SARS-CoV-2 Infection in Captive Hippos (Hippopotamus amphibius), Belgium.

Two adult female hippos in Zoo Antwerp who were naturally infected with SARS-CoV-2 showed nasal discharge for a few days. Virus was detected by immunocytochemistry and PCR in nasal swab samples and by PCR in faeces and pool water. Serology was also positive. No treatment was necessary.

Seroprevalence of pestivirus infections is low in Belgian small ruminant flocks and is significantly associated with the presence of cattle.

A study was implemented to estimate the pestivirus seroprevalence in sheep and goats in Belgium, to identify circulating species and to check for a potential association between seropositivity of small ruminants and presence of cattle in the same farm. It was based on the testing of serum samples and bulk tank milk samples (BTM) collected in sheep and goat flocks in 2018-2019 all over the country. 7460 serum samples collected from 410 flocks were tested by a commercial ELISA able to detect antibodies (Ab) against Border Disease Virus (BDV), and Bovine Viral Diarrhea Virus (BVDV). BTM samples (n = 144) were collected from dairy flocks in November 2019 and tested with the same Ab ELISA. ELISA positive serum samples were also tested by virus neutralization test (VNT) for neutralizing antibodies against BDV, BVDV-type1 and BVDV-type2. Virological tests (RT-PCR) were performed on pools of serum samples from pestivirus-exposed flocks with at least two seropositive animals and on all Antibody-positive BTM samples. Information about serum and milk samples (identification, test results, farm of origin and location, presence of cattle) were gathered in animal-level and farm-level databases. Based on this study, the apparent animal seroprevalence for pestiviruses in small ruminant flocks in Belgium in 2018 was estimated to be 0.87 % (95 % C.I. [0.68 %-1.11 %]). The prevalence of flocks exposed to pestivirus (i.e. with at least one seropositive animal) was estimated to be 8.5 % (95 % C.I. [6.4 % - 11.6 %]). In exposed flocks, the average within-flock seroprevalence was 9.9 %. In dairy sheep and goats, the estimated proportion of exposed flocks in 2019, based on the detection of pestivirus antibodies in the bulk tank milk, was 9.7 % [5.9 %-15.7 %]. All PCR tests were negative, indicating the likely absence of active pestivirus circulation in these flocks. Although the observed pestivirus seroprevalence was found to be low in Belgian small ruminants, this study also showed, based on VNT results, that they are exposed to several pestivirus species: BDV, BVDV-1 and BVDV-2. 22.4 % of the farms included in the serological survey were holding both a small ruminant flock and a cattle herd, hence with a potential risk of contact between the two species. There was a significant positive association between pestivirus seropositivity in the sheep/goat flocks and the presence of a cattle herd in the same farm (OR = 2.42 (95 %C.I. [1.18-4.94]) but this association was not found for Ab-positive BTM in dairy flocks.

Semi-quantitative risk assessment by expert elicitation of potential introduction routes of African swine fever from wild reservoir to domestic pig industry and subsequent spread during the Belgian outbreak (2018-2019).

Since the introduction in Georgia in 2007 of an African swine fever (ASF) genotype 2 virus strain, the virus has rapidly spread to both Western European and Asian countries. It now constitutes a major threat for the global swine industry. The ongoing European transmission cycle has been related to the 'wild boar habitat' with closed transmission events between wild boar populations and incidental spillovers to commercial and non-commercial (backyard) pig holdings. During the epidemic in Belgium, only wild boar were infected and although the introduction route has not yet been elucidated, the 'human factor' is highly suspected. While ASF was successfully contained in a small region in the Southern part of Belgium without affecting domestic pigs, the risk of spillover at the wild/domestic interface remains poorly assessed. In this study, we used a semi-quantitative method, involving national and international experts, to assess the risk associated with different transmission routes for ASF introduction from wild boar to domestic pig holdings and subsequent dissemination between holdings in the Belgian epidemiological context. Qualitative responses obtained by our questionnaire were numerically transformed and statistically processed to provide a semi-quantitative assessment of the occurrence of the hazard and a ranking of all transmission routes. 'Farmer', 'bedding material', 'veterinarian' and 'professionals from the pig sector' were considered as the most important transmission routes for ASF introduction from the wild reservoir to pig holdings. 'Animal movements', 'farmer', 'veterinarian', 'iatrogenic', 'animal transport truck' and 'animal care equipment' were considered as the most important transmission routes posing a risk of ASF spread between pig holdings. Combined with specific biosecurity checks in the holdings, this assessment helps in prioritizing risk mitigation measures against ASF introduction and further spread in the domestic pig industry, particularly while the ASF situation in Western Europe is worsening.

Seroprevalence of border disease virus and other pestiviruses in sheep in Algeria and associated risk factors.

BACKGROUND: Border disease virus (BDV) is a pestivirus responsible for significant economic losses in sheep industry. The present study was conducted between 2015 and 2016 to determine the flock seroprevalence of the disease in Algeria and to identify associated risk factors. 56 flocks from nine departments were visited and 689 blood samples were collected from adult sheep between 6 and 24 months of age (n = 576) and from lambs younger than 6 months (n = 113). All samples were tested by RT-PCR as well as by Ag-ELISA, to detect Persistently Infected (PI) animals. Serum samples from adults were tested by Ab-ELISA (Enzyme Linked Immuno-Sorbent Assay), to detect specific antibodies against pestivirus and 197 of them were further characterized by VNT (virus neutralization test) for the detection of neutralizing antibodies specific for BDV and for Bovine virus diarrhea virus (BVDV-1 and BVDV-2). RESULTS: No PI animals were found among the 689 sheep tested. 144/197 sera were positive in VNT for BDV, and 2 sera were strongly positive BVDV-2. Fifty-five flocks (98%) had at least one seropositive animal and the apparent within-flock seroprevalence was estimated to be 60.17% (95% C.I.: 52.96-66.96). The true seroprevalence based on estimated sensitivity and specificity of the Ab-ELISA was 68.20% (95% C.I.; 60.2-76.3). Several risk factors were identified as linked to BDV such as climate, landscape, flock management and presence of other ruminant species in the farm. CONCLUSION: These high seroprevalence rates suggest that BDV is widespread and is probably endemic all over the country. Further studies are needed to detect and isolate the virus strains circulating in the country and understand the distribution and impact of pestiviruses in the Algerian livestock.

Serological monitoring on milk and serum samples in a BVD eradication program: A field study in Belgium showing antibody ELISA performances and epidemiological aspects.

In a cross-sectional field study involving 51 cattle herds in Belgium, 3159 serum samples and 557 individual milk samples were collected and tested by four different commercial antibody (Ab) ELISAs on serum and two Ab ELISAs on milk. A virus neutralization test (VNT) was performed on serum samples with discording ELISA results and on all samples from non-vaccinating herds. An epidemiological survey was carried out in the same herds to collect information about herd characteristics, management practices, BVD vaccination and BVD infection status. The objective of the study was to evaluate the performances of the Ab ELISAs relatively to the VNT, to assess the possibility of using pooled samples and to give recommendations regarding serological monitoring of BVD-free herds in the context of the Belgian national BVD eradication program which started early 2015. Depending on the assays, for ELISAs on serum, the diagnostic sensitivity (DSe) was estimated to be between 93.0 and 98.7% and the diagnostic specificity (DSp) between 94.3% and 99.1%. For the two ELISAs on milk, the DSe were 91.3% and 96.7% and the DSp 94.0% and 100% respectively and the Cohen's agreement coefficients between serum and milk samples were 0.75 and 0.85. Positive serum and milk samples diluted in negative samples to mimic different pool sizes were not detected by all ELISAs at dilutions above 1:5 or 1:10, leading to the conclusion that the testing of pooled samples should be used cautiously for serological monitoring and only with ELISAs with high sensitivity. The epidemiological analysis and the seroprevalence study, based on a general estimating equation model, showed that several factors had a significant influence on overall animal seroprevalence and within-herd seroprevalence such as age class, herd size, BVD herd infection status, BVD vaccination of young and/or adult cattle and the number of stables in the farm. This study showed that the best performances obtained with commercial Ab ELISAs are observed on individual serum samples, which should therefore be the preferred matrix to monitor BVD-free herds in the context of the Belgian eradication program. By regularly testing a limited number of samples from young (6-18 months) unvaccinated cattle it is possible to confirm the BVD-free herd status or to detect a recent infection.

Epidemiological analyses of African swine fever in the European Union (November 2017 until November 2018).

This update on the African swine fever (ASF) outbreaks in the EU demonstrated that out of all tested wild boar found dead, the proportion of positive samples peaked in winter and summer. For domestic pigs only, a summer peak was evident. Despite the existence of several plausible factors that could result in the observed seasonality, there is no evidence to prove causality. Wild boar density was the most influential risk factor for the occurrence of ASF in wild boar. In the vast majority of introductions in domestic pig holdings, direct contact with infected domestic pigs or wild boar was excluded as the route of introduction. The implementation of emergency measures in the wild boar management zones following a focal ASF introduction was evaluated. As a sole control strategy, intensive hunting around the buffer area might not always be sufficient to eradicate ASF. However, the probability of eradication success is increased after adding quick and safe carcass removal. A wider buffer area leads to a higher success probability; however it implies a larger intensive hunting area and the need for more animals to be hunted. If carcass removal and intensive hunting are effectively implemented, fencing is more useful for delineating zones, rather than adding substantially to control efficacy. However, segments of fencing will be particularly useful in those areas where carcass removal or intensive hunting is difficult to implement. It was not possible to demonstrate an effect of natural barriers on ASF spread. Human-mediated translocation may override any effect of natural barriers. Recommendations for ASF control in four different epidemiological scenarios are presented.

Evaluation of 16 commercial antibody ELISAs for the detection of bovine viral diarrhea virus-specific antibodies in serum and milk using well-characterized sample panels.

We performed a thorough fit-for-purpose evaluation of commercial ELISAs for the detection of bovine viral diarrhea virus (BVDV)-specific antibodies in serum and in milk by testing 2 panels of well-characterized serum and milk samples. Sixteen ELISAs from 9 different manufacturers, available on the Belgian market at the time of our study, were assessed for their diagnostic and analytical sensitivity (DSe and ASe, respectively), diagnostic specificity (DSp), and repeatability relative to the virus neutralization (VN) test considered to be the gold standard assay. Using serum as a matrix, DSe was much lower for competitive (c)ELISAs (min. 45%, max. 65%) than for indirect (i)ELISAs (min. 85%, max. 100%), partly because of the lower detection of positive samples from vaccinated animals included in the panel. ASe was also better for iELISAs; DSp was >95% for all but 2 ELISAs. Repeatability, expressed as coefficients of variation (CV) of optical densities, was generally good, although 3 ELISAs had a mean CV >10%. With milk samples, as observed for serum, DSe was lower for cELISAs (min. 57%, max. 75%) than for iELISAs (min. 61%, max. 89%), and DSp was high for all ELISAs (min. 94%, max. 100%). Both DSe and ASe were lower when testing milk samples compared to serum samples. These results confirm that serologic monitoring of BVDV-free herds should be performed using serum samples of unvaccinated animals to avoid interference of vaccination and to maximize the chance of detecting seroconversion linked to BVDV infection. Further investigations using a larger collection of field samples are recommended.

Genetically stable infectious Schmallenberg virus persists in foetal envelopes of pregnant ewes.

Schmallenberg virus (SBV) is a recently emerged vector-borne virus, inducing congenital defects in bovines, ovines and caprines. Here we have shown that infectious SBV is capable of persisting until the moment of birth in the foetal envelopes of ewes infected with SBV-infectious serum at day 45 (1/5 positive) and 60 (4/6 positive) of gestation. This persistence of at least 100 days is a new aspect of the SBV pathogenesis that could help to explain how SBV overwinters the cold season in temperate climate zones. Furthermore, sequencing of the M segment shows that the persisting virus in the foetal envelopes is genetically stable since only a few mutations compared to the inoculum were found. This supports the hypothesis that persisting virus could start the infection of new hosts. Finally, neutralization tests showed that infectious SBV present in the foetal envelopes at birth can be neutralized by the humoral immunity present in the infected ewes.

Age-Dependent Differences in Pseudorabies Virus Neuropathogenesis and Associated Cytokine Expression.

The severity of clinical symptoms induced by pseudorabies virus (PRV) infection of its natural host is inversely related to the age of the pig. During this study, 2- and 15-week-old pigs were inoculated with PRV strain NIA3. This resulted in important clinical disease, although the associated morbidity and mortality were lower in older pigs. Quantitative PCR analysis of viral DNA in different organs confirmed the general knowledge on PRV pathogenesis. Several new findings and potential explanations for the observed age-dependent differences in virulence, however, were determined from the study of viral and cytokine mRNA expression at important sites of neuropathogenesis. First, only limited viral and cytokine mRNA expression was detected in the nasal mucosa, suggesting that other sites may serve as the primary replication site. Second, PRV reached the trigeminal ganglion (TG) and brain stem rapidly upon infection but, compared to 2-week-old pigs, viral replication was less pronounced in 15-week-old pigs, and the decrease in viral mRNA expression was not preceded by or associated with an increased cytokine expression. Third, extensive viral replication associated with a robust expression of cytokine mRNA was detected in the olfactory bulbs of pigs from both age categories and correlated with the observed neurological disease. Our results suggest that age-dependent differences in PRV-induced clinical signs are probably due to enhanced viral replication and associated immunopathology in immature TG and the central nervous system neurons of 2-week-old pigs and that neurological disease is related with extensive viral replication and an associated immune response in the olfactory bulb. IMPORTANCE: It is well known that alphaherpesvirus infections of humans and animals result in more severe clinical disease in newborns than in older individuals and that this is probably related to differences in neuropathogenesis. The underlying mechanisms, however, remain unclear. Pseudorabies virus infection of its natural host, the pig, provides a suitable infection model to study this more profoundly. We show here that the severe neurological disease observed in 2-week-old pigs does not appear to be related to a hampered innate immune response but is more likely to reflect the immature development state of the trigeminal ganglia (TG) and central nervous system (CNS) neurons, resulting in an inefficient suppression of viral replication. In 15-week-old pigs, viral replication was efficiently suppressed in the TG and CNS without induction of an extensive immune response. Furthermore, our results provide evidence that neurological disease could, at least in part, be related to viral replication and associated immunopathology in the olfactory bulb.

Comparison of PRRSV Nucleic Acid and Antibody Detection in Pen-Based Oral Fluid and Individual Serum Samples in Three Different Age Categories of Post-Weaning Pigs from Endemically Infected Farms.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of an economically important disease in swine. Since it has been shown that PRRSV and PRRSV specific antibodies can be detected in oral fluid, many different aspects have been studied to show that oral fluid could be a worthy alternative diagnostic sample to serum for monitoring and surveillance of this disease. Thorough field evaluations are however missing to convincingly show its usefulness under representative field conditions. METHODOLOGY: Pen-based oral fluid samples and serum samples from all individual pigs in the corresponding pens were collected from post-weaning pigs of three different age categories in eight endemically PRRSV infected farms and one PRRSV free farm in Belgium. All samples were tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and ELISA to detect PRRSV RNA and PRRSV specific antibodies, respectively. RESULTS: While the relative specificity of PRRSV detection by qRT-PCR in pen-based oral fluid compared to serum collected from individual pigs was high in all age categories (>90%), the relative sensitivity decreased with the age of the pigs (89, 93 and 10% in 8-12w, 16-20w and 24-28w old pigs, respectively). The latter correlated with a lower percentage of PRRSV positive pigs in serum/pen in the different age categories (55, 29 and 6%, respectively). Irrespective of the age category, pen-based oral fluid samples were always found PCR positive when at least 30% of the individual pigs were positive in serum. PRRSV specific antibody detection in oral fluid by ELISA showed a 100% relative sensitivity to detection in serum since oral fluid samples were always positive as soon as one pig in the pen was positive in serum. On the other hand, two false positive oral fluid samples in 11 pens without serum positive pigs were found, resulting in a relative specificity of 82%. Indications are however present that the oral fluid result indicated the correct infection status but the absence of a golden standard test makes it difficult to define definitive test characteristics. CONCLUSIONS: Overall it can be concluded that oral fluid seems to be a useful matrix for diagnosis of PRRSV under field conditions and that differences in kinetics of PRRSV and PRRSV specific antibody detection in oral fluid and serum of individual pigs can also be reflected in pen-based oral fluid results.

Reconstruction of the Schmallenberg virus epidemic in Belgium: Complementary use of disease surveillance approaches.

Schmallenberg virus (SBV) emerged across Europe in 2011 and Belgium was among the first countries affected. In this study, published findings are combined with new data from veterinary surveillance networks and the Belgian reference laboratory for SBV at the Veterinary and Agrochemical Research centre (CODA-CERVA) to reconstruct the epidemic in Belgium. First retrospective cases of SBV were reported by veterinarians that observed decreased milk yield and fever in dairy cattle in May 2011. The number of SBV suspicions subsequently increased in adult cattle in August 2011. That month, first SBV positive pools of Culicoides were detected and extensive virus circulation occurred in Belgium during late summer and autumn 2011. As a consequence, most pregnant ruminants were infected and their fetuses exposed to the virus. This resulted in an outbreak of abortions, still-births and malformed new-borns observed between January and April 2012. The number of cases drastically diminished in 2012-2013, although multiple lines of evidence obtained from cross-sectional serological surveys, analyses on aborted foetuses, sentinel herd surveillance and surveillance of SBV in vectors prove that SBV was still circulating in Belgium at that time. Virus circulation was then probably strongly reduced in 2013-2014, while increasing evidence indicates its recirculation in 2014-2015 in Belgium. Based on the experience gathered with the closely related Akabane virus, recurrent outbreaks of congenital events can be expected for a long period. Vaccination of seronegative animals before the first mating could be used to prevent the deleterious effects of SBV. During this epidemic, different surveillance approaches including syndromic surveillance, sentinel herd surveillance, cross-sectional seroprevalence studies and pathogen surveillance in vectors have proven their utility and should be considered to continue in the future.

Clinical problems due to encephalomyocarditis virus infections in two pig herds.

BACKGROUND: Infections with encephalomyocarditis virus may cause myocarditis and sudden death in young pigs and reproduction disorders in sows. The presence of encephalomyocarditis virus infected rodents is considered a major risk factor for transmission of the virus to pigs. There is currently no effective treatment. Tightening up biosecurity, applying effective rodent control and reducing stress are the main control measures. CASE PRESENTATION: Two farrow-to-finish herds suffering from problems with sudden death are presented. In herd A, suckling piglets from 3 to 12 days old were dying acutely whereas in herd B, piglets at the end of the nursery period (8-10 weeks) were showing identical problems. A presumptive diagnosis of encephalomyocarditis virus infection was made because typical lesions were observed in some of the affected pigs. These lesions were not always present in pigs dying acutely or in some cases the lesions were very subtle. Therefore other causes had to be ruled out based upon clinical history, clinical signs and diagnostic tests. A conclusive diagnosis was finally established by showing encephalomyocarditis virus in heart tissue using conventional gel-based polymerase chain reaction tests. The real-time PCR test that gave initially negative result was further optimized to avoid false negative results. CONCLUSIONS: Typical lesions are not always present in piglets infected with encephalomyocarditis virus, indicating the importance of examining multiple animals. Problems in suckling piglets may occur in affected herds without reproductive problems in sows. Transmission routes of EMCV in swine are not fully understood. A stand-empty period following thorough cleaning and disinfection is recommended for controlling EMC virus infections.

Persistence of the protective immunity and kinetics of the isotype specific antibody response against the viral nucleocapsid protein after experimental Schmallenberg virus infection of sheep.

Schmallenberg virus (SBV) is an Orthobunyavirus that induces abortion, stillbirths and congenital malformations in ruminants. SBV infection induces a long lasting seroconversion under natural conditions. The persistence of the protective immunity and the isotype specific antibody response upon SBV infection of sheep has however not been studied in detail. Five sheep were kept in BSL3 facilities for more than 16 months and subjected to repeated SBV infections. Blood was regularly sampled and organs were collected at euthanasia. The presence of SBV RNA in serum and organs was measured with quantitative real-time PCR. The appearance and persistence of neutralizing and SBV nucleoprotein (N) isotype specific antibodies was determined with virus neutralization tests (VNT) and ELISAs. The primo SBV infection protected ewes against clinical signs, viraemia and virus replication in organs upon challenge infections more than 15 months later. Production of neutralizing SBV specific antibodies was first detected around 6 days post primo-inoculation with VNT and correlated with the appearance of SBV-N specific IgM antibodies. These IgM antibodies remained present for 2 weeks. SBV-N specific IgG antibodies were first detected between 10 and 21 dpi and reached a plateau at 28 dpi. This plateau remained consistently high and no significant decrease in titre was found over a period of more than 1 year. Similar results were found for the neutralising antibody response. In conclusion, the SBV specific IgM response probably eliminates SBV from the blood and the protective immunity induced by SBV infection protects sheep against reinfection for at least 16 months.

Experimental Infection of Sheep at 45 and 60 Days of Gestation with Schmallenberg Virus Readily Led to Placental Colonization without Causing Congenital Malformations.

BACKGROUND: Main impact of Schmallenberg virus (SBV) on livestock consists in reproductive disorders, with teratogenic effects, abortions and stillbirths. SBV pathogenesis and viral placental crossing remain currently poorly understood. Therefore, we implemented an experimental infection of ewes, inoculated with SBV at 45 or 60 days of gestation (dg). METHODOLOGY: "Mourerous" breed ewes were randomly separated in three groups: eight and nine ewes were subcutaneously inoculated with 1 ml of SBV infectious serum at 45 and 60 dg, respectively (G45 and G60). Six other ewes were inoculated subcutaneously with sterile phosphate buffer saline as control group. All SBV inoculated ewes showed RNAemia consistent with previously published studies, they seroconverted and no clinical sign was reported. Lambs were born at term via caesarian-section, and right after birth they were blood sampled and clinically examined. Then both lambs and ewes were euthanatized and necropsied. PRINCIPAL FINDINGS/SIGNIFICANCE: No lambs showed any malformation suggestive of SBV infection and none of them had RNAemia or anti-SBV antibodies prior to colostrum uptake. Positive SBV RNA detection in organs was rare in both G45 and G60 lambs (2/11 and 1/10, respectively). Nevertheless most of the lambs in G45 (9/11) and G60 (9/10) had at least one extraembryonic structure SBV positive by RTqPCR. The number of positive extraembryonic structures was significantly higher in G60 lambs. Time of inoculation (45 or 60 dg) had no impact on the placental colonization success rate but affected the frequency of detecting the virus in the offspring extraembryonic structures by the time of lambing. SBV readily colonized the placenta when ewes were infected at 45 or 60 dg but infection of the fetuses was limited and did not lead to congenital malformations.

Experimental Schmallenberg virus infection of pigs.

Schmallenberg virus (SBV) is a newly emerged virus responsible for an acute non-specific syndrome in adult cattle including high fever, decrease in milk production and severe diarrhea. It also causes reproductive problems in cattle, sheep and goat including abortions, stillbirths and malformations. The role of pigs in the epidemiology of SBV has not yet been evaluated while this could be interesting seen their suggested role in the epidemiology of the closely related Akabane virus. To address this issue, four 12 week old seronegative piglets were subcutaneously infected with 1 ml of SBV infectious serum (FLI) and kept into contact with four non-infected piglets to examine direct virus transmission. Throughout the experiment blood, swabs and feces samples were collected and upon euthanasia at 28 dpi different organs (cerebrum, cerebellum, brain stem, lung, liver, iliac lymph nodes, kidney and spleen) were sampled. No clinical impact was observed and all collected samples tested negative for SBV in rRT-PCR. Despite the absence of viremia and virus transmission, low and short lasting amounts of neutralizing antibodies were found in 2 out of 4 infected piglets. The limited impact of SBV infection in pigs was further supported by the absence of neutralizing anti-SBV antibodies in field collected sera from indoor housed domestic pigs (n=106). In conclusion, SBV infection of pigs can induce seroconversion but is ineffective in terms of virus replication and transmission indicating that pigs have no obvious role in the SBV epidemiology.

Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the detection of genotype A strains of small ruminant lentiviruses.

Small ruminant lentiviruses (SRLV) infect sheep and goats. Diagnosis of SRLV infection mostly relies on serological testing but more recently, also PCR is regarded as a useful complementary tool in SRLV diagnosis. The goal of this study was to develop and validate a quantitative PCR capable to detect a broad range of SRLV strains from genotype A, including strains circulating in Belgium. The developed q(RT)-PCR targets a region of the gag gene and showed to be highly sensitive and specific with a limit of detection of 6 DNA and 40 RNA copies/reaction respectively. SRLV sequences could be detected in lung samples and leukocytes pellets. The q(RT)-PCR identified SRLV positive animals in Belgian sheep flocks, but also SRLV isolates and samples from Scotland, The Netherlands, Spain, Portugal, UK, Iceland, Finland and USA were found positive. Samples known to contain 'CAEV like' SRLV from France and Spain were not identified as positive. Combined serological and PCR analysis of a limited number (n=35) of Belgian sheep underlined the usefulness of the described PCR as a complementary diagnostic tool since 3 seronegative animals were found positive by the PCR. In conclusion, the validated q(RT)-PCR shows excellent analytical characteristics and is capable to detect SRLV strains belonging to genotype A from various countries.

Diagnosis of Schmallenberg virus infection in malformed lambs and calves and first indications for virus clearance in the fetus.

Since mid-December 2011, samples from malformed lambs and calves are sent to CODA-CERVA in Belgium for diagnosis of Schmallenberg virus (SBV), a novel Orthobunyavirus that was first detected by researchers of the Friedrich-Loeffler-Institut (FLI, Germany) in German cattle in autumn 2011 and was later shown to be involved in congenital malformations in lambs, goat kids and calves. Surprisingly, by making use of real time RT-PCR (rRT-PCR) assays developed by the FLI, presence of SBV RNA could only be confirmed in part of the SBV suspected newborns examined. To investigate possible causes for non-confirmation by rRT-PCR, a comparative analysis between different organs and tissues (cerebrum, cerebellum, brain stem, spinal cord, thymus, spleen, lymph nodes, meconium) originating from respectively 90 and 81 malformed lambs and calves was undertaken. Furthermore, thoracic fluids of respectively 55 malformed lambs and calves were examined by a virus neutralization test (VNT) to evaluate the presence of neutralizing anti-SBV antibodies in these animals. Our results show that among the different organs tested by rRT-PCR, brain stem material is the most appropriate tissue for SBV detection while it could also be detected in all other tissues but to a more variable degree. The VNT test showed that 95% of the malformed lambs were positive for anti-SBV neutralizing antibodies while this was only the case for 44% of malformed calves. These immunological data suggest that a humoral immune response could assist in the clearance of SBV from the fetus during gestation and that SBV specific antibody testing should be considered together with rRT-PCR analysis for confirmation of SBV infection.