Innate immune gene expression differentiates the early avian intestinal response between Salmonella and Campylobacter.

Salmonella enterica serovar Typhimurium and Campylobacter jejuni are major human pathogens, yet colonise chickens without causing pathology. The aim of this study was to compare intestinal innate immune responses to both bacterial species, in a 4-week-old broiler chicken model. Challenged and control birds were sacrificed and tissue samples taken for histopathology and RNA extraction. No significant clinical or pathological changes were observed in response to infection with either bacterial species. Expression of selected genes involved in pathogen detection and the innate immune response were profiled in caecal tissues by quantitative real-time PCR. TLR4 and TLR21 gene expression was transiently increased in response to both bacterial species (P<0.05). Significant increases in TLR5 and TLR15 gene expression were detected in response to S. Typhimurium but not to C. jejuni. Transient increases of proinflammatory cytokine (IL6 and IFNG) and chemokine (IL8 and K60) genes increased as early as 6h in response to S. Typhimurium. Minimal cytokine gene expression was detected in response to C. jejuni after 20h. IL8 gene expression however, was significantly increased by 24-fold (P<0.01). The differential expression profiles of innate immune genes in both infection models shed light on the tailored responses of the host immune system to specific microbes. It is further evidence that innate regulation of these responses is an important prerequisite to preventing development of disease.

Comparative in vivo infection models yield insights on early host immune response to Campylobacter in chickens.

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian beta-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter.

The PhoQ-activating potential of antimicrobial peptides contributes to antimicrobial efficacy and is predictive of the induction of bacterial resistance.

Antimicrobial peptides (AMPs) are among the leading candidates to replace antibiotics which have been rendered ineffective by the evolution of resistant bacterial strains. Concerns do exist, however, that the therapeutic administration of AMPs may also select for resistant strains but with much more dire consequences, as these peptides represent an endogenous and essential component of host immune defense. The recent demonstration that AMPs function as ligands for the bacterial sensory kinase PhoQ for the initiation of virulence and adaptive responses lends credence to these concerns. While the ability to serve as PhoQ ligands suggests that the therapeutic administration of AMPs could (i) exacerbate infections by promoting bacterial virulence and (ii) select resistant mutants by encouraging adaptive behaviors, it also provides a rational basis for AMP selection and optimization. Here, we demonstrate that derivatives of a representative AMP have differential abilities to serve as PhoQ ligands and that this correlates with the ability to induce bacterial adaptive responses. We propose that PhoQ-activating potential is a logical parameter for AMP optimization and introduce a novel strategy for the treatment of minimal bactericidal concentration data that permits the discrimination and quantification of the contributions of PhoQ-activating potential and direct antimicrobial activity to net antimicrobial efficiency.

Effect of cytolethal distending toxin of Campylobacter jejuni on adhesion and internalization in cultured cells and in colonization of the chicken gut.

Campylobacter jejuni produces cytolethal distending toxin (CDT) that causes host cells to arrest during their cell cycle and that is involved in the pathogenesis of inflammatory diarrhea in humans. To assess the role of CDT in adherence and invasion of different cultured host cells (HeLa and HD-11) and in colonization of the chicken intestine, the genes of C jejuni NCTC11168 encoding the toxin subunits (cdtA, cdtB, and cdtC) were inactivated by insertional mutagenesis. No significant difference was found in adhesion of the wild-type C. jejuni and the isogenic mutants to HeLa and HD-11 cells. All of the mutants exhibited a decrease (>10-fold) in the ability to invade HeLa cells, but no significant difference was noticed for HD-11 cells. The ability of mutants to colonize birds either directly or by horizontal transfer was unchanged. These data indicated that although the production of cytotoxin does not play a role in the adherence to either human or avian cells, it may play a role in the invasion, survival, or both of C. jejuni in human cells, which are more susceptible to C. jejuni internalization. The CDT also does not seem to play a role in the colonization of poultry.