Neutralizing Antibody to Calf Diarrhea Coronavirus in Various Animal Species in Japan

Coronavirus infections associated with respiratory, intestinal or other diseases have been reported in a number of animal species, including man, cattle, pig, dog, cat, mouse, rat, turkey, and chicken (1-3,5,6,8, 10, 12, 13). Recently coronaviruses have been divided by immunofluorescence into two distinct groups on the basis of antigenic cross-reactivity (9), although coronaviruses, as a group, display complex serologic variability (5). In this paper we report on the presence of neutralizing antibodies against calf diarrhea coronavirus (6, 12) in serum specimens from various animal species. Calf diarrhea coronavirus passaged in bovine embryonic kidney cells (6) was kindly supplied by Dr. C.A. Mebus, University of Nebraska, U.S.A. In our laboratory the virus was shown to replicate readily and induce a cytopathic effect in cultures of a continuous cell line, BEK-l, derived from bovine embryonic kidney (4). In the present study the virus was used at the 7th passage level in BEK-l cells. The neutralization test was carried out by the serum dilution method in II X 100-mm tube cultures of BEK-l cells. The growth medium was Eagle's minimum essential medium (MEM) containing 10% tryptose phosphate broth (TPB) and 10% inactivated calf serum, and the maintenance medium was MEM containing 10% TPB, 0.05% yeast extract, 0.5% sodium glutamate and 0.1 % glucose. Serial fourfold dilutions of the serum inactivated at 56 C for 30 min were made in maintenance medium and mixed with equal volumes of maintenance medium containing 200 TCIDso of virus per 0.1 ml. The serum-virus mixtures were incubated at 37 C for 1 hr before inoculation ofO.l-ml volumes into two tube cultures per serum dilution. The tests were read after incubation in a roller drum at 37 C for 4 days. The antibody titer was expressed as the reciprocal of the highest serum dilution which showed complete neutralization in at least one of the two tubes. Titers of 2 or higher were taken as positive. A total of 306 serum samples were collected from 12 animal species in various parts of Japan during the period from 1975 to 1977 (Table 1). These samples were stored at -20 C and inactivated at 56 C for 30 min before being tested. The results of neutralization tests on these serum samples are summarized 10