Genotyping of Cryptosporidium spp. isolated from young domestic ruminants in some targeted areas of India

Abstract

Faecal samples (363) from kids, lambs, calves and buffalo calves of below 3 months of age were collected from various parts of India and screened microscopically for Cryptosporidium oocysts using modified Ziehl–Neelsen method of staining. Microscopically positive samples (20) were genotyped by PCR amplification of the partial 18S rRNA region and subsequent digestion by SspI, VspI and MboII restriction enzymes. Based on the PCR-RFLP patterns of 18S rRNA, all the 20 samples were found positive for C. parvum. All the positive samples were also used for amplification of partial actin gene of Cryptosporidium spp. For further confirmation of the species of Cryptosporidium, amplified 818 bp partial actin gene of 3 representative isolates was cloned and sequenced. The sequence and phylogenetic analysis of PCR-positive samples confirmed the presence of C. parvum. Thus, actin gene can also be used for specific molecular diagnosis of Cryptosporidium spp., in addition to 18S rRNA. These findings also indicated that young domestic ruminants can be a potent source of cryptosporidial infection for humans and animals in India.

Keywords

• 18S rRNA
• Actin gene
• Cryptosporidium parvum
• Genotyping
• India