Serial inoculation of sheep with two bluetongue virus types

Groups of sheep inoculated with bluetongue virus type 4 were challenged at various intervals after inoculation (from seven to 70 days) with bluetongue virus type 3. Examination of the clinical and serological response showed that animals were protected from challenge with a second bluetongue virus for up to 14 days after the inoculation of the first virus type. An adoptive transfer experiment in monozygotic sheep involving both antibody and T lymphocytes was carried out. Only partial protection was observed against heterologous virus challenge, indicating that although the T cell response has a cross-protective component, antibody is not involved. These observations indicate that current vaccination procedures should be reappraised, particularly in terms of revaccination with multiple bluetongue virus type.     

Laboratory evaluation of a living attenuated vaccine against bluetongue type 20 virus

Living attenuated bluetongue Type 20 virus vaccine was tested in 9 to 12 month-old Australian Merino sheep, held in air conditioned, insect-free accommodation. The vaccine appeared avirulent and immunogenic and protected against infection with a second dose of homologous vaccine virus. No enhancement of virulence or significant change in immunogenicity was observed when the vaccine was passaged 3 times through sheep without antibody to bluetongue virus.     

Clinical and serological response of sheep to serial challenge with different bluetongue virus types

A group of British sheep was infected with bluetongue virus 5 (BTV5) and subsequently challenged with the same virus type. Protection from this challenge and a homotypic BTV neutralising antibody response were observed. A second group of sheep was infected serially with three different BTV types. Animals previously exposed to BTV4 and BTV3 were found to be resistant to challenge by BTV6. Animals infected with BTV4 and challenged with BTV3 were shown to produce a transient heterotypic neutralising antibody response to a number of types. Although the level of this heterotypic response diminished with time, after challenge with BTV6 these animals developed a similar broad heterotypic response. The nature of this response and its implications in terms of observed protection merit consideration in future vaccine design and evaluation of field survey work.     

Bluetongue and related viruses in Nigeria: Experimental infection of West African dwarf sheep with Nigeria strains of the viruses of epizootic haemorrhagic disease of deer and bluetongue

West African dwarf sheep were inoculated by the subcutaneous route with epizootic haemorrhagic disease of deer (EHD) virus or bluetongue (BT) virus. No clinical disease was observed following primary EHD or BT infection, or subsequent challenge with either homologous or heterologous virus. However, viraemia was detected in non-immune sheep exposed to BT virus, but not in BT- or EHD-immune sheep challenged with either virus, or in non-immune sheep exposed to primary EHD virus infection. Complement fixing antibodies developed against both EHD and BT viruses following the primary infection with either virus, or subsequent challenge with homologous or heterologous virus. Following a primary infection, virus-neutralising (VN) antibodies developed only against the inoculated virus, while the detection of VN antibodied to both viruses followed the challenge of an EHD- or BT-immuned sheep with either the homologous or heterologous virus. These findings further support previous reports of a relationship between EHD and BT viruses. between EHD and BT viruses.     

In vitro immune serum-mediated protection of pig monocytes against African swine fever virus

Serum samples from pigs that had recovered from infection with a Dominican Republic isolate of African swine fever virus (ASFV) were mixed with dilutions of the virus, then assayed in microcultures of normal pig mononuclear leukocytes to determine whether the samples contained antibodies that protected monocytes against the virus. Protection was determined by the difference in titer (log10) between virus mixed with healthy pig serum and virus mixed with immune pig serum, using 50% cytopathogenic effect end points; protection was expressed as an immune serum-protection index. After addition of virus-serum mixtures to mononuclear leukocyte microcultures, a time-dependent decrease in protective index and production of infectious virus (determined by use of yield reduction assays) were observed. Protective effects were associated with the immunoglobulin fraction of serum, were rapidly lost on dilution, and were independent of complement. Antibody was most protective for the homologous Dominican Republic isolate of ASFV, with decreased protection against Lisbon '60 ASFV, and no protection against foot-and-mouth disease virus or bluetongue virus. Low concentrations of protective antibody were found during the acute viremic phase in infected pigs; antibody increased to maximal concentrations as the viremia decreased.     

Immune responses of sheep to louping-ill virus vaccine

The immune responses of sheep to single and double doses of commercially available louping-ill virus vaccine were examined. The susceptibility to challenge of sheep which had been vaccinated but showed a poor response was also investigated. Two injections of vaccine were required to provoke an adequate antibody response and maximum titres were obtained when there was an interval of two to eight weeks between injections. After challenge, viraemia could not be detected in animals with an antibody titre of 20 although increase in the concentration of humoral antibodies indicated that infection had occurred. Vaccinated but seronegative sheep and vaccinated animals with an antibody titre of 10 were also clinically resistant to the challenge, although circulation of virus was demonstrated. That vaccination had sensitised those animals to viral antigen was evident from the reduced viraemias, the early rise in humoral antibody titres and subsequent protection afforded compared to unvaccinated control animals. Thus, animals with minimal antibody titres after vaccination are protected, but it is recommended that vaccines eliciting the highest possible antibody responses will be the most useful under field conditions.     

A monovalent attenuated serotype 2 bluetongue virus vaccine confers homologous protection in sheep

An outbreak of bluetongue caused by bluetongue virus serotype 2 virus in certain Mediterranean countries during 1999/2000, presented an opportunity to produce a monovalent type 2 vaccine. Since no data have been published previously on the protection conferred by the current live attenuated bluetongue vaccine strains used in the polyvalent vaccine, a challenge experiment was performed to determine the degree of homologous protection induced by the type 2 vaccine strain. The standard vaccine dose of 5 x 10(4) pfu of vaccine conferred 99.7% protection against clinical disease and no viraemia was detected in the vaccinates.     

Virological, clinical and serological responses of sheep infected with tissue culture adapted bluetongue virus serotypes 10, 11, 13 and 17

Sheep were experimentally infected with cloned strains of tissue culture adapted bluetongue virus (BTV) serotypes 10, 11, 13 and 17. All the infected animals developed viremia by Day 2 or 3 post-inoculation (P.I.) and reached maximum viremia on Day 7 P.I. The viremia lasted for 2 to 3 weeks. Animals infected with the different serotypes showed mild clinical bluetongue (BT) responses, characterized by pyrexia and leukopenia, which coincided with the peak of viremia. Antibodies appeared by Day 10 P.I. and reached maximum by Day 28 P.I. There was a temporal relationship between the increase in neutralizing antibody titer, the drop in titer and clearance of virus from the peripheral circulation. Recovery from primary infection protected the animals against secondary challenge with homologous virus.     

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.     

Vaccination of sheep with cell culture grown orf virus

Orf virus, derived from contagious pustular dermatitis (scabby mouth) lesions in sheep, was adapted to cell culture and subsequently evaluated as a potential vaccine for sheep. The traditional vaccine virus, prepared from the infected scabs of orf virus lesions in sheep, was used to vaccinate sheep by scratching with an applicator (mounted pins) dipped in virus. Less than 10 TCID50 (50% tissue culture infectious doses) of virus was required to produce large lesions (greater than 5 mm diameter) which developed during a period of 10 to 14 d prior to onset of healing which was complete by 28 to 30 d. A serum neutralising antibody response was also detected and protection against challenge by application of virulent virus to abraded skin was demonstrated in that challenge lesions developed and healed more quickly (14 d against 30 d). However, cell culture-adapted virus required more than 10(5) TCID50 to induce even small lesions (less than 2 mm diameter). An antibody response could not be detected and no evidence of protection against challenge with virulent virus was demonstrated. In contrast, a recent field isolate has yielded a cell culture-adapted virus preparation that readily infects sheep, produces large lesions, detectable antibody and protects against challenge. This isolate is distinct from the traditional vaccine strain on the basis of restriction enzyme analysis but provides cross-protection in sheep inmmunisation and challenge studies. These results demonstrate that a cell culture produced scabby mouth vaccine is feasible.     

An inactivated parainfluenza virus type 3 vaccine: the influence of vaccination regime on the response of calves and their subsequent resistance to challenge.

Calves maintained in insolated pens were vaccinated with an inactivated parainfluenza virus type (3) (pi3) vaccine usingparenteral and local route singly and in combination. The calves were subsequently monitored for serum antibody response and challenged intranasally with live virus to assess the protection derived from vaccination. Calves receiving one subcutaneous dose of vaccine in oil adjuvant produced a marked antibody response and were partially protected against challenge. Those receiving two successive subcutaneous doses produced a much greater antiboyd response and were completely protected against challenge. One intranasal dose of aqueous vaccine failed elicit a significant serum antibody response or protection against challenge. However, there was some evidence that intranasal vaccination following a single subcutaneous vaccination produced more effective immunity than one subcutaneous dose alone. Thus a vaccination regime was established which protected calves against experimental challenge and which could thefore be used in the field to assess the role of Pi3 virus in calf respiratory disease.     

Serological reactions in sheep and cattle experimentally infected with three Australian isolates of bluetongue virus

Serums from 103 sheep and 24 cattle experimentally infected with one of 3 serotypes of bluetongue virus isolated in Australia were tested for antibody to bluetongue virus in the serum neutralisation test and the agar gel diffusion precipitin test. Antibody to bluetongue virus was first detected by these tests 8 to 10 days after intravenous infection in 4 sheep that were bled daily for serum analysis. The agar gel diffusion test failed to detect antibody in 28% (29/103) of sheep which had seroconverted in the serum neutralisation test. A further 7% (7/103) of sheep serums were negative in both tests 14 to 22 d after infection. Both tests detected antibody to bluetongue virus in all cattle serums by 10 days after detection of viraemia. In comparison with the intravenous route of infection, extended prepatent periods for the commencement of viraemia resulting from intradermal, subcutaneous and intrauterine routes of infection in the cattle caused corresponding delays in the detection of antibody. For example, one cow that was infected by intrauterine inoculation did not become viraemic until 22 d after inoculation and antibody was not detected until 32 d after inoculation.     

The effect of dose, route and virulence of bovine herpesvirus 1 vaccine on experimental respiratory disease in cattle.

Three experiments were conducted with calves in which, following intramuscular or intranasal vaccination with virulent or attenuated bovine herpesvirus 1, calves were protected against bovine herpesvirus 1 -- Pasteurella haemolytica challenge. Calves receiving low doses of vaccine had lower levels of antibody and greater evidence of virus replication upon challenge than those receiving higher doses. In contrast 11/13 unvaccinated controls had fibrino-purulent pneumonia following challenge. The immune response developed later in younger calves and those given low doses of vaccine. Neutralizing antibodies to bovine herpes-virus 1 were not found in nasal secretions, but were present in serum seven days after vaccination. Bovine herpesvirus 1 was isolated before challenge from nasal secretions of calves vaccinated intranasally or intramuscularly with virulent virus but not those vaccinated intramuscularly with vaccine virus. It was concluded that both routes of vaccination with either virulent or attenuated bovine herpesvirus 1 provided protection from challenge with homologous or heterologous bovine herpesvirus 1 and that live vaccines should contain at least 10(3) plaque forming units/dose for effective immunization.     

Failure of an Actinomyces pyogenes vaccine to protect sheep against an intravenous challenge

The immunity conferred by an A. pyogenes bacterin-toxoid was evaluated in sheep, using an intravenous challenge system. Three sheep were vaccinated and 3 served as controls. The vaccinated sheep were not protected against pyogenic conditions. High antitoxin levels were induced by vaccination but could not be associated with protection against infection. Antibacterial antibody levels elicited with initial vaccination dropped progressively with the 2nd and 3rd vaccinations. Nevertheless, these antibodies did not seem to be necessary for protection against A. pyogenes conditions.     

Efficacy of swine influenza A virus vaccines against an H3N2 virus variant

We compared the efficacy of 3 commercial vaccines against swine influenza A virus (SIV) and an experimental homologous vaccine in young pigs that were subsequently challenged with a variant H3N2 SIV, A/Swine/Colorado/00294/2004, selected from a repository of serologically and genetically characterized H3N2 SIV isolates obtained from recent cases of swine respiratory disease. The experimental vaccine was prepared from the challenge virus. Four groups of 8 pigs each were vaccinated intramuscularly at both 4 and 6 wk of age with commercial or homologous vaccine. Two weeks after the 2nd vaccination, those 32 pigs and 8 nonvaccinated pigs were inoculated with the challenge virus by the deep intranasal route. Another 4 pigs served as nonvaccinated, nonchallenged controls. The serum antibody responses differed markedly between groups. After the 1st vaccination, the recipients of the homologous vaccine had hemagglutination inhibition (HI) titers of 1:640 to 1:2560 against the challenge (homologous) virus. In contrast, even after 2nd vaccination, the commercial-vaccine recipients had low titers or no detectable antibody against the challenge (heterologous) virus. After the 2nd vaccination, all the groups had high titers of antibody to the reference H3N2 virus A/Swine/Texas/4199-2/98. Vaccination reduced clinical signs and lung lesion scores; however, virus was isolated 1 to 5 d after challenge from the nasal swabs of most of the pigs vaccinated with a commercial product but from none of the pigs vaccinated with the experimental product. The efficacy of the commercial vaccines may need to be improved to provide sufficient protection against emerging H3N2 variants.     

Comparison of alum-absorbed or non-alum-absorbed oil emulsion vaccines containing either pilate or non-pilate Bacteroides nodosus cells in inducing and maintaining resistance of sheep to experimental foot rot

Highly pilate (P) or non-pilate (NP) cells of Bacteroides nodosus were compounded into oil emulsion (O) either with or without prior absorption onto alum (A). The abilities of these four preparations (referred to as PAO, NPAO, PO and NPO vaccines) to stimulate antibody production and to protect sheep from foot rot were compared. Two injections of PAO vaccine protected sheep against homologous challenge 12 weeks after the second dose by PO, NPO and NPAO vaccines were less effective. Sheep were protected against homologous challenge for 14 weeks after a single dose of PAO vaccine and for 22 weeks after three doses; an ameliorative effect was still evident 40 weeks after the third dose. Protection against challenge with two heterologous strains was demonstrated at six weeks after three doses of vaccine. A numerical system of scoring the lesions also confirmed that foot rot in vaccinated sheep challenged outside the 'protective' period of the vaccine was somewhat less severe than in controls. PAO vaccine induced much higher and more persistent titres of agglutinins than the other vaccines tested. There was a relationship between agglutinin titres and resistance to homologous challenge.     

Immunologic response of sheep to inactivated and virulent bluetongue virus

Humoral and cellular immune responses of sheep to inactivated and virulent bluetongue virus (BTV) were studied. All sheep inoculated with inactivated BTV developed BTV group-specific nonneutralizing antibodies, as determined by agar-gel immunodiffusion. The development of group-specific, nonneutralizing, complement-fixing antibodies was variable and appeared to be dependent on immunizing BTV serotype, sheep breed, and individual variation. Virus-neutralizing antibodies were never detected after inoculation with the inactivated BTV. In vitro lymphocyte stimulation to BTV soluble antigen was observed with cells from all inoculated Warhill sheep and with cells from 1 of 3 inoculated Suffolk cross sheep. Complement-fixation titers did not appear to correlate with the degree of protection observed, ie, duration of postchallenge-exposure viremia. The development of postchallenge-exposure neutralizing antibody titer was inversely correlated to protective immunity. The development of a response to BTV antigen in the lymphocyte-stimulation test associated most closely with protection. Warhill sheep were afforded better protection, by inoculation with inactivated BTV, to live virus challenge exposure than were the Suffolk cross sheep. Approximately 30% of the inoculated Suffolk cross sheep responded to challenge exposure with intensified clinical signs of blue-tongue, compared with the challenge-exposed control sheep of the same breed.     

Comparison of competitive ELISA, indirect ELISA and standard AGID tests for detecting blue-tongue virus antibodies in cattle and sheep

The performances of a competitive enzyme-linked immunosorbent assay (ELISA) using a group specific monoclonal antibody against bluetongue virus, an indirect ELISA and the standard agar gel immunodiffusion (AGID) test were compared in the detection of serum antibody against bluetongue virus. Test sera consisted of 1300 bovine, 530 ovine and 160 carpine samples from bluetongue-free areas of Canada, 605 bovine and ovine field samples from the USA and Barbados and 464 samples from 79 cattle and sheep experimentally infected with 19 South African and five USA serotypes of bluetongue virus. The diagnostic specificity of the competitive ELISA, as determined for the bluetongue virus-free cattle sera was superior (99.92 per cent) to that of the indirect ELISA (99.85 per cent) and the AGID (99.0 per cent). The specificities of the competitive ELISA for sheep (99.63 per cent) and goats (100.0 per cent) sera were also higher than those of the AGID test. The performance of the ELISA tests was similar whether a gamma-ray-irradiated (2.0 Mrad) or a non-irradiated bluetongue virus antigen preparation was used. The competitive ELISA results for bovine field sera from endemic areas demonstrated a relatively low level of agreement (92.04 per cent) with AGID test results, with 9.7 per cent false negatives. The possible presence in these sera of antibody to cross-reacting antigens or to other orbiviruses, eg, epizootic haemorrhagic disease virus, which react in the AGID but not in the competitive ELISA may account for this lack of agreement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vaccines for bluetongue

Isolation of 8 serotypes of bluetongue virus (BTV) in Australia has led to widespread debate on how to prepare for an outbreak of bluetongue disease and the type of vaccine best suited to control bluetongue in Australia. This article describes the vaccine options under consideration by research workers and animal health administrators. The most widely discussed options are live attenuated virus, killed virus and virus-like particles (VLP) generated by recombinant baculoviruses. Attenuated virus vaccines are cheap and easy to produce and are administered in a single dose. They replicate in sheep without causing significant clinical effects and provide protection against challenge with virulent virus of the same serotype. The possibility that insects could acquire vaccine virus by feeding on vaccinated animals and transmit it to sheep or cattle cannot be eliminated. This poses a risk because attenuated viruses are teratogenic if ewes are infected in the first half of pregnancy. In addition, vaccine virus replication in insects and ruminants may lead to a reversion to virulence. Killed virus vaccines have been shown to be efficacious in small laboratory trials and cannot be transmitted to other animals in the field, but are significantly more expensive to produce than attenuated viruses and require at least 2 doses with adjuvant to elicit an immune response. More work is needed to properly assess their effectiveness and determine their cost of production. Recombinant VLP contain the 4 major structural proteins of BTV but no nucleic acid. VLP are relatively easy to isolate, but it is unlikely that the purification methods currently used in laboratories will be adapted for use commercially. Despite the enthusiasm of recent years, little commercial progress appears to have been made. Although scientific research in Australia and overseas has provided a number of options for development of bluetongue vaccines, the decisions on which to use in an outbreak are complex and will require, not only consideration of factors discussed here, but also agreement from industry and government.