Construction And Characterization Of SprT Gene Deleted Mutant Strain Of Riemerella Anatipestifer

Riemerella anatipestifersis is a bacterial disease of domestic ducks, geese, turkey and other poultry caused by Riemerella anatipestifer. It owns series of serotypes but lacks cross-protection between serotypes. In addition, the drug resistance makes it difficult to control. So, to strengthen the research of pathogenic mechanism of the Riemerella anatipestifer will be of great significance to control the the disease.The Por secretion system is named T9 SS, which distributes in the bacteroidetes members mediates the motility of bacteroidetes bacteria and pathogenic mechanism. Bioinformatics analysis shows that RA-YM genome owns complete Por secretion system coding genes. The sprT gene is critical component of the Por secretion system. So, our research constructed sprT gene（RAYM₀3924）deletion mutant strain and made a series of studies on its biological characteristics as follows.1 Construction and identification of sprT gene deletion mutant strain Amplified upstream and downstream homology arms of sprT gene using RA-YM genome. Connect upstream and downstream homology arms and spectinomycin gene together by using overlapping PCR method, and then connected it to plasmid p RE112. The recombination plasmid was transformed into the E.coli X7213. In E.coli X7213, which contains p RE-LSR as donor strain, RA-YM as receptor strain. After transconjugation, select spectacular resistant colonies on the plate, and then using the PCR method to identify whether there has been exchange of homology arms. We constructed sprT gene deletion strain successfully in the end.2 The biological characteristics of sprT gene deletion mutant strain Comparison growth characteristics and biochemical characteristics of RA-YM sprT gene deletion mutant strain with the parent strain RA-YM, we found that the growth rate of sprT gene deletion mutant strain was faster than that of the parent RA-YM strain. Biochemical experiments found that the main biochemical characteristics of the parent strain and the sprT gene deletion mutant strain did not change, while the sprT gene deletion mutant strain did not degrad gelatin. In respect of hypertonic saline, the hypertonic saline tolerance concentration of the sprT gene deletion mutant strain was 1.88% as well as the parent RA-YM strain. The Choline salts tolerance concentration of the sprT gene deletion mutant strain was 23.4 μg/m L, while the parent RA-YM strain was 46.9 μg/m L. Serum survival rate of sprT gene deletion mutant strain in 12.5% normal duck serum was higher than that of the parent RA-YM strain. Analysis of gelatin zymography proved that the sprT gene deletion mutant strain can not secret gelatinase, which showed that some proteins can not be secreted because of the deleted sprT gene.3 The pathogenicity of sprT gene deletion mutant strain and the parent RA-YM strain Comparison the virulence of ducklings aged 10 days between the sprT gene deletion mutant strain and the parent RA-YM strain. All the ducklings were infected through muscle. The LD50 value of parent RA-YM strain was 3.82×104CFU, while the LD50 value of the sprT gene deletion mutant strain was 1.61×109CFU. It showed that the virulence of the sprT gene deletion mutant strain fell about 4.21 × 104 times. It showed that 24 h after infected, the blood, liver and spleen tissue bacterial content of the sprT gene deletion mutant strain was significantly lower than that of the parent RA-YM strain; 48 h after infected, the heart, liver, spleen and brain tissue bacterial content of the sprT gene deletion mutant strain was significantly lower than that of the parent RA-YM strain. It showed that the tissue invasiveness of the sprT gene deletion mutant strain was lower than that of the parent RA-YM strain.The research found that the virulence of the sprT gene deletion mutant strain was significantly decreased. It showed that the secreted factors through Por SS were important virulence factors which deserves further investigation.