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. 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.

FIG. 1

Phylogenetic relationships of Cryptosporidium parasites to other apicomplexan parasites (A) and to each other (B).

FIG. 2

Molecular diagnosis of Cryptosporidium parasites by a nested PCR-RFLP procedure based on SSU rRNA gene sequences. (A) Specific detection of Cryptosporidium spp. by nested PCR. Lane 1, molecular weight markers; lane 2, C. parvum bovine genotype; lane 3, C. parvum human genotype; lane 4, C. muris; lane 5, C. serpentis; lane 6, C. baileyi; lane 7, Eimeria papillata; lane 8, Eimeria nieschulzi; lane 9, Giardia duodenalis; lane 10, negative control. The upper panel contained the primary PCR products, and the lower panel contained the secondary PCR products. (B) Species diagnosis of Cryptosporidium parasites by SspI digestion of the nested PCR products. For lane contents see above. (C) Differentiation of two genotypes of C. parvum by VspI digestion of the nested PCR products. For lane contents see above.