Molecular Identification Of Klebsiella Pneumoniae And Expression Of Immune Genes In Infected Spotted Gar Lepisosteus Oculatus

Spotted gar(Lepisosteus oculatus)is a lower holostean,which has not undergone the teleost genome duplication(TGD).Therefore,spotted gar genomes are conserved in terms of content and size,and are closer to the human genome than those of teleost fish,serving as a bridge between teleost fish and tetrapods.In recent years,spotted gar has provided important resources for studying the evolution of immune genes as a biological model.In this study,pathogens were accidentally isolated from diseased spotted gar and identified as Klebsiella pneumoniae.In order to understand the pathogenicity of isolated strains,different concentrations of pathogenic bacteria were injected into healthy spotted gar,and the symptoms of spotted gar were observed and recorded in half a month after treatment.After 8 days post infection,fish displayed disease symptoms,including apparent swelling and hemorrhage of liver,spleen and other internal organs and K.pneumoniae can be isolated again in spotted gar.Drug sensitivity test showed that the K.pneumoniae strain was sensitive to streptomycin and gentamicin,but was relatively resistant to amoxicillin,rifampicin and erythromycin.We identified the virulence and resistance genes contained in the purified strains by PCR and analyzed the biosynthetic gene clusters of potential secondary metabolites of the strains using online software.In addition,we infected healthy spotted gar with the isolated strains and do histopathological analysis after 2,4 and 8 days infection.We found that the spleen was swollen and the sections showed a large reduction of lymphocytes and a large increase of red pulp area.Based on the special evolutionary status of spotted gar,we obtained partial c DNA sequences of tumor necrosis factor ligand and receptor genes(TNFSF14,TNFRSF1 A,TNFRSF1B and TNFRSF14)from spotted gar genome data uploaded by NCBI.Their full length was amplified by SMART-RACE method,and their gene structure,amino acid sequence alignment,evolutionary relationship,syntenic and 3D structure were analyzed by bioinformatics techniques.The full-length c DNA of TNFRSF1 A,TNFRSF1B and TNFRSF14 were 723,1915,1863 and 1383 bp,encoding 240,403,446 and 197 amino acids(aa),respectively.Furthermore,TNFSF14,TNFRSF1 A,TNFRSF1B and TNFRSF14 were constitutively expressed in the kidney,gills,liver,intestine,and spleen of spotted gar.Among them,TNFRSF14 gene was first reported in fish,while TNFSF14 gene was only reported in zebrafish and fugu.In order to explore the relationship between bacteria and host immunity,we further analyzed the expression of immune-related genes after spotted gar infection with KPY01.We sampled spleen,kidney and gills 2,4 and 8 days after infection to analyze expression of immune genes including TNFSF14,TNFRSF1 A,TNFRSF1B and TNFRSF14,IL-8,IL-10,IL-12 A,IL-18 and IFN-γ.The expression of IL-10,IL-12 A,IL-18 and interferon IFN-γ in spleen,gill and kidney were significantly up-regulated after infection,suggesting that these immune genes play an important role in host immune defense against bacterial infection.In this study,K.pneumoniae was isolated from lower holostean spotted gar,and its symptoms,virulence,drug resistance and potential secondary metabolite synthesis clusters were analyzed and predicted.In order to provide the basis and reference for the effective examination and prevention of diseases caused by K.pneumoniae and the immune response mechanism of fish responding to K.pneumoniae infection,we conducted pathological sections and investigated the expression of host immune genes after infection.