Molecular characterization of Cryptosporidium isolates from pre-weaned calves in western France in relation to age

Abstract

Eighteen pre-weaned female calves from a single beef cattle herd in western France were sampled weekly from birth to 21/2 months of age in order to characterize Cryptosporidium oocyst output. 182 fecal samples were screened for the presence of oocysts after concentration using immunofluorescence analysis. DNA was extracted from positive samples and a PCR-RFLP protocol, with the restriction enzyme SspI and MboII, to amplify the partial SSU rRNA gene was performed. For the subtyping of Cryptosporidium parvum, a gp60 PCR was carried out. All animals excreted oocysts at at least one sampling date and 80% of the calves presented a mild diarrhea at at least one occasion, with no mortality. The prevalence of excretion reached 94% when calves were 17-23 days of age. The mean number of oocysts at the peak of excretion (10-16 days) was 5 × 10(5) oocysts per gram of feces. PCR-RFLP analysis was successful for 61 of 84 positive samples: 14 were identified as C. parvum, 15 as Cryptosporidium bovis, and 22 as Cryptosporidium ryanae. Ten mixed infections with all combinations of these species were also identified. Calves excreted the following Cryptosporidium species: C. parvum between 7 and 27 days of age, C. bovis between 11 and 38 days and C. ryanae from 19 to 72 days. The IIaA15G2R1 zoonotic subtype of C. parvum subtype was the only subtype identified. We observed the presence of different Cryptosporidium species depending on the age of the animals. This study showed that C. parvum and C. bovis can infect beef calf neonates at similar levels of oocyst excretion with or without clinical signs and that C. parvum isolates had zoonotic potential.

Keywords: Cryptosporidium; Mixed infections; PCR-RFLP; Pre-weaned calves; gp60.