Study On The Intestinal Microorganisms Of Grass Carp Antagonise Aeromonas Hydrophila

Bacterial hemorrhagic disease for aquatic animals caused by Aeromonas hydrophila is a a serious fish disease which had the high incidence rate,wide endemic areas,and caused great harm to fish.Nowadays,the use of drug and antibiotics is still the main mean of prevention and treatment of disease occurrence in fishery production.Antibiotics are the most effective way to control diseases in aquaculture,but an indiscriminate use of antibiotics as preventative and curative measures for diseases has resulted in antimicrobial resistance among pathogenic bacteria,and degraded environmental conditions,and can cause a serious impact on the safety of environment and food.A viable alternative to antibiotics would therefore be an important venture and for this reason the development of new antagonistic bacteria products that could be licensed for aquaculture use is receiving considerable interest.Antagonism is a natural phenomenon common among microbes,it refers to the growth inhibition between microbes in the same niche.Antagonistic bacteria can inhibit or kill pathogenic bacteria to achieve the purpose of disease control.At present,aquatic probiotics and antagonistic bacteria are mainly derived from terrestrial animals.These bacteria can not play an effective role in water and the intestines of aquatic animals.Therefore,it is imperative to screening autochthonous antagonistic bacteria from the gut of aquatic animals.In this study,a strain of antagonistic bacteria was isolated from healthy grass carp,which showed significant inhibition effect against A.hydrophila on NB agar plates.And the colonization ability and disease resistance of the antagonistic bacteria were further studied.Moreover,the metabolites of the antagonistic were separated and analyzed.The achievements of this research are as fellows:1.A strain of antagonistic bacteria was isolated from healthy grass carp,which showed significant inhibition effect against A.hydrophila on NB agar plates,with an inhibition zone of 9 mm in radius.This antagonistic bacteria belonged to Pseudomonas plecoglossicida.And the strain J-1showed no probable pathogenic in injection assays.2.The agar-plate culture method was used to testing the colonization ability to gut of grass carp.Grass carp with antagonistic bacteria bait after 6 days fed with normal diet.We quantified the abundance of the antagonistic bacteria J-1,and found that the strain J-1 still maintain a high level within 21 days.And fishes model(newborn organisms)were generated via treatment with sterile water containing antibiotics.We quantified the abundance of the J-1and transit marker(Escherichia coli),and found that the strain J-1 still maintain a high level unlike the of E.coli droped fast;Eighteenth days the number of antagonistic bacteria J-1 was about 10~5 CFU/per individual,the number of E.coli in the intestine of grass carp eighth days down to about 10~2 CFU/per individual.3.The in vivo experiments were adopted to verifying that the strain J-1 can inhibit A.hydrophila.This strain J-1 dietary supplement could increase the survival rates of grass carp challenged with A.hydrophila,the protective rate was 45.4%.We quantified the abundance of the J-1 and A.hydrophila,and found that antagonistic bacteria J-1 could inhibiting the growth of A.hydrophila in the gut of grass carp.In this study,mRNA changes of the above cytokine genes in liver,spleen and kidney were detected after oral administration of the antagonistic strain J-1.Our results showed that this antagonistic strain significantly down-regulated IL-1?and TNF-?in liver and kidney(P<0.05),while TGF-?was significantly up-regulated in kidney(P<0.05).Additionally,the expression of these cytokine genes were basically maintained similar levels to the control group in spleen.In general,there was no inflammatory response and this strain J-1 have little effect on cytokine expression.4.We get some crude extract by ethyl acetate from supernatant of bacteria fluid.This crude extract can inhibit the growth of A.hydrophila.We found that there were many kinds of active substances produced by the antagonistic bacteria J-1,including phenazine.And the specific components need to be further separated.These results suggest that this antagonistic strain P.plecoglossicida could colonize to grass carp intestinal and inhibit A.hydrophila in vitro and in vivo.Hence,P.plecoglossicida was suitable candidates for development as biological control agents of bacterial hemorrhagic disease...