Campylobacter jejuni is the leading bacterial cause of human enteritis in developed countries. Chicken is the major animal reservoir of C. jejuni and a powerful infection model for human campylobacteriosis. No commercial vaccine against C. jejuni is available to date. The high affinity iron acquisition mediated through enterobactin (Ent), a small siderophore, plays a critical role in the colonization of C. jejuni in the intestine. Recently, an innovative Ent conjugate vaccine has been demonstrated to induce high-level of Ent-specific antibodies in rabbits; the Ent-specific antibodies displayed potent binding ability to Ent and inhibited Ent-dependent growth of C. jejuni. In this study, using specific-pathogen-free (SPF) chickens, we performed three trials to evaluate the immunogenicity of the Ent conjugate vaccine and its efficacy to control C. jejuni colonization in the intestine. The purified Ent was conjugated to the carrier keyhole limpet hemocyanin (KLH). Intramuscular immunization of chickens with the Ent–KLH conjugate for up to three times did not affect the body weight gain, the development of major immune organs and the gut microbiota. In the first two trials, immunizations of chickens with different regimens (two or three times of vaccination) consistently induced strong Ent-specific immune response when compared to control group. Consistent with the high-level of systemic anti-Ent IgG, C. jejuni colonization was significantly reduced by 3–4 log₁₀ units in the cecum in two independent vaccination trials. The third trial demonstrated that single Ent–KLH vaccination is sufficient to elicit high level of systemic Ent-specific antibodies, which could persist for up to eight weeks in chickens. Taken together, the Ent–KLH conjugate vaccine could induce high-level of Ent-specific antibodies in chickens and confer host protection against C. jejuni colonization, which provides a novel strategy for Campylobacter control in poultry and humans.

Enterobactin (Ent) is a highly conserved and important siderophore for the growth of many Gram-negative bacterial pathogens. Therefore, targeting Ent for developing innovative intervention strategies has attracted substantial research interest in recent years. Recently, we developed a novel Ent conjugate vaccine that has been demonstrated to be effective for controlling Gram-negative pathogens using both in vitro and in vivosystems. In particular, active immunization of chickens with the Ent conjugate vaccine elicited strong immune responses and significantly reduced intestinal colonization of Campylobacter jejuni, the leading foodborne bacterial pathogen. Given that hyperimmune egg yolk immunoglobulin Y (IgY) has been increasingly recognized as a promising and practical non-antibiotic approach for passive immune protection against pathogens in livestock, in this study, we assessed the efficacy of oral administration of broiler chickens with the anti-Ent hyperimmune egg yolk powder to control C. jejuni colonization in the intestine. However, supplementation of feed with 2% (w/w) of anti-Ent egg yolk powder failed to reduce C. jejuni colonization when compared to the control group. Consistent with this finding, the ELISA titers of the specific IgY in cecum, ileum, duodenum, gizzard, and serum contents were similar between the two groups throughout the trial. Chicken intestinal microbiota also did not change in response to the egg yolk powder treatment. Subsequently, to examine ex vivo stability of the egg yolk IgY, the chicken gizzard and duodenum contents from two independent sources were spiked with the egg yolk antibodies, incubated at 42 °C for different lengths of time, and subjected to ELISA analysis. The specific IgY titers were dramatically decreased in gizzard contents (up to 2048-fold) but were not changed in duodenum contents. Collectively, oral administration of broiler chickens with the anti-Ent egg yolk powder failed to confer protection against intestinal colonization of C. jejuni, which was due to instability of the IgY in gizzard contents as demonstrated by both in vivo and ex vivo evidence.

Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is an important infectious disease in chickens and a major cause of mortality in young chicks. Therefore, protecting young chickens from colibacillosis is important for improving welfare and productivity in the poultry industry. Recently, we developed a novel enterobactin (Ent) conjugate vaccine that could induce high titers of anti-Ent immunoglobulin Y (IgY) in chicken serum and consequently mitigate the organ lesions caused by APEC infection. Considering that maternal immunization is a practical approach to confer instant immune protection to the hatchlings, in this study, we immunized breeder hens with the Ent conjugate vaccine and evaluated the maternal immune protection on the progenies challenged with APEC. Three doses of the vaccine induced high titers of anti-Ent IgY in the hens (about 16- and 64-fold higher than the control group in the sera and egg yolks, respectively), resulting in an eight-fold of increase in anti-Ent IgY in the sera of progenies. However, the anti-Ent maternal immunity did not display significant protection against APEC challenge in the young chicks as there was no significant difference in APEC load (in liver, lung, and spleen) or organ lesions (in heart, liver, spleen, lung, and air sac) between the vaccinated and control groups. In future studies, the APEC infection model needs to be optimized to exhibit proper pathogenicity of APEC, and the maternal immunization regimen can be further improved to boost the maternally derived anti-Ent IgY in the hatchlings.