Investigation of Campylobacter jejuni and Campylobacter coli colonisation of commercial free-range chickens

Campylobacter spp. are a leading cause of human gastroenteritis worldwide. Most infections are caused by C. jejuni, followed by C. coli. Chickens are considered a natural reservoir of Campylobacter spp. with most outbreaks associated with the consumption of poultry products contaminated with these bacteria at slaughter. Changing consumer awareness of issues associated with animal welfare and well-being is driving a move away from intensive poultry production to free-range systems. As a consequence of this recent shift, there is a need for greater understanding of the epidemiology of C. jejuni and C. coli colonisation and genetic diversity in relation to meat production on free-range poultry farms in Australia. Currently, there is limited information on this, and no specific strategies are applied on free-range farms to prevent Campylobacter colonisation of poultry. This study aimed to address these important knowledge gaps by investigating C. jejuni and C. coli colonisation of chickens in commercial free-range broiler farms in New South Wales, Australia through targeted isolation of C. jejuni and C. coli from chicken faeces. Potential sources of C. jejuni and C. coli on farms were also investigated by culturing these bacteria from samples taken from the production environment. The genetic relatedness of isolates was assessed to evaluate modes of transmission.
Fresh chicken faecal/caecal droppings (n=1,265) and environmental samples (n=471) were collected from 18 free-range broiler flocks at weekly intervals for three weeks after placement. Faecal/caecal droppings (n=120) were also collected from the five breeder farms which supplied the broiler chicks. Samples were used for Campylobacter isolation using standard methods (ISO 10272:2006). A combination of MALDI-TOF and PCR methods was used to identify and speciate the C. jejuni and C. coli isolates. The C. jejuni and C. coli isolates were genotyped with a flaA-HRM PCR assay to evaluate genetic diversity within and between the sampled flocks. These data were also used to evaluate potential sources of the C. jejuni and C. coli genotypes isolated from chickens. C. jejuni and C. coli genes homologous which encode antigens known to induce immune responses that significantly reduce Campylobacter colonisation of chickens, were characterised by PCR amplification and DNA sequencing.
Campylobacter spp. were isolated from 526 (28%) samples in this study. Forty-one and 26 flaA-HRM genotypes were identified for the C. jejuni (n=406) and C. coli (n=145) isolates, respectively. C. jejuni and C. coli were isolated from the production environment prior to chick placement. C. jejuni and C. coli were first detected in free-range broiler faeces as early as 15 and 10 days of rearing, respectively. Typically, once a few broiler chicks in the flock were positive for C. jejuni or C. coli, all sampled broilers within the same flock were later found to be colonised with multiple genotypes of C. jejuni and/or C. coli within one week. Very few C. jejuni and C. coli flaA-HRM genotypes (n=3) were shared between free-range broiler chicks and their parental breeder flocks.
Four genes, katA, cadF, peb1A and cjaA, encoding protective antigens were found to be present in the genomes of the dominant C. jejuni and C. coli flaA-HRM genotypes identified in this study. These conserved genes were expressed in both prokaryotic and eukaryotic systems (Escherichia coli cells and Vero cells). Different levels of protein expression in each system were observed for each antigen. In E. coli cells, the expression of KatA was highest, while Peb1A expression was lowest. In contrast, the expression of KatA was the low and Peb1A was high in Vero cells.
The results of the current study have enhanced the understanding of the timing, potential sources, and genetic diversity of Campylobacter colonisation in free-range broiler farms. There was minimal evidence to indicate the spread of Campylobacter by vertical transmission between layers and broiler chickens. Rather, the results suggested some birds initially acquired Campylobacter spp. from the production environment soon after placement. Subsequently, horizontal transmission was the major route of colonisation, leading to the rapid spread of Campylobacter within the free-range broiler flocks in this study.
The results of this study suggest that any intervention in the commercial free-range chicken meat production industry to prevent Campylobacter transmission, such as enhanced biosecurity measures, would need to be implemented early in the broiler growth stage, at the farm level, to be effective. Vaccination was identified as a potential future control method, as genes encoding antigens known to provide significant protection from colonisation were characterised and shown to have high sequence identity, in the isolates from this study. These antigens could underpin the future development of a multivalent vaccine for C. jejuni and C. coli.