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.

Use of an in vitro culture system to detect Theileria equi strains from infected equids and/or reservoirs.

A horse erythrocyte culture technique, partly modifying that originally developed by Holman, was used to detect the presence of Theileria equi strains in 12 horse and 2 mule blood samples. The animals were placed into four groups on the basis of their case history and laboratory test results: the mules and two horses were considered as infected and included in the 'recent infection' group, four horses with a history of past infection were included in the 'past infection' group and four animals subjected to anti-theileria treatment formed the 'treated animals' group. The final group consisted of two horses with an unknown history of infection. Ten T. equi strains were isolated and adapted in vitro from the fourteen animals tested: nine of these originated from the horse samples and one from mule blood. This is the first time that a T. equi strain isolated from a mule has been adapted in erythrocyte cultures. All strains isolated from horses showed growth and in vitro adaptation with a parasitaemia peak of over 10%. Following freezing and reculturing, adapted strains showed growth after a quiescence period of eight days. The method proved to be effective for culturing and replicating field T. equi strains from horses and mules. The technique was also able to identify carriers of infection which were negative on microscopic examination, indirect immunofluorescence and complement fixation.

Evaluation of an indirect ELISA for the detection of Brucella antibodies in cow's milk.

An indirect ELISA was developed by the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' (IZS A&M) for the detection of Brucella antibodies in cow's milk. Specific monoclonal antibody was used against a bovine IgG(1) epitope and complies with European Commission requirements. The test accuracy was evaluated on milk samples from the regions of Abruzzo and Molise in Italy. The negative samples came from 1,250 officially brucellosis-free herds from the Molise region (Italy). The positive samples were taken from three herds in the Abruzzo and Molise regions where animals positive to the official tests were present and Brucella abortus was isolated. Test specificity was 99.8% (with a confidence interval [CI] of 99.6%-99.9%), while sensitivity was 100% (CI 91.2%-100%). The probability of detecting antibodies in positive milk samples was higher than 50% up to a dilution of 1:256 in negative milk. The probability of identifying an infected herd in the dairy cattle population. Under study was 88.6% (CI 73.9%-95.3%).

Serological response in cattle and sheep following infection or vaccination with bluetongue virus.

Data from various experimental and field studies were compiled and analysed to evaluate the serological response in sheep and cattle against different bluetongue (BT) virus (BTV) vaccine combinations (Onderstepoort Biological Products, South Africa); the accuracy of diagnostic procedures commonly used for detecting BTV antibodies was also assessed. Using the competitive enzyme-linked immunosorbent assay (c-ELISA) (IZSA&M, Teramo, Italy) and the virus neutralisation (VN) test, antibody responses were evaluated under the following vaccination regimes: monovalent modified-live vaccine against BTV-2 in cattle and sheep, monovalent modified-live vaccine against BTV-9 in sheep, and bivalent modified-live vaccine against BTV-2 and BTV-9 in cattle and sheep. The data were compared to serological results observed in cattle and sheep infected with Italian field strains of BTV-2 or BTV-9. The c-ELISA consistently detected antibodies earlier than the VN test in both livestock species and against all BTV serotypes. The highest and most rapid antibody responses were observed in sheep infected in the field. In cattle and in sheep, high VN titres were detected using monovalent vaccines, while bivalent vaccines initiated lower antibody titres that developed more slowly.

Vaccination of cattle using monovalent modified-live vaccine against bluetongue virus serotype 2: innocuity, immunogenicity and effect on pregnancy.

The immunogenicity, innocuity and possible teratogenic effects of the monovalent modified-live vaccine against bluetongue (BT) virus (BTV) serotype 2, manufactured by Onderstepoort Biological Products in South Africa, was evaluated in cows. Twenty-one cows, 14 of which were at different stages of gestation, were vaccinated with 2 ml of monovalent vaccine; two served as unvaccinated controls. After immunisation, 16 vaccinated and the 2 unvaccinated controls were kept in the field; the remaining 5 pregnant cows were maintained in an insect-proof stable with a controlled environment. Blood samples were taken from field cattle once a week for two months and from the stable cattle three times a week. All samples were screened for the presence of BTV and for BT antibody using the competitive enzyme-linked immunosorbent assay (c-ELISA) and the virus neutralisation (VN) test. Intravenous egg inoculation, followed by two blind passages in Vero cells, was used to isolate BTV-2 from ethylene-diaminetetra-acetic acid (EDTA) blood samples and virus titres in viraemic animals were determined. After immunisation, 9 of the cows developed a viraemia which commenced on day 7 post vaccination (pv) and lasted for three weeks. The virus titres were never higher than 10(2.8)TCID50/ml with the highest titre observed on day 14 pv. None of the vaccinated animals developed clinical symptoms that could be attributed to BTV; after three weeks all animals showed a serological response to BTV-2. In the c-ELISA, antibodies were detected from day 7 pv while in the VN test, antibodies were observed from day 21 pv. All pregnant cows completed their gestation: 13 gave birth to healthy calves, while one of those in the field group, vaccinated at the six months gestation, delivered a calf with prosencephalic hypoplasia, possibly developed during foetal organogenesis prior to vaccination. Fourteen months after immunisation the stabled cows were challenged subcutaneously by administering 2x10(6.8)TCID50 BTV-2 Italian isolate. A third group of 4 cows was also inoculated with the BTV-2 Italian field isolate, as described for the second group and was used as the unvaccinated positive control group. Vaccinated cows had a detectable viraemia only on day 14 pv and virus titres were very low. Virus titres never exceeded 10(2.3)TCID50/ml, while the unvaccinated group developed a long and intense viraemia, peaking on day 14 pv with a titre of 1.18x10(4). It is concluded that the BTV-2 modified-live vaccine used in this study was a harmless and effective immunogen that did not cross the placental membrane.

Serological surveillance of bluetongue virus in cattle, sheep and goats in Albania.

The recent spread of the bluetongue (BT) virus (BTV) in the Mediterranean Basin encouraged numerous countries to undertake entomological and serological surveillance programmes to identify affected areas and control the infection. Hitherto, no data on the presence and diffusion of BTV in Albania were available. Between October and November 2002 serum samples from 857 cattle and 870 sheep and goats were collected by the Albanian Veterinary Services in 15 districts, some bordering Yugoslavia, Macedonia and Greece, and others along the Adriatic coast. At the Albanian Veterinary Research Institute the samples were tested for the presence of BTV antibodies using a competitive enzyme-linked immunosorbent assay (c-ELISA) (bluetongue antibody test kit, IZS A&M, Teramo); in Italy, the virus neutralisation (VN) test was used to confirm the ELISA results and determine the serotype of BTV circulating. Overall seroprevalence was 18.9% in cattle and 4.4% in sheep and goats; seropositive animals occurred in all districts surveyed. The highest prevalence of BT was observed in the Tirana District, with 61% of the cattle and 20% of the sheep and goat populations BT-positive. The VN test confirmed the c-ELISA results revealing the presence of antibodies against BTV serotype 9.

Bluetongue laboratory diagnosis: a ring test to evaluate serological results using a competitive ELISA kit.

The occurrence of bluetongue (BT) in Italy prompted an increase in disease surveillance. Thus a competitive enzyme-linked immunosorbent assay (c-ELISA) to detect immunoglobulins to BT virus (BTV) was developed and distributed amongst 27 laboratories comprising the Italian veterinary diagnostic laboratories network to screen field sera. This ring test enabled comparison of the results and the evaluation of the reproducibility of the method. The c-ELISA developed by the National Reference Centre for Exotic Diseases (c-ELISA-IZSA&M) was compared also against a commercially available c-ELISA. In addition, results obtained by the Centre of Athens Veterinary Institutions are presented.