Isolation And Identification Of Vibrio Harveyi In Cultured Litopenaeus Vannamei And Its Prevention And Treatment

The identification and pathogenicity of bacterium which was isolated from the diseased Litopenaeus vannamei with muscle whitish disease were studied.Then we researched effects of dietary chitosans with different molecular weight on growth and non-specific defence of Litopenaeus vannamei,finally discussed pharmacoknetics of enroflxacin and its metabolite ciprofloxacin in Litopenaeus vannamei.The results as follows:1.In September 2007,an epizootic occurred among cultered Litopenaeus vannamei (Penaeus vannamei Boone)in qiong hai,China.The diseased Litopenaeus vannamei exhibited whitish muscle in abdomen or opacitymuscles,and the pleopods turned red,gills were light yellow and hepatopancreas necrosis.Samples were taken aseptically from the hepatopancreas and muscle and put into LBN(LB medium supplement with 2% NaCl)paltes,which were incubated at 28℃for 24h.A predominant strain,which was designated as hai3,was isolated from the muscle of the infected shrimp.Pathogenicity assays revealed that hai3 was virulent to Litopenaeus vannamei by intraperitoneal injection challenge.The LD50 of hai3 to Litopenaeus vannamei calculated as 8.12×103CFU/shrimp to the 10±1g weight Litopenaeus vannamei.Strain hai3 was a Gramnegative,and curved bacterium with single polar flagellum.It could grow on TCBS medium and produced yellow colonies.Strain hai3 was similar to Vibrio harveyi inmorphological,most of the physiological,biochemical characterisics and 16S rDNA sequence.Strain hai3 was identified to be V.harveyi.Drugs sensiticity test showed that strain hai3 were highly senstivie to enroflxacin and norfloxacin, etc.The efficacy of Florfenicol,Thiamphenicol,Flumequine,oxytetracycline,Enrofloxacin Hydrochloride and Enrofloxacin Base against strain hai3 was evaluated.The results showed that the minimal inhibitoryconcentration(MIC)of Florfenicol,Thiamphenicol,Flumequine,oxytetracycline, Enroflox Acin Hydrochlorideand Enrofloxacin Base was 2μg/mL,64μg/mL,8μg/mL, 0.25μg/mL,0.5μg/mL and 0.5μg/mL.Portions of muscles,gilles,hepatopancreas,midguts and hearts from the experiments on Litopenaeus vannamei injected with strain hai3,desc- ribed the external sympoms of diseased shrimps.There were distinctively histopathology-cal changes in muscles,gilles,midguts and hearts,especially hepatopancreas.These histop-athological changes included celluar purescence,desquamation and tissues degeneration as well as bacteria centralization or decentralization in some tissues. Some mitochondrial cristae broke up; mitochondrial membrane and nuclear menmbrane partially swelled, broke up and finally became unclear.2.A feeding experiment was conducted to examine the effects of dietary different molecular weight chitosan on body weight, non-specific immunity and disease resistance for Litopenaeus vannamei. The basal diet was supplemented with 0,1,3,5 and 10 g·kg-1 1×104 molecular weight chitosan(MWC) and 1,3,5 and 10g·kg-1 1×104 molecular weight chitosan (MWC),respectively,in order to formulate nine experimental diets.Each diet was fed to triplicate groups of shrimps in bucket of plastics(1.5m3),and each bucket was stocked with 40 shimps(with initial weight of 5±1 g).Shimp were fed three times daily (8:00-8:30,15:00-15:30 and 22:30-23:00) to apparent satiation for 4 weeks. The water temperature fluctuated from 27.5 to 30.5℃, the salinity from 30 to33%o and dissolved oxygen content was approximatel 5mg·g-1 during the experimental period.After 1 week, results showed that body weight and Vibrio harveyi challenge were no significantly affected by dietary chitosan(P>0.05). After 4 weeks,the results of the feeding experiment showed that there were no significant differences in the body weight between the treatments(P>0.05).The examination of some non-specific immunological activities in serum revealed that there were significant differences(P<0.05)in phenoloxidas (PO) acticvity and lysozyme activity,whereas between 1×104 MWC and 3×104 MWC there were no significant(P>0.05).The total superoxide dismutase(SOD) activity was no significant differences(P>0.05) between the treatments.The results of Vibrio harveyi challenge showed that the shimps fed 3g·kg-1 1×104 MWC and 3g·kg-1 3×104 MWC had significantly lower cumulative mortality(43% and 47%,respectively) than the control group(67%).It is therefore suggested that whether oral administration of 1×104 MWC or 3×104 MWC at an optimal level of 3g·kg-1 for 4 weeks effectively improved Vibrio harveyi resistance and enhanced immune activity of the shrimp ingeneral.3.This experiment dealed with the pharmacokinetic study of Enrofloxacin following oral administration carried out in Litopenaeus vannamei(Penaeus vannamei Boone). Hemolymph and hepatopancreas and muscle concentration of Enrofloxacin were examined by high performance liquid chromatogram(HPLC).The pharmacyoknetic study showed that the concentration-time curve of enrofloxacin in hemolymph after oral admi-nistration can be described by a two-compartment open model. The main pharmaco-knetics parameters were as follows:t 1/2α,t1/2β,UC 0-t,AUC0-∞,K12,K21,CLB,3.167h, 37.277h,188.887 mg/L-h,190.362 mg/L-h,0.071/h,0.0211/h,0.158 L/h/kg.The paharmaco-kinetic parameters of the profile between enrofloxacin comcentration and time in muscle and hepatopancreas were analyzed by statiscical moment theory. The main pharmaco-knetics parameters were as follows:ti/2Z75.863h and207.592h,AUC0.655 L/h/kg and 0.043 L/h/kg.Ciprofloxacin,the metabolite of enrofloxacin can be detected in all three tissues.The values of main pharmacoknetics parameters Cmax (μg/mL),AUC mg/L·h,t1/2Z (h) were as followed:0.451,7.423 and 12.432for hemolymph,respectively.3.803,47.843 and 24.647 for hepatopancreas,respectively.0.038,0.937 and 11.854,mg/L·h for muscle, respectively.15 days after oral administration,the enrofloxacin concentration in all tissues lower than0.100μg/g.Base on the date above,we suggested dosage regimen of enrofloxa-cin is that Litopenaeus vannamei can take enrofloxacin twice dayly by oral administration(30mg/kg).