysteine Protease Inhibitor Rescues Mice from a Lethal Cryptosporidium parvum Infection

    PubMed Central

    Nath-Chowdhury, Milli; Sajid, Mohammed; Marcus, Victoria; Mashiyama, Susan T.; Sakanari, Judy; Chow, Eric; Mackey, Zachary; Land, Kirkwood M.; Jacobson, Matthew P.; Kalyanaraman, Chakrapani; McKerrow, James H.; Arrowood, Michael J.; Caffrey, Conor R.

    2013-01-01

    Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis. PMID:24060869

  9. Genome-wide upstream motif analysis of Cryptosporidium parvum genes clustered by expression profile

    PubMed Central

    2013-01-01

    Background There are very few molecular genetic tools available to study the apicomplexan parasite Cryptosporidium parvum. The organism is not amenable to continuous in vitro cultivation or transfection, and purification of intracellular developmental stages in sufficient numbers for most downstream molecular applications is difficult and expensive since animal hosts are required. As such, very little is known about gene regulation in C. parvum. Results We have clustered whole-genome gene expression profiles generated from a previous study of seven post-infection time points of 3,281 genes to identify genes that show similar expression patterns throughout the first 72 hours of in vitro epithelial cell culture. We used the algorithms MEME, AlignACE and FIRE to identify conserved, overrepresented DNA motifs in the upstream promoter region of genes with similar expression profiles. The most overrepresented motifs were E2F (5′-TGGCGCCA-3′); G-box (5′-G.GGGG-3′); a well-documented ApiAP2 binding motif (5′-TGCAT-3′), and an unknown motif (5′-[A/C] AACTA-3′). We generated a recombinant C. parvum DNA-binding protein domain from a putative ApiAP2 transcription factor [CryptoDB: cgd8_810] and determined its binding specificity using protein-binding microarrays. We demonstrate that cgd8_810 can putatively bind the overrepresented G-box motif, implicating this ApiAP2 in the regulation of many gene clusters. Conclusion Several DNA motifs were identified in the upstream sequences of gene clusters that might serve as potential cis-regulatory elements. These motifs, in concert with protein DNA binding site data, establish for the first time the beginnings of a global C. parvum gene regulatory map that will contribute to our understanding of the development of this zoonotic parasite. PMID:23895416

  10. Genome-wide upstream motif analysis of Cryptosporidium parvum genes clustered by expression profile.

    PubMed

    Oberstaller, Jenna; Joseph, Sandeep J; Kissinger, Jessica C

    2013-07-29

    There are very few molecular genetic tools available to study the apicomplexan parasite Cryptosporidium parvum. The organism is not amenable to continuous in vitro cultivation or transfection, and purification of intracellular developmental stages in sufficient numbers for most downstream molecular applications is difficult and expensive since animal hosts are required. As such, very little is known about gene regulation in C. parvum. We have clustered whole-genome gene expression profiles generated from a previous study of seven post-infection time points of 3,281 genes to identify genes that show similar expression patterns throughout the first 72 hours of in vitro epithelial cell culture. We used the algorithms MEME, AlignACE and FIRE to identify conserved, overrepresented DNA motifs in the upstream promoter region of genes with similar expression profiles. The most overrepresented motifs were E2F (5'-TGGCGCCA-3'); G-box (5'-G.GGGG-3'); a well-documented ApiAP2 binding motif (5'-TGCAT-3'), and an unknown motif (5'-[A/C] AACTA-3'). We generated a recombinant C. parvum DNA-binding protein domain from a putative ApiAP2 transcription factor [CryptoDB: cgd8_810] and determined its binding specificity using protein-binding microarrays. We demonstrate that cgd8_810 can putatively bind the overrepresented G-box motif, implicating this ApiAP2 in the regulation of many gene clusters. Several DNA motifs were identified in the upstream sequences of gene clusters that might serve as potential cis-regulatory elements. These motifs, in concert with protein DNA binding site data, establish for the first time the beginnings of a global C. parvum gene regulatory map that will contribute to our understanding of the development of this zoonotic parasite.

  11. Characterization of an Intestinal Epithelial Cell Receptor Recognized by the Cryptosporidium parvum Sporozoite Ligand CSL

    PubMed Central

    Langer, Rebecca C.; Schaefer, Deborah A.; Riggs, Michael W.

    2001-01-01

    The protozoan parasite Cryptosporidium parvum is a leading cause of diarrhea in humans and neonatal calves. The absence of approved parasite-specific drugs, vaccines, and immunotherapies for cryptosporidiosis relates in part to limited knowledge on the pathogenesis of zoite attachment and invasion. We recently reported that the C. parvum apical complex glycoprotein CSL contains a zoite ligand for intestinal epithelial cells which is defined by monoclonal antibody (MAb) 3E2. In the present study, the host cell receptor for CSL was characterized. For these studies, a panel of epithelial and mesenchymal cell lines was examined for permissiveness to C. parvum and the ability to bind CSL. Cells of epithelial origin were significantly more permissive and bound significantly greater quantities of CSL than cells of mesenchymal origin. Caco-2 intestinal cells were selected from the epithelial panel for further characterization of the CSL receptor. Immunoelectron microscopy demonstrated that CSL bound initially to the surface of Caco-2 cells and was rapidly internalized. The molecule bound by CSL was identified as an 85-kDa Caco-2 cell surface protein by radioimmunoprecipitation and CSL affinity chromatography. Sporozoite incubation with the isolated 85-kDa protein reduced binding of MAb 3E2. Further, attachment and invasion were significantly inhibited when sporozoites were incubated with the 85-kDa protein prior to inoculation onto Caco-2 cells. These observations indicate that the 85-kDa protein functions as a Caco-2 cell receptor for CSL. CSL also bound specifically to intestinal epithelium from calves, indicating receptor expression in a second important host species. Molecular characterization of the CSL receptor may lead to novel avenues for disrupting ligand-receptor interactions in the pathogenesis of C. parvum infection. PMID:11179341

  12. Pseudo-Second-Order Calcium-Mediated Cryptosporidium parvum Oocyst Attachment to Environmental Biofilms

    PubMed Central

    Luo, Xia; Jedlicka, Sabrina

    2016-01-01

    ABSTRACT Cryptosporidium parvum oocysts are able to infect a wide range of mammals, including humans, via fecal-oral transmission. The remobilization of biofilm-associated C. parvum oocysts back into the water column by biofilm sloughing or bulk erosion poses a threat to public health and may be responsible for waterborne outbreaks; thus, the investigation of C. parvum attachment mechanisms to biofilms, particularly the physical and chemical factors controlling oocyst attachment to biofilms, is essential to predict the behavior of oocysts in the environment. In our study, biofilms were grown in rotating annular bioreactors using prefiltered stream water (0.2-μm retention) and rock biofilms (6-μm retention) until the mean biofilm thickness reached steady state. Oocyst deposition followed a calcium-mediated pseudo-second-order kinetic model. Kinetic parameters (i.e., initial oocyst deposition rate constant and total number of oocysts adhered to biofilms at equilibrium) from the model were then used to evaluate the impact of water conductivity on the attachment of oocysts to biofilms. Oocyst deposition was independent of solution ionic strength; instead, the presence of calcium enhanced oocyst attachment, as demonstrated by deposition tests. Calcium was identified as the predominant factor that bridges the carboxylic functional groups on biofilm and oocyst surfaces to cause attachment. The pseudo-second-order kinetic profile fit all experimental conditions, regardless of water chemistry and/or lighting conditions. IMPORTANCE The cation bridging model in our study provides new insights into the impact of calcium on the attachment of C. parvum oocysts to environmental biofilms. The kinetic parameters derived from the model could be further analyzed to elucidate the behavior of oocysts in commonly encountered complex aquatic systems, which will enable future innovations in parasite detection and treatment technologies to protect public health. PMID:27793825

  13. Stochastic modeling of Cryptosporidium parvum to predict transport, retention, and downstream exposure

    NASA Astrophysics Data System (ADS)

    Drummond, J. D.; Boano, F.; Atwill, E. R.; Li, X.; Harter, T.; Packman, A. I.

    2016-12-01

    Rivers are a means of rapid and long-distance transmission of pathogenic microorganisms from upstream terrestrial sources. Thus, significant fluxes of pathogen loads from agricultural lands can occur due to transport in surface waters. Pathogens enter streams and rivers in a variety of processes, notably overland flow, shallow groundwater discharge, and direct inputs from host populations such as humans and other vertebrate species. Viruses, bacteria, and parasites can enter a stream and persist in the environment for varying amounts of time. Of particular concern is the protozoal parasite, Cryptosporidium parvum, which can remain infective for weeks to months under cool and moist conditions, with the infectious state (oocysts) largely resistant to chlorination. In order to manage water-borne diseases more effectively we need to better predict how microbes behave in freshwater systems, particularly how they are transported downstream in rivers and in the process interact with the streambed and other solid surfaces. Microbes continuously immobilize and resuspend during downstream transport due to a variety of processes, such as gravitational settling, attachment to in-stream structures such as submerged macrophytes, and hyporheic exchange and filtration within underlying sediments. These various interactions result in a wide range of microbial residence times in the streambed and therefore influence the persistence of pathogenic microbes in the stream environment. We developed a stochastic mobile-immobile model to describe these microbial transport and retention processes in streams and rivers that also accounts for microbial inactivation. We used the model to assess the transport, retention, and inactivation of C. parvum within stream environments, specifically under representative flow conditions of California streams where C. parvum exposure can be at higher risk due to agricultural nonpoint sources. The results demonstrate that the combination of stream reach

  14. Efficacy of Pyrvinium Pamoate against Cryptosporidium parvum Infection In Vitro and in a Neonatal Mouse Model▿

    PubMed Central

    Downey, Autumn S.; Chong, Curtis R.; Graczyk, Thaddeus K.; Sullivan, David J.

    2008-01-01

    No effective approved drug therapy exists for Cryptosporidium infection of immunocompromised patients. Here we investigated the nonabsorbed anthelmintic drug pyrvinium pamoate for inhibition of the growth of the intestinal protozoan parasite Cryptosporidium parvum. The concentration of pyrvinium that effected 50% growth inhibition in human enterocytic HCT-8 cells by a quantitative alkaline phosphatase immunoassay was 354 nM. For comparison, in the same assay, 50% growth inhibition was obtained with 711 μM paromomycin or 27 μM chloroquine. We used a neonatal mouse model to measure the anti-Cryptosporidium activity of pyrvinium pamoate in vivo. Beginning 3 days after infection, pyrvinium at 5 or 12.5 mg/kg of body weight/day was administered to the treatment group mice for 4 or 6 consecutive days. Nine days after infection, the mice were sacrificed, and drug efficacy was determined by comparing the numbers of oocysts in the fecal smears of treated versus untreated mice. The intensities of trophozoite infection in the ileocecal intestinal regions were also compared using hematoxylin-and-eosin-stained histological slides. We observed a >90% reduction in infection intensity in pyrvinium-treated mice relative to that in untreated controls, along with a substantial reduction in tissue pathology. Based on these results, pyrvinium pamoate is a potential drug candidate for the treatment of cryptosporidiosis in both immunocompetent and immunocompromised individuals. PMID:18591280

  15. Symptomatic and asymptomatic secondary transmission of Cryptosporidium parvum following two related outbreaks in schoolchildren.

    PubMed

    Johansen, Ø H; Hanevik, K; Thrana, F; Carlson, A; Stachurska-Hagen, T; Skaare, D; Robertson, L J

    2015-06-01

    Two related outbreaks (in 2009 and 2012) of cryptosporidiosis in Norwegian schoolchildren during a stay at a remote holiday farm provided us with a natural experiment to investigate possible secondary transmission of Cryptosporidium parvum IIa A19G1R1. After the children had returned home, clinical data and stool samples were obtained from their household contacts. Samples were investigated for the presence of Cryptosporidium oocysts by immunofluorescence antibody test. We found both asymptomatic and symptomatic infections, which are likely to have been secondary transmission. Laboratory-confirmed transmission rate was 17% [4/23, 95% confidence interval (CI) 7·0-37·1] in the 2009 outbreak, and 0% (95% CI 0-16·8) in the 2012 outbreak. Using a clinical definition, the probable secondary transmission rate in the 2012 outbreak was 8% (7/83, 95% CI 4·1-16·4). These findings highlight the importance of hygienic and public health measures during outbreaks or individual cases of cryptosporidiosis. We discuss our findings in light of previous studies reporting varying secondary transmission rates of Cryptosporidium spp.

  16. Leaching of Salmonella Senftenberg and Cryptosporidium Parvum in Intact Clay Columns

    NASA Astrophysics Data System (ADS)

    Bech, T. B.; Forslund, A.; Dalsgaard, A.; Jacobsen, O.; Jacobsen, C. S.

    2008-12-01

    Manure application on land has been associated with both environmental and public health problems, even when management is within the current guidelines. Outbreaks of infection have been associated with water or food, including processed fruits and vegetables, contaminated with animal manure. A wide range of pathogenic microorganisms can be found in animal waste, including bacteria, protozoan, and viruses. When animal waste is disposed on agricultural land different factors will influence the risk for contaminating the groundwater. 1) Animal waste application method, rate, volume and frequency will have an effect on contamination. 2) Survival of the pathogens in the soil will e.g. depend on soil water content, temperature and pH. Salmonella species can survive up to 332 days and Cryptosporidium species can remain viable for several years in the soil environment. In the present study we compared the transport between the pathogenic bacteria S. senftenberg and the pathogenic protozoan C. parvum in intact clay columns. Furthermore, we compared the effect from surface and sub-surface manure application on the transport potential. 15 intact clay columns were placed in an outdoor multi-column lysimeter for 36 days. Manure inoculated with S. senftenberg, C. parvum and chloride was added to the soil surface or injected 8 cm into the columns. Drainage water was collected from the soil columns and DNA was extracted to quantify S. senftenberg and C. parvum by quantitative PCR. In addition S. senftenberg was enumerated by plate counting. Acid yellow was applied to selected columns to visualize the pathway down through the soil column. The highest concentration of S. senftenberg was in the first drainage sample ranging from 100-10000 CFU/ml. Breakthrough curves for chloride and S. senftenberg indicates the importance of preferential flow as well as a faster transport for the bacteria compared to chloride. C. parvum is retained to a higher degree in the soil but is still found

  17. [Development of an IMS-qPCR method for detection of Cryptosporidium parvum in water].

    PubMed

    Gao, Shan-Shan; Wu, Shao-Qiang; Luo, Jing; Wang, Cheng-Min; Zhang, Min; Zhao, Bao-Hua; He, Hong-Xuan

    2013-06-01

    To develop a detection method for Cryptosporidium parvum oocysts from water samples, which combined immunomagnetic separation (IMS) with Taqman real-time PCR (qPCR). Conditions of separation and enrichment of IMS method by using specific streptavidin magnetic beads coated with monoclonal antibodies Cp23 directed against C. parvum oocysts were optimized. Special primers of PCR and Taqman probes were designed referring to the 18S rDNA gene of C. parvum (GenBank Accession No. AB513881.1). The conserved genes were amplified from genomic DNA of C.parvum, and then cloned into Peasy-T1 vector. Tenfold dilutions of positive plasmids (10(4)-10(8) copy/microl) were used to construct a standard curves by Taqman qPCR. The specificity of the assay was determined using genomic DNA of C. baileyi, Toxoplasma gondii, C. canis and Escherichia coli. The sensitivity of this assay was evaluated by analyzing 10-fold serially dilutions of plasmids ranging from 10(0) to 10(8) copy/microl. Both IMS-qPCR assay and indirect immunofluorescent-antibody assay (IFA) were applied to detect 50 water samples collected from the dairy cattle farms in Hebei. The optimal incubation concentration and time of antibody Cp23 were 20 ng/ml and 30 min, respectively, and the catching time was 30 min, the recovery rate was more than 95%. PCR product was 272 bp, and identified by restriction enzyme digestion and nucleotide sequencing. There was a good linear relationship between the standard plasmids and Ct value (correlation r2 = 0.996 1) of the Taqman qPCR. No cross-reactivity was observed with C. baileyi, T. gondii, C. canis and E. coli. The sensitivity of C. parvum-specific assay was 10 copy/microl. Compared with IFA as golden standard method, the specificity and sensitivity of IMS-qPCR for 50 water samples was 100%(18/18) and 93.8% (30/32), respectively. The IMS-qPCR assay can be used to specifically detect C. parvum oocysts in water samples.

  18. Role of CpSUB1, a subtilisin-like protease, in Cryptosporidium parvum infection in vitro.

    PubMed

    Wanyiri, Jane W; Techasintana, Patsharaporn; O'Connor, Roberta M; Blackman, Michael J; Kim, Kami; Ward, Honorine D

    2009-04-01

    The apicomplexan parasite Cryptosporidium is a significant cause of diarrheal disease worldwide. Previously, we reported that a Cryptosporidium parvum subtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity in C. parvum lysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in the C. parvum genome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA from C. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of approximately 64 kDa and approximately 48 kDa, for C. parvum lysates and proteins "shed" during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 by C. parvum lysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity in C. parvum and for processing of gp40/15. Importantly, the recombinant prodomain inhibited C. parvum infection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.

  19. Viability and fate of Cryptosporidium parvum and Giardia lamblia in tubular anaerobic digesters.

    PubMed

    Kinyua, Maureen N; Trimmer, John; Izurieta, Ricardo; Cunningham, Jeffrey; Ergas, Sarina J

    2016-06-01

    In many developing countries where pathogenic diseases of animal waste origin, such as giardiasis and cryptosporidiosis, are often prevalent, facilities are limited to treat livestock waste. However, household-scale anaerobic digesters are currently being promoted for bioenergy production from livestock manure. Since the effluent is often used as a fertilizer for food crops, it is critical to understand the effect of environmental conditions within household-scale digesters on the viability of Cryptosporidium parvum oocysts and Giardia lamblia cysts. In this study, key environmental parameters affecting (oo)cyst inactivation were measured in four tubular anaerobic digesters, which are a type of household-scale digester promoted for treatment of swine waste in rural Costa Rica. Interviews and participant observations were used to understand digester operation and maintenance procedures. Ambient temperatures (21-24°C), near-neutral pH, total ammonia nitrogen (TAN) concentrations<250 mg/L and hydraulic retention times (HRTs) between 23 and 180 days were observed. Laboratory (oo)cysts inactivation studies were performed in bench-scale digesters, which were maintained under conditions similar to those observed in the field. Apparent first-order inactivation rate coefficients for Giardia lamblia and Cryptosporidium parvum were 0.155 ± 0.041 and 0.054 ± 0.006 day(-1), respectively. Temperature and volatile fatty acids were the main factors contributing to Cryptosporidium parvum and Giardia lamblia inactivation. A mathematical model was developed that predicts the concentration of (oo)cysts in the liquid effluent of tubular digesters like those observed in Costa Rica. A mathematical model was developed that predicts the concentration of (oo)cysts in the liquid effluent of tubular digesters like those observed in Costa Rica. Two dimensionless groups can be used to predict the performance of the digesters for inactivating pathogens; both dimensionless groups depend upon

  20. Simultaneous detection of Giardia lamblia and Cryptosporidium parvum (oo)cysts in soil using immunomagnetic separation and direct fluorescent antibody staining.

    PubMed

    Orlofsky, Ezra; Gillor, Osnat; Melli, Ann; Miller, Woutrina; Wuertz, Stefan; Bernstein, Nirit; Shapiro, Karen

    2013-09-01

    An improved approach for simultaneous detection of Cryptosporidium parvum and Giardia lamblia (oo)cysts in soil is described. Recoveries>70% were obtained for concentrations>55 and 21 (oo)cysts g(-1) for C. parvum and G. lamblia, respectively. The limits of detection were determined to be<5 (oo)cysts g(-1) soil.

  1. Risk of Handling as a Route of Exposure to Infectious Waterborne Cryptosporidium parvum Oocysts via Atlantic Blue Crabs (Callinectes sapidus)▿

    PubMed Central

    Graczyk, Thaddeus K.; McOliver, Cynthia; Silbergeld, Ellen K.; Tamang, Leena; Roberts, Jennifer D.

    2007-01-01

    Commercial Atlantic blue crabs (Callinectes sapidus) were exposed to 2.0 × 104 infectious waterborne oocysts of Cryptosporidium parvum. The study demonstrated that blue crabs can transfer C. parvum oocysts to persons involved in handling or preparing crabs and that they may contaminate other surfaces or products during storage. PMID:17449680

  2. A QSAR model of benzoxazole derivatives as potential inhibitors for inosine 5`-monophosphate dehydrogenase from Cryptosporidium parvum

    PubMed Central

    Teotia, Pratibha; Prakash Dwivedi, Surya; Dwivedi, Neeraja

    2016-01-01

    Cryptosporidium parvum is the common enteric protozoan pathogen causing cryptosporidiosis in human. Available drugs to treat cryptosporidiosis are ineffective and there is yet no vaccine against C. parvum. Therefore, it is of interest to design an improved yet effective drug against C. parvum. Here, we docked benzoxazole derivatives (collected from literature) with inosine 5`- monophosphate dehydrogenase (IMPDH) from Cryptosporidium parvum using the program AutoDock 4.2. The docked protein - inhibitor complex structure was optimized using molecular dynamics simulation for 5 ps with the CHARMM-22 force field using NAMD (NAnoscale Molecular Dynamics program) incorporated in visual molecular dynamics (VMD 1.9.2) and then evaluating the stability of complex structure by calculating RMSD values. NAMD is a parallel, object-oriented molecular dynamics code designed for high-performance simulation of large biomolecular systems. A quantitative structure activity relationship (QSAR) model was built using energy-based descriptors as independent variable and pIC50 value as dependent variable of fifteen known benzoxazole derivatives with C. parvum IMPDH protein, yielding correlation coefficient r2 of 0.7948. The predictive performance of QSAR model was assessed using different cross-validation procedures. Our results suggest that a ligand-receptor binding interaction for inosine 5`-monophosphate dehydrogenase using a QSAR model is promising approach to design more potent inosine 5`-monophosphate dehydrogenase inhibitors prior to their synthesis. PMID:28149045

  3. Parasites and malignancies, a review, with emphasis on digestive cancer induced by Cryptosporidium parvum (Alveolata: Apicomplexa)

    PubMed Central

    Benamrouz, S.; Conseil, V.; Creusy, C.; Calderon, E.; Dei-Cas, E.; Certad, G.

    2012-01-01

    The International Agency for Research on Cancer (IARC) identifies ten infectious agents (viruses, bacteria, parasites) able to induce cancer disease in humans. Among parasites, a carcinogenic role is currently recognized to the digenetic trematodes Schistosoma haematobium, leading to bladder cancer, and to Clonorchis sinensis or Opisthorchis viverrini, which cause cholangiocarcinoma. Furthermore, several reports suspected the potential association of other parasitic infections (due to Protozoan or Metazoan parasites) with the development of neoplastic changes in the host tissues. The present work shortly reviewed available data on the involvement of parasites in neoplastic processes in humans or animals, and especially focused on the carcinogenic power of Cryptosporidium parvum infection. On the whole, infection seems to play a crucial role in the etiology of cancer. PMID:22348213

  4. Parasites and malignancies, a review, with emphasis on digestive cancer induced by Cryptosporidium parvum (Alveolata: Apicomplexa).

    PubMed

    Benamrouz, S; Conseil, V; Creusy, C; Calderon, E; Dei-Cas, E; Certad, G

    2012-05-01

    The International Agency for Research on Cancer (IARC) identifies ten infectious agents (viruses, bacteria, parasites) able to induce cancer disease in humans. Among parasites, a carcinogenic role is currently recognized to the digenetic trematodes Schistosoma haematobium, leading to bladder cancer, and to Clonorchis sinensis or Opisthorchis viverrini, which cause cholangiocarcinoma. Furthermore, several reports suspected the potential association of other parasitic infections (due to Protozoan or Metazoan parasites) with the development of neoplastic changes in the host tissues. The present work shortly reviewed available data on the involvement of parasites in neoplastic processes in humans or animals, and especially focused on the carcinogenic power of Cryptosporidium parvum infection. On the whole, infection seems to play a crucial role in the etiology of cancer.

  5. Evaluation of Immunomagnetic Separation for Recovery of Cryptosporidium parvum and Giardia duodenalis from High-Iron Matrices

    PubMed Central

    Yakub, Gary P.; Stadterman-Knauer, Kathleen L.

    2000-01-01

    In this study we examined the recovery of Cryptosporidium parvum and Giardia duodenalis from matrices containing various concentrations of dissolved iron. The organisms were recovered by using the immunomagnetic separation-immunofluorescent assay method, and the levels of recovery were compared to the dissolved iron concentrations. The levels of recovery of C. parvum decreased sharply at dissolved iron concentrations greater than 4 mg/liter, while the levels of recovery of G. duodenalis decreased sharply at concentrations greater than 40 mg/liter. PMID:10919831

  6. Detection of Cryptosporidium parvum and Cyclospora cayetanensis infections among people living in a slum area in Kathmandu valley, Nepal.

    PubMed

    Bhattachan, Balkrishna; Sherchand, Jeevan Bahadhur; Tandukar, Sarmila; Dhoubhadel, Bhim Gopal; Gauchan, Leesa; Rai, Ganesh

    2017-09-07

    The aim of this study is to determine the prevalence of Cyclospora cayetanensis and Cryptosporidium parvum infections among people living a slum in Kathmandu valley, Nepal. Ten different parasites were detected in the stool samples; the prevalence of any parasite was in 27.1% (71/262). The prevalence of C. cayetanensis and C. parvum were 14.1% (10/71) and 5.6% (4/71), respectively. This study showed high prevalence of intestinal parasitic infections along with the coccidian parasites in the slum area of Kathmandu Valley.

  7. Transport of Cryptosporidium parvum oocysts in soil columns following applications of raw and separated liquid slurries.

    PubMed

    Petersen, Heidi H; Enemark, Heidi L; Olsen, Annette; Amin, M G Mostofa; Dalsgaard, Anders

    2012-09-01

    The potential for the transport of viable Cryptosporidium parvum oocysts through soil to land drains and groundwater was studied using simulated rainfall and intact soil columns which were applied raw slurry or separated liquid slurry. Following irrigation and weekly samplings over a 4-week period, C. parvum oocysts were detected from all soil columns regardless of slurry type and application method, although recovery rates were low (<1%). Soil columns with injected liquid slurry leached 73 and 90% more oocysts compared to columns with injected and surface-applied raw slurries, respectively. Among leachate samples containing oocysts, 44/72 samples yielded viable oocysts as determined by a dye permeability assay (DAPI [4',6'-diamidino-2-phenylindole]/propidium iodide) with the majority (41%) of viable oocysts found in leachate from soil columns with added liquid slurry. The number of viable oocysts was positively correlated (r = 0.63) with the total number of oocysts found. Destructively sampling of the soil columns showed that type of slurry and irrigation played a role in the vertical distribution of oocysts, with more oocysts recovered from soil columns added liquid slurry irrespective of the irrigation status. Further studies are needed to determine the effectiveness of different slurry separation technologies to remove oocysts and other pathogens, as well as whether the application of separated liquid slurry to agricultural land may represent higher risks for groundwater contamination compared to application of raw slurry.

  8. Zoonotic linkage and variation in Cryptosporidium parvum from patients in the United Kingdom.

    PubMed

    Chalmers, Rachel M; Smith, Richard P; Hadfield, Stephen J; Elwin, Kristin; Giles, Michaela

    2011-05-01

    Relationships between patient exposure risks and variation within the Cryptosporidium parvum 60 kDa glycoprotein (GP60) gene were explored in samples isolated from human cases of cryptosporidiosis (n=69) in England and Wales. GP60 family IIa predominated (n=56), followed by IId (n=9). One case was IIc, a newly named genotype IIcA5G3j, and isolates from three cases did not amplify with the GP60 primers. Cases with GP60 family IIa were more likely than IId to have visited a farm, or had contact with farm animals or with their faeces in the 2 weeks prior to illness. Within GP60 family IIa, genotypes IIaA15G2R1 and IIaA17G1R1 predominated (22 cases each); nine other IIa genotypes accounted for 12 cases. The IId genotypes were mainly IIdA17G1 and IIdA18G1 (3 each). Cases with IIaA17G1R1 were particularly linked to zoonotic exposures: visiting a farm or having farm animal contact in the 2 weeks prior to illness. These findings provide further evidence of zoonotic pathways for the transmission of C. parvum isolates.

  9. Transport of Cryptosporidium parvum Oocysts in Soil Columns following Applications of Raw and Separated Liquid Slurries

    PubMed Central

    Petersen, Heidi H.; Enemark, Heidi L.; Olsen, Annette; Amin, M. G. Mostofa

    2012-01-01

    The potential for the transport of viable Cryptosporidium parvum oocysts through soil to land drains and groundwater was studied using simulated rainfall and intact soil columns which were applied raw slurry or separated liquid slurry. Following irrigation and weekly samplings over a 4-week period, C. parvum oocysts were detected from all soil columns regardless of slurry type and application method, although recovery rates were low (<1%). Soil columns with injected liquid slurry leached 73 and 90% more oocysts compared to columns with injected and surface-applied raw slurries, respectively. Among leachate samples containing oocysts, 44/72 samples yielded viable oocysts as determined by a dye permeability assay (DAPI [4′,6′-diamidino-2-phenylindole]/propidium iodide) with the majority (41%) of viable oocysts found in leachate from soil columns with added liquid slurry. The number of viable oocysts was positively correlated (r = 0.63) with the total number of oocysts found. Destructively sampling of the soil columns showed that type of slurry and irrigation played a role in the vertical distribution of oocysts, with more oocysts recovered from soil columns added liquid slurry irrespective of the irrigation status. Further studies are needed to determine the effectiveness of different slurry separation technologies to remove oocysts and other pathogens, as well as whether the application of separated liquid slurry to agricultural land may represent higher risks for groundwater contamination compared to application of raw slurry. PMID:22706058

  10. Optimization of benzoxazole-based inhibitors of Cryptosporidium parvum inosine 5′-monophosphate dehydrogenase

    PubMed Central

    Gorla, Suresh Kumar; Kavitha, Mandapati; Zhang, Minjia; Chin, James En Wai; Liu, Xiaoping; Striepen, Boris; Makowska-Grzyska, Magdalena; Kim, Youngchang; Joachimiak, Andrzej; Hedstrom, Lizbeth; Cuny, Gregory D.

    2013-01-01

    Cryptosporidium parvum is an enteric protozoan parasite that has emerged as a major cause of diarrhea, malnutrition and gastroenteritis as well as posing a potential bioterrorism threat. C. parvum synthesizes guanine nucleotides from host adenosine in a streamlined pathway that relies on inosine 5′-monophosphate dehydrogenase (IMPDH). We have previously identified several parasite-selective C. parvum IMPDH (CpIMPDH) inhibitors by high-throughput screening. In this paper, we report the structure-activity relationship (SAR) for a series of benzoxazole derivatives with many compounds demonstrating CpIMPDH IC50 values in the nanomolar range and > 500-fold selectivity over human IMPDH (hIMPDH). Unlike previously reported CpIMPDH inhibitors, these compounds are competitive inhibitors versus NAD+. The SAR study reveals that pyridine and other small heteroaromatic substituents are required at the 2-position of the benzoxazole for potent inhibitory activity. In addition, several other SAR conclusions are highlighted with regard to the benzoxazole and the amide portion of the inhibitor, including preferred stereochemistry. An x-ray crystal structure of a representative E•IMP•inhibitor complex is also presented. Overall, the secondary amine derivative 15a (Q67) demonstrated excellent CpIMPDH inhibitory activity (IC50 = 0.5 ± 0.1 nM) and moderate stability (t1/2 = 44 min) in mouse liver microsomes. Compound 73, the racemic version of 15a, also displayed superb antiparasitic activity in a Toxoplasma gondii strain that relies on CpIMPDH (EC50 = 20 ± 20 nM), and selectivity versus a wild-type T. gondii strain (200-fold). No toxicity was observed (LD50 > 50 μM) against a panel of four mammalian cells lines. PMID:23668331

  11. Inactivation of Cryptosporidium parvum oocysts using medium- and low-pressure ultraviolet radiation.

    PubMed

    Craik, S A; Weldon, D; Finch, G R; Bolton, J R; Belosevic, M

    2001-04-01

    The effect of ultraviolet radiation from low- and medium-pressure mercury arc lamps on Cryptosporidium parvum oocysts was studied using a collimated beam apparatus. Experiments were conducted using parasites suspended in both filtered surface water and phosphate buffered laboratory water. Inactivation of oocysts was measured as reduction in infectivity using a CD-1 neonatal mouse model and was found to be a non-linear function of UV dose over the range of germicidal doses tested (0.8-119 mJ/cm2). Oocyst inactivation increased rapidly with UV dose at doses less than 25 mJ/cm2 with two and three log-units inactivation at approximately 10 and 25 mJ/cm2, respectively. The cause of significant leveling-off and tailing in the UV inactivation curve at higher doses was not determined. Maximum measured oocyst inactivation ranged from 3.4 to greater than 4.9 log-units and was dependent on different batches of parasites. Water type and temperature, the concentration of oocysts in the suspension, and the UV irradiance did not have significant impacts on oocyst inactivation. When compared on the basis of germicidal UV dose, the oocysts were equally sensitive to low- and medium-pressure UV radiation. With respect to Cryptosporidium, both low- and medium-pressure ultraviolet radiation are attractive alternatives to conventional chemical disinfection methods in drinking water treatment.

  12. Infectivity of Cryptosporidium parvum oocysts after storage of experimentally contaminated apples.

    PubMed

    Macarisin, Dumitru; Santín, Mónica; Bauchan, Gary; Fayer, Ronald

    2010-10-01

    Irrigation water and washing water have been inferred to be associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to determine whether apples experimentally contaminated with Cryptosporidium oocysts represent a food safety concern. Laser scanning confocal microscopy revealed no morphological changes in Cryptosporidium parvum oocysts attached to apples after 6 weeks of cold storage, suggesting that oocysts might remain viable and possibly infectious during prolonged storage. Mice were fed apple peels from experimentally contaminated apples to determine whether oocysts had remained infectious on apples stored for 4 weeks. All mice developed cryptosporidiosis. To evaluate the strength of oocyst attachment to apples, washing methods that have been reported to be helpful for recovery of oocysts from various foodstuffs were evaluated, except that the intensity of washing was increased in the present study. None of the tested washing methods succeeded in completely removing oocysts from the apple peel. The most efficient removal (37.5%) was achieved by rigorous manual washing in water with a detergent and by agitation in an orbital shaker with Tris-sodium dodecyl sulfate buffer. Glycine and phosphate-buffered saline buffers had no effect on oocyst removal. Scanning electron microscopy revealed that some oocysts were attached in deep natural crevices in the apple exocarp and others were attached to the smooth surface of the peel. Some oocysts were closely associated with what appeared to be an amorphous substance with which they might have been attached to the apple surface.

  13. Evaluation of DNA encoding acidic ribosomal protein P2 of Cryptosporidium parvum as a potential vaccine candidate for cryptosporidiosis.

    PubMed

    Benitez, Alvaro; Priest, Jeffrey W; Ehigiator, Humphrey N; McNair, Nina; Mead, Jan R

    2011-11-15

    The Cryptosporidium parvum acidic ribosomal protein P2 (CpP2) is an important immunodominant marker in C. parvum infection. In this study, the CpP2 antigen was evaluated as a vaccine candidate using a DNA vaccine model in adult C57BL/6 IL-12 knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding CpP2 (CpP2-DNA) cloned into the pUMVC4b vector induced a significant anti-CpP2 IgG antibody response that was predominantly of the IgG1 isotype. Compared to control KO mice immunized with plasmid alone, CpP2-immunized mice demonstrated specific in vitro spleen cell proliferation as well as enhanced IFN-γ production to recombinant CpP2. Further, parasite loads in CpP2 DNA-immunized mice were compared to control mice challenged with C. parvum oocysts. Although a trend in reduction of infection was observed in the CpP2 DNA-immunized mice, differences between groups were not statistically significant. These results suggest that a DNA vaccine encoding the C. parvum P2 antigen is able to provide an effective means of eliciting humoral and cellular responses and has the potential to generate protective immunity against C. parvum infection but may require using alternative vectors or adjuvant to generate a more potent and balanced response.

  14. Environmental load of Cryptosporidium parvum oocysts from cattle manure in feedlots from the central and western United States.

    PubMed

    Atwill, Edward R; Pereira, Maria Das Gracas C; Alonso, L Herrera; Elmi, Cyrus; Epperson, William B; Smith, Robert; Riggs, Walter; Carpenter, Linda V; Dargatz, David A; Hoar, Bruce

    2006-01-01

    The first step in assessing the risk of water contamination by Cryptosporidium parvum oocysts from feedlot cattle (Bos taurus) production systems is to quantify the number of C. parvum oocysts present in the fecal material deposited by feedlot cattle. Our primary objective for this project was to estimate the daily environmental load of C. parvum oocysts in fecal material deposited by feedlot cattle from across the central and western USA. Our secondary goal was to genotype isolates of C. parvum from feedlot cattle to help facilitate proper identification of mammalian sources of waterborne C. parvum. Based on 5274 fecal samples from 22 feedlots in seven states (California, Washington, Colorado, Oklahoma, Texas, Nebraska, and South Dakota), we estimated a point prevalence of C. parvum of 0.99 to 1.08% in fecal material from feedlot pens from a wide range of climates and a diverse range of feedlot management systems. On average, fresh fecal material from throughout feedlot systems (recent arrivals to nearing slaughter) contained about 1.3 to 3.6 oocysts/g feces, which roughly translates to about 2.8 x 10(4) to 1.4 x 10(5) oocysts/animal per day.

  15. Real-time, sensitive electrical detection of Cryptosporidium parvum oocysts based on chemical vapor deposition-grown graphene

    NASA Astrophysics Data System (ADS)

    It Wong, Jen; Wang, Lu; Shi, Yumeng; Palacios, Tomás; Kong, Jing; Dong, Xiaochen; Ying Yang, Hui

    2014-02-01

    Cryptosporidium parvum is a common intestinal parasitic protozoan that causes gastroenteritis in man and animals. It poses high risks to drinking water supply because of its ubiquitous distribution in water and their oocysts are resistant to harsh environment conditions. In this work, we demonstrated the use of large-size chemical vapor deposition (CVD) grown graphene films configured as field-effect device for rapid electrical detection of Cryptosporidium parvum oocysts (Cp. oocysts). The presence of Cp. oocysts causes the change in the transport characteristics of the antibody-functionalized graphene device, which can be measured in terms of the dependence of the drain current on the sweep of the gate voltage or the real-time drain current data under a constant gate voltage. The high sensor sensitivity of 25 oocysts per milliliter solution and good specificity were evaluated, indicating it a promising candidate for detecting waterborne pathogens in water quality control.

  16. CpABC, a Cryptosporidium parvum ATP-binding cassette protein at the host–parasite boundary in intracellular stages

    PubMed Central

    Perkins, Margaret E.; Riojas, Ynolde A.; Wu, Teresa W.; Le Blancq, Sylvie M.

    1999-01-01

    The intracellular parasite Cryptosporidium parvum develops inside a vacuole at the apex of its epithelial host cell. The developing parasite is separated from the host cell cytoplasm by a zone of attachment that consists of an extensively folded membranous structure known as the feeder organelle. It has been proposed that the feeder organelle is the site of regulation of transport of nutrients and drugs into the parasite. In this report, we localize an ≈200-kDa integral membrane protein, CpABC, from Cryptosporidium parvum to the host–parasite boundary, possibly the feeder organelle. The predicted amino acid sequence of CpABC has significant structural similarity with the cystic fibrosis conductance regulator and the multidrug resistance protein subfamily of ATP-binding cassette proteins. This is an example of a parasite-encoded transport protein localized at the parasite–host interface of an intracellular protozoan. PMID:10318953

  17. Effect of cyanuric acid on the inactivation of Cryptosporidium parvum under hyperchlorination conditions.

    PubMed

    Murphy, Jennifer L; Arrowood, Michael J; Lu, Xin; Hlavsa, Michele C; Beach, Michael J; Hill, Vincent R

    2015-06-16

    Cyanuric acid (CYA) is a chlorine stabilizer used in swimming pools to limit UV degradation of chlorine, thus reducing chlorine use and cost. However, CYA has been shown to decrease the efficacy of chlorine disinfection. In the event of a diarrheal incident, CDC recommends implementing 3-log10 inactivation conditions for Cryptosporidium (CT value = 15 300 mg·min/L) to remediate pools. Currently, CYA's impact on Cryptosporidium inactivation is not fully determined. We investigated the impact of multiple concentrations of CYA on C. parvum inactivation (at 20 and 40 mg/L free chlorine; average pH 7.6; 25 °C). At 20 mg/L free chlorine, average estimated 3-log10 CT values were 17 800 and 31 500 mg·min/L with 8 and 16 mg/L CYA, respectively, and the average estimated 1-log10 CT value was 76 500 mg·min/L with 48 mg/L CYA. At 40 mg/L free chlorine, 3-log10 CT values were lower than those at 20 mg/L, but still higher than those of free chlorine-only controls. In the presence of ∼100 mg/L CYA, average 0.8- and 1.4-log10 reductions were achieved by 72 h at 20 and 40 mg/L free chlorine, respectively. This study demonstrates CYA significantly delays chlorine inactivation of Cryptosporidium oocysts, emphasizing the need for additional pool remediation options following fecal incidents.

  18. Pomegranate (Punica granatum) peel is effective in a murine model of experimental Cryptosporidium parvum.

    PubMed

    Al-Mathal, Ebtisam M; Alsalem, Afaf M

    2012-07-01

    Cryptosporidiosis, a major health issue for neonatal calves, is caused by the parasite Cryptosporidium parvum, which is highly resistant to drug treatments. To date, many anti-parasitic drugs have been tested, but only a few have been shown to be partially effective in treating cryptosporidiosis. Previous studies have indicated that pomegranate (Punica granatum) possesses anti-plasmodium, anti-cestode, and anti-nematode activities. Therefore, the aim of this study was to evaluate the effect of P. granatum peel on suckling mice infected with experimental C. parvum. At 4days of age, 72 neonatal albino mice were randomly divided into five groups: G1: healthy controls, G2: infected/untreated controls, G3: uninfected/distilled water-treated, G4: uninfected/P. granatum peel-treated, and G5: infected/P. granatum peel-treated. Mice were experimentally-infected by oral administration of 1×10(3)C. parvum oocysts per animal. On day 7 post-inoculation (pi), treated mice received an aqueous suspension of P. granatum peel orally (3g/kg body weight). The presence of diarrhea, oocyst shedding, and weight gain/loss, and the histopathology of ileal sections were examined. Infected mice treated with the P. granatum peel suspension showed improvement in all parameters examined. Additionally, these mice did not exhibit any clinical symptoms and no deaths occurred. Oocyst shedding was very significantly reduced in the P. granatum-treated mice by day 14 pi (P<.05), and was completely eliminated by day 28 pi. The mean weight gain of the P. granatum-treated mice was significantly higher than that of the infected/untreated controls throughout the study (P<.01). Histopathological analysis of ileal sections further supported the clinical and parasitological findings. The histological architecture of villi from the P. granatum-treated mice on day 14 pi showed visible improvement in comparison with the infected/untreated controls, including renewed brush borders, reduced numbers of C. parvum

  19. Cryptosporidium parvum genotype IIa and Giardia duodenalis assemblage A in Mytilus galloprovincialis on sale at local food markets.

    PubMed

    Giangaspero, Annunziata; Papini, Roberto; Marangi, Marianna; Koehler, Anson V; Gasser, Robin B

    2014-02-03

    To date, there has been no study to establish the genotypic or subgenotypic identities of Cryptosporidium and Giardia in edible shellfish. Here, we explored the genetic composition of these protists in Mytilus galloprovincialis (Mediterranean mussel) purchased from three markets in the city of Foggia, Italy, from May to December 2012. Samples from the digestive glands, gills and haemolymph were tested by nested PCR, targeting DNA regions within the 60 kDa glycoprotein (gp60) gene of Cryptosporidium, and the triose-phosphate isomerase (tpi) and β-giardin genes of Giardia. In total, Cryptosporidium and Giardia were detected in 66.7% of mussels (M. galloprovincialis) tested. Cryptosporidium was detected mostly between May and September 2012. Sequencing of amplicons showed that 60% of mussels contained Cryptosporidium parvum genotype IIa (including subgenotypes A15G2R1, IIaA15G2 and IIaA14G3R1), 23.3% Giardia duodenalis assemblage A, and 6.6% had both genetic types. This is the first report of these types in fresh, edible shellfish, particularly the very commonly consumed M. galloprovincialis from highly frequented fish markets. These genetic types of Cryptosporidium and Giardia are known to infect humans and thus likely to represent a significant public health risk. The poor observance of hygiene rules by vendors, coupled to the large numbers of M. galloprovincialis sold and the eating habits of consumers in Italy, call for more effective sanitary measures pertaining to the selling of fresh shellfish in street markets.

  20. Cloning and Characterization of the Acidic Ribosomal Protein P2 of Cryptosporidium parvum, a New 17-Kilodalton Antigen▿

    PubMed Central

    Priest, Jeffrey W.; Kwon, James P.; Montgomery, Joel M.; Bern, Caryn; Moss, Delynn M.; Freeman, Amanda R.; Jones, Cara C.; Arrowood, Michael J.; Won, Kimberly Y.; Lammie, Patrick J.; Gilman, Robert H.; Mead, Jan R.

    2010-01-01

    Cryptosporidium infection is commonly observed among children and immunocompromised individuals in developing countries, but large-scale outbreaks of disease among adults have not been reported. In contrast, outbreaks of cryptosporidiosis in the United States and Canada are increasingly common among patients of all ages. Thus, it seems likely that residents of regions where Cryptosporidium is highly endemic acquire some level of immunity, while residents of the developed world do not. A new immunodominant Cryptosporidium parvum antigen in the 15- to 17-kDa size range was identified as the Cryptosporidium parvum 60S acidic ribosomal protein P2 (CpP2). We developed a recombinant protein-based enzyme-linked immunosorbent assay for serologic population surveillance for antibodies that was 89% sensitive and 92% specific relative to the results of the large-format Western blot assay. The human IgG response is directed almost exclusively toward the highly conserved, carboxy-terminal 15 amino acids of the protein. Although IgG antibody cross-reactivity was documented with sera from patients with acute babesiosis, the development of an anti-CpP2 antibody response in our Peru study population correlated better with Cryptosporidium infection than with infection by any other parasitic protozoan. In Haiti, the prevalence of antibodies to CpP2 plateaus at 11 to 20 years of age. Because anti-CpP2 IgG antibodies were found only among residents of countries in the developing world where Cryptosporidium infection occurs early and often, we propose that this response may be a proxy for the intensity of infection and for acquired immunity. PMID:20410328

  1. Cryptosporidium parvum Among Coprolites from La Cueva de los Muertos Chiquitos (600-800 CE), Rio Zape Valley, Durango, Mexico.

    PubMed

    Morrow, Johnica J; Reinhard, Karl J

    2016-08-01

    :  In the present study, 90 coprolites from La Cueva de los Muertos Chiquitos (CMC) were subjected to enzyme-linked immunosorbent assay (ELISA) tests for 3 diarrhea-inducing protozoan parasites, Entamoeba histolytica , Giardia duodenalis , and Cryptosporidium parvum , to determine whether these parasites were present among the people who utilized this cave 1,200-1,400 yr ago. These people, the Loma San Gabriel, developed as a culture out of the Archaic Los Caracoles population and lived throughout much of present-day Durango and Zacatecas in Mexico. The Loma San Gabriel persisted through a mixed subsistence strategy of hunting-gathering and agricultural production. The results of ELISA testing were negative for both E. histolytica and G. duodenalis across all coprolites. A total of 66/90 (∼73% prevalence) coprolites tested positive or likely positive for C. parvum . The high prevalence of C. parvum among CMC coprolites contributes to our growing knowledge of the pathoecology among the Loma San Gabriel who utilized CMC. Herein, we report the successful recovery of C. parvum coproantigens from prehistoric coprolites. The recovery of these coproantigens demonstrates the existence of C. parvum in Mesoamerica before European contact in the 1400s.

  2. Induction of murine immune responses by DNA encoding a 23-kDa antigen of Cryptosporidium parvum.

    PubMed

    Ehigiator, Humphrey N; Romagnoli, Pablo; Priest, Jeffrey W; Secor, W Evan; Mead, Jan R

    2007-09-01

    Cp23 has been identified as one of the immunodominant antigens involved in the immune response to Cryptosporidium parvum infection. Thus, in this study, Cp23 antigen was investigated as a vaccine candidate using the DNA vaccine model in adult interleukin-12 (IL-12) knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding Cp23 (Cp23-DNA) cloned into the pUMVCb4 vector induced a significant anti-Cp23 immunoglobulin G1 (IgG1) and IgG2a antibody response and specific in vitro spleen cell proliferation to recombinant Cp23 as compared to control mice. Long-term memory responses were also detected after administration of the Cp23-DNA vaccine. Furthermore, Cp23-DNA vaccination induced a 50-60% reduction in oocysts shedding, indicating a partial protection against C. parvum infection in IL-12 KO mice. However, it is possible that this protective response was nonspecific because mice immunized with vector only also exhibited lower oocyst shedding than the naive controls. These results suggest that DNA encoding for immunodominant C. parvum antigens may provide an effective means of eliciting humoral and cellular responses and possibly in generating protective immunity against C. parvum infections in mammals.

  3. Prevalence of Cryptosporidium parvum infection in children along the Texas-Mexico border and associated risk factors.

    PubMed

    Leach, C T; Koo, F C; Kuhls, T L; Hilsenbeck, S G; Jenson, H B

    2000-05-01

    We examined the epidemiology of Cryptosporidium parvum in children aged 6 months to 13 years living in 1) colonias along the border (n = 105), 2) a clinic in an urban border community (n = 65), and 3) clinics in a large urban nonborder area (n = 109). Serum IgG and IgA anticryptosporidial antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Overall, 70.2% (196/279) of subjects had detectable C. parvum antibodies. Prevalence rates were higher (93/105 [89%]) in the colonias and urban border community (53/65 [82%]) compared to the urban nonborder community (50/109 [46%]). Within colonias, independent risk factors for C. parvum infection included consumption of municipal water instead of bottled water, older age, and lower household income. Children living along the Texas-Mexico border have a higher rate of infection with C. parvum compared to children living in a large nonborder urban area. Within colonias, C. parvum infection was associated with source of water supply, age, and socioeconomic status.

  4. Obtaining hyperimmune anti-Cryptosporidium parvum ovine colostrum. A study of the humoral immune response in immunized sheep.

    PubMed

    Martín-Gómez, S; Alvarez-Sánchez, M A; Rojo-Vázquez, F A

    2006-01-01

    Three ewes were immunized five times over a 2-month period prior to giving birth by intramuscular injection, oral administration and intramammary infusion of antigen and viable or freeze-dried Cryptosporidium parvum oocyst solution emulsified with Freund's complete and incomplete adjuvant. Two animals served as controls and another two as adjuvant controls. Serum was collected at first immunization and thereafter every 2 to 4 weeks. Colostrum and milk were collected as well. All samples were assayed for C. parvum-specific antibodies using an enzyme-linked immunosorbent assay methodology, and Western blotting was used to recognize the C. parvum antigens. Hyperimmunization resulted in a progressive and significant increase in specific anti-C. parvum serum IgG, IgA and IgM titres, with the highest values noted at the point of lambing. Titres decreased slightly in milk, although they were in all cases higher than those in the control animals. Moreover, some 30 bands of C. parvum were recognized.

  5. Transport, fate, and infectivity of Cryptosporidium parvum oocysts released from manure and leached through macroporous soil

    NASA Astrophysics Data System (ADS)

    Boyer, Douglas G.; Kuczynska, Ewa; Fayer, Ron

    2009-09-01

    A major mode of transmission of Cryptosporidium parvum, a widespread waterborne pathogen, is via contaminated drinking and recreational waters. Oocyst transport to surface water can occur by deposition of manure directly in the water or by wash off in surface runoff. Oocyst transport to groundwater is less straightforward and requires that the oocysts move through soil and bedrock to reach the water table. The purpose of this study was to determine the relative concentration and infectivity of C. parvum oocysts released from manure and leached through columns of undisturbed, macroporous karst soil. Modeling the fate of oocysts in this system over time can provide baseline data for evaluating real world events. Substantially more oocysts leached from undisturbed soil columns than disturbed soil columns. Oocyst survival studies using BALB/c neonatal suckling mice showed that about 85% of oocysts were infective at the beginning of leaching experiments. The oocyst infectivity decreased to about 20% after 12 weeks of leaching from soil columns maintained at 10°C. Cool (10°C) temperatures appear to increase survivability and maintain infectivity of many oocysts for 3 months or longer. Cool temperatures also appear to increase rates of release of oocysts from manure and leaching through soil. This study demonstrated that leaching is an important mechanism of oocyst transport in karst soils where infiltration capacities are high and long, continuous macropores exist. Karst groundwater systems might be especially vulnerable to contamination by leached oocysts, because of the prevalence of shallow soils and rapid groundwater movement. Oocysts leaching from soils into the epikarst could accumulate and remain viable for months until hydrological conditions are right for flushing the oocysts into the conduit flow system.

  6. Composition and conformation of Cryptosporidium parvum oocyst wall surface macromolecules and their effect on adhesion kinetics of oocysts on quartz surface.

    PubMed

    Liu, Yuanyuan; Kuhlenschmidt, Mark S; Kuhlenschmidt, Theresa B; Nguyen, Thanh H

    2010-08-09

    We characterized the composition and conformation of Cryptosporidium parvum ( C. parvum ) oocyst wall surface macromolecules and studied their effect on interactions between C. parvum oocyst and quartz surface. Proteinase K and mixed glycosidases were used to modify C. parvum oocyst surface macromolecules. The peptides released by proteinase K and carbohydrates hydrolyzed by mixed glycosidases were respectively analyzed with liquid chromatography/nanoelectrospray ionization tandem mass spectrometry (LC-MS/MS) and phenol-sulfuric acid assay to determine the composition of C. parvum oocyst wall surface macromolecules. Surface potential and polarity of the untreated and proteinase treated C. parvum oocysts revealed information about the conformation of oocyst wall surface macromolecules. The results illustrated that C. parvum oocyst wall is covered by a fluffy layer of glycoproteins. Adhesion kinetics of untreated and proteinase K treated C. parvum oocysts on quartz surface were studied in a radial stagnation point flow cell over a wide range of ionic strength to investigate the effect of C. parvum oocyst wall surface macromolecules on oocysts-quartz interactions. The adhesion rate coefficient of proteinase K treated C. parvum oocysts significantly decreased compared to that of untreated oocysts. This observation indicated that the fluffy layer on C. parvum oocysts wall leads to weaker van der Waals interaction and stronger steric repulsion.

  7. Expression of Cryptosporidium parvum thioredoxin peroxidase in COS-7 cells confers radioprotection.

    PubMed

    Hong, Semie; Kim, Jae-Hwan; Yoon, Sejoung; Kim, Kyoungjin; Sim, Seobo; Park, Woo-Yoon; Yu, Jae-Ran

    2016-04-01

    Cryptosporidium parvum is one of the most radioresistant organisms identified to date. In a previous study, we found that thioredoxin peroxidase (CpTPx) was significantly upregulated in this species following exposure to high dose (10 kGy) of γ-irradiation. To assess the potential of CpTPx to confer radioprotection in mammalian cells, it was expressed in COS-7 African green monkey kidney cells (CpTPx-COS7). For comparison, the thioredoxin peroxidase of Cryptosporidium muris (CmTPx) was also expressed in these cells (CmTPx-COS7 cells), which has been confirmed to have lesser antioxidant activity than CpTPx in the previous study. Notably, the survival rates of CpTPx-COS7 cells were significantly higher (12-22%) at 72 h after 8 Gy irradiation than CmTPx-COS7 or non-transfected COS-7 (ntCOS-7) counterparts. In addition, CpTPx revealed a 50% of ROS reduction in irradiated CpTPx-COS7 cells, while γ-H2AX DNA damage marker expression was not significantly changed. Furthermore, the amount of apoptosis only increased to about 120% after 2-8 Gy irradiation compared to 200-300% increase observed in ntCOS-7 cells. CmTPx was shown to have antioxidant and DNA damage protection activities; however, these activities were always lower than those of CpTPx. These results suggest that the potent antioxidant and protective activities of CpTPx are well conserved in this cell-based system and that CpTPx contributed to the radioprotection of mammalian cells through its exceptional antioxidant activity.

  8. Surface complexation of carboxylate adheres Cryptosporidium parvum oocysts to the hematite-water interface

    USGS Publications Warehouse

    Gao, X.; Metge, D.W.; Ray, C.; Harvey, R.W.; Chorover, J.

    2009-01-01

    The interaction of viable Cryptosporidium parvum ??ocysts at the hematite (??-Fe2O3)-water interface was examined over a wide range in solution chemistry using in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Spectra for hematite-sorbed ??ocysts showed distinctchangesin carboxylate group vibrations relative to spectra obtained in the absence of hematite, indicative of direct chemical bonding between carboxylate groups and Fe metal centers of the hematite surface. The data also indicate that complexation modes vary with solution chemistry. In NaCl solution, ??ocysts are bound to hematite via monodentate and binuclear bidentate complexes. The former predominates at low pH, whereas the latter becomes increasingly prevalent with increasing pH. In a CaCl2 solution, only binuclear bidentate complexes are observed. When solution pH is above the point of zero net proton charge (PZNPC) of hematite, ??ocyst surface carboxylate groups are bound to the mineral surface via outer-sphere complexes in both electrolyte solutions. ?? 2009 American Chemical Society.

  9. Confirmed detection of Cyclospora cayetanesis, Encephalitozoon intestinalis and Cryptosporidium parvum in water used for drinking.

    PubMed

    Dowd, Scot E; John, David; Eliopolus, James; Gerba, Charles P; Naranjo, Jaime; Klein, Robert; López, Beatriz; de Mejía, Maricruz; Mendoza, Carlos E; Pepper, Ian L

    2003-09-01

    Human enteropathogenic microsporidia (HEM), Cryptosporidium parvum, Cyclospora cayetanesis, and Giardia lamblia are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of HEM (Encephalitozoon intestinalis and Enterocytozoon bieneusi) and Cyclospora cayetanesis have not been fully elucidated due to lack of sensitive and specific environmental screening methods. The present study was undertaken with recently developed methods, to screen various water sources used for public consumption in rural areas around the city of Guatemala. Water concentrates collected in these areas were subjected to community DNA extraction followed by PCR amplification, PCR sequencing and computer database homology comparison (CDHC). All water samples screened in this study had been previously confirmed positive for Giardia spp. by immunofluorescent assay (IFA). Of the 12 water concentrates screened, 6 showed amplification of microsporidial SSU-rDNA and were subsequently confirmed to be Encephalitozoon intestinalis. Five of the samples allowed for amplification of Cyclospora 18S-rDNA; three of these were confirmed to be Cyclospora cayetanesis while two could not be identified because of inadequate sequence information. Thus, this study represents the first confirmed identification of Cyclospora cayetanesis and Encephalitozoon intestinalis in source water used for consumption. The fact that the waters tested may be used for human consumption indicates that these emerging protozoa may be transmitted by ingestion of contaminated water.

  10. Surface complexation of carboxylate adheres Cryptosporidium parvum oocysts to the hematite-water interface.

    PubMed

    Gao, Xiaodong; Metge, David W; Ray, Chittaranian; Harvey, Ronald W; Chorover, Jon

    2009-10-01

    The interaction of viable Cryptosporidium parvum oocysts at the hematite (alpha-Fe2O3)-water interface was examined over a wide range in solution chemistry using in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Spectra for hematite-sorbed oocysts showed distinct changes in carboxylate group vibrations relative to spectra obtained in the absence of hematite, indicative of direct chemical bonding between carboxylate groups and Fe metal centers of the hematite surface. The data also indicate that complexation modes vary with solution chemistry. InNaCl solution, oocysts are bound to hematite via monodentate and binuclear bidentate complexes. The former predominates at low pH, whereas the latter becomes increasingly prevalent with increasing pH. In a CaCl2 solution, only binuclear bidentate complexes are observed. When solution pH is above the point of zero net proton charge (PZNPC) of hematite, oocyst surface carboxylate groups are bound to the mineral surface via outer-sphere complexes in both electrolyte solutions.

  11. Evaluating the Transport of Bacillus subtilis Spores as a Potential Surrogate for Cryptosporidium parvum Oocysts.

    PubMed

    Bradford, Scott A; Kim, Hyunjung; Headd, Brendan; Torkzaban, Saeed

    2016-02-02

    The U.S. Environmental Protection Agency has recommended the use of aerobic spores as an indicator for Cryptosporidium oocysts when determining groundwater under the direct influence of surface water. Surface properties, interaction energies, transport, retention, and release behavior of B. subtilis spores were measured over a range of physicochemical conditions, and compared with reported information for C. parvum oocysts. Interaction energy calculations predicted a much larger energy barrier and a shallower secondary minimum for spores than oocysts when the solution ionic strength (IS) equaled 0.1, 1, and 10 mM, and no energy barrier when the IS = 100 mM. Spores and oocysts exhibited similar trends of increasing retention with IS and decreasing Darcy water velocity (qw), and the predicted setback distance to achieve a six log removal was always larger for spores than oocysts. However, low levels of observed spore and oocyst release significantly influenced the predicted setback distance, especially when the fraction of reversibly retained microbes (Frev) was high. An estimate for Frev was obtained from large release pulses of spore and oocyst when the IS was reduced to deionized water. The value of Frev always increased with qw, whereas an opposition trend for Frev with IS was observed for spores (decreasing) and oocysts (increasing).

  12. Molecular cloning and characterization of a M17 leucine aminopeptidase of Cryptosporidium parvum.

    PubMed

    Kang, J-M; Ju, H-L; Sohn, W-M; Na, B-K

    2011-05-01

    Leucine aminopeptidases (LAPs) are a group of metalloexopeptidases that catalyse the sequential removal of amino acids from the N-termini of polypeptides or proteins. They play an important role in regulating the balance between catabolism and anabolism in living cells. LAPs of apicomplexa parasitic protozoa have been intensively investigated due to their crucial roles in parasite biology as well as their potentials as drug targets. In this study, we identified an M17 leucine aminopeptidase of Cryptosporidium parvum (CpLAP) and characterized the biochemical properties of the recombinant protein. Multiple sequence alignment of the deduced amino acid sequence of CpLAP with those of other organisms revealed that typical amino acid residues essential for metal binding and active-site formation in M17 LAPs were well conserved in CpLAP. Recombinant CpLAP shared similar biochemical properties such as optimal pH, stability at neutral pHs, and metal-binding characteristics with other characterized LAPs. The enzyme showed a marked preference for Leu and its activity was effectively inhibited by bestatin. These results collectively suggest that CpLAP is a typical member of the M17 LAP family and may play an important role in free amino acid regulation in the parasite.

  13. Inactivation of Cryptosporidium parvum oocysts with ozone and monochloramine at low temperature.

    PubMed

    Driedger, A M; Rennecker, J L; Mariñas, B J

    2001-01-01

    The rate of Cryptosporidium parvum inactivation decreased with decreasing temperature (1-20 degrees C) for ozone and for monochloramine applied alone as well as after pre-treatment with ozone. Synergy was observed at all temperatures studied for the ozone/monochloramine sequential disinfection scheme. The synergistic effect was found to increase with decreasing temperature. The inactivation rate with monochloramine after ozone pre-treatment was 5 times faster at 20 degrees C and 22 times faster at 1 degree C than the corresponding post-lag phase rates of inactivation with monochloramine at these temperatures when no ozone pre-treatment was applied. The CT required for achieving 2-logs of inactivation ranged from 11,400 mg min l-1 at 20 degrees C to 64,600 mg min l-1 at 1 degree C when monochloramine was applied alone. In contrast, the CT required for an overall sequential inactivation of 2-logs ranged from 721 mg min l-1 at 20 degrees C to 1350 mg min l-1 at 1 degree C when applying monochloramine after ozone pre-treatment. The presence of excess ammonia in the monochloramine solutions was not responsible for the synergy observed in ozone/monochloramine sequential disinfection.

  14. In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum.

    PubMed Central

    Arrowood, M J; Jaynes, J M; Healey, M C

    1991-01-01

    Cryptosporidium parvum is a protozoan parasite that causes mild to severe diarrheal disease in animals and humans. There are currently no effective chemotherapeutic agents available for the treatment of cryptosporidiosis. Recent studies have described small, naturally occurring antimicrobial lytic peptides with antiprotozoal activities. In the present study, the anticryptosporidial activities of three synthetic lytic peptides were determined in an in vitro sporozoite susceptibility assay. Sporozoite viability was assessed microscopically by the uptake of the vital dyes fluorescein diacetate and propidium iodide. Sporozoite viability was reduced by 93.5% following a 60-min exposure to 10 microM Hecate-1 at 37 degrees C. Shiva-10 reduced sporozoite viability by approximately 74.0% after a 60-min exposure at 100 microM and 37 degrees C. The cecropin-b analog SB-37 reduced sporozoite viability by 6.0% following a 60-min exposure at 100 microM and 37 degrees C. A control peptide showed no anticryptosporidial activity. PMID:1708975

  15. Depletion of Cryptosporidium parvum Oocysts from Contaminated Sewage by Using Freshwater Benthic Pearl Clams (Hyriopsis schlegeli)

    PubMed Central

    Yagita, Kenji; Izumiyama, Shinji; Endo, Takuro; Itoh, Yasoo

    2012-01-01

    The freshwater benthic pearl clam, Hyriopsis schlegeli, was experimentally exposed to Cryptosporidium parvum oocysts, and it was verified that the oocysts were eliminated predominantly via the fecal route, retaining their ability to infect cultured cells (HCT-8). The total fecal oocyst elimination rate was more than 90% within 5 days after exposure to the oocysts. H. schlegeli was able to survive in the final settling pond of a sewage plant for long periods, as confirmed by its pearl production. In the light of these findings, the clam was placed in the final settling pond in a trial to test its long-term efficacy in depleting oocysts contaminating the pond water. The number of clams placed was set to ensure a theoretical oocyst removal rate of around 50%, and the turbidity and the density of feed microbes in the overflow trough water of the pond were about 35% and 40 to 60% lower, respectively, than in the control water throughout the year. It was found that the clam feces containing oocysts were sufficiently heavy for them to settle to the bottom of the pond, despite the upward water flow. From these results, we concluded that efficient depletion of oocysts in the sewage water of small or midscale sewage treatment plants can be achieved by appropriate placement of H. schlegeli clams. PMID:22904053

  16. A novel Cryptosporidium parvum antigen, CP2, preferentially associates with membranous structures.

    PubMed

    O'Hara, Steven P; Yu, Jae-Ran; Lin, Jim Jung-Ching

    2004-03-01

    The present study addresses the cloning and characterization of a Cryptosporidium parvum antigen, CP2. Sequencing of cDNA and genomic clones revealed a novel gene capable of coding a message of 2,136 nucleotides flanked by 28 and 140 nucleotides of the 5'- and 3'-noncoding regions, respectively. The deduced amino acid sequence suggests that CP2 is a secreted and/or membrane protein. Immunofluorescence microscopy detected CP2 enrichment in sporozoites that subsequently appeared to encase type I meronts in infected HCT-8 cells. Immunogold electron microscopy revealed that CP2 consistently localized to membranous structures throughout development. In addition, progression from macrogametocyte to sporulated oocyst revealed CP2 initially at the periphery of amylopectin-like granules, in the cytoplasm and discrete vesicles, the parasitophorous vacuole, on the surface of sporozoites, and finally on the parasitophorous vacuole membrane (PVM). The observed expression pattern suggests that CP2 may be involved in the invasion process and/or PVM integrity.

  17. Depletion of Cryptosporidium parvum oocysts from contaminated sewage by using freshwater benthic pearl clams (Hyriopsis schlegeli).

    PubMed

    Izumi, Toshihiko; Yagita, Kenji; Izumiyama, Shinji; Endo, Takuro; Itoh, Yasoo

    2012-10-01

    The freshwater benthic pearl clam, Hyriopsis schlegeli, was experimentally exposed to Cryptosporidium parvum oocysts, and it was verified that the oocysts were eliminated predominantly via the fecal route, retaining their ability to infect cultured cells (HCT-8). The total fecal oocyst elimination rate was more than 90% within 5 days after exposure to the oocysts. H. schlegeli was able to survive in the final settling pond of a sewage plant for long periods, as confirmed by its pearl production. In the light of these findings, the clam was placed in the final settling pond in a trial to test its long-term efficacy in depleting oocysts contaminating the pond water. The number of clams placed was set to ensure a theoretical oocyst removal rate of around 50%, and the turbidity and the density of feed microbes in the overflow trough water of the pond were about 35% and 40 to 60% lower, respectively, than in the control water throughout the year. It was found that the clam feces containing oocysts were sufficiently heavy for them to settle to the bottom of the pond, despite the upward water flow. From these results, we concluded that efficient depletion of oocysts in the sewage water of small or midscale sewage treatment plants can be achieved by appropriate placement of H. schlegeli clams.

  18. Impact of bathers on levels of Cryptosporidium parvum oocysts and Giardia lamblia cysts in recreational beach waters.

    PubMed

    Sunderland, Deirdre; Graczyk, Thaddeus K; Tamang, Leena; Breysse, Patrick N

    2007-08-01

    Recreational beach water samples collected on weekends and weekdays during 11 consecutive summer weeks were tested for potentially viable Cryptosporidium parvum oocysts and Giardia lamblia cysts using the multiplexed fluorescence in situ hybridization (FISH) method. The levels of oocysts and cysts on weekends were significantly higher than on the weekdays (P<0.01). Concentrations of oocysts in weekend samples (n=27) ranged from 2 to 42 oocysts/L (mean: 13.7 oocysts/L), and cyst concentration ranged from 0 to 33 cysts/L (mean: 9.1 cysts/L). For the samples collected on weekdays (n=33), the highest oocyst concentration was 7 oocysts/L (mean: 1.5 oocysts/L), and the highest cyst concentration was 4 cysts/L (mean: 0.6 cysts/L). The values of water turbidity were significantly higher on weekends than on weekdays, and were correlated with the number of bathers and concentration of C. parvum oocysts and G. lamblia cysts (P<0.04). The study demonstrated positive relationships between number of bathers and levels of waterborne C. parvum oocysts and G. lamblia cysts in recreational beach water. It is essential to test recreational waters for Cryptosporidium and Giardia when numbers of bathers are greatest, or limit the number of bathers in a recreational beach area.

  19. Evaluation of a magnetic microsphere antibody detection assay for the investigation of exposure to Cryptosporidium parvum and Cryptosporidium hominis

    EPA Science Inventory

    Anti-Cryptosporidium IgA and IgG are useful markers of exposure to Cryptosporidium in human populations, but detection in saliva may be difficult. To evaluate a magnetic microsphere assay for detection of anti-Cryptosporidium IgA and IgG in saliva, recombinant sporozoite gp15 (1...

  20. Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler’s Diarrhea

    PubMed Central

    Shin, Ji-Hun; Lee, Sang-Eun; Kim, Tong Soo; Ma, Da-Won; Chai, Jong-Yil; Shin, Eun-Hee

    2016-01-01

    This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler’s diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×103 oocysts for C. parvum, >1×104 cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples. PMID:27853120

  1. The Cryptosporidium parvum ApiAP2 gene family: insights into the evolution of apicomplexan AP2 regulatory systems.

    PubMed

    Oberstaller, Jenna; Pumpalova, Yoanna; Schieler, Ariel; Llinás, Manuel; Kissinger, Jessica C

    2014-07-01

    We provide the first comprehensive analysis of any transcription factor family in Cryptosporidium, a basal-branching apicomplexan that is the second leading cause of infant diarrhea globally. AP2 domain-containing proteins have evolved to be the major regulatory family in the phylum to the exclusion of canonical regulators. We show that apicomplexan and perkinsid AP2 domains cluster distinctly from other chromalveolate AP2s. Protein-binding specificity assays of C. parvum AP2 domains combined with motif conservation upstream of co-regulated gene clusters allowed the construction of putative AP2 regulons across the in vitro life cycle. Orthologous Apicomplexan AP2 (ApiAP2) expression has been rearranged relative to the malaria parasite P. falciparum, suggesting ApiAP2 network rewiring during evolution. C. hominis orthologs of putative C. parvum ApiAP2 proteins and target genes show greater than average variation. C. parvum AP2 domains display reduced binding diversity relative to P. falciparum, with multiple domains binding the 5'-TGCAT-3', 5'-CACACA-3' and G-box motifs (5'-G[T/C]GGGG-3'). Many overrepresented motifs in C. parvum upstream regions are not AP2 binding motifs. We propose that C. parvum is less reliant on ApiAP2 regulators in part because it utilizes E2F/DP1 transcription factors. C. parvum may provide clues to the ancestral state of apicomplexan transcriptional regulation, pre-AP2 domination. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. The Cryptosporidium parvum ApiAP2 gene family: insights into the evolution of apicomplexan AP2 regulatory systems

    PubMed Central

    Oberstaller, Jenna; Pumpalova, Yoanna; Schieler, Ariel; Llinás, Manuel; Kissinger, Jessica C.

    2014-01-01

    We provide the first comprehensive analysis of any transcription factor family in Cryptosporidium, a basal-branching apicomplexan that is the second leading cause of infant diarrhea globally. AP2 domain-containing proteins have evolved to be the major regulatory family in the phylum to the exclusion of canonical regulators. We show that apicomplexan and perkinsid AP2 domains cluster distinctly from other chromalveolate AP2s. Protein-binding specificity assays of C. parvum AP2 domains combined with motif conservation upstream of co-regulated gene clusters allowed the construction of putative AP2 regulons across the in vitro life cycle. Orthologous Apicomplexan AP2 (ApiAP2) expression has been rearranged relative to the malaria parasite P. falciparum, suggesting ApiAP2 network rewiring during evolution. C. hominis orthologs of putative C. parvum ApiAP2 proteins and target genes show greater than average variation. C. parvum AP2 domains display reduced binding diversity relative to P. falciparum, with multiple domains binding the 5′-TGCAT-3′, 5′-CACACA-3′ and G-box motifs (5′-G[T/C]GGGG-3′). Many overrepresented motifs in C. parvum upstream regions are not AP2 binding motifs. We propose that C. parvum is less reliant on ApiAP2 regulators in part because it utilizes E2F/DP1 transcription factors. C. parvum may provide clues to the ancestral state of apicomplexan transcriptional regulation, pre-AP2 domination. PMID:24957599

  3. Impact of zooplankton grazing on the excystation, viability, and infectivity of the protozoan pathogens Cryptosporidium parvum and Giardia lamblia.

    PubMed

    Connelly, S J; Wolyniak, E A; Dieter, K L; Williamson, C E; Jellison, K L

    2007-11-01

    Very little is known about the ability of the zooplankton grazer Daphnia pulicaria to reduce populations of Giardia lamblia cysts and Cryptosporidium parvum oocysts in surface waters. The potential for D. pulicaria to act as a biological filter of C. parvum and G. lamblia was tested under three grazing pressures (one, two, or four D. pulicaria grazers per 66 ml). (Oo)cysts (1 x 10(4) per 66 ml) were added to each grazing bottle along with the algal food Selenastrum capricornutum (6.6 x 10(4) cells per 66 ml) to stimulate normal grazing. Bottles were rotated (2 rpm) to prevent settling of (oo)cysts and algae for 24 h (a light:dark cycle of 16 h:8 h) at 20 degrees C. The impact of D. pulicaria grazing on (oo)cysts was assessed by (i) (oo)cyst clearance rates, (ii) (oo)cyst viability, (iii) (oo)cyst excystation, and (iv) oocyst infectivity in cell culture. Two D. pulicaria grazers significantly decreased the total number of C. parvum oocysts by 52% and G. lamblia cysts by 44%. Furthermore, two D. pulicaria grazers significantly decreased C. parvum excystation and infectivity by 5% and 87%, respectively. Two D. pulicaria grazers significantly decreased the viability of G. lamblia cysts by 52%, but analysis of G. lamblia excystation was confounded by observed mechanical disruption of the cysts after grazing. No mechanical disruption of the C. parvum oocysts was observed, presumably due to their smaller size. The data provide strong evidence that zooplankton grazers have the potential to substantially decrease the population of infectious C. parvum and G. lamblia in freshwater ecosystems.

  4. The Effect of the Anionic Surfactant Aerosol-80 on the Transport of Cryptosporidium parvum Oocysts through Soil

    NASA Astrophysics Data System (ADS)

    Jacobson, A. R.; Powelson, D.; Darnault, C.

    2012-12-01

    Transport of the pathogenic protozoan Cryptosporidium parvum through soils threatens ground and surface waters. C. parvum may be introduced into soils in the manure of infected calves. The presence of other chemicals in the soil applied as or with amendments, may affect the transport of the C. parvum oocysts. Surfactants, which are used in many herbicide formulations, decrease water tension and may disrupt the air-water interface where oocysts are thought to accumulate. We investigate the effect of the anionic surfactant Aerosol-80, at two concentrations, on the transport of C. parvum oocysts by unsaturated flow through "undisturbed" soil columns from Illinois and Utah. Following each experiment oocysts in the leachate and distributed throughout the soil profile are quantified by real time PCR. We find that the presence of the surfactant accelerates the transport of the oocysts through preferential flow paths. On the other hand, when connected macropores are not present in the soils, the presence of the surfactant retards the transport of the oocysts through the soil matrix by straining oocyst-surfactant-Ca flocs. Surfactant efficacy is affected by soil type.

  5. Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair.

    PubMed

    Oguma, K; Katayama, H; Mitani, H; Morita, S; Hirata, T; Ohgaki, S

    2001-10-01

    UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.

  6. Adhesion of Cryptosporidium parvum and Giardia lamblia to solid surfaces: the role of surface charge and hydrophobicity.

    PubMed

    Dai, X; Boll, J; Hayes, M E; Aston, D E

    2004-04-15

    Adhesion of Cryptosporidium parvum and Giardia lamblia to four materials of different surface charge and hydrophobicity was investigated. Glass beads were used with and without three polymer coatings: aminosilines (A0750), fluorosilines (T2494), an amino cationic polymer. Surface charge density and hydrophobicity of the beads were characterized by measuring the zeta potential (ZP) and the contact angle, respectively. Adhesion was derived from batch experiments where negatively charged (oo)cysts were mixed with the beads and recovery was determined by counting (oo)cysts remaining in suspension using a flow cytometer. Experimental results clearly show that adhesion to solid surfaces of C. parvum is different from G. lamblia. Adhesion of C. parvum to positively charged, hydrophilic beads (82% recovery relative to control) indicated that surface charge was the more important factor for C. parvum, dominating any hydrophobic effects. Adhesion of G. lamblia cysts to negatively charged, hydrophobic beads (0% recovery relative to control) indicated that although hydrophobicity and surface charge both played a role in the adhesion of G. lamblia to solid surfaces, hydrophobicity was more important than surface charge.

  7. Attempts to protect severe combined immunodeficient (scid) mice with antibody enriched for reactivity to Cryptosporidium parvum surface antigen-1.

    PubMed

    Tatalick, L M; Perryman, L E

    1995-07-01

    Cryptosporidium parvum is a protozoal pathogen which infects the gastrointestinal epithelium of mammals causing diarrhoea, the duration and severity of which is determined by the immunocompetency of the host. Currently, there is no effective treatment or prevention. We evaluated the ability of surface antigen-1 (SA-1), defined as those antigens recognized by neutralizing mAb 17.41, to elicit a protective antibody response when used as an immunogen. A SA-1 enriched fraction was obtained by immunoaffinity chromatography and was used to immunize a naive Holstein calf. SA-1 immune serum from this calf detected C. parvum epitopes to a 1:10,000 dilution in a dot blot assay, and sporozoite surface epitopes at a 1:10,000 dilution in a live immunofluorescence assay. Western blot analysis showed that SA-1 immune bovine serum recognized a similar pattern of C. parvum antigens as the defining mAb 17.41. Oral passive transfer of SA-1 immune bovine serum did not protect severe combined immunodeficient (scid) mice or suckling BALB/c mice from initial infection with C. parvum, or terminate a persistent infection in scid mice.

  8. Electron microscopic observation of the early stages of Cryptosporidium parvum asexual multiplication and development in in vitro axenic culture.

    PubMed

    Aldeyarbi, Hebatalla M; Karanis, Panagiotis

    2016-02-01

    The stages of Cryptosporidium parvum asexual exogenous development were investigated at high ultra-structural resolution in cell-free culture using transmission electron microscopy (TEM). Early C. parvum trophozoites were ovoid in shape, 1.07 × 1.47 μm(2) in size, and contained a large nucleus and adjacent Golgi complex. Dividing and mature meronts containing four to eight developing merozoites, 2.34 × 2.7 μm(2) in size, were observed within the first 24h of cultivation. An obvious peculiarity was found within the merozoite pellicle, as it was composed of the outer plasma membrane with underlying middle and inner membrane complexes. Further novel findings were vacuolization of the meront's residuum and extension of its outer pellicle, as parasitophorous vacuole-like membranes were also evident. The asexual reproduction of C. parvum was consistent with the developmental pattern of both eimerian coccidia and Arthrogregarinida (formerly Neogregarinida). The unique cell-free development of C. parvum described here, along with the establishment of meronts and merozoite formation, is the first such evidence obtained from in vitro cell-free culture at the ultrastructural level.

  9. Evidence that Cryptosporidium parvum Populations Are Panmictic and Unstructured in the Upper Midwest of the United States

    PubMed Central

    Herges, Grant R.; Widmer, Giovanni; Clark, Mark E.; Khan, Eakalak; Giddings, Catherine W.; Brewer, Matt

    2012-01-01

    Cryptosporidium parvum is a zoonotic protozoan parasite that causes cryptosporidiosis, an infectious diarrheal disease primarily affecting humans and neonatal ruminants. Understanding the transmission dynamics of C. parvum, particularly the specific contributions of zoonotic and anthroponotic transmission, is critical to the control of this pathogen. This study used a population genetics approach to better understand the transmission of C. parvum in the Upper Midwest United States. A total of 254 C. parvum isolates from cases of human cryptosporidiosis in Minnesota and Wisconsin and diarrheic calves in Minnesota, Wisconsin, and North Dakota were genotyped at eight polymorphic loci. Isolates with a complete profile from all eight loci (n = 212) were used to derive a multilocus genotype (MLT), which was used in population genetic analyses. Among the 94 MLTs identified, 60 were represented by a single isolate. Approximately 20% of isolates belonged to MLT 2, a group that included both human and cattle isolates. Population analyses revealed a predominantly panmictic population with no apparent geographic or host substructuring. PMID:22983961

  10. An Outbreak of Cryptosporidium parvum across England & Scotland Associated with Consumption of Fresh Pre-Cut Salad Leaves, May 2012

    PubMed Central

    McKerr, Caoimhe; Adak, Goutam K.; Nichols, Gordon; Gorton, Russell; Chalmers, Rachel M.; Kafatos, George; Cosford, Paul; Charlett, Andre; Reacher, Mark; Pollock, Kevin G.; Alexander, Claire L.; Morton, Stephen

    2015-01-01

    Background We report a widespread foodborne outbreak of Cryptosporidium parvum in England and Scotland in May 2012. Cases were more common in female adults, and had no history of foreign travel. Over 300 excess cases were identified during the period of the outbreak. Speciation and microbiological typing revealed the outbreak strain to be C. parvum gp60 subtype IIaA15G2R1. Methods Hypothesis generation questionnaires were administered and an unmatched case control study was undertaken to test the hypotheses raised. Cases and controls were interviewed by telephone. Controls were selected using sequential digit dialling. Information was gathered on demographics, foods consumed and retailers where foods were purchased. Results Seventy-four laboratory confirmed cases and 74 controls were included in analyses. Infection was found to be strongly associated with the consumption of pre-cut mixed salad leaves sold by a single retailer. This is the largest documented outbreak of cryptosporidiosis attributed to a food vehicle. PMID:26017538

  11. Revisiting the reference genomes of human pathogenic Cryptosporidium species: reannotation of C. parvum Iowa and a new C. hominis reference

    PubMed Central

    Isaza, Juan P.; Galván, Ana Luz; Polanco, Victor; Huang, Bernice; Matveyev, Andrey V.; Serrano, Myrna G.; Manque, Patricio; Buck, Gregory A.; Alzate, Juan F.

    2015-01-01

    Cryptosporidium parvum and C. hominis are the most relevant species of this genus for human health. Both cause a self-limiting diarrhea in immunocompetent individuals, but cause potentially life-threatening disease in the immunocompromised. Despite the importance of these pathogens, only one reference genome of each has been analyzed and published. These two reference genomes were sequenced using automated capillary sequencing; as of yet, no next generation sequencing technology has been applied to improve their assemblies and annotations. For C. hominis, the main challenge that prevents a larger number of genomes to be sequenced is its resistance to axenic culture. In the present study, we employed next generation technology to analyse the genomic DNA and RNA to generate a new reference genome sequence of a C. hominis strain isolated directly from human stool and a new genome annotation of the C. parvum Iowa reference genome. PMID:26549794

  12. Revisiting the reference genomes of human pathogenic Cryptosporidium species: reannotation of C. parvum Iowa and a new C. hominis reference.

    PubMed

    Isaza, Juan P; Galván, Ana Luz; Polanco, Victor; Huang, Bernice; Matveyev, Andrey V; Serrano, Myrna G; Manque, Patricio; Buck, Gregory A; Alzate, Juan F

    2015-11-09

    Cryptosporidium parvum and C. hominis are the most relevant species of this genus for human health. Both cause a self-limiting diarrhea in immunocompetent individuals, but cause potentially life-threatening disease in the immunocompromised. Despite the importance of these pathogens, only one reference genome of each has been analyzed and published. These two reference genomes were sequenced using automated capillary sequencing; as of yet, no next generation sequencing technology has been applied to improve their assemblies and annotations. For C. hominis, the main challenge that prevents a larger number of genomes to be sequenced is its resistance to axenic culture. In the present study, we employed next generation technology to analyse the genomic DNA and RNA to generate a new reference genome sequence of a C. hominis strain isolated directly from human stool and a new genome annotation of the C. parvum Iowa reference genome.

  13. An Outbreak of Cryptosporidium parvum across England & Scotland Associated with Consumption of Fresh Pre-Cut Salad Leaves, May 2012.

    PubMed

    McKerr, Caoimhe; Adak, Goutam K; Nichols, Gordon; Gorton, Russell; Chalmers, Rachel M; Kafatos, George; Cosford, Paul; Charlett, Andre; Reacher, Mark; Pollock, Kevin G; Alexander, Claire L; Morton, Stephen

    2015-01-01

    We report a widespread foodborne outbreak of Cryptosporidium parvum in England and Scotland in May 2012. Cases were more common in female adults, and had no history of foreign travel. Over 300 excess cases were identified during the period of the outbreak. Speciation and microbiological typing revealed the outbreak strain to be C. parvum gp60 subtype IIaA15G2R1. Hypothesis generation questionnaires were administered and an unmatched case control study was undertaken to test the hypotheses raised. Cases and controls were interviewed by telephone. Controls were selected using sequential digit dialling. Information was gathered on demographics, foods consumed and retailers where foods were purchased. Seventy-four laboratory confirmed cases and 74 controls were included in analyses. Infection was found to be strongly associated with the consumption of pre-cut mixed salad leaves sold by a single retailer. This is the largest documented outbreak of cryptosporidiosis attributed to a food vehicle.

  14. Infection of immunocompetent mice with acid-water-pretreated Cryptosporidium parvum results in weight loss, and intestinal (structural and physiological) alterations.

    PubMed

    Garza, Armandina; Castellanos-Gonzalez, Alejandro; Castenallos-Gonzalez, Alejandro; Griffiths, Jeffrey; Robinson, Prema

    2008-02-01

    Cryptosporidiosis, caused by Cryptosporidium, causes self-limited diarrhea in normal hosts but may cause life-threatening diarrhea in immunocompromised persons. Cryptosporidium-induced manifestations, including weight-loss and intestinal physiological alterations are not noted in adult immunocompetent mice. So far, studies that have been used to test the therapeutic efficacy of drugs have been performed using various immunocompromised animal models. There is an urgent need of an immunocompetent small animal model that portrays Cryptosporidium-induced manifestations. In the current studies, we have compared two Cryptosporidium parvum pretreatment methods, we have hence used sodium hypochlorite or acidic water to treat Cryptosporidium parvum, followed by infection by oral gavage in adult immunocompetent C57BL6 mice. We demonstrated manifestations such as weight loss, intestinal structural and physiological alterations such as intestinal, villi blunting, and glucose malabsorption (as studied by the Ussing chamber technique) only in response to infection with C. parvum that has been treated with acidic water and not with sodium hypochlorite. These novel studies reveal that acidic water treatment of C. parvum results in manifestations of cryptosporidiosis in otherwise resistant immunocompetent mice. The current studies open up possibilities of using the normal immunocompetent mice model to test therapeutic drugs against cryptosporidiosis.

  15. Identification and Characterization of Cryptosporidium parvum Clec, a Novel C-Type Lectin Domain-Containing Mucin-Like Glycoprotein

    PubMed Central

    Bhalchandra, Seema; Ludington, Jacob; Coppens, Isabelle

    2013-01-01

    Cryptosporidium species are waterborne apicomplexan parasites that cause diarrheal disease worldwide. Although the mechanisms underlying Cryptosporidium-host cell interactions are not well understood, mucin-like glycoproteins of the parasite are known to mediate attachment and invasion in vitro. We identified C. parvum Clec (CpClec), a novel mucin-like glycoprotein that contains a C-type lectin domain (CTLD) and has orthologs in C. hominis and C. muris. CTLD-containing proteins are ligand-binding proteins that function in adhesion and signaling and are present in a wide range of organisms, from humans to viruses. However, this is the first report of a CTLD-containing protein in protozoa and in Apicomplexa. CpClec is predicted to be a type 1 membrane protein, with a CTLD, an O-glycosylated mucin-like domain, a transmembrane domain, and a cytoplasmic tail containing a YXXϕ sorting motif. The predicted structure of CpClec displays several characteristics of canonical CTLD-containing proteins, including a long loop region hydrophobic core associated with calcium-dependent glycan binding as well as predicted calcium- and glycan-binding sites. CpClec expression during C. parvum infection in vitro is maximal at 48 h postinfection, suggesting that it is developmentally regulated. The 120-kDa mass of native CpClec is greater than predicted, most likely due to O-glycosylation. CpClec is localized to the surface of the apical region and to dense granules of sporozoites and merozoites. Taken together, these findings, along with the known functions of C. parvum mucin-like glycoproteins and of CTLD-containing proteins, strongly implicate a significant role for CpClec in Cryptosporidium-host cell interactions. PMID:23817613

  16. Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum.

    PubMed

    Rochelle, Paul A; Marshall, Marilyn M; Mead, Jan R; Johnson, Anne M; Korich, Dick G; Rosen, Jeffrey S; De Leon, Ricardo

    2002-08-01

    In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all

  17. Coupled factors influencing the transport and retention of Cryptosporidium parvum oocysts in saturated porous media.

    PubMed

    Kim, Hyunjung N; Walker, Sharon L; Bradford, Scott A

    2010-02-01

    The coupled role of solution ionic strength (IS), hydrodynamic force, and pore structure on the transport and retention of viable Cryptosporidium parvum oocyst was investigated via batch, packed-bed column, and micromodel systems. The experiments were conducted over a wide range of IS (0.1-100 mM), at two Darcy velocities (0.2 and 0.5 cm/min), and in two sands (median diameters of 275 and 710 microm). Overall, the results suggested that oocyst retention was a complex process that was very sensitive to the solution IS, the Darcy velocity, and the grain size. Increasing IS led to enhanced retention of oocysts in the column, which is qualitatively consistent with predictions of Derjaguin-Landau-Verwey-Overbeek theory. Conversely, increasing velocity and grain size resulted in less retention of oocysts in the column due to the difference in the fluid drag force and the rates of mass transfer from the liquid to the solid phase and from high to low velocity regions. Oocyst retention was controlled by a combined role of low velocity regions, weak attractive interactions, and/or steric repulsion. The contribution of each mechanism highly depended on the solution IS. In particular, micromodel observations indicated that enhanced oocyst retention occurred in low velocity regions near grain-grain contacts under highly unfavorable conditions (IS=0.1 mM). Oocyst retention was also found to be influenced by weak attractive interactions (induced by the secondary energy minimum, surface roughness, and/or nanoscale chemical heterogeneity) when the IS=1 mM. Reversible retention of oocysts to the sand in batch and column studies under favorable attachment conditions (IS=100 mM) was attributed to steric repulsion between the oocysts and the sand surface due to the presence of oocyst surface macromolecules. Comparison of experimental observations and theoretical predictions from classic filtration theory further supported the presence of this weak interaction due to steric repulsion.

  18. Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica.

    PubMed

    Madison-Antenucci, S; Relich, R F; Doyle, L; Espina, N; Fuller, D; Karchmer, T; Lainesse, A; Mortensen, J E; Pancholi, P; Veros, W; Harrington, S M

    2016-11-01

    Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays.

  19. Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica

    PubMed Central

    Relich, R. F.; Doyle, L.; Espina, N.; Fuller, D.; Karchmer, T.; Lainesse, A.; Mortensen, J. E.; Pancholi, P.; Veros, W.; Harrington, S. M.

    2016-01-01

    Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis. Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays. PMID:27535690

  20. The Cryptosporidium parvum C-Type Lectin CpClec Mediates Infection of Intestinal Epithelial Cells via Interactions with Sulfated Proteoglycans

    PubMed Central

    Ludington, Jacob G.

    2016-01-01

    The apicomplexan parasite Cryptosporidium causes significant diarrheal disease worldwide. Effective anticryptosporidial agents are lacking, in part because the molecular mechanisms underlying Cryptosporidium-host cell interactions are poorly understood. Previously, we identified and characterized a novel Cryptosporidium parvum C-type lectin domain-containing mucin-like glycoprotein, CpClec. In this study, we evaluated the mechanisms underlying interactions of CpClec with intestinal epithelial cells by using an Fc-tagged recombinant protein. CpClec-Fc displayed Ca2+-dependent, saturable binding to HCT-8 and Caco-2 cells and competitively inhibited C. parvum attachment to and infection of HCT-8 cells. Binding of CpClec-Fc was specifically inhibited by sulfated glycosaminoglycans, particularly heparin and heparan sulfate. Binding was reduced after the removal of heparan sulfate and following the inhibition of glycosaminoglycan synthesis or sulfation in HCT-8 cells. Like CpClec-Fc binding, C. parvum attachment to and infection of HCT-8 cells were inhibited by glycosaminoglycans and were reduced after heparan sulfate removal or inhibition of glycosaminoglycan synthesis or sulfation. Lastly, CpClec-Fc binding and C. parvum sporozoite attachment were significantly decreased in CHO cell mutants defective in glycosaminoglycan synthesis. Together, these results indicate that CpClec is a novel C-type lectin that mediates C. parvum attachment and infection via Ca2+-dependent binding to sulfated proteoglycans on intestinal epithelial cells. PMID:26975991

  1. Biotin- and Glycoprotein-Coated Microspheres as Surrogates for Studying Filtration Removal of Cryptosporidium parvum in a Granular Limestone Aquifer Medium

    PubMed Central

    Blaschke, A. P.; Toze, S.; Sidhu, J. P. S.; Ahmed, W.; van Driezum, I. H.; Sommer, R.; Kirschner, A. K. T.; Cervero-Aragó, S.; Farnleitner, A. H.; Pang, L.

    2015-01-01

    Members of the genus Cryptosporidium are waterborne protozoa of great health concern. Many studies have attempted to find appropriate surrogates for assessing Cryptosporidium filtration removal in porous media. In this study, we evaluated the filtration of Cryptosporidium parvum in granular limestone medium by the use of biotin- and glycoprotein-coated carboxylated polystyrene microspheres (CPMs) as surrogates. Column experiments were carried out with core material taken from a managed aquifer recharge site in Adelaide, Australia. For the experiments with injection of a single type of particle, we observed the total removal of the oocysts and glycoprotein-coated CPMs, a 4.6- to 6.3-log10 reduction of biotin-coated CPMs, and a 2.6-log10 reduction of unmodified CPMs. When two different types of particles were simultaneously injected, glycoprotein-coated CPMs showed a 5.3-log10 reduction, while the uncoated CPMs displayed a 3.7-log10 reduction, probably due to particle-particle interactions. Our results confirm that glycoprotein-coated CPMs are the most accurate surrogates for C. parvum; biotin-coated CPMs are slightly more conservative, while unmodified CPMs are markedly overly conservative for predicting C. parvum removal in granular limestone medium. The total removal of C. parvum observed in our study suggests that granular limestone medium is very effective for the filtration removal of C. parvum and could potentially be used for the pretreatment of drinking water and aquifer storage recovery of recycled water. PMID:25888174

  2. DEVELOPMENT OF A CT EQUATION TAKING INTO CONSIDERATION THE EFFECT OF LOT VARIABILITY ON THE INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH OZONE

    EPA Science Inventory

    Cryptosporidium parvum oocysts are prevalent in surface water and ground water under the influence of surface water, and are difficult to inactivate using free chlorine, the most common disinfectant currently used for treating drinking water. In contrast, it has been shown...

  3. DEVELOPMENT OF A CT EQUATION TAKING INTO CONSIDERATION THE EFFECT OF LOT VARIABILITY ON THE INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH OZONE

    EPA Science Inventory

    Cryptosporidium parvum oocysts are prevalent in surface water and ground water under the influence of surface water, and are difficult to inactivate using free chlorine, the most common disinfectant currently used for treating drinking water. In contrast, it has been shown...

  4. Processes affecting the transport of Cryptosporidium parvum and other persistent pathogens in surface- and ground-waters

    NASA Astrophysics Data System (ADS)

    Packman, A. I.; Lau, B. L.; Harter, T.; Atwill, E. R.

    2007-12-01

    Waterborne diseases are transmitted through numerous environmental pathways, and their migration is strongly mediated by interaction with a wide variety of sediments and other natural materials during transport. Here we provide an overview of factors that affect the fate of persistent water-borne pathogens, focusing particularly on the zoonotic pathogen Cryptosporidium parvum as an example. While individual microbial cells are both small and have low specific gravity, suggesting that they should be highly mobile and remain suspended for long periods of time, attachment to a variety of background materials can substantially reduce pathogen mobility. Cryptosporidium oocysts readily associate with both inorganic and organic particles, resulting in the formation of aggregates. This process tends to increase the effective settling velocity of C. parvum in surface waters. Similarly, pathogens readily become associated with the solid matrix during transport in groundwater, resulting in removal by filtration. However, this process is reversible with C. parvum, resulting in a slow long-term release following the initial deposition. Pathogens also become associated with biofilms, which are surface-attached communities of microorganisms in a gelatinous matrix. The presence of biofilms increases the immobilization and retention of Cryptosporidium on solid surfaces. All of these processes influence pathogen transmission in surface waters such as rivers and water-supply canals. In these environments, pathogens can be immobilized by deposition into stable sediment beds by a combination of gravitational sedimentation and advection into pore waters followed by subsurface filtration. Association with background suspended matter tends to increase pathogen deposition by sedimentation, and the presence of benthic (sedimentary) biofilms also tends to increase pathogen retention. For pathogens that remain viable for long periods of time in natural aquatic systems, as is the case with

  5. Infectivity to experimental rodents of Cryptosporidium parvum oocysts from Siberian chipmunks (Tamias sibiricus) originated in the People's Republic of China.

    PubMed

    Matsui, T; Fujino, T; Kajima, J; Tsuji, M

    2000-05-01

    We isolated Cryptosporidium parvum-type oocysts from naturally infected siberian chipmunks which originated in the People's Republic of China and examined the infectivity to rodents as experimental animals. The naturally infected chipmunks did not show any clinical symptoms. The oocysts were 4.8 x 4.2 microm on average in size. They were ovoid and morphologically similar to the C. parvum oocysts isolated from human and cattle. Experimental rodents were inoculated with 1.6 x 10(6) original oocysts each. SCID mice began to shed oocysts on day 7 and the OPG value was 10(5) from 50 days. The oocysts were found from ICR mice on days 13 and 16 by only sugar flotation method, however, any oocysts were not detected from the rats, guinea pigs and rabbits until 30 days. Two infected SCID mice were necropsied on days 100 and 102 and examined for coccidian organisms. Merozoites and oocysts were found in the low part of jejunum and ileum, however, no parasites were detected in the stomach. Consequently, it was considered that the present species was C. parvum and was probably genotype 2 from result of infectivity to rodents.

  6. Capture of water-borne colloids in granular beds using external electric fields: improving removal of Cryptosporidium parvum.

    PubMed

    Kulkarni, Pramod; Dutari, Gabriel; Weingeist, David; Adin, Avner; Haught, Roy; Biswas, Pratim

    2005-03-01

    Suboptimal coagulation in water treatment plants often results in reduced removal efficiency of Cryptosporidium parvum oocysts by several orders of magnitude (J. AWWA 94(6) (2002) 97, J. AWWA 93(12) (2001) 64). The effect of external electric field on removal of C. parvum oocysts in packed granular beds was studied experimentally. A cylindrical configuration of electrodes, with granular media in the annular space was used. A negative DC potential was applied to the central electrode. No coagulants or flocculants were used and filtration was performed with and without application of an electric field to obtain improvement in removal efficiency. Results indicate that removal of C. parvum increased from 10% to 70% due to application of field in fine sand media and from 30% to 96% in MAGCHEM media. All other test particles (Kaolin and polystyrene latex microspheres) used in the study also exhibited increased removal in the presence of an electric field. Single collector efficiencies were also computed using approximate trajectory analysis, modified to account for the applied external electric field. The results of these calculations were used to qualitatively explain the trends in the experimental observations.

  7. Efficacy of the solar water disinfection method in turbid waters experimentally contaminated with Cryptosporidium parvum oocysts under real field conditions.

    PubMed

    Gómez-Couso, H; Fontán-Saínz, M; Sichel, C; Fernández-Ibáñez, P; Ares-Mazás, E

    2009-06-01

    To investigate the efficacy of the solar water disinfection (SODIS) method for inactivating Cryptosporidium parvum oocysts in turbid waters using 1.5 l polyethylene terephthalate (PET) bottles under natural sunlight. All experiments were performed at the Plataforma Solar de Almería, located in the Tabernas Desert (Southern Spain) in July and October 2007. Turbid water samples [5, 100 and 300 nephelometric turbidity units (NTU)] were prepared by addition of red soil to distilled water, and then spiked with purified C. parvum oocysts. PET bottles containing the contaminated turbid waters were exposed to full sunlight for 4, 8 and 12 h. The samples were then concentrated by filtration and the oocyst viability was determined by inclusion/exclusion of the fluorogenic vital dye propidium iodide. Results After an exposure time of 12 h (cumulative global dose of 28.28 MJ/m(2); cumulative UV dose of 1037.06 kJ/m(2)) the oocyst viabilities were 11.54%, 25.96%, 41.50% and 52.80% for turbidity levels of 0, 5, 100 and 300 NTU, respectively, being significantly lower than the viability of the initial isolate (P < 0.01). SODIS method significantly reduced the potential viability of C. parvum oocysts on increasing the percentage of oocysts that took up the dye PI (indicator of cell wall integrity), although longer exposure periods appear to be required than those established for the bacterial pathogens usually tested in SODIS assays. SODIS.

  8. Use of a common laboratory glassware detergent improves recovery of Cryptosporidium parvum and Cyclospora cayetanensis from lettuce, herbs and raspberries.

    PubMed

    Shields, Joan M; Lee, Michelle Minjung; Murphy, Helen R

    2012-02-01

    The success of any protocol designed to detect parasitic protozoa on produce must begin with an efficient initial wash step. Cryptosporidium parvum and Cyclospora cayetanensis oocysts were seeded onto herbs, lettuces and raspberries, eluted with one of four wash solutions and the recovered number of oocysts determined via fluorescent microscopy. Recovery rates for fluorescein thiosemicarbazide labeled C. parvum oocysts seeded onto spinach and raspberries and washed with de-ionized water were 38.4 ± 10.1% and 34.9 ± 6.2%, respectively. Two alternative wash solutions viz. 1M glycine, pH 5.5 and a detachment solution were tested also using labeled C. parvum seeded spinach and raspberries. No statistically significant difference was noted in the recovery rates. However, a wash solution containing 0.1% Alconox, a laboratory glassware detergent, resulted in a significant improvement in oocyst recovery. 72.6 ± 6.6% C. parvum oocysts were recovered from basil when washed with 0.1% Alconox compared to 47.9 ± 5.8% using detachment solution. Also, C. cayetanensis oocysts were seeded onto lettuces, herbs and raspberries and the recovery using de-ionized water were compared to 0.1% Alconox wash: basil 17.5 ± 5.0% to 76.1 ± 14.0%, lollo rosso lettuce 38.3 ± 5.5% to 72.5 ± 8.1%, Tango leaf lettuce 45.9 ± 5.4% to 71.1 ± 7.8% and spring mix (mesclun) 39.8 ± 0.7% to 80.2 ± 11.3%, respectively. These results suggest that the use of Alconox in a wash solution significantly improves recovery resulting in the detection of these parasitic protozoa on high risk foods. Published by Elsevier B.V.

  9. Disinfection of drinking water contaminated with Cryptosporidium parvum oocysts under natural sunlight and using the photocatalyst TiO2.

    PubMed

    Méndez-Hermida, Fernando; Ares-Mazás, Elvira; McGuigan, Kevin G; Boyle, Maria; Sichel, Cosima; Fernández-Ibáñez, Pilar

    2007-09-25

    The results of a batch-process solar disinfection (SODIS) and solar photocatalytic disinfection (SPCDIS) on drinking water contaminated with Cryptosporidium are reported. Cryptosporidium parvum oocyst suspensions were exposed to natural sunlight in Southern Spain and the oocyst viability was evaluated using two vital dyes [4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)]. SODIS exposures (strong sunlight) of 8 and 12h reduced oocyst viability from 98% (+/-1.3%) to 11.7% (+/-0.9%) and 0.3% (+/-0.33%), respectively. SODIS reactors fitted with flexible plastic inserts coated with TiO2 powder (SPCDIS) were found to be more effective than those which were not. After 8 and 16 h of overcast and cloudy solar irradiance conditions, SPCDIS reduced oocyst viability from 98.3% (+/-0.3%) to 37.7% (+/-2.6%) and 11.7% (+/-0.7%), respectively, versus to that achieved using SODIS of 81.3% (+/-1.6%) and 36.0% (+/-1.0%), respectively. These results confirm that solar disinfection of drinking water can be an effective household intervention against Cryptosporidium contamination.

  10. Characterization of a > 900,000-M(r) Cryptosporidium parvum sporozoite glycoprotein recognized by protective hyperimmune bovine colostral immunoglobulin.

    PubMed Central

    Petersen, C; Gut, J; Doyle, P S; Crabb, J H; Nelson, R G; Leech, J H

    1992-01-01

    Cryptosporidium parvum, a zoonotic Apicomplexan pathogen, causes profound diarrhea, malnutrition, and dehydration in patients with AIDS. A less severe, self-limited disease occurs in immunocompetent individuals, particularly children, animal handlers, and residents of the developing world. Very little is known about the biology of the organism, the pathophysiology of the disease process, or the mechanism of protective immunity. There is no effective therapy for cryptosporidiosis, but hyperimmune bovine colostrum raised against Cryptosporidium oocysts and sporozoites has ameliorated infection and disease in some patients with AIDS, and a variety of monoclonal antibodies, as well as hyperimmune bovine colostrum, have significantly reduced cryptosporidial infection of mice and calves. We report here the identification and initial characterization of a > 900,000-M(r) Cryptosporodium sporozoite glycoprotein (GP900) that is a prominent antigen recognized by protective hyperimmune bovine colostral immunoglobulin. Three of six murine anticryptosporidial monoclonal antibodies reacted with GP900, indicating that the molecule is highly immunogenic in mice as well as in cows. GP900 is Triton X-100 soluble and N glycosylated. Western blotting of the N-deglycosylated protein, detected with antibodies eluted from recombinant clones expressing a partial GP900 fusion protein, suggested that the polypeptide backbone of the glycoprotein has an M(r) of < 190,000. GP900 is encoded by a single-copy gene that resides on the largest Cryptosporidium chromosome. Images PMID:1452347

  11. Effect of storage media, temperature, and time on preservation of Cryptosporidium parvum oocysts for PCR analysis.

    PubMed

    Lalonde, L F; Gajadhar, A A

    2009-03-23

    The effect of storage media, temperature, and time on suitability of oocysts for use in subsequent molecular studies was examined. Cryptosporidium parvum oocysts were stored for 3, 6, 9, or 12 months in sterile dH(2)O, 70 or 95% ethanol, (room temperature [RT], 4, -20, and -70 degrees C), 10% formalin (RT and 4 degrees C), PBS, TE buffer, antibiotic-antimycotic (A-A) solution (4, -20 and -70 degrees C), 2% sulphuric acid, 2.5% potassium dichromate (4 degrees C), and gDNA from 10(4) oocysts was extracted in triplicate and subjected to PCR. To determine the effect of storage media on PCR sensitivity, gDNA from 10(4), 10(2), and 10(0) oocysts stored for 15 months in the media listed above at RT or 4 degrees C was also extracted in triplicate and subjected to PCR. At RT, ethanol was suitable for up to 15 months, while gDNA from oocysts stored in dH(2)O amplified inconsistently after 3 months. At 4 degrees C, all tested media except dH(2)O and formalin were suitable for storage of 10(4) oocysts up to 15 months, but only 70% ethanol, A-A solution, 2% sulphuric acid and 2.5% potassium dichromate supported amplification of gDNA from fewer than 100 oocysts. At -20 degrees C, 95% ethanol, PBS, or TE were suitable for up to 9 months, while 70% ethanol and A-A solution were effective up to 12 months, and gDNA from oocysts stored in dH(2)O was inconsistently amplified after 6 months. Storage at -70 degrees C for up to 12 months was effective regardless of media type. Oocysts stored in formalin at RT or 4 degrees C could not be amplified by PCR despite washing prior to gDNA extraction. To maintain gDNA quality suitable for PCR, it is recommended that coccidian oocysts be stored at -70 degrees C in dH(2)O, ethanol, PBS, TE or A-A solution, at 4 degrees C in A-A or ethanol, or at RT in ethanol where refrigerated storage is unavailable.

  12. Preferential Flow and Transport of Cryptosporidium Parvum Oocysts Through Vadose Zone: Experiments and Modeling

    NASA Astrophysics Data System (ADS)

    Darnault, C. J.; Darnault, C. J.; Garnier, P.; Kim, Y.; Oveson, K.; Jenkins, M.; Ghiorse, W.; Baveye, P.; Parlange, J.; Steenhuis, T.

    2001-12-01

    Oocysts of the protozoan Cryptosporidium parvum, when they contaminate drinking water supplies, can cause outbreaks of Cryptosporidiosis, a common waterborne disease. Of the different pathways by which oocysts can wind up in drinking water, one has received very little attention to date; because soils are often considered to be perfect filters, the transport of oocysts through the subsoil to groundwater by preferential flow is generally ignored. To evaluate its significance, three set of laboratory experiments investigated transport of oocysts through vadose zone. Experiment set I was carried out in a vertical 50 cm-long column filled with silica sand, under conditions known to foster fingered flow. Experiment set II investigates the effect of gas-water interfaces by modifying the hydrodynamical conditions in the sand columns with water-repellent sand barriers. Experiment III involved undisturbed soil columns subjected to macropores flow. The sand and soil columns were subjected to artificial rainfall and were allowed to reach steady-state. At that point, feces of contaminated calves were applied at the surface, along with a known amount of KCl to serve as tracer, and rainfall was continued at the same rate. The breakthrough of oocysts and Cl-, monitored in the effluent, demonstrate the importance of preferential flow - fingered flow and macropore flow - on the transport of oocysts through vadose zone. Peak oocyst concentrations were not appreciably delayed, compared to Cl-, and in some cases, occurred even before the Cl- peak. However, the numbers of oocysts present in the effluents were still orders of magnitude higher than the 5 to 10 oocysts per liter that are considerable sufficient to cause cryptosporidiosis in healthy adults. The transport of oocysts was simulated based on a partitioning the soil profile in both a distribution zone and a preferential zone, In particular, the model simulates accurately the markedly asymmetric breakthrough patterns, and the

  13. Calf-level risk factors for neonatal diarrhea and shedding of Cryptosporidium parvum in Ontario dairy calves.

    PubMed

    Trotz-Williams, Lise A; Wayne Martin, S; Leslie, Kenneth E; Duffield, Todd; Nydam, Daryl V; Peregrine, Andrew S

    2007-11-15

    This work was conducted to investigate calf-level factors that influence the risk of neonatal diarrhea and shedding of Cryptosporidium parvum oocysts in calves, on dairy farms in Ontario with histories of calf diarrhea or cryptosporidiosis. Fecal samples were collected weekly for 4 weeks from each of 1045 calves under 30 days of age on 11 dairy farms in south-western Ontario during the summer of 2003 and the winter of 2004. A questionnaire designed to gather information on calf-level management factors was administered on farm for each calf in the study. Samples were examined for C. parvum oocysts by microscopy, and a subset of specimens was also tested for enterotoxigenic Escherichia coli, Salmonella, bovine rotavirus and bovine coronavirus. The consistency of each sample was scored and recorded at the time of collection in order to assess the presence or absence of diarrhea. In addition, a blood sample was taken from each calf upon enrollment in the study, for assessment of maternal antibody transfer and for polymerase chain reaction testing for persistent bovine viral diarrhea virus infection. Using the GLLAMM function in Stata 9.0, multilevel regression techniques were employed to investigate associations between management practices and the risk of C. parvum shedding or diarrhea. C. parvum oocysts were detected in the feces of 78% of the 919 calves from which all four fecal samples had been collected. Furthermore, 73% of the 846 calves for which all four fecal consistency scores had been recorded were diarrheic at the time of collection of at least one sample. Significant predictors of the calf-level risk of C. parvum shedding included the use of calf diarrhea prophylaxis in pregnant cows, and the type of maternity facilities in which the calves were born. Factors associated with an increased risk of diarrhea were leaving the calf with the dam for more than an hour after birth, and the birth of a calf in the summer as opposed to winter. Calves shedding C

  14. Presence of Cryptosporidium parvum and Giardia lamblia in water samples from Southeast Asia: towards an integrated water detection system.

    PubMed

    Kumar, Thulasi; Abd Majid, Mohamad Azlan; Onichandran, Subashini; Jaturas, Narong; Andiappan, Hemah; Salibay, Cristina C; Tabo, Hazel A L; Tabo, Norbel; Dungca, Julieta Z; Tangpong, Jitbanjong; Phiriyasamith, Sucheep; Yuttayong, Boonyaorn; Polseela, Raxsina; Do, Binh Nhu; Sawangjaroen, Nongyao; Tan, Tian-Chye; Lim, Yvonne A L; Nissapatorn, Veeranoot

    2016-01-13

    Access to clean and safe drinking water that is free from pathogenic protozoan parasites, especially Cryptosporidium parvum and Giardia lamblia that cause gastrointestinal illness in humans, is still an issue in Southeast Asia (SEA). This study is the first attempt to detect the aforementioned protozoan parasites in water samples from countries in SEA, using real-time polymerase chain reaction (qPCR) assays. A total of 221 water samples of 10 l each were collected between April and October 2013 from Malaysia (53), Thailand (120), the Philippines (33), and Vietnam (15). A physicochemical analysis was conducted. The water samples were processed in accordance with the US Environmental Protection Agency's methods 1622/1623.1, microscopically observed and subsequently screened using qPCR assays. Cryptosporidium oocysts were detected in treated water samples from the Philippines (1/10), with a concentration of 0.06 ± 0.19 oocyst/L, and untreated water samples from Thailand (25/93), Malaysia (17/44), and the Philippines (11/23), with concentrations ranging from 0.13 ± 0.18 to 0.57 ± 1.41 oocyst/L. Giardia cysts were found in treated water samples from the Philippines (1/10), with a concentration of 0.02 ± 0.06 cyst/L, and in untreated water samples from Thailand (20/93), Vietnam (5/10), Malaysia (22/44), and the Philippines (16/23), with concentrations ranging from 0.12 ± 0.3 to 8.90 ± 19.65 cyst/L. The pathogens C. parvum and G. lamblia were detected using using qPCR assays by targeting the 138-bp fragment and the small subunit gene, respectively. C. parvum was detected in untreated water samples from the Philippines (1/23) and Malaysia (2/44), whilst, G. lamblia detected was detected in treated water samples from the Philippines (1/10) and in untreated water samples from Thailand (21/93), Malaysia (12/44), and the Philippines (17/23). Nitrate concentration was found to have a high positive correlation with (oo)cyst (0.993). The presence of

  15. Detection of the Cryptosporidium parvum "human" genotype in a dugong (Dugong dugon).

    PubMed

    Morgan, U M; Xiao, L; Hill, B D; O'Donoghue, P; Limor, J; Lal, A; Thompson, R C

    2000-12-01

    The Cryptosporidium "human" genotype was identified in a paraffin-embedded tissue section from a dugong (Dugong dugon) by 2 independent laboratories. DNA sequencing and polymerase chain reaction/restriction fragment length polymorphism analysis of the 18S ribosomal RNA gene and the acetyl CoA synthethase gene clearly identified the genotype as that of the Cryptosporidium variant that infects humans. This is the first report of the human Cryptosporidium genotype in a nonprimate host.

  16. Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR

    PubMed Central

    Kaucner, Christine; Stinear, Timothy

    1998-01-01

    We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable Cryptosporidium parvum oocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample concentrates with simultaneous detection of viable C. parvum oocysts, Giardia cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-μl packed pellet volumes of concentrates from creek and river water samples. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water samples. Sample volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable Giardia cysts increased from 24% by IF microscopy to 69% by RT-PCR. Viable C. parvum oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable Cryptosporidium spp. in four samples by IF microscopy (14%), suggesting that Cryptosporidium species other than C. parvum were present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology. PMID:9572946

  17. Cryptosporidium parvum: identification of a new surface adhesion protein on sporozoite and oocyst by screening of a phage-display cDNA library.

    PubMed

    Yao, Longquan; Yin, Jigang; Zhang, Xichen; Liu, Quan; Li, Jianhua; Chen, Lifeng; Zhao, Yueping; Gong, Pengtao; Liu, Chengwu

    2007-04-01

    Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host interaction and the molecular mechanisms involved in the pathogenesis are unknown. In this study we described a novel phage display method to identify surface adhesion proteins of C. parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum expressed on the surface of T7 phage was screened with intestinal epithelial cells (IECs) from the newborn Cryptosporidium-free Holstein calves. Proteins that selectively and specifically bound to IECs were then enriched using a multi-step panning procedure. Two proteins of C. parvum were selected, one was previously reported (p23), which was an important surface adhesion protein; the other was a novel surface adherence protein (CP12). Sequence analysis showed that CP12 has a N-terminal signal peptide, a transmembrane region, a N-glycosylation site, a casein kinase II phosphorylation site and two N-myristoylation sites. Immunofluorescence assay (IFA) using antibody specific for rCP12 demonstrated that the antibody can specifically bind the surface of sporozoite and oocyst, especially apical region of sporozoite. The surface localization of CP12 and its involvement in the host-parasite interaction suggest that it may serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.

  18. Spinacia oleracea L. leaf stomata harboring Cryptosporidium parvum oocysts: A potential threat for food safety

    USDA-ARS?s Scientific Manuscript database

    Scientific literature documents the prevalence of Cryptosporidium oocysts in irrigation waters and on fresh produce. In the present study spinach leaves were experimentally exposed to Cryptosporidium oocysts which were subsequently irrigated with clean water daily for 5 days. As determined by confoc...

  19. Long-term health effects after resolution of acute Cryptosporidium parvum infection: a 1-year follow-up of outbreak-associated cases.

    PubMed

    Stiff, Rhianwen E; Davies, Angharad P; Mason, Brendan W; Hutchings, Hayley A; Chalmers, Rachel M

    2017-10-06

    We describe a longitudinal study carried out in an adult outbreak-associated cohort to investigate health effects, including post-infectious irritable bowel syndrome, occurring after resolution of acute Cryptosporidium parvum infection. New symptoms self-reported up to 12 months included: weight loss (31 %), abdominal pain (38 %), diarrhoea (33 %), eye pain (9 %), joint pain (33 %), fatigue (22 %) and symptoms consistent with irritable bowel syndrome (IBS) (28 %). Two people were medically diagnosed with IBS. This study describes for the first time sequelae reported by patients up to 12 months after infection with C. parvum, which appear to be similar to those described with C. hominis.

  20. Comparison of immunofluorescence assay and immunomagnetic electrochemiluminescence in detection of Cryptosporidium parvum oocysts in karst water samples.

    PubMed

    Kuczynska, Ewa; Boyer, Douglas G; Shelton, Daniel R

    2003-04-01

    Immunofluorescence assay (IFA) and immunomagnetic electrochemiluminescence (IM-ECL) were used for comparison of the percent recovery of Cryptosporidium parvum in environmental water samples obtained from a spring draining a karst basin. The monoclonal antibodies to C. parvum, isotype IgG3 were used for optimization of the IM-ECL protocol. The combination of biotinylated and TAG-labeled anti-C. parvum antibodies with the streptavidin beads gave a linear regression slope for log ECL vs. log fresh oocysts of 0.79 (from 5 to 5,000 oocysts), which indicates a constant ECL signal per oocyst. Standard curves gave a dynamic range of 5 to 5,000 oocysts/ml (fresh) and 10 to 100,000 cells/ml (4-month-old oocysts) with the maximum limit of linear detection higher than 100,000. The linear slope of 4-month-old oocysts decreased to 0.62, which indicates that ECL signal is a function of oocyst age. The experiment associated with bead storage time shows that even after 4 months of storage of the biotinylated antibodies, the complex retains the ability for binding the oocysts and generating the ECL signal. Based on the IFA results in the experiment evaluating different protocols for oocysts recovery from karst water samples, the most efficient protocol involved dispersion, followed by flotation and immunomagnetic separation (IMS) (24% recovery). The ECL results obtained in that experiment were very similar to the results obtained in the IFA method, which indicates that the IM-ECL method is accurate. Results of the IFA in the study of the prevalence of C. parvum in the groundwater showed that oocysts were present in 78% of 1 L water samples with average number of oocysts of 6.4+/-5.5 and ranged from 0 (13 samples) to 23.3 (2 samples). The ECL signal generated from these water samples ranged from 3,771 to 622 (average 1,620+/-465). However, the background value estimated in groundwater samples with low number of oocysts detected by IFA was highly variable and elevated (from 3,702 to

  1. Removal and fate of Cryptosporidium parvum, Clostridium perfringens and small-sized centric diatoms (Stephanodiscus hantzschii) in slow sand filters.

    PubMed

    Hijnen, Wim A M; Dullemont, Yolanda J; Schijven, Jack F; Hanzens-Brouwer, Anke J; Rosielle, Martine; Medema, Gertjan

    2007-05-01

    The decimal elimination capacity (DEC) of slow sand filtration (SSF) for Cryptosporidium parvum was assessed to enable quantitative microbial risk analysis of a drinking water production plant. A mature pilot plant filter of 2.56m(2) was loaded with C. parvum oocysts and two other persistent organisms as potential surrogates; spores of Clostridium perfringens (SCP) and the small-sized (4-7microm) centric diatom (SSCD) Stephanodiscus hantzschii. Highly persistent micro-organisms that are retained in slow sand filters are expected to accumulate and eventually break through the filter bed. To investigate this phenomenon, a dosing period of 100 days was applied with an extended filtrate monitoring period of 150 days using large-volume sampling. Based on the breakthrough curves the DEC of the filter bed for oocysts was high and calculated to be 4.7log. During the extended filtrate monitoring period the spatial distribution of the retained organisms in the filter bed was determined. These data showed little risk of accumulation of oocysts in mature filters most likely due to predation by zooplankton. The DEC for the two surrogates, SCP and SSCD, was 3.6 and 1.8log, respectively. On basis of differences in transport behaviour, but mainly because of the high persistence compared to the persistence of oocysts, it was concluded that both spores of sulphite-reducing clostridia (incl. SCP) and SSCD are unsuited for use as surrogates for oocyst removal by slow sand filters. Further research is necessary to elucidate the role of predation in Cryptosporidium removal and the fate of consumed oocysts.

  2. The fine structure of sexual stage development and sporogony of Cryptosporidium parvum in cell-free culture.

    PubMed

    Aldeyarbi, Hebatalla M; Karanis, Panagiotis

    2016-05-01

    The sexual stages and new oocysts development of Cryptosporidium parvum were investigated in a cell-free culture system using transmission electron microscopy (TEM). Sexual development was extremely rapid after inoculation of oocysts into the medium. The process began within 1/2-12 h and was completed with new oocyst formation 120 h post-inoculation. The macrogamonts were bounded by two membranes and had amylopectin granules and two distinct types of wall-forming bodies. The microgamonts had a large nucleus showing lobe projections and condensation of chromatin, giving rise to peripherally budding microgametes. The microgametes contained a large area of granular substance containing groups of microtubules surrounding the electron-dense nucleus. In some instances, the dividing microgamy was observed in cell-free cultures with no preceding merogonic process. Fertilization was observed with the bullet-shaped microgamete penetrating an immature macrogamont at 24 and 216 h. The new thin- and thick-walled oocysts had a large residuum with polysaccharide granules and sporogony noted inside these oocysts. Novel immature four-layer walled thick oocysts with irregular knob-like protrusions on the outer layer resembling the immature Eimeria oocysts were also observed. The present study confirms the gametogony and sporogony of C. parvum in cell-free culture and describes their ultra-structure for the first time.

  3. Stress-induced Hsp70 gene expression and inactivation of Cryptosporidium parvum oocysts by chlorine-based oxidants.

    PubMed

    Bajszár, George; Dekonenko, Alexander

    2010-03-01

    Our research on the mechanisms of action of chlorine-based oxidants on Cryptosporidium parvum oocysts in water revealed a dual-phase effect: (i) response to oxidative stress, which was demonstrated by induced expression of the Hsp70 heat shock gene, and (ii) oocyst inactivation as a result of long-term exposure to oxidants. The relative biocidal effects of sodium hypochlorite (bleach) and electrolytically generated mixed oxidant solution (MOS) on C. parvum oocysts were compared at identical free chlorine concentrations. Oocyst inactivation was determined by quantitative reverse transcription-PCR (qRT-PCR) amplification of the heat-induced Hsp70 mRNA and compared with tissue culture infectivity. According to both assays, within the range between 25 and 250 mg/liter free chlorine and with 4 h contact time, MOS exhibits a higher efficacy in oocyst inactivation than hypochlorite. Other RNA-based viability assays, aimed at monitoring the levels of beta-tubulin mRNA and 18S rRNA, showed relatively slow decay rates of these molecules following disinfection by chlorine-based oxidants, rendering these molecular diagnostic viability markers inappropriate for disinfection efficacy assessment.

  4. Stress-Induced Hsp70 Gene Expression and Inactivation of Cryptosporidium parvum Oocysts by Chlorine-Based Oxidants▿

    PubMed Central

    Bajszár, George; Dekonenko, Alexander

    2010-01-01

    Our research on the mechanisms of action of chlorine-based oxidants on Cryptosporidium parvum oocysts in water revealed a dual-phase effect: (i) response to oxidative stress, which was demonstrated by induced expression of the Hsp70 heat shock gene, and (ii) oocyst inactivation as a result of long-term exposure to oxidants. The relative biocidal effects of sodium hypochlorite (bleach) and electrolytically generated mixed oxidant solution (MOS) on C. parvum oocysts were compared at identical free chlorine concentrations. Oocyst inactivation was determined by quantitative reverse transcription-PCR (qRT-PCR) amplification of the heat-induced Hsp70 mRNA and compared with tissue culture infectivity. According to both assays, within the range between 25 and 250 mg/liter free chlorine and with 4 h contact time, MOS exhibits a higher efficacy in oocyst inactivation than hypochlorite. Other RNA-based viability assays, aimed at monitoring the levels of β-tubulin mRNA and 18S rRNA, showed relatively slow decay rates of these molecules following disinfection by chlorine-based oxidants, rendering these molecular diagnostic viability markers inappropriate for disinfection efficacy assessment. PMID:20118357

  5. Hydrologic and vegetative removal of Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii Surrogate microspheres in coastal wetlands.

    PubMed

    Hogan, Jennifer N; Daniels, Miles E; Watson, Fred G; Oates, Stori C; Miller, Melissa A; Conrad, Patricia A; Shapiro, Karen; Hardin, Dane; Dominik, Clare; Melli, Ann; Jessup, David A; Miller, Woutrina A

    2013-03-01

    Constructed wetland systems are used to reduce pollutants and pathogens in wastewater effluent, but comparatively little is known about pathogen transport through natural wetland habitats. Fecal protozoans, including Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii, are waterborne pathogens of humans and animals, which are carried by surface waters from land-based sources into coastal waters. This study evaluated key factors of coastal wetlands for the reduction of protozoal parasites in surface waters using settling column and recirculating mesocosm tank experiments. Settling column experiments evaluated the effects of salinity, temperature, and water type ("pure" versus "environmental") on the vertical settling velocities of C. parvum, G. lamblia, and T. gondii surrogates, with salinity and water type found to significantly affect settling of the parasites. The mesocosm tank experiments evaluated the effects of salinity, flow rate, and vegetation parameters on parasite and surrogate counts, with increased salinity and the presence of vegetation found to be significant factors for removal of parasites in a unidirectional transport wetland system. Overall, this study highlights the importance of water type, salinity, and vegetation parameters for pathogen transport within wetland systems, with implications for wetland management, restoration efforts, and coastal water quality.

  6. Hydrologic and Vegetative Removal of Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii Surrogate Microspheres in Coastal Wetlands

    PubMed Central

    Hogan, Jennifer N.; Daniels, Miles E.; Watson, Fred G.; Oates, Stori C.; Miller, Melissa A.; Conrad, Patricia A.; Shapiro, Karen; Hardin, Dane; Dominik, Clare; Melli, Ann; Jessup, David A.

    2013-01-01

    Constructed wetland systems are used to reduce pollutants and pathogens in wastewater effluent, but comparatively little is known about pathogen transport through natural wetland habitats. Fecal protozoans, including Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii, are waterborne pathogens of humans and animals, which are carried by surface waters from land-based sources into coastal waters. This study evaluated key factors of coastal wetlands for the reduction of protozoal parasites in surface waters using settling column and recirculating mesocosm tank experiments. Settling column experiments evaluated the effects of salinity, temperature, and water type (“pure” versus “environmental”) on the vertical settling velocities of C. parvum, G. lamblia, and T. gondii surrogates, with salinity and water type found to significantly affect settling of the parasites. The mesocosm tank experiments evaluated the effects of salinity, flow rate, and vegetation parameters on parasite and surrogate counts, with increased salinity and the presence of vegetation found to be significant factors for removal of parasites in a unidirectional transport wetland system. Overall, this study highlights the importance of water type, salinity, and vegetation parameters for pathogen transport within wetland systems, with implications for wetland management, restoration efforts, and coastal water quality. PMID:23315738

  7. Serum and colostrum antibody responses induced by jet-injection of sheep with DNA encoding a Cryptosporidium parvum antigen.

    PubMed

    Jenkins, M; Kerr, D; Fayer, R; Wall, R

    1995-12-01

    In an effort to generate high titer colostrum for immunotherapy of cryptosporidiosis, a study was conducted to test the efficacy of immunizing sheep with recombinant plasmid DNA (pCMV-CP15/60) encoding epitopes of 15 and 60 kDa surface antigens of Cryptosporidium parvum sporozoites. The plasmid DNA was used to immunize preparturient ewes at three dose levels by jet-injection into either hind limb muscle (IM) or mammary tissue (IMAM). Regardless of route of injection, a dose-dependent anti-CP15/60 immunoglobulin response was observed in sera and colostrum from sheep immunized with pCMV-CP15/60 plasmid DNA. High titer antibody responses were observed in one of three animals per group receiving an IM injection of 100 or 1000 micrograms pCMV-CP15/60. IMAM immunization with 100 or 1000 micrograms pCMV-CP15/60 plasmid DNA elicited higher titer colostrum responses and more consistent serum responses compared to IM injections. A negligible serum and colostrum anti-CP15/60 response was observed in ewes injected IM with 10 micrograms pCMV-CP15/60 or 1000 micrograms control plasmid DNA. Immunoblotting of native C. parvum sporozoite/oocyst protein with hyperimmune serum and colostrum corroborated the increased titers against CP15/60 antigen. Serum and colostrum antibodies from pCMV-CP15/60-immunized sheep were eluted from native CP15 protein and bound a surface antigen of C. parvum sporozoites as indicated by indirect immunofluorescence staining.

  8. Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts.

    PubMed Central

    Jenkins, M B; Anguish, L J; Bowman, D D; Walker, M J; Ghiorse, W C

    1997-01-01

    The ability to determine inactivation rates of Cryptosporidium parvum oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite in watersheds. We compared a dye permeability assay, which tests the differential uptake of the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) by the oocysts (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992), with an in vitro excystation assay, which tests their ability to excyst and, thus, their metabolic potential and potential for infectivity (J.B. Rose, H. Darbin, and C.P. Gerba, Water Sci. Technol. 20:271-276, 1988). Formaldehyde-fixed (killed) oocysts and untreated oocysts were permeabilized with sodium hypochlorite and subjected to both assays. The results of the dye permeability assays were the same, while the excystation assay showed that no excystation occurred in formaldehyde-fixed oocysts. This confirmed that oocyst wall permeability, rather than metabolic activity potential, was the basis of the dye permeability viability assessment. A previously developed protocol (L. J. Anguish and W. C. Ghiorse, Appl. Environ. Microbiol. 63:724-733, 1997) for determining viability of oocysts in soil and sediment was used to examine further the use of oocyst permeability status as an indicator of oocyst viability in fecal material stored at 4 degrees C and in water at various temperatures. Most of the oocysts in fresh calf feces were found to be impermeable to the fluorochromes. They were also capable of excystation, as indicated by the in vitro excystation assay, and were infective, as indicated by a standard mouse infectivity assay. The dye permeability assay further showed that an increase in the intermediate population of oocysts permeable to DAPI but not to PI occurred over time. There was also a steady population of oocysts permeable to both dyes. Further experiments with purified oocysts suspended in

  9. Method for detection and enumeration of Cryptosporidium parvum oocysts in feces, manures, and soils.

    PubMed

    Kuczynska, E; Shelton, D R

    1999-07-01

    Eight concentration and purification methods were evaluated to determine percentages of recovery of Cryptosporidium parvum oocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 10(2), 10(3), 10(4), and 10(5) oocysts g of bovine feces-1. The percentages of recovery ranged from 10.8% (25 oocysts g-1) to 17.0% (10(4) oocysts g-1) (r2 = 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces-1. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (10(4) oocysts g-1). The percentages of recovery were highest for bovine feces (17. 0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO4), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris-0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay

  10. Method for Detection and Enumeration of Cryptosporidium parvum Oocysts in Feces, Manures, and Soils

    PubMed Central

    Kuczynska, Ewa; Shelton, Daniel R.

    1999-01-01

    Eight concentration and purification methods were evaluated to determine percentages of recovery of Cryptosporidium parvum oocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 102, 103, 104, and 105 oocysts g of bovine feces−1. The percentages of recovery ranged from 10.8% (25 oocysts g−1) to 17.0% (104 oocysts g−1) (r2 = 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces−1. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (104 oocysts g−1). The percentages of recovery were highest for bovine feces (17.0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO4), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris–0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay

  11. The effect of cyanuric acid on the disinfection rate of Cryptosporidium parvum in 20-ppm free chlorine.

    PubMed

    Shields, Joan M; Arrowood, Michael J; Hill, Vincent R; Beach, Michael J

    2009-03-01

    Cyanuric acid is used to stabilize free chlorine to reduce photodegradation in outdoor swimming pools. While there have been numerous studies examining its effect on the disinfection rates of bacteria and viruses, it is not known whether cyanuric acid can significantly impact the effectiveness of hyperchlorination for inactivating Cryptosporidium oocysts present in fecally-contaminated swimming pools. This study examined the effect of cyanuric acid on the disinfection rate of Cryptosporidium parvum under swimming pool hyperchlorination conditions (20 mg/ml free chlorine). When 50 mg/L cyanuric acid was present there was a 0.70-log10 reduction in oocyst viability after 10 hours as compared to a 3.7-log10 reduction without cyanuric acid. Aids to remediation, such as decreasing the pH to enhance the germicidal efficiency of the free chlorine and doubling the amount of free chlorine residual, were still unable to achieve a 3-log10 reduction. Current public health recommendations for hyperchlorination and pool remediation are insufficient for pools using cyanurate-stabilized chlorine to achieve a three log inactivation of the parasite.

  12. Fluorescent microspheres as surrogates in evaluating the efficacy of riverbank filtration for removing Cryptosporidium parvum oocysts and other pathogens

    USGS Publications Warehouse

    Harvey, Ronald W.; Metge, David W.; Sheets, Rodney A.; Jasperse, Jay

    2011-01-01

    A major benefit of riverbank filtration (RBF) is that it provides a relatively effective means for pathogen removal. There is a need to conduct more injection-and-recovery transport studies at operating RBF sites in order to properly assess the combined effects of the site heterogeneities and ambient physicochemical conditions, which are difficult to replicate in the lab. For field transport studies involving pathogens, there is considerable interest in using fluorescent carboxylated microspheres (FCM) as surrogates, because they are chemically inert, negatively charged, easy to detect, available in a wide variety of sizes, and have been found to be nonhazardous in tracer applications. Although there have been a number of in-situ studies comparing the subsurface transport behaviors of FCM to those of bacteria and viruses, much less is known about their suitability for investigations of protozoa. Oocysts of the intestinal protozoan pathogen Cryptosporidium spp are of particular concern for many RBF operations because of their ubiquity and persistence in rivers and high resistance to chlorine disinfection. Although microspheres often have proven to be less-than-ideal analogs for capturing the abiotic transport behavior of viruses and bacteria, there is encouraging recent evidence regarding use of FCM as surrogates for C. parvum oocysts. This chapter discusses the potential of fluorescent microspheres as safe and easy-to-detect surrogates for evaluating the efficacy of RBF operations for removing pathogens, particularly Cryptosporidium, from source waters at different points along the flow path.

  13. Bioaccumulation and elimination of Cryptosporidium parvum oocysts in experimentally exposed Eastern oysters (Crassostrea virginica) held in static tank aquaria.

    PubMed

    Willis, Jessica E; McClure, J T; McClure, Carol; Spears, Jonathan; Davidson, Jeff; Greenwood, Spencer J

    2014-03-03

    A variety of human enteropathogens, including viruses, bacteria, and parasites, have been shown to bioaccumulate in suspension-feeding bivalve shellfish. Cryptosporidium parvum is a zoonotic protozoan parasite that has been detected in many shellfish species within both fecally contaminated and clean oyster growing areas across the globe. For this study, C. parvum oocysts (1000 and 10,000) were spiked into 10 L of water in static tank systems housing Crassostrea virginica. Oysters were either held in the contaminated aquaria for 7 days of exposure or were exposed for 24h and subsequently placed in a clean static tank system for the remainder of the trial. Individual oysters, fecal material, and tank water were analyzed for oocysts up to 7 days post-exposure via direct immunofluorescence. Oysters held under chronic exposure conditions gradually accumulated oocysts (1.5 or 34.4 oocysts/oyster/day for low or high dose exposure groups, respectively) between days 1 and 7, with an exponential uptake in oocysts observed within the first 24h post-exposure (mean uptake of 29.6 or 241.9 oocysts/oyster, respectively). Oysters that were transferred to clean water after 24h were capable of slowly depurating oocysts, following a linear trend. During chronic exposure trials 48-49% of the total spiked inoculum was recovered from oyster tissue, whereas 4.8-5.9% and 38-40% was recovered from tank water and from fecal material at day 7, respectively. In acute exposure trials, 30-31% of the total tank inoculum was found in oysters, suggesting that chronically exposed oysters were likely re-filtering some oocysts. Examinations of oyster fecal material from acute exposures revealed that 72-82% of oocysts recovered were already excreted at the time of oyster transfer (day 1), with only 18-28% being excreted during the static depuration phase. These data support that although most C. parvum oocysts are removed by C. virginica oysters within 24h, elimination after this point occurs slowly

  14. Pilot-Scale Pulsed UV Light Irradiation of Experimentally Infected Raspberries Suppresses Cryptosporidium parvum Infectivity in Immunocompetent Suckling Mice.

    PubMed

    Le Goff, L; Hubert, B; Favennec, L; Villena, I; Ballet, J J; Agoulon, A; Orange, N; Gargala, G

    2015-12-01

    Cryptosporidium spp., a significant cause of foodborne infection, have been shown to be resistant to most chemical food disinfectant agents and infective for weeks in irrigation waters and stored fresh vegetal produce. Pulsed UV light (PL) has the potential to inactivate Cryptosporidium spp. on surfaces of raw or minimally processed foods or both. The present study aimed to evaluate the efficacy of PL on viability and in vivo infectivity of Cryptosporidium parvum oocysts present on raspberries, a known source of transmission to humans of oocyst-forming apicomplexan pathogens. The skin of each of 20 raspberries was experimentally inoculated with five 10-μl spots of an oocyst suspension containing 6 × 10(7) oocysts per ml (Nouzilly isolate). Raspberries were irradiated by PL flashes (4 J/cm(2) of total fluence). This dose did not affect colorimetric or organoleptic characteristics of fruits. After immunomagnetic separation from raspberries, oocysts were bleached and administered orally to neonatal suckling mice. Seven days after infection, mice were euthanized, and the number of oocysts in the entire small intestine was individually assessed by immunofluorescence flow cytometry. Three of 12 and 12 of 12 inoculated mice that received 10 and 100 oocysts isolated from nonirradiated raspberries, respectively, were found infected. Four of 12 and 2 of 12 inoculated mice that received 10(3) and 10(4) oocysts from irradiated raspberries, respectively, were found infected. Oocyst counts were lower in animals inoculated with 10(3) and 10(4) oocysts from irradiated raspberries (92 ± 144 and 38 ± 82, respectively) than in animals infected with 100 oocysts from nonirradiated raspberries (35,785 ± 66,221, P = 0.008). PL irradiation achieved oocyst reductions of 2 and 3 log for an inoculum of 10(3) and 10(4) oocysts, respectively. The present pilot-scale evaluation suggests that PL is an effective mode of decontamination for raspberries and prompts further applicability

  15. Biotin- and Glycoprotein-Coated Microspheres as Surrogates for Studying Filtration Removal of Cryptosporidium parvum in a Granular Limestone Aquifer Medium.

    PubMed

    Stevenson, M E; Blaschke, A P; Toze, S; Sidhu, J P S; Ahmed, W; van Driezum, I H; Sommer, R; Kirschner, A K T; Cervero-Aragó, S; Farnleitner, A H; Pang, L

    2015-07-01

    Members of the genus Cryptosporidium are waterborne protozoa of great health concern. Many studies have attempted to find appropriate surrogates for assessing Cryptosporidium filtration removal in porous media. In this study, we evaluated the filtration of Cryptosporidium parvum in granular limestone medium by the use of biotin- and glycoprotein-coated carboxylated polystyrene microspheres (CPMs) as surrogates. Column experiments were carried out with core material taken from a managed aquifer recharge site in Adelaide, Australia. For the experiments with injection of a single type of particle, we observed the total removal of the oocysts and glycoprotein-coated CPMs, a 4.6- to 6.3-log10 reduction of biotin-coated CPMs, and a 2.6-log10 reduction of unmodified CPMs. When two different types of particles were simultaneously injected, glycoprotein-coated CPMs showed a 5.3-log10 reduction, while the uncoated CPMs displayed a 3.7-log10 reduction, probably due to particle-particle interactions. Our results confirm that glycoprotein-coated CPMs are the most accurate surrogates for C. parvum; biotin-coated CPMs are slightly more conservative, while unmodified CPMs are markedly overly conservative for predicting C. parvum removal in granular limestone medium. The total removal of C. parvum observed in our study suggests that granular limestone medium is very effective for the filtration removal of C. parvum and could potentially be used for the pretreatment of drinking water and aquifer storage recovery of recycled water. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Source water assessment and nonpoint sources of acutely toxic contaminants: A review of research related to survival and transport of Cryptosporidium parvum

    NASA Astrophysics Data System (ADS)

    Walker, Mark J.; Montemagno, Carlo D.; Jenkins, Michael B.

    1998-12-01

    Amendments to the Safe Drinking Water Act (PL-930123) in 1996 required that public water supply managers identify potential sources of contamination within contributing areas. Nonpoint sources of acutely toxic microbial contaminants, such as Cryptosporidium parvum, challenge current approaches to source identification and management as a first step toward developing management plans for public water supply protection. Little may be known about survival and transport in the field environment, prescribed practices may not be designed to manage such substances, and infective stages may be present in vast numbers and may resist water treatment and disinfection processes. This review summarizes research related to survival and transport of C. parvum oocysts, as an example of an acutely toxic contaminant with nonpoint sources in animal agriculture. It discusses ∥1) significance of infected domesticated animals as potential sources of C. parvum, (2) laboratory and field studies of survival and transport, and (3) approaches to source control in the context of public health protection.

  17. Deposition of Cryptosporidium parvum oocysts on natural organic matter surfaces: microscopic evidence for secondary minimum deposition in a radial stagnation point flow cell.

    PubMed

    Liu, Yuanyuan; Janjaroen, Dao; Kuhlenschmidt, Mark S; Kuhlenschmidt, Theresa B; Nguyen, Thanh H

    2009-02-03

    A radial stagnation point flow (RSPF) system combined with a microscope was used to determine the deposition kinetics of Cryptosporidium parvum oocysts on quartz surfaces and silica surfaces coated with Suwannee River natural organic matter (SRNOM) in solutions with different ionic strengths. Microscopic evidence of C. parvum oocysts entrapped in the secondary minimum energy well was presented to show that among the entrapped C. parvum oocysts some were washed away by the radial flow and some were able to transfer to deep primary minima and become irreversibly deposited. Experimental data were compared with simulation results obtained by the convective-diffusion equation and Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The experimental results suggested that surface charge heterogeneity led to a higher attachment efficiency at low ionic strength. In addition, the maximum attachment efficiency was less than 1 at high ionic strength due to steric interaction.

  18. Does the use of tubular digesters to treat livestock waste lower the risk of infection from Cryptosporidium parvum and Giardia lamblia?

    PubMed

    Kinyua, Maureen N; Wald, Ileana; Camacho-Céspedes, Fabricio; Izurieta, Ricardo; Haas, Charles N; Ergas, Sarina J

    2016-10-01

    Worldwide, high incidences of cryptosporidiosis and giardiasis are attributed to livestock waste. Quantitative microbial risk assessment can be used to estimate the risk of livestock related infections from Cryptosporidium parvum and Giardia lamblia. The objective of this paper was to assess the occupational and public health risks associated with management of raw and anaerobically digested livestock waste in two rural communities in Costa Rica based on fomite, soil and crop contamination and livestock waste management exposure pathways. Risks related to cattle waste were greater than swine waste due to cattle shedding more (oo)cysts. Cryptosporidium parvum also posed a greater risk than Giardia lamblia in all exposure pathways due to livestock shedding high loads of Cryptosporidium parvum oocysts and oocysts' lower inactivation rates during anaerobic digestion compared with Giardia lamblia cysts. The risk of infection from exposure to contaminated soil and crops was significantly lower for a community using tubular anaerobic digesters to treat livestock waste compared to a community where the untreated waste was applied to soil. The results indicate that treatment of livestock waste in small-scale tubular anaerobic digesters has the potential to significantly decrease the risk of infection below the World Health Organization's acceptable individual annual risk of infection (10(-4)).

  19. An evaluation of primers amplifying DNA targets for the detection of Cryptosporidium spp. using C. parvum HNJ-1 Japanese isolate in water samples.

    PubMed

    Leetz, Anna Susanne; Sotiriadou, Isaia; Ongerth, Jerry; Karanis, Panagiotis

    2007-09-01

    The performance of polymerase chain reaction (PCR) procedures for the detection of Cryptosporidium parvum HNJ-1 strain (genotype II) oocysts purified from mice using published protocols was evaluated. Oocysts were concentrated from fecal samples of infected severe combined immunodeficiency (SCID) mice by sucrose flotation and were then purified by immunomagnetic separation method. The genotype of C. parvum was established as type II by restriction fragment length polymorphism (RFLP) analysis. Water samples were spiked with different numbers of oocysts, determined by limiting dilution. Genomic DNA was extracted and used for PCR assays targeting various Cryptosporidium species genes (Beta-Tubulin, COWP, 70 kDa HSP, SSU rRNA, ITS1, TRAP-C1 and TRAP-C2 gene). DNA from oocyst numbers of more than 1 x 10(4) was detected using each of the primers. However, when using lower oocyst numbers, the tools based on 9 of the 16 different primer assays gave sufficient results. Assays using the remaining seven primers gave less than satisfactory results. A new primer set, named VKSS-F1/2 and VKSS-R1/2, that target the 18 SSU rRNA gene of C. parvum was constructed and applied. The VKSS-F1/2 and VKSS-R1/2 assays amplified DNA isolated from spiked samples in 206 of 211 trials (97.6%). This illustrates the difficulty of detecting low numbers of Cryptosporidium spp. oocysts by molecular methods when working with environmental samples.

  20. Evaluation of the BD MAX Enteric Parasite Panel for the detection of Cryptosporidium parvum/hominis, Giardia duodenalis and Entamoeba histolytica.

    PubMed

    Perry, Michael D; Corden, Sally A; Lewis White, P

    2017-08-01

    Conventional laboratory detection methods for gastrointestinal parasites are time consuming, require considerable technical expertise and may suffer from poor analytical sensitivity. This study sought to evaluate the automated BD MAX Enteric Parasite Panel (EPP) for the detection of Cryptosporidium parvum/hominis, Entamoeba histolytica and Giardia duodenalis.Methodolgy. A total of 104 known positive samples (43 Cryptosporidium parvum/hominis and 61 G. duodenalis), 15 simulated samples (E. histolytica and other Entamoeba species) and 745 patient stool samples, submitted for enteric pathogen culture and microscopy, were inoculated into BD MAX EPP sample buffer tubes (SBTs). All specimens were blinded and tested within 7 days of SBT inoculation using the BD MAX EPP assay with results compared to those generated by microscopy.Results/Key findings. Combining the results from the known positive samples and anonymously tested patient samples, the sensitivity of the BD MAX EPP assay was 100 % for both Cryptosporidium spp. and G. duodenalis. Specificities of 99.7 and 98.9 % were calculated for the detection of Cryptosporidium spp. and G. duodenalis respectively. Insufficient clinical specimen data was available to determine the performance of the assay for E. histolytica detection. The findings of this study indicate that the BD MAX EPP is suitable for the detection of Cryptosporidium parvum/hominis and G. duodenalis from clinical specimens with reduced hands-on time and complexity compared to microscopy. Results for the detection of E. histolytica were promising although further work is required to evaluate the assay for the detection of this pathogen.

  1. Cryptosporidium parvum vaccine candidates are incompletely modified with O-linked-N-acetylgalactosamine or contain N-terminal N-myristate and S-palmitate.

    PubMed

    Haserick, John R; Klein, Joshua A; Costello, Catherine E; Samuelson, John

    2017-01-01

    Cryptosporidium parvum (studied here) and Cryptosporidium hominis are important causes of diarrhea in infants and immunosuppressed persons. C. parvum vaccine candidates, which are on the surface of sporozoites, include glycoproteins with Ser- and Thr-rich domains (Gp15, Gp40, and Gp900) and a low complexity, acidic protein (Cp23). Here we used mass spectrometry to determine that O-linked GalNAc is present in dense arrays on a glycopeptide with consecutive Ser derived from Gp40 and on glycopeptides with consecutive Thr derived from Gp20, a novel C. parvum glycoprotein with a formula weight of ~20 kDa. In contrast, the occupied Ser or Thr residues in glycopeptides from Gp15 and Gp900 are isolated from one another. Gly at the N-terminus of Cp23 is N-myristoylated, while Cys, the second amino acid, is S-palmitoylated. In summary, C. parvum O-GalNAc transferases, which are homologs of host enzymes, densely modify arrays of Ser or Thr, as well as isolated Ser and Thr residues on C. parvum vaccine candidates. The N-terminus of an immunodominant antigen has lipid modifications similar to those of host cells and other apicomplexan parasites. Mass spectrometric demonstration here of glycopeptides with O-glycans complements previous identification C. parvum O-GalNAc transferases, lectin binding to vaccine candidates, and human and mouse antibodies binding to glycopeptides. The significance of these post-translational modifications is discussed with regards to the function of these proteins and the design of serological tests and vaccines.

  2. Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum *

    PubMed Central

    Matsubayashi, Makoto; Teramoto-Kimata, Isao; Uni, Shigehiko; Lillehoj, Hyun S.; Matsuda, Haruo; Furuya, Masaru; Tani, Hiroyuki; Sasai, Kazumi

    2013-01-01

    The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis. PMID:24085304

  3. BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

    EPA Science Inventory

    An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

  4. BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

    EPA Science Inventory

    An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

  5. Removals of cryptosporidium parvum oocysts and cryptosporidium-sized polystyrene microspheres from swimming pool water by diatomaceous earth filtration and perlite-sand filtration.

    PubMed

    Lu, Ping; Amburgey, James E; Hill, Vincent R; Murphy, Jennifer L; Schneeberger, Chandra L; Arrowood, Michael J; Yuan, Tao

    2017-06-01

    Removal of Cryptosporidium-sized microspheres and Cryptosporidium parvum oocysts from swimming pools was investigated using diatomaceous earth (DE) precoat filtration and perlite-sand filtration. In pilot-scale experiments, microsphere removals of up to 2 log were obtained with 0.7 kg·DE/m(2) at a filtration rate of 5 m/h. A slightly higher microsphere removal (2.3 log) was obtained for these DE-precoated filters when the filtration rate was 3.6 m/h. Additionally, pilot-scale perlite-sand filters achieved greater than 2 log removal when at least 0.37 kg/m(2) of perlite was used compared to 0.1-0.4 log removal without perlite both at a surface loading rate of 37 m/h. Full-scale testing achieved 2.7 log of microspheres and oocysts removal when 0.7 kg·DE/m(2) was used at 3.6 m/h. Removals were significantly decreased by a 15-minute interruption of the flow (without any mechanical agitation) to the DE filter in pilot-scale studies, which was not observed in full-scale filters. Microsphere removals were 2.7 log by perlite-sand filtration in a full-scale swimming pool filter operated at 34 m/h with 0.5 kg/m(2) of perlite. The results demonstrate that either a DE precoat filter or a perlite-sand filter can improve the efficiency of removal of microspheres and oocysts from swimming pools over a standard sand filter under the conditions studied.

  6. Cryptosporidium parvum and Cyclospora cayetanensis: a review of laboratory methods for detection of these waterborne parasites.

    PubMed

    Quintero-Betancourt, Walter; Peele, Emily R; Rose, Joan B

    2002-05-01

    Cryptosporidium and Cyclospora are obligate, intracellular, coccidian protozoan parasites that infest the gastrointestinal tract of humans and animals causing severe diarrhea illness. In this paper, we present an overview of the conventional and more novel techniques that are currently available to detect Cryptosporidium and Cyclospora in water. Conventional techniques and new immunological and genetic/molecular methods make it possible to assess the occurrence, prevalence, virulence (to a lesser extent), viability, levels, and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne oocysts. These steps have been optimized to such an extent that low levels of naturally occurring Cryptosporidium oocysts can be efficiently recovered from water. The filtration systems developed in the US and Europe trap oocysts more effectively and are part of the standard methodologies for environmental monitoring of Cryptosporidium oocysts in source and treated water. Purification techniques such as immunomagnetic separation and flow cytometry with fluorescent activated cell sorting impart high capture efficiency and selective separation of oocysts from sample debris. Monoclonal antibodies with higher avidity and specificity to oocysts in water concentrates have significantly improved the detection and enumeration steps. To date, PCR-based detection methods allow us to differentiate the human pathogenic Cryptosporidium parasites from those that do not infect humans, and to track the source of oocyst contamination in the environment. Cell culture techniques are now used to examine oocyst viability. While fewer studies have focused on Cyclospora cayetanensis, the parasite has been successfully detected in drinking water and wastewater using current methods to recover Cryptosporidium oocysts. More research is needed for monitoring of Cyclospora in the environment

  7. Point-of-Use Removal of Cryptosporidium parvum from Water: Independent Effects of Disinfection by Silver Nanoparticles and Silver Ions and by Physical Filtration in Ceramic Porous Media.

    PubMed

    Abebe, Lydia S; Su, Yi-Hsuan; Guerrant, Richard L; Swami, Nathan S; Smith, James A

    2015-11-03

    Ceramic water filters (CWFs) impregnated with silver nanoparticles are a means of household-level water treatment. CWFs remove/deactivate microbial pathogens by employing two mechanisms: metallic disinfection and physical filtration. Herein we report on the independent effects of silver salt and nanoparticles on Cryptosporidium parvum and the removal of C. parvum by physical filtration in porous ceramic filter media. Using a murine (mouse) model, we observed that treatment of oocysts with silver nitrate and proteinate-capped silver nanoparticles resulted in decreased infection relative to untreated oocysts. Microscopy and excystation experiments were conducted to support the disinfection investigation. Heat and proteinate-capped silver-nanoparticle treatment of oocysts resulted in morphological modifications and decreased excystation rates of sporozoites. Subsequently, disk-shaped ceramic filters were produced to investigate the transport of C. parvum. Two factors were varied: sawdust size and clay-to-sawdust ratio. Five disks were prepared with combinations of 10, 16, and 20 mesh sawdust and sawdust percentage that ranged from 9 to 11%. C. parvum removal efficiencies ranged from 1.5 log (96.4%) to 2.1 log (99.2%). The 16-mesh/10% sawdust had the greatest mean reduction of 2.1-log (99.2%), though there was no statistically significant difference in removal efficiency. Based on our findings, physical filtration and silver nanoparticle disinfection likely contribute to treatment of C. parvum for silver impregnated ceramic water filters, although the contribution of physical filtration is likely greater than silver disinfection.

  8. Inhibition of Calcium-Dependent Protein Kinase 1 (CDPK1) In Vitro by Pyrazolopyrimidine Derivatives Does Not Correlate with Sensitivity of Cryptosporidium parvum Growth in Cell Culture

    PubMed Central

    Kuhlenschmidt, Theresa B.; Rutaganira, Florentine U.; Long, Shaojun; Tang, Keliang; Shokat, Kevan M.; Kuhlenschmidt, Mark S.

    2015-01-01

    Cryptosporidiosis is a serious diarrheal disease in immunocompromised patients and malnourished children, and treatment is complicated by a lack of adequate drugs. Recent studies suggest that the natural occurrence of a small gatekeeper residue in serine threonine calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium parvum might be exploited to target this enzyme and block parasite growth. Here were explored the potency with which a series of pyrazolopyrimidine analogs, which are selective for small gatekeeper kinases, inhibit C. parvum CDPK1 and block C. parvum growth in tissue culture in vitro. Although these compounds potently inhibited kinase activity in vitro, most had no effect on parasite growth. Moreover, among those that were active against parasite growth, there was a very poor correlation with their 50% inhibitory concentrations against the enzyme. Active compounds also had no effect on cell invasion, unlike the situation in Toxoplasma gondii, where these compounds block CDPK1, prevent microneme secretion, and disrupt cell invasion. These findings suggest that CPDK1 is not essential for C. parvum host cell invasion or growth and therefore that it is not the optimal target for therapeutic intervention. Nonetheless, several inhibitors with low micromolar 50% effective concentrations were identified, and these may affect other essential targets in C. parvum that are worthy of further exploration. PMID:26552986

  9. Transport of Cryptosporidium parvum in porous media: Long-term elution experiments and continuous time random walk filtration modeling

    NASA Astrophysics Data System (ADS)

    Cortis, Andrea; Harter, Thomas; Hou, Lingling; Atwill, E. Robert; Packman, Aaron I.; Green, Peter G.

    2006-12-01

    Complex transport behavior other than advection-dispersion, simple retardation, and first-order removal has been observed in many biocolloid transport experiments in porous media. Such nonideal transport behavior is particularly evident in the late time elution of biocolloids at low concentrations. Here we present a series of saturated column experiments that were designed to measure the breakthrough and long-term elution of Cryptosporidium parvum in medium sand for a few thousand pore volumes after the initial source of oocysts was removed. For a wide range of ionic strengths, I, we consistently observe slower-than-Fickian, power law tailing. The slope of the tail is flatter for higher I. At very high ionic strength the slope decays to a rate slower than t-1. To explain this behavior, we propose a new filtration model based on the continuous time random walk (CTRW) theory. Our theory upscales heterogeneities at both the pore-scale geometry of the flow field and the grain surface physicochemical properties that affect biocolloid attachment and detachment. Pore-scale heterogeneities in fluid flow are shown to control the breakthrough of a conservative tracer but are shown to have negligible effect on oocyst transport. In our experiments, C. parvum transport is dominated by the effects of physicochemical heterogeneities. The CTRW model provides a parsimonious theory of nonreactive and reactive transport. The CTRW filtration process is controlled by three parameters, Λ, β, and c, which are related to the overall breakthrough retardation (R = 1 + Λ), the slope of the power law tail (β), and the transition to a slower than t-1 decay (c).

  10. Transport and fate of Cryptosporidium parvum oocysts in intermittent sand filters.

    PubMed

    Logan, A J; Stevik, T K; Siegrist, R L; Rønn, R M

    2001-12-01

    The transport potential of Cryptosporidiim parvum (C. parvum) through intermittent. unsaturated, sand filters used for water and wastewater treatment was investigated using a duplicated. 2(3) factorial design experiment performed in bench-scale, sand columns. Sixteen columns (dia = 15 cm, L = 61 cm) were dosed eight times daily for up to 61 days with 65,000 C. parvum oocysts per liter at 15 degrees C. The effects of water quality, media grain size, and hydraulic loading rates were examined. Effluent samples were tested for pH, turbidity, and oocyst content. C. parvum effluent concentrations were determined by staining oocysts on polycarbonate filters and enumerating using epifluorescent microscopy. At completion, the columns were dismantled and sand samples were taken at discrete depths within the columns. These samples were washed in a surfactant solution and the oocysts were enumerated using immunomagnetic separation techniques. The fine-grained sand columns (d50 = 0.31 mm) effectively removed oocysts under the variety of conditions examined with low concentrations of oocysts infrequently detected in the effluent. Coarse-grained media columns (d = 1.40 mm) yielded larger numbers of oocysts which were commonly observed in the effluent regardless of operating conditions. Factorial design analysis indicated that grain size was the variable which most affected the oocyst effluent concentrations in these intermittent filters. Loading rate had a significant effect when coarse-grained media was used and lesser effect with fine-grained media while the effect of feed composition was inconclusive. No correlations between turbidity, pH, and effluent oocyst concentrations were found. Pore-sizc calculations indicated that adequate space for oocyst transport existed in the filters. It was therefore concluded that processes other than physical straining mechanisms are mainly responsible for the removal of C. pavum oocysts from aqueous fluids in intermittent sand filters used

  11. Evaluating the transport of bacillus subtilis spores as a potential surrogate for Cryptosporidium parvum Oocysts

    USDA-ARS?s Scientific Manuscript database

    The USEPA has recommended the use of aerobic spores as an indicator for Cryptosporidium oocysts when determining groundwater under the direct influence of surface water. Surface properties, interaction energies, transport, retention, and release behavior of B. subtilis spores were measured over a r...

  12. Effects of Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in mice

    USDA-ARS?s Scientific Manuscript database

    Cryptosporidium is an opportunistic protozoan parasite of humans and animals worldwide, causes diarrheal disease that is typically self-limiting in immunocompetent hosts but often life-threatening to immunocompromised individuals. However, there is a lack of completely efficient therapy available. P...

  13. Infiltration and Transport of Bromide and Cryptosporidium parvum in Vegetated, Tilted Soil Box Experiments

    NASA Astrophysics Data System (ADS)

    Harter, T.; Atwill, E. R.; Hou, L.; Carle, B. M.

    2005-12-01

    In this paper we develop a conceptual model of the physics of flow and transport in packed, tilted, and vegetated soil boxes during and immediately after simulated rainfall events and apply it to 54 experiments implemented for three different soils at three different slopes and two different rainfall rates. Using an inverse modeling procedure, we show that a significant amount of the subsurface outflow from the soil boxes is due to macropore flow. The effective hydraulic properties of the macropore space were obtained by calibration of a simple two-domain flow and transport model that accounts for coupled flow in the matrix and in the macropores of the soils. While the macropore hydraulic properties are highly variable, linear mixed effects ( LME) modeling showed significant association with soil bulk density and with the rainfall rate. Macropore flow is shown to be responsible for both, tracer (bromide) and C. parvum transport through the soil into the underlying pore space observed during the 4 hours experiments. Over a 20 cm thick soil horizon, the soil attenuation rate for C. parvum due to straining in the soil matrix and due to filtration to the macropore surfaces is 0.6 (half an order of magnitude). The LME and logistic regression models developed from the soil box experiments provide a basis for estimating macropore hydraulic properties and the risk of C. parvum transport through shallow soils from bulk density, precipitation, and total subsurface flow rate information.

  14. Cryptosporidium parvum: determination of ID₅₀ and the dose-response relationship in experimentally challenged dairy calves.

    PubMed

    Zambriski, J A; Nydam, D V; Wilcox, Z J; Bowman, D D; Mohammed, H O; Liotta, J L

    2013-10-18

    The objectives were to determine the median infective dose (ID₅₀) of Cryptosporidium parvum and to describe the dose-response relationship including associated clinical illness in experimentally challenged dairy calves. Within the first 24h of life, 27 test calves were experimentally challenged with C. parvum oocysts and 3 control calves were sham dosed. Test calves received 1 of 8 possible doses (25, 50, 100, 500, 1 × 10(3), 1 × 10(4), 1 × 10(5), and 1 × 10(6) oocysts). All 27 test calves developed diarrhea. Fecal oocyst shedding occurred in 25 (92.6%) test calves and in 0 control calves. The 2 non-shedding test calves both received 25 oocysts. There was an inverse relationship between dose and time to onset of fecal oocyst shedding (P=0.005). There was no relationship found between dose and duration (P=0.2) or cessation (P=0.3) of fecal oocyst shedding. In addition, there was not a significant relationship between log-dose and the log-peak oocysts (P=0.2) or log-total oocysts (P=0.5) counted/g of feces across the dose groups. There was a positive dose-response relationship between log-dose and diarrhea (P=0.01). However, when controlling for other factors, such as onset and cessation of fecal oocyst shedding, dose was not a significant predictor of diarrhea (P=0.5). Onset and cessation of fecal oocyst shedding were found to be the best predictors of diarrhea (P=0.0006 and P=0.04, respectively). The ID₅₀ for fecal oocyst shedding was 5.8 oocysts, for diarrhea was 9.7 oocysts, and for fecal oocyst shedding with diarrhea was 16.6 oocysts. Given that the ID₅₀ of C. parvum is far less than would be excreted into the environment by a naturally infected calf, prevention and control of cryptosporidiosis is a formidable challenge.

  15. Using ultrafiltration to concentrate and detect Bacillus anthracis, Bacillus atrophaeus subspecies globigii, and Cryptosporidium parvum in 100-liter water samples.

    PubMed

    Lindquist, H D Alan; Harris, Stephanie; Lucas, Sasha; Hartzel, Margaret; Riner, Diana; Rochele, Paul; Deleon, Ricardo

    2007-09-01

    A strategy that uses ultrafiltration (UF) to concentrate microorganisms from water samples has been developed and tested. This strategy was tested using 100-liter water samples with volume reduction achieved through ultrafiltration and recycling the microorganisms of interest through a retentate vessel, rather than returning them to the sample container, where they might pose an incremental hazard to sample takers or the environment. Three protocols based on this strategy were tested. The first protocol entailed sample volume reduction and collection of the final reduced sample. The second and third protocols both incorporated pretreatment of the filter and fluid lines with a solution to prevent microorganisms from adhering. In the second protocol, the filter was back flushed with a surfactant solution to recover microorganisms. The third protocol used recirculation of a surfactant solution to recover microorganisms. Tests were undertaken using 100-liter water samples spiked with approximately 100 or 1000 microorganisms (1 or 10 per liter). Test microorganisms included Bacillus anthracis Sterne strain, Bacillus atrophaeus subsp. globigii, and Cryptosporidium parvum. The first protocol had significantly lower recovery than the other two. Back flushing resulted in higher recovery than forward flushing, but the difference was not statistically significant.

  16. Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts.

    PubMed Central

    Peeters, J E; Mazás, E A; Masschelein, W J; Villacorta Martiez de Maturana, I; Debacker, E

    1989-01-01

    Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable. PMID:2764564

  17. Second outbreak of infection with a rare Cryptosporidium parvum genotype in schoolchildren associated with contact with lambs/goat kids at a holiday farm in Norway.

    PubMed

    Lange, H; Johansen, O H; Vold, L; Robertson, L J; Anthonisen, I L; Nygard, K

    2014-10-01

    In March 2012, a second outbreak of Cryptosporidium parvum affected children following a stay at a holiday farm in Norway; the first outbreak occurred in 2009. We studied a cohort of 145 schoolchildren who had visited the farm, of which 40 (28%) were cases. Cryptosporidium oocysts were detected in faecal samples from humans, goat kids and lambs. Molecular studies revealed C. parvum subtype IIa A19G1R1 in all samples including human samples from the 2009 outbreak. A dose-response relationship was found between the number of optional sessions with animals and illness, increasing from two sessions [risk ratio (RR) 2·7, 95% confidence interval (CI) 0·6-11·5] to six sessions (RR 8·0, 95% CI 1·7-37·7). The occurrence of two outbreaks 3 years apart, with the same subtype of C. parvum, suggests that the parasite is established in the farm's environment. We recommend greater emphasis on hand hygiene and routines related to animal contact.

  18. Bobel-24 Activity against Cryptosporidium parvum in Cell Culture and in a SCID Mouse Model▿

    PubMed Central

    Rueda, Cristina; Fenoy, Soledad; Simón, Fernando; del Aguila, Carmen

    2008-01-01

    The anticryptosporidial activity of Bobel-24 (2,4,6-triiodophenol) was studied for the first time, resulting in a reduction of the in vitro growth of Cryptosporidium of up to 99.6%. In a SCID mouse model of chronic cryptosporidiosis, significant differences (P < 0.05) in oocyst shedding were observed in animals treated with 125 mg/kg/day. These results merit further investigation of Bobel-24 as a chemotherapeutic option for cryptosporidiosis. PMID:18160525

  19. Dynamics of cytokines and immunoglobulins serum profiles in primary and secondary Cryptosporidium parvum infection: usefulness of Luminex® xMAP technology.

    PubMed

    Codices, Vera; Martins, Catarina; Novo, Carlos; de Sousa, Bruno; Lopes, Ângela; Borrego, Miguel; Matos, Olga

    2013-01-01

    Infection by Cryptosporidium parvum triggers a complex array of innate and adaptive cell mediated immune response, playing an important role in controlling the infection. To date, there are no studies applying the Luminex® xMAP technology to determine profiles of cytokines and immunoglobulins in the context of an infection by C. parvum. In this study, we analyzed these immune mediators in the serum of immunocompetent mice inoculated with C. parvum oocysts, using Luminex, to understand how the immune system responds to an infection by this parasite. Animal sera were also analyzed by ELISA to determine the expressed immunoglobulin isotype profile, and compare the obtained trend with data obtained by Luminex. Specific-pathogen-free BALB/C mice were inoculated with oocysts of C. parvum at days 0 and 22. Peripheral blood was aseptically collected from sacrificed mice on several time points, and immune mediators were evaluated in serum samples. Infection was confirmed by the presence of C. parvum DNA in feces by a nested-PCR assay (60-kDa glycoprotein). Luminex results showed predominance in the secretion of IgG1 and IgG2a, confirmed by ELISA, which also showed that IgG1 is the major immunoglobulin isotype produced during the infection. The analysis of cytokines suggests a preferential Th(1) over the Th(2) response, with increased production of TNF-α, IFN-γ and GM-CSF. This work contributed to a better understanding of the immune response to the infection by C. parvum, as well as demonstrated the advantage of Luminex® xMAP technology to study immune mediators, using small sample volumes. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Induced Susceptibility of Host Is Associated with an Impaired Antioxidant System Following Infection with Cryptosporidium parvum in Se-Deficient Mice

    PubMed Central

    Wang, Chengmin; Wu, Yanyun; Qin, Jianhua; Sun, Haoxue; He, Hongxuan

    2009-01-01

    Background Susceptibility or resistance to infection with Cryptosporidium parvum (C.parvum) correlates with Selenium (Se) deficiency in response to infection. Both adult Se-adequate and Se-deficient mouse models of cryptosporidiosis were used to study the cell-mediated immune response during the course of C. parvum infection. Methodology/Principal Findings Blood samples from mouse models were used for Se status. The concentration of MDA, SOD, GPx and CAT in blood has revealed that lower Se level exist in Se-deficient mice. Mesenteric lymph node (MLN) lymphocytes from both mouse models were proliferated after ex vivo re-stimulation with C. parvum sporozoite antigen. The study of the cytokine profiles from the supernatant of proliferated MLN cells revealed that Se-adequate mice produced higher levels of Th1 (IFN-γ and IL-2) and moderate amounts of Th2 (IL-4) cytokines throughout the course of infection. Whereas, MLN cells from Se-deficient mice produced lower levels of IFN-γ, IL-2 and IL-4 cytokines. The counts of total white cell and CD3, CD4, CD8 cell in Se-adequate were higher than that in Se-deficient mice. Significance These results suggest that Cell immunity is affected by Se status after infection with C.parvum from kinetic changes of different white cells and cytokine. In conclusion, induced susceptibility of host is associated with an impaired antioxidant system following infection with C.parvum in C57BL/6 Selenium deficient mice. PMID:19247447

  1. Seroprevalence of Cryptosporidium parvum infection of dairy cows in three northern provinces of Thailand determined by enzyme-linked immunosorbent assay using recombinant antigen CpP23.

    PubMed

    Inpankaew, T; Jittapalapong, S; Phasuk, J; Pinyopanuwut, N; Chimnoi, W; Kengradomkit, C; Sunanta, C; Zhang, G; Aboge, G O; Nishikawa, Y; Igarashi, I; Xuan, X

    2009-06-01

    Cryptosporidium parvum is the most frequent parasitic agent that causes diarrhoea in AIDS patients in Thailand. Cryptosporidiosis outbreaks in humans may be attributed to contamination of their drinking water from infected dairy pastures. A 23-kDa glycoprotein of C. parvum (CpP23) is a sporozoite surface protein that is geographically conserved among C. parvum isolates. This glycoprotein is a potentially useful candidate antigen for the diagnosis of cryptosporidiosis by enzyme-linked immunosorbent assay. Therefore, we investigated the seroprevalence of C. parvum infection in dairy cows in northern Thailand using an ELISA based on recombinant CpP23 antigen. Sera were randomly collected from 642 dairy cows of 42 small-holder farmers, which had the top three highest number of the dairy cows' population in Northern Thailand, that included Chiang Mai, Chiang Rai and Lumpang provinces. The overall seroprevalence of the infection was 4.4%, and the seropositive rates for the three provinces were 3.3% in Chiang Mai, 5.1% in Chiang Rai and 3% in Lumpang. These results suggest that cattle could play a role in zoonotic cryptosporidiosis in Thailand.

  2. Fulminant Cryptosporidiosis after Near-Drowning: a Human Cryptosporidium parvum Strain Implicated in Invasive Gastrointestinal Adenocarcinoma and Cholangiocarcinoma in an Experimental Model

    PubMed Central

    Benamrouz, Sadia; Guyot, Karine; Mouray, Anthony; Chassat, Thierry; Flament, Nicolas; Delhaes, Laurence; Coiteux, Valerie; Delaire, Baptiste; Praet, Marleen; Cuvelier, Claude; Gosset, Pierre; Dei-Cas, Eduardo; Creusy, Colette

    2012-01-01

    In the present work, we report the characterization of a Cryptosporidium parvum strain isolated from a patient who nearly drowned in the Deule River (Lille, France) after being discharged from the hospital where he had undergone allogeneic stem cell transplantation. After being rescued and readmitted to the hospital, he developed fulminant cryptosporidiosis. The strain isolated from the patient's stools was identified as C. parvum II2A15G2R1 (subtype linked to zoonotic exposure) and inoculated into SCID mice. In this host, this virulent C. parvum isolate induced not only severe infection but also invasive gastrointestinal and biliary adenocarcinoma. The observation of adenocarcinomas that progressed through all layers of the digestive tract to the subserosa and spread via blood vessels confirmed the invasive nature of the neoplastic process. These results indicate for the first time that a human-derived C. parvum isolate is able to induce digestive cancer. This study is of special interest considering the exposure of a large number of humans and animals to this waterborne protozoan, which is highly tumorigenic when inoculated in a rodent model. PMID:22247151

  3. A fast method for detecting Cryptosporidium parvum oocysts in real world samples

    NASA Astrophysics Data System (ADS)

    Stewart, Shona; McClelland, Lindy; Maier, John

    2005-04-01

    Contamination of drinking water with pathogenic microorganisms such as Cryptosporidium has become an increasing concern in recent years. Cryptosporidium oocysts are particularly problematic, as infections caused by this organism can be life threatening in immunocompromised patients. Current methods for monitoring and analyzing water are often laborious and require experts to conduct. In addition, many of the techniques require very specific reagents to be employed. These factors add considerable cost and time to the analytical process. Raman spectroscopy provides specific molecular information on samples, and offers advantages of speed, sensitivity and low cost over current methods of water monitoring. Raman spectroscopy is an optical method that has demonstrated the capability to identify and differentiate microorganisms at the species and strain levels. In addition, this technique has exhibited sensitivities down to the single organism detection limit. We have employed Raman spectroscopy and Raman Chemical Imaging, in conjunction with chemometric techniques, to detect small numbers of oocysts in the presence of interferents derived from real-world water samples. Our investigations have also indicated that Raman Chemical Imaging may provide chemical and physiological information about an oocyst sample which complements information provided by the traditional methods. This work provides evidence that Raman imaging is a useful technique for consideration in the water quality industry.

  4. Computer-Assisted Laser Scanning and Video Microscopy for Analysis of Cryptosporidium parvum Oocysts in Soil, Sediment, and Feces

    PubMed Central

    Anguish, L. J.; Ghiorse, W. C.

    1997-01-01

    A computer-assisted laser scanning microscope equipped for confocal laser scanning and color video microscopy was used to examine Cryptosporidium parvum oocysts in two agricultural soils, a barnyard sediment, and calf fecal samples. An agar smear technique was developed for enumerating oocysts in soil and barnyard sediment samples. Enhanced counting efficiency and sensitivity (detection limit, 5.2 x 10(sup2) oocysts(middot)g [dry weight](sup-1)) were achieved by using a semiautomatic counting procedure and confocal laser scanning microscopy to enumerate immunostained oocysts and fragments of oocysts in the barnyard sediment. An agarose-acridine orange mounting procedure was developed for high-resolution confocal optical sectioning of oocysts in soil. Stereo images of serial optical sections revealed the three-dimensional spatial relationships between immunostained oocysts and the acridine orange-stained soil matrix material. In these hydrated, pyrophosphate-dispersed soil preparations, oocysts were not found to be attached to soil particles. A fluorogenic dye permeability assay for oocyst viability (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992) was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4(prm1),6-diamidino-2-phenylindole. Comparison of conventional color epifluorescence and differential interference contrast images on one video monitor with comparable black-and-white laser-scanned confocal images on a second monitor allowed for efficient location and interpretation of fluorescently stained oocysts in the soil matrix. This multi-imaging procedure facilitated the interpretation of the viability assay results by overcoming the uncertainties caused by matrix interference and background fluorescence. PMID:16535523

  5. Detection of Infectious Cryptosporidium parvum Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Integrated Cell Culture-PCR

    PubMed Central

    Di Giovanni, George D.; Hashemi, F. Helen; Shaw, Nancy J.; Abrams, Felicia A.; LeChevallier, Mark W.; Abbaszadegan, Morteza

    1999-01-01

    A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected. PMID:10427030

  6. Development of Cryptosporidium parvum-Induced Gastrointestinal Neoplasia in Severe Combined Immunodeficiency (SCID) Mice: Severity of Lesions Is Correlated with Infection Intensity

    PubMed Central

    Certad, Gabriela; Creusy, Colette; Ngouanesavanh, Tramy; Guyot, Karine; Gantois, Nausicaa; Mouray, Anthony; Chassat, Thierry; Flament, Nicolas; Fleurisse, Laurence; Pinon, Anthony; Delhaes, Laurence; Dei-Cas, Eduardo

    2010-01-01

    We reported previously that Cryptosporidium parvum was able to induce intestinal tumors in severe combined immunodeficiency (SCID) mice treated with corticoids. To further characterize this Cryptosporidium-induced cell transformation, SCID mice treated with dexamethasone were challenged with C. parvum oocysts, and euthanatized sequentially after infection for histologic examination. Ki-67 was used as a marker of cellular proliferation. Our previous results were confirmed, and it was also found that mice receiving higher inocula (106–107) experienced more severe neoplastic development. Additionally, neoplastic changes were observed not only in the caecum but also in the stomach and duodenum of some animals. Interestingly, SCID mice (6/6) inoculated with 105–107 oocysts showed high grade intraepithelial neoplasia or adenomas with high grade dysplasia in the caecum after Day 46 post-infection (PI). Immunohistochemistry for Ki-67 staining indicated the neoplastic process associated to cryptosporidiosis, and evidenced the first immunohistochemical alterations at early stages of the process, even at 3 weeks PI. PMID:20134002

  7. Influence of colostrum deprivation and concurrent Cryptosporidium parvum infection on the colonization and persistence of Escherichia coli O157 : H7 in young lambs.

    PubMed

    La Ragione, R M; Best, A; Clifford, D; Weyer, U; Johnson, L; Marshall, R N; Marshall, J; Cooley, W A; Farrelly, S; Pearson, G R; Woodward, M J

    2006-07-01

    Escherichia coli O157 : H7 and Cryptosporidium parvum infections of man have been associated with direct contact with small ruminants. Colostrum protects neonates against gastrointestinal pathogens, and orphan lambs, which are common on petting farms, may be deprived of this protection. In a recent study, it was demonstrated that high shedding of E. coli O157 : H7 by an 8-week-old goat kid was associated with coincidental C. parvum infection. Furthermore, both pathogens were co-located in the distal gastrointestinal tract. It was hypothesized that colostrum deprivation and pre-infection with C. parvum predisposed young ruminants to colonization and increased shedding of E. coli O157 : H7. To test this, 21 lambs 5 weeks of age were divided into four groups as follows: (A) colostrum-deprived and inoculated with E. coli O157 : H7, (B) colostrum-deprived and inoculated with C. parvum and then E. coli O157 : H7, (C) conventionally reared and inoculated with E. coli O157 : H7, (D) conventionally reared and inoculated with C. parvum and then E. coli O157 : H7. C. parvum was detected between 8 and 12 days post-inoculation in most of the infected lambs. At 24 h post-inoculation with E. coli O157 : H7, all lambs were shedding between 5 x 10(4) and 5 x 10(7) c.f.u. E. coli O157 : H7 per gram of faeces. E. coli O157 : H7 was shed in higher numbers in the groups pre-inoculated with C. parvum, whether conventionally reared or colostrum-deprived. Interestingly, for the colostrum-deprived lambs on day 3, a significant difference in shedding of E. coli O157 : H7 was observed (P = 0.038), with the lambs inoculated with E. coli alone yielding higher counts than those pre-inoculated with C. parvum. From day 15 onwards, shedding of E. coli O157 : H7 was highest from the colostrum-deprived C. parvum-infected lambs, then (in descending order of shedding) the colostrum-deprived lambs, the conventionally reared lambs infected with C. parvum, and the conventionally reared animals. In total

  8. Comparison of different solar reactors for household disinfection of drinking water in developing countries: evaluation of their efficacy in relation to the waterborne enteropathogen Cryptosporidium parvum.

    PubMed

    Gómez-Couso, H; Fontán-Sainz, M; Navntoft, C; Fernández-Ibáñez, P; Ares-Mazás, E

    2012-11-01

    Solar water disinfection (SODIS) is a type of treatment that can significantly improve the microbiological quality of drinking water at household level and therefore prevent waterborne diseases in developing countries. Cryptosporidium parvum is an obligate protozoan parasite responsible for the diarrhoeal disease cryptosporidiosis in humans and animals. Recently, this parasite has been selected by the WHO as a reference pathogen for protozoan parasites in the evaluation of household water treatment options. In this study, the field efficacy of different static solar reactors [1.5 l transparent plastic polyethylene terephthalate (PET) bottles as well as 2.5 l borosilicate glass and 25 l methacrylate reactors fitted with compound parabolic concentrators (CPC)] for solar disinfection of turbid waters experimentally contaminated with C. parvum oocysts was compared. Potential oocyst viability was determined by inclusion/exclusion of the fluorogenic vital dye propidium iodide. The results demonstrate that static solar reactors fitted with CPCs are an excellent alternative to the conventional SODIS method with PET bottles. These reactors improved the efficacy of the SODIS method by enabling larger volumes of water to be treated and, in some cases, the C. parvum oocysts were rendered totally unviable, minimising the negative effects of turbidity.

  9. Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targeting hsp70 mRNA ▿

    PubMed Central

    Liang, Zhanbei; Keeley, Ann

    2011-01-01

    Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure for Cryptosporidium detection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection of Cryptosporidium with oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and Salmonella enterica serovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed that Salmonella cells most efficiently relieved binding of RNA. With the inclusion of Salmonella during extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102 oocysts g−1 of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104 C. parvum oocysts g−1 soil for sandy, loamy, and clay samples, respectively. PMID:21803904

  10. Prevalence of Cryptosporidium parvum/hominis, Entamoeba histolytica and Giardia lamblia among Young Children with and without Diarrhea in Dar es Salaam, Tanzania

    PubMed Central

    Tellevik, Marit G.; Moyo, Sabrina J.; Blomberg, Bjørn; Hjøllo, Torunn; Maselle, Samuel Y.; Langeland, Nina; Hanevik, Kurt

    2015-01-01

    Background Although enteroparasites are common causes of diarrheal illness, few studies have been performed among children in Tanzania. This study aimed to investigate the prevalence of Cryptosporidium parvum/hominis, Entamoeba histolytica and Giardia lamblia among young children in Dar es Salaam, Tanzania, and identify risk factors for infection. Methodology/Principal Findings We performed an unmatched case-control study among children < 2 years of age in Dar es Salaam, recruited from August 2010 to July 2011. Detection and identification of protozoans were done by PCR techniques on DNA from stool specimens from 701 cases of children admitted due to diarrhea at the three study hospitals, and 558 controls of children with no history of diarrhea during the last month prior to enrollment. The prevalence of C. parvum/hominis was 10.4% (84.7% C. hominis), and that of G. lamblia 4.6%. E. histolytica was not detected. The prevalence of Cryptosporidium was significantly higher in cases (16.3%) than in controls (3.1%; P < 0.001; OR = 6.2; 95% CI: 3.7–10.4). G. lamblia was significantly more prevalent in controls (6.1%) than in cases (3.4%; P = 0.027; OR = 1.8; 95% CI: 1.1–3.1). Cryptosporidium infection was found more often in HIV-positive (24.2%) than in HIV-negative children (3.9%; P < 0.001; OR = 7.9; 95% CI: 3.1–20.5), and was also associated with rainfall (P < 0.001; OR = 2.41; 95% CI: 1.5–3.8). Among cases, stunted children had significantly higher risk of being infected with Cryptosporidium (P = 0.011; OR = 2.12; 95% CI: 1.2–3.8). G. lamblia infection was more prevalent in the cool season (P = 0.004; OR = 2.2; 95% CI: 1.3–3.8), and more frequent among cases aged > 12 months (P = 0.003; OR = 3.5; 95% CI: 1.5–7.8). Among children aged 7–12 months, those who were breastfed had lower prevalence of G. lamblia infection than those who had been weaned (P = 0.012). Conclusions Cryptosporidium infection is common among young Tanzanian children with diarrhea

  11. GIS-based analysis of the fate of waste-related pathogens Cryptosporidium parvum, Giardia lamblia and Escherichia coli in a tropical canal network.

    PubMed

    Diallo, Mamadou B C; Anceno, Alfredo J; Tawatsupa, Benjawan; Tripathi, Nitin K; Wangsuphachart, Voranuch; Shipin, Oleg V

    2009-03-01

    Urban canals play a major socio-economic role in many tropical countries and, particularly, Thailand. One of the overlooked functions that they perform is a significant attenuation of waste-related pathogens posing considerable health risk, as well as pollution attenuation in general. The study dealt with a comparison of three canals receiving: (i) municipal, (ii) mainly industrial and (iii) mainly agricultural wastewater, listed in order of progressively decreasing organic loading. The occurrence and fate of waterborne Cryptosporidium parvum, Giardia lamblia and Escherichia coli were monitored in the canals by both real-time PCR and conventionally for 12 months. The pathogens are etiological agents of an estimated 38% and 47% of diarrhea cases worldwide and in Thailand, respectively. The geographic information system (GIS) was used to evaluate and map point and, particularly, non-point pollution sources which allowed differentiating the canal sections in terms of predominant pathogen sources. The flowthrough canals, which can be viewed as waste stabilization ponds, were found to be efficiently removing the pathogens at the following generalized specific rates: 0.3 (C. parvum), 1.2 (G. lamblia), 1.8 (E. coli) log10/km.d in the dry season. The rates decreased in the rainy season for E. coli and G. lamblia, but increased for C. parvum which indicated different removal mechanisms. Data suggest that E. coli and G. lamblia were mainly removed through sedimentation and sunlight (UV) irradiation, while the likely mechanism for C. parvum was predation. Overall, the specific pathogen removal rates positively correlated with the canal organic loading rates in the rainy season. As an important result, an estimate of the municipal pollution mitigation by over 2280 km canals in the Greater Bangkok suggests that concomitant to the pathogens at least 36-95 tons of BOD5 is being removed daily, thereby saving the receiving Chao Phraya River and Bight of Bangkok, by far exceeding

  12. Oral immunization with attenuated Salmonella enterica serovar Typhimurium encoding Cryptosporidium parvum Cp23 and Cp40 antigens induces a specific immune response in mice.

    PubMed

    Benitez, Alvaro J; McNair, Nina; Mead, Jan R

    2009-09-01

    Attenuated Salmonella enterica serovar Typhimurium vaccine strain SL3261 was used as an antigen delivery system for the oral immunization of mice against two Cryptosporidium parvum antigens, Cp23 and Cp40. Each antigen was subcloned into the pTECH1 vector system, which allows them to be expressed as fusion proteins with highly immunogenic fragment C of tetanus toxin under the control of the anaerobically inducible nirB promoter. The recombinant vector was introduced into Salmonella Typhimurium vaccine strain SL3261, and the stable soluble expression of the chimeric protein was evaluated and confirmed by Western blotting with polyclonal C. parvum antisera. Mice were inoculated orally with a single dose of SL3261/pTECH-Cp23 or Cp40, respectively, and plasmid stability was demonstrated both in vitro and in vivo. Specific serum immunoglobulin G (IgG) antibodies against the Cp23 or Cp40 antigen were detected by enzyme-linked immunosorbent assay 35 days after immunization. Also, serum IgA and mucosal (feces) IgA antibodies were detected in 30% of the mice immunized with Cp23. In addition, prime-boosting with Cp23 and Cp40 DNA vaccine vectors followed by Salmonella immunization significantly increased antibody responses to both antigens. Our data show that a single oral inoculation with recombinant S. Typhimurium SL3261 can induce specific antibody responses to the Cp23 or Cp40 antigen from C. parvum in mice, suggesting that recombinant Salmonella is a feasible delivery system for a vaccine against C. parvum infection.

  13. Use of a Sentinel System for Field Measurements of Cryptosporidium parvum Oocyst Inactivation in Soil and Animal Waste

    PubMed Central

    Jenkins, M. B.; Walker, M. J.; Bowman, D. D.; Anthony, L. C.; Ghiorse, W. C.

    1999-01-01

    A small-volume sentinel chamber was developed to assess the effects of environmental stresses on survival of sucrose-Percoll-purified Cryptosporidium parvum oocysts in soil and animal wastes. Chambers were tested for their ability to equilibrate with external chemical and moisture conditions. Sentinel oocysts were then exposed to stresses of the external environment that affected their viability (potential infectivity), as indicated by results of a dye permeability assay. Preliminary laboratory experiments indicated that temperatures between 35 and 50°C and decreases in soil water potential (−0.003 to −3.20 MPa) increased oocyst inactivation rates. The effects of two common animal waste management practices on oocyst survival were investigated on three dairy farms in Delaware County, N.Y., within the New York City watershed: (i) piling wastes from dairy youngstock (including neonatal calves) and (ii) spreading wastes as a soil amendment on an agricultural field. Sentinel containers filled with air-dried and sieved (2-mm mesh) youngstock waste or field soil were wetted and inoculated with 2 million oocysts in an aqueous suspension and then placed in waste piles on two different farms and in soil within a cropped field on one farm. Controls consisted of purified oocysts in either phosphate-buffered saline or distilled water contained in sealed microcentrifuge tubes. Two microdata loggers recorded the ambient temperature at each field site. Sentinel experiments were conducted during the fall and winter (1996 to 1997) and winter (1998). Sentinel containers and controls were removed at 2- to 4-week intervals, and oocysts were extracted and tested by the dye permeability assay. The proportions of potentially infective oocysts exposed to the soil and waste pile material decreased more rapidly than their counterpart controls exposed to buffer or water, indicating that factors other than temperature affected oocyst inactivation in the waste piles and soil. The effect

  14. Presence of Cryptosporidium scrofarum, C. suis and C. parvum subtypes IIaA16G2R1 and IIaA13G1R1 in Eurasian wild boars (Sus scrofa).

    PubMed

    García-Presedo, Ignacio; Pedraza-Díaz, Susana; González-Warleta, Marta; Mezo, Mercedes; Gómez-Bautista, Mercedes; Ortega-Mora, Luis Miguel; Castro-Hermida, José Antonio

    2013-09-23

    The aim of the present study was to identify the species of Cryptosporidium infecting Eurasian wild boars (Sus scrofa) in Galicia (NW, Spain). A sampling of 209 wild boars shot in different game preserves was carried out during the hunting season in 2009-2010. All samples were examined for Cryptosporidium infection, using both immunological and molecular tools. Cryptosporidium oocysts in faecal samples were identified using a direct immunofluorescence technique with monoclonal antibodies (DFA). The presence of Cryptosporidium DNA was determined using nested PCR involving amplification of a fragment of the small-subunit (SSU) ribosomal RNA gene (SSU rRNA). A total of 35 (16.7%) samples tested positive with both techniques. However, sequencing was only possible in 27 samples. Cryptosporidium scrofarum, Cryptosporidium suis and Cryptosporidium parvum oocysts were identified in 19, 5 and 3 of the samples, respectively. Moreover, C. scrofarum was detected as a dominant species infecting all age groups (juveniles, sub adults and adults). Sequence analyses of the glycoprotein (GP60) gene revealed the presence of C. parvum subtypes IIaA16G2R1 in 2 juveniles and IIaA13G1R1 in 1 sub adult wild boar. These species and subtypes have previously been described in human patients, indicating that isolates from asymptomatic wild boars might have zoonotic potential. This is the first report of the presence of C. scrofarum, C. suis and C. parvum subtypes IIaA16G2R1 and IIaA13G1R1 in wild boars (S. scrofa) in Spain.

  15. First molecular characterization of Cryptosporidium and Giardia from bovines (Bos taurus and Bubalus bubalis) in Sri Lanka: unexpected absence of C. parvum from pre-weaned calves

    PubMed Central

    2014-01-01

    Background The genetic characterization of Cryptosporidium and Giardia has important implications for investigating their epidemiology and underpins their control. We undertook the first molecular epidemiological survey of domestic bovids in selected regions of Sri Lanka to establish whether they excreted Cryptosporidium and/or Giardia with zoonotic potential. Methods Faecal samples were collected from dairy calves (n = 340; Bos taurus; < 3 months of age; weekly sampling for six weeks) and water buffaloes (n = 297; Bubalus bubalis; <6 months and ≥6 months of age; one sampling) from seven different farms in Sri Lanka. Genomic DNAs were extracted from individual faecal samples and then tested for the presence of parasite DNA using a PCR-based mutation scanning-targeted sequencing-phylogenetic approach, employing genetic markers within the small subunit of nuclear ribosomal RNA and 60 kDa glycoprotein genes (designated pSSU and pgp60, respectively) for Cryptosporidium, and within the triose phosphate isomerise (ptpi) gene for Giardia. Results Based on pSSU sequence data, C. bovis, C. ryanae and six new genotypes that were genetically similar but not identical to C. andersoni (n = 1), C. bovis (n = 1), C. ryanae (n = 3) and C. suis (n = 1) were recorded in cattle. For pSSU, two other, new genotypes were defined in water buffalo, which were genetically most similar to Cryptosporidium genotypes recorded previously in this host species in other countries including Australia. Consistent with the findings for pSSU, no species or genotypes of Cryptosporidium with zoonotic potential were detected using pgp60. Based on ptpi sequence data, G. duodenalis assemblages A and E were detected in four and 137 samples from cattle, respectively, and assemblage E in two samples from water buffaloes. Conclusions The present study showed that C. parvum, the most commonly reported zoonotic species of Cryptosporidium recognised in bovine calves globally, was not

  16. Pyruvate : NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum: a biochemical relic linking pyruvate metabolism in mitochondriate and amitochondriate protists.

    PubMed

    Rotte, C; Stejskal, F; Zhu, G; Keithly, J S; Martin, W

    2001-05-01

    Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2). The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.

  17. Quantitative polymerase chain (QPCR) reaction using the MIMIC approach to estimate Cryptosporidium parvum oocysts, an intestinal pathogen, in municipal water treatment sludge samples.

    PubMed

    Udeh, P; Veenstra, J; Abraham, A J; John, G H

    2000-04-01

    An accurate estimation of the number of Cryptosporidium parvum oocysts in water treatment plant sludge was determined using the Quantitative Polymerase Chain Reaction (QPCR) method. Approximately 8x10(6)purified viable oocysts were spiked into raw water and treated by conventional water treatment methods. The settled sludge was collected and the DNA extracted. The QPCR Mimic produced two competing products that were 300 and 435 base pairs in size. The log ratio of the products were used in the standard curve to determine a final estimation of oocysts in the sludge sample. The final number of oocysts in the sludge sample was estimated at 258 oocyst per two litres of treated water. This is the first time sludge from a water treatment process has been tested for presence of C. parvum oocysts, which is a known contaminant of drinking water. The QPCR method can be used to test other sludge samples and help estimate the sanitary risks associated with using sludge to fertilize agricultural lands. Copyright 2000 Academic Press.

  18. Leaching of Cryptosporidium parvum Oocysts, Escherichia coli, and a Salmonella enterica Serovar Typhimurium Bacteriophage through Intact Soil Cores following Surface Application and Injection of Slurry▿

    PubMed Central

    Forslund, Anita; Markussen, Bo; Toenner-Klank, Lise; Bech, Tina B.; Jacobsen, Ole Stig; Dalsgaard, Anders

    2011-01-01

    Increasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching of Salmonella enterica serovar Typhimurium bacteriophage 28B, Escherichia coli, and Cryptosporidium parvum oocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148. C. parvum oocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching of E. coli was delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, but E. coli could be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenous E. coli was not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions. PMID:21948848

  19. Leaching of Cryptosporidium parvum oocysts, Escherichia coli, and a Salmonella enterica serovar Typhimurium bacteriophage through intact soil cores following surface application and injection of slurry.

    PubMed

    Forslund, Anita; Markussen, Bo; Toenner-Klank, Lise; Bech, Tina B; Jacobsen, Ole Stig; Dalsgaard, Anders

    2011-11-01

    Increasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching of Salmonella enterica serovar Typhimurium bacteriophage 28B, Escherichia coli, and Cryptosporidium parvum oocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148. C. parvum oocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching of E. coli was delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, but E. coli could be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenous E. coli was not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions.

  20. Binding and activation of human and mouse complement by Cryptosporidium parvum (Apicomplexa) and susceptibility of C1q- and MBL-deficient mice to infection.

    PubMed

    Petry, Franz; Jakobi, Vera; Wagner, Swen; Tessema, Tesfaye Sisay; Thiel, Steffen; Loos, Michael

    2008-07-01

    Cryptosporidium parvum is a protozoan parasite (Apicomplexa) that causes gastrointestinal disease in animals and humans. Whereas immunocompetent hosts can limit the infection within 1 or 2 weeks, immunocompromised individuals develop a chronic, life-threatening disease. The importance of the adaptive cellular immune response, with CD4+ T-lymphocytes being the major players, has been clearly demonstrated. Several non-adaptive immune mechanisms have been suggested to contribute to the host defence, such as interferon-gamma (IFN-gamma) from NK cells, certain chemokines, beta-defensins and pro-inflammatory cytokines, but the influence of the complement systems has been less well studied. We analysed the in vitro binding and activation of the human and mouse complement systems and tested the susceptibility to infection in complement-deficient mouse strains. We found that C. parvum can activate both the classical and lectin pathways, leading to the deposition of C3b on the parasite. Using real-time PCR, parasite development could be demonstrated in adult mice lacking mannan-binding lectin (MBL-A/C-/-) but not in mice lacking complement factor C1q (C1qA-/-) or in wild type C57BL/6 mice. The contribution of the complement system and the lectin pathway in particular to the host defence against cryptosporidiosis may become apparent in situations of immunodeficiency such as HIV infections or in early childhood.

  1. THE EFFICACY OF THREE MEDICINAL PLANTS; GARLIC, GINGER AND MIRAZID AND A CHEMICAL DRUG METRONIDAZOLE AGAINST CRYPTOSPORIDIUM PARVUM: II-HISTOLOGICAL CHANGES.

    PubMed

    Abouel-Nour, Mohamed F; El-Shewehy, Dina Magdy M; Hamada, Shadia F; Morsy, Tosson A

    2016-04-01

    Cryptosporidiosis parvum is a zoonotic protozoan parasite infects intestinal epithelial cells of man and animals causing a major health problem. This study was oriented to evaluate the protective and curative capacity of garlic, ginger and mirazid in comparison with metronidazole drug (commercially known) against Cryptosporidium in experimental mice. Male Swiss Albino mice experimentally infected with C. parvum were treated with medicinal plants extracts (Ginger, Mirazid, and Garlic) as compared to chemical drug Metronidazole. Importantly, C. parvum-infected mice treated with ginger, Mirazid, garlic and metronidazole showed a complete elimination in shedding oocysts by 9th day PI. The reduction and elimination of shedding oocysts in response to the treatments might be attributable to a direct effect on parasite growth in intestines, sexual phases production and/or the formation of oocysts. The results were evaluated histopathological examination of ideum section of control mice (uninfected, untreated) displayed normal architecture of the villi. Examiination of infected mice ileum section (infected, untreated) displayed histopathological alterations from uninfected groups. Examination of ileum section prepared from mice treated with garlic, ginger, mirazid, and metronidazole displayed histopathological alterations from that of the control groups, and showed marked histologic correction in the pattern with the four regimes used in comparison to control mice. Garlic successfully eradicated oocysts of infected mice from stool and intestine. Supplementation of ginger to infected mice markedly corrected elevation in the inflammatory risk factors and implied its potential antioxidant, anti-inflammatory and immunomodulatory capabilities. Infected mice treated with ginger, mirazid, garlic and metronidazole showed significant symptomatic improvements during treatment.

  2. Two-year monitoring of Cryptosporidium parvum and Giardia lamblia occurrence in a recreational and drinking water reservoir using standard microscopic and molecular biology techniques.

    PubMed

    Helmi, Karim; Skraber, Sylvain; Burnet, Jean-Baptiste; Leblanc, Laurence; Hoffmann, Lucien; Cauchie, Henry-Michel

    2011-08-01

    Starting in 2006, a monitoring of Giardia lamblia and Cryptosporidium parvum occurrence was conducted for 2 years in the largest drinking water reservoir of Luxembourg (Esch-sur-Sûre reservoir) using microscopy and qPCR techniques. Parasite analyses were performed on water samples collected from three sites: site A located at the inlet of the reservoir, site B located 18 km downstream site A, at the inlet of the drinking water treatment plant near the dam of the reservoir and site C where the finished drinking water is injected in the distribution network. Results show that both parasites are present in the reservoir throughout the year with a higher occurrence of G. lamblia cysts compared to C. parvum oocysts. According to our results, only 25% of the samples positive by microscopy were confirmed by qPCR. (Oo)cyst concentrations were 10 to 100 times higher at site A compared to site B and they were positively correlated to the water turbidity and negatively correlated to the temperature. Highest (oo)cyst concentrations were observed in winter. In contrast, no relationship between the concentrations of (oo)cysts in the reservoir and rain events could be established. Though a correlation has been observed between both parasites and faecal indicators in the reservoir, some discrepancies highlight that the latter do not represent a reliable tool to predict the presence/absence of these pathogenic protozoa. In summer 2007, the maximal risk of parasite infection per exposure event for swimmers in the reservoir was estimated to be 0.0015% for C. parvum and 0.56% for G. lamblia. Finally, no (oo)cysts could be detected in large volumes of finished drinking water.

  3. Structure of a CutA1 divalent-cation tolerance protein from Cryptosporidium parvum, the protozoal parasite responsible for cryptosporidiosis

    DOE PAGES

    Buchko, Garry W.; Robinson, Howard; Abendroth, Jan; ...

    2015-05-01

    Cryptosporidiosis is an infectious disease caused by protozoan parasites from the Cryptosporidium species. Infection is associated with mild to severe diarrhea that usually resolves spontaneously in healthy human adults, but may lead to severe complications in young children and in immunocompromised patients. The genome of Cryptosporidium parvum contains a gene, CUTA_CRYPI, that may play a role in regulating the intracellular concentration of copper, a toxic element if left unchecked. Here we report the crystal structure for this CutA1 protein, Cp-CutA1, is reported at 2.0 Å resolution (4E98). As observed for other CutA1 structures, the 117-residue protein is a trimer withmore » a core ferrodoxin-like fold. Circular dichroism spectroscopy shows little unfolding of Cp-CutA1 up to 353 K. This robustness is corroborated by ¹H-¹⁵N HSQC spectra at 333 K that is characteristic of a folded protein, suggesting NMR spectroscopy may be a useful tool to further probe the function of the CutA1 proteins. While robust, Cp-CutA1 is not as stable as the homologous protein from a hyperthermophile, perhaps due to a wide β-bulgein β2 that protrudes P48 and S49 outside the β-sheet.« less

  4. Structure of a CutA1 divalent-cation tolerance protein from Cryptosporidium parvum, the protozoal parasite responsible for cryptosporidiosis

    SciTech Connect

    Buchko, Garry W.; Robinson, Howard; Abendroth, Jan; Clitfon, Mathew C.; Zhang, Yanfeng; Hewitt, Stephen N.; Staker, Bart L.; Van Voorhis, Wesley C.; Mylera, Peter J.

    2015-05-01

    Cryptosporidiosis is an infectious disease caused by protozoan parasites from the Cryptosporidium species. Infection is associated with mild to severe diarrhea that usually resolves spontaneously in healthy human adults, but may lead to severe complications in young children and in immunocompromised patients. The genome of Cryptosporidium parvum contains a gene, CUTA_CRYPI, that may play a role in regulating the intracellular concentration of copper, a toxic element if left unchecked. Here we report the crystal structure for this CutA1 protein, Cp-CutA1, is reported at 2.0 Å resolution (4E98). As observed for other CutA1 structures, the 117-residue protein is a trimer with a core ferrodoxin-like fold. Circular dichroism spectroscopy shows little unfolding of Cp-CutA1 up to 353 K. This robustness is corroborated by ¹H-¹⁵N HSQC spectra at 333 K that is characteristic of a folded protein, suggesting NMR spectroscopy may be a useful tool to further probe the function of the CutA1 proteins. While robust, Cp-CutA1 is not as stable as the homologous protein from a hyperthermophile, perhaps due to a wide β-bulgein β2 that protrudes P48 and S49 outside the β-sheet.

  5. Comparing the efficacy of chlorine, chlorine dioxide, and ozone in the inactivation of Cryptosporidium parvum in water from Parana State, Southern Brazil.

    PubMed

    Pereira, Juliana Tracz; Costa, Adriana Oliveira; de Oliveira Silva, Márcia Benedita; Schuchard, Wagner; Osaki, Silvia Cristina; de Castro, Edilene Alcântara; Paulino, Rosangela Clara; Soccol, Vanete Thomaz

    2008-12-01

    In the present work, assays were performed to compare the efficacy of hypochlorous acid, chlorine dioxide, and ozone in the inactivation of Cryptosporidium oocyst in public water supply from Brazilian South conditions. Experiments were carried out in samples containing 2 x 10(4) oocysts/ml of C. parvum purified from feces of experimentally contaminated calves. An in vitro excystation method was used to evaluate oocysts' viability and to determine the inactivation rates of hypochlorous acid at 2 ppm, chlorine dioxide at 1, 2, and 5 ppm, and ozone at the doses of 0.18, 0.24, 0.36, 0.48, and 1.44 mg/l. By using hypochlorous acid, the maximum inactivation rate obtained was 49.04% after 120 min. Chlorine dioxide at 5 ppm inactivated 90.56% of oocysts after 90 min of contact. Ozone was the most effective product, rendering an inactivation of 100% with the concentration of 24 mg/l. Resistance of Cryptosporidium to the usual disinfectants and the need for more effective water treatments to prevent waterborne diseases in Brazil are discussed in this manuscript.

  6. Application of Quantitative Real-Time Reverse Transcription-PCR in Assessing Drug Efficacy against the Intracellular Pathogen Cryptosporidium parvum In Vitro

    PubMed Central

    Cai, Xiaomin; Woods, Keith M.; Upton, Steve J.; Zhu, Guan

    2005-01-01

    We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (CT[P18S]) and host cells (CT[H18S]), and evaluates the relative expression between parasite and host rRNA levels (i.e., ΔCT = CT[P18S] − CT[H18S]) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[P]) and the normalized relative level of rRNA (ΔΔCT) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in ∼3 h) in dead parasites is faster than that of DNA (99% in ∼24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC50 values for nitazoxanide and paromomycin were 0.30 to 0.45 μg/ml and 89.7 to 119.0 μg/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit C. parvum development in vitro (MIC50 = 15.8 mM), suggesting that the recently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets. PMID:16251280

  7. Evaluation of Four RNA Extraction Methods for Gene Expression Analyses of Cryptosporidium parvum and Toxoplasma gondii Oocys

    EPA Science Inventory

    Cryptosporidium spp. and Toxoplasma gondii are important coccidian parasites that have caused waterborne and foodborne disease outbreaks worldwide. Techniques like subtractive hybridization, microarrays, and quantitative reverse transcriptase real-time polymerase chain reaction (...

  8. Evaluation of Four RNA Extraction Methods for Gene Expression Analyses of Cryptosporidium parvum and Toxoplasma gondii Oocys

    EPA Science Inventory

    Cryptosporidium spp. and Toxoplasma gondii are important coccidian parasites that have caused waterborne and foodborne disease outbreaks worldwide. Techniques like subtractive hybridization, microarrays, and quantitative reverse transcriptase real-time polymerase chain reaction (...

  9. Reducing gut effects from Cryptosporidium parvum infection in dairy calves through prophylactic glucagon-like peptide 2 therapy or feeding of an artificial sweetener.

    PubMed

    Connor, E E; Wall, E H; Bravo, D M; Evock-Clover, C M; Elsasser, T H; Baldwin, R L; Santín, M; Vinyard, B T; Kahl, S; Walker, M P

    2017-04-01

    Glucagon-like peptide 2 (GLP-2) therapy was shown previously to reduce inflammation-related gut damage from coccidiosis in dairy calves, and feeding of artificial sweetener stimulates GLP-2 secretion from intestinal L cells. The purpose of this study was to determine whether GLP-2 treatment or artificial sweetener feeding beginning 1 wk before an experimental inoculation with the coccidian parasite Cryptosporidium parvum can reduce infection-related intestinal damage in Holstein bull calves. Newborn calves were assigned to 1 of 4 treatment groups of 6 calves each, including noninfected control calves injected s.c. every 12 h with control buffer (CON), infected control calves injected s.c. every 12 h with control buffer (INF), infected calves injected s.c. every 12 h with 50 µg/kg of body weight of GLP-2 (GLP2), and infected calves injected s.c. every 12 h with control buffer and supplemented in the diet with Sucram (Pancosma, Geneva, Switzerland) at 400 mg/kg of dry matter of milk replacer (SUC). Treatments were initiated on d 1, and calves in INF, GLP2, and SUC were orally dosed on d 8 with 12,500 C. parvum oocysts. Fecal scores were recorded daily, plasma was collected on d 1, 8, 12, 15, and 18 to evaluate markers of inflammation, and fecal samples were collected on d 1, 8, and every other day thereafter to determine the presence of oocysts. Calves were euthanized on d 18 for collection of intestinal tissues and histological and gene expression analyses. Relative to CON, calves in INF exhibited an increase in diarrhea severity, increased plasma serum amyloid A concentration on d 15 and 18, reduced intestinal villus height, increased villus apoptosis and crypt cell proliferation, and increased intestinal mRNA expression of MARVELD2 and GPX2. However, calves in SUC and GLP2 had reduced diarrhea severity and fecal C. parvum oocyst shedding, reduced plasma serum amyloid A concentration on d 15 and 18, and, depending on the intestinal segment, increased villus height

  10. Speeding up the solar water disinfection process (SODIS) against Cryptosporidium parvum by using 2.5l static solar reactors fitted with compound parabolic concentrators (CPCs).

    PubMed

    Gómez-Couso, H; Fontán-Sainz, M; Fernández-Ibáñez, P; Ares-Mazás, E

    2012-12-01

    Water samples of 0, 5, and 100 nephelometric turbidity units (NTU) spiked with Cryptosporidium parvum oocysts were exposed to natural sunlight in 2.5l static borosilicate solar reactors fitted with two different compound parabolic concentrators (CPCs), CPC1 and CPC1.89, with concentration factors of the solar radiation of 1 and 1.89, respectively. The global oocyst viability was calculated by the evaluation of the inclusion/exclusion of the fluorogenic vital dye propidium iodide and the spontaneous excystation. Thus, the initial global oocyst viability of the C. parvum isolate used was 95.3 ± 1.6%. Using the solar reactors fitted with CPC1, the global viability of oocysts after 12h of exposure was zero in the most turbid water samples (100 NTU) and almost zero in the other water samples (0.3 ± 0.0% for 0 NTU and 0.5 ± 0.2% for 5 NTU). Employing the solar reactors fitted with CPC1.89, after 10h exposure, the global oocyst viability was zero in the non-turbid water samples (0 NTU), and it was almost zero in the 5 NTU water samples after 8h of exposure (0.5 ± 0.5%). In the most turbid water samples (100 NTU), the global viability was 1.9 ± 0.6% after 10 and 12h of exposure. In conclusion, the use of these 2.5l static solar reactors fitted with CPCs significantly improved the efficacy of the SODIS technique as these systems shorten the exposure times to solar radiation, and also minimize the negative effects of turbidity. This technology therefore represents a good alternative method for improving the microbiological quality of household drinking water in developing countries. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Inhibitory Activities of Epidermal Growth Factor Receptor Tyrosine Kinase-Targeted Dihydroxyisoflavone and Trihydroxydeoxybenzoin Derivatives on Sarcocystis neurona, Neospora caninum, and Cryptosporidium parvum Development

    PubMed Central

    Gargala, G.; Baishanbo, A.; Favennec, L.; François, A.; Ballet, J. J.; Rossignol, J.-F.

    2005-01-01

    Several gene sequences of parasitic protozoa belonging to protein kinase gene families and epidermal growth factor (EGF)-like peptides, which act via binding to receptor tyrosine kinases of the EGF receptor (EGFR) family, appear to mediate host-protozoan interactions. As a clue to EGFR protein tyrosine kinase (PTK) mediation and a novel approach for identifying anticoccidial agents, activities against Sarcocystis neurona, Neospora caninum, and Cryptosporidium parvum grown in BM and HCT-8 cell cultures of 52 EGFR PTK inhibitor isoflavone analogs (dihydroxyisoflavone and trihydroxydeoxybenzoine derivatives) were investigated. Their cytotoxicities against host cells were either absent, mild, or moderate by a nitroblue tetrazolium test. At concentrations ranging from 5 to 10 μg/ml, 20 and 5 analogs, including RM-6427 and RM-6428, exhibited an in vitro inhibitory effect of ≥95% against at least one parasite or against all three, respectively. In immunosuppressed Cryptosporidium parvum-infected Mongolian gerbils orally treated with either 200 or 400 mg of agent RM-6427/kg of body weight/day for 8 days, fecal microscopic oocyst shedding was abolished in 6/10 animals (P of <0.001 versus untreated controls) and mean shedding was reduced by 90.5% (P of <0.0001) and 92.0% (P of <0.0001), respectively, higher levels of inhibition than after nitazoxanide (200 mg/kg/day for 8 days) or paromomycin (100 mg/kg/day for 8 days) treatment (55.0%, P of <0.001, and 17.5%, P of >0.05, respectively). After RM-6427 therapy (200 mg/kg/day for 8 days), the reduction in the ratio of animals with intracellular parasites was nearly significant in ileum (P = 0.067) and more marked in the biliary tract (P < 0.0013) than after nitazoxanide or paromomycin treatment (0.05 < P < 0.004). RM-6428 treatment at a regimen of 400 mg/kg/day for 12 days inhibited oocyst shedding, measured using flow cytometry from day 4 (P < 0.05) to day 12 (P < 0.02) of therapy, when 2/15 animals had no shedding (P

  12. Die-off rates of Cryptosporidium parvum oocysts in a swine lagoon and in a spray field

    USDA-ARS?s Scientific Manuscript database

    Background: Because of several large-scale outbreaks of cryptosporidiosis in humans, Cryptosporidium has become a public health concern. Commercial swine operations apply large volumes of effluent from lagoons to spray fields as a waste management practice. This effluent is a source of Cryptosporidi...

  13. Transport of MS2 phage, Escherichia coli, Clostridium perfringens, Cryptosporidium parvum, and Giardia intestinalis in a gravel and a sandy soil.

    PubMed

    Hijnen, Wim A M; Brouwer-Hanzens, Anke J; Charles, Katrina J; Medema, Gertjan J

    2005-10-15

    To define protection zones around groundwater abstraction wells and safe setback distances for artificial recharge systems in watertreatment, quantitative information is needed about the removal of microorganisms during soil passage. Column experiments were conducted using natural soil and water from an infiltration site with fine sandy soil and a river bank infiltration site with gravel soil. The removal of phages, bacteria, bacterial spores, and protozoan (oo)-cysts was determined at two velocities and compared with field data from the same sites. The microbial elimination rate (MER) in both soils was generally >2 log, but MER in the gravel soil was higher than that in the fine sandy soil. This was attributed to enhanced attachment, related to higher metal-hydroxides content. From the high sticking efficiencies (>1) and the low influence of flow rate on MER it was deduced that straining played a significant role in the removal of Escherichia coli and Cryptosporidium parvum oocysts in the gravel soil. Lower removal of oocysts than the 4-5 times smaller E. coli and spores in the fine sand indicates that the contribution of straining is variable and needs further attention in transport models. Thus, simple extrapolation of grain size and particle size to the extent of microbial transport underground is inappropriate. Finally, the low MER of indigenous E. coli and Clostridium perfringens observed in the soil columns as well as under field conditions and the second breakthrough peak found for Cryptosporidium and spores in the fine sandy soil upon a change in the feedwater pH indicate a significant role of detachment and retardation to microbial transport and the difficulty of extrapolation of quantitative column test results to field conditions.

  14. Intra-Species Genetic Diversity and Clonal Structure of Cryptosporidium parvum in Sheep Farms in a Confined Geographical Area in Northeastern Spain

    PubMed Central

    Ramo, Ana; Monteagudo, Luis V.; Del Cacho, Emilio; Sánchez-Acedo, Caridad

    2016-01-01

    A multilocus fragment typing approach including eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene was used to investigate the intra-farm and intra-host genetic diversity of Cryptosporidium parvum in sheep farms in a confined area in northeastern Spain. Genomic DNA samples of 113 C. parvum isolates from diarrheic pre-weaned lambs collected in 49 meat-type sheep farms were analyzed. Loci exhibited various degrees of polymorphism, the finding of 7–9 alleles in the four most variable and discriminatory markers (ML2, Cgd6_5400, Cgd6_3940, and GP60) being remarkable. The combination of alleles at the twelve loci identified a total of 74 multilocus subtypes (MLTs) and provided a Hunter-Gaston discriminatory index of 0.988 (95% CI, 0.979−0.996). The finding that most MLTs (n = 64) were unique to individual farms evidenced that cryptosporidial infection is mainly transmitted within sheep flocks, with herd-to-herd transmission playing a secondary role. Limited intra- host variability was found, since only five isolates were genotypically mixed. In contrast, a significant intra-farm genetic diversity was seen, with the presence of multiple MLTs on more than a half of the farms (28/46), suggesting frequent mutations or genetic exchange through recombination. Comparison with a previous study in calves in northern Spain using the same 12-loci typing approach showed differences in the identity of major alleles at most loci, with a single MLT being shared between lambs and calves. Analysis of evolutionary descent by the algorithm eBURST indicated a high degree of genetic divergence, with over 41% MLTs appearing as singletons along with a high number of clonal complexes, most of them linking only two MLTs. Bayesian Structure analysis and F statistics also revealed the genetic remoteness of most C. parvum isolates and no ancestral population size was chosen. Linkage analysis evidenced a prevalent pattern of clonality within the parasite population. PMID:27176718

  15. Disinfection and toxicological assessments of pulsed UV and pulsed-plasma gas-discharge treated-water containing the waterborne protozoan enteroparasite Cryptosporidium parvum.

    PubMed

    Hayes, Jennifer; Kirf, Dominik; Garvey, Mary; Rowan, Neil

    2013-09-01

    We report for the first time on the comparative use of pulsed-plasma gas-discharge (PPGD) and pulsed UV light (PUV) for the novel destruction of the waterborne enteroparasite Cryptosporidium parvum. It also describes the first cyto-, geno- and ecotoxicological assays undertaken to assess the safety of water decontaminated using PPGD and PUV. During PPGD treatments, the application of high voltage pulses (16 kV, 10 pps) to gas-injected water (N2 or O2, flow rate 2.5L/min) resulted in the formation of a plasma that generated free radicals, ultraviolet light, acoustic shock waves and electric fields that killed ca. 4 log C. parvum oocysts in 32 min exposure. Findings showed that PPGD-treated water produced significant cytotoxic properties (as determined by MTT and neutral red assays), genotoxic properties (as determined by comet and Ames assays), and ecotoxic properties (as determined by Microtox™, Thamnotox™ and Daphnotox™ assays) that are representative of different trophic levels in aquatic environment (p<0.05). Depending in part on the type of injected gas used, PPGD-treated water became either alkaline (pH ≤ 8.58, using O2) or acidic (pH ≥ 3.21, using N2) and contained varying levels of reactive free radicals such as ozone (0.8 mg/L) and/or dissociated nitric and nitrous acid that contributed to the observed disinfection and toxicity. Chemical analysis of PPGD-treated water revealed increasing levels of electrode metals that were present at ≤ 30 times the tolerated respective values for EU drinking water. PUV-treated water did not exhibit any toxicity and was shown to be far superior to that of PPGD for killing C. parvum oocysts taking only 90 s of pulsing [UV dose of 6.29 μJ/cm(2)] to produce a 4-log reduction compared to a similar reduction level achieved after 32min PPGD treatment as determined by combined in vitro CaCo-2 cell culture-qPCR.

  16. Comparison of transport and attachment behaviors of Cryptosporidium parvum oocysts and oocyst-sized microspheres being advected through three minerologically different granular porous media

    USGS Publications Warehouse

    Mohanram, Arvind; Ray, Chittaranjan; Harvey, Ronald W.; Metge, David W.; Ryan, Joseph N.; Chorover, Jon; Eberl, D.D.

    2010-01-01

    In order to gain more information about the fate of Cryptosporidium parvum oocysts in tropical volcanic soils, the transport and attachment behaviors of oocysts and oocyst-sized polystyrene microspheres were studied in the presence of two soils. These soils were chosen because of their differing chemical and physical properties, i.e., an organic-rich (43–46% by mass) volcanic ash-derived soil from the island of Hawaii, and a red, iron (22–29% by mass), aluminum (29–45% by mass), and clay-rich (68–76% by mass) volcanic soil from the island of Oahu. A third agricultural soil, an organic- (13% by mass) and quartz-rich (40% by mass) soil from Illinois, was included for reference. In 10-cm long flow-through columns, oocysts and microspheres advecting through the red volcanic soil were almost completely (98% and 99%) immobilized. The modest breakthrough resulted from preferential flow-path structure inadvertently created by soil-particle aggregation during the re-wetting process. Although a high (99%) removal of oocysts and microsphere within the volcanic ash soil occurred initially, further examination revealed that transport was merely retarded because of highly reversible interactions with grain surfaces. Judging from the slope of the substantive and protracted tail of the breakthrough curve for the 1.8-μm microspheres, almost all (>99%) predictably would be recovered within ∼4000 pore volumes. This suggests that once contaminated, the volcanic ash soil could serve as a reservoir for subsequent contamination of groundwater, at least for pathogens of similar size or smaller. Because of the highly reversible nature of organic colloid immobilization in this soil type, C. parvum could contaminate surface water should overland flow during heavy precipitation events pick up near-surface grains to which they are attached. Surprisingly, oocyst and microsphere attachment to the reference soil from Illinois appeared to be at least as sensitive to changes in pH as

  17. Comparison of transport and attachment behaviors of Cryptosporidium parvum oocysts and oocyst-sized microspheres being advected through three minerologically different granular porous media

    USGS Publications Warehouse

    Mohanram, A.; Ray, C.; Harvey, R.W.; Metge, D.W.; Ryan, J.N.; Chorover, J.; Eberl, D.D.

    2010-01-01

    In order to gain more information about the fate of Cryptosporidium parvum oocysts in tropical volcanic soils, the transport and attachment behaviors of oocysts and oocyst-sized polystyrene microspheres were studied in the presence of two soils. These soils were chosen because of their differing chemical and physical properties, i.e., an organic-rich (43-46% by mass) volcanic ash-derived soil from the island of Hawaii, and a red, iron (22-29% by mass), aluminum (29-45% by mass), and clay-rich (68-76% by mass) volcanic soil from the island of Oahu. A third agricultural soil, an organic- (13% by mass) and quartz-rich (40% by mass) soil from Illinois, was included for reference. In 10-cm long flow-through columns, oocysts and microspheres advecting through the red volcanic soil were almost completely (98% and 99%) immobilized. The modest breakthrough resulted from preferential flow-path structure inadvertently created by soil-particle aggregation during the re-wetting process. Although a high (99%) removal of oocysts and microsphere within the volcanic ash soil occurred initially, further examination revealed that transport was merely retarded because of highly reversible interactions with grain surfaces. Judging from the slope of the substantive and protracted tail of the breakthrough curve for the 1.8-??m microspheres, almost all (>99%) predictably would be recovered within ~4000 pore volumes. This suggests that once contaminated, the volcanic ash soil could serve as a reservoir for subsequent contamination of groundwater, at least for pathogens of similar size or smaller. Because of the highly reversible nature of organic colloid immobilization in this soil type, C. parvum could contaminate surface water should overland flow during heavy precipitation events pick up near-surface grains to which they are attached. Surprisingly, oocyst and microsphere attachment to the reference soil from Illinois appeared to be at least as sensitive to changes in pH as was observed

  18. Intra-Species Genetic Diversity and Clonal Structure of Cryptosporidium parvum in Sheep Farms in a Confined Geographical Area in Northeastern Spain.

    PubMed

    Ramo, Ana; Monteagudo, Luis V; Del Cacho, Emilio; Sánchez-Acedo, Caridad; Quílez, Joaquín

    2016-01-01

    A multilocus fragment typing approach including eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene was used to investigate the intra-farm and intra-host genetic diversity of Cryptosporidium parvum in sheep farms in a confined area in northeastern Spain. Genomic DNA samples of 113 C. parvum isolates from diarrheic pre-weaned lambs collected in 49 meat-type sheep farms were analyzed. Loci exhibited various degrees of polymorphism, the finding of 7-9 alleles in the four most variable and discriminatory markers (ML2, Cgd6_5400, Cgd6_3940, and GP60) being remarkable. The combination of alleles at the twelve loci identified a total of 74 multilocus subtypes (MLTs) and provided a Hunter-Gaston discriminatory index of 0.988 (95% CI, 0.979-0.996). The finding that most MLTs (n = 64) were unique to individual farms evidenced that cryptosporidial infection is mainly transmitted within sheep flocks, with herd-to-herd transmission playing a secondary role. Limited intra- host variability was found, since only five isolates were genotypically mixed. In contrast, a significant intra-farm genetic diversity was seen, with the presence of multiple MLTs on more than a half of the farms (28/46), suggesting frequent mutations or genetic exchange through recombination. Comparison with a previous study in calves in northern Spain using the same 12-loci typing approach showed differences in the identity of major alleles at most loci, with a single MLT being shared between lambs and calves. Analysis of evolutionary descent by the algorithm eBURST indicated a high degree of genetic divergence, with over 41% MLTs appearing as singletons along with a high number of clonal complexes, most of them linking only two MLTs. Bayesian Structure analysis and F statistics also revealed the genetic remoteness of most C. parvum isolates and no ancestral population size was chosen. Linkage analysis evidenced a prevalent pattern of clonality within the parasite population.

  19. Effect of ferric oxyhydroxide grain coatings on the transport of bacteriophage PRD1 and Cryptosporidium parvum oocysts in saturated porous media

    USGS Publications Warehouse

    Abudalo, R.A.; Bogatsu, Y.G.; Ryan, J.N.; Harvey, R.W.; Metge, D.W.; Elimelech, M.

    2005-01-01

    To test the effect of geochemical heterogeneity on microorganism transport in saturated porous media, we measured the removal of two microorganisms, the bacteriophage PRD1 and oocysts of the protozoan parasite Cryptosporidium parvum, in flow-through columns of quartz sand coated by different amounts of a ferric oxyhydroxide. The experiments were conducted over ranges of ferric oxyhydroxide coating fraction of ?? = 0-0.12 for PRD1 and from ?? = 0-0.32 for the oocysts at pH 5.6-5.8 and 10-4 M ionic strength. To determine the effect of pH on the transport of the oocysts, experiments were also conducted over a pH range of 5.7-10.0 at a coating fraction of ?? = 0.04. Collision (attachment) efficiencies increased as the fraction of ferric oxyhydroxide coated quartz sand increased, from ?? = 0.0071 to 0.13 over ?? = 0-0.12 for PRD1 and from ?? = 0.059 to 0.75 over ?? = 0-0.32 for the oocysts. Increasing the pH from 5.7 to 10.0 resulted in a decrease in the oocyst collision efficiency as the pH exceeded the expected point of zero charge of the ferric oxyhydroxide coatings. The collision efficiencies correlated very well with the fraction of quartz sand coated by the ferric oxyhydroxide for PRD1 but not as well for the oocysts. ?? 2005 American Chemical Society.

  20. Evaluation of the solar water disinfection process (SODIS) against Cryptosporidium parvum using a 25-L static solar reactor fitted with a compound parabolic collector (CPC).

    PubMed

    Fontán-Sainz, María; Gómez-Couso, Hipólito; Fernández-Ibáñez, Pilar; Ares-Mazás, Elvira

    2012-02-01

    Water samples of 0, 5, and 30 nephelometric turbidity units (NTU) spiked with Cryptosporidium parvum oocysts were exposed to natural sunlight using a 25-L static solar reactor fitted with a compound parabolic collector (CPC). The global oocyst viability was calculated by the evaluation of the inclusion/exclusion of the fluorogenic vital dye propidium iodide and the spontaneous excystation. After an exposure time of 8 hours, the global oocyst viabilities were 21.8 ± 3.1%, 31.3 ± 12.9%, and 45.0 ± 10.0% for turbidity levels of 0, 5, and 30 NTU, respectively, and these values were significantly lower (P < 0.05) that the initial global viability of the isolate (92.1 ± 0.9%). The 25-L static solar reactor that was evaluated can be an alternative system to the conventional solar water disinfection process for improving the microbiological quality of drinking water on a household level, and moreover, it enables treatment of larger volumes of water (> 10 times).

  1. Evaluation of the Solar Water Disinfection Process (SODIS) Against Cryptosporidium parvum Using a 25-L Static Solar Reactor Fitted with a Compound Parabolic Collector (CPC)

    PubMed Central

    Fontán-Sainz, María; Gómez-Couso, Hipólito; Fernández-Ibáñez, Pilar; Ares-Mazás, Elvira

    2012-01-01

    Water samples of 0, 5, and 30 nephelometric turbidity units (NTU) spiked with Cryptosporidium parvum oocysts were exposed to natural sunlight using a 25-L static solar reactor fitted with a compound parabolic collector (CPC). The global oocyst viability was calculated by the evaluation of the inclusion/exclusion of the fluorogenic vital dye propidium iodide and the spontaneous excystation. After an exposure time of 8 hours, the global oocyst viabilities were 21.8 ± 3.1%, 31.3 ± 12.9%, and 45.0 ± 10.0% for turbidity levels of 0, 5, and 30 NTU, respectively, and these values were significantly lower (P < 0.05) that the initial global viability of the isolate (92.1 ± 0.9%). The 25-L static solar reactor that was evaluated can be an alternative system to the conventional solar water disinfection process for improving the microbiological quality of drinking water on a household level, and moreover, it enables treatment of larger volumes of water (> 10 times). PMID:22302852

  2. Structure of a CutA1 divalent-cation tolerance protein from Cryptosporidium parvum, the protozoal parasite responsible for cryptosporidiosis

    PubMed Central

    Buchko, Garry W.; Abendroth, Jan; Clifton, Matthew C.; Robinson, Howard; Zhang, Yanfeng; Hewitt, Stephen N.; Staker, Bart L.; Edwards, Thomas E.; Van Voorhis, Wesley C.; Myler, Peter J.

    2015-01-01

    Cryptosporidiosis is an infectious disease caused by protozoan parasites of the Cryptosporidium genus. Infection is associated with mild to severe diarrhea that usually resolves spontaneously in healthy human adults, but may lead to severe complications in young children and in immunocompromised patients. The genome of C. parvum contains a gene, CUTA_CRYPI, that may play a role in regulating the intracellular concentration of copper, which is a toxic element in excess. Here, the crystal structure of this CutA1 protein, Cp-CutA1, is reported at 2.0 Å resolution. As observed for other CutA1 structures, the 117-residue protein is a trimer with a core ferrodoxin-like fold. Circular dichroism spectroscopy shows little, in any, unfolding of Cp-CutA1 up to 353 K. This robustness is corroborated by 1H–15N HSQC spectra at 333 K, which are characteristic of a folded protein, suggesting that NMR spectroscopy may be a useful tool to further probe the function of the CutA1 proteins. While robust, Cp-CutA1 is not as stable as the homologous protein from a hyperthermophile, perhaps owing to a wide β-bulge in β2 that protrudes Pro48 and Ser49 outside the β-sheet. PMID:25945704

  3. Cryptosporidiosis outbreak in visitors of a UK industry-compliant petting farm caused by a rare Cryptosporidium parvum subtype: a case-control study.

    PubMed

    Utsi, L; Smith, S J; Chalmers, R M; Padfield, S

    2016-04-01

    A case-control study was conducted to investigate an outbreak of 46 cases of cryptosporidiosis in visitors to a petting farm in England. Details of exposures on the farm were collected for 38 cases and 39 controls, recruited through snowball sampling. Multivariable logistic regression identified that cases were 5·5 times more likely than controls to have eaten without washing their hands [95% confidence interval (CI) 1·51-19·9, P = 0·01] and 10 times less likely to report being informed of risk of infection on arrival (odds ratio 0·10, 95% CI 0·01-0·71, P = 0·02). An uncommon Cryptosporidium parvum gp60 subtype (IIaA19G1R1) was identified in a lamb faecal sample and all subtyped cases (n = 22). We conclude that lack of verbal advice and non-compliance with hand washing are significantly associated with a risk of cryptosporidiosis on open farms. These findings highlight the public health importance of effectively communicating risk to petting farm visitors in order to prevent future outbreaks of zoonotic infections.

  4. Activities of dl-α-Difluoromethylarginine and Polyamine Analogues against Cryptosporidium parvum Infection in a T-Cell Receptor Alpha-Deficient Mouse Model▿

    PubMed Central

    Yarlett, Nigel; Waters, W. Ray; Harp, James A.; Wannemuehler, Michael J.; Morada, Mary; Bellcastro, Josephine; Upton, Steve J.; Marton, Laurence J.; Frydman, Benjamin J.

    2007-01-01

    The in vivo effectiveness of a series of conformationally restricted polyamine analogues alone and selected members in combination with dl-α-difluoromethylarginine against Cryptosporidium parvum infection in a T-cell receptor alpha-deficient mouse model was tested. Polyamine analogues were selected from the extended bis(ethyl)-sym-homospermidine or bis(ethyl)-spermine backbone having cis or trans double bonds at the center of the molecule. The cis isomers were found to have significantly greater efficacy in both preventing and curing infection in a mouse model than the trans polyamine analogues when tested in a T-cell receptor alpha-deficient mouse model. When tested in combination with dl-α-difluoromethylarginine, the cis-restricted analogues were found to be more effective in preventing oocyst shedding. This study demonstrates the potential of polyamine analogues as anticryptosporidial agents and highlights the presence of multiple points in polyamine synthesis by this parasite that are susceptible to inhibition resulting in growth inhibition. PMID:17242149

  5. Influence of organic matter on the transport of Cryptosporidium parvum oocysts in a ferric oxyhydroxide-coated quartz sand saturated porous medium

    USGS Publications Warehouse

    Abudalo, R.A.; Ryan, J.N.; Harvey, R.W.; Metge, D.W.; Landkamer, L.

    2010-01-01

    To assess the effect of organic matter on the transport of Cryptosporidium parvum oocysts in a geochemically heterogeneous saturated porous medium, we measured the breakthrough and collision efficiencies of oocysts as a function of dissolved organic matter concentration in a flow-through column containing ferric oxyhydroxide-coated sand. We characterized the surface properties of the oocysts and ferric oxyhydroxide-coated sand using microelectrophoresis and streaming potential, respectively, and the amount of organic matter adsorbed on the ferric oxyhydroxide-coated sand as a function of the concentration of dissolved organic matter (a fulvic acid isolated from Florida Everglades water). The dissolved organic matter had no significant effect on the zeta potential of the oocysts. Low concentrations of dissolved organic matter were responsible for reversing the charge of the ferric oxyhydroxide-coated sand surface from positive to negative. The charge reversal and accumulation of negative charge on the ferric oxyhydroxide-coated sand led to increases in oocyst breakthrough and decreases in oocyst collision efficiency with increasing dissolved organic matter concentration. The increase in dissolved organic matter concentration from 0 to 20 mg L-1 resulted in a two-fold decrease in the collision efficiency. ?? 2009 Elsevier Ltd.

  6. Cryptosporidium parvum in calves: kinetics and immunoblot analysis of specific serum and local antibody responses (immunoglobulin A [IgA], IgG, and IgM) after natural and experimental infections.

    PubMed Central

    Peeters, J E; Villacorta, I; Vanopdenbosch, E; Vandergheynst, D; Naciri, M; Ares-Mazás, E; Yvoré, P

    1992-01-01

    Fecal and serum anti-Cryptosporidium parvum immunoglobulin A (IgA), IgM, and IgG were monitored by an enzyme-linked immunosorbent assay after experimental and natural infection of calves with C. parvum. Although all experimentally infected calves showed high levels of colostral antibodies in the feces, they acquired C. parvum infection. Three of five animals died. Calves which acquired natural infection showed only diarrhea. Levels of colostral coproantibodies dropped quickly. Experimental infection was followed by a rise in local anti-C. parvum IgM levels from day 5 postinfection (p.i.). IgM peaked at day 14 p.i. and then disappeared quickly. Anti-C. parvum IgA levels rose between days 7 and 14 p.i. and decreased slowly. Rising levels of coproantibodies coincided with falling oocyst output. Fecal anti-C. parvum IgG levels rose slightly during oocyst output, and IgG disappeared 3 weeks p.i. Similar kinetics were established in naturally infected calves. Although fecal anti-C. parvum IgA levels declined slowly, reinfections were established 5, 7, and 14 weeks after the primary contact. Serum anti-C. parvum IgG levels rose during maximal oocyst excretion, whereas serum anti-C. parvum IgA levels peaked later than did local IgA levels. Challenge reinfection of naturally infected calves at day 112 was not followed by clinical signs or oocyst output or by a secondary antibody response. Sequential Western immunoblotting with fecal extracts revealed up to 32 different parasite antigens. Convalescent-phase sera recognized up to 23 antigens. Fecal IgA reacted intensely with antigens with relative molecular weights (M(r)) of approximately 11,000 and 15,000. These antigens were not recognized by convalescent-phase serum IgG. Both local IgA and serum IgG also showed strong reactions with 23,000- and 44,000-M(r) antigens and with several antigens of between 66,200 and 200,000 M(r). Most bands remained detectable for at least 16 weeks p.i. Images PMID:1587597

  7. Influence of organic carbon loading, sediment associated metal oxide content and sediment grain size distributions upon Cryptosporidium parvum removal during riverbank filtration operations, Sonoma County, CA

    USGS Publications Warehouse

    Metge, D.W.; Harvey, R.W.; Aiken, G.R.; Anders, R.; Lincoln, G.; Jasperse, J.

    2010-01-01

    This study assessed the efficacy for removing Cryptosporidium parvum oocysts of poorly sorted, Fe- and Al-rich, subsurface sediments collected from 0.9 to 4.9 and 1.7-13.9??m below land surface at an operating riverbank filtration (RBF) site (Russian River, Sonoma County, CA). Both formaldehyde-killed oocysts and oocyst-sized (3????m) microspheres were employed in sediment-packed flow-through and static columns. The degree of surface coverage of metal oxides on sediment grain surfaces correlated strongly with the degrees of oocyst and microsphere removals. In contrast, average grain size (D50) was not a good indicator of either microsphere or oocyst removal, suggesting that the primary mechanism of immobilization within these sediments is sorptive filtration rather than physical straining. A low specific UV absorbance (SUVA) for organic matter isolated from the Russian River, suggested that the modest concentration of the SUVA component (0.8??mg??L-1) of the 2.2??mg??L-1 dissolved organic carbon (DOC) is relatively unreactive. Nevertheless, an amendment of 2.2??mg??L-1 of isolated river DOC to column sediments resulted in up to a 35.7% decrease in sorption of oocysts and (or) oocyst-sized microspheres. Amendments (3.2????M) of the anionic surfactant, sodium dodecyl benzene sulfonate (SDBS) also caused substantive decreases (up to 31.9 times) in colloid filtration. Although the grain-surface metal oxides were found to have a high colloid-removal capacity, our study suggested that any major changes within the watershed that would result in long-term alterations in either the quantity and (or) the character of the river's DOC could alter the effectiveness of pathogen removal during RBF operations.

  8. Pathogen and chemical transport in the karst limestone of the Biscayne aquifer: 3. Use of microspheres to estimate the transport potential of Cryptosporidium parvum oocysts

    USGS Publications Warehouse

    Harvey, R.W.; Metge, D.W.; Shapiro, A.M.; Renken, R.A.; Osborn, C.L.; Ryan, J.N.; Cunningham, K.J.; Landkamer, Lee L.

    2008-01-01

    The vulnerability of a municipal well in the Northwest well field in southeastern Florida to potential contamination by Cryptosporidium parvum oocysts was assessed in a large-scale, forced-gradient (convergent) injection and recovery test. The field study involved a simultaneous pulse introduction of a nonreactive tracer (SF6, an inert gas) and oocyst-sized (1.6, 2.9, and 4.9 ??m diameter) carboxylated polystyrene microspheres into karst limestone of the Biscayne aquifer characterized by a complex triple (matrix, touching-vug, and conduit) porosity. Fractional recoveries 97 m down gradient were inversely related to diameter and ranged from 2.9% for the 4.9 ??m microspheres to 5.8% for 1.6 ??m microspheres. Their centers of mass arrived at the pumping well approximately threefold earlier than that of the nonreactive tracer SF6 (gas), underscoring the need for use of colloid tracers and field-scale tracer tests for these kinds of evaluations. In a modified triaxial cell using near in situ chemical conditions, 2.9 and 4.9 ??m microspheres underestimated by fourfold to sixfold the attachment potential of the less electronegative 2.9-4.1 ??m oocysts in the matrix porosity of limestone core samples. The field and laboratory results collectively suggested that it may take 200-300 m of transport to ensure even a 1-log unit removal of oocysts, even though the limestone surfaces exhibited a substantive capability for their sorptive removal. The study further demonstrated the utility of microspheres as oocyst surrogates in field-scale assessments of well vulnerability in limestone, provided that differences in attachment behaviors between oocysts and microspheres are taken into account. Copyright 2008 by the American Geophysical Union.

  9. Interactions of Cryptosporidium parvum, Giardia lamblia, Vaccinal Poliovirus Type 1, and Bacteriophages φX174 and MS2 with a Drinking Water Biofilm and a Wastewater Biofilm▿

    PubMed Central

    Helmi, Karim; Skraber, Sylvain; Gantzer, Christophe; Willame, Raphaël; Hoffmann, Lucien; Cauchie, Henry-Michel

    2008-01-01

    Biofilms colonizing surfaces inside drinking water distribution networks may provide a habitat and shelter to pathogenic viruses and parasites. If released from biofilms, these pathogens may disseminate in the water distribution system and cause waterborne diseases. Our study aimed to investigate the interactions of protozoan parasites (Cryptosporidium parvum and Giardia lamblia [oo]cysts) and viruses (vaccinal poliovirus type 1, φX174, and MS2) with two contrasting biofilms. First, attachment, persistence, and detachment of the protozoan parasites and the viruses were assessed with a drinking water biofilm. This biofilm was allowed to develop inside a rotating annular reactor fed with tap water for 7 months prior to the inoculation. Our results show that viable parasites and infectious viruses attached to the drinking water biofilm within 1 h and persisted within the biofilm. Indeed, infectious viruses were detected in the drinking water biofilm up to 6 days after the inoculation, while viral genome and viable parasites were still detected at day 34, corresponding to the last day of the monitoring period. Since viral genome was detected much longer than infectious particles, our results raise the question of the significance of detecting viral genomes in biofilms. A transfer of viable parasites and viruses from the biofilm to the water phase was observed after the flow velocity was increased but also with a constant laminar flow rate. Similar results regarding parasite and virus attachment and detachment were obtained using a treated wastewater biofilm, suggesting that our observations might be extrapolated to a wide range of environmental biofilms and confirming that biofilms can be considered a potential secondary source of contamination. PMID:18281435

  10. Interactions of Cryptosporidium parvum, Giardia lamblia, vaccinal poliovirus type 1, and bacteriophages phiX174 and MS2 with a drinking water biofilm and a wastewater biofilm.

    PubMed

    Helmi, Karim; Skraber, Sylvain; Gantzer, Christophe; Willame, Raphaël; Hoffmann, Lucien; Cauchie, Henry-Michel

    2008-04-01

    Biofilms colonizing surfaces inside drinking water distribution networks may provide a habitat and shelter to pathogenic viruses and parasites. If released from biofilms, these pathogens may disseminate in the water distribution system and cause waterborne diseases. Our study aimed to investigate the interactions of protozoan parasites (Cryptosporidium parvum and Giardia lamblia [oo]cysts) and viruses (vaccinal poliovirus type 1, phiX174, and MS2) with two contrasting biofilms. First, attachment, persistence, and detachment of the protozoan parasites and the viruses were assessed with a drinking water biofilm. This biofilm was allowed to develop inside a rotating annular reactor fed with tap water for 7 months prior to the inoculation. Our results show that viable parasites and infectious viruses attached to the drinking water biofilm within 1 h and persisted within the biofilm. Indeed, infectious viruses were detected in the drinking water biofilm up to 6 days after the inoculation, while viral genome and viable parasites were still detected at day 34, corresponding to the last day of the monitoring period. Since viral genome was detected much longer than infectious particles, our results raise the question of the significance of detecting viral genomes in biofilms. A transfer of viable parasites and viruses from the biofilm to the water phase was observed after the flow velocity was increased but also with a constant laminar flow rate. Similar results regarding parasite and virus attachment and detachment were obtained using a treated wastewater biofilm, suggesting that our observations might be extrapolated to a wide range of environmental biofilms and confirming that biofilms can be considered a potential secondary source of contamination.

  11. Pathogen and chemical transport in the karst limestone of the Biscayne aquifer: 3. Use of microspheres to estimate the transport potential of Cryptosporidium parvum oocysts

    NASA Astrophysics Data System (ADS)

    Harvey, Ronald W.; Metge, David W.; Shapiro, Allen M.; Renken, Robert A.; Osborn, Christina L.; Ryan, Joseph N.; Cunningham, Kevin J.; Landkamer, Lee

    2008-08-01

    The vulnerability of a municipal well in the Northwest well field in southeastern Florida to potential contamination by Cryptosporidium parvum oocysts was assessed in a large-scale, forced-gradient (convergent) injection and recovery test. The field study involved a simultaneous pulse introduction of a nonreactive tracer (SF6, an inert gas) and oocyst-sized (1.6, 2.9, and 4.9 μm diameter) carboxylated polystyrene microspheres into karst limestone of the Biscayne aquifer characterized by a complex triple (matrix, touching-vug, and conduit) porosity. Fractional recoveries 97 m down gradient were inversely related to diameter and ranged from 2.9% for the 4.9 μm microspheres to 5.8% for 1.6 μm microspheres. Their centers of mass arrived at the pumping well approximately threefold earlier than that of the nonreactive tracer SF6 (gas), underscoring the need for use of colloid tracers and field-scale tracer tests for these kinds of evaluations. In a modified triaxial cell using near in situ chemical conditions, 2.9 and 4.9 μm microspheres underestimated by fourfold to sixfold the attachment potential of the less electronegative 2.9-4.1 μm oocysts in the matrix porosity of limestone core samples. The field and laboratory results collectively suggested that it may take 200-300 m of transport to ensure even a 1-log unit removal of oocysts, even though the limestone surfaces exhibited a substantive capability for their sorptive removal. The study further demonstrated the utility of microspheres as oocyst surrogates in field-scale assessments of well vulnerability in limestone, provided that differences in attachment behaviors between oocysts and microspheres are taken into account.

  12. Influence of organic carbon loading, sediment associated metal oxide content and sediment grain size distributions upon Cryptosporidium parvum removal during riverbank filtration operations, Sonoma County, CA.

    PubMed

    Metge, D W; Harvey, R W; Aiken, G R; Anders, R; Lincoln, G; Jasperse, J

    2010-02-01

    This study assessed the efficacy for removing Cryptosporidium parvum oocysts of poorly sorted, Fe- and Al-rich, subsurface sediments collected from 0.9 to 4.9 and 1.7-13.9 m below land surface at an operating riverbank filtration (RBF) site (Russian River, Sonoma County, CA). Both formaldehyde-killed oocysts and oocyst-sized (3 microm) microspheres were employed in sediment-packed flow-through and static columns. The degree of surface coverage of metal oxides on sediment grain surfaces correlated strongly with the degrees of oocyst and microsphere removals. In contrast, average grain size (D(50)) was not a good indicator of either microsphere or oocyst removal, suggesting that the primary mechanism of immobilization within these sediments is sorptive filtration rather than physical straining. A low specific UV absorbance (SUVA) for organic matter isolated from the Russian River, suggested that the modest concentration of the SUVA component (0.8 mg L(-1)) of the 2.2 mg L(-1) dissolved organic carbon (DOC) is relatively unreactive. Nevertheless, an amendment of 2.2 mg L(-1) of isolated river DOC to column sediments resulted in up to a 35.7% decrease in sorption of oocysts and (or) oocyst-sized microspheres. Amendments (3.2 microM) of the anionic surfactant, sodium dodecyl benzene sulfonate (SDBS) also caused substantive decreases (up to 31.9 times) in colloid filtration. Although the grain-surface metal oxides were found to have a high colloid-removal capacity, our study suggested that any major changes within the watershed that would result in long-term alterations in either the quantity and (or) the character of the river's DOC could alter the effectiveness of pathogen removal during RBF operations. Published by Elsevier Ltd.

  13. An evaluation of a laser scanning device for the detection of Cryptosporidium parvum in treated water samples.

    PubMed

    Rushton, P; Place, B M; Lightfoot, N F

    2000-04-01

    A laser scanning device, the ChemScan RDI (Chemunex, Paris, France), was compared with manual fluorescence microscopy for the detection of oocysts of Cryptosporidium. Pairs of filters were spiked with approximately 100 oocysts. Over 24 h at least 1000 l of treated water was passed through the filters, then concentrated deposits were subjected to an immunomagnetic separation (IMS) protocol described by the manufacturer (Dynal, Oslo, Norway) and examination by fluorescence microscopy, or an IMS protocol (Chemunex) and detection by ChemScan laser scanning. Subsequently a set of five 1-ml samples containing oocysts over a range of concentrations, including a negative control, were examined blind by the two methods (stage two). In stage 1 the average recovery rates were estimated to be 49% (manual fluorescence microscopy) and 73% (ChemScan). The average ratio of ChemScan to manual fluorescence microscopy counts was 1.54 (range 1.08-2.36). In stage 2, statistical comparison of all but one set of results showed there was no significant difference between methods. Differences for the high count sample may possibly have been caused by duplicate counting of oocysts by manual fluorescence microscopy.

  14. A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli: Its application to a 12 kDa antigen from Cryptosporidium parvum.

    PubMed

    Costa, Sofia J; Silva, Pedro; Almeida, André; Conceição, Antónia; Domingues, Lucília; Castro, António

    2013-01-01

    The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimize recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunization. The H tag (of only 1 kDa) efficiently triggered a CP12-specific immune response, and it also improved the immunization procedure without requiring co-administration of adjuvants. Moreover, polyclonal antibodies raised against the HCP12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP12 antigen.

  15. Solar Radiation Induces Non-Nuclear Perturbations and a False Start to Regulated Exocytosis in Cryptosporidium parvum

    PubMed Central

    King, Brendon J.; Hoefel, Daniel; Ee Wong, Pao; Monis, Paul T.

    2010-01-01

    Stratospheric ozone depletion, climate warming and acidification of aquatic ecosystems have resulted in elevated levels of solar radiation reaching many aquatic environments with an increased deleterious impact on a wide range of living organisms. While detrimental effects on living organisms are thought to occur primarily through DNA damage, solar UV can also damage cellular proteins, lipids and signalling pathways. Cryptosporidium, a member of the eukaryotic phylum Apicomplexa, contain numerous vesicular secretory organelles and their discharge via regulated exocytosis is essential for the successful establishment of infection. Using flow cytometric techniques we demonstrate that solar UV rapidly induces sporozoite exocytosis resulting in a significant reduction in the ability of sporozoites to attach and invade host cells. We found that solar UV induced sporozoite membrane depolarization, resulting in reduced cellular ATP and increased cytosolic calcium. These changes were accompanied by a reduction in the internal granularity of sporozoites, indicative of apical organelle discharge, which was confirmed by analysis of sporozoites with an exocytosis-sensitive dye. The precise timing of apical organelle discharge in the presence of a compatible host cell is critical for sporozoite attachment and invasion. Our results demonstrate for the first time how solar UV radiation can interfere with exocytosis, a fundamental cellular process in all eukaryotic cells. We contend that not only may the forecast increases in solar radiation in both aquatic and terrestrial environments significantly affect members of the Apicomplexa, solar UV-induced membrane depolarizations resulting in cytosolic calcium perturbation may affect a wider range of eukaryotic organisms through antagonistic effects on a myriad of calcium dependant cellular functions. PMID:20668710

  16. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

    PubMed

    Laude, A; Valot, S; Desoubeaux, G; Argy, N; Nourrisson, C; Pomares, C; Machouart, M; Le Govic, Y; Dalle, F; Botterel, F; Bourgeois, N; Cateau, E; Leterrier, M; Le Pape, P; Morio, F

    2016-02-01

    Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice.

  17. First description of Cryptosporidium hominis GP60 genotype IkA20G1 and Cryptosporidium parvum GP60 genotypes IIaA18G3R1 and IIaA15G2R1 in foals in Brazil.

    PubMed

    Inácio, Sandra Valéria; Widmer, Giovanni; de Brito, Roberta Lomonte Lemos; Zucatto, Anaiza Simão; de Aquino, Monally Conceição Costa; Oliveira, Bruno César Miranda; Nakamura, Alex Akira; Neto, Luiz da Silveira; Carvalho, João Gabriel Balizardo; Gomes, Jancarlo Ferreira; Meireles, Marcelo Vasconcelos; Bresciani, Katia Denise Saraiva

    2017-01-15

    The present study focuses on Cryptosporidium infections of foals in Brazil. A total of 92 animals of different breeds from 11 farms in the vicinity of Araçatuba in the state of São Paulo, were examined. According to PCR targeting the 18S rRNA gene, Cryptosporidium sp. DNA was detected in 21.7% (20/92) of foals. Good quality 18S rRNA, actin, HSP70 and gp60 genes nPCR amplicons were obtained from five fecal samples. PCR amplification and sequencing of a fragment of the GP60 sporozoite surface glycoprotein gene revealed C. parvum genotypes IIaA18G3R1, IIaA15G2R1. Interestingly, we also detected in two foals a GP60 genotype related to the human parasite C. hominis. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. COMPARISON OF FILTRATION METHODS FOR PRIMARY RECOVERY OF CRYPTOSPORIIDUM PARVUM FROM WATER

    EPA Science Inventory

    Waterborne disease outbreaks from contaminated drinking water have been linked to the protozoan parasite, Cryptosporidium parvum. To improve monitoring for this agent, the USEPA developed Method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 i...

  19. COMPARISON OF FILTRATION METHODS FOR PRIMARY RECOVERY OF CRYPTOSPORIIDUM PARVUM FROM WATER

    EPA Science Inventory

    Waterborne disease outbreaks from contaminated drinking water have been linked to the protozoan parasite, Cryptosporidium parvum. To improve monitoring for this agent, the USEPA developed Method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 i...

  20. Synthesis and modulation properties of imidazo[4,5-b]pyridin-7-one and indazole-4,7-dione derivatives towards the Cryptosporidium parvum CpABC3 transporter.

    PubMed

    Zeinyeh, Waël; Xia, Hexue; Lawton, Philippe; Radix, Sylvie; Marminon, Christelle; Nebois, Pascal; Walchshofer, Nadia

    2010-06-01

    The syntheses of new N-polysubstituted imidazo[4,5-b]pyridine-7-one (IP, 5 and 8a-8f) and indazole-4,7-dione (ID, 9 and 10) derivatives are described. The binding affinity of IP and ID towards the recombinant Nucleotide Binding Domain NBD1 of Cryptosporidium parvum CpABC3 was evaluated by intrinsic fluorescence quenching. IP induced a moderate quenching of the intrinsic fluorescence of H6-NBD1 whereas IDs 9 and 10 showed a binding affinity comparable to the ATP analogue TNP-ATP. In addition, 8d, 8e and 10 were shown to be competitive inhibitors of the ATPase activity, but with low affinity. These compounds could thus act like some flavonoid derivatives, which can partly overlap both the nucleotide-binding site and the adjacent hydrophobic steroid-binding region of mammalian P-glycoproteins.

  1. Stimulation of innate immunity in newborn kids against Cryptosporidium parvum infection-challenge by intranasal/per-oral administration of liposomal formulation of N-L18-norAbu-GMDP adjuvant.

    PubMed

    Turánek, J; Kasná, A; Koudela, B; Ledvina, M; Miller, A D

    2005-11-01

    The effects of a liposomal preparation of lipophilic immunomodulator beta-D-GlcNstearoyl-(1-4)-norMurNAc-L-Abu-D-isoGln (N-L18-norAbu-GMDP) were investigated on resistance to Cryptosporidium parvum infection in neonatal kids. The liposomal preparation was administered subcutaneously or intranasally/orally (i.n./p.o.) twice at doses of 100 microg, 200 microg, or 1000 microg per kid pre-infection challenge. The treatment schemes were (i) 72 and 24 h pre-infection challenge, (ii) 24 h pre-infection challenge and 24 h post-infection challenge (oral inoculation with 1 x 10(7) oocysts of C. parvum in 5 ml of PBS). Administration of liposomal N-L18-norAbu-GMDP by i.n./p.o. route at the cumulative dose of 2000 microg per kid 72 and 24 h pre-infection challenge, lead to substantially increased clearance of coccidian parasites from various parts of the intestine. On the basis of histological examination, the distribution of cryptosporidia in the intestine and the severity of the infection, treated kids were classified on day 5 as having a strong reduction in infection in comparison to the control group (P < 0.05). No cryptosporidia were found on the mucosal surface of treated kids by day 10, while the intestines of the control kids were still infected. All doses and routes of administration were judged effective with respect to suppression of cryptosporidia infections.

  2. Genetic Diversity of Cryptosporidium spp. in Captive Reptiles

    PubMed Central

    Xiao, Lihua; Ryan, Una M.; Graczyk, Thaddeus K.; Limor, Josef; Li, Lixia; Kombert, Mark; Junge, Randy; Sulaiman, Irshad M.; Zhou, Ling; Arrowood, Michael J.; Koudela, Břetislav; Modrý, David; Lal, Altaf A.

    2004-01-01

    The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium. Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards. PMID:14766569

  3. Genetic diversity of Cryptosporidium spp. in captive reptiles.

    PubMed

    Xiao, Lihua; Ryan, Una M; Graczyk, Thaddeus K; Limor, Josef; Li, Lixia; Kombert, Mark; Junge, Randy; Sulaiman, Irshad M; Zhou, Ling; Arrowood, Michael J; Koudela, Bretislav; Modrý, David; Lal, Altaf A

    2004-02-01

    The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium: Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.

  4. New developments in Cryptosporidium research.

    PubMed

    Ryan, Una; Hijjawi, Nawal

    2015-05-01

    Cryptosporidium is an enteric parasite that is considered the second greatest cause of diarrhoea and death in children after rotavirus. Currently, 27 species are recognised as valid and of these, Cryptosporidium hominis and Cryptosporidium parvum are responsible for the majority of infections in humans. Molecular and biological studies indicate that Cryptosporidium is more closely related to gregarine parasites rather than to coccidians. The identification of gregarine-like gamont stages and the ability of Cryptosporidium to complete its life cycle in the absence of host cells further confirm its relationship with gregarines. This opens new avenues into the investigation of pathogenesis, epidemiology, treatment and control of Cryptosporidium. Effective drug treatments and vaccines are not yet available due, in part, to the technical challenges of working on Cryptosporidium in the laboratory. Whole genome sequencing and metabolomics have expanded our understanding of the biochemical requirements of this organism and have identified new drug targets. To effectively combat this important pathogen, increased funding is essential.

  5. First molecular characterization of Cryptosporidium in Yemen.

    PubMed

    Alyousefi, N A; Mahdy, M A K; Lim, Y A L; Xiao, L; Mahmud, R

    2013-05-01

    Cryptosporidium is a protozoan parasite of humans and animals and has a worldwide distribution. The parasite has a unique epidemiology in Middle Eastern countries where the IId subtype family of Cryptosporidium parvum dominates. However, there has been no information on Cryptosporidium species in Yemen. Thus, this study was conducted in Yemen to examine the distribution of Cryptosporidium species and subtype families. Fecal samples were collected from 335 patients who attended hospitals in Sana'a city. Cryptosporidium species were determined by PCR and sequence analysis of the 18 s rRNA gene. Cryptosporidium parvum and C. hominis subtypes were identified based on sequence analysis of the 60 kDa glycoprotein (gp60) gene. Out of 335 samples, 33 (9.9%) were positive for Cryptosporidium. Of them, 97% were identified as C. parvum whilst 1 case (3%) was caused by C. hominis. All 7 C. parvum isolates subtyped belonged to the IIaA15G2R1 subtype. The common occurrence of the zoonotic IIa subtype family of C. parvum highlights the potential occurrence of zoonotic transmission of cryptosporidiosis in Yemen. However, this postulation needs confirmation with future molecular epidemiological studies of cryptosporidiosis in both humans and animals in Yemen.

  6. Use of carboxylated microspheres to assess transport potential of Cryptosporidium parvum oocysts at the Russian River water supply facility, Sonoma County, California

    USGS Publications Warehouse

    Metge, D.W.; Harvey, R.W.; Anders, R.; Rosenberry, D.O.; Seymour, D.; Jasperse, J.

    2007-01-01

    Carboxylated microspheres were employed as surrogates to assess the transport potential of Cryptosporidium parvumoocysts during forced- and natural-gradient tests conducted in July and October 2004. The tests involved poorly-sorted, near-surface sediments where groundwater is pumped from an alluvial aquifer underlying the Russian River, Sonoma County, CA. In an off channel infiltration basin and within the river, a mixture (2-, 3-, and 5- ??m diameters) of fluorescently-labeled carboxylated microspheres and bromide tracers were used in two injection and recovery test to assess sediment removal efficiency for the microspheres. Bottom sediments varied considerably in their filtration efficiency for Cryptosporidium.

  7. Effects of sediment-associated extractable metals, degree of sediment grain sorting, and dissolved organic carbon upon cryptosporidium parvum removal and transport within riverbank filtration sediments, Sonoma County, California

    USGS Publications Warehouse

    Metge, D.W.; Harvey, R.W.; Aiken, G.R.; Anders, R.; Lincoln, G.; Jasperse, J.; Hill, M.C.

    2011-01-01

    Oocysts of the protozoan pathogen Cryptosporidium parvum are of particular concern for riverbank filtration (RBF) operations because of their persistence, ubiquity, and resistance to chlorine disinfection. At the Russian River RBF site (Sonoma County, CA), transport of C. parvum oocysts and oocyst-sized (3 ??m) carboxylate-modified microspheres through poorly sorted (sorting indices, ??1, up to 3.0) and geochemically heterogeneous sediments collected between 2 and 25 m below land surface (bls) were assessed. Removal was highly sensitive to variations in both the quantity of extractable metals (mainly Fe and Al) and degree of grain sorting. In flow-through columns, there was a log-linear relationship (r2 = 0.82 at p < 0.002) between collision efficiency (??, the probability that colloidal collisions with grain surfaces would result in attachment) and extractable metals, and a linear relationship (r2 = 0.99 at p < 0.002) between ?? and ??1. Collectively, variability in extractable metals and grain sorting accounted for ???83% of the variability in ?? (at p < 0.0002) along the depth profiles. Amendments of 2.2 mg L-1 of Russian River dissolved organic carbon (DOC) reduced ?? for oocysts by 4-5 fold. The highly reactive hydrophobic organic acid (HPOA) fraction was particularly effective in re-entraining sediment-attached microspheres. However, the transport-enhancing effects of the riverine DOC did not appear to penetrate very deeply into the underlying sediments, judging from high ?? values (???1.0) observed for oocysts being advected through unamended sediments collected at ???2 m bls. This study suggests that in evaluating the efficacy of RBF operations to remove oocysts, it may be necessary to consider not only the geochemical nature and size distribution of the sediment grains, but also the degrees of sediment sorting and the concentration, reactivity, and penetration of the source water DOC. ?? 2011 American Chemical Society.

  8. UV Inactivation of Cryptosporidium hominis as Measured in Cell Culture

    PubMed Central

    Johnson, Anne M.; Linden, Karl; Ciociola, Kristina M.; De Leon, Ricardo; Widmer, Giovanni; Rochelle, Paul A.

    2005-01-01

    The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts. PMID:15870378

  9. [Cryptosporidium: phylogeny and taxonomy].

    PubMed

    Chacín-Bonilla, Leonor

    2007-03-01

    Members of the genus Cryptosporidium in the phylum Apicomplexa were long thought to be closely related to the coccidia. However, despite strong morphological similarities to these organisms, Cryptosporidium has notable differences with them and similarities with the gregarine protozoa. On the basis of phylogenetic analysis of molecular data, some authors place Cryptosporidium at the basis of the phylum Apicomplexa, others consider species of this genus to be phylogenetically too distant from the coccidia and do not include them in this group of protozoa, and others think that Cryptosporidium is closely related to gregarines. The taxonomy of this genus and the naming of species are undergoing rapid change due to the new and increasing molecular information. Molecular characterization of oocysts using polymerase chain reaction based procedures has not only a major impact on resolving the taxonomy of Cryptosporidium at the species level but also on the molecular epidemiology of cryptosporidiosis. Today, it is recognized that this genus is a phenotypically and genotypically heterogeneous assemblage of largely morphologically identical species and genotypes. Fourteen Cryptosporidium species and 21 C. parvum genotypes are currently recognized. Phylogenetic analyses have shown that genetically related hosts often have related forms of Cryptosporidium. Application of molecular techniques to taxonomy and epidemiology is helping to characterize new and existing species and determine the sources of the parasites that will facilitate the identification of sources of water-borne cryptosporidiosis.

  10. METHODS FOR DETECTION OF CRYPTOSPORIDIUM SP. AND GIARDIA SP.

    EPA Science Inventory

    There have been several waterborne outbreaks of giardiasis caused by infection with Giardia lamblia, and cryptosporidiosis, caused by infection with Cryptosporidium parvum. These outbreaks have created a need to detect these organisms in source and finished drinking water. The pr...

  11. METHODS FOR DETECTION OF CRYPTOSPORIDIUM SP. AND GIARDIA SP.

    EPA Science Inventory

    There have been several waterborne outbreaks of giardiasis caused by infection with Giardia lamblia, and cryptosporidiosis, caused by infection with Cryptosporidium parvum. These outbreaks have created a need to detect these organisms in source and finished drinking water. The pr...

  12. Effect of dissolved organic carbon on the transport and attachment behaviors of Cryptosporidium parvum oocysts and carboxylate-modified microspheres advected through temperate humic and tropical volcanic agricultural soil

    USGS Publications Warehouse

    Mohanram, Arvind; Ray, Chittaranjan; Metge, David W.; Barber, Larry B.; Ryan, Joseph N.; Harvey, Ronald W.

    2012-01-01

    Transport of Cryptosporidium parvum oocysts and microspheres in two disparate (a clay- and Fe-rich, volcanic and a temperate, humic) agricultural soils were studied in the presence and absence of 100 mg L–1 of sodium dodecyl benzene sulfonate (SDBS), and Suwannee River Humic Acid (SRHA) at pH 5.0–6.0. Transport of carboxylate-modified, 1.8 μm microspheres in soil columns was highly sensitive to the nature of the dissolved organic carbon (DOC), whereas oocysts transport was more affected by soil mineralogy. SDBS increased transport of microspheres from 48% to 87% through the tropical soil and from 43% to 93% in temperate soil. In contrast, SRHA reduced transport of microspheres from 48% to 28% in tropical soil and from 43% to 16% in temperate soil. SDBS also increased oocysts transport through the temperate soil 5-fold, whereas no oocyst transport was detected in tropical soil. SRHA had only a nominal effect in increasing oocysts transport in tropical soil, but caused a 6-fold increase in transport through the temperate soil. Amendments of only 4 mg L–1 SRHA and SDBS decreased oocyst hydrophobicity from 66% to 20% and from 66% to 5%, respectively. However, SDBS increased microsphere hydrophobicity from 16% to 33%. Soil fines, which includes clays, and SRHA, both caused the oocysts zeta potential (ζ) to become more negative, but caused the highly hydrophilic microspheres to become less negatively charged. The disparate behaviors of the two colloids in the presence of an ionic surfactant and natural organic matter suggest that microspheres may not be suitable surrogates for oocysts in certain types of soils. These results indicate that whether or not DOC inhibits or promotes transport of oocysts and microspheres in agricultural soils and by how much, depends not only on the surface characteristics of the colloid, but the nature of the DOC and the soil mineralogy.

  13. Effect of dissolved organic carbon on the transport and attachment behaviors of Cryptosporidium parvum oocysts and carboxylate-modified microspheres advected through temperate humic and tropical volcanic agricultural soil.

    PubMed

    Mohanram, Arvind; Ray, Chittaranjan; Metge, David W; Barber, Larry B; Ryan, Joseph N; Harvey, Ronald W

    2012-02-21

    Transport of Cryptosporidium parvum oocysts and microspheres in two disparate (a clay- and Fe-rich, volcanic and a temperate, humic) agricultural soils were studied in the presence and absence of 100 mg L(-1) of sodium dodecyl benzene sulfonate (SDBS), and Suwannee River Humic Acid (SRHA) at pH 5.0-6.0. Transport of carboxylate-modified, 1.8 μm microspheres in soil columns was highly sensitive to the nature of the dissolved organic carbon (DOC), whereas oocysts transport was more affected by soil mineralogy. SDBS increased transport of microspheres from 48% to 87% through the tropical soil and from 43% to 93% in temperate soil. In contrast, SRHA reduced transport of microspheres from 48% to 28% in tropical soil and from 43% to 16% in temperate soil. SDBS also increased oocysts transport through the temperate soil 5-fold, whereas no oocyst transport was detected in tropical soil. SRHA had only a nominal effect in increasing oocysts transport in tropical soil, but caused a 6-fold increase in transport through the temperate soil. Amendments of only 4 mg L(-1) SRHA and SDBS decreased oocyst hydrophobicity from 66% to 20% and from 66% to 5%, respectively. However, SDBS increased microsphere hydrophobicity from 16% to 33%. Soil fines, which includes clays, and SRHA, both caused the oocysts zeta potential (ζ) to become more negative, but caused the highly hydrophilic microspheres to become less negatively charged. The disparate behaviors of the two colloids in the presence of an ionic surfactant and natural organic matter suggest that microspheres may not be suitable surrogates for oocysts in certain types of soils. These results indicate that whether or not DOC inhibits or promotes transport of oocysts and microspheres in agricultural soils and by how much, depends not only on the surface characteristics of the colloid, but the nature of the DOC and the soil mineralogy.

  14. Cryptosporidium Pathogenicity and Virulence

    PubMed Central

    Bouzid, Maha; Chalmers, Rachel M.; Tyler, Kevin M.

    2013-01-01

    Cryptosporidium is a protozoan parasite of medical and veterinary importance that causes gastroenteritis in a variety of vertebrate hosts. Several studies have reported different degrees of pathogenicity and virulence among Cryptosporidium species and isolates of the same species as well as evidence of variation in host susceptibility to infection. The identification and validation of Cryptosporidium virulence factors have been hindered by the renowned difficulties pertaining to the in vitro culture and genetic manipulation of this parasite. Nevertheless, substantial progress has been made in identifying putative virulence factors for Cryptosporidium. This progress has been accelerated since the publication of the Cryptosporidium parvum and C. hominis genomes, with the characterization of over 25 putative virulence factors identified by using a variety of immunological and molecular techniques and which are proposed to be involved in aspects of host-pathogen interactions from adhesion and locomotion to invasion and proliferation. Progress has also been made in the contribution of host factors that are associated with variations in both the severity and risk of infection. Here we provide a review comprised of the current state of knowledge on Cryptosporidium infectivity, pathogenesis, and transmissibility in light of our contemporary understanding of microbial virulence. PMID:23297262

  15. FINGERPRINTING OF C. PARVUM BY MATRIX ASSISTED LASER DESORPTION IONIZATION MASS SPECTROMETRY

    EPA Science Inventory

    The oocysts of Cryptosporidium parvum, an enteric protozoan pathogen, are responsible for the worst microbial waterborne outbreak of gastroenteritis in recent history. The 1993 outbreak in Milwaukee, WI, sickened approximately 403,000 individuals, resulting in the hospitalizatio...

  16. FINGERPRINTING OF C. PARVUM BY MATRIX ASSISTED LASER DESORPTION IONIZATION MASS SPECTROMETRY

    EPA Science Inventory

    The oocysts of Cryptosporidium parvum, an enteric protozoan pathogen, are responsible for the worst microbial waterborne outbreak of gastroenteritis in recent history. The 1993 outbreak in Milwaukee, WI, sickened approximately 403,000 individuals, resulting in the hospitalizatio...

  17. USE OF LONG ACTING STEROID METHYLPREDNISOLONE ACETATE FOR THE PROLONGATION OF CRYPTOSPORIDIUM SP. IN MICE

    EPA Science Inventory

    Current protocols for Cryptosporidium sp. propagation in mice vary according to the strain and age of mouse as well as the species of Cryptosporidium. Cryptosporidium muris is a natural parasite of mice, but only neonatal mice are susceptible to C. parvum infection. Published C...

  18. USE OF LONG ACTING STEROID METHYLPREDNISOLONE ACETATE FOR THE PROLONGATION OF CRYPTOSPORIDIUM SP. IN MICE

    EPA Science Inventory

    Current protocols for Cryptosporidium sp. propagation in mice vary according to the strain and age of mouse as well as the species of Cryptosporidium. Cryptosporidium muris is a natural parasite of mice, but only neonatal mice are susceptible to C. parvum infection. Published C...

  19. COMPARISON OF TISSUE CULTURE AND ANIMAL MODELS FOR ASSESSMENT OF CRYPTOSPRIDIUM PARVUM INFECTION

    EPA Science Inventory

    Data from three different disinfection studies using both cell culture and mouse infectivity to assess Cryptosporidium parvum inactivation were evaluated in a total of 35 comparison including process controls and treated samples. C. parvum infectivity in the in vitro FDM-MPN assa...

  20. COMPARISON OF TISSUE CULTURE AND ANIMAL MODELS FOR ASSESSMENT OF CRYPTOSPRIDIUM PARVUM INFECTION

    EPA Science Inventory

    Data from three different disinfection studies using both cell culture and mouse infectivity to assess Cryptosporidium parvum inactivation were evaluated in a total of 35 comparison including process controls and treated samples. C. parvum infectivity in the in vitro FDM-MPN assa...

  1. Molecular Characterization of Isolates of Waterborne Cryptosporidium spp. Collected during an Outbreak of Gastroenteritis in South Burgundy, France

    PubMed Central

    Dalle, Frédéric; Roz, Pascale; Dautin, Guillaume; Di-Palma, Marc; Kohli, Evelyne; Sire-Bidault, C.; Fleischmann, Marie George; Gallay, Anne; Carbonel, Sylvia; Bon, Fabienne; Tillier, Claude; Beaudeau, Pascal; Bonnin, Alain

    2003-01-01

    In September 2001, a waterborne outbreak of gastroenteritis occurred in eastern France. Of 31 fecal samples from symptomatic individuals, 19 tested positive for Cryptosporidium with two PCRs targeting the Hsp70 and the 18S rRNA genes of Cryptosporidium. Sequencing of the PCR fragments produced sequences identical to that of Cryptosporidium parvum genotype 1. PMID:12791906

  2. Molecular Surveillance of Cryptosporidium spp. in Raw Wastewater in Milwaukee: Implications for Understanding Outbreak Occurrence and Transmission Dynamics

    PubMed Central

    Zhou, Ling; Singh, Ajaib; Jiang, Jianlin; Xiao, Lihua

    2003-01-01

    Six Cryptosporidium spp. were found in 50 of 179 Milwaukee wastewater samples collected weekly over a year. Of the eight subtypes of Cryptosporidium hominis and Cryptosporidium parvum present, allele Ib was found in 14 of 16 samples, and its sequence was identical to that of the subtype in human samples from the 1993 Milwaukee outbreak of cryptosporidiosis. PMID:14605176

  3. Molecular characterization of isolates of waterborne Cryptosporidium spp. collected during an outbreak of gastroenteritis in South Burgundy, France.

    PubMed

    Dalle, Frédéric; Roz, Pascale; Dautin, Guillaume; Di-Palma, Marc; Kohli, Evelyne; Sire-Bidault, C; Fleischmann, Marie George; Gallay, Anne; Carbonel, Sylvia; Bon, Fabienne; Tillier, Claude; Beaudeau, Pascal; Bonnin, Alain

    2003-06-01

    In September 2001, a waterborne outbreak of gastroenteritis occurred in eastern France. Of 31 fecal samples from symptomatic individuals, 19 tested positive for Cryptosporidium with two PCRs targeting the Hsp70 and the 18S rRNA genes of CRYPTOSPORIDIUM: Sequencing of the PCR fragments produced sequences identical to that of Cryptosporidium parvum genotype 1.

  4. Molecular Genotyping of Viable Cryptosporidium Oocysts

    EPA Science Inventory

    Cryptosporidium is a chlorination-resistant protozoan parasite that causes a self-limiting diarrheal disease in the immunocompetent or severe chronic diarrhea in the immunocompromised. Two species, C. parvum and C. hominis, cause most cases of cryptosporidiosis in humans, while C...

  5. Infectivity of the cervine genotype of Cryptosporidium

    USDA-ARS?s Scientific Manuscript database

    Cryptosporidiosis is a diarrheal disease of humans and animals caused by parasites in the genus Cryptosporidium, a genus comprising 19 valid species and 40 genotypes. Most human infections are caused by C. hominis and C. parvum. To a lesser extent infections with C. meleagridis, C. felis, C. canis, ...

  6. Molecular Genotyping of Viable Cryptosporidium Oocysts

    EPA Science Inventory

    Cryptosporidium is a chlorination-resistant protozoan parasite that causes a self-limiting diarrheal disease in the immunocompetent or severe chronic diarrhea in the immunocompromised. Two species, C. parvum and C. hominis, cause most cases of cryptosporidiosis in humans, while C...

  7. Molecular characterization of Cryptosporidium in children in Oyo State, Nigeria: implications for infection sources.

    PubMed

    Ayinmode, Adekunle Bamidele; Fagbemi, Benjamin Olakunle; Xiao, Lihua

    2012-01-01

    A study was conducted to detect and identify Cryptosporidium spp. in 43 children from Oyo State, Nigeria. Using nested polymerase chain reaction, 11.6% of the children were identified as positive for Cryptosporidium spp. Restriction fragment length polymorphism analysis and DNA sequencing of the PCR products showed the presence of three subtype families of Cryptosporidium hominis (two isolates of Ia and one isolate of Ib) and Cryptosporidium parvum (two isolates of IIc), all anthroponotic in nature. This study identified a high diversity of Cryptosporidium subtypes and clearly suggested that anthroponotic rather than zoonotic transmission played a more important role in the epidemiology of Cryptosporidium in the studied area.

  8. Surveillance Systems for Waterborne Cryptosporidium: US EPA method 1523 and Beyond

    EPA Science Inventory

    Waterborne cryptosporidiosis remains a significant public health concern in countries around the world. Many species and genotypes of Cryptosporidium contaminate drinking water sources, but C. parvum and C. hominis remain the two predominant species known to cause waterborne dis...

  9. Removal Efficiencies and Attachment Coefficients for Cryptosporidium in Sandy Alluvial Riverbank Sediment

    EPA Science Inventory

    Riverbank filtration has been shown to be effective at removing viable Cryptosporidium parvum oocysts and, therefore, drinking water systems that employ riverbank filtration may receive additional treatment credits beyond that which they can obtain using traditional engineering a...

  10. Literature Reference for Cryptosporidium spp. (Applied and Environmental Microbiology. 1999. 65(9): 3936–3941)

    EPA Pesticide Factsheets

    Procedures are described for analysis of animal samples using tissue culture techniques that may be adapted for assessment of solid, particulate, liquid and water samples contaminated with Cryptosporidium parvum.

  11. MALDI-MIS INVESTIGATIONS OF DRINKING WATER PATHOGENS--GIARDIA AND CRYPTOSPORIDIUM

    EPA Science Inventory

    The protozoan parasites, Cryptosporidium parvum and Giardia lamblia, have been responsible for numerous waterborne outbreaks of gastrointestinal illness in the United States. The 1993 cryptosporidiosis outbreak in Milwaukee affected approximately 400,000 people and resulted in o...

  12. SPECIES AND GENUS DIFFERENTIATION OF PARASITES (GIARDIA AND CRYPTOSPORIDIUM) BY MALDI - MASS SPECTROMETRY

    EPA Science Inventory

    The protozoan parasites, Cryptosporidium parvum and Giardia lamblia, have been responsible for numerous waterborne outbreaks of gastrointestinal illness in the United States. The 1993 cryptosporidiosis outbreak in Milwaukee affected approximately 400,000 people and resulted in o...

  13. SPECIES AND GENUS DIFFERENTIATION OF PARASITES (GIARDIA AND CRYPTOSPORIDIUM) BY MALDI - MASS SPECTROMETRY

    EPA Science Inventory

    The protozoan parasites, Cryptosporidium parvum and Giardia lamblia, have been responsible for numerous waterborne outbreaks of gastrointestinal illness in the United States. The 1993 cryptosporidiosis outbreak in Milwaukee affected approximately 400,000 people and resulted in o...

  14. MALDI-MIS INVESTIGATIONS OF DRINKING WATER PATHOGENS--GIARDIA AND CRYPTOSPORIDIUM

    EPA Science Inventory

    The protozoan parasites, Cryptosporidium parvum and Giardia lamblia, have been responsible for numerous waterborne outbreaks of gastrointestinal illness in the United States. The 1993 cryptosporidiosis outbreak in Milwaukee affected approximately 400,000 people and resulted in o...

  15. Surveillance Systems for Waterborne Cryptosporidium: US EPA method 1523 and Beyond

    EPA Science Inventory

    Waterborne cryptosporidiosis remains a significant public health concern in countries around the world. Many species and genotypes of Cryptosporidium contaminate drinking water sources, but C. parvum and C. hominis remain the two predominant species known to cause waterborne dis...

  16. Removal Efficiencies and Attachment Coefficients for Cryptosporidium in Sandy Alluvial Riverbank Sediment

    EPA Science Inventory

    Riverbank filtration has been shown to be effective at removing viable Cryptosporidium parvum oocysts and, therefore, drinking water systems that employ riverbank filtration may receive additional treatment credits beyond that which they can obtain using traditional engineering a...

  17. Genetic Diversity of Cryptosporidium spp. within a Remote Population of Soay Sheep on St. Kilda Islands, Scotland

    PubMed Central

    Connelly, L.; Craig, B. H.; Jones, B.

    2013-01-01

    This is the first report to characterize the genotypes and subtypes of Cryptosporidium species infecting a geographically isolated population of feral Soay sheep (Ovis aries) on Hirta, St. Kilda, Scotland, during two distinct periods: (i) prior to a population crash and (ii) as host numbers increased. Cryptosporidium DNA was extracted by freeze-thawing of immunomagnetically separated (IMS) bead-oocyst complexes, and species were identified following nested-PCR-restriction fragment length polymorphism (RFLP)/PCR sequencing at two Cryptosporidium 18S rRNA loci. Two hundred fifty-five samples were analyzed, and the prevalent Cryptosporidium species in single infections were identified as C. hominis (11.4% of all samples tested), C. parvum (9%), C. xiaoi (12.5%), and C. ubiquitum (6.7%). Cryptosporidium parvum was also present with other Cryptosporidium species in 27.1% of all samples tested. Cryptosporidium parvum- and C. hominis-positive isolates were genotyped using two nested-PCR assays that amplify the Cryptosporidium glycoprotein 60 gene (GP60). GP60 gene analysis showed the presence of two Cryptosporidium genotypes, namely, C. parvum IIaA19G1R1 and C. hominis IbA10G2. This study reveals a higher diversity of Cryptosporidium species/genotypes than was previously expected. We suggest reasons for the high diversity of Cryptosporidium parasites within this isolated population and discuss the implications for our understanding of cryptosporidiosis. PMID:23354707

  18. Distribution and clinical manifestations of Cryptosporidium species and subtypes in HIV/AIDS patients in Ethiopia.

    PubMed

    Adamu, Haileeyesus; Petros, Beyene; Zhang, Guoqing; Kassa, Hailu; Amer, Said; Ye, Jianbin; Feng, Yaoyu; Xiao, Lihua

    2014-04-01

    Cryptosporidiosis is an important cause for chronic diarrhea and death in HIV/AIDS patients. Among common Cryptosporidium species in humans, C. parvum is responsible for most zoonotic infections in industrialized nations. Nevertheless, the clinical significance of C. parvum and role of zoonotic transmission in cryptosporidiosis epidemiology in developing countries remain unclear. In this cross-sectional study, 520 HIV/AIDS patients were examined for Cryptosporidium presence in stool samples using genotyping and subtyping techniques. Altogether, 140 (26.9%) patients were positive for Cryptosporidium spp. by PCR-RFLP analysis of the small subunit rRNA gene, belonging to C. parvum (92 patients), C. hominis (25 patients), C. viatorum (10 patients), C. felis (5 patients), C. meleagridis (3 patients), C. canis (2 patients), C. xiaoi (2 patients), and mixture of C. parvum and C. hominis (1 patient). Sequence analyses of the 60 kDa glycoprotein gene revealed a high genetic diversity within the 82 C. parvum and 19 C. hominis specimens subtyped, including C. parvum zoonotic subtype families IIa (71) and IId (5) and anthroponotic subtype families IIc (2), IIb (1), IIe (1) and If-like (2), and C. hominis subtype families Id (13), Ie (5), and Ib (1). Overall, Cryptosporidium infection was associated with the occurrence of diarrhea and vomiting. Diarrhea was attributable mostly to C. parvum subtype family IIa and C. hominis, whereas vomiting was largely attributable to C. hominis and rare Cryptosporidium species. Calf contact was identified as a significant risk factor for infection with Cryptosporidium spp., especially C. parvum subtype family IIa. Results of the study indicate that C. parvum is a major cause of cryptosporidiosis in HIV-positive patients and zoonotic transmission is important in cryptosporidiosis epidemiology in Ethiopia. In addition, they confirm that different Cryptosporidium species and subtypes are linked to different clinical manifestations.

  19. Phylogenetic Analysis of Cryptosporidium Parasites Based on the Small-Subunit rRNA Gene Locus

    PubMed Central

    Xiao, Lihua; Escalante, Lillian; Yang, Chunfu; Sulaiman, Irshad; Escalante, Anannias A.; Montali, Richard J.; Fayer, Ronald; Lal, Altaf A.

    1999-01-01

    Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed. PMID:10103253

  20. COMPARING METHODS FOR ESTIMATING {ITAL CRYPTOSPORIDIUM} SPP. OOCYST CONCENTRATIONS IN WATER

    EPA Science Inventory

    {ital Cryptosporidium parvum} in drinking water is a threat to public health. Estimating the parasite burden of {ital C. parvum} in water to be treated for use as drinking water constitutes an integral element to developing strategies for protecting public health in a cost effec...

  1. BIALLELIC POLYMORPHISM IN THE INTRON REGION OF B-TUBULIN GENE OF CRYPTOSPORIDIUM PARASITES

    EPA Science Inventory

    Nucleotide sequencing of polymerase chain reaction-amplified intron region of the Cryptosporidium parvum B-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is...

  2. AGING OF CRYPTOSPORIDIUM PARVUUM OOCYSTS STUDIED BY MALDI-TOF MS

    EPA Science Inventory

    Cryptosporidium parvum is a protozoan parasite, and it causes a potentially fatal gastrointestinal illness. This water borne pathogen has been the subject of several high profile disease outbreaks in the US and abroad. C. parvum presents challenges for both compliance monitorin...

  3. BIALLELIC POLYMORPHISM IN THE INTRON REGION OF B-TUBULIN GENE OF CRYPTOSPORIDIUM PARASITES

    EPA Science Inventory

    Nucleotide sequencing of polymerase chain reaction-amplified intron region of the Cryptosporidium parvum B-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is...

  4. AGING OF CRYPTOSPORIDIUM PARVUUM OOCYSTS STUDIED BY MALDI-TOF MS

    EPA Science Inventory

    Cryptosporidium parvum is a protozoan parasite, and it causes a potentially fatal gastrointestinal illness. This water borne pathogen has been the subject of several high profile disease outbreaks in the US and abroad. C. parvum presents challenges for both compliance monitorin...

  5. Cryptosporidium ryanae n.sp. (Apicomplexa:Cryptosporidiidae)in cattle (Bos Taurus)

    USDA-ARS?s Scientific Manuscript database

    A new species, Cryptosporidium ryanae, is described from cattle. Oocysts of C. ryanae, previously identified as the Cryptosporidium deer-like genotype and recorded as such in GenBank (AY587166, EU203216, DQ182597, AY741309, and DQ871345), are morphologically similar to those of C. parvum and C. bovi...

  6. Wide distribution of Cryptosporidium bovis and the deer-like genotype in bovines

    USDA-ARS?s Scientific Manuscript database

    We recently reported that on 14 dairy farms from Vermont to Florida ~85% of pre-weaned dairy calves were infected with zoonotic Cryptosporidium parvum whereas only 1-2% of post-weaned calves and 1-2 year-old heifers were infected with this species. Cryptosporidium bovis and the deer-like genotype w...

  7. Giardia Cysts and Cryptosporidium Oocysts in Membrane-Filtered Municipal Wastewater Used for Irrigation▿

    PubMed Central

    Lonigro, A.; Pollice, A.; Spinelli, R.; Berrilli, F.; Di Cave, D.; D'Orazi, C.; Cavallo, P.; Brandonisio, O.

    2006-01-01

    A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system. PMID:17056696

  8. Molecular identification of Giardia and Cryptosporidium from dogs and cats.

    PubMed

    Sotiriadou, Isaia; Pantchev, Nikola; Gassmann, Doreen; Karanis, Panagiotis

    2013-01-01

    The aim of the present study was to diagnose the presence of Giardia cysts and Cryptosporidium oocysts in household animals using nested polymerase chain reaction (PCR) and sequence analysis. One hundred faecal samples obtained from 81 dogs and 19 cats were investigated. The Cryptosporidium genotypes were determined by sequencing a fragment of the small subunit (SSU) rRNA gene, while the Giardia Assemblages were determined through analysis of the glutamate dehydrogenase (GDH) locus. Isolates from five dogs and two cats were positive by PCR for the presence of Giardia, and their sequences matched the zoonotic Assemblage A of Giardia. Cryptosporidium spp. isolated from one dog and one cat were both found to be C. parvum. One dog isolate harboured a mixed infection of C. parvum and Giardia Assemblage A. These findings support the growing evidence that household animals are potential reservoirs of the zoonotic pathogens Giardia spp. and Cryptosporidium spp. for infections in humans.

  9. Molecular epidemiology of Cryptosporidium in livestock animals and humans in the Ismailia province of Egypt.

    PubMed

    Helmy, Yosra A; Krücken, Jürgen; Nöckler, Karsten; von Samson-Himmelstjerna, Georg; Zessin, Karl-H

    2013-03-31

    The zoonotic potential of Cryptosporidium was studied in one of the most densely populated provinces of Egypt regarding livestock and people. In a representative survey, faecal samples from cattle, buffalo and stool samples from diarrhoeic children (<10 years) were investigated. Parameters assumed to be related to cryptosporidiosis were recorded for animals and children. Animal samples (804) were examined by the Copro-antigen RIDA(®)QUICK test, followed by PCRs targeting the 18S rDNA and gp60 genes for antigen-positive and 10% randomly selected negative samples. All 165 human samples were tested by both methods. The overall estimated prevalence of Cryptosporidium in ruminants was 32.2%, without significant difference between animal species. PCR identified 65.7% Cryptosporidium parvum, 11.8% Cryptosporidium ryanae, 4.1% Cryptosporidium bovis, and combinations of C. parvum plus C. ryanae (11.2%), C. parvum plus C. bovis (5.3%) and of C. parvum plus Cryptosporidium andersoni (1.8%), also without significant differences in species occurrence between cattle and buffalos. The human Cryptosporidium spp. prevalence was 49.1%, of which 60.5% were Cryptosporidium hominis, 38.2% C. parvum and 1.2% C. parvum plus C. bovis. Analysis of gp60 variants allocated C. parvum found in animals to the zoonotic subtype family IIa (18.9%, subtype IIaA15G1R1 only) and to IId (81.1%, mostly IIdA20G1). In humans 50% were classified as subtype family IIa (IIaA15G1R1 and IIaA15G2R1) and 50% were IIdA20G1. C. andersoni occurred only in cattle older than 1 year. In contrast, mono-infections with one of the three single Cryptosporidium species and the three combinations with C. parvum were more prevalent in cattle and buffaloes younger than 1 year, particularly in those younger than 3 months, and were predominantly subtype family IId. In human samples no Cryptosporidium were identified in children younger than 7 months. Neither place of residence nor the source of drinking-water had measurable

  10. CONCENTRATION AND DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN SURFACE WATER SAMPLES BY METHOD 1622 USING ULTRAFILTRATION AND CAPSULE FILTRATION

    EPA Science Inventory

    The protozoan parasite cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the USEPA developed Method 1622 for isolation and detection of cryptosporidim oocysts in water. ...

  11. CONCENTRATION AND DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN SURFACE WATER SAMPLES BY METHOD 1622 USING ULTRAFILTRATION AND CAPSULE FILTRATION

    EPA Science Inventory

    The protozoan parasite cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the USEPA developed Method 1622 for isolation and detection of cryptosporidim oocysts in water. ...

  12. RAPID COMMUNICATION: A combined travelling wave dielectrophoresis and electrorotation device: applied to the concentration and viability determination of Cryptosporidium

    NASA Astrophysics Data System (ADS)

    Goater, Andrew D.; Burt, Julian P. H.; Pethig, Ronald

    1997-09-01

    We describe a microelectrode device, fabricated using photolithography and laser ablation, that combines the electrokinetic effects of travelling wave dielectrophoresis and electrorotation. Here it has been used to concentrate and then assay the viability of Cryptosporidium parvum oocysts.

  13. Synthesis, in Vitro Evaluation and Cocrystal Structure of 4-Oxo-[1]benzopyrano[4,3-c]pyrazole Cryptosporidium parvum Inosine 5′-Monophosphate Dehydrogenase (CpIMPDH) Inhibitors

    PubMed Central

    2015-01-01

    Cryptosporidium inosine 5′-monophosphate dehydrogenase (CpIMPDH) has emerged as a therapeutic target for treating Cryptosporidium parasites because it catalyzes a critical step in guanine nucleotide biosynthesis. A 4-oxo-[1]benzopyrano[4,3-c]pyrazole derivative was identified as a moderately potent (IC50 = 1.5 μM) inhibitor of CpIMPDH. We report a SAR study for this compound series resulting in 8k (IC50 = 20 ± 4 nM). In addition, an X-ray crystal structure of CpIMPDH·IMP·8k is also presented. PMID:25474504

  14. Cryptosporidium species in Australian wildlife and domestic animals.

    PubMed

    Ryan, Una; Power, Michelle

    2012-11-01

    Cryptosporidium is an important enteric parasite that is transmitted via the fecal-oral route, water and food. Humans, wildlife and domestic livestock all potentially contribute Cryptosporidium to surface waters. Most species of Cryptosporidium are morphologically indistinguishable and can only be identified using molecular tools. Over 24 species have been identified and of these, 7 Cryptosporidium species/genotypes are responsible for most human cryptosporidiosis cases. In Australia, relatively few genotyping studies have been conducted. Six Cryptosporidium species (C. hominis, C. parvum, C. meleagridis, C. fayeri, C. andersoni and C. bovis) have been identified in humans in Australia. However, little is known about the contribution of animal hosts to human pathogenic strains of Cryptosporidium in drinking water catchments. In this review, we focus on the available genotyping data for native, feral and domestic animals inhabiting drinking water catchments in Australia to provide an improved understanding of the public health implications and to identify key research gaps.

  15. Fine targeting of purine salvage in Cryptosporidium parasites.

    PubMed

    Hyde, John E

    2008-08-01

    The apicomplexan pathogen Cryptosporidium parvum poses major logistical problems in the search for effective drug treatments. These treatments are required urgently because this parasite can cause severe disease and death in immunocompromised individuals and young children. In a recent study, the dependence of Cryptosporidium parasites on a single salvage pathway that leads to essential purine derivatives has been exploited and inhibitors have been identified that selectively target a key enzyme in this salvage process, inosine 5'-monophosphate dehydrogenase.

  16. Genomics meets transgenics in search of the elusive Cryptosporidium drug target.

    PubMed

    Striepen, Boris; Kissinger, Jessica C

    2004-08-01

    Cryptosporidium is an important pathogen of humans, and a challenging model for the laboratory. The parasite genome sequence, accessible through a comprehensive database, now provides exciting opportunities for urgently needed advances. Comparative genomics, combined with the genetic system in the related parasite Toxoplasma gondii, outlines a detailed Cryptosporidium parvum metabolic map and facilitates cell biological analyses. New targets for Cryptosporidium drug and vaccine development can be identified and validated based on this approach.

  17. Occurrence of Cryptosporidium sp. oocysts in fecal and water samples in Austria.

    PubMed

    Hassl, A; Benyr, G; Sommer, R

    2001-10-22

    Oocysts of Cryptosporidium spp. were detected and differentiated by a modular arranged gene amplification procedure in various samples, mostly human stool, feces of herpetotaxa, and water, in different locations of South and Eastern Austria. Cryptosporidium parvum was found in stool samples of immunocompromised persons, in reptile feces, and in water samples. The presence of Cryptosporidium in an area is probably associated with high human population densities since water from protected sources in sparsely inhabited areas is rarely contaminated.

  18. Cryptosporidium taxonomy: recent advances and implications for public health.

    PubMed

    Xiao, Lihua; Fayer, Ronald; Ryan, Una; Upton, Steve J

    2004-01-01

    There has been an explosion of descriptions of new species of Cryptosporidium during the last two decades. This has been accompanied by confusion regarding the criteria for species designation, largely because of the lack of distinct morphologic differences and strict host specificity among Cryptosporidium spp. A review of the biologic species concept, the International Code of Zoological Nomenclature (ICZN), and current practices for Cryptosporidium species designation calls for the establishment of guidelines for naming Cryptosporidium species. All reports of new Cryptosporidium species should include at least four basic components: oocyst morphology, natural host specificity, genetic characterizations, and compliance with the ICZN. Altogether, 13 Cryptosporidium spp. are currently recognized: C. muris, C. andersoni, C. parvum, C. hominis, C. wrairi, C. felis, and C. cannis in mammals; C. baïleyi, C. meleagridis, and C. galli in birds; C. serpentis and C. saurophilum in reptiles; and C. molnari in fish. With the establishment of a framework for naming Cryptosporidium species and the availability of new taxonomic tools, there should be less confusion associated with the taxonomy of the genus Cryptosporidium. The clarification of Cryptosporidium taxonomy is also useful for understanding the biology of Cryptosporidium spp., assessing the public health significance of Cryptosporidium spp. in animals and the environment, characterizing transmission dynamics, and tracking infection and contamination sources.

  19. Molecular characterization and assessment of zoonotic transmission of Cryptosporidium from dairy cattle in West Bengal, India.

    PubMed

    Khan, Shahbaz Manzoor; Debnath, Chanchal; Pramanik, Amiya Kumar; Xiao, Lihua; Nozaki, Tomoyoshi; Ganguly, Sandipan

    2010-07-15

    Few studies in the past have examined the genetic diversity and zoonotic potential of Cryptosporidium in dairy cattle in India. To assess the importance of these animals as a source of human Cryptosporidium infections, fecal samples from 180 calves, heifers and adults and 51 farm workers on two dairy farms in West Bengal, India were genotyped by PCR-RFLP analysis of the 18S rRNA gene of Cryptosporidium followed by DNA sequencing of the PCR products. Phylogenetic analysis was carried out on the DNA sequences obtained in the study and those available in GenBank. The overall prevalence of Cryptosporidium in cattle was 11.7% though the infection was more prevalent in younger calves than in adult cattle. The occurrence of Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae and Cryptosporidium andersoni in cattle followed an age-related pattern. A Cryptosporidium suis-like genotype was also detected in a calf. Farm workers were infected with Cryptosporidium hominis, C. parvum and a novel C. bovis genotype. These findings clearly suggest that there is a potential risk of zoonotic transmission of Cryptosporidium infections between cattle and humans on dairy farms in India. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  20. Cryptosporidium enteritis

    MedlinePlus

    Proper sanitation and hygiene, including handwashing, are important measures for preventing this illness. Certain water filters can also reduce risk by filtering out the cryptosporidium eggs. However, ...

  1. Molecular characterization of Cryptosporidium isolates from humans in Equatorial Guinea.

    PubMed

    Blanco, María Alejandra; Iborra, Asunción; Vargas, Antonio; Nsie, Eugenia; Mbá, Luciano; Fuentes, Isabel

    2009-12-01

    The aim of the study was to perform a molecular characterization of clinical isolates of Cryptosporidium species from Equatorial Guinea. Standard laboratory methods were used to identify 35 cryptosporidiosis cases among 185 patients. PCR-RFLP successfully identified 34 Cryptosporidium species from these 35 cases, comprising C. parvum (52.9%), C. hominis (44.1%) and C. meleagridis (2.9%); over 90% of the species were isolated from HIV-positive patients. This is the first report of the molecular characterization of Cryptosporidium species isolated from humans in Equatorial Guinea and shows that zoonotic and anthroponotic transmission is present in this country.

  2. Cryptosporidium and Giardia removal by secondary and tertiary wastewater treatment.

    PubMed

    Taran-Benshoshan, Marina; Ofer, Naomi; Dalit, Vaizel-Ohayon; Aharoni, Avi; Revhun, Menahem; Nitzan, Yeshayahu; Nasser, Abidelfatah M

    2015-01-01

    Wastewater disposal may be a source of environmental contamination by Cryptosporidium and Giardia. This study was conducted to evaluate the prevalence of Cryptosporidium oocysts and Giardia cysts in raw and treated wastewater effluents. A prevalence of 100% was demonstrated for Giardia cysts in raw wastewater, at a concentration range of 10 to 12,225 cysts L(-1), whereas the concentration of Cryptosporidium oocysts in raw wastewater was 4 to 125 oocysts L(-1). The removal of Giardia cysts by secondary and tertiary treatment processes was greater than those observed for Cryptosporidium oocysts and turbidity. Cryptosporidium and Giardia were present in 68.5% and 76% of the tertiary effluent samples, respectively, at an average concentration of 0.93 cysts L(-1) and 9.94 oocysts L(-1). A higher detection limit of Cryptosporidium oocysts in wastewater was observed for nested PCR as compared to immune fluorescent assay (IFA). C. hominis was found to be the dominant genotype in wastewater effluents followed by C. parvum and C. andersoni or C. muris. Giardia was more prevalent than Cryptosporidium in the studied community and treatment processes were more efficient for the removal of Giardia than Cryptosporidium. Zoonotic genotypes of Cryptosporidium were also present in the human community. To assess the public health significance of Cryptosporidium oocysts present in tertiary effluent, viability (infectivity) needs to be assessed.

  3. UTILITY OF SURROGATES FOR MEASURING CRYPTOSPORIDIUM OOCYSTS INFECTIVITY

    EPA Science Inventory

    The water industry must assess whether Cryptosporidium oocysts detected in source and finished water are viable and/or infectious. Initial approaches measuring the infectious nature of C. parvum oocysts have focused on in vitro excystation and in vitro vital dye staining. Recen...

  4. DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN SOURCE AND FINISHED WATERS

    EPA Science Inventory

    Numerous waterborne outbreaks of cryptosporidiosis have occurred with the most notable being the 1993 episode in Milwaukee. As a result, the past decade has seen a massive effort expended on the development of methods to detect Cryptosporidium parvum oocysts in source and finish...

  5. UTILITY OF SURROGATES FOR MEASURING CRYPTOSPORIDIUM OOCYST INFECTIVITY

    EPA Science Inventory

    The water industry must assess whether Cryptosporidium oocysts detected in source and finished water are viable and/or infectious. Initial approaches measuring the infectious nature of C. parvum oocysts have focused on in vitro excystation and in vitro vital dye staining. Recen...

  6. Unique Cryptosporidium Population in HIV-Infected Persons, Jamaica

    PubMed Central

    Gatei, Wangeci; Barrett, Donnett; Lindo, John F.; Eldemire-Shearer, Denise; Cama, Vitaliano

    2008-01-01

    A cryptosporidiosis survey showed the presence of Cryptosporidium hominis, C. parvum, C. canis, and C. felis in 25, 7, 1, and 1 HIV-positive persons from Jamaica, respectively; 1 person had both C. hominis and C. felis. Multilocus sequence typing indicated the presence of a homogeneous but geographically distinct C. hominis population in Jamaica. PMID:18439378

  7. CRYPTOSPORIDIUM VIRULENCE DETERMINANTS-ARE WE THERE YET? (R828035)

    EPA Science Inventory

    Exposure to Cryptosporidium parvum in healthy individuals results in transient infection that may be asymptomatic or can result in self-limited diarrhoea. In contrast, acquired immune deficiency syndrome patients with cryptosporidiosis can experience severe manifestatio...

  8. TESTING METHODS FOR DETECTION OF CRYPTOSPORIDIUM SPP. IN WATER SAMPLES

    EPA Science Inventory

    A large waterborne outbreak of cryptosporidiosis in Milwaukee, Wisconsin, U.S.A. in 1993 prompted a search for ways to prevent large-scale waterborne outbreaks of protozoan parasitoses. Methods for detecting Cryptosporidium parvum play an integral role in strategies that lead to...

  9. DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN SOURCE AND FINISHED WATERS

    EPA Science Inventory

    Numerous waterborne outbreaks of cryptosporidiosis have occurred with the most notable being the 1993 episode in Milwaukee. As a result, the past decade has seen a massive effort expended on the development of methods to detect Cryptosporidium parvum oocysts in source and finish...

  10. CRYPTOSPORIDIUM VIRULENCE DETERMINANTS-ARE WE THERE YET? (R828035)

    EPA Science Inventory

    Exposure to Cryptosporidium parvum in healthy individuals results in transient infection that may be asymptomatic or can result in self-limited diarrhoea. In contrast, acquired immune deficiency syndrome patients with cryptosporidiosis can experience severe manifestatio...

  11. TESTING METHODS FOR DETECTION OF CRYPTOSPORIDIUM SPP. IN WATER SAMPLES

    EPA Science Inventory

    A large waterborne outbreak of cryptosporidiosis in Milwaukee, Wisconsin, U.S.A. in 1993 prompted a search for ways to prevent large-scale waterborne outbreaks of protozoan parasitoses. Methods for detecting Cryptosporidium parvum play an integral role in strategies that lead to...

  12. UTILITY OF SURROGATES FOR MEASURING CRYPTOSPORIDIUM OOCYST INFECTIVITY

    EPA Science Inventory

    The water industry must assess whether Cryptosporidium oocysts detected in source and finished water are viable and/or infectious. Initial approaches measuring the infectious nature of C. parvum oocysts have focused on in vitro excystation and in vitro vital dye staining. Recen...

  13. UTILITY OF SURROGATES FOR MEASURING CRYPTOSPORIDIUM OOCYSTS INFECTIVITY

    EPA Science Inventory

    The water industry must assess whether Cryptosporidium oocysts detected in source and finished water are viable and/or infectious. Initial approaches measuring the infectious nature of C. parvum oocysts have focused on in vitro excystation and in vitro vital dye staining. Recen...

  14. Cryptosporidium sp. infections in green turtles, Chelonia mydas, as a potential source of marine waterborne oocysts in the Hawaiian Islands

    USGS Publications Warehouse

    Graczyk, T.K.; Balazs, G.H.; Work, T.M.; Aguirre, A.A.; Ellis, D.M.; Murakawa, Shawn K. K.; Morris, Robert

    1997-01-01

    For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum.

  15. Cryptosporidium sp. Infections in Green Turtles, Chelonia mydas, as a Potential Source of Marine Waterborne Oocysts in the Hawaiian Islands

    PubMed Central

    Graczyk, T. K.; Balazs, G. H.; Work, T.; Aguirre, A. A.; Ellis, D. M.; Murakawa, S.; Morris, R.

    1997-01-01

    For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum. PMID:16535658

  16. REMOVAL OF CRYPTOSPORIDIUM BY IN-LINE FILTRATION AS A FUNCTION OF OOCYST AGE AND PRESERVATION METHOD

    EPA Science Inventory

    This study examined the impacts of oocyst preservation method and age on the removal of seeded Cryptosporidium oocysts by in-line filtration. An existing study has investigated the infectivity of Cryptosporidium parvum as a function of preservation method and oocyst age. Simila...

  17. Cryptosporidium sp. infections in green turtles, Chelonia mydas, as a potential source of marine waterborne oocysts in the Hawaiian Islands

    USGS Publications Warehouse

    Graczyk, T.K.; Balazs, G.H.; Work, Thierry M.; Aguirre, A.A.; Ellis, D.M.; Murakawa, Shawn K. K.; Morris, Robert

    1997-01-01

    For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum.

  18. REMOVAL OF CRYPTOSPORIDIUM BY IN-LINE FILTRATION AS A FUNCTION OF OOCYST AGE AND PRESERVATION METHOD

    EPA Science Inventory

    This study examined the impacts of oocyst preservation method and age on the removal of seeded Cryptosporidium oocysts by in-line filtration. An existing study has investigated the infectivity of Cryptosporidium Parvum as a function of preservation method and oocyst age. Simila...

  19. REMOVAL OF CRYPTOSPORIDIUM BY IN-LINE FILTRATION AS A FUNCTION OF OOCYST AGE AND PRESERVATION METHOD

    EPA Science Inventory

    This study examined the impacts of oocyst preservation method and age on the removal of seeded Cryptosporidium oocysts by in-line filtration. An existing study has investigated the infectivity of Cryptosporidium Parvum as a function of preservation method and oocyst age. Simila...

  20. REMOVAL OF CRYPTOSPORIDIUM BY IN-LINE FILTRATION AS A FUNCTION OF OOCYST AGE AND PRESERVATION METHOD

    EPA Science Inventory

    This study examined the impacts of oocyst preservation method and age on the removal of seeded Cryptosporidium oocysts by in-line filtration. An existing study has investigated the infectivity of Cryptosporidium parvum as a function of preservation method and oocyst age. Simila...

  1. Zoonotic Cryptosporidium spp. and Enterocytozoon bieneusi in pet chinchillas (Chinchilla lanigera) in China.

    PubMed

    Qi, Meng; Luo, Nannan; Wang, Haiyan; Yu, Fuchang; Wang, Rongjun; Huang, Jianying; Zhang, Longxian

    2015-10-01

    Cryptosporidium and Enterocytozoon bieneusi are the most prevalent protist pathogens responsible for inducing human and animal diseases worldwide. The aim of the present work was to determine the occurrence of Cryptosporidium spp. and E. bieneusi in pet chinchillas in China. One hundred forty fecal samples were collected from four cities: Beijing, Zhengzhou, Anyang and Guiyang. They were then examined with PCR amplification of the small subunit ribosomal RNA (SSU rRNA) of Cryptosporidium spp. and the internal transcribed spacer (ITS) of the ribosomal RNA of E. bieneusi. The infection rates for Cryptosporidium spp. and E. bieneusi were 10.0% and 3.6%, respectively. Sequence analysis of SSU rRNA gene products identified two Cryptosporidium spp., Cryptosporidium ubiquitum (n=13) and Cryptosporidium parvum (n=1). Subtyping with the 60-kDa glycoprotein (gp60) gene showed that all C. ubiquitum isolates belonged to zoonotic subtype family XIId, while the subtype of the C. parvum isolate could not be identified. Two E. bieneusi genotypes were identified in five samples, zoonotic genotypes BEB6 (n=3) and D (n=2). This is the first report of C. ubiquitum and C. parvum, and E. bieneusi in chinchillas. This result indicates that pet chinchillas may be a potential source of human infection with Cryptosporidium spp. and E. bieneusi.

  2. Cryptosporidium Species and Subtypes and Clinical Manifestations in Children, Peru

    PubMed Central

    Cama, Vitaliano A.; Bern, Caryn; Roberts, Jacqueline; Cabrera, Lilia; Sterling, Charles R.; Ortega, Ynes; Gilman, Robert H.

    2008-01-01

    To determine whether clinical manifestations are associated with genotypes or subtypes of Cryptosporidium spp., we studied a 4-year longitudinal birth cohort of 533 children in Peru. A total of 156 infection episodes were found in 109 children. Data from first infections showed that C. hominis was associated with diarrhea, nausea, vomiting, general malaise, and increased oocyst shedding intensity and duration. In contrast, C. parvum, C. meleagridis, C. canis, and C. felis were associated with diarrhea only. C. hominis subtype families were identified (Ia, Ib, Id, and Ie); all were associated with diarrhea. Ib was also associated with nausea, vomiting, and general malaise. All C. parvum specimens belonged to subtype family IIc. Analysis of risk factors did not show associations with specific Cryptosporidium spp. genotypes or subtypes. These findings strongly suggest that Cryptosporidium spp. and subtypes are linked to different clinical manifestations in children. PMID:18826821

  3. Cryptosporidium oocysts in mussels (Mytilus edulis) from Normandy (France).

    PubMed

    Li, Xunde; Guyot, Karine; Dei-Cas, Eduardo; Mallard, Jean-Paul; Ballet, Jean Jacques; Brasseur, Philippe

    2006-05-01

    Cultured mussels (Mytilus edulis) were collected seasonally during one year from three sites on the Northwestern coastal area of Normandy (France). Flesh, gills and innerwater were examined for Cryptosporidium oocyst detection using immunomagnetic separation and immunofluorescence assay. Oocysts were present in all samples for all sites and seasons and flesh was the most contaminated part. Oocyst rates were apparently related with seasonal rain precipitation variations. Molecular analysis revealed that oocysts belonged to the species Cryptosporidium parvum (formerly genotype 2 or ). Oocyst infectivity was assessed by oral administration to suckling NMRI-mice, and developmental stages were observed in only one mouse infected with oocysts from one location. The detection of potentially infectious C. parvum oocysts of likely cattle-breeding origin in cultured edible mussels confirms their resistance to sea environments, and underlines the potential risk of food-borne infection. This work reports for the first time the presence of infectious Cryptosporidium oocysts in shellfish from France.

  4. Common occurrence of Cryptosporidium hominis in horses and donkeys.

    PubMed

    Jian, Fuchun; Liu, Aiqin; Wang, Rongjun; Zhang, Sumei; Qi, Meng; Zhao, Wei; Shi, Yadong; Wang, Jianling; Wei, Jiujian; Zhang, Longxian; Xiao, Lihua

    2016-09-01

    Extensive genetic variation is observed within the genus Cryptosporidium and the distribution of Cryptosporidium species/genotypes in humans and animals appears to vary by geography and host species. To better understand the genetic diversity of Cryptosporidium spp. in horses and donkeys, we characterized five horse-derived and 82 donkey-derived Cryptosporidium isolates from five provinces or autonomous regions (Sichuan, Gansu, Henan, Inner Mongolia and Shandong) in China at the species/genotype and subtype levels. Three Cryptosporidium species/genotypes were identified based on the analysis of the SSU rRNA gene, including Cryptosporidium parvum (n=22), the Cryptosporidium horse genotype (n=4), and Cryptosporidium hominis (n=61). The identification of C. hominis was confirmed by sequence analysis of the HSP70 and actin genes. Subtyping using sequence analysis of the 60kDa glycoprotein gene identified 21 C. parvum isolates as subtype IIdA19G1, the four horse genotype isolates as subtypes VIaA15G4 (n=2) and VIaA11G3 (n=2), and the 61 C. hominis isolates as IkA16G1 (n=59) and IkA16 (n=2). The common finding of C. hominis reaffirms the heterogeneity of Cryptosporidium spp. in horses and donkeys and is possibly a reflection of endemic transmission of C. hominis in these animals. Data of the study suggest that horses and donkeys as companion animals may potentially transmit Cryptosporidium infections to humans. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Molecular detection of Cryptosporidium spp. infections in water buffaloes from northeast Thailand.

    PubMed

    Inpankaew, Tawin; Jiyipong, Tawisa; Wongpanit, Kannika; Pinyopanuwat, Nongnuch; Chimnoi, Wissanuwat; Kengradomkij, Chanya; Xuan, Xuenan; Igarashi, Ikuo; Xiao, Lihua; Jittapalapong, Sathaporn

    2014-02-01

    The objectives of this study were to determine the individual and herd-level prevalence and genotype of Cryptosporidium and to identify putative risk factors associated with Cryptosporidium spp. infections in water buffaloes in northeast Thailand. Fecal samples from 600 water buffaloes of 287 farms in six provinces were collected and tested using DMSO-modified acid-fast staining and polymerase chain reaction. The overall prevalence of Cryptosporidium infections in buffaloes was 5.7 and 8.7% among individual animals and herds, respectively. The provinces with highest infected Cryptosporidium were located in the Sakon Nakhon Basin in the northern part of the region. In addition, higher herd prevalence was observed among farms with more than five buffaloes (30%) than those with five or less animals (16.2%). Thirty (88.2%) of the 34 Cryptosporidium-positive samples were Cryptosporidium parvum and four (11.8%) were Cryptosporidium ryanae.

  6. Detection of Cryptosporidium and Identification to the Species Level by Nested PCR and Restriction Fragment Length Polymorphism

    PubMed Central

    Coupe, Stephane; Sarfati, Claudine; Hamane, Samia; Derouin, Francis

    2005-01-01

    Cryptosporidiosis is an emerging protozoan disease associated with large waterborne outbreaks. Diagnosis relies on microscopic examination of stools, but this method cannot identify the infecting species of Cryptosporidium. We have developed a test based on nested PCR and restriction fragment length polymorphism (RFLP) that offers simple identification of Cryptosporidium hominis, Cryptosporidium parvum, and most other human infective species in stool samples. Purified C. parvum oocysts were used for PCR development. Extracted DNA was amplified by nested PCR targeting a 214-bp fragment of the 18S RNA gene. Enzymatic restriction sites were identified by bioinformatic analysis of all published Cryptosporidium 18S rRNA sequences. Experiments with spiked stool samples gave an estimated PCR detection limit of one oocyst. Specificity was assessed by testing 68 stool samples from patients with microscopically proven cryptosporidiosis and 31 Cryptosporidium-negative stools. Sixty-seven (98.5%) of the 68 stool samples from patients with microscopically proven cryptosporidiosis and 2 of the other stool samples were positive by PCR and could be genotyped. RFLP analysis identified 36 C. hominis, 19 C. parvum, 8 Cryptosporidium meleagridis, and 6 Cryptosporidium felis or Cryptosporidium canis samples. Species determination in 26 PCR-positive cases was in full agreement with DNA sequencing of the 18S rRNA hypervariable region. The excellent sensitivity of PCR, coupled with the accuracy of RFLP for species identification, make this method a suitable tool for routine diagnosis and genotyping of Cryptosporidium in stools. PMID:15750054

  7. Cryptosporidium: Treatment

    MedlinePlus

    ... Cryptosporidium Tracking in the United States CryptoNet Healthy Water Sites Healthy Water Drinking Water Healthy Swimming Global ... and HIV-Infected Children [PDF - 384 pages] Healthy Water Sites Healthy Water Drinking Water Healthy Swimming Global ...

  8. Occurrence of Giardia and Cryptosporidium in pigs on Prince Edward Island, Canada.

    PubMed

    Budu-Amoako, Ebo; Greenwood, Spencer J; Dixon, Brent R; Barkema, Herman W; Hurnik, Daniel; Estey, Chelsie; McClure, J T

    2012-02-28

    In a cross-sectional study of 633 pigs from 21 herds on Prince Edward Island, Canada (PEI), the prevalence of infection with Cryptosporidium and Giardia, and the genotypes and species of isolates were determined in order to establish the zoonotic potential of pigs in this region. As determined by direct immunofluorescence microscopy (DFA), 18 herds (86%) and 163 animals (26%; 95% CI: 22-29%) tested positive for Cryptosporidium, while just 3 herds (14%) and 6 animals (1%; 95% CI: 0.4-2%) tested positive for Giardia. Cryptosporidium spp. isolates were detected in 39% (95% CI: 34-44%) of weanlings (1-3 months of age) and 9% (95% CI: 6-13) of sows (>8months of age). Molecular characterization using the 18S rDNA and HSP70 gene fragments revealed the presence of Cryptosporidium sp. pig genotype II, C. suis, C. parvum, and Cryptosporidium sp. mouse genotype. Among the 113 isolates of Cryptosporidium spp. successfully genotyped, pig genotype II (61%) predominated, with C. suis (36%) being the next most prominant isolate. C. parvum (2%; two isolates) and Cryptosporidium sp. mouse genotype (0.9%; one isolate) were only occasionally isolated. The only two Cryptosporidium-positive genotyped isolates from sows included one each of C. suis and Cryptosporidium sp. pig genotype II. All but one of the six Giardia positive isolates were detected in weanling pigs. None of the Giardia-positive isolates was amenable to PCR. This study demonstrates that Cryptosporidium spp. are highly prevalent in pigs on PEI, Canada, are found mostly in weanlings (1-3 months of age). Furthermore, the pigs are primarily infected by the host-specific genotypes and species, Cryptosporidium sp. pig genotype II and C. suis, whereas the zoonotic C. parvum is rare. Giardia duodenalis is only occasionally found in pigs. These findings suggest that domestic pigs on PEI, Canada, likely do not pose a significant health risk to humans from these parasites.

  9. A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China.

    PubMed

    Liu, Xuehan; He, Tingmei; Zhong, Zhijun; Zhang, Hemin; Wang, Rongjun; Dong, Haiju; Wang, Chengdong; Li, Desheng; Deng, Jiabo; Peng, Guangneng; Zhang, Longxian

    2013-10-01

    Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60×3.99 μm (n=50). The isolate was genetically analyzed using the partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype. © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Identification of Cryptosporidium Species in Fish from Lake Geneva (Lac Léman) in France

    PubMed Central

    Certad, Gabriela; Dupouy-Camet, Jean; Gantois, Nausicaa; Hammouma-Ghelboun, Ourida; Pottier, Muriel; Guyot, Karine; Benamrouz, Sadia; Osman, Marwan; Delaire, Baptiste; Creusy, Colette; Viscogliosi, Eric; Aliouat-Denis, Cecile Marie; Follet, Jérôme

    2015-01-01

    Cryptosporidium, a protozoan parasite that can cause severe diarrhea in a wide range of vertebrates including humans, is increasingly recognized as a parasite of a diverse range of wildlife species. However, little data are available regarding the identification of Cryptosporidium species and genotypes in wild aquatic environments, and more particularly in edible freshwater fish. To evaluate the prevalence of Cryptosporidiumspp. in fish from Lake Geneva (Lac Léman) in France, 41 entire fish and 100 fillets (cuts of fish flesh) were collected from fishery suppliers around the lake. Nested PCR using degenerate primers followed by sequence analysis was used. Five fish species were identified as potential hosts of Cryptosporidium: Salvelinus alpinus, Esox lucius, Coregonus lavaretus, Perca fluviatilis, and Rutilus rutilus. The presence of Cryptosporidium spp. was found in 15 out of 41 fish (37%), distributed as follows: 13 (87%) C. parvum, 1 (7%) C. molnari, and 1 (7%) mixed infection (C. parvum and C. molnari). C. molnari was identified in the stomach, while C. parvum was found in the stomach and intestine. C. molnari was also detected in 1 out of 100 analyzed fillets. In order to identify Cryptosporidium subtypes, sequencing of the highly polymorphic 60-kDa glycoprotein (gp60) was performed. Among the C. parvum positive samples, three gp60 subtypes were identified: IIaA15G2R1, IIaA16G2R1, and IIaA17G2R1. Histological examination confirmed the presence of potential developmental stages of C. parvum within digestive epithelial cells. These observations suggest that C. parvum is infecting fish, rather than being passively carried. Since C. parvum is a zoonotic species, fish potentially contaminated by the same subtypes found in terrestrial mammals would be an additional source of infection for humans and animals, and may also contribute to the contamination of the environment with this parasite. Moreover, the risk of human transmission is strengthened by the

  11. Identification of Cryptosporidium Species in Fish from Lake Geneva (Lac Léman) in France.

    PubMed

    Certad, Gabriela; Dupouy-Camet, Jean; Gantois, Nausicaa; Hammouma-Ghelboun, Ourida; Pottier, Muriel; Guyot, Karine; Benamrouz, Sadia; Osman, Marwan; Delaire, Baptiste; Creusy, Colette; Viscogliosi, Eric; Dei-Cas, Eduardo; Aliouat-Denis, Cecile Marie; Follet, Jérôme

    2015-01-01

    Cryptosporidium, a protozoan parasite that can cause severe diarrhea in a wide range of vertebrates including humans, is increasingly recognized as a parasite of a diverse range of wildlife species. However, little data are available regarding the identification of Cryptosporidium species and genotypes in wild aquatic environments, and more particularly in edible freshwater fish. To evaluate the prevalence of Cryptosporidiumspp. in fish from Lake Geneva (Lac Léman) in France, 41 entire fish and 100 fillets (cuts of fish flesh) were collected from fishery suppliers around the lake. Nested PCR using degenerate primers followed by sequence analysis was used. Five fish species were identified as potential hosts of Cryptosporidium: Salvelinus alpinus, Esox lucius, Coregonus lavaretus, Perca fluviatilis, and Rutilus rutilus. The presence of Cryptosporidium spp. was found in 15 out of 41 fish (37%), distributed as follows: 13 (87%) C. parvum, 1 (7%) C. molnari, and 1 (7%) mixed infection (C. parvum and C. molnari). C. molnari was identified in the stomach, while C. parvum was found in the stomach and intestine. C. molnari was also detected in 1 out of 100 analyzed fillets. In order to identify Cryptosporidium subtypes, sequencing of the highly polymorphic 60-kDa glycoprotein (gp60) was performed. Among the C. parvum positive samples, three gp60 subtypes were identified: IIaA15G2R1, IIaA16G2R1, and IIaA17G2R1. Histological examination confirmed the presence of potential developmental stages of C. parvum within digestive epithelial cells. These observations suggest that C. parvum is infecting fish, rather than being passively carried. Since C. parvum is a zoonotic species, fish potentially contaminated by the same subtypes found in terrestrial mammals would be an additional source of infection for humans and animals, and may also contribute to the contamination of the environment with this parasite. Moreover, the risk of human transmission is strengthened by the

  12. Cryptosporidium species in sheep and goats from Papua New Guinea.

    PubMed

    Koinari, M; Lymbery, A J; Ryan, U M

    2014-06-01

    Species of Cryptosporidium are extensively recognised as pathogens of domesticated livestock and poultry, companion animals, wildlife, and are a threat to public health. Little is known of the prevalence of Cryptosporidium spp. in humans, domesticated animals or wildlife in Papua New Guinea (PNG). The aim of the present study was to screen sheep and goats for Cryptosporidium using molecular tools. A total of 504 faecal samples were collected from sheep (n=276) and goats (n=228) in village, government and institutional farms in PNG. Samples were screened by nested PCR and genotyped at the 18S rRNA and at the 60kDa glycoprotein (gp60) loci. The overall prevalences were 2.2% for sheep (6/278) and 4.4% (10/228) for goats. The species/genotypes identified were Cryptosporidium hominis (subtype IdA15G1) in goats (n=6), Cryptosporidium parvum (subtypes IIaA15G2R1and IIaA19G4R1) in sheep (n=4) and in goats (n=2), Cryptosporidium andersoni (n=1) and Cryptosporidium scrofarum (n=1) in sheep, Cryptosporidium xiao (n=1) and Cryptosporidium rat genotype II (n=1) in goats. This is the first report of Cryptosporidium spp. identified in sheep and goats in PNG. Identification of Cryptosporidium in livestock warrants better care of farm animals to avoid contamination and illness in vulnerable population. The detection of zoonotic Cryptosporidium in livestock suggests these animals may serve as reservoirs for human infection. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Prevalence of species and genotypes of Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States.

    PubMed

    Fayer, Ronald; Santín, Mónica; Trout, James M; Greiner, Ellis

    2006-01-30

    The prevalence of Cryptosporidium species in 1-2-year-old heifers was determined for 571 animals on 14 dairy farms in seven states on the East Coast of the United States. A fecal specimen collected directly from each heifer was processed to concentrate oocysts that were then examined by polymerase chain reaction (PCR). For every PCR-positive specimen the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified by PCR from heifers on 13 of 14 farms. On all except four farms groups of heifers were housed in a barn or in large covered pens. Others were pastured. From many of the same farms an earlier study reported that 41% of 393 pre-weaned calves and 26.2% of 447 post-weaned calves were infected. In the present study, 11.9% of 571 heifers were infected with Cryptosporidium, 0.7% with Cryptosporidium parvum, the zoonotic species. Of 68 PCR-positive specimens characterized by gene sequencing 1, 4, 10, 24, and 29 calves were infected with Cryptosporidium suis, Cryptosporidium parvum, Cryptosporidium deer-like genotype, Cryptosporidium bovis, and Cryptosporidium andersoni, respectively. These findings demonstrate a lower prevalence of infection in 1-2-year-old dairy cattle than in younger cattle as well as a change in the diversity of species present. Consequently, the risk of humans acquiring infection with C. parvum from exposure to feces from yearling and older cattle appears much lower than from exposure to pre-weaned calves.

  14. Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens.

    PubMed

    Morgan-Ryan, Una M; Fall, Abbie; Ward, Lucy A; Hijjawi, Nawal; Sulaiman, Irshad; Fayer, Ronald; Thompson, R C Andrew; Olson, M; Lal, Altaf; Xiao, Lihua

    2002-01-01

    The structure and infectivity of the oocysts of a new species of Cryptosporidium from the feces of humans are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts. and measure 4.4-5.4 microm (mean = 4.86) x 4.4-5.9 microm (mean = 5.2 microm) with a length to width ratio 1.0-1.09 (mean 1.07) (n = 100). Oocysts were not infectious for ARC Swiss mice, nude mice. Wistar rat pups, puppies, kittens or calves, but were infectious to neonatal gnotobiotic pigs. Pathogenicity studies in the gnotobiotic pig model revealed significant differences in parasite-associated lesion distribution (P = 0.005 to P = 0.02) and intensity of infection (P = 0.04) between C. parvum and this newly described species from humans. In vitro cultivation studies have also revealed growth differences between the two species. Multi-locus analysis of numerous unlinked loci, including a preliminary sequence scan of the entire genome demonstrated this species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further support for its species status. Based on biological and molecular data, this Cryptosporidium infecting the intestine of humans is proposed to be a new species Cryptosporidium hominis n. sp.

  15. Cryptosporidium fayeri n. sp. (Apicomplexa: Cryptosporidiidae) from the Red Kangaroo (Macropus rufus).

    PubMed

    Ryan, Una M; Power, Michelle; Xiao, Lihua

    2008-01-01

    The morphology and infectivity of the oocysts of a new species of Cryptosporidium from the faeces of the red kangaroo (Macropus rufus) are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts, and measure 4.5-5.1 microm (mean=4.9) x 3.8-5.0 microm (mean=4.3 microm) with a length to width ratio 1.02:1.18 (mean 1.14) (n=50). Oocysts were not infectious for neonate ARC Swiss mice. Multi-locus analysis of numerous unlinked loci demonstrated this species to be distinct (90.64%-97.88% similarity) from C. parvum. Based on biological and molecular data, this Cryptosporidium infecting marsupials is proposed to be a new species Cryptosporidium fayeri n. sp.

  16. Occurrence of Cryptosporidium in a wastewater treatment plant in North Germany.

    PubMed

    Ajonina, Caroline; Buzie, Christopher; Ajonina, Irene U; Basner, Alexander; Reinhardt, Heiko; Gulyas, Holger; Liebau, Eva; Otterpohl, Ralf

    2012-01-01

    Cryptosporidium parvum is one of the most common human parasitic protozoa and is responsible for many waterborne outbreaks in several industrialized countries. The oocyst, which is the infective form, is known to be highly resistant to wastewater treatment procedures and represents a potential hazard to human populations through contaminated raw or treated wastewater. In this investigation, the occurrence of Cryptosporidium in wastewater samples was monitored and removal efficiency was assessed. Treated (effluent) and untreated (influent) wastewater samples were collected seasonally over a period of 2 years. Oocysts were repeatedly detected in influent and effluent samples collected from the treatment plant during all sampling seasons, with a mean concentration of 782 oocysts/L. The seasonal distribution showed that oocysts are predominant during autumn and winter. Molecular analyses via the small (18S) subunit of rRNA amplification and subsequent sequencing with an objective of characterizing the oocysts revealed that Cryptosporidium parvum was the dominant Cryptosporidium parasite present in wastewater.

  17. Cryptosporidium and Giardia as foodborne zoonoses.

    PubMed

    Smith, H V; Cacciò, S M; Cook, N; Nichols, R A B; Tait, A

    2007-10-21

    Cryptosporidium and Giardia are major causes of diarrhoeal disease in humans, worldwide and are major causes of protozoan waterborne diseases. Both Cryptosporidium and Giardia have life cycles which are suited to waterborne and foodborne transmission. There are 16 'valid'Cryptosporidium species and a further 33+ genotypes described. Parasites which infect humans belong to the Giardia duodenalis "type", and at least seven G. duodenalis assemblages are recognised. Cryptosporidium parvum is the major zoonotic Cryptosporidium species, while G. duodenalis assemblages A and B have been found in humans and most mammalian orders. In depth studies to determine the role of non-human hosts in the transmission of Cryptosporidium and Giardia to humans are required. The use of harmonised methodology and standardised and validated molecular markers, together with sampling strategies that provide sufficient information about all contributors to the environmental (oo)cyst pool that cause contamination of food and water, are recommended. Standardised methods for detecting (oo)cysts in water are available, as are optimised, validated methods for detecting Cryptosporidium in soft fruit and salad vegetables. These provide valuable data on (oo)cyst occurrence, and can be used for species and subspecies typing using appropriate molecular tools. Given the zoonotic potential of these organisms, epidemiological, source and disease tracking investigations involve multidisciplinary teams. Here, the role of the veterinarian is paramount, particularly in understanding the requirement for adopting comprehensive sampling strategies for analysing both sporadic and outbreak samples from all potential non-human contributors. Comprehensive sampling strategies increase our understanding of parasite population biology and structure and this knowledge can be used to determine what level of discrimination is required between isolates. Genetic exchange is frequent in C. parvum populations, leading to

  18. Cryptosporidium Priming Is More Effective than Vaccine for Protection against Cryptosporidiosis in a Murine Protein Malnutrition Model

    PubMed Central

    Bartelt, Luther A.; Bolick, David T.; Kolling, Glynis L.; Zaenker, Edna I.; Lara, Ana M.; Noronha, Francisco Jose; Cowardin, Carrie A.; Moore, John H.; Turner, Jerrold R.; Warren, Cirle A.; Buck, Gregory A.; Guerrant, Richard L.

    2016-01-01

    Cryptosporidium is a major cause of severe diarrhea, especially in malnourished children. Using a murine model of C. parvum oocyst challenge that recapitulates clinical features of severe cryptosporidiosis during malnutrition, we interrogated the effect of protein malnutrition (PM) on primary and secondary responses to C. parvum challenge, and tested the differential ability of mucosal priming strategies to overcome the PM-induced susceptibility. We determined that while PM fundamentally alters systemic and mucosal primary immune responses to Cryptosporidium, priming with C. parvum (106 oocysts) provides robust protective immunity against re-challenge despite ongoing PM. C. parvum priming restores mucosal Th1-type effectors (CD3+CD8+CD103+ T-cells) and cytokines (IFNγ, and IL12p40) that otherwise decrease with ongoing PM. Vaccination strategies with Cryptosporidium antigens expressed in the S. Typhi vector 908htr, however, do not enhance Th1-type responses to C. parvum challenge during PM, even though vaccination strongly boosts immunity in challenged fully nourished hosts. Remote non-specific exposures to the attenuated S. Typhi vector alone or the TLR9 agonist CpG ODN-1668 can partially attenuate C. parvum severity during PM, but neither as effectively as viable C. parvum priming. We conclude that although PM interferes with basal and vaccine-boosted immune responses to C. parvum, sustained reductions in disease severity are possible through mucosal activators of host defenses, and specifically C. parvum priming can elicit impressively robust Th1-type protective immunity despite ongoing protein malnutrition. These findings add insight into potential correlates of Cryptosporidium immunity and future vaccine strategies in malnourished children. PMID:27467505

  19. An IC-PCR method for detection of Cryptosporidium and Giardia in natural surface waters in Finland.

    PubMed

    Rimhanen-Finne, Ruska; Hörman, Ari; Ronkainen, Pilvi; Hänninen, Marja Liisa

    2002-08-01

    We developed an immunocapture-based polymerase chain reaction (PCR) assay for simultaneous detection of Cryptosporidium parvum oocysts and Giardia intestinalis cysts in surface water. Using primer pairs Cry9/Cry15 and LaxA/LaxB for Cryptosporidium and Gdh1/Gdh4 for Giardia, the sensitivity of the entire detection procedure (dealing with concentration, separation, DNA purification and PCR amplification) was at the level of 50-100 oocysts and cysts. Of 54 surface water samples, 4 were positive for Cryptosporidium and 1 for Giardia. Cryptosporidium and Giardia were detected for the first time in surface water in Finland.

  20. Giardia and Cryptosporidium in cetaceans on the European Atlantic coast.

    PubMed

    Reboredo-Fernández, Aurora; Ares-Mazás, Elvira; Martínez-Cedeira, José A; Romero-Suances, Rafael; Cacciò, Simone M; Gómez-Couso, Hipólito

    2015-02-01

    The occurrence of Giardia and Cryptosporidium was investigated in cetacean specimens stranded on the northwestern coast of Spain (European Atlantic coast) by analysis of 65 samples of large intestine from eight species. The parasites were identified by direct immunofluorescence antibody test (IFAT) and by PCR amplification of the β-giardin gene, the ITS1-5.8S-ITS2 region and the SSU-rDNA gene of Giardia and the SSU-rDNA gene of Cryptosporidium. Giardia and Cryptosporidium were detected in 7 (10.8 %) and 9 samples (13.8 %), respectively. In two samples, co-infection with both parasites was observed. Giardia duodenalis assemblages A, C, D and F, and Cryptosporidium parvum were identified. This is the first report of G. duodenalis in Balaenoptera acutorostrata, Kogia breviceps and Stenella coeruleoalba and also the first report of Cryptosporidium sp. in B. acutorostrata and of C. parvum in S. coeruleoalba and Tursiops truncatus. These results extend the known host range of these waterborne enteroparasites.

  1. Epidemiology of Cryptosporidium infection in cattle in China: a review

    PubMed Central

    Gong, Chao; Cao, Xue-Feng; Deng, Lei; Li, Wei; Huang, Xiang-Ming; Lan, Jing-Chao; Xiao, Qi-Cheng; Zhong, Zhi-Jun; Feng, Fan; Zhang, Yue; Wang, Wen-Bo; Guo, Ping; Wu, Kong-Ju; Peng, Guang-Neng

    2017-01-01

    The present review discusses the findings of cryptosporidiosis research conducted in cattle in China and highlights the currently available information on Cryptosporidium epidemiology, genetic diversity, and distribution in China, which is critical to understanding the economic and public health importance of cryptosporidiosis transmission in cattle. To date, 10 Cryptosporidium species have been detected in cattle in China, with an overall infection rate of 11.9%. The highest rate of infection (19.5%) was observed in preweaned calves, followed by that in juveniles (10.69%), postweaned juveniles (9.0%), and adult cattle (4.94%). The dominant species were C. parvum in preweaned calves and C. andersoni in postweaned, juvenile, and adult cattle. Zoonotic Cryptosporidium species (C. parvum and C. hominis) were found in cattle, indicating the possibility of transmission between humans and cattle. Different cattle breeds had significant differences in the prevalence rate and species of Cryptosporidium. This review demonstrates an age-associated, breed-associated, and geographic-related occurrence of Cryptosporidium and provides references for further understanding of the epidemiological characteristics, and for preventing and controlling the disease. PMID:28098070

  2. Detection of viable Cyptosporidium parvum in soil by reverse transcription real time PCR targeting hsp70 mRNA

    EPA Science Inventory

    Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive proce...

  3. Detection of viable Cyptosporidium parvum in soil by reverse transcription real time PCR targeting hsp70 mRNA

    EPA Science Inventory

    Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive proce...

  4. Prevalence and molecular characterization of Cryptosporidium in goats across four provincial level areas in China.

    PubMed

    Mi, Rongsheng; Wang, Xiaojuan; Huang, Yan; Zhou, Peng; Liu, Yuxuan; Chen, Yongjun; Chen, Jun; Zhu, Wei; Chen, Zhaoguo

    2014-01-01

    This study assessed the prevalence, species and subtypes of Cryptosporidium in goats from Guangdong Province, Hubei Province, Shandong Province, and Shanghai City of China. Six hundred and four fecal samples were collected from twelve goat farms, and the overall infection rate was 11.4% (69/604). Goats infected with Cryptosporidium were found in eleven farms across four provincial areas, and the infection rate ranged from 2.9% (1/35) to 25.0% (9/36). Three Cryptosporidium species were identified. Cryptosporidium xiaoi (45/69, 65.2%) was the dominant species, followed by C. parvum (14/69, 20.3%) and C. ubiquitum (10/69, 14.5%). The infection rate of Cryptosporidium spp. was varied with host age and goat kids were more susceptible to be infected than adult goats. Subtyping C. parvum