Veterinary Microbiology, 6 (1981) 143-155 143 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands INHIBITORS OF BOVINE PARVOVIRUS, CORONAVIRUS AND ROTAVIRUS IN PRECOLOSTRAL AND FETAL BOVINE SERA S. TOKUHISA, Y. INABA, K. SATO, Y. MIURA, H. AKASHI, K. SATODA and M. MATUMOTO* National Institute of Animal Health, Tsukuba, Ibaraki (Japan) *Kitasato Institute, Minato-ku, Tokyo (Japan) (Accepted 12 January 1981) ABSTRACT Tokuhisa, S., Inaba, Y., Sato, K., Miura, Y., Akashi, H., Satoda, K. and Matumoto, M., 1981. Inhibitors of bovine parvovirus, coronavirus and rotavirus in precolostral and fetal bovine sera. Vet. Microbiol., 6: 143-155. Eleven, 11 and 2 of 11 precolostral sera from normal calves and 13, 14 and 9 of 14 sera from normal fetuses, 7 to 10 months of gestation, neutralized bovine parvovirus, coronavirus and rotavirus, respectively. When assayed by single radial immunodiffusion, all the sera contained IgG at 360 to 1400 mg/dl, and some of them had much smaller amounts of IgM or IgA. Most of the neutralizing activities against bovine coronavirus and rotavirus were readily inactivated by treatment with acetone or 2-mercaptoethanol. Some sera frectionated by Sephadex G-200 gel filtration or starch block electrophoresis had neutralizing activities against bovine parvovirus or coronavirus in fractions containing no detectable amounts of immunoglobulins. These observations seem to indicate the presence of substance(s), other than immunoglobulins, capable of inhibiting replication of bovine parvovirus, coronavirus or rotavirus. The chemical nature and the mode of action of the inhibitors await elucida- tion. INTRODUCTION Transplacental transfer of maternal immunoglobulins does not occur in cattle, and the newborn animal acquires maternal antibodies by ingesting colostrum (Brambell, 1970; Solomon, 1971). On the other hand, bovine fetuses develop the ability to produce antibodies upon antigenic stimulation relatively early in gestation (Solomon, 1971; Osburn, 1973; Schultz, 1973; Schultz et al., 1973). Many workers have reported the presence of antibodies against various viruses in sera from colostrum-deprived calves and fetuses (Kniazeffet al., 1967; Dunne et al., 1973; Homer et al., 1973; Hubbert et al., 1973; Schultz et al., 1973; Miura et al., 1974; Rossi and Kiesel, 1974; Kurogi et al., 1975). On the other hand, there have been reported studies suggesting the presence of virus-neutralizing substances, other than immunoglobulins, in 0378-1135/81/0000-0000/$ 02.50 1981 Elsevier Scientific Publishing Company 144 bovine sera (McFerran, 1962 a, b; Kanamitsu et al., 1967; Doggett et al., 1968; Urasawa et al., 1968 a, b; 1969). Recently we have reported that high percentages of normal precolostral and fetal calf sera neutralize bovine parvovirus, coronavirus and rotavirus (Sato et al., 1980). In the present study, experiments were carried out to relate these neutralizing activities to immunoglobulins in the sera, and the results obtained suggested that some precolostral and fetal calf sera might contain substances, other than immunoglobulins, capable of inhibiting the replication of these bovine viruses. MATERIALS AND METHODS Sera. Serum samples were obtained by jugular puncture from normal new- born calves before or after ingestion of colostrum and from some of their dams in 1973 and 1974, and by cardiac puncture from normal fetuses, 7 to 10 months of gestation, in 1979. The sera were stored at -20C and inactivated at 56C for 30 min before use. Viruses. The K-71 strain of bovine parvovirus was originally isolated from an aborted fetus (Sugimura, et al., 1974) and had undergone 5 passages in primary cultures of bovine kidney (BK) cells when used in the present study. The Lincoln strain of bovine rotavirus (Mebus et al., 1971) was used at the 7th passage level in BK cell cultures. Calf diarrhea coronavirus (Mebus et al., 1973) was found in our laboratory to replicate with cytopathic effect in cultures of the BEK-1 cell line derived from bovine embryonic kidney (Inaba et al., 1976). In the present study the virus was used at the 7th BEK-1 passage. Neutralization tests. Neutralization tests with bovine parvovirus and rotavirus were carried out in tube cultures of BK cells and those with bovine coronavirus in tube cultures of BEK-1 cells. The medium for cell growth was Eagles minimum essential medium (MEM) containing 10% tryptose phosphate broth, 10% bovine serum and antibiotics, and the maintenance medium was MEM containing 10% tryptose phosphate broth, 0.5% sodium glutamate and 0.05% yeast extract. Cultures were prepared in 11 X 100 mm tubes by seeding 0.5-ml volumes of growth medium containing 2.5 X l0 s cells/ml and incubating at 37C for 4 days. Serial twofold dilutions of the serum or fractionated materials were made with maintenance medium, and each dilution was mixed with an equal volume of maintenance medium con- taining 200 TCIDs0 per 0.1 ml of virus. The vir