| Coinfection or secondary bacterial infection followed with viral infection were common in the aquaculture industry.However,there was lack of mechanism investigation on the virus/bacteria coinfection,which seriously restricts the formulation of effective prevention and control measures.Epidemiological investigation found that CyHV-2infection was usually accompanied by a coinfection of bacteria,suggesting that coinfection with CyHV-2 and bacteria may be associated with the high mortality of gill bleeding disease.Thus,based on the epidemiological investigation of hemorrhagic disease,we analyzed the pathogenic effects of viral/bacterial coinfection as well as the kinetic characterization of pathogens.We further investigated the pathogenic mechanism by histopathology and transcriptome analysis,which would help to develop effective control measures against CyHV-2.The main findings are as follows:1.Epidemiological investigation of CyHV-2 and secondary bacterial infections in cultured gibel carpTypical diseased gibel carp,Carassius auratus gibelio were collected from the diseased pond,and CyHV-2/bacterial infection were determined.The results showed that the amount of CyHV-2 DNA copies in the tissues of spleen or kidney sampled from the diseased fish was around 108～1012copies/μg DNA.Meanwhile bacteria were also isolated from liver,spleen and（or）kidney.The bacteria identified in our investigation included Aeromonas,Acinetobacter,Bacillus,Enterobacter,Vibrio,etc.,most of which could be found in the normal intestinal flora.In agreement with the epidemic investigation results,the bacterial species of secondary infection after artificial infection with CyHV-2 under aseptic condition are normal intestinal flora.These results indicate that CyHV-2 can lead bacterial secondary infection acute infection.2.Pathogenicity and pathogenic kinetics investigation of coinfection with CyHV-2 and bacteria through experimental infection.The pathogenicity of coinfection with CyHV-2 and AH combined with virus and bacteria titer were analyzed through CyHV-2/AH artificial infection in gibel carp.The results showed that the mortality in the group of AH secondary infection at 3 d after CyHV-2 infection（CyHV-2-AH-3d）was highest.Interestingly,no mortality was observed in the group of AH secondary infectionatn 14d after CyHV-2 infection.Correspondingly,CyHV-2titer at the 3d after CyHV-2 infection increased sharply,and the titer of virus and bacteria in the group of CyHV-2-AH-3d was significantly higher than any other coinfected or single infected groups.Both of the virus and bacteria titer were significantly lower than that in the group infected with AH or CyHV-2 alone.These results indicated that there is synergistic between CyHV-2 and AH in the specific coinfection pattern,of bacterial secondary infection at 3d after CyHV-2 infection.Conversely,there is antagonistic between CyHV-2 and AH in the coinfection pattern of bacterial secondary infection at 14d after CyHV-2 infection3.Pathological and molecular mechanism of bacterial secondary infection after CyHV-2infection.The pathological mechanism of coinfection was studied by intestinal histopathological section.Compared with other groups,the intestinal tissue showed obvious lesions and the intestinal mucosa was seriously damaged at the 3 d after CyHV-2infection,which indicated that CyHV-2 infection created conditions for intestinal bacterial translocation.The molecular mechanism of coinfection was analyzed by spleen transcriptomics analysis.Compared with control group,the expression level of MHCI pathway was significantly up-regulated at 3d after CyHV-2 infection with the downregulation of the expression level of MHCII gene.In addition,the expression level of intestinal mucosal immunoglobulin production pathway related genes including MHCII,TCR,AID,CCR9and CCL25 gene were significantly down-regulated.These results indicated that CyHV-2infection activated host anti-viral immunity and weakened fish antigen presentation and phagocytosis pathway which leads to the translocation of intestinal bacteria and its clearance in spleen. |
