Assessing the export trade risk of bluetongue virus serotypes 4 and 8 in France.

Bluetongue (BT) causes an economic loss of $3 billion every year in the world. After two serious occurrences of BT (bluetongue virus [BTV] occurrence in 2006 and 2015), France has been controlling for decades, but it has not been eradicated. As the largest live cattle export market in the world, France is also one of the major exporters of breeding animals and genetic materials in the world. The biosafety of its exported cattle and products has always been a concern. The scenario tree quantitative model was used to analyze the risk of BTV release from French exported live cattle and bovine semen. The results showed that with the increase in vaccination coverage rates, the risk decreased. If the vaccine coverage is 0%, the areas with the highest average risk probability of BTV-4 and BTV-8 release from exported live cattle were Haute-Savoie and Puy-de-Dôme, and the risk was 2.96 × 10-4 and 4.25 × 10-4 , respectively. When the vaccine coverage was 90%, the risk probability of BTV-4 and BTV-8 release from exported live cattle was 2.96 × 10-5 and 4.24 × 10-5 , respectively. The average probability of BTV-8 release from bovine semen was 1.09 × 10-10 . Sensitivity analysis showed that the probability of false negative polymerase chain reaction (PCR) test and the probability of BT infection in the bull breeding station had an impact on the model. The identification of high-risk areas and the discovery of key control measures provide a reference for decision makers to assess the risk of French exports of live cattle and bovine semen.

Bluetongue Virus Infection of Goats: Re-Emerged European Serotype 8 vs. Two Atypical Serotypes.

In recent years, numerous atypical Bluetongue virus (BTV) strains have been discovered all around the world. Atypical BTV strains are phylogenetically distinct from the classical BTV serotypes 1-24 and differ in terms of several biological features. For the first time, the atypical strains BTV-25-GER2018 and BTV-33-MNG3/2016 as well as the re-emerged classical strain BTV-8-GER2018 were evaluated comparatively in a pathogenesis study in goats-the natural host of atypical BTV. A substantial number of in-contact animals were included in this study to detect potential contact transmissions of the virus. After infection, EDTA blood, ocular, nasal and oral swab samples as well as serum were collected regularly and were used for virological and serological analyses, respectively. Our study showed differences in the immunological reaction between the two atypical BTV strains (no group-specific antibody detection) and the classical BTV strain BTV-8-GER2018 (group-specific antibody detection). Furthermore, we observed an increase in the total WBC count (neutrophils and lymphocytes) in goats infected with the atypical BTV strains. No horizontal transmission was seen for all three strains. Our study suggests that the atypical BTVs used in the trial differ from classical BTVs in their immunopathogenesis. However, no evidence of direct contact transmission was found.

Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls.

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

Origin of Bluetongue Virus Serotype 8 Outbreak in Cyprus, September 2016.

In September 2016, clinical signs, indicative of bluetongue, were observed in sheep in Cyprus. Bluetongue virus serotype 8 (BTV-8) was detected in sheep, indicating the first incursion of this serotype into Cyprus. Following virus propagation, Nextera XT DNA libraries were sequenced on the MiSeq instrument. Full-genome sequences were obtained for five isolates CYP2016/01-05 and the percent of nucleotide sequence (% nt) identity between them ranged from 99.92% to 99.95%, which corresponded to a few (2-5) amino acid changes. Based on the complete coding sequence, the Israeli ISR2008/13 (98.42-98.45%) was recognised as the closest relative to CYP2016/01-05. However, the phylogenetic reconstruction of CYP2016/01-05 revealed that the possibility of reassortment in several segments: 4, 7, 9 and 10. Based on the available sequencing data, the incursion BTV-8 into Cyprus most likely occurred from the neighbouring countries (e.g., Israel, Lebanon, Syria, or Jordan), where multiple BTV serotypes were co-circulating rather than from Europe (e.g., France) where a single BTV-8 serotype was dominant. Supporting this hypothesis, atmospheric dispersion modelling identified wind-transport events during July-September that could have allowed the introduction of BTV-8 infected midges from Lebanon, Syria or Israel coastlines into the Larnaca region of Cyprus.

Failure to Remove Bluetongue Serotype 8 Virus (BTV-8) From in vitro Produced and in vivo Derived Bovine Embryos and Subsequent Transmission of BTV-8 to Recipient Cows After Embryo Transfer.

The behavior of BTV-8 in cattle is different from most other serotypes not only with regards to clinical signs but certainly with respect to virus transmission (transplacental, contact). Therefore, the possibility of virus transmission by means of embryo transfer was examined by in vitro exposure of in vitro produced and in vivo derived bovine blastocysts to BTV-8 followed by different washing protocols, including longer exposure times (up to 120 s) to 0.25% trypsin at room temperature or at 37°C. None of the washing protocols used was successful in removing the viral genome completely from the in vitro produced and in vivo derived embryos as was demonstrated by real-time PCR. Moreover, BTV-8 virus was transmitted to recipient cows after embryo transfer of in vivo derived BTV8-exposed embryos, which had been subjected to routine decontamination as recommended by IETS, consisting of 5 washes in PBS followed by a double treatment of 0.25% trypsin for 45s at 37°C, and an additional 5 washes in PBS with 2% FCS. This study clearly demonstrates the necessity of vigorous application of the directives for screening of potential donors and the collected embryos, especially in regions with BTV-8, to prevent transmission of the disease.

A comparison of different vaccination schemes used in sheep combining inactivated bluetongue vaccines against serotypes 4 and 8.

Eight different vaccination schemes using four commercially available inactivated Bluetongue vaccines against serotypes 4 and 8 in three different combinations (setting 1-3) were tested under field conditions for their ability to generate a measurable immune response in sheep. Animals of setting 1 (groups A-D) were simultaneously vaccinated using either individual injections at different locations (groups A & D) or double injection by a twin-syringe (groups B & C). For both application methods, a one-shot vaccination (groups C & D) was compared to a boosted vaccination (groups A & B). Sheep of setting 2 (groups E-G) were vaccinated in an alternating, boosted pattern at fortnightly intervals starting with serotype 4 (groups E & F) or vice versa (group G). Group H of setting 3 was vaccinated simultaneously and vaccines were injected individually as a one-shot application. Each group consisted of 30 sheep. The immunogenic response was tested in all sheep (n = 240) by ELISA (IDScreen®Bluetongue Competition), while serum neutralisation tests were performed in five to six sheep from each group (n = 45). All vaccine combinations were well tolerated by all sheep. Of all vaccines and schemes described, the simultaneous double injected boosted vaccination of setting 1 (group B) yielded the highest median serotype-specific titres 26 weeks after the first vaccination (afv) and 100% seropositive animals (ELISA) one year afv. In setting 1, there were no relevant significant differences in the immunogenic response between simultaneously applied vaccines at different sites or at the same injection site. Importantly, a one-shot vaccination induced comparable immunogenicity to a boosted injection half a year afv. Low serotype-specific neutralising antibody levels were detected in settings 2 and 3 and are attributed to diverse factors which may have influenced the measured immunogenicity.

Presence of Antibodies against Bluetongue Virus (BTV) in Sheep 5 to 7.5 Years after Vaccination with Inactivated BTV-8 Vaccines.

Thirty-six female sheep, previously vaccinated against Bluetongue virus serotype 8 (BTV-8) using inactivated vaccines, were included in this field study. In Germany, vaccination was compulsory in 2008 and 2009, voluntary in 2010 and early 2011, and later, was prohibited in 2011. Due to their age, eighteen sheep had been vaccinated for two or more consecutive years, while a further eighteen animals had only been vaccinated once or not at all. The sheep were blood sampled five (n = 31) to 7.5 years (n = 5) after their last vaccination. All serum samples (n = 36) were tested for BTV group-specific antibodies by an ELISA (IDScreen® Bluetongue Competition assay, ID Vet). In five of the animals, the BTV-8 serotype-specific antibody titers were measured by serum neutralization (SN). The majority of sheep that were vaccinated annually for two or more years showed a positive ELISA (14/18 sheep) and a SN (two of two sheep) result 5 years after their last vaccination. Most of the sheep vaccinated fewer than twice showed a negative ELISA result 5 to 7.5 years after their last vaccination (13/18 animals). The three animals in this group tested by SN showed one negative and two positive results. This short communication is the first to describe the presence of BTV antibodies in sheep 5 to 7.5 years after vaccination with inactivated BTV-8 vaccines.

BTV antibody longevity in cattle five to eight years post BTV-8 vaccination.

The Bluetongue virus serotype -8 (BTV-8) epizootic in Germany (2006-2008) was successfully eradicated, essentially by the massive application of commercially available inactivated BTV-8 vaccines. While a six-year antibody longevity of BTV antibodies post BTV-8 vaccination in cattle has been described previously, our study investigated the BTV-8-vaccine antibodies in cattle for up to eight years. In total, 157 bovine serum samples were analysed for the presence of group-specific BTV antibodies in both a commercial cELISA, and a BTV-8- specific serum neutralization test. A robust number of cattle were seropositive for group- and serotype-specific neutralising antibodies for five or more years. In selected animals, born and vaccinated in 2009 or later, the presence of BTV antibodies for up to eight years post BTV-8 vaccination could be confirmed. Our data also show, that booster vaccination prolonged the antibody longevity of vaccine-induced antibodies and the number of serologically positive cattle.

Circulation of bluetongue virus 8 in French cattle, before and after the re-emergence in 2015.

Bluetongue virus serotype 8 (BTV-8) re-emerged in Central France in August 2015. The viral strain identified is nearly identical to the one that circulated during the 2006/2009 massive outbreak throughout Europe. To address the question of an undetected BTV-8 circulation on the French territory, a serological study was conducted on young cattle along a transect of seven departments, three of them located in areas where the virus presence had been confirmed by RT-PCR by winter 2015/2016. Sera from 2,565 animals were collected during the winters preceding and following the re-emergence, with 414 animals being sampled in each of the two consecutive years. All samples were tested by competitive ELISA (IDVet) and, when enough serum was available, ELISA-positive samples were confirmed by seroneutralization tests. In areas with infected holdings, seropositive animals were found before the re-emergence (N = 14 of 511), significantly more on the following year (N = 17 of 257), and eight animals (N = 158) seroconverted over 2015. Seropositive animals were also detected as early as winter 2014/2015 in one department without known infected holdings (N = 12 of 150), and in winter 2015/2016 in three of them (N = 21 of 555), where seven animals (N = 154) seroconverted over 2015. These results suggest that BTV-8 may have spread at low levels before the re-emergence, even in areas considered virus-free. Unfortunately, whole blood from the seropositive animals was not available to definitely confirm the virus presence by RT-PCR.

Serological status for BTV-8 in French cattle prior to the 2015 re-emergence.

Undetected in Europe since 2010, bluetongue virus serotype 8 (BTV-8) re-emerged in August 2015 in Central France. To gain insight into the re-emergence on the French territory, we estimated the seroprevalence in cattle before the detection of BTV-8 in 2015, in areas differentially affected by the current outbreak. A retrospective survey based on the analysis of stored sera was thus conducted in the winter preceding the re-emergence in seven French departments including the one where the virus was first detected. A total of 10,066 sera were retrieved from animals sampled in 444 different herds in winter 2014/15. Between-herd seroprevalence revealed the presence of seropositive animals in almost all herds sampled (97.4%). The animal-level seroprevalence averaged at 44%, with a strong age pattern reflecting the cumulative exposure to both natural infection and to vaccination. A multivariable analysis allowed separating the respective effects of both exposures. A higher proportion of seropositivity risk was attributed to vaccination (67.4%) than to exposure to natural infection (24.2%). The evolution of seroprevalence induced by the two main risk factors in 74 mainland departments was reconstructed between the vaccination ban (2013) and the re-emergence (2015). We showed a striking decrease in seroprevalence with time after the vaccination ban, due to population renewal, which could have facilitated virus transmission leading to the current outbreak situation.

European Bluetongue Serotype 8: Disease Threat Assessment for U.S. Sheep.

Bluetongue virus (BTV) is an orbivirus transmitted by biting midges (Culicoides spp.) that can result in moderate to high morbidity and mortality primarily in sheep and white-tailed deer. Although only 5 serotypes of BTV are considered endemic to the United States, as many as 11 incursive serotypes have been detected in livestock and wildlife in the past 16 years. Introductions of serotypes, with unknown virulence and disease risk, are constant threats to US agriculture. One potential incursive serotype of particular concern is the European strain of BTV-8, which was introduced into Northern Europe in 2006 and caused unprecedented livestock disease and mortality during the 2006-2007 vector seasons. To assess disease risk of BTV-8 in a common white-faced American sheep breed, eight Polled Dorset yearlings were experimentally infected and monitored for clinical signs. Viremia and viral tissue distribution were detected and quantified by real-time qRT-PCR. Overall, clinical disease was moderate with no mortality. Viremia reached as high as 9.7 log10 particles/mL and persisted at 5 logs or higher through the end of the study (28 days). Virus distribution in tissues was extensive with the highest mean titers at the peak of viremia (day 8) in the kidney (8.38 log10 particles/mg) and pancreas (8.37 log10 particles/mg). Virus persisted in tissues of some sheep at 8 logs or higher by day 28. Results of this study suggest that should BTV-8 emerge in the United States, clinical disease in this common sheep breed would likely be similar in form, duration, and severity to what is typically observed in severe outbreaks of endemic serotypes, not the extraordinary disease levels seen in Northern Europe. In addition, a majority of exposed sheep would be expected to survive and act as significant BTV-8 reservoirs with high titer viremias for subsequent transmission to other livestock and wildlife populations.

A one-year follow-up of antibody response in cattle and sheep after vaccination with serotype 8- and serotype 1-inactivated BT vaccines.

Sixteen sheep and 18 cattle were followed up during 1 year to estimate the duration of immunity induced by inactivated bluetongue virus serotype 8 (BTV-8) vaccines (sheep and cattle) and a bluetongue virus serotype 1 (BTV-1) vaccine (cattle) under field conditions using cELISA and seroneutralization test (SNT). Four sheep never seroconverted. Those that seroconverted were all seronegative by BTV-8 SNT at the date of last sampling [378 days post-vaccination (dpv)]. Eight sheep were still positive by competitive ELISA (cELISA) 378 dpv. All the cattle seroconverted. At the end of the study, eight and 11 cattle were still positive by BTV-8 SNT and cELISA, respectively (335 dpv); and nine were still positive by BTV-1 SNT (301 dpv).

The development of an accelerated reverse-transcription loop mediated isothermal amplification for the serotype specific detection of bluetongue virus 8 in clinical samples.

In 2006 bluetongue virus serotype 8 (BTV 8) was identified for the first time in the Netherlands causing a major epidemic in sheep and cattle that quickly spread to neighbouring Belgium, Germany and beyond to France and the UK. This resulted in severe animal health and welfare problems as well as substantial economic losses to the agrifood industries of these countries. Given that the early diagnosis of BTV infection 'in-the-field' is extremely useful to its subsequent management and control, this study was established to design a novel, sensitive and rapid nucleic acid diagnostic test for the serotype-specific detection of BTV 8, which could be used without the use of advanced laboratory support and equipment. Primers for the detection of BTV 8 were based on genome segment 2 of the virus, the VP2 gene. The assay was assessed using a full panel of BTV reference strains and clinical samples. Positive amplification was observed using a fluorescent detection reagent. The sensitivity of the RT-LAMP assay was 102 copies of RNA. The assay did not amplify the closely related orbivirus EHDV. This novel RT-LAMP offers a sensitive, specific and rapid method of detecting BTV 8. The approach is inexpensive and easy to use and could potentially be used in a 'pen-side' setting 'in the field' or by smaller less well-equipped laboratories in developing countries.

Bluetongue in Spain: from the first outbreak to 2012.

Outbreaks of bluetongue disease have occurred in Spain six times and have been caused by the following serotypes of bluetongue virus (BTV), in chronological order: BTV10, BTV2, BTV4, BTV1 and BTV8. Serotypes BTV1, BTV2 and BTV4 may have entered the country in Culicoides transported by wind; BTV8 via infected animal movements; and BTV10 across the Portuguese border. The evolution of each serotype has been different: BTV1, BTV4 and BTV10 spread throughout mainland Spain; BTV2 did not spread from the Balearic Islands to the Iberian Peninsula; and BTV8 has proven very poor at spreading throughout mainland Spain. The significant economic impact of the disease has led authorities to adopt control and eradication measures, which have evolved as new diagnostic tools and vaccines have become available. This review describes BTV infection in Spain, and it focuses on the clinical disease produced by each serotype, the Culicoides species which were present at what time, the origin of the virus and the control measures adopted. In the field, it has proven necessary to vaccinate livestock against each new BTV serotype as it arrived. Therefore, future eradication strategies should focus on developing polyvalent vaccines and vaccines that allow the differentiation of infected and vaccinated animals. As of 1 January 2013, the Iberian Peninsula is considered a restricted area for BTV1, and a small zone in southern Spain is a restricted area for BTV4, which includes the little BTV8 restricted area. Serotypes BTV1 and BTV4 were detected in sentinel animals in January and November and in March 2012, respectively. The last BTV8 positive animal was detected in November 2010, which implies that in the coming months, Spain may be declared free of BTV8.

Evidence of transplacental transmission of bluetongue virus serotype 8 in goats.

During the incursion of bluetongue virus (BTV) serotype 8 in Europe, an increase in the number of abortions in ruminants was observed. Transplacental transmission of BTV-8 in cattle and sheep, with subsequent foetal infection, is a feature of this specific bluetongue serotype. In this study, BTV-8 ability to cross the placental barrier at the beginning of the second third of pregnancy and at the end of pregnancy was investigated in goats in two separate experiments. In the first experiment, nine goats were experimentally infected with BTV-8 at 61 days of pregnancy. Foetuses were collected 21 dpi. BTV-8 was evidenced by real time RT-PCR and by viral isolation using blood from the umbilical cord and the spleens of 3 out of the 13 foetuses. All dams were viraemic (viral isolation) at the moment of sampling of the foetuses. Significant macroscopic or histological lesions could not be observed in foetuses or in their infected dams (notably at the placenta level). In the second experiment, 10 goats were infected with BTV-8 at 135 days of pregnancy. Kids were born by caesarean section at the programmed day of birth (15 dpi). BTV-8 could not be detected by rt-RT-PCR in blood or spleen samples from the kids. This study showed for the first time that BTV-8 transplacental transmission can occur in goats that have been infected at 61 days of pregnancy, with infectious virus recovered from the caprine foetuses. The observed transmission rate was quite high (33%) at this stage of pregnancy. However, it was not possible to demonstrate the existence of BTV-8 transplacental transmission when infection occurred at the end of the goat pregnancy.

Experimental infection of white-tailed deer (Odocoileus virginianus) with Northern European bluetongue virus serotype 8.

Bluetongue (BT) is an insect-transmitted, economically important disease of domestic and wild ruminants. Although only five of the 26 reported bluetongue virus (BTV) serotypes are considered endemic to the USA, 10 exotic serotypes have been isolated primarily in the southeastern region of the country since 1999. For an exotic BTV serotype to become endemic there must be susceptible animal species and competent vectors. In the USA, sheep and white-tailed deer (WTD) are the primary sentinel livestock and wildlife species, respectively. In 2006, BTV-8 was introduced into Northern Europe and subsequently overwintered, causing unprecedented livestock disease and mortality during the 2006-2007 vector seasons. To assess the risk of the European strain of BTV-8 to North American WTD, and understand the role they could play after a similar introduction, eight bluetongue-seronegative WTD were inoculated with BTV-8. Body temperatures and clinical signs were recorded daily. Blood samples were analyzed for BTV RNA with quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR), serum analyzed for BTV antibodies by cELISA, and tissues taken for histopathology and qRT-PCR. All eight deer became infected and developed moderate to severe clinical disease from days 8 to 15. Peak viremia was from day 7 to 10 with detectable titers through the end of the study (28 days) in most deer. Serum antibody was detected by day 6, peaked by day 10 and continued through day 28. We conclude that North American WTD are highly susceptible to BTV-8 and would act as clinical disease sentinels and amplifying hosts during an outbreak.