Genotype and subtype analyses of Cryptosporidium isolates from cattle in Hungary

Seventy-nine faecal samples from calves with diarrhoea were collected on 52 farms from different counties in Hungary to investigate the species, genotypes and subtypes of Cryptosporidium in calves. Oocysts from faecal sample collected from each animal were concentrated using sucrose gradient centrifugation and examined by immunofluorescence assay (IFA). Genomic DNA was extracted from microscopically positive samples and nested PCR was performed to amplify the partial SSU rRNA and GP60 genes of Cryptosporidium that were subsequently digested by SspI, VspI and MboII restriction enzymes to determine the Cryptosporidium species and genotype present. Cryptosporidium parvum was detected in 21 samples while the Cryptosporidium deer-like genotype was found in another sample. The sequence and phylogenetic analysis of 21 isolates of the GP60 PCR products showed that the most common C. parvum subtype is IIaA16G1R1. Interestingly, two isolates were found to contain the C. parvum allele IId in addition to identifying another containing a new C. parvum IIa A18G1R1 subgenotype firstly described in the C. parvum complex. These findings suggest that cattle can be a source of cryptosporidial infections for humans and animals in Hungary. This is the first published description of Cryptosporidium subgenotyping in Hungary.