Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA).

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

Evaluation of Humoral Response and Protective Efficacy of an Inactivated Vaccine Against Peste des Petits Ruminants Virus in Goats.

Four goats were inoculated with an inactivated peste des petits ruminants virus (PPRV) vaccine. Three unvaccinated goats were kept as controls. After 36 days, the four goats were revaccinated. The immune response was monitored by virus neutralization test showing that two doses of the vaccine were able to stimulate strong immune response in all the vaccinated animals. The vaccinated goat and the controls were challenged with virulent PPRV intranasally. After PPRV challenge, the three control goats showed fever, viremia and virus excretion through mucosal surfaces, whereas the vaccinated goats were fully protected against PPRV infection and replication.

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.

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 indirect ELISA for the detection of antibody in milk from sheep experimentally infected with Brucella melitensis biovar 3.

An indirect ELISA was evaluated for the detection of Brucella antibodies in milk (m-ELISA) from sheep experimentally infected with B. melitensis biovar 3. At the end of the second reproductive cycle (13 months post infection), the milk of 22 lactating sheep was tested using the m-ELISA. Sera from the same sheep were also tested for Brucella antibodies using the Rose Bengal test (RBT) and the complement fixation test (CFT). The first serum sampling after parturition showed 100% sensitivity in both the RBT and the CFT (confidence interval [CI] 94-100%), but in subsequent samplings the sensitivity of the RBT decreased to 73% (CI 55-85%). Similarly, the sensitivity of the CFT decreased two months after the first sampling, when respective sensitivities of 95% (CI 81-98%) and 81% (CI 61-93%) were recorded for the final two samplings. The sensitivity of the m-ELISA decreased initially (68% on the third sampling, CI 50-81%), but then increased to 95% (CI 81-98%) for the final sampling. When disease prevalence in a flock is below 5%, the estimated probability of not detecting an infected flock through m-ELISA bulk milk testing is over 25%. Under field conditions in Italy (average sheep flock size of 70), the probability that the infection will not be detected is over 25% when four (or less) infected milking sheep are present in the flock. The results show that the m-ELISA is not a reliable screening test for bulk milk samples when the prevalence of brucellosis in a sheep flock is low.

The persistence of Brucella melitensis in experimentally infected ewes through three reproductive cycles.

The authors studied the persistence of infection in 46 ewes experimentally infected with Brucella melitensis biovar 3 and monitored through three subsequent reproductive cycles. The entire experimental period lasted for 151 weeks. Infection of ewes and elimination of Brucella in milk, or its presence in vaginal discharges, persisted throughout the duration of the trial, as demonstrated by recurrent elimination of Brucella in milk and vaginal discharges. Brucella melitensis was recovered from the tissues of one ewe killed at the end of the trial. The strain was recovered from vaginal swabs and milk following parturition in the third reproductive cycle from an ewe that had aborted in the first cycle but was not pregnant in the second cycle. From a public health point of view, the periodical recovery of Brucella from the milk during the entire trial period illustrated that brucellosis in sheep remains a continuous occupational risk and a significant public health problem for consumers of fresh milk and milk products. That risk may persist for at least 3 years following the initial infection of the flock. Lamb antibody titres became negative in all lambs within 5 months after birth. This suggested that serological tests on lambs may have no practical diagnostic significance if performed during the first 5 months of life. Nevertheless, the birth of three infected lambs suggested that the phenomenon of latent carrier state may represent another way for B. melitensis to persist in a flock.

The use of homologous antigen in the serological diagnosis of brucellosis caused by Brucella melitensis.

In the European Union the serological diagnosis of brucellosis caused by Brucella melitensis is performed using the heterologous antigen of B. abortus S99. The possible higher sensitivity or ability of an early detection of antibodies by a homologous antigen may prove very useful in the final phases of an eradication programme. Results obtained in sheep experimentally infected by B. melitensis biovar 3 were compared using B. abortus S99, B. melitensis M1, M2 and M3 antigens in the Rose Bengal plate test (RBPT), the complement fixation test (CFT) and an enzyme-linked immunosorbent assay (ELISA) test. Forty-six sheep from an officially brucellosis-free flock were experimentally infected intraconjunctivally with B. melitensis biovar 3. Prior to infection, all animals were tested first against Brucella antibodies, weekly for 2 months post-infection (PI) and then monthly for a further 7 months. All sera were tested against the antigens listed above using RBPT, CFT and ELISA. Using a Bayesian approach, test sensitivities were estimated and compared. Their ability for the early detection of antibodies was evaluated through a regression model based on a logit response model, using the number of days PI as the independent variable and the logit of the fraction of positive animals as the dependent variable. No significant differences were detected among the various antigens used, either in terms of sensitivity or in terms of antibody kinetics; however, the CFT was significantly less sensitive than the RBPT and ELISA and it also showed a lower rate of increase of percentage positive animals (beta-coefficient of regression analysis).

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%).

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.

Field vaccination of sheep with bivalent modified-live vaccine against bluetongue virus serotypes 2 and 9: effect on milk production.

In response to complaints of the potential side-effects of the bivalent live-modified vaccine used to control the spread of bluetongue (BT) virus (BTV) serotypes 2 and 9 in Italy, a study was conducted to determine the effects of immunisation on milk production. Thirty-four Comisana cross-bred sheep were vaccinated with the bivalent BTV-2/BTV-9 modified-live vaccine produced by Onderstepoort Biological Products in South Africa; six animals served as unvaccinated controls. All animals were bled twice a week for two months and the presence and titres of BTV in the blood determined. The somatic cell count, pH, fat, protein and lactose content of the milk, as well as the quantity of the milk produced, were also measured. Vaccine virus was isolated from vaccinated animals between day 3 and day 20 post vaccination (pv) with peak titres observed on days 3 and 6 pv for BTV-2 and BTV-9, respectively. Milk production declined in the vaccinated group between days 8 and 14 pv, with the greatest decrease on day 9 pv. No differences were observed in the somatic cell count and pH, or in the milk fat, protein and lactose content.

Virological and serological response of sheep following field vaccination with bivalent modified-live vaccine against bluetongue virus serotypes 2 and 9.

A group of 44 sheep was vaccinated with the bivalent modified-live vaccine against bluetongue virus (BTV) serotype 2 (BTV-2) and BTV-9 to evaluate viraemia and antibody kinetics. Blood samples were taken from the sheep three times a week for two months and screened for the presence of BTV and for 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 and BTV-9 from the ethylene-diaminetetra-acetic acid (EDTA) blood samples; virus titres were also determined in the viraemic animals. BTV was detected in the blood of 39 sheep between day 3 and day 24 post vaccination (pv). Viraemia peaked on day 7 pv with average titres of 10(5.3)TCID50/ml. Antibodies were first detected in the c-ELISA on day 6 pv and by day 16, all sheep were seropositive. Only 36 of the 44 inoculated sheep developed virus-neutralising antibodies against both BTV-2 and BTV-9 while 4 were positive to BTV-2 only; neutralising antibodies were not detected in the 4 remaining animals. Antibody titres were very low and unstable and often bordered on the negative/positive threshold.

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.

Health management of large transhumant animal populations and risk of bluetongue spread to disease-free areas.

Transhumance, or seasonal grazing, in central Italy is a husbandry practice that is over two thousand years old. It involves the seasonal movement of sheep, goats and cattle from the southern lowlands of mainly the Puglia and Lazio regions, to summer pastures in the mountains of Abruzzo and Molise. Bluetongue (BT) made its appearance in Italy in 2000. In the early summer of 2001, disease was present in three regions: Sardinia, Sicily and Calabria. Neither an effective surveillance system nor a vaccination campaign had been implemented. Movement of ruminants to the disease-free regions of Abruzzo and Molise was therefore banned. The Italian Veterinary Services had to meet the challenge of the movement of ruminants from surveillance to disease-free zones, given the impossibility of stopping transhumance. The General Directorate of Veterinary Public Health, Food and Nutrition of the Ministry of Health developed a plan for both the Puglia and Abruzzo regions based on serological, virological and entomological surveillance. The plan was implemented between May and June 2001 when 7,000 animals moved from the Puglia surveillance zone to the infection-free summer pastures. In the early summer of 2002, eight regions were infected (Sardinia, Sicily, Calabria, Basilicata, Puglia, Campania, Lazio and Tuscany). Simultaneously, a nationwide surveillance system and a vaccination campaign, were implemented in infected regions. In the provinces where vaccination was compulsory, deviation from the animal movement ban was allowed if at least 80% of susceptible stock had been vaccinated. However, this objective was not achieved in the provinces of Rome and Viterbo (Lazio) where a large transhumant population was present and where sporadic virus circulation had been detected. A specific control plan to allow transhumance from Lazio to Abruzzo, Marche and Umbria was designed and implemented to increase the number of animals that could be moved. Between May and June 2002, authorisation was granted to move 28,000 head, whereas prohibition of movement was ordered for 12,000 sheep (belonging to 21 flocks). Regional authorities financed feeding, watering and housing for these animals. Transhumance did not spread infection to disease-free areas either in 2001 or in 2002.

Kinetics of the antibody response in ewes experimentally infected with Brucella melitensis biovar 3.

The authors evaluated the kinetics of antibody response in 46 ewes coming from officially brucellosis free flocks that were experimentally infected with Brucella melitensis biovar 3, and monitored through three subsequent reproductive cycles. In this study, results of Rose Bengal test (RBT) and complement fixation test (CFT) were considered. Test results of 2nd and 3rd reproductive cycle show a peak in the antibody production at parturition, followed by a drop in the following months. The peak at parturition is significantly lower in the 3rd reproductive cycle compared to the 2nd. The drop in antibody production observed after parturition of the 3rd reproductive cycle is significantly higher than that observed after parturition of the 2nd reproductive cycle. Nevertheless, the infection can still be revealed at flock level after three years post infection.