Eukaryotic Expression Of OmpA Gene And Assessment Of Efficacy Against Chlamydia Psittaci Infection Via Different Routes

Chlamydia psittaci(C.psittaci)is an intracellular bacterium and also an important zoonoses.Ducks and pigeons are two major reserviors of C.psittaci..Clinically,C.psittaci infection is manifest as persistent,repeated or asymptomatic,which hurdles healthy breeder flocks and sustainable poultry industry.In order to elucidate the correlation among C.psittaci infection,ovdict disorders and reducted egg production of breeder ducks,the pathogen was isolated and identified from diseased ducks;the major outer membrane protein(MOMP)was expressed and optimized using eukaryotic expression system;Finally,immune protections were evaluated against C.psittaci attack using different antigen combinations and different immune routes in breeder ducks.1.Isolation and identification of C.psittaci in breeder ducks:Clinical samples were collected from breeder ducks with egg production and low fertilization of Zhejiang Branch Breeder Duck Company affiliated with Wen’s Group,the pathogens were isolated using SPF embryonated eggs and Hela cells while the agent identification was targeted to Chlamydia 23S r RNA gene and omp A gene using Taqman probe-q PCR).Afterwards,positive C.psittaci was determined from diseased follicles of breeder ducks and the isolate was determined to be genotype A,C.psittaci Guangdong＿strain＿19.Post inoculation into embryonated chickens,ELD50 was determined to be 190 IFU EB.Finally,breeder ducks developed hemorrhagic ovarians,follicles,ruptured follicles and yolk peritonitis post artificial inoculation with 10~6 IFU of C.psittaci EB.2.Construction of eukaryotic expression vector and expression of MOMP:Firstly,omp A genes were cloned into a Baculovirus transfer vector(p Fast Bac-omp A),and then transformed into E.coli DH10Bac.Subsequently,the recombinant plasmids were amplified and the recombinant bacmids were extracted.Secondly,the recombinant bacmids were used to transfect insect cells(Sf9)and the recombinant baculovirus was reconstructed to proliferate for 3 passages.The third passages were identified to yield MOMP and react with His-tagged monoclonal antibody.by Western-blot.Lastly,the eukaryotic expression of recombinant MOMP protein was purified using 50%Ni-NTA resin.3.Effect of antigen combinations and inoculation routes on efficacy combating C.psittaci infection:In present study,the recombinant MOMP and five series of recombinant Pmp Gs were mixed with chitosan and Vibrio Cholerae Ghost adjuvant as the vaccine preparations.Breeder ducks received MOMP vaccine or Pmp Gs vaccine via intramuscular injection or intranasal immunization,and then were assessed immune efficacy post challenge with C.psittaci isolate.The results showed that high specific Ig G antibodies,lymphocyte proliferation,and increasing secretions of IFN-γand IL-12 were generated in the birds inoculated with MOP vaccine via intranasal drip or intramuscular injection.More important,both lesions of ovaries and follicles were reduced dramatically.Compared to MOMP vaccine,Pmp Gs vaccine produced comparable immunity via injection route.Both MOMP and Pmp Gs were vaccine candidate against C.psittaci infection.In summary,the isolated C.psittaci was closely related to the reduced egg production of breeder ducks.The eukaryotic expression of MOMP was able to induce good humoral-and cellular immunity via intramuscular injection and intranasal immunization and the eukaryotic expressed MOMP as vaccine candiate is a novel approach combating infection of C.psittaci.