Evidence for the presence of bluetongue virus in Kosovo between 2001 and 2004

In 2001, clinical cases of bluetongue were observed in Kosovo, and in that year and in 2003 and 2004, serum samples were collected from cattle and small ruminants and tested for antibodies to bluetongue virus. The results provide evidence that bluetongue virus was not present in Kosovo before the summer of 2001, but that the virus circulated subclinically among the cattle and sheep populations of Kosovo in 2002, 2003 and 2004.

Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain

[EN] Background: Culicoides (Diptera: Ceratopogonidae) biting midges are vectors for a diversity of pathogens including bluetongue virus (BTV) that generate important economic losses. BTV has expanded its range in recent decades, probably due to the expansion of its main vector and the presence of other autochthonous competent vectors. Although the Canary Islands are still free of bluetongue disease (BTD), Spain and Europe have had to face up to a spread of bluetongue with disastrous consequences. Therefore, it is essential to identify the distribution of biting midges and understand their feeding patterns in areas susceptible to BTD. To that end, we captured biting midges on two farms in the Canary Islands (i) to identify the midge species in question and characterize their COI barcoding region and (ii) to ascertain the source of their bloodmeals using molecular tools.Methods: Biting midges were captured using CDC traps baited with a 4-W blacklight (UV) bulb on Gran Canaria and on Tenerife. Biting midges were quantified and identified according to their wing patterns. A 688 bp segment of the mitochondrial COI gene of 20 biting midges (11 from Gran Canaria and 9 from Tenerife) were PCR amplified using the primers LCO1490 and HCO2198. Moreover, after selected all available females showing any rest of blood in their abdomen, a nested-PCR approach was used to amplify a fragment of the COI gene from vertebrate DNA contained in bloodmeals. The origin of bloodmeals was identified by comparison with the nucleotide-nucleotide basic alignment search tool (BLAST). Results: The morphological identification of 491 female biting midges revealed the presence of a single morphospecies belonging to the Obsoletus group. When sequencing the barcoding region of the 20 females used to check genetic variability, we identified two haplotypes differing in a single base. Comparison analysis using the nucleotide-nucleotide basic alignment search tool (BLAST) showed that both haplotypes belong to Culicoides obsoletus, a potential BTV vector. As well, using molecular tools we identified the feeding sources of 136 biting midges and were able to confirm that C. obsoletus females feed on goats and sheep on both islands.Conclusions: These results confirm that the feeding pattern of C. obsoletus is a potentially important factor in BTV transmission to susceptible hosts in case of introduction into the archipelago. Consequently, in the Canary Islands it is essential to maintain vigilance of Culicoides-transmitted viruses such as BTV and the novel Schmallenberg virus.

Bluetongue disease: impact of the 2008 vaccination on fertility in supervised dairy herds

In June 2008 the compulsary nationwide vaccination against BTV-8 (Bluetongue virus serotype 8) was started. After a short time, several owners complained about undesirable effects of the vaccination on fertility and milk quality. Data from 47 dairy farms, regularly supervised by herd health practitioners, were analysed in order to clarify a possible connection between vaccination and fertility. Both vaccinations given each cow for basic immunization were evaluated according to their effects on conception rate and pregnancy. In model calculations the first vaccination had no significant effect on the first service conception rate (FCR), the all service conception rate (ACR) and on the abortion rate. The second vaccination led to a significantly reduced FCR when the cow was inseminated within 20 days of being vaccinated and to a significantly worse ACR when inseminated 10 days before or after vaccination. However, these individually established reductions of the insemination rate had only little influence on overall data.

Virological and pathological findings in Bluetongue virus serotype 8 infected sheep

Twenty-seven sheep of the four most common Swiss breeds and the English breed Poll Dorset were experimentally infected with a northern European field strain of bluetongue virus serotype 8 (BTV-8). Animals of all breeds developed clinical signs, viremia and pathological lesions, demonstrating that BTV-8 is fully capable of replicating and inducing bluetongue disease (BT) in the investigated sheep. Necropsy performed between 10 and 16 days post-infectionem (d.p.i.) revealed BT-typical hemorrhages, effusions, edema, erosions and activation of lymphatic tissues. Hemorrhages on the base of the Arteria pulmonalis and the left Musculus papillaris subauricularis were frequently present. Histology confirmed the macroscopical findings. Using a score system, clinical manifestation and pathology were found to be significantly related. Furthermore, clinical signs and fever were shown to be indicative for the concurrent presence of high amounts of viral ribonucleic acid (RNA) in blood. Spleen, lung, lymph nodes and tonsils from all animals were analyzed regarding viral RNA loads and infectivity using real-time reverse transcriptase PCR (rRT-PCR) and virus isolation in cell culture, respectively. The highest amount of viral RNA was detected in spleen and lung and rRT-PCR revealed to be a more sensitive method for virus detection compared to virus isolation. A long-term follow-up was performed with three sheep showing that BTV-8 viral RNA in blood was present up to 133 d.p.i. and in certain tissues even on 151 d.p.i. No significant breed-related differences were observed concerning clinicopathological picture and viremia, and the Swiss sheep were as susceptible to BTV-8 infection as Poll Dorset sheep, demonstrating a remarkably high virulence of BTV-8 for indigenous sheep breeds.

[Investigation of abortions and other animal health problems in relation to vaccination against Bluetongue virus in 2009]

By the distribution of a questionnaire between all Swiss cattle practitioners it was possible to investigate abortions and other animal health problems related to Bluetongue vaccination 2009. The questionnaire helped to obtain plausibility and timely relation of the reported disorders. 58 abortions in cattle and different herd health problems could be examined. Because there is no possibility to show that a vaccination itself leads to an abortion the results of proven causes of abortions prior and after Bluetongue vaccination were compared regarding their diagnosis. Due to the fact that diagnosis and solving rate of abortions did not differ before and after vaccination, the vaccination itself cannot be responsible for the abortions. Evaluation of different herd health problems showed that Bluetongue vaccination was not responsible for these disorders which often existed already prior to vaccination. Herd health problems generally have multifactorial causes what makes it difficult to asses the effect of Bluetongue vaccination in some cases.

Humoral response to 2 inactivated bluetongue virus serotype-8 vaccines in South American camelids

BACKGROUND: Bluetongue virus serotype 8 (BTV-8) has caused disease in domestic ruminants in several countries of northern Europe since 2006. In 2008 a mass-vaccination program was launched in most affected countries using whole virus inactivated vaccines. OBJECTIVE: To evaluate 2 inactivated vaccines (Bovilis BTV 8; BTVPUR AlSap8) for immunogenicity and safety against BTV-8 in South American camelids (SAC) in a field trial. ANIMALS: Forty-two SAC (25 Alpacas, 17 Llamas) aged between 1 and 16 years. METHODS: The animals were vaccinated twice at intervals of 21 days. They were observed clinically for adverse local, systemic, or both reactions throughout the trial. Blood samples collected on days 0, 14, 21, 43, and 156 after vaccination were tested for the presence of BTV-8 virus by real time-polymerase chain reaction and of specific antibodies by competitive ELISA and a serum neutralization test. RESULTS: All vaccinated animals developed antibodies to BTV-8 after the 2nd administration of the vaccine. No adverse effects were observed except for moderate local swellings at the injection site, which disappeared within 21 days. Slightly increased body temperatures were only observed in the first 2 days after vaccination. The BTV was not detected in any of the samples analyzed. CONCLUSIONS AND CLINICAL IMPORTANCE: The administration of the 2 inactivated commercial vaccines was safe and induced seroconversion against BTV-8 in all vaccinated animals. The results of this study suggest that 2 doses injected 3 weeks apart is a suitable vaccination regimen for SAC.

Serological survey of bluetongue virus serotype-8 infection in South American camelids in Switzerland (2007-2008)

BACKGROUND: Outbreak of bluetongue virus serotype-8 (BTV-8) infection in domestic ruminants in Northern Europe. OBJECTIVE: To investigate the South American camelids' (SAC) susceptibility to BTV-8 infection, their role in the epidemiology of the disease, and the use of currently available serological screening tests in SAC in an endemic region. ANIMALS: Three hundred and fifty-four unvaccinated and 27 vaccinated SAC (170 llamas, 201 alpacas), ranging in age from 1 month to 17 years between June and August 2008. The SAC originated from 44 herds throughout the country, representing 10% of the Swiss SAC population. METHODS: Prospective, observational study of a convenience sample of SAC. Serum samples were analyzed with 2 serological screening tests. When results diverged, a 3rd ELISA was carried out for confirmation (ID Screen Bluetongue Competition ELISA kit). RESULTS: All sera from the 354 unvaccinated animals were negative in the endemic region. Reliable seroconversion was observed after administration of 2 doses of vaccine. CONCLUSIONS AND CLINICAL IMPORTANCE: This study suggests a low susceptibility of SAC to BTV-8 despite the presence of the virus in the cattle and small ruminant population, indicating that SAC do not play a major role in the epidemiology of BTV-8. Furthermore, these results indicate that commercially available serological tests for BTV-8 can be used in SAC.

[Livestock owners with specifically increased disease awareness for Bluetongue: A new approach to disease surveillance]

For the first time in Switzerland, specifically trained livestock owners were included in a national disease surveillance program by the Federal Veterinary Office. A questionnaire on data about clinical and epidemiological aspects of Bluetongue Disease (BT) as well as on herd management was completed by 26 sheep owners three months after they had attended a training course about BT. The control group, consisted of 264 randomly selected sheep and cattle owners who had not visited a training course. Results showed that disease awareness for BT after attending the training course was considerably increased. This was especially evident in the better knowledge of the participants about the great number of possible symptoms. Training courses with the objective of increased disease awareness of livestock owners are an efficient, cost-effective instrument in control programs for exotic diseases.

Survey of bluetongue virus infection in free-ranging wild ruminants in Switzerland.

BACKGROUND In 2006, bluetongue virus serotype 8 (BTV-8) was detected for the first time in central Europe. Measures to control the infection in livestock were implemented in Switzerland but the question was raised whether free-ranging wildlife could be a maintenance host for BTV-8. Furthermore Toggenburg orbivirus (TOV), considered as a potential 25th BTV serotype, was detected in 2007 in domestic goats in Switzerland and wild ruminants were considered a potential source of infection. To assess prevalences of BTV-8 and TOV infections in wildlife, we conducted a serological and virological survey in red deer, roe deer, Alpine chamois and Alpine ibex between 2009 and 2011. Because samples originating from wildlife carcasses are often of poor quality, we also documented the influence of hemolysis on test results, and evaluated the usefulness of confirmatory tests. RESULTS Ten out of 1,898 animals (0.5%, 95% confidence interval 0.3-1.0%) had detectable antibodies against BTV-8 and BTV-8 RNA was found in two chamois and one roe deer (0.3%, 0.1-0.8%). Seroprevalence was highest among red deer, and the majority of positive wild animals were sampled close to areas where outbreaks had been reported in livestock. Most samples were hemolytic and the range of the optical density percentage values obtained in the screening test increased with increasing hemolysis. Confirmatory tests significantly increased specificity of the testing procedure and proved to be applicable even on poor quality samples. Nearly all samples confirmed as positive had an optical density percentage value greater than 50% in the ELISA screening. CONCLUSIONS Prevalence of BTV-8 infection was low, and none of the tested animals were positive for TOV. Currently, wild ruminants are apparently not a reservoir for these viruses in Switzerland. However, we report for the first time BTV-8 RNA in Alpine chamois. This animal was found at high altitude and far from a domestic outbreak, which suggests that the virus could spread into/through the Alps. Regarding testing procedures, hemolysis did not significantly affect test results but confirmatory tests proved to be necessary to obtain reliable prevalence estimates. The cut-off value recommended by the manufacturer for the screening test was applicable for wildlife samples.

Development of a novel marker vaccine platform for protection against Bluetongue Virus (BTV)

Bluetongue virus (BTV) is an economically important member of the genus Orbivirus and closely related to African horse sickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV). Currently, 26 different serotypes of BTV are known. The virus is transmitted by blood-feeding Culicoides midges and causes disease (bluetongue [BT]) in ruminants. In 2006/2007, BTV serotype 8 (BTV-8) caused widespread outbreaks of BT amongst livestock in Europe, which were eventually controlled employing a conventionally inactivated BTV vaccine. However, this vaccine did not allow the discrimination of infected from vaccinated animals (DIVA) by the commonly used VP7 cELISA. RNA replicon vectors based on propagation-incompetent recombinant vesicular stomatitis virus (VSV) represent a novel vaccine platform that combines the efficacy of live attenuated vaccines with the safety of inactivated vaccines. Our goal was to generate an RNA replicon vaccine for BTV-8, which is safe, efficacious, adaptable to emerging orbivirus infections , and compliant with the DIVA principle. The VP2, VP5, VP3 and VP7 genes encoding the BTV-8 capsid proteins, as well as the non-structural proteins NS1 and NS3 were inserted into a VSV vector genome lacking the essential VSV glycoprotein (G) gene. Infectious virus replicon particles (VRP) were produced on a transgenic helper cell line providing the VSV G protein in trans. Expression of antigens in vitro was analysed by immunofluorescence using monoclonal and polyclonal antibodies. In a pilot study, sheep were immunized with two different VRP-based vaccine candidates, one comprising the BTV-8 antigens VP2, VP5, VP3, VP7, NS1, and NS3, the other one containing antigens VP3, VP7, NS1, and NS3. Control animals received VRPs containing an irrelevant antigen. Virus neutralizing antibodies and protection after BTV-8 challenge were evaluated and compared to animals immunized with the conventionally inactivated vaccine. Full protection was induced only when the two antigens VP2 and VP5 were included in the vaccine. To further evaluate if VP2 alone, a combination of VP2 and VP5 or VP5 alone were necessary for complete protection, we performed a second animal trial. Interestingly, VP2 as well as the combination of VP2 and VP5 but not VP5 alone conferred full protection in terms of neutralizing antibodies, and protection from clinical signs and viremia after BTV-8 challenge. These results show that the VSV replicon system represents a safe, efficacious and DIVA-compliant vaccine against BTV as well as a possible platform for protection against other Orbiviruses, such as AHSV and EHDV.

Prevalence of antibodies against bluetongue virus serotype 8 in bulk-tank milk samples from dairy cattle herds located in risk areas for bluetongue virus transmission after a vaccination programme in Switzerland.

Switzerland had been affected by the bluetongue virus serotype 8 (BTV-8) epidemic in Europe in the years 2007 to 2009. After three years of mandatory vaccination and comprehensive surveillance, Switzerland showed to be free of BTV-8 in 2012. In the future Elisa testing of bulk-tank milk (BTM) samples as a very sensitive and cost-effective method should be used for the surveillance of all serotypes of BTV. To determine the prevalence of seropositive herds, BTM from 240 cattle herds was sampled in July 2012. The results showed an apparent seroprevalence of 98.7% in the investigated dairy herds. Most plausible, the high prevalence was caused by the vaccination campaigns rather than by infections with BTV-8. In the outbreak the cumulative number of BTV-8 cases in Switzerland had been 75.Thus it is very likely that the used inactivated vaccines induced long-term antibody titres. Due to the high seroprevalence, investigating for BT-antibodies cannot be used for early recognition of a new introduction of BTV at the moment. Nonetheless, testing of BTM samples is appropriate for an annual evaluation of the seroprevalence and especially as an instrument for early recognition for incursions as soon as the antibody prevalence declines.To determine this decline the BTM testing scheme should be conducted each year as described in this work.

Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection.

Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.

Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus

The core of bluetongue virus (BTV) is a multienzyme complex composed of two major proteins (VP7 and VP3) and three minor proteins (VP1, VP4, and VP6) in addition to the viral genome. The core is transcriptionally active and produces capped mRNA from which all BTV proteins are translated, but the relative role of each core component in the overall reaction process remains unclear. Previously we showed that the 76-kDa VP4 protein possesses guanylyltransferase activity, a necessary part of the RNA capping reaction. Here, through the use of highly purified (>95%) VP4 and synthetic core-like particles containing VP4, we have investigated the extent to which this protein is also responsible for other activities associated with cap formation. We show that VP4 catalyzes the conversion of unmethylated GpppG or in vitro-produced uncapped BTV RNA transcripts to m7GpppGm in the presence of S-adenosyl-l-methionine. Analysis of the methylated products of the reaction by HPLC identified both methyltransferase type 1 and type 2 activities associated with VP4, demonstrating that the complete BTV capping reaction is associated with this one protein.