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No recent information is available on the specificity of current M. synoviae (Ms) and M. gallisepticum (Mg) serological tests. In this study the performance of a currently available Mg and Ms Rapid Plate Agglutination (RPA) test, and three Mg, three Ms and three Mg/Ms combination ELISAs were evaluated on SPF sera that were obtained from days (D) 0-28 after M. gallinarum, M. imitans or M. gallinaceum inoculation, after sham inoculation and without inoculation. Tracheal swabs for mycoplasma culture were obtained before inoculation (D0), 7 and 28 days post inoculation (d.p.i.) in all groups except the sham inoculated group. The different mycoplasma species colonized well. In the early stage after inoculation (7-14 d.p.i.) with heterologous mycoplasma species, the specificity varied from 85% to 100% in the Mg RPA test and from 70% to 85% in the Ms RPA test. The specificity of both Mg and Ms RPA test was 100% in the sham inoculated samples and ruled out the effect of sham medium. In the late stage (21-28 d.p.i.) specificity was 100% for both RPA tests. The test specificity was 100% for seven ELISAs except for two combination ELISAs where a specificity of 95% was found in the late stage after inoculation. However, this was not significantly different from the specificity of all other tests in the late stage of these groups. These results show that it is not advisable to establish Mg and Ms seromonitoring programmes on the Mg and Ms RPA test alone as other mycoplasma species frequently occur in poultry.

Poultry can become infected with avian influenza viruses (AIV) via (in) direct contact with infected wild birds. Free‐range chicken farms in the Netherlands were shown to have a higher risk for introduction of low pathogenic avian influenza (LPAI) virus than indoor chicken farms. Therefore, during outbreaks of highly pathogenic avian influenza (HPAI), free‐range layers are confined indoors as a risk mitigation measure. In this study, we characterized the seasonal patterns of AIV introductions into free‐range layer farms, to determine the high‐risk period. Data from the LPAI serological surveillance programme for the period 2013–2016 were used to first estimate the time of virus introduction into affected farms and then assess seasonal patterns in the risk of introduction. Time of introduction was estimated by fitting a mathematical model to seroprevalence data collected longitudinally from infected farms. For the period 2015–2016, longitudinal follow‐up included monthly collections of eggs for serological testing from a cohort of 261 farms. Information on the time of introduction was then used to estimate the monthly incidence and seasonality by fitting harmonic and Poisson regression models. A significant yearly seasonal risk of introduction that lasted around 4 months (November to February) was identified with the highest risk observed in January. The risk for introduction of LPAI viruses in this period was on average four times significantly higher than the period of low risk around the summer months. Although the data for HPAI infections were limited in the period 2014–2018, a similar risk period for introduction of HPAI viruses was observed. The results of this study can be used to optimize risk‐based surveillance and inform decisions on timing and duration of indoor confinement when HPAI viruses are known to circulate in the wild bird population.

Associations between animal health and performance, and the host’s microbiota have been recently established. In poultry, changes in the intestinal microbiota have been linked to housing conditions and host development, but how the intestinal microbiota respond to environmental changes under farm conditions is less well understood. To gain insight into the microbial responses following a change in the host’s immediate environment, we monitored four indoor flocks of adult laying chickens three times over 16 weeks, during which two flocks were given access to an outdoor range, and two were kept indoors. To assess changes in the chickens’ microbiota over time, we collected cloacal swabs of 10 hens per flock and performed 16S rRNA gene amplicon sequencing. The poultry house (i.e., the stable in which flocks were housed) and sampling time explained 9.2 and 4.4% of the variation in the microbial community composition of the flocks, respectively. Remarkably, access to an outdoor range had no detectable effect on microbial community composition, the variability of microbiota among chickens of the same flock, or microbiota richness, but the microbiota of outdoor flocks became more even over time. Fluctuations in the composition of the microbiota over time within each poultry house were mainly driven by turnover in rare, rather than dominant, taxa and were unique for each flock. We identified 16 amplicon sequence variants that were differentially abundant over time between indoor and outdoor housed chickens, however none were consistently higher or lower across all chickens of one housing type over time. Our study shows that cloacal microbiota community composition in adult layers is stable following a sudden change in environment, and that temporal fluctuations are unique to each flock. By exploring microbiota of adult poultry flocks within commercial settings, our study sheds light on how the chickens’ immediate environment affects the microbiota composition.

The zoonotic origin of the SARS-CoV-2 pandemic is still unknown. Animal experiments have shown that non-human primates, cats, ferrets, hamsters, rabbits and bats can be infected by SARS-CoV-2. In addition, SARS-CoV-2 RNA has been detected in felids, mink and dogs in the field. Here, we describe an in-depth investigation of outbreaks on 16 mink farms and humans living or working on these farms, using whole genome sequencing. We conclude that the virus was initially introduced from humans and has evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period several weeks prior to detection. At the moment, despite enhanced biosecurity, early warning surveillance and immediate culling of infected farms, there is ongoing transmission between mink farms with three big transmission clusters with unknown modes of transmission. We also describe the first animal to human transmissions of SARS-CoV-2 in mink farms.One sentence summarySARS-CoV-2 transmission on mink farms.

In April 2020, respiratory disease and increased mortality were observed in farmed mink on two farms in the Netherlands. In both farms, at least one worker had been found positive for SARS-CoV-2. Necropsies of the mink revealed interstitial pneumonia, and organ and swab samples tested positive for SARS-CoV-2 RNA by qPCR. Variations in viral genomes point at between-mink transmission on the farms and lack of infection link between the farms. Inhalable dust in the mink houses contained viral RNA, indicating possible exposure of workers.

No recent information is available on the specificity of current

In this study, we explore the circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands. Surveillance data collected between 2006 and 2016 was used to evaluate subtype diversity, spatiotemporal distribution and genetic relationships between wild bird and poultry viruses. We observed close species-dependent associations among hemagglutinin and neuraminidase subtypes. Not all subtypes detected in wild birds were found in poultry, suggesting transmission to poultry is selective and likely depends on viral factors that determine host range restriction. Subtypes commonly detected in poultry were in wild birds most frequently detected in mallards and geese. Different temporal patterns in virus prevalence were observed between wild bird species. Virus detections in domestic ducks coincided with the prevalence peak in wild ducks, whereas virus detections in other poultry types were made throughout the year. Genetic analysis of the surface genes demonstrated that most poultry viruses were related to locally circulating wild bird viruses, but no direct spatiotemporal link was observed. Results indicate prolonged undetected virus circulation and frequent reassortment events with local and newly introduced viruses within the wild bird population. Increased knowledge on LPAI virus circulation can be used to improve surveillance strategies.

This study reports the results of diagnostic and molecular typing methods for 18

Poultry can become infected with low pathogenic avian influenza (LPAI) viruses via (in)direct contact with infected wild birds or by transmission of the virus between farms. This study combines routinely collected surveillance data with genetic analysis to assess the contribution of between‐farm transmission to the overall incidence of LPAI virus infections in poultry. Over a 10‐year surveillance period, we identified 35 potential cases of between‐farm transmission in the Netherlands, of which 10 formed geographical clusters. A total of 21 LPAI viruses were isolated from nine potential between‐farm transmission cases, which were further studied by genetic and epidemiological analysis. Whole genome sequence analysis identified close genetic links between infected farms in seven cases. The presence of identical deletions in the neuraminidase stalk region and minority variants provided additional indications of between‐farm transmission. Spatiotemporal analysis demonstrated that genetically closely related viruses were detected within a median time interval of 8 days, and the median distance between the infected farms was significantly shorter compared to farms infected with genetically distinct viruses (6.3 versus 69.0 km; p

Analysis of low pathogenic avian influenza (LPAI) viruses circulating in the Netherlands in a previous study revealed associations of specific hemagglutinin (HA) and neuraminidase (NA) subtypes with wild bird or poultry hosts. In this study, we identified putative host associations in LPAI virus internal proteins. We show that LPAI viruses isolated from poultry more frequently carried the allele A variant of the nonstructural protein (NS) gene, compared to wild bird viruses. We determined the susceptibility of chickens to wild bird&ndash, associated subtypes H3N8 and H4N6 and poultry-associated subtypes H8N4 and H9N2, carrying either NS allele A or B, in an infection experiment. We observed variations in virus shedding and replication patterns, however, these did not correlate with the predicted wild bird- or poultry-associations of the viruses. The experiment demonstrated that LPAI viruses of wild bird-associated subtypes can replicate in chickens after experimental infection, despite their infrequent detection in poultry. Although the NS1 protein is known to play a role in immune modulation, no differences were detected in the limited innate immune response to LPAI virus infection. This study contributes to a better understanding of the infection dynamics of LPAI viruses in chickens.

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any ‘gold standard’. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability.

Introduction of highly pathogenic avian influenza (HPAI) virus A(H5N8) into Europe prompted animal and human health experts to implement protective measures to prevent transmission to humans. We describe the situation in 2016 and list public health measures and recommendations in place. We summarise critical interfaces identified during the A(H5N1) and A(H5N8) outbreaks in 2014/15. Rapid exchange of information between the animal and human health sectors is critical for a timely, effective and efficient response.

To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3-100.0%). The ELISA showed a high repeatability (cv=6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats.

This study aimed at estimating the Schmallenberg virus (SBV) seroprevalence in dairy heifers, non-dairy adult cattle, sheep and goats in the Netherlands after cessation of SBV transmission at the end of 2011. Archived serum samples from ruminants submitted to the GD Animal Health Service for monitoring purposes between November 2011 and March 2012 were selected and tested for presence of SBV-specific antibodies using an in-house ELISA. Animal seroprevalences were estimated at 63.4% in dairy heifers, 98.5% in adult non-dairy cattle, 89.0% in sheep and 50.8% in goats. Multivariable analyses were carried out to describe the relationship between potential risk factors and the ELISA outcome S/P%. The overall SBV seroprevalence in ruminants and ruminant herds in the Netherlands at the end of 2011 was high, with considerable differences between species and farm types. No gradient spatial pattern in final seroprevalence could be detected and therefore no suggestions about the site of introduction and spread of SBV in the Netherlands in 2011 could be made. In dairy heifers, it was shown that S/P% increased with age. In sheep, S/P% was lower in animals located in the coastal area. Whether herds were located near the German border did not affect the S/P% in sheep nor in dairy heifers. An attempt was made to gain insight in the spatiotemporal introduction of SBV in the Netherlands in 2011, by testing sheep serum samples from 2011. A seroprevalence of about 2% was found in samples from April, June and July 2011, but the ELISA positive samples could not be confirmed in a virus neutralization test. A clear increase in seroprevalence started at August 2011. From mid-August 2011 onwards, seropositive samples were confirmed positive by virus neutralization testing. This indicated the start of the epidemic, but without a clear spatial pattern.

Seroprevalence was highest in the eastern part of the country, bordering Germany, where the virus was first identified., Infections with Schmallenberg virus (SBV) are associated with congenital malformations in ruminants. Because reporting of suspected cases only could underestimate the true rate of infection, we conducted a seroprevalence study in the Netherlands to detect past exposure to SBV among dairy cattle. A total of 1,123 serum samples collected from cattle during November 2011–January 2012 were tested for antibodies against SBV by using a virus neutralization test; seroprevalence was 72.5%. Seroprevalence was significantly higher in the central-eastern part of the Netherlands than in the northern and southern regions (p

Genetic analyses of highly pathogenic avian influenza A(H5N8) virus from the Netherlands, and comparison with strains from Europe, South Korea, and Japan, showed a close relation. Data suggest the strains were probably carried to the Netherlands by migratory wild birds from Asia, possibly through overlapping flyways and common breeding sites in Siberia.

At the end of 2011, a new Orthobunyavirus was discovered in Germany and named Schmallenberg virus (SBV). In the Netherlands malformations in new-born ruminants were made notifiable from the 20th of December 2011. After a notification, malformed new-borns were necropsied and brain tissue was sampled for reverse transcription-polymerase chain reaction (RT-PCR). In addition, blood samples from mothers of affected new-borns were tested for antibodies in a virus neutralization test (VNT). The aim of this study was to summarize and evaluate the diagnostic data obtained and to gain insight into the possible regional differences. In total 2166 brains were tested: 800 from lambs, 1301 from calves and 65 from goat kids. Furthermore 1394 blood samples were tested: 458 from ewes, 899 from cows and 37 from goats. Results showed that 29% of the lamb brains, 14% of the calf brains, and 9% of the goat kid brains were RT-PCR positive. The number of malformed and RT-PCR positive lambs decreased over time while the number of malformed and RT-PCR positive calves increased. In the VNT 92% of the ewes, 96% of the cows and 43% of the goats tested positive. Combining RT-PCR and VNT results, 18% of all farms tested positive in both the RT-PCR and VNT. The relative sensitivity and specificity of the RT-PCR are 19% and 97% respectively, and of the VNT 99% and 6%. The results show a widespread exposure to SBV and the regional evaluation seems to indicate an introduction of SBV in the central/eastern part.

Since December 2011, an epizootic of congenital malformations, designated as arthrogryposis-hydranencephaly syndrome (AHS), has been seen in ruminants in several countries in Western Europe (Van den Brom and others 2012). The disease has been associated with a new orthobunyavirus, named Schmallenberg virus (SBV) (Hoffmann and others 2012). After SBV was detected in brain tissue from malformed lambs by PCR on December 15, 2011, the Dutch Ministry of Economic Affairs, Agriculture and Innovation made congenital malformations in ruminants notifiable. Farmers and veterinarians are obliged to notify and submit malformed newborns to the Animal Health Service (GD-Deventer) for autopsy examination. Until May 15, 2012, over 600 lambs and over 1200 calves had been examined, and many of them showed AHS which is likely to be caused by an intrauterine infection with SBV. Brain tissue samples of all malformed newborns were tested by PCR at the Central Veterinary Institute (CVI). However, SBV could only be confirmed in 22 per cent of the lambs and 19 per cent of the calves that were investigated until May 15, 2012. Initially, a real-time PCR protocol targeting the L-segment was used. Since February 17, 2012, a real-time PCR protocol targeting the S-segment was used, since the latter PCR demonstrated a higher analytical and diagnostic sensitivity as indicated by the Friedrich Loeffler Institute in Germany and Dijkman and others (2012). Both protocols were kindly provided by the Friedrich Loeffler Institute in Germany. Persistent infection in Akabane virus-infected foetuses has not been reported, and the virus cannot be easily isolated from affected full-term calves or lambs because of the presence of neutralising (NT) …

Since the beginning of November 2014, nine outbreaks of highly pathogenic avian influenza virus (HPAIV) A(H5N8) in poultry have been detected in four European countries. In this report, similarities and differences between the modes of introduction of HPAIV A(H5N1) and A(H5N8) into Europe are described. Experiences from outbreaks of A(H5N1) in Europe demonstrated that early detection to control HPAIV in poultry has proven pivotal to minimise the risk of zoonotic transmission and prevention of human cases.

Phylogenetic analysis of highly pathogenic avian influenza A(H5N8) virus strains causing outbreaks in Dutch poultry farms in 2014 provides evidence for separate introduction of the virus in four outbreaks in farms located 16-112 km from each other and for between-farm transmission between the third and fourth outbreak in farms located 550 m from each other. In addition, the analysis showed that all European and two Japanese H5N8 virus strains are very closely related and seem to originate from a calculated common ancestor, which arose between July and September 2014. Our findings suggest that the Dutch outbreak virus strain 'Ter Aar' and the first German outbreak strain from 2014 shared a common ancestor. In addition, the data indicate that the Dutch outbreak viruses descended from an H5N8 virus that circulated around 2009 in Asia, possibly China, and subsequently spread to South Korea and Japan and finally also to Europe. Evolution of the virus seemed to follow a parallel track in Japan and Europe, which supports the hypothesis that H5N8 virus was exchanged between migratory wild waterfowl at their breeding grounds in Siberia and from there was carried by migrating waterfowl to Europe.

Background At the end of 2011, a new orthobunyavirus, tentatively named Schmallenberg virus (SBV), was discovered in Germany. This virus has since been associated with clinical signs of decreased milk production, watery diarrhoea and fever in dairy cows, and subsequently also with congenital malformations in calves, lambs and goat kids. In affected countries, initial surveillance for the infection was based on examination of malformed progeny. These suspicions were followed up by real-time reverse transcription polymerase chain reaction (RT-PCR) on brain tissue. For epidemiological purposes, a serological assay was, however, needed. Results A virus neutralisation test (VNT) was developed and optimized, and subsequently evaluated. This VNT has a specificity of >99% and the sensitivity is likely also very close to 100%. The assay is highly repeatable and reproducible. The final assay was used to test for antibodies in cows, ewes and does from herds known to be infected or suspected to be so. Targets for sampling in these herds were the mothers of malformed offspring. In herds with an RT-PCR confirmed SBV infection, more than 94% (190 out of 201) of the ewes and 99% (145 out of 146) of the cows were seropositive. In herds with suspicion of SBV infection based on birth of malformed offspring only (no or negative RT-PCR), more than 90% (231 out of 255) of the ewes and 95% (795 out of 834) of the cows were seropositive. In goats, on the other hand, only a low number of seropositives was found: overall 36.4%, being 16 out of 44 goats tested. Conclusions Given the characteristics of this VNT, it can be used at a relative high throughput for testing of animals for export, surveillance, screening and research purposes, but can also be used as a confirmation test for commercially available enzyme-linked immunosorbent assays (ELISA’s) and for (relative) quantification of antibodies. Suspicions of SBV infections that were confirmed by RT-PCR were almost always confirmed by serology in cows. Due to individual registration and identification of cows and calves, affected offspring could almost always be traced back to the mother. Ewes on the other hand were not always the mothers of affected lambs, but were in many cases herd mates with unaffected lambs. This indicated a high within-herd seroprevalence of antibodies against SBV.