Isolation And Identification Pathogen Of Litopenaeus Vannamei Molecular Cloning Of Crustin-like Gene And Its MRNA Expression With Vibrio Parahaemolyticus Challenge

Five hundreds Litopenaeus vannamei shrimps from Nanjiang Biotechnology CompanyLimited in Hainan province were used for pathogenic bacteria isolation identification, growthcurve, challenge assays, and drug sensitive test which was isolated in the diseased L.vannameiin this study. RT-PCR was used to clone the coding region sequence of Cructin-Like （CL）gene in L.vannamei. DNAstar, Clustalx, MEGA5.0, PROSITE, TMpred and SignalP wereused to analyse the structure and function of CL gene and CL protein. The mRNA expressionpattern of CL gene in hepatopancreas was studied by challenge assays and Real-Timequantitative PCR （qRT-PCR） at different times. It would lay a foundation for the further studythe function of CL gene and rearing new resistant varieties.1. The analysis of the HL19The pathogenic bacterium was identified as Vibro harveyi based on the morphologiccharacters, physiological and biochemical identification, and16S rRNA sequence analysisfrom the pond of diease one-month-old shrimps. According to the growth curve including2hadjustment phase（0-2h）,7h exponential phase（3-9h）,10h stationary phase(10^-19 h), and5hfor decline phase（20-24h）, we found V.harveyi growing fast. The maximum concentration is4.05×10¹²CFU/ml at18h after incubate the TSA. There were sensitive to13kinds of drugsincluding Doxycycline, Norfloxacin, Ceftazidine, Rocephin, SMZ-TMP, Tetracycline and soon. But there were no sensitive to penicillin as Benzathinepenicillin G, Oxacillin,Carbenicillin, and Cefradine. The minimum mortality is0%and it is the group of1×10⁶CFU/ml after injection. And the maximum mortality is100%and it is the group of1×10¹¹CFU/ml after injection. The LD50is4.66×10⁸CFU/ml.The morphologic characteristics of colonies and physiology and biochemistry aredifferent from V. harveyi, based on the identification, isolation, growth curve, challengeassays, and drug sensitive test of HL19, because of the variability between individuals.HL19’s pathogenic is not high, but it growth fast. Penicillins as benzathinepenicillin G,Oxacillin, Carbenicillin, and Cefradine are not suitable using as drugs，for penicillin drugsabuse causing HL19penicillins resistant. Doxycycline, Norfloxacin, Ceftazidine, Rocephin, SMZ-TMP, and Tetracycline which are suitable used as drugs to avoid infection during theearly stage of L.vannamei to disease control.2. The analysis of the XG409The isolate was identified as Vibro parahaemolyticus based on the morphologiccharacters, physiological and biochemical identification, and16S rRNA sequence analysisfrom three two-month-old shrimps. According to the growth curve including8h exponentialphase （0-8h）,9h stationary phase （9-17h）, and7h for decline phase （18-24h）, we foundV.parahaemolyticus growing very fast. There were sensitive to14kinds of drugs Norfloxacin,SMZ-TMP, Ceftazidine, Trocephin and so on. But there were no sensitive to Penicillin asBenzathinepenicillin G, Oxacillin, Carbenicillin, and Ccefradine. The minimum mortality is0%and it is the group of injection of1×10⁴CFU/ml. And the maximum mortality is100%andit is the group of injection of1×10⁹CFU/ml. The LD50is4.66×10⁶CFU/ml.The identification and isolation, growth curve, challenge assays, and drug sensitive testof XG409, we found the morphologic characteristics of colonies and physiology andbiochemistry is same as V.parahaemolyticus. XG409grows very fast, because of itspropagation fast and strong adaptability. XG409’s pathogenic is high. In the concentration of1×10⁹CFU/ml, all of shrimps were dead and most of the shrimps were died within48h. Ifyou can not timely treatment, it will be probably caused a large of death. There were sensitiveto Penicillins, because of Penicillin drugs abuse causing HL19Penicillins resistant or drugitself. But there were sensitive to Paraxin, Tobramycin, Doxycycline, Norfloxacin, Zanocin,Furazolidone，Doxycycline，Ceftriaxone and SMZ which are suited for using as drugs to helpgrowing at the later stage of L.vannamei to disease control.3. Molecular cloning and analysis of CL geneRT-PCR was used to clone the coding region sequence of CL gene in L.vannamei.DNAstar, Clustalx, MEGA5.0, PROSITE, TMpred and SignalP were used to analyse thestructure and function of CL gene and CL protein. CL gene in L.vannamei （Accession number:JQ824114） were501bp including a33bp5′UTR （1-33bp）,24bp3′UTR （478bp-501bp）and a444bp CDS which encoded147amino acids with the initial code ATG and the terminalcode TGA. Amino acids squence was consisting of a signal peptide, a single transmembranehelix, and a WAP domain.According to sequence of similarity analysis, Crustin gene sequence is different amongdifferent species which might play different roles.4. Real-time quantitative analysis of the CL geneThe expression of the CL gene showed the most significant changes in hepatopancreas.The general trend was that the expression of CL gene goes down at first, and then up after vibrio challenge. The expression of CL gene was lower significantly than that in control at4hours after challenge. It rose gradually and back to the level of CL gene expression beforeinjection at6hours after challenge. In the control the expression of CL gene remained thesame level. The fact that bacterial infection can change expression of the CL gene and thatthey appeared to be expressed naturally at higher levels in immune-related organs maysuggest that they are of L.vannamei against V.parahaemolyticus bacterial infections.