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.     

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.     

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.     

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.     

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.     

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.     

A wild circulation: High presence of Porcine circovirus 3 in different mammalian wild hosts and ticks

Porcine circovirus 3 (PCV‐3) has emerged as a potential threat for swine industry, being consistently reported in the presence of several clinical signs all around the world. Recently, its presence in wild boar has been demonstrated at high prevalence. This evidence is surprising since the lower density of wild populations might not be expected to sustain such efficient viral transmission. Porcine circoviruses were proven to exhibit a certain plasticity in the host tropism and were detected in unrelated species, like mice, dogs and ruminants. However, if this scenario applies also to wild animals remains to be established. Therefore, this study aimed to investigate the presence of PCV‐3 in wild ungulates other than wild boar and in related hematophagous ectoparasites. One hundred and nine animals were sampled from different hilly and mountain areas of Friuli Venezia Giulia, including 9 chamois (Rupicapra rupicapra), 17 red deer (Cervus elaphus), 4 mouflons (Ovis musimon), 50 roe deer (Capreolus capreolus) and 29 wild boars (Sus scrofa). Additionally, host‐matched ectoparasites were collected when present. Porcine circovirus 3 was diagnosed using molecular techniques and sequencing. This study results confirmed the high PCV‐3 occurrence in wild boar and reported for the first time its presence, at low prevalence, in chamois and roe deer. Moreover, two ticks (Ixodes ricinus), one of which non‐engorged, collected from PCV‐3 negative roe deer, tested PCV‐3 positive. The genetic characterization of some of the strains collected from non‐swine hosts allowed to prove that, albeit clearly part of PCV‐3 species, they were genetically unique, demonstrating the absence of among‐samples contamination and thus confirming the actual presence of PCV‐3 genome in these new hosts. Therefore, this study highlights an unexpected broad PCV‐3 distribution and circulation in the wild, rising further questions on porcine circoviruses infectious cycle, epidemiology and origin, which will deserve additional investigations.     

Evidence of extensive renewed Schmallenberg virus circulation in Belgium during summer of 2016 – increase in arthrogryposis‐hydranencephaly cases expected

A seroprevalence study carried out between June and September 2016 in the Belgian sheep population showed a significant increase in overall (from 25% to 62%) and between‐herd (from 60% to 96%) seroprevalence against Schmallenberg virus (SBV) during this period, indicating the most extensive recirculation of SBV since its original emergence in 2011. SBV recirculation was confirmed by the detection of SBV RNA‐positive Culicoides obsoletus complex midges collected in the region of Antwerp in August 2016, reaching a minimum infection rate of 3%. The recirculation of SBV in the largely unprotected ruminant population during summer 2016 will likely cause an increase in the number of arthrogryposis‐hydranencephaly cases in newborn ruminants during the coming months.     

Bluetongue Dynamics in French Wildlife: Exploring the Driving Forces

Bluetongue (BT) was monitored in wildlife in France during two consecutive years corresponding to contrasting incidence rates in livestock: in 2008 at the peak of domestic outbreaks and in 2009 when very few outbreaks were observed. The disease status of 2 798 ruminants comprising 837 red deer (Cervus elaphus) was explored using ELISA test on serum and real‐time RT‐PCR test on blood or spleen. A large proportion of red deer were seropositive and positive to RT‐PCR in 2008, but also in 2009 (seroprevalence: 47.1% and 24.3%), suggesting that red deer could maintain infection when domestic incidence was negligible. By contrast, low seroprevalence (<3%) and few RT‐PCR positive results were observed in other wild ruminant species, which rather appeared thus as dead‐end hosts. The risk factors of bluetongue circulation during the periods of high (2008) and low (2009) domestic incidence were explored in red deer using logistic mixed models. In this species, prevalence has been mainly influenced by the initial peak of BT in livestock, but also by environmental factor such as elevation and edge density between forest and pastures. Surprisingly, cattle density has a negative influence on prevalence in red deer, possibly due to the protective effect of cattle regarding midges' bites and/or to still unexplained factors dealing with the host/midge interface. To our knowledge, this study is the first attempt at measuring the effect of landscape and wildlife/domestic interface on BT prevalence in wildlife in Europe.     

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.     

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.     

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 (IDV et) 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 .     

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.     

Schmallenberg Virus beyond Latitude 65°N

Extensive and rapid spread of Schmallenberg virus (SBV) in Sweden was detected by consecutive serological bulk milk surveys conducted before and after the vector season of 2012. Whereas <0.2% of cattle herds tested positive in a first survey in spring 2012, SBV‐specific antibodies were detected in almost 75% of 723 bulk milk samples randomly collected all over the country 6 months later, beyond the 65th northern latitude, and with an observed spatial distribution suggesting multiple introductions of the virus. Circulation of virus was later confirmed by the detection of SBV in malformed lambs and calves starting from November 2012 and January 2013, respectively. These observations suggest SBV circulation starting from July 2012, with a peak in transmission between August and October. A local heterogeneity of within‐herd seroprevalence was found, indicating that SBV‐naïve animals remain also in highly infected areas enabling the re‐emergence of the infection in the coming vector season.     

Spatial–temporal Trends and Factors Associated with the Bluetongue Virus Seropositivity in Large Game Hunting Areas from Southern Spain

An epidemiological study was carried out to determine the spatial–temporal trends and risk factors potentially involved in the seropositivity to bluetongue virus (BTV) in hunting areas with presence of red deer (Cervus elaphus). A total of 60 of 98 (61.2%; CI_95%: 51.6–70.9) hunting areas sampled presented at least one seropositive red deer. Antibodies against BTV were detected in juvenile animals during the hunting seasons 2007/2008 to 2013/2014 in 15 of 98 (15.3%) hunting areas, which indicates an uninterrupted circulation of BTV in this period. A multivariate logistic regression model showed that the red deer density at hunting area level (>22 individuals/km²), the annual abundance of Culicoides imicola (>1.4 mosquitoes/sampling) and the goat density at municipality level (>24.1 individuals/km²) were factors significantly associated with BTV seropositivity in hunting areas. Control measures against BTV in the studied area include vaccination programmes in wild and domestic ruminants, movement control in areas with high densities and abundance of red deer and C. imicola, respectively. Considering the potential risk of BTV re‐emergence, red deer should be included in the BT surveillance programmes in regions where these species share habitats with livestock.     

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.     

Detection of bovine coronavirus in nasal swab of non‐captive wild water deer,Korea

Bovine coronavirus (BCoV) is a causative agent of respiratory and enteric diseases in cattle and calves. BCoV infection was also evident in captive wild ruminants. Recently, water deer are recognized as the most common wildlife to approach farmhouses and livestock barns in Korea. Therefore, we investigated 77 nasal swab samples from non‐captive wild water deer (Hydropotes inermis ) between November 2016 and September 2017 and identified three samples positive for coronavirus, indicating potential for respiratory shedding. The full genomic sequences of the water deer coronavirus were closely related to BCoV (>98%). Therefore, effective biosecurity system in bovine farms would be necessary to prevent contact between farm ruminants and free‐ranging wild water deer.     

Rift Valley Fever in Kruger National Park: Do Buffalo Play a Role in the Inter‐Epidemic Circulation of Virus?

Rift Valley fever (RVF) is a zoonotic mosquito‐borne virus disease of livestock and wild ruminants that has been identified as a risk for international spread. Typically, the disease occurs in geographically limited outbreaks associated with high rainfall events and can cause massive losses of livestock. It is unclear how RVF virus persists during inter‐epidemic periods but cryptic cycling of the virus in wildlife populations may play a role. We investigated the role that free‐living African buffalo (Syncerus caffer caffer) might play in inter‐epidemic circulation of the virus and looked for geographic, age and sex patterns of Rift Valley fever virus (RVFV) infection in African buffalo. Buffalo serum samples were collected (n = 1615) in Kruger National Park (KNP), South Africa, during a period of 1996–2007 and tested for antibodies to RVF. We found that older animals were more likely to be seropositive for anti‐RVFV antibody than younger animals, but sex was not correlated with the likelihood of being anti‐RVFV antibody positive. We also found geographic variation within KNP; herds in the south were more likely to have acquired anti‐RVFV antibody than herds farther north – which could be driven by host or vector ecology. In all years of the study between 1996 and 2007, we found young buffalo (under 2 years of age) that were seropositive for anti‐RVFV antibody, with prevalence ranging between 0 and 27% each year, indicating probable circulation. In addition, we also conducted a 4‐year longitudinal study on 227 initially RVFV seronegative buffalo to look for evidence of seroconversion outside known RVF outbreaks within our study period (2008–2012). In the longitudinal study, we found five individuals that seroconverted from anti‐RVFV antibody negative to anti‐RVFV antibody positive, outside of any detected outbreak. Overall, our results provide evidence of long‐term undetected circulation of RVFV in the buffalo population.     

HoBi‐like is the most prevalent ruminant pestivirus in Northeastern Brazil

The ruminant pestiviral species BVDV ‐1, BVDV ‐2 and BDV , along with the putative species HoBi‐like, may cause substantial economic losses in cattle, sheep and goats. Brazil's large size, variable biomes and wide range of ruminant animal production within different geographic regions suggest that the presence and prevalence of ruminant pestivirus may differ by regions within Brazil. This study investigated the genetic diversity of ruminant pestiviruses and determined the frequency of active infections within two states of the Northeast Region of Brazil, Maranhão and Rio Grande do Norte. Serum samples from 16,621 cattle and 2,672 small ruminants from 569 different herds residing in this region were tested by RT ‐PCR followed by DNA sequencing. Seventeen positive cattle were detected (0.1%) from fifteen different herds (2.64%). All isolates were classified as HoBi‐like pestiviruses based on phylogenetic analysis. All small ruminant samples tested negative. The findings presented herein suggest that the Northeast Region of Brazil has a uniquely high prevalence of HoBi‐like viruses. The increasing reports of HoBi‐like viruses detected in cattle in the field suggest that natural infection with these viruses may be more widespread than previously thought. The identification of HoBi‐like viruses as the most prevalent type of ruminant pestivirus circulating in the Northeast Region of Brazil indicates the need for both continued monitoring and determination of the extent of economic losses associated with HoBi‐like virus infections. In addition, it must be taken into account in the choice of diagnostic tests and in vaccine formulations.     

Monitoring of the West Nile Virus epidemic in Spain between 2010 and 2011

West Nile virus (WNV) is a mosquito‐transmitted flavivirus recognized as an emerging and re‐emerging pathogen in different countries. This study describes the monitoring of the first WNV epidemic in Spain between 2010 and 2011. Between September and December 2010, 36 outbreaks of WNV in horses were reported in three different provinces of Andalusia (southern Spain), with no apparent spread outside this area. The temporal distribution and the clinical signs observed during the WNV epidemic in Spain were, in general, similar to those reported in Europe and in the Mediterranean Basin. Morbidity, mortality and fatality rate in the affected herds were 4.6, 1.4 and 35.3%, respectively. Thirty‐six of 75 (47.4%) suspected herds investigated presented at least one IgM seropositive animal. The individual seroprevalence in unvaccinated animals from the infected holdings was 51.7%. RNA WNV lineage 1 virus was confirmed from blood and cerebrospinal fluid samples in a lethally infected horse. The entomological survey showed that the most abundant mosquito species detected in the affected area was Culex pipiens. A cross‐sectional study was carried out in non‐suspected herds between April 2010 and February 2011 in the affected area. The individual seroprevalence was 11.0%, and six of the 38 herds sampled (15.8%) presented at least one seropositive animal. The results showed active WNV circulation several months before the first outbreak was reported in horses. The seropositivity found in municipalities where clinical cases were not reported indicates a higher geographical dissemination of the virus. Significantly higher seroprevalences were detected in areas close to Morocco. Furthermore, 90 wild ruminants were tested for the presence of antibodies against WNV, but the results were all negative.