Analysis Of The Causative Pathogens Of Acute Hepatopancreatic Necrosis Disease (AHPND) In Litopenaeus Vannamei Anditsprevention And Control

Acute hepatopancreatic necrosis disease（AHPND）has strong pathogenicity,high mortality and wide spreading,is currently one of themost serious diseases in shrimp farming.Since 2009,AHPNDoccurred frequentlyin the main shrimp producing areas in the coastal areas of our country.The failed pond was counted over 80%,and has the deeply impact on Chinese shrimp industry,resulting in mass economic losses.The purpose of this study is analysis of five strains of potential pathogenic bacteria isolated from diseased shrimp,through the artificial infection experiment with the method of oral bacteria.Four strains of bacteria were identified as pathogens for AHPND.Meanwhile,a study on the relationship between the four kinds of disinfectants with the concentration,killing time and killing rate of the four pathogens,finally screened out an efficient,green disinfectant called as polyhexamethylene guanidine hydrochloride（PHMG）.Using candidate highly effective antibiotic drug "HPP-1"（20%florfenicol）and PHMG to establisha quantitative control technique to vibrios in shrimp culture system,the prevention and treatment experiments of AHPND were conducted in shrimp farms.The results were reported as below.1、Virulence analysis of five bacterialstrains to AHPNDFive strains（PV130715A,PV130903 A,PV140731A,PV150526 A,PV140821A）were isolated from shrimp affected AHPND in Zhejiang,Jiangsu,Shandong,Hebei and Tianjin Province.We design the artificial infection experiment with the method of oral bacteria 3d（once a day）at5.89⁵8915.94 cell/g to analysis the pathogenicity of the five bacterial strains,the cumulative mortality of Litopenaeus vannamei and the infection symptoms were obsevered.The results showed that four strains bacteria could cause AHPND infection with typical signs.We have isolated four strains（PV130903B,PV140731 B,PV150526B,PV140821B）from the artificial infected shrimp,theirsequence homology was 100%to PV130903 A,PV140731A,PV150526 A,PV140821A,though physiological and biochemical and 16 S rDNA molecular identification.Determination of PV130903 A and PV130903 B for the same strains,identified as Vibrio parahaemolyticus;PV140731A and PV140731 B for the same strains,identified as Vibrio harveyi;PV150526A and PV150526 B for the same strains,identified as Vibrio alginolyticus;PV140821A and PV140821 B for the same strain,identified as Vibrio sp.Ex25.According to the Koch rule,symptoms and pathologic features,we confirmed that Vibrio parahaemolyticus,Vibrio harveyi,Vibrio alginolyticusand Vibrio sp.Ex25 were the pathogens of AHPND.The LD50 of the four strains was 1.8×10⁵cfu/g,8.5×10⁴cfu/g,2.3×10⁵cfu/g,2.9×10⁵cfu/g。In the case of Litopenaeus vannamei,the pathogenicity of Vibrio harveyi was stronger than that of Vibrio parahaemolyticus,Vibrio alginolyticusand Vibrio sp.Ex25.PV130715 A is Aeromonas hydrophila,was not the pathogen of AHPND.2、Study on the effect of several disinfectants on the pathogenic bacteria of AHPNDWe have isolated Vibrio parahaemolyticus,V.harveyi,V.alginolyticus,V.sp.Ex25 from the diseased shrimp,and designed the qualitative and quantitative germicidal experiment on them.In which,PHMG,hydrogen peroxide,povidone-iodine and chlorine dioxide were chosen to determine the concentration,killing rate and killing time on the four kinds of pathogenic bacteria.The results show that,in terms of bactericidal concentration within 24 h,the concentration of 0.5μl/L PHMG could kill V.parahaemolyticus,V.harveyi and V.sp.Ex25,1μl/L PHMG could kill the four pathogenic bacteria mentioned above;0.5μl/L H₂O₂ could only kill V.sp.Ex25,1μl/L H₂O₂ could kill all the four bacteria;6μl/L povidone-iodine（PVPI）could kill V.parahaemolyticus and V.sp.Ex25,8μl/L PVPI could kill all the four bacteria;and 2μl/L chlorine dioxide（ClO₂）could kill all the bacteria;In terms of sterilizing time by 10 minutes,the lowest concentration of PHMG and H₂O₂ could kill all the bacteria were 1μl/L;In killing V.parahaemolyticus,V.harveyi,and V.sp.Ex25,the lowest concentration of ClO₂ was 2μl/L;10μl/L PVPI could kill the four bacteria.By 1h,2μl/L ClO₂ could completely kill the four bacteria;by 2h,1μl/L PHMG and H₂O₂ could completely kill the four bacteria.It should be pointed out that PHMG and PVPI showed continuously sterilizing ability,the sterilizing rate of PHMG and PVPI gradually increased as time goes on.On contrast,the sterilizing rate of H₂O₂ and ClO₂ decreased after reaching their peak value,the higher of the concentration,the shorter of the peak time.The bactericidal ability of these four disinfectants was PHMG>H₂O₂>ClO₂>PVPI.In consideration of bactericidal concentration,germicidal efficacy,duration as well as the cost,it was proved that PHMG has the advantages of safety,green non-pollution,efficiency and stamina in aquaculture,and having a good application prospect in the future.3、Effects of the candidate drugs on vibrios and their application in shrimp cultureIn this paper,we used a combination of two methods to kill vibrios in 12 shrimp farms located at Tianjin,Hebei,Jiangsu,Zhejiang,Shandong provinces.One is using specific oral drugs to kill the vibrios in the hepatopancreas.And another one is using PHMG to kill the vibrio in the pond water.The results show that the total amount of vibrio in the aquaculture ponds decreased 80.84% on4 d under 0.5μl/L concentration of PHMG and the 4d got the highest rate;the total amount of vibrios in the culture ponds decreased 85.67% on 3d under 1μl/L concentration of PHMG and the 3d got the highest rate;the total amount of vibrios in the culture ponds decreased 88.60% on the3 d under 2μl/L concentration of PHMG and the 3d got the highest rate in the prevention experiment.The total amount of vibrios in the culture ponds decreased 65.25% on 3d under 0.5μl/L concentration of PHMG and the 3d got the highest rate;the total amount of vibrios in the culture ponds decreased 62.29% on 3d under 1μl/L concentration of PHMG and the 3d got the highest rate;the total amount of vibrios in the culture ponds decreased 87.05% on the3 d under 2μl/L concentration of PHMG and the 3d got the highest rate in the treatment experiment.Applying one time using 0.5²μl/L concentration of PHMG,till 11 d the total amount of vibrios in water could maintain below the initial value.With the specific oral drugs "HPP-1"（dose 6g/kg,continuous feeding 5d）,the total amount of vibrios of test groups in the hepatopancreas was straightdown;The total amount of vibrios in the hepatopancreas decreased more than 99% on the 3d;From the beginning of the 6d,the total amount of vibrios of test groups showed slow growth,the total amount of vibrios until 15 dwas still lower than the initial amount value.The harvestingof the AHPND prevention experiment suggested that 11 earthen ponds and 9 greenhouses were not infected by AHPND and the morbidity was nil;In the earthen ponds,we harvested 128kg/mu（24 pics per kg,FCR 1.03）;304.5kg/mu（54 picsper kg FCR 1.02）;In the greenhouses,we harvested 767kg/mu（52 picsper greenhouse,FCR 1.06）;In control groups,all of the five earthen ponds were finally infected by AHPND;while 6 of the 8 greenhouses were infected by AHPND and the morbidity was 75%.Folliwing the treatment experiment of AHPND,it illustrated that186 ponds were recovered among 217 diseased ponds,the cure rate was 86.92%.In the earthen ponds,we harvested 109³70kg/mu（FCR1.02¹.05）;In the greenhouses,we harvested 567kg/mu（FCR 1.08）;In the intensive ponds,we harvested 2109.75kg/mu（FCR 0.97）;In the industrial ponds,we harvested 7.9kg/m2（FCR1.18）.All of the control groups were infected by AHPND.The prevention and treatment experiment suggests that the combination of special oral drugs "HPP-1" and disinfectant PHMG could kill vibrios in both hepatopancreas and water body,to quantitatively maintain the vibrio acount.This method can effectively reduce the morbidity and improve the cure rate.It also has very important value to control AHPND,and gaving good guidance for healthy shrimp farming.