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Numerous source attribution studies for foodborne pathogens based on epidemiological and microbiological methods are available. These studies provide empirical data for modelling frameworks that synthetize the quantitative evidence at our disposal and reduce reliance on expert elicitations. Here, we develop a statistical model within a Bayesian estimation framework to integrate attribution estimates from expert elicitations with estimates from microbial subtyping and case-control studies for sporadic infections with four major bacterial zoonotic pathogens in the Netherlands (Campylobacter, Salmonella, Shiga toxin-producing E. coli [STEC] O157 and Listeria). For each pathogen, we pooled the published fractions of human cases attributable to each animal reservoir from the microbial subtyping studies, accounting for the uncertainty arising from the different typing methods, attribution models, and year(s) of data collection. We then combined the population attributable fractions (PAFs) from the case-control studies according to five transmission pathways (domestic food, environment, direct animal contact, human-human transmission and travel) and 11 groups within the foodborne pathway (beef/lamb, pork, poultry meat, eggs, dairy, fish/shellfish, fruit/vegetables, beverages, grains, composite foods and food handlers/vermin). The attribution estimates were biologically plausible, allowing the human cases to be attributed in several ways according to reservoirs, transmission pathways and food groups. All pathogens were predominantly foodborne, with Campylobacter being mostly attributable to the chicken reservoir, Salmonella to pigs (albeit closely followed by layers), and Listeria and STEC O157 to cattle. Food-wise, the attributions reflected those at the reservoir level in terms of ranking. We provided a modelling solution to reach consensus attribution estimates reflecting the empirical evidence in the literature that is particularly useful for policy-making and is extensible to other pathogens and domains., (© 2022 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Soil-transmitted helminths, such as Ascaris lumbricoides , are the most prevalent parasites globally. Optimal anthelmintic treatment for A. lumbricoides in endemically infected communities is challenged by several host-related and environmental factors influencing infection acquisition. We assessed the risk of A. lumbricoides (re)infection after treatment in a Venezuelan rural community. Individual merthiolate-iodine-formaldehyde-fixed faecal samples were collected from 224 persons before a single-dose pyrantel treatment and at 1, 3, 6, 9 and 15 months after treatment. Effects of age, sex and socioeconomic status (SES) on A. lumbricoides prevalence, eggs/gram faeces (EPG) and infection (re)acquisition were assessed using both generalised linear mixed-effects models and survival analysis. Pre-treatment A. lumbricoides prevalence was 39.7%. Higher prevalence was associated with younger age and lower SES. Higher EPG values were observed among young children. Median time to A. lumbricoides infection was six months after treatment: at 1, 3, 6, 9 and 15 months post-treatment, cumulative incidence was 6.7%, 18.9%, 34.6%, 42.2%, and 52.6%, respectively. Younger age, lower SES, and pre-treatment A. lumbricoides infection status showed significantly elevated hazard ratios. Mass drug administration protocols would benefit from considering these factors in selective treatment strategies and possibly more than just annual or biannual treatments in the target population.

The use of antimicrobials in agricultural, veterinary and medical practice exerts selective pressure on environmental microbiota, promoting the emergence and spread of antimicrobial resistance (AMR), a global concern for the One Health Initiative Task Force (OHITF). Honeybees have been studied as bioindicators of AMR in the environment, but little is known about beehive products like honey and pollen. The aim of this study was to assess the prevalence of AMR genes (ARGs) in beehive products and investigated their origins. Specifically, possible associations between ARGs, microbiota and other characteristics of different honey and pollen samples, including country of origin, flower type, type of commercial distribution and environmental factors, such as land use, weather and composition of the environment surrounding the beehives were investigated. We found that beehive products harboured ARGs conferring resistance to β-lactams, macrolides, (fluoro)quinolones and polymyxins. Most samples possessed resistance to multiple antimicrobial classes, with honey and pollen showing similar ARG profiles. Even if Lactobacillus and Acinetobacter genera were common in the microbial communities of both honey and pollen, Bacillus, Clostridium, and Bombella defined honey microbiota, while Pseudomonas and Vibrio were enriched in pollen. ErmB and bla TEM-1 co-occurred with Lactobacillus and Fructobacillus, while positive associations between β-lactams and macrolides and anthropogenic environments (i.e. industrial and commercial areas and non-irrigated arable lands) were found. Altogether, our findings suggest that ARGs in honey and pollen might originate from the honeybee foraging environment, and that the beehive products can be used as bioindicators of the AMR environmental contamination., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Biosecurity in poultry farms represents the first line of defense against the entry and spread of pathogens that may have animal health, food safety, and economic consequences. The aim of this study was to assess biosecurity compliance in poultry farms located in a densely populated poultry area in North East Italy. A total of 259 poultry farms (i.e., broilers, turkeys, and layers) were surveyed between 2018 and 2019 using standardized checklists, and differences in biosecurity compliance between the poultry sectors and years (only for turkey farms) were tested for significance. Among the three sectors, turkey farms showed the highest compliance. Farm hygiene, infrastructure condition, cleaning and disinfection tools, and procedures were the biosecurity measures most complied with. Some deficiencies were observed in the cleanliness of the farm hygiene lock in broiler farms, as well as the presence of the house hygiene lock in broiler and layer farms and an adequate coverage of built-up litter in turkey and broiler farms. In conclusion, this study highlighted a generally high level of biosecurity in the visited poultry farms (probably due to the stringent national regulation and the integration of the poultry industry) and identified some measures that still need to be improved.

RESEARCH HIGHLIGHTSSulfamethoxazole and ampicillin resistance are the highest in E. coli isolates of both broilers and turkeys.Short-term effects of antibiotic use on resistance are seen more often than long-term effects.Historical penicillin use is associated with penicillin resistance in broilers.There is an association between historical use and resistance to trimethoprim in turkeys.

In the past few years, microwave induced plasma optical emission spectrometry (MIP-OES) has generated great interest as an alternative technique to inductively coupled plasma-based techniques due to its lower operational cost. Since MIP-OES suffers from severe matrix effects due to easily ionizable elements (EIEs) (Na, Ca, etc.), it is unclear whether this technique could be employed for elemental bioavailability studies in soils and sediments since the main extractant solutions employed in such works may contain high levels of these elements. Thus, the aim of this work was to evaluate the feasibility of MIP-OES as a detector for such applications. To this end, the influence of different extractant solutions (0.25 mol L -1 MgCl 2 , 0.25 mol L -1 CaCl 2 , 0.10 mol L -1 acetic acid, 0.05 mol L -1 Na 2 EDTA, 0.25 mol L -1 NaNO 3 , 0.25 mol L -1 NaOAc/HOAc and 0.10 mol L -1  NH 2 OH·HCl) on the analyte emission of several elements (As, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Pb, Rh, Se, Sr and Zn) was investigated. Results were compared to those obtained using a reference solution made of 5% w w -1 HNO 3 solution. For saline extractant solutions, both the optimum nebulizer gas flow rate (Q g ) and analyte emission were modified with regard to the reference solution. In general, the optimum Q g was reduced by between 0.1 and 0.2 L min -1 for both ionic and atomic lines. Under optimum Q g conditions, analyte emission was supressed by saline solutions except for atomic lines with an upper electronic state below 4 eV, which were enhanced. The magnitude of matrix effects was strongly dependent on EIE ionization energy. The lower the ionization energy, the greater the matrix effects were registered. No measurable matrix effects were registered on both Q g and analyte emission within experimental uncertainties for NH 2 OH·HCl and acetic acid extractant solutions. Experimental data suggest that matrix effects were related to changes in plasma characteristics and the analyte excitation/ionization mechanism. To mitigate matrix effects and improve long-term MIP-OES performance, internal standardization using either Rh (343.489 nm and 369.236 nm) or OH molecular emission band (308.958 nm) was required. This calibration methodology was successfully applied to the study of the elemental bioavailability in soil samples from a vineyard affected by copper-based fungicides and sediment samples from an area affected by mining waste., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Autologous hematopoietic cell transplantation (auto-HCT) may be performed in multiple myeloma (MM) patients relapsing after a previous auto-HCT. For those without an adequate dose of stored stem cells, remobilization is necessary. This retrospective study included patients who, following disease relapse after the first auto-HCT(s), underwent stem cell remobilization and auto-HCT performed using these cells. There were 305 patients, 68% male, median age at salvage auto-HCT was 59 years. The median time to relapse after the first-line penultimate auto-HCT(s) was 30.6 months, the median follow-up after salvage auto-HCT 31 months. The 2- and 4-year non-relapse mortality (NRM) after the salvage auto-HCT was 5 and 9%, the relapse incidence 56 and 76%, respectively. Overall survival (OS) after 2 and 4 years was 76 and 52%, progression-free survival (PFS) 39 and 15%. In multivariable analysis an increasing interval between the penultimate auto-HCT and relapse was associated with better OS and PFS, later calendar year of salvage auto-HCT with better OS. In conclusion, salvage auto-HCT performed with cells remobilized after a previous auto-HCT was associated with acceptable NRM. The leading cause of failure was disease progression of MM, which correlated with a shorter interval from the penultimate auto-HCT to the first relapse., (© 2022. The Author(s).)

Background: The impact of infections with tick-borne pathogens (TBPs) other than Borrelia burgdorferi (s.l.) and tick-borne encephalitis virus (TBEV) on public health in Europe remains unclear. Our goal is to evaluate whether the presence of these TBPs in ticks can be associated with self-reported health complaints., Methods: We enrolled individuals who were bitten by I. ricinus between 2012 and 2015 and collected their relevant demographic and clinical information using a self-administered online questionnaire. A total of 4163 I. ricinus ticks sent by the participants were subject to molecular analyses for detection of specific TBPs. Associations between the presence of TBPs in ticks and self-reported complaints and symptoms were evaluated by means of a stepwise approach using a generalized linear model (GLM)., Results: Of 17 self-reported complaints and symptoms significant in the univariate analyses, 3 had a highly significant association (P < 0.01) with at least one TBP in the multivariate analysis. Self-reported Lyme borreliosis was significantly associated (P < 0.001) with B. burgdorferi (s.l.) infection. Facial paralysis was associated (P < 0.01) with infection with B. miyamotoi, N. mikurensis and R. helvetica. Finally, a significant association (P < 0.001) was found between nocturnal sweating and A. phagocytophilum., Conclusions: We found associations between the presence of TBPs in ticks feeding on humans and self-reported symptoms. Due to the subjective nature of such reports and the fact that infection was determined in the ticks and not in the patient samples, further prospective studies utilizing diagnostic modalities should be performed before any clinical outcome can be causally linked to infection with TBPs., (© 2022. The Author(s).)

Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative treatment for patients with myeloid/lymphoid neoplasm (MLN) with FGFR1 rearrangement, but data on overall results are limited. We report on the largest series of patients (n = 22) with FGFR1-rearranged MLN undergoing allo-HCT. Distribution according to cytogenetic subtype was: t(8;13) in 11 cases, t(8;22) in 7 cases, t(6;8) in 2 cases, and other (n = 2). Over a third of patients displayed a chronic myeloproliferative (MPN) phenotype, another third showed MPN features with concomitant lymphoma or acute leukemia, and the remaining ones presented as acute leukemia. After a median follow-up of 4.1 years from transplant, the estimated 5-year survival rate, progression-free survival, non-relapse mortality and relapse incidence was 74%, 63%, 14% and 23%, respectively. Causes of death were relapse/progression (n = 4), graft-versus-host disease (n = 2) and organ toxicity (n = 1). Six patients experienced disease relapse at a median of 6.1 months (range: 2.3-119.6). Two of them achieved complete remission with ponatinib or pemigatinib and were alive at 34.5 and 37 months from relapse, respectively. These data highlight the significant curative potential of allo-HCT in this aggressive disease. Maintenance with tyrosine kinase inhibitors may be a promising approach, at least in cases with detectable residual disease after transplant., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Minimizing antimicrobial use (AMU) in livestock is needed to control antimicrobial resistance (AMR). In the Netherlands, the livestock sector reduced AMU by almost 70 % since 2009, but this reduction stagnated in recent years. With only therapeutic AMU allowed, it is clear that besides socio-economic and behavioral factors, also the farm technical characteristics influence the conditions under which farmers need AMU. These characteristics pertain to farm management, including biosecurity, vaccination schemes, nutrition, micro-climate and husbandry practices. Identifying farm-related risk factors for AMU is needed to control AMR in a sustainable and pragmatic way. This need, often concerns the overall contribution of seemingly related (rather than individualized) factors. Here, risk factors for AMU in pig and calf farms were determined using two approaches: a typical risk factor analysis based on generalized estimating equations (GEEs) or hierarchical mixed-effects models and a multiblock partial least-squares regression analysis. These methods were applied to longitudinal data from two previous studies, i.e. a panel study and an intervention study involving 36 multiplier pig farms and 51 veal calf farms in the Netherlands, respectively. The multiblock analysis allowed us to quantify the importance of each factor and their respective block (i.e. farm management domain). For pigs, factors related to internal biosecurity had the highest impact on AMU, while for calves, these were mainly related to micro-climate. Structural characteristics, such as farm size and production type, followed in importance for both sectors. While both methods provided similar outcomes, the multiblock approach provided further insights by grouping and comparing factors believed to be inter-related., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The oncogenic potential of viral infections is well established and documented for many years already. However, the contribution of (commensal) bacteria and parasites to the development and progression of cancers has only recently gained momentum, resulting in a rapid growth of publications on the topic. Indeed, various bacteria and parasites have been suggested to play a role in the development of gastrointestinal cancer in particular. Therefore, an overview of the current epidemiological knowledge on the association between infections with bacteria and parasites and cancers of the gastrointestinal tract is needed. In this review, we summarized the methodological characteristics and main results of epidemiological studies investigating the association of 10 different bacteria ( Bacteroides fragilis , Campylobacter spp., Clostridium spp., Enterococcus faecalis , Escherichia coli , Fusobacterium nucleatum , Porphyromonas gingivalis , non-typhoidal Salmonella , Salmonella Typhi, and Streptococcus spp.) and three parasites ( Cryptosporidium spp., Schistosoma spp., and Strongyloides stercoralis ) with gastrointestinal cancer. While the large body of studies based on microbiome sequencing provides valuable insights into the relative abundance of different bacterial taxa in cancer patients as compared to individuals with pre-malignant conditions or healthy controls, more research is needed to fulfill Koch's postulates, possibly making use of follow-up data, to assess the complex role of bacterial and parasitic infections in cancer epidemiology. Studies incorporating follow-up time between detection of the bacterium or parasite and cancer diagnosis remain valuable as these allow for estimation of cause-effect relationships., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Duijster, Franz, Neefjes and Mughini-Gras.)

The public health measures implemented to control coronavirus disease 2019 (COVID-19) may influence also other infectious diseases. Using national laboratory surveillance data, we assessed the impact of the COVID-19 pandemic on human salmonellosis in the Netherlands until March 2021. Salmonellosis incidence decreased significantly after March 2020: in the second, third and fourth quarters of 2020, and in the first quarter of 2021, the incidence decreased by 55%, 57%, 47% and 37%, respectively, compared to the same quarters of 2016-2019. The decrease was strongest among travel-related cases (94%, 84%, 79% and 93% in the aforementioned quarters, respectively). Other significant changes were: increased proportion of cases among older adults and increased proportion of invasive infections, decreased proportion of trimethoprim resistance and increased proportion of serovar Typhimurium monophasic variant vs. Enteritidis. This led to decreased contributions of laying hens and increased contributions of pigs and cattle as sources of human infections. The observed changes probably reflect a combination of reduced exposure to Salmonella due to restrictions on international travels and gatherings, closure of dine-in restaurants, catering and hospitality sectors at large and changes in healthcare-seeking and diagnostic behaviours.

Allogeneic hematopoietic cell transplantation (allo-HCT) is increasingly used in older myelofibrosis (MF) patients, but its risk/benefit ratio compared to non-transplant approaches has not been evaluated in this population. We analyzed the outcomes of allo-HCT in 556 MF patients aged ≥65 years from the EBMT registry, and determined the excess mortality over the matched general population of MF patients ≥65 years managed with allo-HCT (n = 556) or conventional drug treatment (n = 176). The non-transplant cohort included patients with intermediate-2 or high risk DIPSS from the Spanish Myelofibrosis Registry. After a median follow-up of 3.4 years, the estimated 5-year survival rate, non-relapse mortality (NRM), and relapse incidence after transplantation was 40%, 37%, and 25%, respectively. Busulfan-based conditioning was associated with decreased mortality (HR: 0.7, 95% CI: 0.5-0.9) whereas the recipient CMV+/donor CMV- combination (HR: 1.7, 95% CI: 1.2-2.4) and the JAK2 mutated genotype (HR: 1.9, 95% CI: 1.1-3.5) predicted higher mortality. Busulfan-based conditioning correlated with improved survival due to less NRM, despite its higher relapse rate when compared with melphalan-based regimens. Excess mortality was higher in transplanted patients than in the non-HCT cohort in the first year of follow-up (ratio: 1.93, 95% CI: 1.13-2.80), whereas the opposite occurred between the fourth and eighth follow-up years (ratio: 0.31, 95% CI: 0.18-0.53). Comparing the excess mortality of the two treatments, male patients seemed to benefit more than females from allo-HCT, mainly due to their worse prognosis with non-transplant approaches. These findings could potentially enhance counseling and treatment decision-making in elderly transplant-eligible MF patients., (© 2021 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the disease with the highest economic loss for the broiler industry. However, studies focusing on the prevalence and population structure of APEC in the broiler production pyramid are scarce. Here, we used genotyping and serotyping data to elucidate the APEC population structure and its changes in different broiler production stages along with whole-genome sequencing (WGS) in a subset of APEC isolates to determine transmission patterns amongst dominant APEC sequence types (STs) and characterize them in detail. Comparison of genotypes encountered in both APEC and avian fecal E. coli (AFEC) provided further insights. Overall, APEC-related mortality, as the proportion of the total sampled mortality in the broiler production, was high (35%), while phylogroup C and serogroup O78 were predominant amongst APEC isolates. We found a low (34.0%) and high (53.3%) incidence of colibacillosis in chicks and end-cycle broilers, respectively, which may be related to a shift in APEC genotypes, suggesting a trend from commensalism to pathogenicity across different broiler production stages. Despite considerable APEC genotypic diversity, there was substantial genotype overlap (40.9%, overall) over the production stages and convergence of STs to the four clusters. Within these clusters, WGS data provided evidence of clonal transmission events and revealed an enriched virulence and resistance APEC repertoire. More specifically, sequenced APEC were assigned to defined pathotypes based on their virulence gene content while the majority (86%) was genotypically multi-drug resistant. Interestingly, WGS-based phylogeny showed that a subset of APEC, which are cephalosporin-resistant, may originate directly from cephalosporin-resistant AFEC. Finally, exploration of the APEC plasmidome indicated that the small fraction of the APEC virulome carried by IncF plasmids is pivotal for the manifestation of the APEC pathotype; thus, plasmid exchange can promote pathogenicity in strains that are at the edge of the commensal and pathogenic states., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Apostolakos, Laconi, Mughini-Gras, Yapicier and Piccirillo.)

Objectives: While colon cancer (CC) risk is associated with several lifestyle-related factors, including physical inactivity, smoking and diet, the contribution of occupation to CC morbidity remains largely unclear. Growing evidence indicates that gastrointestinal infections like salmonellosis could contribute to CC development. We performed a nationwide registry study to assess potential associations between occupation (history) and CC, including also those occupations with known increased exposure to gastrointestinal pathogens like Salmonella. METHODS: Person-level occupational data for all residents in The Netherlands were linked to CC diagnosis data. Differences in the incidence of (overall, proximal and distal) CC among occupational sectors and risk groups were tested for significance by calculating standardised incidence ratios (SIRs) with 95% CIs using the general population as reference group. Effects of gender, age, exposure duration and latency were also assessed., Results: Significant differences in CC incidence were observed only for a few occupational sectors, including the manufacturing of rubber and plastics, machinery and leather, the printing sector and the information service sector (SIRs 1.06-1.88). No elevated risk of CC was observed among people with increased salmonellosis risk through occupational exposure to live animals, manure or among those working in the sale of animal-derived food products (SIRs 0.93-0.95, 0.81-0.95 and 0.93-1.09 for overall, proximal and distal CC, respectively)., Conclusions: The results of this study suggest that occupation in itself provides a relatively small contribution to CC incidence. This is consistent with previous studies where a similar degree of variation in risk estimates was observed. The lack of an association with the high-risk occupations for salmonellosis might be due to higher levels of physical activity, a known protective factor for CC and other diseases, of people working in the agricultural sector, which might outweigh the potential Salmonella -associated risk of CC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Salmonella Infantis is a poultry-adapted Salmonella enterica serovar that is increasingly reported in broilers and is also regularly identified among human salmonellosis cases. An emerging S. Infantis mega-plasmid (pESI), carrying fitness, virulence and antimicrobial resistance genes, is also increasingly found. We investigated the prevalence, genetic characteristics and risk factors for (pESI-carrying) S. Infantis in broilers. Faecal samples from 379 broiler flocks (in 198 farms with ≥3000 birds) in the Netherlands were tested. A questionnaire about farm characteristics was also administered. Sampling was performed in July 2018-May 2019, three weeks before slaughter. Fourteen flocks (in 10 farms) were S. Infantis-positive, resulting in a 3.7 % flock-level and 5.1 % farm-level prevalence. Based on multi-locus sequence typing (MLST), all isolates belonged to sequence type 32. All but one isolate carried a pESI-like mega-plasmid. Core-genome MLST showed considerable heterogeneity among the isolates, even within the same farm, with a few small clusters detected. The typical pESI-borne multi-resistance pattern to aminoglycosides, sulphonamide and tetracycline (93 %), as well as trimethoprim (71 %), was found. Additionally, resistance to (fluoro)quinolones based on gyrA gene mutations was detected. S. Infantis was found more often in flocks using salinomycin as coccidiostat, where flock thinning was applied or litter quality was poor, whereas employing external cleaning companies, wheat in feed, and vaccination against infectious bronchitis, were protective. Suggestive evidence for vertical transmission from hatcheries was found. A heterogeneous (pESI-carrying) S. Infantis population has established itself in Dutch broiler flocks, calling for further monitoring of its spread and a comprehensive appraisal of control options., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

National burden of foodborne disease (FBD) studies are essential to establish food safety as a public health priority, rank diseases, and inform interventions. In recent years, various countries have taken steps to implement them. Despite progress, the current burden of disease landscape remains scattered, and researchers struggle to translate findings to input for policy. We describe the current knowledge base on burden of FBDs, highlight examples of well-established studies, and how results have been used for decision-making. We discuss challenges in estimating burden of FBD in low-resource settings, and the experience and opportunities deriving from a large-scale research project in these settings. Lastly, we highlight the role of international organizations and initiatives in supporting countries to develop capacity and conduct studies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Plastic pollution in aquatic environments, particularly microplastics (<5 mm), is an emerging health threat. The buoyancy, hydrophobic hard surfaces, novel polymer carbon sources and long-distance transport make microplastics a unique substrate for biofilms, potentially harbouring pathogens and enabling antimicrobial resistance (AMR) gene exchange. Microplastic concentrations, their polymer types and the associated microbial communities were determined in paired, contemporaneous samples from the Dutch portion of the river Rhine. Microplastics were collected through a cascade of 500/100/10 μm sieves; filtrates and surface water were also analysed. Microplastics were characterized with infrared spectroscopy. Microbial communities and selected virulence and AMR genes were determined with 16S rRNA-sequencing and qPCR. Average microplastic concentration was 213,147 particles/m 3 ; polyamide and polyvinylchloride were the most abundant polymers. Microbial composition on 100-500 μm samples differed significantly from surface water and 10-100 μm or smaller samples, with lower microbial diversity compared to surface water. An increasingly 'water-like' microbial community was observed as particles became smaller. Associations amongst specific microbial taxa, polymer types and particle sizes, as well as seasonal and methodological effects, were also observed. Known biofilm-forming and plastic-degrading taxa (e.g. Pseudomonas) and taxa harbouring potential pathogens (Pseudomonas, Acinetobacter, Arcobacter) were enriched in certain sample types, and other risk-conferring signatures like the sul1 and erm(B) AMR genes were almost ubiquitous. Results were generally compatible with the existence of taxon-selecting mechanisms and reduced microbial diversity in the biofilms of plastic substrates, varying over seasons, polymer types and particle sizes. This study provided updated field data and insights on microplastic pollution in a major riverine environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Antimicrobials are commonly used in conventional livestock production and manure is widely applied to agricultural lands as fertilizer. This practice raises questions regarding the effects of fertilization on (i) soil microbiota composition and (ii) spread of antimicrobials and antimicrobial resistance (AMR) in the environment. This study was conducted in a high-density farming area of Northern Italy and aimed at assessing the impact of (dairy cattle, chickens and swine) manure application on soil microbiome, antimicrobial concentrations and antimicrobial resistance gene (ARG) abundance. We found the microbial community composition in manure to be different and less diverse than in soil, with manure application altering only marginally the soil microbiome. Exceptions were the phyla Firmicutes, Tenericutes and Cloacimonetes, which significantly enriched in fertilized soil. Of the antimicrobials investigated, only flumequine concentrations increased after manure application, albeit non-significantly. ARGs were more abundant in manure, with ermA, ermB, bla OXA-1 and oqxA being significantly enriched in fertilized soil. Positive correlations between oqxA and qnrS abundances and flumequine concentrations were observed, together with the co-occurrence of some ARGs and microbial taxa (e.g. oqxA correlated with Acidobacteria and Gemmatimonadetes). This study showed that manure application has little effect on soil microbiome but may contribute to the dissemination of specific ARGs into the environment. Moreover, flumequine residues seem to enhance the emergence of oqxA and qnrS in soil., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Laboratory data increasingly suggest that Salmonella infection may contribute to colon cancer (CC) development. Here, we examined epidemiologically the potential risk of CC associated with salmonellosis in the human population. We conducted a population-based cohort study using four health registries in Denmark. Person-level demographic data of all residents were linked to laboratory-confirmed non-typhoidal salmonellosis and to CC diagnoses in 1994-2016. Hazard ratios (HRs) for CC (overall/proximal/distal) associated with reported salmonellosis were estimated using Cox proportional hazard models. Potential effects of serovar, age, sex, inflammatory bowel disease and follow-up time post-infection were also assessed. We found no increased risk of CC ≥1 year post-infection (HR 0.99; 95% confidence interval (CI) 0.88-1.13). When stratifying by serovar, there was a significantly increased risk of proximal CC ≥1 year post-infection with serovars other than Enteritidis and Typhimurium (HR 1.40; 95% CI 1.03-1.90). CC risk was significantly increased in the first year post-infection (HR 2.08; 95% CI 1.48-2.93). The association between salmonellosis and CC in the first year post-infection can be explained by increased stool testing around the time of CC diagnosis. The association between proximal CC and non-Enteritidis/non-Typhimurium serovars is unclear and warrants further investigation. Overall, this study provides epidemiological evidence that notified Salmonella infections do not contribute significantly to CC risk in the studied population.

Objectives: To determine the contributions of several animal and environmental sources of human campylobacteriosis and identify source-specific risk factors., Methods: 1417 Campylobacter jejuni/coli isolates from the Netherlands in 2017-2019 were whole-genome sequenced, including isolates from human cases (n = 280), chickens/turkeys (n = 238), laying hens (n = 56), cattle (n = 158), veal calves (n = 49), sheep/goats (n = 111), pigs (n = 110), dogs/cats (n = 100), wild birds (n = 62), and surface water (n = 253). Questionnaire-based exposure data was collected. Source attribution was performed using core-genome multilocus sequence typing. Risk factors were determined on the attribution estimates., Results: Cases were mostly attributed to chickens/turkeys (48.2%), dogs/cats (18.0%), cattle (12.1%), and surface water (8.5%). Of the associations identified, never consuming chicken, as well as frequent chicken consumption, and rarely washing hands after touching raw meat, were risk factors for chicken/turkey-attributable infections. Consuming unpasteurized milk or barbecued beef increased the risk for cattle-attributable infections. Risk factors for infections attributable to environmental sources were open water swimming, contact with dog faeces, and consuming non-chicken/turkey avian meat like game birds., Conclusions: Poultry and cattle are the main livestock sources of campylobacteriosis, while pets and surface water are important non-livestock sources. Foodborne transmission is only partially consistent with the attributions, as frequency and alternative pathways of exposure are significant., (Copyright © 2020. Published by Elsevier Ltd.)

Objectives: To determine spatial clustering and risk factors for occurrence and intensity of infection for soil-transmitted helminthiasis (STH), namely Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis and hookworms in a Venezuelan rural community., Methods: MIF-fixed faecal samples were individually collected for STH testing. The Getis-Ord statistic was used to determine significant STH clustering within 25/50/100 m radiuses around houses. Individual- and house-level factors associated with STH occurrence and intensity of infection were determined using generalized estimating equations., Results: Significant clusters of "wormy" houses for one or multiple parasites were found at distances of 25-50 m around 13 houses. Risk factors differed between occurrence and intensity of infection. Overcrowding in the house increased occurrence of S. stercoralis, T. trichiura and hookworm infections, while poor housing conditions increased A. lumbricoides infection risk. Overcrowding, poor faecal disposal system, economic dependency and lack of basic services differentially influenced the STHs. The "wormy" houses were mainly those built with waste materials, under economic dependency and lacking indoor water supply., Conclusions: STH distribution in a community is clustered, with significant hotspots of STH occurrence and intensity of infection and different associated risk factors. Targeting the "wormy" houses is expected to affect STH morbidity more efficiently., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020. Published by Elsevier Ltd.)

Campylobacter jejuni and C. coli, the primary agents of human bacterial gastroenteritis worldwide, are widespread in surface water. Several animal sources contribute to surface water contamination with Campylobacter, but their relative contributions thus far remained unclear. Here, the prevalence, genotype diversity, and potential animal sources of C. jejuni and C. coli strains in surface water in the Netherlands were investigated. It was also assessed whether the contribution of the different animal sources varied according to surface water type (i.e. agricultural water, surface water at discharge points of wastewater treatment plants [WWTPs], and official recreational water), season, and local livestock (poultry, pig, ruminant) density. For each surface water type, 30 locations spread over six areas with either high or low density of poultry, ruminants, or pigs, were sampled once every season in 2018-2019. Campylobacter prevalence was highest in agricultural waters (77%), and in autumn and winter (74%), and lowest in recreational waters (46%) and in summer (54%). In total, 76 C. jejuni and 177 C. coli water isolates were whole-genome sequenced. Most C. coli water isolates (78.5%) belonged to hitherto unidentified clones when using the seven-locus sequence type (ST) scheme, while only 11.8% of the C. jejuni isolates had unidentified STs. The origin of these isolates, as defined by core-genome multi-locus sequence typing (cgMLST), was inferred by comparison with Campylobacter strain collections from meat-producing poultry, laying hens, adult cattle, veal calves, small ruminants, pigs, and wild birds. Water isolates were mainly attributed to wild birds (C. jejuni: 60.0%; C. coli: 93.7%) and meat-producing poultry (C. jejuni: 18.9%; C. coli: 5.6%). Wild bird contribution was high among isolates from recreational waters and WWTP discharge points, and in areas with low poultry (C. coli) or high ruminant (C. jejuni) densities. The contribution of meat-producing poultry was high in areas with high density of poultry, springtime, agricultural waters and WWTP discharge points. While wild birds and poultry were the main contributors to Campylobacter contamination in surface water, their contribution differed significantly by water type, season, and local poultry and ruminant densities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Background: In dogs, infections with Giardia duodenalis are mainly caused by assemblages C and D, but also by the potentially zoonotic assemblages A and B. The aims of this study were to assess differences in assemblages (i) between dogs living mainly in close proximity to humans (synanthropic dogs) versus dogs living mainly among other dogs, (ii) between samples of dogs with or without loose stool, and (iii) related to the amount of cysts shedding., Methods: One hundred eighty-nine qPCR Giardia positive fecal samples of dogs originating from four groups (household, sheltered, hunting, and dogs for which a veterinarian sent a fecal sample to a diagnostic laboratory) were used for genotyping. For this, multi-locus genotyping of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase and genotyping of SSU rDNA gene fragments were performed. Fecal consistency was scored (loose or non-loose stool), and cysts per gram of feces were determined with qPCR., Results: Assemblage D was the most prevalent in all groups, followed by the other canid assemblage C. Also, mixed C/D was common. In two (synanthropic) household dogs, the potentially zoonotic assemblage AI was present. Although occurrence of assemblage AI in household dogs was not significantly different from dogs living among other dogs (sheltered and hunting dogs), it was significantly higher compared to dogs for which a sample was sent to a diagnostic laboratory. Dogs with assemblage D shed significantly more cysts than dogs with other assemblages (except for mixed C/D results) or dogs in which no assemblage could be determined. None of the assemblages was significantly associated with loose stool., Conclusion: Not only do dogs mainly shed the canid Giardia duodenalis assemblages D and/or C, the numbers of cysts per gram for the canid assemblage D were also higher than for the potential zoonotic assemblage AI. Based on the assemblages shed by dogs, the risk to public health posed by dogs is estimated to be low, even though the dogs that shed AI were synanthropic household dogs. Loose stool in infected dogs was not associated with any particular Giardia assemblage.

The role of environmental transmission of typically foodborne pathogens like Shiga toxin-producing Escherichia coli (STEC) O157 is increasingly recognized. To gain more insights into spatially restricted risk factors that play a role in this transmission, we assessed the spatial association between sporadic STEC O157 human infections and the exposure to livestock (i.e. small ruminants, cattle, poultry, and pigs) in a densely populated country: the Netherlands. This was done for the years 2007-2016, using a state-of-the-art spatial analysis method in which hexagonal areas with different sizes (90, 50, 25 and 10 km 2 ) were used in combination with a novel probability of exposure metric: the population-weighted number of animals per hexagon. To identify risk factors for STEC O157 infections and their population attributable fraction (PAF), a spatial regression model was fitted using integrated nested Laplace approximation (INLA). Living in hexagonal areas of 25, 50 and 90 km 2 with twice as much population-weighted small ruminants was associated with an increase of the incidence rate of human STEC O157 infections in summer (RR of 1.09 [95%CI;1.01-1.17], RR of 1.17 [95%CI;1.07-1.28] and RR of 1.13 [95%CI;1.01-1.26]), with a PAF of 49% (95%CI;8-72%). Results suggest exposure to small ruminants to be a risk factor, although no evidence on the mode of transmission is provided. Therefore, the underlying mechanisms warrant further investigation and could offer new targets for control. The newly proposed exposure metric has potential to improve existing spatial modeling studies on infectious diseases related to livestock exposure, especially in densely populated countries like the Netherlands., Competing Interests: The authors declare no conflicts of interest relevant to this study., (©2020 The Authors.)

Salmonella spp. infection has shown to have oncogenic transformative effects and thereby increases the risk of certain cancers. For Campylobacter spp., similar effects have been demonstrated. Risk factor identification may allow for timely diagnosis and preventive treatment. To substantiate the oncogenic potential of Salmonella and Campylobacter spp., this study compared the incidence of extrahepatic biliary tract cancer (BTC) in patients with diagnosed Salmonella or Campylobacter spp. infection with BTC incidence in the Netherlands. National infectious diseases surveillance records of patients diagnosed with a laboratory-confirmed Salmonella or Campylobacter spp. infection during 1999-2016 were linked to the Netherlands Cancer Registry. Incidence of BTC in Salmonella and Campylobacter spp. patients was compared to the incidence of BTC in the general population using Standardized Incidence Ratios (SIRs). In total, 16,252 patients were diagnosed with Salmonella spp. and 27,668 with Campylobacter spp. infection. Nine patients developed BTC at a median of 46 months (13-67) after Salmonella spp. infection and seven at a median of 60 months (18-138) after Campylobacter spp. infection. SIR of BTC in salmonellosis patients was 1.53 (95% CI 0.70-2.91). In patients aged <60 years, the SIR was 1.74 (95% CI 0.36-5.04). For campylobacteriosis patients, the SIR was 0.97 (95% CI 0.39-2.00). Even though Salmonella or Campylobacter spp. infection was not significantly associated with increased BTC risk in this cohort, it remains extremely important to study potential risk factors for cancer to facilitate screening and ultimately improve prognosis of cancer patients.

Observed multiple adverse effects of livestock production have led to increasing calls for more sustainable livestock production. Quantitative analysis of adverse effects, which can guide public debate and policy development in this area, is limited and generally scattered across environmental, human health, and other science domains. The aim of this study was to bring together and, where possible, quantify and aggregate the effects of national-scale livestock production on 17 impact categories, ranging from impacts of particulate matter, emerging infectious diseases and odor annoyance to airborne nitrogen deposition on terrestrial nature areas and greenhouse gas emissions. Effects were estimated and scaled to total Dutch livestock production, with system boundaries including feed production, manure management and transport, but excluding slaughtering, retail and consumption. Effects were expressed using eight indicators that directly express Impact in the sense of the Drivers-Pressures-State-Impact-Response framework, while the remaining 14 express Pressures or States. Results show that livestock production may contribute both positively and negatively to human health with a human disease burden (expressed in disability-adjusted life years) of up to 4% for three different health effects: those related to particulate matter, zoonoses, and occupational accidents. The contribution to environmental impact ranges from 2% for consumptive water use in the Netherlands to 95% for phosphorus transfer to soils, and extends beyond Dutch borders. While some aggregation across impact categories was possible, notably for burden of disease estimates, further aggregation of disparate indicators would require normative value judgement. Despite difficulty of aggregation, the assessment shows that impacts receive a different contribution of different animal sectors. While some of our results are country-specific, the overall approach is generic and can be adapted and tuned according to specific contexts and information needs in other regions, to allow informed decision making across a broad range of impact categories., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Listeriosis is a foodborne illness characterized by a relatively low morbidity, but a large disease burden due to the severity of clinical manifestations and the high case fatality rate. Increased listeriosis notifications have been observed in Europe since the 2000s. However, the reasons for this increase are largely unknown, with the sources of sporadic human listerioris often remaining elusive. Here we inferred the relative contributions of several putative sources of Listeria monocytogenes strains from listerioris patients in Northern Italy (Piedmont and Lombardy regions), using two established source attribution models (i.e. 'Dutch' and 'STRUCTURE') in comparative fashion. We compared the Multi-Locus Sequence Typing and Multi-Virulence-Locus Sequence Typing profiles of strains collected from beef, dairy, fish, game, mixed foods, mixed meat, pork, and poultry. Overall, 634 L. monocytogenes isolates were collected from 2005 to 2016. In total, 40 clonal complexes and 51 virulence types were identified, with 36% of the isolates belonging to possible epidemic clones (i.e. genetically related strains from unrelated outbreaks). Source attribution analysis showed that 50% of human listerioris cases (95% Confidence Interval 44-55%) could be attributed to dairy products, followed by poultry and pork (15% each), and mixed foods (15%). Since the contamination of dairy, poultry and pork products are closely linked to primary production, expanding actions currently limited to ready-to-eat products to the reservoir level may help reducing the risk of cross-contamination at the consumer level., Competing Interests: Declaration of competing interest Declarations of conflict of interest: none., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

CSF biomarkers, including total-tau, neurofilament light chain (NfL) and amyloid-β, are increasingly being used to define and stage Alzheimer's disease. These biomarkers can be measured more quickly and less invasively in plasma and may provide important information for early diagnosis of Alzheimer's disease. We used stored plasma samples and clinical data obtained from 4444 non-demented participants in the Rotterdam study at baseline (between 2002 and 2005) and during follow-up until January 2016. Plasma concentrations of total-tau, NfL, amyloid-β40 and amyloid-β42 were measured using the Simoa NF-light® and N3PA assays. Associations between biomarker plasma levels and incident all-cause and Alzheimer's disease dementia during follow-up were assessed using Cox proportional-hazard regression models adjusted for age, sex, education, cardiovascular risk factors and APOE ε4 status. Moreover, biomarker plasma levels and rates of change over time of participants who developed Alzheimer's disease dementia during follow-up were compared with age and sex-matched dementia-free control subjects. During up to 14 years follow-up, 549 participants developed dementia, including 374 cases with Alzheimer's disease dementia. A log2 higher baseline amyloid-β42 plasma level was associated with a lower risk of developing all-cause or Alzheimer's disease dementia, adjusted hazard ratio (HR) 0.61 [95% confidence interval (CI), 0.47-0.78; P < 0.0001] and 0.59 (95% CI, 0.43-0.79; P = 0.0006), respectively. Conversely, a log2 higher baseline plasma NfL level was associated with a higher risk of all-cause dementia [adjusted HR 1.59 (95% CI, 1.38-1.83); P < 0.0001] or Alzheimer's disease [adjusted HR 1.50 (95% CI, 1.26-1.78); P < 0.0001]. Combining the lowest quartile group of amyloid-β42 with the highest of NfL resulted in a stronger association with all-cause dementia [adjusted HR 9.5 (95% CI, 2.3-40.4); P < 0.002] and with Alzheimer's disease [adjusted HR 15.7 (95% CI, 2.1-117.4); P < 0.0001], compared to the highest quartile group of amyloid-β42 and lowest of NfL. Total-tau and amyloid-β40 levels were not associated with all-cause or Alzheimer's disease dementia risk. Trajectory analyses of biomarkers revealed that mean NfL plasma levels increased 3.4 times faster in participants who developed Alzheimer's disease compared to those who remained dementia-free (P < 0.0001), plasma values for cases diverged from controls 9.6 years before Alzheimer's disease diagnosis. Amyloid-β42 levels began to decrease in Alzheimer's disease cases a few years before diagnosis, although the decline did not reach significance compared to dementia-free participants. In conclusion, our study shows that low amyloid-β42 and high NfL plasma levels are each independently and in combination strongly associated with risk of all-cause and Alzheimer's disease dementia. These data indicate that plasma NfL and amyloid-β42 levels can be used to assess the risk of developing dementia in a non-demented population. Plasma NfL levels, although not specific, may also be useful in monitoring progression of Alzheimer's disease dementia., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

In Venezuela, areas endemic for schistosomiasis are of low transmission, with low parasite loads. Immunological tests often lack specificity and cannot differentiate past from present infections. Molecular tests are an alternative, although validation studies in endemic areas are needed. The aim of this study was to determine the performance of parasitological, immunological and molecular tests for the diagnosis of Schistosoma mansoni infection in low-transmission settings. A cross-sectional study was carried out in a rural community located in a schistosomiasis-endemic area of Venezuela to determine the prevalence and diagnostic performance of the Kato-Katz (KK) technique, Circumoval Precipitin Test (COPT), ELISA based on soluble egg antigen (ELISA-SEA) with and without treatment with sodium metaperiodate (ELISA-SEA-SMP), and PCR for amplification of the 121 bp highly repeated sequence of Schistosoma mansoni in faeces, urine and serum samples. The highest prevalence rates were obtained with ELISA-SEA (38.7%), COPT (33.3%), ELISA-SEA-SMP (31.5%), PCR on faeces (21.6%), and KK (17.1%), whereas PCR-based prevalence in urine was 6.2% and no positivity was detected in serum samples. Results showed that ELISA-SEA is the best method for the diagnosis of both current and former infections and that PCR on faeces is the best method for detecting recent transmission. The use of different tests that complement one another also allowed for a better diagnosis of Schistosoma mansoni infection, revealing a relatively high prevalence (33.8%) of schistosomiasis in a community of low transmission., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Antimicrobial treatment protocols for foals with sepsis that do not improve clinically often are adjusted based on bacteriological and antimicrobial susceptibility testing results from samples collected at hospital admission., Objectives: To evaluate whether hospitalization for ≥48 hours affects bacteriological and antimicrobial susceptibility testing results., Animals: Two-hundred sixty-seven foals <30 days of age admitted to a neonatal intensive care unit and diagnosed with sepsis., Methods: Medical records were reviewed retrospectively to identify foals with sepsis and positive bacteriological cultures. Results from samples collected at hospital admission were compared to those collected ≥48 hours after admission. Logistic regression for clustered data and exact logistic regression were used for statistical analysis., Results: Three-hundred fifty-three unique bacterial isolates were obtained from 231 foals at hospital admission and 92 unique bacterial isolates were obtained from 57 foals after ≥48 hours of hospitalization. Relative isolation frequency after ≥48 hours of hospitalization increased for Acinetobacter spp., 0.6% versus 3.3% (odds ratio [OR], 7.63; 95% confidence interval [CI], 1.28-45.45); Enterococcus spp., 4.8% versus 19.6% (OR, 5.37; 95% CI, 2.64-10.90); Klebsiella spp., 5.1% versus 10.9% (OR, 2.27; 95% CI, 1.05-4.89); Pseudomonas spp., 3.0% versus 7.6% (OR, 3.49; 95% CI, 3.49-240.50); and Serratia spp., 3.0% versus 5.4% (OR, 20.23; 95% CI, 2.20-186.14). Bacteria isolated after ≥48 hours of hospitalization were less susceptible to all tested antimicrobial drugs, except for imipenem., Conclusions and Clinical Importance: Decreased antimicrobial susceptibility of bacteria isolated after ≥48 hours of hospitalization provides a rationale for repeated bacteriological culture and susceptibility testing in hospitalized foals with sepsis., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Staphylococcus pseudintermedius is a common pathogen in dogs and methicillin resistance has emerged over recent decades. According to the current guidelines, S. pseudintermedius displaying oxacillin resistance should be reported as resistant to all β-lactams., Objectives: To identify possible associations between β-lactam resistance levels and clonal complexes (CCs) and/or staphylococcal cassette chromosome mec (SCCmec) types in methicillin-resistant S. pseudintermedius (MRSP)., Methods: MICs of oxacillin, penicillin, ampicillin, amoxicillin/clavulanic acid and cefalotin were determined by broth microdilution for 86 clinical canine MRSP isolates from Denmark and the Netherlands. PCR and sequencing were used for SCCmec typing and MLST., Results: Isolates belonged to CC71 (n = 36), CC258 (n = 33), CC45 (n = 11), CC68 (n = 1) and five singleton STs. SCCmecII-III was exclusively found in CC71 and SCCmecIV was significantly associated with CC258. SCCmecV and non-typeable SCCmec types occurred in 4 and 14 isolates, respectively. SCCmecIV was associated with lower MICs of oxacillin (<2 mg/L), ampicillin (<8 mg/L) and amoxicillin/clavulanic acid (<4 mg/L) and with susceptibility to cefalotin (<4 mg/L). All isolates harbouring SCCmecV were susceptible to cefalotin as well., Conclusions: SCCmec types were associated with different CCs and with either high- or low-level resistance to different β-lactams. The finding of amoxicillin/clavulanic acid (20%) and cefalotin (70%) in vitro susceptibility across all CCs might have clinical implications, since amoxicillin/clavulanic acid and first-generation cephalosporins are first-choice antibiotics for treatment of S. pseudintermedius infections. Pharmacokinetic/pharmacodynamic and clinical outcome studies are warranted to evaluate the in vivo efficacy of these β-lactams for treatment of MRSP infections., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Suboptimal animal welfare may affect natural immunity, rendering animals more susceptible to environmentally conditioned diseases, including those requiring antimicrobial treatment, which may promote antimicrobial resistance (AMR) in bacterial populations. Herewith, we tested the hypothesis that conventionally raised turkeys have higher levels of AMR in indicator Escherichia coli bacteria, but lower levels of natural immunity, as compared to turkeys reared under organic conditions. Litter and serum samples were collected from 28 conventional and 4 organic turkey farms: E. coli isolates from litter were tested for resistance to 14 antimicrobials, while 3 parameters of natural immunity (i.e., lysozyme, hemolytic complement levels, and serum bactericidal activity) were assessed in the sera. Resistant E. coli isolates were identified in both conventional and organic farms but generally more frequently in conventional farms. High rates of resistance to ampicillin (96%), tetracycline (95%), streptomycin (82%), sulfamethoxazole (80%), ciprofloxacin (73%), and trimethoprim (71%), as well as high rates of multiresistance, were observed in conventional farms. Organically raised turkeys had significantly higher levels of lysozyme and serum bactericidal activity than conventional turkeys, and these levels were also higher in turkeys housed in farms where AMR frequency was lower. Findings support the hypothesis that conventional farming conditions may affect turkeys' natural immunity, rendering the animals more susceptible to environmentally conditioned diseases requiring antimicrobial treatment, which would in turn promote AMR. Reducing AMR in turkey farming is therefore more likely to be successful when considering animal welfare as an option to reduce the need of antimicrobial use., (Copyright © 2019. Published by Elsevier Inc.)

Presence of extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase (pAmpC)-producing Escherichia coli (ESBL/pAmpC-EC) in humans and animals is alarming due to the associated risks of antibiotic therapy failure. ESBL/pAmpC-EC transmission between the human and animal compartments remains controversial. Using cefotaxime-supplemented (selective) media, we recently showed high sample prevalence of ESBL/pAmpC-EC in an integrated broiler chain [i.e. Parent Stock (PS), offspring broilers and their carcasses]. Here, we used a different approach. In parallel with the selective isolation, samples were processed on non-selective media. E. coli isolates were tested for ESBL/pAmpC-production and those found positive were genotyped. For carcasses, total E. coli were enumerated. This approach enabled us to estimate prevalence at the isolate level, which mirrors ESBL/pAmpC-EC colonisation levels. We showed that although present in many animals, ESBL/pAmpC-EC were overall subdominant to intestinal E. coli, indicating that high sample prevalence is not associated with high levels of resistance in individual hosts. This is a relevant aspect for risk assessments, especially regarding the immediate exposure of farm personnel. An exception was a particularly dominant B2/bla CMY-2 lineage in the gut of imported PS chicks. This predominance obscured presence of latent genotypes, however bias towards particular ESBL/pAmpC-EC genotypes from the selective method or underestimation by the non-selective approach did not occur. At the slaughterhouse, we showed a link between total E. coli and ESBL/pAmpC-EC on carcasses. Mitigation strategies for reducing consumers' exposure should aim at suppressing ESBL/pAmpC-EC in the broiler gut as well as controlling critical points in the processing line., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Embryonated Toxocara eggs in the environment are considered to be the most important source of human toxocariasis. These eggs, however, are also a source of infection for the definitive and paratenic hosts. Most available literature focuses on Toxocara canis in dogs, or other canids. There are, however, other Toxocara species with their accompanying definitive hosts. Results from studies that modelled patent infections in dogs, cats and foxes show that although dogs are a very important source for environmental contamination with Toxocara eggs, other sources cannot be ignored. For a good understanding of the relative contributions of different definitive hosts to environmental contamination with Toxocara eggs, computational models should be built and fed with data from different fields of expertise as is discussed in this paper., (© 2020 Elsevier Ltd All rights reserved.)

Elucidating the genetic basis of phenotypic variation in response to different environments is key to understanding how populations evolve. Facultatively sexual rotifers can develop adaptive responses to fluctuating environments. In a previous evolution experiment, diapause-related traits changed rapidly in response to two selective regimes (predictable vs unpredictable) in laboratory populations of the rotifer Brachionus plicatilis. Here, we investigate the genomic basis of adaptation to environmental unpredictability in these experimental populations. We identified and genotyped genome-wide polymorphisms in 169 clones from both selective regimes after seven cycles of selection using genotyping by sequencing (GBS). Additionally, we used GBS data from the 270 field clones from which the laboratory populations were established. This GBS dataset was used to identify candidate SNPs under selection. A total of 76 SNPs showed divergent selection, three of which are candidates for being under selection in the particular unpredictable fluctuation pattern studied. Most of the remaining SNPs showed strong signals of adaptation to laboratory conditions. Furthermore, a genotype-phenotype association approach revealed five SNPs associated with two key life-history traits in the adaptation to unpredictability. Our results contribute to elucidating the genomic basis for adaptation to unpredictable environments and lay the groundwork for future evolution studies in rotifers.

Purpose: Complementary and alternative medicine (CAM) use increases in cancer patients, including adult patients with diffuse gliomas., Methods: Questionnaires addressing CAM use were distributed to adult patients with gliomas of WHO grades II-IV and ECOG performance score of 0-2 during hospital visits and filled in anonymously. The study was conducted in nine centers in France from May 2017 to May 2018. Descriptive cohort analyses and comparative analyses according to gender, age, WHO grade, and recurrent versus newly diagnosed disease were conducted., Results: Two hundred twenty-seven questionnaires were collected; 135 patients (59%) were male. Median age was 48 years, 105 patients (46%) declared having glioblastoma, 99 patients (43%) declared having recurrent disease. Hundred-three patients (45%) had modified their alimentary habits after the glioma diagnosis. At the time of the questionnaire, 100 patients (44%) were on complementary treatment, mainly vitamins and food supplements, and 73 patients (32%) used alternative medicine approaches, mainly magnetism and acupuncture. In total, 154 patients (68%) declared using at least one of these approaches. Expenditures exceeding 100 € per month were reported by users in 14% for modification of alimentary habits, in 25% for complementary treatment, and in 18% for alternative medicines. All approaches were commonly considered as improving quality of life and experienced as efficient, notably those associated with more expenditures., Conclusions: CAM are frequently used by glioma patients in France. Underlying needs and expectations, as well as potential interactions with tumor-specific treatments, and financial and quality of life burden, should be discussed with patients and caregivers.

Background: The aim of this study was to assess potential associations between Giardia duodenalis infection in dogs, as determined by three diagnostic tests, and dog's group of origin, fecal consistency, age, sex, neuter status, and co-infections with other gastrointestinal parasites., Methods: Fecal samples from 1291 dogs from four groups (household, shelter, hunting and clinical dogs) were tested with qPCR, rapid enzyme immunochromatographic assay (IDEXX SNAP ® Giardia), and direct immunofluorescence (DFA, Merifluor) for presence of G. duodenalis. Moreover, fecal samples were tested with centrifugation sedimentation flotation (CSF) coproscopical analysis for presence of gastrointestinal parasites. Associations were expressed as odds ratios (ORs)., Results: Several significant associations were found, of which a few were consistent for all three tests and Giardia positivity in general (positive with at least one of these tests). Dogs older than one year were significantly less likely to test positive for Giardia than younger dogs. Group-housed dogs, especially hunting dogs, were significantly more likely to test positive for Giardia compared to household and clinical dogs. A consistently significant association with Trichuris appeared to be driven by the high prevalence in hunting dogs. Although there was no significant association between loose stool and Giardia infection in the overall population, household dogs were significantly more likely to test Giardia-positive when having loose stool. Overall, Giardia-positive dogs with loose stool shed significantly more cysts, both determined semi-quantitatively with CSF and quantitatively by qPCR, than positive dogs with no loose stool. When other gastrointestinal parasites were present, significantly fewer cysts were detected with CSF, but this was not confirmed with qPCR., Conclusion: Giardia is the most common gastrointestinal parasite in Dutch dogs, except for hunting dogs, in which Trichuris and strongyle-type eggs (hookworms) prevailed. Giardia infection was not significantly associated with loose stool, except for household dogs. Young dogs and group-housed dogs were significantly more often Giardia-positive. These associations were consistent across diagnostic tests. Young dogs, clinical dogs and dogs with loose stool shed Giardia cysts in the highest numbers. If another gastrointestinal parasite was present lower numbers of cysts were observed by microscope (CSF), but not with a molecular method (qPCR).

With increased interest in source attribution of foodborne pathogens, there is a need to sort and assess the applicability of currently available methods. Herewith we reviewed the most frequently applied methods for source attribution of foodborne diseases, discussing their main strengths and weaknesses to be considered when choosing the most appropriate methods based on the type, quality, and quantity of data available, the research questions to be addressed, and the (epidemiological and microbiological) characteristics of the pathogens in question. A variety of source attribution approaches have been applied in recent years. These methods can be defined as top-down, bottom-up, or combined. Top-down approaches assign the human cases back to their sources of infection based on epidemiological (e.g., outbreak data analysis, case-control/cohort studies, etc.), microbiological (i.e., microbial subtyping), or combined (e.g., the so-called 'source-assigned case-control study' design) methods. Methods based on microbial subtyping are further differentiable according to the modeling framework adopted as frequency-matching (e.g., the Dutch and Danish models) or population genetics (e.g., Asymmetric Island Models and STRUCTURE) models, relying on the modeling of either phenotyping or genotyping data of pathogen strains from human cases and putative sources. Conversely, bottom-up approaches like comparative exposure assessment start from the level of contamination (prevalence and concentration) of a given pathogen in each source, and then go upwards in the transmission chain incorporating factors related to human exposure to these sources and dose-response relationships. Other approaches are intervention studies, including 'natural experiments,' and expert elicitations. A number of methodological challenges concerning all these approaches are discussed. In absence of an universally agreed upon 'gold' standard, i.e., a single method that satisfies all situations and needs for all pathogens, combining different approaches or applying them in a comparative fashion seems to be a promising way forward., (Copyright © 2019 Mughini-Gras, Kooh, Fravalo, Augustin, Guillier, David, Thébault, Carlin, Leclercq, Jourdan-Da-Silva, Pavio, Villena, Sanaa and Watier.)

The IncK plasmid group can be divided into two separate lineages named IncK1 and IncK2. IncK2 is found predominantly in poultry while IncK1 was reported in various mammals, including animals and humans. The physiological basis of this distinction is not known. In this manuscript we examined fitness cost of IncK1 and IncK2 plasmids at 37 and 42°C, which resembles mammalian and chicken body temperatures, respectively. We analyzed conjugation frequency, plasmid copy number and plasmid fitness cost in direct competition. Additionally, we measured levels of σ-32 in Escherichia coli carrying either wild type or conjugation-deficient IncK plasmids. The results show that IncK2 plasmids have a higher conjugation frequency and lower copy number at 42°C compared to IncK1. While the overall fitness cost to the host bacterium of IncK2 plasmids was higher than that of IncK1, it was not affected by the temperature while the fitness cost of IncK1 was shown to increase at 42°C compared to 37°C. These differences correlate with an increased expression of σ-32, a regulator of heat-shock protein expression, in E. coli with IncK2 compared to cells containing IncK1. This effect was not seen in cells containing conjugation deficient plasmids. Therefore, it is hypothesized that the assembly of the functional T4S may lead to these increased levels of σ-32. Increased activation of CpxR at 42°C may explain why IncK2 plasmids, and not IncK1, are predominantly found in chicken isolates., (Copyright © 2019 Rozwandowicz, Brouwer, Mughini-Gras, Wagenaar, Gonzalez-Zorn, Mevius and Hordijk.)

Antimicrobial use (AMU) in humans and animals facilitates the emergence of antimicrobial resistance (AMR). With increasing AMR being recognised as a major global threat for public health, responsible AMU is strongly advocated in both human and veterinary medicine. Knowledge on factors influencing antimicrobial prescribing behaviour of companion animal veterinarians is needed to promote responsible AMU in companion animals and to improve compliance with current legislation and guidelines. The present study aimed to quantitively investigate attitudes and perceptions of companion animal veterinarians towards AMU and AMR and to identify associations with demographic characteristics as possible explanatory variables. A self-administered questionnaire was developed based upon an earlier qualitative interview study, and 1608 potential participants (i.e. practising companion animal veterinarians) were invited. The questionnaire included questions addressing general descriptives of the respondents and questions with 6-point Likert scale statements, to assess attitudes towards AMU, AMR, factors influencing antimicrobial prescribing, and possible options to support responsible AMU. The response rate was 32% (22% when complete questionnaires considered). Categorical Principal Component Analysis (CATPCA) was conducted on 76 Likert scale questions. This resulted in a final model with 37 questions explaining 38.7% of the variance of the question scores, with three underlying dimensions ("attitudinal profiles"). Additionally, general descriptives were added to the CATPCA as possible explanatory variables. The first dimension, related to "social responsibility" was positively associated with veterinarians working in clinics dedicated to companion animals, with veterinarians working in a referral clinic, and with more experienced veterinarians. The second dimension was related to "scepticism", which was positively associated with being a male veterinarian and with more experienced veterinarians. The third dimension was related to "risk avoidance", especially regarding surgical procedures, and was negatively associated with veterinarians working in clinics in urban areas and with veterinarians working part-time. Antimicrobial prescribing behaviour was self-reported to be well considered, and respondents did not see economic drivers as important influencing factors. The unwillingness of owners and financial constraints were perceived as important barriers for performing further diagnostics. To improve AMU, a multifaceted approach, taking differences between companion animal veterinarians (e.g., in experience and gender) and differences in work situation (e.g., full-time versus part-time) into account, should be directed at companion animal veterinarians and owners. Moreover, a joint and comprehensive effort of several stakeholders, like veterinary nurses, guideline developers, pharmaceutical industry, and providers of diagnostics, is needed to optimise AMU in companion animals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

BackgroundThe epidemiology of carriage of extended-spectrum beta-lactamase-producing (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) in the general population is unknown.AimIn this observational study, the prevalence and risk factors for intestinal ESBL-E and CPE carriage in the Dutch general population were determined. ESBL-E were characterised.MethodsFrom 2014 to 2016, ca 2,000 residents were invited monthly to complete a questionnaire and provide a faecal sample, which was tested for ESBL-E. The first 1,758 samples were also tested for CPE. Risk factors for ESBL-E carriage were identified by multivariable logistic regression analysis. ESBL-E isolates underwent whole genome sequencing.ResultsOf 47,957 individuals invited, 4,177 (8.7%) completed the questionnaire and provided a faecal sample. ESBL-E were detected in 186 (4.5%) individuals, resulting in an adjusted prevalence of 5.0% (95% confidence interval (CI):3.4-6.6%). Risk factors were: born outside the Netherlands (odds ratio (OR): 1.99; 95% CI: 1.16-4.54), eating in restaurants > 20 times/year (OR: 1.70; 95% CI: 1.04-2.76), antibiotic use < 6 months ago (OR: 2.05; 95% CI: 1.05-4.03), swimming in sea/ocean < 12 months ago (OR: 1.63; 95% CI: 1.11-2.39), travelling to Africa (OR: 3.03; 95% CI: 1.23-7.46) or Asia (OR: 2.00; 95% CI: 1.02-3.90) < 12 months ago, and not changing kitchen towels daily (OR: 2.19; 95% CI: 1.24-3.87). The last had the largest population attributable risk (PAR) (47.5%). Eighty-four of 189 (44.4%) ESBL-E isolates carried bla CTX-M-15 . Escherichia coli isolates belonged to 70 different sequence types (ST)s, of which ST131 (42/178 isolates; 23.6%) was most prevalent. Associations were observed between IncFIA plasmids and ST131 and bla CTX-M-27 , and between IncI1 and ST88 and bla CTX-M-1 . No CPE were detected.ConclusionsThe prevalence of ESBL-E carriage in the Netherlands' community-dwelling population is 5.0%. Identified risk factors were mostly travelling (particularly to Asia and Africa) and kitchen hygiene. CPE were not detected.

Objectives: Occupational exposure to animals and foods thereof is a poorly characterised risk factor for salmonellosis and campylobacteriosis, the main causes of bacterial gastroenteritis in the Western world. We performed a population-based registry study in the Netherlands to assess whether differences exist in the incidence of reported salmonellosis and campylobacteriosis cases among occupational groups, and whether they can be explained by differences in the magnitude of exposure to these pathogens, as defined by serology., Methods: Person-level occupational data for all Dutch residents were linked to lab-confirmed salmonellosis and campylobacteriosis data, and to serological data from a previous national serosurvey. SIRs for salmonellosis and campylobacteriosis among occupational sectors and specific high-risk occupations were calculated based on the total employed population. Moreover, Salmonella and Campylobacter seroincidence rates were compared among sectors and high-risk occupations., Results: Occupational exposure to live animals or manure and working in the sale of animal-derived food products were associated with significantly increased risks of salmonellosis (SIR 1.55-1.82) and campylobacteriosis (SIR 1.36-1.65). Moreover, incidences were significantly higher in specific industrial sectors, as well as healthcare and social work sectors. Mean seroincidence rates ranged from 1.28 to 2.30 infections/person-year for Campylobacter , and from 0.36 to 0.99 for Salmonella , with only slightly higher rates for people in high-risk occupations., Conclusions: Significant differences in reported salmonellosis and campylobacteriosis incidence exist among occupational sectors, with the highest incidence in those persons occupationally exposed to live animals. These differences are only partially reflected in the serology., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Antimicrobial usage (AMU) in livestock plays a key role in the emergence and spread of antimicrobial resistance. Analysis of AMU data in livestock is therefore relevant for both animal and public health., Objectives: To assess AMU in 470 broiler and 252 turkey farms of one of Italy's largest poultry companies, accounting for around 30% of national poultry production, to identify trends and risk factors for AMU., Methods: Antimicrobial treatments administered to 5827 broiler and 1264 turkey grow-out cycles in 2015-17 were expressed as DDDs for animals per population correction unit (DDDvet/PCU). A retrospective analysis was conducted to examine the effect of geographical area, season and prescribing veterinarian on AMU. Management and structural interventions implemented by the company were also assessed., Results: AMU showed a 71% reduction in broilers (from 14 to 4 DDDvet/PCU) and a 56% reduction in turkeys (from 41 to 18 DDDvet/PCU) during the study period. Quinolones, macrolides and polymyxins decreased from 33% to 6% of total AMU in broilers, and from 56% to 32% in turkeys. Broiler cycles during spring and winter showed significantly higher AMU, as well as those in densely populated poultry areas. Different antimicrobial prescribing behaviour was identified among veterinarians., Conclusions: This study evidenced a decreasing trend in AMU and identified several correlates of AMU in broilers and turkeys. These factors will inform the design of interventions to further reduce AMU and therefore counteract antimicrobial resistance in these poultry sectors., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chronological age alone is not a sufficient measure of the true physiological state of the body. The aims of the present study were to: (1) quantify biological age based on a physiological biomarker composite model; (2) and evaluate its association with death and age-related disease onset in the setting of an elderly population. Using structural equation modeling we computed biological age for 1699 individuals recruited from the first and second waves of the Rotterdam study. The algorithm included nine physiological parameters (c-reactive protein, creatinine, albumin, total cholesterol, cytomegalovirus optical density, urea nitrogen, alkaline phosphatase, forced expiratory volume and systolic blood pressure). We assessed the association between biological age, all-cause mortality, all-cause morbidity and specific age-related diseases over a median follow-up of 11 years. Biological age, compared to chronological age or the traditional biomarkers of age-related diseases, showed a stronger association with all-cause mortality (HR 1.15 vs. 1.13 and 1.10), all-cause morbidity (HR 1.06 vs. 1.05 and 1.03), stroke (HR 1.17 vs. 1.08 and 1.04), cancer (HR 1.07 vs. 1.04 and 1.02) and diabetes mellitus (HR 1.12 vs. 1.01 and 0.98). Individuals who were biologically younger exhibited a healthier life-style as reflected in their lower BMI (P < 0.001) and lower incidence of stroke (P < 0.001), cancer (P < 0.01) and diabetes mellitus (P = 0.02). Collectively, our findings suggest that biological age based on the biomarker composite model of nine physiological parameters is a useful construct to assess individuals 65 years and older at increased risk for specific age-related diseases.

Background: Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC), plasmid-mediated AmpC-producing E coli (pAmpC-EC), and other bacteria are resistant to important β-lactam antibiotics. ESBL-EC and pAmpC-EC are increasingly reported in animals, food, the environment, and community-acquired and health-care-associated human infections. These infections are usually preceded by asymptomatic carriage, for which attributions to animal, food, environmental, and human sources remain unquantified., Methods: In this population-based modelling study, we collected ESBL and pAmpC gene data on the Netherlands population for 2005-17 from published datasets of gene occurrences in E coli isolates from different sources, and from partners of the ESBL Attribution Consortium and the Dutch National Antimicrobial Surveillance System. Using these data, we applied an established source attribution model based on ESBL-EC and pAmpC-EC prevalence and gene data for humans, including high-risk populations (ie, returning travellers, clinical patients, farmers), farm and companion animals, food, surface freshwater, and wild birds, and human exposure data, to quantify the overall and gene-specific attributable sources of community-acquired ESBL-EC and pAmpC-EC intestinal carriage. We also used a simple transmission model to determine the basic reproduction number (R 0 ) in the open community., Findings: We identified 1220 occurrences of ESBL-EC and pAmpC-EC genes in humans, of which 478 were in clinical patients, 454 were from asymptomatic carriers in the open community, 103 were in poultry and pig farmers, and 185 were in people who had travelled out of the region. We also identified 6275 occurrences in non-human sources, including 479 in companion animals, 4026 in farm animals, 66 in wild birds, 1430 from food products, and 274 from surface freshwater. Most community-acquired ESBL-EC and pAmpC-EC carriage was attributed to human-to-human transmission within or between households in the open community (60·1%, 95% credible interval 40·0-73·5), and to secondary transmission from high-risk groups (6·9%, 4·1-9·2). Food accounted for 18·9% (7·0-38·3) of carriage, companion animals for 7·9% (1·4-19·9), farm animals (non-occupational contact) for 3·6% (0·6-9·9), and swimming in freshwater and wild birds (ie, environmental contact) for 2·6% (0·2-8·7). We derived an R 0 of 0·63 (95% CI 0·42-0·77) for intracommunity transmission., Interpretation: Although humans are the main source of community-acquired ESBL-EC and pAmpC-EC carriage, the attributable non-human sources underpin the need for longitudinal studies and continuous monitoring, because intracommunity ESBL-EC and pAmpC-EC spread alone is unlikely to be self-maintaining without transmission to and from non-human sources., Funding: 1Health4Food, Dutch Ministry of Economic Affairs, and the EU's Horizon-2020 through One-Health European Joint Programme., (Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Objectives: This study aimed to (i) determine risk factors for enteropathogen co-infections, (ii) determine whether enteropathogen co-infections influence gastroenteritis risk, and (iii) determine whether enteropathogen co-infection occurred randomly in preschool children., Methods: A monthly-repeated cross-sectional survey in Dutch children aged 0-48 months was conducted during October 2012 to October 2014. A total of 981 stool samples were collected along with questionnaires collecting data on gastrointestinal symptoms and potential risk factors; 822 samples were successfully tested for 19 enteropathogens using real-time multiplex PCRs. Logistic regression analysis assessed co-infections in relation to gastroenteritis and potential risk factors., Results: In all, 598/822 (72.7%) stool samples tested positive for at least one enteropathogen, of which 290 (48.5%) were positive for two or more enteropathogens. Risk factors for two or more enteropathogen co-infections were young age (<12 months, OR 1.9, 95% CI 1.1-3.3; 13-36 months, OR 1.7, 95% CI 1.1-2.5, versus 37-48 months), day-care attendance (OR 1.8, 95% CI 1.3-2.5), households with three or more children versus those with one child (OR 1.7, 95% CI 1.1-2.8). Stool samples collected in spring less often had two or more enteropathogens versus summer (OR 0.4, 95% CI 0.2-0.7). Food allergy was a risk factor for three or more enteropathogen co-infections (OR 3.2, 95% CI 1.1-8.9). The frequency of co-infection was higher than expected for norovirus GI/norovirus GII, Clostridium difficile/norovirus GI, C. difficile/rotavirus, astrovirus/Dientamoeba fragilis, atypical enteropathogenic Escherichia coli/adenovirus, typical enteropathogenic E. coli/adenovirus, and enteroaggregative E. coli/astrovirus. No co-infection was associated with increased gastroenteritis risk., Conclusions: Risk factors for enteropathogen co-infections were identified and specific enteropathogens co-occurred significantly more often than expected by chance. Enteropathogen co-infections were not associated with increased gastroenteritis risk, calling into question their clinical relevance in preschool children., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)