Evaluation of Cryptosporidium parvum genotyping techniques.

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 Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.     

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.     

Molecular Fingerprinting of Cryptosporidium Oocysts Isolated during Water Monitoring

We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.     

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.     

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.     

Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.     

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.     

Comparative Sensitivity of PCR Primer Sets for Detection of Cryptosporidium parvum

Improved methods for detection of Cryptosporidium oocysts in environmental and clinical samples are urgently needed to improve detection of cryptosporidiosis. We compared the sensitivity of 7 PCR primer sets for detection of Cryptosporidium parvum. Each target gene was amplified by PCR or nested PCR with serially diluted DNA extracted from purified C. parvum oocysts. The target genes included Cryptosporidium oocyst wall protein (COWP), small subunit ribosomal RNA (SSU rRNA), and random amplified polymorphic DNA. The detection limit of the PCR method ranged from 10³ to 10⁴ oocysts, and the nested PCR method was able to detect 10⁰ to 10² oocysts. A second-round amplification of target genes showed that the nested primer set specific for the COWP gene proved to be the most sensitive one compared to the other primer sets tested in this study and would therefore be useful for the detection of C. parvum.     

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.     

Host-Adapted Cryptosporidium spp. in Canada Geese (Branta canadensis)

The prevalence and distribution of Cryptosporidium spp. in the fecal droppings of the free-living waterfowl Canada geese were examined at 13 sites in Ohio and Illinois. On the basis of the analysis of the small-subunit rRNA gene by PCR, followed by restriction fragment length polymorphism analysis and DNA sequencing, 49 (23.4%) of 209 fecal specimens collected from 10 sites (76.9%) were positive for Cryptosporidium spp. The following five Cryptosporidium species and genotypes were identified: Cryptosporidium goose genotype I (in 36 specimens), Cryptosporidium goose genotype II (in 9 specimens), Cryptosporidium duck genotype (in 1 specimen), Cryptosporidium parvum (in 4 specimens), and C. hominis (in 2 specimens). Cryptosporidium goose genotype I was the most prevalent parasite and was found at all five Cryptosporidium-positive sites in Ohio and at four of five positive sites in Illinois, followed by Cryptosporidium goose genotype II, which was found at two of five positive sites in Ohio and at four of five positive sites in Illinois. Cryptosporidium goose genotype II was detected for the first time, and it is phylogenetically related to goose genotype I and the duck genotype. All three genotypes have not so far been reported in humans, and their pathogenicity in geese has not been determined. Only 10.2% of the Cryptosporidium-positive specimens had C. parvum and C. hominis. The results of this study indicate that Canada geese might only serve as accidental carriers of cryptosporidia infectious to humans and probably play a minor role in the animal-to-human transmission cycle of the pathogen.     

The CpA135 gene as a marker to identify Cryptosporidium species infecting humans

Of the 22 species currently recognized as valid in the Cryptosporidium genus, C. parvum and C. hominis account for most cases of human infections worldwide. However, C. meleagridis, C. canis, C. felis, C. suis, C. muris, as well as the cervine, rabbit and monkey Cryptosporidium genotypes, have also been recognized as the etiologic cause of both sporadic and epidemic cryptosporidiosis in humans. Molecular methods are necessary to distinguish species and genotypes of Cryptosporidium, due to the lack of reliable morphological variations. The aim of this work was to determine the genetic polymorphisms in a fragment of the A135 gene in isolates of C. parvum, C. hominis, C. meleagridis, C. canis, C. muris, C. andersoni and the Cryptosporidium cervine genotype. Primers were designed on conserved regions identified on a multiple alignment of the C. parvum, C. hominis and C. muris sequences, the three species for which information is available at the genome level. PCR amplification and direct sequencing of a 576 bp fragment revealed the presence of numerous single nucleotide polymorphisms (SNPs) among the species/genotype tested. The genetic variability was exploited to design a PCR-RFLP assay useful for a rapid identification of the most important human pathogens in the genus Cryptosporidium.     

Comparison of Single- and Multilocus Genetic Diversity in the Protozoan Parasites Cryptosporidium parvum and C. hominis

The genotyping of numerous isolates of Cryptosporidium parasites has led to the definition of new species and a better understanding of the epidemiology of cryptosporidiosis. A single-locus genotyping method based on the partial sequence of a polymorphic sporozoite surface glycoprotein gene (GP60) has been favored by many for surveying Cryptosporidium parvum and C. hominis populations. Since genetically distinct Cryptosporidium parasites recombine in nature, it is unclear whether single-locus classifications can adequately represent intraspecies diversity. To address this question, we investigated whether multilocus genotypes of C. parvum and C. hominis cluster according to the GP60 genotype. C. hominis multilocus genotypes did not segregate according to this marker, indicating that for this species the GP60 sequence is not a valid surrogate for multilocus typing methods. In contrast, in C. parvum the previously described “anthroponotic” genotype was confirmed as a genetically distinct subspecies cluster characterized by a diagnostic GP60 allele. However, as in C. hominis, several C. parvum GP60 alleles did not correlate with distinct subpopulations. Given the rarity of some C. parvum GP60 alleles in our sample, the existence of additional C. parvum subgroups with unique GP60 alleles cannot be ruled out. We conclude that with the exception of genotypically distinct C. parvum subgroups, multilocus genotyping methods are needed to characterize C. parvum and C. hominis populations. Unless parasite virulence is controlled at the GP60 locus, attempts to find associations within species or subspecies between GP60 and phenotype are unlikely to be successful.The lack of variable morphological traits to identify oocysts from different Cryptosporidium species has driven the development of numerous genotyping methods to survey the diversity in this genus. Genetic markers such as single-nucleotide polymorphisms (24), restriction fragment length polymorphisms (7, 34), random amplification methods (17, 20), conformational polymorphisms (11), simple sequence repeats (3, 10), and DNA sequence polymorphisms (6, 36) have been used to type Cryptosporidium oocysts excreted by humans and animals and oocysts recovered from the environment. This effort has led to a deeper understanding of the taxonomy of the genus Cryptosporidium and the epidemiology of cryptosporidiosis in humans and livestock. As a result of this work, two species responsible for a majority of human infections, Cryptosporidium parvum and C. hominis, were identified (21) and our understanding of the taxonomy of the genus was refined (35).The application of genetic markers to define species, i.e., reproductively isolated populations, is straightforward. At this taxonomic level, all genotypes cosegregate and the choice of marker will have little impact on the outcome, provided that the marker, or combination thereof, is sufficiently polymorphic. The classical example is the variable region of the small-subunit rRNA gene which has been used, as in other taxa, to define many Cryptosporidium species. For studying intraspecies polymorphism, the choice of genotyping methods needs to take into consideration the potential for genetic recombination. This is clearly the case for species such as those belonging to the genus Cryptosporidium, which are known to undergo an obligatory sexual cycle during which genetically dissimilar haplotypes can recombine (28).Among the many markers that have been applied in epidemiological surveys of C. parvum and C. hominis, a variable fragment of the gene encoding a sporozoite surface glycoprotein (8, 26) has been particularly popular. As a result of the widespread adoption of this marker, variously named GP60, cpgp40/15, or gp40, numerous alleles have been identified and deposited in GenBank. The analysis of this continuously growing collection of GP60 sequences has led to the identification of groups of related sequences (18, 27, 32, 33). In an attempt to simplify the comparison of GP60 genotypes among different laboratories, a GP60 nomenclature distinguishing the main groups of alleles has been created (26) and later refined (27).The desire to streamline the genotyping of large numbers of Cryptosporidium isolates collected during surveys has led to the widespread adoption of the GP60 genotype as the only marker for defining intraspecies groups. Since this approach is not compatible with the reassortment of unlinked loci, the classification of isolates on the basis of the GP60 genotype, or any other single marker, needs to be evaluated. Within a recombining population, no single genetic marker can a priori be expected to serve as a surrogate for other loci or multilocus genotypes (MLGs), and any apparent clustering of isolates is dependent on the marker. To investigate the validity of the GP60 genotyping method as commonly applied to the classification of C. parvum and C. hominis isolates, the GP60 genotype was added to a previously described 9-locus genotype (29) and a diversified collection of 10-locus genotypes was examined for intraspecies clusters. We show that, with the exception of some GP60 alleles apparently restricted to human C. parvum, neither C. parvum nor C. hominis GP60 alleles define subspecies genotypes. These results are discussed in the context of ongoing research to better understand the population structure of these parasites and identify genotypes associated with virulence traits.     

Genetic Characterization and Transmission Cycles of Cryptosporidium Species Isolated from Humans in New Zealand

Little is known about the genetic characteristics, distribution, and transmission cycles of Cryptosporidium species that cause human disease in New Zealand. To address these questions, 423 fecal specimens containing Cryptosporidium oocysts and obtained from different regions were examined by the PCR-restriction fragment length polymorphism technique. Indeterminant results were resolved by DNA sequence analysis. Two regions supplied the majority of isolates: one rural and one urban. Overall, Cryptosporidium hominis accounted for 47% of the isolates, with the remaining 53% being the C. parvum bovine genotype. A difference, however, was observed between the Cryptosporidium species from rural and urban isolates, with C. hominis dominant in the urban region, whereas the C. parvum bovine genotype was prevalent in rural New Zealand. A shift in transmission cycles was detected between seasons, with an anthroponotic cycle in autumn and a zoonotic cycle in spring. A novel Cryptosporidium sp., which on DNA sequence analysis showed a close relationship with C. canis, was detected in two unrelated children from different regions, illustrating the genetic diversity within this genus.     

Failure To Differentiate Cryptosporidium parvum from C. meleagridis Based on PCR Amplification of Eight DNA Sequences

In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of the Cryptosporidium genome were evaluated for the detection of C. parvum, the agent of human cryptosporidiosis, and C. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridis couldn’t be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.     

Targeting Single-Nucleotide Polymorphisms in the 18S rRNA Gene To Differentiate Cyclospora Species from Eimeria Species by Multiplex PCR

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3′ end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.     

First Molecular Characterization of Cryptosporidium spp. Infecting Buffalo Calves in Brazil

With the aim of determining the occurrence of Cryptosporidium spp., 222 fecal samples were collected from Murrah buffalo calves aged up to 6 mo. Fecal DNA was genotyped with a nested polymerase chain reaction targeting the 18S rRNA gene and sequencing of the amplified fragment. Nested 18S PCR was positive for 48.2% of the samples. Sequence analysis showed that the most frequent species in these animals was Cryptosporidium ryanae, which was present in buffalo calves as young as 5 d. The zoonotic species Cryptosporidium parvum was detected in one animal. An uncommon Cryptosporidium 18S genotype was found in buffaloes.     

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.     

Cryptosporidium in wild placental mammals

Cryptosporidium species are common parasites of wild placental mammals. Recent parasitological studies combined with molecular genotyping techniques have been providing valuable new insight into the host specificity and potential transmission of various Cryptosporidium species/genotypes among animals and between these animals and humans. Although Cryptosporidium in wild animals may possess a potential public health problem due to oocyst contamination in the environment, studies at various regions of the world have indicated a strong host-adaptation by these parasites and a limited potential of cross-species transmission of cryptosporidiosis among placental mammals, suggesting that these animals are probably not a major reservoir for human infection. However, Cryptosporidium species/genotypes in placental animals have been reported occasionally in humans. Therefore, public health significance of some Cryptosporidium species in wild placental mammals, such as the cervine genotype, should not be overlooked and should be further studied.     

Identification of Cryptosporidium Species and Genotypes in Scottish Raw and Drinking Waters during a One-Year Monitoring Period

We analyzed 1,042 Cryptosporidium oocyst-positive slides (456 from raw waters and 586 from drinking waters) of which 55.7% contained 1 or 2 oocysts, to determine species/genotypes present in Scottish waters. Two nested PCR-restriction fragment length polymorphism (RFLP) assays targeting different loci (1 and 2) of the hypervariable region of the 18S rRNA gene were used for species identification, and 62.4% of samples were amplified with at least one of the PCR assays. More samples (577 slides; 48.7% from raw water and 51.3% from drinking water) were amplified at locus 1 than at locus 2 (419 slides; 50.1% from raw water and 49.9% from drinking water). PCR at loci 1 and 2 amplified 45.4% and 31.7% of samples containing 1 or 2 oocysts, respectively. We detected both human-infectious and non-human-infectious species/genotype oocysts in Scottish raw and drinking waters. Cryptosporidium andersoni, Cryptosporidium parvum, and the Cryptosporidium cervine genotype (now Cryptosporidium ubiquitum) were most commonly detected in both raw and drinking waters, with C. ubiquitum being most common in drinking waters (12.5%) followed by C. parvum (4.2%) and C. andersoni (4.0%). Numerous samples (16.6% total; 18.9% from drinking water) contained mixtures of two or more species/genotypes, and we describe strategies for unraveling their identity. Repetitive analysis for discriminating mixtures proved useful, but both template concentration and PCR assay influenced outcomes. Five novel Cryptosporidium spp. (SW1 to SW5) were identified by RFLP/sequencing, and Cryptosporidium sp. SW1 was the fourth most common contaminant of Scottish drinking water (3%).The protozoan parasite Cryptosporidium has been implicated in numerous waterborne and food-borne outbreaks of cryptosporidiosis (3, 6, 16, 17, 18). Currently, there are 22 valid Cryptosporidium species: Cryptosporidium hominis, infecting mainly humans; C. parvum, in humans and numerous other mammals, including cattle; C. andersoni, C. bovis (previously bovine genotype B), and C. ryanae (previously deer-like genotype) in cattle; C. xiaoi (previously bovis-like genotype) in sheep; C. muris in mice; C. felis in cats; C. suis (previously pig genotype I) in pigs; C. wrairi in guinea pigs; C. canis in dogs; C. meleagridis and C. baileyi in birds; C. galli in finches and chickens; C. fayeri (previously marsupial genotype I) and C. macropodum (previously marsupial genotype II) in various species of marsupials; C. fragile in toads; C. varanii (previously C. saurophilum) in lizards and snakes; C. serpentis in snakes; C. scophthalmi and C. molnari in fish (20); and C. ubiquitum (previously Cryptosporidium cervine genotype) in a wide variety of host species, including white-tailed deer, sheep, cattle, goat, mouse, various species of rodents, and humans (4). In addition, there are over 60 Cryptosporidium genotypes, which differ significantly in their molecular sequences but, as yet, have not been ascribed species status (13, 29).Genetic analyses reveal that at least eight species (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. suis, C. muris, and C. ubiquitum) and seven Cryptosporidium genotypes (C. hominis monkey, C. andersoni-like, and Cryptosporidium chipmunk I, skunk, horse, rabbit, and pig genotype II) are associated with human disease (1, 9, 22), but C. parvum and C. hominis remain the most common species infecting humans. Environmental contamination with oocysts of Cryptosporidium species that are not infectious to susceptible human hosts contributes to the difficulties in assessing the risk to public health from waterborne oocysts.Oocysts occur at low densities in water (16, 17, 21), and molecular methods which can genotype small numbers of organisms reliably and reproducibly from water concentrates are required to determine which species occur, and with what frequency, in water. We used our standardized, maximized freezing and thawing method for DNA extraction (10) and our procedure for retrieving oocysts from Cryptosporidium water monitoring slides to maximize DNA extraction for PCR-restriction fragment length polymorphism (RFLP) analysis (11, 12, 19) in this study.We undertook a 1-year survey to identify the species and genotypes of Cryptosporidium oocysts detected in the Scottish Water (SW) Routine Cryptosporidium Monitoring Programme to gain information on the occurrence and diversity of Cryptosporidium oocysts in drinking water sources and drinking waters in order to determine predominant types in water catchment areas and monitor variations in oocyst population distribution over a 1-year period with a view to adding value to current assessments of risk to human health.     

Identification of Cryptosporidium felis in a Cow by Morphologic and Molecular Methods

Apicomplexan Cryptosporidium parasites infect a wide range of vertebrate hosts. While some species are limited to a single host group, such as Cryptosporidium baileyi, which infects chickens, other species of this genus, such as C. parvum, infect a wide range of mammalian species from mice to humans. During an investigation of Cryptosporidium infection in cattle on a farm in northern Poland, we identified an infection caused by C. felis, in addition to known infections with C. muris and C. parvum. This new infection was identified based on the size of the oocysts (mean size, 4.3 ± 0.4 μm; range, 3.5 to 5.0 μm), as well as by analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding demonstrates the complex host specificity and circulation in the environment of Cryptosporidium species.