IntroductionThis study describes a quantitative Rapid Risk Assessment Tool (RRAT) for zoonotic signals, such as those evaluated within the Dutch Signalling Forum for Zoonoses (SO-Z). This multidisciplinary committee systematically collects signals of potentially (re-)emerging zoonotic pathogens at the human-animal interface. Signals from surveillance activities and field observations are discussed to assess risks and determine whether escalation, further investigation, or advice to professionals and policymakers is warranted. While qualitative risk assessment criteria are available, a quantitative RRAT is needed to strengthen the risk assessment procedure.ObjectiveTo develop a RRAT to support experts in deciding whether zoonotic signals warrant upscaling. It also highlights the relative importance of signals, enabling more transparent and consistent risk communication.MethodBased on a scientific literature review, quantitative risk factors were selected to score signals assessed by the SO-Z between July 2011 and December 2025. These factors included pathogen characteristics, geographic spread, animal involvement, human health impact, and signal dynamics. Using SMOTE (Synthetic Minority Over-sampling Technique) and XGBoost (Extreme Gradient Boosting) analysis, we identified non-trivial patterns underlying expert decisions to upscale signals. These patterns were subsequently translated into a classification tool.ResultsA total of 443 SO-Z signals were analysed, of which 48 were escalated. Distribution of herds, pathogen characteristics, geographic origin, companion animal involvement, and signal repetition were associated with increased likelihood of upscaling. To distinguish multiple levels of association for upscaling, a RRAT was developed with three threshold values: A) most sensitive threshold (100% sensitivity and 24% specificity); B) most discriminating threshold (69% sensitivity and 72% specificity); C) most specific threshold (31% sensitivity and 94% specificity).ConclusionsThe RRAT is quick and easy to use. However, it does not replace experts’ judgments; rather, it supports them by providing data-driven insights for upscaling zoonosis control measures and enabling more transparent risk communication.

Echinococcus multilocularis and Echinococcus granulosus sensu lato are the causative agents of two major zoonotic diseases, alveolar echinococcosis and cystic echinococcosis, respectively, and are ranked among the four most important foodborne parasites worldwide. Although interest in food contamination by taeniid eggs is increasing, the estimation of the number of eggs present in food items is still lacking. Using E. multilocularis eggs isolated from fecal samples of experimentally infected red foxes, an average of 5261 (±1253) mitochondrial DNA copies per egg was estimated by digital PCR (dPCR). Based on this estimation, 47 taeniid DNA samples previously obtained from different food items and identified as positive for E. multilocularis, E. granulosus sensu stricto or Taenia species, were submitted to dPCR to estimate the number of eggs present. In 95.7% of the samples, the contamination was estimated to be caused by one to five eggs, with only one egg in 83.0% of the samples. As detection of such low-level contamination requires sensitive detection methods, a performance comparison between microscopy and molecular methods was conducted using 15 pellets obtained from lettuce wash residues spiked with 0 to 63 E. multilocularis eggs. Two operators performed blinded microscopy, followed by DNA extraction from the examined pellets for real-time PCR detection and then an estimation of the number of eggs by dPCR. A higher sensitivity was obtained with real-time PCR (88.5%) compared to microscopy (40%) in samples containing one to five eggs. The correlation between the number of eggs spiked into the samples and the estimated egg number was strong using dPCR (r = 0.99, p < 0.01), while only moderate with microscopy (r = 0.67, p < 0.05). Estimation of the number of taeniid eggs in food samples by dPCR provides the first quantitative basis for exposure assessment and is important for evaluating the risk of human foodborne infections, with future work needed to address egg viability.

Human alveolar echinococcosis is a notifiable parasitic infectious disease in most European countries; however, in practice, it is under-reported by national health systems. To fill this knowledge gap, data on the number, incidence, and trend of cases in Europe were extracted through a systematic review approach from both the scientific and grey literature, covering 1997-2023. This systematic review identified 4207 human alveolar echinococcosis cases from 28 of the 40 European countries investigated. Historically endemic Austria, France, Germany, and Switzerland accounted for 2864 (68·08%) of 4207 cases documented in Europe, and Lithuania, Poland, and Slovakia represented an additional 887 (21·08%) cases. Based on incidence rates and trends detected in this study, two main epicentres were seen in countries in the Alpine and the Baltic areas. The mean annual incidence from 1997 to 2023 throughout Europe was 0·063 cases per 100 000 people and in EU member states was 0·060 cases per 100 000 people. Data collected during this period suggest that alveolar echinococcosis is emerging in almost every country where this neglected parasitic infectious disease has been detected.

Livestock farm emissions have been linked to respiratory health, potentially mediated through alterations in the respiratory microbiota. We investigated the associations between occupational and environmental livestock farm exposures and changes in the upper respiratory tract (URT) microbiome. Nasopharyngeal (NP) and oropharyngeal (OP) swabs were collected from goat farmers (91 NP and 96 OP samples) and rural residents (956 NP and 954 OP samples), and microbial community composition was characterised using 16S rRNA gene amplicon sequencing. First, we compared the respiratory microbiome of goat farmers and residents to assess the effects of occupational exposure. Next, we evaluated how varying levels of environmental livestock exposure influenced the microbiome of rural residents. Goat farmers exhibited higher NP microbial diversity but lower OP diversity compared to the residents. NP samples from farmers had higher relative abundances of the genera Sphingomonas, Pseudomonas and Jeotgalicoccus, and lower abundances of Corynebacterium, Lawsonella and Nocardioides. In the OP of goat farmers, Rothia was the most significantly increased genus. Residential exposure levels - assessed through livestock density (pigs, cattle, poultry, and goats) and modelled livestock-related microbial emissions - were not associated with overall microbiome diversity or composition. However, increasing livestock exposure was associated with subtle taxonomic shifts, particularly in the NP. Our findings highlight the pronounced influence of occupational exposure on the URT microbiome and the more subtle effects of environmental exposure, both of which may have implications for respiratory health.

Cystic and alveolar echinococcosis are severe zoonotic diseases characterized by long asymptomatic periods lasting months or years. Viable Echinococcus spp. eggs released into the environment through the feces of canids can infect humans through accidental ingestion via hand-to-mouth contact or consumption of contaminated food or water. Both Echinococcus multilocularis and Echinococcus granulosus sensu lato are considered as foodborne parasites. However, when considering possible pathways of human infection, it appears that food and waterborne related variables do not significantly increase the risk of infection. Providing evidence-based data for the presence of DNA and, potentially, eggs in fresh produce is crucial in understanding foodborne transmission of Echinococcus spp. to humans. Two multicenter and multicountry studies were conducted within the One Health EJP framework to estimate the proportion of lettuces and berries contaminated by E. multilocularis, E. granulosus sensu lato, and other taeniid DNAs from a total of 12 European countries, Tunisia and Pakistan. A total of 1117 lettuces, 71 others vegetables, 300 strawberries, 130 blueberries and 50 others berries samples were collected and analysed by washing, sequential sieving and real-time PCRs. E. multilocularis DNA was detected in 1.2 % (7/ 570) of lettuce samples tested from the seven European endemic countries (Denmark, France, Germany, Latvia, the Netherlands, Poland and Switzerland) and in 2 % (2/100) from Pakistan. E. granulosus sensu lato DNA was identified in 1.3 % of lettuces (9/695) collected in five European endemic countries (France, Italy, Latvia, Poland and Portugal) and in 12 % (9/75) and 4 % (4/100) from Tunisia and Pakistan, respectively. All E. granulosus sensu lato samples were identified as E. granulosus sensu stricto (20/22), except for two identified as E. canadensis (2/22) from Latvia and Pakistan. Regarding berries, E. multilocularis DNA was detected in 5.4 % (n = 11/202) of strawberries, 7.3 % (6/82) of blueberries from the seven European endemic countries and 56 % (14/25) of blueberries from Pakistan. High contamination rates of E. granulosus sensu stricto were found outside of Europe, with 12.0 % (3/25) in blueberries from Pakistan and 81.3 %. (13/16) in strawberries from Tunisia. The total contamination rate of all taeniid species DNA in lettuces (5.3 %; 59/1117), others vegetables (5.6 %; 4/71) and berries (12.1 %; 58/480) suggests that the transfer of taeniid eggs from carnivore feces to food is not uncommon. Although we assume that eggs are the source of the DNA detected in this study, the viability of such eggs is unknown. The detection of Echinococcus species in lettuces and berries suggests a potential risk of foodborne human infection. The relative contribution of this risk remains to be estimated. Further studies on food and environmental contamination are necessary to cover different epidemiological contexts and social habits, leading to a better understanding of human infections by Echinococcus spp. eggs.

BackgroundZoonotic influenza, including infections with avian and swine influenza A viruses (swIAV), is a notifiable disease in the Netherlands. Human cases infected with swIAV have previously been rarely detected in the Netherlands.AimWe aimed to describe detection and characterisation of Eurasian avian-like swIAV infections in humans in the Netherlands 2020-2023.MethodsThe Dutch National Influenza Center coordinates different activities to monitor respiratory infections and circulating human influenza viruses. This monitoring includes sentinel surveillance in general practitioner practices, community participatory surveillance and characterisation of influenza viruses received from diagnostic laboratories. A subset of the specimens positive for influenza A virus from the monitoring activities are sent for further characterisation. We characterised swIAV from human patients using whole genome sequencing, tested the viruses for antiviral susceptibility and in haemagglutination inhibition assays for antigenic characterisation and compared them with previous detections from humans and pigs.ResultsAvian-like swine influenza virus was detected in three persons presenting with mild respiratory symptoms, and all recovered fully. Only one patient had close contact with pigs shortly before the start of symptoms. Sequence analyses of the viruses showed clustering with swAIV from pigs in a recently initiated surveillance system on pig farms.ConclusionsThese human cases show that swIAV viruses with zoonotic potential are enzootic in the Netherlands. Finding them by coincidence suggests human infections might occur more frequently than noticed.

BackgroundSwine influenza has a considerable impact on pig populations and poses a pandemic threat to humans. However, little is known about the influenza A viruses circulating among pigs in the Netherlands.AimWe piloted a surveillance programme aimed at enabling swine influenza A virus (swIAV) surveillance in the Netherlands: investigated prevalence, genomic characteristics and recent evolution of circulating swIAV variants and compared them with relevant human and swine influenza viruses from the Netherlands and other European countries.MethodsWe collected and tested respiratory samples from pigs (2019-2023) for swIAV, characterised the viruses with molecular and virological methods and shared molecular data of swine and relevant human influenza A viruses in a national platform.ResultsWe detected swIAV throughout the year in 342 (42%) of 824 respiratory samples from 90 farms. Complete genome sequencing identified 73 H1N1, 51 H1N2 and one H3N2 viruses. Phylogenetic analyses identified viruses from each of the three H1 swine lineages (1A/B/C) and four subclades. Viruses from the 1A lineage clustered into three subgroups with distinct antigenic properties, which seemed descendent from separate introductions of human seasonal A(H1N1)pdm09 viruses. Phenotypically, no reduced susceptibility to existing antiviral drugs oseltamivir and zanamivir was found.ConclusionWe provided insights into swIAVs in pigs in the Netherlands, including antiviral susceptibility and antigenic differences. It highlighted occasional virus transmission between humans and pigs. Sharing swIAV data at a national level will be continued to reduce influenza burden in swine and support identification and characterisation of emerging swIAVs with zoonotic potential.

IntroductionLiteracy about zoonoses can contribute people adapt their behaviour to minimize zoonotic risks. In this study, associations between sociodemographic factors and zoonotic risk-averse attitudes were explored.ObjectiveTo determine factors significantly associated with literacy about zoonoses across sociodemographic groups to inform targeted interventions aiming at improving awareness and zoonotic risk-avoidance behaviours.MethodData was collected in 2022 using an online survey of a nationally representative sample of residents in the Netherlands. A multivariable logistic regression analysis, accounting for multiple hypothesis testing, was applied to assess whether there were significant associations between socio-demographic factors and attitudes towards zoonosis prevention.ResultsA total of 2039 respondents completed the survey. People who were female, older, highly educated and those who searched for information about zoonoses, were relatively more likely to report behaviours favourable to the prevention of zoonoses. However, people with limited language and computer skills and immunocompromised people were significantly more likely to report risky behaviours. There were no significant associations found for pregnant women, dog and cat owners, those with an intermediate level of education and those who do have contact with farm animals.ConclusionCertain sociodemographic groups display significantly riskier attitudes towards zoonoses. These groups provide targets where to improve literacy about zoonoses. This also implies that there is room for improvement in literacy about zoonoses, particularly among immunocompromised people and people with limited language and limited computer skills.

The global spread of Monkeypox virus (MPXV) clade IIb in 2022/2023 raised concerns about spillback into new animal reservoirs. Experimental inoculation of rats with MPXV resulted in skin lesions and viral shedding in the respiratory tract and skin. These findings suggest a potential role for rats in MPXV transmission. ### Competing Interest Statement The authors have declared no competing interest.

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections. However, most non-heated meat products contain salt and additives, which affect T. gondii viability. It was our aim to develop an in vitro method to substitute the mouse bioassay for determining the effect of salting on T. gondii viability. Two sheep were experimentally infected by oral inoculation with 6.5 × 104 oocysts. Grinded meat samples of 50 g were prepared from heart, diaphragm, and four meat cuts. Also, pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution, and digests were inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for up to four weeks and parasite growth was monitored by assessing Cq-values using the T. gondii qPCR on cell culture supernatant in intervals of one week and ΔCq-values determined. Additionally, 500 μL of each digest from the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFNγ KO mice. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. From all untreated meat samples positive ΔCq-values were obtained in the in vitro assay, indicating presence and multiplication of viable parasites. This was in line with the mouse bioassay, with the exception of a negative mouse bioassay on one heart sample. Samples supplemented with 0.6 %-1.2 % NaCl showed positive ΔCq-values over time. The frozen sample and the sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values, which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less.

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Background: Clostridioides difficile is a leading cause of infectious diarrhea in both humans and livestock. In particular, C. difficile strains belonging to sequence type (ST) 11 are common enteropathogens. The aim of this study was to determine the presence and genetic relatedness of C. difficile types in dairy cattle and calves. Method: Dutch dairy farms were visited between February and December 2021. Feces was collected from adult dairy cattle and calves of two age categories ( <4 weeks and 4 weeks -4 months). Fecal samples were also requested from dairy farmers, family members and employees. Fecal samples were cultured in an enrichment medium for 10 -15 days and subcultured on solid media for capillary PCR ribotyping and whole genome sequencing. Results: C. difficile was detected on 31 out of 157 (19.8%) dairy farms. The highest prevalence was found in calves <4 weeks (17.5%). None of the 99 human samples collected were positive. Thirty-seven cultured isolates belonged to 11 different PCR ribotypes (RT) of which RT695 (56.8%) and RT078/126 (16.2%) were most abundant. In the database of the Netherlands National Expertise Centre for C. difficile infections (CDI, >10.000 patient isolates), RT695 was found in only two patients with hospital -onset CDI, diagnosed in 2020 and 2021. Sequence analysis of 21 C. difficile RT695 from cattle revealed that all isolates belonged to clade 5, ST11 and contained genes encoding toxin A, toxin B and binary toxin. RT695 strains carried antimicrobial resistance genes typically found in clade 5 C. difficile . Groups of genetically related RT695 isolates were found between dairy farms, whereas identical strains were only present in individual farms. Conclusions: C. difficile was found in similar to 20% of dairy farms with a predominance of the relatively unknown RT695. Isolates of RT695 belonged to the same clade and sequence type as RT078/126, which is recognized as an important zoonotic type.

Background: In the Netherlands, living in proximity to goat farms has been consistently associated with an increased incidence of community-acquired pneumonia (CAP). The cause remains largely unknown though airborne microbial agents could play a role.Objective: The aim of this study is to explore micro-organisms present in goats that can cause human pneumonia.Methods: An extensive literature review was conducted to identify all micro-organisms detected in goats that are associated with human pneumonia. Additionally, the identified micro-organisms were prioritized using a self-developed scoring system and expert opinion.Results: Through extensive literature review, 4309 references describing 302 different micro-organisms in goats or on goat farms were identified. Additional searches and reviews for human respiratory disease caused by each of these micro-organisms yielded a final list of 76 bacteria, 7 viruses, 7 fungi, and 6 protozoa. They were assigned scores based on pneumonia type, diagnosis of respiratory disease, patient immune status, and evidence strength. Based on these scores, the most likely potential causal micro-organisms included Moraxella spp. Chlamydia psittaci, Staphylococcus aureus, and Streptococcus pneumoniae. Subsequently, the list of micro-organisms was reviewed by external experts on their perceived likelihood of the organism causing this CAP.Conclusion: Results of this literature study can give insight into the possible causes of pneumonia. Nonetheless, no unambiguous conclusion on the actual cause of the increased CAP risk around goat farms can be drawn solely based on these results.

De afgelopen decennia is het risico op nieuwe of opnieuw opduikende infectieziekten toegenomen. In een niet-immune populatie kan dit voor grote medische, maatschappelijke en economische problemen zorgen. Driekwart van de nieuwe ziekteverwekkers komt van (wilde) dieren. Wanneer deze pathogenen vervolgens ook van mens tot mens overdraagbaar zijn, kan dit tot een pandemie leiden, zoals bij COVID-19 is gebeurd. Huisartsen kunnen als een van de eersten humane signalen van nieuwe infectieziekten oppikken. Daarmee spelen ze een belangrijke rol bij de vroegtijdige opsporing en bestrijding ervan.

Toxoplasmosis caused by the protozoan parasite Toxoplasma gondii occurs worldwide. Infections range from asymptomatic to life-threatening. T. gondii infection is acquired either via bradyzoites in meat or via oocysts in the environment, but the relative importance of these path ways and the different sources remains unclear. In this study, possible risk factors for toxoplasmosis in the Netherlands were investigated. A case-control study was conducted including persons with recent infection and individuals with a negative test result for IgM and IgG for T. gondii between July 2016 and April 2021. A total of 48 cases and 50 controls completed the questionnaire. Food history and environmental exposure were compared using logistic regression. Consumption of different meats was found to be associated with recent infection. In the multivariable model, adjusted for age, gender, and pregnancy, consumption of large game meat (adjusted odds ratio (aOR) 8.2, 95% confidence interval 1.6-41.9) and sometimes (aOR 4.1, 1.1-15.3) or never (aOR 15.9, 2.2-115.5) washing hands before food preparation remained. These results emphasize the value of the advice to be careful with the consumption of raw and undercooked meat. Good hand hygiene could also be promoted in the prevention of T. gondii infection.

Leptospirosis is a zoonosis caused by the spirochete Leptospira spp. It is often not clear why certain areas appear to be hotspots for human leptospirosis. Therefore, a predictive risk map for the Netherlands was developed and assessed, based on a random forest model for human leptospirosis incidence levels with various environmental factors and rat density as variables. Next, it was tested whether misclassifications of the risk map could be explained by the prevalence of Leptospira spp. in brown rats. Three recreational areas were chosen, and rats (≥25/location) were tested for Leptospira spp. Concurrently, it was investigated whether Leptospira spp. prevalence in brown rats was associated with Leptospira DNA concentration in surface water, to explore the usability of this parameter in future studies. Approximately 1 L of surface water sample was collected from 10 sites and was tested for Leptospira spp. Although the model predicted the locations of patients relatively well, this study showed that the prevalence of Leptospira spp. infection in rats may be an explaining variable that could improve the predictive model performance. Surface water samples were all negative, even if they had been taken at sites with a high Leptospira spp. prevalence in rats.

Toxoplasma gondii is a zoonotic parasite of importance to both human and animal health. The parasite has various transmission routes, and the meat of infected animals appears to be a major source of human infections in Europe. We aimed to estimate T. gondii prevalence in a selection of animal host species. A systematic literature review resulting in 226 eligible publications was carried out, and serological data were analyzed using an age-dependent Bayesian hierarchical model to obtain estimates for the regional T. gondii seroprevalence in livestock, wildlife, and felids. Prevalence estimates varied between species, regions, indoor/outdoor rearing, and types of detection methods applied. The lowest estimated seroprevalence was observed for indoor-kept lagomorphs at 4.8% (95% CI: 1.8–7.5%) and the highest for outdoor-kept sheep at 63.3% (95% CI: 53.0–79.3%). Overall, T. gondii seroprevalence estimates were highest within Eastern Europe, whilst being lowest in Northern Europe. Prevalence data based on direct detection methods were scarce and were not modelled but rather directly summarized by species. The outcomes of the meta-analysis can be used to extrapolate data to areas with a lack of data and provide valuable inputs for future source attribution approaches aiming to estimate the relative contribution of different sources of T. gondii human infection.

Introduction: Longitudinal studies investigating air pollution from livestock farms and respiratory health effects in neighboring residents are lacking. The aim of this study was to assess the relationship between livestock farm exposures and lung function decline over a 7-year period in people living in livestock-dense areas. Methods: Spirometry was performed in 2014/2015 and 2021/2022 for 847 adults (28-80 years). We analyzed the annual rate of change in FEV1, FVC, FEV1/FVC, PEF and MMEF in relation to the annual-average livestock-emitted endotoxin concentration at the home address, which was predicted by dispersion modeling at baseline. Data analysis was performed using generalized additive models with a non-linear term for endotoxin exposure. Models were adjusted for age, sex, height, BMI, education level, smoking history, atopy, and growing up on a livestock farm. Results: Endotoxin exposure was not associated with the annual rate of change in lung function (p>0.05). Subjects with a farm childhood had larger annual decreases in FEV1 (-5.63 ml/y, p=0.018) and MMEF (-11.15 ml/s per year, p=0.032), compared to those who did not grow up on a farm. Models stratified for atopy revealed that the association between accelerated FEV1 decline and farm childhood was more pronounced in individuals without atopy. Conclusions: Early-life farm exposures were associated with accelerated lung function decline in non-atopic adults. As growing up on a farm was associated with increased livestock exposure later in life (e.g. visiting farms), further investigation of current exposures is needed. Air pollutants other than livestock-emitted endotoxin could also play a role.

Aims The aim of our study was to investigate the virulence and resistance of STEC from small ruminants farms in The Netherlands. Moreover, the potential transmission of STEC between animals and humans on farms was evaluated. Methods and results From 182 farms, in total, 287 unique STEC isolates were successfully recovered from animal samples. In addition, STEC was isolated from eight out of 144 human samples. The most detected serotype was O146:H21; however, among other serotypes also O26:H11, O157:H7, and O182:H25 isolates were present. Whole genome sequencing covering all human isolates and 50 of the animal isolates revealed a diversity of stx(1), stx(2), and eae sub-types and an additional 57 virulence factors. The assessed antimicrobial resistance phenotype, as determined by microdilution, was concordant with the genetic profiles identified by WGS. WGS also showed that three of the human isolates could be linked to an animal isolate from the same farm. Conclusions The obtained STEC isolates showed great diversity in serotype, virulence, and resistance factors. Further analysis by WGS allowed for an in-depth assessment of the virulence and resistance factors present and to determine the relatedness of human and animal isolates.