| Mandarin fish(Siniperca chuatsi)is a traditional and valuable fish in China.With the increase of intensive cultivation,the increase of density and the deterioration of water environment,the disease problem of mandarin fish is becoming more and more serious.At present,it has become the main factor that threatens the healthy development of Chinese mandarin fish aquaculture.Septicemia caused by Aeromonas hydrophila often results in mass death of mandarin fish,which has caused the huge economic loss to the farmer.In this study,the pathogenicity of A.hydrophila to mandarin fish was studied,and a LAMP method was developed for the detection of A.hydrophila.The transcriptome analysis and the expression analysis of immune related genes at different time points of mandarin fish infected with A.hydrophila were analyzed to provide scientific basis for the healthy and sustainable development of the culture industry of mandarin fish.In this study,the pathogenicity of bacterial diseases that occurred in the breeding and production of mandarin fish in Jiangsu Province was studied,and the phenotypic characteristics,pathogenicity,homology of 16S rRNA and gyrB genes of the isolated bacteria were tested.The results showed that A.hydrophila was an important pathogen to the culture of mandarin fish,and the LD50 of A.hydrophila to mandarin fish was 1.6×106 CFU/mL;The strain was rod-shaped,blunt at both ends and coverd with a large number of fimbriae on its surface;A.hydrophila has 14 virulence genes,including aerA,act,ahp,fla,alt,lip,ast,ahpB,eprCAI,hlyA,lafA,acg,gcaT and ompAI;A.hydrophila had protease,lipase,lecithin,amylase,gelatinase activities and ? hemolytic activity,but not urease activities.The gill,liver,spleen and head kidney of the diseased mandarin fish showed that the cells were loosely arranged and appeared vacuoles and necrosis.A Loop-mediated isothermal amplification(LAMP)method was developed for the rapid detection of pathogenic A.hydrophila using aerolysin gene(aerA)as a molecular target primer.The results showed that a ladder-shaped band was amplified from the A.hydrophila(G3),and showed a positive reaction with SYBR Green I staining,while other control strains were negative.The results indicated that the LAMP method had good specificity for A.hydrophila detection,and the sensitivity was 4.6×101 CFU/mL.The detection method has the characteristics of strong specificity,high sensitivity and convenient use,and can be applied to the detection of aquatic animal diseases caused by A.hydrophila.In order to better reveal the complex relationship between the mandarin fish and A.hydrophila,transcriptome sequencing was performed in the head kidney tissues of mandarin fish infected with A.hydrophila 24 hours later.A total of 53,040 Unigene genes were obtained,and the gene expression maps of head kidney tissues of the mandarin fish and uninfected group were compared and analyzed.A total of 529 differentially expressed genes(DEGs)were obtained,including 254 up-regulated genes and 272 down-regulated genes.The differentially expressed genes were analyzed for GO enrichment,and 29 types of GO were found,including biostimulation,immune system regulation,immune response and cytokine-induced response.The differentially expressed genes were enriched by KEGG,and the immunologic-related KEGG pathway was found to include endophagocytosis,phagosome,mitogen-activated protein kinase and transforming growth factor ? pathway.In addition,seven immune-related DEGs,including IL-6,were selected randomly by qRT-PCR for further verification,and the results were consistent with the RNA-seq data.Transcriptome analysis will help us understand the immune and defense mechanisms of mandarin fish at the whole transcriptome level,and will provide a theoretical basis for the prevention of septicemia caused by A.hydrophila.In order to further clarify the immune response of mandarin fish infected with A.hydrophila,the expression levels of six immune related genes(MHC?,TCR-?,TNF-?,CC Chemokine 3,IL-8 and Hepcidin)were detected by qRT-PCR at 6 h,12 h,24 h,48 h and 72 h after A.hydrophila infection.The results showed that the expression of six immune related genes in gill,spleen and head kidney of mandarin fish was significantly up-regulated at 6-48 h after A.hydrophila infection,and reached the normal level at 72 h.The results showed that the expression levels of these immune-related genes were rapidly up-regulated in the early stage of mandarin fish infected with A.hydrophila.These immune-related genes participate in the path of resisting pathogen invasion and play an immune protective role on the body itself.The results provide a theoretical basis for the defense of mandarin fish diseases and healthy breeding. |
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