红尾皇冠鱼(Aequidens rivulatus)病原无乳链球菌的分离、鉴定与特性分析

为查明天津地区养殖红尾皇冠鱼(Aequidens rivulatus)大量死亡的原因,取患病红尾皇冠鱼进行细菌分离,从病鱼肾脏中分离到优势菌株071901,人工感染试验证实其对红尾皇冠鱼有较强的致病性;采用形态学观察、生理生化特性、16S r RNA基因序列系统发育树分析及cfb(GBS-specific gene cfb,CAMP factor)基因检测对菌株071901进行鉴定。结果显示,菌株071901为革兰氏阳性球菌,生化特性与链球菌属的无乳链球菌(Streptococcus agalactiae)相近,基于16S r RNA基因序列的系统发育分析将菌株071901与无乳链球菌聚为一支,以071901为模板特异性扩增无乳链球菌的cfb基因,也得到了预期的900 bp的核酸片段,进一步证实菌株071901为无乳链球菌。对30种抗生素的药敏结果显示,菌株071901对红霉素、阿奇霉素等19种药物敏感,对多粘霉素、罗红霉素、呋喃唑酮等11种药物耐药。     

红尾皇冠鱼(Aequidens rivulatus)病原无乳链球菌的分离、鉴定与特性分析

为查明天津地区养殖红尾皇冠鱼(Aequidens rivulatus)大量死亡的原因,取患病红尾皇冠鱼进行细菌分离,从病鱼肾脏中分离到优势菌株071901,人工感染试验证实其对红尾皇冠鱼有较强的致病性;采用形态学观察、生理生化特性、16S r RNA基因序列系统发育树分析及cfb(GBS-specific gene cfb,CAMP factor)基因检测对菌株071901进行鉴定。结果显示,菌株071901为革兰氏阳性球菌,生化特性与链球菌属的无乳链球菌(Streptococcus agalactiae)相近,基于16S r RNA基因序列的系统发育分析将菌株071901与无乳链球菌聚为一支,以071901为模板特异性扩增无乳链球菌的cfb基因,也得到了预期的900 bp的核酸片段,进一步证实菌株071901为无乳链球菌。对30种抗生素的药敏结果显示,菌株071901对红霉素、阿奇霉素等19种药物敏感,对多粘霉素、罗红霉素、呋喃唑酮等11种药物耐药。     

鲢腐败希瓦氏菌株的16S rRNA鉴定

从患病鲢(Hypophthalmichthys molitrix)体表分离获得1株革兰氏阴性细菌,菌株编号为BD1-5,通过细菌形态学观察、革兰氏染色镜检、16S r RNA序列分析以及构建系统发育树分析等进行鉴定研究。结果表明,患病白鲢体表皮肤溃烂,尤其尾部溃烂严重;对该病例主要组织进行镜检未见明显寄生虫;分子生物学检测结果显示鲤春病毒血症病毒(Spring viremia of carp virus, SVCV)、鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus Ⅱ, Cy HV-Ⅱ)和锦鲤疱疹病毒(Koi herpesvirus, KHV)均为阴性;采用PCR方法获得该分离菌株16S r RNA序列片段大小为1 416 bp,与Shewanella putrefaciens(Gen Bank NO.:KX185698)序列相似性为99.72%;构建系统发育树分析显示,该菌株与Shewanella putrefaciens(Gen Bank NO.:KY817253)等自然聚为一枝。综合菌株形态及16S r RNA序列分析,最终推测该分离株为腐败希瓦氏菌(S. putrefaciens),属于革兰氏阴性杆菌。研究结果为证明腐败希瓦氏菌(S. putrefaciens)是鲢养殖过程中一种潜在致病源提供理论依据和参考。     

罗非鱼无乳链球菌巢式PCR检测方法的建立

根据无乳链球菌(Streptococcus agalactiae)cfb基因序列,设计、合成2对引物,优化扩增条件,建立了快速高灵敏度鉴别无乳链球菌的巢式PCR方法。结果显示:使用该方法对罗非鱼(Oreochromis mossambicus)血液样品进行检测,可检测到活菌浓度为8.7×104CFU/m L的无乳链球菌。对采自广西地区受无乳链球菌感染的19份罗非鱼样品进行检测,18份可获得目的片段,扩增到的序列均为无乳链球菌cfb基因序列,检测准确度达到94.7%。     

中国七种水生动物源无乳链球菌的分子特征及其对斑马鱼的致病性

为了解中国水生动物源无乳链球菌的分子流行特征,揭示其传播和流行规律,本实验对分离得到并鉴定的10株7种水生动物源无乳链球菌通过分子血清型、多位点序列分型(MLST)分型、毒力基因型和前噬菌体分型等方法进行分子分型;其次,通过斑马鱼评价7种水生动物源无乳链球菌的致病性。分子血清型分析结果表明,10株无乳链球菌可分为3种血清型,即Ⅰa、Ⅰb和Ⅲ型;MLST分型结果表明,Ⅰa型无乳链球菌均为ST7型,Ⅰb无乳链球菌均是ST261型,只有Ⅲ型无乳链球菌是ST739型。进一步分型结果表明,10株无乳链球菌可分为3种毒力基因型和4种前噬菌体基因型。根据4种分型结果可知,10株水生动物源无乳链球菌可分为4种类型,其中虎纹蛙源无乳链球菌具有独立的分子血清型、MLST型、毒力基因型和前噬菌体基因型,即Ⅲ-ST739-V1-P3;罗非鱼源无乳链球菌的基因型有3种,即Ⅰa-ST7-V2-P1、Ⅰa-ST7-V2-P2和Ⅰa-ST7-V3-P4;红尾皇冠鱼、鳙和罗非鱼源无乳链球菌的基因型相同:Ⅰa-ST7-V2-P2;卵形鲳鲹、宝石鲈和罗非鱼源无乳链球菌具有相同的基因型:Ⅰa-ST7-V2-P1;鲮和罗非鱼源无乳链球菌的基因型相同,即Ⅰb-ST261-V3-P4。致病性研究表明,7种水生动物源无乳链球菌对斑马鱼均有强致病性。研究表明,两栖类虎纹蛙源无乳链球菌和鱼源无乳链球菌的基因型明显不同,它们之间遗传变异较大,因此,无乳链球菌在两栖类和鱼类之间相互传播的可能性较小。鱼源无乳链球菌有3种基因型,且这3种基因型均在罗非鱼中流行,这表明无乳链球菌在鱼类中相互传播的可能性较大,尤其是在罗非鱼与其他鱼类之间。     

嗜水气单胞菌引致的金钱鱼细菌性疾病

为了分离鉴定实验室养殖的金钱鱼暴发性疾病的病原,利用传统病原分离的方法,从病鱼肝脏分离得到一株G–短杆菌(Ah201416),对其进行电镜观察和生理生化鉴定,对病鱼组织进行病理切片分析,并根据科赫法则,用分离株对健康金钱鱼进行人工感染。结果显示,该菌株电镜下观察细菌大小为0.8~1.0μm×1.0~2.0μm(宽×长),无芽孢和荚膜,生理生化特性与嗜水气单胞菌基本一致;16S r RNA序列(登录号:KR006248)与Gen Bank中嗜水气单胞菌(Aeromonas hydrophila,Ah ATCC7966)的16S r RNA基因序列的同源性高达99%,系统进化树与Ah聚为一支。病理切片分析发现,发病金钱鱼与正常金钱鱼相比,鳃小片细胞不同程度地脱落,伴有白细胞浸润;肠绒毛结构消失,肌肉层疏松明显;肾脏中肾小管细胞脱落,管腔中有坏死细胞,并出现不同程度的颗粒变性;肝脏细胞形态不规则,伴有血细胞浸润等病理损伤。人工感染实验表明,其对健康金钱鱼96h的半数致死剂量(LD50)为7.35×107 CFU/m L。研究表明,发病金钱鱼中分离的Ah201416菌株为嗜水气单胞菌,丰富了金钱鱼细菌性病原的研究,此结果为金钱鱼细菌性疾病的防治和养殖业的健康发展提供了重要的理论依据。     

一株梭鲈源无乳链球菌的分离、鉴定及分子分型分析

为查明梭鲈(Lucioperca lucioperca)凸眼病病原,从患病梭鲈病灶眼、肝、脾和肾分离纯化病原菌,进行了生理生化特性测定及16S rRNA基因序列分析,并开展人工感染试验,测定其血清型及MLST分型,并利用纸片扩散法进行药敏特性分析。结果显示:分离菌株(命名为SL1701株)为本次引发梭鲈病害的致病菌,Ib-ST261型。回归感染实验证实,分离得到的细菌SL1701对健康梭鲈具有非常强的致病性,4.5×105CFU/m L时可使80%受感染梭鲈死亡,并可复制出自然发病鱼的症状。菌株SL1701革兰氏染色为阳性链球菌,γ溶血,生理生化特性与普通无乳链球菌基本一致,16S rRNA基因序列与无乳链球菌(Streptococcus agalactiae)同源性最高,综合判定分离菌株为无乳链球菌(Streptococcus agalactiae)。药敏试验结果显示:分离菌株SL1701对氟苯尼考、氟罗沙星等17种抗生素高度敏感,对青霉素、磷霉素等8种抗生素耐药。结果表明,分离菌株SL1701为Ib-ST261型无乳链球菌,养殖时可选用氟苯尼考及氟罗沙星等药物进行防控。     

西伯利亚鲟停乳链球菌的分离、鉴定与致病性

从患暴发性流行病的两伯利亚鲟(Acipenser baerii)肝脏和心脏中各分离到1株细菌,分离纯化后获得2个分离株,编号分别为AeBF070904、AbHT070912,对分离菌进行了菌株鉴定、致病性分析及药敏实验.分别应用常规生理生化鉴定、全自动细菌测定卡API 20 STREP和ID 32STREP进行检测,结果表明,2个分离株均为停乳链球菌(Streptococcus dysgalactiae).对2个分离株的16S rRNA基因进行PCR扩增和测序,并与GenBank中收录的链球菌16S rRNA基因进行序列分析并构建系统进化树,结果显示,2个分离株的16S rRNA基因序列相同,与停乳链球菌同源性最高达97.3%,在系统进化树上与停乳链球菌聚为一簇,进一步确认2个分离株均为停乳链球菌.从人工感染后发病鱼的内脏组织再分离的细菌特性与原感染菌相同,确认停乳链球菌是西伯利亚鲟的致病菌.2个分离株对两伯利亚鲟、杂交鲟及剑尾鱼均有致死毒性,37℃培养的细菌毒力比28℃培养的细菌毒力强.2个分离株均对青霉素、诺氟沙星等7种药物敏感;对头孢唑啉、庆大霉素等2种药物耐受;对红霉素巾等敏感;对卡那霉素等8种药物菌株之间出现差异.     

罗非鱼无乳链球菌的分子鉴定

对罗非鱼致病菌株TL60829NA及其人工感染后分离菌株TL60829NA1、TL60829NA2应用原核生物16S rRNA基因通用引物进行分子分类学鉴定.对这些菌株进行16srRNA基因的克隆、序列分析,核酸序列同源性分析表明,TL60829NA及其人工感染后分离菌株TL60829NA1、TL60829NA2为同一种细菌.其中,TL60829NA2与GenBank登陆号为DQ303183的无乳链球菌(Streptococcus agalaciate)菌株ATCC13813序列同源性最高(99.8%).同时,通过与常引起罗非鱼链球菌病的S.agalaciate、S.iniae代表菌株16srRNA基因构建的发育进化树表明,该菌株及其人工感染后分离菌株与S.agalaciate代表菌株构成一个进化分枝,而4株S.iniae代表菌株的16srRNA基因则构成另一个分枝.本研究证实了从发病罗非鱼肝脏组织中分离到的致病性链球菌为无乳链球菌.     

我国罗非鱼源新型无乳链球菌的分离、鉴定及其分子特征

2014年海南省文昌市多个养殖场的罗非鱼出现暴发性疾病,患病罗非鱼表现出体色发黑、打转游动、眼球突出或混浊等典型的链球菌病症状。从患病罗非鱼的肝、肾、脾、眼球和脑等组织中分离到19株病原菌,即TC-1、TC-2、BL1441~BL1448和WT1451~WT1459。通过形态观察、生理生化特征和16S r RNA基因序列分析等方法对病原菌进行鉴定,结果表明,这些病原菌均为无乳链球菌。溶血试验结果表明,TC-1、TC-2和BL1441~BL1448菌株为β-溶血性无乳链球菌,而WT1451~WT1459菌株为不溶血无乳链球菌。进一步通过MLST、分子血清型和毒力相关基因检测等技术对这些分离菌株进行遗传特征分析,结果表明TC-1、TC-2和BL1441~BL1448菌株是常见的Ia-ST7型,其毒力基因型为bac+-bca+-bib A+-cfb+-hyl B+-iag A+-fbs B+-lmb–-scp B–-cyl E+-gbs20186–。而WT1451~WT1459菌株则是Ib-ST261型,其毒力基因型为bac–-bca–-bib A+-cfb+-hyl B+-iag A+-fbs B+-lmb–-scp B–-cyl E–-gbs20186+。将分离菌株BL1441和WT1451分别对罗非鱼进行攻毒试验,结果表明,WT1451菌株是强毒株,当其攻毒剂量为4.5×103CFU/m L时,罗非鱼累积死亡率可达85%。本研究将为我国罗非鱼无乳链球菌的流行病学、疫苗研制以及疾病防控等研究奠定基础。     

Identification and molecular typing of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> isolated from pond-cultured tilapia in China

Bacteria strains with strong virulence were isolated from pond-cultured tilapia in China. They were identified as Streptococcus agalactiae by biochemical assays, and confirmed by 16S ribosomal RNA (rRNA) and group B Streptococcus (GBS)-specific gene cfb analyses. Multiplex polymerase chain reaction (PCR) assay of the alpha C protein (ACP) gene and capsular polysaccharide antigen (cps) gene was employed to identify their molecular serotype (MS). Amplification of the ACP gene produced a 400-bp C alpha protein gene (bca) fragment, suggesting that these isolates belong to MS Ia, Ib or II; amplification of cps produced a 790-bp amplicon, indicating that they belong to MS Ia/III-3. An additional PCR based on nucleotide difference in the cps H–I region of MS Ia and III further suggested that the isolates belong to serotype MS Ia. Moreover, multi-locus sequence typing (MLST) indicated that these strains were of sequence type 7 (ST-7). These results showed that isolates from different regions of China shared the same MS and ST. However, none of the isolated ST-7 GBS corresponded to the capsular serotype, suggesting that these fish GBS possessed specific molecular characteristics not present in human or other animals. Data from this study will facilitate the understanding of epidemiology and nosogenesis of tilapia GBS and the establishment of effective disease prevention methods.     

Comprehensive Investigation of Streptococcosis Outbreaks in Cultured Nile Tilapia,Oreochromis niloticus,and Red Tilapia,Oreochromis sp., of Thailand

Streptococcosis causes economic losses due to mass mortality at all culturing stages in Nile tilapia, Oreochromis niloticus, and red tilapia, Oreochromis sp., farming throughout Thailand. Diseased tilapia collected from outbreak areas during 2003–2012 were examined using histopathological, biochemical, and molecular tools. Infected fish showed clinical signs of septicemia, and bacteria were found in visceral organs. All gram‐positive cocci isolates were negative to catalase and oxidase, and exhibited β‐hemolysis; however, they possessed various biochemical profiles. PCR amplification of the 16S rRNA gene was used for 165 samples, and resulted in identification of 143 (86.67%) with Streptococcus agalactiae and 14 (8.48%) with Streptococcus iniae, and 8 (4.85%) with mixed infection. High similarity (≥98%) of 16S rRNA gene sequences to the reference strain S. agalactiae (accession no. EF092913) and S. iniae ATCC29178 type strain was observed in the typing of S. agalactiae and S. iniae from Thai farmed tilapia. This investigation documented that at least two species of streptococcal bacteria, S. agalactiae and S. iniae, were involved in tilapia streptococcal infection in Thailand. The molecular recognition of the etiologic agents showed that S. agalactiae was the dominant species that cause disease in all culture areas, whereas S. iniae were discovered only in cases from the northeastern and central regions.     

Development of Loop‐mediated Isothermal Amplification (LAMP) for the Rapid Detection of Streptococcus agalactiae in Tilapia,Oreochromis niloticus

Streptococcus agalactiae is one of the major causative agents of tilapia streptococcosis, which has caused severe economic losses in aquaculture. Rapid and accurate detection of the pathogen is necessary to limit losses because of this disease. In this study, a loop‐mediated isothermal amplification (LAMP) assay was developed for the detection of S. agalactiae. Firstly, a set of four primers was designed to target the cfb gene of S. agalactiae. Then, using Bst DNA polymerase, the LAMP assay was performed at 65 C for 60 min and terminated at 80 C for 10 min in a simple water bath. Positive or negative results could be observed by direct visual inspection. There were no cross reactions with other bacterial species, indicating high specificity of the LAMP assay. The sensitivity of the LAMP assay for detecting S. agalactiae was about 20 cells per reaction. Moreover, the developed closed‐tube step has greatly improved the LAMP system. The LAMP method was also applied to detect S. agalactiae in infected tilapia tissue, demonstrating usefulness in diagnostics. Overall, this study showed that the cfb‐based LAMP assay was an effective method to detect S. agalactiae rapidly.     

Cytotoxicity of Streptococcus agalactiae secretory protein on tilapia cultured cells

Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.     

Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia

Ninety‐three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Rüppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S. agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S. agalactiae; genotyping of selected S. agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S. agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.     

<Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> from tilapia (<Emphasis Type="Italic">Oreochromis</Emphasis> sp.) transmitted to a new host,bighead carp (<Emphasis Type="Italic">Aristichthys nobilis</Emphasis>), in China

Several outbreaks of Streptococcus agalactiae infection of bighead carp (Aristichthys nobilis) were observed in China. The molecular epidemiology and pathogenicity of S. agalactiae in bighead carp and tilapia (Oreochromis sp.) is poorly understood. In the present study, we identified S. agalactiae strains isolated from diseased bighead carp using the API 20 Strep kit and 16S rDNA sequencing and determined whether these strains came from tilapia. Of the 46 identified S. agalactiae strains, 24 strains were successfully isolated from diseased bighead carps, 20 S. agalactiae strains were isolated from tilapia, and two S. agalactiae strains were isolated from tiger frog (Hoplobatrachus chinensis). The results of molecular typing, including multilocus sequence typing, molecular serotyping, surface protein gene detection, and virulence-related gene detection showed that the 44 strains from bighead carp and tilapia were highly similar, whereas different from tiger frog GBS strains. Remarkably, the bighead carp strain Hn1404 showed high virulence in bighead carp and zebrafish. Moreover, this strain was pathogenic to Nile tilapia (Oreochromis niloticus). In addition, comparative genomic analysis showed that isolate Hn1404 had a close relationship with the bighead carp and tilapia S. agalactiae strains. All the analyses of the genetic characteristics of bighead carp and tilapia strains showed that tilapia S. agalactiae strains could be transmitted to other fish species such as bighead carp.     

凡纳滨对虾细菌性红体病病原的分子特征与耐药性

为探明引起凡纳滨对虾细菌性红体病的病原,从病虾肝胰脏分离得到10株优势菌,经回归感染实验证实其为引起此次红体病的病原菌.Vitek与16S rRNA序列分析显示,分离株均为副溶血弧菌.基于dnaE-gyrB-recA-dtdS-pntA-pyrC-tnaA的多位点序列分型(multilocus sequence typing,MLST)表明,这些菌株形成3个新序列型(ST),其中1株为ST413,7株为ST414,2株为ST415;ST413包含新等位基因型recA-166与tnaA-121,ST414则含有新等位基因型gyrB-219.MLST结果提示,这些副溶血弧菌分离株并非来自单一克隆,呈现出一定水平的分子多样性.但这些菌株均含有大流行群(PG)的分子标记toxRS与VPA1168,并具有相同的毒力基因构成(tlh+ tdhtrh-T3SS1+T3SS2-)与耐药谱,其tdh与trh的缺失并未影响细菌对凡纳滨对虾的致病力.综上所述,引起此次凡纳滨对虾红体病的10株副溶血弧菌可能为PG的不同变异株.     

Genotypic diversity among Shewanella spp. collected from freshwater fish

Different Shewanella species are isolated both from healthy and from diseased fish. To date, contemporary methods do not provide sufficient insight to determine species and detail differentiation between tested strains. Bacteria isolated from cultured (n = 33), wild (n = 12) and ornamental (n = 6) fish, as well as several reference strains, were tested by 16S rRNA gene sequencing, ERIC‐PCR and pulsed‐field gel electrophoresis (PFGE) assays. Our study indicates that isolates collected from freshwater fish were genetically diverse. Based on 16S rRNA gene sequences, bacteria were clustered into groups S. putrefaciens, S. xiamenensis and S. oneidensis. Some isolates were classified only to genus Shewanella; thus, 16S rRNA gene analyses were not enough to determine the species. ERIC‐PCR revealed 49 different genotype profiles indicating that the method might be useful for differentiation of Shewanella isolates irrespectively to species identification, contrary to PFGE which is not suitable for Shewanella typing.