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Bovine Viral Diarrhea Virus (BVDV) is a common viral disease in cattle, causing economic losses in naive herds where the virus is introduced. In the Netherlands, a BVDV control program has been in place since 1998, evolving from voluntary to mandatory participation for dairy herds since April 2018. Participation in the BVDV control program is not mandatory for non-dairy farms. The aim of this study was to determine risk factors for new introduction of BVDV into certified BVDV-free herds in the context of a national BVDV control program in dairy herds. In this retrospective case-control study, survey data were collected from 149 dairy farms that lost their BVDV-free status (case herds) and 148 matched dairy farms that maintained their BVDV-free status (control herds) between 2018 and 2021. The survey captured information about potential risk factors and herd characteristics in the 2 years leading up to the loss of BVDV-free status (case herds, virus detection in at least one animal or when seroconversion was detected) or remaining BVDV-free (control herds). Descriptive statistics and logistic regression with a backward selection and elimination procedure were used to identify potential risk factors associated with losing BVDV-free status. Risk factors were quantified as an Odds Ratio (OR) with the associated 95% confidence interval (CI). The risk factor with the highest OR for losing BVDV-free status was purchasing cattle from herds without BVDV-free status (OR 1.25, CI 1.10-1.41), followed by the farmer having another profession that resulted in contact with other cattle (OR 1.25, CI 1.06-1.47), housing young calves and adult cows in the same barn (OR 1.22, CI 1.08-1.38), having a permanent employee on the farm (OR 1.17, CI 1.04-1.31), having a group calving pen (OR 1.16, CI 1.03-1.32), escaped cattle from other farms that mingled with own cattle (OR 1.16, CI 1.01-1.33), and nearest distance to a non-dairy farm (OR 1.15, CI 1.03-1.28). Although the BVDV status of most dairy herds can be checked in an open register, approximately half of the farmers indicated that they purchased cattle from BVDV-free herds while they were actually purchasing from non-BVDV-free farms. Farmers should be stimulated to actively check the true BVDV status of the herd from which cattle are purchased to further reduce the risk of introduction. In addition, indirect contact with cattle from other farms through either the farmer or other on-farm staff should be avoided. It is strongly advised to work in these situations with proper biosecurity measures such as changing boots and coveralls. The results can be used to improve BVDV control programs to further decrease the prevalence., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Since 1998, notifiable bluetongue virus (BTV) serotypes 1-4, 6, 8, 9, 11, and 16 have been reported in Europe. In August 2006, a bluetongue (BT) outbreak caused by BTV serotype 8 began in northwestern Europe. The Netherlands was declared BT-free in February 2012, and annual monitoring continued. On September 3, 2023, typical BT clinical manifestations in sheep were notified to the Netherlands Food and Product Safety Consumer Authority. On September 6, we confirmed BTV infection through laboratory diagnosis; notifications of clinical signs in cattle were also reported. We determined the virus was serotype 3 by whole-genome sequencing. Retrospective analysis did not reveal BTV circulation earlier than September. The virus source and introduction route into the Netherlands remains unknown. Continuous monitoring and molecular diagnostic testing of livestock will be needed to determine virus spread, and new prevention strategies will be required to prevent BTV circulation within the Netherlands and Europe.

Within the European Union, infectious cattle diseases are categorized in the Animal Health Law. No strict EU regulations exist for control, evidence of disease freedom, and surveillance of diseases listed other than categories A and B. Consequently, EU member states follow their own varying strategies for disease control. The aim of this study was to provide an overview of the control and eradication programs (CPs) for six cattle diseases in the Netherlands between 2009 and 2019 and to highlight characteristics specific to the Dutch situation. All of these diseases were listed as C,D or E in the New Animal Health Law. In the Netherlands, CPs are in place for six endemic cattle diseases: bovine viral diarrhea, infectious bovine rhinotracheitis, salmonellosis, paratuberculosis, leptospirosis, and neosporosis. These CPs have been tailored to the specific situation in the Netherlands: a country with a high cattle density, a high rate of animal movements, a strong dependence on export of dairy products, and a high-quality data-infrastructure. The latter specifically applies to the dairy sector, which is the leading cattle sector in the Netherlands. When a herd enters a CP, generally the within-herd prevalence of infection is estimated in an initial assessment. The outcome creates awareness of the infection status of a herd and also provides an indication of the costs and time to achieve the preferred herd status. Subsequently, the herd enrolls in the control phase of the CP to, if present, eliminate the infection from a herd and a surveillance phase to substantiate the free or low prevalence status over time. The high-quality data infrastructure that results in complete and centrally registered census data on cattle movements provides the opportunity to design CPs while minimizing administrative efforts for the farmer. In the CPs, mostly routinely collected samples are used for surveillance. Where possible, requests for proof of the herd status are sent automatically. Automated detection of risk factors for introduction of new animals originating from a herd without the preferred herd status i.e., free or unsuspected, is in place using centrally registered data. The presented overview may inspire countries that want to develop cost-effective CPs for endemic diseases that are not (yet) regulated at EU level., Competing Interests: IS-B, MM, MW, LD, HW, KB, TD, AR, AV, and GS are employed by Royal GD, which runs most of the CPs described in this paper. All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Santman-Berends, Mars, Weber, van Duijn, Waldeck, Biesheuvel, van den Brink, Dijkstra, Hodnik, Strain, de Roo, Veldhuis and van Schaik.)

Two vector-borne infections have emerged and spread throughout the north-western part of Europe in the last decade: Bluetongue virus serotype-8 (BTV-8) and the Schmallenberg virus (SBV). The objective of the current study was to compare three statistical methods when applied in a syndromic surveillance context for the early detection of emerging diseases in cattle in the Netherlands. Since BTV-8 and SBV both have a negative effect on milk production in dairy cattle, routinely collected bulk milk recordings were used to compare the three statistical methods in their potential to detect drops in milk production during a period of seven years in which BTV-8 and SBV emerged. A Cusum algorithm, Bayesian disease mapping model, and spatiotemporal cluster analysis using the space-time scan statistic were performed and their performance in terms of sensitivity and specificity was compared. Spatiotemporal cluster analysis performed best for early detection of SBV in cattle in the Netherlands with a relative sensitivity of 71% compared to clinical surveillance and 100% specificity in a year without major disease outbreaks. Sensitivity to detect BTV-8 was low for all methods. However, many alerts of reduced milk production were generated several weeks before the week in which first clinical suspicions were reported. It cannot be excluded that these alerts represent the actual first signs of BTV-8 infections in cattle in the Netherlands thus leading to an underestimation of the sensitivity of the syndromic surveillance methods relative to the clinical surveillance in place., (Copyright © 2020 Veldhuis, Swart, Brouwer-Middelesch, Stegeman, Mars and van Schaik.)

The outcomes of a voluntary bovine viral diarrhoea virus (BVDV) control programme that has been in place in the Netherlands since 1997 were analysed. This 'BVDV-free' programme was studied in dairy herds in the period 1 August 2007 to 1 August 2013. The programme was based on a test and cull approach at the herd level, after which the BVDV status was monitored by testing young stock for antibodies against BVDV or by antigen testing of newborn calves. One of the challenges of the programme was that, without any legislation or subsidies, farmers had to be motivated to pay all costs involved, with eradication of BVDV from their farm as the only incentive. During the study period, the percentage of dairy farms with a 'BVDV-free' status in the Netherlands increased from 13% to 24%, while the prevalence of active BVDV infections in Dutch dairy herds decreased. This may be related to the increasing number of participants in the 'BVDV-free' programme., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In the Netherlands, the feasibility of a national control program for infectious bovine rhinotracheitis (IBR) is discussed. The aim of this program would be to achieve freedom from BoHV1 circulation (the causal agent of IBR), in the Dutch cattle population. When IBR would be eradicated, maintaining the free status is essential and insight in the probability of introduction of IBR through cattle imports is crucial. Values for input parameters such as the number of imports per country of origin, herd level prevalence and probability that a random imported animal per age category was either acutely or latently infected with IBR were quantified. A stochastic simulation model was built to predict the basic risk and the efficacy of four risk mitigating scenarios were evaluated. These scenarios involved testing prior to import, import restrictions and vaccination. The model output predicted that IBR infected animals are imported regularly. In an IBR free situation, 571 (5th and 95th percentile: 431-781) cattle herds will be newly infected. Latent infections account for most newly infected herds (77%). When the virus in the imported latently infected animal does not reactivate, subsequent impact of such infections remains limited. The model predicted that most of the herds infected by introduction of acutely infected animals would be veal herds. The scenario in which imports were only allowed from status 9 or 10 countries combined with testing cattle that originated from status 9 countries was most effective in reduction of the import risk to 70 herds per year. The scenario in which vaccination of calves was combined with testing of older cattle was estimated to reduce the number of newly infected herds to 82 per year. The stakeholders classified the latter scenario as most realistic because this scenario was deemed both feasible and rather effective. This study did not evaluate the impact of introduction of IBR in the cattle population, which might differ depending on the type of infection (acute vs. latent) and the herd type in which the virus is introduced. Moreover, when making the final decision about the optimal intervention, the economic perspective should also be taken into account. This study predicted that introduction of IBR will remain a risk for the Dutch cattle population after virus circulation is eliminated from the Netherlands. The import risk is reduced most in scenarios in which testing and vaccination are combined., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Many countries have implemented control programmes aiming to eradicate Bovine Viral Diarrhoea Virus (BVDV). After obtaining the free status, a risk of re-introduction of the virus through import may remain. Therefore the risk of introduction of BVDV through cattle imports in the Netherlands was quantified and the effectiveness of subsequent intervention measures was assessed. Data, literature and expert opinion were used to estimate values for input parameters to feed a stochastic simulation model. The probability that BVDV was imported was differentiated into persistently infected (PI) cattle, trojan cows that transmitted the virus vertically resulting in a PI foetus (TR) and transient infected cattle (TI). The import risk was stratified to beef, dairy, small scale, suckler, trade, veal and young stock herds. The intervention scenarios that were evaluated consisted of virus testing, a combination of virus testing and antibody testing in pregnant cows, abolishment of imports from high risk countries (i.e. countries with a BVDV prevalence >15%) and a combination of import restrictions and testing prior to import. Each year, 334 (5th and 95th percentile: 65-902) Dutch cattle herds were estimated to be infected with BVDV through import. Veal herds account for most infections associated with import (87%), whereas in the other herd types, only 9 beef, 6 dairy, 2 small scale, 16 suckler, 10 trade and 2 young stock herds are infected through imports per year. Import of PI cattle is the most important risk for introduction in veal herds, while import of TR cows is the main source of BVDV introduction in dairy, small scale and suckler herds. With the intervention scenarios, the number of BVDV infected herds in the Netherlands could be reduced to 81 and 58 herds per year when respectively virus testing or a combination of virus and antibody testing was applied or to 108 herds when import from high risk countries was abolished. With the scenario in which both import from high risk countries was abolished combined with virus and antibody testing, the number of BVDV infected herds could be reduced to 17 herds per year. The risk assessment showed that BVDV is regularly imported in the Netherlands. The import risk can effectively be reduced by implementing diagnostic testing prior to import and only import cattle with a favourable result, eventually combined with certain trade restrictions., (Copyright © 2017. Published by Elsevier B.V.)

Two years after the introduction of the Schmallenberg virus in north-western Europe, it is unknown whether the virus is still circulating in countries that were the first to be confronted with it. When the population-level immunity declines in Europe, reintroduction or the re-emergence of SBV in Europe might eventually result in an outbreak of similar magnitude of that seen in 2011-2012. The Netherlands was part of the primary outbreak region of SBV in 2011. The aim of this study was to determine whether SBV circulated amongst dairy herds in the Netherlands in 2013, and if so, to which extent. For this purpose, the presence of SBV-specific antibodies in naive cattle was investigated. A total of 394 dairy farms were sampled between October and December 2013 by collecting five serum samples per herd. Antibodies were detected in 1.1% [95% confidence interval (CI): 0.7-1.7)] of the animals. All seropositive animals were single reactors per herd and were at least 8 months old at sampling. As these results were inconclusive in demonstrating freedom of SBV circulation, a more in-depth investigation was initiated to provide more insight: an additional sample of 20 youngstock within the same age category (including the five initially sampled animals) was collected from 17 of the 21 positive herds and tested for SBV-specific antibodies. This resulted in 9 antibody-positive test results of 316 samples. Again, the positive samples were single reactors within the sample obtained from each farm, which is unlikely given the characteristics of SBV. Therefore, assuming the single reactors as false-positive, this survey showed with 95% confidence that the maximum possible prevalence of herds with SBV circulation in the Netherlands was <1% in 2013., (© 2015 Blackwell Verlag GmbH.)

Bovine viral diarrhea virus (BVDV) is an important endemic infection. However, no information was available on whether it would be economically beneficial to implement a national control program in the Netherlands. Therefore, a stochastic simulation model was developed in which control scenarios were added to compare the epidemiological and economic consequences of BVDV control in Dutch dairy herds in the next 10 yr. In the epidemiological part of the model, herds could be classified as susceptible, infectious, recovered, or vaccinated. The outputs of the epidemiological module served as input for the economic module. Net costs that could be attributed to bovine viral diarrhea consisted of production losses, costs for testing, and culling persistently infected cattle in the present voluntary Dutch BVDV control program and costs for vaccination. Four different control scenarios were simulated, involving testing and culling of persistently infected (based on serum or ear-notch testing), and monitoring BVDV statuses and vaccination and were derived from BVDV control programs that are currently executed in Europe. The costs and benefits of BVDV control in the current situation and in each of the simulated control scenarios were evaluated assuming an annual discount rate of 2%. The model estimated a mean BVDV herd prevalence of 18.0% in 2014 and showed a slightly decreasing prevalence over time. The outputs seemed realistic for the present situation in the Netherlands when compared with actual survey data. The average annual net costs associated with bovine viral diarrhea were estimated at €27.8 million for the dairy industry. Two control scenarios were beneficial in controlling BVDV during the study period (between 2015 and 2025). In the scenario where tracing and removing of PI animals and monitoring of the subsequent status was obligatory, the benefit to cost (B/C) ratio was 1.5 (€1.5 benefit for each invested euro). In the scenario in which the BVDV status of all herds was determined, followed by voluntary measures before control measures became obligatory, the B/C ratio was 1.1. The B/C ratio of the scenarios included could be even higher when it was assumed that nondairy herds participated in the control program as well. The model provided the opportunity to compare the effect of voluntary and mandatory control scenarios on the BVDV prevalence and costs and benefits relative to the current situation in the Netherlands. The model was used to support policy makers in their decisions about a BVDV control program., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Schmallenberg virus (SBV), a novel orthobunyavirus that rapidly spread throughout north-western Europe in 2011, caused congenital malformations in lambs and goat kids (Van den Brom et al., 2012) and newborn calves (Hoffmann et al., 2012). The impact of the SBV epidemic seemed limited however, in terms of the number of affected herds with malformed offspring (European Food Safety Authority, 2012b). Nevertheless, little is known with regard to the overall within-herd impact of SBV infection. The objective of the current study was to quantify the impact of the 2011 SBV epidemic on the productivity of dairy cattle in the Netherlands and the district of Kleve, Germany. For the Netherlands, several multilevel multivariable statistical models were applied on eight productivity parameters regarding milk production, reproductive performance and mortality. All four fertility parameters analysed were slightly but significantly reduced between August 1st and November 1st 2011 compared to the reference period in 2009-2010. Between August 15th and September 19th 2011, the average loss in milk production per cow was -0.26kg (95% CI: -0.30; -0.22) per day in dairy herds, compared to the reference period (p<0.001). The total loss per cow in a subgroup of dairy herds that notified malformations in newborn calves during the mandatory notification period in the Netherlands was -0.43kg (95% CI: -0.59; -0.28) per day (p<0.001). For Germany, a study was carried out in the district of Kleve, situated in the state of North Rhine-Westphalia near the Dutch border. Data on milk yield, two fertility parameters and the number of rendered calves in this specific region were analysed. There was a small but significant increase in the number of secondary and third inseminations between August 1st and November 1st 2011, indicating reduced fertility. No significant change in calf mortality was observed in the assumed SBV period. Milk production at district level did not seem to be affected by SBV in August and September 2011. SBV had no or limited impact on mortality rates, which was as expected given the relatively mild expression of SBV in adult cows and the low incidence of notified malformations in newborn calves. Our results indicate that SBV had a limited impact on productivity of dairy cattle. However, the total economic impact of SBV on the ruminant industry not only consists of productivity caused losses; it is expected that international trade restrictions formed a larger part of the total economic impact., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

In 2011, Schmallenberg virus (SBV), a novel member of the Simbu serogroup, genus Orthobunyavirus, was identified as the causative agent of a disease in ruminants in Europe. Based on the current knowledge on arthropods involved in the transmission of Simbu group viruses, a role of both midges and mosquitoes in the SBV transmission cycle cannot be excluded beforehand. The persistence of SBV in mosquitoes overwintering at SBV-affected farms in the Netherlands was investigated. No evidence for the presence of SBV in 868 hibernating mosquitoes (Culex, Anopheles, and Culiseta spp., collected from January to March 2012) was found. This suggests that mosquitoes do not play an important role, if any, in the persistence of SBV during the winter months in northwestern Europe., (© 2013 The Royal Entomological Society.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Epizootic outbreaks of congenital malformations in sheep are rare and have, to the best of our knowledge, never been reported before in Europe. This paper describes relevant preliminary findings from the first epizootic outbreak of ovine congenital malformations in the Netherlands. Between 25 November and 20 December 2011, congenital malformations in newborn lambs on sheep farms throughout the country were reported to the Animal Health Service in Deventer. Subsequently, small ruminant veterinary specialists visited these farms and collected relevant information from farmers by means of questionnaires. The deformities varied from mild to severe, and ewes were reported to have given birth to both normal and deformed lambs; both male and female lambs were affected. Most of the affected lambs were delivered at term. Besides malformed and normal lambs, dummy lambs, unable to suckle, were born also on these farms. None of the ewes had shown clinical signs during gestation or at parturition. Dystocia was common, because of the lambs' deformities. Lambs were submitted for post-mortem examination, and samples of brain tissue were collected for virus detection. The main macroscopic findings included arthrogryposis, torticollis, scoliosis and kyphosis, brachygnathia inferior, and mild-to-marked hypoplasia of the cerebrum, cerebellum and spinal cord. Preliminary data from the first ten affected farms suggest that nutritional deficiencies, intoxication, and genetic factors are not likely to have caused the malformations. Preliminary diagnostic analyses of precolostral serum samples excluded border disease virus, bovine viral diarrhoea virus, and bluetongue virus. In December 2011, samples of brain tissue from 54 lambs were sent to the Central Veterinary Institute of Wageningen University Research, Lelystad. Real-time PCR detected the presence of a virus, provisionally named the Schmallenberg virus, in brain tissue from 22 of the 54 lambs, which originated from seven of eight farms that had submitted lambs for post-mortem examination. This Schmallenberg virus was first reported in Germany and seems to be related to the Shamonda, Aino, and Akabane viruses, all of which belong to the Simbu serogroup of the genus Orthobunyavirus of the family Bunyaviridae. These preliminary findings suggest that the Schmallenberg virus is the most likely cause of this epizootic of ovine congenital malformations, which is the first such outbreak reported in Europe.

At the end of August and the first two weeks of September 2011 dozens of veterinary practitioners reported to GD Veekijker (Animal Health Service) several dairy herds with cows with sudden decreased milk production, watery diarrhea and sometimes fever. In the beginning these reports came from the Eastern region of the Netherlands, after that also from the other three regions. The percentages of diseased herds per veterinary practice varied from a few till dozens per cent. Extensive bacteriological, virological and parasitological testing of the feces of sick cows did not reveal an infectious cause of the clinical problems. Recently, 50 stored blood samples of clinically diseased cattle were tested for the Schmallenbergvirus using a PCR, and 36% (18/50) tested positive. A large group of control cows (n=115) was also tested with the PCR and all cattle tested negative. Likely the Schmallenbergvirus was the primary cause of the clinical symptoms in the Dutch dairy herds. Further research will be done to confirm this.

In cattle, Coxiella burnetii infections are generally asymptomatic but can also be associated with reproductive disorders. Metritis is considered as one of the symptoms of C. burnetii infections, but reliable information is lacking. Therefore, information on the presence of C. burnetii in the uterine content of cows with metritis is important to increase our knowledge on this pathogen. In this study, the uterine content of 45 dairy cows with metritis belonging to 12 herds was tested for C. burnetii with a real-time PCR assay. Only one uterine sample tested PCR (highly) positive, all other samples were PCR negative. The PCR positive cow tested also positive for antibodies. Three other cows from other herds tested antibody positive. The bulk milk (BM) samples of these 12 herds were tested by real-time PCR assay and antibody-ELISA. Six BM samples (50%) were positive in PCR and 10 (83%) were positive in ELISA. Culturing the uterus samples by bacteriology, the most frequently cultured bacteria were arcanobacterium (n=24), E. coli (n=16), other streptococci (n=10), Streptococcus uberis (n=8) and Streptococcus dysgalactiae (n=5). This study indicates that C. burnetii is not an important cause for metritis in dairy herds, although apparently C. burnetii was or had been present in most of these herds., (Copyright © 2010 Elsevier B.V. All rights reserved.)

After the introduction of bluetongue virus serotype 8 (BTV-8) in western Europe in 2006, an indirect ELISA for detection of serogroup-specific antibodies against BTV in serum samples was validated for individual milk samples by the Central Veterinary Institute and the Animal Health Service in the Netherlands (Kramps et al., 2008). In order to develop a cost-effective monitoring tool, we now have evaluated this ELISA also for use in bulk milk. Therefore, bulk milk samples and individual milk samples were collected from 92 herds in the affected southern region in the Netherlands in 2007, before the start of the vaccination campaign. In addition, bulk milk samples collected from 88 herds before the bluetongue introduction in 2006 ("historically negative" samples) have been tested. With these results ROC analyses were performed and herd specificity and herd sensitivity of the bulk milk ELISA were estimated. All "historically negative" bulk milk samples were negative in the ELISA, with a mean S/P ratio of 10 ± 0.8%. The herd sensitivity and herd specificity of the ELISA in bulk milk samples depend on the cut-off that is chosen. In order to detect a within-herd-prevalence of 1%, the optimal cut-off S/P ratio 13% was found. A few herds with one or two milk-positive animals would then be missed. The specificity will be 100%. A within-herd-prevalence of 10% can be detected with 100% sensitivity at a cut-off S/P ratio of 96%. In conclusion, the indirect ELISA in bulk milk samples is a very specific and sensitive test which can be implemented in monitoring and surveillance systems in unvaccinated populations., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested. Compared to the ELISA for sera, the relative specificity and sensitivity of the ELISA for milk samples is 96.5% and 98.9%, respectively when using a S/P% cut-off value of 50% as advised by the manufacturer. The optimal cut-off value was found at S/P% of 90% revealing an optimal specificity (99.0%) combined with an optimal sensitivity (98.1%). Titres in positive individual milk samples ranged from 1 to 2048 with a peak titre of 128. Bulk milk samples contained antibodies with titres ranging from 64 to 512. The ELISA for milk samples was found to be a reliable and robust test. This diagnostic tool is very useful, and may replace the ELISA for serum samples as first choice in order to get insight into the status of lactating individual animals and therewith of the entire herd with respect to BTV infection.

The protozoan parasite Cryptosporidium is found world-wide and can cause disease in both humans and animals. To study the zoonotic potential of Cryptosporidium in The Netherlands we isolated this parasite from the faeces of infected humans and cattle and genotyped those isolates for several different markers. The overall genotyping results showed: for humans isolates, 70% Cryptosporidium hominis, 19% Cryptosporidium parvum, 10% a combination of C. hominis and C. parvum, and 1% Cryptosporidium felis; and for cattle isolates 100% C. parvum. Analysis of the genetic variants detected for the HSP70, ML1 and GP60 markers showed: for human isolates, one C. hominis and two C. parvum variants (C. parvum and C. parvum NL) for HSP70, one C. hominis and five C. parvum variants (C1, C2, C3, and C2 NL1 and C2 NL2) for ML1, four C. hominis (mainly IbA10G2) and four C. parvum variants (mainly IIaA15G2R1) for GP60; and the cattle isolates only C. parvum (not C. parvum NL1) for HSP70, C1 and C2 for ML1, and 17 different IIa sub-types (mainly IIaA15G2R1) for GP60. Molecular epidemiological analysis of the human data showed a C. hominis peak in autumn. The majority (80%) of the human cases were children aged between 0 and 9 years and >70% of these were caused by C. hominis. Patients >25 years of age were infected mainly with C. parvum. We conclude that C. hominis IbA10G2 is found at high frequencies in autumn in humans and not in cattle. The high prevalence of C. parvum IIaA15G2R1 in both humans and cattle indicates that cattle may be a reservoir for this sub-type in The Netherlands.

Q fever, a zoonosis caused by the gram-negative bacterium Coxiella burnetii, occurs worldwide and affects both humans and animals. Ruminants are considered to be the main source of infection of humans, with the main route of infection being through inhalation of the organism of fine-particle aerosols. Abortion is the main clinical sign in ruminants. During and after abortion, large quantities of the bacterium are shed via the placenta and other vaginal secretions. The bacterium may also be present in faeces and milk. The bacterium can survive for a long time in the environment after shedding and can be spread over long distances. Seroprevalence among cattle is rather high in the Netherlands and in many other countries. Infection is diagnosed by detecting antibodies against the bacterium or the bacterium itself by means of a PCR method. The efficacy of using antibiotics or vaccines for treatment or prevention of the disease in cattle is still unclear and there are currently no effective disease control programmes.

In The Netherlands, a voluntary bovine virus diarrhoea virus (BVDV)-free certification programme was started in 1997. After an intake procedure in which all cattle are tested for the presence of BVD virus, a herd obtains the status "BVD-virus-free". To maintain this status a monitoring procedure is executed to verify absence of BVDV circulation in the herd. Several diagnostic tests are used: RT-PCR in bulk milk and pooled blood samples, antigen-ELISA (Ag-ELISA) and antibody ELISA in individual blood samples. Sensitivity and specificity of these tests are discussed. In addition, a diagnostic quick scan has been introduced, consisting of a combination of bulk milk tests for virus and antibody, and antibody tests in samples from young stock. Preliminary results are presented.

A total of 15,822 cattle aged 3 years and older, belonging to 378 randomly selected herds, were tested for paratuberculosis using an absorbed enzyme-linked immunosorbent assay (ELISA); 3.3% tested positive. This percentage was lowest for the group of cattle aged 3-4 years (2.3%) and highest for cattle with the age of 5-6 years (4.5%). The mean Sample to Positive (S/P) ratio of seropositive cattle vaccinated against paratuberculosis was higher (0.75 +/- 0.33) than that of seropositive, non-vaccinated cattle (0.58 +/- 0.26). Faecal samples of 422 ELISA-positive cattle were cultured for the presence of Mycobacterium avium subsp. paratuberculosis, 12% of these were contaminated. The percentage of non-contaminated samples with positive culture results was 17.3%, with a substantial difference between vaccinated (1.7%) and non-vaccinated cattle (20.2%). Of the positive cultures, the number of colonies varied from 1-10 (22% of cultures), 11-100 (22%), to more than 100 (55%). The percentage of ELISA-positive, non-vaccinated cattle tested culture-positive was positively correlated with the magnitude of the S/P ratio. This percentage varied from 12% (S/P ratio 0.3-0.5) to 58% (S/P ratio > 1.1), a result that might have implications for interpretation of the test. In this study, the percentage of ELISA-positive cattle with positive faecal culture results was limited and these individuals were mostly moderate to heavy shedders.

In a cattle herd problems were noticed during 1.5 years, characterised by emaciation and loss of milk production with lower fat- and protein%, tenesmus, central nervous symptoms (aggression, circling and blindness), diarrhoea, eczema solare and death. Clinical, laboratory and post mortem examinations did not reveal a specific cause at first. An intoxication was suspected based on the clinical symptoms, the course of the disease in sick animals and severe liver cirrhosis found in a sudden death cow: The animals were fed grassilage derived from an air force base on which an overwhelming amount of tansy ragwort (Senecio jacobaea) was present. On subsequent microscopic examination of the livers of 11 slaughtered clinically healthy animals all of these showed fibrosis in varying extension. Cirrhosis and fibrosis of the liver and the clinical symptoms are all features of chronic pyrrolyzidine alkaloidosis. Tansy ragwort poisoning has not been diagnosed in the Netherlands for years. The rise in cases is due to more extensive use of land.

To assess the efficacy of a live glycoprotein E-negative bovine herpesvirus 1 (BHV1) vaccine to reduce transmission of BHV1 in cattle, a randomised, double-blind, placebo-controlled field trial including 84 herds was conducted in the Netherlands. The incidence of BHV1 infections during 17 months was monitored by detecting antibodies against BHV1 glycoprotein E. In the placebo-treated group 214 seroconversions in 3985 paired sera, and in the vaccinated group 67 seroconversions in 3601 paired sera were detected. Based on these data, the transmission ratio R(0) was estimated for each treatment, using the maximum likelihood approach and the martingale approach. In placebo-treated herds R(0) was 2.5 (CI 1.4-3.1) using maximum likelihood and 2.8 (S.E. 0.4) using the martingale approach. In the vaccinated group these estimations were 1.2 (CI 0.5-1.5) and 1.5 (S.E. 0.4) respectively. The vaccinated and placebo-treated group differed significantly in transmission of BHV1. These results suggest that the use of this live gE-negative BHV1 vaccine will reduce the incidence and transmission of BHV1 infections in the field.

We examined whether antibodies against bovine herpesvirus (BHV) 5 cross-react with BHV1 antigens and whether they could interfere with BHV1 eradication programmes. Six calves were experimentally infected with different doses of BHV5 strain N569; homologous antibodies were first detectable on day 11 post infection; they cross-reacted in a BHV1 virus neutralisation test, in a BHV1-glycoprotein (g)-B blocking ELISA and in a BHV1-gE ELISA, but not in a BHV1-gE blocking ELISA. This study indicates that, in ongoing BHV1 eradication programmes, based on vaccines that lack gE, BHV5 infections may not lead to false-positive serological reactions in case cattle are tested for BHV1-gE antibodies by the BHV1-gE blocking ELISA; antibodies against BHV5 may be differentiated from antibodies against BHV1. The BHV1-gE blocking ELISA may, therefore, offer opportunities for the serological differentiation between BHV1 and BHV5 infections.

Two hundred and thirty-seven of 2052 cattle which had not been vaccinated against bovine herpesvirus 1 (BHV-1) were seropositive in a glycoprotein B (gB)-blocking ELISA, but seronegative in a glycoprotein E (gE)-blocking ELISA. In order to detect whether they were latently infected with BHV-1, 10 of them were treated with corticosteroids in an attempt to reactivate putatively latent virus. After successive treatments with dexamethasone and prednisolone, no virus excretion was detected and they showed no increase in antibody titres. In contrast, one gE-seropositive animal re-excreted BHV-1 and had a four-fold increase in antibody titre after the corticosteroid treatments. After slaughter, no BHV-1 DNA could be detected with a sensitive PCR in samples of the trigeminal, cervical and sacral ganglia and spinal cords of the gE-seronegative cattle.

A small scale transmission experiment was performed with bovine herpesvirus 1 (BHV1) in a cattle population under field conditions. 10 calves were housed under strict hygienic conditions, with a distance of 4m between each calf. Five calves were experimentally infected with BHV1, two calves with strain Harberink and three with strain Lam, respectively. Experimentally infected calves were placed at 4 m distance from five susceptible sentinel calves. Airborne transmission to sentinel calves was detected using virus isolation and BHV1 specific polymerase chain reactions in samples of nasal fluids, and BHV1 specific antibodies in serum samples. Strain Harberink was hardly transmitted to sentinel calves, whereas strain Lam was transmitted to all sentinels. Estimating the rate of transmission per day, the total number of calves infected by one (strain Lam) infected calf was 1.18. Comparing this estimated transmission ratio between cattle at a distance of 4 m to the estimated transmission ratio R of BHV1 in susceptible commingled cattle reported before, the effect of the factor distance on the transmission ratio could be calculated. Extrapolating these results, a distance of 4.4 m between cattle populations would be necessary to reduce transmission for this strain to R<1.

An experiment was performed to develop a model to study the impact of stress on responsiveness to infection with bovine herpesvirus 1 (BHV1) in veal calves. Social isolation after previous group-housing was used as a putatively stressful treatment. Group-housed specific pathogen-free veal calves (n=8) were experimentally infected with BHV1 at the age of 12 weeks. Half of the calves were socially isolated at the time of infection. Clinical, virological and serological responses to BHV1, and adreno-cortical reactivity to exogenous ACTH were examined. In comparison with group-housed calves, calves socially isolated at the time of infection showed a diminished clinical and fever response, and delayed viral excretion after primary infection with BHV1. Four weeks after social isolation, basal cortisol levels before, and the integrated cortisol response after administration of a low dose of ACTH, were significantly depressed in socially isolated calves. The results suggest that social isolation in veal calves influences the response to an experimental BHV1 infection. A possible mechanism is discussed.

Three identical transmission experiments were successively performed to quantitatively evaluate the possible transmission of a gE-negative bovine herpesvirus 1 (BHV1) vaccine strain among cattle. After intranasal inoculation, the vaccine virus was excreted in high titers in nasal fluids. However, the vaccine virus was transmitted to only one sentinel in one experiment, and not to any of the 10 sentinel cattle in the other two experiments. Based on these observations, it can be concluded that the expected number of cases per vaccine-inoculated animal, i.e. the transmission ratio R(0) of the vaccine strain, is significantly below 1. The R(0) was estimated to be 0.14. After intramuscular inoculation, shedding of vaccine virus was not detected. Therefore, we concluded that it is highly unlikely that this live gE-negative BHV1 vaccine strain will perpetuate in the cattle population.

To study possible reactivation and to quantify subsequent transmission of a live gE-negative bovine herpesvirus 1 (BHV1) vaccine strain in cattle populations, four experiments were performed. Two groups of cattle were each tested twice for the possibility of reactivation. Inoculation with a gE-negative BHV1 vaccine was done either intramuscularly or intranasally and treatment with corticosteroids in an attempt to reactivate vaccine virus, was done after 6 or 11 weeks, and again after 6 months. To quantify transmission of vaccine virus following possible reactivation, each cattle was housed together with one susceptible contact-cattle. Contact-infections were monitored using virus shedding and antibody responses. After corticosteroid treatments, re-excretion of virus was never detected in cattle that had been inoculated with the gE-negative BHV1 vaccine strain. Contact cattle did not shed gE-negative BHV1, nor mounted any antibody response against BHV1. In contrast, positive control cattle, inoculated intranasally with wild-type BHV1, re-excreted virus in high titers in nasal fluids and transmitted the virus to contact cattle. Based on these results, the transmission ratio R(0) of the vaccine strain was zero. We concluded that it is highly unlikely that the live gE-negative BHV1 vaccine strain will be re-excreted after possible reactivation, and consequently, it is even less likely that reactivated vaccine virus will spread in the cattle population.

To control the diseases caused by bovine herpesvirus 1 (BHV1), bovine respiratory syncytial virus (BRSV), and bovine virus diarrhoea virus (BVDV), it is crucial to know their modes of transmission. The purpose of this study was to determine whether these viruses can be transmitted by air to a substantial extent. Calves were housed in two separate isolation stables in which a unidirectional airflow was maintained through a tube in the wall. In one stable, three of the five calves were experimentally infected with BHV1 and later with BRSV. In the BVDV experiment, two calves persistently infected with BVDV (PI-calves) instead of experimentally infected calves, were used as the source of the virus. In all the calves infections were monitored using virus and antibody detection. Results showed that all the three viruses were transmitted by air. BHV1 spread to sentinel calves in the adjacent stable within three days, and BRSV within nine days, and BVDV spread to sentinel calves probably within one week. Although airborne transmission is possibly not the main route of transmission, these findings will have consequences for disease prevention and regulations in control programmes.

The purpose of this study was to determine whether individual milk samples can replace serum samples for the detection of bovine herpesvirus 1 (BHV1) glycoprotein E (gE)-specific antibodies. Serum and milk samples were collected at the same time from cattle in BHV1-free herds, cattle in unvaccinated herds, and cattle in herds that were vaccinated twice with a BHV1 marker vaccine. The samples were tested in two gE enzyme-linked immunosorbent assay (ELISA) systems. In comparison to serum, the results showed that the gE-blocking ELISA was highly sensitive for testing milk samples (0.96). In contrast, the gE ELISA was less sensitive (0.79). The specificities of the gE-blocking ELISA and the gE ELISA for testing milk samples were very high (1.00 and 0.99, respectively). The presented results indicate that individual milk samples, which can be collected relatively easily and inexpensively, can be used instead of individual serum samples in the gE-blocking ELISA for the screening of cattle for BHV1 gE antibodies.

A method is described to evaluate pharmacologic influence on bovine tracheal mucus transport in vitro. In this model, cholinergic agonist methacholine stimulated transport velocity. Methacholine at concentration of 10(-6)M increased velocity by 13.6%, whereas 10(-5)M increased velocity by 26%. Stimulation was inhibited by atropine.

A 12-year-old Dutch warmblood mare was examined because it had suffered colic intermittently for a few years and had lost weight in the previous two months. Palpation per rectum revealed a large firm mass in the left sublumbar region; the mass was classified post mortem as an adrenocortical carcinoma. The basal plasma cortisol concentration (at 10.00) of the mare was 94 nmol/litre, within the normal range. As in another case of adrenocortical neoplasm, a functional tumour could not be demonstrated. Only one of the 21 horses with a neoplasm of the pituitary-adrenocortical axis examined by the authors, had the tumour in the adrenal gland.