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

Abstract

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.

Highly sensitive non‐isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species, genotypes and subgenotypes infective to humans, and its implications

“…Recent studies have demonstrated the utility of SSCP for the rapid and cost effective analysis of nucleotide variation in pgp60 ($350-450 bp) [18][19][20][21] and other loci [5]. These investigations have shown consistent differentiation among subgenotypes differing by as little as a single-nucleotide substitution in the TCA microsatellite region, reinforcing the high-mutation detection capacity of this analytical approach for amplicons in this size range [5,[18][19][20][21]. However, the mutation detection capacity of SSCP can decrease substantially for larger amplicons [6].…”

“…Genomic DNA from ruminant faecal samples was isolated using the PowerSoil kit (MoBio), according to manufacturer's instructions. The genomic DNA samples had been classified to species based on PCR-based SSCP and/or sequencing of loci in the small subunit (SSU) and second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and/or gp60 [18][19][20]24].…”

“…The gp60 gene is an attractive marker for population genetic studies, as it is present as a single copy in the nuclear genome (thus being suited to PCR-based REF and sequencing) and displays substantial intraspecific variability, particularly in a TCA (perfect and imperfect) microsatellite region [14]. Numerous population genetic studies of gp60 sequence data using phylogenetic analyses have demonstrated that, although this gene exhibits substantial variability, particular sequence types consistently cluster into genotypic 'families' with strong support [14,[17][18][19]. According to the original nomenclature proposed by Strong et al [14], these genotypic families are designated Ia, Ib, Id to If for C. hominis and IIa to IIk for C. parvum.…”

“…According to the original nomenclature proposed by Strong et al [14], these genotypic families are designated Ia, Ib, Id to If for C. hominis and IIa to IIk for C. parvum. Additional sequence variability in the TCA microsatellite region of the gene is also often used to further define or differentiate genetic subgenotypes within genotypic families [17][18][19].…”

Comparison of techniques for DNA extraction and agarose gel staining of DNA fragments using samples of Cryptosporidium

“…Differentiating Cryptosporidium species and diagnosing C. parvum genotypes is impossible using microscopy due to the morphological similarities between the oocysts of the various species and their subtypes (Jex and Gasser 2008;Fayer 2010;Das et al 2011). Therefore, molecular tools are being increasingly used to detect and differentiate between Cryptosporidium species and subtypes (Xiao 2010).…”