Abstract
Bovine coronavirus (BCV) has been previously detected in the enteric and respiratory tracts of cattle and is specifically associated with enteritis and diarrhoea in neonatal calves. Two diagnostic tests, an ELISA and an immunogold EM technique for the detection of BCV in faeces, were developed, optimised and compared with direct EM, immunosorbent EM and haemadsorption-elution-haemagglutination (HEHA). The immunogold EM technique was found to be the most sensitive test followed by the ELISA, HEHA, immunosorbent EM and direct EM. An IF test for detecting BCV in the respiratory tract and a neutralization test for quantifying anti-BCV antibody titres in serum and milk were also developed. Using the immunogold EM technique BCV was demonstrated in 39 of 123 field samples of bovine diarrheic faeces. From 25 of these samples 2 isolates were successfully adapted to grow in HRT 18 cells following initial isolation in bovine fetal tracheal organ culture. These, and three other strains of BCV and a human coronavirus (HCV) strain obtained from other laboratories, were compared in immunofluorescence (IF), haemagglutination inhibition (HAI) and neutralization tests. Polyclonal antisera against these 6 viruses were raised in rabbits. No significant differences between viruses were detected by IF incorporating homologous and heterologous antisera but HCV could be distinguished from the bovine coronaviruses in a cross neutralization test. In this test all BCV isolates were determined to be of one serotype. In the HAI test however, the HCV strain was distinguishable from the 5 BCV strains and differences between the BCV strains were shown. Two monoclonal antibodies prepared against one of the BCV strains distinguished the HCV from the BCV strains in all three tests. These monoclonal antibodies did not distinguish between the 5 BCV strains in the IF or HAI test but did so in the neutralization test. The various strains were also compared at the molecular level using the Western blotting technique. This technique showed no significant differences between the molecular weights or serological reactivity of the structural proteins of these strains. Experimental infection of a gnotobiotic calf with BCV resulted in diarrhoea and fever, but no clinical evidence of disease was seen when 4 conventionally reared colostrum fed calves and 4 gnotobiotic lambs were similarly infected. The oral infection of suckling mice with BCV produced diarrhoea in some animals but a full investigation is required to optimise this model. A prospective epidemiological survey on one farm was carried out and showed Cryptosporidium and BCV to be associated with diarrhoea. Additionally this survey showed that the detection of BCV in the respiratory tract was associated significantly with respiratory symptoms.