Isolation And Identification Of Prote?s Mirabilis From Diarrhea Lamb And Development Of Recombinant Lactococc?s Lactis-expressing Prote?s Mirabilis OmpA Oral Vaccine

In the spring of 2015,thelambs in a large-scale sheep farm of Tengzho? in Shandong province showed severe diarrhea,and some lambs died.Fo?r 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 Prote?s mirabilis(P.mirabilis).The res?lts 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 H?(China),PPB3(Malaysia),and BAB-199(India),which showed the closest relationship with strain H?(China).The homology between the 4isolates was 100%,which was j?dged as the same strain.The res?lts of bacterial growth c?rve,dr?g sensitivity test,toxicity test,toxin test,and swarming behavior showed that the isolatereached the stable stage of growth at abo?t 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 circ?lar motionon theLB plates containing agar 1.5% and 2%.The res?lts of this st?dy provided technical s?pports for the prevention and control ofsheepProte?s mirabilis disease.The test was constr?cted a recombinant plasmid pNZ8149-ompA containing sheep P.mirabilis o?ter membrane protein A(ompA)gene,and the recombinant plasmid was electro-transformed into Lactococc?s 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 imm?nofl?orescence assay and flow cytometry.BALB/c mice were perf?sed with recombinant L.lactisand theintestinal fl?shing fl?id of d?odenal,jej?n?m,ile?m,and intestinal tract of the mo?se were collected after oral administration of 1,2,3,4,5,6,7 d.Detection of intestinal colonization by L.lactis in plate colony co?nting method.Specific bands appeared at 49 kDa detected by western blot;Green fl?orescence were detected on the s?rface of recombinant L.lactis by indirect imm?nofl?orescence assay;Recombinant L.lactis s?rface fl?orescence intensity was stronger than the empty plasmid control gro?p detected by Flow cytometry.Recombinant L.lactis can s?rvive 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 d?oden?m,jej?n?m,ile?m and cec?mof first days were 15.23%,24.19%,48.57%,40.07%,respectively.The res?lt showed that the sheep P.mirabilis ompA can be expressed on the s?rface of L.lactisstably,and the recombinant bacteria with good colonization ability in mo?se intestine with the colonization capacity of ile?m> cec?m > jej?n?m > d?oden?m.Taishan Pin?s massoniana pollen polysaccharides(TPPPS)were ?sed as adj?vants to examine theimm?nomod?latory effects.Res?lts showed that the p?re recombinant L.lactis vaccine significantly ind?ced the prod?ction of specific IgA and IgG,IL-2,IL-4,IFN-?,and T lymphocyte proliferation,and the imm?nized mice exhibited significant resistance to P.mirabilis colonization.Notably,the TPPPS adj?vant vaccines ind?ced higher levels of imm?ne responses than the p?reL.lactis.Overall,theL.lactis as a vaccine vehicle combined with TPPPSadj?vantprovides afeasible method on preventing P.mirabilis infection.