Contaminated environment plays an important role in the transmission of Campylobacter, directly to humans or indirectly via farm animals. Emission from broiler farms could play an important role in environmental contamination. However, systematic investigations about different emission routes from broiler farms and their impact are still scarce. Moreover, the role of viable but nonculturable (VBNC) state in environmental transmission is not studied up to now.

Therefore, in this study broiler farms will intensively be investigated over a time. Samples from animals and their environment inside the barn will be collected simultaneously to different samples in the farms´ surrounding with focus on as well as a direct entry/ immission and a direct emission. These processes will be recorded over a longer time period. Besides the cultivation and quantification of Campylobacter also VBNC state will be detected and quantified.

Isolates from the barn will be compared to isolates from the farms´ environment via NGS and epidemiological relationships will be evaluated. Moreover, the tenacity and pathogenic potential of isolated environmental Campylobacter including their VBNC state will be analysed in a laboratory approach.

In the end, the relevance of emissions from broiler farms for a direct transmission to humans as well as a recurring contamination of broiler flocks from the environment will be evaluated.

Campylobacter can survive in biofilms in the unfavourable conditions outside the host and it is suggested that Campylobacter is rather a secondary coloniser of pre-existing biofilms. However, information on mechanisms of biofilm production of Campylobacter and options of biofilm reduction as well as places containing Campylobacter biofilms in the food productions chains are still scarce. Therefore, presence and places of biofilms within the farm and commercial poultry meat production as well as transcriptomic changes during biofilm formation will be elucidated. Further, environmental isolates will be included in our studies to investigate their potential in biofilm formation compared to chicken and human-derived isolates.

Different strategies to reduce Campylobacter biofilms will be investigated: i) As biofilm formation of many bacteria is regulated by Quorum sensing processes, the impact of Quorum quencher in reducing Campylobacter of mono- and multi-species biofilms will be investigated. ii) The extracellular polymeric substance of Campylobacter biofilms also consists of extracellular DNAs (eDNA). As the C. jejuni eDNase, encoded by cje1441, is able to reduce Campylobacter mono-species biofilms their efficiency in reducing Campylobacter from multi-species biofilms will be analysed. iii) The impact of further intervention strategies e.g. bacteriocins and organic acids on biofilm formation and reduction will be analysed. The most promising strategies will be implemented in commercial production stages and efficiency determined.

In addition, the role of Quorum quenching compounds in pathogenicity will be investigated to elucidate further therapeutical approaches.

Altogether, this sub-project contributes to clarify regulation of biofilm formation in C. jejuni and possible biofilm reduction strategies at environmental conditions and in commercial poultry meat production, which could result in lower cross-contamination of food products, thereby improving human health.

C. jejuni exhibits outstanding genetic diversity primarily due to frequent natural transformation. Routine monitoring of antibiotic resistance in Campylobacter performed in the National Reference Laboratory for Campylobacter revealed that a high percentage of isolates from food are resistant against multiple antimicrobials. Extracellular DNA can serve as nutrient source, matrix for biofilm formation, repair of genetic damage and genome variation, leading to improved adaptation and survival of pathogens. In this sub-project, we intend to address the impact of horizontal gene transfer on genetic diversity in C. jejuni and intend to open up strategies for reduction of genetic diversity/ survival by inhibiting DNA binding and/ or uptake. This strategy might be applicable to reduce resistance spreading, biofilm formation, survival in the environment and colonisation in the host. In particular, a single cell assay will be applied for the monitoring of DNA uptake in C. jejuni, which allows for the characterisation of competence development. In addition, mutants defective in natural transformation will be tested (i) for their ability to form biofilms, (ii) for their colonisation capacity in vivo and (iii) their adaptive potential with and without selection pressure, (iv) their survival/ tenacity in the environment.

Established control strategies against Campylobacter spp. infections in livestock are focusing mainly on improved biosecurity measures, vaccination trials and non-specific nutritional approaches.

However, these measures alone result so far only in an insufficient reduction of Campylobacter infections both of the farm animals and subsequently the consumers. Therefore, in this individual project (IP) various non biosecurity-based measures of keeping and housing conditions and their combinations will be evaluated for their impact on Campylobacter reduction on herd level and on individual animal level. Initially, these management factors will be evaluated in experimental Campylobacter infection experiments of chickens. Investigated factors will be: i) different stocking densities, ii) two slowly growing genetics/ breeds compared to fast growing breeds, each combined with tailored diet, iii) continuous decontamination of the air and the barn surfaces due to vaporisation of essential oils, iv) litter with decontaminating characteristics and with a reduced humidity. For this purpose, an experimental animal model with broilers will be established in the Toolbox (Z) as the in vivo platform Z1. This model is based on “Seeder Birds” which are orally infected with either one (for monovalent infection) or three (for trivalent infection) well-characterised C. jejuni strains (and in the trivalent infection beside two C. jejuni strains also one C. coli strain). Effects were evaluated quantitatively and qualitatively on group and animal levels. Induced VBNC state will be also monitored in some trials. Successful measures, combinations of that and combinations of measures successfully evaluated in the experimental infections and measures already known to be able to effectively reduce Campylobacter will be later implemented and evaluated under practical conditions in poultry farms.

This sub-project aims at finding the optimal combination of the presently available measures to effectively reduce Campylobacter in chickens. This approach of a successful synergistic large scale arrangement shall reduce Campylobacter on broiler carcasses in order to improve human health in a sustainable way and according to the idea of a “One- Health” approach.

The system will be tested under controlled conditions and in field trials. The effect on Campylobacter colonisation, resistance and pathogenicity will be monitored. This sub-project aims in closing the gap of effective measures for use in commercial broiler production and livestock farming.

Even though primary production is regarded as the prominent step to tackle Campylobacter, intervention measures should be applied at all steps of the chicken meat production chain to enable a maximum reduction of Campylobacter on chicken meat.

For this, we will identify options to optimise the slaughter technology and hygienic standards to reduce Campylobacter contamination/ cross-contamination.

In close collaboration with IPs of research complex A, we will apply intervention measures that were established and tested in the chicken models on farm and slaughter level to generate data under industry conditions. These data enable us to evaluate and adapt the risk intervention model that was established based on data from IPs of research complex A and calculated by IP10. Based on that, a best practice manual to reduce Campylobacter during rearing, slaughter and processing will be created and disseminated to poultry industry.

The intestinal microbiota of conventional laboratory mice mediate strict colonisation resistance against C. jejuni. Therefore, the identification of intestinal bacteria and metabolites causing colonisation resistance combating C. jejuni and C. coli within the murine gut lumen will allow to develop novel therapeutic and/ or preventive strategies to complement biosafety measures directed against Campylobacter colonisation in farm animals and to abolish its transmission via established infection routes. Thus, we will investigate the gut microbiota, intestinal metabolites and corresponding host immune responses in mice applying metabolomic and microbiomic approaches. Given that our murine infection models mimic key features of human campylobacteriosis, investigation of the intestinal and systemic immune responses will be excellently suited to unravel immunopathogenic properties of C. coli in vivo for the first time. The collaborative experimental work within this unique consortium provides the exceptional opportunity to transfer corresponding findings directly into practice. Therefore, synthetic metabolites and/ or viable bacterial or fungal species conferring colonisation resistance against C. jejuni and/ or C. coli in mice will be evaluated for their preventive properties against colonisation of chicken, for practical transfer into food processing procedures and for suppression of enterocyte invasion or human infection in respective model systems established by the expert research partners. Finally, our murine infection models will be provided to the consortium in order to validate preventive or therapeutic measures and to provide data furthering the development to the pharmaceutical or product level.

Zoonotic infections with Campylobacteraceae, the most common bacterial diarrhoeal pathogens, represent a serious problem in human medicine. C. jejuni as prototype but also others such as C. coli and C. concisus belong to these clinically relevant bacteria, with repeatedly occurring outbreaks and cases of fatal septicaemia. For this purpose, we want to extend our basic knowledge of the pathogenesis of Campylobacter associated gastrointestinal diseases. We will investigate the underlying mechanisms that contribute to the development of diarrhoea and inflammation. For this purpose, molecular and electrophysiological studies will be performed on human epithelial cell lines (HT-29/B6), epithelial cell-immune cell-cocultures, mouse infection models, and human intestinal biopsies. Preventive therapies will be studied using these experimental systems which includes vitamins (vitamin D), trace elements (zinc), dietary components (short-chain fatty acids), plant components (quercetin), whey components (lactoferrin), and traditional drugs (berberine), all of which have been identified to stabilise epithelial barrier function. The main focus is on multimodal protective strategies against C. jejuni in humans and farm animals on the basis of molecular barrier research.

We are interested in the cellular biology of two Campylobacter subspecies, C. jejuni and C. coli, using polarized epithelial cell models in vitro. We hypothesize that yet unknown virulence factors exist in both pathogens. Our major goal is to identify and characterize these novel virulence factors. We are also aiming to uncover host factors interacting with these bacterial effectors and to study how these interactions manipulate normal cellular signal transduction functions to cause disease development. We are focusing on HtrA-independent bacterial proteases, for which we want to study the epidemiology and develop small molecule inhibitors. Our studies take advantage of powerful technologies including electron microscopy, confocal microscopy and live cell imaging as well as proteomics-based and cellular signal transduction approaches. Aim is to develop a new pharmaceutical intervention product, which could help to reduce the Campylobacter load in animals and the burden of infections in humans.

A baseline risk model will be conducted that explains the contribution of the selected risk factors to the human campylobacteriosis cases. Consortium’s study data will be complemented by publicly available data as well as by data from previous studies. Secondly, the impact of measures with the following objectives will be investigated by use of risk intervention models: (a) reducing the prevalence of Campylobacter in poultry flocks, (b) reducing the bacterial load of colonised chickens, (c) reducing the release of Campylobacter spp. from broiler farms to the environment, and (d) reducing the proportion of contaminated carcasses. At last, these approaches will be combined by constructing a general risk intervention model and will be validated by a concerted field study including selected interventions on farm and slaughterhouse level.

Identification of genomic signatures for host specificity to cattle, pig and chicken of C. jejuni and C. coli and its distribution in the host-specialists as well as in the host-generalists will be determined by applying a k-mer approach to whole genome sequence data of selected strains. The strains will also be characterised according to their phylogenetic relationships and their virulence profile and an evaluation on genomes of Campylobacter isolated from humans will be performed. The findings in this sub-project will help to improve the source attribution in human Campylobacter infections and may help to identify and interrupt zoonotic infect-chains.