【作者】 苏杭；

【导师】 房文红；

【作者基本信息】 四川农业大学 ， 水产养殖， 2010， 硕士

【摘要】 本文从发病濒死的养殖凡纳滨对虾(Penaeus vannamei Boone)病灶部位分离出优势菌株并对其致病性和分类地位进行研究。探索饲喂不同分子量壳聚糖对凡纳滨对虾体重和非特异性免疫力影响,以及分析盐酸恩诺沙星及其代谢产物环丙沙星在凡纳滨对虾体内药代动力学,为凡纳滨对虾病原菌病防治提供科学依据。结果如下：1.2009年7月,海南省琼海市椰林村某凡纳滨对虾养殖场养殖的凡纳滨对虾发生严重病害,累计死亡率高。病虾的主要症状表现为步足变红,鳃丝变黄,腹部肌肉有坏死,肝胰腺糜烂性坏死等。从病虾肌肉中分离纯化得到优势菌株。人工感染试验证实该菌株对凡纳滨对虾有较强的致病性,对体重为10g±1g的凡纳滨对虾的半数致死量为8.12×103cfu/mL。通过形态学和生理生化特征以及细菌16S rDNA序列研究确定,该病原菌为哈维氏弧菌(Vibrio harveyi)。该菌对恩诺沙星、氯霉素、四环素、丙氟哌酸、氟哌酸及复方新诺明敏感。选用6种抗生素对所分离到的致病菌株的最小抑菌浓度(MIC)试验表明盐酸恩诺沙星MIC最低为0.25μg/mL。经试验室注射感染试验虾,通过组织病理学观察表明,病虾的鰓、肝胰腺、中肠、胃、围心腔和肌肉的微观结构发生了明显的病理变化,其中尤以肝胰腺最为严重,主要表现为细胞坏死、脱落,细胞肿胀,细胞核边聚,线粒体肿胀、变性,严重时整个细胞呈溶解状态。由此引起的机体功能衰竭可能是病虾死亡的主要原因。2.以初始体重(5±1)g的凡纳滨对虾为研究对象,探讨饲料中分别添加1万分子量壳聚糖和3万分子量壳聚糖对其体重和非特异性免疫力的影响。对照组投喂相应的基础饲料,1万分子量壳聚糖组试验饲料在其基础饲料中分别添加1g/kg、3g/kg、5 g/kg、10 g/kg的1万分子量壳聚糖,3万分子量壳聚糖组试验饲料在其基础饲料中分别添加1 g/kg、3 g/kg、5 g/kg、10 g/kg的3万分子量壳聚糖；配制出9种基本等氮等能的实验饲料。实验在1m3的圆桶中进行,随机分为9组,每组90尾,试验期28d。饲喂7天后每个桶分别随机抽取30尾试验虾测定体重并进行所分离到的哈维氏弧菌攻毒实验。饲喂28天后测定试验虾的体重,并分别从每个桶抽取部分试验虾的血淋巴进行血清中非特异性免疫指标的分析,同时每个桶取30尾试验虾进行攻毒试验。实验结果表明,凡纳滨对虾摄食7d后各个处理间体重和抗所分离到的哈维氏弧菌的能力差异不显著(P>0.05)。凡纳滨对虾摄食28d后,各个处理间体重差异不显著(P>0.05)；各个处理间酚氧化酶活力和溶菌酶活力差异显著(P<0.05),超氧化物歧化酶活力差异不显著(P>0.05)。用所分离到的哈维氏弧菌攻毒实验结果表明,摄食28d后,3g/kg的1万分子量壳聚糖组和3 g/kg的3万分子量壳聚糖组的14d累积死亡率(分别为43%和47%)显著低于空白对照组(67%)(P<0.05)。因此,饲料中添加适宜含量的壳聚糖能在一定程度上提高凡纳滨对虾抵抗本次分离到的哈维氏弧菌的能力,但是与壳聚糖的添加量和饲喂时间有关；同时壳聚糖对凡纳滨对虾体重没有正面影响。3.采用高效液相色谱法通过凡纳滨对虾(15±2g)口灌30mg/kg恩诺沙星研究在其体内药代动力学。在水温29.5±1℃下,凡纳滨对虾口灌恩诺沙星后,血液中药物浓度2h后到达峰值,并迅速向组织中分布,血药经时过程符合一级吸收二室开放模型,主要药动学参数为：分布半衰期(t 1/2α),消除半衰期(t1/2β),0-t时间曲线下面积(AUC 0-t),0-∞时间的曲线下面积(AUC 0-∞),一室到二室转运一级消除常数(K12)、二室到一室转运一级消除常数(K21),总体清除率(CLs)分别为3.167h、37.277h、188.887 mg/L·h、190.362 mg/L·h、0.071/h、0.0211/h、0.158 L/h/kgo肌肉和肝胰腺中恩诺沙星浓度与时间关系的药动学参数采用统计矩原理分析,半衰期(tl/2z)分别为75.863h和207.592h,总体清除率(CLz)分别为0.655 L/h/kg和0.043 L/h/kg。在试验虾血淋巴、肝胰腺和肌肉3种组织均能检测到恩诺沙星的代谢产物环丙沙星,主要参数：血药峰浓度(Cmax)为0.451μg/mL、3.803μg/mL、0.038μg/mL。AUC为7.423 mg/L·h、47.843 mg/L·h、0.937 mg/L·h, t1/2z为12.432h、24.647h、11.854h。给药后15d时,血淋巴、肝胰腺和肌肉中恩诺沙星含量都已低于0.10μg/g,恩诺沙星及其代谢产物环丙沙星在凡纳滨对虾体内消除较慢。根据本实验结果,预测以30mg/kg的剂量盐酸恩诺沙星一次性口灌给药凡纳滨对虾,可以维持2d以上的药效作用,以此剂量隔日连续使用3次,对于凡纳滨对虾细菌感染的防治效果比较好。更多还原

【Abstract】 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).更多还原