Estimation of French cattle herd immunity against bluetongue serotype 8 at the time of its re-emergence in 2015.

BACKGROUND: From 2006 to 2010, France experienced two bluetongue epidemics caused by serotype 1 (BTV-1) and 8 (BTV-8) which were controlled by mass vaccination campaigns. After five years without any detected cases, a sick ram was confirmed in August 2015 to be infected by a BTV-8 strain almost identical to that circulating during the previous outbreak. By then, part of the French cattle population was expected to be still protected, since bluetongue antibodies are known to last for many years after natural infection or vaccination. The objective of this study was to estimate the proportion of cattle in France still immune to BTV-8 at the time of its re-emergence in 2015. RESULTS: We used BTV group-specific cELISA results from 8525 cattle born before the vaccination ban in 2013 and 15,799 cattle born after the ban. Samples were collected from January to April 2016 to estimate seroprevalence per birth cohort. The overall seroprevalence in cattle at national and local levels was extrapolated from seroprevalence results per birth cohort and their respective proportion at each level. To indirectly assess pre-immune status of birth cohorts, we computed prevalence per birth cohort on infected farms in autumn 2015 using 1377 RT-PCR results. These revealed limited BTV circulation in 2015. Seroprevalence per birth cohort was likely to be connected to past exposure to natural infection and/or vaccination with higher seroprevalence levels in older animals. A seroprevalence of 95% was observed for animals born before 2008, of which > 90% were exposed to two compulsory vaccination campaigns in 2008-2010. None of the animals born before 2008 were found to be infected, unlike 19% of the young cattle which had never been vaccinated. This suggests that most ELISA-positive animals were pre-immune to BTV-8. We estimated that 18% (from 12% to 32% per département) of the French cattle population was probably pre-immune in 2015. CONCLUSIONS: These results strongly suggest a persistence of antibodies for at least 5-6 years after natural infection or vaccination. The herd immunity of the French cattle population probably limited BTV circulation up to 2015, by which time more than 80% of cattle were naive.

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.

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.

Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador.

Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador.

Complete genome sequence of bluetongue virus serotype 4 that emerged on the French island of Corsica in December 2016.

In November 2016, sheep located in the south of Corsica island exhibited clinical signs suggestive of bluetongue virus (BTV) infection. Laboratory analyses allowed to isolate and identify a BTV strain of serotype 4. The analysis of the full viral genome showed that all the 10 genomic segments were closely related to those of the BTV-4 present in Hungary in 2014 and involved in a large BT outbreak in the Balkan Peninsula. These results together with epidemiological data suggest that BTV-4 has been introduced to Corsica from Italy (Sardinia) where BTV-4 outbreaks have been reported in autumn 2016. This is the first report of the introduction in Corsica of a BTV strain previously spreading in eastern Europe.

Experimental infection of sheep, goats and cattle with a bluetongue virus serotype 4 field strain from Bulgaria, 2014.

In 2014, a new bluetongue virus serotype 4 (BTV-4) strain was detected in southern Greece and spread rapidly throughout the Balkan Peninsula and adjacent countries. Within half a year, more than 7,068 outbreaks were reported in ruminants, particularly in sheep. However, the reported morbidity and case fatality rates in ruminants varied. The pathogenesis of a Bulgarian BTV-4 strain isolated from sheep during the BTV-4 epizootic was studied in different species. Therefore, four sheep, three goats and three cattle were experimentally infected with the isolate BTV-4/BUL2014/15 and monitored for clinical signs up to several weeks. Serum and whole-blood samples were collected at regular intervals and subjected to serological and virological analyses. In this context, BTV-4-specific real-time RT-PCR assays were developed. The infection kinetics were similar to those known for other traditional BTV serotypes, and only mild BT-like clinical signs were observed in goats and sheep. In cattle, no obvious clinical signs were observed, except a transient increase in body temperature. The study results contrast with the severe clinical signs reported in sheep experimentally infected with an African BTV-4 strain and with the reports of BT-like clinical signs in a considerable proportion of different ruminant species infected with BTV-4 in the Balkan region and Italy. The discrepancies between the results of these animal trials and observations of BTV-4 infection in the field may be explained by the influence of various factors on the manifestation of BT disease, such as animal breed, fitness and virus strain, as described previously.

Bluetongue virus serotype 27: Experimental infection of goats, sheep and cattle with three BTV-27 variants reveal atypical characteristics and likely direct contact transmission BTV-27 between goats.

Bluetongue virus (BTV) hitherto consisted of 26 recognized serotypes, of which all except BTV-26 are primarily transmitted by certain species of Culicoides biting midges. Three variants of an additional 27th bluetongue virus serotype (BTV-27v01-v03) were recently detected in asymptomatic goats in Corsica, France, 2014-2015. Molecular characterization revealed genetic differences between the three variants. Therefore, in vivo characteristics were investigated by experimental infection of a total of 15 goats, 11 sheep and 4 cattle with any one of the three variants in separated animal trials. In goat trials, BTV-naïve animals of the same species were kept in a facility where direct contact was unhindered. Of the 15 inoculated goats, 13 and 14 animals were found positive for BTV-RNA and antibodies (Ab), respectively, until the end of the experiments. Surprisingly, BTV-Ab levels as measured with ELISA and neutralization test (SNT) were remarkably low in all seropositive goats. Virus isolation from whole-blood was possible at the peak of viremia until 49 dpi. Moreover, detection of BTV-27v02-RNA and Ab in one contact goat indicated that-similar to BTV-26-at least one of three BTV-27 variants may be transmitted by contact between goats. In the field, BTV-27 RNA can be detected up to 6 months in the whole-blood of BTV-27-infected Corsican goats. In contrast, BTV RNA was not detected in the blood of cattle or sheep. In addition, BTV-27 Abs were not detected in cattle and only a transient increase in Ab levels was observed in some sheep. None of the 30 animals showed obvious BT-like clinical signs. In summary, the phenotypes observed for BTV-27v01-v03 phenotypes correspond to a mixture of characteristics known for BTV-25 and 26.

Re-Emergence of Bluetongue Virus Serotype 8 in France, 2015.

At the end of August 2015, a ram located in central France (department of Allier) showed clinical signs suggestive of BTV (Bluetongue virus) infection. However, none of the other animals located in the herd showed any signs of the Bluetongue disease. Laboratory analyses identified the virus as BTV serotype 8. The viro and sero prevalence intraherd were 2.4% and 8.6% in sheep and 18.3% and 42.9% in cattle, respectively. Phylogenetic studies showed that the sequences of this strain are closely related to another BTV-8 strain that has circulated in France in 2006-2008. The origin of the outbreak is unclear but it may be assumed that the BTV-8 has probably circulated at very low prevalence (possibly in livestock or wildlife) since its first emergence in 2007-2008.

Exposure of Wildlife to the Schmallenberg Virus in France (2011-2014): Higher, Faster, Stronger (than Bluetongue)!

The Schmallenberg virus (SBV) has recently emerged in Europe, causing losses to the domestic livestock. A retrospective analysis of serodata was conducted in France for estimating seroprevalence of SBV among six wildlife species from 2011-2012 to 2013-2014, that is during the three vector seasons after the emergence of the SBV in France. Our objective was to quantify the exposure of wildlife to SBV and the potential protective effect of elevation such as previously observed for bluetongue. We also compared the spatiotemporal trends between domestic and wild animals at the level of the departments. We tested 2050 sera using competitive ELISA tests. Individual and population risk factors were further tested using general linear models among 1934 individuals. All populations but one exhibited positive results, seroprevalence up to 30% being observed for all species. The average seroprevalence did not differ between species but ranged from 0 to 90% according to the area and period, due to the dynamic pattern of infection. Seroprevalence was on average higher in the lowlands compared to areas located up to 800 m. Nevertheless, seroprevalence above 50% occurred in areas located up to 1500 m. Thus, contrary to what had been observed for bluetongue during the late 2000s in the same areas, SBV could spread to high altitudes and infect all the studied species. The spatial spread of SBV in wildlife did not fully match with SBV outbreaks reported in the domestic livestock. The mismatch was most obvious in mountainous areas where outbreaks in wildlife occurred on average one year after the peak of congenital cases in livestock. These results suggest a much larger spread and vector capacity for SBV than for bluetongue virus in natural areas. Potential consequences for wildlife dynamics are discussed.

Development of a Double-Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies.

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time-consuming and require BSL3 facilities. VP2 protein is considered the major serotype-defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV-8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti-VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti-VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti-VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.

Development of a Luminex-Based DIVA Assay for Serological Detection of African Horse Sickness Virus in Horses.

African horse sickness (AHS) is considered a fatal re-emergent vector-borne disease of horses. In the absence of any effective treatment for AHS, vaccination remains the most effective form of disease control. The new generation of vaccines, such as one based on purified, inactivated AHS virus (AHSV, serotype 4), which does not induce antibodies against non-structural protein 3 (NS3), enables the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA assays). As detecting AHS in AHSV-free countries may lead to restrictions on international animal movements and thereby cause significant economic damage, these DIVA assays are crucial for reducing movement restrictions. In this article, we describe a Luminex-based multiplex assay for DIVA diagnosis of AHS, and we validate it in a duplex format to detect antibodies against structural protein 7 (VP7) and NS3 in serum samples from horses vaccinated with inactivated AHSV4 vaccine or infected with a live virus of the same serotype. Results of the Luminex-based assay for detecting anti-NS3 antibodies showed good positive correlation with results from an in-house enzyme-linked immunosorbent assay (ELISA). Thus, the Luminex-based technique described here may allow multiplex DIVA antibody detection in a single sample in less than 2 h, and it may prove adaptable for the development of robust, multiplex serological assays.

Likely Introduction Date of Schmallenberg Virus into France According to Monthly Serological Surveys in Cattle.

To estimate the date of introduction of Schmallenberg virus (SBV) into France, the prevalence of antibodies against the virus was determined monthly in cattle from two northern departments from August 2011 to April 2012. Seropositive cattle were detected from October 2011 in both departments with a prevalence of 55.6% in the westernmost department (Meurthe-et-Moselle) and of 12.7% in the easternmost department (Manche). Schmallenberg virus seroprevalence then increased rapidly to high levels.

Emergence of Bluetongue Virus Serotype 1 in French Corsica Island in September 2013.

Since 2000, French Corsica Island has been exposed to the emergence of three different BT virus (BTV) serotypes: serotype 2 in 2000 and 2001, serotype 4 in 2003 and serotype 16 in 2004. Between 2005 and August 2013, no outbreaks have been reported in the French Island. At the beginning of September 2013, sheep located in the south of the island showed clinical signs suggestive of BTV infection. Laboratory analyses identified the virus as BTV serotype 1. Phylogenetic studies showed that the sequences of this strain are closely related to the BTV-1 strain that was circulating in the Mediterranean basin and in Sardinia in 2012.

Full-Genome Sequencing of Four Bluetongue Virus Serotype 11 Viruses.

Recently, a contamination incident was described in which the challenge inoculum used in a bluetongue virus serotype 8 (BTV-8) vaccination trial was contaminated with a BTV-11 virus that was closely related to the Belgian BTV-11 virus from 2008. This study reports the first complete genome sequences of four BTV-11 viruses: the BTV-11 contaminant, BTV-11 reference strain, BTV-11 vaccine strain and a recently isolated BTV-11 field strain from Martinique. Full-genome analysis showed that these viruses belong to serotype 11/nucleotype A and cluster together with other western topotype bluetongue viruses. Detailed comparisons of the genomes further indicated that the contaminant was derived from the BTV-11 reference strain, as they were distinguished by a single synonymous nucleotide substitution. The previously reported partial sequence of genome segment 2 of the Belgian BTV-11 was found to be identical to that of the BTV-11 vaccine strain, indicating that it most likely was the BTV-11 vaccine strain. These findings also suggest that the BTV-11 contaminant and the Belgian BTV-11 are not the same viruses. Finally, comparison of the reference and vaccine strain did not allow determining the amino acid substitutions that contribute to the attenuated phenotype.

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).

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.

Evaluation of bluetongue virus (BTV) decontamination techniques for caprine embryos produced in vivo.

The objective of this study was to investigate methods of decontaminating early goat embryos that had been infected in vitro with bluetongue virus (BTV). Embryos were isolated from in vivo-fertilized BTV-free goats. Zona pellucida (ZP)-intact 8 to 16 cell embryos were cocultured for 36 h in an insert over a Vero cell monolayer infected with BTV serotype 8. The embryos were then treated with one of five different washing procedures. The treatment standard (TS) comprised phosphate-buffered saline (PBS) + 0.4% BSA (five times over for 10 s), Hank's +0.25% trypsin (twice for 45 s), and then PBS + 0.4% BSA again (five times for 10 s). The four other washing procedures all included the same first and last washing steps with PBS but without BSA (five times for 10 s) and with PBS + 0.4% BSA (five times for 10 s), respectively. The intermediate step varied for each washing procedure. Treatment 1 (T1): 0.25% trypsin (twice for 45 s). Treatment 2 (T2): 0.25% trypsin (twice for 60 s). Treatment 3 (T3): 0.5% trypsin (twice for 45 s). Treatment 4 (T4): 1% hyaluronidase (once for 5 min). After washing, the embryos were transferred and cocultured with BTV indicator Vero cell monolayers for 6 h, to detect any cytopathic effects (CPE). The effectiveness of the different washing techniques in removing the virus was evaluated by RT-qPCR analysis. The TS, T1, T3, and T4 trypsin or hyaluronidase treatments did not eliminate BTV; Treatment 2 eliminated the virus from in vitro infected goat embryos.

Segmental configuration and putative origin of the reassortant orbivirus, epizootic hemorrhagic disease virus serotype 6, strain Indiana.

In 2006, an exotic reassortant orbivirus, epizootic hemorrhagic disease virus serotype 6 (EHDV-6) [strain (Indiana)], was first detected in the United States. To characterize the reassortment configuration of this virus and to conclusively determine the parental virus of each RNA segment, the complete genome of EHDV-6 (Indiana) was sequenced, in addition to the genomes of representative EHDV-6 and EHDV-2 isolates. Based on genomic comparisons to all other EHDV serotypes, we determined that EHDV-6 (Indiana) originated from a reassortment event between the Australian prototype strain of EHDV-6 (CSIRO 753) and the North American topotype of EHDV-2 (Alberta). Additionally, phylogenetic analysis of all EHDV-6 (Indiana) isolates detected in the United States from 2006 to 2010 suggests that the virus may be undergoing continual reassortment with EHDV-2 (Alberta). In 2010, EHDV-6 (CSIRO 753) was detected in Guadeloupe, demonstrating that the parental virus of the reassortment event is circulating in the Caribbean.

Bluetongue virus serotype 8: abortion and transplacental transmission in cattle in the Burgundy region, France, 2008-2009.

During the incursion of bluetongue virus (BTV) serotype 8 in France in 2007, an increase in the number of abortions in cattle was observed, but the cause was not clearly established. A survey of all the reported cases of abortion in cattle from November 2008 to April 2009 was conducted in the Nièvre district (Burgundy region) to determine the percentage of abortions as a result of BTV-8 and to study factors that could have played a role in BTV-8 transplacental transmission. BTV-8 was present in 16% of the fetuses or newborn calves that died within 48 h, from 780 dams. Dams inseminated before the BTV epizootic peak recorded from July to September 2008 were more likely to have BTV-positive abortions (OR=5.7, P<0.001) and those vaccinated in May or June 2008 were less likely to have BTV-positive abortions (OR=0.3, P=0.01 and OR=0.4, P=0.001, respectively). The gestational month was not a predictor of BTV abortion. In blood or spleen, fetuses/calves from RT-PCR-positive dams had significantly higher RNA concentrations than fetuses/calves from RT-PCR-negative dams. Of the 128 dams that had BTV-positive fetuses or calves, 60% were RT-PCR-negative. BTV-8-positive fetuses/calves were significantly more frequent (n=42 vs n=21, P=0.082) amongst those showing clinical signs or lesions suggestive of cerebral damage.