Campylobacter is the major bacterial cause of gastroenteritis. Transmission occurs via contaminated food, water, animals and environments. Most infections are attributed to poultry, but cannot be explained with foodborne transmission alone. Campylobacter is found in animals and water, where its DNA is threatened by invading DNA from bacteriophages or plasmids. Like many bacteria it has an adaptive immune system (CRISPR-Cas) that protects its DNA against this threat, by recognizing and inactivating invading DNA. .

Approach/working method

As bacteriophages and plasmids are often specific to a particular source, CRISPR offers the possibility to link the acquired spacers to specific environments. In PINNACLE, we will investigate how CRISPR dynamics are related to environmental niche adaptation and recalibrate source attribution models. 

(Expected) results

Our outcomes will provide better estimates of the (non-food) sources of Campylobacter, facilitating novel interventions to reduce its disease burden.

Amount granted: € 364,916.00.

• dr. B. (Birgitta) Duim
• dr. A.L. (Aldert) Zomer

• Indra Bergval (RIVM)

Cryptosporidium is a unicellular parasite that can cause intestinal inflammation and diarrhea in humans and animals. Approximately 70.000 persons are infected with this parasite in the Netherlands every year. Infections can be acquired via contact with the feces of infected humans or animals, the consumption of contaminated food, or via contaminated surface water. The source of the infection is often unknown, which complicates the prevention of new infections and outbreaks and limits insight in the spread of this pathogen in the Netherlands.

Aim

In this project, new typing methods will be applied to gain insight into the types of Cryptosporidium infecting humans and animals. 

(Expected) results

This information will aid source identification and contact tracing, outbreak control and prevention.

Amount granted: € 149,965.00.

• dr. E.R. (Rolf) Nijsse
• prof. dr. A. (Andrea) Gröne

• Kees van der Ark

A main focus of the COVRIN project was to reinforce collaboration and integration of research activities on SARS-CoV-2. The aim of this project was to integrate coronavirus research activities of all project partners. The project had two main operational objectives:

1. To identify the drivers for the emergence and spread of SARS-CoV-2.
2. To generate data and build models for risk assessment of SARS-CoV-2.

Activities to connect with stakeholders and avoid overlaps with other projects were a key focus and the project was split into four key research activities:

1. Research on detection of SARS-CoV2 in animal species and the environment.
2. Research on SARS-CoV2 molecular and biological characterisation.
3. SARS-CoV2 surveillance and risk assessment, focused on the animal human interface.
4. Coronavirus preparedness.

Project Outputs and Outcomes

• Key outputs
  • Shared molecular tests
  • Shared immunoassays
  • Shared cell line and animal models
• Expected implementations
  • Improved reference laboratory testing procedures
  • Improved risk modelling approaches
• Expected impact of the research
  • Preparedness research
  • Risk assessments and control research
• Societal and policy impact
  • Improved risk assessment
  • Improved health risks control

Amount granted: € 2,000,000.00 

• Wim van der Poel (WBVR)

Het gebruik van antibiotica in de veehouderij is een belangrijke oorzaak van antibioticaresistentie in dieren en mensen. Alhoewel het antibioticagebruik in dieren in Nederland de laatste jaren aanzienlijk is verminderd is het niet duidelijk of dit effect heeft gehad op de mate van resistentie onder humane ziekteverwekkers zoals Campylobacter of Salmonella.
Dit project zal kwantitatieve kennis opleveren over de relatie tussen antibioticagebruik in de veehouderij en resistentie bij zoönotische infecties bij de mens door het uitvoeren van analyses van bestaande surveillance data. Daarnaast wordt onderzocht of er overdracht van resistentiegenen plaatsvindt door whole-genome-sequencing data te vergelijken van bacteriën afkomstig van dieren, voedselproducten en mensen.
De resultaten van dit project zullen indicaties opleveren voor toekomstige beleidsdoelstellingen over verdere reductie van antibioticagebruik in de veehouderij op basis van meetbare impact op resistentie bij humane infecties.

• Eelco Franz (RIVM)

The project has mapped the existing knowledge gaps. The work started by mapping existing data and establishing a joint data-sharing platform for the project partners. Data was included from a broad range of reservoirs and sources, including those that are not traditionally part of existing monitoring and surveillance activities, e.g. pets (incl. reptiles), wildlife, and environmental sources.

The work also focused on cataloging, evaluating and advancing existing methods for source attribution and developing methods for the critical assessment of source attribution models. Novel approaches for source attribution were explored, developed and assessed. Investigation of existing approaches included microbial subtyping, meta-analysis of case-control studies and outbreak data, and risk-assessment-based methods. The source attribution estimates focused on three pathogens (Salmonella, Campylobacter, and STEC) and AMR.

Project Outputs and Outcomes

DiSCoVeR project has collected comprehensive and standardised datasets for the target pathogens (Salmonella (n=145,000 isolates; s=4,185 sequences), Campylobacter (n=5,361 isolates/sequences), STEC (n=7,552 isolates; s=3,418), and ESBL (n=10,674 isolates)) and made the phenotypic and metadata open accessible through Zenodo. The sequence data (WGS) will be available at ENA through an umbrella project. We believe that these multi-country datasets compiled of isolate data and sequences from humans and various animal and food sources are quite unique and could be analysed by other methods and by other researchers to provide novel insights to the epidemiology of foodborne hazards in EU MS.

DiSCoVeR project has made a critical and systematic assessment of existing source attribution models and developed new phenotypic and genomic-based attribution models for pathogen and antimicrobial resistance.

Based on the results, DiSCoVeR project has provided recommendations on the translation of results from source attribution models in to actions, emphasising that Salmonella in pigs and pork and Campylobacter in broilers and chicken meat stand out as areas, where targeted future control and intervention could be implemented to reduce the burden of human infections significantly. The project has also evaluated current surveillance and control activities in a One Health context and made recommendations for improvement by seeking out (further) collaboration and identifying possible surveillance synergies within the environmental/ecosystem pillar of One Health, as surveillance and research focus currently is very much oriented towards human health and food-producing animals. Most of these recommendations are considered relevant for risk managers/decision makers such as ECDC and EFSA, national food, veterinary, and environmental authorities, and farmer and consumer organisations. These recommendations have been presented to and discussed with stakeholders, including ECDC, EFSA, EURLs, and the EC.

Finally, DiSCoVeR project has evaluated transfer of the source attribution approaches as an opportunity to strengthen the institutional capacity building for One Health by promoting increased network-collaboration and training within integrated surveillance, source attribution approaches, burden of disease methodologies, risk assessment, and system thinking.

Amount granted: € 2,956,833.00. 

• dr. A.L. (Aldert) Zomer

• Tine Hald (DTU)

• Eelco Franz (RIVM)

If, how and why does antimicrobial resistance spread between animals, the environment and humans?  These questions drive the project 'DiSSeMINATE - Drivers of Selection and Spread of Mobile Genetic Elements INvolved in antimicrobial resistance'. ‘DiSSeMINATE’ is a public-private partnership of the UMC Utrecht, the Wageningen University and Research (WUR), the Institute for Risk Assessment Sciences of the University of Utrecht (IRAS), the Dutch National Institute for Public Health and the Environment (RIVM), and two private partners (VION Food and Cargill).

Antimicrobials are life-saving medicines that are used to treat bacterial infections. Bacteria however can acquire resistance against antimicrobials. This endangers the efficacy of antimicrobials which makes infectious diseases increasingly difficult to treat. Genes which confer resistance to antimicrobials are often not limited to individual bacterial strains but can be exchanged between bacterial strains and species as part of so-called mobile genetic elements. Antimicrobial resistance genes that are part of mobile genetic elements can swiftly spread through different environments.

In DiSSeMINATE, the aim is to increase the understanding of the spread of mobile genetic elements that carry antimicrobial resistance genes by collecting and analyzing relevant samples of humans, the environment and animals, with or without feed additives, and changes in feed as introduced by regionally produced products in circular farming projects. Novel bioinformatics methods will be developed and in vitro assays to determine transfer events and rates.

The project will result in novel methods to analyse the spread of antimicrobial resistance genes through mobile gene flow and in estimated rates of transmission. Finally, the risks and contributions of livestock feeding regimes, feed additives and livestock production chains to the spread of mobile genetic elements carrying antimicrobial resistance genes to humans will be estimated by epidemiological modelling.
 

Amount granted: € 1,000,000.00.

• dr. J.G. (Annelies) Kers

Aanleiding

Plasticvervuiling in aquatische omgevingen vormt een opkomende bedreiging voor de gezondheid. Met name microplastics (<5mm) die industrieel worden vervaardigd of ontstaan als plastic afval uit elkaar valt. Microplastics hebben een goed drijfvermogen en een hydrofoob hard oppervlakte. Dat maakt ze tot een uniek substraat voor biofilm vormende micro-organismen. Hierdoor kunnen ze mogelijk pathogenen herbergen, of antimicrobiële resistentie (AMR) gen uitwisseling bevorderen.

Doel en onderzoeksopzet

Een van de belangrijkste en langste rivieren in Europa is de Rijn. Op twee plekken in de Rijn zijn watermonsters genomen. Microplastics zijn verzameld met 500 µm, 100 µm en 10 µm zeven. Via infraroodspectroscopie is de concentratie en het type microplastic bepaald. De microbiële kolonisatie en AMR genen zijn bepaald via 16S rRNA-sequencing and qPCR.

Resultaten

Microplastics komen algemeen voor in de Rijn; de gemiddelde microplastic concentratie was 213.147 deeltjes/m3 water. Polyamide en polyvinylchloride waren de meest voorkomende polymeren. De samenstelling van de microbiële kolonisatie op de 100-500 µm plasticmonsters verschilde significant van het oppervlaktewater en de kleinere plasticmonsters. Er werd lagere diversiteit waargenomen op grotere plastic deeltjes. Hoe kleiner de microplastics waren, hoe meer ‘waterachtig’ de microbiële samenstelling werd. Daarnaast had drijvend plastic een latere diversiteit dan gesedimenteerd plastic. De diversiteit was hoger in de winter dan in de zomer, alhoewel de microplastic concentraties in de zomer hoger waren. De microplastics bevatte microbiële taxa die bekend staan om hun biofilmvorming (Gammaproteobacteria and Betaproteobacteria) en plastic afbraak (Pseudomonas). De microplastics bevatte ook microbiële taxa met potentiële pathogenen (Pseudomonas, Acinetobacter en Arcobacter). AMR-genen werden alomtegenwoordig gedetecteerd. Deze resultaten tonen aan dat microplastics als karakteristiek substraat dienen voor microbiële kolonisatie.

• prof. dr. A.M. (Ana Maria) de Roda Husman

Since the 1950s, antimicrobials have been increasingly used in modern intensive livestock production systems. Besides preventive and therapeutic use, antimicrobials were used as growth promoters in the European Union (EU) until their ban in 2006. This was preceded by decades of growing evidence and concerns about the public health impact of widespread veterinary antimicrobial use (AMU) and associated increasing antimicrobial resistance (AMR). As AMU in livestock favors AMR development in bacterial populations as it does in humans, the public health risks of veterinary AMU are threefold: 1) resistant bacteria can pass onto humans via direct contact with animals; 2) these bacteria can pass on via food of either animal origin or cross-contaminated during production; 3) these bacteria can spread into the environment via farm runoff or unprocessed manure used as fertilizer. Moreover, large volumes of antimicrobial residues in manure cause further environmental exposure and potential selective pressure. In 2007, the Netherlands was the largest veterinary antimicrobial consumer per biomass unit of animal production among 10 EU countries. Together with the discovery of large reservoirs of methicillin-resistant S. aureus (MRSA) and Extended-Spectrum Beta-Lactamase (ESBL)-producing bacteria in Dutch livestock, this led to considerable socio-political pressure, with the government imposing 20%, 50% and 70% AMU reductions in livestock in 2011, 2013 and 2015, respectively. After an initial rapid AMU reduction (56% in 2013), mostly attributable to replacing group treatments, adopting herd health and treatment plans, guidelines, benchmarking systems and transparency in prescriptions, a 58% AMU reduction was reported in 2015, indicating that a 70% of higher AMU reduction would require more fundamental changes in the livestock production systems rather than in prescribing procedures alone. Indeed some structural differences in AMU still exist between Dutch livestock farms and overall AMU remains high in a subset of them, mainly because of the highly intensive nature of the Dutch farming industry. Moreover, in recent years AMU reduction has levelled off despite further reduction is sought after, particularly in broilers, weaned piglets and veal calves, whose groups now account for most AMU in Dutch livestock. An important counter argument is the increased economic burden placed on the farmers and eventually on the consumers through further restriction to veterinary AMU when this is needed for therapeutic purposes. A rise in livestock production costs would be unbearable in the highly competitive international agricultural market wherein the Dutch livestock industry relies heavily on exports. Providing the right conditions for incentivizing further AMU reduction in Dutch livestock requires an assessment of the potential impact on AMU and associated (negative or positive) financial effects of the available interventions aiming at keeping livestock healthy, on the principle that every infection prevented is an opportunity for no treatment. Policy makers and livestock producers would then be able to consider supporting the implementation of a specific intervention instead of another based their cost-effectiveness. These interventions include: (i) infection control (i.e. enhanced farm biosecurity and hygiene standards), (ii) animal husbandry practices (i.e. enhanced farm management, e.g. low-stock density farming, all-in/all-out production systems, rearing of slow-growing breeds, etc.), (iii) vaccination (for bacterial diseases, but also viral diseases often complicated by secondary bacterial infections). Indeed, there is still no quantitative evidence for the impact of these interventions on AMU, and even less evidence for their sustainability. Consequently, comprehensive recommendations to farmers about which interventions are most cost-effective and would best suit their specific situations in relation to the public health needs remain rather vague (e.g. as general statements like ‘increased vaccination’ or ‘improved biosecurity’) or are based on individual veterinarians’ personal experience and opinion. As a collaboration of two leading institutions in animal and public health in the Netherlands (Veterinary Medicine Faculty of Utrecht University and the RIVM) and using both existing and newly collected data, we will quantify in a scenario-based modelling framework the impact of different biosecurity/hygiene standards, vaccination schemes and husbandry practices on AMU reduction (overall and for specific antimicrobials) in broilers, weaned piglets and veal calves, including their cost-effectiveness. Determining the impact and feasibility of these interventions will provide livestock producers and policy makers with a management tool to set targets and draw plans for the implementation of those interventions with the highest potential for AMU reduction, and so decreasing AMR in a rational and sustainable way.

• prof. dr. J.A. (Jaap) Wagenaar
• prof. dr. J.A. (Arjan) Stegeman
• dr. P. (Panagiotis) Mallioris

Over 20% of the global cancer burden is attributable to infectious agents, including bacteria. This is best established for Helicobacter pylori infection and gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma, and for Salmonella Typhi and gallbladder carcinoma in chronic typhoid carriers. Bacteria can contribute to cancer through inflammation, induction of DNA damage by toxins and manipulation of host cell signaling pathways during their infectious cycle. This is also true for the major foodborne pathogen Salmonella, which can manipulate host cell signaling pathways to enforce bacterial uptake, intracellular survival and egress, providing a step forward in the cancer formation cascade. Salmonella would contribute to cancer mainly under conditions of long-lasting infections and/or when significant numbers of pre-transformed cells are present in the intestine, as demonstrated by experiments evidencing colon carcinoma formation after S. Typhimurium infection in genetically predisposed (APC+/-) mice. Globally, an estimated ~94 million people are infected with Salmonella each year, but most cases remain unreported, as they often present with mild and self-limiting symptoms that do not require medical attention. In the Netherlands, about 1,500-2,000 lab-confirmed salmonellosis cases (~75% of which caused by S. Typhimurium and S. Enteritidis), representing the most severe cases occurring in the population (as Salmonella testing is usually performed after 1-2 weeks of illness), and 10,000-15,000 colon cancer diagnoses (of which ~30% leading to death), are reported annually. We have recently showed in a nationwide epidemiological study a ~3-fold increased colon cancer risk in patients with a history of (severe) Salmonella Enteritidis infection as compared to the general Dutch population. However, people acquire multiple mild Salmonella infections throughout life, leaving open the question of whether these infections may contribute to colon cancer as well. Moreover, as S. Typhimurium is virtually ubiquitous and S. Enteritidis is essentially foodborne, a putative oncogenic effect of S. Typhimurium is more likely to be evidenced by looking at repeated mild infections that do not necessarily lead to clinical disease. Whether also these infections contribute to colon cancer remains unknown. Moreover, we found that the tumors of patients with a history of severe Salmonella infection were mainly of low grade, suggesting that by virtue of their history, these tumors may differ from those developed without a contribution from Salmonella, a finding that would have direct implications for treatment. By building on current evidence and combining knowledge and methods from epidemiology, immunology, pathology and cell biology, this project will explore the association between repeated mild Salmonella infections and colon cancer, i.e. whether their cumulative effect is comparable to a single severe infection. Whilst we recently obtained support from the Dutch Cancer Society to unravel the molecular mechanisms of cancer formation following Salmonella or Campylobacter infection and to repeat the aforementioned epidemiological study in a Danish cohort, the present project will look at other crucial aspects of the role of Salmonella infection in colon cancer development. Specifically, we will determine whether: 1) people at high risk of frequent exposure to Salmonella (because of particular occupations, eating habits or patterns of contact with animals) are also at high risk of colon cancer; 2) repeated low-dose exposure to Salmonella promotes cancer formation in vivo; 3) infection pressure by Salmonella, as defined by serology, in colon cancer patients differs from that of non-colon cancer patients; 4) tumors from patients with a history of Salmonella infection or frequently exposed to Salmonella have distinctive features. This will provide evidence from different angles to claim a causal relation between recurrent mild Salmonella infections and colon cancer risk, providing new impetus to competent authorities to strengthen current Salmonella-control efforts in the food production chain, as well as to increase public awareness on the importance of, e.g., food safety practices and kitchen hygiene, as preventing Salmonella infection would go far beyond ‘just’ preventing food poisoning per se. Indeed, the societal implications of the oncogenic potential of Salmonella can drastically redefine its disease burden and the perception of foodborne infections in general. Other interventions to be considered include invitation of salmonellosis patients and those repeatedly exposed to Salmonella in the National Colon Cancer Screening Program for early detection of the disease. This project has therefore the potential to contribute to less foodborne infections and less cancer in an innovative and significant way.

• Eelco Franz (RIVM)
• Sjaak Neefjes (LUMC)
• Andra Borst (VUmc)
• Michael Schaapveld (NKI)
• Wilfrid va Pelt (RIVM)
• Janneke Duijster (LUMC)

Antimicrobial resistance (AMR) is a growing European and global health problem in both humans and animals, leading to limited or poor treatment options for many diseases. Containment of AMR spread is part of the European Commission action plan against the rising threats from AMR. However, consensus estimates on actual exposure risk, sources, transmission routes and disease burden is scarce. A major reason for the lack of well-developed analyses might be the complexity of these approaches and their data requirements, and their interdisciplinary nature. Crucial to progression on this theme is the development of modelling methodology and the systematic integration of data. Currently much microbiological, molecular and epidemiological data is being gathered on AMR development, prevalence, spread, transmission, risks, etc. in the veterinary, food and human domain. However, assessment of the importance of different transmission routes and quantifying public health effects (i.e. disease burden) associated with AMR represent major knowledge gaps. In order to make full use of all available data, to check consistency, and to find consensus estimates, both problems (attribution and disease burden) require an integrated framework. In different WPs this project will generate genomic information to feed into transmission models (WP1), will establish estimates for the relation between antimicrobial use and the development of resistance (WP2), develop transmission models for AMR spread between and within animal and human population, establish risk assessment models to estimate the risk of spread through the food chain (WP3), and will construct a methodological framework for estimating the disease burden of AMR (WP5). The extreme diverse nature of the data available and generated in this project requires innovative methods to effectively integrate this data into final estimates for source attribution, risks and disease burden. To that end we will employ machine learning techniques (WP4) and Bayesian Evidence Synthesis (WP6). To this end innovative modelling techniques such as machine learning and Bayesian evidence synthesis will be employed. This project is expected to deliver harmonized modelling approaches and methodologies that are clearly beyond the state-of-the-art and will change the way of approaching the AMR problem to an integrative approach.

• dr. M.C.J. (Martin) Bootsma

• Eelco Franz (RIVM)
• Tine Hald (DTU)
• Aline de Koeijer (WBVR)
• Thomas Sellhorst (BfR)
• Christine Muller-Graf (BfR)
• Annemarie Kaesbohrer (DfR)
• Luca Busani (ISS)
• Annalisa Pantosti (ISS)
• Robin Simons (APHA)
• Emma Snary (APHA)
• Madelaine Norstrom (NVI)
• Camilla Sekse (NVI)
• Michel-Yves Mistou (ANSES)
• Pascal Sanders (ANSES)
• Sophie Granier (ANSES)

Problem description
Pathogenic bacteria often influence host cell biology as part of their infectious cycle to manipulate host tissues and immunity. Such manipulation can support cell transformation and, as we showed, can contribute to the multistep process towards cancer. Yet, there are only a few recognized bacteria contributing to cancer: Salmonella Typhi associated with gallbladder carcinoma, and Helicobacter associated with gastric cancer and MALT lymphoma. The association between other bacteria and cancer have not been evaluated.

Research direction
Since bacteria do not leave a genetic imprint, it is difficult to show their direct contribution to cancer. Our approach is to combine epidemiology and cell biology to understand the oncogenic potential of two major foodborne pathogens: non- typhoid Salmonella and Campylobacter. In the Netherlands, human Salmonella and Campylobacter infections are usually laboratory confirmed upon medical consultation after 1-2 weeks of illness. Confirmed cases are reported to health authorities and we plan to link these data with the cancer registry to determine whether patients with a history of salmonellosis have an increased risk for colon cancer. Moreover, laboratory experiments will be performed to understand the molecular basis of bacterially induced cell transformation. By integrating epidemiology with cell biology, we will have the unique opportunity to perform studies that move from correlation to causality.

Aim
We aim to test whether two major foodborne bacterial pathogens contribute to colon cancer formation and to determine the molecular basis of this association. In addition, we aim to test whether frequent, mild Salmonella infections that do not result in long episodes of illness also represent an “environmental factor” contributing to colon cancer.

Plan of investigation
Our earlier work showed how S. Typhi contributes to gallbladder carcinoma, and that patients with a history of Salmonella Enteritidis infection have a 3-fold increased risk of cancer in the ascending and transverse parts of the colon in the Netherlands. We now propose to:
1. Repeat the epidemiological study on the association between non-typhoid salmonellosis and colon cancer using a Danish cohort. This is important as an independent validation and will increase the power of the analyses.
2. Study the mechanism of oncogenic imprinting of Salmonella infection.
3. Perform laboratory experiments to test whether repeated low-dose Salmonella infections have a similar effect on cell transformation as a single high-dose infection (doses are related to reported rates of infection). Indeed, serological studies indicate that, on average, people acquire mild Salmonella infections every 3.5 years throughout life. This may then represent an environmental factor contributing to colon cancer. We will test this on organoids and MEFs, and will also be modelled to add further rationale. We will also test whether antibodies for Salmonella in the serum of colon cancer patients provide indications for such association, and whether an association between occupation at high risk of exposure to Salmonella and colon cancer exists.
4. Study the biology of Campylobacter cytolethal distending toxin Cdt that is reported to generate DNA double stranded breaks. We will generate the toxin, and test for its effects on DNA break formation and repair. In addition, we will test whether the toxin can induce transformation of (pre-transformed) MEFs in soft agar assays and colon organoids in depletion studies and histochemistry (as we have done before).
5. We will perform an epidemiological study using the Dutch as well as the Danish Registries to test whether Campylobacter infection increases the risk of colon cancer.

Expected outcome
This project will consolidate evidence on the relationship between severe food poisoning by Salmonella and colon cancer. It will also reveal whether Salmonella infection is an environmental factor contributing to colon cancer. This project will also determine the contribution of Campylobacter infection to colon cancer. The unique combination of epidemiological and cell biological aspects allows us to elucidate the role of foodborne bacterial infections as cancer-inducing agents. These infections are largely preventable though basic hygiene measures and infected patients may benefit from being invited to participate in early cancer screening programs. This will be possible only when the relationship between bacteria and cancer will be thoroughly understood and accepted, which is what we aim to provide with this proposal.

• Eelco Franz (RIVM)
• Sjaak Neefjes (LUMC)
• Andra Borst (VUmc)
• Michael Schaapveld (NKI)
• Wilfrid va Pelt (RIVM)
• Janneke Duijster (LUMC)

Campylobacteriosis is the primary zoonosis in Europe, causing over 1.6 M cases and €76 M costs annually in the Netherlands alone (~17 M people). Most cases are caused by Campylobacter jejuni and C. coli, which are widespread in livestock and wildlife, providing many ways for human exposure beyond just food. Despite all the research and control efforts in the food chain, there is no significant decrease in human campylobacteriosis. Up to 80% of human campylobacteriosis cases can be attributed to the poultry reservoir, but only ~40% of poultry-borne cases are attributable to poultry consumption; thus, many poultry-borne Campylobacter strains infect humans via other routes. Campylobacter is often found in environmental sources like surface water, indicating recent contamination with (animal) fecal material, but its environmental routes are still largely unexplored. While Campylobacter survives poorly outside the host, there are some environmentally adapted strains that play a key role in the transmission between animals and humans via the environment. Surface water is a ‘sink’ that collects Campylobacter strains from different hosts whose relative contributions are largely unknown, though wild birds are thought to play a major role. However, the devastating H7N7 bird flu epidemic hitting the Netherlands in 2003 showed that even without a huge drop in poultry consumption, the massive poultry culling and closure of poultry abattoirs to contain the epidemic was associated with a 44-50% drop in human campylobacteriosis where these measures were enforced, suggesting a major role of the environment in human exposure to poultry-borne strains. Moreover, while studies show that poultry and wild birds are the most important contributors to Campylobacter surface water pollution, their contributions vary with the size of the poultry production. The environment may also act as a source of Campylobacter (re)colonization in livestock. As the environment seems to be a key player in Campylobacter epidemiology and the non-foodborne side of campylobacteriosis receives little attention despite its potential to provide new targets for Campylobacter control and research, this project will discern the origins and spread of Campylobacter strains contaminating the environment and will determine their contribution to human campylobacteriosis morbidity, as well as the underlying (non-foodborne) transmission routes. As a collaboration of 4 leading institutes in public, animal and environmental health in the Netherlands (RIVM, CVI, UU and Alterra WUR), we will collect and type with a gene-by-gene approach (whole-genome multilocus sequence typing, wg-MLST) over 1200 C. jejuni/coli isolates representative of the Dutch eco-epidemiological situation from human cases, surface water (agricultural ditches, recreational waters, wastewater outlets), animals (broilers, layers, beef/dairy cattle, sheep/goats, pigs, pets), and wild birds (Anseriformers, Charadriformes, Columbiformes, Suliformes) using both well-established surveillance systems and ad hoc sampling schemes. We will: (i) characterize genotypically the strains circulating in surface water, animals and humans; (ii) quantify the contribution of different wild bird, farm and companion animals to Campylobacter pollution in different surface water types, seasons and areas with varying human, livestock and wild bird densities; (iii) quantify the contribution of different animal reservoirs and surface water to human campylobacteriosis in different seasons and areas with varying human, livestock and wild bird densities to estimate both the fraction of human cases attributable to the environment and the relation between human cases and reservoir density; (iv) determine the evolutionary history and relations of Campylobacter strains in the environment, humans and animals to understand their diversity and ecology, identify source-specific genetic markers, examine the role of the environment in the emergence of pathogenic strains, and perform source attribution at a high resolution; (v) conduct combined source attribution and case-control analyses to identify risk exposures for human campylobacteriosis of environmental origin and its underlying transmission routes. By depicting the epidemiology of non-foodborne campylobacteriosis, this project will allow for the delineation of more holistic control strategies, including those preventing Campylobacter dissemination into the environment. Besides standard scientific outputs, the results of this project will be translated into practical action points and advices for regulatory authorities on how to tackle environmental campylobacteriosis. The project members host national/international Campylobacter reference labs and participate since many years in advisory groups for the Dutch Ministries of Health and Economic Affairs, as well as the poultry industry, so through these forums knowledge gained in this project will be translated into interventions.

• prof. dr. J.A. (Jaap) Wagenaar
• dr. B. (Birgitta) Duim
• dr. A.L. (Aldert) Zomer

• Eelco Franz (RIVM)
• Wilfrid van Pelt (RIVM)
• Hetty Blaak (RIVM)
• Ciska Schets (RIVM)
• Ralf Buij (WUR Alterra)
• Miriam Koene (WBVR)