In fact, discrepancies and limitations of these markers for Cryptosporidium typing have been reported. Hunter and colleagues [37] described the difficulty in interpreting the presence of different subtypes in outbreak setting and Proteasome activity Widmer [38] reported that gp60 might not be a reliable

marker of C. parvum and C. hominis population structure. The ten novel loci, described in this study, showed excellent discriminatory power and consistency to assess phylogenetic relationships at the species and infra-species levels. These findings suggest that these loci could be alternative valuable genotyping and subtyping targets for Cryptosporidium. RG-7388 price However, their stability should be assessed in an extensive collection of isolates from different subtype families and geographical locations to validate their discriminatory power. Conclusions In this study, comparative genomics were used to identify putative C. parvum and C. hominis species-specific genes. Despite the fact that the majority of the predicted genes were common to both species and some to C. meleagridis, experimental evidence was found for one specific gene for each species. The ten novel genetic loci studied showed an interesting polymorphism. In fact, sequence Cell Cycle inhibitor analysis of PCR products revealed multiple SNPs, the majority

of which were species-specific. These SNPs were stable and consistent across Cryptosporidium species and subtypes. These results showed

that the ten novel genetic loci can potentially be used to assess the phylogenetic distance and relationships at the species and infra-species level of human infective Cryptosporidium isolates. In addition, the paired SNP analysis was found to be a good strategy to assess the genetic divergence of the isolates tested. Methods Reciprocal Blast was used to identify genes with high sequence variability between C. parvum and C. hominis. This is a variant of Blast (Basic local alignment search tool), originally described by Altschul and colleagues [39] and is a common computational tool for predicting putative orthologs http://​www.​ncbi.​nlm.​nih.​gov/​blast/​blast_​overview.​shtml. Subsequently, each of the ~ 3900 genes of C. parvum and C. hominis was assigned a similarity score. Only sequences new which returned genes with less than 10% sequence similarity from the other genome were considered. These coding sequences are putatively species-specific genes. A secondary screen was performed as follows: each gene was individually tested using Blastn algorithm http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi to confirm specificity and reveal any sequence similarity to genes from other Cryptosporidium species. Furthermore, orthology queries were performed using CryptoDB database. Whenever a gene showed sequence similarity, it was eliminated from the selection. This secondary screen increased the prediction stringency.