%0 Journal Article %J Animals %D 2023 %T SARS-CoV-2 Infection in Captive Hippos (Hippopotamus amphibius), Belgium %A Francis Vercammen %A Ann Brigitte Cay %A Sophie Gryseels %A Nadège Balmelle %A Léa Joffrin %A Koenraad Van Hoorde %A Bavo Verhaegen %A Elisabeth Mathijs %A Rianne Van Vredendaal %A Tanmay Dharmadhikari %A Koen Chiers %A Tim Van Olmen %A Gianfilippo Agliani %A Judith Van den Brand %A Herwig Leirs %B Animals %V 13 %8 Jan-01-2023 %G eng %N 2 %R 10.3390/ani13020316 %0 Journal Article %J Viruses %D 2022 %T Porcine Reproductive and Respiratory Syndrome virus (PRRSv): A Cross-Sectional Study on ELISA Seronegative, Multivaccinated Sows %A Jorian Fiers %A Marylène Tignon %A Ann Brigitte Cay %A Xavier Simons %A Maes, Dominiek %K immunology %K non-responder %K PRRSv %K sow %K Vaccination %X

Vaccination against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) is widely used to control clinical disease, but the effectiveness appears in some cases to be suboptimal. Field reports have stated the presence of routinely PRRSv-vaccinated but ELISA seronegative sows: the ELISA non-responders. The real extent of this phenomenon (prevalence–origin–consequences) was not yet investigated. In this study, the prevalence of ELISA non-responders was assessed by measuring PRRSv-specific antibodies in 1400 sows, originating from 70 PRRSv-vaccinating sow herds, using IDEXX ELISA (ELISA 1) and CIVTEST E/S ELISA (ELISA 2). Neutralizing antibodies (NAbs) were quantified in a virus neutralization assay. Univariable logistic regression was used to identify herd risk factors for the presence of ELISA non-responders. The global prevalence of non-responders varied from 3.5% (ELISA 1) to 4.1% (ELISA 2), the herd-level prevalence was 40% and the within-herd prevalence ranged from 5% to 20% (ELISA 1) and from 5% to 30% (ELISA 2). The ELISA non-responders had significantly lower NAbs than the ELISA responders. Herds using the combination of one modified live vaccine and one killed vaccine had a significantly reduced risk of having ELISA non-responders. A first assessment of the prevalence and possible consequences of ELISA non-responders has been provided by this study. The clinical importance, origin and underlying immunological mechanisms warrant further research.

%B Viruses %V 14 %8 31/08/2022 %G eng %N 9 %R 10.3390/v14091944 %0 Journal Article %J Viruses %D 2022 %T Porcine Reproductive and Respiratory Syndrome virus (PRRSv): A Cross-Sectional Study on ELISA Seronegative, Multivaccinated Sows. %A Jorian Fiers %A Marylène Tignon %A Ann Brigitte Cay %A Xavier Simons %A Maes, Dominiek %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K cross-sectional studies %K Enzyme-Linked Immunosorbent Assay %K Female %K Porcine Reproductive and Respiratory Syndrome %K Porcine respiratory and reproductive syndrome virus %K Swine %K Vaccines, Inactivated %K Viral Vaccines %X

Vaccination against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) is widely used to control clinical disease, but the effectiveness appears in some cases to be suboptimal. Field reports have stated the presence of routinely PRRSv-vaccinated but ELISA seronegative sows: the ELISA non-responders. The real extent of this phenomenon (prevalence-origin-consequences) was not yet investigated. In this study, the prevalence of ELISA non-responders was assessed by measuring PRRSv-specific antibodies in 1400 sows, originating from 70 PRRSv-vaccinating sow herds, using IDEXX ELISA (ELISA 1) and CIVTEST E/S ELISA (ELISA 2). Neutralizing antibodies (NAbs) were quantified in a virus neutralization assay. Univariable logistic regression was used to identify herd risk factors for the presence of ELISA non-responders. The global prevalence of non-responders varied from 3.5% (ELISA 1) to 4.1% (ELISA 2), the herd-level prevalence was 40% and the within-herd prevalence ranged from 5% to 20% (ELISA 1) and from 5% to 30% (ELISA 2). The ELISA non-responders had significantly lower NAbs than the ELISA responders. Herds using the combination of one modified live vaccine and one killed vaccine had a significantly reduced risk of having ELISA non-responders. A first assessment of the prevalence and possible consequences of ELISA non-responders has been provided by this study. The clinical importance, origin and underlying immunological mechanisms warrant further research.

%B Viruses %V 14 %8 2022 Aug 31 %G eng %N 9 %R 10.3390/v14091944 %0 Journal Article %J Transboundary and Emerging Diseases %D 2021 %T 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) %A Mauroy, Axel %A Depoorter, Pieter %A Saegerman, Claude %A Ann Brigitte Cay %A Nick De Regge %A Maria‐Eleni Filippitzi %A Claude Fischer %A Martine Laitat %A Maes, Dominiek %A Kevin Morelle %A Nauwynck, Hans %A Xavier Simons %A Thierry van den Berg %A Van Huffel, Xavier %A Thiry, Etienne %A Dewulf, Jeroen %B Transboundary and Emerging Diseases %V 68 %8 Jan-09-2021 %G eng %N 5 %R 10.1111/tbed.14067 %0 Journal Article %J Curr Issues Mol Biol %D 2021 %T Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution. %A Laura Van Poelvoorde %A Mathieu Gand %A Marie-Alice Fraiture %A Sigrid C.J. De Keersmaecker %A Bavo Verhaegen %A Koenraad Van Hoorde %A Ann Brigitte Cay %A Nadège Balmelle %A Philippe Herman %A Nancy Roosens %K COVID-19 %K COVID-19 Nucleic Acid Testing %K Diagnostic Tests, Routine %K Evolution, Molecular %K Humans %K RNA, Viral %K Roc Curve %K SARS-CoV-2 %X

The worldwide emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2019 has highlighted the importance of rapid and reliable diagnostic testing to prevent and control the viral transmission. However, inaccurate results may occur due to false negatives (FN) caused by polymorphisms or point mutations related to the virus evolution and compromise the accuracy of the diagnostic tests. Therefore, PCR-based SARS-CoV-2 diagnostics should be evaluated and evolve together with the rapidly increasing number of new variants appearing around the world. However, even by using a large collection of samples, laboratories are not able to test a representative collection of samples that deals with the same level of diversity that is continuously evolving worldwide. In the present study, we proposed a methodology based on an in silico and in vitro analysis. First, we used all information offered by available whole-genome sequencing data for SARS-CoV-2 for the selection of the two PCR assays targeting two different regions in the genome, and to monitor the possible impact of virus evolution on the specificity of the primers and probes of the PCR assays during and after the development of the assays. Besides this first essential in silico evaluation, a minimal set of testing was proposed to generate experimental evidence on the method performance, such as specificity, sensitivity and applicability. Therefore, a duplex reverse-transcription droplet digital PCR (RT-ddPCR) method was evaluated in silico by using 154 489 whole-genome sequences of SARS-CoV-2 strains that were representative for the circulating strains around the world. The RT-ddPCR platform was selected as it presented several advantages to detect and quantify SARS-CoV-2 RNA in clinical samples and wastewater. Next, the assays were successfully experimentally evaluated for their sensitivity and specificity. A preliminary evaluation of the applicability of the developed method was performed using both clinical and wastewater samples.

%B Curr Issues Mol Biol %V 43 %8 2021 Nov 06 %G eng %N 3 %R 10.3390/cimb43030134 %0 Journal Article %J Animals (Basel) %D 2021 %T Towards Efficient Early Warning: Pathobiology of African Swine Fever Virus "Belgium 2018/1" in Domestic Pigs of Different Age Classes. %A Jutta Pikalo %A Marie-Eve Schoder %A Julia Sehl-Ewert %A Angele Breithaupt %A Ann Brigitte Cay %A Coline Lhoëst %A Willem Van Campe %A Laurent Mostin %A Paul Deutschmann %A Hanna Roszyk %A Beer, Martin %A Blome, Sandra %A Marylène Tignon %X

African swine fever (ASF) is one of the most important and devastating viral diseases in wild boar and domestic pigs worldwide. In the absence of vaccines or treatment options, early clinical detection is crucial and requires a sound knowledge of disease characteristics. To provide practitioners and state veterinarians with detailed information, the objective of the present study was to characterize the ASF virus (ASFV) isolate "Belgium 2018/1" in subadult and weaning domestic pigs. To this end, two animal trials were performed. Trial A included eight subadult domestic pigs and trial B five weaner pigs. In general, clinical signs and pathological lesions were in line with previous studies utilizing highly virulent ASF genotype II viruses. However, in trial A, four subadult domestic pigs survived and recovered, pointing to an age-dependent outcome. The long-term fate of these survivors remains under discussion and would need further investigation.

%B Animals (Basel) %V 11 %8 2021 Sep 04 %G eng %N 9 %R 10.3390/ani11092602 %0 Journal Article %J Viruses %D 2021 %T WGS- versus ORF5-Based Typing of PRRSV: A Belgian Case Study. %A Frank Vandenbussche %A Elisabeth Mathijs %A Marylène Tignon %A Tamara Vandersmissen %A Ann Brigitte Cay %X

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most widespread and economically devastating diseases in the swine industry. Typing circulating PRRSV strains by means of sequencing is crucial for developing adequate control strategies. Most genetic studies only target the highly variable open reading frame (ORF) 5, for which an extensive database is available. In this study, we performed whole-genome sequencing (WGS) on a collection of 124 PRRSV-1 positive serum samples that were collected over a 5-year period (2015-2019) in Belgium. Our results show that (nearly) complete PRRSV genomes can be obtained directly from serum samples with a high success rate. Analysis of the coding regions confirmed the exceptionally high genetic diversity, even among Belgian PRRSV-1 strains. To gain more insight into the added value of WGS, we performed phylogenetic cluster analyses on separate ORF datasets as well as on a single, concatenated dataset (CDS) containing all ORFs. A comparison between the CDS and ORF clustering schemes revealed numerous discrepancies. To explain these differences, we performed a large-scale recombination analysis, which allowed us to identify a large number of potential recombination events that were scattered across the genome. As PRRSV does not contain typical recombination hot-spots, typing PRRSV strains based on a single ORF is not recommended. Although the typing accuracy can be improved by including multiple regions, our results show that the full genetic diversity among PRRSV strains can only be captured by analysing (nearly) complete genomes. Finally, we also identified several vaccine-derived recombinant strains, which once more raises the question of the safety of these vaccines.

%B Viruses %V 13 %8 2021 Dec 02 %G eng %N 12 %R 10.3390/v13122419 %0 Journal Article %J Transboundary and Emerging Diseases %D 2020 %T The African swine fever virus isolate Belgium 2018/1 shows high virulence in European wild boar. %A Jutta Pikalo %A Marie-Eve Schoder %A Julia Sehl %A Angele Breithaupt %A Marylène Tignon %A Ann Brigitte Cay %A Anna Maria Gager %A Melina Fischer %A Beer, Martin %A Blome, Sandra %K African Swine Fever Virus %K Belgium %K histopathology %K viral distribution %K virulence %K wild boar %X

African swine fever (ASF) is one of the most important and complex viral diseases in domestic pigs and wild boar. Over the last decade, the disease has spread to several European and Asian countries and is now one of the major threats to profitable pig production worldwide. One of the more recently affected western countries is Belgium. To date, only wild boar are affected in a rather defined area in the Luxembourg region close to France, Luxembourg and Germany. While detailed sequence analyses were recently performed, biological characterization was still pending. Here, we report on the experimental inoculation of four sub-adult wild boar to further characterize the virus and its distribution in different tissues. After oronasal inoculation with the virus strain 'Belgium 2018/1', all animals developed an acute and severe disease course with typical pathomorphological and histopathological lesions. Organs and blood samples were positive in qPCR, haemadsorption test and antigen lateral flow devices (LFD). Virus and viral genome were also detected in genitals and accessory sex glands of two boars. There were no antibodies detectable in commercial antibody ELISAs, antibody LFDs and indirect immunoperoxidase tests. Thus, the genotype II ASF virus isolate 'Belgium 2018/1' showed a highly virulent phenotype in European wild boar similar to parental viruses like Armenia 2007 and other previously characterized ASFV strains. The study also provided a large set of well-characterized sample materials for test validation and assay harmonization.

%B Transboundary and Emerging Diseases %8 2020 Feb 03 %G eng %R 10.1111/tbed.13503 %0 Journal Article %J J Virol Methods %D 2020 %T Evaluation of seven commercial African swine fever virus detection kits and three Taq polymerases on 300 well-characterized field samples. %A Marie-Eve Schoder %A Marylène Tignon %A Linden, Annick %A Vervaeke, Muriel %A Ann Brigitte Cay %K Actins %K African Swine Fever %K African Swine Fever Virus %K Animals %K Belgium %K Capsid Proteins %K DNA, Viral %K Molecular Diagnostic Techniques %K Reagent Kits, Diagnostic %K Real-Time Polymerase Chain Reaction %K Sensitivity and Specificity %K Sus scrofa %K Swine %K Taq Polymerase %X

African swine fever virus (ASFV) is a complex double stranded DNA virus, responsible for a highly infectious and fatal disease in pigs and boars and for important deterioration of animal welfare. Over the last decade, the disease spread to several European and Asian countries causing unprecedented dramatic economic losses in pig industry. In the absence of a vaccine, affected countries rely on trustful diagnostic tests and adapted testing policies to set up control programs to fight against the disease. In this study, we evaluated the sensitivity and specificity of seven commercially available ASFV real-time PCR detection kits and three Taq polymerases on 300 well-characterized wild boar samples collected in Belgium during the 2018-2019 outbreak. This study confirms that all commercial kits and two Taq polymerases are suitable for ASFV detection in diagnostic laboratories. Furthermore, the use of endogenous controls is emphasized when testing field samples harvested on carcasses in an advanced stage of decomposition, in order to avoid false negative results.

%B J Virol Methods %V 280 %8 2020 06 %G eng %R 10.1016/j.jviromet.2020.113874 %0 Journal Article %J Emerg Infect Dis %D 2019 %T Comparative Analysis of Whole-Genome Sequence of African Swine Fever Virus Belgium 2018/1. %A Jan H Forth %A Marylène Tignon %A Ann Brigitte Cay %A Leonie F Forth %A Höper, Dirk %A Blome, Sandra %A Beer, Martin %X

We analyzed the whole-genome sequence of African swine fever virus Belgium 2018/1. The strain fits into the European genotype II (>99.98% identity). The high-coverage sequence revealed 15 differences compared with an improved virus African swine fever virus Georgia 2007/1 sequence. However, in the absence of genetic markers, no spatial or temporal correlations could be defined.

%B Emerg Infect Dis %V 25 %8 2019 Jun 17 %G eng %N 6 %R 10.3201/eid2506.190286 %0 Journal Article %J Emerg Infect Dis %D 2019 %T Phylogeographic Analysis of African Swine Fever Virus, Western Europe, 2018. %A Mutien Garigliany %A Desmecht, Daniel %A Marylène Tignon %A Cassart, Dominique %A Christophe Lesenfant %A Julien Paternostre %A Rosario Volpe %A Ann Brigitte Cay %A Thierry van den Berg %A Linden, Annick %X

In September 2018, African swine fever in wild boars was detected in Belgium. We used African swine fever-infected spleen samples to perform a phylogenetic analysis of the virus. The causative strain belongs to genotype II, and its closest relatives are viruses previously isolated in Ukraine, Belarus, Estonia, and European Russia.

%B Emerg Infect Dis %V 25 %8 2019 01 %G eng %N 1 %R 10.3201/eid2501.181535 %0 Journal Article %J Transbound Emerg Dis %D 2019 %T Summer 2018: African swine fever virus hits north-western Europe. %A Linden, Annick %A Alain Licoppe %A Rosario Volpe %A Julien Paternostre %A Christophe Lesenfants %A Cassart, Dominique %A Mutien Garigliany %A Marylène Tignon %A Thierry van den Berg %A Desmecht, Daniel %A Ann Brigitte Cay %B Transbound Emerg Dis %V 66 %8 2019 01 %G eng %N 1 %R 10.1111/tbed.13047 %0 Journal Article %J Viruses %D 2018 %T Comparative Analysis of Different Serological and Molecular Tests for the Detection of Small Ruminant Lentiviruses (SRLVs) in Belgian Sheep and Goats. %A Michiels, Rodolphe %A Van Mael, Eva %A Quinet, Christian %A Nadjah Radia Adjadj %A Ann Brigitte Cay %A Nick De Regge %X

Countries rely on good diagnostic tests and appropriate testing schemes to fight against economically important small ruminant lentivirus (SRLV) infections. We undertook an extensive comparative analysis of seven commercially available serological tests and one in-house real-time PCR (qPCR) detecting genotype A and B strains using a large panel of representative Belgian field samples and samples from experimentally infected sheep and goats. ELISAs generally performed well and detected seroconversion within three weeks post experimental infection. Two enzyme-linked immunosorbent assays (ELISAs) (Elitest and IDscreen kits) showed the highest sensitivities (>96%) and specificities (>95%) in both species, and their combined use allowed to correctly identify the infection status of all animals. Individual agar gel immunodiffusion (AGIDs) kits lacked sensitivity, but interestingly, the combined use of both kits had a sensitivity and specificity of 100%. qPCRs detected SRLV infection before seroconversion at two weeks post infection and showed a specificity of 100%. Sensitivity however remained suboptimal at 85%. These results allow to propose a faster and cheaper diagnostic testing strategy for Belgium by combining a first ELISA screening, followed by confirmation of positive samples in AGID and/or a second ELISA. Since genotypes A and B strains are predominant in many countries, these results are interesting for other countries implementing SRLV control programs.

%B Viruses %V 10 %8 2018 12 08 %G eng %N 12 %R 10.3390/v10120696 %0 Journal Article %J Vet Microbiol %D 2018 %T Sensitivity of African swine fever virus (ASFV) to heat, alkalinity and peroxide treatment in presence or absence of porcine plasma. %A I.D. Kalmar %A Ann Brigitte Cay %A Marylène Tignon %K Abattoirs %K African Swine Fever %K African Swine Fever Virus %K Animals %K Antacids %K Hot Temperature %K Hydrogen Peroxide %K plasma %K Swine %K Virus Inactivation %X

African swine fever virus (ASFV) is a highly resistant viraemic virus with devastating socio-economic impact. Its present epidemiology in Eastern Europe and Russia warrants increased biosecurity measures in Western Europe. This includes proactive precautions on traffic of pork products within and between areas that are officially free from ASF. Namely, delayed notification of clinical signs or introduction of a low-virulent strain in ASF-free areas could result in presence of ASFV in veterinary inspected pork and pork by-products. The present study evaluated sensitivity of ASFV to physical and chemical processing conditions that can be applied on abattoir collected blood for production of spray dried porcine plasma (SDPP). Standard endpoint dilution assays were used to determine the sensitivity of Vero-cell adapted Lisbon/60 strain ASFV to heat treatment (H) at alkaline conditions (A) with or without peroxide (P). Time (T) dependent inactivation was evaluated in presence or absence of porcine plasma. HAPT-treatment at H = 48 °C, A = pH 10.2 and P = 20.6 or 102.9 mM HO during 10 min (T) inactivated (95LCL) 3.35, respectively, 4.17 log TCID ASFV/ml plasma. In absence of plasma, 6.99 log-inactivation was reached within 5 min. Implementation of HAPT-treatment on plasma from ASFV-free areas provides an additional safety hurdle for derived blood products in the unlikely event that blood from few undetected infected pigs would contaminate pooled veterinary inspected blood. Such an additional processing step in the production of SDPP is thus a valuable precautionary measure to overcome a potential biosecurity-break that may arise during the high-risk phase between transboundary introduction of ASFV and first notification of the disease.

%B Vet Microbiol %V 219 %8 2018 Jun %G eng %R 10.1016/j.vetmic.2018.04.025 %0 Journal Article %J Prev Vet Med %D 2018 %T Seroprevalence and risk factors related to small ruminant lentivirus infections in Belgian sheep and goats. %A Michiels, Rodolphe %A Van Mael, Eva %A Quinet, Christian %A Sarah Welby %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Arthritis-Encephalitis Virus, Caprine %K Belgium %K Goat Diseases %K Goats %K Lentivirus Infections %K prevalence %K Risk Factors %K Seroepidemiologic Studies %K Sheep %K Sheep Diseases %K Visna-maedi virus %X

Maedi-Visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are two prototype members of the group of small ruminant lentiviruses (SRLVs). Both result in progressive and persistent infections of sheep and goats that impact animal health and cause economic losses. In Belgium, the sheep and goat sector is small and consists mostly of hobbyist farmers keeping few animals. A voluntary control program however exists, but less than 2% of the farmers participate to the program. The current lack of SRLV seroprevalence data and knowledge on risk factors related to SRLV seropositivity in this hobbyist sector makes it difficult to evaluate the risk of SRLV transmission from non-certified to SRLV free certified farms. We performed a nationwide SRLV seroprevalence study based on a stratified sampling proportional to the number of sheep and goat holders per province. Randomly selected sheep and goat owners were invited to participate and subject to a short questionnaire to collect information about flock size, animal health condition, age, flock constitution and housing conditions. Samples were collected from maximum 7 animals per farm and tested in a commercial ELISA. In total, we received samples from 87 sheep and 76 goat farms. Sheep flocks showed an overall seroprevalence of 9% (CI : 5-15) and a between-herd seroprevalence of 17% (CI :11-27). Seroprevalence at animal level in goat flocks was 6% (CI : 3-12) and the between-herd seroprevalence was 13% (CI : 7-23). Multiple sheep and goat breeds were found SRLV seropositive. Answers provided during the questionnaire confirmed the mostly hobbyist nature of the sector and showed that more than 65% of sheep and goat farmers had never heard of the disease. The only risk factor found to be related to SRLV seroprevalence was flock size. Herds of more than 10 goats had significantly higher chance to harbor seropositive animals (OR: 4.36; CI: 1.07; 17.73). In conclusion, it was shown that participants to the SRLV free certification program are at risk for reintroduction of the disease in their herds since SRLVs are present on about 15%-20% of non-certified farms. Except from flock size, no clear risk factors were found that are helpfull to identify flocks at risk. Greater effort should be made to inform sheep and goat farmers about the existence and consequences of this disease in order to promote the voluntary control program and further reduce the disease prevalence.

%B Prev Vet Med %V 151 %8 2018 Mar 01 %G eng %R 10.1016/j.prevetmed.2017.12.014 %0 Journal Article %J BMC Vet Res %D 2018 %T Seroprevalence of border disease virus and other pestiviruses in sheep in Algeria and associated risk factors. %A Naouel Feknous %A Jean-Baptiste Hanon %A Marylène Tignon %A Khaled, Hamza %A Abdallah Bouyoucef %A Ann Brigitte Cay %K Algeria %K Animals %K Border Disease %K Border disease virus %K Enzyme-Linked Immunosorbent Assay %K Female %K Male %K Pestivirus %K Pestivirus Infections %K Real-Time Polymerase Chain Reaction %K Risk Factors %K Seroepidemiologic Studies %K Sheep %X

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.

%B BMC Vet Res %V 14 %8 2018 Nov 12 %G eng %N 1 %R 10.1186/s12917-018-1666-y %0 Journal Article %J J Virol %D 2017 %T Age-Dependent Differences in Pseudorabies Virus Neuropathogenesis and Associated Cytokine Expression. %A Verpoest, Sara %A Ann Brigitte Cay %A Favoreel, Herman %A Nick De Regge %K Age factors %K Animals %K Brain Stem %K Cytokines %K DNA, Viral %K Female %K Gene Expression %K Herpesvirus 1, Suid %K Olfactory Bulb %K Pseudorabies %K Swine %K Swine Diseases %K Trigeminal Ganglion %K virulence %X

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.

%B J Virol %V 91 %8 2017 Jan 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/27852848?dopt=Abstract %R 10.1128/JVI.02058-16 %0 Journal Article %J J Virol Methods %D 2017 %T Characterization of three commercial ELISA kits for detection of BOHV-1 gE specific antibodies in serum and milk samples and applicability of bulk milk for determination of herd status. %A Marylène Tignon %A Miet De Baere %A Jean-Baptiste Hanon %A Goolaerts, Annelies %A Houtain, Jean-Yves %A Delooz, Laurent %A Ann Brigitte Cay %X

Vaccination of animals with gE-deleted vaccine strains (gE- marker vaccines) and differential detection of vaccinated vs infected animals with antibody ELISA targeting the gE or the gB proteins have been proved to be useful tools in programs for control and eradication of the bovine herpesvirus 1 (BoHV-1) responsible for infectious bovine rhinotracheitis (IBR), a major pathogen of cattle. The diagnostic sensitivity (DSe) and specificity (DSp) of three commercial gE ELISA kits from IDEXX, IDVet and CIV-HIPRA were compared for serum and milk matrices. Limiting the analysis to 198 individual with concordant ELISA results in serum (91 naïve, 37 vaccinated and 70 infected) the DSe of gE kits was estimated to 0,97 for IDEXX, 0,93 for CIV-HIPRA and 0,53 for IDVet using milk samples and the DSp to 0,95 for IDEXX, 1,00 for IDVet and CIV-HIPRA. The applicability of gE ELISA for individual or bulk milk testing as an additional tool in control programs dedicated to the certification and control of vaccinated herds was evaluated. Two of the three evaluated gE ELISA kits presented substantial to good agreement individual milk and serum samples. The bulk-tank milk also proved to be suitable for the detection of BoHV-1 in vaccinated herds provided that gE prevalence is superior to 10% as false negative results are often observed at lower gE herd prevalence. This limitation could be reduced to 8% of prevalence when a prior concentration step was applied to bulk milk samples.

%B J Virol Methods %V 245 %P 66-72 %8 2017 Jul %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28363451?dopt=Abstract %R 10.1016/j.jviromet.2017.03.015 %0 Journal Article %J Open Vet J %D 2017 %T Encephalomyocarditis virus in a captive Malayan tapir (). %A Vercammen, Francis %A Bosseler, Leslie %A Marylène Tignon %A Ann Brigitte Cay %X

A 5-month-old female captive Malayan tapir () died suddenly without preceding symptoms. Gross necropsy revealed numerous white circular and linear foci in the myocard. Differential diagnosis all turned out negative, except for encephalomyocarditis virus. Histopathology revealed mineralisation of myocardial cells and interstitial infiltration of lymphocytes, plasma cells and less neutrophils. Encephalomyocarditis virus was detected by PCR. Although encephalomyocarditis virus occurs in many mammals, this is the first published description of this virus in a Malayan tapir.

%B Open Vet J %V 7 %P 100-103 %8 2017 %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/28616390?dopt=Abstract %R 10.4314/ovj.v7i2.4 %0 Journal Article %J J Vet Diagn Invest %D 2017 %T 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. %A Jean-Baptiste Hanon %A Miet De Baere %A De la Ferté, Camille %A Sophie Roelandt %A Yves Van der Stede %A Ann Brigitte Cay %X

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.

%B J Vet Diagn Invest %P 1040638717724839 %8 2017 Aug 01 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28803517?dopt=Abstract %R 10.1177/1040638717724839 %0 Journal Article %J Transboundary and Emerging Diseases %D 2017 %T Evidence of extensive renewed Schmallenberg virus circulation in Belgium during summer of 2016 - increase in arthrogryposis-hydranencephaly cases expected %A Charlotte Sohier %A I. Deblauwe %A T. Van Loo %A Jean-Baptiste Hanon %A Ann Brigitte Cay %A Nick De Regge %X

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.

%B Transboundary and Emerging Diseases %V 64 %8 Jan-08-2017 %G eng %N 4 %R 10.1111/tbed.12655 %0 Journal Article %J J Gen Virol %D 2017 %T Genetically stable infectious Schmallenberg virus persists in foetal envelopes of pregnant ewes. %A Poskin, Antoine %A Martinelle, Ludovic %A Yves Van der Stede %A Saegerman, Claude %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Antibodies, Viral %K Base Sequence %K Bunyaviridae Infections %K Chorion %K Female %K Orthobunyavirus %K Placenta %K Pregnancy %K RNA, Viral %K Sequence Analysis, RNA %K Sheep %K Sheep Diseases %X

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.

%B J Gen Virol %V 98 %P 1630-1635 %8 2017 Jul %G eng %N 7 %1 https://www.ncbi.nlm.nih.gov/pubmed/28699878?dopt=Abstract %R 10.1099/jgv.0.000841 %0 Journal Article %J Virulence %D 2017 %T Reduced virulence of a pseudorabies virus isolate from wild boar origin in domestic pigs correlates with hampered visceral spread and age-dependent reduced neuroinvasive capacity. %A Verpoest, Sara %A Valerie Redant %A Ann Brigitte Cay %A Favoreel, Herman %A Nick De Regge %X

Morbidity and mortality associated with pseudorabies virus (PRV) infection are dependent on the age of the pig and the virulence of the strain. PRV strains circulating in wild boar are considered to be low virulent, but no mechanistic explanation for their reduced virulence is available. Here infection of 2- and 15-week-old domestic pigs with the PRV wild boar strain BEL24043 did not induce clinical symptoms in 15-week-old pigs, but resulted in important neurological and respiratory disease in 2-week-old piglets. A detailed study of the (neuro) pathogenesis and associated cytokine mRNA expression showed that the reduced virulence of the wild boar strain, compared to what was previously reported for the virulent domestic NIA3 strain, is due to a severely hampered spread to visceral organs in pigs of both age categories and to an efficient suppression of viral replication at primary replication sites of 15-week-old pigs and to a lesser extent in those of 2-week-old piglets. The age-dependent difference in induced symptoms seems to be due to an immature development state of the immune and/or nervous system in 2-week-old pigs. An extensive viral replication associated with a robust expression of cytokine-related mRNA was found in the olfactory bulb of 2-week-old piglets, correlating with observed neurological disease. Neuroinvasion also occurred via the trigeminal route in 2-week-old pigs, but viral replication was efficiently suppressed in the trigeminal ganglion in the presence of a moderate induction of cytokine-related mRNA. Viral replication in the peripheral and central nervous system of 15-week-old pigs was limited and efficiently suppressed.

%B Virulence %P 1-14 %8 2017 Sep 05 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28873002?dopt=Abstract %R 10.1080/21505594.2017.1368941 %0 Journal Article %J Transbound Emerg Dis %D 2017 %T Resurgence of Schmallenberg Virus in Belgium after 3 Years of Epidemiological Silence. %A Delooz, L %A Saegerman, C %A Quinet, C %A Petitjean, T %A Nick De Regge %A Ann Brigitte Cay %X

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.

%B Transbound Emerg Dis %V 64 %P 1641-1642 %8 2017 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/27485019?dopt=Abstract %R 10.1111/tbed.12552 %0 Journal Article %J Transbound Emerg Dis %D 2017 %T Three Different Routes of Inoculation for Experimental Infection with Schmallenberg Virus in Sheep. %A Martinelle, L %A Poskin, A %A dal Pozzo, F %A Laurent Mostin %A Willem Van Campe %A Ann Brigitte Cay %A Nick De Regge %A Saegerman, C %K Administration, Intranasal %K Animals %K Bunyaviridae Infections %K Female %K Injections, Intradermal %K Injections, Subcutaneous %K Lymph Nodes %K Orthobunyavirus %K Sheep %K Sheep Diseases %K Spleen %K Vaccination %X

Schmallenberg virus (SBV) is an emerging Orthobunyavirus affecting European domestic ruminants. In this study, three groups of ewes (n = 3) were inoculated with 1 ml of an SBV infectious serum, via the subcutaneous (SC), intradermal (ID) or intranasal (IN) route. The ewes were monitored for 10 days and no clinical signs were reported. IN inoculation failed to generate any detectable RNAemia. SC and ID inoculation induced typical SBV RNAemia and seroconversion upon day 6 post-inoculation in 3/3 and 2/3 sheep, respectively. In all the animals that showed RNAemia, the viral genome could be detected in spleen and mesenteric lymph nodes. Both the SC and ID routes seem suitable to properly reproduce field conditions, as comparable observations were reported regarding RNAemia, seroconversion and viral genome detection in organs.

%B Transbound Emerg Dis %V 64 %P 305-308 %8 2017 Feb %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25891033?dopt=Abstract %R 10.1111/tbed.12356 %0 Journal Article %J Research in Veterinary Science %D 2017 %T Unchanged Schmallenberg virus seroprevalence in the Belgian sheep population after the vector season of 2014 and 2015 despite evidence of virus circulation %A Charlotte Sohier %A Michiels, Rodolphe %A Kapps, Elena %A Van Mael, Eva %A Quinet, Christian %A Ann Brigitte Cay %A Nick De Regge %B Research in Veterinary Science %V 114 %8 Jan-10-2017 %G eng %R 10.1016/j.rvsc.2017.04.011 %0 Journal Article %J J Gen Virol %D 2017 %T Us3 and Us9 proteins contribute to the stromal invasion of bovine herpesvirus 1 in the respiratory mucosa. %A Zhao, Jing %A Poelaert, Katrien C K %A Steukers, Lennert %A Favoreel, Herman W %A Li, Yewei %A Chowdhury, Shafiqul I %A van Drunen Littel-van den Hurk, Sylvia %A Ann Brigitte Cay %A Nauwynck, Hans J %X

Bovine herpesvirus 1 (BHV-1) infection may lead to conjunctivitis, upper respiratory tract problems, pneumonia, genital disorders and abortion. BHV-1 is able to spread quickly in a plaque-wise manner and invade by breaching the basement membrane (BM) barrier in the respiratory mucosa. BHV-1 Us3, a serine/threonine kinase, induces a dramatic cytoskeletal reorganization and BHV-1 Us9, a tail-anchored membrane protein, is required for axonal transport of viruses in neurons. In this study, we investigated the role of Us3 and Us9 during BHV-1 infection in the respiratory mucosa. First, we constructed and characterized BHV-1 Us3 null, Us9 null and revertant viruses. Then, we analysed the viral replication and plaque size (latitude) in Madin-Darby bovine kidney (MDBK) cells and the respiratory mucosa as well as viral penetration depth underneath the BM of the respiratory mucosa when inoculated with these recombinant viruses. Knockout of Us3 resulted in a 1 log10 reduction in viral titre and plaque size (latitude) in MDBK cells and the trachea mucosa. There were no defects in the cell-to-cell spread observed for BHV-1 Us9 null virus. Both BHV-1 Us3 null and Us9 null viruses showed a significant reduction of plaque penetration underneath the BM; however, penetration was not completely inhibited. In conclusion, the current findings demonstrated that Us3 and Us9 play an important role in the invasion of BHV-1 through the BM of the respiratory mucosa, which shows the way forward for research-based attenuation of viruses in order to make safer and better-performing vaccines.

%B J Gen Virol %8 2017 May 18 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28516841?dopt=Abstract %R 10.1099/jgv.0.000764 %0 Journal Article %J J Gen Virol %D 2016 %T Age- and strain-dependent differences in the outcome of experimental infections of domestic pigs with wild boar pseudorabies virus isolates. %A Verpoest, Sara %A Ann Brigitte Cay %A Willem Van Campe %A Laurent Mostin %A Sarah Welby %A Favoreel, Herman %A Nick De Regge %K Animals %K Disease Models, Animal %K Disease Transmission, Infectious %K Europe %K Herpesvirus 1, Suid %K Pseudorabies %K Sus scrofa %K Swine %K Swine Diseases %K Treatment Outcome %X

Although pseudorabies virus (PRV) has been eradicated in domestic swine in many countries, its presence in wild boars remains a threat for a reintroduction into the currently unprotected swine population. To assess the possible impact of such a reintroduction in a naive herd, an in vivo infection study using two genetically characterized wild boar PRV isolates (BEL24043 and BEL20075) representative for wild boar strains circulating in south-western and central Europe and the virulent NIA3 reference strain was performed in 2- and 15-week-old domestic pigs. Our study revealed an attenuated nature of both wild boar strains in 15-week-old pigs. In contrast, it showed the capacity of strain BEL24043 to induce severe clinical symptoms and mortality in young piglets, thereby confirming that the known age dependency of disease outcome after PRV infection also holds for wild boar isolates. Despite the absence of clinical disease in 15-week-old sows, both wild boar PRV strains were able to induce seroconversion, but to a different extent. Importantly, differences in infection and transmission capacity of both strains were observed in 15-week-old sows. Strain BEL24043 induced a more prolonged and disseminated infection than strain BEL20075 and was able to spread efficiently to contact animals, indicative of its capacity to induce a sustained infection. In conclusion, it was shown that a reintroduction of a wild boar isolate into the domestic swine population could have serious economic consequences due to the induction of clinical symptoms in piglets and by jeopardizing the PRV-negative status.

%B J Gen Virol %V 97 %P 487-495 %8 2016 Feb %G eng %N 2 %1 https://www.ncbi.nlm.nih.gov/pubmed/26589961?dopt=Abstract %R 10.1099/jgv.0.000347 %0 Journal Article %J Porcine Health Manag %D 2016 %T Clinical problems due to encephalomyocarditis virus infections in two pig herds. %A Vansteenkiste, Klaas %A Van Limbergen, Tommy %A Decaluwé, Ruben %A Marylène Tignon %A Ann Brigitte Cay %A Maes, Dominiek %X

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.

%B Porcine Health Manag %V 2 %P 19 %8 2016 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28405445?dopt=Abstract %R 10.1186/s40813-016-0036-z %0 Journal Article %J PLoS One %D 2016 %T 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. %A Nick De Regge %A Ann Brigitte Cay %K Animals %K Antibodies, Viral %K Belgium %K farms %K nucleic acids %K Porcine Reproductive and Respiratory Syndrome %K Porcine respiratory and reproductive syndrome virus %K Saliva %K Sensitivity and Specificity %K Swine %K Weaning %X

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.

%B PLoS One %V 11 %P e0166300 %8 2016 %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/27820859?dopt=Abstract %R 10.1371/journal.pone.0166300 %0 Journal Article %J J Gen Virol %D 2016 %T Pseudorabies virus isolates from domestic pigs and wild boars show no apparent in vitro differences in replication kinetics and sensitivity to interferon-induced antiviral status. %A Verpoest, Sara %A Ann Brigitte Cay %A Favoreel, Herman %A Nick De Regge %K Animals %K Antiviral Agents %K Cells, Cultured %K Herpesvirus 1, Suid %K Interferons %K Sus scrofa %K Viral Plaque Assay %K Virus Replication %X

Pseudorabies virus is the causative agent of Aujeszky's disease. Domestic pigs and wild boars are its natural hosts, and strains circulating within both populations differ in their capacity to induce clinical disease. Cell biological and molecular explanations for the observed differences in virulence are, however, lacking. Different virulence determinants that can be assessed in vitro were determined for five domestic swine strains, four wild boar strains and the NIA3 reference strain. Replication kinetics and plaque formation capacity in continuous swine testicular cells and different primary porcine cell lines were highly similar for isolates from both populations. Treatment of these cell lines with IFNα, IFNγ or a combination of both provoked similar plaque-reducing effects for all strains. In conclusion, our results indicate that isolates from domestic swine and wild boar differ neither in intrinsic replication and dissemination capacity nor in sensitivity to antiviral effects of IFNs.

%B J Gen Virol %V 97 %P 473-9 %8 2016 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/26590089?dopt=Abstract %R 10.1099/jgv.0.000348 %0 Journal Article %J Vet Microbiol %D 2016 %T Reconstruction of the Schmallenberg virus epidemic in Belgium: Complementary use of disease surveillance approaches. %A Poskin, Antoine %A Théron, Léonard %A Jean-Baptiste Hanon %A Saegerman, Claude %A Vervaeke, Muriel %A Yves Van der Stede %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Animals, Wild %K Belgium %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K Ceratopogonidae %K Computer Simulation %K Orthobunyavirus %K Population Surveillance %K Reverse Transcriptase Polymerase Chain Reaction %K Seroepidemiologic Studies %X

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.

%B Vet Microbiol %V 183 %P 50-61 %8 2016 Feb 01 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26790935?dopt=Abstract %R 10.1016/j.vetmic.2015.11.036 %0 Journal Article %J Med Vet Entomol %D 2015 %T Culicoides monitoring in Belgium in 2011: analysis of spatiotemporal abundance, species diversity and Schmallenberg virus detection. %A Nick De Regge %A De Deken, R %A Fassotte, C %A Losson, B %A Deblauwe, I %A Madder, M %A Vantieghem, P %A Tomme, M %A Smeets, F %A Ann Brigitte Cay %K Animals %K Belgium %K Bunyaviridae Infections %K Ceratopogonidae %K Insect Vectors %K Orthobunyavirus %K polymerase chain reaction %K Population Density %K Species Specificity %X

In 2011, Culicoides (Diptera: Ceratopogonidae) were collected at 16 locations covering four regions of Belgium with Onderstepoort Veterinary Institute (OVI) traps and at two locations with Rothamsted suction traps (RSTs). Quantification of the collections and morphological identification showed important variations in abundance and species diversity between individual collection sites, even for sites located in the same region. However, consistently higher numbers of Culicoides midges were collected at some sites compared with others. When species abundance and diversity were analysed at regional level, between-site variation disappeared. Overall, species belonging to the subgenus Avaritia together with Culicoides pulicaris (subgenus Culicoides) were the most abundant, accounting for 80% and 96% of all midges collected with RSTs and OVI traps, respectively. Culicoides were present during most of the year, with Culicoides obsoletus complex midges found from 9 February until 27 December. Real-time reverse-transcription polymerase chain reaction screening for Schmallenberg virus in the heads of collected midges resulted in the first detection of the virus in August 2011 and identified C. obsoletus complex, Culicoides chiopterus and Culicoides dewulfi midges as putative vector species. At Libramont in the south of Belgium, no positive pools were identified.

%B Med Vet Entomol %V 29 %P 263-75 %8 2015 Sep %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/25761054?dopt=Abstract %R 10.1111/mve.12109 %0 Journal Article %J PLoS One %D 2015 %T Detection and Isolation of Swine Influenza A Virus in Spiked Oral Fluid and Samples from Individually Housed, Experimentally Infected Pigs: Potential Role of Porcine Oral Fluid in Active Influenza A Virus Surveillance in Swine. %A Decorte, Inge %A Mieke Steensels %A Bénédicte Lambrecht %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Chick Embryo %K Influenza A Virus, H1N1 Subtype %K Influenza A Virus, H3N2 Subtype %K Real-Time Polymerase Chain Reaction %K Reverse Transcriptase Polymerase Chain Reaction %K Saliva %K Swine %X

BACKGROUND: The lack of seasonality of swine influenza A virus (swIAV) in combination with the capacity of swine to harbor a large number of co-circulating IAV lineages, resulting in the risk for the emergence of influenza viruses with pandemic potential, stress the importance of swIAV surveillance. To date, active surveillance of swIAV worldwide is barely done because of the short detection period in nasal swab samples. Therefore, more sensitive diagnostic methods to monitor circulating virus strains are requisite.

METHODS: qRT-PCR and virus isolations were performed on oral fluid and nasal swabs collected from individually housed pigs that were infected sequentially with H1N1 and H3N2 swIAV strains. The same methods were also applied to oral fluid samples spiked with H1N1 to study the influence of conservation time and temperature on swIAV infectivity and detectability in porcine oral fluid.

RESULTS: All swIAV infected animals were found qRT-PCR positive in both nasal swabs and oral fluid. However, swIAV could be detected for a longer period in oral fluid than in nasal swabs. Despite the high detectability of swIAV in oral fluid, virus isolation from oral fluid collected from infected pigs was rare. These results are supported by laboratory studies showing that the PCR detectability of swIAV remains unaltered during a 24 h incubation period in oral fluid, while swIAV infectivity drops dramatically immediately upon contact with oral fluid (3 log titer reduction) and gets lost after 24 h conservation in oral fluid at ambient temperature.

CONCLUSIONS: Our data indicate that porcine oral fluid has the potential to replace nasal swabs for molecular diagnostic purposes. The difficulty to isolate swIAV from oral fluid could pose a drawback for its use in active surveillance programs.

%B PLoS One %V 10 %P e0139586 %8 2015 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/26431039?dopt=Abstract %R 10.1371/journal.pone.0139586 %0 Journal Article %J J Vet Diagn Invest %D 2015 %T Diagnosis of the Lelystad strain of Porcine reproductive and respiratory syndrome virus infection in individually housed pigs: comparison between serum and oral fluid samples for viral nucleic acid and antibody detection. %A Decorte, Inge %A Willem Van Campe %A Laurent Mostin %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Antibodies, Viral %K Enzyme-Linked Immunosorbent Assay %K Housing, Animal %K Mouth %K Porcine Reproductive and Respiratory Syndrome %K Porcine respiratory and reproductive syndrome virus %K Reverse Transcriptase Polymerase Chain Reaction %K Swine %X

There has been a developing interest in the use of oral fluid for the diagnosis of different pathogens such as Porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV and PRRSV-specific antibodies have been shown to be present in oral fluid samples, but the correlation between diagnostic results in oral fluid and serum samples has been insufficiently addressed. Studies investigating this correlation focused on boars older than 6 months and type 2 strains, but it is known that the outcome of a PRRSV infection is age and strain dependent. To address this gap, the current study reports on the detection of PRRSV and PRRSV-specific antibodies in serum and oral fluid samples collected over a 6-week period after an experimental infection of 8-week-old individually housed pigs with Lelystad virus, the type 1 prototype strain. Quantitative reverse transcription polymerase chain reaction analysis showed that significantly more serum samples were PRRSV RNA-positive than oral fluid until 5 days postinfection (dpi). Between 7 and 21 dpi, PRRSV RNA detection was similar in both samples but higher detection rates in oral fluid were found from 28 dpi. Compared with existing literature, this highlights that detection rates at particular time points postinfection might vary in function of strain virulence and animal age and provides useful information for the interpretation of pen-based oral fluid results. An excellent agreement between the oral fluid and serum enzyme-linked immunosorbent assay results was observed at every time point, further supporting the usefulness of oral fluid as a diagnostic sample for antibody detection.

%B J Vet Diagn Invest %V 27 %P 47-54 %8 2015 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25525137?dopt=Abstract %R 10.1177/1040638714561252 %0 Journal Article %J J Vet Diagn Invest %D 2015 %T European interlaboratory comparison of Schmallenberg virus (SBV) real-time RT-PCR detection in experimental and field samples: The method of extraction is critical for SBV RNA detection in semen. %A Schulz, Claudia %A van der Poel, Wim H M %A Ponsart, Claire %A Ann Brigitte Cay %A Steinbach, Falko %A Zientara, Stéphan %A Beer, Martin %A Hoffmann, Bernd %K Animals %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K Europe %K Orthobunyavirus %K Real-Time Polymerase Chain Reaction %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Viral %K semen %K Sensitivity and Specificity %X

Molecular methods for the detection of Schmallenberg virus (SBV) RNA were rapidly developed after the emergence of this novel orthobunyavirus in Europe. The SBV epizootic wave has declined, but infectious SBV in SBV RNA-positive semen remains a possible risk for the distribution of SBV. However, the abilities of SBV molecular detection methods used at European laboratories have not yet been assessed, to our knowledge. The performances of extraction and real-time reverse transcription polymerase chain reaction (RT-qPCR) methods used at 27 German and 17 other European laboratories for SBV RNA detection in the matrices of whole blood, serum, tissue homogenate, RNA eluates, and bovine semen were evaluated in 2 interlaboratory trials with special emphasis on semen extraction methods. For reliable detection of viral genome in bovine semen samples, highly effective extraction methods are essential to cope with the potential inhibitory effects of semen components on PCR results. All methods used by the 44 laboratories were sufficiently robust to detect SBV RNA with high diagnostic sensitivity (100%) and specificity (95.8%) in all matrices, except semen. The trials demonstrated that the published recommended semen extraction methods (Hoffmann et al. 2013) and a combination of TRIzol LS with an alternative extraction kit have a considerably higher diagnostic sensitivity to detect SBV RNA in semen up to a detection limit of Cq ≤35 compared to other extraction methods used. A thorough validation of extraction methods with standardized semen batches is essential before their use for SBV RNA detection in bovine semen.

%B J Vet Diagn Invest %V 27 %P 422-30 %8 2015 Jul %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/26185122?dopt=Abstract %R 10.1177/1040638715593798 %0 Journal Article %J PLoS One %D 2015 %T Experimental Infection of Sheep at 45 and 60 Days of Gestation with Schmallenberg Virus Readily Led to Placental Colonization without Causing Congenital Malformations. %A Martinelle, Ludovic %A Poskin, Antoine %A Dal Pozzo, Fabiana %A Nick De Regge %A Ann Brigitte Cay %A Saegerman, Claude %K Animals %K Animals, Newborn %K Antibodies, Viral %K Bunyaviridae Infections %K Cercopithecus aethiops %K Congenital Abnormalities %K Female %K Gestational Age %K Infectious Disease Transmission, Vertical %K Male %K Orthobunyavirus %K Placenta %K Pregnancy %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Viral %K Sheep %K Sheep Diseases %K Time Factors %K Vero Cells %X

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.

%B PLoS One %V 10 %P e0139375 %8 2015 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/26418420?dopt=Abstract %R 10.1371/journal.pone.0139375 %0 Journal Article %J Transbound Emerg Dis %D 2015 %T Follow-up of the Schmallenberg Virus Seroprevalence in Belgian Cattle. %A Estelle Méroc %A Poskin, A %A Van Loo, H %A Van Driessche, E %A Czaplicki, G %A Quinet, C %A Riocreux, F %A Nick De Regge %A Ann Brigitte Cay %A Thierry van den Berg %A Hooyberghs, J %A Yves Van der Stede %K Animals %K Belgium %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K cross-sectional studies %K Follow-Up Studies %K Orthobunyavirus %K Risk Factors %K Seasons %K Seroepidemiologic Studies %X

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.

%B Transbound Emerg Dis %V 62 %P e80-4 %8 2015 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/24330658?dopt=Abstract %R 10.1111/tbed.12202 %0 Journal Article %J Vet Res %D 2015 %T 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. %A Poskin, Antoine %A Verite, Stephanie %A Comtet, Loïc %A Yves Van der Stede %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Bunyaviridae Infections %K Enzyme-Linked Immunosorbent Assay %K Female %K Immunity, Innate %K Neutralization Tests %K Nucleocapsid Proteins %K Orthobunyavirus %K Real-Time Polymerase Chain Reaction %K RNA, Viral %K Sheep %K Sheep Diseases %X

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.

%B Vet Res %V 46 %P 119 %8 2015 Oct 15 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26472116?dopt=Abstract %R 10.1186/s13567-015-0260-6 %0 Journal Article %J Infect Ecol Epidemiol %D 2015 %T Serologic screening for 13 infectious agents in roe deer (Capreolus capreolus) in Flanders. %A Tavernier, Paul %A Sys, Stanislas U %A Kris De Clercq %A Ilse De Leeuw %A Ann Brigitte Cay %A Miet De Baere %A Nick De Regge %A David Fretin %A Virginie Roupie %A Govaerts, Marc %A Heyman, Paul %A Vanrompay, Daisy %A Yin, Lizi %A Kalmar, Isabelle %A Vanessa Suin %A Bernard Brochier %A Alexandre Dobly %A Stéphane De Craeye %A Sophie Roelandt %A Goossens, Els %A S. Roels %X

INTRODUCTION: In order to investigate the role of roe deer in the maintenance and transmission of infectious animal and human diseases in Flanders, we conducted a serologic screening in 12 hunting areas.

MATERIALS AND METHODS: Roe deer sera collected between 2008 and 2013 (n=190) were examined for antibodies against 13 infectious agents, using indirect enzyme-linked immunosorbent assay, virus neutralisation, immunofluorescence, or microagglutination test, depending on the agent.

RESULTS AND DISCUSSION: High numbers of seropositives were found for Anaplasma phagocytophilum (45.8%), Toxoplasma gondii (43.2%) and Schmallenberg virus (27.9%), the latter with a distinct temporal distribution pattern following the outbreak in domestic ruminants. Lower antibody prevalence was found for Chlamydia abortus (6.7%), tick-borne encephalitis virus (5.1%), Neospora caninum (4.8%), and Mycobacterium avium subsp paratuberculosis (4.1%). The lowest prevalences were found for Leptospira (1.7%), bovine viral diarrhoea virus 1 (1.3%), and Coxiella burnetii (1.2%). No antibodies were found against Brucella sp., bovine herpesvirus 1, and bluetongue virus. A significant difference in seroprevalence between ages (higher in adults >1 year) was found for N. caninum. Four doubtful reacting sera accounted for a significant difference in seroprevalence between sexes for C. abortus (higher in females).

CONCLUSIONS: Despite the more intensive landscape use in Flanders, the results are consistent with other European studies. Apart from maintaining C. abortus and MAP, roe deer do not seem to play an important role in the epidemiology of the examined zoonotic and domestic animal pathogens. Nevertheless, their meaning as sentinels should not be neglected in the absence of other wild cervid species.

%B Infect Ecol Epidemiol %V 5 %P 29862 %8 2015 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26609692?dopt=Abstract %R 10.3402/iee.v5.29862 %0 Journal Article %J Vector Borne Zoonotic Dis %D 2014 %T Autochthonous tick-borne encephalitis virus-seropositive cattle in Belgium: a risk-based targeted serological survey. %A Sophie Roelandt %A Vanessa Suin %A Riocreux, Flavien %A Lamoral, Sophie %A Van der Heyden, Sara %A Yves Van der Stede %A Bénédicte Lambrecht %A Ann Brigitte Cay %A Bernard Brochier %A S. Roels %A Steven Van Gucht %K Animals %K Antibodies, Viral %K Arachnid Vectors %K Belgium %K Cattle %K Cattle Diseases %K cross-sectional studies %K Encephalitis Viruses, Tick-Borne %K Encephalitis, Tick-Borne %K Female %K Humans %K Ixodes %K mice %K risk %K Sentinel Surveillance %K Seroepidemiologic Studies %K Zoonoses %X

The risk of tick-borne encephalitis virus (TBEV) introduction into Belgium remains high, and the presence of infected wildlife in Belgium is suspected. Domestic animals can serve as excellent sentinels for TBEV surveillance to install an early warning surveillance component for this emerging zoonotic disease of public health importance. In a targeted, risk-based and cross-sectional sampling design, serological screening was performed on Belgian cattle (n=650), selected from the 2010 Belgian national cattle surveillance serum bank. All samples were subjected to a gold standard TBEV seroneutralization test (SNT), based on the rapid fluorescent focus inhibition test (RFFIT) protocol. Seventeen bovines were seropositive (titer >1/15) and six had borderline results (1/10 < titer < 1/15). The accuracy of the RFFIT-SNT was confirmed in a mouse inoculation test. The overall bovine TBEV seroprevalence in the targeted area was estimated between 2.61% and 4.29%. This confirms for the first time the presence of infected foci in Belgium. Further surveillance in cattle, other sentinels, ticks, and humans at risk is recommended to further determine the location and size of endemic foci and the risk for public health.

%B Vector Borne Zoonotic Dis %V 14 %P 640-7 %8 2014 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/25229702?dopt=Abstract %R 10.1089/vbz.2014.1576 %0 Journal Article %J BMC Vet Res %D 2014 %T Detection of total and PRRSV-specific antibodies in oral fluids collected with different rope types from PRRSV-vaccinated and experimentally infected pigs. %A Decorte, Inge %A Van Breedam, Wander %A Yves Van der Stede %A Nauwynck, Hans J %A Nick De Regge %A Ann Brigitte Cay %K Animals %K Antibodies, Viral %K cannabis %K Cotton Fiber %K Female %K Immunoglobulin Isotypes %K Nylons %K Polyesters %K Porcine Reproductive and Respiratory Syndrome %K Porcine respiratory and reproductive syndrome virus %K Saliva %K specimen handling %K Swine %K Viral Vaccines %X

BACKGROUND: Oral fluid collected by means of ropes has the potential to replace serum for monitoring and surveillance of important swine pathogens. Until now, the most commonly used method to collect oral fluid is by hanging a cotton rope in a pen. However, concerns about the influence of rope material on subsequent immunological assays have been raised. In this study, we evaluated six different rope materials for the collection of oral fluid and the subsequent detection of total and PRRSV-specific antibodies of different isotypes in oral fluid collected from PRRSV-vaccinated and infected pigs.

RESULTS: An initial experiment showed that IgA is the predominant antibody isotype in porcine saliva. Moreover, it was found that synthetic ropes may yield higher amounts of IgA, whereas all rope types seemed to be equally suitable for IgG collection. Although IgA is the predominant antibody isotype in porcine oral fluid, the PRRSV-specific IgA-based IPMA and ELISA tests were clearly not ideal for sensitive detection of PRRSV-specific IgA antibodies. In contrast, PRRSV-specific IgG in oral fluids was readily detected in PRRSV-specific IgG-based IPMA and ELISA tests, indicating that IgG is a more reliable isotype for monitoring PRRSV-specific antibody immunity in vaccinated/infected animals via oral fluids with the currently available tests.

CONCLUSIONS: Since PRRSV-specific IgG detection seems more reliable than PRRSV-specific IgA detection for monitoring PRRSV-specific antibody immunity via oral fluids, and since all rope types yield equal amounts of IgG, it seems that the currently used cotton ropes are an appropriate choice for sample collection in PRRSV monitoring.

%B BMC Vet Res %V 10 %P 134 %8 2014 Jun 17 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/24938323?dopt=Abstract %R 10.1186/1746-6148-10-134 %0 Journal Article %J Transbound Emerg Dis %D 2014 %T Distinction between persistent and transient infection in a bovine viral diarrhoea (BVD) control programme: appropriate interpretation of real-time RT-PCR and antigen-ELISA test results. %A Jean-Baptiste Hanon %A Yves Van der Stede %A Antonissen, A %A Mullender, C %A Marylène Tignon %A Thierry van den Berg %A Ann Brigitte Cay %K Animals %K Antibodies, Viral %K Antigens, Viral %K Belgium %K Bovine Virus Diarrhea-Mucosal Disease %K Cattle %K Diarrhea Virus 1, Bovine Viral %K Diarrhea Viruses, Bovine Viral %K Enzyme-Linked Immunosorbent Assay %K Follow-Up Studies %K Real-Time Polymerase Chain Reaction %K Retrospective Studies %K RNA, Viral %X

Control of bovine viral diarrhoea (BVD) in Belgium is currently implemented on a voluntary basis at herd level and mainly relies on detection and culling of persistently infected (PI) animals. The present field study was conducted during the winter of 2010/2011 to assess the performances of diagnostic assays used in the testing scheme for BVD as proposed by the two Belgian regional laboratories. Individual blood samples were collected from 4972 animals, and individual samples from the same herd were pooled (maximum of 30 individual samples per pool) and screened for the presence of Bovine Viral Diarrhoea Virus (BVDV)-specific RNA using a commercial real-time RT-PCR test (ADIAGENE). Individual samples from positive pools were then tested in parallel with the same RT-PCR test and with an antigen-capture ELISA test (IDEXX) to detect viremic animals. This study demonstrated that individual results differed according to the type of assay used (P < 0.001): 140 animals (2.8%) were positive by RT-PCR and 72 (1.4%) by antigen-ELISA. A second blood sample was taken 40 days later from 74 PCR positive animals to detect persistent viremia: 17 (23%) of these were still PCR positive and considered to be PI and the 57 that no longer tested positive were assumed to be transiently infected (TI) animals. All PI animals were positive also by antigen-ELISA at both time points. Among TI animals, 10 (16%) were positive by antigen-ELISA at the first but none at the second sampling. A highly significant difference in cycle threshold (Ct ) values obtained by RT-PCR was observed between PI and TI animals. ROC analysis was performed to establish thresholds to confirm with high probability that an animal is PI, based on the result of RT-PCR test performed on a single individual blood sample.

%B Transbound Emerg Dis %V 61 %P 156-62 %8 2014 Apr %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/23009318?dopt=Abstract %R 10.1111/tbed.12011 %0 Journal Article %J Transbound Emerg Dis %D 2014 %T Distribution of Schmallenberg virus and seroprevalence in Belgian sheep and goats. %A Estelle Méroc %A Nick De Regge %A Riocreux, F %A Ann Brigitte Cay %A Thierry van den Berg %A Yves Van der Stede %K Animals %K Belgium %K Bunyaviridae Infections %K Epidemics %K Goat Diseases %K Goats %K Orthobunyavirus %K Seroepidemiologic Studies %K Sheep %K Sheep Diseases %X

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

%B Transbound Emerg Dis %V 61 %P 425-31 %8 2014 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/23305427?dopt=Abstract %R 10.1111/tbed.12050 %0 Journal Article %J Vet J %D 2014 %T Dose-dependent effect of experimental Schmallenberg virus infection in sheep. %A Poskin, A %A Martinelle, L %A Laurent Mostin %A Willem Van Campe %A dal Pozzo, F %A Saegerman, C %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Bunyaviridae Infections %K Cattle %K Enzyme-Linked Immunosorbent Assay %K Female %K Orthobunyavirus %K polymerase chain reaction %K Sheep %K Sheep Diseases %K Viremia %X

Schmallenberg virus (SBV) is an orthobunyavirus affecting European domestic ruminants. In this study, the dose-dependent effect of experimental infection of sheep with SBV was evaluated. Four groups of three ewes were each inoculated subcutaneously with 1 mL of successive 10-fold dilutions of an SBV infectious serum. The ewes were monitored for 10 days, but no clinical signs were observed. The number of productively infected animals within each group, as evidenced by viraemia, seroconversion and viral RNA in the organs, depended on the inoculated dose, indicating that a critical dose has to be administered to obtain a homogeneous response in infected animals under experimental conditions. In the productively infected animals, no statistical differences between the different inoculation doses were found in the duration or quantity of viral RNA circulating in blood, nor in the amount of viral RNA present in virus positive lymphoid organs.

%B Vet J %V 201 %P 419-22 %8 2014 Sep %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/24954869?dopt=Abstract %R 10.1016/j.tvjl.2014.05.031 %0 Journal Article %J Transbound Emerg Dis %D 2014 %T Epidemiology and genetic characterization of equine infectious anaemia virus strains isolated in Belgium in 2010. %A Ann Brigitte Cay %A Marylène Tignon %K Animals %K Base Sequence %K Belgium %K Equine Infectious Anemia %K Horses %K Infectious Anemia Virus, Equine %K Molecular Sequence Data %K Phylogeny %K Romania %K Transportation %K United Kingdom %X

In January 2010, the United Kingdom notified cases of equine infectious anaemia (EIA) in two horses introduced from Belgium. The animals came from one assembly centre in Romania and had transited through Belgium with 16 other horses. Nine of them, bought by a Belgian horse breeder, were investigated in Belgium and revealed one additional EIA-positive animal. Afterwards, the Belgian Federal Agency for the Safety of the Food Chain (FASFC) organized a serological EIA survey of the horses introduced into Belgium from Romania between 2007 and 2009. Among the 95 horses identified, six additional serological positive cases were found that had been introduced into Belgium in 2008 (n = 4) and in 2009 (n = 2). The survey was extended to the horses in contact with the positive cases, but all contact animals were negative, indicating the absence of transmission. Virological examination performed on tissue samples collected from two seropositive animals demonstrated the presence of viral DNA of EIA virus. Phylogenetic analysis based on the sequences of EIA virus gag gene clustered the Belgian isolates with Romanian strains isolated in 2009. The presumption of a common Belgian origin could be rejected.

%B Transbound Emerg Dis %V 61 %P 464-8 %8 2014 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/23173784?dopt=Abstract %R 10.1111/tbed.12031 %0 Journal Article %J Vet Microbiol %D 2014 %T Experimental Schmallenberg virus infection of pigs. %A Poskin, Antoine %A Willem Van Campe %A Laurent Mostin %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Bunyaviridae Infections %K Feces %K Orthobunyavirus %K Swine %X

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.

%B Vet Microbiol %V 170 %P 398-402 %8 2014 Jun 04 %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/24679959?dopt=Abstract %R 10.1016/j.vetmic.2014.02.026 %0 Journal Article %J European Journal of Wildlife Research %D 2014 %T Isolation and characterization of pseudorabies virus from a wolf (Canis lupus) from Belgium %A Verpoest, Sara %A Ann Brigitte Cay %A Bertrand, Olivier %A Saulmont, Marc %A Nick De Regge %K Belgium %K pseudorabies virus %K wolf %B European Journal of Wildlife Research %V 6010574930126969143277316113815614693318912315733 %8 Jan-02-2014 %G eng %N 1 %R 10.1007/s10344-013-0774-z %0 Journal Article %J Vet Rec %D 2014 %T Limited interlaboratory comparison of Schmallenberg virus antibody detection in serum samples. %A van der Poel, W H M %A Ann Brigitte Cay %A Zientara, S %A Steinbach, F %A Valarcher, J F %A Bøtner, A %A Mars, M H %A Hakze-van der Honing, R %A Schirrmeier, H %A Beer, M %K Animals %K Antibodies, Viral %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K Enzyme-Linked Immunosorbent Assay %K Europe %K Neutralization Tests %K Orthobunyavirus %K Sensitivity and Specificity %K Sheep %K Sheep Diseases %X

Eight veterinary institutes in seven different countries in Europe participated in a limited interlaboratory comparison trial to evaluate laboratory performances of Schmallenberg virus (SBV) antibody detection in serum. Seven different sheep sera and three different cattle sera were circulated, and all participating institutes were asked to test these sera using SBV antibody detection assay(s) in place in their laboratories. All laboratories within the trial performed a virus neutralisation test (VNT) as well as one or two ELISAs on all samples, and swiftly detected SBV antibodies using these assays. VNT was more sensitive in detecting SBV antibodies than several of the used ELISA assays. Based on the test results, one cattle and one sheep SBV antibody-positive serum were selected to serve as reference sera, which now can be supplied to other laboratories on request.

%B Vet Rec %V 174 %P 380 %8 2014 Apr 12 %G eng %N 15 %1 http://www.ncbi.nlm.nih.gov/pubmed/24591480?dopt=Abstract %R 10.1136/vr.102180 %0 Journal Article %J Vet Microbiol %D 2014 %T Molecular characterization of Belgian pseudorabies virus isolates from domestic swine and wild boar. %A Verpoest, Sara %A Ann Brigitte Cay %A Nick De Regge %K Animals %K Belgium %K Disease Reservoirs %K Genotype %K Herpesvirus 1, Suid %K Phylogeny %K Pseudorabies %K Sus scrofa %K Swine %K Swine Diseases %X

Aujeszky's disease is an economically important disease in domestic swine caused by suid herpesvirus 1, also called pseudorabies virus (PRV). In several European countries, including Belgium, the virus has successfully been eradicated from the domestic swine population. The presence of PRV in the wild boar population however poses a risk for possible reintroduction of the virus into the domestic pig population. It is therefore important to assess the genetic relatedness between circulating strains and possible epidemiological links. In this study, nine historical Belgian domestic swine isolates that circulated before 1990 and five recent wild boar isolates obtained since 2006 from Belgium and the Grand Duchy of Luxembourg were genetically characterized by restriction fragment length polymorphism (RFLP) analysis and phylogenetic analysis. While all wild boar isolates were characterized as type I RFLP genotypes, the RFLP patterns of the domestic swine isolates suggest that a shift from genotype I to genotype II might have occurred in the 1980s in the domestic population. By phylogenetic analysis, Belgian wild boar isolates belonging to both clade A and B were observed, while all domestic swine isolates clustered within clade A. The joint phylogenetic analysis of both wild boar and domestic swine strains showed that some isolates with identical sequences were present within both populations, raising the question whether these strains represent an increased risk for reintroduction of the virus into the domestic population.

%B Vet Microbiol %V 172 %P 72-7 %8 2014 Aug 06 %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/24908275?dopt=Abstract %R 10.1016/j.vetmic.2014.05.001 %0 Journal Article %J PLoS One %D 2014 %T Schmallenberg virus circulation in culicoides in Belgium in 2012: field validation of a real time RT-PCR approach to assess virus replication and dissemination in midges. %A Nick De Regge %A Madder, Maxime %A Deblauwe, Isra %A Losson, Bertrand %A Fassotte, Christiane %A Demeulemeester, Julie %A Smeets, François %A Tomme, Marie %A Ann Brigitte Cay %K Animals %K Belgium %K Bunyaviridae Infections %K Ceratopogonidae %K Insect Vectors %K Orthobunyavirus %K Real-Time Polymerase Chain Reaction %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Viral %K Virus Replication %X

Indigenous Culicoides biting midges are suggested to be putative vectors for the recently emerged Schmallenberg virus (SBV) based on SBV RNA detection in field-caught midges. Furthermore, SBV replication and dissemination has been evidenced in C. sonorensis under laboratory conditions. After SBV had been detected in Culicoides biting midges from Belgium in August 2011, it spread all over the country by the end of 2011, as evidenced by very high between-herd seroprevalence rates in sheep and cattle. This study investigated if a renewed SBV circulation in midges occurred in 2012 in the context of high seroprevalence in the animal host population and evaluated if a recently proposed realtime RT-PCR approach that is meant to allow assessing the vector competence of Culicoides for SBV and bluetongue virus under laboratory conditions was applicable to field-caught midges. Therefore midges caught with 12 OVI traps in four different regions in Belgium between May and November 2012, were morphologically identified, age graded, pooled and tested for the presence of SBV RNA by realtime RT-PCR. The results demonstrate that although no SBV could be detected in nulliparous midges caught in May 2012, a renewed but short lived circulation of SBV in parous midges belonging to the subgenus Avaritia occured in August 2012 at all four regions. The infection prevalence reached up to 2.86% in the south of Belgium, the region where a lower seroprevalence was found at the end of 2011 than in the rest of the country. Furthermore, a frequency analysis of the Ct values obtained for 31 SBV-S segment positive pools of Avaritia midges showed a clear bimodal distribution with peaks of Ct values between 21-24 and 33-36. This closely resembles the laboratory results obtained for SBV infection of C. sonorensis and implicates indigenous midges belonging to the subgenus Avaritia as competent vectors for SBV.

%B PLoS One %V 9 %P e87005 %8 2014 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/24466312?dopt=Abstract %R 10.1371/journal.pone.0087005 %0 Journal Article %J Vet Microbiol %D 2014 %T Study of the virulence of serotypes 4 and 9 of African horse sickness virus in IFNAR(-/-), Balb/C and 129 Sv/Ev mice. %A de la Grandière, Maria Ana %A Dal Pozzo, Fabiana %A Marylène Tignon %A Zonta, William %A Thiry, Damien %A Mauroy, Axel %A Elisabeth Mathijs %A Ann Brigitte Cay %A Saegerman, Claude %A Thiry, Etienne %K African Horse Sickness %K African Horse Sickness Virus %K Animals %K Female %K Horses %K Interferon-alpha %K mice %K Mice, Inbred BALB C %K Mice, Knockout %K Receptor, Interferon alpha-beta %K RNA, Double-Stranded %K Serogroup %K virulence %X

African horse sickness virus (AHSV) is a double-stranded RNA virus which belongs to the family Reoviridae, genus Orbivirus. Recent studies have focused on the interferon-α/β receptor knock-out mice (IFNAR(-/-)) as a small animal laboratory for the development of AHSV vaccines. The aim of this work was to study in vivo the virulence of two strains of AHSV and to compare the outcome of the infection of three mouse strains. To address this, AHSV serotypes 4 (AHSV-4) and 9 (AHSV-9) were inoculated subcutaneously (SC) and intranasally (IN) in two immunocompetent mouse strains (Balb/C and 129 Sv/Ev (129 WT)) as well as IFNAR(-/-) mice (on 129 Sv/Ev genetic background). In IFNAR(-/-) mice, fatality up to 50% was measured and significantly more clinical signs were observed in comparison with SC inoculated immunocompetent mice. The observed clinical signs were significantly more severe after AHSV-4 infection, in particular in immunocompetent mice inoculated by IN route. Considering RNAemia, significantly higher viral loads were measured following AHSV-4 infection. In the organs of 129 WT inoculated by IN route, significantly higher viral loads were detected after AHSV-4 infection. Together the results support a higher virulence for AHSV-4 compared to AHSV-9 and a higher clinical impact following infections in IN inoculated mice, at least in the investigated strains. The study also brought indirect evidences for type I IFN involvement in the control of AHSV infection.

%B Vet Microbiol %V 174 %P 322-332 %8 2014 Dec 05 %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/25458420?dopt=Abstract %R 10.1016/j.vetmic.2014.10.006 %0 Journal Article %J Prev Vet Med %D 2014 %T A survey on biosecurity and management practices in selected Belgian cattle farms. %A Sarrazin, Steven %A Ann Brigitte Cay %A Laureyns, Jozef %A Dewulf, Jeroen %K Animal Distribution %K Animal Husbandry %K Animals %K Belgium %K Cattle %K Cattle Diseases %K Communicable Disease Control %K Data collection %K Surveys and Questionnaires %X

The shift from cure towards prevention in veterinary medicine involves the implementation of biosecurity, which includes all measures preventing pathogens from entering a herd and reducing the spread of pathogens within a herd. In Belgium no studies have considered the implementation of biosecurity measures in the daily management of cattle farms. Therefore the aim of the study was to map the current application of biosecurity measures in Belgian cattle farms in the prevention of disease transmission within and between farms. Between March 2011 and April 2013 the data were collected as part of a larger cross-sectional study, conducted to identify risk factors for reinfection with BVDV in cattle herds assumed free from BVDV. Questionnaire data from 33 dairy farms, 16 beef farms and 25 mixed (dairy and beef cattle) farms were analyzed using a combination of a linear scoring system, a categorical principal component analysis and a two-step cluster analysis to differentiate these farms based on their biosecurity levels and visit frequencies. Further enhancement of preventive measures considering external and internal biosecurity was still possible for each farm, as none of the farms obtained an overall high biosecurity level. Three groups of cattle farms were differentiated with a biosecurity level varying from low to high-medium, of which the group with the lowest biosecurity level mainly consisted of mixed farms. Animal-to-animal contacts with cattle from other herds were frequently possible as only 12% of the farmers purchasing cattle quarantined purchased animals at least three weeks and contacts over fences on pasture were possible in 70% of the herds. Basic biosecurity measures such as farm-specific protective clothing and boots were present in the majority of the farms, but they were insufficiently or incorrectly used. Cattle farms were very often visited by professional visitors of which the herd veterinarian, the AI technician and the cattle salesman most frequently entered the farm. It can be concluded that few biosecurity measures were undertaken by Belgian cattle farmers, thereby exposing themselves to the risk of disease transmission within and between farms. Especially in regions with a high cattle density, small distances to neighbouring farms and high frequencies of professional visits, a farm-specific preventive strategy should be developed, thereby using the facilities often already present on the farm.

%B Prev Vet Med %V 117 %P 129-39 %8 2014 Nov 01 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25156945?dopt=Abstract %R 10.1016/j.prevetmed.2014.07.014 %0 Journal Article %J Vet J %D 2014 %T Virulence comparison and quantification of horizontal bovine viral diarrhoea virus transmission following experimental infection in calves. %A Sarrazin, S %A Dewulf, J %A Elisabeth Mathijs %A Laureyns, J %A Laurent Mostin %A Ann Brigitte Cay %K Animals %K Belgium %K Bovine Virus Diarrhea-Mucosal Disease %K Cattle %K Diarrhea Virus 1, Bovine Viral %K Diarrhea Virus 2, Bovine Viral %K virulence %X

Bovine viral diarrhoea virus (BVDV) causes persistent infections by infecting the fetus of susceptible animals during gestation. These persistently infected (PI) animals are important sources of infection. On the contrary, transiently infected (TI) animals are believed to be less important, but transient infections with a severe BVDV-2 strain can spread explosively. To assess the importance of TI cattle in the epidemiology of BVDV, two experimental infections were performed to determine basic reproduction ratios (R0). In each experiment three calves were infected via intranasal inoculation and housed together with seven susceptible animals. Two strains isolated in Belgium were used, a virulent BVDV-1b and a virulent BVDV-2a field isolate, resulting in an R0 of 0.25 (95% CI 0.01; 1.95) and 0.24 (95% CI 0.01; 2.11), respectively. A PI animal was then introduced to the remaining uninfected animals and produced an R of +∞ (95% CI 1.88; +∞). These results support the suggestion that TI animals, compared to PI animals, contribute only a limited amount to BVDV spread. Additionally, the severe clinical symptoms observed in the field with these isolates could not be reproduced during these experiments, suggesting that other factors besides strain virulence influence the clinical manifestations evoked by BVDV.

%B Vet J %V 202 %P 244-9 %8 2014 Nov %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/25201251?dopt=Abstract %R 10.1016/j.tvjl.2014.07.010 %0 Journal Article %J Vet Rec %D 2013 %T Can horses be clinically screened for West Nile Fever? %A van Galen, G %A Calozet, L %A Leblond, A %A Tritz, P %A dal Pozzo, F %A Porter, S R %A Ann Brigitte Cay %A Amory, H %A Saegerman, C %K Animals %K Case-Control Studies %K Diagnosis, Differential %K Disease Outbreaks %K Europe %K Female %K Horse Diseases %K Horses %K Male %K Mass Screening %K Nervous System Diseases %K Sentinel Surveillance %K West Nile Fever %B Vet Rec %V 172 %P 101 %8 2013 Jan 26 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/23292842?dopt=Abstract %R 10.1136/vr.101267 %0 Journal Article %J J Virol Methods %D 2013 %T Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the detection of genotype A strains of small ruminant lentiviruses. %A Nick De Regge %A Ann Brigitte Cay %K Animals %K Belgium %K Goat Diseases %K Goats %K Lentivirus %K Lentivirus Infections %K Molecular Diagnostic Techniques %K Molecular Sequence Data %K Real-Time Polymerase Chain Reaction %K RNA, Viral %K Sensitivity and Specificity %K Sequence Analysis, DNA %K Sheep %K Sheep Diseases %K Veterinary Medicine %X

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.

%B J Virol Methods %V 194 %P 250-7 %8 2013 Dec %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/24045043?dopt=Abstract %R 10.1016/j.jviromet.2013.09.001 %0 Journal Article %J Vet Microbiol %D 2013 %T Diagnosis of Schmallenberg virus infection in malformed lambs and calves and first indications for virus clearance in the fetus. %A Nick De Regge %A Thierry van den Berg %A Georges, Laura %A Ann Brigitte Cay %K Abortion, Veterinary %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Brain Stem %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K Congenital Abnormalities %K Female %K Neutralization Tests %K Orthobunyavirus %K Pregnancy %K Pregnancy Complications, Infectious %K Real-Time Polymerase Chain Reaction %K Sheep %K Sheep Diseases %K Sheep, Domestic %K Stillbirth %X

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.

%B Vet Microbiol %V 162 %P 595-600 %8 2013 Mar 23 %G eng %N 2-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/23265245?dopt=Abstract %R 10.1016/j.vetmic.2012.11.029 %0 Journal Article %J Vet J %D 2013 %T Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR. %A Decorte, Inge %A Yves Van der Stede %A Nauwynck, Hans %A Nick De Regge %A Ann Brigitte Cay %K Animals %K Excipients %K Porcine respiratory and reproductive syndrome virus %K Real-Time Polymerase Chain Reaction %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Viral %K Saliva %K specimen handling %K Swine %X

This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser.

%B Vet J %V 197 %P 224-8 %8 2013 Aug %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/23489844?dopt=Abstract %R 10.1016/j.tvjl.2013.02.001 %0 Journal Article %J Transbound Emerg Dis %D 2013 %T Large-scale cross-sectional serological survey of Schmallenberg virus in Belgian cattle at the end of the first vector season. %A Estelle Méroc %A Poskin, A %A Van Loo, H %A Quinet, C %A Van Driessche, E %A Delooz, L %A Isabelle Behaeghel %A Riocreux, F %A Hooyberghs, J %A Nick De Regge %A Ann Brigitte Cay %A Thierry van den Berg %A Yves Van der Stede %K Animals %K Belgium %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K cross-sectional studies %K Enzyme-Linked Immunosorbent Assay %K Female %K Orthobunyavirus %K Seasons %K Seroepidemiologic Studies %K Serologic Tests %X

A cross-sectional survey was conducted in the Belgian cattle population after the first period of infection of the emerging Schmallenberg virus. A total number of 11 635 cattle from 422 herds sampled between 2 January and 7 March 2012 were tested for the presence of Schmallenberg-specific antibodies using an ELISA kit. Between-herd seroprevalence in cattle was estimated at 99.76% (95% CI: 98.34-99.97) and within-herd seroprevalence at 86.3% (95% CI: 84.75-87.71). An Intraclass Correlation Coefficient of 0.3 (P < 0.001) was found, indicating that the correlation between two animals within a herd with respect to their serological status was high. Those results corroborate the conclusion that the Schmallenberg virus was widespread in Belgium during winter 2011. Seroprevalence was shown to be statistically associated to the animal's age (P < 0.0001): with 64.9% (95% CI: 61.34-68.3) estimated for the 6-12 months of age, 86.79% (95% CI: 84.43-88.85) for the 12-24 months of age and 94.4% (95% CI: 93.14-95.44) for the animals older than 24 months. Based on the results of the described serological survey, we can conclude that after the first Schmallenberg virus episode, almost every Belgian cattle has already been in contact with the virus. In consequence, the vast majority of the host animals should have developed post infection protective immunity against the virus.

%B Transbound Emerg Dis %V 60 %P 4-8 %8 2013 Feb %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/23206240?dopt=Abstract %R 10.1111/tbed.12042 %0 Journal Article %J Prev Vet Med %D 2013 %T Serological and virological BVDV prevalence and risk factor analysis for herds to be BVDV seropositive in Belgian cattle herds. %A Sarrazin, Steven %A Veldhuis, Anouk %A Estelle Méroc %A Vangeel, Ilse %A Laureyns, Jozef %A Dewulf, Jeroen %A Ann Brigitte Cay %A Piepers, Sofie %A Hooyberghs, Jozef %A Ribbens, Stefaan %A Yves Van der Stede %K Animals %K Antibodies, Viral %K Antigens, Viral %K Belgium %K Bovine Virus Diarrhea-Mucosal Disease %K Cattle %K cross-sectional studies %K Diarrhea Viruses, Bovine Viral %K Enzyme-Linked Immunosorbent Assay %K Logistic Models %K prevalence %K Risk Factors %K Seroepidemiologic Studies %K Surveys and Questionnaires %K Vaccination %X

Bovine viral diarrhoea virus (BVDV) is a worldwide spread virus that most commonly infects cattle and can cause considerable economic losses. To determine the prevalence of BVDV in Belgium, a cross-sectional study was performed between November 2009 and March 2010. Young stock aged between 6 and 12 months from 773 randomly selected Belgian cattle herds were tested for BVDV-specific antibodies and antigen. With a target and maximum of 10 animals per sampled herd, a total of 5246 animals were selected. Additionally a questionnaire including different herd management topics and questions about participation in animal health programmes, including BVDV, was sent to 1100 Belgian cattle herds, including the 773 herds for BVDV testing. This paper focuses on results regarding these 773 herds. The true prevalence of BVDV-specific antibodies and antigen at herd level was respectively 47.4% and 4.4%, while at animal level this was respectively 32.9% and 0.3%. In 44.4% of the herds where BVDV-specific antibodies were detected at least 60% of the sampled young stock was BVDV seropositive. Interestingly, 83.4% of these farmers stated not to have suffered from problems related to BVDV. Moreover, only 8.4% of all farmers who completed the questionnaire (n=895) reported problems possibly related to BVDV the past 3 years. This demonstrates that farmers are often unaware of the presence of BVDV in their herd. Risk factors for a herd to be BVDV seropositive were identified by means of a multivariable logistic regression model. Large herds were significantly more likely to be BVDV seropositive (OR=1.004, p<0.01). The interaction between "Antigen positive animal detected in this study" and "BVDV vaccination in 2009" was significant (p<0.01). In non-vaccinating herds, the detection of antigen positive animals was significantly associated with BVDV seropositive herds (OR=13.8, p<0.01). In herds with no antigen positive animals detected, vaccination resulted in a significant risk factor to be BVDV seropositive compared to non-vaccinating herds (OR=3.4, p<0.01). Herds reporting BVDV-related problems the past 3 years were more likely to be BVDV seropositive (OR=1.9, p<0.05). This relation became non-significant (OR=1.8, p=0.08) when only a subset of herds with no vaccination of animals <12 months was taken into account. The results of the current study suggest an active circulation of BVDV in a considerable number of Belgian cattle herds.

%B Prev Vet Med %V 108 %P 28-37 %8 2013 Jan 01 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/22878124?dopt=Abstract %R 10.1016/j.prevetmed.2012.07.005 %0 Journal Article %J PLoS One %D 2013 %T Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies. %A Bréard, Emmanuel %A Lara, Estelle %A Comtet, Loïc %A Viarouge, Cyril %A Doceul, Virginie %A Desprat, Alexandra %A Vitour, Damien %A Pozzi, Nathalie %A Ann Brigitte Cay %A Nick De Regge %A Pourquier, Philippe %A Schirrmeier, Horst %A Hoffmann, Bernd %A Beer, Martin %A Sailleau, Corinne %A Zientara, Stéphan %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Bunyaviridae Infections %K Cattle %K Enzyme-Linked Immunosorbent Assay %K Europe %K Fluorescent Antibody Technique, Indirect %K Gene Expression %K Neutralization Tests %K Nucleocapsid Proteins %K Orthobunyavirus %K Reagent Kits, Diagnostic %K Recombinant Proteins %K Reproducibility of Results %K Roc Curve %K Sheep %X

A newly developed Enzym Like Immuno Sorbant Assay (ELISA) based on the recombinant nucleocapsid protein (N) of Schmallenberg virus (SBV) was evaluated and validated for the detection of SBV-specific IgG antibodies in ruminant sera by three European Reference Laboratories. Validation data sets derived from sheep, goat and bovine sera collected in France and Germany (n = 1515) in 2011 and 2012 were categorized according to the results of a virus neutralization test (VNT) or an indirect immuno-fluorescence assay (IFA). The specificity was evaluated with 1364 sera from sheep, goat and bovine collected in France and Belgium before 2009. Overall agreement between VNT and ELISA was 98.9% and 98.3% between VNT and IFA, indicating a very good concordance between the different techniques. Although cross-reactions with other Orthobunyavirus from the Simbu serogroup viruses might occur, it is a highly sensitive, specific and robust ELISA-test validated to detect anti-SBV antibodies. This test can be applied for SBV sero-diagnostics and disease-surveillance studies in ruminant species in Europe.

%B PLoS One %V 8 %P e53446 %8 2013 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/23335964?dopt=Abstract %R 10.1371/journal.pone.0053446 %0 Journal Article %J Transbound Emerg Dis %D 2012 %T Detection of Schmallenberg virus in different Culicoides spp. by real-time RT-PCR. %A Nick De Regge %A Deblauwe, I %A De Deken, R %A Vantieghem, P %A Madder, M %A Geysen, D %A Smeets, F %A Losson, B %A Thierry van den Berg %A Ann Brigitte Cay %K Animals %K Belgium %K Bunyaviridae Infections %K Cattle %K Cattle Diseases %K Ceratopogonidae %K Insect Vectors %K Orthobunyavirus %K Reverse Transcriptase Polymerase Chain Reaction %K Seasons %X

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.

%B Transbound Emerg Dis %V 59 %P 471-5 %8 2012 Dec %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/23025501?dopt=Abstract %R 10.1111/tbed.12000 %0 Journal Article %J Theriogenology %D 2012 %T Impact of a novel inactivated PRRS virus vaccine on virus replication and virus-induced pathology in fetal implantation sites and fetuses upon challenge. %A Karniychuk, U U %A Saha, D %A Vanhee, M %A Geldhof, M %A Cornillie, P %A Ann Brigitte Cay %A Nick De Regge %A Nauwynck, H J %K Animals %K Antibodies, Viral %K Female %K Fetal Diseases %K Fetus %K Gestational Age %K Placenta %K Porcine Reproductive and Respiratory Syndrome %K Porcine respiratory and reproductive syndrome virus %K Pregnancy %K Pregnancy Complications, Infectious %K Sus scrofa %K Swine %K Vaccination %K Vaccines, Inactivated %K Viral Vaccines %K Viremia %X

Preventing congenital infection is important for the control of porcine reproductive and respiratory syndrome (PRRS). Recently, in our laboratory, an inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine has been developed. Promising results in young pigs encouraged us to test the vaccine potency to prevent congenital infection. In the present study, the performance of this experimental inactivated vaccine was investigated in pregnant gilts. An advanced protocol was used to test the PRRSV vaccine efficacy. This protocol is based on recent insights in the pathogenesis of congenital PRRSV infections. Three gilts were vaccinated with an experimental PRRSV 07V63 inactivated vaccine at 27, 55, and 83 days of gestation. Three unvaccinated gilts were included as controls. At 90 days of gestation, all animals were intranasally inoculated with 10(5) tissue culture infectious dose 50 (TCID(50)) of PRRSV 07V63. Twenty days postchallenge animals were euthanized and sampled. The vaccinated gilts quickly developed virus neutralizing (VN) antibodies starting from 3 to 7 days postchallenge (1.0 to 5.0 log2). In contrast, the unvaccinated gilts remained negative for VN antibodies after challenge. The vaccinated gilts had shorter viremia than the control gilts. Gross pathology (mummification) was observed in 8% of the fetuses from vaccinated gilts and in 15% of the fetuses from unvaccinated gilts. The number of fetuses with severe microscopic lesions in the fetal implantation sites (a focal detachment of the trophoblast from the uterine epithelium; a focal, multifocal, or full degeneration of the fetal placenta) was lower in the vaccinated (19%) versus unvaccinated (45%) gilts (P < 0.05). The number of PRRS-positive cells in the fetal placentae was higher in unvaccinated versus vaccinated gilts (P < 0.05). In contrast, the number of PRRS-positive cells in the myometrium/endometrium was higher in vaccinated versus unvaccinated gilts (P < 0.05). Fifty-seven percent of the fetuses from the vaccinated gilts and 75% of the fetuses from the unvaccinated gilts were PRRSV-positive. In conclusion, implementation of the novel experimental inactivated PRRSV vaccine primed the VN antibody response and slightly reduced the duration of viremia in gilts. It also reduced the number of virus-positive fetuses and improved the fetal survival, but was not able to fully prevent congenital PRRSV infection. The reduction of fetal infection and pathology is most probably attributable to the vaccine-mediated decrease of PRRSV transfer from the endometrium to the fetal placenta.

%B Theriogenology %V 78 %P 1527-37 %8 2012 Oct 15 %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/22980086?dopt=Abstract %R 10.1016/j.theriogenology.2012.06.015 %0 Journal Article %J Vet Microbiol %D 2011 %T Epidemiology of Pestivirus infection in wild ungulates of the French South Alps. %A Martin, Claire %A Letellier, Carine %A Ann Brigitte Cay %A Gauthier, Dominique %A Jean, Nicolas %A Shaffii, Anahita %A Saegerman, Claude %K Age factors %K Animals %K Animals, Wild %K Antibodies, Viral %K Enzyme-Linked Immunosorbent Assay %K Female %K France %K Male %K Neutralization Tests %K Pestivirus %K Pestivirus Infections %K Risk Factors %K Ruminants %K Seroepidemiologic Studies %X

Inter-species transmission is often incriminated in the epidemiology of Pestivirus diseases. The purpose of this study was to investigate the prevalence of Pestivirus in some mountain wild ungulates and to determine their role in Pestivirus transmission, as mountain pastures are a place where cohabitations between wild and domestic ungulates are particularly high. Between 2003 and 2007, a longitudinal epidemiological study was carried out on hunted ungulates in the French Hautes-Alpes department. Pestivirus-specific antibodies against p80 protein (also named NS3) common to all Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV) were found in 45.9% (95% confidence interval [CI95%]: 40.5-51.3%) of the 343 tested chamois (Rupicapra rupicapra). In addition, mouflons (Ovis gmelinii musimon) were shown for the first time to be strongly infected (61.1%; CI95%: 38.6-83.6) by a Pestivirus. These serological ELISA results were confirmed by comparative virus neutralization tests, performed on seven Pestivirus strains by using 15 seropositive samples. The highest antibody titers were directed against 2 BDV strains (Av and 33s strains), rather than BDV-4, a strain responsible for Pyrenean-chamois epizooties. Virus neutralization tests confirm a BDV circulation in wild ungulates in the French South Alps. However, no Pestivirus RNA was detected by reverse-transcriptase polymerase chain reaction in serum and spleen samples from seronegative animals and no virus was isolated from those samples either. Efforts should be made to improve the protocol in order to be able to isolate and characterize the local strain. Finally, the oldness (age) and femaleness (gender) increase the risk of seroconversion in chamois.

%B Vet Microbiol %V 147 %P 320-8 %8 2011 Jan 27 %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/20709472?dopt=Abstract %R 10.1016/j.vetmic.2010.07.010 %0 Journal Article %J Vector Borne Zoonotic Dis %D 2011 %T Tick-borne encephalitis virus seropositive dog detected in Belgium: screening of the canine population as sentinels for public health. %A Sophie Roelandt %A Heyman, Paul %A De Filette, Marina %A Vene, Sirkka %A Yves Van der Stede %A Ann Brigitte Cay %A Tavernier, Paul %A Alexandre Dobly %A De Bosschere, Hendrik %A Vyt, Philip %A Meersschaert, Carole %A S. Roels %K Animals %K Antibodies, Viral %K Belgium %K Dog Diseases %K Dogs %K Encephalitis Viruses, Tick-Borne %K Encephalitis, Tick-Borne %K Enzyme-Linked Immunosorbent Assay %K Female %K Humans %K Neutralization Tests %K public health %K Reagent Kits, Diagnostic %K Sentinel Surveillance %X

Tick-borne encephalitis virus (TBEV) is an important emerging tick-borne viral infection of humans and dogs in Europe. Currently, TBEV surveillance is virtually nonexistent in Belgium, which is considered nonendemic. A commercial enzyme-linked immunosorbent assay (ELISA) was adapted for the detection of TBEV-specific IgG-antibodies in canine sera. Serum samples of Belgian dogs were obtained from three diagnostic laboratories from Northern (n=688) and Southern Belgium (n=192). ELISA-positive and borderline samples were subjected to a TBEV rapid fluorescent focus inhibition confirmation test. One dog was confirmed TBEV seropositive. Several ELISA-positive and borderline sera underwent seroneutralization and hemagglutinin inhibition tests to rule out West Nile and Louping Ill viruses, but tested negative. The clinical history of the seropositive dog could not explain beyond doubt where and when TBEV infection was acquired. Further surveillance is necessary to determine whether this dog remains a single travel-related case or whether it represents an early warning of a possible future emergence of TBEV.

%B Vector Borne Zoonotic Dis %V 11 %P 1371-6 %8 2011 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/21919722?dopt=Abstract %R 10.1089/vbz.2011.0647 %0 Journal Article %J Rev Sci Tech %D 2010 %T Influence of the incubation temperature and the batch components on the sensitivity of an enzyme-linked immunosorbent assay to detect Aujeszky's disease virus glycoprotein E (gE). %A Ann Brigitte Cay %A Yves Van der Stede %K Animals %K Enzyme-Linked Immunosorbent Assay %K Herpesvirus 1, Suid %K Pseudorabies %K Quality Control %K Reference Values %K Sensitivity and Specificity %K Temperature %K Viral Envelope Proteins %X

Although licensed batches of an enzyme-linked immunosorbent assay (ELISA) for Aujeszky's disease virus (ADV) were used, and the assays were performed within an ISO/IEC 17025 accredited quality control system, certain routine runs of the ADV ELISA were not validated using the quality system criteria, even when all technical parameters were controlled. Incubation at different temperatures and batch composition were identified as parameters that could result in non-validated assays/runs. Therefore, the effect of incubation temperature and batch composition on the analytical sensitivity of the ELISA was investigated. The World Organisation for Animal Health (OIE) standard reference serum ADV1 was diluted 1:8 and tested in 94 different glycoprotein E ELISA runs performed with different batches and different incubation temperatures. The incubation temperature and batch components had a significant influence on the qualitative result for the OIE standard reference serum. An incubation temperature of at least 22 degrees C was recommended, based on the results of this analysis. Which of the batch components caused these differences in sensitivity was not investigated further.

%B Rev Sci Tech %V 29 %P 565-71 %8 2010 Dec %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/21309455?dopt=Abstract %0 Journal Article %J Ann.Méd.Vét %D 2010 %T Revue du risque zoonotique, de l'impact vétérinaire et de la surveillance des virus influenza porcins dans le cadre de l'émergence du virus pandémique influenza A/H1N1 (2009).36859 %A S. Cardoen %A Etienne Thiry %A Van Reeth,K. %A Ann Brigitte Cay %A Dewulf,J. %A Hooyberghs,J. %A P. Houdart %A Saegerman,C. %A Dirk Berkvens %A Goubau,P. %A Desmecht,D. %A Bernard Brochier %A Maes,D. %A G. Czaplicki %A Castryk,F. %A Thierry van den Berg %K 2009 %K de %K ET %K INFLUENZA %K LE %K Surveillance %K VIRUS %X Not available %B Ann.Méd.Vét %V x %8 0/0/2010 %G eng %1 38105 %0 Journal Article %J Rev Sci Tech %D 2007 %T Uncertainty of measurement for competitive and indirect ELISAs. %A Toussaint, J F %A Assam, P %A Ann Brigitte Cay %A Dekeyser, F %A K Knapen %A Hein Imberechts %A Goris, N %A Molenberghs, G %A Mintiens, K %A Kris De Clercq %K Analysis of Variance %K Animals %K Data Interpretation, Statistical %K Diagnosis, Differential %K Enzyme-Linked Immunosorbent Assay %K Evaluation Studies as Topic %K Foot-and-Mouth Disease %K Foot-and-Mouth Disease Virus %K Logistic Models %K Predictive Value of Tests %K Quality Control %K Reference Values %K Reproducibility of Results %K Sensitivity and Specificity %K uncertainty %X

A method for the estimation of the uncertainty of measurements for Gaussian outcomes of enzyme-linked immunosorbent assay (ELISA) is described using competitive and indirect foot and mouth disease (FMD) ELISAs. Assay repeatability was determined by random effects analysis of variance, and the normality of the residuals was checked. The standard errors of the individual predicted values were transformed into confidence intervals around the corresponding observed values and further transformed into probabilities of being above/below a cut-off. Logistic regression models were subsequently used to interpolate probability values for the whole range of possible assay values. The uncertainty of measurement of a test result was finally defined as the probability of not observing the same qualitative test result when retesting the same sample. For the competitive ELISA any sample with a percent inhibition 4% above the cut-off value had an uncertainty level (probability of a negative result in the case of retest) below 5%. In the indirect ELISA with a cut-off OD of 0.1, the uncertainty was below 5% for any sample with a normalised OD value above 0.22.

%B Rev Sci Tech %V 26 %P 649-56 %8 2007 Dec %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/18293613?dopt=Abstract %0 Journal Article %J Vet Microbiol %D 2006 %T Absence of viral envelope proteins in equine herpesvirus 1-infected blood mononuclear cells during cell-associated viremia. %A van der Meulen, Karen %A Ann Brigitte Cay %A Pensaert, Maurice %A Nauwynck, Hans %K Animals %K Cytotoxicity, Immunologic %K Herpesviridae Infections %K Herpesvirus 1, Equid %K Histocompatibility Antigens Class I %K Horse Diseases %K Horses %K Leukocytes, Mononuclear %K Viral Envelope Proteins %K Viremia %X

In vitro studies demonstrated that most equine herpesvirus 1 (EHV-1)-infected peripheral blood mononuclear cells (PBMC) do not expose viral envelope proteins on their surface. This protects them against antibody-dependent lysis. We examined whether viral envelope proteins are also undetectable on infected PBMC during cell-associated viremia. Further, surface expression of major histocompatibility complex (MHC)-I was examined, since MHC-I assists in making infected cells recognizable for cytotoxic T-lymphocytes (CTL). Four ponies, previously exposed to EHV, and two ponies that had no contact with EHV before, were inoculated with EHV-1. PBMC were collected at different time points up to 28 days post inoculation. Ninety-eight percent of the infected PBMC did not show viral envelope proteins on their surface. Moreover, infected PBMC without surface expression only produced immediate early and, at least, one early protein, ICP22, but not late envelope proteins gB and gM. This indicates that surface expression of viral envelope proteins is absent, simply because the PBMC are in an early phase of infection. The percentage of infected PBMC showing surface expression of MHC-I was similar as observed in non-infected PBMC from the same ponies (80-100%). Therefore, inefficient recognition of EHV-1-infected PBMC by CTLs does not arise from absent surface expression of MHC-I.

%B Vet Microbiol %V 113 %P 265-73 %8 2006 Mar 31 %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/16387454?dopt=Abstract %R 10.1016/j.vetmic.2005.11.048 %0 Journal Article %J Arch Virol %D 1993 %T Characterization of structural and non-structural proteins of hog cholera virus by means of monoclonal antibodies. %A Gaetan Muyldermans %A Ann Brigitte Cay %A De Smet, A %A F. Koenen %A Hamers, R %K Animals %K Antibodies, Monoclonal %K Blotting, Western %K Cattle %K Cell Line %K Classical swine fever virus %K Cross Reactions %K Diarrhea Viruses, Bovine Viral %K Electrophoresis, Polyacrylamide Gel %K Epitopes %K Glycoproteins %K Molecular Weight %K Radioimmunoprecipitation Assay %K Viral Nonstructural Proteins %K Viral Structural Proteins %X

A panel of 15 monoclonal antibodies, produced against the hog cholera virus, were characterized by radioimmunoprecipitation assays. Using this panel, we were able to identify 4 sets of monoclonal antibodies precipitating each a different viral protein with relative molecular weight of 40, 46, 120 kDa, respectively, and a protein complex containing 15, 16, 27, and 55 kDa polypeptides which were further characterized. One monoclonal antibody recognized an antigenic determinant at the C-terminal cleavage product of the non-structural p 125 of BVDV. The 40 kDa protein was precipitated from the pelleted virions, indicating its structural importance. On the contrary the 46 kDa protein could only be precipitated from the cell lysate and not from the pelleted virions. The glycosylated 15/16 kDa-55 kDa proteins form a disulfide linked heterodimer on the virus particle with a relative molecular weight of 65 kDa.

%B Arch Virol %V 131 %P 405-17 %8 1993 %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/7688508?dopt=Abstract %0 Journal Article %J Arch Virol %D 1993 %T Polymerase chain reaction-mediated cloning and in vitro translation of the genes coding for the structural proteins of hog cholera virus. %A Gaetan Muyldermans %A San Gabriel, M C %A Ann Brigitte Cay %A De Smet, A %A Hamers, R %K Base Sequence %K Classical swine fever virus %K Cloning, Molecular %K Molecular Sequence Data %K Pestivirus %K polymerase chain reaction %K Protein Biosynthesis %K Viral Structural Proteins %X

After amplification by PCR, the 5' region of the genome of hog cholera virus (HCV) strain Alfort 187 was cloned and sequenced. The nucleotide and deduced amino acid sequences were compared with the ones of other pestiviruses. By in vitro translation experiments we were able to demonstrate the protease activity of the p 20 protein of HCV.

%B Arch Virol %V 132 %P 429-35 %8 1993 %G eng %N 3-4 %1 http://www.ncbi.nlm.nih.gov/pubmed/8397504?dopt=Abstract %0 Journal Article %J Arch Virol %D 1993 %T Production and characterization of monoclonal antibodies against hog cholera virus (Alfort 187 strain). %A Ann Brigitte Cay %A Gaetan Muyldermans %A De Smet, A %A Hamers, R %A F. Koenen %K Animals %K Antibodies, Monoclonal %K Antibodies, Viral %K Antibody Specificity %K Belgium %K Classical Swine Fever %K Classical swine fever virus %K Disease Outbreaks %K Hybridomas %K mice %K Mice, Inbred BALB C %K Neutralization Tests %K Swine %B Arch Virol %V 131 %P 185-92 %8 1993 %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/8328912?dopt=Abstract %0 Journal Article %J Vet Rec %D 1992 %T Evaluation of the complex trapping blocking-ELISA in a serological survey during the Belgian classical swine fever epizootic in 1990. %A F. Koenen %A Ann Brigitte Cay %A Lefebvre, J %A Desmet, A %K Animals %K Antibodies, Viral %K Belgium %K Classical Swine Fever %K Classical swine fever virus %K Disease Outbreaks %K Enzyme-Linked Immunosorbent Assay %K Evaluation Studies as Topic %K Neutralization Tests %K Reagent Kits, Diagnostic %K Reproducibility of Results %K Sensitivity and Specificity %K Swine %B Vet Rec %V 131 %P 396 %8 1992 Oct 24 %G eng %N 17 %1 http://www.ncbi.nlm.nih.gov/pubmed/1455586?dopt=Abstract %0 Journal Article %J Vet Microbiol %D 1989 %T Comparative analysis of monoclonal antibodies against pestiviruses: report of an international workshop. %A Ann Brigitte Cay %A Chappuis, G %A Coulibaly, C %A Dinter, Z %A Edwards, S %A Greiser-Wilke, I %A Gunn, M %A Have, P %A Hess, G %A Juntti, N %K Animals %K Antibodies, Monoclonal %K Antibodies, Viral %K Cell Line %K Cross Reactions %K Fluorescent Antibody Technique %K Immunoenzyme Techniques %K mice %K Pestivirus %X

Thirty-three pestivirus strains were grown in cell culture and characterized by immunostaining with 19 monoclonal antibodies (MAbs) raised against hog cholera virus (HCV), with 42 MAbs against bovine viral diarrhoea virus (BVDV) and with 13 MAbs against border disease virus (BDV). Seven MAbs reacted with all pestivirus strains tested, eight MAbs detected only the seven HCV strains, three detected only the 16 BVDV strains. No MAb was found that was specific for BDV. BVDV and BDV strains were broadly cross-reactive with the MAbs, indicating a close relationship between these two species, whereas HCV strains were characterized as distinct from BVDV and BDV.

%B Vet Microbiol %V 20 %P 123-9 %8 1989 Jun %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/2549680?dopt=Abstract %0 Journal Article %J Arch Virol %D 1989 %T High titre Hog Cholera virus production on Cytodex 3 microcarrier cultures. %A Ann Brigitte Cay %A De Smet, A %A Dubois, N %A F. Koenen %K Animals %K Cells, Cultured %K Classical swine fever virus %K Dextrans %K Virus Cultivation %X

In an attempt to produce Hog Cholera virus (HCV) preparations of high titre, optimal growth and trypsinization conditions of PK-15 microcarrier cell cultures were defined. Infecting a PK-15 Cytodex 3 microcarrier culture with HCV increased the yield of virus more than 10 times compared with conventional monolayer culture in Roux flasks.

%B Arch Virol %V 105 %P 113-8 %8 1989 %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/2470336?dopt=Abstract %0 Generic %D 0 %T Assessment of the presence of non-responding, seronegative sows after vaccination against Porcine Reproductive and Respiratory Syndrome (PRRS) virus %A Jorian Fiers %A Marylène Tignon %A Ann Brigitte Cay %A Maes, Dominiek %G eng %0 Generic %D 0 %T Assessment of the presence of non-responding, seronegative sows after vaccination against Porcine Reproductive and Respiratory Syndrome (PRRS) virus %A Jorian Fiers %A Marylène Tignon %A Ann Brigitte Cay %A Maes, Dominiek %G eng %0 Generic %D 0 %T Presence of seronegative sows after routine vaccination against Porcine Reproductive and Respiratory Syndrome (PRRS) %A Jorian Fiers %A Xavier Simons %A Marylène Tignon %A Ann Brigitte Cay %A Maes, Dominiek %G eng %0 Generic %D 0 %T Responses of PRRSv vaccination in piglets born from PRRSv vaccinated, ELISA responding and non-responding sows %A Jorian Fiers %A Marylène Tignon %A Ann Brigitte Cay %A Maes, Dominiek %G eng