The Role Of Lipopolysaccharide Biosynthesis Related Gene LpsA In CH-1 Strain Of Riemerella Anatipestifer

Riemerella anatipestifer is the mainly causative agent of serious septicaemia in domestic ducks, geese, turkeys and various other domestic and wild birds. It mainly causes the so-called anatipestifer syndrome in ducks characterized by fibrinous pericarditis, perihepatitis, air sacculitis, and meningitis and diarrhoea, and consequently to enormous economic losses in the poultry industry worldwide. There are various serotypes in Riemerella anatipestifer, however, the vaccines for each serotype have not provided significant cross-protection among R. anatipestifer serotypes. There has been little work on the molecular basis of the pathogenesis of R. anatipestifer. It is difficult for the prevention and treatment of R.anatipestifer infection in poultry in production.Lipopolysaccharide (LPS), also known as endotoxin, is a main pathogenic factor of most bacteria. At present, it has been paid widely attention to the chemical structure, synthesis, genetic regulation, pathogenic mechanism and immunity of lipopolysaccharide. The structure of lipopolysaccharide have been charaterized in several species of bacteria, such as Escherichia Coli, Salmonella, Pseudomonas aeruginosa. The more clearly recognize to the structure, the better it provides new approach to the prevention and treatment of diseases. At present, as far as R.anatipestifer is concerned, the structure, synthesis and genetic regulation of LPS have not been characterized yet. In order to research the pathogenic mechanism of R. anatipestifer, this study constructed a mutant strain, in which the gene for modifying the synthesis of LPS in R. anatipestifer. In the study, we preliminarily research on relationship between synthesis of LPS and pathogenicity.The lpsA gene encodes a mofified protein for the synthesis of LPS. The results of the gene function prediction shows:the modified protein was acted as glycosyl transferase in synthesis process of lipopolysaccharide in R.anatipestifer. The transfering enzyme belongs to the superfamily of Glycosyltransferase GTB type. In this study, we constructed successfully R.anatipestifer CH-1 △lpsA by homologous recombination through a recombination suicide vector. Amplification of about 600bp DNA fragment of lpsA gene of RA CH-1 as homologous arms and a spectinomycin resistance cassette by PCR using the primers respectively. The three fragments were then spliced together in vitro by overlap extension and then was then ligated to suicide vector pYA4278. The constructed recombinant plasmid was transformed into RA CH-1 strain by filter-membrane combined transfermation menthod through double parents. The transconjugants in which the lpsA gene replaced by spectinomycin resistance cassette, then the lpsA mutant strain had been generated. The analysis of biological characteristics both the mutant and parental strains, including determination of growth curve, API and Biolog biochemical identification, medicine sensitive experiment, genetic stability, cell adhesion and aggressivity and LD50 determination, etc. Using various extraction method to extract the lipopolysaccharide of two strain, then analysis of the structures of lipopolysaccharide differences by SDS-PAGE and silver stain. Finally, Rabbit polyclonal antibodies were prepared anti lipopolysaccharide extracted with hot phenol water method. The agglutination test was done the mutant and parent strain with polyclonal antiserum prepared.The experimental results showed that:when the lpsA gene was deleted, the growth and virulence of the R.anatipestifer have not been significantly changed. Hower, it had some influence on the biochemical characteristics and antibiotic resistance of mutant strain. The mutant could not ferment and use some carbon source, such as acidified glucose, D-fructose, D-galactose, D-trehalose, etc. At the same time, it was more sensitive to some antibiotics, such as Sulfisoxazole, Florfenicol, Norfloxacin, etc. According to the results of preliminary studies the lipopolysaccharide band of the mutant and the parent strains by SDS-PAGE, silver stain, there was no obvious difference between them. The rabbit polyclonal antiserum prepared with the lipopolysaccharide of R.anatipestifer strains can produce agglutination. Conclusion:There is little impact on the growth or virulence for R.anatipestifer strains when the lpsA gene was mutant. The lipopolysaccharide silver stain band is similar between the mutant lpsA gene and the parental strain RA CH-1. The antigenicity of lipopolysaccharide had no any change. The gene is involved in process of carbohydrate metabolism process of bacteria. However, it should be further reaearched on the gene function in the process of lipopolysaccharide synthesis and modification in R.anatipestifer.