Genotyping of Cryptosporidium species reveals prevalence of zoonotic C. parvum subtype in bovine calves of north India

Abstract

Faecal samples of diarrhoeic bovine calves (below 2 months of age) collected from government and privately owned dairy farms of north Indian states (Uttar Pradesh, Uttarakhand and Bihar) were stained by modified Zeihl-Neelsen stain and examined microscopically for the presence or absence of Cryptosporidium oocysts. Partial 18S rRNA of 25 Cryptosporidium positive samples were amplified by nested PCR and subsequently digested by SspI, VspI and MboII restriction enzymes to determine the Cryptosporidium species. Based on the PCR-RFLP patterns of 18S rRNA, all the 25 samples were identified as positive for C. parvum. For further confirmation, partial 18S rRNA gene of 4 representative samples was cloned and sequenced. The sequence and phylogenetic analysis of PCR-positive specimens also confirmed the presence of C. parvum. For subtyping of C. parvum, GP60 gene was amplified and sequenced. The sequence analysis of the glycoprotein (GP60) gene revealed that all the 4 isolates belonged to IIdA15G1R1 zoonotic subtype of C. parvum. These findings further confirmed that cattle and buffalo calves can be a potent source of cryptosporidial infection for humans and animals.