Screening Of Transposon Insertion Mutant Of Riemerella Anatipestifer And The Biological Characterization Of The M949₁360 Gene

Riemerella anatipestifer （RA） is a Gram-negative, rod-shaped bacterium that causes septicemic and exudative diseases in domestic ducks, geese, turkeys and wild birds, which also called as duck infectious serositis. This infectious disease leads to high mortality and morbidity that is the major economic losses in the duck industry worldwide. Lipopolysaccharide （LPS）, as an important component of Gram-negative bacteria, can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. Using monoclonal antibody against R. anatipestifer serotype 1 LPS （anti-LPS MAb）, we screened a library of random Tn4351 transposon mutants constructed from RA CH3.Then, a LPS mutant strain RAA604 which lost the reactivity with anti-LPS MAb was identified using an indirect ELISA assay. Furthermore, the pathogenicity and biological characteristics of the mutant strain were investigated.First, we prepared monoclonal antibody （MAb） against R. anatipestifer LPS. BALB/c mice were immunized three times subcutaneously with inactivated R. anatipestifer CH3 at a dose of 10* CFU. Hybridoma technique was performed and single clones were screened using an indirect ELISA, in which plates were coated with purified CH3 LPS. Positive clone 1C1 was selected for MAb production. The subtype of 1C1 was detected as IgG3 and the ascites antibody titer was 1:3200. Previous study has constructed 1462 random transposon mutants library of RA CH3, then we screened the library by using anti-LPS MAb 1C1 to obtain LPS defective mutant strain RAA604 in an indirect ELISA. Southern blot analysis and inverse PCR assay helped us to confirm that the mutant strain RAA604 was a single Tn4351 insertion and the M949₁360 gene was inactivated by the insertion of transposon. BLAST search shows that the M9491360 gene shares 98%-100%identity with the R. anatipestifer strains of DSM 15868, RA-GD, ATCC 11845 = DSM 15868, RA-CH-2,17,153, Yb2 and RA-CH-1.Next, the expression of Tn4351-disrupted M949₁360 gene can not be detected in the mutant strain RA△604 by RT-PCR. There were no significant changes in the gene expression levels of upstream M949₁359 gene and downstream M949₁361 gene. The result indicated that deletion of M949₁360 gene had no polar effect on the expression of its down stream gene in mutant strain RA△604. The complementation strain cRA604 was constructed so that it can help us understand more about the inactivated M949₁360 gene. Using silver staining analyses of LPS, we found that the LPS pattern of the mutant strain RA△604 displayed two bands at 15 kDa and 10 kDa, and the 18 kDa band was absent compared with wild-type strain CH3. Western blot showed thatthe wild type strainCH3 displayed the binding band with the anti-LPS MAb 1C1, whereas the mutant strain RA△604 lost the reactivity. The complementation strain cRA604 recovered the reactivity with anti-LPS MAb 1C1. The mutant strain RAA604showed no significant influence on the bacterial growth but decreased bacterial viability by Live/dead BacLight Bacterial Viability staining.Furthermore, both in vivo and in vitro experiments were used to determine the function of the M949₁360 gene in the bacterial pathogenicity. In vivo, according to the consequence of LD50, the virulence of mutant strain RA△604 was attenuated more than 200 times in compared with R. Anatipestifer CH3. Within 5 days post infection of RA△604, the bacterial loads in the blood was significantly decreased with time, compared with R. Anatipestifer CH3 infected. These implied that M949₁360 gene is an important virulence gene in R. anatipestifer. In vitro, the mutant strain RA△604 had lower levels of adherence and invasion to Vero cells as compared with R. anatipestifer CH3. Adherence of bacteria to target cells of a susceptible host is considered the first step of successful colonization and has been associated with bacterial virulence. Serum bactericidal test showed that mutant strain RAA604 resist complement pathway to let bacterial survival from the duck serum. The M949₁360 gene was supposed play a role in regulation of complement pathway. Moreover, immunization with attenuated bacteria of RA△604 protected ducks from R. anatipestifer serotype 1 WJ4 challenge, the protection rate was 85.7%. These results indicated that the M949₁360 gene plays a role in the bacterial pathogenicity and the mutant strain RAA604 could be used as a potential live vaccine candidate.In conclusion, the M949₁360 gene is involved in LPS biosynthesis and pathogenicity. This study provides a new basis for the future prevention and control strategies against R. anatipestifer infection.