Subtype analysis of Cryptosporidium parvum isolates from calves on farms around Belgrade, Serbia and Montenegro, using the 60 kDa glycoprotein gene sequences.

Cryptosporidium parvum is composed of multiple subtypes, which appear to be zoonotic or anthroponotic. Therefore, in order to understand the molecular epidemiology of this species and to control the zoonotic transmission, it is essential to identify the isolates at the subtype level. In the present study, 60.2% of 103 examined dairy calves from 10 farms in the Belgrade area were positive for Cryptosporidium infection by PCR, and all of them were identified as C. parvum by PCR-RFLP of SSUrRNA and COWP genes. Eighteen C. parvum isolates selected randomly from 9 positive farms were classified by molecular phylogenetic analysis of the 60 kDa glycoprotein gene sequences into 3 subtype allele families, IIa and IId, which are known to be zoonotic, and a new family IIj. Furthermore 10 isolates in IIa were classified into 3 subtypes IIaA16G1R1b, IIaA18G1R1, IIaA20G1R1, 2 in IId into IIdA18G1b, and 6 in IIj into IIjA16R2 and IIjA17R2. The last 2 subtypes in IIa and 2 subtypes in IIj were new subtypes. These results suggest that C. parvum isolates harboured in calves in the Belgrade area have zoonotic potential and C. parvum is genetically diverse only in a limited area. This is the first molecular epidemiological report about Cryptosporidium infection in Serbia and Montenegro.