In vitro characteristics of Lactobacillus spp. strains isolated from the chicken digestive tract and their role in the inhibition of Campylobacter colonization.

Campylobacter jejuni/coli infections are the leading cause of bacterial diarrheal illnesses in humans. Many epidemiological studies indicate that improperly prepared meat from chickens that carry a high load of Campylobacter in their intestinal tracts is the key source of human infections. LAB, mainly members of the Lactococcus and Lactobacillus genera, increasingly have been tested as vehicles for the delivery of heterologous bacterial or viral antigens to animal mucosal immune systems. Thus, the objective of this study was to isolate, identify, and characterize Lactobacillus spp. strains isolated from chickens bred in Poland. Their ability to decrease the level of bird gut colonization by C. jejuni strain was also analyzed. First, the influence of the different chicken rearing systems was evaluated, especially the effect of diets on the Lactobacillus species that colonize the gut of chickens. Next, selected strains were analyzed in terms of their anti-Campylobacter activity in vitro; potential probiotic traits such as adhesion properties, bile and low pH tolerance; and their ability to grow on a defined carbon source. Given that improperly prepared chicken meat is the main source of human infection by Campylobacter, the selected strains were also assessed for their ability to inhibit Campylobacter colonization in the bird's intestine. These experiments revealed enormous physiological diversity among the Lactobacillus genus strains. Altogether, our results showed that L. plantarum strains isolated from the digestive tracts of chickens bred in Poland displayed some probiotic attributes in vitro and were able to decrease the level of bird gut colonization by C. jejuni strain. This suggests that they can be employed as vectors to deliver Campylobacter immunodominant proteins to the bird's immune system to strengthen the efficacy of in ovo vaccination.

Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis.

Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein - CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type C. jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analyzed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ Lactic Acid Bacteria (LAB) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to deliver heterologous antigens to the bird immune system. Additionally, the analysis of the structure and immunogenicity of the generated rCjaAD hybrid protein showed that the CjaA antigen can be considered as a starting point to construct multiepitope anti-Campylobacter vaccines.

Chicken Anti-Campylobacter Vaccine - Comparison of Various Carriers and Routes of Immunization.

Campylobacter spp, especially the species Campylobacter jejuni, are important human enteropathogens responsible for millions of cases of gastro-intestinal disease worldwide every year. C. jejuni is a zoonotic pathogen, and poultry meat that has been contaminated by microorganisms is recognized as a key source of human infections. Although numerous strategies have been developed and experimentally checked to generate chicken vaccines, the results have so far had limited success. In this study, we explored the potential use of non-live carriers of Campylobacter antigen to combat Campylobacter in poultry. First, we assessed the effectiveness of immunization with orally or subcutaneously delivered Gram-positive Enhancer Matrix (GEM) particles carrying two Campylobacter antigens: CjaA and CjaD. These two immunization routes using GEMs as the vector did not protect against Campylobacter colonization. Thus, we next assessed the efficacy of in ovo immunization using various delivery systems: GEM particles and liposomes. The hybrid protein rCjaAD, which is CjaA presenting CjaD epitopes on its surface, was employed as a model antigen. We found that rCjaAD administered in ovo at embryonic development day 18 by both delivery systems resulted in significant levels of protection after challenge with a heterologous C. jejuni strain. In practice, in ovo chicken vaccination is used by the poultry industry to protect birds against several viral diseases. Our work showed that this means of delivery is also efficacious with respect to commensal bacteria such as Campylobacter. In this study, we evaluated the protection after one dose of vaccine given in ovo. We speculate that the level of protection may be increased by a post-hatch booster of orally delivered antigens.