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PubMed2003

A novel multiplex polymerase chain reaction approach for detection of four human infective Cryptosporidium isolates: Cryptosporidium parvum, types H and C, Cryptosporidium canis, and Cryptosporidium felis in fecal and soil samples.

A nested multiplex polymerase chain reaction (PCR) approach was adopted for the simultaneous detection of 4 human infective genotypes of the protozoan parasite Cryptosporidium. Specific PCR primers were designed for the heat shock protein 70 gene of 2 genotypes of Cryptosporidium parvum (human and bovine types), Cryptosporidium canis, and Cryptosporidium felis. These 4 genotypes have all been found in human fecal samples. The primers amplified DNA fragments of specific sizes, each representing a unique genotype. The limit of detection of the method was found to vary between 10 and 100 oocysts per 1 ml fecal material. There appeared to be no cross-reactivity with other organisms commonly present in feces and soil, and the approach has a high specificity. The rapid identification of various human infective Cryptosporidium isolates is a part of the authors' long-term aim of determining the routes of infection with oocysts and thereby increase their epidemiological understanding of Cryptosporidium infection in humans and animals.

Animals↗

PubMed2000

Prevalence of Giardia sp. Cryptosporidium parvum and Cryptosporidium andersoni (syn. C. muris) [correction of Cryptosporidium parvum and Cryptosporidium muris (C. andersoni)] in 109 dairy herds in five counties of southeastern New York.

A cross-sectional study was undertaken to determine the prevalence of Giardia sp. (G. duodenalis group), Cryptosporidium parvum and Cryptosporidium andersoni (C. muris) [corrected] in dairy cattle in three different age groups, and to evaluate the association of age and season with prevalence. One hundred and nine dairy farms, from a total of 212 farms, in five counties of southeastern New York volunteered to participate. On these farms, 2943 fecal samples were collected from three defined age groups. The farms were randomly assigned for sampling within the four seasons of the year. Each farm was visited once during the study period from March 1993 to June 1994 to collect fecal samples. Demographic data on the study population was collected at the time of sampling by interviewing the farm owner or manager. At collection, fecal samples were scored as diarrheic or non-diarrheic, and each condition was later related to positive or negative infection with these parasites. Fecal samples were processed using a quantitative centrifugation concentration flotation technique and enumerated using bright field and phase contrast microscopy. In this study, the overall population prevalence for Giardia sp. was 8.9%; C. parvum, 0.9%; and C. muris, 1.1%. When considering animals most at the risk of infection (those younger than 6 months of age) Giardia sp. and C. parvum was found in 20.1 and 2.4% of the animals, respectively. Giardia sp. and C. muris were found in all age groups. There was no significant seasonal pattern of infection for any of these parasites.

Age Factors↗

PubMed2001

Unusual cryptosporidium species recovered from human faeces: first description of Cryptosporidium felis and Cryptosporidium 'dog type' from patients in England.

DNA was extracted from faecal samples collected from 1680 patients in which Cryptosporidium oocysts were recognised by light microscopy. DNA from faeces from five of these patients failed to amplify by PCR three gene fragments--the Cryptosporidium oocyst wall protein (COWP) gene, the thrombospondin-related adhesive protein of Cryptosporidium-1 (TRAP-C1) gene and the thrombospondin-related adhesive protein of Cryptosporidium-2 (TRAP-C2) gene--with primers designed from C. parvum sequences. However, DNA from these five patients did amplify cryptosporidial 18S rDNA gene fragments and a heat-shock protein (HSP70) gene fragment was also amplified from four of them. The purpose of this study was to characterise further the Cryptosporidium associated with infection in these patients. DNA sequence analysis of 18S rDNA genes showed that four of these patients were infected by C. felis, and the remaining one by an as yet un-named Cryptosporidium species designated the 'dog type' (C. dt). Infection by C. felis was further confirmed in all four patients by DNA sequence analysis of the HSP70 gene. Oocysts present in all five samples reacted strongly with two anti-cryptosporidial oocyst monoclonal antibodies, except for the C. dt, which was tested with only one of the antibodies. Two of the patients infected by C. felis had underlying illness; one 8-year-old male had an undefined severe inherited underlying condition, and the second patient, a 32-year-old male, was HIV positive. Two of the remaining three patients (two females aged 1 and 2 years, respectively) were apparently immunocompetent (one infected with C. felis and one with the C. dt). No information was obtained for the fifth patient. The patient infected by C. dt had a recent history of travel to Africa. This is the first report of infection with these two Cryptosporidium species in immunocompetent patients, and in any patient in the UK.

Adult↗

PubMed1999

The identification of Cryptosporidium species and Cryptosporidium parvum directly from whole faeces by analysis of a multiplex PCR of the 18S rRNA gene and by PCR/RFLP of the Cryptosporidium outer wall protein (COWP) gene.

A multiplex polymerase chain reaction (PCR) procedure to amplify 18S rRNA gene fragments has been developed. Amplified DNA fragments of the expected size were obtained which were specific for Cryptosporidium parvum and Cryptosporidium wrairi (422 bp), Cryptosporidium baileyi (11106 bp) and Cryptosporidium muris (1346 bp). Criptosporidium parvum and C. wrairi can be distinguished using a PCR/restriction fragment length polymorphism (RFLP) analysis of the Cryptosporidium outer wall protein (COWP) gene, and these two techniques were applied to DNA extracted from whole faeces using a simple and rapid procedure. Cryptosporidium parvum DNA was detected in the faeces of 72 humans and 24 calves where cryptosporidial oocysts were demonstrated using conventional light microscopy. The specific DNA fragments were not amplified using extracts of material containing other lower eukaryotic parasites.

Animals↗

PubMed2006

Characterisation of small double stranded RNA molecule in Cryptosporidium hominis, Cryptosporidium felis and Cryptosporidium meleagridis.

Coding regions of double stranded RNA molecules from 3 human faecal samples containing Cryptosporidium hominis, C. felis and C. meleagridis were characterised by sequencing and compared with that previously obtained for C. parvum. Sequences outside the coding regions were also obtained. Overall similarities of between 86% and 92% and between 86% and 93% were observed in the nucleotide and amino acid sequences respectively between these species. These larger sequences will allow further molecular tools for detection, identification and characterisation of Cryptosporidium spp.

Animals↗

PubMed2002

Prevalence of Giardia and Cryptosporidium and characterization of Cryptosporidium spp. isolated from wildlife, human, and agricultural sources in the North Saskatchewan River Basin in Alberta, Canada.

The environmental distribution of Giardia spp. and Cryptosporidium spp. is dependent upon human, agricultural, and wildlife sources. The significance of each source with regard to the presence of parasites in the environment is unknown. This 2-year study examined parasite prevalence in human sewage influent, wildlife, and agricultural sources associated with the North Saskatchewan River Basin in Alberta, Canada. Fecal samples were collected from cow-calf, dairy, and hog operations in the watershed area. Sewage-treatment facilities were sampled bimonthly during the 2-year study, and wildlife scat was collected at locations along tributaries of the North Saskatchewan River. All samples were analyzed for the presence of Giardia and Cryptosporidium, using sucrose-gradient separation followed by immunofluorescent microscopy. Giardia and Cryptosporidium were detected in all three sources. The lowest prevalence of both Giardia (3.28%) and Cryptosporidium (0.94%) was found in wildlife, with 6 of 19 species testing positive. Sewage influent had the highest prevalence of Giardia (48.80%) and Cryptosporidium parvum-like oocysts (5.42%); however, the concentration of both parasites was minimal compared with the concentration detected in cattle feces. Cow-calf sources contained the highest concentration of Giardia (mean 5800/g feces, P < 0.01), and dairy sources contained the highest concentration of C. parvum-like oocysts (mean 295/g feces, P < 0.01). Although prevalence and concentration are higher in cattle feces than in sewage, the Giardia and Cryptosporidium in animal manure do not have direct access to water draining into the North Saskatchewan River. PCR-based characterization of rDNA from isolates of Cryptosporidium collected from Alberta human, pig, calf, mature steer, dog, cat, and beaver hosts revealed distinct genetic differences that may reflect host specificity.

Animals↗

PubMed2006

Factors associated with shedding of Cryptosporidium parvum versus Cryptosporidium bovis among dairy cattle in New York State.

OBJECTIVE: To isolate and speciate Cryptosporidium DNA from fecal samples obtained from dairy cattle in New York State and identify factors associated with whether cattle were shedding Cryptosporidium parvum versus Cryptosporidium bovis. DESIGN: Cross-sectional study. SAMPLE POPULATION: 115 fecal samples positive for DNA coding for the Cryptosporidium 18S rRNA gene from dairy cattle in New York State. PROCEDURES: A PCR assay was used to amplify DNA from fecal samples; amplification products were submitted for bidirectional DNA sequencing. Logistic regression was used to test for associations between various host factors and Cryptosporidium spp. RESULTS: 70 of the 115 (61%) fecal samples were found to have C parvum DNA, 42 (37%) were determined to have C bovis DNA, and 3 (3%) were found to have C parvum deer-type DNA. The presence of diarrhea at the time of fecal sample collection, oocyst count, and breed were associated with whether cattle were infected with C parvum or C bovis, with animals more likely to be infected with C parvum if they had diarrhea, had a high oocyst count, or were Holsteins. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that C parvum and C bovis can be isolated from dairy cattle in New York State and that various factors affect whether cattle infected with Cryptosporidium spp are infected with C parvum or C bovis. Findings also lend credence to the theory that C bovis may be more host adapted and thus less pathogenic to dairy cattle than C parvum.

Animals↗

PubMed2005

Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies.

In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the beta-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.

Animals↗

PubMed1999

Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species.

To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidium isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus Cryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most Cryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiate C. wrairi and C. meleagridis from some of the C. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.

Animals↗

PubMed2026

Detection and genomic characterization of cryptosporidium parvum virus 1 (CSpV1): A potential biomarker for Cryptosporidium parvum detection in bovine calves.

Cryptosporidium parvum is a ubiquitous enteric parasite that infects a diverse range of vertebrate species. The detection of C. parvum can be confounded when oocysts are intermittently shed below an assay's limit of detection, yielding a false-negative result. We therefore investigated the utility of Cryptosporidium parvum virus 1 (CSpV1), a putative symbiont of Cryptosporidium parvum, as a surrogate target for detecting the parasite in bovine calves. Using real-time polymerase chain reaction (qPCR), we tested 422 samples for Cryptosporidium spp., C. parvum-associated targets, and CSpV1. Among the 189 samples positive for at least one target, CSpV1 was detected in 24 (12.70%) samples without concurrent detection of Cryptosporidium. Additionally, we analyzed CSpV1 genomic sequences to ascertain its value as an epidemiological biomarker. Evaluation of dsRNA1 amino acid sequences identified country-associated patterns, suggesting potential utility for geographic distribution analyses. These findings suggest that CSpV1 may serve as a biological signature of C. parvum and support further investigation into its usefulness as an adjunct molecular target.

Biomarker↗

PubMed2000

The identification and characterisation of an unusual genotype of Cryptosporidium from human faeces as Cryptosporidium meleagridis.

An unusual genotype of Cryptosporidium was identified in the faeces of six human patients by PCR/RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene. Conventional microscopy showed oocysts indistinguishable in size from those of Cryptosporidium parvum, which reacted with two different commercially available anti-oocyst monoclonal antibodies. The isolates were further characterised by PCR/RFLP analysis of the thrombospondin-related adhesive protein of Cryptosporidium-1 (TRAP-C1) genes as well as by DNA sequencing of the COWP and the TRAP-C1 gene fragments and of two regions of the 18S rRNA gene. Sequence analysis of the COWP, TRAP-C1, and 18S rRNA gene fragments confirmed that this genotype is genetically distinct from C. parvum. 18S rRNA gene sequences were found to be identical to those published for Cryptosporidium meleagridis.

Amino Acid Sequence↗

PubMed2006

Interaction of Cryptosporidium hominis and Cryptosporidium parvum with primary human and bovine intestinal cells.

Cryptosporidiosis in humans is caused by the zoonotic pathogen Cryptosporidium parvum and the anthroponotic pathogen Cryptosporidium hominis. To what extent the recently recognized C. hominis species differs from C. parvum is unknown. In this study we compared the mechanisms of C. parvum and C. hominis invasion using a primary cell model of infection. Cultured primary bovine and human epithelial intestinal cells were infected with C. parvum or C. hominis. The effects of the carbohydrate lectin galactose-N-acetylgalactosamine (Gal/GalNAc) and inhibitors of cytoskeletal function and signal transduction mechanisms on entry of the parasites into host cells were tested. HCT-8 cells (human ileocecal adenocarcinoma cells) were used for the purpose of comparison. Pretreatment of parasites with Gal/GalNAc inhibited entry of C. parvum into HCT-8 cells and primary bovine cells but had no effect on entry of either C. parvum or C. hominis into primary human cells or on entry of C. hominis into HCT-8 cells. Both Cryptosporidium species entered primary cells by a protein kinase C (PKC)- and actin-dependent mechanism. Staurosporine, in particular, attenuated infection, likely through a combination of PKC inhibition and induction of apoptosis. Diversity in the mechanisms used by Cryptosporidium species to infect cells of different origins has important implications for understanding the relevance of in vitro studies of Cryptosporidium pathogenesis.

Animals↗

PubMed2005

Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species.

A study was undertaken to compare the performance of five different molecular methods (available in four different laboratories) for the identification of Cryptosporidium parvum and Cryptosporidium hominis and the detection of genetic variation within each of these species. The same panel of oocyst DNA samples derived from faeces (n=54; coded blindly) was sent for analysis by: (i) DNA sequence analysis of a fragment of the HSP70 gene; (ii) DNA sequence analysis and the ssrRNA gene in laboratory 1; (iii) single-strand conformation polymorphism analysis of part of the ssrRNA; (iv) SSCP analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA region in laboratory 2; (v) 60 kDa glycoprotein (gp60) gene sequencing with prior species determination using PCR with restriction fragment length polymorphism analysis of the ssrRNA gene in laboratory 3; and (vi) multilocus genotyping at three microsatellite markers in laboratory 4. For detecting variation within C. parvum and C. hominis, SSCP analysis of ITS-2 was considered to have superior utility and determined 'subgenotypes' in samples containing DNA from both species. SSCP was also most cost effective in terms of time, cost and consumables. Sequence analysis of gp60 and microsatellite markers ML1, ML2 and 'gp15' provided good comparators for the SSCP of ITS-2. However, applicability of these methods to other Cryptosporidium species or genotypes and to environmental samples needs to be evaluated. This trial provided, for the first time, a direct comparison of multiple methods for the genetic characterisation of C. parvum and C. hominis samples. A protocol has been established for the international distribution of samples for the characterisation of Cryptosporidium. This can be applied in further evaluation of molecular methods by investigation of a larger number of unrelated samples to establish sensitivity, typability, reproducibility and discriminatory power based on internationally accepted methods for evaluation of microbial typing schemes.

Adolescent↗

PubMed2006

Heavy cryptosporidial infections in children in northeast Brazil: comparison of Cryptosporidium hominis and Cryptosporidium parvum.

Cryptosporidium is an important cause of infectious diarrhoea worldwide, but little is known about the course of illness when infected with different species. Over a period of 5 years, Cryptosporidium was identified in the stools of 58 of 157 children prospectively followed from birth in an urban slum (favela) in northeast Brazil. Forty isolates were available for quantification and 42 for speciation (24 Cryptosporidium hominis and 18 C. parvum). Children with C. hominis shed significantly more oocysts/ml of stool (3.5 x 10(6) vs. 1.7 x 10(6)perml; P=0.001), and oocyst counts were higher among symptomatic children (P=0.002). Heavier C. parvum shedding was significantly associated with symptoms (P=0.004), and symptomatic C. parvum-infected children were significantly more likely than asymptomatic children to be lactoferrin-positive (P=0.004). Height-for-age (HAZ) Z-scores showed significant declines within 3 months of infection for children infected with either C. hominis (P=0.028) or C. parvum (P=0.001). However, in the 3-6 month period following infection, only C. hominis-infected children continued to demonstrate declining HAZ score and asymptomatic children showed even greater decline (P=0.01). Cryptosporidium hominis is more common than C. parvum in favela children and is associated with heavier infections and greater growth shortfalls, even in the absence of symptoms.

Animals↗

PubMedDate not supplied

Comparison of the host ranges and antigenicity of Cryptosporidium parvum and Cryptosporidium wrairi from guinea pigs.

Oocysts of a Cryptosporidium isolate from guinea pigs were not infectious for adult mice, but were infectious for two of three newborn calves and for suckling mice. However, oocysts isolated from calves or mice infected with guinea pig Cryptosporidium were not infectious for guinea pigs. Four isolates of C. parvum from calves were incapable of infecting weanling guinea pigs. Microscopic examination of tissue from the colon and cecum of suckling guinea pigs inoculated with C. parvum revealed sparse infection of some pups. These host range studies and previously described differences in 125I-labeled oocyst surface protein profiles between Cryptosporidium sp. from guinea pigs and C. parvum suggest they are distinct species. We propose the name Cryptosporidium wrairi be retained. Studies with monoclonal antibodies indicate that C. wrairi and C. parvum are antigenically related.

Animals↗

PubMed2006

Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy.

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ x cm-2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4',6'-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ x cm-2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ x cm-2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ x cm-2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.

Animals↗

PubMed2005

Cryptosporidium infection in livestock and first identification of Cryptosporidium parvum genotype in cattle feces in Taiwan.

Fecal survey by modified Ziehl-Neelsen (MZN) method and immunofluorescence assay (IFA) of Cryptosporidium infection in cattle and goats in Taiwan showed a prevalence of 37.6% (173/460) and 35.8% (44/123), respectively. In addition to the calves, adult cattle were also found to be shedding Cryptosporidium oocyst. No significant difference was observed between diarrheic and non-diarrheic cattle feces with regard to the presence of Cryptosporidium oocyst. Two groups of oocysts with different diameter sizes, possibly indicating two different species, were observed in the cattle feces. By PCR analysis using primers directed against the 18S rRNA gene, followed by sequencing of the amplicon, we were able to confirm that one of the oocyst species belong to that of the bovine genotype of Cryptosporidium parvum. This is the first identification of a genotype of C. parvum oocyst in the feces of cattle in Taiwan.

Animals↗

PubMed2002

Successful in vitro cultivation of Cryptosporidium andersoni: evidence for the existence of novel extracellular stages in the life cycle and implications for the classification of Cryptosporidium.

The present study describes the complete development of all life cycle stages of Cryptosporidium andersoni in the HCT-8 cell line. The in vitro cultivation protocols were the same as those used for the successful growth of all life cycle stages of Cryptosporidium parvum (Int. J. Parasitol. 31 (2001) 1048). Under these culture conditions, C. andersoni grew and proliferated rapidly with the completion of the entire life cycle within 72h post-infection. The developmental stages of C. andersoni are larger than those of C. parvum enabling easier identification of life cycle stages including a previously unrecognised extracellular stage. The presence of this extracellular stage was further confirmed following its isolation from the faeces of infected cattle using a laser microdissection technique. This stage was present in large numbers and some of them were seen undergoing syzgy. Extraction of DNA from the extracellular stage, followed by polymerase chain reaction-restriction fragment length polymorphism and sequencing of the 18S rDNA confirmed that this is a stage in the life cycle of C. andersoni. In vitro, extracellular stages were always observed moving over the HCT-8 cells infected with C. andersoni. Comparative observations with C. parvum also confirmed the presence of extracellular stages. Extracellular stages were recovered from in vitro culture after 5 days post-infection with the cattle genotype of C. parvum and from infected mice. At least two morphologically different stages (stages one and two) were purified from mice after 72h of infection. The presence and morphological characterisation of extracellular developmental stages in the life cycle of Cryptosporidium confirms its relationship to gregarines and provides important implications for our understanding of the taxonomic and phylogenetic affinities of the genus Cryptosporidium. The growth of C. andersoni in cell culture now provides a means of studying its development, metabolism, and behaviour as well as testing its response to different therapeutic agents.

Animals↗