| As the largest breed of freshwater fish in China,grass carp(Ctenopharyngodon idellus)has a large scale of cultivation in Hubei Province and throughout the country.With the increase of breeding density and breeding scale,disease outbreaks are inevitable.Among them,bacterial gill-rot disease of grass carp caused by Flavobacterium cloumnare is an important disease that damages the grass carp breeding industry,causing great economic losses every year.In the past few years,the use of antibiotics to prevent columnaris disease had led to the emergence of multi-drug resistant strains.Immunostimulants having small side effects,specific cellular responses,and prevention of specific pathogens,have been used in aquaculture.Related studies have shown that bacterial exopolysaccharides(EPS)were able to enhance the immune response of fish and could confer protection against pathogen infections.It is a novel polysaccharide immunostimulant that needs to be developed.At present,most of the EPS produced by marine and polar microorganisms were developed.EPS extracted from aquatic animal pathogens are expected to become novel immunostimulants due to their specific immunogenicity.Therefore,this study was conducted on grass carp,with EPS of F.columnare as an immune stimulant to explore it effects on the immune function.Main results for this study were as follows:1.The preparation of EPS from F.columnare.EPS was obtained by fermentation of a F.columnare Pf1 isolated from Pelteobagrus fulvidraco in the laboratory.We obtained the high purity of EPS by method of removing protein with trichloroacetic acid,alcohol precipitation,distilled water dialysis and PEG20000 concentration.The concentration of EPS is 1.2 mg/m L by method of phenol-sulfuric acid.2.The study on effects of EPS on serum enzymes and relative percent survival(RPS).The alkaline phosphatase(AKP)and Super oxide Dismutase(SOD)enzyme activity in the serum were measured by Elisa,and the results showed that the fish stimulated with EPS per tail was significantly increased the enzyme activity than that of the control group after challenge of F.columnare.Before infection,AKP enzyme activity was significantly lowerthan that of control group;4d after infection,AKP enzyme activity reached a peak,which was significantly higher than that of control group.2d after infection,SOD enzyme activity reached a peak,which was significantly higher than that of control group.The survival rate of F.columnare on fish with injection of EPS,which was 85% respectively,was far higher than that of the control group,which was 55%.Relative percent survival of F.columnare EPS on grass carp was 67%.It is thus indicated that EPS produced by F.columnare can be used as an immunostimulant.3.The study on effects of EPS on immune related genes in the grass carp.Real-time PCR detection results showed that grass carp injected with 0.2mg EPS per tail was significantly up-regulated the m RNA expression levels of the interleukin-1(IL-1),major histocompatibility complex I(MHC I)and tumor necrosis factor-?(TNF-?)gene in the Hepatopancreas,spleen and head kidney than that of the control group after challenge of F.columnare in 4 d.The expression of short pentraxin(PTX)in the Hepatopancreas was tended to stabilization,while in spleen and head kidney,it changed like the other three genes.The m RNA expression levels of four gene appeared different variation trends in the trunk kidney.The results indicated that EPS produced by Flavobacterium columnar could enhance the inflammatory response,humoral immunity and antigen presentation ability of the fish.The results show that EPS of F.columnare could improve grass carp's immune response against F.columnare.Injection of EPS,the m RNA expression levels of the IL-1,MHC I,PTX and TNF-? were significantly up-regulated after challenge of F.columnare,producing and secreting more AKP and SOD,and significantly increasing the survival rate of grass carp. |
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