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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. 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...

  4. Detection of viable Cryptosporidium parvum oocysts by PCR.

    PubMed Central

    Wagner-Wiening, C; Kimmig, P

    1995-01-01

    PCR was used to detect and specifically identify a gene fragment from Cryptosporidium parvum. An 873-bp region of a 2,359-bp DNA fragment encoding a repetitive oocyst protein of C. parvum was shown to be specifically amplified in C. parvum. An excystation protocol before DNA extraction allowed the differentiation between live and dead Cryptosporidium parvum oocysts. PMID:8534121

  5. Infection status of pigs with Cryptosporidium parvum

    PubMed Central

    Yu, Jae-Ran

    2004-01-01

    To investigate the infection status of pigs with Cryptosporidium parvum, 589 fecal samples were collected from pigs raised at farm in Chungcheongbuk-do and Chungcheongnam-do. Of the 589 pig fecal samples, 62 (10.5%) were positive for C. parvum. The area showing the highest positive rate was Dangjin-gun, Chungcheongnam-do (14.0%), and the lowest (0%) Salmi-myon, Chungcheongbuk-do. The positive rate of C. parvum in Judok-eup increased from 12.7% in the winter to 22.1% in the summer. The results of this study suggest that the pigs may be a source of human C. parvum infection. PMID:15060340

  6. Cryptosporidium parvum: From foal to veterinary students.

    PubMed

    Galuppi, R; Piva, S; Castagnetti, C; Sarli, G; Iacono, E; Fioravanti, M L; Caffara, M

    2016-03-30

    This paper describes the transmission of a zoonotic subtype of Cryptosporidium parvum between two foals hospitalized in an Equine Perinatology Unit (EPU) linked to an outbreak of cryptosporidiosis in veterinary students. Fecal specimens of 36 mares (105 samples) and 28 foals (122 samples) were subjected to Ziehl-Neelsen staining, nested PCR of 18S rDNA. Two foals tested positive for Cryptosporidium; PCR restriction fragment length polymorphism (PCR-RFLP) analysis and subtyping by nested PCR of the 60kDa glycoprotein (gp60) gene revealed C. parvum subtype IIdA23G1. The introduction of Cryptosporidium into the EPU is suspected to be in a foal showing no initial clinical signs that tested positive for C. parvum during an asymptomatic phase. A second foal, hospitalized afterwards for perinatal asphyxia syndrome complicated with failure of passive transfer and sepsis, showed severe watery diarrhea after 4 days of hospitalization and was positive for the same subtype. During this period, six students attending the EPU complained of abdominal pain and diarrhea and were positive for the same subtype of C. parvum. To the authors' knowledge, this is the first description of this subtype in foals and the first report of evidence of zoonotic transmission of cryptosporidiosis from foals to human. PMID:26921039

  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. A highly divergent 33 kDa Cryptosporidium parvum antigen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies comparing the genome sequences of Cryptosporidium parvum with C. hominis identified a number of highly divergent genes that might reflect positive selection for host specificity. In the present study, a C. parvum sequence, namely cgd8-5370, whose amino acid sequence differs appreci...

  9. Gene expression during excystation of Cryptosporidium parvum oocysts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study describes transcription of mRNA from genes encoding metabolic or structural proteins during excystation of Cryptosporidium parvum oocysts. RNA was harvested from C. parvum oocysts before excystation, and at 5, 10, and 15 min during excystation. Subtractive cDNA libraries were pre...

  10. Biology of Cryptosporidium parvum in pigs: from weaning to market.

    PubMed

    Guselle, N J; Appelbee, A J; Olson, M E

    2003-04-01

    Cryptosporidium parvum is commonly identified as infecting domestic livestock and humans. Prevalence of C. parvum in pigs has been reported, however, the duration and infection pattern of naturally acquired Cryptosporidium infections in pigs has not been reported. This study was undertaken to investigate the age of oocyst shedding and duration of natural Cryptosporidium parvum infections in pigs from weaning to market weight. Fecal samples were collected from weaned Yorkshire-Landrace piglets (n=33) twice per week until Cryptosporidium oocysts were detected. Upon oocyst detection, fecal samples were collected three times per week and pigs were monitored throughout the study for diarrhea and examined after concentration and immunofluroescent staining. Cryptosporidium isolates were genotyped by polymerase chain reaction to amplify the HSP70 gene which was subsequently sequence analyzed. All 33 pigs shed oocysts some time during the study. The mean age of initial oocyst detection was 45.2 days post-weaning with the mean duration of infection 28.7 days. Mean number of Cryptosporidium oocysts was low and declined to zero prior to study completion. Episodes of diarrhea were not associated with oocyst excretion. Genetic sequences were obtained for 10 of the pigs. All of the 10 isolates aligned as the Cryptosporidium parvum 'pig' genotype. This study demonstrates that the age and duration of oocyst shedding in pigs infected with C. parvum porcine genotype is different from other livestock species. PMID:12651214

  11. 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

  12. A HAPPY Map of Cryptosporidium parvum

    PubMed Central

    Piper, Michael B.; Bankier, Alan T.; Dear, Paul H.

    1998-01-01

    We have constructed a HAPPY map of the apicomplexan parasite Cryptosporidium parvum. We have placed 204 markers on the 10.4-Mb genome, giving an average marker spacing of ∼50 kb, with an effective resolution of ∼40 kb. HAPPY mapping (an in vitro linkage technique based on screening approximately haploid amounts of DNA by the polymerase chain reaction) is fast and accurate and is not subject to the distortions inherent in cloning, meiotic recombination, or hybrid cell formation. In addition, little genomic DNA is needed as a substrate, and the AT content of the genome is largely immaterial, making it an ideal method for mapping otherwise intractable parasite genomes. The map, covering all eight chromosomes, consists of 10 linkage groups, each of which has been chromosomally assigned. We have verified the accuracy of the map by several methods, including the construction of a >140-kb PAC contig on chromosome VI. Less than 1% of our markers detect non-rDNA duplicated sequences. PMID:9872984

  13. 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. PMID:8885883

  14. THE INFECTIVITY OF CRYPTOSPORIDIUM PARVUM IN HEALTHY VOLUNTEERS

    EPA Science Inventory

    Background. Small numbers of Cryptosporidium parvum oocysts can contaminate even treated drinking water, and ingestion of oocysts can cause diarrheal disease in normal as well as immunocompromised hosts. Since the number of organisms necessary to cause infection in humans is unkn...

  15. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. PROBES FOR THE SPECIFIC DETECTION OF CRYPTOSPORIDIUM PARVUM

    EPA Science Inventory

    A probe set, consisting of two synthetic oligonucleotides each tagged with a fluorescent reporter molecule, has been developed for specific detection of Cryptosporidium parvum.Each probe strand detects ribosomal RNA from a range of isolates of this species, and the combination is...

  18. [Cryptosporidium parvum Gastroenteritis in a Patient with Renal Transplantation].

    PubMed

    Çetinkaya, Ülfet; Dursun, İsmail; Kuk, Salih; Şahin, İzzet; Yazar, Süleyman

    2015-09-01

    In this study, a case who starting abundant watery diarrhea on the 14th day of renal transplantation is presented. Stool sample was analyzed for Cryptosporidium spp. by carbol fuchsin staining method, copro-ELISA and nested polimeraze chain reaction (PCR). From sample found positive by Carbol-fuchsin staining method and Copro-ELISA, DNA sequence analysis was performed, gel-purified from amplicon obtained by nested PCR. As a result of DNA sequence analysis was determined to be Cryptosporidium parvum. Although C. parvum is a rare causative agent of gastroenteritis it can be cause serious clinical diarrhea solid organ transplantation patient. As a result, also C.parvum must be considered as a causative agent of diarrhea occurring after organ transplantation. PMID:26470932

  19. 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

    2016-01-01

    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. PMID:26439535

  20. 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...

  1. Cryptosporidium parvum, a potential cause of colic adenocarcinoma

    PubMed Central

    Certad, Gabriela; Ngouanesavanh, Tramy; Guyot, Karine; Gantois, Nausicaa; Chassat, Thierry; Mouray, Anthony; Fleurisse, Laurence; Pinon, Anthony; Cailliez, Jean-Charles; Dei-Cas, Eduardo; Creusy, Colette

    2007-01-01

    Background Cryptosporidiosis represents a major public health problem. This infection has been reported worldwide as a frequent cause of diarrhoea. Particularly, it remains a clinically significant opportunistic infection among immunocompromised patients, causing potentially life-threatening diarrhoea in HIV-infected persons. However, the understanding about different aspects of this infection such as invasion, transmission and pathogenesis is problematic. Additionally, it has been difficult to find suitable animal models for propagation of this parasite. Efforts are needed to develop reproducible animal models allowing both the routine passage of different species and approaching unclear aspects of Cryptosporidium infection, especially in the pathophysiology field. Results We developed a model using adult severe combined immunodeficiency (SCID) mice inoculated with Cryptosporidium parvum or Cryptosporidium muris while treated or not with Dexamethasone (Dex) in order to investigate divergences in prepatent period, oocyst shedding or clinical and histopathological manifestations. C. muris-infected mice showed high levels of oocysts excretion, whatever the chemical immunosuppression status. Pre-patent periods were 11 days and 9.7 days in average in Dex treated and untreated mice, respectively. Parasite infection was restricted to the stomach, and had a clear preferential colonization for fundic area in both groups. Among C. parvum-infected mice, Dex-treated SCID mice became chronic shedders with a prepatent period of 6.2 days in average. C. parvum-inoculated mice treated with Dex developed glandular cystic polyps with areas of intraepithelial neoplasia, and also with the presence of intramucosal adenocarcinoma. Conclusion For the first time C. parvum is associated with the formation of polyps and adenocarcinoma lesions in the gut of Dex-treated SCID mice. Additionally, we have developed a model to compare chronic muris and parvum cryptosporidiosis using SCID mice

  2. Incorporation of exogenous uracil by Cryptosporidium parvum in vitro.

    PubMed Central

    Upton, S J; Tilley, M; Mitschler, R R; Oppert, B S

    1991-01-01

    Oocysts of Cryptosporidium parvum were used to infect Madin-Darby bovine kidney cells. Cultures were incubated in a reduced-oxygen atmosphere in candle jars or in a 5% CO2-95% air atmosphere. At 72 h, parasites were quantitated microscopically and found to be enhanced 5.5-fold in the reduced-oxygen atmosphere. Using candle jars, we then determined that C. parvum was amenable to [3H]uracil incorporation assays and easily quantitated with this method. Images PMID:2056042

  3. 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. PMID:25542170

  4. Cryptosporidium parvum and Cryptosporidium andersoni infection in naturally infected cattle of northwest Iran

    PubMed Central

    Mirzai, Yousef; Yakhchali, Mohammad; Mardani, Karim

    2014-01-01

    The protozoan intestinal parasite Cryptosporidium commonly infects cattle throughout the world and Iran. The present study was undertaken to determine the abundance and associated risk factors of Cryptosporidium infection in cattle herds of northwestern Iran. A total number of 246 fecal samples from 138 (56.1%) diarrheic (D) and 108 (43.9%) non-diarrheic (ND) cattle were randomly collected and examined by fecal smears stained with Ziehl-Neelsen. For molecular specification, DNA was extracted from collected Cryptosporidium oocysts and a fragment of 1325 bp in size from 18S rRNA gene was amplified. The overall prevalence of Cryptosporidium infection was 22.3% (55/246). The prevalence of Cryptosporidium infection in examined calves less than 6 month-old was significantly higher than adult cattle. C. parvum and C. andersoni were identified in 20.3% (50/246) and 2.03% (5/246) of examined cattle, respectively. The highest prevalence of C. parvum infection was found in D calves < 6 month-old (13.4%, 33/246), while C. andersoni was only detected in ND cattle (8.9%, 22/246). There was significant difference in the prevalence between male than female cattle. There was no significant difference between prevalence and seasons of investigation. It was concluded that C. parvum was the prevalent species in younger animals compared to older ones as a potentially zoonotic agent in the region. PMID:25568693

  5. Detection of Cryptosporidium parvum and Cryptosporidium hominis in human patients in Cairo, Egypt.

    PubMed

    Abd El Kader, Nour M; Blanco, María-Alejandra; Ali-Tammam, Marwa; Abd El Ghaffar, Abd El Rahman B; Osman, Ahmed; El Sheikh, Nabila; Rubio, José Miguel; de Fuentes, Isabel

    2012-01-01

    Cryptosporidium is a significant cause of diarrheal disease in developing and industrialized nations. Cryptosporidium hominis and Cryptosporidium parvum are the main agents of cryptosporidiosis in humans. In Egypt, very little is known about genetic structure of Cryptosporidium spp. Therefore, this study was designed to examine samples from sporadic cases of cryptosporidiosis in Egyptians in order to identify the species involved in infection as well as the transmission dynamics and distribution of the parasite in the Great Cairo area. A total of 391 human faecal samples were collected, between May 2008 and March 2009, from ten public hospitals in Great Cairo. Initial screening by immunochromatographic detection kit "the Stick Crypto-Giardia; Operon" showed 23 possible positive cases. Twenty of them were confirmed by microscopic examination. PCR was performed by amplification of the oocyst wall protein (COWP) gene where 18 out of 23 samples were positive, one not detected by microscopy. Cryptosporidium genotyping was performed by RFLP analysis of PCR products of the diagnosis PCR. Only 15 samples rendered a digestion pattern. The genotyping distribution was nine cases showing C. hominis genotype, three showing C. parvum genotype and three showing mixed infection by C. hominis and C. parvum. The data showed an elevated prevalence of C. hominis (80.0%), the most anthroponotic species, suggesting a human-human transmission. Furthermore, the presence of up to 40% of samples infected with C. parvum shows that further investigations are required to determine the subgenotypes of C. parvum to clarify the mode of transmission in order to improve the control measures. PMID:21607688

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 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. PMID:26176919

  8. Genetic modification of the diarrheal pathogen Cryptosporidium parvum

    PubMed Central

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

    2015-01-01

    Recent studies into the global causes of severe diarrhea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrheal pathogen after rotavirus1–3. Diarrheal disease is estimated to be responsible for 10.5% of overall child mortality4. Cryptosporidium is also an opportunistic pathogen in the context of HIV-AIDS and organ transplantation5,6. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger, malnourished children and immunocompromised patients7,8. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes lack of continuous culture, facile animal models, and molecular genetic tools3,9. Here we describe an experimental framework to genetically modify this important human pathogen. We establish and optimize transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we develop a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium CRISPR/Cas9 system, and in vivo selection for aminoglycoside resistance. We derive reporter parasites suitable for in vitro and in vivo drug screening, and we evaluate the basis of drug susceptibility by gene knock out. We anticipate the ability to genetically engineer the 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. PMID:26176919

  9. Comparison of Cryptosporidium parvum and Cryptosporidium wrairi by reactivity with monoclonal antibodies and ability to infect severe combined immunodeficient mice.

    PubMed Central

    Chrisp, C E; Mason, P; Perryman, L E

    1995-01-01

    Twenty-three monoclonal antibodies raised to Cryptosporidium parvum and 12 raised to C. wrairi reacted with equal intensity with the heterologous species. Despite demonstration of a close immunologic relationship between these two species, C. wrairi did not induce persistent infection in severe combined immunodeficient mice as did C. parvum. PMID:7806379

  10. Environmental inactivation of Cryptosporidium parvum oocysts in waste stabilization ponds.

    PubMed

    Reinoso, Roberto; Bécares, Eloy

    2008-11-01

    The survival of Cryptosporidium parvum oocysts in a waste stabilization pond system in northwestern Spain and the effects of sunlight and the depth and type of pond on oocyst viability were evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). All tested factors had significant effects (P < 0.01) over time on C. parvum oocyst viability. Sunlight exposure was the most influential factor for oocyst inactivation. A 40% reduction was observed after 4 days exposure to sunlight conditions compared with dark conditions. The type of pond also caused a significant reduction in C. parvum oocyst viability (P < 0.01). Inactivation rates reflected that the facultative pond was the most aggressive environment for oocysts placed both at the surface (presence of sunlight) and at the bottom (absence of sunlight) of the pond, followed by the maturation pond and the anaerobic pond. The mean inactivation rates of oocysts in the ponds ranged from 0.0159 to 0.3025 day(-1). PMID:18345476

  11. 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...

  12. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  14. 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...

  15. 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...

  16. 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.

  17. EFFECTS OF OZONE, CHLORINE DIOXIDE, CHLORINE, AND MONOCHLORAMINE ON CRYPTOSPORIDIUM PARVUM OOCYST VIABILITY

    EPA Science Inventory

    Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. xcystation and mouse infectivity were comparatively evaluated to assess oocyst viability. zone and chlorine dioxide more effectively inactivated oocysts than chlorine an...

  18. 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 ...

  19. Rapid extraction of DNA From Escherichia coli and Cryptosporidium parvum for use in PCR.

    PubMed

    Higgins, J A; Jenkins, M C; Shelton, D R; Fayer, R; Karns, J S

    2001-11-01

    The Xtra Amp tube, Isocode paper, Instagene matrix, and PrepMan matrix methods were evaluated for their ability to rapidly extract PCR-quality DNAs from Escherichia coli O157:H7 and Cryptosporidium parvum. All methods provided satisfactory DNA from E. coli, and the Xtra Amp and Instagene reagents provided satisfactory DNA from C. parvum. PMID:11679362

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. 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...

  2. A novel multiplex PCR coupled with Luminex assay for the simultaneous detection of Cryptosporidium spp., Cryptosporidium parvum and Giardia duodenalis.

    PubMed

    Li, Wei; Zhang, Nan; Gong, Pengtao; Cao, Lili; Li, Jianhua; Su, Libo; Li, Shuhong; Diao, Yumei; Wu, Kang; Li, He; Zhang, Xichen

    2010-10-11

    Cryptosporidium parvum and Giardia duodenalis are the most frequently identified enteric parasites associated with diarrhea-causing disease outbreaks, and many non-parvum species of Cryptosporidium also can replicate and cause illness in mammals including humans. In this study, we describe a novel multiplex PCR coupled with Luminex assay for the identification of Cryptosporidium spp., C. parvum and G. duodenalis in a rapid manner. The multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was developed using three pairs of biotinylated primers which amplify 424, 223 and 267 bp products from the U1 small nuclear ribonucleoprotein (U1 snr) gene, 18S rRNA gene of Cryptosporidium and the beta-giardin gene of Giardia, respectively. The genus and species-specific capture probes linked to carboxylated Luminex microspheres hybridized to the multiplex PCR amplicons to enhance sensitivity and specificity. The conditions of multiplex PCR and Luminex hybridization reaction were optimized to enable the minimum detection limits of 5x10(-6), 5x10(-6), and 5x10(-6) ng DNAs (corresponding approximately to 0.1 oocyst/cyst). The Luminex approach proved to be 100% specific and accurate by testing a total of 240 fecal samples compared with microscopic examination of fecal smears and further modified acid-fast staining or iodine-staining observation. The established assay offers the potential for rapid detection of Cryptosporidium spp., C. parvum and G. duodenalis in fecal and environmental samples. PMID:20594647

  3. Effect of high hydrostatic pressure on Cryptosporidium parvum infectivity.

    PubMed

    Slifko, T R; Raghubeer, E; Rose, J B

    2000-09-01

    The incidence of foodborne disease outbreaks caused by contaminated low-pH fruit juices is increasing. With recent mandatory pasteurization of apple juice and the industry's concerns of food safety, fruit juice processors are showing more interest in alternative nonthermal technologies that can kill >99.99% of microbial pathogens present in foods. The association of the coccidian protozoan, Cryptosporidium, with diarrheal disease outbreaks from contaminated tap water and fruit juice raises a safety concern in the food and beverage industries. The objective of this study was to evaluate the effects of high hydrostatic pressure (HHP) on C. parvum oocysts. Oocysts were suspended in apple and orange juice and HHP treated at 5.5 x 10(8) Pa (80,000 psi) for 0, 30, 45, 60, 90, and 120 s. Oocyst viability was assessed by excystation using bile salts and trypsin while the cell culture foci detection method was used to assess infectivity. Results indicated that HHP inactivated C. parvum oocysts by at least 3.4 log10 after 30 s of treatment. No infectivity was detected in samples exposed to > or =60 s of HHP and >99.995% inactivation was observed. This study demonstrated that HHP efficiently rendered the oocysts nonviable and noninfectious after treatment at 5.5 x 10(8) Pa. PMID:10983803

  4. Cryptosporidium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Of nearly 25 named species and numerous genotypes of Cryptosporidium, two are of special importance relative to human health and food safety: Cryptosporidium hominis and Cryptosporidium parvum, the former with a predilection for humans and the latter a promiscuous species. Genetic tools have been es...

  5. Fecundity of Cryptosporidium parvum is correlated with intracellular levels of the viral symbiont CPV.

    PubMed

    Jenkins, M C; Higgins, J; Abrahante, J E; Kniel, K E; O'Brien, C; Trout, J; Lancto, C A; Abrahamsen, M S; Fayer, R

    2008-07-01

    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 fecundity of two isolates of the parasite, namely C. parvum Beltsville (B) and C. parvum Iowa (I). Dairy calves infected with 10(6)C. parvum-B excreted 5-fold more oocysts compared with calves infected with the same number of C. parvum-I oocysts. The increased fecundity of the former strain was corroborated by semi-quantitative PCR assay of DNA isolated from cell cultures infected with either C. parvum-B or C. parvum-I. Quantitative reverse transcriptase-PCR analysis of viral RNA revealed a 3-fold greater number of CPV in C. parvum-B compared with C. parvum-I oocysts. These findings may indicate a role for CPV in fecundity and possibly virulence of C. parvum. PMID:18096164

  6. Prevalence of infection with Cryptosporidium parvum and Cyclospora cayetanensis among international travellers

    PubMed Central

    Jelinek, T; Lotze, M; Eichenlaub, S; Loscher, T; Nothdurft, H

    1997-01-01

    Background—Cryptosporidium parvum and Cyclospora cayetanensis are recognised as possible pathogens of traveller's diarrhoea. 
Aims—To identify the prevalence of C parvum and Cyc cayetanensis in travellers returning from developing countries. 
Patients—Nine hundred and seventy eight stool samples were taken from 795 patients returning from developing countries. 
Methods—Microscopy (iron-haematoxylin stain, SAF concentration, modified acid fast stain) and a commercially available enzyme linked immunosorbent assay (ELISA) kit for the detection of Cryptosporidium antigen in stool. 
Results—Of the 795 patients in the study, 469 suffered from diarrhoea. Infection with Cyc cayetanensis could be detected in five subjects (1.1%) by acid fast stain, and 13 patients (2.8%) were infected with C parvum. On evaluation, the antigen capture ELISA turned out to be clearly less sensitive for detection of C parvum than microscopy. All patients with either C parvum or Cyc cayetanensis infection suffered from watery diarrhoea. 
Conclusions—C parvum and Cyc cayetanensis are not major causes of diarrhoea in international travellers. In cases of persistent watery diarrhoea, however, these pathogens should be taken into account in the differential diagnosis. 

 Keywords: diarrhoea; Cryptosporidium parvum; Cyclospora cayetanensis PMID:9462213

  7. Application of RT-PCR to study in vitro development of Cryptosporidium parvum and its viral symbiont CPV

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cryptosporidium parvum and C. hominis contain a double-stranded RNA viral symbiont termed CPV. Our research seeks to find a role for CPV in the pathogenicity and development of C. parvum. Cell cultures were infected with C. parvum sporozoites, and extracted at various times post-infection for DNA ...

  8. 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...

  9. 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.

  10. Effects of Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in mice.

    PubMed

    Del Coco, Valeria F; Sparo, Mónica D; Sidoti, Alicia; Santín, Mónica; Basualdo, Juan Angel; Córdoba, María Alejandra

    2016-08-01

    Cryptosporidium is an opportunistic protozoan parasite of humans and animals worldwide and 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. Probiotics have attracted the attention as potential antiparasite compounds against protozoa involved in intestinal infections. This study investigated the effects of administration of probiotic Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in immunosuppressed mice. Effects on C. parvum infection at the intestinal mucosa were studied and scored at each portion of the gut. It was demonstrated that Ef CECT 7121 interfered with C. parvum infection when both probiotic and parasite were present in the same intestinal location suggesting that Ef CECT 7121 supplementation can alleviate the negative effects of C. parvum infection. PMID:27193238

  11. Inactivation kinetics of Cryptosporidium parvum oocysts in a swine waste lagoon and spray field.

    PubMed

    Jenkins, Michael B; Liotta, Janice L; Bowman, Dwight D

    2013-04-01

    Because of outbreaks of cryptosporidiosis in humans, some Cryptosporidium spp. have become a public health concern. Commercial swine operations can be a source of this protozoan parasite. Although the species distribution of Cryptosporidium is likely dominated by Cryptosporidium suis , a fraction may be comprised of other Cryptosporidium species infectious to humans such as Cryptosporidium parvum . To better understand the survival dynamics of Cryptosporidium spp., oocysts associated with swine operations, 2 experiments were performed to determine die-off rates of C. parvum oocysts in a swine waste lagoon (2009 and 2010) and its spray field (2010 and 2011). Sentinel chambers containing a lagoon effluent suspension of C. parvum oocysts were submerged in the lagoon, and triplicate chambers were removed over time; oocysts were extracted and assayed for viability. For comparative purposes, inactivation rates of Ascaris suum eggs contained in sentinel chambers were also determined. For 2 spray field experiments, air-dried and sieved surface soil was placed in sentinel chambers, hydrated, and inoculated with a lagoon effluent suspension of C. parvum oocysts. Sentinel chambers and control oocysts in PBS contained in microcentrifuge tubes were buried 1.5 cm below the soil surface in 3 blocks. Triplicate chambers and controls were removed over time; oocysts were extracted and assayed for viability. Based on the first order decay equation, days to reach 99% die-off (T(99)) were determined. T(99)-values determined for the 2 lagoon experiments were 13.1 and 20.1 wk, respectively. A T(99)-value for C. parvum in the spray field was significantly longer at 38.0 wk than the control oocysts in PBS at 29.0 wk. The waste lagoon and spray field system of manure management at this large-scale farrowing operation appeared to reduce the load of C. parvum oocysts before they can be hydrologically transported off the operation and reduces their likelihood of contaminating surface waters

  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. PMID:25481361

  13. Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

    PubMed

    Yang, Zhengtao; Fu, Yunhe; Gong, Pengtao; Zheng, Jingtong; Liu, Li; Yu, Yuqiang; Li, Jianhua; Li, He; Yang, Ju; Zhang, Xichen

    2015-08-01

    Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4. PMID:26048276

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. APPLICATION OF RAMAN SPECTROSCOPY TO ANALYSE THE CRYPTOSPORIDIUM PARVUM OOCYST WALL AND SUTURE CONSTITUENTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The protozoan parasite Cryptosporidium parvum, associated with diarrheal disease in humans, livestock, companion animals, and wildlife, infects drinking water. Fecal contamination is the ultimate source of the oocyst, found in surface waters throughout the United States. The parasite has so far sho...

  18. Transport, Fate, and Infectivity of Cryptosporidium parvum Oocysts Released from Manure and Leached through Macroporous Soil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  19. INACTIVATION OF CRYPTOSPORIDIUM PARVUM OOCYSTS WITH OZONE AND MONOCHLORAMINE AT LOW TEMPERATURE. (R826830)

    EPA Science Inventory

    The rate of Cryptosporidium parvum inactivation decreased with decreasing temperature (1¯20°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/m...

  20. 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,...

  1. 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...

  2. 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...

  3. A RAPID METHOD FOR PRODUCTION OF HIGH NUMBERS OF PURIFIED CRYPTOSPORIDIUM PARVUM OOCYSTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most procedures that have been described for purifying Cryptosporidium parvum oocysts are designed to either identify the parasites in clinical specimens or isolate oocysts from a small volume of feces from infected animals. The present study describes a rapid method for purifying high numbers of C...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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)...

  6. 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

  7. 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.

  8. 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. PMID:15353844

  9. Cryptosporidium parvum and Enterocytozoon bieneusi in American Mustangs and Chincoteague ponies.

    PubMed

    Wagnerová, Pavla; Sak, Bohumil; McEvoy, John; Rost, Michael; Sherwood, Dawn; Holcomb, Kevin; Kváč, Martin

    2016-03-01

    The prevalence of Cryptosporidium and microsporidia in feral horses, which have minimal contact with livestock and humans, is not currently known. We report the findings of a study on Cryptosporidium and microsporidia in 34 Mustangs and 50 Chincoteague ponies in the USA. Fecal samples were screened for presence of Cryptosporidium spp. by analysis of the small-subunit rRNA (SSU) and 60-kDa glycoprotein (gp60) genes, and Enterocytozoon bieneusi and Encephalitozoon spp. by analysis of the ribosomal internal transcribed spacer region (ITS). Cryptosporidium spp. and E. bieneusi were detected in 28/84 (33.3%) and 7/84 (8.3%) samples, respectively. Sequence analysis of SSU and ITS revealed the presence of Cryptosporidium parvum (n = 20) and E. bieneusi genotype horse 1 (n = 7), respectively. Subtyping of C. parvum isolates at the gp60 locus showed the presence of subtype IIaA17G2R1 in Mustangs and subtypes IIaA13G2R1 and IIaA15G2R1 in Chincoteague ponies. Enterocytozoon bieneusi genotype horse 1 was detected in Mustangs (n = 2) and Chincoteague ponies (n = 5). No Cryptosporidium or E. bieneusi positive animals had diarrhea. The finding that Mustangs and Chincoteague ponies are host to the zoonotic pathogen C. parvum suggests that their infrequent contact with humans and livestock is sufficient to maintain transmission; however, we should also consider the possibility that C. parvum is an established parasite of Mustangs and Chincoteague ponies that persists in these animals independently of contact with humans or livestock. PMID:26688100

  10. 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...

  11. 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...

  12. Quantitative Evaluation of Infectivity Change of Cryptosporidium parvum after Gamma Irradiation

    PubMed Central

    Lee, Soo-Ung; Joung, Mikyo; Nam, Taekyoung; Park, Woo-Yoon

    2009-01-01

    Cryptosporidium parvum is a well-known waterborne and opportunistic intracellular protozoan parasite that causes diarrheal illness. In this study, we quantitatively investigated reduction of the infectivity of C. parvum after gamma irradiation and repair of the infectivity during incubation time after irradiation. C. parvum oocysts were subjected to gamma irradiation at various doses (1, 5, 10, and 25 kGy), and the in vitro infectivity was measured by real-time PCR every day up to 7 days after irradiation. The in vitro infectivity of C. parvum on human ileocecal adenocarcinoma cells (HCT-8) was effectively reduced (> 2 log10) by irradiation at 10 kGy or more. However, in the experiment to find out repair of the infectivity, recovery was not noted until day 7 post-incubation. PMID:19290085

  13. Identification of Cryptosporidium parvum Dihydrofolate Reductase Inhibitors by Complementation in Saccharomyces cerevisiae

    PubMed Central

    Brophy, Victoria Hertle; Vasquez, John; Nelson, Richard G.; Forney, John R.; Rosowsky, Andre; Sibley, Carol Hopkins

    2000-01-01

    There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium parvum. Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested against C. parvum. To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae. We expressed the DHFR genes of C. parvum, Plasmodium falciparum, Toxoplasma gondii, Pneumocystis carinii, and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinant C. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. parvum DHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis. PMID:10722506

  14. Detection of Cryptosporidium parvum DNA in human feces by nested PCR.

    PubMed Central

    Balatbat, A B; Jordan, G W; Tang, Y J; Silva, J

    1996-01-01

    Cryptosporidium parvum is a coccidian protozoan that causes diarrhea in humans, often chronic and severe in patients with AIDS. Conventionally, diagnosis is made by concentration of stools followed by acid-fast staining (AF) or immunofluorescent staining. The threshold of detection in human stool specimens by these methods may require the presence of 50,000 (immunofluorescent staining) to 500,000 (AF) oocysts per g of stool. In this study, a nested PCR assay was developed to detect C. parvum DNA directly from stool specimens. After extraction of DNA from formalinized stool, a 400-bp fragment of C. parvum DNA was amplified with two 26-mer outer primers. The amplicon from this reaction was amplified with a second primer pair. With these nested primers, a 194-bp DNA fragment was amplified and confirmed as C. parvum DNA by internal probing with an enzyme-linked chemiluminescence system. This PCR-based test allowed the detection of 500 oocysts per g of stool or 100 ng of C. parvum DNA. Studies indicate that the primers utilized are specific for the DNA of C. parvum. DNA sequences were also detected in stool specimens from 4 of 28 patients previously reported negative by AF. In summary, a rapid, sensitive, and specific assay for the detection of C. parvum directly from stool specimens has been developed. This test has the potential for detecting asymptomatic infection, monitoring the response to therapy, and detecting the organism in environmental sources. PMID:8784586

  15. Intracellular levels of the viral symbiont CPV in Cryptosporidium parvum correlate with fecundity of the parasite in dairy calves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous reports have cited differences in clinical signs and oocyst output among strains of Cryptosporidium parvum. The purpose of this study was to determine if levels of the C. parvum intracellular viral symbiont CPV correlated with observed clinical and parasitological differences. Calves infe...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. USE OF MICRO ASSAY TECHNOLOGY TO COMPARE GENE EXPRESSION OF MICE INFECTED OR NOT INFECTED WITH CRYPTOSPORIDIUM PARVUM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cryptosporidium parvum is a protozoan parasite that is a frequent cause of diarrheal disease in young calves. In laboratory models of infection, infant mice are susceptible to C. parvum infection, while normal adult mice are not. Various immunodeficient strains of mice are, however, susceptible to...

  18. 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. PMID:26827859

  19. Identification of Cryptosporidium parvum Active Chemical Series by Repurposing the Open Access Malaria Box

    PubMed Central

    Bessoff, Kovi; Spangenberg, Thomas; Foderaro, Jenna E.; Jumani, Rajiv S.; Ward, Gary E.

    2014-01-01

    The apicomplexan parasites Cryptosporidium parvum and Cryptosporidium hominis are major etiologic agents of human cryptosporidiosis. The infection is typically self-limited in immunocompetent adults, but it can cause chronic fulminant diarrhea in immunocompromised patients and malnutrition and stunting in children. Nitazoxanide, the current standard of care for cryptosporidiosis, is only partially efficacious for children and is no more effective than a placebo for AIDS patients. Unfortunately, financial obstacles to drug discovery for diseases that disproportionately affect low-income countries and technical limitations associated with studies of Cryptosporidium biology impede the development of better drugs for treating cryptosporidiosis. Using a cell-based high-throughput screen, we queried the Medicines for Malaria Venture (MMV) Open Access Malaria Box for activity against C. parvum. We identified 3 novel chemical series derived from the quinolin-8-ol, allopurinol-based, and 2,4-diamino-quinazoline chemical scaffolds that exhibited submicromolar potency against C. parvum. Potency was conserved in a subset of compounds from each scaffold with varied physicochemical properties, and two of the scaffolds identified exhibit more rapid inhibition of C. parvum growth than nitazoxanide, making them excellent candidates for further development. The 2,4-diamino-quinazoline and allopurinol-based compounds were also potent growth inhibitors of the related apicomplexan parasite Toxoplasma gondii, and a good correlation was observed in the relative activities of the compounds in the allopurinol-based series against T. gondii and C. parvum. Taken together, these data illustrate the utility of the Open Access Malaria Box as a source of both potential leads for drug development and chemical probes to elucidate basic biological processes in C. parvum and other apicomplexan parasites. PMID:24566188

  20. 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

  1. Coinfection by Cryptosporidium parvum and porcine circovirus type 2 in weaned pigs.

    PubMed

    Núñez, A; McNeilly, F; Perea, A; Sánchez-Cordón, P J; Huerta, B; Allan, G; Carrasco, L

    2003-06-01

    Routine histopathological diagnosis of one representative 3-month-old pig from a group suffering from diarrhoea revealed a massive degree of parasitation by Cryptosporidium parvum, with a concomitant infection by porcine circovirus type 2 (PCV2), that was confirmed by immunohistochemical procedures. The areas of intestine where parasites were more numerous presented abundant PCV2 infected cells in mucosa and submucosa. The concurrence of C. parvum, a rare primary intestinal pathogen in post-weaning and growing pigs, and PCV2 infections suggest an increased susceptibility as a result of an immunosuppression state. PMID:12864903

  2. Expression of P23 of Cryptosporidium parvum in Toxoplasma gondii and evaluation of its protective effects.

    PubMed

    Shirafuji, Hiroaki; Xuan, Xuenan; Kimata, Isao; Takashima, Yasuhiro; Fukumoto, Shinya; Otsuka, Haruki; Nagasawa, Hideyuki; Suzuki, Hiroshi

    2005-04-01

    In this study, P23 of Cryptosporidium parvum sporozoites, an immunodominant surface protein, was stably expressed in Toxoplasma gondii (Tg/P23) and its protective effects were evaluated in a mouse model. The molecular weight and antigenic property of P23 expressed by Tg/P23 were similar to those of the native P23. Mice immunized with lysed Tg/P23 tachyzoites produced specific neutralizing antibodies against C. parvum. These findings indicate that the T. gondii vector may provide a new tool for the production of a recombinant vaccine against cryptosporidiosis in animals. PMID:15986633

  3. Comparison of techniques for detecting antigens of Giardia lamblia and Cryptosporidium parvum in faeces.

    PubMed Central

    Tee, G H; Moody, A H; Cooke, A H; Chiodini, P L

    1993-01-01

    AIM--To compare the use of commercial monoclonal antibody test systems--the Giardia CEL IF test and the Crypto CEL IF test--for the detection of Giardia lamblia and Cryptosporidium parvum antigens in faeces with conventional techniques. METHODS--Sensitivity and specificity were evaluated using preparations of cysts of G lamblia and purified oocysts of C parvum. Evaluation of 59 random faecal samples passing through the Department of Clinical Parasitology, Hospital for Tropical Diseases, London, was carried out for both organisms. RESULTS--The fluorescence staining techniques proved more sensitive than other tests routinely used for diagnosis. PMID:8331181

  4. In Vitro Interactions of Asian Freshwater Clam (Corbicula fluminea) Hemocytes and Cryptosporidium parvum Oocysts

    PubMed Central

    Graczyk, T. K.; Fayer, R.; Cranfield, M. R.; Conn, D. B.

    1997-01-01

    Corbicula fluminea hemocytes phagocytosed infectious oocysts of Cryptosporidium parvum in vitro. After 15, 30, 60, 90, and 120 min of incubation, averages of 35.8, 58.0, 69.7, 77.7, and 81.6% of the oocysts were phagocytosed by 24.3, 70.0, 78.5, 87.3, and 93.0% of the hemocytes, respectively. A single clam can retain by phagocytosis an average of 1.84 x 10(sup6) oocysts per ml of hemolymph. C. fluminea bivalves can serve as biological indicators of contamination of wastewaters and agricultural drainages with Cryptosporidium. PMID:16535656

  5. Cryptosporidium parvum oocysts in zebra mussels (Dreissena polymorpha): evidence from the St Lawrence River.

    PubMed

    Graczyk, T K; Marcogliese, D J; de Lafontaine, Y; Da Silva, A J; Mhangami-Ruwende, B; Pieniazek, N J

    2001-03-01

    Molluscan shellfish can recover and concentrate environmentally derived waterborne pathogens and can be used for the sanitary assessment of water quality. Oocysts of Cryptosporidium parvum (genotype 1) were identified in zebra mussels (Dreissena polymorpha) from the St. Lawrence River, Quebec. Approximately 67 oocysts/ml of hemolymph and 129 oocysts/g of soft tissue were recovered. The adjusted concentration of oocysts per gram of tissue was 2.2 x 10(2), and approximately 4.4 x 10(2) oocysts were recovered from a single mussel. Zebra mussels can serve as biological indicators of waterborne contamination with Cryptosporidium. PMID:11293571

  6. Cryptosporidium parvum IId family: clonal population and dispersal from Western Asia to other geographical regions

    PubMed Central

    Wang, Rongjun; Zhang, Longxian; Axén, Charlotte; Bjorkman, Camilla; Jian, Fuchun; Amer, Said; Liu, Aiqin; Feng, Yaoyu; Li, Guoquan; Lv, Chaochao; Zhao, Zifang; Qi, Meng; Dong, Haiju; Wang, Helei; Sun, Yanru; Ning, Changshen; Xiao, Lihua

    2014-01-01

    In this study, 111 Cryptosporidium parvum IId isolates from several species of animals in China, Sweden, and Egypt were subtyped by multilocus sequence typing (MLST). One to eleven subtypes were detected at each of the 12 microsatellite, minisatellite, and single nucleotide polymorphism (SNP) loci, forming 25 MLST subtypes. Host-adaptation and significant geographical segregation were both observed in the MLST subtypes. A clonal population structure was seen in C. parvum IId isolates from China and Sweden. Three ancestral lineages and the same RPGR sequence were shared by these isolates examined. Therefore, the present genetic observations including the higher nucleotide diversity of C. parvum IId GP60 sequences in Western Asia, as well as the unique distribution of IId subtypes (almost exclusively found in Asia, Europe, and Egypt) and in combination with the domestication history of cattle, sheep, and goats, indicated that C. parvum IId subtypes were probably dispersed from Western Asia to other geographical regions. More population genetic structure studies involving various C. parvum subtype families using high-resolution tools are needed to better elucidate the origin and dissemination of C. parvum in the world. PMID:24572610

  7. Cryptosporidium parvum Induces B7-H1 Expression in Cholangiocytes by Downregulating MicroRNA-513

    PubMed Central

    Gong, Ai-Yu; Zhou, Rui; Hu, Guoku; Liu, Jun; Sosnowska, Danuta; Drescher, Kristen M.; Dong, Haidong; Chen, Xian-Ming

    2009-01-01

    Expression of B7 costimulatory molecules represents an important compartment of immune response of epithelial cells following microbial infection. We reported here that the protozoan parasite Cryptosporidium parvum induced B7-H1 expression in cultured human cholangiocytes. Induced expression of B7-H1 was identified in cells after exposure to infective C. parvum parasite or parasite lysate. Interestingly, microRNA-513 (miR-513) level was reduced in cells after exposure to C. parvum, resulting in a relief of 3′-untranslated region-mediated translational suppression of B7-H1. Overexpression of miR-513 through transfection of miR-513 precursor inhibited C. parvum-induced B7-H1 protein expression. Moreover, enhanced apoptotic cell death was identified in activated human T cells following co-culture with C. parvum-infected cholangiocytes. The apoptosis of activated T cells was partially blocked by a neutralizing antibody to B7-H1 or transfection of cholangiocytes with miR-513 precursor. These data suggest a role of miR-513 in regulating B7-H1 expression by cholangiocytes in response to C. parvum infection. PMID:19916867

  8. In vitro infection of Cryptosporidium parvum to four different cell lines.

    PubMed

    Yu, J R; Choi, S D; Kim, Y W

    2000-06-01

    To determine a suitable condition for in vitro infection model of Cryptosporidium parvum, four different cell lines, AGS, MDCK, HCT-8 and Caco-2, were used as host cell lines which were cultured at various concentrations of added supplements. These supplement include fetal bovine serum (FBS), sodium choleate, ascorbic acid, folic acid, calcium pantothenate, para-aminobenzoic acid and pyruvate and their effects on the cell lines which were infected with C. parvum were evaluated. The results of this study showed that the AGS cell line was most susceptible to C. parvum whereas the Caco-2 cells appeared to be least susceptible to C. parvum. In regards to the serum condition, 10% FBS was suitable for the growth of AGS and HCT-8 cells, and 1% FBS was good for the growth of the MDCK cells when they were inoculated with C. parvum. Vitamins had a positive effect on the AGS cells, and pyruvate also showed positive effects on all of the cell lines except for Caco-2. Modified medium for each cell line was prepared by adding appropriate amounts of each supplement which resulted in the highest parasite infection number. Modified media increased the number of parasites infected on AGS cells to 2.3-fold higher when compared to the control media. In this study, we found that the AGS cell line was a suitable host model for evaluating C. parvum in vitro study and the media contents for the optimal infection conditions were suggested. PMID:10905066

  9. Enhancing Cryptosporidium parvum recovery rates for improved water monitoring.

    PubMed

    Pavli, Pagona; Venkateswaran, Sesha; Bradley, Mark; Bridle, Helen

    2016-01-01

    Water monitoring is essential to ensure safe drinking water for consumers. However existing methods have several drawbacks, particularly with regard to the poor recovery of Cryptosporidium due to the inability to efficiently elute Cryptosporidium oocysts during the established detection process used by water utilities. Thus the development of new inexpensive materials that could be incorporated into the concentration and release stage that would control Cryptosporidium oocysts adhesion would be beneficial. Here we describe improved filter performance following dip-coating of the filters with a "bioactive" polyacrylate. Specifically 69% more oocysts were eluted from the filter which had been coated with a polymer than on the naked filter alone. PMID:26009471

  10. Molecular typing of Cryptosporidium parvum associated with a diarrhoea outbreak identifies two sources of exposure

    PubMed Central

    MATTSSON, J. G.; INSULANDER, M.; LEBBAD, M.; BJÖRKMAN, C.; SVENUNGSSON, B.

    2008-01-01

    SUMMARY An outbreak of cryptosporidiosis associated with exposure to outdoor swimming-pool water affected an estimated 800–1000 individuals. PCR products were obtained from faecal specimens from 30 individuals who tested positive for Cryptosporidium oocysts. RFLP and sequencing analyses showed that all individuals were infected with Cryptosporidium parvum. Among the infected individuals, five had just swum in an adjacent indoor pool during the same period, and had no identified contact with individuals linked to the outdoor pool. With the use of subgenotyping based on analysis of three mini- and microsatellite loci, MS1, TP14, and GP15, we could identify two sources of exposure. One subtype was associated with the outdoor pool and another with the indoor pool. These data demonstrate that the use of mini- and microsatellite loci as markers for molecular fingerprinting of C. parvum isolates are valuable in the epidemiological investigation of outbreaks. PMID:17961283

  11. 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.

  12. 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

  13. 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

  14. Prevalence and molecular epidemiology of Cryptosporidium parvum in dairy calves in Punjab (India).

    PubMed

    Joute, J R; Gill, J P S; Singh, B B

    2016-09-01

    Cryptosporidium parvum is an important zoonotic protozoan parasite that infects the gastrointestinal tract of vertebrate animals and man. The current study was contemplated for molecular detection of Cryptosporidium species prevalent in dairy calves in Punjab, India. A total of 302 faecal samples were screened by modified Ziehl-Neelsen staining technique for the detection of Cryptosporidium oocysts. Molecular characterisation was done using PCR followed by sequence analysis of the representative isolates. An overall prevalence of 26.15 % was obtained with the highest prevalence obtained in 0-30 day old calves in both diarrhoeic and non-diarrhoeic animals. PCR analysis revealed the expected bands at 1,325 and 835 bp from all the isolates for primary and secondary/nested PCR respectively. Ten representative samples were sequenced in both directions. Phylogenetic analysis revealed the presence of C. parvum in all the samples. The high rate of calves infected with C. parvum can act as a great source of zoonotic cryptosporidiosis which indicates a potential risk of zoonotic transmission from animal to human beings in Punjab (India). PMID:27605777

  15. Detection of Cryptosporidium parvum in raw milk by PCR and oligonucleotide probe hybridization.

    PubMed Central

    Laberge, I; Ibrahim, A; Barta, J R; Griffiths, M W

    1996-01-01

    Cryptosporidium spp. are potential contaminants of food. Suspected cases of food-borne cryptosporidiosis are rarely confirmed because of the limited numbers of oocysts in the samples and the lack of sensitive detection methods adaptable to food. PCR was investigated as a means of overcoming this problem. A PCR assay was designed for the specific amplification of a previously sequenced portion of an oocyst protein gene fragment of Cryptosporidium parvum (N. C. Lally, G. D. Baird, S. J. McQuay, F. Wright, and J. J. Oliver, Mol. Biochem. Parasitol. 56:69-78, 1992) and compared with the primer set of Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688-694, 1991). The PCR products were hybridized with digoxigenin-labeled internal probes and detected by chemiluminescence to enhance sensitivity. The two sets of primers were compared with regard to their sensitivity and specificity by using a variety of human and animal isolates of C. parvum and related parasites. Both assays enabled the detection of 1 to 10 oocysts in 20 ml of artificially contaminated raw milk. The assay based on the PCR set and probe of Laxer et al. detected DNAs from Eimeria acervulina and Giardia intestinalis. The new assay has good specificity for C. parvum bovine isolates and hence has a better potential for monitoring the prevalence of C. parvum in raw milk and other environmental samples. PMID:8795214

  16. 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...

  17. Cryptosporidium parvum and Giardia intestinalis in calf diarrhoea in Sweden.

    PubMed

    Björkman, C; Svensson, C; Christensson, B; de Verdier, K

    2003-01-01

    The objective of this study conducted in 75 herds was to investigate the presence and significance of Criptosporidium parvum and Giardia intestinalis in Swedish dairy calves in comparison with rotavirus, coronavirus and Escherichia coli K99+. The farmers were asked to collect faecal samples from each heifer calf that had diarrhoea between birth and 90 days of age, and also from a healthy calf of the same age. In total, 270 samples were collected and analysed. C. parvum, either alone or together with G. intestinalis and/or rotavirus, was detected in 16 (11%) and 6 (5%) of the samples from diarrhoeic and healthy calves, respectively. Even though a higher proportion of diarrhoeic calves shed C. parvum, the difference between the groups was not statistically significant (p = 0.067), possibly due to the low number of positive samples. G. intestinalis was found in 42 (29%) of the diarrhoea samples and in 29 (23%) of the samples from healthy calves. Rotavirus and coronavirus were demonstrated in 24% and 3% of the diarrhoea samples, respectively, whereas E. coli K99+ was only found in samples from 2 healthy calves. C. parvum and G. intestinalis were found in samples from calves 7 to 84 days of age and during all seasons. The results confirm that C. parvum is present in Swedish dairy herds and might have clinical significance. G. intestinalis was the most common agent found but the importance of this parasite remains unclear. Both parasites have suggested zoonotic potential and thus warrant further attention. In addition, rotavirus is a major pathogen in neonatal enteritis in Sweden, whereas coronavirus and E. coli K99+ seem to be of less importance. PMID:15074627

  18. Putative cis-Regulatory Elements Associated with Heat Shock Genes Activated During Excystation of Cryptosporidium parvum

    PubMed Central

    Lara, Ana M.; Serrano, Myrna; Sheth, Nihar; Buck, Gregory

    2010-01-01

    Background Cryptosporidiosis is a ubiquitous infectious disease, caused by the protozoan parasites Cryptosporidium hominis and C. parvum, leading to acute, persistent and chronic diarrhea worldwide. Although the complications of this disease can be serious, even fatal, in immunocompromised patients of any age, they have also been found to lead to long term effects, including growth inhibition and impaired cognitive development, in infected immunocompetent children. The Cryptosporidium life cycle alternates between a dormant stage, the oocyst, and a highly replicative phase that includes both asexual vegetative stages as well as sexual stages, implying fine genetic regulatory mechanisms. The parasite is extremely difficult to study because it cannot be cultured in vitro and animal models are equally challenging. The recent publication of the genome sequence of C. hominis and C. parvum has, however, significantly advanced our understanding of the biology and pathogenesis of this parasite. Methodology/Principal Findings Herein, our goal was to identify cis-regulatory elements associated with heat shock response in Cryptosporidium using a combination of in silico and real time RT-PCR strategies. Analysis with Gibbs-Sampling algorithms of upstream non-translated regions of twelve genes annotated as heat shock proteins in the Cryptosporidium genome identified a highly conserved over-represented sequence motif in eleven of them. RT-PCR analyses, described herein and also by others, show that these eleven genes bearing the putative element are induced concurrent with excystation of parasite oocysts via heat shock. Conclusions/Significance Our analyses suggest that occurrences of a motif identified in the upstream regions of the Cryptosporidium heat shock genes represent parts of the transcriptional apparatus and function as stress response elements that activate expression of these genes during excystation, and possibly at other stages in the life cycle of the parasite

  19. Arginine reduces Cryptosporidium parvum infection in undernourished suckling mice involving both nitric oxide synthase and arginase

    PubMed Central

    Castro, Ibraim C.; Oliveira, Bruna B.; Slowikowski, Jacek J.; Coutinho, Bruna P.; Siqueira, Francisco Júlio W.S.; Costa, Lourrany B.; Sevilleja, Jesus Emmanuel; Almeida, Camila A.; Lima, Aldo A.M.; Warren, Cirle A.; Oriá, Reinaldo B.; Guerrant, Richard L.

    2011-01-01

    Objective This study investigated the role of L-arginine supplementation to undernourished and Cryptosporidium parvum-infected suckling mice. Methods The following regimens were initiated on the 4th day of life and given subcutaneously daily: either 200mM of L-arginine or PBS for the C. parvum-infected controls. L-arginine-treated mice were grouped to receive either 20mM of NG-nitroarginine-methyl-ester (L-NAME) or PBS. Infected mice received orally 106 excysted-C. parvum oocysts on day 6 and were euthanized on day 14th at the infection peak. Results L-arginine improved weight gain compared to the untreated infected controls. L-NAME profoundly impaired body weight gain as compared to all other groups. Cryptosporidiosis was associated with ileal crypt hyperplasia, villus blunting, and inflammation. L-arginine improved mucosal histology following infection. L-NAME abrogated these arginine-induced improvements. Infected control mice showed an intense arginase expression, which was even greater with L-NAME. L-arginine reduced parasite burden, an effect that was reversed by L-NAME. C. parvum infection increased urine NO3-/NO2- concentration when compared to uninfected controls, which was increased by L-arginine supplementation, an effect that was also reversed by L-NAME. Conclusion These findings show a protective role of L-arginine during C. parvum infection in undernourished mice with involvement of arginase I and nitric oxide synthase enzymatic actions. PMID:22261576

  20. 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. PMID:27377768

  1. SHP-2 Mediates Cryptosporidium parvum Infectivity in Human Intestinal Epithelial Cells

    PubMed Central

    Varughese, Eunice A.; Kasper, Susan; Anneken, Emily M.; Yadav, Jagjit S.

    2015-01-01

    The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention. PMID:26556238

  2. Three sample preparation protocols for polymerase chain reaction based detection of Cryptosporidium parvum in environmental samples.

    PubMed

    Kostrzynska, M; Sankey, M; Haack, E; Power, C; Aldom, J E; Chagla, A H; Unger, S; Palmateer, G; Lee, H; Trevors, J T; De Grandis, S A

    1999-02-01

    Cryptosporidium parvum is a protozoan parasite responsible for an increasing number of outbreaks of gastrointestinal illness worldwide. In this report, we describe development of sample preparation protocols for polymerase chain reaction (PCR)-based detection of C. parvum in fecal material and environmental water samples. Two of these methods were found adequate for isolation of Cryptosporidium DNA from filtered water pellet suspensions. The first involved several filtration steps, immunomagnetic separation and freeze-thaw cycles. The second method involved filtration, addition of EnviroAmp lysis reagent, freeze-thaw cycles and precipitation of the DNA with isopropanol. Using nested PCR, we detected 100 oocysts/ml of filtered water pellet suspension, with either of the above sample preparation procedures. Nested PCR increased sensitivity of the assay by two to three orders of magnitude as compared to the primary PCR. The detection limit for seeded fecal samples was 10-fold higher than for filtered environmental water pellet suspension. Nested PCR results showed 62.4 and 91.1% correlation with immunofluorescence assay (IFA) for fecal samples and filtered environmental water pellet suspensions, respectively. This correlation decreased to 47.2% and 44.4%, respectively, when only IFA positive samples were analyzed. However, in fecal samples contaminated with a high number (> 10(5)/g) of C. parvum oocysts, this correlation was 100%. PMID:10076632

  3. 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. PMID:26173708

  4. Development of a nested-PCR assay for the detection of Cryptosporidium parvum in finished water.

    PubMed

    Monis, P T; Saint, C P

    2001-05-01

    A nested-PCR assay, incorporating an internal positive control, was developed for Cryptosporidium monitoring in finished water. This assay was capable of reproducibly detecting 8 oocysts in spiked-filtered water samples collected from 5 South Australian water treatment plants. The RT-PCR assay of Kaucner and Stinear (Appl. Environ. Microbiol. 64(5) (1998) 1743) was also evaluated for the detection of Cryptosporidium parvum. Initially, under our experimental conditions, a detection level of 27 oocysts was achieved for spiked reagent water samples. This level was improved to 5 oocysts by modification of the method. Untreated South Australian source waters concentrated by calcium carbonate flocculation were found to be highly inhibitory to the RT-PCR assay. Concentration of similar samples using Envirochek filters appeared to eliminate PCR inhibition. While both methods possessed similar sensitivities the nested-PCR assay was more reproducible, more cost effective, simpler to perform and could detect both viable and non-viable intact Cryptosporidium parvum oocysts, which is an important consideration for plant operators. These factors make the nested-PCR assay the method of choice for screening large numbers of potable water samples, where a reliable low level of detection is essential. PMID:11329665

  5. 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. PMID:26939237

  6. Perturbation of the intestinal microbiota of mice infected with Cryptosporidium parvum.

    PubMed

    Ras, Refaat; Huynh, Kevin; Desoky, Enas; Badawy, Ahmed; Widmer, Giovanni

    2015-07-01

    Understanding the interaction between the intestinal microbiota (microbiome) and enteric pathogens is of interest in the development of alternative treatments that do not rely on chemotherapy and do not lead to drug resistance. We undertook research in a rodent model of cryptosporidiosis to assess whether the bacterial gut microbiota is impacted by infection with the protozoan pathogen Cryptosporidium parvum. The profile of the faecal bacterial microbiota in infected and uninfected animals was compared using 16S amplicon sequencing. In four independent experiments, the intestinal microbiota of infected mice differed from that of uninfected animals, regardless of the C. parvum isolate used to infect mice. The use of replicated treatment groups demonstrated that microbiota divergence between treatments was driven by the infection and did not result from spontaneous changes in the intestinal ecosystem unrelated to the infection. Microbiota perturbation induced by C. parvum appeared to be reversible, as we observed a tendency for the phylogenetic distance between infected and uninfected mice to diminish after mice cleared the infection. As mice infected with C. parvum do not develop diarrhoea, these observations indicate that microbiota perturbation results from other mechanisms than an accelerated movement of gut content. PMID:25913477

  7. Transport of Cryptosporidium parvum Oocysts through Vegetated Buffer Strips and Estimated Filtration Efficiency

    PubMed Central

    Atwill, Edward R.; Hou, Lingling; Karle, Betsy M.; Harter, Thomas; Tate, Kenneth W.; Dahlgren, Randy A.

    2002-01-01

    Vegetated buffer strips were evaluated for their ability to remove waterborne Cryptosporidium parvum from surface and shallow subsurface flow during simulated rainfall rates of 15 or 40 mm/h for 4 h. Log10 reductions for spiked C. parvum oocysts ranged from 1.0 to 3.1 per m of vegetated buffer, with buffers set at 5 to 20% slope, 85 to 99% fescue cover, soil textures of either silty clay (19:47:34 sand-silt-clay), loam (45:37:18), or sandy loam (70:25:5), and bulk densities of between 0.6 to 1.7 g/cm3. Vegetated buffers constructed with sandy loam or higher soil bulk densities were less effective at removing waterborne C. parvum (1- to 2-log10 reduction/m) compared to buffers constructed with silty clay or loam or at lower bulk densities (2- to 3-log10 reduction/m). The effect of slope on filtration efficiency was conditional on soil texture and soil bulk density. Based on these results, a vegetated buffer strip comprised of similar soils at a slope of ≤20% and a length of ≥3 m should function to remove ≥99.9% of C. parvum oocysts from agricultural runoff generated during events involving mild to moderate precipitation. PMID:12406745

  8. 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. PMID:23145570

  9. Cryptosporidium parvum: functional complementation of a parasite transcriptional coactivator CpMBF1 in yeast.

    PubMed

    Zhu, G; LaGier, M J; Hirose, S; Keithly, J S

    2000-12-01

    We report here the identification of a novel multiprotein bridging factor type 1 from the apicomplexan Cryptosporidium parvum (CpMBF1), one of the opportunistic pathogens in AIDS patients. In slime molds, insects, and humans, MBF1-regulated systems have been associated with cell differentiation, which indicates that CpMBF1 could be responsible for the activation of similar systems in C. parvum during its complex life cycle. Because of the difficulties and high cost in obtaining sufficient and purified C. parvum material for molecular and biochemical analyses, well-characterized yeast genetic systems may be useful for investigating the functions of C. parvum genes. In this study, the function of CpMBF1 as an interconnecting element between a DNA-binding regulator and TATA-box-binding protein (TBP) was confirmed using a yeast complementation assay. Under conditions of histidine starvation, an MBF1-deficient strain of Saccharomyces cerevisiae was unable to activate the HIS3 gene, which encodes imidazoleglycerol-phosphate dehydratase (IGPDH), and thus became sensitive to 3-amino triazole, an inhibitor of this enzyme. Upon introduction of parasite CpMBF1 into S. cerevisiae, 3-amino triazole resistance of the MBF1-deficient strain was restored to wild-type levels, and Northern blot analysis revealed that CpMBF1 was able to activate HIS3 transcription in response to histidine starvation. PMID:11162372

  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. PCR slippage across the ML-2 microsatellite of the Cryptosporidium MIC1 locus enables development of a PCR assay capable of distinguishing the zoonotic Cryptosporidium parvum from other human infectious Cryptosporidium species.

    PubMed

    Webber, M A; Sari, I; Hoefel, D; Monis, P T; King, B J

    2014-08-01

    Cryptosporidium are ubiquitous and significant enteropathogens of all classes of vertebrates and a major cause of human morbidity and mortality worldwide. Of the 24 recognized species, the zoonotic Cryptosporidium parvum and the host-specific Cryptosporidium hominis cause the majority of cases of human cryptosporidiosis. Here, we report on structural and transcriptional variability between C. parvum and C. hominis at the MIC1 locus, which encodes a microneme localized thrombospondin-like domain containing protein previously demonstrated to be critical for host cell infection by C. parvum. We demonstrate, using reverse transcription quantitative PCR with the aid of genomic data from the EuPathDB site, that the transcribed product in C. hominis is both truncated and significantly down-regulated in the sporozoite. We hypothesize that CpMIC1 may be a genetic factor involved in facilitating the wider host range of C. parvum in comparison with the specific host range of C. hominis. Furthermore, we show that the presence of a microsatellite (ML-2) within the C. parvum MIC-1 locus enables the development of a PCR marker that can rapidly distinguish the zoonotic C. parvum from C. hominis and other significant human infectious Cryptosporidium species due to reproducible PCR slippage across the ML-2 microsatellite. Additionally, we demonstrate that this locus is tightly linked to the GP60 locus, a locus commonly used in the genetic characterization of C. parvum and C. hominis isolates. This marker should provide a robust and additional tool to aid in the rapid identification of C. parvum from other Cryptosporidium species. PMID:23954136

  14. Influence of Surface Characteristics on the Stability of Cryptosporidium parvum Oocysts

    PubMed Central

    Butkus, Michael A.; Bays, J. Timothy; Labare, Michael P.

    2003-01-01

    Microelectrophoresis is a common technique for probing the surface chemistry of the Cryptosporidium parvum oocyst. Results of previous studies of the electrophoretic mobility of C. parvum oocysts in which microelectrophoresis was used are incongruent. In this work we demonstrated that capillary electrophoresis may also be used to probe the surface characteristics of C. parvum oocysts, and we related the surface chemistry of C. parvum oocysts to their stability in water. Capillary electrophoresis results indicated that oocysts which were washed in a phosphate buffer solution had neutrally charged surfaces. Inactivation of oocysts with formalin did not influence their electrophoretic mobility, while oocyst populations that were washed in distilled water consisted of cells with both neutral and negative surface charges. These results indicate that washing oocysts in low-ionic-strength distilled water can impart a negative charge to a fraction of the oocysts in the sample. Rapid coagulation experiments indicated that oocysts did not aggregate in a 0.5 M NaCl solution; oocyst stability in the salt solution may have been the result of Lewis acid-base forces, steric stabilization, or some other factor. The presence of sucrose and Percoll could not be readily identified on the surface of C. parvum oocysts by attenuated total reflectance-Fourier transform infrared spectroscopy, suggesting that these purification reagents may not be responsible for the stability of the uncharged oocysts. These findings imply that precipitate enmeshment may be the optimal mechanism of coagulation for removal of oocysts in water treatment systems. The results of this work may help elucidate the causes of variation in oocyst surface characteristics, may ultimately lead to improved removal efficiencies in full-scale water treatment systems, and may improve fate and transport predictions for oocysts in natural systems. PMID:12839749

  15. Neutrophils do not mediate the pathophysiological sequelae of Cryptosporidium parvum infection in neonatal piglets.

    PubMed

    Zadrozny, Leah M; Stauffer, Stephen H; Armstrong, Martha U; Jones, Samuel L; Gookin, Jody L

    2006-10-01

    Cryptosporidium parvum is a minimally invasive protozoal pathogen of intestinal epithelium that results in villus atrophy, mucosal lipid peroxidation, diarrhea, and diminished barrier function. Influx of neutrophils is a consistent feature of human and animal cryptosporidiosis, and yet their contribution to the pathological sequelae of infection has not been investigated. Accordingly, we used an established neonatal piglet model of C. parvum infection to examine the role of neutrophils in disease pathogenesis by inhibiting their recruitment and activation in vivo using a monoclonal anti-CD18 antibody. Infected piglets were treated daily with anti-CD18 or isotype control immunoglobulin G and euthanized at peak infection, at which time neutrophil infiltrates, lipid peroxidation, severity of infection, and intestinal barrier function were quantified. C. parvum infection resulted in a significant increase in mucosal neutrophil myeloperoxidase activity that was prevented by treatment of piglets with anti-CD18 antibody. Neutrophil recruitment was dependent on mucosal superoxide formation (prevented by treatment of infected piglets with superoxide dismutase). Neutrophils did not contribute to peroxynitrite formation or peroxidative injury of C. parvum-infected mucosa and had no impact on the severity of epithelial infection, villus atrophy, or diarrhea. The presence of neutrophils in C. parvum-infected mucosa was associated with enhanced barrier function that could not be attributed to mucosal elaboration of prostaglandins or stimulation of their synthesis. These studies are the first to demonstrate that neutrophilic inflammation arising in response to infection by a noninvasive epithelial pathogen results in physiologic rather than pathological effects in vivo. PMID:16988224

  16. Neutrophils Do Not Mediate the Pathophysiological Sequelae of Cryptosporidium parvum Infection in Neonatal Piglets

    PubMed Central

    Zadrozny, Leah M.; Stauffer, Stephen H.; Armstrong, Martha U.; Jones, Samuel L.; Gookin, Jody L.

    2006-01-01

    Cryptosporidium parvum is a minimally invasive protozoal pathogen of intestinal epithelium that results in villus atrophy, mucosal lipid peroxidation, diarrhea, and diminished barrier function. Influx of neutrophils is a consistent feature of human and animal cryptosporidiosis, and yet their contribution to the pathological sequelae of infection has not been investigated. Accordingly, we used an established neonatal piglet model of C. parvum infection to examine the role of neutrophils in disease pathogenesis by inhibiting their recruitment and activation in vivo using a monoclonal anti-CD18 antibody. Infected piglets were treated daily with anti-CD18 or isotype control immunoglobulin G and euthanized at peak infection, at which time neutrophil infiltrates, lipid peroxidation, severity of infection, and intestinal barrier function were quantified. C. parvum infection resulted in a significant increase in mucosal neutrophil myeloperoxidase activity that was prevented by treatment of piglets with anti-CD18 antibody. Neutrophil recruitment was dependent on mucosal superoxide formation (prevented by treatment of infected piglets with superoxide dismutase). Neutrophils did not contribute to peroxynitrite formation or peroxidative injury of C. parvum-infected mucosa and had no impact on the severity of epithelial infection, villus atrophy, or diarrhea. The presence of neutrophils in C. parvum-infected mucosa was associated with enhanced barrier function that could not be attributed to mucosal elaboration of prostaglandins or stimulation of their synthesis. These studies are the first to demonstrate that neutrophilic inflammation arising in response to infection by a noninvasive epithelial pathogen results in physiologic rather than pathological effects in vivo. PMID:16988224

  17. 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. PMID:25687523

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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....

  1. 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...

  2. 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...

  3. A Unique Hexokinase in Cryptosporidium parvum, an Apicomplexan Pathogen Lacking the Krebs Cycle and Oxidative Phosphorylation

    PubMed Central

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

    2014-01-01

    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 (IC50 = 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. PMID:25216472

  4. 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

  5. [The impact of nematode invasions on the pattern of Cryptosporidium parvum infection in wild rodents].

    PubMed

    Kuliś-Małkowska, Karolina

    2007-01-01

    Fragmentation of the environment by natural barriers (lakes, mountain ranges) and human activities (towns, major roads, agriculture) can lead to isolated subpopulations of hosts. The study was carried out in Mazury Lake District in North-East of Poland, the region rich in forests, lakes, rivers and canals, which are able to create passable such barriers. Population of bank voles (Myodes glareolus) and yellow-necked mice (Apodemus flavicollis)--dominant woodland rodents--showed local differences in helminth communities in fragmented forest habitat. The sites were chosen on the basis of the similarity of their habitat structure and type, and isolation from one another. The impact of nematode (Heligmosomoididae) infections on co-occurrence and dynamic of Cryptosporidium parvum infection was studied in both rodent species Myodes glareolus (n=781) and Apodemusflavicollis (n=302) from three different habitats. Presented results clearly revealed that natural nematode invasion could facilitate the presence of chronic infections of Cryptosporidium parvum in wild rodent populations. Also, the intrinsic (host sex and year) as well as extrinsic (season and year of study) factors have obvious effect on dynamics of infections with both groups of parasites. However, there are also some evidences that steroids hormones associated with stress and reproduction may mediate trade-offs between physiology and immune function and can affect co-occurrence of both groups of parasites. PMID:18075159

  6. 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

  7. Role of Immunoglobulin A Monoclonal Antibodies against P23 in Controlling Murine Cryptosporidium parvum Infection

    PubMed Central

    Enriquez, F. Javier; Riggs, Michael W.

    1998-01-01

    Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvum infection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer’s patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvum oocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvum infection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72.7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murine C. parvum

  8. 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

  9. 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%).

  10. Fate of Cryptosporidium parvum oocysts within soil, water, and plant environment.

    PubMed

    McLaughlin, Stephen J; Kalita, Prasanta K; Kuhlenschmidt, Mark S

    2013-12-15

    Vegetative Filter Strips (VFS) have long been used to control the movement of agricultural nutrients and prevent them from reaching receiving waters. Earlier studies have shown that VFS also dramatically reduce both the kinetics and extent of Cryptosporidium parvum (C. parvum) oocysts overland transport. In this study, we investigated possible mechanisms responsible for the ability of VFS to reduce oocyst overland transport. Measurement of the kinetics of C. parvum adhesion to individual sand, silt, and clay soil particles revealed that oocysts associate over time, albeit relatively slow, with clay but not silt or sand particles. Measurement of oocyst overland transport kinetics, soil infiltration depth, distance of travel, and adhesion to vegetation on bare and vegetated soil surfaces indicate that oocysts move more slowly, and penetrate the soil profile to a greater extent on a vegetated surface than on a bare soil surface. Furthermore, we demonstrate a small fraction of the oocysts become attached to vegetation at the soil-vegetation interface on VFS. These results suggest VFS function to reduce oocyst overland transport by primarily decreasing oocyst surface flow enough to allow penetration within the soil profile followed by subsequent adhesion to or entrapment within clay particle aggregates, and to a lesser extent, adhesion to the surface vegetation. PMID:24157412

  11. 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. PMID:27627637

  12. Cryptosporidium parvum infections in a cohort of veterinary students in Sweden.

    PubMed

    Kinross, P; Beser, J; Troell, K; Axén, C; Silverlås, C; Björkman, C; Lebbad, M; Winiecka-Krusnell, J; Lindh, J; Löfdahl, M

    2015-10-01

    In March 2013, a veterinary student tested positive for Cryptosporidium; four classmates reported similar gastrointestinal symptoms. We aimed to identify source(s) and risk factors for Cryptosporidium infection in university persons symptomatic between 21 January and 14 April 2013. Sixty-four (79%) students from a cohort of 81 fourth-year veterinary students completed questionnaires, identifying 13 cases; four were Cryptosporidium parvum GP60 subtype IIaA16G1R1b, two were IIdA24G1, seven did not submit stool samples. Thirteen cases attended the university's field clinic before symptom onset (13/37 attendees, 35%); 11 visited at least one of four farms where students recalled seeing calves with diarrhoea. C. parvum subtype IIaA16G1R1b was identified in calves at one of the farms. Entering pens of calves with diarrhoea [relative risk (RR) 7·6, 95% confidence interval (CI) 1·7-33·5] and eating in clinic cars (RR 9·1, 95% CI 1·3-65·8) were associated with being a case. Washing hands at least twice per farm visit (0 cases, P = 0·03) was protective. This outbreak investigation was notable for rapid and effective collaboration between public health, veterinary and environmental sectors, leading to swift identification of a microbiological and epidemiological link between cases, infected calves and their farms. We recommend frequent hand-washing using proper technique and dissuasion from eating in clinic cars to minimize possible exposure to contaminated surfaces. PMID:25633822

  13. 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

  14. Cryptosporidium parvum induces an endoplasmic stress response in the intestinal adenocarcinoma HCT-8 cell line.

    PubMed

    Morada, Mary; Pendyala, Lakhsmi; Wu, Gang; Merali, Salim; Yarlett, Nigel

    2013-10-18

    Invasion of human intestinal epithelial cells (HCT-8) by Cryptosporidium parvum resulted in a rapid induction of host cell spermidine/spermine N(1)-acetyltransferase 1 (hSSAT-1) mRNA, causing a 4-fold increase in SSAT-1 enzyme activity after 24 h of infection. In contrast, host cell SSAT-2, spermine oxidase, and acetylpolyamine oxidase (hAPAO) remained unchanged during this period. Intracellular polyamine levels of C. parvum-infected human epithelial cells were determined, and it was found that spermidine remained unchanged and putrescine increased by 2.5-fold after 15 h and then decreased after 24 h, whereas spermine decreased by 3.9-fold after 15 h. Concomitant with these changes, N(1)-acetylspermine and N(1)-acetylspermidine both increased by 115- and 24-fold, respectively. Increased SSAT-1 has previously been shown to be involved in the endoplasmic reticulum (ER) stress response leading to apoptosis. Several stress response proteins were increased in HCT-8 cells infected with C. parvum, including calreticulin, a major calcium-binding chaperone in the ER; GRP78/BiP, a prosurvival ER chaperone; and Nrf2, a transcription factor that binds to antioxidant response elements, thus activating them. However, poly(ADP-ribose) polymerase, a protein involved in DNA repair and programmed cell death, was decreased. Cumulatively, these results suggest that the invasion of HCT-8 cells by C. parvum results in an ER stress response by the host cell that culminates in overexpression of host cell SSAT-1 and elevated N(1)-acetylpolyamines, which can be used by a parasite that lacks ornithine decarboxylase. PMID:23986438

  15. Effect of organic acids and hydrogen peroxide on Cryptosporidium parvum viability in fruit juices.

    PubMed

    Kniel, Kalmia E; Sumner, Susan S; Lindsay, David S; Hackney, Cameron R; Pierson, Merle D; Zajac, Anne M; Golden, David A; Fayer, Ronald

    2003-09-01

    Cryptosporidium parvum has historically been associated with waterborne outbreaks of diarrheal illness. Foodborne cryptosporidiosis has been associated with unpasteurized apple cider. Infectious oocysts are shed in the feces of common ruminants like cattle and deer in and near orchards. In this study, the ability of organic acids and hydrogen peroxide (H2O2) added to fruit juice to inhibit the survival of C. parvum was analyzed. Oocyst viability was analyzed by a cell culture infectivity assay with the use of a human ileocecal cell line (HCT-8) whose infectivity pattern is similar to that for human oral infectivity. Cell monolayers were infected with 10(6) treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized through immunohistochemistry with 100 microscope fields per monolayer being counted. In vitro excystation assays were also used to evaluate these treatments. Organic acids and H2O2 were added to apple cider, orange juice, and grape juices on a weight/volume basis. Malic, citric, and tartaric acids at concentrations of 1 to 5% inhibited C. parvum's infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025 to 3% H2O2 were evaluated. The addition of 0.025% H2O2 to each juice resulted in a >5-log reduction of C. parvum infectivity as determined with a most-probable-number-based cell culture infectivity assay. As observed with differential interference contrast and scanning electron microscopy, reduced infectivity may be mediated through effects on the oocyst wall that are caused by the action of H2O2 or related oxygen radicals. The addition of low concentrations of H2O2 can represent a valuable alternative to pasteurization. PMID:14503720

  16. 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

  17. 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. PMID:15870337

  18. 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

  19. Overland and near-surface transport of Cryptosporidium parvum from vegetated and nonvegetated surfaces.

    PubMed

    Trask, Jennifer R; Kalita, Prasanta K; Kuhlenschmidt, Mark S; Smith, Ronald D; Funk, Ted L

    2004-01-01

    Understanding microbial pathogen transport patterns in overland flow is important for developing best management practices for limiting microbial transport to water resources. Knowledge about the effectiveness of vegetative filter strips (VFS) to reduce pathogen transport from livestock confinement areas is limited. In this study, overland and near-surface transport of Cryptosporidium parvum has been investigated. Effects of land slopes, vegetation, and rainfall intensities on oocyst transport were examined using a tilting soil chamber with two compartments, one with bare ground and the other with brome (Bromus inermis Leyss.) vegetation. Three slope conditions (1.5, 3.0, and 4.5%) were used in conjunction with two rainfall intensities (25.4 and 63.5 mm/h) for 44 min using a rainfall simulator. The vegetative surface was very effective in reducing C. parvum in surface runoff. For the 25.4 mm/h rainfall, the total percent recovery of oocysts in overland flow from the VFS varied from 0.6 to 1.7%, while those from the bare ground condition varied from 4.4 to 14.5%. For the 63.5 mm/h rainfall, the recovery percentages of oocysts varied from 0.8 to 27.2% from the VFS, and 5.3 to 59% from bare-ground conditions. For all slopes and rainfall intensities, the total (combining both surface and near-surface) recovery of C. parvum oocysts was considerably less from the vegetated surface than those from the bare-ground conditions. These results indicate that the VFS can be a best management practice for controlling C. parvum in runoff from animal production facilities. PMID:15224935

  20. 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

  1. Specific serum and local antibody responses against Cryptosporidium parvum during medication of calves with halofuginone lactate.

    PubMed Central

    Peeters, J E; Villacorta, I; Naciri, M; Vanopdenbosch, E

    1993-01-01

    Fecal and serum anti-Cryptosporidium parvum immunoglobulin A (IgA), IgM, and IgG were monitored by an enzyme immunoassay in C. parvum-infected calves after medication with halofuginone lactate. In a first experiment, four groups of five 1-day-old colostrum-fed calves were inoculated with 10(6) oocysts of C. parvum. They were medicated with 0, 30, 60, or 120 micrograms of halofuginone lactate per kg from days 2 to 8 postinfection (p.i.). Unmedicated calves passed large numbers of oocysts between 3 and 14 days p.i. Treatment with 30 micrograms/kg did not completely inhibit oocyst output during medication, whereas 60 and 120 micrograms/kg did. The latter groups passed only a reduced number of oocysts when the drug was withdrawn. In a second experiment, 3- to 6-day-old colostrum-fed calves were divided into three groups of 16 or 17 animals each. All animals had acquired C. parvum infection before arrival at the fattening unit. They were medicated with 0, 60, or 120 micrograms/kg for 7 days beginning on the day of arrival. Unmedicated calves passed large numbers of oocysts from 0 to 21 days. Medication stopped oocyst output at day 7, but some of the calves again passed low numbers of oocysts 7 days after withdrawal of the drug. Experimental infection of unmedicated calves was followed by a rise in local anti-C. parvum IgA and IgM titers. Rising coproantibody levels coincided with falling oocyst output. In halofuginone-medicated and experimentally infected calves, only specific anti-C. parvum IgM levels rose during the first 5 days p.i. Specific IgA levels increased in association with oocyst output after withdrawal of the drug in the 60- and 120-micrograms/kg groups. In naturally infected calves, on the other hand, both specific IgA and IgM levels rose further during medication. Although titers were lower than in unmedicated controls, no significant differences were observed. Both medicated and unmedicated calves were equally protected from a challenge with 10

  2. 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

  3. COMPARISON OF IMMUNOFLUORESCENT ANTIBODY ASSAY (IFA) AND IMMUNOMAGNETIC ELECTROCHEMILUMINESCENCE (IM-ECL) IN DETECTION OF CRYPTOSPORIDIUM PARVUM IN KARST WATER SAMPLES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunofluorescent antibody assay (IFA) and immunomagnetic electrochemiluminescence (IM-ECL) were used for comparison of the percent of recovery of Cryptosporidium parvum in environmental water samples obtained from a spring draining a karst basin. The monoclonal antibodies to C. parvum, isotype IgG3...

  4. Prevalence of Cryptosporidium parvum infections in weaned piglets and fattening porkers in Kanagawa Prefecture, Japan.

    PubMed

    Izumiyama, S; Furukawa, I; Kuroki, T; Yamai, S; Sugiyama, H; Yagita, K; Endo, T

    2001-02-01

    Fecal samples from 232 weaned piglets (1 and 3 months old) and 252 fattening porkers (6 months old) in 8 stock-raising farms located in Kanagawa Prefecture, Japan, from June 1998 to June 2000 were examined to determine the prevalence of Cryptosporidium infection. Detection of oocysts was performed using the ethyl acetate fecal concentration method and immunofluorescent staining. C. parvum oocysts were identified in 77 (33.2%) 1-3 months old weaned piglets from four farms. The odds of excreting among 1-3 months old piglets were more than 100 times greater than among 6 months old porkers (95% confidence interval: 17-902). This strongly suggests that weaned piglets are important reservoirs of pathogenic microbes whose potential contamination of drinking water has epidemiological implications for human health. PMID:11326125

  5. In vitro effect on Cryptosporidium parvum of short-term exposure to cathelicidin peptides.

    PubMed

    Giacometti, Andrea; Cirioni, Oscar; Del Prete, Maria Simona; Skerlavaj, Barbara; Circo, Raffaella; Zanetti, Margherita; Scalise, Giorgio

    2003-04-01

    Two laboratory methods, a cell culture system and double fluorogenic staining, were used to study the viability and infective ability of Cryptosporidium parvum sporozoites and oocysts after short-term exposure to four cathelicidin peptides. The compounds, SMAP-29, BMAP-28, PG-1 and Bac7(1-35), exerted a strong cytotoxic effect on sporozoites, but did not affect the viability and function of oocysts consistently. Overall, in the sporozoite series, a percentage of the viable population decreased rapidly to less than detectable levels after 15 and 60 min exposure to the peptides at concentrations of 100 and 10 micro g/mL, respectively. In the oocyst series, no compound produced complete inhibition of parasite growth: 60-85% of the oocyst population was viable after 180 min exposure at 100 micro g/mL. SMAP-29 exerted the highest activity against both sporozoites and oocysts. PMID:12654759

  6. Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall.

    PubMed

    Chatterjee, Anirban; Banerjee, Sulagna; Steffen, Martin; O'Connor, Roberta M; Ward, Honorine D; Robbins, Phillips W; Samuelson, John

    2010-01-01

    Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls. PMID:19949049

  7. miR-27b Targets KSRP to Coordinate TLR4-Mediated Epithelial Defense against Cryptosporidium parvum Infection

    PubMed Central

    Zhou, Rui; Gong, Ai-Yu; Eischeid, Alex N.; Chen, Xian-Ming

    2012-01-01

    Cryptosporidium is a protozoan parasite that infects the gastrointestinal epithelium and causes a diarrheal disease. Toll-like receptor (TLR)- and NF-κB-mediated immune responses from epithelial cells, such as production of antimicrobial peptides and generation of reactive nitrogen species, are important components of the host's defense against cryptosporidial infection. Here we report data demonstrating a role for miR-27b in the regulation of TLR4/NF-κB-mediated epithelial anti-Cryptosporidium parvum responses. We found that C. parvum infection induced nitric oxide (NO) production in host epithelial cells in a TLR4/NF-κB-dependent manner, with the involvement of the stabilization of inducible NO synthase (iNOS) mRNA. C. parvum infection of epithelial cells activated NF-κB signaling to increase transcription of the miR-27b gene. Meanwhile, downregulation of KH-type splicing regulatory protein (KSRP) was detected in epithelial cells following C. parvum infection. Importantly, miR-27b targeted the 3′-untranslated region of KSRP, resulting in translational suppression. C. parvum infection decreased KSRP expression through upregulating miR-27b. Functional manipulation of KSRP or miR-27b caused reciprocal alterations in iNOS mRNA stability in infected cells. Forced expression of KSRP and inhibition of miR-27b resulted in an increased burden of C. parvum infection. Downregulation of KSRP through upregulating miR-27b was also detected in epithelial cells following LPS stimulation. These data suggest that miR-27b targets KSRP and modulates iNOS mRNA stability following C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general. PMID:22615562

  8. 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. PMID:22706058

  9. Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits.

    PubMed

    Takashima, Yasuhiro; Xuan, Xuenan; Kimata, Isao; Iseki, Motohiro; Kodama, Yoshikatsu; Nagane, Noriko; Nagasawa, Hideyuki; Matsumoto, Yasunobu; Mikami, Takeshi; Otsuka, Haruki

    2003-04-01

    In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits. PMID:12760641

  10. Murine infection model for maintenance and amplification of Cryptosporidium parvum oocysts.

    PubMed Central

    Petry, F; Robinson, H A; McDonald, V

    1995-01-01

    Propagation of Cryptosporidium parvum is problematic because in vitro development of the parasite is poor and animals are only briefly susceptible as neonates. At present oocysts of the parasite are usually procured by passage in neonatal sheep or cattle. In the present study, large numbers of oocysts of C. parvum could be isolated following infection of dexamethasone-treated adult C57BL/6 mice. The amount of immunosuppressive drug and the regimen of administration were critical for successful maintenance of the parasite, however. Routinely, 10 mice (age, 8 to 12 weeks) were injected four times on alternate days with 1.0 mg of dexamethasone, and the last injection was given on the same day as oral inoculation with 10(6) oocysts. By using a simplified procedure for oocyst purification from mouse feces, approximately 10(9) oocysts were obtained. This model is inexpensive and comparatively safe to handle, and the numbers of animals inoculated can be varied to obtain the required number of oocysts. Thus, this murine infection model would be a suitable alternative to the use of neonatal calves or sheep for efficient oocyst propagation. PMID:7665672

  11. Cryptosporidium parvum induces SIRT1 expression in host epithelial cells through downregulating let-7i

    PubMed Central

    Xie, Hongguan; Lei, Ningfei; Gong, Ai-Yu; Chen, Xian-Ming; Hu, Guoku

    2015-01-01

    Epithelial cells along human gastrointestinal mucosal surface express pathogen-recognizing receptors and actively participate in the regulation of inflammatory reactions in response to microbial infection. The NAD-dependent deacetylase sirtuin-1 (SIRT1), one member of the sirtuin family of proteins and an NAD-dependent deacetylase has been implicated in the regulation of multiple cellular processes, including inflammation, longevity, and metabolism. In this study, we demonstrated that infection of cultured human biliary epithelial cells (H69 cholangiocytes) with a parasitic protozoan, Cryptosporidium parvum, induced SIRT1 expression at the protein level without a change in SIRT1 mRNA content. Using real-time PCR and Northern blot analyses, we found that C. parvum infection decreased the expression of let-7i in infected H69 cells. Down-regulation of let-7i caused relief of miRNA-mediated translational suppression of SIRT1 and consequently, resulted in an increased SIRT1 protein level in infected H69 cell cultures. Moreover, gain- and loss-of-function studies revealed that let-7i could modulate NF-κB activation through modification of SIRT1 protein expression. Thus, our data suggest that let-7i regulates SIRT1 expression in human biliary epithelial cells in response to microbial challenge, suggesting a new role of let-7i in the regulation of NF-κB-mediated epithelial innate immune response. PMID:24862934

  12. Simplified methods for obtaining purified oocysts from mice and for growing Cryptosporidium parvum in vitro.

    PubMed

    Meloni, B P; Thompson, R C

    1996-10-01

    Seven- to 8-day-old Arc/Swiss mice were infected with 100,000-120,000 Cryptosporidium parvum oocysts. At 8 days postinfection (PI) the jejunum, ileum, cecum, colon, and rectum were removed. Using a simple extraction procedure and purification by Ficoll gradient centrifugation, we rountinely obtained between 3-6 million and up to 15 million purified oocysts per mouse. For in vitro cultivation, purified oocysts were pretreated in a low pH (2.5-3) 0.5% trypsin solution for 20 min, resuspended in supplemented RPMI-1640 containing glucose 0.1 g (5.55 mM), sodium bicarbonate 0.3 g, bovine bile 0.02 g, folic acid 25 micrograms, 4-aminobenzoic acid 100 micrograms, calcium pantothenate 50 micrograms, ascorbic acid 875 micrograms, penicillin G 10,000 U and streptomycin 0.01 g per 100 ml, and 1% fetal bovine serum (pH 7.4 before filtration), and used to inoculate confluent monolayers of the human adenocarcinoma cell line HCT-8. Incubation was in a candle jar at 37 C. We tested numerous supplements to RPMI-1640, different pHs, and atmospheric conditions and found the parameters described above produced the greatest parasite numbers in vitro. We obtained significantly superior growth of C. parvum grown in HCT-8 cells using the conditions described above than in culture conditions described previously. PMID:8885885

  13. Recovery of Waterborne Cryptosporidium parvum Oocysts by Freshwater Benthic Clams (Corbicula fluminea)

    PubMed Central

    Graczyk, Thaddeus K.; Fayer, Ronald; Cranfield, Michael R.; Conn, David Bruce

    1998-01-01

    Asian freshwater clams, Corbicula fluminea, exposed for 24 h to 38 liters of water contaminated with infectious Cryptosporidium parvum oocysts (1.00 × 106 oocysts/liter; approximately 1.9 × 105 oocysts/clam) were examined (hemolymph, gills, gastrointestinal [GI] tract, and feces) on days 1, 2, 3, 7, and 14 postexposure (PE). No oocysts were detected in the water 24 h after the contamination event. The percentage of oocyst-containing clams varied from 20 to 100%, depending on the type of tissue examined and the technique used—acid-fast stain (AFS) or immunofluorescent antibody (IFA). The oocysts were found in clam tissues and feces on days 1 through 14 PE; the oocysts extracted from the tissues on day 7 PE were infectious for neonatal BALB/c mice. Overall, the highest number of positive samples was obtained when gills and GI tracts were processed with IFA (prevalence, 97.5%). A comparison of the relative oocyst numbers indicated that overall, 58.3% of the oocysts were found in clam tissues and 41.7% were found in feces when IFA was used; when AFS was used, the values were 51.9 and 48.1%, respectively. Clam-released oocysts were always surrounded by feces; no free oocysts or oocysts disassociated from fecal matter were observed. The results indicate that these benthic freshwater clams are capable of recovery and sedimentation of waterborne C. parvum oocysts. To optimize the detection of C. parvum oocysts in C. fluminea tissue, it is recommended that gill and GI tract samples be screened with IFA (such as that in the commercially available MERIFLUOR test kit). PMID:9464376

  14. Local Peroxynitrite Formation Contributes to Early Control of Cryptosporidium parvum Infection

    PubMed Central

    Gookin, Jody L.; Allen, Jessica; Chiang, Sophia; Duckett, Laurel; Armstrong, Martha U.

    2005-01-01

    In intestinal inflammation, mucosal injury is often exacerbated by the reaction of NO with neutrophil-derived superoxide to form the potent oxidant peroxynitrite. Peroxynitrite also has antimicrobial properties that aid in the killing mechanism of macrophages and neutrophils. Cryptosporidium parvum parasitizes intestinal epithelium, resulting in loss of epithelial cells and mucosal inflammation. Synthesis of NO is significantly increased and arises from the induced expression of inducible nitric oxide synthase (iNOS) by the infected epithelium. Inhibition of iNOS results in intensified epithelial parasitism and oocyst excretion. We hypothesized that formation of peroxynitrite is restricted to sites of iNOS expression by the epithelium and contributes to host defense in C. parvum infection. Accordingly, the location and biological effects of peroxynitrite formation were examined in neonatal piglets infected with C. parvum. Infected piglets were treated daily with a selective iNOS inhibitor [L-N6-(1-iminoethyl)-lysine] or one of two peroxynitrite scavengers [5,10,15,20-tetrakis(N-methyl-4′-pyridyl)porphyrinato iron(III) or uric acid] or received vehicle. At peak infection, peroxynitrite formation was restricted to sites of iNOS expression by parasitized epithelium and lamina propria of the apical villi. Peroxynitrite formation was dependent on iNOS activity and was inhibited by treatment with peroxynitrite scavengers. Scavengers increased the number of intracellular parasites and the number of infected epithelial cells present per villus and significantly exacerbated oocyst excretion. Recovery from infection was not delayed by ongoing treatment with scavenger. The present results are the first to demonstrate an in vivo role for peroxynitrite formation in acute mucosal defense against a noninvasive intestinal epithelial pathogen. PMID:15972479

  15. Local peroxynitrite formation contributes to early control of Cryptosporidium parvum infection.

    PubMed

    Gookin, Jody L; Allen, Jessica; Chiang, Sophia; Duckett, Laurel; Armstrong, Martha U

    2005-07-01

    In intestinal inflammation, mucosal injury is often exacerbated by the reaction of NO with neutrophil-derived superoxide to form the potent oxidant peroxynitrite. Peroxynitrite also has antimicrobial properties that aid in the killing mechanism of macrophages and neutrophils. Cryptosporidium parvum parasitizes intestinal epithelium, resulting in loss of epithelial cells and mucosal inflammation. Synthesis of NO is significantly increased and arises from the induced expression of inducible nitric oxide synthase (iNOS) by the infected epithelium. Inhibition of iNOS results in intensified epithelial parasitism and oocyst excretion. We hypothesized that formation of peroxynitrite is restricted to sites of iNOS expression by the epithelium and contributes to host defense in C. parvum infection. Accordingly, the location and biological effects of peroxynitrite formation were examined in neonatal piglets infected with C. parvum. Infected piglets were treated daily with a selective iNOS inhibitor [L-N6-(1-iminoethyl)-lysine] or one of two peroxynitrite scavengers [5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron(III) or uric acid] or received vehicle. At peak infection, peroxynitrite formation was restricted to sites of iNOS expression by parasitized epithelium and lamina propria of the apical villi. Peroxynitrite formation was dependent on iNOS activity and was inhibited by treatment with peroxynitrite scavengers. Scavengers increased the number of intracellular parasites and the number of infected epithelial cells present per villus and significantly exacerbated oocyst excretion. Recovery from infection was not delayed by ongoing treatment with scavenger. The present results are the first to demonstrate an in vivo role for peroxynitrite formation in acute mucosal defense against a noninvasive intestinal epithelial pathogen. PMID:15972479

  16. TLR4 PROMOTES CRYPTOSPORIDIUM PARVUM CLEARANCE IN A MOUSE MODEL OF BILIARY CRYPTOSPORIDIOSIS

    PubMed Central

    O’Hara, Steven P.; Tietz Bogert, Pamela S.; Chen, Xianming; LaRusso, Nicholas F.

    2011-01-01

    Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, express multiple toll-like receptors (TLRs) and thus have the capacity to recognize and respond to microbial pathogens. In previous work we demonstrated that TLR4, which is activated by gram-negative lipopolysaccharide (LPS), is upregulated in cholangiocytes in response to infection with Cryptosporidium parvum in vitro and contributes to NFkB activation. Here, using an in vivo model of biliary cryptosporidiosis we addressed the functional role of TLR4 in C. parvum infection dynamics and hepatobiliary pathophysiology. We observed that C57BL mice clear the infection by three weeks post-infection. In contrast, parasites were detected in bile and stool in TLR4 deficient mice at four weeks post-infection. The liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST), and the proinflammatory cytokines Tumor Necrosis Factor (TNF)-α, Interferon (IFN)-γ, and Interleukin (IL)-6 peaked at one to two weeks postinfection and normalized by four weeks in infected C57BL mice. C57BL mice also demonstrated increased cholangiocyte proliferation (PCNA staining) at one-week post-infection, which was resolved by two weeks post-infection. In contrast, TLR4 deficient mice showed persistently elevated serum ALT and AST, elevated hepatic IL-6 levels, and histological evidence of hepatocyte necrosis, increased inflammatory cell infiltration, and cholangiocyte proliferation through four weeks post-infection. These data suggest that a TLR4-mediated response is required for efficient eradication of biliary C. parvum infection in vivo, and lack of this pattern recognition receptor contributes to an altered inflammatory response and an increase in hepatobiliary pathology. PMID:21506806

  17. 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. PMID:11317885

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

    PubMed Central

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

    2011-01-01

    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. PMID:21968447

  19. 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.

  20. 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

  1. 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. PMID:26042636

  2. Effect of tillage and rainfall on transport of manure-applied Cryptosporidium parvum oocysts through soil.

    PubMed

    Ramirez, Norma E; Wang, Ping; Lejeune, Jeff; Shipitalo, Martin J; Ward, Lucy A; Sreevatsan, Srinand; Dick, Warren A

    2009-01-01

    Most waterborne outbreaks of cryptosporidiosis have been attributed to agricultural sources due to the high prevalence of Cryptosporidium oocysts in animal wastes and manure spreading on farmlands. No-till, an effective conservation practice, often results in soil having higher water infiltration and percolation rates than conventional tillage. We treated six undisturbed no-till and six tilled soil blocks (30 by 30 by 30 cm) with 1 L liquid dairy manure containing 10(5) C. parvum oocysts per milliliter to test the effect of tillage and rainfall on oocyst transport. The blocks were subjected to rainfall treatments consisting of 5 mm or 30 mm in 30 min. Leachate was collected from the base of the blocks in 35-mL increments using a 64-cell grid lysimeter. Even before any rain was applied, approximately 300 mL of water from the liquid manure (30% of that applied) was transported through the no-till soil, but none through the tilled blocks. After rain was applied, a greater number and percentage of first leachate samples from the no-till soil blocks compared to the tilled blocks tested positive for Cryptosporidium oocysts. In contrast to leachate, greater numbers of oocysts were recovered from the tilled soil, itself, than from the no-till soil. Although tillage was the most important factor affecting oocyst transport, rainfall timing and intensity were also important. To minimize transport of Cryptosporidium in no-till fields, manure should be applied at least 48 h before heavy rainfall is anticipated or methods of disrupting the direct linkage of surface soil to drains, via macropores, need to be used. PMID:19875795

  3. Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 1: development and optimization of methods.

    PubMed

    Cook, N; Paton, C A; Wilkinson, N; Nichols, R A B; Barker, K; Smith, H V

    2006-06-15

    No standard method is available for detecting protozoan parasites on foods such as soft fruit and salad vegetables. We report on optimizing methods for detecting Cryptosporidium parvum on lettuce and raspberries. These methods are based on four basic stages: extraction of oocysts from the foodstuffs, concentration of the extract and separation of the oocysts from food materials, staining of the oocysts to allow their visualization, and identification of oocysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Oocyst staining is also performed using proprietary reagents. The performance parameters of the extraction steps were extensively optimized, using artificially contaminated samples. The fully developed methods were tested several times to determine their reliability. The method to detect C. parvum on lettuce recovered 59.0+/-12.0% (n=30) of artificially contaminated oocysts. The method to detect C. parvum on raspberries recovered 41.0+/-13.0% (n=30) of artificially contaminated oocysts. PMID:16529835

  4. 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

  5. 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. PMID:16292678

  6. 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. PMID:27098916

  7. Efficacy of Nitazoxanide against Cryptosporidium parvum in Cell Culture and in Animal Models

    PubMed Central

    Theodos, Cynthia M.; Griffiths, Jeffrey K.; D’Onfro, Jennifer; Fairfield, Alexandra; Tzipori, Saul

    1998-01-01

    Nitazoxanide (NTZ), a drug currently being tested in human clinical trials for efficacy against chronic cryptosporidiosis, was assessed in cell culture and in two animal models. The inhibitory activity of NTZ was compared with that of paromomycin (PRM), a drug that is partially effective against Cryptosporidium parvum. A concentration of 10 μg of NTZ/ml (32 μM) consistently reduced parasite growth in cell culture by more than 90% with little evidence of drug-associated cytotoxicity, in contrast to an 80% reduction produced by PRM at 2,000 μg/ml (3.2 mM). In contrast to its efficacy in vitro, NTZ at either 100 or 200 mg/kg of body weight/day for 10 days was ineffective at reducing the parasite burden in C. parvum-infected, anti-gamma-interferon-conditioned SCID mice. Combined treatment with NTZ and PRM was no more effective than treatment with PRM alone. Finally, NTZ was partially effective at reducing the parasite burden in a gnotobiotic piglet diarrhea model when given orally for 11 days at 250 mg/kg/day but not at 125 mg/kg/day. However, the higher dose of NTZ induced a drug-related diarrhea in piglets that might have influenced its therapeutic efficacy. As we have previously reported, PRM was effective at markedly reducing the parasite burden in piglets at a dosage of 500 mg/kg/day. Our results indicate that of all of the models tested, the piglet diarrhea model most closely mimics the partial response to NTZ treatment reported to occur in patients with chronic cryptosporidiosis. PMID:9687390

  8. Effects of select medium supplements on in vitro development of Cryptosporidium parvum in HCT-8 cells.

    PubMed

    Upton, S J; Tilley, M; Brillhart, D B

    1995-02-01

    Surface-sterilized oocysts of Cryptosporidium parvum were applied to subconfluent monolayers of human adenocarcinoma (HCT-8) cells grown on coverslips in six-well cluster plates. Parasite-infected cultures were then incubated in RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, and antibiotics at 37 degrees C in a 5% CO2-95% air incubator for 2 h to allow sporozoites to excyst and enter cells. After cultures were washed free of debris, fresh cell culture media containing select supplements were added and cultures were reincubated. Parasite growth was assessed 66 h later by counting the number of parasite developmental stages in 25 random x 100 oil fields by Nomarski interference-contrast microscopy. Four vitamin supplements, calcium pantothenate, L-ascorbic acid, folic acid, and 4-(para)-aminobenzoic acid, each resulted in a significant increase in parasite numbers in vitro. The addition of insulin and the sugars glucose, galactose, and maltose also had a positive effect on parasite growth, although the effect was less pronounced than with any of the vitamins. Using the above information, we developed a supplemental medium formulation consisting of RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES, 50 mM glucose, and 35 micrograms of ascorbic acid, 1.0 micrograms of folic acid, 4.0 micrograms of 4-aminobenzoic acid, 2.0 micrograms of calcium pantothenate, 0.1 U of insulin, 100 U of penicillin G, 100 micrograms of streptomycin, and 0.25 microgram of amphotericin B (Fungizone) per ml (pH 7.4). The growth of c. parvum in this medium was found to be enhanced approximately 10-fold compared with that in control medium without additional glucose, insulin, or vitamins. PMID:7714194

  9. 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.

  10. Genotyping and subtyping Cryptosporidium parvum and Giardia duodenalis carried by flies on dairy farms in Henan, China

    PubMed Central

    2014-01-01

    Background Cryptosporidium and Giardia are important causes of diarrhea diseases in humans and animals worldwide, and both of them are transmitted by the fecal–oral route, either by direct contact or by the ingestion of contaminated food or water. The role of flies in the mechanical transmission of Cryptosporidium and Giardia has been receiving increasing attention. To date, no information is available in China about the occurrence of Cryptosporidium and Giardia in flies. We here investigated Cryptosporidium and Giardia in flies on dairy farms in Henan Province, China, at the genotype and subtype levels. Methods Eight hundred flies were randomly collected from two dairy farms from July 2010 to September 2010 and were divided evenly into 40 batches. The fly samples were screened for the presence of Cryptosporidium and Giardia with nested PCR. Cryptosporidium was genotyped and subtyped by analyzing the DNA sequences of small subunit rRNA (SSU rRNA) and 60-kDa glycoprotein (gp60) genes, respectively. The identity of Giardia was determined by sequence analyzing of the triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and β-giardin (bg) genes. Results Forty batches of flies had 10% of contamination with Cryptosporidium or Giardia, with a mixed infection of the two parasites in one batch of flies. The Cryptosporidium isolates were identified as C. parvum at the SSU rRNA locus, and all belonged to subtype IIdA19G1 at the gp60 locus. The Giardia isolates were all identified as assemblage E of G. duodenalis at the tpi, gdh, and bg loci. One novel subtype of assemblage E was identified based on the gdh and bg loci. Conclusions This is the first molecular study of Cryptosporidium and Giardia in flies identified at both genotype and subtype levels. SSU rRNA and gp60 sequences of C. parvum in flies was 100% homologous with those derived from humans, suggesting flies act as an epidemiological vector of zoonotic cryptosporidiosis. The variable PCR efficiencies

  11. Differential Gene Expression and Protein Localization of Cryptosporidium parvum Fatty Acyl-CoA Synthetase Isoforms.

    PubMed

    Guo, Fengguang; Zhang, Haili; Payne, Harold Ross; Zhu, Guan

    2016-03-01

    Cryptosporidium parvum is unable to synthesize fatty acids de novo, but possesses three long-chain fatty acyl-CoA synthetase (CpACS) isoforms for activating fatty acids. We have recently shown that these enzymes could be targeted to kill the parasite in vitro and in vivo. Here, we demonstrated that the CpACS genes were differentially expressed during the parasite life cycle, and their proteins were localized to different subcellular structures by immunofluorescence and immuno-electron microscopies. Among them, CpACS1 displayed as an apical protein in sporozoites and merozoites, but no or little presence during the intracellular merogony until the release of merozoites, suggesting that CpACS1 probably functioned mainly during the parasite invasion and/or early stage of intracellular development. Both CpACS2 and CpACS3 proteins were present in all parasite life cycle stages, in which CpACS2 was present in the parasite and the parasitophorous vacuole membranes (PVM), whereas CpACS3 was mainly present in the parasite plasma membranes with little presence in the PVM. These observations suggest that CpACS2 and CpACS3 may participate in scavenging and transport of fatty acids across the PVM and the parasite cytoplasmic membranes, respectively. PMID:26411755

  12. Comparison of oocyst shedding and the serum immune response to Cryptosporidium parvum in cattle and pigs.

    PubMed

    Quílez, J; Ares-Mazás, E; Sánchez-Acedo, C; del Cacho, E; Clavel, A; Causapé, A C

    1996-01-01

    A comparison was made between oocyst shedding and the presence of specific serum IgG antibodies to Cryptosporidium parvum in 108 bovines and 90 pigs. Oocysts were detected by a commercial immunofluorescence assay in feces from 26.8% of bovines and 34.4% of pigs, whereas positive titers as determined by an indirect fluorescent antibody method were found in sera from 12.9% and 48.9% of the respective animals. Infection was significantly most frequent in suckling calves (82.7%) and weaned piglets (87.5%). By contrast, the numbers of seropositives were highest in weaned calves (17.1%) and fattening pigs (76.6%). The results of coprological and serological analysis corresponded in 65.7% of bovines and 56.7% of pigs. When used to diagnose the shedding of cryptosporidial oocysts, the detection of specific IgG antibodies had a sensitivity ranging from 10.3% (cattle) to 58.1% (pigs) and a specificity of 86.1% (cattle) and 55.9% (pigs). PMID:8832734

  13. Seasonal Retention and Release of Cryptosporidium parvum Oocysts by Environmental Biofilms in the Laboratory ▿

    PubMed Central

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

    2010-01-01

    Cryptosporidium is a genus of waterborne protozoan parasites that causes significant gastrointestinal disease in humans. These parasites can accumulate in environmental biofilms and be subsequently released to contaminate water supplies. Natural microbial assemblages were collected each season from an eastern Pennsylvania stream and used to grow biofilms in laboratory microcosms in which influx, efflux, and biofilm retention were determined from daily oocyst counts. For each seasonal biofilm, oocysts attached to the biofilm quickly during oocyst dosing. Upon termination of oocyst dosing, the percentage of oocysts retained within the biofilm decreased to a new steady state within 5 days. Seasonal differences in biofilm retention of oocysts were observed. The spring biofilm retained the greatest percentage of oocysts, followed (in decreasing order) by the winter, summer, and fall biofilms. There was no statistically significant correlation between the percentage of oocysts attached to the biofilm and (i) any measured stream water quality parameter (including temperature, pH, conductivity, and dissolved organic carbon concentration) or (ii) experimental temperature. Seasonal differences in oocyst retention persisted when biofilms were tested with stream water from a different season. These data suggest that seasonal variation in the microbial community and resulting biofilm architecture may be more important to oocyst transport in this stream site than water quality. The biofilm attachment and detachment dynamics of C. parvum oocysts have implications for public health, and the drinking water industry should recognize that the potential exists for pathogen-free water to become contaminated during the distribution process as a result of biofilm dynamics. PMID:20023100

  14. 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.

  15. 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. PMID:15384722

  16. 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. PMID:17873076

  17. 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. PMID:26587578

  18. 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.

  19. 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

  20. 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

    2015-01-01

    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

  1. 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

  2. Survival of Infectious Cryptosporidium parvum Oocysts in Seawater and Eastern Oysters (Crassostrea virginica) in the Chesapeake Bay

    PubMed Central

    Fayer, Ronald; Graczyk, Thaddeus K.; Lewis, Earl J.; Trout, James M.; Farley, C. Austin

    1998-01-01

    Oocysts of Cryptosporidium parvum placed in artificial seawater at salinities of 10, 20, and 30 ppt at 10°C and at 10 ppt at 20°C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20°C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters, Crassostrea virginica, were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of Cryptosporidium oocysts and Giardia cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. Giardia was not detected by this method from any of the 360 oysters examined. Presumptive identification of Cryptosporidium oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of C. parvum. These findings demonstrate for the first time that oysters in natural waters harbor infectious C. parvum oocysts and can serve as mechanical vectors of this pathogen. PMID:9501446

  3. Evaluation and optimization of a reusable hollow fiber ultrafilter as a first step in concentrating Cryptosporidium parvum oocysts from water.

    PubMed

    Kuhn, R C; Oshima, K H

    2001-08-01

    Experiments with a small-scale hollow fiber ultrafiltration system (50,000 MWCO) was used to characterize the filtration process and identify conditions that optimize the recovery of Cryptosporidium parvum oocysts from 2 L samples of water. Seeded experiments were conducted using deionized water as well as four environmental water sources (tap, ground, Arkansas river, and Rio Grande river; 0-30.9NTU). Optimal and consistent recovery of spiked oocysts was observed (68-81%), when the membrane was sanitized with a 10% sodium dodecyl sulfate (SDS) solution and then blocked with 5% fetal bovine serum (FBS). PMID:11456179

  4. Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1

    PubMed Central

    Zhang, Zhongsheng; Ojo, Kayode K.; Johnson, Steven M.; Larson, Eric T.; He, Penqing; Geiger, Jennifer A.; Castellanos-Gonzalez, Alejandro; White, A. Clinton; Parsons, Marilyn; Merritt, Ethan A.; Maly, Dustin J.; Verlinde, Christophe L. M. J.; Van Voorhis, Wesley C.; Fan, Erkang

    2012-01-01

    Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50 nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues. PMID:22795629

  5. Comparison of In Vitro Cell Culture and a Mouse Assay for Measuring Infectivity of Cryptosporidium parvum

    PubMed Central

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

    2002-01-01

    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

  6. Efficacy of gaseous chlorine dioxide as a sanitizer against Cryptosporidium parvum, Cyclospora cayetanensis, and Encephalitozoon intestinalis on produce.

    PubMed

    Ortega, Ynes R; Mann, Amy; Torres, Maria P; Cama, Vitaliano

    2008-12-01

    The efficacy of gaseous chlorine dioxide to reduce parasite and bacterial burden in produce was studied. Basil and lettuce leaves were inoculated with Cryptosporidium parvum and Cyclospora cayetanensis oocysts, Encephalitozoon intestinalis spores, and a cocktail of two isolates of nalidixic acid-resistant Escherichia coli O157:H7. The inoculated samples were then treated for 20 min with gaseous chlorine dioxide at 4.1 mg/liter. Cryptosporidium had a 2.6 and 3.31 most-probable-number log reduction in basil and lettuce, respectively. Reduction of Encephalitozoon in basil and lettuce was 3.58 and 4.58 CFU/g respectively. E. coli loads were significantly reduced (2.45 to 3.97 log), whereas Cyclospora sporulation was not affected by this treatment. PMID:19244892

  7. Efficacy of levulinic acid-sodium dodecyl sulfate against Encephalitozoon intestinalis, Escherichia coli O157:H7, and Cryptosporidium parvum.

    PubMed

    Ortega, Ynes R; Torres, Maria P; Tatum, Jessica M

    2011-01-01

    Foodborne parasites are characterized as being highly resistant to sanitizers used by the food industry. In 2009, a study reported the effectiveness of levulinic acid in combination with sodium dodecyl sulfate (SDS) in killing foodborne bacteria. Because of their innocuous properties, we studied the effects of levulinic acid and SDS at various concentrations appropriate for use in foods, on the viability of Cryptosporidium parvum and Encephalitozoon intestinalis. The viability of Cryptosporidium and E. intestinalis was determined by in vitro cultivation using the HCT-8 and RK-13 cell lines, respectively. Two Escherichia coli O157:H7 isolates were also used in the present study: strain 932 (a human isolate from a 1992 Oregon meat outbreak) and strain E 0018 (isolated from calf feces). Different concentrations and combinations of levulinic acid and SDS were tested for their ability to reduce infectivity of C. parvum oocysts (10(5)), E. intestinalis spores (10(6)), and E. coli O157:H7 (10(7)/ml) when in suspension. Microsporidian spores were treated for 30 and 60 min at 20 ± 2°C. None of the combinations of levulinic acid and SDS were effective at inactivating the spores or oocysts. When Cryptosporidium oocysts were treated with higher concentrations (3% levulinic acid-2% SDS and 2% levulinic acid-1% SDS) for 30, 60, and 120 min, viability was unaffected. E. coli O157:H7, used as a control, was highly sensitive to the various concentrations and exposure times tested. SDS and levulinic acid alone had very limited effect on E. coli O157:H7 viability, but in combination they were highly effective at 30 and 60 min of incubation. In conclusion, Cryptosporidium and microsporidia are not inactivated when treated for various periods of time with 2% levulinic acid-1% SDS or 3% levulinic acid-2% SDS at 20°C, suggesting that this novel sanitizer cannot be used to eliminate parasitic contaminants in foods. PMID:21219777

  8. 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

    2014-02-01

    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. PMID:24334090

  9. Efficacy of wash solutions in recovering Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from basil.

    PubMed

    Chandra, Venessa; Torres, Maria; Ortega, Ynés R

    2014-08-01

    Parasitic diseases can be acquired by ingestion of contaminated raw or minimally processed fresh produce (herbs and fruits). The sensitivity of methods used to detect parasites on fresh produce depends in part on the efficacy of wash solutions in removing them from suspect samples. In this study, six wash solutions (sterile E-Pure water, 3% levulinic acid-3% sodium dodecyl sulfate, 1 M glycine, 0.1 M phosphate-buffered saline, 0.1% Alconox, and 1% HCl-pepsin) were evaluated for their effectiveness in removing Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from basil. One hundred or 1,000 oocysts of these parasites were inoculated onto the adaxial surfaces of 25 g of basil leaves, placed in stomacher bags, and stored for 1 h at 21°C or 24 h at 4°C. Leaves were hand washed in each wash solution for 1 min. DNA was extracted from the wash solutions and amplified using PCR for the detection of all parasites. Oocysts inoculated at a concentration of 1,000 oocysts per 25 g of basil were detected in all wash solutions. At an inoculum concentration of 100 oocysts per 25 g, oocysts were detected in 18.5 to 92.6% of the wash solutions. The lowest variability in recovering oocysts from basil inoculated with 100 oocysts was observed in 1% HCl-pepsin wash solution. Oocyst recovery rates were higher at 1 h than at 24 h postinoculation. Unlike most bacteria, parasites cannot be enriched; therefore, an optimal recovery process for oocysts from suspected foods is critical. The observations in this study provide guidance concerning the selection of wash solutions giving the highest retrieval of parasite oocysts. PMID:25198596

  10. Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 2: validation.

    PubMed

    Cook, N; Paton, C A; Wilkinson, N; Nichols, R A B; Barker, K; Smith, H V

    2006-06-15

    We report the results of interlaboratory collaborative trials of methods to detect oocysts of the protozoan parasite Cryptosporidium parvum on lettuce and raspberries. The trials involved eight expert laboratories in the United Kingdom. Samples comprised 30 g lettuce, and 60 g raspberries. Lettuce samples were artificially contaminated at three levels: low (8.5-14.2 oocysts), medium (53.5-62.6 oocysts), and high (111.3-135.0 oocysts). Non-contaminated lettuce samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated lettuce samples) of 89.6%, and a specificity (correct identification of non-contaminated samples) of 85.4%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 30.4%. The method was just as reproducible between laboratories, as repeatable within a laboratory. Raspberry samples were artificially contaminated at three levels: low (8.5-26.8 oocysts), medium (29.7-65.7 oocysts), and high (53.9-131.3 oocysts). Non-contaminated raspberry samples were also tested. The method had an overall sensitivity (correct identification of all artificially contaminated raspberry samples) of 95.8%, and a specificity (correct identification of non-contaminated samples) of 83.3%. The total median percentage recovery (from all artificially contaminated samples) produced by the method was 44.3%. The method was just as reproducible between laboratories, as repeatable within a laboratory. The results of the collaborative trial indicate that these assays can be used effectively in analytical microbiological laboratories. PMID:16546283

  11. Detection of Giardia lamblia and Cryptosporidium parvum Antigens in Human Fecal Specimens Using the ColorPAC Combination Rapid Solid-Phase Qualitative Immunochromatographic Assay

    PubMed Central

    Garcia, Lynne S.; Shimizu, Robyn Y.

    2000-01-01

    The ColorPAC Giardia/Cryptosporidium (Becton Dickinson) is a solid-phase qualitative immunochromatographic assay that detects and distinguishes between Giardia lamblia and Cryptosporidium parvum in human stool. Agreement between the Alexon-Trend ProSpecT Giardia Rapid EIA and the ColorPAC assay was 166 of 172 (96.5%). Agreement between the Alexon-Trend ProSpecT Cryptosporidium Rapid EIA and the ColorPAC assay was 169 of 171 (98.8%). No cross-reactions were seen with other parasites or human cells. PMID:10699038

  12. 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. PMID:25888174

  13. 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

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

    PubMed

    Ludington, Jacob G; Ward, Honorine D

    2016-05-01

    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 Ca(2+)-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 Ca(2+)-dependent binding to sulfated proteoglycans on intestinal epithelial cells. PMID:26975991

  15. Activity of DL-alpha-Difluoromethylarginine and Polyamine Analogues against Cryptosporidium parvum Infection in a T-Cell Receptor Alpha-Deficient Mouse Model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The in vivo effectiveness of a series of conformationally restricted polyamine analogs alone and in combination with DL-alpha-difluoromethylarginine (DFMA) towards a T-cell receptor-alpha deficient mouse model infection of Cryptosporidium parvum was tested. Polyamine analogues were constructed from ...

  16. Evaluation of recombinant P23 protein as a vaccine for passive immunization of newborn calves against Cryptosporidium parvum.

    PubMed

    Askari, N; Shayan, P; Mokhber-Dezfouli, M R; Ebrahimzadeh, E; Lotfollahzadeh, S; Rostami, A; Amininia, N; Ragh, M J

    2016-05-01

    Cryptosporidiosis is a zoonotic protozoan disease that affects the gastrointestinal tract of animals and humans. Diarrhoea as the most important indication of the infection leads to high economic losses in livestock industries and is a life threatening infection in immunocompromised individuals. In the absence of the effective drugs, vaccine has an effective role in the prevention of infection. For this purpose we developed a vaccine utilizing recombinant P23 protein and immunized pregnant cows four times from 70 days to parturition every 2 weeks. After parturition, each calf received his dam colostrum and challenged with 1 × 10(7) Cryptosporidium parvum oocysts at 12 h of age. Results showed that in contrast with the control group, the antibody titre in the sera and first milking colostra of the immunized cows significantly increased and calves fed hyperimmune colostrum did not show cryptosporidiosis signs. Moreover, enriched colostrum not only reduced significantly the amount of oocyst excretion but also delayed its onset. Our study showed that recombinant P23 protein could be used for passive immunization of newborn calves against Cryptosporidium parvum. PMID:27012710

  17. Molecular characterization of Cryptosporidium parvum detected in Japanese black and Holstein calves in Iwate Prefecture and Tanegashima Island, Kagoshima Prefecture, Japan

    PubMed Central

    AITA, Junya; ICHIKAWA-SEKI, Madoka; KINAMI, Aiko; YAITA, Seiko; KUMAGAI, Yoshihiro; NISHIKAWA, Yoshifumi; ITAGAKI, Tadashi

    2015-01-01

    Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans. PMID:25819544

  18. Molecular characterization of Cryptosporidium parvum detected in Japanese black and Holstein calves in Iwate Prefecture and Tanegashima Island, Kagoshima Prefecture, Japan.

    PubMed

    Aita, Junya; Ichikawa-Seki, Madoka; Kinami, Aiko; Yaita, Seiko; Kumagai, Yoshihiro; Nishikawa, Yoshifumi; Itagaki, Tadashi

    2015-08-01

    Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans. PMID:25819544

  19. Modeling Cryptosporidium parvum oocyst inactivation and bromate in a flow-through ozone contactor treating natural water.

    PubMed

    Kim, Jae-Hong; Elovitz, Michael S; von Gunten, Urs; Shukairy, Hiba M; Mariñas, Benito J

    2007-01-01

    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 was coupled with an empirical ozone decay model and a one-dimensional axial dispersion reactor (ADR) model to represent the performance of a lab-scale flow-through ozone bubble-diffuser contactor. Dissolved ozone concentration, bromate concentration (in flow-through experiments only), hydroxyl radical exposure and C. parvum oocyst survival were measured in batch and flow-through experiments performed with filtered Ohio River water. The model successfully represented ozone concentration and C. parvum oocyst survival ratio in the flow-through reactor using parameters independently determined from batch and semi-batch experiments. Discrepancies between model prediction and experimental data for hydroxyl radical concentration and bromate formation were attributed to unaccounted for reactions, particularly those involving natural organic matter, hydrogen peroxide and carbonate radicals. Model simulations including some of these reactions resulted in closer agreement between predictions and experimental observations for bromate formation. PMID:17123571

  20. HIV-1 Tat Protein Suppresses Cholangiocyte Toll-Like Receptor 4 Expression and Defense against Cryptosporidium parvum

    PubMed Central

    O’Hara, Steven P.; Small, Aaron J.; Gajdos, Gabriella B.; Badley, Andrew D.; Chen, Xian-Ming; LaRusso, Nicholas F.

    2009-01-01

    Biliary cryptosporidiosis is associated with acquired immunodeficiency syndrome (AIDS) cholangiopathy and occurs almost exclusively in adult patients with AIDS. Infection of biliary epithelial cells (cholangiocytes) with Cryptosporidium parvum induces Toll-like receptor (TLR) 4 expression and stimulates a TLR-dependent response against infection. Here, we tested whether human immunodeficiency virus type 1 (HIV-1) Tat affects TLR expression and, hence, anti–C. parvum defense responses. Using an in vitro model of human biliary cryptosporidiosis, we found that recombinant Tat protein increased TLR4 mRNA expression in both uninfected and C. parvum–infected cholangiocytes. Conversely, Tat decreased TLR4 protein levels and suppressed C. parvum–induced TLR4 protein expression. Using actinomycin to inhibit transcription, we found that Tat increased the half-life of TLR4 mRNA from ~25 to 60 min, and RNA gel-shift assays demonstrated direct binding of Tat to TLR4 mRNA. In vitro transcription/translation studies suggested that Tat does not affect transcription but does decrease TLR4 translation. Importantly, more parasites were found in Tat-treated cells than in control cells 48h after infection. These findings suggest that Tat inhibits cholangiocyte TLR4protein expression through translational inhibition. These events appear to diminish the ability of cholangiocytes to initsiate an innate immune response to C. parvum. We suggest that these findings may contribute to the unusual susceptibility of HIV-infected individuals to biliary cryptosporidiosis. PMID:19265483

  1. Effectiveness of Standard UV Depuration at Inactivating Cryptosporidium parvum Recovered from Spiked Pacific Oysters (Crassostrea gigas)▿

    PubMed Central

    Sunnotel, O.; Snelling, W. J.; McDonough, N.; Browne, L.; Moore, J. E.; Dooley, J. S. G.; Lowery, C. J.

    2007-01-01

    When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 × 106 oocysts liter −1) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis. PMID:17574996

  2. Long-term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels (Mytilus galloprovincialis).

    PubMed

    Tamburrini, A; Pozio, E

    1999-05-01

    Transmission of infectious oocysts of Cryptosporidium parvum via surface- and drinking-water supplies has been reported and many surface waters flow into the sea, potentially causing runoff of animal-infected faeces. Eating raw mussels is a common practice in many countries, increasing the public's risk of acquiring enteric pathogens. The aims of the present study were to estimate how long C. parvum oocysts remain infectious in artificial seawater, to determine if the oocysts are retained in mussel tissues (Mytilus galloprovincialis), and how long they maintain their infectivity. Oocysts were incubated in artificial seawater at 6-8 degrees C under moderate oxygenation and the infectivity of oocysts was tested five times, over a 12 month period after incubation in seawater, in BALB/c mice. Each pup was inoculated per os with 10(5) oocysts and killed 5 days p.i. Oocysts remained infectious for 1 year. Forty mussels held in an aquarium containing artificial seawater filtered out more than 4 x 10(8) oocysts in a 24 h period. Oocysts were detected in the gill washing up to 3 days p.i., in the haemolymph up to 7 days p.i., and in the intestinal tract up to 14 days p.i. Oocysts collected from the gut of mussels 7 and 14 days p.i. were observed to have infected mice. These results suggest that C. parvum oocysts can survive in seawater for at least 1 year and can be filtered out by benthic mussels, retaining their infectivity up to 14 days, so seawater and molluscs are a potential source of C. parvum infection for humans. PMID:10404265

  3. 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. PMID:22094179

  4. 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. PMID:19128883

  5. 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

  6. 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

  7. [1N,12N]Bis(Ethyl)-cis-6,7-Dehydrospermine: a New Drug for Treatment and Prevention of Cryptosporidium parvum Infection of Mice Deficient in T-Cell Receptor Alpha

    PubMed Central

    Waters, W. R.; Frydman, B.; Marton, L. J.; Valasinas, A.; Reddy, V. K.; Harp, J. A.; Wannemuehler, M. J.; Yarlett, N.

    2000-01-01

    Cryptosporidium parvum infection of T-cell receptor alpha (TCR-α)-deficient mice results in a persistent infection. In this study, treatment with a polyamine analogue (SL-11047) prevented C. parvum infection in suckling TCR-α-deficient mice and cleared an existing infection in older mice. Treatment with putrescine, while capable of preventing infection, did not clear C. parvum from previously infected mice. These findings provide further evidence that polyamine metabolic pathways are targets for new anticryptosporidial chemotherapeutic agents. PMID:10991882

  8. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Inactivation kinetics of Cryptosporidium parvum oocysts in swine waste lagoon and spray field

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Because of outbreaks of cryptosporidiosis in humans, Cryptosporidium has become a public health concern. Commercial swine operations can be a source of this protozoan parasite. Although the species distribution of Cryptosporidium is likely dominated by C. suis, a fraction may be comprised of the zoo...

  11. NF-kappaB p65-Dependent Transactivation of miRNA Genes following Cryptosporidium parvum Infection Stimulates Epithelial Cell Immune Responses

    PubMed Central

    Liu, Jun; Gong, Ai-Yu; Drescher, Kristen M.; Chen, Xian-Ming

    2009-01-01

    Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelium and causes diarrheal disease worldwide. Innate epithelial immune responses are key mediators of the host's defense to C. parvum. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and are involved in regulation of both innate and adaptive immune responses. Using an in vitro model of human cryptosporidiosis, we analyzed C. parvum-induced miRNA expression in biliary epithelial cells (i.e., cholangiocytes). Our results demonstrated differential alterations in the mature miRNA expression profile in cholangiocytes following C. parvum infection or lipopolysaccharide stimulation. Database analysis of C. parvum-upregulated miRNAs revealed potential NF-κB binding sites in the promoter elements of a subset of miRNA genes. We demonstrated that mir-125b-1, mir-21, mir-30b, and mir-23b-27b-24-1 cluster genes were transactivated through promoter binding of the NF-κB p65 subunit following C. parvum infection. In contrast, C. parvum transactivated mir-30c and mir-16 genes in cholangiocytes in a p65-independent manner. Importantly, functional inhibition of selected p65-dependent miRNAs in cholangiocytes increased C. parvum burden. Thus, we have identified a panel of miRNAs regulated through promoter binding of the NF-κB p65 subunit in human cholangiocytes in response to C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general. PMID:19997496

  12. The human immunodeficiency virus type 1 tat protein enhances Cryptosporidium parvum-induced apoptosis in cholangiocytes via a Fas ligand-dependent mechanism.

    PubMed

    O'Hara, Steven P; Small, Aaron J; Nelson, Jeremy B; Badley, Andrew D; Chen, Xian-Ming; Gores, Gregory J; Larusso, Nicholas F

    2007-02-01

    While Cryptosporidium parvum infection of the intestine has been reported in both immunocompetent and immunocompromised individuals, biliary infection is seen primarily in adult AIDS patients and is associated with development of AIDS cholangiopathy. However, the mechanisms of pathogen-induced AIDS cholangiopathy remain unclear. Since we previously demonstrated that the Fas/Fas ligand (FasL) system is involved in paracrine-mediated C. parvum cytopathicity in cholangiocytes, we also tested the potential synergistic effects of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat)-mediated FasL regulation on C. parvum-induced apoptosis in cholangiocytes by semiquantitative reverse transcription-PCR, immunoblotting, immunofluorescence analysis, and immunogold electron microscopy. H69 cells do not express CXCR4 and CCR5, which are receptors required for direct HIV-1 viral infection. However, recombinant biologically active HIV-1-associated Tat protein increased FasL expression in the cytoplasm of cholangiocytes without a significant increase in apoptosis. We found that C. parvum-induced apoptosis was associated with translocation of intracellular FasL to the cell membrane surface and release of full-length FasL from infected H69 cells. Tat significantly (P < 0.05) increased C. parvum-induced apoptosis in bystander cells in a dose-dependent manner. Moreover, Tat enhanced both C. parvum-induced FasL membrane translocation and release of full-length FasL. In addition, the FasL neutralizing antibody NOK-1 and the caspase-8 inhibitor Z-IETD-fmk both blocked C. parvum-induced apoptosis in cholangiocytes. The data demonstrated that HIV-1 Tat enhances C. parvum-induced cholangiocyte apoptosis via a paracrine-mediated, FasL-dependent mechanism. Our results suggest that concurrent active HIV replication, with associated production of Tat protein, and C. parvum infection synergistically increase cholangiocyte apoptosis and thus jointly contribute to

  13. 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

  14. 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. PMID:26935529

  15. 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. PMID:23315738

  16. Immunogenicity of orally administrated recombinant Lactobacillus casei Zhang expressing Cryptosporidium parvum surface adhesion protein P23 in mice.

    PubMed

    Geriletu; Xu, Rihua; Jia, Honglin; Terkawi, Mohamad Alaa; Xuan, Xuenan; Zhang, Heping

    2011-05-01

    Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal diseases in both humans and animals. What is more, there is no promising strategy for controlling cryptosporidiosis. In this study, the P23 immunodominant surface protein of C. parvum sporozoites was stably expressed in the Lactobacillus casei Zhang strain and its immunogenicity was evaluated in a mouse model. The molecular weight (23 kDa) and immunogenicity of p23 gene expressed by L. casei Zhang were similar to that of the native P23 protein. Oral immunization with control L. casei Zhang and recombinant L. casei Zhang-p23 activated the mucosal immune system to elicit serum immunoglobulin G (IgG) and mucosal IgA in mice. Furthermore, the expression of cytokines such as IL-4, IL-6, and IFN-γ in splenocytes of mice was detected by real-time PCR after oral immunization. P23-specific immunocyte activation was also verified. These findings indicate that the live L. casei Zhang vector may be a new tool for the production of mucosal vaccines against cryptosporidiosis in animals. PMID:21336991

  17. Deposition of Cryptosporidium parvum oocysts in porous media: a synthesis of attachment efficiencies measured under varying environmental conditions.

    PubMed

    Park, Yeonjeong; Atwill, E Robert; Hou, Lingling; Packman, Aaron I; Harter, Thomas

    2012-09-01

    An extensive set of column experiments was performed with freshly harvested Cryptosporidium parvum oocysts to evaluate the effects of solution chemistry, surface coatings, interactions with other suspended particles, and pore fluid velocity on the fate and transport of this widely occurring waterborne pathogen in sandy porous media. We synthesized our data set with a comprehensive literature survey of similar experiments, to compute attachment (collision) efficiencies (α) used in colloid filtration theory (CFT) using three models for the single collector efficiency (η) across a wide range of experimental conditions. Most prior experiments have observed the transport of surface-treated, sterile C. parvum oocyst in porous media. Our column data confirm for freshly harvested oocysts that the presence of iron coatings on the sand medium and the presence of suspended illite clay drastically enhance oocyst deposition. Increasing ionic strength and decreasing pH also systematically enhance the attachment efficiency. Attachment efficiency decreases only at a very high ionic strength, most likely as a result of steric repulsion and possibly other changes in oocyst surface properties. Attachment efficiencies vary with fluid flow rate but without showing specific trends. We found that the computed attachment efficiency across all reported experiments could be reliably estimated using a regression model based on parameters related to ionic strength and pH. The regression model performed better with the Nelson-Ginn η model and Tufenkji-Elimelech η model than with the Rajagopalan-Tien η model. When CFT is used in environmental assessments, the proposed regression model provides a practical estimator for attachment efficiencies of C. parvum oocyst deposition in porous media for a variety of environmental conditions unfavorable to attachment. PMID:22861686

  18. Binding mode of inhibitors and Cryptosporidium parvum IMP dehydrogenase: A combined ligand- and receptor-based study.

    PubMed

    Li, R-J; Wang, Y-L; Wang, Q-H; Huang, W-X; Wang, J; Cheng, M-S

    2015-01-01

    A combined ligand- and target-based approach was used to analyse the interaction models of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase (CpIMPDH) with selective inhibitors. First, a ligand-based pharmacophore model was generated from 20 NAD(+) competitive CpIMPDH inhibitors with the HipHop module. The characteristic of the NAD(+) binding site of CpIMPDH was then described, and the binding modes of the representative inhibitors were studied by molecular docking. The combination of the pharmacophore model and the docking results allowed us to evaluate the pharmacophore features and structural information of the NAD(+) binding site of CpIMPDH. This research supports the proposal of an interaction model inside the NAD(+) binding site of CpIMPDH, consisting of four key interaction points: two hydrophobic-aromatic groups, a hydrophobic-aliphatic group and a hydrogen bond donor. This study also provides guidance for the design of more potent CpIMPDH inhibitors for the treatment of Cryptosporidium infections. PMID:25978645

  19. Cloning and expression of a cDNA encoding epitopes shared by 15- and 60-kilodalton proteins of Cryptosporidium parvum sporozoites.

    PubMed Central

    Jenkins, M C; Fayer, R; Tilley, M; Upton, S J

    1993-01-01

    A cDNA (CP15/60) encoding epitopes of Cryptosporidium parvum 15- and 60-kDa sporozoite proteins was isolated and expressed in Escherichia coli toward the goal of developing an immunogen for producing high-titer anticryptosporidial colostrum. Antisera prepared in rats to native C. parvum 15-kDa protein and used to identify the CP15/60 bacteriophage clone recognized both 15- and 60-kDa in vitro translation products derived from sporozoite RNA. Antisera specific for recombinant CP15/60 antigen recognized native 15- and 60-kDa C. parvum sporozoite proteins by immunoblotting and identified both surface and internal antigens on C. parvum sporozoites by immunofluorescence staining. Northern (RNA) and Southern blot hybridization experiments using sporozoite RNA and DNA indicated that CP15/60 DNA is transcribed as a single 1.4-kb RNA species from a single-copy gene. Recombinant CP15/60 antigen was recognized by hyperimmune colostrum from cows immunized with C. parvum oocyst-sporozoite protein and by convalescent-phase sera from C. parvum-infected calves. Images PMID:7684726

  20. 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

    2014-03-01

    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

  1. 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

  2. NF-kappaB-mediated expression of iNOS promotes epithelial defense against infection by Cryptosporidium parvum in neonatal piglets.

    PubMed

    Gookin, Jody L; Chiang, Sophia; Allen, Jessica; Armstrong, Martha U; Stauffer, Stephen H; Finnegan, Colleen; Murtaugh, Michael P

    2006-01-01

    Cryptosporidium sp. parasitizes intestinal epithelium, resulting in enterocyte loss, villous atrophy, and malabsorptive diarrhea. We have shown that mucosal expression of inducible nitric oxide (NO) synthase (iNOS) is increased in infected piglets and that inhibition of iNOS in vitro has no short-term effect on barrier function. NO exerts inhibitory effects on a variety of pathogens; nevertheless, the specific sites of iNOS expression, pathways of iNOS induction, and mechanism of NO action in cryptosporidiosis remain unclear. Using an in vivo model of Cryptosporidium parvum infection, we have examined the location, mechanism of induction, specificity, and consequence of iNOS expression in neonatal piglets. In acute C. parvum infection, iNOS expression predominated in the villous epithelium, was NF-kappaB dependent, and was not restricted to infected enterocytes. Ongoing treatment of infected piglets with a selective iNOS inhibitor resulted in significant increases in villous epithelial parasitism and oocyst excretion but was not detrimental to maintenance of mucosal barrier function. Intensified parasitism could not be attributed to attenuated fluid loss or changes in epithelial proliferation or replacement rate, inasmuch as iNOS inhibition did not alter severity of diarrhea, piglet hydration, Cl- secretion, or kinetics of bromodeoxyuridine-labeled enterocytes. These findings suggest that induction of iNOS represents a nonspecific response of the epithelium that mediates enterocyte defense against C. parvum infection. iNOS did not contribute to the pathogenic sequelae of C. parvum infection. PMID:16123198

  3. 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

  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. Evaluation of the Triage Micro Parasite Panel for Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in Patient Stool Specimens

    PubMed Central

    Sharp, Susan E.; Suarez, Clarisa A.; Duran, Yolanda; Poppiti, Robert J.

    2001-01-01

    A study comparing the Triage Micro Parasite Panel (Biosite Diagnostics, Inc., San Diego, Calif.) to conventional O&P examination (O&P) was performed using patient fecal specimens. Five hundred twenty-three stool samples were compared. Nineteen specimens were found to be positive by Triage, and 29 were found to be positive by O&P. Seven specimens were positive for Giardia lamblia, four were positive for Entamoeba histolytica/E. dispar, and three were positive for Cryptosporidium parvum as determined by both methods. There was one false positive by Triage (C. parvum) and four false negatives by O&P (two G. lamblia, one E. histolytica/E. dispar, and one C. parvum). The Triage test accurately detected all 18 specimens that contained one of the three organisms that it was designed to detect. The Triage test is a rapid, easy-to-use enzyme immunoassay for the detection of G. lamblia, E. histolytica/E. dispar, and C. parvum in fresh or fresh-frozen fecal specimens. These data suggest that the Triage test can be used as a screen for the immediate testing of stool specimens for these three pathogenic parasites. If Triage test results are negative, O&P can be performed if parasitic infections other than G. lamblia, E. histolytica/E. dispar, or C. parvum are suspected. PMID:11136793

  6. A Cellular Micro-RNA, let-7i, Regulates Toll-like Receptor 4 Expression and Contributes to Cholangiocyte Immune Responses against Cryptosporidium parvum Infection*

    PubMed Central

    Chen, Xian-Ming; Splinter, Patrick L.; O'Hara, Steven P.; LaRusso, Nicholas F.

    2007-01-01

    Toll-like receptors (TLRs) are important pathogen recognition molecules and are key to epithelial immune responses to microbial infection. However, the molecular mechanisms that regulate TLR expression in epithelia are obscure. Micro-RNAs play important roles in a wide range of biological events through post-transcriptional suppression of target mRNAs. Here we report that human biliary epithelial cells (cholangiocytes) express let-7 family members, micro-RNAs with complementarity to TLR4 mRNA. We found that let-7 regulates TLR4 expression via post-transcriptional suppression in cultured human cholangiocytes. Infection of cultured human cholangiocytes with Cryptosporidium parvum, a parasite that causes intestinal and biliary disease, results in decreased expression of primary let-7i and mature let-7 in a MyD88/NF-κB-dependent manner. The decreased let-7 expression is associated with C. parvum-induced up-regulation of TLR4 in infected cells. Moreover, experimentally induced suppression or forced expression of let-7i causes reciprocal alterations in C. parvum-induced TLR4 protein expression, and consequently, infection dynamics of C. parvum in vitro. These results indicate that let-7i regulates TLR4 expression in cholangiocytes and contributes to epithelial immune responses against C. parvum infection. Furthermore, the data raise the possibility that micro-RNA-mediated post-transcriptional pathways may be critical to host-cell regulatory responses to microbial infection in general. PMID:17660297

  7. MicroRNA-98 and let-7 Regulate Expression of Suppressor of Cytokine Signaling-4 in Biliary Epithelial Cells in Response to Cryptosporidium parvum Infection

    PubMed Central

    Hu, Guoku; Zhou, Rui; Liu, Jun; Gong, Ai-Yu; Chen, Xian-Ming

    2010-01-01

    Expression of the cytokine-inducible Src homology 2 protein (CIS) and suppressors of cytokine signaling proteins (SOCS) represents an important element of host cell reactions in response to pathogen infection. We previously demonstrated that Cryptosporidium parvum infection downregulates miR-98 and let-7 to induce CIS expression in biliary epithelial cells. We reported here that downregulation of miR-98 and let-7 also coordinates epithelial expression of SOCS4 following C. parvum infection. Targeting of SOCS4 3'-untranslated region by miR-98 or let-7 resulted in translational repression. Functional manipulation of miR-98 caused reciprocal alterations in SOCS4 protein expression. Transfection of miR-98 precursor abolished C. parvum-stimulated SOCS4 upregulation. Moreover, expression of SOCS4 in epithelial cells showed an inhibitory effect on phosphorylation of signal transducers and activators of transcription proteins induced by C. parvum. These data suggest an important role for miRNAs in the coordinated regulation of CIS/SOCS expression in epithelial cells in response to C. parvum infection. PMID:20486857

  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. 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

    2015-11-01

    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. PMID:26398590

  10. Cholangiocyte Myosin IIB Is Required for Localized Aggregation of Sodium Glucose Cotransporter 1 to Sites of Cryptosporidium parvum Cellular Invasion and Facilitates Parasite Internalization ▿

    PubMed Central

    O'Hara, Steven P.; Gajdos, Gabriella B.; Trussoni, Christy E.; Splinter, Patrick L.; LaRusso, Nicholas F.

    2010-01-01

    Internalization of the obligate intracellular apicomplexan parasite, Cryptosporidium parvum, results in the formation of a unique intramembranous yet extracytoplasmic niche on the apical surfaces of host epithelial cells, a process that depends on host cell membrane extension. We previously demonstrated that efficient C. parvum invasion of biliary epithelial cells (cholangiocytes) requires host cell actin polymerization and localized membrane translocation/insertion of Na+/glucose cotransporter 1 (SGLT1) and of aquaporin 1 (Aqp1), a water channel, at the attachment site. The resultant localized water influx facilitates parasite cellular invasion by promoting host-cell membrane protrusion. However, the molecular mechanisms by which C. parvum induces membrane translocation/insertion of SGLT1/Aqp1 are obscure. We report here that cultured human cholangiocytes express several nonmuscle myosins, including myosins IIA and IIB. Moreover, C. parvum infection of cultured cholangiocytes results in the localized selective aggregation of myosin IIB but not myosin IIA at the region of parasite attachment, as assessed by dual-label immunofluorescence confocal microscopy. Concordantly, treatment of cells with the myosin light chain kinase inhibitor ML-7 or the myosin II-specific inhibitor blebbistatin or selective RNA-mediated repression of myosin IIB significantly inhibits (P < 0.05) C. parvum cellular invasion (by 60 to 80%). Furthermore ML-7 and blebbistatin significantly decrease (P < 0.02) C. parvum-induced accumulation of SGLT1 at infection sites (by approximately 80%). Thus, localized actomyosin-dependent membrane translocation of transporters/channels initiated by C. parvum is essential for membrane extension and parasite internalization, a phenomenon that may also be relevant to the mechanisms of cell membrane protrusion in general. PMID:20457792

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Irrigation water has been associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to experimentally determine if apples contaminated with waterborne oocysts of Cryptosporidium might represent a food saf...

  12. Evaluating the transport of bacillus spores as a potential surrogate for Cryptosporidium parvum Oocysts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Effects of Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Cryptosporidium parvum pig genotype II diagnosed in pigs from the state of Rio de Janeiro, Brazil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pigs may represent a source of Cryptosporidium sp. infection to humans. The objective of this study was to identify the species present in pigs from the State of Rio de Janeiro, Brazil, and verify what risks pigs represent in transmission of human cryptosporidiosis, since there is no such informati...

  15. Influence of temperature on Cryptosporidium parvum oocyst infectivity in river water samples as detected by tissue culture assay.

    PubMed

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

    2002-06-01

    Cryptosporidium parvum oocysts were stored in 1-ml aliquots of filtered river water at -20, 4, 10, and 21-23 C in the dark. Oocysts were also added to filter-sterilized river water samples and stored at 21-23 C. The infectivity of oocysts stored under different conditions was assayed at weekly intervals through infection of human adenocarcinoma ileocecal (HCT-8) cell monolayers. Wells containing between 10 and 100 foci of infection were enumerated by immunofluorescent microscopy, and the number of infective oocysts was calculated. No infectious oocysts were detected after 1 wk at -20 C. The number of infective oocysts stored at 4 C decreased 5-fold, and the number of those stored at 10 C decreased 2.5-fold after 14 wk. The infectivity of oocysts stored in potassium dichromate (positive control) at 4 C decreased 2-fold over 14 wk. The number of infective oocysts in filter-sterilized and non-filter-sterilized river water stored at 21-23 C decreased by 3.3 and 2.6 log units, respectively, over 12 wk, and no foci of infection were detected at 14 wk. The results show that as temperature increased from 4 to 23 C, the duration of oocyst infectivity decreased. PMID:12099446

  16. Functional characterization of a fatty acyl-CoA binding protein (ACBP) from the apicomplexan Cryptosporidium parvum

    PubMed Central

    Zeng, Bin; Cai, Xiaomin; Zhu, Guan

    2006-01-01

    SUMMARY We have identified and conducted functional analysis of a fatty acyl-CoA binding protein (ACBP) gene from the opportunistic protist Cryptosporidium parvum. The CpACBP1 gene encodes a protein of 268 aa that is 3X larger than the typical ACBP proteins (i.e., ∼90 aa) of humans and animals. Sequence analysis indicated that CpACBP1 consists of an N-terminal ACBP domain (∼90 aa) and a C-terminal ankrin repeat sequence (∼170 aa). The entire CpACBP1 ORF was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 was palmitoyl-CoA. RT-PCR, Western blotting and immuno-labeling analyses clearly showed that the CpACBP1 gene was mainly expressed in the intracellular developmental stages and the level increases during the parasite development. Immunofluorescence microscopy shows that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in the lipid remodeling in the PVM or the transport of fatty acids across the membrane. PMID:16849800

  17. 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

  18. Bovine antibody against Cryptosporidium parvum elicits a circumsporozoite precipitate-like reaction and has immunotherapeutic effect against persistent cryptosporidiosis in SCID mice.

    PubMed Central

    Riggs, M W; Cama, V A; Leary, H L; Sterling, C R

    1994-01-01

    Control of cryptosporidiosis is currently hampered by the absence of drugs or vaccines proven consistently effective against Cryptosporidium parvum. On the basis of observations that anti-C. parvum antibody has therapeutic effect against cryptosporidiosis, cows were immunized with C. parvum to produce hyperimmune colostral antibody. An antibody-rich fraction was prepared and differentiated from control (nonhyperimmune) antibody by enzyme-linked immunosorbent assay, immunofluorescence assay, immunoelectron microscopy, and in vitro neutralizing titer against DEAE-cellulose-isolated C. parvum sporozoites. Oocyst, purified sporozoite, and merozoite antigens recognized by hyperimmune antibody were defined by Western blot (immunoblot). Hyperimmune antibody recognized antigens common to oocysts, sporozoites, and merozoites, as well as stage-specific antigens. Upon incubation with hyperimmune antibody, sporozoites underwent distinct morphologic changes characterized by progressive formation and eventual release of membranous sporozoite surface antigen-antibody complexes, similar to the malaria circumsporozoite precipitate reaction. The infectivity of sporozoites having undergone this reaction was neutralized. The reaction was minimal or absent on sporozoites incubated with control antibody. To determine therapeutic effect in vivo, persistent C. parvum infection was established in adult severe combined immune-deficient (SCID) mice by oral inoculation with 10(7) oocysts. At 5 weeks postinfection, infected mice were treated for 10 days with hyperimmune or control antibody by inclusion in drinking water and daily gavage. Fecal oocyst shedding and infection scores in the gastrointestinal tract and gall bladder/common bile duct in hyperimmune antibody-treated mice were significantly lower than those in the control antibody-treated mice. Hyperimmune bovine antibody prepared against C. parvum may provide a first-generation therapy for control of cryptosporidiosis. Additionally

  19. NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.

    PubMed

    Muñoz-Caro, Tamara; Lendner, Matthias; Daugschies, Arwid; Hermosilla, Carlos; Taubert, Anja

    2015-10-01

    Cryptosporidium parvum causes a zoonotic infection with worldwide distribution. Besides humans, cryptosporidiosis affects a wide range of animals leading to significant economic losses due to severe enteritis in neonatal livestock. Neutrophil extracellular trap (NET) formation has been demonstrated as an important host effector mechanism of PMN acting against several invading pathogens. In the present study, C. parvum-mediated NET formation was investigated in human and bovine PMN in vitro. We here demonstrate that C. parvum sporozoites indeed trigger NET formation in a time-dependent manner. Thereby, the classical characteristics of NETs were demonstrated by co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO). A significant reduction of NET formation was measured following treatments of PMN with NADPH oxidase-, NE- and MPO-inhibitors, confirming the key role of these enzymes in C. parvum-induced NETs. Additionally, sporozoite-triggered NETosis revealed as dependent on intracellular Ca(++) concentration and the ERK 1/2 and p38 MAPK-mediated signaling pathway. Moreover, sporozoite-triggered NET formation led to significant parasite entrapment since 15% of the parasites were immobilized in NET structures. Consequently, PMN-pre-exposed sporozoites showed significantly reduced infectivity for epithelial host cells confirming the capability of NETs to prevent active parasite invasion. Besides NETs, we here show that C. parvum significantly up-regulated CXCL8, IL6, TNF-α and of GM-CSF gene transcription upon sporozoite confrontation, indicating a pivotal role of PMN not only in the bovine and human system but most probably in other final hosts for C. parvum. PMID:26026247

  20. Survival of Cryptosporidium parvum oocysts in the presence of hydrated lime.

    PubMed

    Zintl, A; Keogh, B; Ezzaty-Mirhashemi, M; De Waal, T; Scholz, D; Mulcahy, G

    2010-03-01

    To investigate the effects of hydrated lime on the survival of Cryptosporidium oocysts, the percentage viability of oocysts was assessed using fluorescent in situ hybridisation. In the absence of lime and with lime at a concentration of 1 per cent, there was a gradual decline in oocyst viability during the 10-day trial. Although the addition of 5 or 10 per cent lime caused the total number of oocysts to decrease, there appeared to be an increase in the proportion of potentially viable oocysts. PMID:20208077

  1. Intra-Species Diversity and Panmictic Structure of Cryptosporidium parvum Populations in Cattle Farms in Northern Spain

    PubMed Central

    Ramo, Ana; Quílez, Joaquín; Monteagudo, Luis; Del Cacho, Emilio; Sánchez-Acedo, Caridad

    2016-01-01

    The intra-herd and intra-host genetic variability of 123 Cryptosporidium parvum isolates was investigated using a multilocus fragment typing approach with eleven variable-number tandem-repeat (VNTR) loci and the GP60 gene. Isolates were collected from intensively farmed diarrheic pre-weaned calves originating from 31 dairy farms in three adjoining regions in northern Spain (País Vasco, Cantabria and Asturias). The multilocus tool demonstrated an acceptable typeability, with 104/123 samples amplifying at all twelve loci. The ML2, TP14, GP60 and the previously un-described minisatellite at locus cgd2_3850 were the most discriminatory markers, while others may be dismissed as monomorphic (MSB) or less informative (CP47, ML1 and the novel minisatellites at loci Cgd1_3670 and Cgd6_3940). The 12-satellite typing tool provided a Hunter-Gaston index (HGDI) of 0.987 (95% CI, 0.982–0.992), and differentiated a total of 70 multilocus subtypes (MLTs). The inclusion of only the four most discriminatory markers dramatically reduced the number of MLTs (n: 44) but hardly reduced the HGDI value. A total of 54 MLTs were distinctive for individual farms, indicating that cryptosporidiosis is an endemic condition on most cattle farms. However, a high rate of mixed infections was detected, suggesting frequent meiotic recombination. Namely, multiple MLTs were seen in most farms where several specimens were analyzed (90.5%), with up to 9 MLTs being found on one farm, and individual specimens with mixed populations being reported on 11/29 farms. Bayesian Structure analysis showed that over 35% of isolates had mixed ancestry and analysis of evolutionary descent using the eBURST algorithm detected a high rate (21.4%) of MLTs appearing as singletons, indicating a high degree of genetic divergence. Linkage analysis found evidence of linkage equilibrium and an overall panmictic structure within the C. parvum population in this discrete geographical area. PMID:26848837

  2. 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.

  3. Commercial Assay for Detection of Giardia lamblia and Cryptosporidium parvum Antigens in Human Fecal Specimens by Rapid Solid-Phase Qualitative Immunochromatography

    PubMed Central

    Garcia, Lynne S.; Shimizu, Robyn Y.; Novak, Susan; Carroll, Marilyn; Chan, Frank

    2003-01-01

    The ImmunoCard STAT! Cryptosporidium/Giardia rapid assay (Meridian Bioscience, Inc.) is a solid-phase qualitative immunochromatographic assay that detects and distinguishes between Giardia lamblia and Cryptosporidium parvum in aqueous extracts of human fecal specimens (fresh, frozen, unfixed, or fixed in 5 or 10% formalin or sodium acetate-acetic acid-formalin). By using specific antibodies, antigens specific for these organisms are isolated and immobilized on a substrate. After the addition of appropriate reagents, a positive test is detected visually by the presence of a gray-black color bar (regardless of the intensity) next to the organism name printed on the test device. A control is included in the device. Steps include tube preparation (buffer, patient specimen, conjugates A and B), testing (addition of sample onto the test device), and visual reading (total time, 12 min). Test performance was evaluated with known positive and negative stool specimens (170 specimens positive for Giardia and 231 specimens negative for Giardia) (85 specimens positive for Cryptosporidium and 316 specimens negative for Cryptosporidium); they were tested with trichrome, iron-hematoxylin, or modified acid-fast stains or the Meridian Bioscience, Inc., Giardia/Cryptosporidium Merifluor combination reagent; specimens with discrepant results were retested by using the Merifluor combination reagent. On the basis of the results of the reference methods, the sensitivities, specificities, and positive and negative predictive values were as follows: for G. lamblia, 93.5, 100, 100, and 95.5%, respectively; for C. parvum, 98.8, 100, 100, and 99.7%, respectively. False-negative results for G. lamblia were obtained with specimens with low parasite numbers (n = 7) or specimens containing trophozoites only (n = 3); one specimen with a false-negative result contained numerous cysts. The one specimen false negative for C. parvum was confirmed to be positive by immunofluorescence. No cross

  4. Aging of Cryptosporidium parvum oocysts in river water and their susceptibility to disinfection by chlorine and monochloramine.

    PubMed

    Chauret, C; Nolan, K; Chen, P; Springthorpe, S; Sattar, S

    1998-12-01

    Cryptosporidium parvum oocysts were aged in waters from both the St. Lawrence River and the Ottawa River. In situ survival experiments were carried out by incubating the oocysts in either dialysis cassettes or microtubes floated into an overflow tank. A significant portion of the oocysts survived in the test waters for several weeks. Oocyst survival in the St. Lawrence River was better in membrane-filtered (0.2 microm-pore diameter) water than in unfiltered water, suggesting that biological antagonism may play a role in the environmental fate of the parasite. Oocysts aged in river waters under in situ conditions and control oocysts kept refrigerated in synthetic water (100 ppm as CaCO3); pH 7.0) were subjected to the same disinfection protocol. Aged oocysts were at least as resistant as, if not more resistant than, the control oocysts to disinfection. This indicates that the oocysts surviving in the water environment may be just as difficult to inactivate by potable water disinfection as freshly shed oocysts. Therefore, water treatment should not be based on the assumption that environmental oocysts may be more easily inactivated than freshly shed oocysts. First-order kinetics die-off rates varied from one river to another (from 0.013 to 0.039 log(10).day(-1)) and from one experiment to another with water from the same river collected at different times. Calculation of the die-off rates based on either in vitro excystation or in vitro excystation in combination with total counts (overall die-off rates) showed that the assessment of oocyst viability by microscopic methods must account for the total oocyst loss observed during long-term inactivation assays of river waters. PMID:10383227

  5. Role of collector alternating charged patches on transport of Cryptosporidium parvum oocyst in a patchwise charged heterogeneous micromodel

    SciTech Connect

    Liu, Yuanyuan; Zhang, Changyong; Hu, Dehong; Kuhlenschmidt, Mark S.; Kuhlenschmidt, Theresa B.; Mylon, Steven E.; Kong, Rong; Bhargava, Rohit; Nguyen, Thanh H.

    2013-02-04

    The role of collector surface charge heterogeneity on transport of Cryptosporidium parvum oocyst and carboxylate microsphere in 2-dimensional micromodels was studied. The cylindrical silica collectors within the micromodels were coated with 0, 10, 20, 50 and 100% Fe2O3 patches. The experimental values of average single collector removal efficiencies (η) of the Fe2O3 patches and on the entire collectors were determined. In the presence of significant (>3500 kT) Derjaguin–Landau–Verwey–Overbeek (DLVO) energy barrier between the microspheres and the silica collectors at pH 5.8 and 8.1, the values of η determined for Fe2O3 patches were significantly less (p < 0.05, t-test) than that obtained for collectors coated entirely with Fe2O3. However, η on Fe2O3 patches for microspheres at pH 4.4 and for oocysts at pH 5.8 and 8.1, where the DLVO energy barrier was relatively small (ca. 200-360 kT), were significantly greater (p < 0.05, t-test) than that on the collectors coated entirely with Fe2O3. The dependence of η determined for Fe2O3 patches on the DLVO energy barrier indicated the importance of periodic favorable and unfavorable electrostatic interactions between colloids and collectors with alternating Fe2O3 and silica patches. Differences between experimentally determined η and that predicted by a patchwise geochemical heterogeneous model was observed, but can be explained by the model’s lack of consideration for the spatial distribution of charge heterogeneity on the collector surface and colloid migration on patchwise heterogeneous collectors.

  6. 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

  7. 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

  8. 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

  9. 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

  10. Oleylphosphocholine (OlPC) arrests Cryptosporidium parvum growth in vitro and prevents lethal infection in interferon gamma receptor knock-out mice

    PubMed Central

    Sonzogni-Desautels, Karine; Renteria, Axel E.; Camargo, Fabio V.; Di Lenardo, Thomas Z.; Mikhail, Alexandre; Arrowood, Michael J.; Fortin, Anny; Ndao, Momar

    2015-01-01

    Cryptosporidium parvum is a species of protozoa that causes cryptosporidiosis, an intestinal disease affecting many mammals including humans. Typically, in healthy individuals, cryptosporidiosis is a self-limiting disease. However, C. parvum can cause a severe and persistent infection that can be life-threatening for immunocompromised individuals, such as AIDS patients. As there are no available treatments for these patients that can cure the disease, there is an urgent need to identify treatment options. We tested the anti-parasitic activity of the alkylphosphocholine oleylphosphocholine (OlPC), an analog of miltefosine, against C. parvum in in vitro and in vivo studies. In vitro experiments using C. parvum infected human ileocecal adenocarcinoma cells (HCT-8 cells) showed that OlPC has an EC50 of 18.84 nM. Moreover, no cell toxicity has been seen at concentrations ≤50 μM. C57BL/6 interferon gamma receptor knock-out mice, were infected by gavage with 4000 C. parvum oocysts on Day 0. Oral treatments, with OlPC, miltefosine, paromomycin or PBS, began on Day 3 post-infection for 10 days. Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30. Doses of 30 and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction. Mice treated with 10 mg/kg/day of miltefosine resulted in 50% survival at Day 30. In contrast, control mice, treated with PBS or 100 mg/kg/day of paromomycin, died or had to be euthanized between Days 6 and 13 due to severe illness. Results of parasite burden were obtained by qPCR and cross-validated by both flow cytometry of stool oocysts and histological sections of the ileum. Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients. PMID:26441906