Characteristic Research And Separation Of Several Bacterial Pathogens In Fish, Grass Carp And Silver Carp

Grass carp,silver carp,as China's traditional fine varieties of freshwater aquaculture,has been welcomed by the overwhelming majority of the fishmen and consumers.Especially in recent years, the demand for market diversification makes grass carp,silver carp demand increase every year, prices rose steadily,and the Green Grass carp feed breeding's low cost,high efficiency,silver carp's feeding on the natural food organisms in water,both well satisfied people's needs on natural food. Therefore,grass carp,silver carp now are more and more widely cultured.However,as with the development of aquaculture intensive cultivation,the farming methods of increasing stocking density,artificial diets fed significantly increased,the quality of aquaculture water also has been deteriorating.With the people's awareness of preventing and curing diseases backward,aquatic disease research lags behind,thus a variety of bacterial diseases,such as bacterial gill-rot disease, skin disease and Chek enteritis disease.etc.They have seriously affected the healthy culture and constrainted the increase of its yield and fishemen's incomes,which also becomed a bottleneck which impedes grass carp,silver carp scale aquaculture developmentThis paper researched grass carp,silver carp's several serious bacterial disease,made separation, identification and the determination of the characteristics of the pathogens,and the sensitivity tests of commonly used antibiotics and Chinese herbal medicine on the grass carp bacterial gill-rot, enteritis,Chek Paper Pathogen and silver carp Klebsiella pneumoniae drug,the study also compared the effects of the use of formalin-inactivated Pseudomonas fluorescens manufactured by immersion vaccine,vaccination and oral health microencapsulated vaccine to species of grass carp.1.Characteristic identification and separation of grass carp's bacterial gill-rot,enteritis,and red skin disease pathogensIsolated respectively 2(CLS-01,CLS-02),1(CCY-01)and 2(CCP-01,CCP-02)purification bacteria from grass carps which suffered from gill-rot disease,enteritis disease and red skin disease. After the morphology,staining characteristics,hemolytic and physicochemical identification,these bacteria were respectively columnar flexor nuisance bacilli(Flexibacter columnaris),intestinal point Aeromonas(Aeromonaspunctata fintestinalis)and Pseudomonas fluorescens (Pseudo-monasfluorescens).Rsult of the pathogenic test showed the 3 isolated bacteria made separately the mortality rate of grass carp reach 100%,95%,100%in 96h,48h,and 96h,and the isolated bacteria recycled from dead grass carp were same as natural bodies.The sensitivity test of Chinese and western medicine showed that in addition to ampicillin sodium,Kushen,Verbenaceae, Bupleurum,Andrographis,Daqingye,herbs Compound 3,not sensitive compound 6(CCP-01 Mr Wong,berberine,and Kam grass and skeletal grass,CLS-01 on the bones and tendons grass,not sensitive Houttuynia cordata),the 3 isolated bacteria showed different degrees of sensitivity to other antibiotics and Chinese herbal compound medicine.Of them CLS-1 to ofloxacin(50mg/L),CCY-01 to enrofloxacin(50mg/L)and norfloxacin(50mg/L)were extremely sensitive,as well as the 3 isolated bacteria to gallnut,Chinese herbal compoundl,compound 2,compound 5,Compound 9. The minimum inhibitory concentration of nrofloxacin,florfenicol,gallnut and baicalin to three kinds of bacteria(CLS-01,CCY-01,CCP-01)were respectively(0.391mg / L,0.049mg/L,78.1mg/L), (0.977mg/L,0.4883mg/L,31.250mg/L),(0.781mg/mL,1.563mg/mL,0.391mg/mL),the minimal bactericidal concentration were respectively(1.563mg/mL,6.250mg/mL,25000mg/mL);(1.562mg/L, 0.195mg/L,3.125mg/L),(3.906mg/L,3.9062mg/L,125.000mg/L),(3.125mg/mL,6.25mg/mL, 1.563mg/mL),(15.625mg/mL,25.000mg/mL,100.000mg/mL).The results showed that enrofloxacin,florfenicol,gallnut and baicalin had a strong antibacterial and germicidal effect on the 3 aquatic pathogens above.The drug would be safe and effective if used in a proper concentration.2 Characteristic identification and separation of Silver carp's gill-rot disease pathogensThis research separated pathogen from the disease silver carp's gill and liver.The pathogen is a gram-negative bacteria,a capsule,Cuduan rod,the size of about 0.3-0.4×0.5-0.8μm,single-existent or two adjacent:fermentation of glucose produced gas and acid,positive for lactose,maltose, mannitol,sucrose,citric acid salt,urea;negative for ornithine,peptone water,alcohol Mao Wei, phenylalanine,H2S and strong positive for VP test,it turned out to be Klebsiella pneumoniae after bacterium identification.The regression tests in which separated bacteria were injected in the bottom of pectoral fin were all presented with symptoms similar to the natural incidence.Disk diffusion and double dilution method were used to test the sensitivity of the commonly used antibody western medicine to this bacterium and to determine the minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC);the sensitivity of the commonly used Chinese medicine to this bacterium was tested by the drilling method.The results show that:the selected 17 kinds of antibody western medicine all had different levels of inhibition on the isolated bacterium (KPCQC01),of them ofloxacin(50mg/L),ciprofloxacin hydrochloride(50mg/l),Connaught sparfloxacin Star(50mg/l),SMZ:TMZ(100mg/L),florfenicol(50mg/L)were extremely sensitive to this bacterium,but norfloxacin,ciprofloxacin hydrochloride and florfenic's antibacterial activity were weaker than ofloxacin and compound SMZ;trimethoprim(100mg/L),ampicillin sodium (0.2mg/L)were highly sensitive;cotrimoxazole(100mg/L),gentamicin sulfate(00,400IU/ml), Thiamphenicol(50mg/L),chloramphenicol(50mg/L)moderately sensitive;2 chlorine preparations, sulfamethoxazole(100 mg/l),Midecamycin(50mg/L),oxytetracycline(50 mg/L),roxithromycin (50mg/L)inhibition effect was not significant;the continual effect of ofloxacin and compound SMZ: TMZ2 drugs' inhibition to this bacterium were better in 48h,and the minimum inhibitory concentration and the minimum bactericidal concentrations were respectively 0.5mg/L and 1.0mg/L and 6.25mg/L and 400mg/L.Of the Selected 12 kinds of Chinese herb medicine,gallnut was extremely sensitive to the isolated bacterium(KPCQC01);while honeysuckle,Houttuynia cordata, and Yu baicalin were highly sensitive;the bacterium was not sensitive to Huanglian,Kam grass and gentian;11 groups of the Chinese herbal compound medicine had obvious inhibition effect on this bacterium,of which compound 9 was the best,whose diameter of the inhibition zone was 29.7mm.3 check the immune effects of grass carp in red skin diseaseInactivated grass carp's red skin pathogen by 0.5%formalin,then made separately into immersion vaccine,vaccination and oral microencapsulated vaccine,and thenused the corresponding vaccine to soak(a few bacteria 1.5×10~7 cfu/ml the vaccine solution)3 to 5 cm fry grass carp,inject 0.2ml/tail(for the number of bacteria 1.5×10~8 cfu/ml the vaccine)to10 g-grass carp and to feed 16g/ Group·every time microencapsulated vaccine in the mixed diet(containing 2%microencapsulated vaccine)to 10g- grass carp.After immunization,analyzed the prevent results of grass carp's red skin disease by detecting the experiment carp'serum and adopting the use of poison attack poison method in the first21 d,35d,49d,63d,77d.The results showed that all the 3 three immune system channels were effective.The average antibody titers(agglutination titer)of the Whole-cell vaccine immunization,injection immunization and microencapsulated vaccine oral immunization were respectively 1:19,1:190,1:62;the Pseudomonas fluorescens infection showed that 3 kinds of immune paths all had good protection effects,and the immune protection rates reached respectively 41.6% and 40.0%,83.4%and 80%,58.4%and 53.3%.