Multi-locus analysis of human infective Cryptosporidium species and subtypes using ten novel genetic loci

Abstract

Background: Cryptosporidium is a protozoan parasite that causes diarrheal illness in a wide range of hosts including humans. Two species, C. parvum and C. hominis are of primary public health relevance. Genome sequences of these two species are available and show only 3-5% sequence divergence. We investigated this sequence variability, which could correspond either to sequence gaps in the published genome sequences or to the presence of species-specific genes. Comparative genomic tools were used to identify putative species-specific genes and a subset of these genes was tested by PCR in a collection of Cryptosporidium clinical isolates and reference strains.

Results: The majority of the putative species-specific genes examined were in fact common to C. parvum and C. hominis. PCR product sequence analysis revealed interesting SNPs, the majority of which were species-specific. These genetic loci allowed us to construct a robust and multi-locus analysis. The Neighbour-Joining phylogenetic tree constructed clearly discriminated the previously described lineages of Cryptosporidium species and subtypes.

Conclusions: Most of the genes identified as being species specific during bioinformatics in Cryptosporidium sp. are in fact present in multiple species and only appear species specific because of gaps in published genome sequences. Nevertheless SNPs may offer a promising approach to studying the taxonomy of closely related species of Cryptosporidia.

Figure 1

Amplification of Cryptosporidium DNA from clinical isolates and reference strains. A: amplification of 266 bp of Cgd2_80 gene, B: amplification of 368 bp of Chro.50330 gene. Both Cryptosporidium species and all isolates were PCR positive. MW: molecular weight, 1: Cp2, 2: Cp3, 3: Cp4, 4: Ch2, 5:Ch3, 6: Ch4, 7: Iowa, 8: Moredun, 9: TU502, NTC: non template control.

Figure 2

Phylogenetic Tree based on the gene sequences of 10 new loci and the COWP gene sequence. The trees were constructed using Neighbour-Joining algorithm of MEGA software. A: Phylogenetic tree constructed using C. parvum, C. hominis and C. cuniculus sequences. B: Phylogenetic tree with C. meleagridis as an out-group.