Characterising the microbial communities associated with the water distribution system of a broiler farm and their role in Campylobacter infection

Campylobacters are zoonotic pathogens with extensive animal and environmental reservoirs in spite of their fastidious nature. Campylobacter jejuni is the main bacterial cause of human gastroenteritis worldwide. Epidemiological studies have identified chicken meat as the main vehicle for human infection in industrialised countries. In some countries in the developing world, however, there is little information about Campylobacter prevalence in poultry and poultry meat. The dynamics of Campylobacter colonisation of chickens remain poorly understood. It is agreed that in most cases Campylobacter is transmitted to chickens horizontally from the farm environment. Different sources have been recognised, but their actual contribution to Campylobacter epidemiology remains a matter of debate. Water has rarely been identified as a possible source of Campylobacter contamination of chickens, but represents an understudied source. Research suggests that natural microbial communities might promote the survival of Campylobacter in environmental reservoirs by enabling incorporation into biofilms or interaction with protozoa. The aim of this study was to improve our understanding of Campylobacter ecology and epidemiology at the chicken farm level. The main objectives were to unravel the microbial communities associated with the drinking water system of broiler farms and their dynamics through the rearing cycle; to investigate the aerobic survival of the pathogen in co-culture with Pseudomonas environmental isolates and Acanthamoeba polyphaga; and to study Campylobacter prevalence in chicken farms in Uganda. A longitudinal study was conducted in a commercial broiler farm in the UK during a whole rearing cycle. The purpose of this study was to gain an understanding about the microbial communities that inhabit the drinking water system of the broiler farm, and their implication in Campylobacter ecology in the chicken farm. 16S and 18S rRNA profiling of bulk water and biofilm samples were carried out over a seven-week production cycle period. The same samples were screened for the presence of Campylobacter by culture-dependent methods and molecular techniques. Analysis of 16S and 18S rRNA profiles suggested that microbial communities in the water distribution system are niche specific. Biofilm and bulk water samples harboured distinct communities. Moreover, bulk water communities inside the broiler house were significantly different from those sampled from the source water (R=0.88, p<0.05). Inside the broiler house, microbial communities were found to vary across the rearing cycle. Similar changes in the dynamics of prokaryotic and eukaryotic communities were observed. Bacterial communities were dominated by the phylum Proteobacteria with a shift to Firmicutes towards the end of the cycle, mainly due to an increase in relative abundance of the genera Staphylococcus and Lactobacillus. Eukaryotic communities included a diversity of yeasts, fungi and protozoa. Campylobacter spp. were not detected on the UK broiler farm by culture-dependent techniques. However, the emerging gastrointestinal pathogen Helicobacter pullorum was isolated by culture towards the end of the rearing cycle. Another emerging pathogen C. ureolyticus was detected by both 16S rRNA amplicon sequencing and PCR in water samples and chicken faeces. This is the first report of C. ureolyticus in association with poultry. 16S rRNA reads belonging to the genus Campylobacter were also found in the sequencing data towards the end of the rearing cycle suggesting that the pathogen could be in a viable but non-culturable state in the water distribution system. Little information is available on the epidemiology of campylobacteriosis in Africa. In Uganda Campylobacter infections have been reported in children, and gorillas have been identified as carriers but no information is available on Campylobacter prevalence in chickens. Studies in neighbouring countries, however, have shown high Campylobacter prevalence in poultry. A pilot study, visiting three small-scale farms around Fort Portal, was conducted to investigate the prevalence of Campylobacter in chickens in W. Uganda. Through a combination of culture-dependent and culture-independent techniques Campylobacter was detected in all three farms with high prevalence. Mixed presence of C. jejuni and C. coli in the same farm were observed, suggesting that chickens and chicken meat can be a source of human infection in Uganda. Pseudomonas spp. and protozoa of the genus Acanthamoeba are ubiquitous organisms that commonly co-localise in water and in the farm environment with Campylobacter. In vitro assays were performed to investigate whether they could enhance C. jejuni survival under atmospheric conditions. Co-culture assays indicated that certain Pseudomonas ssp. and strains were able to enhance Campylobacter survival with an increase in viable cell recovery ranging from 0.8 LOG to 4.5 LOG depending on the strain after 24 hours of aerobic incubation, while others showed no interactive effect or even reduced Campylobacter survival. A. polyphaga was shown to internalize C. jejuni, protecting it from the oxygen in the atmosphere and thus enhancing its survival with an increase in viable cell recovery of 2 LOG. These studies have further shown the importance of chicken farms in Campylobacter ecology. It has been demonstrated that microorganisms that are capable of enhancing Campylobacter survival in vitro are found in the water distribution system of the farm studied in UK. The characterisation of these communities for a whole rearing cycle has led to the detection of emerging human pathogens in broiler chicken production showing how doing exploratory work targeting whole microbial communities can inform epidemiology.