Study On Immunoprotective Efficacy Of Formalin-inactivated Vibrio Harveyi Vaccine And Iridovirus Disease Of Hybrid Grouper Epinephelus Bruneus (♀) × E. Lanceolatus (♂)

In order to control Vibrio harveyi disease of marine fish,formalin-inactivated V.harveyi strain ML01 Vaccine was produced and the immunoprotective efficacy was evaluated.The vaccine with or without astragalus polysaccharides（APS）and taraxacum polysaccharides were used to immunize olive flounder（Paralichthys olivaceus）through intraperitoneal injection.The immunological protective efficacy was analyzed by detecting specific serum antibody titers against V.harveyi cells and the expression of MHCⅠ,MHCⅡ,TLR2,HSP70,IL-6,NF-κB,CD4,CD40,NKEF in head-kidney and spleen with RT-PCR after immunization.The SOD,ACP,AKP activity of immunized fish was also investigated.On the 28 days post immunization（dpi）,P.olivaceus were challenged by ten fold LD₅₀0 of V.harveyi and the death of P.olivaceus was recorded.The results showed that all immune parameters,including SOD,ACP,AKP,serum antibody titer,immune related gene expression and relative percentage survival,were enhanced in all treatment groups.The study showed that the formalin-inactivated V.harveyi vaccine can reduce the death rate of the infected flounder,and the protective efficacy can be improved by astragalus polysaccharides and taraxacum polysaccharides.“Helong grouper”is the hybrid progeny produced by female longtooth grouper（Epinephelus bruneus）and male giant grouper（E.lanceolatus）.As a new breed of hybrid grouper,there is no report on diseases of Helong grouper at home and abroad.In July 2017,more than 4000 individuals of 2-year-old Helong grouper on a fish farm in Shandong province suddenly died and the cumulative mortality was as high as 80%within 10 days.A field survey found that the water temperature was 28°C and the salinity was 31 during epidemics.The appearance of diseased fish was normal.However,they were unresponsive,with poor vitality and death at the bottom.Clinical examination and necropsy revealed that the diseased fish showed normal gills.The stomach and intestines were empty,but no significant inflammation.Some parts of the liver were hemorrhagic.The spleen and kidney were severely swollen and crispy.The kidney usually were pale.The histopathology by hematoxylin and eosin staining indicated that tissues of diseased fish were severely damaged.The hematopoietic tissues in the spleen and head kidney of diseased fish were necrosis.A large number of basophilic,homogeneous cytoplasmic,swollen cells with a diameter of 10—15μm were observed in these tissues.Focal necrosis were also found in liver tissue.The cells of glomerulus in kidney were hydropic degeneration.The epithelial cells in secondary lamellar were exfoliation.Some pillar cells ruptured to produce lamellar telangiectasis in diseased fish.The structure of the myocardium was disordered and degenerate,accompanying necrosis of myocardial cells.In ultra-thin tissue sections of spleen and head kidney,there are a large number of iridovirus-like particles with a diameter of 130—150 nm in the cytoplasm of the swollen cells.The cell membrane of swollen cells invaginated and formed many dendritic sulcus structures.The mitochondria of cells in diseased fish swelled and vacuolated,and a large number of longitudinal cristae appeared.The mitochondria in the myocardial cells were degenerated into myelin figures.Using specific PCR primers,severe infections of red seabream iridovirus（RSIV）were detected in the spleen,head kidney,kidney,and liver tissues of diseased fish,which was consistent with electron microscopy observations.The complete coding sequence of the major capsid protein（MCP）gene of the virus was determined to be 1362 bp in length.According to Blast,this sequence is identical to the corresponding sequence of four RSIV strains that infects brown-marbled grouper（E.fuscoguttatus）,orange-spotted grouper（E.coioides）,red seabream（Pagrus major）,and greater amberjack（Seriola dumerili）.That means the similarity of MCP gene of the five iridovirus is 100%.Based on the complete coding sequence of MCP gene,a phylogenetic tree of 19 species（isolates）of iridovirus was constructed.The tree showed that the iridovirus infected with the Helong grouper belongs to the RSIV clusters of genus Megalocytivirus,family Iridovirus.This research not only confirms that RSIV can naturally infect Helong grouper,reveals a new susceptible host of RSIV,but also provides an important reference for the diagnosis and control of hybrid grouper virus disease.