Antigenicity and immunogenicity of equine influenza vaccines containing a Carbomer adjuvant.

Equine influenza vaccines containing inactivated whole virus and Carbomer adjuvant stimulated higher levels and longer lasting antibody to haemagglutinin in ponies than vaccines of equivalent antigenic content containing aluminium phosphate adjuvants. Five months after the third dose of vaccine containing Carbomer adjuvant, ponies were protected against clinical disease induced by an aerosol of virulent influenza virus (A/equine/Newmarket/79, H3N8). In contrast ponies which received vaccine containing aluminium phosphate adjuvant were susceptible to infection and disease. There was an inverse correlation between prechallenge levels of antibody detected by single radial haemolysis (SRH) and duration of virus excretion, pyrexia and coughing. All ponies with antibody levels equivalent to SRH zones of > or = 154 mm2 were protected against infection and all those with levels > or = 85 mm2 were protected from disease.

Duration of protective efficacy of equine influenza immunostimulating complex/tetanus vaccines.

Seven previously untreated five-month-old New Forest ponies received two doses of equine influenza immunostimulating complex vaccines, one with and one without an immunopurified tetanus toxoid component, given by deep intramuscular injection six weeks apart, followed by a booster dose without tetanus toxoid five months later. Fifteen months after the third dose of vaccine, the ponies were challenged by exposure to an aerosol of influenza A/Equine 2/Sussex/89 (H3N8), a virus isolated from a recent outbreak of influenza A/equine 2 in Britain. The challenge produced severe clinical signs of influenza (pyrexia and coughing) in five unvaccinated control ponies. Four of the vaccinated ponies were completely protected against clinical disease, and two of these were also protected against infection as demonstrated by their lack of an antibody response after challenge. No coughing was recorded among the vaccinated ponies, and only three of the seven vaccinated ponies experienced a transient mild pyrexia. The mean duration and severity of the pyrexia among the vaccinated ponies was significantly less (P < 0.01) than among the controls, and the excretion of virus was almost eliminated, thus demonstrating the protective efficacy of the vaccines 15 months after vaccination. Monitoring of tetanus antitoxin antibodies showed that protective levels (> or = 0.01/iu/ml) were maintained for at least 20 months after vaccination.

Responses of ponies to equid herpesvirus-1 ISCOM vaccination and challenge with virus of the homologous strain.

An experimental (ISCOM) vaccine previously shown to protect hamsters from lethal challenge with equid herpesvirus-1 (EHV-1), was tested in horses. Vaccination with EHV-1 ISCOMs induced serum antibodies to the major virus glycoproteins gp10, 13, 14, 17, 18 and 21/22a, whereas antibody responses to gp2 were weak or absent. High levels of virus neutralising antibody of long duration were induced, but did not prevent challenge infection with virus of the homologous strain. However, in the vaccinated ponies there was a significant reduction in clinical signs, nasal virus excretion and cell associated viraemia compared with age-matched unvaccinated controls. There was a strong correlation between pre-challenge levels of serum virus neutralising antibody and the duration and total amount of virus excreted from the nasopharynx.

Experimental infection of ponies with equine influenza (H3N8) viruses by intranasal inoculation or exposure to aerosols.

Infection of seronegative Welsh mountain ponies was established by intranasal instillation or exposure to nebulised aerosol of egg grown H3N8 viruses. Pyrexia and coughing were noted following intranasal instillation and high titres of virus were recovered from the nasopharynx. Exposure to aerosol resulted in more severe clinical signs characterised by high temperatures, dyspnoea, anorexia and coughing; lower levels of virus were recovered from the nasopharynx. The severity of clinical signs and the kinetics of virus shedding were dose-related with the minimal infectious dose being 10(2)EID50/ml when ponies were exposed to aerosols produced by nebulisation of 20ml allantoic fluid. Full clinical signs only developed when ponies were exposed to a dose of 10(6)EID50/ml. It was concluded that exposure to nebulised aerosols of egg grown H3N8 viruses was a more reliable method of inducing clinical influenza than intranasal inoculation and would be more suitable for challenge studies.

Antibody isotype responses in the serum and respiratory tract to primary and secondary infections with equine influenza virus (H3N8).

Serum antibody (IgGab, IgM and IgA) responses to primary and secondary infection with influenza A/equine/Newmarket/79 (H3N8) by nebulised aerosol were compared with local (nasopharyngeal and tracheal) antibody responses in ponies. Circulating IgGab antibody was of long duration after primary infection, whereas IgM responses were short-lived after both primary and secondary infections. The antigenic stimulation of secondary infection with equine influenza was sufficient to induce elevations of serum IgM and IgA in the presence of high levels of circulating IgGab. These results support the potential of virus-specific IgM measurement for the detection of recent exposure to virus in horses which have high levels of circulating IgGab. Unlike serum IgGab, nasal and tracheal wash antibody of this isotype did not show long duration after primary infection, but local antibody memory was demonstrated by anamnestic responses on rechallenge. Nasopharyngeal IgA developed later than IgGab and IgM, and was more durable.

Duration of circulating antibody and immunity following infection with equine influenza virus.

The duration of immunity as measured by virological, serological and clinical responses following infection with influenza A/equine/Newmarket/79 (H3N8) was assessed in repeated challenge experiments in which ponies were infected by exposure to aerosols of infectious virus. Previous infection stimulated complete clinical protection which persisted for at least 32 weeks as demonstrated by the absence of febrile responses and coughing in two groups of ponies infected 16 weeks or 32 weeks after the first infection. Partial clinical protection persisted for over a year as demonstrated by the absence of coughing and a reduction in the number of febrile responses in a group of ponies infected 62 weeks after their first infection. These results contrasted with those observed in immunologically naive control ponies which developed pyrexia, dyspnoea and nasal discharge and coughing. The kinetics of virus specific antibody production in primary and secondary infections with equine influenza were studied by the single radial haemolysis test and a radioisotopic antiglobulin binding assay which measured virus specific IgGab antibody isotype. Antibody to the haemagglutinin, as measured by the single radial haemolysis test, declined rapidly after primary infection whereas the IgGab responses to whole virus antigens persisted for longer. The single radial haemolysis test was therefore particularly useful for the detection of antibody responses in multiple infections or exposures to influenza antigens. The radioisotopic antiglobulin binding assay was more sensitive for identifying infections which had occurred more than six months previously, as evidenced by anamnestic IgGab responses in ponies with low levels of antibody before rechallenge.

Serological and virological investigations of an equid herpesvirus 1 (EHV-1) abortion storm on a stud farm in 1985.

An extensive outbreak of EHV-1 abortions occurred on a stud farm in England in 1985. Of the 67 pregnant mares present on the stud farm, 31 were challenged with EHV-1, resulting in the loss of 22 fetuses or foals. Laboratory investigations revealed that the spread of the virus closely followed movement of apparently healthy mares (during the incubation period of the infection). During the outbreak mares were challenged 1-4 months before the expected foaling date. There was no relationship between the gestational age at the time of challenge and the subsequent outcome of infection in terms of abortion. The period between the estimated date of infection and abortion varied between 9 days and 4 months. Virus was isolated from the nasopharynx of 3 apparently healthy foals born during the outbreak.

A study of the host range and distribution of antibody to Akabane virus (genus bunyavirus, family Bunyaviridae) in Kenya.

Serum neutralizing antibody to Akabane virus (genus bunyavirus, family Bunyaviridae) was found in a high proportion (50-95%) of cattle sampled in Kenya, while sheep and goats had fewer positive (13-33%). Camel and horse sera also contained antibody to the virus (70% and 50% respectively). The antibody was found in animals from the high altitude temperature type of grasslands, drier bushed and wooded grasslands and the semi-desert. No arthrogryposis nor hydranencephaly has been encountered in Kenya which might be related to this widespread virus infection. A wide range of Kenyan wild ruminants had antibody to Akabane virus in their sera, as also did zebra.

Microneutralisation systems for use with different strains of peste des petits ruminants virus and rinderpest virus.

Comparative studies were made to determine the most suitable microtitration system for assaying strains of peste des petits ruminants virus (PPRV) and rinderpest virus (RV). Infectivity titres did not differ significantly when assayed in either calf kidney, sheep kidney or Vero cells. However, cytopathic effects were much easier to detect in the latter making them the cell of choice. Addition of small amounts of virus to preformed cell monolayers in microplates with the subsequent addition of maintenance medium give higher infectivity titres than when cell suspension was added to virus, although the latter is more convenient for routine use. The titres of PPRV and neutralising antibodies assayed in tubes and microplates were not significantly different. Simultaneous screening of sera at a 1 in 20 dilution against both PPRV and RV gave a higher incidence of positives against homologous as opposed to heterologous virus.

Re-emergence of rinderpest as a threat in East Africa since 1979.

Following the success of the JP15 scheme and subsequent annual vaccination campaigns, East Africa was virtually free of rinderpest after the mid 1960s and the disease was considered beaten. However, economic difficulties have recently reduced the expensively maintained vaccine cover and the disease has reappeared throughout much of the region. In 1979 rinderpest was diagnosed in cattle in north eastern Uganda and caused considerable losses until finally brought under control in 1981. No field outbreaks of the disease in cattle have been seen in Kenya but there is serological evidence that the virus has recently infected unvaccinated sheep and goats and wild ungulates in that country. In 1982 rinderpest was confirmed in the laboratory as the cause of death of large numbers of buffaloes in northern Tanzania and implicated as the cause of a rinderpest-like disease of cattle which is reported to be still active in that area. Substantial aid is essential for further control and research if the virus is not again to become endemic in the region.

Role of wildebeest fetal membranes and fluids in the transmission of malignant catarrhal fever virus.

Malignant catarrhal fever virus was not isolated from samples of fetal membranes or fluid collected from 93 calving wildebeest (Connochaetes taurinus) in Kenya Maasailand. Cell-free strains of malignant catarrhal fever virus were very rapidly inactivated when exposed to the sun under field conditions, at least 3.0 log10 units/25 microliter being lost per hour at midday. It is suggested that wildebeest fetal membranes and fluids act as visual markers for areas of pasture which are particularly heavily contaminated with malignant catarrhal fever virus in oculonasal secretions of wildebeest calves. It is possible that starting to graze cattle one to two hours later each morning may be a useful measure for helping to protect cattle from malignant catarrhal fever in areas where they are forced to share pastures with calving wildebeest.

Antibodies in carrier wildebeest to the lymphoproliferative herpesvirus of malignant catarrhal fever.

Six types of antibody to malignant catarrhal fever virus (MCFV) were measured in 132 sera collected from Wildebeest in Kenya Masailand. The titre of all types of antibody declined slowly with increasing age of the wildebeest. A significantly greater proportion of wildebeest calves had higher titres of antibodies to MCFV early antigens, IgM antibodies to MCFV late antigens and complement-fixing antibodies, than did older animals. One seronegative calf, reared in isolation without colostrum, became seropositive 4 1/2 weeks after birth but did not show any clinical signs indicative of MCFV infection. Similarities between MCFV infection of wildebeest calves and other inapparent infections with lymphoproliferative herpesviruses are discussed.

Neutralising antibodies to rinderpest virus in wild animal sera collected in Kenya between 1970 and 1981.

Four hundred and twenty five sera were collected from 18 species of wild mammals in Kenya between 1970 and 1981. Neutralising activity to rinderpest virus (RV) was detected in 35 samples from 13 species. This activity appeared to be specific antibody to RV since it did not neutralise the virus of peste des petits ruminants. It was associated with the serum globulin fraction and it blocked the staining of a rabbit immunofluorescent antibody conjugate to RV. Positive sera were not restricted to any particular area of Kenya. It is possible that strains of RV with low pathogenicity and low transmissibility are still present in wildlife in East Africa, a fact which must be considered when studying the epidemiology and control of rinderpest.

Detection of rinderpest virus antigens in vitro and in vivo by direct immunofluorescence.

Cell-culture attenuated and virulent strains of rinderpest virus (RV) were inoculated on to bovine kidney cell cultures. A direct immunofluorescent antibody test detected RV antigens in cell cultures within one to three days after inoculation whereas RV cytopathic effects usually took three to nine days to develop. Cells containing RV antigens were also detected in impression smears and frozen sections of tissues collected from RV infected animals at post mortem examination, and in smears of lymph node biopsies taken from cattle with clinical rinderpest. These techniques may offer additional methods for rapid diagnosis of rinderpest.

Microtitre techniques for the assay of rinderpest virus and neutralising antibody.

Microtitre techniques were compared with conventional tube techniques for their ability to assay rinderpest virus and neutralising antibody to the virus. The microtitre technique was as sensitive and reliable for assaying the virus as the recommended tube technique, using cell suspensions. Both of these methods, however, were less sensitive than tube titrations on preformed cell monolayers. The microtitre test was as sensitive as the tube test for detecting and assaying virus neutralising antibody and more robust in that it was less sensitive to variations in virus dose.

Influence of temperature on the growth in cell culture of malignant catarrhal fever virus.

The growth characteristics of malignant catarrhal fever (MCF) virus in bovine thyroid cultures were affected by incubation temperature. The cytopathic effect at 37 degrees C was predominantly syncytial and little or no cell-free virus could be detected. At 32 degrees to 34 degrees C foci of rounded refractile cells were observed, and this was accompanied by an increase in the amount of cell-free virus found in culture fluids. Growth curve studies with one low passage isolate of MCF virus showed that optimum yields of cell-free virus were obtained at 32 degrees to 34 degrees C and survival curves at 32 degrees and 37 degrees C indicated that this was a result of the relatively short half life of the virus at the higher temperature. A number of other benefits resulted from the use of lowered incubation temperature and these are discussed with reference to in vitro work with the virus.

Microelisa test for detecting antibodies to rinderpest virus antigens.

A microplate enzyme-linked immunosorbent assay (ELISA) was developed which detected antibodies to a soluble antigen prepared from sonicated rinderpest virus-infected cells. The ELISA detected titres of antibody to the virus in the sera of cattle 3 weeks after immunisation with tissue culture rinderpest virus vaccine which were similar to those detected by the virus neutralisation test. The ELISA test shows potential as a rapid and economic technique for screening large numbers of sera for antibody to rinderpest virus.