Laboratory tests for evaluating the level of attenuation of bluetongue virus.

One of the most important steps when preparing a live attenuated vaccine is the assessment of the level of attenuation in target animals. It is costly and time consuming as it requires, on each occasion, a large number of susceptible animals and contained accommodation. This study assessed the consistency of the bovine foetal aorta endothelial (BFA) cell line and newborn mice for evaluating the attenuation level of BTV4, BTV9 and BTV16 Italian field isolates. Following serial passages in BHK(21c13) or Vero cell cultures, BTV attenuated clones demonstrated a reduced replication capability in the BFA cells compared to the homologous virulent strains. Similarly, following intracerebral inoculation, the attenuated clones were completely innocuous to newborn mice contrary to the homologous virulent strains which killed all animals within 10 days. Vaccines produced with the BTV9 or BTV4 attenuated clones were safe, immunogenic and capable of preventing clinical symptoms and viraemia in sheep following challenge with homologous virulent virus. The two assays may be valuable indicators of the gradual changes occurring in the BTV population leading to virus attenuation, they can predict the safety of a BTV attenuated vaccine and, in turn, reduce the number of sheep and cattle required to assess the level of attenuation attained.

An inactivated vaccine for the control of bluetongue virus serotype 16 infection in sheep in Italy.

Because no suitable products are at the moment available to safely control the spread of BTV-16 in Europe, an inactivated vaccine was produced from the reference field isolate of bluetongue virus serotype 16. One group of six sheep was vaccinated subcutaneously with the inactivated vaccine twice, on days 0 and 28, whereas a second group of eight sheep was inoculated with saline solution and used as mock-vaccinated control animals. Seventy-eight days after the first vaccination, all sheep were inoculated subcutaneously with a suspension containing 10(6.3) TCID(50) of a virulent reference BTV-16 isolate. Apart from a transient inflammatory reaction at the injection site, no adverse effects were reported following vaccination. All vaccinated animals developed high titres (7.3-9.3log(2)(ED50%/50 microl)) of virus-specific neutralising antibodies and were resistant to challenge with BTV-16. Conversely, following challenge, control animals developed hyperthermia and long lasting high-titre viraemia.

Rapid detection of bluetongue virus in blood and organ samples using a capture enzyme-linked immunosorbent assay.

An antigen capture ELISA for bluetongue (BT) virus was developed using tissue culture supernatant to identify different BT virus (BTV) serotypes 1, 2, 4, 9 and 16, which have been incriminated in the current epidemic in the Mediterranean Basin. To obtain a positive result and after amplification in tissue culture, the minimum amount of infecting virus required was 100 TCID(50). Results from the antigen capture ELISA were compared with conventional methods for virus isolation and identification, such as virus amplification on embryonated chicken eggs (ECEs), followed by tissue culture and the direct immunofluorescence test. The sensitivity and specificity of the ELISA in infected tissue culture supernatant using homogenates of BTV-positive ovine and bovine organs and blood, without a previous step in ECEs, were 100%. The assay was also applied to homogenates of chicken embryo tissues, which had been infected with different BTV serotypes. This method enabled detection of the virus with 100% sensitivity and specificity rates, also using amplification in ECEs. Furthermore, among the various embryo tissues tested, liver was found to be the most suitable for use with ELISA. In experimentally infected ovine blood samples, the ELISA revealed the presence of the virus. Given the high sensitivity and specificity obtained with the BTV serotypes in this trial, the method should greatly facilitate BT diagnosis.

Use of the complement fixation and brucellin skin tests to identify cattle vaccinated with Brucella abortus strain RB51.

In the European Union, RB51 vaccine can be used only under strictly controlled conditions for the immunisation of cattle at risk of infection with Brucella abortus. A test is therefore necessary to distinguish vaccinated from unvaccinated animals. The complement fixation test with RB51 antigen (RB51-CFT), dot-blot and gamma-interferon used to identify vaccinated animals have been described, but sensitivity of the tests has been poor and positivity transient after calfhood vaccination. To avail of a rapid and accurate diagnostic tool, the authors produced, controlled and evaluated an experimental brucellin prepared from strain RB51 (RB51 brucellin). The potency of this brucellin was evaluated in guinea-pigs sensitised with RB51 and compared with a commercially available brucellin. Both allergens produced similar biological activity in guinea-pigs. The RB51 brucellin skin test was performed in 10 cattle 414 days after calfhood vaccination with RB51 when they were negative to the RB51-CFT. The skin test revealed 60% sensitivity (with a confidence interval of 95%, CI 30.8%-83.3%) and 100% specificity (CI 60.7%-100%). These findings limit the use of the skin test only for screening to detect RB51 vaccinated herds, not individual animals. Nevertheless, following intradermal inoculation of RB51 brucellin, a transient antibody increase to the RB51-CFT was observed, from day 9 to day 20 post inoculation with RB51 brucellin. This transient antibody increase, when evaluated in parallel with the RB51 brucellin skin test results, enables detection of individual vaccinated animals (sensitivity 100%; CI 76.2%-100%).

Efficacy and safety studies on an inactivated vaccine against bluetongue virus serotype 2.

An inactivated vaccine was produced from an Italian field isolate of bluetongue virus serotype 2 (BTV-2) with a titre of 10(7.8)TCID50/ml. The virus was purified through a molecular cut cassette membrane, inactivated with beta-propriolactone and emulsified with ISA 206 (Seppic) adjuvant. The vaccine was then tested for sterility, toxicity and safety in laboratory and target animals according to European Pharmacopoeia standards. Immunogenicity was assessed by inoculating subcutaneously 10 sheep and 10 goats each with 2 ml of the vaccine and 10 bovines each with 5 ml of the vaccine. A booster dose was inoculated after 14 days and no side-effects were reported following vaccination. Fourteen days after the booster dose, all vaccinated animals developed virus neutralising (VN) bluetongue (BT) antibody titres that on day 60 post vaccination ranged between 1/20 and 1/1 280. After one year, goats still had high VN antibody titres. Sheep were challenged 138 days after vaccination by subcutaneously inoculating 1 ml of 10(5.6)TCID50/ml of an Italian field isolate of BTV serotype 2; four unvaccinated animals were also inoculated and used as controls. Starting from day 6 post challenge, control animals developed a fever, with temperature ranging from 39.9 degrees C to 40.6 degrees C and lasting 48 h on average. BTV-2 was also isolated from the blood of control animals between days 4 and 20 post challenge. Conversely, neither fever nor viraemia were detected in the vaccinated animals that were challenged. A new trial with a larger number of animals, including all target species, has been planned and is in progress.