Tracking And Detecting Of A Litopenaeus Vannamei Pathogen In A Shrimp Farm And Application Of Denitrifying Bacteria In Shrimp Culture

1.Tracking and detecting of a Litopenaeus vannamei pathogen in a shrimp farmLitopenaeus vannamei is one of the main farmed shrimp in China.Along with the heavey demand of the shrimp product in the market in recent 10 years,intensive culture of shrimp is becoming a main culture method.However,in high density shrimp pond,the remnants of feed and faeces of shrimp could produce large amont of toxicant such as.NH4+-N and NO2--N,which were hamful to the shrimp by reduce the immunity and induce the outbreak of disease.In this paper we had tracked and detected the variation of NH4+-N and NO2--N content according the national detection standard,analyzed the composition of microorganisms and the variation of total vibrio sp in pond water and in the tissue of hepatopancreas and intestinal track by plate count method.Also the virulence assay of the vibrio were carried on.shrimp pathogens including vibrio,enterocytozoon hepatopenaei(EHP)and four viral pathogens(WSSV?IMNV?CMNV and IHHNV)were detected using molecular biological detect methods under a regular sampling pattern.The results show that the concentration of ammonia-N and nitrogen-N in the water of the indoor culture were fluctuating heavly with peak value at 4.9mg/L and higher than those in the pond culture(<1 mg/L).Concentration of nitrogen-N rose rapidly after 60 d of putting the shrimp seed in pond,whent it could reach 8.3 mg/L in indoor pond,while it was only lower than 0.4 mg/L in ourdoor pond;The results of the pathogen test showed that the shrimp show EHP positive in 40 d after putting the shrimp seed in pond indoor culture,while in the outside pond it wers only partially weak positive detected out during 40-60 d.All the shrimp were slight infected by VAHPND.The main species of vibrio identified by 16 sr DNA sequencing were Vibrio parahaemolyticus,V.lginolyticus,V.owensii? V.vulnificus,V.harveyi,The peak concentrion of vibrio in the indoor pond was 104CFU/m L and the outdoor pond was only 103 CFU/m L.The occurrence of AHPND may be related to the high concentration of ammonia and nitrous nitrogen in the environment,and it is needed further study.2.The efficiency and denitrification conditions of a denitrifier isolated from a tilapia recirculating aquaculture systemA bacterial strain with denitrification activity was isolated from a tilapia(Oreochromis Niloticus)recirculating aquaculture system and name as DZYC02.Research were used to determine the effects of carbon source on the denitrifying efficacy of DZYC02 strain.Also sodium citrate and ammonia were used as carbon source and nitrogen source to determine the effects of salinity,p H and C:N on the denitrifying efficacy.The results showed that the 24h-denitrifying rate was 100% for ammonia(NH4+-N)and 48h-denitrifying rate was 100% for nitrite(NO2--N)under the condition of sodium citrate being used as the carbon source,C:N ?15,initial p H5~7 and salinity between 0~15.Zebra fish(Danio rerio)were used to evaluate the biosafety of strain DZYC02 by immersion challenge,and the results showed that strain DZYC02 appear better safety features for fish.Bacterial identification by both 16 S r DNA sequencing and Biolog microbes identification system show that strain DZYC02 was a Klebsiella pneumoniae.The results of this study could provide theoretical support for the utilization of microbial denitrification technology in recirculating aquaculture system.3.The efficiency of two denitrifier used in shrimp cultureTwo denitrifier strain Halomonas.sp and K.pneumoniae,which were isolated from healthy shrimp pond tilapia respectively,were fermented and used in biofloc shrimp culture system.The efficiency of two denitrifier to shrimp health were evaluated by detecting the concentration of ammonia nitrogen and nitrate nitrogen in the water body,the body growth rate,survival rate and the nospecific immue parameter including total protein,activity of superoxide dismutase(SOD),lysozyme and phenol oxidase(PO)in the serum.The results show that adding both strain Halomonas.sp and K.pneumoniae to the shrimp culture system could reduce the concentration of ammonia nitrogen and nitrate nitrogen.The concetration of ammonia nitrogen in group Halomonas.sp with surcrose was 4.5 mg/L,which was remarkably lower thant that in the control group(7.5mg/L)on the day of fifth of the experiment.And on the day of 20 th,the concentration of nitrate nitrogen in group Halomonas.sp with surcrose was only 5mg/L,while it was 22 mg/L in the control.The survival rate and body growth rate in group of Halomonas.sp with surcrose were 60% and 70% respectively,which were significantly higher than those in the control(20% and 40%),moreover,the survial rate was higher than that in all other groups(P<0.05).The use of strain Halomonas sp had no effect on the total protein,activity of SOD,lysozyme and PO of shrimp serum.While using strain of K.pneumoniae coult increase the lysozyme activity of shrimp serum.The experimental results indacted that the use of strain Halomonas sp with concentraton at 103-104CFU/m L combined with surcrose with content at 50-70% of shrimp feed could get the best results.