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 GEM (Gram-positive Enhancer Matrix) 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 CjaAD, which is CjaA presenting CjaD epitopes on its surface, was employed as a model antigen. We found that CjaAD administered in ovo at embryonic development day 18 by both delivery systems resulted in significant levels of protection after challenge with a heterologous Campylobacter 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

Functional and bioinformatics analysis of two Campylobacter jejuni homologs of the thiol-disulfide oxidoreductase, DsbA.

BACKGROUND: Bacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world. METHODS AND RESULTS: Phylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon. CONCLUSIONS: Our results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far

Harde demonstraties versus een gastvrij welkom voor vluchtelingen: Een casestudy over burgerparticipatie omtrent de noodopvang van vluchtelingen en de effecten hiervan op de houding van de burgers.

<p>The diagrams illustrate mean values and standard deviations of AstA activity derived from three experiments; for each experiment the AstA activity were carried out in triplicate. Statistical significance was calculated using Student <i>t</i> test for comparison of independent groups (GraphPad Prism) with reference to the AstA activity in the wild type (WT) strain. P values of P<0.05 were considered statistically significant (*).</p

Autoagglutination of <i>C. jejuni</i> 81116 strains: wild type (WT), <i>cjdsbA1^-</i>, <i>cjdsbA2^-</i>, <i>cjdsbB^-</i> and <i>cjdsbI^-</i> mutants.

<p>Bacterial autoagglutination was monitored as a decrement of turbidity (A) or optical density (B) of bacterial suspension in LB at room temperature after harvesting cells from BA plates. The <i>cjdsbA1^-</i> strain does not autoagglutinate, contrary to the wild type (WT), <i>cjdsbA2^-</i>, <i>cjdsbB^-</i> and <i>cjdsbI^-</i> strains. The figure presents a representative result.</p

Taxonomic distribution of the DsbA DsbB/I and AstA in gamma-Proteobacteria and epsilon-Proteobacteria.

<p>Color boxes indicate the presence of the classical DsbA and DsbB (two inner rings), DsbA1-2, DsbB2, AstA, and DsbI (four outer rings) in a given genome.</p

Alkaline phosphatase PhoX activity in <i>C. jejuni</i> 81116 strains: wild type (WT), <i>cjdsbA1^-</i>, <i>cjdsbA2^-, cjdsbB^-</i> and <i>cjdsbI^-</i> mutants.

<p>The diagrams illustrate mean values and standard deviations of PhoX activity derived from three experiments; for each experiment the PhoX activity were carried out in triplicate. Statistical significance was calculated using Student t test for comparison of independent groups (GraphPad Prism) with reference to the PhoX activity in the wild type (WT) strain. P values of P<0.05 were considered statistically significant (*).</p

Oligonucleotides used in the present study.

<p><b>Bold letters</b> indicate <i>C. jejuni</i> nucleotide sequences; restriction recognition sites introduced for cloning purposes are <u>underlined</u>, complementary fragments of primers Cj872up and Cj872dw are marked with <i>italics</i>. Most primers were based on the <i>C. jejuni</i> 81116 nucleotide sequence, but for some experiments, previously designed primers based on the <i>C. jejuni</i> NCTC or 81–176 nucleotide sequences were used, or primers were designed to introduce point mutations. Their single pair mismatches with <i>C. jejuni</i> 81116 are <u>double underlined</u>.</p><p>Oligonucleotides used in the present study.</p

Homology models of <i>C. jejuni</i> DsbA1 and DsbA2.

<p><i>C. jejuni</i> DsbA1 and DsbA2 (CjDsbA1 and CjDsbA2) models built on <i>E. coli</i> DsbA [EcDsbA (PDB ID: 2ZUP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106247#pone.0106247-Inaba1" target="_blank">[80]</a>)] and DsbL [EcDsbL (PDB ID: 3C7M <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106247#pone.0106247-Grimshaw1" target="_blank">[22]</a>)], experimentally characterized members of the DsbA superfamily. Structural representations are shown in ribbon view (A, D, G and J). Electrostatic surfaces coloured by charge from red, acidic, -1kT to blue, basic, +1kT. The orientation in B, E, H and K follows the orientation in the top row (A, D, G and J) and in C, F, I and L is rotated by 130 degrees around the vertical axis, clockwise.</p