Generation of an inactivated vaccine for avian pathogenic Escherichia coli using microarrays: A more rational approach to inactivated vaccine design.

Background: Escherichia coli remains a major pathogen of poultry. Most vaccines are inactivated and produced empirically. Although inactivated Salmonella vaccines have been produced by culture under conditions of Fe deprivation, no vaccines have been produced which are likely to express all the proteins expressed during infection of antigen-presenting cells. Aim: The aim was to produce a more protective inactivated vaccine by culturing the avian E. coli in a synthetic medium that resembled the environment of the phagolysosome. Methods: Global gene expression in a pathogenic avian O78:K80 strain of E. coli, harvested from infected avian macrophage-like HD11 cells, was compared by microarray with bacteria cultured in a tissue culture medium. A liquid synthetic medium was produced based on the environmental conditions identified to which the bacteria were exposed intracellularly. A bacterin was produced from this strain and its protective ability was assessed in chickens. Results: The changes in E. coli gene expression observed included the use of different electron acceptors and carbon sources such as ethanolamine, ß-glucosides, galactonate, dicarboxylic acids, and amino acids, up-regulation of genes associated with Fe and Mn uptake, and up-regulation of type-1 and curli fimbriae, other adhesion genes and down-regulation of sialic acid synthesis genes. The bacterin produced in the synthetic medium was statistically more protective than a bacterin prepared from bacteria cultured in the nutrient broth when tested in vaccinated chickens challenged with a different virulent E. coli O78:K80 strain. Conclusion: The approach of using gene expression to produce synthetic media for the generation of more effective bacterins could be used for a number of intracellular bacteria pathogens including Enteroinvasive E. coli, Salmonella, and the Pasteurella/Riemerella/Mannheimia group of organisms.

A flagellated motile Salmonella Gallinarum mutant (SG Fla+) elicits a pro-inflammatory response from avian epithelial cells and macrophages and is less virulent to chickens.

Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum (SG) is a non-flagellated bacterium which causes fowl typhoid, a systemic disease associated with high mortality in birds. It has been suggested that the absence of flagella in SG is advantageous in the early stages of systemic infection through absence of TLR-5 activation. In order to investigate this hypothesis in more detail a flagellated and motile SG mutant (SG Fla(+)) was constructed. The presence of flagella increased invasiveness for chicken kidney cells (CKC) while its presence did not alter survival in HD11 macrophages. SG Fla(+) induced higher levels of CXCLi2, IL-6 and iNOS mRNA expression in CKC than the SG parent strain. The expression of genes responsible for immune response mediators in infected HD11 macrophages were not related to the presence of flagella. Mortality rates were lower in birds challenged with SG Fla(+) when compared with the SG parent. SG Fla(+) was recovered from caecal contents which showed pathological changes suggestive of inflammation and suggested increased colonization ability.