| The saliva of vector tick is known as a cocktail of potent pharmacologicallyactive components able to disarm the host haemostatic system and alter theinflammatory and host immune responses. The molecules present in tick salivarepresent a plethora of biological activities which alter the physiology processes at thefeeding site, consequently affecting pathogen transmission. Adaptation of ticks totheir natural hosts has resulted in the ability of ticks to modulate the host immune andhaemostatic response with their saliva, consequently forming the salivary activetransmission module through affecting the vector component to transmit pathogens.Because of the importance of tick saliva in immunity and pathogen transmission, theisolation of the molecules in saliva responsible for pathogen transmission is a logicalstep to understand the role of saliva in blood feeding and pathogen transmission, toidentify and to design potential vaccine candidates based on tick salivary proteins andfinally to pursue the reasonable strategies to control tick borne diseases.Currently, prevention and control of tick borne disease has become a Herculeantask all over the world and no nation can avoid. Accordingly, in the present studies,Ixodes persulcatus was chose for its prominent vector role in transmitting variouspathogens, of which, bacteria, fungus, protozoa and virus was involved. Lymespirochetes were selected to represent common tick borne pathogen and utilized astarget to understand the role of salivary gland in pathogen transmission. Salp15protein, a kind of immunological regulatory proteins, was focus to evaluate itsimmunological effect against Lyme spirochete strains through gene isolation, in vitro expression and immunological assay and experimental transmission. And the potentialapplication of Salp15to prevent tick borne disease were also discussed and proposed.1. Genetic diversity of Salp15coding sequenceSalp15genes exist in Ixodes persulcatus and express during blood suckingprocess. Five cDNA clones encoding Salp15homologues were identified from semi-engorged I. persulcatus, three from larva(Ipers-2Ipers-3Ipers-4), one from nymphand the rest one from adult(Ipers-5). Differential transcription of Salp15in one tickspecies was discovered and reported at first time. Among these Salp15homologues,the overall similarity values of amino acid sequences among the different sequencesrange from53.1to94.4%. The maximum similarity value between amino acidsequences is94.4%between I. persulcatus Ipers-3and I.ricinus iric3, followed by89.1%between I.persulcatus iper-3and I.persulcatus iper-2, then53.1%betweenI.ricinus iric2and I.persulcatus Ipers-2. The differential transcription of Salp15suggested the diversity expression of salp15proteins could occur in the same ticks.2. Fusion expression of Salp15protein in vitroWith the pMal-c4x prokaryotic expression system, recombined plasmid wasconstructed to express the coding regions of Ipers-5together with the necessarytagged protein MBP. The fusion protein expressed and tag protein MBP was removedsuccessfully by factor Xa. The target Salp15Ipers-5were purified by affinitychromatography and identified, which pave a way to the following assays.3. Evaluation of Salp15Ipers-5for its transmission-blocking effect againstBorrelia SpirochetesSusceptive C3H mice were utilized as na ve host for Lyme spirochetes toevaluate the effects on the transmission blocking through intraperitoneal injection andbiting of I. persulcatus positive infected spirochete. Both the two infection methodsresulted in the high infection level in unchallenged C3H mice indicated the highertransmission efficiency of the two methods. When immunized by expressed Salp15 Ipers-5, the transmission efficiency of Lyme spirochetes were dramatically reducedevidenced by the lower infection rate in challenged C3H compared, though a littlehigher transmission efficiency was present in tick bite procedure than that inintraperitoneal injection. The results show immunized with Salp15proteins canreduce the transmission efficiency of I. persulcatus in Lyme transmission cycles,which also indicate the potential application of Salp15to block the transmission ofLyme spirochetes in the future.ConclusionAccording to the present study, the following conclusion would be emphasized.Salp15genes do exist in Ixodes persulcatus and express during blood sucking process.They transcribe differently in the development stages and result in diversity pattern ofSalp15protein. Using plasmid pMal-c4x as backbone, recombined express vector wasconstructed and succeed to express Salp15protein in host Transetta DE3cell asexpected. Immunized with Salp15expressed in vitro to C3H mice would inducedantibodys of Salp15proteins in hosts. Challenged C3H mice exhibits significantlyreduced transmission efficiency evidenced by significant low load of spirochetes inorgans and lower infection rate in C3H mice with both vector tick bite and directintraperitoneal inoculations methods. So Salp15might be a candidate vaccinemolecular to block the transmission of tick borne pathogens in the future. |
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