Comparative Effectiveness of Various Multi-Antigen Vaccines in Controlling Campylobacter jejuni in Broiler Chickens

Abstract

This study was undertaken to evaluate and compare the efficacy of different multi-antigen vaccines, including heat-inactivated, whole lysate, and subunit (outer membrane proteins [OMPs]) C. jejuni vaccines along with the immunostimulant CpG ODN in controlling Campylobacter colonization in chickens. In the first trial, 125 μg of C. jejuni OMPs and 50 μg of CpG ODN were administered individually or in combination, either in ovo to chick embryos or subcutaneously (SC) to one-day-old chicks. In the second trial, different concentrations of C. jejuni antigens (heat-killed, whole lysate, and OMPs) were administered SC to one-day-old chicks. The results of the first trial revealed that SC immunization with the combination of CpG ODN and C. jejuni OMPs elevated interferon (IFN)-γ, interleukin (IL)-1β, and IL-13 gene expression in the spleen, significantly increased serum IgM and IgY antibody levels, and reduced cecal C. jejuni counts by approximately 1.2 log₁₀. In contrast, in ovo immunization did not elicit immune responses or confer protection against Campylobacter. The results of the second trial showed that SC immunization with C. jejuni whole lysate or 200 μg OMPs reduced C. jejuni counts by approximately 1.4 and 1.1 log₁₀, respectively. In conclusion, C. jejuni lysate and OMPs are promising vaccine antigens for reducing Campylobacter colonization in chickens.

Keywords: Campylobacter; CpG ODN; chickens; in ovo; outer membrane protein; vaccine.

Figure 1

Illustration of first experimental design. On embryonic day 18, 136 eggs were randomly divided into four groups (G1–G4), each containing 34 eggs. Embryos of each group were injected intra-amniotic with the assigned immunization: 50 µg CpG ODN or 125 µg C. jejuni OMPs or their combination or PBS (negative control group). On the first day post-hatch, chicks (136) of non-immunized eggs were randomly allocated into four groups (G5–G8) and immunized SC with 50 µg CpG ODN or 125 µg C. jejuni OMPs or their combination or PBS. All chicks received the booster vaccination on day seven, and all groups were challenged with 10⁷ CFUs of C. jejuni on day 14. Bursa of Fabricius and spleens were collected for three successive days (n = 8) post-initial vaccination of either SC or in ovo immunized groups. Blood samples were collected weekly, and the cecal contents were collected on day 35 of age (the end of the experiment).

Figure 2

Illustration of the second experimental design. On the first day post-hatch, chicks were immunized SC with C. jejuni OMPs (50, 125 or 200 μg) or C. jejuni lysate; low (21.5 μg) or high (43 μg) protein, or heat-killed (10⁶ or 10⁷ CFUs of C. jejuni). All chicks received the booster vaccination on day 14 of age. The control group was injected at the same age with PBS only. All groups were challenged with 10⁷ CFUs of C. jejuni on day 15 of age. Blood samples were collected weekly, and the cecal contents were collected at the fifth week at the end of the experiment.

Figure 3

C. jejuni CFUs per gram of the cecal content. After primary immunization in ovo or SC on day one of age and booster immunization on day seven of age, chicks in all groups were orally challenged with 10⁷ CFUs of C. jejuni on day 14 of age. At 35 days of age (21 days post-challenge), cecal contents were collected for Campylobacter enumeration. Bars marked with different letters (a–c) indicate significant differences (p < 0.05) between the groups, while bars marked with the same letter denote no significant differences between the groups. OMPs = outer membrane proteins. SC = subcutaneous. ODN = synthetic single-stranded oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs.

Figure 4

Serum IgY antibody levels. Chicks were immunized in ovo or SC with 125 µg C. jejuni OMPs or 50 µg CpG ODN or their combination or PBS. A booster dose was delivered orally (for those primed in ovo) or SC (for those primed SC) on day seven of age. All the chicks were then orally challenged with 10⁷ CFUs of C. jejuni on day 14 of age. Blood samples were collected weekly from all groups, starting from the first week of age through the fifth week of age, with the sera subsequently separated for measuring the IgY antibody (Ab) levels using ELISA. Bars marked with different letters (a–b) indicate significant differences (p < 0.05) between the groups, while bars marked with the same letter denote no significant differences between the groups. OMPs = outer membrane proteins. SC = subcutaneous. ODN = synthetic single-stranded oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs.

Figure 5

The correlation between C. jejuni CFUs and serum antibody (Ab) levels using Pearson’s r correlation coefficient. No correlation was observed between the serum IgY Ab levels and cecal counts of C. jejuni in the group immunized SC with the combination of 50 µg CpG ODN and 125 µg OMPs at the fifth week of age.

Figure 6

Serum IgM antibody levels. Chicks were immunized in ovo or SC with 125 µg C. jejuni OMPs or 50 µg CpG ODN or their combination or PBS. A booster dose was delivered orally (for those primed in ovo) or SC (for those primed SC) on day seven of age. All the chicks were then orally challenged with 10⁷ CFUs of C. jejuni on day 14 of age. Blood samples were collected weekly from all groups, starting from the first week of age through the fifth week of age, with the sera subsequently separated for measuring the IgM antibody (Ab) levels using ELISA. Bars marked with different letters (a–c) indicate significant differences (p < 0.05) between the groups, while bars marked with the same letter denote no significant differences between the groups. OMPs = outer membrane proteins. SC = subcutaneous. ODN = synthetic single-stranded oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs.

Figure 7

The correlation between C. jejuni CFUs and serum antibody (Ab) levels using Pearson’s r correlation coefficient. No correlation was observed between the serum IgM Ab levels and cecal counts of C. jejuni in the group immunized with the combination of 50 µg CpG ODN and 125 µg OMPs at the fifth week of age.

Figure 8

Relative gene expression of interferon (IFN)-γ (a), interleukin (IL)-13 (b), and IL-1β (c) in the spleen at 24, 48, and 72 h following SC immunization with 125 µg C. jejuni OMPs or 50 µg CpG ODN or their combination or PBS. Data are presented as mean expression (delta CT values) of cytokine mRNA relative to β-actin (housekeeping gene) ± standard error of the mean (SEM). Statistical significance among treatment groups was calculated using 1-way ANOVA followed by Tukey’s comparison test. Bars marked with different letters (a,b) indicate significant differences (p < 0.05) between the groups, while bars marked with the same letter denote no significant differences between the groups. OMPs = outer membrane proteins. SC = subcutaneously. ODN = synthetic single-stranded oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs.

Figure 9

Relative gene expression of interferon (IFN)-γ (a), interleukin (IL)-13 (b), and IL-1β (c) in the spleen at 24, 48, and 72 h following in ovo immunization with 125 µg C. jejuni OMPs or 50 µg CpG ODN or their combination or PBS. Data are presented as mean expression (delta CT values) of cytokine mRNA relative to β-actin (housekeeping gene) ± standard error of the mean (SEM). Statistical significance among treatment groups was calculated using 1-way ANOVA followed by Tukey’s comparison test. Bars marked with different letters (a,b) indicate significant differences (p < 0.05) between the groups, while bars marked with the same letter denote no significant differences between the groups. OMPs = outer membrane proteins. SC = subcutaneously. ODN = synthetic single-stranded oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs.

Figure 10

C. jejuni CFUs per gram of the cecal content. Day-old chicks were immunized SC with different doses of heat-killed or whole lysate or OMPs of C. jejuni and boosted SC with the corresponding vaccine on day 14 of age. On day 15 of age, chicks in all groups were orally challenged with 10⁷ CFUs of C. jejuni. On day 35 of age (20-day post-challenge), cecal contents were collected for Campylobacter enumeration. Bars marked with different letters (a–c) indicate significant differences (p < 0.05) between the groups, while bars marked with the same letter denote no significant differences between the groups. OMPs = outer membrane proteins. SC = subcutaneous.

Figure 11

Serum IgY antibody levels. On days one and fourteen of age, chicks were immunized SC with different doses of C. jejuni OMPs (50, 125 or 200 μg) or C. jejuni lysate (21.5 μg or 43 μg) or heat-killed C. jejuni (10⁶ or 10⁷ CFUs). The control group was injected at the same age with PBS only. On day 15 of age, chickens were challenged orally with 10⁷ CFUs of C. jejuni. Blood samples were collected weekly from all groups, starting from the first week of age through the fifth week of age, with the sera subsequently separated for measuring the IgY antibody (Ab) levels using ELISA. Bars that are marked with different letters (a–c) indicate significant differences (p < 0.05). OMPs = outer membrane proteins. SC = subcutaneous.

Figure 12

Serum IgM antibody levels. On days one and fourteen of age, chicks were immunized SC with different doses of C. jejuni OMPs (50, 125 or 200 μg), C. jejuni lysate (21.5 μg or 43 μg) and heat-killed C. jejuni (10⁶ or 10⁷ CFUs). The control group was injected at the same age with PBS only. On day 15 of age, chickens were challenged orally with 10⁷ CFUs of C. jejuni. Blood samples were collected weekly from all groups, starting from the first week of age through the fifth week of age, with the sera subsequently separated for measuring the IgM antibody (Ab) levels using ELISA. Bars marked with different letters (a–b) indicate significant differences (p < 0.05) between the groups, while bars marked with the same letter denote no significant differences between the groups. OMPs = outer membrane proteins. SC = subcutaneous.