Specific and genotypic identification of <b><i>Cryptosporidium</i></b> from a broad range of host species by nonisotopic SSCP analysis of nuclear ribosomal DNA

Jex, Aaron R.; Ryan, Una; Ng, Josephine; Campbell, Bronwyn E.; Xiao, Lihua; Stevens, Melita; Gasser, Robin B.

doi:10.1002/elps.200600772

Cited by 30 publications

(23 citation statements)

References 53 publications

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“…Parasitol., 10 (4): 127-141, 2015 In accordance, during the present study, 823 bp fragments amplified from the 18S SSU rRNA gene could be noted after nested PCR reaction from buffalo's feces. Previous studies indicated the usefulness of the small subunit (SSU) ribosomal RNA genes as genetic markers for the specific identification of Cryptosporidium having relatively low intraspecific and relatively high interspecific sequence variation (Fayer et al, 2000;Xiao et al, 2004;Jex et al, 2007). Thus, they had been utilized in systematic (phylogenetic) investigations of Cryptosporidium providing the basis for the current classification of members within the genus (Morgan et al, 1999a;Xiao et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Bubaline Isolate of Cryptosporidium Species from Egypt

Abdelrahma¹,

Megeed²,

Hammam³

et al. 2015

Research J. of Parasitology

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Cryptosporidium an apicomplexan parasite has the ability to induce diarrhea in bovines, goats, pigs, dogs and cats worldwide. In this study, buffalo calves fecal samples were examined after staining their smears with Modified Ziehl-Neelsen Stain (MZN). Ileal sections were examined for the detection of pathological changes. Further molecular characterization was done using nested PCR amplification and partial sequence analysis. The detected oocysts were morphologically similar to Cryptosporidium parvum. Light microscopic examination of Cryptosporidium infected ileal Tissue Section (TS) stained with H and E revealed the presence of altered mucosal architecture with congestion of blood vessels, infiltration, sloughing and complete erosion of epithelial cells and shortening, blunting, stunting and atrophy of the intestinal villi. Molecular characterization gave PCR amplicons of 18S SSU rRNA gene products approximately at 823 bp. Sequences proved specified generalized relatedness with 21 species of Cryptosporidium but the nucleotide homogeneity percentage was insufficient to designate species or genotypes. Further bioinformatics analysis showed that resulting Cryptosporidium isolates had the closest match with three isolates. It was implied that the Cryptosporidium isolates is mostly like Cryptosporidium parvum (JX237832.1) previously isolated from buffaloes in Ismailia province.

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Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Bubaline Isolate of Cryptosporidium Species from Egypt

Abdelrahma¹,

Megeed²,

Hammam³

et al. 2015

Research J. of Parasitology

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“…Molecular methods, particularly those based on the PCR, are being utilized increasingly, as they are often more sensitive and more specific than microscopic methods [39][40][41]. Employing genetic markers, such as the small ribosomal subunit (SSU or 18S) and second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA, Cryptosporidium can be accurately identified to and differentiated at the species level [4,[42][43][44]. In addition, molecular methods using more ''variable'' markers allow the detection of population variants (called ''genotypes'' or ''subgenotypes'') within species, thus enabling an insight into the population genetics and epidemiology of cryptosporidiosis.…”

Section: Introductionmentioning

confidence: 99%

Analysis of the genetic diversity within Cryptosporidium hominis and Cryptosporidium parvum from imported and autochtonous cases of human cryptosporidiosis by mutation scanning

Jex

Gasser

2008

Electrophoresis

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The present study investigated sequence variation in part of the 60 kilodalton glycoprotein (pgp60) gene among Cryptosporidium hominis and Cryptosporidium parvum isolates (n=115) from citizens of the UK inferred to have been infected whilst travelling abroad (to 25 countries) or in the UK. The genomic DNA samples from these isolates were subjected to PCR-coupled single-strand conformation polymorphism analysis, followed by targeted sequencing of pgp60. Individual samples were classified to the genotypic and subgenotypic levels based on phylogenetic analysis (Bayesian inference) of pgp60 data, including published sequences for comparison. Based on this analysis, five C. hominis (Ia-If) and four C. parvum (IIa, IIc-IIe) genotypes were identified, equating to 16 and 10 subgenotypes, respectively. Of these genotypes, C. hominis Ib was predominant (n=82). Interestingly, one subgenotype (C. hominis Ib A10G2R2) accounted for the majority of the samples examined and was identified in travellers to 14 countries; the examination of published records suggested that C. hominis Ib A10G2R2 has a global distribution. Numerous new and seemingly rare subgenotypes (eight for C. hominis and six for C. parvum) were also discovered. In conclusion, the present study revealed substantial genetic variation in pgp60 within both C. hominis and C. parvum and emphasizes the need to undertake investigations of human and animal populations in countries for which there is no information on the genetic make-up of Cryptosporidium infecting humans.

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“…Recently, we demonstrated the utility of a nonisotopic SSCP approach for the delineation of 18 different species or genotypes of Cryptosporidium [12], a group of parasitic apicomplexans which commonly infects the gastrointestinal or respiratory tract of a wide range of vertebrates, including fish, amphibians, reptiles, birds and mammals, causing the disease cryptosporidiosis. Human cryptosporidiosis is of major importance globally [13], particularly in immuno-suppressed or -compromised individuals [13][14][15], and usually manifests itself clinically as watery, steatorrhoeic and/or foul-smelling diarrhoea.…”

Section: Introductionmentioning

confidence: 99%

“…Selecting genetic markers with sufficient levels of sequence variability within individual species of Cryptosporidium is critical for such investigations. Although a number of genetic loci (e.g., the small ribosomal subunit and the internal transcribed spacers of nuclear ribosomal RNA/DNA) provide the specific and/or genotypic identification of Cryptosporidium [4,12,31,33,34], the locus currently considered to hold most promise for population genetic investigations is the gp60 gene [3]. This gene encodes the 15 and 45 kDa glycoproteins (GP15 and GP45, respectively), proposed to be integral in sporozoite attachment during the penetration of the parasite into the intestinal host cells [2].…”

Section: Introductionmentioning

confidence: 99%

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A practical and cost‐effective mutation scanning‐based approach for investigating genetic variation in Cryptosporidium

Jex

Whipp²,

Campbell

et al. 2007

Electrophoresis

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In the present study, we used a mutation scanning-targeted sequencing approach to assess variation in part (pgp60) of the 60 kDa glycoprotein (gp60) gene among Cryptosporidium samples from humans in Victoria, Australia. Two nuclear ribosomal loci (the small subunit rRNA gene and the second internal transcribed spacer) were used to identify the samples as Cryptosporidium hominis (n = 74), Cryptosporidium parvum (n = 23) or Cryptosporidium meleagridis (n = 1). In total, nine distinct pgp60 sequences were identified (three C. hominis, five C. parvum and one C. meleagridis). Phylogenetic analyses of the pgp60 sequence data, employing well-defined reference sequences for comparison, allowed the genotypic and subgenotypic classification of samples. The C. hominis samples were classified as Ib A10G2R2, Id A15G1R2, and a new genotype, designated Ib2, was identified subgenotypically as A18G1R4. The C. parvum samples were classified as IIa A18G3R1, IIa A20G3R1, IIa A22G3R1, IIa A23G3R1 and IIc A5G3R2. These findings suggested that the C. hominis metapopulation is largely homogeneous, consisting of a single dominant genotype, Ib A10G2R2, whereas the C. parvum metapopulation is considerably more heterogeneous, with no single dominant genotype. The greater level of genetic heterogeneity found among the C. parvum samples, despite the smaller sample size, may relate to the zoonotic infection pattern of this species, which would be reflective of a greater number of possible infection sources. The present mutation scanning approach, coupled with targeted sequencing of genetically distinct representatives, is a practical, cost-effective tool for large-scale population genetic and epidemiological studies of Cryptosporidium and other eukaryotic organisms.

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Specific and genotypic identification of Cryptosporidium from a broad range of host species by nonisotopic SSCP analysis of nuclear ribosomal DNA

Cited by 30 publications

References 53 publications

Molecular Characterization of Bubaline Isolate of Cryptosporidium Species from Egypt

Molecular Characterization of Bubaline Isolate of Cryptosporidium Species from Egypt

Analysis of the genetic diversity within Cryptosporidium hominis and Cryptosporidium parvum from imported and autochtonous cases of human cryptosporidiosis by mutation scanning

A practical and cost‐effective mutation scanning‐based approach for investigating genetic variation in Cryptosporidium

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