Longitudinal and cross-sectional detection of four bovine enteric viruses by multiplex- reverse transcription polymerase chain reaction: Identification of possible indicator viruses to assess biosecurity level at bovine farms

Abstract

It can be judged that if the detection frequency of prevalent pathogenic viruses decreases, biosecurity has been enhanced. To monitor bovine farm biosecurity levels, one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) for the simultaneous detection of group A rotavirus (RVA), bovine torovirus (BToV), bovine enterovirus (BEV), and bovine coronavirus (BCV) was designed, with the aim of configuring candidates for "viral pathogen indicators". A total of 322 bovine fecal samples were collected from calves aged less than three months at 48 bovine farms in Ibaraki and Chiba prefectures. At farm A, 20 calves were selected and sampled weekly for 12 weeks (184 samples); at farm B, 10 calves were selected and sampled for five weeks (50 samples); and at the rest of the 46 farms, 88 calves were sampled once. The screening on the 358 field samples proved positive for 27 RVA, 4 BToV, 55 BEV, and 52 BCV. In the successive sampling, RVA was detected once but not continuously, whereas BEV and BCV were detected in succession for up to five weeks. The results revealed that RVA was the primary agent among the positive samples obtained from calves aged three weeks or less, while BEV was the primary among those from the older than three weeks old. They can be employed as useful viral pathogen indicators for soundly evaluating biosecurity at bovine farms.

Regression of viral pathogen indicators due to improvement of hygiene protocols on boots in a bovine farm

“…All of those four viruses were also detected in farm A in Ibaraki prefecture [20]. Our previous results revealed that RVA was the primary agent among the positive samples obtained at the age of 3 weeks or below, while BEV was the primary among those obtained at an age over 3 weeks [20]. So in the present study, fecal samplings were separated by the age of calves (3 weeks or below and over 3 weeks).…”

“…Mixed infections usually make the disease worse than a single infection. BCV and BEV were detected continuously for several weeks in the same calves, however, RVA was detected only for several days [5,6,20].…”

“…Total RNA was extracted using ISOGEN-LS (Nippon Gene Co. Ltd., Tokyo, Japan) from supernatants of 10% fecal homogenates and tested for RVA, BToV and BCV genes by one-step multiplex RT-PCR using PrimeScript One Step RT-PCR Kit ver.2 (Takara Bio Inc., Kusatsu, Japan), as described previously [20]. The PCR products were visualized on 1.5% agarose gels stained with ethidium bromide.…”

“…The PCR products were visualized on 1.5% agarose gels stained with ethidium bromide. The estimated sizes of amplified products were 928 base pears (bp) for RVA, 651 bp for BoTV, 483 bp for BEV and 407 bp for BCV [20].…”

“…In the present research, in order to monitor bovine farm biosecurity level, RVA, bovine torovirus (BToV), bovine enterovirus (BEV) and BCV were selected as "viral pathogen indicators", because these viruses account for the etiology of diarrhea, which causes considerable economic loss for bovine farms [3,8,12,16,19], and were prevalent in bovine farms in Japan [20]. All of those four viruses were also detected in farm A in Ibaraki prefecture [20]. Our previous results revealed that RVA was the primary agent among the positive samples obtained at the age of 3 weeks or below, while BEV was the primary among those obtained at an age over 3 weeks [20].…”