Formation Mechanism And Pathogenicity Of AHPND-causing Vibrio Campbellii Based On Conjugative Transfer

Acute hepatopancreatic necrosis disease(AHPND)is a bacterial disease that caused acute mortalities in farmed shrimp,and the mortality of diseased shrimp can reach 100%.The outbreak has caused huge economic losses to the shrimp farming industry in many countries of Southeast Asia and the America,and has been listed as a notifiable aquatic animal disease by the world organization for animal health(OIE).Initially,Vibrio parahaemolyticus carrying the pVA1 plasmid was considered as the only pathogen of this disease,but recent studies have shown that certain species such as V.campbellii,V.harveyi,V.owensii and V.punensis can also cause AHPND.The sequencing analysis of these pathogenic bacteria revealed that they all carried pVA1-type plasmid.According to the OIE manual,the plasmid carried a cluster of genes related to the conjugative transfer.Therefore,it is speculated that the formation of AHPND pathogenic bacteria diversity may be related to the natural transformation of the pVA1-type plasmid.In this study,the pVPGX1-Cm~r plasmid(a pVA1-type plasmid)was transferred from AHPND-causing Vibrio bacteria(Vp2S01-Cm~r)to a non-pathogenic V.campbellii(Vc LMB29)by conjugation transfer experiments at the transfer efficiency of 2.6×10~8(transconjugant/recipient)in 12 h.DNase I treatment did not eliminate the transfer.The recipient V.campbellii acquired the pVA1-type plasmid and was shown to produce PirAB~vpp protein using RT-PCR and SDS-PAGE.Challenge studies using the transconjugant caused100%mortality in the exposed group of Penaeus vannamei.The challenged shrimp,infected with the transconjugant bacteria,showed typical gross signs and histological lesions of AHPND.The transconjugant obtained from conjugation experiment and recipient bacteria(VcLMB29)were used to carry out the challenge experiment of P.vannamei.The hepatopancreas samples of the two groups of infected P.vannamei were taken for transcriptome sequencing analysis.After Illumina sequencing,174.4 G raw data was obtained.After filtering the date,1162603203 clean reads were obtained,which were mapping to the reference genome.The mapping rate of each sample was between 78.63%-90.35%.Analysis of differentially expressed genes(DEGs)in the transconjugant infection group and recipient bacteria infection group revealed that were 32,1100 and 218 different expressed genes at 2 h,8 h and 30 h,respectively.Analysis of these DEGs through the GO enrichment,these DEGs were annotated to biological process and molecular function,including glycolytic process,ADP metabolic process,oxidoreductase activity,transmembrane transport,fatty acid metabolic process,growth factor binding and serine-type endopeptidase activity.The results of KEGG pathway enrichment analysis showed that these samples could be enriched in various pathways such as pentose phosphate pathway,fructose and mannose metabolism,multiple amino acid metabolism,lysosome,serine and threonine metabolism,and protein export.It was found that under the stress of pVA1-type plasmid,it can cause the host to carry out an autoimmune response and regulate related immune genes such as heat shock protein,chitinase,lysozyme and hemocyanin.According to the results of STEM analysis,it can be obtained that all the gene clusters enriched in the samples of the two groups showed significant changes in expression levels at 8 h after infection.The two groups of samples have the same gene clusters,which can be annotated to transmembrane transport,pathogenesis and oxidoreductase activity.In addition,immune-related functions such as regulation of transcription and evasion or tolerance of host defense response were also found in each group.In this study,transcriptome sequencing was used to exclude the effects of bacteria on P.vannamei,and the effect of pVA1-type plasmid on the immune response of P.vannamei was explored,which provided a basis for the study of a pVA1-type plasmid.This study demonstrated the conjugative transfer of an AHPND pVA1-type plasmid.It provides timely information for explaining the increased species of AHPND-causing Vibrio bacteria and will be useful in the development of management strategies leading to the prevention and control of AHPND.The results of the transcriptome provide transcriptome data for pathogenesis and host immune response of AHPND.