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Campylobacteriose is de meest voorkomende bacteriële voedselinfectie in Nederland en pluimvee is daarbij een belangrijke bron van besmetting. Om het aantal voedselinfecties als gevolg van Campylobacter te verminderen is het voorkomen van besmettingen bij vleeskuikens de meest impactvolle manier. Internationaal is de consensus dat het zo goed mogelijk in stand houden van de biosecurity de belangrijkste (en vrijwel enige) manier is voor pluimveehouders om Campylobacter buiten de stal te houden. In eerder uitgevoerd onderzoek leek het toepassen van de HyCare-methode een positieve uitwerking te hebben op het behouden van een Campylobacter negatieve status bij koppels vleeskuikens, echter de resultaten waren gebaseerd op een beperkt aantal bemonsteringen. In 2023 is daarom vervolgonderzoek uitgevoerd op het vleeskuikenbedrijf van MS Schippers in Hapert naar het effect van het toepassen van de HyCare-methode op de Campylobacter status van een koppel vleeskuikens. Gedurende de periode maart-november zijn de vleeskuikenkoppels in drie van de vier op het bedrijf aanwezige stallen, waaronder de HyCare-stal, wekelijks bemonsterd om de Campylobacter status vast te stellen. Om meer inzicht te krijgen in eventuele aanwezigheid van bedrijfs/stalgebonden Campylobacter stammen (herintroductie vanuit de stal en/of directe omgeving), zijn de geïsoleerde Campylobacter stammen verder genetisch onderzocht d.m.v. whole genome sequensing (WGS) en Multilocus sequence typing (MLST). In totaal is de Campylobacter status van de koppels in stal 1 (HyCare-stal), 3 en 5 gedurende vier opeenvolgende rondes opgevolgd. De resultaten lieten geen verschil zien in het aantal Campylobacterbesmettingen tussen de stal volgens het HyCare principe en de twee overige (reguliere) stallen. Daarmee kunnen resultaten uit een eerdere monitoring op dit bedrijf niet worden bevestigd, waarin het aantal besmettingen in de HyCare-stal lager was in vergelijking met de niet-HyCarestal (één van de vier rondes respect

Research was conducted on the concentrations of Campylobacter in wastewater from a Dutch broiler slaughterhouse and the effects of the treatment steps performed at the slaughterhouse before the wastewater was discharged to the municipal sewer system. Subsequently, samples were collected at the regional sewage treatment plant (WWTP) that processes wastewater including that from the poultry slaughterhouse. Both incoming (influent) and treated water was sampled before it was discharged to surface water. The raw wastewater from the poultry slaughterhouse was found to contain very high numbers of Campylobacter throughout the study period, from September 2022 through January 2023, with an average of 6.6 log10 Campylobacter per Liter. The slaughterhouse purification process consisting of coagulation/flocculation followed by a flotation step gave a limited reduction in Campylobacter count of about 0.7 log10. Discharge of the water to the public sewer system accounts for a dilution due to mixing with other wastewater streams. The dilution based on the measured volumes from the slaughterhouse to the WWTP was found to fairly match the measured concentrations of Campylobacter in the influent of the WWTP averaging 4.4 log10/Liter. Subsequently, a strong reduction occurred as a result of the WWTP's treatment processes by an average of 2.6 log10. Despite this reduction, Campylobacter was also still found in much of the sampled volumes of WWTP effluent, with concentrations ranging from <0.5 log10 to 2.5 log10/Liter of water. In addition to the amount of culturable Campylobacter, wastewater was also examined for the presence of viable but non-culturable (VBNC) Campylobacter. VBNC Campylobacter bacteria may still be infectious and potentially form an underrated source of infection for humans or animals. In this study however, no significant amounts of VBNC Campylobacter were found in any of the samples examined. It is plausible that the transformation to this non-culturable form occu, Nederlandse vleeskuikenslachterij en de effecten van de zuiveringsstappen uitgevoerd op de slachterij zelf, voordat het afvalwater werd geloosd op het gemeentelijke rioolstelsel. Vervolgens zijn ook monsters genomen bij de regionale rioolwaterzuiveringsinstallatie die het afvalwater van onder andere de pluimveeslachterij verwerkt. Zowel het inkomende (influent) als het gezuiverde water is bemonsterd, voordat het werd geloosd op het oppervlaktewater. Het ruwe afvalwater van de pluimveeslachterij bleek gedurende de gehele onderzoeksperiode, van september 2022 tot en met januari 2023, zeer hoge concentraties Campylobacter te bevatten, met gemiddeld 6,6 log10 Campylobacter per liter. Het zuiveringsproces van de slachterij bestaande uit coagulatie/flocculatie, gevolgd door een flotatiestap, gaf een beperkte reductie van de concentratie Campylobacter van ongeveer 0,7 log10. Nadat het water werd geloosd op het openbare rioolstelsel vond een verdunning plaats door het samengaan met andere afvalwaterstromen. De verdunning op basis van de verpompte volumes van de slachterij en de rioolwaterzuiveringsinstallatie (rwzi) bleek redelijk overeen te komen met de aangetoonde concentraties van Campylobacter in het influent van de rwzi van gemiddeld 4,4 log10 per liter. Vervolgens vond een sterke reductie plaats als gevolg van de zuiveringsprocessen van de rwzi met gemiddeld 2,6 log10. Ondanks deze reductie werd ook in een groot deel van de bemonsterde volumes van het effluent van de rwzi nog Campylobacter gevonden, met concentraties die varieerden tussen <0,5 log10 tot 2,5 log10 per liter water. In deze studie is het afvalwater niet alleen onderzocht op de aanwezigheid en hoeveelheid kweekbare Campylobacter, maar is er ook gekeken of er levende, maar niet meer kweekbare Campylobacter bacteriën in de watermonsters aanwezig waren, zogenoemde viable but non-culturable (VBNC) Campylobacter. VBNC Campylobacters zijn mogelijk nog steeds infectieus en kunnen een onderschatte bron zijn voor

Campylobacter is de meest voorkomende bacteriële veroorzaker van voedselinfecties bij de mens. Pluimvee is daarbij een zeer belangrijke bron van besmetting. In het belang van de volksgezondheid is het daarom gewenst om in de pluimveesector het aantal Campylobacter besmettingen te verminderen. Een koppel vleeskuikens kan tijdens een ronde besmet worden met Campylobacter door insleep van de bacterie vanuit de omgeving. Of de overdracht van Campylobacter tussen twee opeenvolgende rondes in één stal ook een rol speelt bij de besmetting, is onzeker. In een samenwerking tussen Wageningen University & Research en Van Eck Bedrijfshygiëne B.V. is in 2021 een onderzoek uitgevoerd, met als doel te onderzoeken in hoeverre er levende Campylobacter na het wegladen van een Campylobacter positief koppel, ondanks reiniging en desinfectie, in de stal kan achterblijven en een bron van besmetting kan zijn voor een volgend koppel. Dit is onderzocht door vleeskuikenstallen, na het wegladen van een positief koppel, uitgebreid te bemonsteren tijdens de leegstandsperiode en deze monsters te onderzoeken op aanwezigheid van levende Campylobacter. Hierbij is zowel gekeken naar kweekbare als naar zogenoemde viable but non-culturable (VBNC; ‘levend maar niet kweekbare) Campylobacter. Deze VBNC’s zijn mogelijk nog steeds infectieus en dus in staat om een nieuw koppel vleeskuikens te besmetten. In deze studie is in geen van de stallen van de deelnemende bedrijven kweekbare Campylobacter teruggevonden tijdens de leegstandsperiode. Ook is er geen VBNC-Campylobacter aangetoond in de stalmonsters genomen na ontsmetting. Deze bevindingen laten zien dat het achterblijven van levende Campylobacter in een stal geen grote rol speelt bij de overdracht van Campylobacter tussen koppels vleeskuikens. Denkend aan de grote hoeveelheden Campylobacter die worden uitgescheiden in de stal door een Campylobacter positief koppel, is het gunstig dat er in deze studie aan het eind van de leegstandsperiode geen leven

Wind-supported transport of particle matter (PM) contaminated with excreta from highly pathogenic avian influenza virus (HPAIv)-infected wild birds may be a HPAIv-introduction pathway, which may explain infections in indoor-housed poultry. The primary objective of our study was therefore to measure the nature and quantity of PM entering poultry houses via air-inlets. The air-inlets of two recently HPAIv-infected poultry farms (a broiler farm and a layer farm) were equipped with mosquito-net collection bags. PM was harvested every 5 days for 25 days. Video-camera monitoring registered wild bird visits. PM was tested for avian influenza viruses (AIV), Campylobacter and Salmonella with PCR. Insects, predominantly mosquitoes, were tested for AIV, West Nile, Usutu and Schmallenberg virus. A considerable number of mosquitoes and small PM amounts entered the air-inlets, mostly cobweb and plant material, but no wild bird feathers. Substantial variation in PM entering between air-inlets existed. In stormy periods, significantly larger PM amounts may enter wind-directed air-inlets. PM samples were AIV and Salmonella negative and insect samples were negative for all viruses and bacteria, but several broiler and layer farm PM samples tested Campylobacter positive. Regular wild (water) bird visits were observed near to the poultry houses. Air-borne PM and insects-potentially contaminated with HPAIv or other pathogens-can enter poultry air-inlets. Implementation of measures limiting this potential introduction route are recommended.

We used national registry data on human cases of Francisella tularensis subspecies holarctica infection to assess transmission modes among all 26 autochthonous cases in the Netherlands since 2011. The results indicate predominance of terrestrial over aquatic animal transmission sources. We recommend targeting disease-risk communication toward hunters, recreationists, and outdoor professionals.

Intestinal campylobacteriosis, caused by Campylobacter ingestion, is the most reported zoonosis in the EU; it is societally costly and can lead to more severe sequelae. To reduce Campylobacter infections, biosecurity measures at both farms and slaughterhouses are warranted. However, the potential improvements achieved by these interventions have not been quantified. We used a systems science approach to develop a simulation model, synthesizing information from interviews with stakeholders in the Dutch broiler production industry and the current literature. The model includes both farms and slaughterhouses in a “system of systems,” helping to clarify the complexity of interrelated components of these systems and analyse the impact of various interventions. Insects, transportation crates, farm personnel, and catchers were identified as potential Campylobacter sources and modelled as elements of feedback loops. Insect control, farm hygiene, visitor control, thinning, and transportation control interventions were analysed. The model was shown to accurately describe the seasonality of Campylobacter, which supports its validity. Model simulation revealed that insect control interventions had the strongest impacts, followed by combined farm hygiene and visitor control, and combined thinning and transportation control. Insect control interventions alone reduced the peak percentage of contaminated chickens from 51% to 26% and the peak percentage of highly contaminated (>1000 CFU/g) neck samples of chicken carcasses from 13% to 8%. Implementing all interventions concurrently reduced the peak percentages of contaminated chickens to 5% and highly contaminated chicken neck samples to 2%. These results suggest that multiple biosecurity measures must be implemented to reduce Campylobacter contamination., Policy Analysis

Wind-supported transport of particle matter (PM) contaminated with excreta from highly pathogenic avian influenza virus (HPAIv)-infected wild birds may be a HPAIv-introduction pathway, which may explain infections in indoor-housed poultry. The primary objective of our study was therefore to measure the nature and quantity of PM entering poultry houses via air-inlets. The air-inlets of two recently HPAIv-infected poultry farms (a broiler farm and a layer farm) were equipped with mosquito-net collection bags. PM was harvested every 5 days for 25 days. Video-camera monitoring registered wild bird visits. PM was tested for avian influenza viruses (AIV), Campylobacter and Salmonella with PCR. Insects, predominantly mosquitoes, were tested for AIV, West Nile, Usutu and Schmallenberg virus. A considerable number of mosquitoes and small PM amounts entered the air-inlets, mostly cobweb and plant material, but no wild bird feathers. Substantial variation in PM entering between air-inlets existed. In stormy periods, significantly larger PM amounts may enter wind-directed air-inlets. PM samples were AIV and Salmonella negative and insect samples were negative for all viruses and bacteria, but several broiler and layer farm PM samples tested Campylobacter positive. Regular wild (water) bird visits were observed near to the poultry houses. Air-borne PM and insects—potentially contaminated with HPAIv or other pathogens—can enter poultry air-inlets. Implementation of measures limiting this potential introduction route are recommended.

Intestinal campylobacteriosis, caused by Campylobacter ingestion, is the most reported zoonosis in the EU; it is societally costly and can lead to more severe sequelae. To reduce Campylobacter infections, biosecurity measures at both farms and slaughterhouses are warranted. However, the potential improvements achieved by these interventions have not been quantified. We used a systems science approach to develop a simulation model, synthesizing information from interviews with stakeholders in the Dutch broiler production industry and the current literature. The model includes both farms and slaughterhouses in a “system of systems,” helping to clarify the complexity of interrelated components of these systems and analyse the impact of various interventions. Insects, transportation crates, farm personnel, and catchers were identified as potential Campylobacter sources and modelled as elements of feedback loops. Insect control, farm hygiene, visitor control, thinning, and transportation control interventions were analysed. The model was shown to accurately describe the seasonality of Campylobacter, which supports its validity. Model simulation revealed that insect control interventions had the strongest impacts, followed by combined farm hygiene and visitor control, and combined thinning and transportation control. Insect control interventions alone reduced the peak percentage of contaminated chickens from 51% to 26% and the peak percentage of highly contaminated (>1000 CFU/g) neck samples of chicken carcasses from 13% to 8%. Implementing all interventions concurrently reduced the peak percentages of contaminated chickens to 5% and highly contaminated chicken neck samples to 2%. These results suggest that multiple biosecurity measures must be implemented to reduce Campylobacter contamination.

Equine piroplasmosis (EP) is a tick-borne disease affecting horses, donkeys, mules and zebras, caused by the intracellular apicomplexan protozoa Babesia caballi and Theileria equi. The geographical distribution of EP is closely related to the distribution of its vector tick species belonging to the genera of Dermacentor, Rhipicephalus and Hyalomma. Since the discovery of Dermacentor reticulatus ticks in 2007 and the first reported autochthonous cases in the South of the Netherlands in 2012, no data on the (sero)prevalence of EP in horses in the Netherlands have been reported and it remains unclear whether B. caballi and T. equi have been able to establish themselves in the Netherlands. This study aims to give an update on the current status of EP in horses in the Netherlands using data from serological tests performed in the context of export and screening of 12,881 horses from 2015 through 2020. Horses were categorized as “Dutch,” “Foreign,” or “Unknown” based on microchip number. The overall seroprevalence of EP in Dutch horses was found to be 0.5% (95% exact CI [0.4–0.7]), compared to 1.9% (95% exact CI [1.3–2.6]) in horses in the category “Foreign” and 1.7% (95% exact CI [1.2–2.3]) in horses in the category “Unknown.” In addition, the seroprevalence per country in the category “Foreign” ranged from 0% (0.95% exact CI [0–2.8]) for Ireland to 6.0% (0.95% exact CI [3.5–9.3]) for Spain. In light of the reports on the seroprevalence during the outbreak of autochthonous EP reported in 2012 and on seroprevalences of EP in other countries in Northwestern Europe, the seroprevalence of EP in horses exported from the Netherlands is very low. However, the higher seroprevalence of EP in horses from abroad warrants the need for the monitoring of EP, as tick vectors are present in the Netherlands and the import of horses from endemic areas increases the chances of EP becoming more prevalent in the Netherlands.

We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.

The Salmonella monitoring program, as outlined in the EU Commission regulation 200/2010, asks for repeated sampling in order to ascertain progress in achievement of the EU target. According to Article 2.2.2.2.c of this regulation, the competent authority may decide to do a resample and retest when it has reasons to question the results of initial testing. In the Netherlands, the competent authorities have been resampling and retesting all initial positive samplings for several years because of doubts about false positive initial test results. An analysis of population data in the period 2015–2019 indicates that 48% of initial samplings at the farm were classified as false positive after resampling and retesting by the competent authorities. A qualitative analysis, assessing factors that could be associated with the occurrence of false positives, indicates that cross-contamination during the sampling process by the poultry farmer is probably the most likely source. Cross-contamination of samples during transport from the farm to the laboratory and/or cross-contamination at the laboratory are also considered possible sources. Given the slightly non-optimal system-specificity of the Salmonella monitoring program, there is good reason to make, or consider, standard resampling and retesting of initial positive results by the competent veterinary authorities possible within the EU.

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the “gold standard” in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10 Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was f

In Europe, wild boar populations pose an increasing risk for livestock and humans due to the transmission of animal and zoonotic infectious diseases, such as African swine fever and brucellosis. Brucella suis is widespread among wild boar in many European countries. In The Netherlands the prevalence of B. suis among wild boar has not been investigated so far, despite the high number of pig farms and the growing wild boar population. The Netherlands has a Brucella-free status for the livestock species. The objective of this study is to investigate the presence and distribution of B. suis in wild boars in The Netherlands and to assess the value of the different laboratory tests available for testing wild boars. A total of 2057 sera and 180 tonsils of wild boar were collected between 2010 and 2015. The sera were tested for Brucella antibodies and the tonsils were tested for Brucella spp. B. suis biovar 2 was detected by MLVA/MLST and culture in wild boar from the province of Limburg, while seropositive wild boar were obtained from the provinces of Limburg, Noord Brabant and Gelderland suggesting the northwards spread of B. suis biovar 2. In this paper, we describe the first isolation of B. suis biovar 2 in wild boar in The Netherlands.

In ovo immunization of chicken embryos with live vaccines is an effective strategy to protect chickens against several viral pathogens. We investigated the immune response of chicken embryos to purified recombinant protein. In ovo delivery of Salmonella flagellin to 18-day old embryonated eggs resulted in elevated pro-inflammatory chIL-6 and chIL-8 (CXCL8-CXCLi2) cytokine transcript levels in the intestine but not in the spleen at 24 h post-injection. Analysis of the chicken Toll-like receptor (TLR) repertoire in 19-day old embryos revealed gene transcripts in intestinal and spleen tissue for most chicken TLRs, including TLR5 which recognizes Salmonella flagellin (FliC). The in ovo administration of FliC did not alter TLR transcript levels, except for an increase in intestinal chTLR15 expression. Measurement of the antibody response in sera collected at day 11 and day 21 post-hatch demonstrated high titers of FliC-specific antibodies for the animals immunized at the late-embryonic stage in contrast to the mock-treated controls. The successful in ovo immunization with purified bacterial antigen indicates that the immune system of the chicken embryo is sufficiently mature to yield a strong humoral immune response after single exposure to purified protein. This finding strengthens the basis for the development of in ovo protein-based subunit vaccines.