A Preliminary Study On The Virulence Differences Of VP_AHPND Pathogenic Bacteria Of Acute Hepatopancreatic Necrosis

Litopenaeus vannamei,belongs to the tropical shrimp species with wide salinity,is currently one of the most productive species of shrimp in the world.With the continuous expansion of the scale of aquaculture,various shrimp diseases frequently occur,which seriously endangers the development of the shrimp aquaculture industry.In recent years,acute hepatopancreas necrosis disease（AHPND）has been an important disease of Litopenaeus vannamei,Penaeus chinensis,Penaeus monodon,Eriocheir sinensis and other aquatic animals.It was confirmed that the pathogen of AHPND was a type of Vibrio containing special virulence factors,mainly Vibrio parahaemolyticus(VP_AHPND).The shrimp hepatopancreas showed whitish,hyperemia,and tubule epithelial shedding after VP_AHPND infected.In 2016-2017,AHPND broke out in many areas of Jiangsu province.It was found that the shrimp death in ponds showed acute and subacute states,causing huge economic losses to local farmers.After isolation and identification,6 VP_AHPND strains were screened and purified from different diseased shrimp hepatopancreas in Jiangsu.In order to study the acute and subacute mortality and pathogenesis of L.vannamei after infection,6 strains of VP_AHPND were used to verify the virulence differences in this study.The virulence genes and related immune genes of the strains were carried out at the molecular level to carry out multilocus sequence typing（MLST）by RT-q PCR,q PCR and other technologies.The results are as follows:1.Verification of the virulence difference of AHPND VPAHPND strains in Jiangsu area and MLST multilocus sequence typingAfter the strain was cultured to the logarithmic growth phase,L.vannamei was infected using 1×10⁷ CFU/m L 6 VP_AHPND strains by the dipping method for 96 h.The mortality rate was calculated every 6 h.The death rate of more than 50%within 72 h was determined as acute death,and the death rate did not reach 50%but occurrence death was determined as subacute death.Using MLST to study the 6 strains and the 12strains of VP_AHPND that have been reported in various regions,the results showed that the strains SHY1669,SHY1776,SHY1777,and SHY1833 were identified as acute strains,and the strains SHY1673 and SHY1697 were subacute strains.The MLST results showed that the typing results of strains SHY1669,SHY1673,SHY1697,SHY1776,SHY1777,SHY1833 belonged to ST452,ST882,ST415,ST114,ST919,ST2355,respectively.Meanwhile,ST2355 was a new ST type.The other strains were all known ST types with the highest homology to ST1743.Phylogenetic tree analysis showed that acute and subacute strains showed no significant differences in evolution.In addition,the results showed that acute strains mainly appeared in Asia,while subacute strains appeared in North America and Asia.The above results confirmed that different VP_AHPND strains with different virulence,but they did not show differences in genetic relationship in MLST.2.Analysis of virulence protein expression in acute and subacute strainsGene pirA and pirB have been confirmed as important virulence factors for VP_AHPND.6 VP_AHPND strains were cultured overnight to the logarithmic growth stage.RNA was extracted and reverse transcribed into c DNA.The results showed that the pir A gene expression in the SHY1697 subacute strain was significantly lower than that of the acute strain,which was consistent with the results of the reversal challenge experiment.The strain with the highest lethality rate,SHY1669,had the highest virulence copy quantity.The pir A transcription in 6 h,12 h and 24 h were detected in different VP_AHPND strains.The results showed that the pir A transcription in acute strains was higher than that of subacute strains in the three time periods.Except for the acute strain SHY1669,the virulence factors in acute strains increased with the extension of the culture time and peaked at 24 h.The subacute strain reached its maximum expression at 12 h,but it was still lower than the acute strain infection group.The above results indicate that the mortality rate has a certain relationship with the secretion of virulence factors in VP_AHPND strains.3.Analysis of VPAHPND quantitative and immune-related genes difference after AHPND infection in Litopenaeus vannamThe acute strains SHY1669 and the subacute strain SHY1697 with the largest difference in virulence were selected to infect L.vannamei.The hepatopancreas were taken at 6 h,12 h and 24 h,respectively.RNA was extracted and reverse transcribed to detect the differences in the transcription levels of virulence factors（pir A,pir B）and immune-related genes(SRC kinase、caspase 2（CAS2）,inhibitor of apoptosis（IAP2）and cytochrome P450 in the hepatopancreas at different time periods.The results showed that at 6 h and 12 h,the pir A gene expression in the hepatopancreas of the acute strain infection group was higher than that in the subacute strain infection group at 6 h and 12 h,and was lower than that in the subacute strain infection group at 24 h.The pir A expression in the acute subacute strain infection group was up-regulated and reached its peak at 12 h after challenge,then down-regulated at 24 h.The expression of pir B in the hepatopancreas is similar to pir A,but it was different from the subacute strain infection group in that the pir B expression in the hepatopancreas of subacute strain SHY1697 was up-regulated and peaked at 24 h;while at the acute strain infection group,pir B was significantly down-regulated at 24 h.CAS2 and cytochrome P450 were higher than the subacute strain infection group in the two time periods.The above results indicated that after VP_AHPND infection,the virulence factors colonized in the hepatopancreas,and destroyed the immune system of the prawns,which in turn caused both acute and subacute courses of disease.