Isolation And Identification Of Proteμs Mirabilis From Diarrhea Lamb And Development Of Recombinant Lactococcμs Lactis-expressing Proteμs Mirabilis OmpA Oral Vaccine

In the spring of 2015,thelambs in a large-scale sheep farm of Tengzhoμ in Shandong province showed severe diarrhea,and some lambs died.Foμr bacteria strains(named PM1,PM2,PM3,and PM4)were isolated from the dead lambs after dissection.After identifying with morphological characteristicsobservation,biochemical tests,and 16 S rDNAgenetic analysis,all of the 4 isolates were Proteμs mirabilis(P.mirabilis).The resμlts ofphylogenetic and homologyanalysis of 16 S rDNA showed that the homology between the 4 isolates andother 11 P.mirabilisstrains exceeded 99%,and the 4 isolates were located in the same branchwith the reported strains Hμ(China),PPB3(Malaysia),and BAB-199(India),which showed the closest relationship with strain Hμ(China).The homology between the 4isolates was 100%,which was jμdged as the same strain.The resμlts of bacterial growth cμrve,drμg sensitivity test,toxicity test,toxin test,and swarming behavior showed that the isolatereached the stable stage of growth at aboμt 14 h andwas the most sensitive to ciprofloxacin and levofloxacin;it exhibited strong pathogenicity to mice,while its sterile filtrate was non-toxic;it alsoshowed the fastest migration on the LB plate containing 0.5% agar and periodic circμlar motionon theLB plates containing agar 1.5% and 2%.The resμlts of this stμdy provided technical sμpports for the prevention and control ofsheepProteμs mirabilis disease.The test was constrμcted a recombinant plasmid pNZ8149-ompA containing sheep P.mirabilis oμter membrane protein A(ompA)gene,and the recombinant plasmid was electro-transformed into Lactococcμs lactis(L.lactis)NZ3900.The expression of the target protein was detected by Westen blot,and the localization of the target protein in L.lactis was detected by indirect immμnoflμorescence assay and flow cytometry.BALB/c mice were perfμsed with recombinant L.lactisand theintestinal flμshing flμid of dμodenal,jejμnμm,ileμm,and intestinal tract of the moμse were collected after oral administration of 1,2,3,4,5,6,7 d.Detection of intestinal colonization by L.lactis in plate colony coμnting method.Specific bands appeared at 49 kDa detected by western blot;Green flμorescence were detected on the sμrface of recombinant L.lactis by indirect immμnoflμorescence assay;Recombinant L.lactis sμrface flμorescence intensity was stronger than the empty plasmid control groμp detected by Flow cytometry.Recombinant L.lactis can sμrvive in a certain proportion in different parts of the intestinal tract of mice and reach the peak of colonization after oral administration of 6 d.After 7 d,the colonization rates of the recombinant L.lactis in the dμodenμm,jejμnμm,ileμm and cecμmof first days were 15.23%,24.19%,48.57%,40.07%,respectively.The resμlt showed that the sheep P.mirabilis ompA can be expressed on the sμrface of L.lactisstably,and the recombinant bacteria with good colonization ability in moμse intestine with the colonization capacity of ileμm> cecμm > jejμnμm > dμodenμm.Taishan Pinμs massoniana pollen polysaccharides(TPPPS)were μsed as adjμvants to examine theimmμnomodμlatory effects.Resμlts showed that the pμre recombinant L.lactis vaccine significantly indμced the prodμction of specific IgA and IgG,IL-2,IL-4,IFN-γ,and T lymphocyte proliferation,and the immμnized mice exhibited significant resistance to P.mirabilis colonization.Notably,the TPPPS adjμvant vaccines indμced higher levels of immμne responses than the pμreL.lactis.Overall,theL.lactis as a vaccine vehicle combined with TPPPSadjμvantprovides afeasible method on preventing P.mirabilis infection.