Schmallenberg virus (SBV) is an orthobunyavirus that primarily infects domestic and wild ruminants, in which it can cause symptoms such as transient fever, diarrhea, reduced milk production, congenital malformations, and abortions [1,2,3,4,5,6,7]. Although most infections probably remain subclinical, and while there is no evidence that SBV may be zoonotic, the economic impact of an outbreak can be significant due to reduced productivity, animal losses, veterinary costs, and trade restrictions [6, 8]. Like other orthobunyaviruses, it is a vector-borne virus and has been shown to be transmitted by Culicoides spp. SBV-associated disease is considered an emerging disease and was first discovered in Germany in 2011 during an outbreak that swept through large parts of Europe in two waves during the 2011 and 2012 vector seasons [1, 9].

SBV-associated cases first appeared in the Dutch/German border region, but their exact origins are unclear [1, 9, 10]. SBV is closely related to other orthobunyaviruses in the Simbu serogroup, such as Akabane virus. Akabane virus has previously been found in Africa, Asia, Australia, and the Middle East and it has been suggested, that SBV may have come to Europe on a ship with infected host or vector animals [1]. Antibodies putatively targeting SBV itself have more recently also been found in Africa, Asia (China), and the Middle East (Lebanon) [11,12,13,14].

There have been smaller outbreaks in Europe since 2012, but none of the magnitude of the original ones, and this is likely due to seroconversion of large parts of the animal population [9, 15, 16]. There is evidence, though, that the virus has since become endemic in areas where conditions are favorable for the vectors and where wild ruminants can act as a reservoir [17].

In 2012, there was an unexpected increase in the number of abortions in cattle and sheep in Azerbaijan that was unrelated to infections with Brucella or Chlamydia. Due to the similarities of the overall symptomology observed, SBV, which at that time, had just recently been described, was suspected as a potential cause. This surveillance study was therefore launched to determine if SBV had made it to Azerbaijan and to monitor the situation.

Following the first confirmed cases in October 2012, every Rayonal Veterinary Office (RVO) in Azerbaijan was notified of the outbreak. Subsequently, the RVOs instructed field veterinarians across the country to report any suspected cases and to collect samples to be sent to the Republican Veterinary Laboratory for confirmatory testing. The field veterinarians were responsible for collecting biological specimens from suspected cases. The materials collected included serum and head and neck biopsies of aborted fetuses as well as serum from other affected animals, and these were sent to the Virology Department at the Republican Veterinary Laboratory in Baku for testing. All samples were directly stored and kept at -80 °C without any medium until further processing.

For all tissue samples, an approximately 0.5- to 1-cm2 section of each internal organ biopsy was cut into small pieces using sterile scissors and pooled with other organs from the same animal (up to 5 g total). Samples of the skin or of pooled internal organs were homogenized using a mortar and pestle in 8 mL of 1X PBS. Once homogenized, the slurry was decanted into 15-mL conical tubes and centrifuged at 8,000 rpm for ten minutes. RNA was extracted from 140-µL aliquots of the resulting supernatant using a QIAGEN RNA Mini Kit & Tissue Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions.

To detect viral RNA in the extracts of skin or pooled organ samples of individual animals, a commercially available reverse transcription quantitative PCR (RT-qPCR) assay was used to rapidly diagnose cases. The assay used was the LSI VetMAX™ Schmallenberg Virus - S Gene - TaqMan™ RT-qPCR for the detection of SBV and was used according to the manufacturer’s recommendations with the provided controls. Tests were run using a RotorGene 6000 instrument (QIAGEN, Germany) until 2016, and afterwards using a Bio-Rad CF96 system (Bio-Rad Laboratories, USA). To screen for the presence of antibodies against SBV, the IDEXX Schmallenberg Ab TestTM was used to test sera of individual animals in accordance with the manufacturer’s protocol.

Throughout the study period, several thousand cattle, sheep and goats with suspected infection were tested each year for SBV RNA or antibodies against the virus (Supplement). The first cases of confirmed SBV infections were detected in October 2012, and by the end of that year, 1301 animals (552 cattle, 749 sheep) with antibodies against the virus had been identified in Azerbaijan (Fig. 1A and Supplement). All local cases in these first months were in the south of the country in the rayons Beylaqan, Imishli, Sabirabad and Neftchala, while the animals found with antibodies in the rayons Sumqait (east) and Gandja (west) were imported (Fig. 2). Local cases in the north (Agstafa and Siyazan) were first detected in March and April of 2013. By February 2014, 2696 additional local and imported animals (cattle and sheep) had been reported to have antibodies against SBV, with reports coming from many parts of the country (Fig. 2, Supplement). In the year 2013, viral RNA was detected in 22% of tested cattle, and the antibody prevalence reached 40% (Fig. 1A and B). Animals with antibodies were also detected in the following years, but with a much lower prevalence than between 2012 and 2014 (Figures 1A, 2 and Supplement). Since 2014, no more RT-qPCR-positive cases were found, despite ELISA-positive samples still being detected and a continuing large number of new suspected cases (Fig. 1B). Between the first cases in October 2012 and January 2018, cattle, sheep or goats with antibodies against SBV were found in 42 rayons in Azerbaijan (Fig. 2). The majority of cases were seen in fall and winter (October to February), with only a few or none occurring between May and July (Supplement).

Fig. 1
figure 1

Diagrams showing evidence for SBV per calendar year. Panel A shows the percentage of tested animals that had antibodies against SBV, panel B shows the percentage of tested animals that were positive for SBV RNA, and panel C shows the total numbers of suspected cases over the study period

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Fig. 2
figure 2

A series of maps of Azerbaijan showing the confirmed cases of SBV infection by rayon between July and June. Rayons depicted in blue reported cases in animals with and without import history, while rayons in red reported only cases in imported animals during the same time period. Numbers in or adjacent to colored rayons indicate the calendar month of the first reported case within the corresponding timeframe. The figure was generated using PAINTMAPS.com

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The data capture the Schmallenberg situation in Azerbaijan from late 2012 to early 2018. While the ELISA kit that was used for antibody detection does not distinguish between SBV itself and other Simbu-group viruses, the RT-qPCR results indicate the presence of SBV. According to the manufacturer, the RT-qPCR is specific for SBV, since the primers and probe were selected to bind to areas of the genome that differ from the corresponding regions of Akabane, Simbu and Shamonda viruses due to several mismatches. Since there is also no evidence of any other Simbu-serogroup virus circulating in any of the neighboring countries, the broader region, or Europe at the time of the study, we assume that SBV contributed to the majority, if not all, of the cases that were considered antibody positive.

Surveillance was initiated in October 2012 as a reaction to increasing numbers of abortions in animals that were negative for Brucella or Chlamydia. Thus, it seems that SBV reached Azerbaijan about a year after its first appearance in Europe in 2011. Since all of the tested material was obtained from suspected (symptomatic) cases, we expect that the virus came into the country during the vector season of 2012, leading to the observed initial disease outbreak with a couple of months’ delay. The very first cases likely occurred before October in smaller numbers and were missed [18]. Since most of the initial cases were detected in the south of Azerbaijan in local sheep and cattle, we assume that the virus might have come into the country with infected wild hosts such as gazelles or directly with the Culicoides vector species from Iran. The latter seems more likely, since vectors have been proposed to be the main route of SBV spread in Europe [18, 19]. However, there are no data regarding SBV presence or testing in Iran from that time. The first report of SBV in Iran was based on samples collected between July 2014 and September 2015 from horses in the northeast of the country. The prevalence at that point was determined to be between 5% and 7%, which is very similar to what we detected in Azerbaijan at the same time [20]. An alternative scenario would be a direct importation of viremic animals from Europe. However, it seems that the spread of virus was stalled by the onset of winter so that large parts of the country were initially not affected. As a consequence, there were still plenty of naïve animals to enable the larger 2013/2014 outbreak that affected most of Azerbaijan. This second wave started in the southern and central rayons, with the first confirmed cases being detected in August (Fig. 2). It is unclear if the virus managed to survive the winter in Azerbaijan and/or beyond the southern border or if it was reintroduced early in the vector season and then swept through the country quickly. The south of Azerbaijan has a subtropical climate, with average temperatures higher than those in northern and central Europe, and at the same time, it has more precipitation year-round than southern Europe. Those factors would favor a longer season or even uninterrupted activity of Culicoides vectors, which, in most parts of Europe, have a season ranging roughly from April to October [21]. While some of the cases during the second wave were imports, the majority of confirmed animals had never left the country.

It is likely that the vast majority of susceptible livestock and wildlife in Azerbaijan got infected with SBV between 2012 and 2014 and seroconverted, which in turn explains the relative abrupt end of the outbreak. We found only up to about 40% of cattle and 24% of sheep and goats to be seropositive (Fig. 1A), but it has been shown that testing symptomatic animals does not accurately reflect the seroprevalence in the general animal population, but rather yields an estimate that is too low [18]. After 2014, antibodies against SBV were almost exclusively detected in cattle, and the prevalence in suspected cases was decreasing (Fig. 1A). This could indicate a low level of virus circulation or constant reintroduction, but it most likely reflects positive test results in animals that had contact with the virus during the outbreaks between 2012 and 2014. The loss of clear seasonality among the antibody-positive cases seems to support this interpretation. Despite the detection of antibodies in a portion of the suspicious cases, no viral RNA has been found in any of the tested samples since February 2014. Although only a fraction of the suspected cases are checked this way, this is further evidence that SBV probably has not become endemic in Azerbaijan and/or is not circulating to a significant degree.

Since the outbreaks in Azerbaijan happened one year and two years after the first documented European outbreak and seemed to have come from the south, the origin of the virus remains an interesting question. The virus strain originally detected in northern Europe may have been imported directly or could have made its way along the Mediterranean towards the Middle East. It is also possible that the outbreak in Azerbaijan was predated by an independent introduction of SBV from its original source into the Middle East. Sequencing of the virus strain responsible for the outbreaks in Azerbaijan would be desirable but could not be realized within the boundaries of this study.

Despite the decline in cases positive for viral RNA or antibodies against the virus, we do see a constantly high and even increasing number of suspected cases, especially in cattle (Fig. 1C). Our data indicates that most of these are unlikely to actually be SBV related, and we believe that the high numbers could be explained largely by two factors: first, sensitization to the symptomology and increased awareness coupled with the availability of testing for SBV, and second, an increase in other diseases with similar or partly similar symptomology, such as brucellosis (data not shown).

We conclude that SBV was introduced into a naïve livestock population in Azerbaijan in 2012 and caused two outbreak waves in 2012/2013 and 2013/2014. The virus does not seem to be circulating at this point, but in the absence of a vaccination program and with post-outbreak herd immunity vanishing, reintroduction or re-emergence seems possible in the future.