A CELL CULTURE VACCINE AGAINST BOVINE EPHEMERAL FEVER

SUMMARY A vaccine was prepared from cell culture fluids harvested from the twelfth passage of the 919 strain of bovine ephemeral fever (BEF) virus in Vero cell cultures. Cattle were vaccinated subcutaneously with various combinations of strain 919 virus and adjuvants. Neutralising antibodies were assayed at various times after vaccination and some cattle were challenged by intravenous inoculation with the virulent 417WBC strain of BEF virus. Strain 919 virus of the third and twelfth passage levels in Vero cells produced neither fever, clinical illness nor detectable viraemia in 5 calves inoculated intravenously. Nor could viraemia be detected in 5 heifers receiving vaccine subcutaneously. When the vaccine was administered mixed with aluminium hydroxide adjuvant, the production of neutralising antibodies increased with an increase in the volume of vaccine from 2.5 ml to 10 ml and the response to 2 injections was significantly better than the response to a single injection. The neutralising antibody response was decreased when vaccine was diluted in phosphate buffered saline. The neutralising antibody response following 2 subcutaneous vaccinations with strain 919 virus mixed with aluminium hydroxide adjuvant was higher than that following intravenous inoculation with virulent virus. The vaccine-induced antibodies persisted for at least 12 months, and revaccination at this time led to an increase in the titre of neutralising antibody. Antibodies induced by a single subcutaneous administration of strain 919 virus mixed with Freund's complete adjuvant persisted for at least 40 weeks; those induced by vaccine containing Freund's incomplete adjuvant had virtually disappeared within 16 weeks. All these calves responded to vaccination with aluminium hydroxide-containing vaccine with increases in levels of neutralising antibodies. Of 26 vaccinated calves challenged with virulent BEF virus, 24 remained clinically normal. Two developed brief periods of pyrexia on the seventh day after challenge, but no other clinical signs. One of these calves had a viraemia that was demonstrated only by intravenous inoculation of a susceptible calf. The remaining calf had no detectable viraemia. All of 7 unvaccinated calves developed severe clinical BEF within 5 days of challenge. No disease attributable to the 919 virus occurred in 24 vaccinated pregnant heifers or their newborn calves.     

ATAXIA AND PARALYSIS ASSOCIATED WITH BOVINE EPHEMERAL FEVER INFECTION

SUMMARY Three adult cattle that had been ataxic for 5 to 7 months and a bull that had been paralysed for 24 days following bovine ephemeral fever infection were studied clinically and pathologically. Severe bilaterally symmetrical Wallerian degeneration was demonstrated in the spinal cords of all 4 cattle; in 3 animals an area of primary damage was present in the first cervical segment of the cord and in the other animal it was at the level of the lumbar cord. Pressure on the cord resulting from trauma was considered to be the most likely aetiology because of the similarity of the lesion to other syndromes causing pressure on the cord and the absence of the usual inflammatory changes seen with infectious agents.     

STUDIES ON THE PATHOGENESIS OF BOVINE EPHEMERAL FEVER

A study of the pathogenesis of bovine ephemeral fever confirmed that the major clinical signs were fever lasting no more than 2 days, with increased respiratory rate, dyspnoea and some degree of lameness. Haematological observations revealed a neutrophilia with a left shift and a lymphopaenia at the time of peak clinical reaction. The net result was a slight leucopaenia on the day after this reaction. The most prominent pathological changes involved the lungs and synovial joints. Pulmonary emphysema and alveolar collapse with bronchiolitis, degenerative changes in synovial membranes and increased synovial fluid were observed. Specific fluorescence indicating the presence of BEF viral antigen could be detected at the time of peak clinical response in individual cells in the lungs, spleen and lymph nodes as well as neutrophils. Before and after the peak fever some fluorescence was seen in cells which appeared to be reticular cells in the lymph nodes. Viral isolation in mice could be made from blood, lungs, spleen and lymph nodes over a period of no more than 3 days. It is postulated that viral growth takes place mainly in the reticuloendothelial cells in the lungs, spleen and lymph nodes and not in vascular endothelium or lymphoid cells.     

THE INFECTION OF MERINO SHEEP WITH BOVINE EPHEMERAL FEVER VIRUS

Four Merino sheep inoculated intravenously with bovine blood containing ephemeral fever virus showed no clinical signs of ephemeral fever. Two sheep showed a mild haematological response and developed a neutralising antibody which closely paralled that of a steer inoculated at the same time. Leucocytes harvested from these 2 sheep on days 3 and 4 after inoculation with virus reproduced ephemeral fever when inoculated into susceptible steers whilst those harvested on days 1, 2 and 5 did not. Even though this indicates that EFV can multiply in some sheep when they are inoculated intravenously, it cannot be inferred that natural infection occurs.     

THE EFFECT OF BOVINE EPHEMERAL FEVER VIRUS ON THE BOVINE FOETUS

Six pregnant heifers that were experimentally infected with bovine ephemeral fever virus produced normal calves. Foetuses of 8 heifers that were immune to bovine ephemeral fever were inoculated with bovine ephemeral fever virus. One was aborted after 36 days, but the cause of abortion could not be determined. One was sacrificed after 28 days. The foetus and its membranes were normal and neither virus nor antibody was demonstrated. The other 6 heifers produced normal calves. One calf, 501, inoculated after 160 days gestation, contained high levels of neutralising antibody in serum before ingestion of colostrum. The others, inoculated at gestational ages of 52 days to 157 days showed no evidence of neutralising antibody in blood samples collected at birth.     

THE SUSCEPTIBILITY OF YOUNG AND NEWBORN CALVES TO BOVINE EPHEMERAL FEVER VIRUS

The pathogenicity of a field strain, 417, of bovine ephemeral fever (BEF) virus for newborn and young calves was investigated. Three colostrum-deprived newborn calves inoculated intravenously developed severe clinical disease and viraemia, and produced long-lasting neutralising antibody. The incubation period in these animals was 10 and 11 days, compared with 5 to 7 days for older calves. Two newborn calves which received colostrum from immune dams and 2 which received colostrum from non-immune dams failed to respond clinically to intravenous inoculation with strain 417. The neutralising antibody response of these calves was of short duration. Four calves, 7 to 8 weeks old and lacking detectable neutralising antibody to BEF virus, or having low levels of antibody, did not develop clinical disease when inoculated intravenously. Four calves 12 to 14 weeks of age and free of detectable neutralising antibody to BEF virus developed clinical disease when inoculated with strain 417.     

THE SUSCEPTIBILITY OF CALVES TO INFECTION WITH A STRAIN OF BOVINE EPHEMERAL FEVER VIRUS INOCULATED INTRACEREBRALLY

Three newborn calves were inoculated intracerebrally with bovine ephemeral fever virus strain 525. The 2 that lacked detectable neutralising antibody to bovine ephemeral fever vaccine developed fatal encephalitis after 4 and 7 days respectively. The third calf which had a low level of maternal antibody remained healthy and developed antibody that became undetectable after 6 months. Bovine ephemeral fever virus strain 525 was reisolated from the brains of both dead calves by intracerebral inoculation of suckling mice. Homogenates that were prepared from the brains of the calves failed to produce disease or to induce antibody formation in susceptible calves when inoculated intravenously. Strain 525 of BEF virus has been shown to possess a degree of neurovirulence for laboratory animals that has not been reported for other strains (Tzipori and Spradbrow 1974). Although this strain is unable to produce viraemia in calves after I/V inoculation, the present study shows that strain 525 can multiply in the brain tissues of calves and cause death after I/C inoculation.     

SOME ASPECTS OF THE EPIZOOTIOLOGY OF BOVINE EPHEMERAL FEVER IN AUSTRALIA

Serum samples collected from cattle in Western Australia, Northern Territory, Queensland and New South Wales in 1966 had neutralising antibodies to ephemeral fever virus, although the last major epizootic of ephemeral fever was in 1955-56. The incidence of antibodies ranged from 1.5% in Western Australia to 29.0% in Queens- land, and 57.6% of serums assayed were of low titre (2 to 5). Antibodies were not found in serums collected from cattle in Victoria, South Australia, southern Western Australia or Norfolk Island. After the 1967-68 epizootic the pro-porton of cattle with antibody ranged from 3.1% to 47.6% in herds with antibody in Victoria to 81.8% to 91.7% in herds in Queensland, and 58.2% of serums assayed had antibody titres greater than 45. Cattle with low levels of antibody in 1966 had high levels after the 1967-68 epizootic, although it is not known what pro-portion showed clinical signs of ephemeral fever during the epizootic. Serum samples collected in 1966, and which contained low levels of antibody, were fractionated by gel filtration and the neutralising activity was confined to the 7S globulin fraction. In one cow experimentally infected with ephemeral fever virus, the neutralising activity at 15 days after inoculation was confined to the 19S globulin fraction, in both the 19S and 7S fractions at 22 days but was almost totally confined to the 7S fraction by day 36. The significance of the results is discussed, and it is suggested that ephemeral fever virus remains enzootic in areas of Australia between major epizootics, but the infecting virus may be of low pathogenicity and immunogenicity for cattle, resulting mainly in subclinical infections.