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。
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).
引文
[1]吴后波,潘金培.弧菌属细菌及其所致海水养殖动物疾病[J].中国水产科学,2001,3(1)：5-7.
[2]Celia R, Pitogo-Lavilla, Cecilia M,et al. Occurrence of luminous bacterialdisease of Penaeus monodon larvae in the Philippines[J].Aquaculture,1990,91:1-13.
[3]Johnson H F, Shunk V I.An interesting new species of luminous bacteria[J].Journal of Bacteriology,1963,31:585-592.
[4]Baumann P, Bauman L, Bang S S,et al.Reevaluation of the taxonomy of Vibrio,Beneckea,and Photobacteruim:abolish the genus Beneckes[J].Current microbiology,1980,4:127-132.
[5]Holt J K, Krieg R N, Sneath P H.Isolation of Vibrio harveyi from Diseased Kuruma Prawns Penaeus japonicus[J].Current Microbiology,1996,33:129-132.
[6]东秀珠,蔡妙英.常见细菌系统见手册[M]：科学出版社；2001.
[7]赵晓华,钟英武,陈吉祥.哈维氏弧菌的生物学特性、流行病学及检测技术[J].中国海洋大学学报,2007,37(5)：740-748.
[8]Austin B, Zhang X H.Vibrio harveyi:a significant pathogen of marine vertebrates and invertebrates.under the microscope,2006,1:132-145.
[9]刘问,钱冬,杨国梁,等.南美白对虾虾苗淡化期间发光病病原研究[[J].集美大学学报,2004,12(4)：300-308
[10]陈月忠,钟硕良,周哀.成虾发光病病原体的分离鉴定及防治技术研究[J].中山大学学报,2000,3：218-324.
[11]李军,徐怀恕.中国对虾幼体致病菌哈维氏弧菌的分离及其生物学特性[J].海洋与湖沼,1998,7：352-362.
[12]王保坤,余俊红,李筠,等.花鲈弧菌病病原菌(哈维氏弧菌)的分离与鉴定[J].中国水产科学,2002,9(1)：54-63.
[13]陈献稿,吴淑勤,石存斌,等.斜带石斑鱼病原菌(哈维氏弧菌)的分离与鉴定[J].中国水产科学,2004,4(11)：313-327.
[14]覃映雪,池信才,苏永全,et al.网箱养殖青石斑鱼的溃疡病病原[J].水产学报,2004,28(3)：297-311.
[15]范文辉,王秀华,史成银,等.养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析[J]. 为生物学报,2005,5(45)：665-673.
[16]石存斌,胡学峰,陈献稿,等.两株致病性哈维氏弧菌胞外产物的特性分析[J].水产学报,2007,31(1)：83-94.
[17]Liu P C, Lee K K, Chen S N.Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon [J].Letters in Applied Microbiology 1996,22:413-416.
[18]Liu P C, Lee K K.Cysteine proteinase is a major exotoxin of pathogenic luninous Vibrio harveyi in the tiger prawn,penaeus monodon[J].Letters in Applied Microbiology,1999,28:428-430.
[19]SASoto-Rodriguez, Roque A, Lizarraga-Partida M L.Virulence of luminous vibrios to Artemia franciscana nauplii[J].Dis Aquat Org,2003,53
[20]Montero B A, Austin B.Characterization of extracellular products from an isolate of Vibrio harveyi recovered from diseased post-larval penaeus vannamei(Bonne)[J].Fishdis,1999,22:377-386.
[21]Zhang X H, Meaden P G, Austin B.Duplication of hemolysin genes in a virulent isolate of Vibrio harveyi[J].Appl Environ Microbiol 2001,67:3161-3167.
[22]蔡潭溪,蒋鲁岩,黄克和.FU溶血弧菌的分子生物学研究进展[J].中国人兽共患病杂志,2005,21(10)：914-916.
[23]郑天伦,王国良.海水养殖动物弧菌病防治的研究进展.台湾海峡,2002,21(3)：327-378.
[24]吴后波,潘金培.海水网箱养殖高体鲫弧菌病药物治疗的实验研究[J].热带海洋,2000,29(1)：85-94.
[25]Austin B, Austin D A, Farhat Z.Diagnosis and control of bacterial fish pathongens with emphasis on vibrio harveyi[J].Journal of Ocean University of Qingdao,1999,29(3):489-499.
[26]郝贵杰,沈锦玉,潘晓义,等.32种常用渔药对大黄鱼致病菌哈维氏弧菌的体外抗菌试验[J].中国兽药杂志,2008,42(5)：36-39.
[27]莫照兰,俞勇,王祥红,等.细菌QJ2对对虾病原性哈维氏弧菌的拮抗作用[J].水产学报,2001,25(5)：432-440.
[28]Kontara E K, Merchie G, Lavens P.Improved production of postlarval white shrimp through supplementation of L-ascorbyl-2-polyphosphste in their diet[J].Aquaculture International,1997,5:127-136.
[29]张春晓,麦康森,艾庆辉,等.饲料中添加肽聚糖对大黄鱼生长和非特异性免疫力的影响 [J].水产学报,2008,32(3)：411-4]7.
[30]Alvarez J D, Austin B, Alvarez M A,等.Vibrio harbeyi:a pathogen of penaeid shrimps and fish in Venezuela[J].FIsh Dis,1998,21:313-316.
[31]蒋挺大.壳聚糖[M]：化学工业出版社；2001.
[32]Majeti N V, Ravi K.A review of chitin and chitosan applications[J].Reactive & Functional Polymers,2000,46:1-27.
[33]Seferian G P, Martinez M L.Sulfated chitin and chitosan as novel biomaterials[J].Vaccine,2000,26(1):661-668.
[34]李梦云,陈代文,张克英.壳聚糖的主要生理功能及其在动物生产中的应用[J].饲料工业,2005,26(12)：1-3.
[35]Mourya V K, Inamdar N N.Chitosan-modifications and applications:Opportunities galore.Reactive & Functional Polymers,2008,68:1013-1051.
[36]Prashanth K V H, Tharanathan R N.Chitin/chitosan:modifications and their unlimited application potentiald-an overview.Trends in Food Science & Technology,2007,18:117-131.
[37]王润莲,贾志海,朱晓萍.甲壳素和壳聚糖营养研究进展[J].动物营养学报,2006,18(4)：299-302.
[38]Anderson P D, Siwicki K A, Rumsey L GDuration of Protection against Aeromonas salmonicida in Brook Trout Immunostimulated with Glucan or Chitosan by Injection or Immersion[J].The Progressive Fish-Culturist,1995,56:258-261.
[39]Ayyaru G, Venkatesan A.Immunomodulatory effects of dietary intake of chitin, chitosan and levamisole on the immune system of Cyprinus carpio and control of Aeromonas hydrophila infection in ponds.Aquaculture,2006,255:179-187.
[40]闫大伟,华雪铭,周洪琪.壳聚糖对草鱼生长、抗病性能的影响[J].饲料工业,2007,28：17-18.
[41]华雪铭,闫大伟,周洪琪.壳聚糖通过甲状腺激素对草鱼免疫功能的调节[J].中国水产科学,2008,4：630-635.
[42]翟少伟,王敦.不同源壳聚糖对鲫生长和抗感染能力的影响[J].水产科学,2009,28：451-453.
[43]华雪铭,周洪琪,张冬青,等.壳聚糖和益生菌对暗纹东方纯抗病力和免疫功能的影响[J].水产学报,2004,31(4)：478-486.
[44]Shi W H, Jiann C C.The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei[J].Fish & Shellfish Immunology,2005,19:191-204.
[45]李荣誉,王笃学,崔耀明.兽医药理学[M]：中国农业出版社；2007.
[46]方星星,王群,李建.恩若沙星及其代谢产物环丙沙星在中国对虾体内的药代动力学.水产学报,2004,28：35-41.
[47]钱云云,唐俊,郑宗林,et al.恩若沙星在罗氏沼虾体内的药物代谢动力学[J].动物学杂志,2007,42(5)：62-69.
[48]王吉桥.南美白对虾生物学研究与养殖[M]：海洋出版社；2003.
[49]王兴强,马牲,懂双林.凡纳滨对虾生物学及养殖生态学研究进展[J].海洋湖沼通报,2004,4：94-100
[50]Ward C A L.Practical statistics for experimental biologists [M].Chichester:John Wiley and Sons,1985
[51]叶应妩,王毓三.全国临床检验操作规程(第二版)[M].南京：东南大学出版社1997.
[52]Garrity M G, ed Bergey, smanual of systematic bacteriology(2nd edn)[M]2005. The Proteobacteria New York:Springer-Verlag.
[53]陆宏达,陈海清.罗氏沼虾肌肉白浊病的病原和组织病理[J].中国水产科学2003,2：125-132.
[54]姜兰,邓国成,石存斌,等.罗氏沼虾肌肉白浊病病原研究[J].水生生物学报,2002,26：477-474.
[55]纪荣兴,邹文政,鄢庆枇,等凡纳滨对虾“肌肉白浊病”病原的初步研究[J].集美大学学报,2008,13：210-215.
[56]黄洪敏,邵健忠,项黎新.鱼类免疫增强剂的研究现状与进展[J].水产学报,2005,29(4)：552-559.
[57]陈乃松,魏涛涛,廖奕招.蝇蛆粉和葡聚糖对凡纳滨对虾生长和免疫的影响[J].水产学报,2007,31(6)：771-777.
[58]Ashuda M.Purification and characterization of prophenoloxidase from hemolymph of the silkworm Bombyx mori[J].Arch Biochem Biophy,1971,144:749-762.
[59]Hultmark D.Insect immunity purification and properties of three inducible bactericidal proteins from hemolymph of imm unized pupae of Hylophora ceropia[J].Eurs Biochem,1980,106::7-16.
[60]陈勇,周洪琪,冷向军,等.壳聚糖对异育银鲫生长和消化酶的影响[J].中国水产科 学,2006,13(3)：440-445.
[61]Shiau Y S, Yu P Y.but not chitosan supplementationenhances growth of grass shrimp,penaeus monodon[J].Journal of Nutrition,1998,128:908-912.
[62]Deuchi K, Kanauchi O, Imasato Y, et al. Decreasing effect of chitosan on the apparent dig estibility by rats fed on a high fat diet[J].Bioscience Biotechnology and Biochemistry,1994,58
[63]Anderson P D.Immunostimuant,adjuvants and vaccine in fish:application to aquaculture[J].Annual Rev.of Fish Disease,1992,56:281-307.
[64]Schwartz E S, Levin A R, Singh A.Sustained pectin ingestion delays gastric emptying[J].Gastroenterology,1982,83:812-817.
[65]黄旭熊,周洪琪,宋理平.急性感染对中国明对虾非特异免疫水平的影响[J].水产学报,2007,31(3)：325-330.
[66]陈国福,黄捷,宋晓玲.对虾免疫机能研究概况[J].水产学报,2004,28(2)：209-215.
[67]王秀华,宋晓玲,黄偼.肽聚糖制剂对南美白对虾体液免疫因子的影响[J].中国水产科学,2004,11(1)：27-30.
[68]Fang C, Chang S M, Houng Y, et al.Dietary β-1,3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus[J].Fish & Shellfish Immunology,2003,15:297-310.
[69]Itami T, Asano M, Tokushige K, et al.Enhancement of disease resistance of kuruma shrimp,Penaeusjaponicus,after oral administration of peptidoglycan derived from Bifidobacterium hermophilum [J].Aquaculture,1998,164:277-288
[70]Boonyaratpalin S, Supamattaya K, law W C A, et al.Effects of peptidoglucan(PG) on growth, survival, immune responses,and tolerance to stress in black tiger shrimp, Penaeus Monodon[J].Diseases in Asian Aquaculture,1995,2:469-477.
[71]薛清刚,王文兴.对虾疾病的研究现状与我国虾病泛滥的成因分析[J].海洋科学,1995,4：32-39.
[72]Sung H H, Kou G H, Song L S.Vibrosis resistance induced by glucan treatment in tiger shrimp (Penaeus monodon)[J].Fish Pathology,1994,29:11-17.
[73]Fang W H, Hu L L,Yang X L, et al. Effect of temperature on pharmacokinetics of enrofloxacin in mud crab, Scylla serrata (Forsskal), following oral administration[J].Journal of Fish Diseases,2008,31:171-176.
[74]Lewbar G, Vaden S, Deen J, et al. Pharmacokinetics of enmfloxacin in the red pacu(Colossoma brachypomum)after in tramuscular, oral and bath administration[J].J Vet Pharmacol Therap,1997,20:124-128.
[75]Bowser R P, Wooster A G, Leger J.Pharmacokinetics of enfloxacin in fingerling rainbow trout(Oncorhynchus mykiss)[J].J Vet Pharmacol Therap,1992,15(1):62-71.
[76]Della G R, Di A S, Malvisi J, et al.The disposition of enrofloxacin in seabream (Sparus aurata L) after single intravenous injection or from medicated feef administration[J].Aquaculture,2004,232:53-56.
[77]Stoffregeno D A, Wooster G A.Multiple route and dose pharmacokinetics of enrofloxacin in juvenile Atlantic salmon[J].J Biol Chem,1997,20(111-123)
[78]Martin H.Denfence functions of granulocytes in the ride-back prawnSicyonia ingent[J].Invertebr Pathol,1989,53:335-346.
[79]房文红,郑国兴.肌注和药饵给药下诺氟沙星在南美白对虾血淋巴中药代动力学[J].水生生物学报,2006,30(5)：541-546.
[80]Fang W H, Hu L L, Yang X L, et al. Pharmacokinetics and tissue distribution of enrofloxacin and its metabolite ciprofloxacin in Scylla serrata following oral gavage at two salinities [J].Aquaculture,2007,272:180-187.
[81]房文红,于慧娟,蔡友琼,等.恩诺沙星及其代谢物环丙沙星在欧洲鳗鲡体内的代谢动力学[J].中国水产科学,2007,14(4)：621-625.
[82]曾振灵,冯淇辉.恩诺沙星在猪体内的生物利用度及药物动力学研究[J].中国兽医学报,1996,16(6)：605-612.
[83]Rocca G, Delaa D A, Salvo J.The disposition of enroflocnecin in seabream(Sparw awatal) after single intravenous injection or fron medicated feed administration[J].Aquaculture,2004,232:53-56.
[84]Roque A, Gomez B G.In vitro susceptibility to 15 antibiotics of vibrios isolated from penaeid shrimps in Northwestern Mexico[J].International Journal of Antimicrobial Agents,2004,17:383-387.
[85]Mohney L, BeLL T, Lightenr D.Shrimp antim icrobial testing:1. In vitro suscep tibility of thirteen gram negative bacteria to twelve antimicrobials[J].Aquat. Anim.Health,1992,4(4):257-261.
[86]Intorre L, Cecchini S, Bertini S, et al.Pharmacokinetics,tissue distribution, and metabolosm of flumequine in channel catfish(Ictalurus punctatus)[J].Aquaculture,2000,182:49-59.
[87]赵晶,康世良.兽用喹诺酮类抗菌剂恩诺沙星的药物代谢动力学研究进展[J].黑龙江畜牧兽医,1999,4：38-39.
