Table 1.
Basal diet ingredients and calculated nutrient composition.
Table 2.
Primers and PCR conditions for RTqPCR.
Table 3.
Effect of Campylobacter jejuni challenge on performance parameters.
Fig 1.
Effect of Campylobacter jejuni challenge on Campylobacter colonization kinetics in the ceca, spleen, and liver.
On d14 of age, birds were weight-matched and randomly assigned to two treatments: control and challenge. Birds were orally challenged with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or mock-challenged with 0.5 mL of 0.85% saline. Colonization of the (a) Ceca, (b) Spleen, and (c) Liver was estimated by micro-dilutions method (CFU/g) and then log transformed to log10 CFU/g for statistical analysis. (d) Campylobacter jejuni in the ceca was quantified using SyBr green qPCR with primers targeting C. jejuni mapA gene. Transcript levels were reported as 40—Ct for statistical analysis. Results were expressed as mean + SEM. Non-detectable (ND). *P < 0.05; **P < 0.01 compared with control (n = 6), Student’s t-test.
Fig 2.
Effect of Campylobacter jejuni challenge on serum IgG and bile IgA anti-Campylobacter antibodies.
On d14 of age, birds were weight-matched and randomly assigned to two treatments: control and challenge. Birds were orally challenged with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or mock-challenged with 0.5 mL of 0.85% saline. Specific (a) serum IgY and (b) bile IgA antibodies directed against C. jejuni (Strain A74C) whole cell (WC) antigens were determined by enzyme-linked immunosorbent assay (ELISA). Results were reported as mean + SEM OD 450 values. *P < 0.05; **P < 0.01 compared with control (n = 6), Student’s t-test.
Fig 3.
Effect of Campylobacter jejuni challenge on cecal tonsil CD4+ and CD8+ T lymphocytes, and CD4+:CD8+ cell ratio.
On d14 of age, birds were weight-matched and randomly assigned to two treatments: control and challenge. Birds were orally challenged with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or mock-challenged with 0.5 mL of 0.85% saline. Cecal tonsils were strained to single-cell suspensions (1 × 106 cells) and were incubated with PE-conjugated mouse anti-chicken CD4 and FITC-conjugated mouse anti-chicken CD8 at 1:200 dilution, and unlabeled mouse IgG at 1:500 dilution in a 96-well plate for 20 minutes. (a) CD4+ and CD8+ cells were reported as percentage of gated cells and (b) CD4+/CD8+ ratio was calculated. Results were expressed as mean + SEM. *P < 0.05; **P < 0.01 compared with control (n = 6), Student’s t-test.
Fig 4.
Effect of Campylobacter jejuni challenge on immune gene expression in cecal tonsils at (a) 0 dpi and (c) 1 dpi, and spleen at (b) 0 dpi and (d) 1 dpi.
On d14 of age, birds were weight-matched and randomly assigned to two treatments: control and challenge. Birds were orally challenged with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or mock-challenged with 0.5 mL of 0.85% saline. Transcript levels of genes were determined by real-time RT-qPCR. Results were expressed as the mean + SEM fold change in tissue mRNA levels in the challenge treatment as compared to the mock-challenged control treatment. *P < 0.05; **P < 0.01 compared with control (n = 6), Student’s t-test.
Fig 5.
Effect of Campylobacter jejuni challenge on immune gene expression in cecal tonsils at (a) 3 dpi and (c) 7 dpi, and spleen at (b) 3 dpi and (d) 7 dpi.
On d14 of age, birds were weight-matched and randomly assigned to two treatments: control and challenge. Birds were orally challenged with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or mock-challenged with 0.5 mL of 0.85% saline. Transcript levels of genes were determined by real-time RT-qPCR. Results are expressed as the mean + SEM fold change in tissue mRNA levels in the challenge treatment as compared to the mock-challenged control treatment. *P < 0.05; **P < 0.01 compared with control (n = 6), Student’s t-test.
Fig 6.
Effect of Campylobacter jejuni challenge on immune gene expression in cecal tonsils at (a) 14 dpi and (c) 21 dpi, and spleen at (b) 14 dpi and (d) 21 dpi.
On d14 of age, birds were weight-matched and randomly assigned to two treatments: control and challenge. Birds were orally challenged with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or mock-challenged with 0.5 mL of 0.85% saline. Transcript levels of genes were determined by real-time RT-qPCR. Results are expressed as the mean + SEM fold change in tissue mRNA levels in the challenge treatment as compared to the mock-challenged control treatment. *P < 0.05 **P < 0.01 compared with control (n = 6), Student’s t-test.
Fig 7.
Effect of Campylobacter jejuni challenge on nitric oxide production from adherent splenocyte MNCs stimulated ex vivo.
On d14 of age, birds were weight-matched and randomly assigned to two treatments: control and challenge. Birds were orally challenged with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or mock-challenged with 0.5 mL of 0.85% saline. Adherent splenocyte MNCs (5 x 104 cells) were stimulated with 10 μg/mL of LPS or 20 μg/mL of lysed C. jejuni (CJ). After 48 h of stimulation, NO production was measuring using the Griess assay. Results were expressed as mean + SEM nitrite concentration (μM). Non-detectable (ND). *P < 0.05; **P < 0.01 compared with control (n = 6), Student’s t-test.
Fig 8.
Effect of Campylobacter jejuni challenge on the proliferation of cecal tonsil MNCs stimulated ex vivo.
On d14 of age, birds were weight-matched and randomly assigned to two treatments: control and challenge. Birds were orally challenged with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or mock-challenged with 0.5 mL of 0.85% saline. Cecal tonsil MNCs (5 x 104 cells) were stimulated with 15 μg/mL of Con A or 20 μg/mL of lysed C. jejuni (CJ) on (a) d15 and d17 or Con A (15 μg/mL), CJ (20 μg/mL), 1:10 CJ (2 μg/mL), Salmonella Enteritidis (SE) OMP (20 μg/mL), or 1:10 SE OMP (2 μg/mL) at (b) 7 dpi, (c) 14 dpi, and (d) 21 dpi. The proliferation of MNCs was measured by MTT assay after 72 h of stimulation. Results were expressed as mean + SEM OD 570 values. *P < 0.05; **P < 0.01 compared with control (n = 6), Student’s t-test.