Comparison Of RseP Positive And Negative Strains Of Riemerella Anatipestifer

Riemerella anatipestifer?R.anatipestifer?can infect a variety of animals such as ducks,geese,turkeys,chickens,and pheasants.It mainly infects ducklings aged 2-8weeks,and is one of the most serious bacteria that harm waterfowl industry.Riemerella anatipestifer has a gene similar to E.coli RseP,encoding the metalloproteinase RseP.The RseP protease of Escherichia coli participates in the stress response of the cell envelope by cleaving the anti-?^E protein?RseA?,and plays an important role in bacterial anti-stress.In this paper,based on the detection of RseP gene of R.anatipestif-er by PCR,the RseP protein,bacterial growth,utilization of metal ions,pathogenicity,resistance to adverse factors,etc.in the bacterial protein of genetically negative and positive strains were determined Comparing the differences between the two types of strains will lay a foundation for further research on the function of the RseP gene.1 Detection of Riemerella anatipestifer RsePBy PCR detection of RseP and OmpA genes of Riemerella anatipestifer,17 strai-ns including RA20,RA22,RA24,RA25,RA26,RA27,RA28,RA29,RA30,RA31,RA33,RA38,RA39,RA40,RA41,RA47 and RA48 The bacteria are RseP genes,and the positive rate of the strain is 77.3%?17/22?;6 strains of bacteria such as RA19,RA42,RA43,RA44 and RA46 are negative.Sub-cloning the partial sequence of the RseP gene and constructing a prokaryotic recombinant expression vector pET-32a-RseP,transformed into E.coli BL21?DE3?,i-nduced by IPTG,can express recombinant fusion protein better,and exists as an intra-cellular soluble protein.The mouse anti-recombinant fusion protein serum antibody has good affinity,and the ELISA titer is above 1:6400.Using ultrasonic lysate of Riemerel-la anatipestifer cells as coating antigen?coating antigen protein concentration is100ug/ml?,mouse anti-recombinant fusion protein RseP serum?1:6400?as primary antibody,horseradish peroxide Enzyme-labeled goat anti-mouse IgG?1:4000?was used for indirect ELISA detection of enzyme-labeled secondary antibodies.The OD450values of RA31,RA19 and RA46 strains were 1.311,0.305 and 0.346,respectively,while antigen blank control,negative serum control and serum blank control Respecti-vely 0.278,0.289,0.313.This shows that the OD value of the RseP-negative strain RA31 is greater than that of the negative strains RA19 and RA46.2 Physiological characteristics of Riemerella anatipestiferIn the LB medium,the time for the three strains of RA31,RA19 and RA46 to enter the logarithmic phase and the stable phase is basically the same,but the bacterial concentration of RA31 strains at each detection time point is lower than RA19 and RA46,and the bacterial concentrations of RA19 and RA46 are basically the same.On the chocolate plate,RA19 and RA46 strains grew faster than RA31 strains,and single colonies appeared at 15h of culture;the smear staining of the culture product showed that the staining and morphological characteristics of the 3 strains were consistent within 20h of culture,but the At 25h,the morphology of RA31 strain was different from RA19 and RA46.3 Metal ions affect the growth of Riemerella anatipestiferWe add different concentrations of metal salts to the chocolate medium to determine the effects of calcium,magnesium and zinc ions on bacterial growth.The results showed that RA31,RA19 and RA46 strains grew well on cultures containing calcium and magnesium ions of 50mg/L-250mg/L;RA31 strains had lower tolerance to zinc ions and had higher zinc ion concentrations Almost no growth in the50mg/L environment,while RA19 and RA46 strains can grow well in the environment of 0 to 250mg/L zinc ion concentration.Zinc sulfate was added to LB to determine the growth curve of bacteria under different zinc ion concentration conditions.The results showed that RA31 strain almost did not grow in the environment with zinc ion concentration higher than 10mg/L,while RA19 and RA46strains had zinc ion concentration.When 10mg/L-20mg/L,the growth and reproduction are faster.4 Survival time of Riemerella anatipestifer in stable environmentThe RA31,RA19 and RA46 strains were recovered and inoculated into LB liquid medium,cultured to an OD₆₀₀00 of 0.4,plate counted and placed at 4°C for static storage,and the survival rate on the fourth and seventh days of storage was measured.The survival rates of RA31 strains stored at 4 and 7 days were 45.3%and 10.5%,RA19 was17.1%and 0.40%,and RA46 was 26.0%and 0.10%,respectively.The results showed that the survival rate of RA31 strains stored at 4?was significantly higher than that of RA19 and RA46.5 Pathogenicity testingAfter RA31,RA19 and RA46 strains were recovered and purified,they were inoculated into LB liquid medium.After culturing to OD600 of 1.0,the 10-day-old ducklings were infected by subcutaneous inoculation at the neck,0.2mL/duck,and RA31 strain test ducks were inoculated during the observation period.The mortality rate is 77.8%?7/9?,and the anatomy of non-dead animals showed cellulose exudative inflammation.The animals vaccinated with RA19 and RA46 showed no lack of energy on the first day of inoculation,and returned to normal on the second day.There were no deaths during the observation period.No obvious abnormalities were found after the killing and autopsy after the observation period.6 Riemerella anatipestifer resistance to adverse factorsThe bacteria were inoculated into LB with different pH values.After incubation at37°C for 1 hour,the bacteria were cultured and counted.The results showed that the pH value of the medium gradually decreased from 7.4,and the survival rate of RA19and RA46 strains continued to decrease,while the survival rate of RA31 strain was only There is a significant decrease at pH 4.4.The bacteria were inoculated into the medium containing different concentrations of H₂O₂.After cultivation and bacterial testing,it was found that the strain RA31 can survive under the condition that the concentration of H₂O₂ in the medium is not greater than 3.6mM,while RA19 and RA46 are not greater than 1.6mM Survive.The bacterial culture count results after the mixed treatment of bacteria and healthy duck serum showed that the survival rate of RA31 bacteria was 19.89%,while that of RA19 and RA46 strains was 0.The above results show that the resistance of RA31 strains to adverse factors is stronger than that of RA19 and RA41 strains.