Analysis On Pathogen In An Outbreak Death Of Marsupenaeus Japonicas And Effect Of Multi-probiotics Combination On Enhancement Of Shrimp Immunity

Part I.Analysis on pathogen in an outbreak death of Marsupenaeus japonicusIn order to clarify the cause of an outbreak death occurred in shrimp Marsupenaeus japonicus in Weifang between July to August 2015,seven main shrimp pathogens including White spot syndrome virus(WSSV),Taura syndrome virus(TSV),Yellow head virus(YHV),Infectious hypodermal and hematopoietic necrosis virus(IHHNV),Infectious myonecrosis virus(IMNV),Covert mortality nodavirus(CMNV)and Vibrio alginolyticus that causing acute hepatopancreatic necrosis disease(AHPND)were detected by Polymerase chain reaction(PCR)method.Meanwhile,histopathology of the diseased shrimp were observed.Furthermore,bacterial strains,isolated from the moribund shrimp and identified by the 16 SrDNA gene,were used for the evaluating of virulent by immersion challenge.The results showed that all the three shrimp samples gave strong positive PCR results(an amplicon of 941 bp)with primers WSSV146F2/R2,and two of three sample show weak positive results(an amplicon of 389 bp)with primers IHHNV 389F/R and two of three sample show strong positive results(an amplicon of 167 bp)with primers CMNV-7F2/R2.However the results of PCR amplification for the other four pathogens(TSV,YHV,IMNV and AHPND)show negative.Histopathology results show that there were inclusion body of WSSV in the epithelial tissue of stomach and gill,and the muscle fibres dispersed.Three dominant strains of bacterial microorganisms 2901,2902,2903 were isolated and 16 S rDNA sequencing indicated that they had higher similarity with Grimontia indica(99%)?Alteromonas sp.(99%)and Vibrio alginolyticus(100%)respectively.Challenge test show that LC50 of bacteria 2901,2902,2903 were 9.8 × 107 CFU / mL,1.1 × 108 CFU /mL and 2.3 × 108 CFU / mL,which mean the bacterial microorganisms had lower virulent and may not be the main pathogen of shrimp.Comprehensive analysis suggest that outbreak death of M.japanicus might be caused by coinfection of WSSV,IHHNVand CMNV,in which WSSV infection might play a key role.The results of this study could provide strong support for the clarifying of the cause of outbreak death in shrimp M.japonicus.Part II.Effect of multi-probiotic combination on the enhancement of shrimp immunityIn order to study the effect of environmental microorganism on the prevention and control of AHPND,six single probiotics including Pseudoalteromonas.sp,Halomonas.sp,Bacillus licheniformis,Bacillus subtitles,Leuconostoc mesemteroides,Saccharomyces cerevisiae and two complicatedfit of probiotics,which comprised by Halomonas.sp?B.licheniformis?B.subtitles ?L.mesemteroides ?S.cerevisiae in composite group 1,and comprised by Pseudoalteromonas.sp ? Halomonas.sp ? L.mesemteroides?S.cerevisiae in composite group 2 were added to the water body and feed of shrimp Litopenaeus vannamei respectively.Challenge test were carried out by shrimp pathogen V.Parahaemolyticus on day 15 and the mortality were counted.Also the cultural survival rate of the experimental shrimp was recorded on 30 d of the trial.Serum and tissue from digestive organ(hepatopancreas and intestinal tract)were sampled and on day 15 of the trial for the analysis of the activity of shrimp immunity and digestive ability.Furthermore,the microbial diversity in the intestinal tract and water body was analyzed by miseq sequencing.The results showed that the mortality after being challenged by VPAHPND in groups of B.subtilis,Pseudoalteromonas.sp composite 1,composite 2,B.licheniformis,Halomonas.sp,L.mesemteroides,S.cerevisiae were 37.5%,45.8%,50.0%,41.6%,54.1%,58.3%,62.5% and 70.8% respectively,significantly lower than that in the control(80%)(P < 0.05).The survival rate of the experimental shrimp in the culture ponds was recorded after 30 days,and the survival rates in the groups composite 1,composite 2,B.subtilis,Pseudoalteromonas.sp.,B.licheniformis,Halomonas sp.,L.mesemteroides,and S.cerevisiae were 65.0%,60.4%,54.7%,50%,54.3%,52.8%,49.9% and 53.0% respectively,which were significantly higher than that of control(40.0%)(P < 0.05).The activity of lysozyme ?PO and SOD in serum of shrimp in each experimental groups were analyzed.The results show that activity of PO in groups of composite 2,B.licheniformis,B.subtilis,L.mesemteroide and Pseudoalteromonas.sp.were 69.2,39.2,34.7,65.6 and 56.9 respectively,which were significantly higher than that of control(8.0)(P < 0.05).The LBS activity in group composite 1(9.38)was higher than that in the control(0.4)(P < 0.05).Activity of SOD in serum and AMS in tissue in all the experiment groups were lower than that in the control.Microbial diversity in the cultural water and intestinal tract were analyzed.The results show that the species of micro organism in aquaculture water from all experimental groups and control were similar.The mains species belong to Bacteroidetes,Proteobacteria,Actinobacteria,Cyanobacteria,Verrucomicrobia,Planctomycetes and Chlorobi,in which the domainent class was Bacteroidetes.The component ratio of Bacteroidetes in groups composite 2,composite 1,B.licheniformis,B.subtilis,Pseudoalteromonas.sp,L.mesemteroides were 54.5%,55.0%,34.8%,55.0%,34.0% and 32.5% respectively,which were all lower than control group(69.1%).The species of micro organism in the intestinal tract of shrimp from experimental and control groups were similar,the major class were Proteobacteria,Bacteroidetes,Actinobacteria and Verrucomicrobia,among of which Proteobacteria,Bacteroidetes were the dominant flora,both of them accounted for over 80 percent.The results show that the combination of multi-probiotc had some positive effect in prevetion and control of AHPND.The results from this study could provide some technical support for shrimp culture.