Detection of rotavirus in the faeces of diarrhoeic calves by reverse transcriptase- polymerase chain reaction and silver staining

Abstract

A study was undertaken to detect the presence of rotavirus in the faeces of diarrhoeic calves by RT-PCR and RNA-PAGE. Agar Gel Immune Diffusion was performed to detect the presence of rotaviral antigens in faecal samples using hyperimmune serum raised in rabbits. The protein profile of BRV was analyzed using SDS-PAGE. Attempts were made to isolate BRV from faecal samples in MDBK cell line. One hundred and twenty four faecal samples of diarrhoeic calves were collected from University Livestock Farm, Mannuthy, Veterinary Hospitals of Kerala Agricultural University, some dairy farms in Thrissur district and also from individual farmers in and around Thrissur. Twenty samples each were collected from adult cattle above one year of age with diarrhoea and normal healthy calves. All these samples were screened for the presence of BRV by RNA-PAGE, RT-PCR and AGID. Among 124 faecal samples collected 29 (23.39 per cent) samples were detected as positive by RNA-PAGE. The clustered arrangement of the 11 segments of the genome showed a 4:2:3:2 migration pattern, typical of group A bovine rotavirus. Reverse Transcriptase – Polymerase Chain Reaction could detect BRV in 35 (28.23 per cent) samples. By using AGID, only 16 (12.90 per cent) samples were found positive. Among the various tests employed, RT-PCR was found to be more sensitive in the diagnosis of BRV infections. All the 20 faecal samples from adult cattle with diarrhoea were tested negative by the three methods. Rotavirus could not be detected in the faeces of healthy calves by any of the tests employed. The protein profile of BRV revealed nine polypeptides having molecular weight in the range of 16.5 to 131 kDa. The age-wise distribution of BRV infection in calves was studied. It was found that the occurrence of infection was most common in zero to two weeks of age (39.39 per cent) followed by two to four weeks of age (37.04 per cent). The faecal samples which were found positive by RNA-PAGE was inoculated into MDBK cell line. But the attempts to isolate BRV were found unsuccessful.