Fig. 5

Prey-dependent depletion of C. jejuni in chickens maintained on bile salt supplementation. (a) Schematic of in vivo chickens feeding trial. After 7 days of acclimatization, birds were maintained in a bile salt (0.2%, w/v) containing diet till day 30. Chickens fed with a normal diet without bile salt were kept in control. From day 14 onwards, experimental birds were fed with either C. jejuni or C. jejuni and E. coli at the indicated time points (black circle). On day 35 (open circle), birds were sacrificed, and cecum, its content, and intestinal lavages were collected for the analysis of bacterial load, anti-C. jejuni antibody response, transcriptional profiles of pro-inflammatory cytokines, and cecal tissue histopathological study. The experimental group details were as follows: Group A: C. jejuni only (no bile salt); Group B: C. jejuni + E. coli (no bile salt); Group C: C. jejuni + bile salt and Group D: C. jejuni + E. coli + bile salt. Parallel with this, in vivo feeding with T6SS-negative C. jejuni under similar experimental conditions was performed (n = 9 birds for each group). (b) Cecal load of C. jejuni showing effective clearance of C. jejuni in Group D birds compared to the other experimental groups. No such difference was observed when T6SS-negative C. jejuni was used. (c) Comparative analysis of local antibody (sIgA) responses in the intestinal lavage against C. jejuni indicates a significantly low level of sIgA titer in the birds belonging to Group D compared to the other experimental groups. Reduced sIgA titer in these birds suggests prey-dependent clearance of T6SS-positive C. jejuni in the presence of bile salt. However, no such difference was noticed when the experiment was performed with T6SS-negative C. jejuni. (d) Pro-inflammatory cytokine gene expression profile of caecal tissue collected from different groups of birds indicates low-level expression of IL-1β, IL-8, IL-17A and IL-6 genes in Group D compared to the other experimental groups. (e) Histopathological changes in cecal tissue from different experiment groups. Panels i-iii show higher lesion scores characterized by necrotic lesions (arrow), lymphocytic infiltration (block arrow), disruption in the top layer of epithelium, and unorganized cell boundaries. Panel-iv displays perfectly oriented, continuous, and well-demarked surface epithelium