Post‐epidemic investigation of Schmallenberg virus in wild ruminants in Slovenia

Schmallenberg virus (SBV) is a vector‐borne virus belonging to the genus Orthobunyavirus within the Bunyaviridae family. SBV emerged in Europe in 2011 and was characterized by epidemics of abortions, stillbirths and congenital malformations in domestic ruminants. The first evidence of SBV infection in Slovenia was from an ELISA‐positive sample from a cow collected in August 2012; clinical manifestations of SBV disease in sheep and cattle were observed in 2013, with SBV RNA detected in samples collected from a total of 28 herds. A potential re‐emergence of SBV in Europe is predicted to occur when population‐level immunity declines. SBV is also capable of infecting several wild ruminant species, although clinical disease has not yet been described in these species. Data on SBV‐positive wild ruminants suggest that these species might be possible sources for the re‐emergence of SBV. The aim of this study was to investigate whether SBV was circulating among wild ruminants in Slovenia and whether these species can act as a virus reservoir. A total of 281 blood and spleen samples from wild ruminants, including roe deer, red deer, chamois and European mouflon, were collected during the 2017–2018 hunting season. Serum samples were tested for antibodies against SBV by ELISA; the overall seroprevalence was 18.1%. Seropositive samples were reported from all over the country in examined animal species from 1 to 15 years of age. Spleen samples from the seropositive animals and serum samples from the seronegative animals were tested for the presence of SBV RNA using real‐time RT‐PCR; all the samples tested negative. Based on the results of the seropositive animals, it was demonstrated that SBV was circulating in wild ruminant populations in Slovenia even after the epidemic, as almost half (23/51) of the seropositive animals were 1 or 2 years old.     

Circulation of a Simbu Serogroup Virus,Causing Schmallenberg Virus‐Like Clinical Signs in Northern Jordan

Schmallenberg virus (SBV)‐like clinical cases of abortions in northern Jordan in early 2013, together with the emergence of SBV in Europe in 2011, its rapid spread within the following years and the detection of this virus in Turkey, raised questions about the distribution of SBV or related orthobunyaviruses. To evaluate the occurrence of SBV or related members of the Simbu serogroup of orthobunyaviruses in Jordan, bulk milk (cattle) and serum samples (cattle, sheep and goat) collected in northern Jordan in 2013 were first tested by commercially available SBV antibody ELISAs. Indeed, 3 of 47 bulk milk samples and 57 of 115 serum samples provided positive results, but SBV specificity of the ELISA results could not be confirmed by virus neutralization assays. Instead, subsequent cross‐neutralization tests were able to further investigate the specificity of these antibodies. Here, a significant inhibition of Aino virus was observed. Thus, the causative agent was most likely a Simbu serogroup virus closely related to Aino virus. Consequently, these results confirm that members of this group of virus are not only present in Europe, Africa or Australia, but also in the Middle East.     

Follow‐up of the Schmallenberg Virus Seroprevalence in Belgian Cattle

Schmallenberg virus (SBV), which emerged in Northwestern Europe in 2011, is an arthropod‐borne virus affecting primarily ruminants. Based on the results of two cross‐sectional studies conducted in the Belgian ruminant population during winter 2011–2012, we concluded that at the end of 2011, almost the whole population had already been infected by SBV. A second cross‐sectional serological study was conducted in the Belgian cattle population during winter 2012–2013 to examine the situation after the 2012 transmission period and to analyse the change in immunity after 1 year. A total of 7130 blood samples collected between 1st January and 28 February 2013 in 188 herds were tested for the presence of SBV‐specific antibodies. All sampled herds tested positive and within‐herd seroprevalence was estimated at 65.66% (95% CI: 62.28–69.04). A statistically significant decrease was observed between the beginning and the end of 2012. On the other hand, age‐cohort‐specific seroprevalence stayed stable from 1 year to the other. During winter 2012–2013, calves between 6 and 12 months had a seroprevalence of 20.59% (95% CI: 15.34–25.83), which seems to be an indication that SBV was still circulating at least in some parts of Belgium during summer–early autumn 2012. Results showed that the level of immunity against SBV of the animals infected has not decreased and remained high after 1 year and that the spread of the virus has slowed down considerably during 2012. This study also indicated that in the coming years, there are likely to be age cohorts of unprotected animals.     

Serosurvey for Schmallenberg Virus in Alpine Wild Ungulates

Because Schmallenberg virus (SBV) was first reported in domestic ruminants in Northern Italy in February 2012, we conducted a serosurvey to assess the presence of SBV‐specific antibodies in free‐ranging alpine ruminants. The tested serum samples were from chamois (23) and red deer (352) hunted from 2007 to 2013. All of the serum samples collected through September, 2012, tested negative, whereas a single chamois serum and 21 red deer sera taken during the 2012–2013 hunting season tested positive for the presence of SBV antibodies. Because this serosurvey is suggestive of an active SBV circulation in Alpine wildlife, targeted surveillance should be performed on wild ruminants to monitor the spread of the virus and to assess the epidemiological role of wildlife at the interface with domestic animals.     

Two Years After the Schmallenberg Virus Epidemic in the Netherlands: Does the Virus still Circulate?

Two years after the introduction of the Schmallenberg virus in north‐western Europe, it is unknown whether the virus is still circulating in countries that were the first to be confronted with it. When the population‐level immunity declines in Europe, reintroduction or the re‐emergence of SBV in Europe might eventually result in an outbreak of similar magnitude of that seen in 2011–2012. The Netherlands was part of the primary outbreak region of SBV in 2011. The aim of this study was to determine whether SBV circulated amongst dairy herds in the Netherlands in 2013, and if so, to which extent. For this purpose, the presence of SBV‐specific antibodies in naive cattle was investigated. A total of 394 dairy farms were sampled between October and December 2013 by collecting five serum samples per herd. Antibodies were detected in 1.1% [95% confidence interval (CI): 0.7–1.7)] of the animals. All seropositive animals were single reactors per herd and were at least 8 months old at sampling. As these results were inconclusive in demonstrating freedom of SBV circulation, a more in‐depth investigation was initiated to provide more insight: an additional sample of 20 youngstock within the same age category (including the five initially sampled animals) was collected from 17 of the 21 positive herds and tested for SBV‐specific antibodies. This resulted in 9 antibody‐positive test results of 316 samples. Again, the positive samples were single reactors within the sample obtained from each farm, which is unlikely given the characteristics of SBV. Therefore, assuming the single reactors as false‐positive, this survey showed with 95% confidence that the maximum possible prevalence of herds with SBV circulation in the Netherlands was <1% in 2013.     

Schmallenberg virus in Culicoides Latreille (Diptera: Ceratopogonidae) populations in France during 2011‐2012 outbreak

Following the emergence of the Schmallenberg virus (SBV) in 2011 in Germany and its rapid spread in Europe, Culicoides (Diptera: Ceratopogonidae) collected through the French surveillance network were analysed in order to record the presence of virus genome into species diversity collected, to assess the minimum infectious rates (MIR) and the virus circulation dynamics in Culicoides populations. Two vector activity periods were selected (2011, August to October, 53 sites and 2012, June to October, 35 sites) corresponding to 704 night collections. A total of 29,285 individual midges covering at least 50 species were tested either in pools of maximum 50 females or individually (for Culicoides obsoletus /Culicoides scoticus ) using real‐time RT‐PCR. Nine species were found SBV positive (C. obsoletus , C. scoticus , Culicoides chiopterus , Culicoides dewulfi , Culicoides imicola , Culicoides pulicaris , Culicoides newsteadi , Culicoides lupicaris and Culicoides nubeculosus ) with overall MIR ranging from 0.2% to 4.2%. While the Culicoides nubeculosus laboratory strain is generally considered to have only low vector competence for viruses, interestingly, field‐caught C. nubeculosus specimens were found positive twice for SBV. The first SBV‐positive pool was recorded in August 2011 in north‐eastern France, dating the virus circulation in France 5 months earlier than the first recorded congenital malformations and 2 months earlier than the former recorded date based on retrospective serological data. The MIR were maximum in October 2011, and in July 2012 according to dates of virus arrival in the studied areas. Moreover, our study also showed that virus circulation could be locally intense with infection rate (IR) reaching up to 16% for C. obsoletus /C. scoticus in July 2012 in one site of western France. This retrospective study demonstrates the importance of large‐scale analysis to describe the spatio‐temporal dynamics of virus circulation.     

Schmallenberg Virus in Belgium: Estimation of Impact in Cattle and Sheep Herds

Schmallenberg virus (SBV) emerged during summer 2011. SBV induced an unspecific syndrome in cattle and congenital signs (abortions, stillbirths and malformations) in domestic ruminants. To study the impact of SBV in Belgium, a phone survey was conducted upon September 2012. Hereto two groups of cattle farmers (A and B) and two groups of sheep farmers (C and D) were randomly selected. Farms from groups A (n = 53) and C (n = 42) received SBV‐positive result at RT‐PCR in the Belgian National Reference Laboratory (NRL). Farms from groups B (n = 29) and D (n = 44) never sent suspected samples to NRL for SBV analysis but were however presumed seropositive for SBV after the survey. Questionnaires related to reproduction parameters and clinical signs observed in newborn and adult animals were designed and addressed to farmers. As calculated on a basis of farmers’ observations, 4% of calves in group A and 0.5% in group B were reported aborted, stillborn or deformed due to SBV in 2011–2012. The impact as observed by sheep farmers was substantially higher with 19% of lambs in group C and 11% in group D that were reported aborted, stillborn or deformed due to SBV in 2011–2012. Interestingly, abortions or stillbirths were not clear consequences of SBV outbreak in cattle farms, and the birth of a deformed animal was an essential condition to suspect SBV presence in cattle and sheep farms. This study contributes to a better knowledge of the impact of the SBV epidemic. The results suggest that SBV impacted Belgian herds mostly by the birth of deformed calves, stillborn lambs and deformed lambs. This work also demonstrates that the birth of a deformed calf or lamb was a trigger for the farmer to suspect the presence of SBV and send samples to NRL for further analyses.     

First report of Schmallenberg Virus Infection in Cattle and Midges in Poland

Two outbreaks of Schmallenberg virus (SBV) infection that coincided with the introduction of two bulls imported from France into two herds located in West Pomerania and Silesia provinces in Poland are described in detail. The first SBV real‐time RT‐PCR‐positive result was obtained during routine testing of one of the imported bulls. The second bull and the affected farms were tracked by further investigation. Transmission of SBV into Polish cattle herds where the bulls were imported was confirmed by viral RNA detection in real‐time RT‐PCR, virus isolation followed by immunoperoxidase (IPX) staining and seroconversion. SBV RNA was detected also in Culicoides obsoletus pools caught in a trap located 5 km from one of the outbreaks. Testing nearly 900 samples collected prior to the two outbreaks from the same areas or provinces neighbouring with Germany where SBV cases had previously been detected gave negative results for the presence of SBV or specific antibodies. These cases are the first ones detected in cattle in Poland and provide evidence of recent transmission of the pathogen into the country and involvement of midge vectors.     

Significant re‐emergence and recirculation of Schmallenberg virus in previously exposed dairy herds in Ireland in 2016

Schmallenberg virus (SBV) circulation was investigated in 25 previously exposed dairy herds in Ireland in 2016. A population of 1,550 spring‐2014‐born animals, which had been monitored for SBV infection in 2014 and 2015 as part of a previous SBV surveillance study, were resampled for evidence of SBV infection during 2016. A total of 366 blood samples were collected in the 25 study herds (15 samples per herd) between 3 March 2017 and 10 March 2017 (before the 2017 vector‐active season) and analysed for SBV antibodies using a competitive ELISA kit (IDVet). A total of 256 animals tested seropositive, an AP of 69.9% (95% CI: 65.1–74.4) and TP of 77.7% (95% CI: 72.3%–82.8%) when correcting for imperfect test characteristics. These results demonstrate that a new epidemic of SBV circulation occurred in these previously exposed herds in Ireland in 2016.     

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.     

Distribution of Schmallenberg Virus and Seroprevalence in Belgian Sheep and Goats

A serological survey to detect Schmallenberg virus (SBV)‐specific antibodies by ELISA was organized in the Belgian sheep population to study the seroprevalence at the end of the epidemic. One thousand eighty‐two sheep samples which were collected from 83 herds all over Belgium between November 2011 and April 2012 were tested. The overall within‐herd seroprevalence and the intraclass correlation coefficient were estimated at 84.31% (95% CI: 84.19–84.43) and 0.34, respectively. The overall between‐herd seroprevalence was 98.03% (95% CI: 97.86–98.18). A spatial cluster analysis identified a cluster of six farms with significantly lower within‐herd seroprevalence in the south of Belgium compared with the rest of the population (P = 0.04). It was shown that seroprevalence was associated to flock density and that the latter explained the presence of the spatial cluster. Additionally, 142 goat samples from eight different herds were tested for SBV‐specific antibodies. The within‐herd seroprevalence in goats was estimated at 40.68% (95% CI: 23.57–60.4%). The results of the current study provided evidence that almost every Belgian sheep herd has been in contact with SBV during 2011 and should be taken into consideration as part of comprehensive SBV surveillance and control strategies.     

Schmallenberg virus detection in Culicoides biting midges in Spain: First laboratory evidence for highly efficient infection of Culicoides of the Obsoletus complex and Culicoides imicola

Since Schmallenberg disease was discovered in 2011, the disease rapidly spread across Europe. Culicoides biting midges have been implicated as putative Schmallenberg vectors in Europe. The detection of Schmallenberg virus (SBV) in field collected Culicoides was evaluated through retrospective (2011–2012) collections and captures performed in 2013. This study represents the first detection of SBV in field collected Culicoides in Spain. Infectious midges were detected at the foothills of Pyrenees, Aramunt, in the summer 2012. All the specimens infected with Schmallenberg were of the species Culicoides obsoletus s.s. confirming its putative vector status in Spain. Experimental infection on field collected Culicoides provided evidence of atypical high efficiency for SBV vector infection and transmission potential in local populations of Culicoides imicola and in Culicoides of the Obsoletus complex. However, captured individuals of C. imicola were more susceptible to SBV infection than C. obsoletus s.l. (p  < .001), with an infection ratio of 0.94 and 0.63, respectively. In contrast, a Culicoides nubeculosus colony appeared to be refractory to SBV infection.     

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.     

Serosurvey of Schmallenberg Virus Infections in Sheep and Goat Flocks in Lower Saxony,Germany

Schmallenberg virus (SBV) infections can cause congenital musculoskeletal and vertebral malformations as well as neurological failures in foetuses of several ruminant species if susceptible mother animals were infected during early gestation. Blood samples gained from 17 goat and 64 sheep flocks in Lower Saxony (LS), Germany (January–May 2012), which is located in the core region of the 2011/2012 epidemic were tested for antibodies against SBV by ELISA to detect past exposure to SBV. A SBV‐specific questionnaire was raised in all flocks. The calculated median within‐herd prevalence was 43.8% (min–max: 5.6–93.3%) for goats and 58.7% (min–max: 6.5–100%) for sheep, showing that small ruminants in LS, especially goats, are still at risk of novel SBV infections in the following lambing seasons as not all animals have seroconverted yet. Statistical analysis revealed that goats have a significantly lower risk of SBV infections than sheep which might be explained by different host preferences of Culicoides ssp. as main vectors for SBV and different housing conditions.     

Serological Screening Suggests Presence of Schmallenberg Virus in Cattle,Sheep and Goat in the Zambezia Province,Mozambique

Schmallenberg virus (SBV) is a novel Orthobunyavirus within the family Bunyaviridae belonging to the Simbu serogroup. Schmallenberg virus infects ruminants and has since its discovery in the autumn 2011 been detected/spread to large parts of Europe. Most bunyaviruses are arboviruses, and SBV has been detected in biting midges in different European countries, suggesting that they may play a role in the transmission of the virus. It is not known how SBV was introduced to Europe and if SBV is present in countries outside of Europe. Thus, in this study, we conducted a serological screening for SBV antibodies in cattle (no. 79), sheep (no. 145) and goat (no. 141) in the Zambezia Province in Mozambique during September 2013. The results show a high percentage of antibody‐positive animals. All farms tested had seropositive animals; cattle displayed the highest prevalence with 100% positive animals. Sheep and goat also displayed high number of positive animals with a 43–97% and 72–100% within‐herd seroprevalence, respectively. This initial serological screening suggests that SBV is present on the African continent. However, cross‐reactivity with other members of the Simbu serogroup cannot be ruled out, and further studies are needed to identify and characterize the virus responsible for the antibody‐positive results.     

Serological Study of Exposure to Selected Arthropod‐Borne Pathogens in European Bison (Bison bonasus) in Poland

Bison bonasus is an indigenous species of Central and Eastern Europe with the largest wild population inhabiting Białowieża Primeval Forest; however, free‐living and captive European bison are reared in many countries around the world. Despite that the European bison was rescued from the extinction after the First World War, it remains as endangered species. Changing environment as well as human activity may have contributed to the observed increase of the risk of the emergence and re‐emergence of pathogens. The aim of the survey was to establish the distribution of four pathogens transmitted by arthropods including three arboviruses [Bluetongue disease virus (BTV), Epizootic haemorrhagic disease virus (EHDV) and Schmallenberg virus (SBV)] and a bacteria (Francisella tularensis) in the main populations of European bison in Poland. A total of 251 European bison originating from eight main populations were included in the study and sampled between February 2011 and December 2014. Serum samples originated from chemically immobilized, eliminated or dead by natural causes animals. Additionally, 65 cervids from Białowieża Forest were tested to compare the seroprevalences of other ruminants inhabiting the same environment. The antibodies to SBV and BTV were found in 76.1% and 24.7% of European bison, respectively. In autumn 2012, simultaneous emergence of SBV and BTV in European bison was observed; however, while SBV has spread in all populations scattered around the country, BTV infections were observed only in the north‐eastern part of Poland, where BTV cases have been previously reported in domestic ruminants. European bison age was found to be the only significant risk factor for SBV and BTV seroprevalences; however, this association was connected to the animal size, rather than to the length of exposure. None of the animals tested positive for antibodies against EHDV or F. tularensis. SBV exposure rate of cervids was much lower (35.4%) than in European bison, while BTV seroprevalence was comparable in both groups.     

Natural Immunity of Sheep and Lambs Against the Schmallenberg Virus Infection

Since the first reports of the Schmallenberg disease (SBD) outbreaks in late 2011, the disease has spread across Europe, affecting cattle and sheep farms. While Schmallenberg virus (SBV) causes a mild clinical disease in adults, infection of pregnant females may lead to the production of typical congenital malformations (CMFs) in their offspring. It is speculated that the immunity acquired after a SBV infection is effective in preventing further infections. However, this has not been proven in naturally infected sheep, especially if they are pregnant when reinfected. The aim of this study was to monitor the natural immunity in SBV‐infected sheep. Twenty‐four ewes from the only Spanish farm with a SBV OIE‐notified outbreak were sampled. Subsequently, nine pregnant ewes were inoculated with SBV infectious plasma under controlled conditions. Six of them were euthanized before delivery, and their fetuses were inspected for lesions indicative for the SBV infection. The three remaining ewes were allowed to deliver one lamb each. Inoculation of the lambs was scheduled at approx. 3 months after birth. All samples were analyzed for viral RNA by RT‐PCR, and for antibodies by an indirect ELISA and a virus neutralization test (VNT). The majority of the 24 ewes showed a serological reaction against SBV. The three ewes that were allowed to lamb down demonstrated variable degrees of seroconversion which corresponded to the levels of immune reaction observed in their lambs. Moreover, no viral RNA was detected, no lesions were observed in the fetuses, and no clinical signs were detected in the inoculated animals. These findings suggest that the immunity acquired by sheep following a natural SBV infection could be sufficient to stop SBV reinfection. However, vaccination could be a valuable tool to control SBV infections and associated economic losses as it affords a more uniform and predictable protection at the flock/herd level.     

Schmallenberg Virus in Culicoides Biting Midges in the Netherlands in 2012

A total of 130 pools of Culicoides biting midges collected between May and September 2012 in the Netherlands were assayed for Schmallenberg virus (SBV). The Culicoides midges were caught in the same area as where in 2011 a high proportion of Culicoides pools tested positive for SBV, in majority with a high viral load (C_t values between 20 and 30). Two of a total of 42 pools comprising 50 midges/pool of the Obsoletus complex from the 2012 collection tested weak positive (C_t values: 34.96 and 37.66), indicating a relatively low viral load. On an individual midge level, the proportion of SBV‐infected Culicoides of the Obsoletus complex caught in the same area and in a comparable period of the year was significantly lower in 2012 (0.1% = 1 per 1050 tested) compared with 2011 (0.56% = 13 per 2300 tested).     

Preliminary Survey on the Impact of Schmallenberg Virus on Sheep Flocks in South of Belgium

Between late February and May 2012, a preliminary anonym survey was conducted among sheep farmers in south of Belgium in order to contribute to future estimations of the economic losses caused by Schmallenberg virus (SBV). Based on clinical signs consistent with SBV infection, this survey involved 13 meat sheep flocks considered as positive flocks with subsequent SBV detection by RT‐qPCR [SBV‐positive flocks (PF); total of 961 animals], and 13 meat sheep flocks considered as negative flocks (NF; total of 331 animals). These preliminary results indicated several significant characteristics that were more present in PF than in NF. These include an increased rate of abortions (6.7% in PF versus 3.2% in NF), of lambs born at term but presenting malformations (10.1% in PF versus 2.0% in NF) and of dystocia (10.1% in PF versus 3.4% in NF). Lamb mortality during the first week of life was reported more frequently in PF (8 of 13 PF, 61.5%) than in NF (1 of 13 NF, 7.7%). In PF, the observed prolificacy rate was 2‐fold lower (93%) than expected (186%). The implementation of a survey at larger scale, including a high number of breeders, is necessary to allow a more detailed analysis of the SBV impact in the sheep sector.     

Serosurvey of Schmallenberg Virus Infection in the Highest Goat‐Specialized Region of France

The monitoring of both the spread and clinical impact of Schmallenberg virus (SBV) infection within its full host range is important for the control of the epidemic and potential new outbreaks. In France, a national surveillance plan based on voluntary notifications of congenital malformations in newborn ruminants revealed that goats were the less affected host species. However, seroprevalence studies only targeted sheep and cattle, preventing accurate estimations of the real impact of SBV infection in goats. Here, a serological survey was conducted in the highest goat‐specialized region of France between June 2012 and January 2013. A total of 1490 goat sera from 50 herds were analysed by ELISA. The between‐herd and within‐herd prevalences were estimated at 62% and 13.1%, respectively. Seroprevalence was not uniformly distributed throughout the territory and markedly differed between intensive and extensive herds. The low within‐herd seroprevalence demonstrates that a large fraction of the French goat population remains susceptible to SBV infection.