Studies on the pathogenesis of bovine ephemeral fever. IV: A comparison with the inflammatory events in milk fever of cattle.

The study of ephemeral fever in cattle has defined a range of haematological and biochemical changes in blood which are characteristic of an inflammatory response. One of the clinical signs of ephemeral fever, a temporary paralysis reversible by treatment with calcium borogluconate, is similar to that in milk fever (parturient paresis), a disease of multiparous dairy cows. Three separate groups of cows were studied. Four multiparous cows were observed and sampled repeatedly during calving, three similar cows and one cow calving for the first time in a dairy herd were sampled daily before and after calving; and, in other dairy herds, seven cows with milk fever were sampled during illness. One of the cows under repeated observation during calving developed milk fever. The results showed that all the inflammatory indicators in blood were present in the multiparous cows at calving and that these were essentially similar to those established in ephemeral fever. The similarities in the four cows sampled repeatedly during the periparturient period were: a rectal temperature rise of 1 to 1.2 degrees C; rise in circulating neutrophils to peaks between 5700 and 11200 l-6; disappearance of eosinophils for 1 day; hypocalcaemia (plasma Ca < 2.0 mM l-1); fall of plasma zinc to low levels immediately after calving (plasma Zn < 500 micrograms l-1); fall of inorganic phosphate (plasma P < 0.9 mM l-1); rises in copper (plasma Cu > 1000 micrograms l-1) and plasma fibrin to > 8.75 g l-1. Plasma glucose peaked at calving between 5.7 and 8.9 mM l-1 then fell to levels ranging between 3.4 and 3.8 mM l-1. Plasma iron rose in one cow to 1220 micrograms l-1, was unchanged in one cow and fell in the other two to 440 and 860 micrograms l-1 respectively. The three multiparous cows which were sampled daily and calved normally showed similar haematological, macro and micromineral changes and fibrin response as did the seven milk fever cases. In the periparturient period, milk fever cows differed from multiparous cows calving normally, in degree but not in kind, of inflammatory response. It is postulated that an inflammatory event occurs in the periparturient period of multiparous cows which partially accounts for the falls in plasma calcium. This can precipitate a paralysis and other hypocalcaemic signs similar to that seen in acute ephemeral fever.

Effective vaccination of cattle using the virion G protein of bovine ephemeral fever virus as an antigen.

In a series of experiments, the envelope glycoprotein (G protein) of bovine ephemeral fever virus (BEFV) induced immunity against challenge with virulent virus. Protection correlated with the level of specific serum antibodies to G protein measured by a blocking ELISA test and with the level of neutralizing antibody. The optimum vaccination regimen consisted of two injections given 21 days apart at a dose rate of 0.32 microgram per cow of purified G protein emulsified in the adjuvant Quil A. This schedule conferred immunity for the duration of the preliminary experiment (46 days). Immunity to severe disease, but not to infection, remained for at least 12 months after vaccination, although BEFV could not be reisolated from vaccinated cattle following challenge. Unvaccinated cattle used as controls exhibited typical signs of clinical ephemeral fever and BEFV was recovered from all control animals following challenge.

Studies on the pathogenesis of bovine ephemeral fever in experimental cattle. III. Virological and biochemical data.

In an attempt to define the nature of the response of cattle to ephemeral fever infection, a number of indicators of inflammation were monitored during clinical disease. The total Ca, Zn, Fe, Cu, glucose and phosphate in plasma, together with blood ammonia, were assayed relative to changes in the rectal temperature. CaT levels fluctuated markedly and hypocalcaemia occurred in 4 of 8 cattle. Plasma Zn and Fe values fell while plasma Cu levels rose markedly in all cattle. Mean levels of serum NH3 of 20-30 mumol l-1 rose to a peak value of 56 mumoll-1. Plasma glucose levels rose to a peak of 4.6 +/- 0.5 mMl-1 and the plasma phosphate levels fell from 2.4 +/- 0.1 mMl-1 to 1.17 +/- 0.2 mMl-1 during fever. Values of pCO2 fell from a mean of 46.9 +/- 3.6 mmHg to 36.4 +/- 3.1 mmHg and coincided with a rise in pH. Virus was isolated 73 h (+/- 23) after inoculation and persisted until 130 h (+/- 21). The common role of these parameters in generalised inflammation and ephemeral fever is discussed.

T-helper cell and associated antibody response to synthetic peptides of the E glycoprotein of Murray Valley encephalitis virus.

A battery of 16 synthetic peptides, selected primarily by computer analysis for predicted B- and T-cell epitopes, was prepared from the deduced amino acid sequence of the envelope (E) glycoprotein of Murray Valley encephalitis (MVE) virus. We examined all of the peptides for T-helper (Th)-cell recognition and antibody induction in three strains of mice: C57BL/6, BALB/c, and C3H. Lymphoproliferative and interleukin-2 assays were performed on splenic T cells from mice inoculated with peptides in Freund's incomplete adjuvant or with MVE virus. Several peptides found to contain predicted T-cell epitopes elicited a Th-cell response in at least one strain of mice, usually with a concomitant antibody response. Peptides 145 (amino acids 145 to 169) and 17 (amino acids 356 to 376) were strongly recognized by T cells from all three inbred strains of mice. Peptide 06 (amino acids 230 to 251) primed C57BL/6 mice for Th- and B-cell reactivity with native MVE virus, and T cells from virus-immune mice were stimulated by this peptide. Peptide 06 was recognized by several Th-cell clones prepared from mice immunized with MVE, West Nile, or Kunjin virus. These results indicate that it may be feasible to design synthetic flavivirus peptides that define T-cell epitopes capable of generating a helper cell response for B-cell epitopes involved in protective immunity.

Bovine ephemeral fever.

Ephemeral fever remains a viral disease of considerable importance to many countries including Australia. The virus has been only partly characterised and still awaits final classification. Although BEF virus was first thought to contain 6 structural proteins there is increasing evidence to suggest that it contains the 5 proteins characteristic of the Rhabdoviridae. Although BEF is thought to be arthropod borne, the vector has yet to be identified but it is clear from the distribution of BEF that more than one vector is capable of transmitting the disease. Despite rigorous investigation of the clinical signs and the pathology of ephemeral fever, little progress has been made on the pathogenesis of the disease. This has been partly due to the difficulty of propagating BEF virus in vitro and the inability to define the site of replication. However, there is mounting evidence to suggest that BEF is immunopathologic in nature and that the clinical expression of the disease is influenced by the release of one or more mediators of inflammation. The disease is characterised by a number of haematological and biochemical changes and early and prolonged treatment with phenylbutazone is capable of reversing a number of these changes. The intravenous administration of calcium can now be considered a justifiable addition to the treatment regimen together with prolonged phenylbutazone therapy. The vaccines currently available are prepared from either live attenuated or killed virus and may be less than reliable. There appears to be a need for a reliable, inexpensive, cold-chain independent alternative vaccine.

The effect of anti-inflammatory agents on the clinical expression of bovine ephemeral fever.

The effect of two anti-inflammatory drugs on the development and persistence of clinical signs in cattle experimentally infected with bovine ephemeral fever (BEF) virus was investigated by their administration, either before or after the commencement of fever. A total of 16 cattle was given phenylbutazone sodium (PBZ). The drug prevented fever and other clinical signs in six cattle when given daily during the incubation period, and at 8-h intervals for 5 days when clinical disease might be expected. When treatment with PBZ was deferred until 2-4 h after the commencement of fever, the rectal temperature returned to normal within 4 h in four of six cattle and the development of other clinical signs was suppressed. Clinical signs of ephemeral fever occurred in four untreated cattle infected at the same time. Viraemia, the development of neutralizing antibodies (at 8-11 days), resistance to subsequent challenge with BEF virus, neutrophilia, lymphopenia and a rise in plasma fibrinogen occurred in all BEF-infected animals whether treated or untreated, despite different clinical appearances. The mean peak of plasma fibrinogen in the untreated cattle was 6.9 g l-1; 3.2 g l-1 when treated 2-4 h after fever developed and 3.8 g l-1 when treated from 18-h post-infection. BEF virus was isolated from leucocytes of each of the cattle, but the frequency of isolation was lower in the treated group. The results indicate that treatment with PBZ blocked the host response which produces the clinical signs and did not have an anti-viral effect. In a similar experiment, a long-acting anti-inflammatory drug, flunixin meglumine, failed to prevent BEF or to modify the clinical signs once they had developed, except for the rectal temperature which returned to normal within 2-4 h of the administration of the drug. The efficacy of this drug was not improved by increasing the dosage to two or three times the recommended level.

Flavivirus-specific murine L3T4+ T cell clones: induction, characterization and cross-reactivity.

Murine T cell clones specific for the Kunjin (KUN), West Nile (WN) and Murray Valley encephalitis (MVE) flaviviruses were generated in vitro following priming in vivo. Clones were isolated by limiting dilution and maintained in culture with antigen stimulation and interleukin-2(IL-2). The cells were characterized as having the Thy 1+, L3T4+ and Lyt 2- phenotype by immunofluorescence. All clones proliferated strongly and secreted high levels of IL-2 and IL-3 in response to homologous antigen. Both KUN- and WN-specific clones showed extensive cross-reactivity to KUN and WN antigen, but recognized MVE to a lesser extent. In contrast, MVE-specific clones cross-reacted strongly with both KUN and WN. These data show that antigen-specific, major histocompatibility complex-restricted L3T4+ T cells are generated during flavivirus infection and are cross-reactive for viruses of the same subgroup.

Epidemiology of bovine ephemeral fever in Australia 1981-1985.

Bovine ephemeral fever is an important viral disease of cattle in Australia. The disease occurred each year, principally in summer and autumn, between 1981 and 1985. Queensland and the northern half of New South Wales were areas of greatest activity with only sporadic cases being reported from the Northern Territory and the northern third of Western Australia. Since 1981, the disease has been endemic in an extensive area of eastern Australia and has tended to occur in widely scattered outbreaks rather than the north-south advancing wave form of the epidemics of 1936-37, 1967-68, 1970-71 and 1972-74. The southernmost outbreaks between 1981 and 1985 were well within the limits of these earlier epidemics. The pattern of disease appears to have become seasonally endemic rather than periodically endemic in the northern two-thirds of eastern Australia. Ephemeral fever was not recorded in Victoria, Tasmania, South Australia or the southern part of Western Australia between 1981 and 1985. The disease was most frequently reported in cattle under 3 years of age, but also occurred in older cattle.

Experimental infection of bulls and cows with bluetongue virus serotype 20.

Bluetongue virus serotype 20 (BTV20) was inoculated intradermally and subcutaneously in 4 bulls and by the intrauterine route in 8 nulliparous cows after insemination at oestrus. Viraemia was detected intermittently between 8 and 21 days after inoculation. Virus was isolated from tissue samples of 2 cows and a bull after slaughter at 14 days and from one bull at 28 days. Group reactive and type specific antibodies to BTV20 were demonstrated from 17 to 27 days after infection. No antibodies were detected in the animals slaughtered at 14 days. No clinical signs of disease were seen during the experiment and no gross or histopathological changes referable to BTV20 infection were observed post-mortem. Because of the viraemia and the production of detectable serum antibodies, gametes from these cattle would be excluded from export.

Bluetongue virus serotype 20: experimental infection of pregnant heifers.

Three groups of 4 cows at 84 to 95 days, 100 to 160 days, and 170 to 180 days pregnant were inoculated both intradermally and subcutaneously with bluetongue virus serotype 20 (BTV20). Clinical observations and the viraemic and serological responses of the cows were followed for 9 to 17 weeks after inoculation. Viraemia developed in 9 of the 12 cows and was first detected 4 to 9 days after inoculation. Viraemia was detected for 4 to 21 days and in some animals only intermittently. The titre of the viraemia was obtained in 4 cows and ranged from detectable only, to 10(1) to 10(2.8) 50% tissue culture infecting doses per ml. Both serum neutralising and precipitating antibodies were detected in 11 of the 12 cows within 2 to 8 weeks after inoculation. No clinical responses were seen and one cow (516) did not develop a viraemia or produce detectable antibodies to the virus. The cows, calves and foetuses were necropsied following either parturition or slaughter between 200 and 270 days of pregnancy. No virus isolations were made from a wide range of tissues from the cows, calves or foetuses and no immunoglobulins or serum neutralising antibodies were detected in the serums of precolostral calves or foetuses at necropsy. No gross or histopathological lesions were seen in the cows, calves or foetuses, and there was no evidence that BTV20 crossed the bovine placenta or infected the foetus.

Studies on the pathogenesis of bovine ephemeral fever in sentinel cattle. II. Haematological and biochemical data.

Twenty-two non-lactating dairy cattle from a sentinel herd previously described (St. George, 1985) were monitored daily during an outbreak of ephemeral fever. Nine developed clinical ephemeral fever between 25 December 1981 and 30 January 1982. There were no subclinical infections with bovine ephemeral fever virus in the group. There were, however, subclinical infections with CSIRO Village, Akabane, Aino, Tinaroo and Kimberley viruses as described by St. George et al. (1984). Six of the nine affected cattle showed a neutrophilia with a concurrent lymphopaenia on the day of pyrexia; however, the differential white cell profile had begun to change up to 24 h prior to leucocytosis. Serum carboxypeptidase values fell by 24 h following the febrile response. Plasma fibrinogen rose rapidly in all six cows. The peak concentration (15.6 +/- 2.70 g l-1) occurred 3 days after pyrexia with the highest individual increase being from 6.05 to 19.6 g l-1. Plasma fibrinogen levels remained elevated for at least 7 days. Serum calcium fell significantly during Day 1 of the disease, the mean decline being 0.22 +/- 0.08 mmol l-1. The greatest individual fall was from 2.33 to 1.92 mmol l-1. None of the affected cattle showed any compensatory change in serum magnesium. There was no change in the normal values of creatinine, urea, gamma-GT, AST and alkaline phosphatase. Bovine ephemeral fever virus was isolated from only four of the six cases, whereas specific antibody was detected in all cattle 3-4 days after recovery.

The clinico-pathological effect of bluetongue virus serotype 20 in sheep.

Fifty-four Merino crossbred sheep were inoculated with bluetongue virus serotype 20 (BTV-20) by the intravenous, subcutaneous and intradermal routes. BTV-20 was successfully transmitted by Culicoides (Avaritia) spp. No. 5 to two additional sheep. Clinical and pathological effects were studied. In the artificially infected sheep, clinical signs were observed after an incubation period of 6 to 10 days and consisted of pyrexia, oral and subcutaneous hyperaemia mild oedema of the ears, face and lips, and coronitis. The major internal pathological changes were petechial and ecchymotic haemorrhages in the tunica media of the pulmonary artery near its junction with the heart and mild haemorrhage and mild oedema in the intestines, coronet, lips, cheeks and ears. Viraemia was detected between day 2 and day 14 post inoculation. The two sheep infected by insect transmission were mildly affected and became viraemic between 16 and 19 days after transmission. No deaths occurred and under experimental conditions BTV-20 caused only mild disease in housed sheep. To date there has been no reported outbreak of natural bluetongue infection in Australia. Compared to other serotypes BTV-20 appears to be of low pathogenicity in sheep.