| Riemerella anatipestifer(RA)can infect ducks,geese and other hosts,cause severe exudative inflammation,and cause serious economic losses to the waterfowl breeding industry.The bacterium exists in major duck-raising countries and regions in the world,with complex serotypes and extremely easy to develop drug resistance.Infected animals are characterized by fibrinous perihepatitis,pericarditis and air sacculitis.Complement C5 a is the strongest mediator of anaphylactoxin in the cleavage fragment of complement.It mainly binds to C5 a R to form a signal axis and exert its biological effects.The C5a-C5 a R axis is related to various bacterial infections such as sepsis and meningitis.Blocking the C5a-C5 a R axis can reduce the degree of infection and improve the development of the disease.Based on the cloning and expression of duck C5 a and C5 a R genes and preparation of polyclonal antibodies,this study established an RA duckling infection model,explored the role of C5a-C5 a R axis in RA infection,and provided new ideas for the treatment of duck infectious serositis.1 Cloning and analysis of duck C5 and C5 a R genesThe primers were designed according to the nucleotide sequences of C5 gene and C5 a R of mallard ducks registered on NCBI,and c DNA synthesized by reverse transcription of total RNA from liver tissue of healthy Sichuan ducks at 10 days old was used as a template for PCR amplification,cloning and sequencing to obtain ducks.The similarities between the C5 a R and C5 nucleotide sequences and the mallard C5 a R sequence(XM＿027447482.1)and C5 sequence(XM＿027470663.1)were 99.8% and 99.6%.2 Prokaryotic expression of duck C5 a and C5 a R gene fragments and preparation of polyclonal antibodiesAccording to the amino acid sequence,the nucleotide sequences of duck C5 a and C5 a R genes were artificially synthesized,and a prokaryotic recombinant expression vector was constructed with p ET32 a,transformed into E.coli BL21,and induced with a final concentration of 0.4m M IPTG at 30°C and 180 rpm shaking conditions for 4h,and the results were good Expressed,and the recombinant fusion protein exists in a soluble form in the cell.Centrifuge the induced bacterial solution,collect the bacterial sludge,resuspend it in physiological saline,lyse with ultrasonic wave,and perform SDS-PAGE with the supernatant collected by centrifugation.The gel band containing the target protein is cut out,washed and homogenized as the immune antigen,Immunize New Zealand white rabbits to prepare polyclonal antibodies.Western-blot test results show that the antiserum can bind to the recombinant fusion protein,and the indirect ELISA test serum antibody titer is more than 1:10000.3 The effect of C5 a,C5aR polyclonal antibodies on RA-stimulated duck peripheral whole bloodThe peripheral blood of 10-day-old healthy ducks prepared by anticoagulation with heparin sodium was treated with RA at 37°C and 200 rpm,and the plasma was sampled and centrifuged to detect C5 a and cytokine content.In the experiment,the effect of anti-recombinant protein C5 a and C5 a R antibodies on the content of C5 a and cytokines in plasma was explored.When the peripheral blood of ducks interacted with RA for 2h and 4h,the plasma C5 a content increased significantly(p<0.05);at 4h,the cytokine IL1β increased extremely significantly(p<0.01);at 2h,IL6 and TNFαincreased Highly significant(p<0.05).During the interaction between peripheral blood and bacteria,the anti-recombinant protein C5 a antibody can significantly down-regulate the plasma C5 a and IL1β levels at 4h and the plasma IL6 and TNFαlevels at 2h(p<0.05),and the anti-recombinant protein C5 a R antibody can significantly down-regulate the plasma C5 a and IL1β levels at 4h.Plasma IL1β level and plasma IL6 level at 2h(p<0.05).The above results showed that the expressions of C5 a and cytokines IL1β,IL6 and TNFα in duck peripheral blood were up-regulated after RA stimulation.Anti-recombinant protein C5 a and C5 a R antibodies could decrease the levels of C5 a,IL6,IL1β and TNFα in stimulated whole blood.4 Explore the dosage and time of anti-recombinant protein antibodiesIn 10-day-old healthy ducks experimentally infected with RA,different doses of anti-recombinant protein C5 a and C5 a R antibodies were injected at different times,and the dosage and time of antibody use were screened based on the survival rate of the animals.The results showed that when RA was inoculated subcutaneously in the neck,0.3 m L of anti-recombinant protein C5 a antibody or 0.5 m L of anti-recombinant protein C5 a R antibody was injected subcutaneously into the legs.Within 72 hours of inoculation,100% of the animals survived,and no antibody infection was used.The survival rate of the group was 60%.5 The effect of antibody blocking C5a-C5 a R axis on RA-infected ducklingsAt the same time when RA was inoculated subcutaneously in the neck,the legs were injected subcutaneously with 0.3 m L of anti-recombinant protein C5 a antibody or 0.5 m L of anti-recombinant protein C5 a R antibody/only to establish an antibody blocking C5a-C5 a R axis model.At the same time,healthy control and RA infection groups were set up.Within 72 hours of inoculation with RA,the incidence of animals in the RA infection group,the anti-recombinant protein C5 a antibody treatment group and the anti-recombinant protein C5 a R antibody treatment group were 60%,40% and 40%,and the survival rate was 40% and 100%,respectively And 100%,while the healthy control animals had no morbidity or death.At 24 h,48h and 72 h after inoculation,the gross pathological changes such as fibrinous pericarditis and perihepatitis and the increase of neutrophils in the tissues of the RA infection group were more obvious than those in the antibody treatment group.The above results show that injection of anti-recombinant protein C5 a antibody or C5 a R antibody during artificial inoculation of RA can reduce animal morbidity and mortality,and reduce inflammation and pathological damage.The numbers of bacteria in the peripheral blood of the RA-infected ducks were3340CFU/m L,11060CFU/m L and 182667CFU/m L at 12 h,24h,and 48 h after vaccination,respectively.The C5 a antibody-treated ducks were 1333CFU/m L,7553CFU/m L and 13633CFU/m L,respectively.The ducks in the C5 a R antibody treatment group were 1853 CFU/m L,6494 CFU/m L and 11433 CFU/m L,while the peripheral blood of the healthy control group had no bacterial growth after culture.The above results show that the anti-recombinant protein C5 a antibody or C5 a R antibody can reduce the amount of RA in the peripheral blood of artificially vaccinated ducks.Compared with the healthy control group,the ALT and AST in the serum of the infected ducks in the infected group at 48 h after RA inoculation decreased significantly(p<0.05),and the AST content in the serum of the infected ducks in the infected group at 72 h after RA inoculation decreased significantly(p<0.01).Compared with the infected group,there were no significant changes in duck serum ALT and AST levels in the C5 a R antibody treatment group and C5 a antibody treatment group at 48 h and 72 h after RA vaccination.ELISA detects the content of C5 a and cytokines in duck serum.Compared with the healthy control group,the levels of C5 a and TNFα in the serum of the infected ducks in the 48 h after RA vaccination increased significantly(p<0.01),and the levels of IL1β increased significantly(p<0.05).The ducks in the infected group 72 h after RA vaccination increased significantly The levels of C5 a,IL1β,TNFα,and IL6 in serum increased significantly(p<0.01).Compared with the RA infection group,the serum IL1β,TNFα,and IL6 contents of ducks in the C5 a R antibody treatment group were significantly reduced(p<0.05),and the serum C5 a,IL1β,TNFα,and IL6 contents of the ducks in the C5 a antibody treatment group were significantly decreased(p<0.05).Fluorescence quantitative PCR was used to detect gene m RNA expression in liver tissue.Compared with the healthy control group,the m RNA expression of C5 a R,Sph K1 and TNFα in the liver tissue of the RA-infected ducklings was extremely significantly up-regulated(p<0.01),and the m RNA expression of IL1β was significantly up-regulated(p<0.05).Compared with the RA infection group,the expression of C5 a R,IL1β and TNFα m RNA in the liver tissues of the C5 a antibody treatment group and the C5 a antibody treatment group was significantly down-regulated(p<0.05),while Sph K1 was extremely significantly down-regulated(p<0.01);the above results showed that Anti-recombinant protein C5 a antibody or C5 a R antibody can reduce the expression of C5 a R,IL1β,TNFα and Sph K1 in infected ducks.In this study,in vitro whole blood and animal infection models were used to find that the C5a-C5 a R axis has an important regulatory role in R.anatipestifer infection,and blocking this axis with antibodies can help reduce the degree of infection. |
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