斑点叉尾鮰病原细菌的分离鉴定及系统发育分析

用传统的微生物学分离培养方法从斑点叉尾鮰的鳃、肠和肝等器官共分离到16株可疑病原菌（编号为WJ01~WJ16）,以提取的基因组DNA为模版,扩增出约600bp大小的16S rDNA片段,测序分析表明：与GenBank数据库中的已知序列相比,16株细菌中有9株同源性在98%以上,5株在95%以上,2株为92%。系统发育分析表明这16株细菌明显的聚为3支。     

斑点叉尾鮰肠败血症病原菌的分离与鉴定

近两年来，一种严重的传染性疾病在广西斑点叉尾鮰养殖业中流行，造成鱼苗100％死亡；咸鱼的损失也高达30％～80％不等。为了确定该病病原，本试验对患病斑点叉尾鮰脑、肝脏、脾脏和肾脏组织进行了细菌的分离、鉴定及致病性试验。结果从南宁和合浦两地分离到NN和HP两株G短杆菌，人工感染试验表明，腹腔注射感染可引起健康斑点叉尾鮰100％死亡，浸泡感染可引起30％-55％死亡，并且感染发病的斑点叉尾鮰症状与自然发病症状相似。两株细菌的形态特征、培养特性和生理生化反应指标均与国外报道的鮰爱德华氏菌参考菌株（JCM1680）相同；两株细菌的16SrRNA基因序列与JCM1680菌株16s rRNA基因序列同源性均为99．3％。以上研究证实，NN和HP两株细菌为致病性鮰爱德华氏菌，其引起的传染性疾病为斑点叉尾鮰肠败血症。本研究首次对国内斑点叉尾鮰肠败血症进行病原菌的分离和鉴定，证实了斑点叉尾鮰肠败血症在广西斑点叉尾鮰养殖业中的流行及造成严重经济损失，对国内该病的控制及斑点叉尾鮰健康养殖具有重要的指导意义。     

斑点叉尾鮰暴发性败血症病原的分离与鉴定

从3尾表现出血症状的患病斑点叉尾鮰（lctalurus Punctatus）体内各分离出一株病菌，测定结果显示致其急性死亡的致病菌为温和气单胞菌（Aeromonas sobria），慢性死亡的致病菌分别为嗜水气单胞菌（A．hydrophila）和温和气单胞菌（A．sobria）。药敏试验结果表明：3株菌对广谱抗生素如氨苄青霉素、青霉素、先锋霉素Ⅵ和红霉素等已产生耐药性，而对菌必治、头孢肤肟和复达欣等高度敏感；菌株Jh-1和菌株Yh-3对复方新诺明、四环素、氟哌酸和丁胺卡那霉素高度敏感，而菌株Jh-2却低度敏感；环丙沙星和诺氟沙星对3株致病菌均具较好的抑菌、杀菌作用。     

斑点叉尾鮰暴发海豚链球菌病的研究

2006—2007连续两年广西斑点叉尾鮰网箱养殖暴发一种流行性传染病,该病波及面广,传染性强,死亡率30％～100％。发病症状主要表现为浮头、转圈、狂游及发病后期单边眼睛浑浊、突出及伴有全身大面积弥漫性出血。作者先后从症状较为典型发病班点叉尾鮰分离到CMS003～CMS005、CMS009～CMS012共7株代表性菌株,经人工感染实验表明分离菌株对健康斑点叉尾鮰具有很强的致病力,出现症状与自然发病斑点叉尾鮰症状相同。通过常规形态、生理生化及海豚链球菌（Streptococcus iniae）特异性PCR诊断和16S rRNA基因序列系统发育进化分析方法对分离菌株进行分类鉴定,结果表明分离的7株细菌均为海豚链球菌。研究结果表明,海豚链球菌已成为广西斑点叉尾鮰网箱养殖的高致病性病原菌。     

斑点叉尾鮰人工繁殖实验

我们于1989年开始进行斑点叉尾鮰的人工繁殖试验研究，现将试验情况报告如下。     

红色和黑色斑点叉尾鮰人工繁殖试验

通过人工控制手段,促使红色斑点叉尾鮰与黑色斑点叉尾鮰自行配对、产卵、收集受精卵集中孵化,获得浅黑色的鱼苗;要获得稳定的红鮰鱼苗,必须采取红色斑点叉尾鮰自交的方法。     

我国首次分离出斑点叉尾鮰病毒病病原

日前，水产科学研究院长江所鱼类营养与病害防治研究室研究员曾令兵博士带领的研究团队，利用自己独立建立的斑点叉尾鮰肾脏组织细胞系在国内首次成功分离得到斑点叉尾鮰病毒病病原，填补了国内空白。     

广西地区斑点叉尾鮰人工繁殖试验

从池塘养殖的成鱼中选择亲鱼，培育至3龄以上，自然受精、人工孵化。4月上旬获第1批受精卵，经4d室内孵化。出苗36万尾，培育至7～8cm 32．4万尾，成活率达90％。比湖北地区产苗时间提前1个多月。     

斑点叉尾鮰亲鱼的运输

2000年3月30日,将美国密西西比品系原种斑点叉尾鮰(Ictalurus punctalus)亲鱼,自湖北荆州张家山渔场运往辽宁铁岭三家子渔场.起运亲鱼698尾共1 087kg,经20 h到达目的地,成活率97.9%.     

斑点叉尾鮰的人工繁殖技术

阐述了斑点叉尾的人工繁殖技术，其中包括亲鱼池的条件、亲鱼的选择与培育、亲鱼的产卵以及鱼卵的孵化。     

淡水珍珠蚌致病菌的分离与鉴定

通过珍珠蚌病原体分离、染色、培养、氧化酶试验、克氏双糖铁琼脂培养和葡萄糖微粒管试验及药敏实验卡鉴定,结果显示病蚌的鳃和斧足存在嗜水气单胞菌和温和气单胞菌,内脏巾寄生肠杆菌科敛病菌和G+球菌,鳃部寄生大量致病菌.     

大西洋鲑杀鲑气单胞菌的分离鉴定

从皮肤溃烂的大西洋鲑(Salmon salar)肌肉、肝、肾分离到一致病性的菌株(AB080226),经人工感染实验证实其为该病的病原菌,研究了该菌的形态、生理生化特征并对其16SrDNA序列进行了在线Classifer和Blast分析。结果显示:该菌为革兰氏阴性杆菌,葡萄糖氧化发酵阳性,氧化酶检测阳性,0%NaCl生长;能利用甘露醇,不具运动性,能发酵麦芽糖,葡萄糖产酸阳性,精氨酸双水解酶阳性,苯丙氨酸脱氨酶阴性;该菌属于气单胞菌属的细菌,在系统发育树上与杀鲑气单胞菌形成一个簇群,同源性高达99.9%以上。综合形态学、生理生化特征及16SrDNA序列鉴定其为杀鲑气单胞菌(Aeromonas salmonicida)。     

患病草鱼肠道病原菌的分离鉴定及毒力研究

自环洞庭湖区精养鱼池患病草鱼肠道中分离获得4种病原菌,进行16S rRNA基因和gyrB基因序列测定和在线比对,并对草鱼进行回归感染。随后进行4种病原菌的药敏特征、溶血活性试验,并利用PCR方法检测了6种毒力因子的携带情况。试验结果表明,病原菌分别为温和气单胞菌、异常嗜糖气单胞菌、类志贺邻单胞菌和格氏乳球菌;4种病原菌回归感染草鱼,均能使草鱼患病致死,自死亡的草鱼体内均能分离得到相应感染的病原菌;4种病原菌对多种抗生素具有耐药性,且在脱纤维绵羊血血平板上检测到了温和气单胞菌、异常嗜糖气单胞菌AaB007的溶血活性;PCR结果表明,4种病原菌携带多种毒力因子。研究结果可为环洞庭湖区草鱼精养鱼池健康养殖及病害防治提供科学依据。     

异育银鲫致病性嗜水气单胞菌的分离鉴定与药敏特性研究

对江苏盐城某养殖场患出血病的异育银鲫(Carassius auratus gibelio)进行细菌分离鉴定和药敏试验,为该病的防控提供科学依据.从病死及濒死异育银鲫的肝脏、脾、肾中分离病原菌,经过形态观察、生理生化试验、16S rRNA序列分析,最终进行细菌鉴定,采用纸片扩散法进行药敏实验,通过人工回归感染试验确定分离菌株的致病性.分离的2株细菌(编号为WJ11-3和WJ11-4)均为嗜水气单胞菌(Aeromonas hydrophila),均能导致异育银鲫死亡,并出现与自然条件下基本一致的症状.药物敏感性试验结果显示,嗜水气单胞菌分离株WJ11-3对苯唑西林、林克霉素、万古霉素显示耐药性,对恩诺沙星等28种药物高度敏感;WJ11-4对苯唑西林、林克霉素、新生霉素显示耐药性,对氟苯尼考等28种药物高度敏感.本次异育银鲫的出血病由嗜水气单胞菌感染引起,可选用恩诺沙星、阿洛西林、氧氟沙星、苯唑西林、林克霉素、新生霉素等多种药物进行防治.     

Identification of Two Channel Catfish Stocks,Ictalurus punctatus,Cultivated in Northeast Mexico

The channel catfish farming in Mexico began in the 1970s. One of the most important areas of production is its northeastern region. Traditionally, the channel catfish farmers have attempted to preserve their original genetic stock. After more than 30 yr of production, the genetic relationship between stocks has not been assessed. The goal of this study was to evaluate the genetic differentiation between five channel catfish farm populations, using 13 microsatellite markers. A total of 154 fin clip samples were collected from five channel catfish farms in Tamaulipas. We also included 31 individuals of the commercial strain NWAC103. In each population, we calculated parameter of genetic variability: inbreeding coefficient (F_IS), pairwise differentiation (F_ST), and genetic distance (D_C). For the inference of populations and individual assignment two Bayesian methods were used. Genetic variability parameters were N_A = 9.54–11.08 and A_E = 5.40–6.67. Hardy–Weinberg equilibrium deviations were observed for all farm populations examined, and a deficit of heterozygotes was found. Signals of inbreeding were observed, particularly for the ACU (Acuamex) hatchery. Populations EKA (Empresas Karol) and ACU showed higher genetic differentiation values with respect to the commercial strain (F_ST = 0.107; F_ST = 0.082, respectively). Moderated genetic differences (F_ST = 0.067; D_C = 0.080) were observed between the EKA and ACU. Clustering analysis revealed a well‐defined group between EKA and TPA (El Tiron Parejo). In the Bayesian analysis three groups were defined. The first group was formed by EKA and TPA, the second group included ACU, LAJ (La Lajilla), and PRO (Prodatec). In the third group was the commercial strain. In the assignment of individuals, EKA and ACU were represented by exclusive genotypes. Identification of two genetic stocks, one located in the EKA and the other in the ACU population, will be relevant for future management of these hatcheries. Additionally, the information from microsatellites and the statistical procedures presented will be important tools for genetic monitoring of these farm populations.     

南方鲇溃疡综合症病原菌的分离与鉴定

从南方鲇(Silurus meriordinalis Chen)病鱼体表溃疡部及内脏分离出细菌12株,经人工感染证实其中6株治病,生化鉴定6株为同一种菌即为引起该鱼溃疡综合症的病原菌,命名为DKN-1。该菌为革兰氏染色阴性,兼性厌氧发酵型短杆菌,极生单鞭毛,对除葡萄糖、麦芽糖、甘露糖、海藻糖、甘露醇、水杨苷、七叶苷以外多种糖不利用,氧化酶、过氧化氢酶、DNA酶、脂酶、蛋白酶、精氨酸双水解酶阳性,MR、VP阳性。在以该菌16S rDNA序列和GenBank及RDP数据库内同源性较高的细菌16S rDNA序列构建的系统发育树中,DKN-1与豚鼠气单胞菌(Aeromonas caviae)同源性达到99.6%,综合该菌在形态、生理生化的结果,鉴定其为豚鼠气单胞菌。该病病原对美满霉素、大观霉素、妥布霉素高度敏感。     

牙鲆仔鱼肠道白浊病病原菌的分离鉴定

对工厂化育苗中患肠道白浊病的牙鲆仔鱼进行了病原菌的分离与特性鉴定,从众多患病仔鱼的肠 道中均分离到几乎纯一的细菌,并对仔鱼的分离培养物进行形态特征检查及理化特性鉴定,结果初步表明所 分离鉴定的病原菌为鳗弧菌(Vibrio anguillarum),为牙鲆仔鱼肠道白浊病的致病菌,测定了对常用抗菌类药 物的敏感性,结果表明分离菌株对环丙沙星等11种药物高度敏感,对林可霉素等4种药物中度敏感,对利福 平等3种药物耐药。     

蓄水塘养殖斑点叉尾鱼回成鱼试验

长江水产研究所试验场用于人工繁殖的蓄水塘,面积8亩.每年6月份以前用于鲤、鲫及四大家鱼的人工繁殖蓄水,2001年我们在人工繁殖过后,利用该池进行了斑点叉尾鱼回成鱼养殖试验,取得了明显的经济效益.现将其主要措施总结如下.     

Isolation of a novel hepatic CYP2-related protein from channel catfish, Ictalurus punctatus

Channel catfish (Ictalurus punctatus) have been previously shown to express two major cytochrome P450 (CYP) protein bands that are cross-reactive with anti-CYP2K1 (rainbow trout, Oncorhynchus mykiss) antibodies on Western blots. These proteins appear to be the major constitutive CYPs in channel catfish and show distinct sex- and age-specific variations in expression. Because I. punctatus is an important agricultural and ecological commodity, and because it displays a high degree of resistance to the toxic effects of many pesticides, the molecular and catalytic characteristics of its biotransformation systems are of interest to those in areas of environmental science and aquaculture research. Using a chromatographic method similar to that employed in the purification of other fish CYP2 enzymes, a single CYP2-related protein (CM-HA3) was isolated from channel catfish hepatic microsomes. The isolated protein displays a relative molecular mass of approximately 47 kDa, and a CO-reduced difference spectrum _max of 449.6 nm. The sequence of 15 residues at the amino-terminal of CM-HA3 is 27% identical to both CYP2K1 and CYP2M1 isoforms of rainbow trout. Correlational analysis was employed to characterize potential substrates for this isoform, but no significant relationship was observed with E₂ hydroxylation, testosterone hydroxylation, or 7-ethoxycoumarin O-deethylase activities. These data indicate that CM-HA3 is a CYP2 family protein, with as yet uncharacterized substrate specificities.     

Behavioral Interactions in Juvenile Channel Catfish,Ictalurus punctatus

Behavioral interactions among juvenile channel catfish, "Ictalurus punctatus," were observed, and fixed action patterns were described and anlyzed. Dominant fish engaged regularly in aggressive activities and never in submissive activities, whereas subordinate fish were observed in submissive activities on a regular basis and rarely in those that were aggressive. Activity levels were significantly higher in dominant fish; however, there was no difference in activity levels between pairs of equal and unequal size fish. A factor analysis indicated that there were three general categoris of behavior in juvenile channel catfish. In addition, two behaviors (which appeared to be displacement activities) were functioning in agnostic encounters. Plasma cortisol concentrations measured in sigle fish and pairs of fish in aquaria and individuals from a holding tank indicated that the least amount of stress occurred in fish that were not interacting with any other individuals. Cortisol levels, however, were lower that those associated with stress levels in fish from other studies.