杂交鲟海豚链球菌的分离、鉴定及药物敏感性

【目的】2012年7月,北京市怀柔区一家养殖场的杂交鲟爆发疾病,陆续死鱼。为确定病原,【方法】从具有典型症状的患病鱼的肝、肾和脾中分离获得3株分离菌,编号HRS12718L、HRS12718K和HRS12718S。采用形态特征、理化特性、16S rDNA和海豚链球菌特异性基因逐步鉴定病原菌的种类。取HRS12718K分离株进行人工感染实验,确认病原菌的致病性。开展药物敏感性实验筛选分离株的敏感药物。【结果】结果显示3株分离菌的形态特征和理化特性与从中国其它鱼类分离的海豚链球菌一致。采用16S rDNA序列构建的进化树与海豚链球菌聚为一支,与海豚链球菌的同源性在99.1%以上。HRS12718K分离株对杂交鲟的半致死量LD50为4.42×105CFU/mL,试验感染鱼出现与自然发病鱼相似临床症状。分离株对诺氟沙星、嗯诺沙星、硫酸新霉素、盐酸多西环素和盐酸四环素敏感,尤其对嗯诺沙星敏感。【结论】最终确认引起该养殖场杂交鲟发病的病原为海豚链球菌,建议使用嗯诺沙星进行治疗。     

似鲶高原鳅温和气单胞菌的分离鉴定及其感染的病理损伤

2013年9月,四川雅安某似鲶高原鳅养殖场发生以体表出血、皮肤溃疡、内脏器官肿大、出血与坏死为特点的疾病。从自然发病的似鲶高原鳅肝与肾分离到1株G-短杆菌(XJJ130928),其在BHI平板上28℃培养24 h,形成灰白色、表面光滑、边缘整齐、半透明状微隆的圆形菌落,人工感染证实其为本次似鲶高原鳅发病的病原菌。根据分离菌株的形态学和生理生化检测结果初步判定其为温和气单胞菌Aeromonas sobria;进一步的16S rRNA和gyrB基因序列分析表明,分离株的16S rRNA与gyrB基因序列(GenBank登录号:KF761305与KJ139988)在GenBank中进行Blast比对与A.sobria同源性最高。在以分离株16S rRNA和gyrB基因序列及GenBank中同源性较高的序列构建的系统发育树上,分离株与A.sobria聚为一族,其同源性分别为96.0%~99.0%与94.5%~98.0%,结合生理生化特性鉴定分离菌为A.sobria。该菌对头孢西丁、亚胺培南、强力霉素、氟苯尼考和氧氟沙星等敏感,对磺胺甲基异恶唑、洛美沙星、环丙沙星中度敏感,对氨苄青霉素、链霉素、卡那霉素耐药。组织病理学观察发现,A.sobria感染似鲶高原鳅对多组织器官都造成明显的病理损伤,尤其是肝、肾、脾、肠的损伤较为严重,表现为明显的淤血、出血,变性,坏死及炎症细胞浸润。     

安徽部分湖泊养殖鱼类暴发性出血病的病原分离与鉴定

目的确定引起安徽省部分湖泊养殖鱼类暴发性出血病的病原,为防治该病提供理论依据。方法随机取濒死期鲫鱼和白鲢的肌肉组织分别进行细菌和病毒分离培养,联合采用细菌表型鉴定法和16S rRNA基因序列分析法鉴定分离菌株,并使用分离菌株进行人工感染实验。结果从5尾患病鱼的肌肉组织中分离获得5株细菌,综合分离菌株的形态特征、理化特性和16S rRNA基因序列与系统发育学分析的结果,确定L1菌株为温和气单胞菌、L2菌株为维氏气单胞菌、J1、J2和L3菌株为嗜水气单胞菌。人工感染实验表明这5个气单胞菌分离株均具有较强的致病性。结论气单胞菌是该次鱼类暴发性出血病的病原,水温剧变、水质恶化和缺氧是疾病暴发的诱因,应采取改善环境、加强饲养管理、提高鱼体抵抗病力和及时杀灭病原体的综合措施防控该病。     

中华鳖源致病性产吲哚金黄杆菌分离、鉴定及药敏特性分析

对患病中华鳖(Pelodiscus sinensis)进行病原分离、鉴定及药敏实验, 从患病中华鳖皮肤、肝肾脾重要器官分离纯化病原菌, 经理化特性测定及16S rRNA序列分析对其进行鉴定及人工感染试验, 并利用K-B及二倍稀释法进行药敏特性分析。结果表明分离株J22是为中华鳖腐皮病病原, 其对中华鳖的LD₅₀为3.30×104 CFU/g。J22株理化特性与产吲哚金黄杆菌(Chryseobacterium indologenes)一致, 16S rRNA序列与产吲哚金黄杆菌同源性为99%, 综合判定J22株是产吲哚金黄杆菌。分离株对新霉素、庆大霉素及阿莫西林等12种抗生素高度敏感, 对氟苯尼考及多西环素等抗生素耐药; 二氧化氯、漂白粉及高铁酸钾对分离株消毒效果较好。分离菌株J22是中华鳖病原菌, 养殖时可选用庆大霉素、新霉素或者阿莫西林内服, 配合使用二氧化氯、漂白粉及高铁酸钾等外用进行防控。     

异育银鲫源杀鲑气单胞菌杀鲑亚种的分离鉴定

【目的】分离鉴定江苏省扬州市养殖场异育银鲫患病病原。【方法】采用常规的理化特性和分子生物学的方法,对从濒死异育银鲫肝脏处分离到的菌株YZ-1进行表型生物学、分子生物学及药敏试验的系统研究。【结果】该菌株16S r RNA基因(序列长度1 446 bp,Gen Bank登录号为JX164202)与其它杀鲑气单胞菌16S r RNA基因一致性在99%-100%之间,构建发育树确定该菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.salmonicida)。人工回感可导致异育银鲫死亡。药敏试验结果显示:对头孢呋辛、复方新诺明、恩诺沙星等23种抗生素敏感;对阿米卡星、四环素、大观霉素、头孢拉定等11种抗生素中度敏感;对青霉素G、链霉素、庆大霉素、氟苯尼考、万古霉素等10种抗生素耐药。【结论】研究结果证实引起异育银鲫死亡的病原为杀鲑气单胞菌杀鲑亚种。     

在患CyHV-2病的异育银鲫肠道黏膜中胆固醇、胆汁酸代谢通路基因的差异表达

为了探讨CyHV-2疾病条件下异育银鲫胆汁酸肠肝循环代谢途径主要基因表达活性的变化, 以患CyHV-2病的、正常的异育银鲫肠道黏膜为材料, 提取总RNA, 采用RNA-Seq测序、对单一基因进行注释, 并进行KEGG富集分析、单一基因差异表达分析。结果显示, 肠道黏膜组织有7770个基因显著性差异表达, 其中, 差异表达上调基因数为3335个、差异表达下调的基因数为4435个, 表明CyHV-2病的发生对肠道黏膜组织基因表达产生了重大的影响。病鱼胆固醇、胆汁酸生物合成代谢途径的酶、蛋白质的基因显著性差异表达, 显示肠道黏膜胆固醇、胆汁酸合成代谢受到显著性影响。参与胆固醇、胆汁酸合成代谢调节作用, 以及胆固醇、胆汁酸分泌、吸收、转运等生理过程的蛋白质、酶的基因也是差异表达下调, 胆汁酸肠肝循环有出现代谢障碍的趋势。结果表明, CyHV-2病的发生对肠道黏膜组织基因表达产生了严重影响, 对胆固醇、胆汁酸的合成代谢途径、胆汁酸肠肝循环途径的基因表达产生了严重影响, 将导致病鱼体内胆固醇、胆汁酸量的不足。患CyHV-2病病鱼血清胆汁酸含量下降了99%、胆固醇含量下降了10%, 证实了上述基因差异表达的趋势。     

美洲黑石斑迟缓爱德华氏菌分离、鉴定及致病性研究

养殖美洲黑石斑(Centropristis striata)发生以“内脏白点”为主要临床症状的暴发性死亡, 为明确美洲黑石斑患病原因, 对患病鱼进行了病原分离、鉴定及致病性研究, 以期为防治美洲黑石斑迟缓爱德华氏菌病提供参考依据。患病的美洲黑石斑的症状主要表现为鳃和内脏组织如脾脏及肾脏上布满白点。从患病鱼的病灶处分离纯化到一株优势致病菌株(ZS201807), 通过对该菌株形态特征、生理生化特性和16S rDNA基因测序等综合分析, 鉴定该菌为迟缓爱德华氏菌(Edwardsiella tarda)。人工感染实验证实此菌株可引起健康美洲黑石斑发病并表现出与自然发病相似的症状。利用组织切片和电镜超薄切片技术对患病美洲黑石斑鱼的肝脏、脾脏、鳃丝等6种组织进行组织病理学分析。组织病理结果显示, 脾和肾是感染较严重的主要靶器官, 脾脏组织内大量红细胞浸润, 出现严重瘀血; 鳃丝毛细血管扩张; 肾小管管腔狭窄, 肾小球肿大, 上皮细胞肿胀, 细胞空泡化。超微病理显示, 病鱼脾脏和头肾组织有大量杆状细菌积聚。药敏试验发现该菌对环丙沙星(5 μg/片)、四环素(30 μg/片)、恩诺沙星(5 μg/片)等14种药物敏感; 对青霉素(10 U/片)、阿奇霉素(15 μg/片)、丁胺卡那(30 μg/片)等13种药物耐受。证实此次养殖美洲黑石斑发病死亡的病原菌为迟缓爱德华氏菌。     

光唇鱼源无乳链球菌的分离鉴定及遗传特征

【目的】为查明浙江养殖光唇鱼大量死亡的病原,了解病原的遗传特征。【方法】本工作对患病光唇鱼进行病原分离,结合形态特征、生理生化特性和16S rRNA基因序列同源性,对分离菌株进行鉴定;采用人工回感试验确定其病原性,并对分离株的血清型、多位点序列分型(multilocus sequence typing,MLST)、毒力基因型和表面蛋白抗原基因型等遗传特征进行分析;此外,还测试了菌株的药敏特性。【结果】从患病光唇鱼体中分离得到优势菌株ACRO-0708,为革兰氏阳性球菌,不溶血,分子与生化鉴定为无乳链球菌(Streptococcus agalactiae);人工感染试验证实其对光唇鱼有较强的致病性,LD50为6.47×10~3CFU/g,属于血清型Ⅰb和MLST型ST261,毒力基因型为sip~+bibA~+cfb~+hylB~+iagA~+fbsA~+fbsB~+bac~–bca~–cylE~–scpB~–lmb~–,不携带所检测的6种表面蛋白基因。药敏试验结果显示,对青霉素、氨苄西林等8种药物较敏感,对氯霉素、复方新诺明等7种药物耐药。【结论】引起浙江养殖光唇鱼死亡的病原菌为无乳链球菌,其分子特征与水产动物主要流行的无乳链球菌株具有显著差异,生产中可选用氨苄西林、氟苯尼考等药物进行防治。     

一种新的异育银鲫病原———腐败希瓦氏菌

【目的】江苏盐城一家养殖场的异育银鲫暴发疾病,通过对病原进行研究,旨在为该病的防治提供理论依据和参考。【方法】从病鱼体表病灶和内脏中分离出优势菌株,经人工感染试验证实为病原菌。采用传统的形态、生理生化表型鉴定与16S rDNA序列分析相结合的方法确定菌株的分类地位。运用K-B琼脂法对病原菌株进行药物敏感性测定。【结果】综合菌株形态、生理生化表型以及16S rDNA序列分析的结果,确定该分离株为腐败希瓦氏菌(Shewanella putrefaciens)。回接感染试验证实腐败希瓦氏菌即是导致此次异育银鲫发病死亡的致病原,其半数致死量(LD50)为2.1×103cfu/g。该株腐败希瓦氏菌对吡哌酸、萘啶酸、氟哌酸、氟啶酸、氟苯尼考、利福平、美满霉素、氟罗沙星、恩诺沙星、复达欣、菌必治、先锋Ⅳ、罗红霉素和左氟沙星等抗生素敏感。【结论】首次报道了异育银鲫一种新的病原,说明腐败希瓦氏菌作为一种潜在的新病原也可能会对异育银鲫的养殖造成威胁。     

红鳍东方鲀病原鱼肠道弧菌的生物学特性研究

对引起红鳍东方鲀发病死亡的病原细菌进行了分离和主要生物学特性研究,包括病原性、形态特征、理化特性、16S rRNA基因序列及其系统发育学分析、胞外酶及溶血素活性、K抗原及耐药性等.结果表明,引起红鳍东方纯发病死亡的病原细菌为弧菌属(-Vibrio Pacini 1854)的鱼肠道弧菌(V.ichthyoenteri Ishimaru,et al.1996),2株代表菌株16S rRNA基凶序列(GenBank登录号分别为:EF611424和EF635304)与GenBank数据库中鱼肠道弧菌的同源性在98%-100%,且在构建的MP系统发生树中与鱼肠道弧菌聚为一个分支.分离菌不具有淀粉酶、蛋白酶、脂肪酶、DNA酶、脲酶、明胶酶和卵磷脂酶活性,且在含7%家兔脱纤血液营养琼脂培养基上不溶血.不具有K抗原.人工感染试验中分离菌对红鳍东方纯表现出明显的致病性.药敏试验结果显示,4株分离菌对供试37种抗菌药物中的苯唑青霉素和杆菌肽2种耐药.     

滇池高背鲫和方正银鲫酯酶、乳酸脱氢酶同工酶的比较研究

本研究以方正银鲫(Carassius auratus gibelio Bloch)普通鲫(Carassius auratus)和滇池高背鲫(Carassius sp.)的各种组织器官为材料,进行酯酶(Esterase)和乳酸脱氢酶(LDH)同工酶电泳图谱的分析比较。结果表明:9种不同组织中酯酶同工酶谱带各不相同,有明显的组织特异性。滇池高背鲫的酯酶谱图有3种表型。方正银鲫和滇池高背鲫同一组织的LDH同工酶酶谱也有明显差异。等电聚焦凝胶电泳(T=7.5％,C=5％)的结果又表明这二种鱼的肝脏、脑、卵的酯酶同工酶酶谱及电泳扫描图亦有差异。这些结果揭示滇池高背鲫与方正银鲫至少在生化水平上已有明显的分化,很可能起源于不同的地区,由不同的祖先,独立演化而形成。滇池高背鲫与云南普通鲫的LDH酶谱较为接近,这说明滇池高背鲫最可能起源于云南本地的普通鲫。     

池养条件下银鲫与鲫鱼的生物学特性比较及其在生产上的意义

银鲫在我国分布于黑龙江流域和新疆额尔齐斯河等水域中。在四川省池养条件下,比较了银鲫与本地鲫的形态、食性、生长、繁殖、含肉量、鱼肉营养成分和起水率等生物学特性。形态差别主要在于银鲫体型较高,头略短,侧线鳞和脊椎骨数稍多。食性相似。银鲫生长较本地鲫约快150%。银鲫在2足龄左右第一次性成熟,产卵盛期较本地鲫略晚1个月,性比为(?)34.1%(体长6.1—14.5厘米)—(?)5.95%(体长15.4—18.5厘米)。银鲫的肥满度,含肉量,鱼肉含脂量以及起水率均较本地鲫高。初步讨论了银鲫的某些生物学特性在其养殖和选育上的经济意义。       

Molecular identification of hybrids of the invasive gibel carp Carassius auratus gibelio and crucian carp Carassius carassius in Swedish waters

Both mitochondrial DNA sequence and two nuclear microsatellite markers were used to confirm the identity of the first record of Carassius auratus gibelio in the western (Swedish) Baltic Sea region. A total of 49 fishes were analysed, where 22 were from three Swedish sites connected to the Baltic Sea. The D-loop mitochondrial DNA sequences showed that 16 of 22 Swedish fishes were related to C. a. gibelio. The phylogenetic analysis of these sequences showed that these fish are probably not native, but represent different lineages of C. a. gibelio from China, Japan and Russia. All except three of these 16 fishes had microsatellite alleles suggesting hybridization with Carassius carassius. These findings suggest that a cryptic invasion of C. a. gibelio might be in progress.     

New species of myxosporidians in the genus Myxobolus (Myxosporidia, Myxobolidae) from cyprinid fishes of the Amur River

Five new species are described as follows: M. haematopterus from gills, fins and skin of Cyprinus carpio haematopterus; M. gibelio from connective tissue of gill filaments, fin rays and kidneys of Carassius auratus gibelio; Myxobolus waleckii from gills of Leuciscus waleckii; Myxobolus junchisi from gills, muscles, kidneys and spleen of Cyprinus carpio haematopterus; M. alacaudatus of Cyprinus and Carassius Myxobolus divergens carassii from gills, muscles, fins, and cartilaginous tissue of operculim of Carassius auratus gibelio and Cyprinus carpio haematopterus.     

Genetic diversity and antimicrobial susceptibility of motile aeromonads isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) farms

A total of 22 motile Aeromonas strains were detected in 48 (18.53%) of 259 fish and 6 (10.71%) of 56 water samples obtained from seven commercial rainbow trout ( Oncorhynchus mykiss , Walbaum) farms in the province of Mersin, Turkey. These strains were identified by conventional microbiological techniques and by using an ID32GN system. Of these isolates 20 (91.3%) were identified as Aeromonas hydrophila and 2 (8.7%) as Aeromonas sobria . While 8 of the A. hydrophila strains were isolated from water samples, 12 isolates were from fish samples. Whereas A. hydrophila strains were found in all farms, A. sobria was detected in only two farms. Genetic diversity by arbitrarily primed polymerase chain reaction (AP-PCR) and antimicrobial sensitivity tests were carried out on eight A. hydrophila isolates obtained from water samples, and isolates from seven A. hydrophila and one A. sobria from fish samples. The AP-PCR band patterns of motile aeromonads demonstrated weak similarity to the A. hydrophila reference strain ATCC 7966. Five A. hydrophila strains in the water samples displayed genetic similarity, but three others were different. Aeromonas hydrophila isolates from fish samples possessed slight similarities, and A. sobria was genetically distant to all A. hydrophila strains. An antimicrobial sensitivity test of 16 isolates revealed that 100% were sensitive to gentamicin, 87.5% to sulphamethoxazole–trimethoprim, 62.5% to enrofloxacin, 43.8% to oxytetracycline, 37.5% to neomycin, 18.75% to streptomycin and 6.25% to erythromycin. All isolates were resistant to novobiocin.     

Identification of triploid individuals and clonal lines in Carassius auratus complex using microsatellites

The Carassius auratus complex in natural populations includes diploid triploid and polyploidy individuals. Diploid individuals belong to the species Carassius auratus whereas triploid and polyploidy individuals are from the subspecies Carassius auratus gibelio. Triploid individuals are all female and reproduce clonally by gynogenesis. Therefore the Carassius auratus complex is an ideal system for studying evolution of unisexual reproduction. Identification of triploid individuals and clonal lines is the first step towards understanding of the evolution of unisexual clonal lines. We examined the ability of 10 microsatellites in identifying triploid individuals in 94 individuals from Japan and China. In 40 confirmed triploid individuals and eight confirmed diploid individuals, all triploid and diploid individuals can be identified by genotyping 10 microsatellite, and four triploid clonal lines were identified. Using the 10 microsatellites we genotyped 46 adult individuals (40 females and six males) from a natural population in China and found that all six males were diploid whereas the majority of females (36 of 40) were triploid and three triploid clonal lines were detected. In 18 diploid individuals from China, all individuals showed different genotypes, suggesting there is no diploid clonal line in diploid crucian carp. A phylogenetic analysis of 94 individuals from China and Japan showed that triploid individuals and clonal lines have originated recurrently.     

异育银鲫c型溶菌酶全长cDNA序列的生物信息学分析及其组织表达分析

利用RACE及克隆等方法获得了异育银鲫（Carassius auratus gibelio）c型溶菌酶基因全长cDNA序列．序列分析表明,所克隆的异育银鲫溶菌酶的cDNA全长751 bp,包括溶菌酶基因开放阅读框（ORF）438 bp,5′ 非编码区（UTR）为109 bp和3′ UTR为204 bp．438 bp ORF共编码146个氨基酸,其成熟肽的分子量预测值为14　543.6,理论等电点为8.86．通过ClustalW软件,将异育银鲫和其它多个物种c型溶菌酶的氨基酸序列进行多序列比对发现,所克隆的异育银鲫溶菌酶编码的氨基酸序列中存在c型溶菌酶的活性中心（Glu53和Asp69）,且与活性位点相邻的氨基酸序列高度保守．同时,8个保守的半胱氨酸残基也与其它物种的c型溶菌酶相一致．结合BLASTN分析的结果,可以确认所获得的异育银鲫溶菌酶cDNA序列属于c型溶菌酶．异育银鲫c型溶菌酶和人c型溶菌酶（pdb 1at6_）在蛋白质序列上有50%相似性,其三维（3-D）结构非常类似．通过氨基酸空间位置比较发现,两者具有类似的酶活中心,异育银鲫c型溶菌酶只能形成3个二硫键,比人少1个．荧光定量RT-PCR检测和溶菌酶活性测定显示,异育银鲫头肾和脾脏c型溶菌酶mRNA的表达量约为肝胰脏的2.9 倍和1.7 倍,异育银鲫头肾和脾脏的溶菌酶活性约为肝胰脏的6.2 倍和4倍．     

Identification of natural hybrids of gibel carp Carassius auratus gibelio (Bloch) and crucian carp Carassius carassius (L.) from lower Dyje River floodplain (Czech Republic)

Morphometric and genetic variation were examined in parental Carassius auratus gibelio and Carassius carassius individuals, and their natural hybrids. Of meristic traits, only the number of gill rakers clearly distinguished hybrids (39·4 ± 1·4) from parental C. a. gibelio (48·3 ± 0·7) and C. carassius (28·6 ± 1·1). MtDNA sequences showed that the hybrids were descendants of female C. a. gibelio . Microsatellite analysis confirmed the presence in hybrids of variants typical of C. a. gibelio and C. carassius .