lipid-a and Poultry-Diseases

Lipopolysaccharide (LPS) is a major surface component of avian pathogenic Escherichia coli (APEC), and is a possible virulence factor in avian infections caused by this organism. The contribution of the lpxM gene, which encodes a myristoyl transferase that catalyzes the final step in lipid A biosynthesis, to the pathogenicity of APEC has not previously been assessed. In this study, an isogenic lpxM mutant, E058ΔlpxM, was constructed in APEC O2 strain E058 and then characterized. Structural analysis of lipid A from the parental strain and derived mutant showed that E058ΔlpxM lacked one myristoyl (C14:0) on its lipid A molecules. No differences were observed between the mutant and wild-type in a series of tests including growth rate in different broths and ability to survive in specific-pathogen-free chicken serum. However, the mutant showed significantly reduced invasion and intracellular survival in the avian macrophage HD11 cell line (P<0.05). Nitric oxide production reduction (P<0.05) and cytokine gene expression downregulation (P<0.05 or P<0.01) also showed in HD11 treated with E058ΔlpxM-derived LPS compared with that in cells treated with E058-derived LPS at different times. Compared to the parental strain E058, E058ΔlpxM had a significant reduction in bacterial load in heart (P<0.01), liver (P<0.01), spleen (P<0.01), lung (P<0.05), and kidney (P<0.05) tissues. The histopathological lesions in visceral organs of birds challenged with the wild-type strain were more severe than in birds infected with the mutant. However, the E058ΔlpxM mutant showed a similar sensitivity pattern to the parental strain following exposure to several hydrophobic reagents. These results indicate that the lpxM gene is important for the pathogenicity and biological activity of APEC strain E058.

Topics: Acyltransferases; Animals; Biosynthetic Pathways; Chickens; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Lipid A; Poultry Diseases; Specific Pathogen-Free Organisms; Virulence

The immunisation of mammals with LPS- and lipid A-antigens leads to the production of specific antibodies. In the present study, we describe the generation and preparation of antibodies from egg yolks of immunized chickens. Egg yolk antibodies were raised by immunizing laying hens with five LPS- and three lipid A-preparations from various gram-negative bacteria species in different immunisation protocols. Antibodies from collected egg yolks were extracted and purified by means of several standard methods. The purity of antibody-preparations was measured and compared by SDS-PAGE. Specific antibodies were assayed by two different EIA procedures. It could be shown that the immunisation of hens with LPS- and lipid A-Antigens resulted in the production of specific egg yolk antibodies, regardless of immunisation-scheme and amount of antigen.

Topics: Animals; Antibodies, Bacterial; Antibody Formation; Antibody Specificity; Bacterial Vaccines; Chickens; Egg Yolk; Female; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Immunoglobulins; Lipid A; Lipopolysaccharides; Oviposition; Poultry Diseases

Various preparations of egg yolk antibodies against different endotoxins of gram-negative bacteria were characterized with regard to their immunological properties. To do this, we investigated the reactivity of antibodies against a number of lipopolysaccharides and lipid-A by enzyme-immuno-assays and immunoblot. It could be shown that all antibody preparations contained specific antibodies, reactive with their homologous antigen. Furthermore these antibodies showed cross-reactivity with structural diverse LPS- and lipid A-antigens from different sources. Anti lipid A-antibodies appeared to be highly crossreactive with purified LPS and lipid A from several gram-negative organisms. Egg yolk antibodies raised by immunization with LPS showed cross-reactivity with enterobacterial LPS and only marginal reactivity with both LPS from other gram-negative bacteria and lipid A. The results from immunoblot experiments confirmed our findings from EIA-studies.

Topics: Animals; Antibody Formation; Antibody Specificity; Chickens; Cross Reactions; Egg Yolk; Enterobacteriaceae; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Immunoblotting; Immunoenzyme Techniques; Immunoglobulins; Lipid A; Lipopolysaccharides; Poultry Diseases