Development Of Method For Rapid Diagnosis And Immune Prevention Of Fish Pathogenic Bacteria

A loop-mediated isothermal amplification (LAMP) technique can amplifiedDNAs under an isothermal condition using a set of four specific primers. In thisstudy, we designed three sets of the LAMP primers to construct a multiplex LAMP(mLAMP) method for simultaneously detecting Vibrio harveyi, V. anguillarum, andV. alginolyticus. The results indicated that when the mixture was incubated at62℃for60min for the mLAMP reaction, the mLAMP method can simultaneously detectthe presence of three Vibrios; when the products were digested with restrictionenzyme, the mLAMP method can distinguish three bacterium. The sensitivities ofthe mLAMP method were102to103times higher than the classical PCR methods onthe detection limits. For the specificities of mLAMP detection,32samples wereselected. The results suggested that the mLAMP can specifically amplify the relatedspecies of bacteria, other strains were negatively. Japanese flounder Paralichthysolivaceus as trial animal, the practicability of the mLAMP were evaluated withhealthy and infected tissues and the results indicated that the mLAMP method cansuccessfully detect and identify the infected bacteria. Summarily, the resultssuggested that the mLAMP is a simple and effective method for detection of bacteria.It can be applied for the early diagnosis of bacteria.V. anguillarum, Streptococcus iniae, and V. harveyi is the causative agent thataffects a wide range of aquiculture animals. In this study, we obtained two mutants V.anguillarum C312M, and S. iniae SF1M1, derived from the pathogenic V.anguillarum C312and S. iniae by selection of rifampicin resistance. The resultsindicated that C312M and SF1M1were slower in growth than the wild type C312and SF1, showed less extracellular protease activity and produced a much loweramount of siderophores. Compared to the widetype, the mutants were altered in protein production profile and exhibited a dramatically increased median lethal dose.Safety analysis showed that the mutants were stable in virulence in the absence ofselective pressure. To examine the potential of C312M and SF1M1mutants as liveattenuated vaccine, Japanese flounder Paralichthys olivaceus were vaccinated withthe mutants. When the fish were challenged with C312at1mo post-vaccination,C312M-vaccinated fish exhibited relative percent survival rates of60-84%. Japaneseflounder were vaccinated with SF1M1. At one and two months post-vaccination, thefish were challenged with different serotype (serotype I and II) strain S. iniae. Theresults showed that fish vaccinated with SF1M1exhibited relative percent survivalrates of54-70%. Further analysis showed that C312M-and SF1M1-vaccinated fishproduced specific serum antibodies which enhanced serum bactericidal activity in amanner that is probably complement-dependent. These results indicate that C312Mand SF1M1are highly attenuated in virulence but still retain residual infectivity.C312M and SF1M1are effective vaccine.In this study, we identified13outer membrane proteins (OMPs) from apathogenic V. harveyi strain and analyzed their immunological properties. In vivoimmunogenicity analysis showed that antibodies specific to recombinant proteins ofthe13OMPs were detected in the antiserum of V. harveyi-infected rat. When used assubunit vaccines to immunize Japanese flounder, all OMPs were able to elicit specificserum antibody production in the vaccinated fish; however, only two OMPs (OMP173and OMP214) induced high levels (>70%) of relative percent survival. Pre-incubationof V. harveyi with the antisera of protective OMPs significantly impaired bacterialinfectivity against cultured flounder FG cells, whereas the antisera of non-protectiveOMPs had no apparent effect on infection. Immunofluorescence microscopy revealedspecific binding of OMP173antibodies to whole V. harveyi cells. Passiveimmunization showed that fish received OMP173antiserum before being infectedwith V. harveyi exhibited significantly reduced mortality rate. Finally, treatment of FGcells with OMP173prior to V. harveyi infection protected the cells from bacterialinvasion to a significant extent. Take together, these results indicate that OMP173andOMP214induce protective immunity through production of specific antibodies that block bacterial invasion. Thus, the immunoprotectivity of the OMPs is probablyassociated with functional participations of the OMPs in bacterial infection.