Neutralizing Patterns of Anti‐Bovine Rotavirus (Lincoln) Serum against Cytopathic Bovine Rotaviruses Isolated from Calves in Japan

Abstract

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

Studies on the Growth of Bovine Rotavirus in Cell Cultures*

“…BGM cell line has the advantage of being easy to grow and handle. MURAKAMI et al (1981) obtained 14 cytopathogenic isolates from cases of calf diarrhea. Their technique was closely similar to the one adapted in the present work for isolations from calf feces.…”

Serotypes of bovine rotaviruses distinguished by serum neutralization

“…An established fetal rhesus monkey kidney cell line (MA104) was used throughout this study. Propagation of MA104 cells was performed as previously described (12,17). The maintenance medium adopted for the cells after virus inoculation was of the same composition as the growth medium (12,17) except that it contained 0.11% bovine serum albumin instead of fetal calf serum.…”

“…Fourteen cytopathic bovine rotavirus isolates, including three viruses used for immunization, were isolated from the feces of calves from seven herds affected by acute diarrhea in Japan. Procedures for isolation and identification of the isolates have been previously described (17). All the isolates were passaged more than three times by means of the terminal dilution method and used for this study at passages six to eight on MA104 cells in the presence of 1 ,ug of trypsin per ml (type 1, Sigma Chemical Co., St. Louis, Mo.).…”

“…Purification and immunization of the rotavirus were performed as previously described (17). Briefly, rotavirus propagated on MA104 cells was concentrated by polyethylene glycol (molecular weight, 7,500; Showa Chemicals Co., Tokyo, Japan) and then treated twice with fluorocarbon.…”

“…In a preliminary report, we provided evidence that antiserum against the Lincoln strain of bovine rotavirus showed different neutralizing behaviors toward the rotaviruses isolated from calves with acute diarrhea, and we suggested the presence of different types of bovine rotavirus which can be discriminated by SN (17).…”