Prevalence and molecular characterization of cryptosporidium species in buffalo calves of western Haryana

Abstract

A sum total of 402 faecal samples from buffalo calves belonging to the age group of three months were collected randomly without any clinical symptom from various livestock farms and small holdings in four districts of Haryana. The presence or absence of Cryptosporidium spp. oocysts in faecal samples was confirmed by the standard microscopic examination of the modified Ziehl-Neelsen (MZN) stained faecal smears. The overall prevalence in buffalo calves was found to be 8.7%. The seasonal prevalence study revealed a maximum occurrence of Cryptosporidium infection during rainy followed by autumn, winters and minimum in summer season. Age related prevalence showed highest infection in calves between 16-30 days age which gradually decreased with increase in age. The DNA of representative positive faecal samples was extracted using standard phenol-chloroform-isoamylalcohol (PCI) method and then was subjected to nested PCR. All the extracted DNA were amplified for a 1325 bp primary fragment followed by a 834 bp nested fragment in a nested PCR of 18S rRNA gene, thereby confirming the presence of Cryptosporidium spp. The restricted enzyme digestion pattern (restricted fragment length polymorphism) of the 834 bp nested fragment using the restriction enzymes SspI and VspI confirmed the presence of C. parvum. Sequencing of Cryptosporidium DNA samples were carried out which also proved all samples to be of C. parvum. This is the first report of molecular identification of C. parvum in buffalo calves of Haryana and these Haryana isolates of Cryptosporidium parvum isolated from diarrhoeic buffalo calves are divergently related to rest of the part of the country isolates and indicates the zoonotic potential.