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.     

Molecular phylogeny and evolutionary relationships of Cryptosporidium parasites at the actin locus

To further validate previous observations in the taxonomy of Cryptosporidium parasites, the phylogenetic relationship was analyzed among various Cryptosporidium parasites at the actin locus. Nucleotide sequences of the actin gene were obtained from 9 putative Cryptosporidium species (C. parvum, C. andersoni, C. baileyi, C. felis, C. meleagridis, C. muris, C. saurophilum, C. serpentis, and C. wrairi) and various C. parvum genotypes. After multiple alignment of the obtained actin sequences, genetic distances were measured, and phylogenetic trees were constructed. Results of the analysis confirmed the presence of genetically distinct species within Cryptosporidium and various distinct genotypes within C. parvum. The phylogenetic tree constructed on the basis of the actin sequences was largely in agreement with previous results based on small subunit rRNA, 70-kDa heat shock protein, and Cryptosporidium oocyst wall protein genes. The Cryptosporidium species formed 2 major clades; isolates of C. andersoni, C. muris, and C. serpentis formed the first major group, whereas isolates of all other species, as well as various C. parvum genotypes, formed the second major group. Intragenotype variations were low or absent at this locus.     

Molecular analysis of the 18S rRNA gene of Cryptosporidium serpentis in a wild-caught corn snake (Elaphe guttata guttata) and a five-species restriction fragment length polymorphism- based assay that can additionally discern C. parvum from C. wrairi

An adult wild-caught corn snake (Elaphe guttata guttata) was presented for humane euthanasia and necropsy because of severe cryptosporidiosis. The animal was lethargic and >5% dehydrated but in good flesh. Gastric lavage was performed prior to euthanasia. Histopathologic findings included gastric mucosal hypertrophy and a hemorrhagic erosive gastritis. Numerous 5- to 7-microm-diameter round extracellular organisms were associated with the mucosal hypertrophy. A PCR, acid-fast stains, Giemsa stains, and an enzyme immunoassay were all positive for Cryptosporidium spp. PCR and restriction fragment length polymorphism (RFLP) analysis on gastric lavage and gastric mucosal specimens, and subsequent sequencing of the 18S rRNA gene, enabled a distinct molecular characterization of the infecting organism as Cryptosporidium serpentis. Until recently, studies on snake Cryptosporidium have relied on host specificity and gross and histopathologic observations to identify the infecting species. A multiple alignment of our sequence against recently published sequences of the 18S rRNA gene of C. serpentis (GenBank accession no. AF093499, AF093500, and AF093501 [L. Xiao et al., unpublished data, 1998]) revealed 100% homology with the C. serpentis (Snake) sequence (AF093499) previously described by Xiao et al. An RFLP method to differentiate the five presently sequenced strains of Cryptosporidium at this locus was developed. This assay, which uses SpeI and SspI, complements a previously reported assay by additionally distinguishing the bovine strain of Cryptosporidium from Cryptosporidium wrairi.     

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.     

Cryptosporidium varanii takes precedence over C. saurophilum

We present biological as well as genetic analysis of Cryptosporidium varanii at the 18S rRNA and actin loci and show that it is genetically identical to C. saurophilum. As C. varanii was described prior to C. saurophilum, it takes precedence over C. saurophilum and therefore C. saurophilum should be considered a junior synonym of C. varanii.     

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.     

Phylogenetic relationships of Cryptosporidium parasites based on the 70-kilodalton heat shock protein (HSP70) gene

We have characterized the nucleotide sequences of the 70-kDa heat shock protein (HSP70) genes of Cryptosporidium baileyi, C. felis, C. meleagridis, C. muris, C. serpentis, C. wrairi, and C. parvum from various animals. Results of the phylogenetic analysis revealed the presence of several genetically distinct species in the genus Cryptosporidium and eight distinct genotypes within the species C. parvum. Some of the latter may represent cryptic species. The phylogenetic tree constructed from these sequences is in agreement with our previous results based on the small-subunit rRNA genes of Cryptosporidium parasites. The Cryptosporidium species formed two major clades: isolates of C. muris and C. serpentis formed the first major group, while isolates of C. felis, C. meleagridis, C. wrairi, and eight genotypes of C. parvum formed the second major group. Sequence variations were also observed between C. muris isolates from ruminants and rodents. The HSP70 gene provides another useful locus for phylogenetic analysis of the genus Cryptosporidium.     

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.     

Sequence differences in the diagnostic target region of the oocyst wall protein gene of Cryptosporidium parasites

Nucleotide sequences of the Cryptosporidium oocyst wall protein (COWP) gene were obtained from various Cryptosporidium spp. (C. wrairi, C. felis, C. meleagridis, C. baileyi, C. andersoni, C. muris, and C. serpentis) and C. parvum genotypes (human, bovine, monkey, marsupial, ferret, mouse, pig, and dog). Significant diversity was observed among species and genotypes in the primer and target regions of a popular diagnostic PCR. These results provide useful information for COWP-based molecular differentiation of Cryptosporidium spp. and genotypes.     

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.     

Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'

Although it has been recognized that PCR amplification of mixed templates may generate sequence artifacts, the mechanisms of their formation, frequency and potential elimination have not been fully elucidated. Here evidence is presented for heteroduplexes as a major source of artifacts in mixed-template PCR. Nearly equal proportions of homoduplexes and heteroduplexes were observed after co-amplifying 16S rDNA from three bacterial genomes and analyzing products by constant denaturing capillary electrophoresis (CDCE). Heteroduplexes became increasingly prevalent as primers became limiting and/or template diversity was increased. A model exploring the fate of cloned heteroduplexes during MutHLS-mediated mismatch repair in the Escherichia coli host demonstrates that the diversity of artifactual sequences increases exponentially with the number of both variable nucleotides and of original sequence variants. Our model illustrates how minimization of heteroduplex molecules before cloning may reduce artificial genetic diversity detected during sequence analysis by clone screening. Thus, we developed a method to eliminate heteroduplexes from mixed-template PCR products by subjecting them to ‘reconditioning PCR’, a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product. This simple modification to the protocol may ensure that sequence richness encountered in clone libraries more closely reflects genetic diversity in the original sample.     

Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction fragment length polymorphism analysis, direct sequencing, and cloning

Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.     

Clonal polymerase chain reaction-single-strand conformational polymorphism analysis: an effective approach for identifying cloned sequences

The amplification of target sequences from genomic DNA can result in more than one amplicon sequence being produced even when highly specific primers are used. Here we present a clonal polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) approach for screening cloned amplicons and identifying particular clones prior to sequence determination. Comparison of the PCR-SSCP patterns of the cloned amplicons with the PCR-SSCP patterns observed for the DNA templates from which the clones were derived allows PCR artifacts, different alleles, and even different loci to be differentiated prior to sequencing. Using this approach, the number of clones required for reliable sequence determination is minimized, and complex “mixed” amplicons can be resolved easily, cost-effectively, and reliably.     

Cryptosporidium parvum infection involving novel genotypes in wildlife from lower New York State

Cryptosporidium, an enteric parasite of humans and a wide range of other mammals, presents numerous challenges to the supply of safe drinking water. We performed a wildlife survey, focusing on white-tailed deer and small mammals, to assess whether they may serve as environmental sources of Cryptosporidium. A PCR-based approach that permitted genetic characterization via sequence analysis was applied to wildlife fecal samples (n = 111) collected from September 1996 to July 1998 from three areas in lower New York State. Southern analysis revealed 22 fecal samples containing Cryptosporidium small-subunit (SSU) ribosomal DNA; these included 10 of 91 white-tailed deer (Odocoileus virginianus) samples, 3 of 5 chipmunk (Tamias striatus) samples, 1 of 2 white-footed mouse (Peromyscus leucopus) samples, 1 of 2 striped skunk (Mephitis mephitis) samples, 1 of 5 racoon (Procyon lotor) samples, and 6 of 6 muskrat (Ondatra zibethicus) samples. All of the 15 SSU PCR products sequenced were characterized as Cryptosporidium parvum; two were identical to genotype 2 (bovine), whereas the remainder belonged to two novel SSU sequence groups, designated genotypes 3 and 4. Genotype 3 comprised four deer-derived sequences, whereas genotype 4 included nine sequences from deer, mouse, chipmunk, and muskrat samples. PCR analysis was performed on the SSU-positive fecal samples for three other Cryptosporidium loci (dihydrofolate reductase, polythreonine-rich protein, and beta-tubulin), and 8 of 10 cloned PCR products were consistent with C. parvum genotype 2. These data provide evidence that there is sylvatic transmission of C. parvum involving deer and other small mammals. This study affirmed the importance of wildlife as potential sources of Cryptosporidium in the catchments of public water supplies.     

Phylogenetic analysis of Cryptosporidium isolates from captive reptiles using 18S rDNA sequence data and random amplified polymorphic DNA analysis.

Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P < 0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates.     

Phylogenetic analysis of the hypervariable region of the 18S rRNA gene of Cryptosporidium oocysts in feces of Canada geese (Branta canadensis): evidence for five novel genotypes

To assess genetic diversity in Cryptosporidium oocysts from Canada geese, 161 fecal samples from Canada geese in the United States were analyzed. Eleven (6.8%) were positive for Cryptosporidium spp. following nested PCR amplification of the hypervariable region of the 18S rRNA gene. Nine PCR products from geese were cloned and sequenced, and all nine diverged from previously reported Cryptosporidium 18S rRNA gene sequences. Five sequences were very similar or identical to each other but genetically distinct from that of Cryptosporidium baileyi; two were most closely related to, but genetically distinct from, the first five; and two were distinct from any other sequence analyzed. One additional sequence in the hypervariable region of the 18S rRNA gene isolated from a cormorant was identical to that of C. baileyi. Phylogenetic analysis provided evidence for new genotypes of Cryptosporidium species in Canada geese. Results of this study suggest that the taxonomy of Cryptosporidium species in geese is complex and that a more complete understanding of genetic diversity among these parasites will facilitate our understanding of oocyst sources and species in the environment.     

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.     

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.     

Recombination among multiple mitochondrial pseudogenes from a Passerine genus

PCR products of a fragment of the mitchondrial protein coding subunit 5 of NADH-dehydrogenase (ND5) from eight individuals representing five species of the South American bird genus Conirostrum were cloned. The 130 clones, which were subsequently sequenced, constituted 55 different sequences. Due to the observed differences in substitution patterns 58% of the cloned sequences were identified as pseudogenes. Recombination could be traced in 19% of the inferred nuclear pseudogenes, but this figure probably represents a significant underestimation of the factual recombination events. The nonrecombined pseudogenes consisted of multiple haplotypes found to diverge from 1 to 16% from the mitochondrial gene. The number of mitochondrial nuclear copies and their apparent frequent recombination suggest that pseudogenes constitute a serious potential risk in confounding phylogenetic studies and population genetic analysis.     

我国蛇类常见寄生虫及其对人类健康的影响

目的研究我国蛇类常见寄生虫对人类健康的影响。方法收集和分析国内外蛇类寄生虫对人类健康影响的研究资料。结果我国常见危害人类健康的蛇源寄生虫有6种,即舌形虫、曼氏迭宫绦虫、隐孢子虫、颚口线虫、广州管圆线虫、线中殖孔绦虫,均为人兽共患性寄生虫,对人类健康造成危害。结论通过加强科普宣传,加强科研工作,加强执法监管,规范蛇类养殖产业,可有效防控蛇类常见寄生虫对人类健康的威胁。