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.     

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.     

Analysis of the quality of protection induced by a porcine influenza A vaccine to challenge with an H3N2 virus

Antigenic drift of swine influenza A (H3N2) viruses away from the human A/Port Chalmers/1/73 (H3N2) strain, used in current commercial swine influenza vaccines, has been demonstrated in The Netherlands and Belgium. Therefore, replacement of this human strain by a more recent swine H3N2 isolate has to be considered. In this study, the efficacy of a current commercial swine influenza vaccine to protect pigs against a recent Dutch field strain (A/Sw/Oedenrode/96) was assessed. To evaluate the level of protection induced by the vaccine it was compared with the optimal protection induced by a previous homologous infection. Development of fever, virus excretion, and viral transmission to unchallenged group mates were determined to evaluate protection. The vaccine appeared efficacious in the experiment because it was able to prevent fever and virus transmission to the unchallenged group mates. Nevertheless, the protection conferred by the vaccine was sub-optimal because vaccinated pigs excreted influenza virus for a short period of time after challenge, whereas naturally immune pigs appeared completely protected. The immune response was monitored, to investigate why the vaccine conferred a sub-optimal protection. The haemagglutination inhibiting and virus neutralising antibody responses in sera, the nucleoprotein-specific IgM, IgG, and IgA antibody responses in sera and nasal secretions and the influenza-specific lymphoproliferation responses in the blood were studied. Vaccinated pigs developed the same or higher serum haemagglutination inhibiting, virus neutralising, and nucleoprotein-specific IgG antibody titres as infected pigs but lower nasal IgA titres and lymphoproliferation responses. The lower mucosal and cell-mediated immune responses may explain why protection after vaccination was sub-optimal.     

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.     

Role of neutralising antibody in passive immunity to bluetongue infection

A group of sheep inoculated with serum obtained from sheep which had recovered from bluetongue virus type 3 infection were protected from challenge with the homologous virus type but not from heterologous challenge. Twin lambs which had received colostrum containing virus antibodies were shown to be only partially protected against homologous challenge. A monoclonal antibody directed against the type-determining protein of the virus was also shown to give partial protection against challenge. From this series of experiments it was concluded that antibody has a significant role in protection from bluetongue but that the outcome of challenge will depend on several interacting factors.     

The immunological response to intact and dissociated blue-tongue virus in mice.

Antigenic fractions of bluetongue virus were separated by ultracentrifugation in Tris-buffered CsCl gradients at pH 6, 7 or 8 and the bluetongue virus polypeptide composition of the bands isolated from these gradeints was monitored by polyacrylamide gel slab electrophoresis. The immunological response to these fractions in mice was determined by a haemolytic plaque-forming cell assay, using sheep erythrocytes onto which intact bluetongue virus was adsorbed as lytic indicator cells. Isolated outer layer bluetongue virus polypeptide 2, from gradients at pH 6, and polypeptides 2 and 5, from gradients at pH 7, produced a strong primary IgM plaque-forming cell response. The subviral particles of density 1, 39 g.cm-3 and the bluetongue virus core particles of density 1,42 g.cm-3 also stimulated an IgM response at least as strong as that to intact bluetongue virus of density 1,38 g.cm-3. The isolated bluetongue virus fractions therefore appear to maintain their immunogenic integrity as effectively as those of intact bluetongue virus. The pattern of the immune response to bluetongue virus type 4 is similar to that of type 10.     

Immune response of chicks to oral vaccination against Newcastle disease and fowl pox

The humoral immune response and immunity conferred in chicks were compared following separate and combined oral vaccination with F strain of Newcastle disease virus (NDV) and HP1 strain of fowl pox virus. The haemagglutination inhibition (HI) antibody titre against NDV and passive haemagglutination (PHA) antibody titre against fowl pox virus were comparable in two respective groups. The serum IgG concentration increased significantly after the second vaccination in all the groups. The NDV vaccine induced significantly higher IgG production as compared to fowl pox virus vaccine. There was no significant difference in serum IgG concentration produced by combined vaccine and separate F strain vaccine. The protection afforded by combined and separate vaccinations did not vary significantly against challenge with virulent strains of NDV and fowl pox virus at different stages.     

Production of murine cytotoxic T lymphocytes by bluetongue virus following various immunisation procedures

The induction of bluetongue virus specific cytotoxic T lymphocytes (CTLs) in C3H mice by various live and inactivated bluetongue virus preparations was studied. Live virus preparations were shown to induce good levels of CTLs; however, inactivation of virus preparations either by beta propriolactone or glutaraldehyde induced only a low level response. The use of Freund's adjuvants and double immunisation procedures failed to improve the response of the inactivated preparations. These findings are discussed in relationship to protection from bluetongue disease with various bluetongue virus vaccines.     

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.     

The Efficacy of Two Commercial Feline Rhinotracheitis-Calicivirus-Panleukopenia Vaccines

The efficacy of two commercial feline vaccines was determined by challenging vaccinated and unvaccinated cats sequentially with a virulent feline calicivirus and rhinotracheitis virus. Serological responses to these viruses as well as to panleuk openia virus were also measured. Results show significant protection and satisfactory serological responses are conferred by both vaccines. One vaccine showed significant superiority in protection against feline viral rhinotracheitis.     

Similarity of European porcine reproductive and respiratory syndrome virus strains to vaccine strain is not necessarily predictive of the degree of protective immunity conferred

The objective of this study was to determine the degree of protection conferred by a Lelystad-like modified live virus (MLV) vaccine against a heterologous wild-type porcine reproductive and respiratory syndrome virus (PRRSV) isolate of the same cluster. For this purpose, fourteen 3-week-old piglets were divided into three groups: Group A pigs were vaccinated with a modified live virus vaccine, Group B pigs were used as positive controls, and Group C pigs as negative controls. Twenty-eight days after the last dose of vaccine, all pigs in Groups A and B were inoculated with the Spanish PRRSV strain 5710. To evaluate efficacy, clinical signs were recorded and the presence of challenge virus was determined by virus isolation in blood samples and nasal swabs collected at various time points post-challenge (p.c.) and in tissue samples collected at necropsy 24 days p.c. After challenge, moderate clinical signs were observed in pigs from Groups A and B. In addition, all vaccinated pigs were viremic at least once, although viremia tended to be more sporadic in this group than in Group B pigs. PRRSV was detected in at least one tissue sample from four out of five pigs from Group A and in all pigs from Group B. The results indicate that the protection conferred by the MLV vaccine used in this study against a closely related virulent strain was only partial. The findings suggest that the degree of genetic homology of ORF5 between MLV vaccine and challenge isolate is not a good predictor of vaccine efficacy.     

Vaccines against Aujeszky's disease: evaluation of their efficacy under standardized laboratory conditions

A standardized test was developed to compare the efficacy of Aujeszky's disease virus (ADV) vaccines under laboratory conditions. Per test 3 groups of 6 to 8 sero-negative pigs were used. The first vaccination was done at 10 weeks of age. One group was vaccinated once, another was vaccinated twice and the 3rd served as control. Pigs were challenge exposed to the virulent NIA-3 strain of ADV 12 weeks after the first vaccination. Apart from mortality, average periods of growth arrest, fever and virus shedding after challenge were used as parameters to evaluate vaccine efficacy. Two inactivated and 4 attenuated vaccines were tested. Two attenuated vaccine viruses were excreted after vaccination. Despite maximal standardization, a considerable variation still existed between the experiments in mortality and growth arrest periods of control pigs after challenge. However, the controls were always more severely affected than the vaccinated pigs. All vaccines except one were effective in preventing death after challenge, but none conferred complete protection. Most vaccinated pigs still lost weight, developed fever and shed virus after challenge. Revaccination after 3 or 4 weeks had little effect, particularly with the attenuated vaccines. The results of the present study indicate that 2 of the attenuated vaccines conferred the best protection, 1 attenuated vaccine appeared to be as effective as the 2 inactivated ones, and the 4th attenuated vaccine was least effective.     

Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle

The efficacy of a bivalent inactivated vaccine against bluetongue virus (BTV) serotypes 2 (BTV-2) and 4 (BTV-4) was evaluated in cattle by general and local examination, serological follow-up, and challenge. Thirty-two 4-month-old calves were randomly allocated into 2 groups of 16 animals each. One group was vaccinated subcutaneously (s/c) with two injections of bivalent inactivated vaccine at a 28-day interval, and the second group was left unvaccinated and used as control. Sixty-five days after first vaccination, 8 vaccinated and 8 unvaccinated calves were s/c challenged with 1 mL of 6.2 Log10 TCID50/mL of an Italian field isolate of BTV serotype 2, while the remaining 8 vaccinated and 8 unvaccinated animals were challenged by 1 mL of 6.2 Log10 TCID50/mL of an Italian field isolate of BTV serotype 4. Three additional calves were included in the study and used as sentinels to confirm that no BTV was circulating locally. At the time of the challenge, only one vaccinated animal did not have neutralizing antibodies against BTV-4, while the remaining 15 showed titres of at least 1:10 for either BTV-2 or BTV-4. However, the BTV-2 component of the inactivated vaccine elicited a stronger immune response in terms of both the number of virus neutralization (VN) positive animals and antibody titres. After challenge, no animal showed signs of disease. Similarly, none of the vaccinated animals developed detectable viraemia while bluetongue virus serotype 2 and 4 titres were detected in the circulating blood of all unvaccinated animals, commencing on day 3 post-challenge and lasting 16 days. It is concluded that administration of the bivalent BTV-2 and BTV-4 inactivated vaccine resulted in a complete prevention of detectable viraemia in all calves when challenged with high doses of BTV-2 or BTV-4.     

Active circulation of bluetongue vaccine virus serotype-2 among unvaccinated cattle in central Italy

Several seroconversions occurring in 2002 among sentinel cattle during the bluetongue-vaccination campaign in Lazio and Tuscany (central Italy) led to the suspicion of vaccine-virus circulation. Therefore in 2003, 17 seroconverting sentinel herds were investigated for the characteristics of the virus involved. From these farms, 91 unvaccinated animals and 57 Culicoides pools were tested for the presence of the bluetongue vaccine virus (serotype-2) or other strains. The presence of vaccine virus serotype-2 was confirmed by PCR followed by restriction analysis in the whole blood of 17 unvaccinated sentinel cattle and 12 pools of Culicoides imicola or C. obsoletus. Of the 17 herds, five were positive only for vaccine virus serotype-2, four were positive for other strains and two for both the vaccine and other strains; the remaining premises were virologicaly negative. The vaccine virus serotype-2 also was detected in areas not included in the vaccination campaign.     

Vaccines against Aujeszky's disease: Evaluation of their efficacy under standardized laboratory conditions

Summary

A standardized test was developed to compare the efficacy of Aujeszky's disease virus (ADV) vaccines under laboratory conditions. Per test 3 groups of 6 to 8 sero‐negative pigs were used. The first vaccination was done at 10 weeks of age. One group was vaccinated once, another was vaccinated twice and the 3rd served as control. Pigs were challenge exposed to the virulent NIA‐3 strain of ADV 12 weeks after the first vaccination. Apart from mortality, average periods of growth arrest, fever and virus shedding after challenge were used as parameters to evaluate vaccine efficacy.

Two inactivated and 4 attenuated vaccines were tested. Two attenuated vaccine viruses were excreted after vaccination. Despite maximal standardization, a considerable variation still existed between the experiments in mortality and growth arrest periods of control pigs after challenge. However, the controls were always more severely affected than the vaccinated pigs. All vaccines except one were effective in preventing death after challenge, but none conferred complete protection. Most vaccinated pigs still lost weight, developed fever and shed virus after challenge. Revaccination after 3 or 4 weeks had little effect, particularly with the attenuated vaccines. The results of the present study indicate that 2 of the attenuated vaccines conferred the best protection, I attenuated vaccine appeared to be as effective as the 2 inactivated ones, and the 4th attenuated vaccine was least effective.     

A new bluetongue virus serotype isolated in Kenya.

An apparently new strain of bluetongue virus was first isolated in Kenya in 1965 and since, has been obtained on 7 further occasions from diseased sheep during clinical outbreaks of disease. It proved to be serologically different from the 16 bluetongue virus strains then held at this laboratory. The virus was modified by passage in embryonated hens eggs to produce a live virus strain suitable for inclusion in a polyvalent vaccine. Recent neutralisation tests, carried out with 24 guinea pig immune sera prepared at Pirbright against the currently known World serotypes, have confirmed the earlier results and show that it is different from any of the existing serotypes.     

Evaluation of protection conferred by a vaccination program based on the H120 and CR88 commercial vaccines against a field variant of avian infectious bronchitis virus

Due to serotype variations among different avian infectious bronchitis viruses isolated in Tunisia since 2000, protection of chicks, especially broiler flocks, with Mass H120 vaccine often fails. Therefore, association of CR88 (793B type) with H120 vaccines was used for better response. Challenge experiments were then conducted to evaluate tracheal and renal cross-protection in chickens immunized via nasal and eye drops. Conferred protection was measured by clinical signs and macroscopic lesions observed, based on scores attributed according to their severities. The results showed a low protection conferred by H120 alone, as vaccination did not reduce tracheal and kidney lesions (70% scored as 3) after TN20/00 virus challenge, which also led to 10% mortality. Conversely, the challenge results indicated that the combination of the 2 strains (H120/CR88) allow high protection. Based on the results of the challenge experiments, a vaccination protocol coupling CR88 to H120 was applied for industrial broiler flocks. Clinical observations and serological results confirmed that association of heterologous serotypes (H120 and CR88 vaccines) increased the levels of protection against infectious bronchitis viruses compared with the H120 vaccine given alone.