Isolation, Characterization and Molecular Identification of Cryptosporidium spp. Causing Diarrhea in Young Calves by Multiplex Nested-PCR

Diagnosis of Cryptosporidium species based on morphological characteristics is very limited and has no taxonomic value alone, and therefore the use of molecular methods removes these limitations to some extent.

The aim of this study was to determine the predominant Cryptosporidium genotypes in calves with diarrhea.

Study were conducted in calves aged less than 3 months for a period of 2 years. During the study period, 160 dung samples were collected from neonatal calves and examined first microscopically and then by molecular techniques. Stools were analyzed for the presence of Cryptosporidium oocysts by Sheather's Sugar Flotation Solution followed by Ziel-Neelsen staining method. DNA of parasite was extracted and multiplex nested-PCR protocol basis on the 18srRNA were done to identify three cattle-adapted species (C. andersoni, C. bovis and C. ryanae) plus the zoonotic species C. parvum.

110 fecal samples were collected from livestock in Alborz province and 50 fecal samples were collected from livestock in Shahroud city. Of the 160 animals examined, 90 were female and 70 were male. In total, out of 160 animals examined, 85 cases (53.12%) had diarrhea, of which 55 cases (34.37%) were positive using Ziel-Neelsen staining. Since all positive cases were related to diarrhea samples and related to calves under one month old, a significant relationship was observed between diarrhea status and the presence of this parasite (p < /em><0.05). In terms of seasonal distribution, no difference was observed in the rate of diarrhea and positive parasitic cases. The presence of 305 bp band in all Ziel-Neelsen positive samples confirmed the presence of C. parvum in all samples.

Neonatal calves are more likely to be infected with Cryptosporidium parvum, as confirmed by the present study.