Neutralizing antibody sensitive to 2-mercaptoethanol in cattle infected with bovine ephemeral fever virus.
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Biomedical subjects
Publications and source records attributed to T Omori.
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Previous serolgoical studies strongly suggested Akabane virus to be the etiologic agent of epizootic abortion and congenital arthrogryposis-hydranencephaly in cattle, and this view was further corroborated in this study by the isolation of the virus from an aborted fetus in an epizootic of the disease and from a fetus extracted froma cow which was suggested by serologic tests to have a recent infection with the virus. The latter fetus had histological changes of encephalomyelitis and polymyositis, and specific antigens of Akabane virus was shown by the immunofluorescent technique in brain tissues as well as skeletal muscular tissues. The virus was recovered from various fetal tissues and fluids, and in relatively large amounts from brain, spinal cord, cerebral fluid, skeletal muscles and fetal placenta. The intracranial inoculation of suckling mice, 1-2 days of age, was the most sensitive system for Akabane virus isolation and HmLu-1, a continuous cell line from hamster lung, seemed almost as sensitive as suckling mice.
A cytopathogenic virus was isolated in the primary culture of bovine kidney cells from a nasal swab of affected calves in an outbreak of acute respiratory disease in Japan in 1971. It agglutinated human type O erythrocytes and produced cytoplasmic inclusion bodies. Viral replication was inhibited by 5-iodo-2'-deoxyuridine, indicating that the viral nucleic acid was RNA. The virus was resistant to ether, chloroform, sodium deoxycholate, and acid, and passed readily through Sartorius' membrane filter 100 nm in pore size, but not through the filter 50 nm in pore size. Electron microscopy showed many spherical particles 60 approximately 75 nm in diameter with a double-layered capsid in a sample taken at a buoyant density of 1.34 produced by CaCl equilibrium centrifugation. The virus suspended in 1M MgCl2 solution was stable against heating at 50 degrees C for 30 minutes, but not against freezing at -20 degrees C for 60 minutes. The virus was resistant to, and increased in infectivity after, treatment with 0.063 approximately 1.0% trypsin. These properties were consistent with those established for the reoviruses. Most affected cattle showed a significant rise of antibody titer against reovirus and bovine respiratory syncytial virus, whereas only a few of them presented a serological evidence for recent infection with parainfluenza virus type 3, bovine adenovirus type 7, and bovine parovirus.
In April, 1971, a disease with pyrexia and diarrhea as main symptoms broke out collectively among calves. Fecal samples were collected from calves involved and inoculated into bovine kidney (BK) cell cultures. As a result, the diarrheal feces of one calf were suspected to contain two agents simultaneously. One agent (C-121 E strain) was isolated from the primary infected BK cell culture fluid by terminal dilution passages. It had been predominant in replication and shown a cytopathic effect which gave rise to a granular appearance in the early stage of culture. The other agent (C-121 R strain) was isolated from the primary infected BK cell culture fluid by neutralizing the C-121 E strain contained in this fluid with antiserum against this strain. It caused cytoplasmic inclusion bodies to form. On the basis of their physico-chemical properties, the C-121 E strain was identified as bovine enterovirus and the C-121 R strain as reovirus. Serological tests indicated that some of the affected calves had been infected not only with the two strains isolated, but also with bovine viral diarrhea virus, bovine adenovirus type 7, and bovine parvovirus.
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An attempt was made to cultivate HmLu-1 cells in a rolling round bottle. As a result, the optimum conditions of cultivation were found to consist in the number of cells transplanted per bottle being 1 X 10(8), the volume of growth medium per bottle being 250 ml, and the velocity of rolling being 6 revolutions per hour. It was possible to make a monolayer of cells develop all over the glass surface under these conditions. A preliminary experiment was carried out to clarify the production of virus in the tube culture. In it, the highest virus titer was obtained two days after inoculation of a 4-day-old culture with a 1:100 dilution of stock virus. On the other hand, when the 4-day-old culture cells in the rolling round bottle were inoculated with virus suspension and when 100, 500, or 800 ml of maintenance medium was added to each bottle, there was little difference in virus titer obtained among the culture bottles. Then the virus yield per cell was compared between the rolling round bottle culture method and the stationary square bottle culture method. The highest virus titer was reached two days after virus inoculation, regardless of the culture method. The virus yield was 1.9 times as high in the rolling method as in the stationary method. From the results mentioned above, it was clarified that the rolling round bottle culture method made it possible to obtain a large amount of bovine ephemeral fever virus at a high titer in a labor-saving manner.
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