Campylobacter jejuni is the leading cause of bacterial food-borne infection; chicken meat is its main source. C. jejuni is considered commensal in chickens based on experimental models unrepresentative of commercial production. Here we show that the paradigm of Campylobacter commensalism in the chicken is flawed. Through experimental infection of four commercial breeds of broiler chickens, we show that breed has a significant effect on C. jejuni infection and the immune response of the animals, although these factors have limited impact on the number of bacteria in chicken ceca. All breeds mounted an innate immune response. In some breeds, this response declined when interleukin-10 was expressed, consistent with regulation of the intestinal inflammatory response, and these birds remained healthy. In another breed, there was a prolonged inflammatory response, evidence of damage to gut mucosa, and diarrhea. We show that bird type has a major impact on infection biology of C. jejuni. In some breeds, infection leads to disease, and the bacterium cannot be considered a harmless commensal. These findings have implications for the welfare of chickens in commercial production where C. jejuni infection is a persistent problem.

Colibacillosis is an economically important syndromic disease of poultry caused by extra-intestinal avian pathogenic Escherichia coli (APEC) but the pathotype remains poorly defined. Combinations of virulence-associated genes (VAGs) have aided APEC identification. The intestinal microbiota is a potential APEC reservoir. Broiler chickens are selectively bred for fast, uniform growth. Here we simultaneously investigate intestinal E. coli VAG carriage in apparently healthy birds and characterise systemic E. coli from diseased broiler chickens from the same flocks. Four flocks were sampled longitudinally from chick placement until slaughter. Phylogrouping, macro-restriction pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed on an isolate subset from one flock to investigate the population structure of faecal and systemic E. coli. Early in production, VAG carriage among chick intestinal E. coli populations was diverse (average Simpson's D value  = 0.73); 24.05% of intestinal E. coli (n = 160) from 1 day old chicks were carrying ≥5 VAGs. Generalised Linear models demonstrated VAG prevalence in potential APEC populations declined with age; 1% of E. coli carrying ≥5 VAGs at slaughter and demonstrated high strain diversity. A variety of VAG profiles and high strain diversity were observed among systemic E. coli. Thirty three new MLST sequence types were identified among 50 isolates and a new sequence type representing 22.2% (ST-2999) of the systemic population was found, differing from the pre-defined pathogenic ST-117 at a single locus. For the first time, this study takes a longitudinal approach to unravelling the APEC paradigm. Our findings, supported by other studies, highlight the difficulty in defining the APEC pathotype. Here we report a high genetic diversity among systemic E. coli between and within diseased broilers, harbouring diverse VAG profiles rather than single and/or highly related pathogenic clones suggesting host susceptibility in broilers plays an important role in APEC pathogenesis.

Avian Pathogenic Escherichia coli (APEC) is a major pathogen within the poultry industry. However disease, especially in broiler chickens, may be caused by range of E. coli genotypes that carry few, if any, virulence factors associated with APEC. Furthermore, commensal E. coli in the intestines of healthy birds may carry an array of APEC virulence factors suggesting they have potential to cause disease when opportunity arises. Given the diseases caused by APEC, namely colibacillosis and salpingitis peritonitis syndrome, are syndromic in nature and the great diversity of the strains causing disease we suggest it is wrong to consider disease as the result of a single APEC pathotype. Whilst it is clear certain pathogenic E. coli can be considered as APEC, much of the disease-associated with E. coli in domestic poultry is as much a consequence of increased host susceptibility due to stress, immunosuppression, co-infection, or poor welfare. This leads to more “opportunistic” infections rather than the result of infection with a specific pathotype. As such the current use of the term APEC for all cases of E. coli infection in the chicken is fundamentally flawed.

Chickens are the most common birds on Earth and colibacillosis is among the most common diseases affecting them. This major threat to animal welfare and safe sustainable food production is difficult to combat because the etiological agent, avian pathogenic Escherichia coli (APEC), emerges from ubiquitous commensal gut bacteria, with no single virulence gene present in all disease-causing isolates. Here, we address the underlying evolutionary mechanisms of extraintestinal spread and systemic infection in poultry. Combining population scale comparative genomics and pangenome-wide association studies, we compare E. coli from commensal carriage and systemic infections. We identify phylogroup-specific and species-wide genetic elements that are enriched in APEC, including pathogenicity-associated variation in 143 genes that have diverse functions, including genes involved in metabolism, lipopolysaccharide synthesis, heat shock response, antimicrobial resistance and toxicity. We find that horizontal gene transfer spreads pathogenicity elements, allowing divergent clones to cause infection. Finally, a Random Forest model prediction of disease status (carriage vs. disease) identifies pathogenic strains in the emergent ST-117 poultry-associated lineage with 73% accuracy, demonstrating the potential for early identification of emergent APEC in healthy flocks.

Avian pathogenic Escherichia coli (APEC) are a substantial burden to the global poultry industry. APEC cause a syndromic poultry infection known as colibacillosis, which has been previously associated with broiler chickens over 2 weeks old. We recently reported that the intestinal tract of 1-day-old broilers harbours a rich reservoir of potentially pathogenic E. coli. Prior infections of the reproductive tract of breeders, egg hygiene and transportation all contribute to early colonization of the neonatal gut. Up to one-half of all flock deaths occur in the first week of production, but few data are available describing the contribution of E. coli. In the present study, all dead birds collected on the first daily welfare walk 48 and 72 h after chick placement underwent post-mortem examination. Diseased tissues were selectively cultured for E. coli and isolates subsequently virulotyped using 10 APEC virulence-associated genes (VAGs): astA, iss, irp2, iucD, papC, tsh, vat, cvi, sitA and ibeA. Approximately 70% of birds displayed signs of colibacillosis. Thirty distinct virulence profiles were identified among 157 E. coli. Isolates carried between zero and seven VAGs; ∼ 30% of E. coli isolates carried five to seven VAGs, with 12.7% sharing the same VAG profile (astA, iss, irp2, iucD, tsh, cvi and sitA). Overall, this study demonstrates the significant contribution of E. coli infections to early broiler mortalities. The identification of a diverse E. coli population is unsurprising based on our previous findings. This work emphasizes the need for an effective vaccination programme and provides preliminary data for vaccine production.

bAlthough multiple genotypes of Campylobacter jejuni may be isolated from the same commercial broiler flock, little is known about the infection dynamics of different genotypes within individuals or their colonization sites within the gut. Single experimental infections with C. jejuni M1 (sequence type 137, clonal complex 45) and C. jejuni 13126 (sequence type 21, clonal complex 21) revealed that 13126 colonized the ceca at significantly higher levels. The dissemination and colonization sites of the two C. jejuni strains then were examined in an experimental broiler flock. Two 33-day-old broiler chickens were infected with M1 and two with 13126, and 15 birds were left unchallenged. Cloacal swabs were taken postinfection to determine the colonization and shedding of each strain. By 2 days postinfection (dpi), 8/19 birds were shedding M1 whereas none were shedding 13126. At 8 dpi, all birds were shedding both strains. At 18 dpi, liver and cecal levels of each isolate were quantified, while in 10 birds they also were quantified at nine sites throughout the gastrointestinal (GI) tract. 13126 was found throughout the GI tract, while M1 was largely restricted to the ceca and colon. The livers of 7/19 birds were culture positive for 13126 only. These data show that 13126 has a distinctly different infection biology than strain M1. It showed slower colonization of the lower GI tract but was more invasive and able to colonize at a high level throughout the GI tract. The finding that C. jejuni strains have markedly different infection ecologies within the chicken has implications for control in the poultry industry and suggests that the contamination risk of edible tissues is dependent on the isolate involved.

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A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22238-5