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Enzyme-linked immunosorbent assays were established to detect Breda virus antigen in feces and homologous antibodies of the IgG1, IgM, and IgA isotypes in serum. With the aid of solid-phase immune-electron microscopy, torovirions in fecal material were observed. The course of natural infection was studied in 10 sentinel calves that had been obtained from different farms, and housed together at 1 week of age. They were separated from other cattle until the age of 10 months. Up to the age of 4 months, all calves regularly excreted Breda virus in the feces. Irrespective of the existence of IgG1 isotype maternal antibodies, all calves had early IgM responses in serum, but lack of IgA seroconversion. In 7 calves, antibody titer decreased below detection, whereas 3 calves had an isotype switch, resulting in persistent IgG1 titer. After introduction into the dairy herd at 10 months of age, all calves had diarrhea, and shedding of Breda virus was observed in 8 of them. Seroconversion for all antibody isotypes was observed, indicating lack of mucosal memory. In contrast, coronavirus infection in the presence of maternal antibodies led to isotype switch in all calves but one, and a memory response was observed after introduction into the dairy herd.

Twelve Dutch isolates and the M41 strain of infectious bronchitis virus (IBV), a coronavirus of chickens, were characterized by cross-neutralization and T1 finger-printing to elucidate their evolutionary relationship. The T1 fingerprinting showed that the Dutch isolates formed two clusters. The first cluster contained strains H52, H120, D387, V1259, V1385 and V1397; the estimated sequence homology is 99%. Cluster two comprised strains D207, D274, D212, D1466, D3128 and D3896, which have about 95% sequence homology. The M41 virus did not belong to either cluster. The four different serotypes which arose in the late 1970s belonged to cluster two and appeared to be different from the vaccine strains (H52 and H120) used at that time. This indicates that the strains were newly introduced and could have arisen from a common virus. On the other hand, three recently isolated field strains were genetically closely related to the vaccine strains H120 and H52 (cluster one), suggesting that these live vaccine strains themselves could have given rise to these serologically altered field isolates. The data are relevant to the development of new vaccine strategies.

Two direct blocking enzyme linked immunosorbent assays (ELISA) for the detection of antibodies to Breda virus in sera of cattle were compared. An ELISA with consecutive addition of antigen and test serum to an antibody-coated plate gave higher positive: negative absorbence ratios than an ELISA in which antigen and test serum were added simultaneously. Sera collected from breeding and fattening herds in The Netherlands (n = 1313) and the F.R.G. (n = 716) were tested, and antibodies to Breda virus were demonstrated in 94% of adult cattle. Ninety percent of newborn calves had high levels of maternal antibodies, which waned until the age of 3 months. Active seroconversion occurred between 7 and 24 months in most animals.