Phylogenetic Analysis of Cryptosporidium Parasites Based on the Small-Subunit rRNA Gene Locus

Abstract

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. serpentisbelonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known asCryptosporidium 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.

Detection of Cryptosporidium oocysts in fresh and frozen cattle faeces: comparison of three methods

“…The DNA was eluted into 70 ll of molecular grade water. An 840-bp region of the 18S rRNA gene was amplified by a nested protocol (using 2 ll of template in the primary reaction), which has been shown to detect many species ⁄ genotypes of Cryptosporidium and is highly sensitive, having successfully amplified the DNA from just one oocyst Xiao et al 1999). The amplicons then underwent PCR-restriction fragment length polymorphism (RFLP) by incubation with restriction enzymes VspI and SspI; the sizes of the fragments produced were compared with restriction patterns characteristic of known Cryptosporidium species.…”

Prevalence of Cryptosporidium parvum in dairy calves and GP60 subtyping of diarrheic calves in central Argentina

“…Oocysts were ruptured with three freeze-thaw cycles, and total DNA was extracted with the QIAmp DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the instructions of the manufacturer. For species determination, genomic DNA isolated from oocyst-positive fecal samples were subjected to PCR-RFLP as described in Xiao et al (1999) including the use of an additional restriction enzyme MboII (Feng et al 2007). For subtyping, C. parvum isolates were selected from calves with diarrhea and high infection intensity (scores 3 and 4), and the PCR amplicon of the gene encoding the 60 kDa glycoprotein (GP60) was directly sequenced and subtyped (Alves et al 2003).…”

Occurrence and Genetic Characteristics of Cryptosporidium hominis and Cryptosporidium andersoni in Horses from Southwestern China

“…Cryptosporidium species/genotypes were identified in all of the extracted DNA samples by nested PCR amplification of an $ 830-bp fragment of the small subunit rRNA (SSU rRNA) gene (Xiao et al 1999) (Table S1). The identification of C. hominis was confirmed by PCR and sequence analysis of the approximately 1,950-and 550-bp fragments for the Cryptosporidium oocyst wall protein (COWP) and 70-kDa heat shock protein (HSP70) genes, respectively Xiao et al 2000) (Table S1).…”