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BACKGROUND: Previous studies, performed between 2009-2019, in the Netherlands observed an until now still unexplained increased risk for pneumonia among residents living close to goat farms. Since data were collected in the provinces Noord-Brabant and Limburg (NB-L), an area with relatively high air pollution levels and proximity to large industrial areas in Europe, the question remains whether the results are generalizable to other regions. In this study, a different region, covering the provinces Utrecht, Gelderland, and Overijssel (UGO) with a similar density of goat farms, was included to assess whether the association between goat farm proximity and pneumonia is consistently observed across the Netherlands. METHODS: Data for this study were derived from the Electronic Health Records (EHR) of 21 rural general practices (GPs) in UGO, for 2014-2017. Multi-level analyses were used to compare annual pneumonia prevalence between UGO and data derived from rural reference practices ('control area'). Random-effects meta-analysis (per GP practice) and kernel analyses were performed to study associations of pneumonia with the distance between goat farms and patients' home addresses. RESULTS: GP diagnoses of pneumonia occurred 40% more often in UGO compared to the control area. Meta-analysis showed an association at a distance of less than 500m (~70% more pneumonia compared to >500m) and 1000m (~20% more pneumonia compared to >1000m). The kernel-analysis for three of the four individual years showed an increased risk up to a distance of one or two kilometers (2-36% more pneumonia; 10-50 avoidable cases per 100,000 inhabitants per year). CONCLUSIONS: The positive association between living in the proximity of goat farms and pneumonia in UGO is similar to the previously found association in NB-L. Therefore, we concluded that the observed associations are relevant for regions with goat farms in the entire country.

The emergence of bacterial strains resistant to topical antimicrobials in both human and veterinary medicine has raised concerns over retaining the efficacy of these preparations. Yet, little information is available regarding the use of topical antimicrobials in either sector for planning targeted interventions. This study aims to quantify the use of topical antimicrobials in 44 Dutch companion animal clinics before and during their participation in an antimicrobial stewardship programme (ASP), to explore the effect of the intervention on topical antimicrobial use (AMU). Hence, prescription and clinic animal population data, collected from July 2012 until June 2018 were used. Specifically, the period from July 2012 until June 2015 was defined as pre-intervention period, whereas clinics started to participate in the ASP from March 2016 onwards. As quantification metric, the Defined Daily Dose for Animals (DDDA) was used and a mixed effect times series model with auto-regression was applied to monthly topical AMU data. The intervention effect was modelled using a step function with a change in (linear) time trend and clinic characteristics, as potential determinants of topical AMU, were assessed using a multivariable regression model. A seasonal pattern was identified, in the pre-intervention period, where topical AMU was highest in July-August and lowest in February-March. In addition, total topical AMU appeared to significantly decrease over time in the pre-intervention period and the proportion of dogs in the clinic was positively associated with topical AMU. The intervention effect was significant only for second line and for skin product AMU. This study demonstrates that during participation in an ASP, second line and skin product AMU decreased in Dutch companion animal clinics. Additionally, this study demonstrates the existence of a seasonal effect and a decrease in topical AMU over time already before introduction of a targeted intervention.

The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00-2.49 log CFU/g of manured soil, ranging between 10-5 and 10-4 transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillus and Nocardioides) and manure (Comamonas and Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.

There is an ongoing debate on airborne transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a risk factor for infection. In this study, the level of SARS-CoV-2 in air and on surfaces of SARS-CoV-2 infected nursing home residents was assessed to gain insight in potential transmission routes. During outbreaks, air samples were collected using three different active and one passive air sampling technique in rooms of infected patients. Oropharyngeal swabs (OPS) of the residents and dry surface swabs were collected. Additionally, longitudinal passive air samples were collected during a period of 4 months in common areas of the wards. Presence of SARS-CoV-2 RNA was determined using RT-qPCR, targeting the RdRp- and E-genes. OPS, samples of two active air samplers and surface swabs with Ct-value ≤35 were tested for the presence of infectious virus by cell culture. In total, 360 air and 319 surface samples from patient rooms and common areas were collected. In rooms of 10 residents with detected SARS-CoV-2 RNA in OPS, SARS-CoV-2 RNA was detected in 93 of 184 collected environmental samples (50.5%) (lowest Ct 29.5), substantially more than in the rooms of residents with negative OPS on the day of environmental sampling (n = 2) (3.6%). SARS-CoV-2 RNA was most frequently present in the larger particle size fractions [>4 μm 60% (6/10); 1-4 μm 50% (5/10)

There is an ongoing debate on airborne transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a risk factor for infection. In this study, the level of SARS-CoV-2 in air and on surfaces of SARS-CoV-2 infected nursing home residents was assessed to gain insight in potential transmission routes. During outbreaks, air samples were collected using three different active and one passive air sampling technique in rooms of infected patients. Oropharyngeal swabs (OPS) of the residents and dry surface swabs were collected. Additionally, longitudinal passive air samples were collected during a period of 4 months in common areas of the wards. Presence of SARS-CoV-2 RNA was determined using RT-qPCR, targeting the RdRp- and E-genes. OPS, samples of two active air samplers and surface swabs with Ct-value ≤35 were tested for the presence of infectious virus by cell culture. In total, 360 air and 319 surface samples from patient rooms and common areas were collected. In rooms of 10 residents with detected SARS-CoV-2 RNA in OPS, SARS-CoV-2 RNA was detected in 93 of 184 collected environmental samples (50.5%) (lowest Ct 29.5), substantially more than in the rooms of residents with negative OPS on the day of environmental sampling (n = 2) (3.6%). SARS-CoV-2 RNA was most frequently present in the larger particle size fractions [>4 μm 60% (6/10); 1–4 μm 50% (5/10)

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales have been increasingly isolated from pigs, highlighting their potential for transmission to humans living and/or working within pig farms. As longitudinal data on the prevalence and the molecular characteristics of such isolates from the high-risk farming population remain scarce, we performed a long-term study on 39 Dutch pig farms. Fecal samples from pigs, farmers, family members, and employees were collected during four sampling occasions with a 6-month period. The presence of ESBL-producing Enterobacterales and their molecular characteristics (ESBL gene, plasmid, and sequence types) were determined by standard methods. Data on personal and farm characteristics were collected using questionnaires. ESBL-producing Escherichia coli was present in pigs at least once for 18 of 39 farms and in 17 of 146 farmers, family members, and/or employees. Among these 417 E. coli isolates, bla CTX-M-1 was the most frequently observed ESBL gene in pigs ( n = 261) and humans ( n = 25). Despite the great variety in plasmid (sub)types and E. coli sequence types (STs), we observed genetic similarity between human- and pig-derived isolates in (i) ESBL gene, plasmid (sub)type, and ST, suggesting potential clonal transmission in seven farms, and (ii) only ESBL gene and plasmid (sub)type, highlighting the possibility of horizontal transfer in four farms. Five pig farmers carried ESBL producers repeatedly, of whom two carried an identical combination of gene, plasmid (sub)type, and ST over time. Human ESBL carriage was associated with both presence of ESBL producers in pigs and average number of hours working on the pig farm per week, while prolonged human carriage was observed only incidentally. IMPORTANCE Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli represents a public health hazard due to reduced therapeutic options for the treatment of infections. Although direct contact with pigs is considered a risk factor for human ESBL-producing E. coli carriage through occupational exposure, nationwide data regarding the occurrence of such isolates among pigs and humans living and/or working on farms remain scarce. Therefore, we determined (i) the longitudinal dynamics in prevalence and molecular characteristics of ESBL-producing E. coli in Dutch pig farmers and their pigs over time and (ii) the potential transmission events between these reservoirs based on genetic relatedness and epidemiological associations in longitudinal data. Our data suggesting the possibility of clonal and horizontal dissemination of ESBL-producing Escherichia coli between pigs and pig farmers can be used to inform targeted intervention strategies to decrease the within-farm human exposure to ESBL-producing E. coli.

IntroductionThe urgency of preventing the increase of antimicrobial resistance has been emphasized by international authorities such as the World Health Organization, European Medicines Agency, and World Organization for Animal Health. Monitoring systems capable of reporting antimicrobial consumption data are regarded as a crucial pillar of this fight. The Vet-AMNet system was developed to collect and analyze national antimicrobial consumption data in Portuguese dairy farms to support the veterinary authority in stewardship actions and to assist both veterinarians and farmers in daily decisions related to antimicrobials.MethodsTo evaluate the robustness of the system and other identified critical success factors, it was used to analyze antimicrobial consumption data available from the Dutch dairy cow sector over the period from 2012 to 2020. The data previously used for publications by the Netherlands Veterinary Medicines Institute (SDa) were imported and pre-processed by the Vet-AMNet system according to the SDa's standard operating procedure and the Dutch metrics to measure antimicrobial consumption were calculated.ResultsBy comparing the outputs with the figures generated by the system established in the Netherlands, the Portuguese system was validated. Antimicrobial consumption data from the Dutch dairy sector during the 9-year period will be presented in unpublished graphs and tables, where each molecule's pharmaceutical formulation, pharmacotherapeutic group and line of choice will be related and discussed, illustrating the evolution of sectorial antimicrobial consumption against a background of a strong national antimicrobial policy initiated by public-private cooperation and supported by legislation.

Livestock animals housed in close proximity to humans can act as sources or intermediate hosts facilitating animal-to-human transmission of zoonotic diseases. Understanding virus diversity in livestock is important for identifying potential zoonotic threats and for ensuring animal health and safe livestock production. Here, we report viral metagenomic characterization of chicken feces (N=51) and paired farm dust samples (N=13) using metagenomic deep sequencing. Samples were collected at 4-5 time points in three broiler farms in the Netherlands. Viruses in the Parvoviridae and Picornaviridae families were the most prevalent, detected in all feces and dust samples and in all feces and 85% of dust samples, respectively. Virus composition found in chicken feces and corresponding dust samples were similar. Great genomic diversity was identified in Picornaviridae and 46 sequences from five picornavirus genera (Sicinivirus, Megrivirus, Anatvirus, Gallivirus and Avisvirus) were detected. For calicivirus, Basovirus and an unclassified novel chicken calicivirus were identified in 13 fecal and 1 dust samples. Two distinct types of chicken astroviruses were identified. Phylogenetic analyses of identified virus sequences from Picornaviridae, Astroviridae and Caliciviridae suggested that viral sequences obtained from different farms are often more closely related to each other than global reference sequences, and sequences from feces and paired dust samples also clustered together. Importantly, our sequencing methodology enabled the recover viral genome sequences from farm dusts, allowing the tracking of virus chatter between livestock animals and their farm environment. This study, albeit relative sample size, does expand current knowledge of virus communities in chickens and surrounding dusts. IMPORTANCEChickens may harbor various zoonotic pathogens, some of which can cause severe clinical outcomes in animals and humans. Farm dust can act as vector to facilitate zoonoses transmission. Here, we report the metagenomic characterization of virus communities of chicken feces and paired farm dust samples collected at multiple time points during the production cycle in broiler farms in the Netherlands. Parvoviridae and Picornaviridae were most frequently detected. We also identified novel astrovirus and calicivirus sequences that would inform future virus taxonomy classification. This is the first study to characterize virus communities in farmed chickens and paired farm dust samples. We also describe a dust sequencing strategy that can be adapted for future dust metagenomic characterization. Our study could help setting up a surveillance baseline for tracking virus flow between chickens and their farm environment which could guide zoonotic outbreak preparedness and health risk assessment of farm exposure.

Livestock feces with antimicrobial resistant bacteria reaches the farm floor, manure pit, farm land and wider environment by run off and aerosolization. Little research has been done on the role of dust in the spread of antimicrobial resistance (AMR) in farms. Concentrations and potential determinants of antimicrobial resistance genes (ARGs) in farm dust are at present not known. Therefore in this study absolute ARG levels, representing the levels people and animals might be exposed to, and relative abundances of ARGs, representing the levels in the bacterial population, were quantified in airborne farm dust using qPCR. Four ARGs were determined in 947 freshly settled farm dust samples, captured with electrostatic dustfall collectors (EDCs), from 174 poultry (broiler) and 159 pig farms across nine European countries. By using linear mixed modeling, associations with fecal ARG levels, antimicrobial use (AMU) and farm and animal related parameters were determined. Results show similar relative abundances in farm dust as in feces and a significant positive association (ranging between 0.21 and 0.82) between the two reservoirs. AMU in pigs was positively associated with ARG abundances in dust from the same stable. Higher biosecurity standards were associated with lower relative ARG abundances in poultry and higher relative ARG abundances in pigs. Lower absolute ARG levels in dust were driven by, among others, summer season and certain bedding materials for poultry, and lower animal density and summer season for pigs. This study indicates different pathways that contribute to shaping the dust resistome in livestock farms, related to dust generation, or affecting the bacterial microbiome. Farm dust is a large reservoir of ARGs from which transmission to bacteria in other reservoirs can possibly occur. The identified determinants of ARG abundances in farm dust can guide future research and potentially farm management policy.

Het Ministerie van LNV heeft naar aanleiding van het rapport “Zoönosen in het vizier” een aantal vragen gesteld, met nadruk op de tussen-bedrijfstransmissie. De gestelde vragen hadden betrekking op de mogelijke toekomstige oorzaken van zoönosen, verspreidingsmechanismen van zoönosen, precieze betekenis en berekening van de RH-waarde, beschikbaarheid van RH-waarden voor verschillende diersoort-pathogeen combinaties, en de rol van aanvullend onderzoek in de beantwoording van vragen over deze onderdelen. De gestelde vragen zijn door de werkgroep van deskundigen beantwoord op basis van de expertise, maar zonder volledig literatuuronderzoek. Vervolgens zijn aanvullend een aantal overwegingen gegeven die betrekking hebben op de beperkingen van de geldigheid van de antwoorden op de gestelde vragen. Daarnaast wordt kort ingegaan op recente kennisontwikkelingen die in de nabije toekomst nieuw perspectief zullen of kunnen bieden bij de beantwoording van de gestelde vragen en preventie en management van zoönotische uitbraken in een periode van transitie naar een duurzamere veehouderij.

Livestock feces with antimicrobial resistant bacteria reaches the farm floor, manure pit, farm land and wider environment by run off and aerosolization. Little research has been done on the role of dust in the spread of antimicrobial resistance (AMR) in farms. Concentrations and potential determinants of antimicrobial resistance genes (ARGs) in farm dust are at present not known. Therefore in this study absolute ARG levels, representing the levels people and animals might be exposed to, and relative abundances of ARGs, representing the levels in the bacterial population, were quantified in airborne farm dust using qPCR. Four ARGs were determined in 947 freshly settled farm dust samples, captured with electrostatic dustfall collectors (EDCs), from 174 poultry (broiler) and 159 pig farms across nine European countries. By using linear mixed modeling, associations with fecal ARG levels, antimicrobial use (AMU) and farm and animal related parameters were determined. Results show similar relative abundances in farm dust as in feces and a significant positive association (ranging between 0.21 and 0.82) between the two reservoirs. AMU in pigs was positively associated with ARG abundances in dust from the same stable. Higher biosecurity standards were associated with lower relative ARG abundances in poultry and higher relative ARG abundances in pigs. Lower absolute ARG levels in dust were driven by, among others, summer season and certain bedding materials for poultry, and lower animal density and summer season for pigs. This study indicates different pathways that contribute to shaping the dust resistome in livestock farms, related to dust generation, or affecting the bacterial microbiome. Farm dust is a large reservoir of ARGs from which transmission to bacteria in other reservoirs can possibly occur. The identified determinants of ARG abundances in farm dust can guide future research and potentially farm management policy.

The emergence of antimicrobial resistance (AMR) is one of the biggest health threats globally. In addition, the use of antimicrobial drugs in humans and livestock is considered an important driver of antimicrobial resistance. The commensal microbiota, and especially the intestinal microbiota, has been shown to have an important role in the emergence of AMR. Mobile genetic elements (MGEs) also play a central role in facilitating the acquisition and spread of AMR genes. We isolated Escherichia coli (n = 627) from fecal samples in respectively 25 poultry, 28 swine, and 15 veal calf herds from 6 European countries to investigate the phylogeny of E. coli at country, animal host and farm levels. Furthermore, we examine the evolution of AMR in E. coli genomes including an association with virulence genes, plasmids and MGEs. We compared the abundance metrics retrieved from metagenomic sequencing and whole genome sequenced of E. coli isolates from the same fecal samples and farms. The E. coli isolates in this study indicated no clonality or clustering based on country of origin and genetic markers; AMR, and MGEs. Nonetheless, mobile genetic elements play a role in the acquisition of AMR and virulence genes. Additionally, an abundance of AMR was agreeable between metagenomic and whole genome sequencing analysis for several AMR classes in poultry fecal samples suggesting that metagenomics could be used as an indicator for surveillance of AMR in E. coli isolates and vice versa.

Moisture-damaged buildings are associated with respiratory symptoms and underlying diseases among building occupants, but the causative agent(s) remain a mystery. We first identified specific fungal and bacterial taxa in classrooms with moisture damage in Finnish and Dutch primary schools. We then investigated associations of the identified moisture damage indicators with respiratory symptoms in more than 2700 students. Finally, we explored whether exposure to specific taxa within the indoor microbiota may explain the association between moisture damage and respiratory health. Schools were assessed for moisture damage through detailed inspections, and the microbial composition of settled dust in electrostatic dustfall collectors was determined using marker-gene analysis. In Finland, there were several positive associations between particular microbial indicators (diversity, richness, individual taxa) and a respiratory symptom score, while in the Netherlands, the associations tended to be mostly inverse and statistically non-significant. In Finland, abundance of the Sphingomonas bacterial genus and endotoxin levels partially explained the associations between moisture damage and symptom score. A few microbial taxa explained part of the associations with health, but overall, the observed associations between damage-associated individual taxa and respiratory health were limited.

Plasmid-mediated dissemination of antibiotic resistance among fecal Enterobacteriaceae in natural ecosystems may contribute to the persistence of antibiotic resistance genes in anthropogenically impacted environments. Plasmid transfer frequencies measured under laboratory conditions might lead to overestimation of plasmid transfer potential in natural ecosystems. This study assessed differences in the conjugative transfer of an IncP-1 (pKJK5) plasmid to three natural Escherichia coli strains carrying extended-spectrum beta-lactamases, by filter mating. Matings were performed under optimal laboratory conditions (rich LB medium and 37°C) and environmentally relevant temperatures (25, 15 and 9°C) or nutrient regimes mimicking environmental conditions and limitations (synthetic wastewater and soil extract). Under optimal nutrient conditions and temperature, two recipients yielded high transfer frequencies (5 × 10–1) while the conjugation frequency of the third strain was 1000-fold lower. Decreasing mating temperatures to psychrophilic ranges led to lower transfer frequencies, albeit all three strains conjugated under all the tested temperatures. Low nutritive media caused significant decreases in transconjugants (−3 logs for synthetic wastewater; −6 logs for soil extract), where only one of the strains was able to produce detectable transconjugants. Collectively, this study highlights that despite less-than-optimal conditions, fecal organisms may transfer plasmids in the environment, but the transfer of pKJK5 between microorganisms is limited mainly by low nutrient conditions.

BACKGROUND/AIM: Active mobility may play a relevant role in the assessment of environmental exposures (e.g. traffic-related air pollution, livestock emissions), but data about actual mobility patterns are work intensive to collect, especially in large study populations, therefore estimation methods for active mobility may be relevant for exposure assessment in different types of studies. We previously collected mobility patterns in a group of 941 participants in a rural setting in the Netherlands, using week-long GPS tracking. We had information regarding personal characteristics, self-reported data regarding weekly mobility patterns and spatial characteristics. The goal of this study was to develop versatile estimates of active mobility, test their accuracy using GPS measurements and explore the implications for exposure assessment studies. METHODS: We estimated hours/week spent on active mobility based on personal characteristics (e.g. age, sex, pre-existing conditions), self-reported data (e.g. hours spent commuting per bike) or spatial predictors such as home and work address. Estimated hours/week spent on active mobility were compared with GPS measured hours/week, using linear regression and kappa statistics. RESULTS: Estimated and measured hours/week spent on active mobility had low correspondence, even the best predicting estimation method based on self-reported data, resulted in a R2 of 0.09 and Cohen's kappa of 0.07. A visual check indicated that, although predicted routes to work appeared to match GPS measured tracks, only a small proportion of active mobility was captured in this way, thus resulting in a low validity of overall predicted active mobility. CONCLUSIONS: We were unable to develop a method that could accurately estimate active mobility, the best performing method was based on detailed self-reported information but still resulted in low correspondence. For future studies aiming to evaluate the contribution of home-work traffic to exposure, applying spatial predictors may be appropriate. Measurements still represent the best possible tool to evaluate mobility patterns.

Any antimicrobial use (AMU) in humans and animals selects for antimicrobial resistance (AMR) and responsible AMU should therefore be promoted both in human and veterinary medicine. Insight into current AMU in companion animal clinics is necessary to be able to optimise antimicrobial (AM) prescribing behaviour. The objective of this study was to describe systemic AMU in 44 Dutch companion animal clinics over a 3-year time period (2012-2015), using retrospectively collected data. The number of Defined Daily Doses for Animals (DDDAs) per month and per clinic were calculated from prescription data for total, 1st, 2nd and 3rd choice AMU (classification according to Dutch policy on veterinary AMU). Time trends, seasonality and the influence of potential determinants (e.g., the number of dogs, cats and rabbits per clinic and other clinic characteristics) were explored using statistical modelling. Overall, the findings show that total AMU decreased over time and a shift in used classes of antimicrobials towards more 1st choice AMs was visible. Mean total AMU decreased from 1.82 DDDA/year in 2012-2013 to 1.56 DDDA/year in 2014-2015. Aminopenicillins, with and without clavulanic acid, accounted for the largest group of antimicrobials used; 38.7% (2012-2013), 40.2% (2013-2014) and 39.3% (2014-2015) of total AMU, respectively. Strong seasonal differences in AMU were found, with highest AMU in July-August and lowest in February-March. The distribution of different animal species per clinic appeared to affect AMU as well. In clinics with a larger proportion of dogs, 2nd choice AMU was significantly higher, whereas in clinics with a larger proportion of rabbits, 2nd choice AMU was significantly lower. Despite the decrease of AMU during the study period, there is still room for improvement left, especially with regard to the antimicrobial classes prescribed. According to Dutch classification of veterinary AMU, 1st choice AMs should be used as empirical therapy. A decrease in 2nd (might select for ESBL-producing bacteria) and 3rd choice AMU (i.e. fluoroquinolones and 3rd generation cephalosporins) should be aimed for.

Objectives: Farm-level quantification of antimicrobial usage (AMU) in pig farms. Methods: In a cross-sectional study, AMU data on group treatments administered to a single batch of fattening pigs from birth to slaughter (group treatment data) and antimicrobials purchased during 1 year (purchase data) were collected at 180 pig farms in nine European countries. AMU was quantified using treatment incidence (TI) based on defined (DDDvet) and used (UDDvet) daily doses and defined (DCDvet) and used (UCDvet) course doses. Results: The majority of antimicrobial group treatments were administered to weaners (69.5% of total TIDDDvet) followed by sucklers (22.5% of total TIDDDvet). AMU varied considerably between farms with a median TIDDDvet of 9.2 and 7.1 for a standardized rearing period of 200 days based on group treatment and purchase data, respectively. In general, UDDvet and UCDvet were higher than DDDvet and DCDvet, respectively, suggesting that either the defined doses were set too low or that group treatments were often dosed too high and/or administered for too long. Extended-spectrum penicillins (31.2%) and polymyxins (24.7%) were the active substances most often used in group treatments, with the majority administered through feed or water (82%). Higher AMU at a young age was associated with higher use in older pigs. Conclusions: Collecting farm-level AMU data of good quality is challenging and results differ based on how data are collected (group treatment data versus purchase data) and reported (defined versus used daily and course doses).

From 2007 through 2010, the Netherlands experienced the largest Q fever epidemic ever reported. This study integrates the outcomes of a multidisciplinary research programme on spatial airborne transmission of Coxiella burnetii and reflects these outcomes in relation to other scientific Q fever studies worldwide. We have identified lessons learned and remaining knowledge gaps. This synthesis was structured according to the four steps of quantitative microbial risk assessment (QMRA): (a) Rapid source identification was improved by newly developed techniques using mathematical disease modelling; (b) source characterization efforts improved knowledge but did not provide accurate C. burnetii emission patterns; (c) ambient air sampling, dispersion and spatial modelling promoted exposure assessment; and (d) risk characterization was enabled by applying refined dose–response analyses. The results may support proper and timely risk assessment and risk management during future outbreaks, provided that accurate and structured data are available and exchanged readily between responsible actors.

Background Real-time quantitative PCR (qPCR) is an affordable method to quantify antimicrobial resistance gene (ARG) targets, allowing comparisons of ARG abundance along animal production chains. Objectives We present a comparison of ARG abundance across various animal species, production environments and humans in Europe. AMR variation sources were quantified. The correlation of ARG abundance between qPCR data and previously published metagenomic data was assessed. Methods A cross-sectional study was conducted in nine European countries, comprising 9572 samples. qPCR was used to quantify abundance of ARGs [aph(3′)-III, erm(B), sul2, tet(W)] and 16S rRNA. Variance component analysis was conducted to explore AMR variation sources. Spearman’s rank correlation of ARG abundance values was evaluated between pooled qPCR data and earlier published pooled metagenomic data. Results ARG abundance varied strongly among animal species, environments and humans. This variation was dominated by between-farm variation (pigs) or within-farm variation (broilers, veal calves and turkeys). A decrease in ARG abundance along pig and broiler production chains (‘farm to fork’) was observed. ARG abundance was higher in farmers than in slaughterhouse workers, and lowest in control subjects. ARG abundance showed a high correlation (Spearman’s ρ > 0.7) between qPCR data and metagenomic data of pooled samples. Conclusions qPCR analysis is a valuable tool to assess ARG abundance in a large collection of livestock-associated samples. The between-country and between-farm variation of ARG abundance could partially be explained by antimicrobial use and farm biosecurity levels. ARG abundance in human faeces was related to livestock antimicrobial resistance exposure.

Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.

Objectives The occurrence and zoonotic potential of antimicrobial resistance (AMR) in pigs and broilers has been studied intensively in past decades. Here, we describe AMR levels of European pig and broiler farms and determine the potential risk factors. Methods We collected faeces from 181 pig farms and 181 broiler farms in nine European countries. Real-time quantitative PCR (qPCR) was used to quantify the relative abundance of four antimicrobial resistance genes (ARGs) [aph(3′)-III, erm(B), sul2 and tet(W)] in these faeces samples. Information on antimicrobial use (AMU) and other farm characteristics was collected through a questionnaire. A mixed model using country and farm as random effects was performed to evaluate the relationship of AMR with AMU and other farm characteristics. The correlation between individual qPCR data and previously published pooled metagenomic data was evaluated. Variance component analysis was conducted to assess the variance contribution of all factors. Results The highest abundance of ARG was for tet(W) in pig faeces and erm(B) in broiler faeces. In addition to the significant positive association between corresponding ARG and AMU levels, we also found on-farm biosecurity measures were associated with relative ARG abundance in both pigs and broilers. Between-country and between-farm variation can partially be explained by AMU. Different ARG targets may have different sample size requirements to represent the overall farm level precisely. Conclusions qPCR is an efficient tool for targeted assessment of AMR in livestock-related samples. The AMR variation between samples was mainly contributed to by between-country, between-farm and within-farm differences, and then by on-farm AMU.

Background: There is increasing evidence of associations between residential proximity to livestock farms and respiratory morbidity, but less is known about potential effects on respiratory mortality among residents. Objectives: We aimed to assess potential associations between respiratory mortality and residential proximity to (intensive) livestock farming. Methods: In DUELS, a national census-based cohort, we selected all inhabitants from rural and semi-urban areas of the Netherlands, aged ≥30 years and living at the same address for five years up to baseline (2004). We followed these ∼4 million individuals for respiratory mortality (respiratory system diseases, chronic lower respiratory diseases, pneumonia) from 2005 to 2012. We computed the average number of cattle, pigs, chicken, and mink present in 500 m, 1000 m, 1500 m and 2000 m of each individual's residence in the period 1999–2003. Analyses were conducted using Cox proportional hazards regression, adjusting for potential confounders at individual and neighbourhood level. Results: We found evidence that living up to 2000 m of pig farms was associated with respiratory mortality, namely from chronic lower respiratory diseases, with Hazard Ratios ranging from 1.06 (1.02, 1.10) in people living close to low numbers (

Background: Livestock farms are a reservoir of antimicrobial resistant bacteria from feces. Airborne dust-bound bacteria can spread across the barn and to the outdoor environment. Therefore, exposure to farm dust may be of concern for animals, farmers and neighboring residents. Although dust is a potential route of transmission, little is known about the resistome and bacterial microbiome of farm dust.Objectives: We describe the resistome and bacterial microbiome of pig and poultry farm dust and their relation with animal feces resistomes and bacterial microbiomes, and on-farm antimicrobial usage (AMU). In addition, the relation between dust and farmers' stool resistomes was explored.Methods: In the EFFORT-study, resistomes and bacterial microbiomes of indoor farm dust collected on Electrostatic Dust fall Collectors (EDCs), and animal feces of 35 conventional broiler and 44 farrow-to-finish pig farms from nine European countries were determined by shotgun metagenomic analysis. The analysis also included 79 stool samples from farmers working or living at 12 broiler and 19 pig farms and 46 human controls. Relative abundance of and variation in resistome and bacterial composition of farm dust was described and compared to animal feces and farmers' stool.Results: The farm dust resistome contained a large variety of antimicrobial resistance genes (ARGs); more than the animal fecal resistome. For both poultry and pigs, composition of dust resistomes finds (partly) its origin in animal feces as dust resistomes correlated significantly with fecal resistomes. The dust bacterial microbiome also correlated significantly with the dust resistome composition. A positive association between AMU in animals on the farm and the total abundance of the dust resistome was found. Occupational exposure to pig farm dust or animal feces may contribute to farmers' resistomes, however no major shifts in farmers resistome towards feces or dust resistomes were found in this study.Conclusion: Poultry and pig farm dust resistomes are rich and abundant and associated with the fecal resistome of the animals and the dust bacterial microbiome.

There is continuing debate and public health concern regarding the previously confirmed association between high livestock density and human health. The primary aim of the current study is to assess the prevalence of respiratory and other health problems in a livestock dense area in the Netherlands, based on recent longitudinal health data and a large sample. Analyses are expanded with the investigation of different subgroups of patients with respiratory health problems and the inclusion of various chronic and acute health outcomes, as well as prescribed medication. Prevalence of health symptoms and chronic conditions was assessed for the period 2014&ndash, 2016, based on electronic health records registered in 26 general practices located in areas with intensive livestock farming in the Netherlands (&ldquo, livestock dense area&rdquo, n = 117,459 unique residents in total). These were compared with corresponding health data from general practices (n = 22) in different rural regions with a low density of livestock farms or other major environmental exposures (&ldquo, control area&rdquo, n = 85,796 unique residents in total). Multilevel regression models showed a significantly higher prevalence of pneumonia in the total sample in the livestock dense area, which was also observed among susceptible subgroups of children, the elderly, and patients with chronic obstructive pulmonary disease (COPD). Lower respiratory tract infections, respiratory symptoms, vertigo, and depression were also more common in the livestock dense area compared to the control area. In general, there were no significant differences in chronic conditions such as asthma, COPD, or lung cancer. Prescription rates for broad-spectrum antibiotics were more common among patients with pneumonia in the livestock dense area. Acute respiratory infections and symptoms, but not chronic conditions, were considerably more common in areas with a high livestock density. Identification of causal pathogens on the basis of serological analyses could further elucidate the underlying mechanisms behind the observed health effects.

Background/aim From 2007 through 2010, the Netherlands experienced the largest recorded Q fever outbreak to date. People living closer to Coxiella burnetii infected goat farms were at increased risk for acute Q fever. Time spent outdoors near infected farms may have contributed to exposure to C. burnetii. The aim of this study was to retrospectively evaluate whether hours/week spent outdoors, in the vicinity of previously C. burnetii infected goat farms, was associated with presence of antibodies against C. burnetii in residents of a rural area in the Netherlands. Methods Between 2014-2015, we collected C. burnetii antibody serology and self-reported data about habitual hours/week spent outdoors near the home from 2494 adults. From a subgroup we collected 941 GPS tracks, enabling analyses of active mobility in the outbreak region. Participants were categorised as exposed if they spent time within specified distances (500m, 1000m, 2000m, or 4000m) of C. burnetii infected goat farms. We evaluated whether time spent near these farms was associated with positive C. burnetii serology using spline analyses and logistic regression. Results People that spent more hours/week outdoors near infected farms had a significantly increased risk for positive C. burnetii serology (time spent within 2000m of a C. burnetii abortion-wave positive farm, OR 3.6 (1.2-10.6)), compared to people spending less hours/week outdoors. Conclusions Outdoor exposure contributed to the risk of becoming C. burnetii serology positive. These associations were stronger if people spent more time near C. burnetii infected farms. Outdoor exposure should, if feasible, be included in outbreak investigations.

BACKGROUND/AIM: Active mobility may play a relevant role in the assessment of environmental exposures (e.g. traffic-related air pollution, livestock emissions), but data about actual mobility patterns are work intensive to collect, especially in large study populations, therefore estimation methods for active mobility may be relevant for exposure assessment in different types of studies. We previously collected mobility patterns in a group of 941 participants in a rural setting in the Netherlands, using week-long GPS tracking. We had information regarding personal characteristics, self-reported data regarding weekly mobility patterns and spatial characteristics. The goal of this study was to develop versatile estimates of active mobility, test their accuracy using GPS measurements and explore the implications for exposure assessment studies. METHODS: We estimated hours/week spent on active mobility based on personal characteristics (e.g. age, sex, pre-existing conditions), self-reported data (e.g. hours spent commuting per bike) or spatial predictors such as home and work address. Estimated hours/week spent on active mobility were compared with GPS measured hours/week, using linear regression and kappa statistics. RESULTS: Estimated and measured hours/week spent on active mobility had low correspondence, even the best predicting estimation method based on self-reported data, resulted in a R2 of 0.09 and Cohen's kappa of 0.07. A visual check indicated that, although predicted routes to work appeared to match GPS measured tracks, only a small proportion of active mobility was captured in this way, thus resulting in a low validity of overall predicted active mobility. CONCLUSIONS: We were unable to develop a method that could accurately estimate active mobility, the best performing method was based on detailed self-reported information but still resulted in low correspondence. For future studies aiming to evaluate the contribution of home-work traffic to exposure, applying spatial predictors may be appropriate. Measurements still represent the best possible tool to evaluate mobility patterns.

The association between household air pollution and lower respiratory tract infections (LRTI) among children under five years of age has been well documented; however, the extent to which the microbiome within the indoor environment contributes to this association is uncertain. The home assessment of indoor microbiome (HAIM) study seeks to assess the abundance of indoor microbiota (IM) in the homes of under-five children (U-5Cs) with and without LRTI. HAIM is a hospital- and community-based study involving 200 cases and 200 controls recruited from three children’s hospitals in Ibadan, Nigeria. Cases will be hospital-based patients with LRTI confirmed by a pediatrician, while controls will be community-based participants, matched to cases on the basis of sex, geographical location, and age (±3 months) without LRTI. The abundance of IM in houses of cases and controls will be investigated using active and passive air sampling techniques and analyzed by qualitative detection of bacterial 16SrRNA gene (V3–V4), fungal ITS1 region, and viral RNA sequencing. HAIM is expected to elucidate the relationship between exposure to IM and incidence of LRTI among U-5Cs and ultimately provide evidence base for strategic interventions to curtail the burgeoning burden of LRTI on the subcontinent.

Researchers in the air pollution field are well aware that indoor and outdoor air pollution is associated with multiple disorders, including an increase in respiratory infection-related morbidity and mortality [1, 2]. Investigating associations linking air pollution and COVID-19 spread and prognosis of respiratory disease in COVID-19 patients is therefore timely, relevant and plausible. Over a ten-day period, three papers involving original research associating COVID-19 mortality and air pollution were published [3–5]. These publications attracted considerable attention from international news outlets and on social media [6, 7]. The first study was performed in the USA and associated COVID-19 mortality in 1783 counties with reported long-term (2000–2016) average air pollution levels (fine particulate matter (PM2.5) with ∼1 km2 resolution) [3]. Adjustments on the county level were made for sixteen variables, including population size, ethnicity, hospital beds, number of individuals tested for COVID-19, weather, and socioeconomic and behavioral variables such as income, obesity and smoking. The study reported that an increase of 1 μg·m−3 in PM2.5 was associated with a remarkably large 15% increase in the COVID-19 death rate. An updated analysis, adjusted for three more confounding factors; days since first COVID-19 case reported, population age distribution, and days since issuance of stay-at-home order for each state, saw the risk estimate halved to an 8% increase in death rate [8]., Present studies on the role of air pollution and COVID-19 spread and prognosis in patients do not fulfill quality criteria and are at present not sufficiently informative.

Manure application can spread antimicrobial resistance (AMR) from manure to soil and surface water. This study evaluated the role of the soil texture on the dynamics of antimicrobial resistance genes (ARGs) in soils and sur- rounding surface waters. Six dairy farms with distinct soil textures (clay, sand, and peat) were sampled at different time points after the application of manure, and three representative ARGs sul1, erm(B), and tet(W) were quantified with qPCR. Manuring initially increased levels of erm(B) by 1.5 ± 0.5 log copies/kg of soil and tet (W) by 0.8 ± 0.4 log copies/kg across soil textures, after which levels gradually declined. In surface waters from clay environments, regardless of the ARG, the gene levels initially increased by 2.6 ± 1.6 log copies/L, after which levels gradually declined. The gene decay in soils was strongly dependent on the type of ARG (erm (B) < tet(W) < sul1; half-lives of 7, 11, and 75 days, respectively), while in water, the decay was primarily dependent on the soil texture adjacent to the sampled surface water (clay < peat < sand; half-lives of 2, 6, and 10 days, respectively). Finally, recovery of ARG levels was predicted after 29–42 days. The results thus showed that there was not a complete restoration of ARGs in soils between rounds of manure application. In conclusion, this study demonstrates that rather than showing similar dynamics of decay, factors such as the type of ARG and soil texture drive the ARG persistence in the environment.

International audience; Background High antimicrobial use (AMU) and antimicrobial resistance (AMR) in veal calves remain a source of concern. As part of the EFFORT project, the association between AMU and the abundance of faecal antimicrobial resistance genes (ARGs) in veal calves in three European countries was determined. Methods In 2015, faecal samples of veal calves close to slaughter were collected from farms located in France, Germany and the Netherlands (20 farms in France, 20 farms in the Netherlands and 21 farms in Germany; 25 calves per farm). Standardized questionnaires were used to record AMU and farm characteristics. In total, 405 faecal samples were selected for DNA extraction and quantitative polymerase chain reaction to quantify the abundance (16S normalized concentration) of four ARGs [aph(3’)-III, ermB, sul2 and tetW] encoding for resistance to frequently used antimicrobials in veal calves. Multiple linear mixed models with random effects for country and farm were used to relate ARGs to AMU and farm characteristics. Results A significant positive association was found between the use of trimethoprim/sulfonamides and the concentration of sul2 in faeces from veal calves. A higher weight of calves on arrival at the farm was negatively associated with aph(3’)-III and ermB. Lower concentrations of aph(3’)-III were found at farms with non-commercial animals present. Furthermore, farms using only water for the cleaning of stables had a significantly lower abundance of faecal ermB and tetW compared with other farms. Conclusion A positive association was found between the use of trimethoprim/sulfonamides and the abundance of sul2 in faeces in veal calves. Additionally, other relevant risk factors associated with ARGs in veal calves were identified, such as weight on arrival at the farm and cleaning practices.

BACKGROUND/AIM: From 2007 through 2010, the Netherlands experienced the largest recorded Q fever outbreak to date. People living closer to Coxiella burnetii infected goat farms were at increased risk for acute Q fever. Time spent outdoors near infected farms may have contributed to exposure to C. burnetii. The aim of this study was to retrospectively evaluate whether hours/week spent outdoors, in the vicinity of previously C. burnetii infected goat farms, was associated with presence of antibodies against C. burnetii in residents of a rural area in the Netherlands. METHODS: Between 2014-2015, we collected C. burnetii antibody serology and self-reported data about habitual hours/week spent outdoors near the home from 2494 adults. From a subgroup we collected 941 GPS tracks, enabling analyses of active mobility in the outbreak region. Participants were categorised as exposed if they spent time within specified distances (500m, 1000m, 2000m, or 4000m) of C. burnetii infected goat farms. We evaluated whether time spent near these farms was associated with positive C. burnetii serology using spline analyses and logistic regression. RESULTS: People that spent more hours/week outdoors near infected farms had a significantly increased risk for positive C. burnetii serology (time spent within 2000m of a C. burnetii abortion-wave positive farm, OR 3.6 (1.2-10.6)), compared to people spending less hours/week outdoors. CONCLUSIONS: Outdoor exposure contributed to the risk of becoming C. burnetii serology positive. These associations were stronger if people spent more time near C. burnetii infected farms. Outdoor exposure should, if feasible, be included in outbreak investigations.

Living close to livestock farms has been associated with increased symptoms in patients with chronic obstructive pulmonary disease (COPD). The causes of these effects are still poorly understood. This panel study attempts to assess the acute effects of livestock-related air pollution in patients with COPD living in an area with intensive livestock farming in the Netherlands.Between February 2015 and July 2016, 82 participants took spirometry measurements twice daily (morning and evening) during a 3-month period, resulting in 12,672 FEV1 and PEF records. Participants also kept a diary on respiratory symptoms as well as livestock-related odor annoyance. Daily average ammonia (NH3) (a proxy for livestock-related air pollution) and fine particulate matter (PM10) levels were collected from monitoring stations in the area. Lung function was analyzed as decrements of >10% and >20% from their median as well as absolute values. Self-reported odor annoyance was analyzed as a dichotomous variable. All analyses were done using generalized estimated equations. We adjusted for humidity, temperature, linear trend, and took multiple testing into account.We found an odds ratio of 1.14 95%CI [1.05; 1.25] for decrements >20% in morning FEV1 per interquartile range (12 µg/m3) increase in NH3 concentration (lag 2). Odor annoyance was negatively associated with evening PEF (−4.46 l/min 95%CI [−7.59; −1.33]). Sensitivity analyses showed a stronger effect in participants with worse baseline lung function. No associations with symptoms were found.Our results show acute effects of livestock-related air pollution on lung function in COPD patients living in close proximity to livestock farms. Keywords: Environmental epidemiology, Air pollution, Livestock, COPD

BACKGROUND: A community-wide outbreak of Legionnaires' disease (LD) occurred in Genesee County, Michigan, in 2014 and 2015. Previous reports about the outbreak are conflicting and have associated the outbreak with a change of water source in the city of Flint and, alternatively, to a Flint hospital. OBJECTIVE: The objective of this investigation was to independently identify relevant sources of Legionella pneumophila that likely resulted in the outbreak. METHODS: An independent, retrospective investigation of the outbreak was conducted, making use of public health, health care, and environmental data and whole-genome multilocus sequence typing (wgMLST) of clinical and environmental isolates. RESULTS: Strong evidence was found for a hospital-associated outbreak in both 2014 and 2015: a) 49% of cases had prior exposure to Flint hospital A, significantly higher than expected from Medicare admissions; b) hospital plumbing contained high levels of L. pneumophila; c) Legionella control measures in hospital plumbing aligned with subsidence of hospital A-associated cases; and d) wgMLST showed Legionella isolates from cases exposed to hospital A and from hospital plumbing to be highly similar. Multivariate analysis showed an increased risk of LD in 2014 for people residing in a home that received Flint water or was located in proximity to several Flint cooling towers. DISCUSSION: This is the first LD outbreak in the United States with evidence for three sources (in 2014): a) exposure to hospital A, b) receiving Flint water at home, and c) residential proximity to cooling towers; however, for 2015, evidence points to hospital A only. Each source could be associated with only a proportion of cases. A focus on a single source may have delayed recognition and remediation of other significant sources of L. pneumophila. https://doi.org/10.1289/EHP5663.

Background Elevated endotoxin levels have been measured in ambient air around livestock farms, which is a cause of concern for neighbouring residents. There is clear evidence that occupational exposure to high concentrations of airborne endotoxin causes respiratory inflammation, respiratory symptoms and lung function decline. However, health effects of exposure to low levels of endotoxin are less well described. The aim of this systematic review is to summarize published associations between exposure to relatively low levels of airborne endotoxin and respiratory health endpoints. Methods Studies investigating respiratory effects of measured or modelled exposure to low levels of airborne endotoxin (average

Background: In recent years, ESBL/AmpC-producing Escherichia coli (ESBL/AmpC-EC) have been isolated with increasing frequency from animals, food, environmental sources and humans. With incomplete and scattered evidence, the contribution to the human carriage burden from these reservoirs remains unclear. Objectives: To quantify molecular similarities between different reservoirs as a first step towards risk attribution. Methods: Pooled data on ESBL/AmpC-EC isolates were recovered from 35 studies in the Netherlands comprising >27 000 samples, mostly obtained between 2005 and 2015. Frequency distributions of ESBL/AmpC genes from 5808 isolates and replicons of ESBL/AmpC-carrying plasmids from 812 isolates were compared across 22 reservoirs through proportional similarity indices (PSIs) and principal component analyses (PCAs). Results: Predominant ESBL/AmpC genes were identified in each reservoir. PCAs and PSIs revealed close human-animal ESBL/AmpC gene similarity between human farming communities and their animals (broilers and pigs) (PSIs from 0.8 to 0.9). Isolates from people in the general population had higher similarities to those from human clinical settings, surface and sewage water and wild birds (0.7-0.8), while similarities to livestock or food reservoirs were lower (0.3-0.6). Based on rarefaction curves, people in the general population had more diversity in ESBL/AmpC genes and plasmid replicon types than those in other reservoirs. Conclusions: Our 'One Health' approach provides an integrated evaluation of the molecular relatedness of ESBL/AmpC-EC from numerous sources. The analysis showed distinguishable ESBL/AmpC-EC transmission cycles in different hosts and failed to demonstrate a close epidemiological linkage of ESBL/AmpC genes and plasmid replicon types between livestock farms and people in the general population.

Objectives To explore the effects of using different indicators to quantify antimicrobial usage (AMU) in livestock and compare outcomes with antimicrobial resistance (AMR) data. Methods Three indicators were used to quantify AMU, two indicators in which the denominator varied: defined daily doses per average mass of the animals present per year (DDD/AY) and defined daily doses per population correction unit (DDD/PCU) and one in which the numerator varied: milligrams of active ingredient per PCU (mg/PCU). AMU was compared with antimicrobial resistance data from the national monitoring programme from 2013 to 2018 with the proportion of Escherichia coli isolates fully susceptible to a predefined panel of antimicrobials for the broiler, dairy cattle and pig farming livestock sectors in the Netherlands. Results The ranking of livestock sectors differs between sectors when using different indicators to express AMU. Dairy cattle rank lowest when expressing AMU in DDD/AY, followed by pigs and broilers corresponding to the rankings of the sectors for AMR. When changing the denominator to PCU, the ranking in AMU is reversed: use ranks highest in dairy cattle and lowest broilers. Conclusions Using different denominators in AMU indicators has a major impact on measured use. This might result in misinterpretation of effects of interventions on AMU and the associations of AMU with AMR across animal sectors. From an epidemiological perspective, indicators that take into account time at risk of exposure to antimicrobials are to be preferred and reflect the AMR risk most accurately.

Comprehensive strategies to improve on-farm antimicrobial use (AMU) are needed to contain antimicrobial resistance (AMR). Little is known about farmers' motivating and enabling factors, and about their influence on AMU. In a cross-sectional online survey, Dutch dairy, veal and pig farmers (n=457) reported their on-farm AMU as "Defined Daily Dose Animal" per year (DDDAF) and completed a detailed questionnaire on their view, knowledge and behavior towards AMU and AMR. Exploratory factor analysis (EFA) on the questionnaire items identified four psychological factors labeled as 'referent beliefs', 'perceived risk', 'knowledge', and 'undesired attitude to regulations'. Linear regression was done to explore the relationship between the obtained factors and on-farm AMU across the three animal sectors. Dairy farmers showed the highest factor scores for 'knowledge' and the lowest for 'perceived risk'. 'Knowledge' scores were significantly and inversely related to AMU (P=0.0004). Borderline significant associations with AMU were found for 'perceived risk' and 'undesired attitude to regulations' (negative and positive relationships respectively). There were no apparent differences for these relationships between the three livestock sectors. Behavioral interventions in farmers such as educational campaigns or increased support by veterinarians could empower farmers with more prudent and rational practices, eventually reducing AMU in food animals.

The Sixth Jack Pepys Workshop on Asthma in the Workplace focused on six key themes regarding the recognition and assessment of work-related asthma and airway diseases: (1) cleaning agents and disinfectants (including in swimming pools) as irritants and sensitizers: how to evaluate types of bronchial reactions and reduce risks; (2) population-based studies of occupational obstructive diseases: use of databanks, advantages and pitfalls, what strategies to deal with biases and confounding?; (3) damp environments, dilapidated buildings, recycling processes, and molds, an increasing problem: mechanisms, how to assess causality and diagnosis; (4) diagnosis of occupational asthma and rhinitis: how useful are recombinant allergens (component-resolved diagnosis), metabolomics, and other new tests?; (5) how does exposure to gas, dust, and fumes enhance sensitization and asthma?; and (6) how to determine probability of occupational causality in chronic obstructive pulmonary disease: epidemiological and clinical, confirmation, and compensation aspects. A summary of the presentations and discussion is provided in this proceedings document. Increased knowledge has been gained in each topic over the past few years, but there remain aspects of controversy and uncertainty requiring further research.

In several regions worldwide, the presence of livestock in close proximity to residential areas raises questions about public health implications. The rapid expansion of large‐scale livestock farms, increasingly interwoven with urbanized areas, and its potential impact on neighboring residents' health has hardly been accompanied by any research. The current situation in densely populated livestock farming areas could be regarded as a “natural experiment.” Most scientific and public health initiatives have focused on emerging zoonoses and antimicrobial resistance as potential health threats. In this commentary, we emphasize the importance of respiratory health effects of noninfectious air pollutant emissions from livestock farms., Key Points The rapid expansion of large‐scale livestock farms near residential areas raises questions about public health implicationsA series of recent studies on air pollutant emissions from agriculture emphasize respiratory health risks among neighboring residentsThere is a clear need to firmly embed public health perspectives in the decision‐making process in environmental and agricultural planning

Objectives Patients with chronic obstructive pulmonary disease (COPD) constitute a potentially susceptible group towards environmental exposures such as livestock farm emissions, given their compromised respiratory health status. The primary aim of this study was to examine the association between livestock exposure and comorbidities and coexisting symptoms and infections in COPD patients. Methods Data were collected from 1828 COPD patients (without co-occurring asthma) registered in 23 general practices and living in a rural area with a high livestock density. Prevalence of comorbid diseases/disorders and coexisting symptoms/infections were based on electronic health records from the year 2012. Various indicators of individual exposure to livestock were estimated based on residential addresses, using a geographic information system. Results At least one comorbid disorder was present in 69% of the COPD patients (especially cardiac disorders and depression, while 49% had at least one coexisting symptom and/or infection (especially upper respiratory tract infections, respiratory symptoms and pneumonia). Half of the COPD-patients resided less than 500 m of the nearest farm. Some positive as well as inverse associations were found between the examined outcomes and exposure estimates, although not consistent. Conclusions Despite the high prevalence of coexisting chronic and acute conditions presented in primary care by in COPD patients, this investigation found no convincing evidence for an association with livestock exposure estimates. There is a need for a replication of the present findings in studies with a longitudinal design, on different groups of potentially susceptible patients. Future research should also elucidate the biological plausibility of possible protective effects of exposure.

Background: Living in livestock-dense areas has been associated with health effects, suggesting airborne exposures to livestock farm emissions to be relevant for public health. Livestock farm emissions involve complex mixtures of various gases and particles. Endotoxin, a pro-inflammatory agent of microbial origin, is a constituent of livestock farm emitted particulate matter (PM) that is potentially related to the observed health effects. Quantification of livestock associated endotoxin exposure at residential addresses in relation to health outcomes has not been performed earlier. Objectives: We aimed to assess exposure-response relations for a range of respiratory endpoints and atopic sensitization in relation to livestock farm associated PM10 and endotoxin levels. Methods: Self-reported respiratory symptoms of 12,117 persons participating in a population-based cross-sectional study were analyzed. For 2494 persons, data on lung function (spirometry) and serologically assessed atopic sensitization was additionally available. Annual-average PM10 and endotoxin concentrations at home addresses were predicted by dispersion modelling and land-use regression (LUR) modelling. Exposure-response relations were analyzed with generalized additive models. Results: Health outcomes were generally more strongly associated with exposure to livestock farm emitted endotoxin compared to PM10. An inverse association was observed for dispersion modelled exposure with atopic sensitization (endotoxin: p = .004, PM10: p = .07) and asthma (endotoxin: p = .029, PM10: p = .022). Prevalence of respiratory symptoms decreased with increasing endotoxin concentration at the lower range, while at the higher range prevalence increased with increasing concentration (p .05). Conclusions: Exposure to livestock farm emitted particulate matter is associated with respiratory health effects and atopic sensitization in non-farming residents. Results indicate endotoxin to be a potentially plausible etiologic agent, suggesting non-infectious aspects of microbial emissions from livestock farms to be important with respect to public health. Keywords: Livestock farming, Emissions, Air pollution, Public health, Spatial modelling, Endotoxin

Background Epidemiological research on health effects of livestock exposure in population subgroups with compromised respiratory health is still limited. The present study explored the association between livestock exposure and comorbid/concurrent conditions in patients with overlapping diagnoses of asthma and COPD. Methods Electronic health record data from 23 general practices in the Netherlands were collected from 425 patients diagnosed with both asthma and COPD, living in rural areas with high livestock density (“study area”). Data of 341 patients with the same overlapping diagnoses, living in rural areas with lower livestock density (“control areas”) were obtained from 19 general practices. First, the prevalence of comorbid disorders and symptoms/infections were compared between the study and control area. Second, the examined health outcomes were analyzed in relation to measures of individual livestock exposure. Results Pneumonia was twice as common among patients living in areas with a high livestock density (OR 2.29, 99% CI 0.96–5.47); however, there were generally no statistically significant differences in the investigated outcomes between the study and control area. Significant associations were observed between presence of goats within 1000 m and allergic rhinitis (OR 5.71, 99% CI 1.11–29.3, p

Antimicrobial resistance (AMR) is an increasing threat, both in human and in veterinary medicine. To reduce the selection and spread of AMR, antimicrobial use (AMU) should be optimized, also in companion animals. To be able to optimize AMU, a feasible method to quantify AMU and information on current AMU are needed. Therefore, a method to quantify AMU was developed, using the number of Defined Daily Doses Animal (DDDA). This method was used to explore applied antimicrobial classes and to identify differences in prescribing patterns in time and between veterinary clinics. Antimicrobial procurement data of the years 2012-2014 were collected retrospectively from 100 Dutch veterinary clinics providing care for companion animals. The mean number of DDDAs per clinic per year decreased significantly from 2012 to 2014. A shift in used classes of antimicrobials (AMs) was seen as well, with a significant decrease in use of third choice AMs (i.e., fluoroquinolones and third generation cephalosporins). Large differences in total AMU were seen between clinics ranging from 64-fold in 2012 to 20-fold in 2014. Despite the relative low and decreasing AMU in Dutch companion animal clinics during the study, the substantial differences in antimicrobial prescribing practices between clinics suggest that there is still room for quantitative and qualitative optimization of AMU.

In the Netherlands, an association was found between the prevalence of pneumonia and living near goat and poultry farms in 2007-2013. This association then led to regulatory decisions to restrict the building of new goat farms and to reduce emissions of poultry farms. Confirmation of these results, however, is required because the period of previous analyses overlapped a Q-fever epidemic in 2007-2010. To confirm the association, we performed a population-based study during 2014-2016 based on general practitioner (GP) data. Electronic medical records of 90,183 persons were used to analyze the association between pneumonia and the population living in the proximity (within 500-2000 m distance) of goat and poultry farms. Data were analyzed with three types of logistic regression (with and without GP practice as a random intercept and with stratified analyses per GP practice) and a kernel model to discern the influence of different statistical methods on the outcomes. In all regression analyses involving adults, a statistically significant association between pneumonia and residence within 500 meters of goat farms was found (odds ratio [OR] range over all analyses types: 1.33-1.60), with a decreasing OR for increasing distances. In kernel analyses (including all ages), a population-attributable risk between 6.0 and 7.8% was found for a distance of 2000 meters in 2014-2016. The associations were consistent across all years and robust for mutual adjustment for proximity to other animals and for several other sensitivity analyses. However, associations with proximity to poultry farms are not supported by the present study. As the causes of the elevated pneumonia incidence in persons living close to goat farms remain unknown, further research into potential mechanisms is required for adequate prevention.

EFFORT consortium members=Haitske Graveland (UUVM), Alieda van Essen (WBVR), Bruno Gonzalez-Zorn (UCM), Gabriel Moyano (UCM), Pascal Sanders (ANSES), Claire Chauvin (ANSES), Julie David (ANSES), Antonio Battisti (IZSLT), Andrea Caprioli (IZSLT), Thomas Blaha (TIHO), Katharina Wadepohl (TIHO), Maximiliane Brandt (TIHO), Tine Hald (DTU), Ana Sofia Ribeiro Duarte (DTU), Dariusz Wasyl (NVRI), Magdalena Skar_zynska (NVRI), Magdalena Zaja˛c (NVRI), Andrzej Hoszowski (deceased) (NVRI), Hristo Daskalov (NDRVI), Helmut W. Saatkamp (BEC) and Katharina D. C. Sta¨ rk (SAFOSO).; International audience; Previous studies in food-producing animals have shown associations between antimicrobial use (AMU) and resistance (AMR) in specifically isolated bacterial species. Multi-country data are scarce and only describe between-country differences. Here we investigate associations between the pig faecal mobile resistome and characteristics at the farm-level across Europe.A cross-sectional study was conducted among 176 conventional pig farms from nine European countries. Twenty-five faecal samples from fattening pigs were pooled per farm and acquired resistomes were determined using shotgun metagenomics and the Resfinder reference database, i.e. the full collection of horizontally acquired AMR genes (ARGs). Normalized fragments resistance genes per kilobase reference per million bacterial fragments (FPKM) were calculated. Specific farm-level data (AMU, biosecurity) were collected. Random-effects meta-analyses were performed by country, relating farm-level data to relative ARG abundances (FPKM).Total AMU during fattening was positively associated with total ARG (total FPKM). Positive associations were particularly observed between widely used macrolides and tetracyclines, and ARGs corresponding to the respective antimicrobial classes. Significant AMU-ARG associations were not found for β-lactams and only few colistin ARGs were found, despite high use of these antimicrobial classes in younger pigs. Increased internal biosecurity was directly related to higher abundances of ARGs mainly encoding macrolide resistance. These effects of biosecurity were independent of AMU in mutually adjusted models.Using resistome data in association studies is unprecedented and adds accuracy and new insights to previously observed AMU-AMR associations. Major components of the pig resistome are positively and independently associated with on-farm AMU and biosecurity conditions.

Objectives: To control the emerging threat of antimicrobial resistance, international policy appeals for appropriate monitoring of antimicrobial usage (AMU) at supranational, species and farm level. The aim of this study was to quantify AMU in broilers at farm and flock level in nine European countries. Methods: Antimicrobial treatment data of one flock and purchased antimicrobials over one year were collected at 181 European broiler farms. Afterwards AMU was quantified using treatment incidence (TI) per 100 days based on Defined Daily Dose (DDDvet), Defined Course Dose (DCDvet) or Used Daily Dose (UDDvet) values. Total AMU at flock level was obtained by summing the TIDDDvet of all treatments in the sampled flock (TIDDDvetFl*). Results: The median TIDDDvetFl* was 9.0 (95% CI 5.5-10.8), meaning that broilers were treated with antimicrobials during 9% of their rearing period. TIDDDvetFl* varied considerably within and between countries. However, in every country at least one untreated flock was present. Average TIDDDvetFl* at country level ranged from 3.3 to 36.7. Polymyxins, extended-spectrum aminopenicillins and fluoroquinolones were the most used antimicrobials, accounting for 26%, 26% and 18% of total AMU, respectively. Twenty-six percent of the farms started a treatment on day 1 of production, and 49% of overall AMU was administered within the first week. Conclusions: Results show that rearing broilers without AMU is feasible. However, a huge variation in AMU in terms of amount, moment of administration and antimicrobial classes was observed. This shows that there is still ground to be covered when it comes to AMU on broiler farms.

Exposure to moisture-damaged indoor environments is associated with adverse respiratory health effects, but responsible factors remain unidentified. In order to explore possible mechanisms behind these effects, the oxidative capacity and hemolytic activity of settled dust samples (n = 25) collected from moisture-damaged and non-damaged schools in Spain, the Netherlands, and Finland were evaluated and matched against the microbial content of the sample. Oxidative capacity was determined with plasmid scission assay and hemolytic activity by assessing the damage to isolated human red blood cells. The microbial content of the samples was measured with quantitative PCR assays for selected microbial groups and by analyzing the cell wall markers ergosterol, muramic acid, endotoxins, and glucans. The moisture observations in the schools were associated with some of the microbial components in the dust, and microbial determinants grouped together increased the oxidative capacity. Oxidative capacity was also affected by particle concentration and country of origin. Two out of 14 studied dust samples from moisture-damaged schools demonstrated some hemolytic activity. The results indicate that the microbial component connected with moisture damage is associated with increased oxidative stress and that hemolysis should be studied further as one possible mechanism contributing to the adverse health effects of moisture-damaged buildings.

Contains fulltext : 215642.pdf (Publisher’s version ) (Open Access) BACKGROUND: To curb increasing resistance rates, responsible antimicrobial use (AMU) is needed, both in human and veterinary medicine. In human healthcare, antimicrobial stewardship programmes (ASPs) have been implemented worldwide to improve appropriate AMU. No ASPs have been developed for and implemented in companion animal clinics yet. OBJECTIVES: The objective of the present study was to implement and evaluate the effectiveness of an ASP in 44 Dutch companion animal clinics. The objectives of the ASP were to increase awareness on AMU, to decrease total AMU whenever possible and to shift AMU towards 1st choice antimicrobials, according to Dutch guidelines on veterinary AMU. METHODS: The study was designed as a prospective, stepped-wedge, intervention study, which was performed from March 2016 until March 2018. The multifaceted intervention was developed using previous qualitative and quantitative research on current prescribing behaviour in Dutch companion animal clinics. The number of Defined Daily Doses for Animal (DDDAs) per clinic (total, 1st, 2nd and 3rd choice AMU) was used to quantify systemic AMU. Monthly AMU data were described using a mixed effect time series model with auto-regression. The effect of the ASP was modelled using a step function and a change in the (linear) time trend. RESULTS: A statistically significant decrease of 15% (7%-22%) in total AMU, 15% (5%-24%) in 1st choice AMU and 26% (17%-34%) in 2nd choice AMU was attributed to participation in the ASP, on top of the already ongoing time trends. Use of 3rd choice AMs did not significantly decrease by participation in the ASP. The change in total AMU became more prominent over time, with a 16% (4%-26%) decrease in (linear) time trend per year. CONCLUSIONS: This study shows that, although AMU in Dutch companion animal clinics was already decreasing and changing, AMU could be further optimised by participation in an antimicrobial stewardship programme.