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.     

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 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.     

Studies on the pathogenesis of bovine ephemeral fever in sentinel cattle. I. Virology and serology

Twenty-two sentinel cattle were observed daily during an outbreak of ephemeral fever on a dairy farm in eastern Australia in the summer of 1981–192. Of the 22 cattle, 9 developed clinical ephemeral fever. None developed sub-clinical infection. The pattern of the epidemic was a single index case followed 10 days later by the main epidemic wave which lasted for 7 days. This wave stopped when there were still 14 uninfected susceptible animals remaining in the sentinel group, and when biting flies were very active. Ten isolations of bovine ephemeral fever virus were made in Aedes albopictus tissue cultures from the blood of 5 clinical cases. One hundred and twelve isolations of CSIRO Village virus and one each of Kimberley and Akabane viruses were also made from various members of the sentinel group. There was serological evidence that infections with Tibrogargan, Tinaroo and Aino viruses also occurred in 6 cattle in the observation period. The 13 cattle undergoing a sub-clinical viraemia with CSIRO Village virus, Tibrogargan, Kimberley, Akabane or Aino viruses at the time of the main outbreak, appeared to be temporarily protected against ephemeral fever. However, 9 of the 11 still remaining in the herd were susceptible in a subsequent outbreak of ephemeral fever 2 years later. Evidence is presented that subclinical infections with other arboviruses may limit an ephemeral fever epidemic by providing temporary protection by interference.     

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.     

Failure of sheep to respond to repeated inoculations with an alcelaphine herpesvirus-1-like virus, isolated from a case of malignant catarrhal fever in American cattle.

The replication of an alcelaphine herpesvirus-1-like virus (707K), isolated from a clinical case of malignant catarrhal fever in American cattle, was studied in sheep. Viraemia was not observed in any of the six sheep repeatedly inoculated with the 707K virus or in four steers susceptible to malignant catarrhal fever which were housed together with these sheep for one year. None of the four steers seroconverted and only two of the six inoculated sheep showed a negligible and short-lived seroconversion. The inability of the sheep to seroconvert adequately after repeated inoculations with the 707K virus, and the failure to recover the agent from them suggests that this agent does not replicate in sheep.     

Transmission of a malignant catarrhal fever-like syndrome to sheep: preliminary experiments

The transmission of a malignant catarrhal fever-like syndrome to sheep is reported. Fetal sheep between 40 and 66 days gestation were inoculated intravenously with viable cells either from a red deer with clinical malignant catarrhal fever or from rabbits with the disease. Of the 21 fetuses in the experiment only five were born live and of these four developed clinical signs similar to malignant catarrhal fever in other species and died or were killed 10, 16, 47 and 175 days after birth. The fifth lamb remained unaffected. Histology of the four affected lambs revealed a generalised lymphoproliferation, with T-dependent areas of lymphoid tissues being affected, and an overall paucity of immunoglobulin containing cells. In addition arteritis and interstitial infiltration of many organs by lymphoid cells was present. The infectious agent was not reisolated in rabbits from lambs or passed to red deer housed in the same pen and it is thus considered possible that gene expression of the putative virus was incomplete.     

Infection of sheep and goats with bovid herpesvirus 2

Sheep and goats were shown to be susceptible to experimental infection with bovid herpesvirus 2 (BHV2), administered by either the intradermal or intravenous route. Lesions developing in sheep following intradermal inoculation of virus were similar to those in cattle inoculated intradermally, whereas the lesions in goats resolved without ulceration or scabbing. Disseminated circumscribed skin lesions developed in sheep and goats given BHV2 by the intravenous route. These lesions resolved in four to eight days without significant effect on the skin. BHV2 was isolated from skin lesions of sheep, goats and cattle that were infected intravenously, from sheep and cattle infected intradermally and from the leucocytes of the three species following intravenous inoculation of virus. Latent infection of sheep and goats was demonstrated following corticosteroid treatment 60 days after infection.     

Experimental induction of malignant catarrhal fever in pigs with ovine herpesvirus 2 by intranasal nebulization

Malignant catarrhal fever (MCF), a frequently fatal herpesviral disease primarily of ruminant species, has been sporadically reported in pigs. All cases of naturally occurring porcine MCF reported to date have been linked to ovine herpesvirus 2 (OvHV-2), a gammaherpesvirus in the genus Macavirus carried by sheep. Experimental induction of MCF by aerosolization of the virus in nasal secretions collected from infected sheep has been successful in bison, cattle and rabbits. The goals of this study were to determine the susceptibility of pigs to MCF following experimental intranasal inoculation of OvHV-2, and to characterize the disease. Twelve pigs in four groups were nebulized with 10(5), 10(6), 10(7), or 10(8) DNA copies of OvHV-2 from sheep nasal secretions. Three control pigs were nebulized with nasal secretions from uninfected sheep. Three additional pigs were inoculated intravenously with 10(7) DNA copies of OvHV-2 to evaluate this route of infection with cell-free virus. Seven of twelve intranasally challenged pigs became infected with OvHV-2. Five of these seven, all in higher dose groups, developed MCF. Lesions resembled those reported in natural cases of porcine MCF. The most striking and consistent histological lesions were in trachea, lung, kidney and brain. These comprised mucopurulent tracheitis, interstitial pneumonia, necrotizing arteritis-periarteritis, and nonpurulent meningoencephalitis. No infection was established in the intravenously challenged or control groups. The study showed that MCF can be experimentally induced in pigs by aerosol challenge using sheep nasal secretions containing OvHV-2. Domestic pigs are a natural clinically susceptible host for sheep-associated MCF. They represent a useful, cost-effective model for MCF research.     

Quantitative transmissible gastroenteritis virus shedding patterns in lactating sows.

To test the role of sows in spreading transmissible gastroenteritis (TGE), 11 sows were intravenously, intranasally, or intramammarily inoculated with virulent virus within 5 days of farrowing. Six of the sows were separated from their offspring, and 5 were allowed to nurse their litters. All sows became clinically ill with sign of anorexia, depression, and fever that persisted until postinoculation day 4 or 5. They shed virus through milk, nasal secretions, and feces, with individual variations occurring in degree and duration of shedding in the 1st week after inoculation. Of 40 pigs separately fed milk samples from the 6 inoculated sows, 19 pigs (47.5%) became sick in 24 to 40 hours, and virus was isolated from them at necropsy. Of 43 pigs in the 5 litters that nursed exposed dams, all became sick with typical signs of TGE, and 29 (67.4%) died in 2 to 9 days. Sows given the single intramammary inoculation of virus developed statistically significant higher levels of TGE virus-neutralizing antibodies than did sows inoculated intravenously or intranasally.     

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.     

Sheep-associated malignant catarrhal fever involving 3-5-week-old calves in Saudi Arabia

Between late December 1999 and late April 2000, three locally bred Friesian calves (ageing 25, 28 and 35 days) in a dairy farm, at Al-Ahsa locality of the eastern region of Saudi Arabia showed dullness and inappetence. Their rectal temperatures ranged between 41 and 41.5 degrees C. One to 2 days later and onwards, the calves showed lacrimation, nasal discharge, salivation, oedema of the head, conjunctivitis, exo-ophthalmia and corneal opacity. One calf showed diarrhoea. The superficial lymph nodes were oedematous and swollen. The calves died after 7, 5 and 8 days, respectively, following the onset of the disease. Calves and rabbits were experimentally infected with materials from the naturally infected calves. The rabbits showed fever, mild conjunctivitis and one rabbit showed wet faeces. The experimentally inoculated calves showed rise in temperature and mild symptoms but none of them died. The virus from the naturally infected calves and from the experimentally infected rabbits was identified as malignant catarrhal fever (MCF) virus using both the complement fixation test and the fluorescent antibody test, employing a reference anti-serum against the WC 11 strain of MCF virus. Serological survey for MCF antibodies in cattle, sheep and goats from the affected farm revealed that 54% of the examined animals were positive. The situation of MCF in Saudi Arabia was discussed in relation to sheep and wild game. This paper constitutes the first report of MCF in Saudi Arabia and the Gulf region.     

Sheep‐Associated Malignant Catarrhal Fever Involving 3–5‐Week‐Old Calves in Saudi Arabia

Between late December 1999 and late April 2000, three locally bred Friesian calves (ageing 25, 28 and 35 days) in a dairy farm, at Al‐Ahsa locality of the eastern region of Saudi Arabia showed dullness and inappetence. Their rectal temperatures ranged between 41 and 41.5°C. One to 2 days later and onwards, the calves showed lacrimation, nasal discharge, salivation, oedema of the head, conjunctivitis, exo‐ophthalmia and corneal opacity. One calf showed diarrhoea. The superficial lymph nodes were oedematous and swollen. The calves died after 7, 5 and 8 days, respectively, following the onset of the disease. Calves and rabbits were experimentally infected with materials from the naturally infected calves. The rabbits showed fever, mild conjunctivitis and one rabbit showed wet faeces. The experimentally inoculated calves showed rise in temperature and mild symptoms but none of them died. The virus from the naturally infected calves and from the experimentally infected rabbits was identified as malignant catarrhal fever (MCF) virus using both the complement fixation test and the fluorescent antibody test, employing a reference anti‐serum against the WC 11 strain of MCF virus. Serological survey for MCF antibodies in cattle, sheep and goats from the affected farm revealed that 54% of the examined animals were positive. The situation of MCF in Saudi Arabia was discussed in relation to sheep and wild game. This paper constitutes the first report of MCF in Saudi Arabia and the Gulf region.     

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.     

Bluetongue virus serotypes 20 and 17 infections in sheep: Comparison of clinical and serological responses

The clinical, virological and serological responses of sheep infected with an Australian bluetongue virus (BTV) isolate (serotype 20) were compared to responses in sheep inoculated with an American bluetongue isolate (serotype 17) with which it had shown cross-reactions in serum neutralization tests. In sheep inoculated with BTV 20, clinical signs were very mild and viremia was first detected by day 5; virus was isolated intermittently for a further 2 to 3 days. Neutralizing and precipitating antibodies were first detected in the serum of the sheep between 2 to 3 weeks following inoculation. In contrast, sheep inoculated with BTV 17 showed pyrexia and severe hyperemia of the nasolabial area and oral mucosa from day 7 to 17. Viremia was first detected on day 3 and extended to day 20, while the appearance and titers of serum antibodies was similar in both groups.After challenge with BTV 17 the sheep in both groups remained clinically normal, and virus was not detected in the blood; however, serum neutralizing antibody titers to both viruses increased 2 weeks after challenge and the mean titer of the two groups ranged from 1:250 to 1:640.     

THE COMPARATIVE SENSITIVITY OF SHEEP AND CHICKEN EMBRYOS TO BLUETONGUE VIRUS AND OBSERVATIONS ON VIRAEMIA IN EXPERIMENTALLY INFECTED SHEEP

The virus titre in sheep blood samples received from BT-suspected cases in the field was assayed in sheep and in chicken embryos. These infected blood samples represented 3 different BT virus types: 4, 10 and 16. Three identical experiments were performed, one with each of the 3 different virus types. Ten-fold dilutions of the infected blood samples were prepared and 1 ml of each blood dilution was inoculated IV into series of 10 to 12-month old susceptible sheep; at the same time 0.1 ml of each dilution was inoculated IV into series of 10 to 13-day-old chicken embryos. The virus titre was found to be similar when assayed in the two host systems. There was no correlation between the amount of virus inoculated and the severity of symptoms in the inoculated sheep. The virus content in daily blood samples collected from the experimental sheep was assayed by IV inoculation of CE. Virus was isolated from all the reacting sheep and was detected sometimes as early as 1 day PI and as late as 30 days PI. A high titre of log10 4.0 to 7.0 per 1 ml of blood was recorded during several consecutive days before and after the onset of clinical signs. There seemed to be an inverse ratio between the amount of virus inoculated and the number of days the virus persisted in the bloodstream. The neutralisation index in day 22 serum samples was 3.5 to 4.5. Virus was isolated from some of the reacting sheep on the day that these antibody levels were recorded. Since the comparative simultaneous titrations of BT virus in sheep and in CE yielded similar results, the IV inoculation of CE is advocated as the routine method to be employed for laboratory diagnosis of this disease.     

Clinical, virological and serological response of the West African dwarf sheep to experimental infection with different strains of Rift Valley fever virus.

West African dwarf sheep were inoculated with three different strains of Rift Valley fever virus (RVFV). Using infective mouse serum as the source of virus classical RVFV disease characterised by sudden onset, a sharp but transient febrile response, viraemia, abortions and the development of specific RVFV antibodies in surviving animals was observed. The severity of clinical response was, however, dependent on the strain of virus used, with animals inoculated with Smithburn's neuroadapted strain showing a milder response than those inoculated with either the Nigerian or Lunyo strain. The inoculation of sheep with RVFV infective mouse brain material of the three different strains resulted in a mild febrile response with low level viraemia. Immune sera from sheep inoculated with both the Nigerian and Smithburn's neurotropic strains did not neutralise the Lunyo virus strain in a mouse intracerebral neutralisation test; the reverse, however, was not the case. The findings indicate that the West African dwarf sheep is highly susceptible to RVFV infection and that previous reports of only a mild clinical response following inoculation with the Nigerian strain were due to infective mouse brain rather than infective mouse serum.     

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, γ-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.     

Experimental infection of fetal and newborn Suffolk sheep with scrapie virus

Fetal (n = 21) and newborn (n = 7) Suffolk sheep were inoculated with scrapie virus isolated from other Suffolk sheep. Twenty fetuses, 76 to 109 days of gestational age, were inoculated IM in the neck through the uterine wall and were examined for virus 47 to 322 days later by mouse inoculation. Scrapie virus was not detected before 254 days of age; only traces of virus were detected in 3 of 7 lambs examined thereafter (2 at 254 days of age and 1 at 322 days of age). Virus was limited to the supra-pharyngeal, prescapular, and mesenteric lymph nodes. Seven lambs were inoculated into the palatine tonsils with scrapie virus as newborns (3 to 12 days old) and were examined for virus when they were 147 to 210 days old. Virus was not detected in the lymphoreticular tissues or terminal portion of ileum of any lamb. Failure to find scrapie virus in these lambs and in most lambs inoculated as fetuses might indicate few had became infected. However, if most lambs and fetuses had become infected, the long zero phase of the infection could have accounted for failure to find scrapie virus in many of them examined too soon after inoculation. The limited findings of this study indicate that efforts to demonstrate prenatal or neonatal transmission of scrapie by detecting virus are hampered by the slowness of its replication.     

Teratogenicity of a mutagenised Rift Valley fever virus (MVP 12) in sheep

A 5-fluorouracil mutagenised Rift Valley fever virus strain, which was shown to be attenuated and immunogenic in cattle and sheep, was evaluated for its ability to cause teratogenic effects in pregnant sheep. A group of 50 sheep at various stages of pregnancy was inoculated with the virus and the pregnancies followed to term. There were two abortions and 14% of the lambs produced by vaccinated ewes showed teratogenic effects, the most prevalent being spinal hypoplasia, hydranencephaly, brachygnathia inferior and arthrygryposis. The foetal malformations of the central nervous and musculo-skeletal systems were mostly consistent with those observed in sheep vaccinated with the attenuated Smithburn RVF strain. The teratogenic effects of MVP12 were not seen in previous experiments by other authors as immunisation of sheep took place in the second to third trimester of pregnancy, when the foetal brain tissue has completed most of its cell division.