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.     

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.     

Resurgence of Schmallenberg Virus in Belgium after 3 Years of Epidemiological Silence

In spring 2016, three years after the last reported outbreak of Schmallenberg virus (SBV) in Belgium, an abortion was notified in a two year old Holstein heifer that previously had not been vaccinated against SBV. The autopsy of the eight‐month‐old malformed foetus revealed hydrocephalus, torticollis and arthrogryposis. Foetal brain tissue and blood were found to be SBV‐positive by RT‐PCR and ELISA tests, respectively. Evidencing the circulation of SBV in Belgium in the autumn 2015 is important to anticipate future outbreaks and advise veterinarians about the risks associated with calving, as more bovine foetuses might have been infected.     

First Confirmation of Schmallenberg Virus in Cattle in Spain: Tissue Distribution and Pathology

Between January and June 2013, nine stillborn bovine foetuses with congenital malformations from nine cattle herds located in Salamanca (central Spain) were detected. Necropsy was performed on two calves. Pathological lesions together with molecular genetics and serological results allowed a definitive diagnosis: first confirmation of Schmallenberg virus (SBV) infection in cattle in Spain. SBV was detected in different tissues and organic fluids in both animals including blood, suggesting a possible viraemia. The umbilical cord was also positive for the presence of SBV in both animals. The former tissue provides an easy to obtain sample and might be a sample of choice when necropsy is carried out in the field.     

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.     

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 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.     

Molecular characterization of Peste des petits ruminants viruses in the Marmara Region of Turkey

Recent outbreaks of Peste des petits ruminants (PPR) in the Marmara region of Turkey including the European part of Thrace is important due to its proximity to Europe (Greece and Bulgaria) and the potential threat of spread of PPR into mainland Europe. In order to investigate the circulation of PPRV in the region suspect clinical and necropsy samples were collected from domestic sheep (n = 211) in the Marmara region of Turkey between 2011 and 2012. PPR virus (PPRV) genome was detected in 10.4% (22 out of 211) of sheep samples by real‐time RT‐PCR, and PPR virus was isolated from lungs of two sheep that died from infection. Of the 22 positive samples nine were used for partial N‐gene amplification and sequencing. The phylogenetic analyses indicated that the virus belongs to lineage IV, the same lineage that is circulating in eastern and central part of Turkey since its first official report in 1999. In addition, samples from 100 cattle were collected to investigate potential subclinical circulation of PPRV. However all were found to be negative by real‐time RT‐PCR, and also in serological tests indicating the large ruminants were likely not exposed or infected with the virus. The impact of these findings on the potential threat of spread of PPR to Europe including the first PPR outbreak in Europe in Bulgaria on 23rd June 2018 is discussed.     

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.     

Bluetongue Virus RNA Detection by Real‐Time RT‐PCR in Post‐Vaccination Samples from Cattle

Bluetongue virus serotype 8 (BTV‐8) was responsible for a large outbreak among European ruminant populations in 2006–2009. In spring 2008, a massive vaccination campaign was undertaken, leading to the progressive disappearance of the virus. During surveillance programmes in Western Europe in 2010–2011, a low but significant number of animals were found weakly positive using BTV‐specific real‐time RT‐PCR, raising questions about a possible low level of virus circulation. An interference of the BTV‐8 inactivated vaccine on the result of the real‐time RT‐PCR was also hypothesized. Several studies specifically addressed the potential association between a recent vaccination and BTV‐8 RNA detection in the blood of sheep. Results were contradictory and cattles were not investigated. To enlighten this point, a large study was performed to determine the risks of detection of bluetongue vaccine‐associated RNA in the blood and spleen of cattle using real‐time RT‐PCR. Overall, the results presented clearly demonstrate that vaccine viral RNA can reach the blood circulation in sufficient amounts to be detected by real‐time RT‐PCR in cattle. This BTV‐8 vaccine RNA carriage appears as short lasting.     

Schmallenberg Virus Antibodies Detected in Poland

Between 24 and 30 July 2012 230 adult goats from three western provinces of Poland bordering on Germany (Western Pomerania, Lubuskie and Lower Silesia) were blood‐sampled and tested for antibodies to Schmallenberg virus (SBV) using indirect immunoenzymatic test (ID Screen® Schmallenberg virus indirect, IDvet Innovative Diagnostics). The ELISA test identified 21 seropositive goats – 15 in Western Pomerania (16% of all goats tested in this province), five in Lubuskie (6%) and one in Lower Silesia (2%). Our study demonstrates for the first time the presence of antibodies to SBV in Poland.     

Detection of Schmallenberg virus in different Culicoides spp. by real‐time RT‐PCR

To identify possible vectors of Schmallenberg virus (SBV), we tested pools containing heads of biting midges (Culicoides) that were caught during the summer and early autumn of 2011 at several places in Belgium by real‐time RT‐PCR. Pools of heads originating from following species: C. obsoletus complex, C. dewulfi and C. chiopterus were found positive, strongly indicating that these species are relevant vectors for SBV.     

Development of universal and quadruplex real‐time RT‐PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses

Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA‐PRRSV2), highly pathogenic (HP‐PRRSV2) and NADC30‐like (NL‐PRRSV2)) are currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real‐time RT‐PCR assays using TaqMan‐MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real‐time RT‐PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real‐time RT‐PCR assays were further validated by comparing with a universal PRRSV conventional RT‐PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real‐time RT‐PCR assays and the conventional RT‐PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV‐positive, including 2 samples that were determined to be co‐infected with NL‐PRRSV2 and HP‐PRRSV2 isolates by the quadruplex real‐time RT‐PCR assay. ORF5 sequencing confirmed the real‐time RT‐PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA‐PRRSV2, NL‐PRRSV2 and HP‐PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.     

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.     

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.     

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.     

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 and molecular epidemiology of foot‐and‐mouth disease viruses in agro‐pastoralist livestock herds in the kachia grazing reserve,Nigeria

The Kachia Grazing Reserve (KGR) is located in Kaduna state in north‐western Nigeria and consists of 6 contiguous blocks housing 744 defined households (HH), all engaged in livestock keeping. It is considered as a homogenous epidemiological unit and a defined study area. In 2012, all cattle and sheep of 40 selected HH were sampled to determine sero‐prevalence of antibodies to foot‐and‐mouth disease virus (FMDV) and of FMDV. The overall sero‐prevalence of antibodies to the non‐structural 3ABC protein (NSP‐3ABC ELISA) was 28.9% (380/1,315) (30.6% cattle; 16.3% sheep), and in 4.5% (62/1,380) (5% cattle; 0.6% sheep) of the examined sera FMD viral RNA could be detected by real‐time RT‐PCR (rRT‐PCR). Additionally, in 2012 and 2014 serum, epithelium and probang samples were collected from cattle in reported FMD outbreaks and the causative FMDVs were molecularly characterized. Approximately half (28/59) of the outbreak sera reacted positive in NSP‐3ABC ELISA, and 88% (52/59) of the outbreak sera contained detectable viral RNA. Overall, antibodies against five FMDV serotypes (O, A, SAT1, SAT2 and SAT3) were detected by solid phase competitive ELISA with combinations of two or more serotypes being common. Of the 21 FMDVs that could be isolated 19 were sequenced and 18 were confirmed as SAT2 (lineage VII) while one was characterized as serotype O (EA‐3 topotype). Phylogenetic analysis revealed a close relationship between Nigerian FMDV strains and strains in this region and even with strains in North‐Africa. Our findings indicate that FMD constitutes an endemic health problem to cattle rearing in the agro‐pastoralist community in the KGR and that the KGR is not a closed epidemiological unit. Insight into the local FMDV epidemiology and in the circulating FMDV serotypes/strains is of support to the relevant authorities in Nigeria when considering the need for an FMD control policy to improve animal production in grazing reserves.     

Outbreak investigation and molecular diagnosis of Lumpy skin disease among livestock in Saudi Arabia 2016

Lumpy skin disease (LSD ) is a highly infectious disease of cattle caused by a virus of the Capripoxvirus genus in the family Poxviridae . The disease is a major concern for the dairy industry in Saudi Arabia. In this study, an outbreak of LSD in cattle herds was detected in Saudi Arabia in 2016. LSD outbreak was investigated in five regions of Saudi Arabia: Al‐Hassa, Al‐Sharqia, Al‐Qassim, Riyadh and Al‐Taif during the period from April to July 2016. Tissues from skin nodules were collected to characterize the virus by a real‐time polymerase chain reaction (rt‐PCR ). During this period, 64,109 cattle were examined and morbidity, mortality and case fatality rates were 6%, 0.99% and 16.6%, respectively. The analysis showed 3,852 infected cases and 641 deaths. highest number of infected animals was reported in Al‐Hassa (2,825), followed by Al‐Qassim (547), Riyadh (471), Al‐Sharqia (6) and Al‐Taif (3). The highest morbidity rates were observed in Al‐Qassim (6.8%), Al‐Hassa (6.2%), Riyadh (5.5%) and Al‐Taif (0.96%), while the lowest morbidity rates were recorded in Al‐Sharqia (0.27%). The highest mortality rates were also observed in Al‐Qassim (2.3%), followed by Al‐Hassa (0.97%), Riyadh (0.19%) and lowest in Al‐Sharqia and Taif (0%). LSD virus was detected in all samples (n  = 191) by real‐time PCR analysis. The disease has been observed in the cattle regardless of previous vaccination using the locally Romanian‐pox vaccine; therefore, vaccination programme and vaccine efficacy should be assessed under field conditions.     

Schmallenberg Virus Infection Diagnosis: Results of a German Proficiency Trial

Since Schmallenberg virus (SBV), an orthobunyavirus of the Simbu serogroup, was detected in Central Europe in 2011 for the first time, numerous diagnostic test systems for genome or antibody detection have been established. Therefore, a laboratory proficiency trial with 28 veterinary laboratories was initiated to allow performance evaluations of the different veterinary diagnostic laboratories and the performance of the used assays. A panel of selected sera and bovine semen samples for the analysis by real‐time PCR and an additional set of serum samples for serological analysis were provided. All participants were asked to investigate the samples with the test systems routinely used in their laboratory. While SBV‐genome was reliably detected in serum samples, the sensitivity in semen samples seems to depend on the application of the recommended optimized nucleic acid extraction method (TRIzol^® LS Reagent‐based, Hoffmann et al., 2013, Vet. Microbiol., 167, 289). SBV‐antibody‐positive samples and sera negative for antibodies against Simbu serogroup viruses were in most cases correctly classified by the participants with the used commercial ELISA kits. However, a serum of the panel which contained antibodies against Akabane and Aino viruses, which are closely related to SBV, was repeatedly tested positive by two of four used ELISA kits. However, an excellent diagnostic sensitivity and specificity was achieved using a serum neutralization test. In conclusion, the here described German SBV proficiency test demonstrated that the available test systems allowed reliable SBV diagnostics in standard veterinary laboratories when recommended and approved assays are used.