Genetic diversity of Cryptosporidium spp. in captive reptiles

The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium: Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.     

Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus

Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.     

The identification of Cryptosporidium species (protozoa) in Ifsahan, Iran by PCR-RFLP analysis of the 18S rRNA gene

Cryptosporidium is an important protozoan that cause diarrheal illness in humans and animals. Different species of Cryptosporidium have been reported and it is believed that species characteristics are an important factor to be considered in strategic planning for control. We therefore analyzed oocysts from human and animal isolates of Cryptosporidium by PCR-RFLP to determine strain variation in Isfahan. In total, 642 human fecal samples from children under five years of age, immunocompromised patients, and high risk persons and 480 randomly selected rectal specimens of cows and calves in Isfahan were examined. Microscopic examination showed that 4.7% (30/642) of human samples and 6.2% (30/480) of animal samples were infected with Cryptosporidium. After identification of the samples infected with the parasite, oocysts were purified and their DNA was extracted. We used PCR-RFLP analysis of a 1750-bp region of 18S rRNA gene to identify Cryptosporidium species. The human samples were infected with Cryptosporidium parvum II, C. muris, C. wrairi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520951). The cattle samples were identified as C. parvum II, C. muris, C. wrairi, C. serpentis, C. baileyi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520952). Also we found a new genotype infecting both human and cattle samples (GenBank accession numbers: DQ520950). In addition to demonstrating the widespread occurrence of most species of Cryptosporidium, C. parvum, we also observed extensive polymorphism within species. Furthermore, the occurrence of the same species of parasite in both animal and human samples shows the importance of the animal-human cycle.     

Cryptosporidium in farmed animals: the detection of a novel isolate in sheep.

We describe the discovery of polymorphisms in the Cryptosporidium oocyst wall protein (COWP) gene conferring a novel restriction fragment length polymorphism (RFLP) pattern in 26/60 (43%) isolates from a flock of sheep sampled following a waterborne outbreak of human cryptosporidiosis. The sheep isolates showed identical PCR-RFLP patterns to each other by COWP genotyping but different from those of most currently recognised genotypes, including the major Cryptosporidium parvum genotypes 1 and 2. Sequence analysis of the 550bp amplicon from the COWP gene was compared with a DNA coding region employed in previous studies and showed the novel isolate to differ from other Cryptosporidium species and C. parvum isolates by 7-21%. The sheep-derived isolates were compared at this and further three Cryptosporidium gene loci with isolates from other farmed animals. The loci employed were one in the thrombospondin related adhesive protein (TRAP-C2) gene and two in the 70kDa heat shock protein (HSP70) gene (CPHSP1 and 2). Other animal samples tested in our laboratory were from clinically ill animals and all contained C. parvum genotype 2. The sheep in which the novel isolate was identified were healthy and showed no symptoms of cryptosporidiosis, and the novel sheep isolate could represent a non-pathogenic strain. Our studies suggest that a previously undetected Cryptosporidium sub-type may exist in sheep populations, reflecting the increasingly recognised diversity within the parasite genus.     

Cryptosporidium viatorum n. sp. (Apicomplexa: Cryptosporidiidae) among travellers returning to Great Britain from the Indian subcontinent, 2007-2011

A novel Cryptosporidium genotype was identified, among travellers with gastro-intestinal symptoms returning to Great Britain from the Indian subcontinent, for which we propose the name Cryptosporidium viatorum n. sp. The epidemiology of these cases was distinctly different from those with Cryptosporidium parvum and Cryptosporidium hominis. Of the 10 cases identified involving C. viatorum, most were in the first quarter of the year. One occurred in 2007, one in 2008, three in 2010 and five to end March 2011. The median age was 19 years but most were in the 20-29 years age group and seven were male. The symptoms included diarrhoea, abdominal pain, nausea, vomiting and fever. Compared with cases due to C. hominis and C. parvum, vomiting was reported less often, although the duration of gastro-intestinal symptoms was longer. The cases of C. viatorum were all travellers to the Indian subcontinent, whereas cases of C. hominis and C. parvum were more likely to have travelled elsewhere. Cryptosporidium viatorum isolates had indistinguishable sequences at each of the 70 kDa heat shock protein (HSP70), actin and ssrRNA loci which did not match any published previously and, although phylogenetically most similar to Cryptosporidium fayeri, they were distinct (<98% similarity) at the ssrRNA, HSP70 and actin genes. Morphologically, oocysts were typical of predominantly human-infecting species. Cryptosporidium viatorum n. sp. is proposed and work is warranted to investigate further the public health significance and occurrence elsewhere of this emerging parasite.     

Potassium uptake and retention by Oceanomonas baumannii at low water activity in the presence of phenol

A polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of a 587-bp region of the Cryptosporidium parvum 70-kDa heat shock protein (HSP70) gene was developed for the detection and discrimination of the two major genotypes of C. parvum, genotype 1 and genotype 2. Ten Cryptosporidium isolates from non-immunocompromised people were identified as genotypes 1 and 2 (five each) by DNA sequencing of the 587-bp PCR product. This distinction was also achieved with the combination of two endonucleases, HinfI and ScaI, which generated a specific pattern for each genotype. A thorough screening of published sequences showed that this combination of enzymes could also be used for the discrimination of other species/genotypes of Cryptosporidium, especially Cryptosporidium meleagridis and the 'dog' genotype of C. parvum, both of which are infectious in humans. The PCR, conducted on genotypes 1 and 2 of C. parvum, could detect one oocyst per reaction. This new and sensitive genotyping procedure should be of particular interest when applied to the monitoring of water resources in which low concentrations of parasites usually occur.     

Morphologic, host specificity, and genetic characterization of a European Cryptosporidium andersoni isolate

This study was undertaken in order to characterize a Cryptosporidium muris-like parasite isolated from cattle in Hungary and to compare this strain with other Cryptosporidium species. To date, the large-type oocysts isolated from cattle were considered as C. muris described from several mammals. The size, form, and structure of the oocysts of the Hungarian strain were identical with those described by others from cattle. An apparent difference between the morphometric data of C. muris-like parasites isolated from cattle or other mammals was noted, which is similar in magnitude to the differences between Cryptosporidium meleagridis and Cryptosporidium felis or between Cryptosporidium serpentis and Cryptosporidium baileyi. The cross-transmission experiments confirmed the findings of others, as C. muris-like oocysts isolated from cattle fail to infect other mammals. The sequence of the variable region of small subunit (SSU) rRNA gene of the strain was 100% identical with that of the U.S. Cryptosporidium andersoni and C. andersoni-like isolates from cattle. The difference between the SSU rRNA sequence of bovine strains and C. muris is similar in magnitude to the differences between C. meleagridis and Cryptosporidium parvum anthroponotic genotype or between Cryptosporidium wrairi and C. parvum zoonotic genotype. Our findings confirm that the Cryptosporidium species responsible for abomasal cryptosporidiosis and economic losses in the cattle industry should be considered a distinct species, C. andersoni Lindsay, Upton, Owens, Morgan, Mead, and Blagburn, 2000.     

Morphology is not a reliable tool for delineating species within Cryptosporidium

Within the coccidia, morphological features of the oocyst stage at the light microscope level have been used more than any other single characteristic to designate genus and species. The aim of this study was to conduct morphometric analysis on a range of Cryptosporidium spp. isolates and to compare morphological data between several genotypes of C. parvum and a second species C. canis, as well as a variation within a specific genotype (the human genotype), with genetic data at 2 unlinked loci (18S ribonucleic deoxyribonucleic acid and HSP 70) to evaluate the usefulness of morphometric data in delineating species within Cryptosporidium. Results indicate that morphology could not differentiate between oocysts from C. parvum genotypes and oocysts from C. canis, whereas genetic analysis clearly differentiated between the two. The small size of the Cryptosporidium spp. oocyst, combined with the very limited characters for analysis, suggests that more reliance should be placed on genetic differences, combined with biological variation, when delineating species within Cryptosporidium.     

Cryptosporidium canis n. sp. from domestic dogs.

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.     

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

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

Biallelic polymorphism in the intron region of beta-tubulin gene of Cryptosporidium parasites

Nucleotide sequencing of polymerase chain reaction amplified intron region of the Cryptosporidium parvum beta-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is in agreement with our previous genotyping data based on the thrombospondin-related adhesion protein (TRAP-C2) gene, indicating these genotype characteristics are linked at 2 genetic loci. Characterization of Cryptosporidium muris and Cryptosporidium serpentis has further shown that non-parvum Cryptosporidium parasites have beta-tubulin intron sequences identical to bovine genotype of C. parvum. Thus, results of this study confirm the lineage of 2 genotypes of C. parvum at 2 genetic loci and suggest a need for extensive characterization of various Cryptosporidium spp.     

Genotyping Cryptosporidium parvum with an hsp70 single-nucleotide polymorphism microarray

We investigated the application of an oligonucleotide microarray to (i) specifically detect Cryptosporidium spp., (ii) differentiate between closely related C. parvum isolates and Cryptosporidium species, and (iii) differentiate between principle genotypes known to infect humans. A microarray of 68 capture probes targeting seven single-nucleotide polymorphisms (SNPs) within a 190-bp region of the hsp70 gene of Cryptosporidium parvum was constructed. Labeled hsp70 targets were generated by PCR with biotin- or Cy3-labeled primers. Hybridization conditions were optimized for hybridization time, temperature, and salt concentration. Two genotype I C. parvum isolates (TU502 and UG502), two C. parvum genotype II isolates (Iowa and GCH1), and DNAs from 22 non-Cryptosporidium sp. organisms were used to test method specificity. Only DNAs from C. parvum isolates produced labeled amplicons that could be hybridized to and detected on the array. Hybridization patterns between genotypes were visually distinct, but identification of SNPs required statistical analysis of the signal intensity data. The results indicated that correct mismatch discrimination could be achieved for all seven SNPs for the UG502 isolate, five of seven SNPs for the TU502 isolate, and six of seven SNPs for both the Iowa and GCH1 isolates. Even without perfect mismatch discrimination, the microarray method unambiguously distinguished between genotype I and genotype II isolates and demonstrated the potential to differentiate between other isolates and species on a single microarray. This method may provide a powerful new tool for water utilities and public health officials for assessing point and nonpoint source contamination of water supplies.     

An Evaluation of Molecular Diagnostic Tools for the Detection and Differentiation of Human-Pathogenic Cryptosporidium spp.

ABSTRACT: The performance of 10 commonly used genotyping tools in the detection and differentiation of 7 human-pathogenic Cryptosporidium spp. ( C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. muris and Cryptosporidium pig genotype I) was evaluated. All 3 SU rRNA gene-based tools could amplify the DNA of 7 Cryptosporidium spp. efficiently. However, the tools based on the antigens TRAP-C1, TRAP-C2 and COWP genes, the housekeeping genes HSP70 and DHFR, or a genomic sequence, failed to detect the DNA of C. felis, C. canis, Cryptosporidium pig genotype I, and C. metris. With the exception of 1 tool based on the TRAP-C2 gene, the PCR-RFLP or the PCR sequencing tools evaluated in this study could differentiate C. hominis, C. parvum and C. meleagridis from each other, and 2 SSU rRNA genebased tools could differentiate all 7 Cryptosporidium spp. Thus, a thorough understanding of the strength and weakness of each technique is needed when using molecular diagnostic tool in epidemiological investigations of human cryptosporidiosis.     

Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture.

This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle genotypes) in the HCT-8 cell line. Furthermore, for the first time the complete life cycle was perpetuated in vitro for up to 25 days by subculturing. The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-reinfection cycle of C. parvum. This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro. In addition, thin-walled oocysts of the cattle genotype were infective for ARC/Swiss mice but similar oocysts of the human genotype were not. This culture system will provide a model for propagation of the complete life cycle of C. parvum in vitro.     

Cryptosporidium bovis n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus)

A new species of Cryptosporidium, C. bovis, is described. Oocysts of C. bovis, previously identified as Cryptosporidium genotype Bovine B (GenBank AY120911), are morphologically indistinguishable from those of C. parvum. They are excreted fully sporulated and contain 4 sporozoites, but lack sporocysts. Oocysts measure 4.76-5.35 microm (mean = 4.89 microm) x 4.17-4.76 microm (mean = 4.63 microm), with a length-to-width ratio of 1.06 (n = 50). Oocysts were not infectious for neonatal BALB/ c mice, but were infectious for 2 calves that were previously infected with C. parvum. Oocysts were not infectious for 2 experimentally exposed lambs less than 1 wk of age and were not detected in 42 lambs 2-3 mo of age, but were detected in a 2-wk-old lamb. In an earlier study, 79 of 840 calves on 14 dairy farms in 7 states were found infected with the new species. Most calves were 2-7 mo of age and none exhibited signs of diarrhea. This new species has been found in 10 of 162 calves aged 9 to 11 mo on a beef farm in Maryland. Fragments of the 18S rDNA, HSP-70, and actin genes were amplified by PCR, and purified PCR products were sequenced. Multilocus analysis of the 3 unlinked loci demonstrated the new species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further evidence of species status. Based on these biological and molecular data, we consider this highly prevalent Cryptosporidium that infects primarily postweaned calves to be a new species and propose the name Cryptosporidium bovis n. sp. for this parasite.     

Tracking host sources of Cryptosporidium spp. in raw water for improved health risk assessment

Recent molecular evidence suggests that different species and/or genotypes of Cryptosporidium display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of Cryptosporidium species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of Cryptosporidium parasites impacting water within the region. Cryptosporidium andersoni, Cryptosporidium muskrat genotype II, Cryptosporidium cervine genotype, C. baileyi, C. parvum, Cryptosporidium muskrat genotype I, the Cryptosporidium fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the Cryptosporidium parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of Cryptosporidium parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of Cryptosporidium.     

Morphologic, host specificity, and molecular characterization of a Hungarian Cryptosporidium meleagridis isolate

This study was undertaken in order to characterize Cryptosporidium meleagridis isolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and other Cryptosporidium species. The phenotypic differences between C. meleagridis and Cryptosporidium parvum Hungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts of C. meleagridis were successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location of C. meleagridis was the small intestine, like the location of C. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene of C. meleagridis isolated from turkeys with other Cryptosporidium sequence data in the GenBank database revealed that the Hungarian C. meleagridis sequence is identical to a C. meleagridis sequence recently described for a North Carolina isolate. Thus, C. meleagridis is a distinct species that occurs worldwide and has a broad host range, like the C. parvum zoonotic strain (also called the calf or bovine strain) and Cryptosporidium felis. Because birds are susceptible to C. meleagridis and to some zoonotic strains of C. parvum, these animals may play an active role in contamination of surface waters not only with Cryptosporidium baileyi but also with C. parvum-like parasites.