Unveiling the genetic diversity of bovine Cryptosporidium isolates in North India inferred by 18S rRNA sequences.

This study was conducted to infer the evolutionary patterns and genetic structure of Cryptosporidium species in bovines in north India and their comparison with the global populations. Microscopic examination of 1417 bovine faecal samples collected from the three states of North India revealed Cryptosporidium oocysts in 11.8% (167/1417) of the samples, with animal age identified as a significant risk factor for disease occurrence. Odds of Cryptosporidium infection in 0-3 months and 3-12 months old calves was 3.5 times and 2.9 times higher than adults, respectively. Molecular analysis of these microscopy-positive samples identified four Cryptosporidium species, C. andersoni (n = 20), C. bovis (n = 42), C. parvum (n = 51) and C. ryanae (n = 47) by SspI, VspI and MboII restriction enzyme-based polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and nucleotide sequencing of 18S rRNA partial gene. The 18S rRNA sequences from this study along with sequences of Asian, American, European and Australian isolates for these four species available in NCBI GenBank were analysed for genetic diversity including the identification of haplotypes, haplotype diversity, nucleotide diversity, neutrality indices, gene flow and genetic differentiation. The haplotype network analysis indicated 34 haplotypes present globally which were distinctly classified into four species specific haplogroups. The clustered network of C. bovis, C. ryanae and C. parvum haplogroups are suggestive of sub-structuring within these species, while the reticulated haplotype network patterns of C. parvum haplogroup and to some extent C. ryanae haplogroup are strongly indicative of recurrent mutations/recombinations, or gene flow among the subpopulations. This suggests a long-term adaptability of these two species. The global populations of four Cryptosporidium species also revealed their population expansion. Intra-specifically, no clear genetic differentiation or geographical structuring was observed within all four species. Present study about the risk factors, co-evolutionary patterns and genetic differentiation of the Cryptosporidium species aims at strengthening our understanding of the disease dynamics and its implications on veterinary and public health.