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Recent studies on rotavirus have shown that there are several serotypes in human and avian rotaviruses discriminated by serum neutralization (SN) tests (2,5, 11,13). Although considerable attention has been paid to proving the existence of rotaviral serotypes in other species (4), comparatively little is known about those of bovine rotaviruses. There is only one report that distinguished two bovine rotavirus strains by the hemagglutination inhibition (HI) test (9). Since bovine rotavirus strains do not always possess hemagglutinating properties (3, 9), it is difficult to distinguish serotypes in them by the HI test.The present report gives the results of SN and complement fixation (CF) tests with antiserum against Lincoln strain of bovine rotavirus when these tests were applied to the cytopathic bovine rotaviruses isolated from the feces of diarrheic calves in Japan.The Lincoln strain at the 17th passage in bovine kidney cells (7) was kindly supplied by Dr. M. Kodama, of the authors' institute. It was further passaged three times in a continuous line of fetal rhesus monkey kidney (MA 104) cells in the presence of trypsin (Type 1, Sigma Chemical Co.) at the authors' laboratory.Isolation and electron microscopic detection of rotaviruses were performed in essentially the same manner as described by Babiuk et al (1). Briefly, the saline extracts of feces obtained from diarrheic calves were treated with 10 ,ug/ml of trypsin at 37 C for 30 min and inoculated into eight tube cultures of MA 104 cells washed with Earle's solution, in 0.1 ml of a ten-fold serial dilution. Incubation was carried out in the presence of 1 ,ug/ml of trypsin. The tube culture with intense cytopathic effect (CPE) at the end of the serial dilution was used for more than three passages by the same method as described above. Fourteen cytopathic bovine rotaviruses were isolated from different calves of seven herds suffering from diarrhea, and identified by the CF test and electron microscopy (EM). The propagation of MA 104 cells was performed in the manner previously described (6). Viral titration and the SN test were also carried out with MA 104 cells. For the titration, 0.1 ml of ten-fold serial dilution of each virus was inoculated into four tube cultures washed with Earle's solution. After viral adsorption at 37 C for 1097

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Fourteen cytopathic bovine rotaviruses isolated from naturally affected calves were characterized serologically by serum neutralization. Antisera were prepared in guinea pigs against the three isolates and the Lincoln strain of bovine rotavirus. These four rotaviruses were segregated into at least two distinct serotypes (designated types 1 and 2) by serum neutralization. The remaining 11 isolates were also placed into two groups according to neutralization specificity by using the antisera against types 1 and 2 bovine rotavirus. When colostrum-deprived calves were experimentally inoculated with type 1 or 2 bovine rotavirus, respectively, the calves produced neutralizing antibodies against the inoculated serotype but not against the other serotype. Calf sera obtained from six herds with naturally occurring rotavirus infections possessed neutralizing antibodies against type 1 or 2 or both. Thus, distinct serotypes of bovine rotavirus were demonstrated by serum neutralization. The possible role of serotypes in the epidemiology of rotavirus infection among bovine species is discussed.