Detection of bovine torovirus in fecal specimens of calves with diarrhea from Ontario farms

Abstract

Breda virus (BRV), a member of the genus Torovirus, is an established etiological agent of disease in cattle. BRV isolates have been detected in the stools of neonatal calves with diarrhea in both Iowa and Ohio and in several areas of Europe. However, this virus has been reported only once in Canada. Therefore, a study was performed to determine the extent to which bovine torovirus is present in calves with diarrhea from farms in southern Ontario. A total of 118 fecal samples from symptomatic calves and 43 control specimens from asymptomatic calves were examined by electron microscopy (EM) and reverse transcription-PCR (RT-PCR) for the presence of torovirus. Torovirus RNA was detected in 43 of the 118 diarrheic samples (36.4%) by RT-PCR with primers designed in the conserved 3' end of the torovirus genome. By EM, torovirus particles were observed in 37 of the 118 specimens (31.4%). All but one of these samples were also positive by RT-PCR. The incidence of torovirus in the asymptomatic control specimens by RT-PCR was only 11.6%. To establish the identity of the particles observed in the diarrheic specimens, five of the amplicons from samples positive by both RT-PCR and EM were cloned and sequenced. Nucleotide sequence analysis revealed that the bovine torovirus found in southern Ontario manifests between 96 and 97% sequence identity to the BRV type 1 strain found in Iowa. This study shows that bovine torovirus is a common virus in the fecal specimens of calves with diarrhea from farms in southern Ontario and thus may be an important pathogen of cattle.

FIG. 1

Electron micrograph of a calf fecal specimen showing torus- and crescent-shaped bovine torovirus particles. Bar, 100 nm.

FIG. 2

Gel electrophoresis of RT-PCR products from five torovirus-positive fecal specimens. Lanes marked − and R represent a negative control (ddH₂O) and a bovine rotavirus sample, respectively. A 100-bp ladder was used as the molecular size marker.

FIG. 3

Alignment of the nucleotide sequence from the 3′ end of the BRV-1 (Iowa strain) genome with those of five bovine torovirus-positive samples from diarrheic calves in southern Ontario (a to e) and the BEV genome. Nucleotides identical to the consensus sequence are shown as dots.