爱德华氏菌常规PCR检测体系的建立

根据NCBI公布的爱德华氏菌(Edwardsiella ictaluri)磷酸丝氨酸转氨酶(phosphoserine transaminase,serC)的基因序列,设计一对特异性引物,建立了可快速而准确地检测爱德华氏菌的PCR方法,并对患病鱼组织进行了检测。研究结果表明,使用设计的引物能够扩增出与预计大小一致的124 bp的特异性片段,具有较好的检测特异性,对靶标DNA的检测灵敏度为50 pg/反应,对靶标细菌的检测灵敏度为56 cfu/反应。样品的检测结果与实际发病情况一致,表明成功建立了爱德华氏菌常规PCR检测方法,可用于爱德华氏菌病的诊断。     

实时定量PCR测定爱德华氏菌隐蔽质粒的拷贝数

为测定爱德华氏菌(Edwardsiella ictaluri)隐蔽质粒的拷贝数,并探究不同基因组制备方式及定量策略对质粒拷贝数测定的影响,分别采用试剂盒和水煮法制备爱德华氏菌基因组,通过绝对定量和相对定量PCR测定两种不同总DNA制备方式下的隐蔽质粒拷贝数,同时测定试剂盒对染色体DNA和质粒DNA的回收率。结果显示,以试剂盒提取的总DNA测得的pEI1和p EI2拷贝数绝对定量和相对定量分别为3.63±0.30、5.04±0.18和4.22±0.15、5.13±0.50,显著低于以水煮法测得的pEI1和p EI2拷贝数的绝对定量11.84±0.80、11.70±0.25和相对定量13.85±1.64、11.90±0.97。回收率结果显示,试剂盒对染色体DNA的回收率(45.8±4.1)%,显著高于对质粒pEII的回收率(25.1±0.5)%和pEI2的回收率(31.3±1.7)%。结果表明:通过实时定量PCR测定爱德华氏菌隐蔽质粒的拷贝数时,基因组的制备以水煮法为宜,爱德华氏菌隐蔽质粒pEI1和pEI2均属于中拷贝质粒。     

迟缓爱德华氏菌SYBR Green I实时荧光定量PCR检测方法的建立及其应用

根据克隆得到的迟缓爱德华氏菌gyrB基因序列设计并合成一对特异性引物,通用性和特异性检测结果显示所设计的引物具有良好的种间特异性和种内通用性,构建含gyrB基因的重组质粒作为标准品,经过反应体系优化后建立了检测迟缓爱德华氏菌的SYBR Green I实时荧光定量PCR检测方法。结果显示,该方法线性关系良好,在Tm为63℃时,扩增产物的熔解曲线仅有一个单特异峰,扩增所得标准曲线为y=-3.32x 39.38,相关系数为0.998,扩增效率为1.00,最低能检测到60个拷贝。应用建立的方法对人工感染的大菱鲆病样进行了检测,三个被检样品均呈阳性反应,证明该方法具有较好的适用性。实验结果表明所建立的实时荧光定量PCR方法具有特异、敏感、快速、定量的优点,可用于迟缓爱德华氏菌病的快速检测。     

黄颡鱼鲇爱德华氏菌的分离鉴定及其致病性研究

从患头顶溃疡症的黄颡鱼(Pelteobagrus fulvidraco)内脏组织中分离出致病力强的菌株JZ086,对该菌进行了形态学、生理生化特性的测定,并进行了Biolog全自动微生物分析系统及16S rDNA序列鉴定。结果显示:人工感染实验证实该菌为黄颡鱼的病原菌,其半数致死量(LD50)为1.7×105CFU。Biolog全自动微生物分析系统鉴定结果显示该菌为鲇爱德华氏菌(Edwardsiella ictaluri)。16S rDNA序列分析显示该菌与鲇爱德华氏菌的亲缘关系最近,同源性高达99%;系统进化树中与鲇爱德华氏菌(AB050826)自然聚为一支,二者遗传距离约为0.002。鉴定结果确认JZ086为鲇爱德华氏菌。药物敏感性试验结果显示,该菌对所测试的15种药物都敏感,其中对氨苄西林、环丙沙星、左氧氟沙星、妥布霉素等尤其敏感。     

(鱼师)鱼诺卡氏菌SYBR Green Ⅰ实时荧光定量PCR检测方法的建立与应用

根据GenBank中(鱼师)鱼诺卡氏菌16S-23S rRNA基因序列设计并合成一对特异性引物,经反应体系优化后建立了检测(鱼师)鱼诺卡氏菌的SYBR Green Ⅰ实时荧光定量PCR方法.结果显示,该方法线性关系良好,标准曲线的相关系数为0.998;溶解曲线分析显示产物为单一的特异峰;检测灵敏度可达10-6 μg/μL的DNA含量,与嗜水气单胞菌、大肠杆菌、金黄色葡萄球菌、副溶血弧菌、麦氏弧菌不发生交叉反应,具有良好的特异性.应用建立的方法在(鱼师)鱼诺卡氏菌病爆发时期,对16份鱼体组织、养殖水体、饲料等样品进行了检测,结果7份为阳性,与细菌分离、培养检查结果100％相符.既能检测发病鱼,又能检出未发病且已感染的病鱼,对病害的早期防控体现应用价值.结果表明,建立的实时荧光定量PCR方法具有特异、敏感、快速、定量等优点,可用于鱼类致病(鱼师)鱼诺卡氏菌的快速检测.     

鮰爱德华氏菌灭活疫苗免疫斑点叉尾鮰后外周血免疫指标的变化

在水温(25±1)℃下,给平均体质量(80±5)g的斑点叉尾鮰Ictalurus punctatus腹腔注射福尔马林灭活的鮰爱德华氏菌(Formalin-killed Edwardsiella ictaluri,FKE)后,第2、4、6、8、14、21,和28d测定外周血液免疫指标,并在免疫后第28天进行攻毒试验。结果显示:注射鮰爱德华氏菌灭活菌苗后,斑点叉尾鮰外周血红细胞和白细胞数量显著升高,白细胞分类组成变化显著,吞噬细胞的吞噬活性与凝集抗体效价显著上升。第4d免疫组红细胞和白细胞达峰值2.52×106/μL和4.67×105/μL,极显著高于对照组(P<0.01);单核细胞、中性粒细胞、吞噬细胞分类百分比和吞噬指数均在第4d达到峰值,分别为22.3%、5.67%、39.3%和4.33,极显著高于对照组(P<0.01);淋巴细胞分类百分比、凝集抗体效价在第21d达到峰值,分别为54.33%和1:341.33,极显著高于对照组(P<0.01)。攻毒试验结果表明:免疫组相对免疫保护率为64.3%。福尔马林灭活的鮰爱德华氏菌免疫后,斑点叉尾鮰获得较强的抗鮰爱德华氏菌感染保护能力,为进一步研究斑点叉尾鮰肠道败血症的免疫奠定基础。     

一株血鹦鹉迟缓爱德华氏菌的分离鉴定及药敏试验

为确定天津市某养殖场血鹦鹉(Cichlasoma var.)发病死亡的原因,并对其进行有效的预防与治疗,本试验对患病的血鹦鹉进行病原的分离鉴定、回归感染及药敏试验.从病灶处分离出一株疑似病原菌命名为XYW-1,经人工感染后具有致病性,临床症状与自然发病症状一致,半致死浓度为4.75×105 CFU/mL;经生化鉴定和1...     

中华鳖爱德华菌病病原菌的分离鉴定及致病因子研究

采用API 20E系列生化鉴定及16S rDNA和gyrB基因序列同源性分析方法,对从患病中华鳖(Trionyx sinen-sis)肝脏中分离到的一株细菌TL5m进行了鉴定,并通过人工感染试验,对该菌株进行了毒力检测;此外分别提取该TL5m株的主要致病因子外膜蛋白、脂多糖和胞外产物,对中华鳖进行毒力和免疫保护率试验。结果显示:菌株TL5m的API 20E鉴定编码为4544000,99.9%为迟钝爱德华氏菌(Edwardsiella tarda);其16SrDNA序列和gyrB基因序列(GenBank登录号分别:EF121756和GU563803)与迟钝爱德华氏菌的同源性最高(分别为94%和98%);菌株TL5m对中华鳖的半数致死量LD50为2.45×106 CFU/ind。药敏感结果显示菌株对磷霉素、菌必治、头孢孟多、头孢噻吩、壮观霉素高度敏感。外膜蛋白攻毒剂量60μg/ind和脂多糖攻毒剂量400μg/ind时,对中华鳖的致死率都为33.3%,胞外产物对中华鳖的LD50为31.73μg/ind;全菌灭活苗、胞外产物、外膜蛋白和脂多糖的免疫保护率分别为75%、62.5%、25%和87.5%。结果表明,发病中华鳖的病原菌为迟钝爱德华氏菌,其分泌的胞外产物对中华鳖具有较高毒力;提取的脂多糖对中华鳖遭受迟钝爱德华氏菌攻击具有较高免疫保护率。     

养殖澳洲宝石鱼迟缓爱德华氏菌的分离鉴定及致病基因的检测

从患病澳洲宝石鱼体内分离到一株致病菌(编号Et4),对该菌的生化特性及致病基因进行检测,并进行了药敏和人工感染实验。结果显示:菌株Et4的生化鉴定结果与迟缓爱德华氏菌(Edwardsiella tarda)标准菌株(AY77513)一致;其16S rRNA序列,经同源性比对与迟缓爱德华氏菌核苷酸相似度最高,达99%;根据迟缓爱德华氏菌的致病基因Ⅲ型分泌系统装置蛋白esaV基因序列设计引物,进行PCR扩增,得到708 bp序列,该序列与迟缓爱德华氏菌的esaV基因序列相似性达99.3%,说明菌株Et4具有esaV基因。菌株Et4对环丙沙星等较敏感,对其它药物中度或不敏感,具有较强的致病力(LD50=3.74×104CFU/mL),从病鱼中可以重新分离出此菌。综合形态学、生化特性、16S rDNA序列及致病基因序列鉴定其为迟缓爱德华氏菌。     

鰤鱼诺卡氏菌SYBR GreenⅠ实时荧光定量PCR检测方法的建立与应用

根据GenBank中蛳鱼诺卡氏菌16S-23S rRNA基因序列设计并合成一对特异性引物,经反应体系优化后建立了检测蛳鱼诺卡氏菌的SYBR GreenⅠ实时荧光定量PCR方法。结果显示,该方法线性关系良好,标准曲线的相关系数为0.998;溶解曲线分析显示产物为单一的特异峰;检测灵敏度可达10 6μg/μL的DNA含量,与嗜水气单胞菌、大肠杆菌、金黄色葡萄球菌、副溶血弧菌、麦氏弧菌不发生交叉反应,具有良好的特异性。应用建立的方法在蛳鱼诺卡氏菌病爆发时期,对16份鱼体组织、养殖水体、饲料等样品进行了检测,结果 7份为阳性,与细菌分离、培养检查结果 100%相符。既能检测发病鱼,又能检出未发病且已感染的病鱼,对病害的早期防控体现应用价值。结果表明,建立的实时荧光定量PCR方法具有特异、敏感、快速、定量等优点,可用于蛳鱼类致病鱼诺卡氏菌的快速检测。     

Susceptibility of five species of fish to Edwardsiella ictaluri

Abstract. Five species of fingerling fish, channel catfish, Ictalurus punctatus , tilapia, Sarotherodon aureus , golden shiner, Notemigonus crysoleucas , largemouth bass, Micropterus salmoides and bighead carp, Aristichthys nobilis , were tested to determine their susceptibility to the bacterium, Edwardsiella ictaluri , at 26°C, Channel catfish demonstrated high susceptibility to E. ictaluri as 100% of those fish injected with 1.5 × 10³ cells died within 10 days. Tilapia demonstrated slight susceptibility to the pathogen while golden shiner, bighead carp and largemouth bass were not susceptible. E. ictaluri was isolated from a higher percentage of peritoneal cavities, livers and kidneys of channel catfish than of other species. Sequential growth of E. ictaluri in the liver of channel catfish is described.     

Recovery of Edwardsiella ictaluri from danio (Danio devario)

A disease outbreak in the danio (Danio devario) resulted in the isolation of Edwardsiella ictaluri as the etiologic agent. This isolate was compared with typical E. ictaluri and found to give identical reactions. It was also pathogenic for channel catfish. This is the second isolation of E. ictaluri from non-ictalurid fish.     

Organization and sequence of four flagellin-encoding genes of Edwardsiella ictaluri

Edwardsiella ictaluri , the cause of enteric septicaemia in channel catfish ( Ictalurus punctatus ), is motile by means of peritrichous flagella. We determined the complete flagellin gene sequences and their organization in E. ictaluri by sequencing genomic segments from a λ-ZAP phage genomic library of E. ictaluri . Four flagellin genes ( fliC1, fliC2, fliC3 and fliC4 ) are arranged in tandem within 6 kb in the E. ictaluri genome. Each flagellin-coding sequence is preceded by a σ²⁸ recognition site consensus sequence. The predicted amino acid sequences of all four flagellin proteins (between 36 and 37.5 kDa) are similar in the N-terminal (1–160 aa) and C-terminal (last 74 aa) portions and are divergent in the central portion of the proteins. Proteins encoded by flC1, fliC2 and fliC3 are more similar to each other (88–90% aa identity) than to the protein encoded by fliC4 (76–78% aa identity). basic local alignment search tool analysis of GenBank sequences showed that all flagellin aa sequences are more similar to those of Serratia marcescens (72–74% identity) than to those of Edwardsiella tarda (≤64% identity). Primary determination of E. ictaluri flagellin gene sequences facilitate advanced studies on the role of flagella in host–pathogen interaction.     

An assessment of the antigenic homogeneity of Edwardsiella ictaluri using monoclonal antibody

Abstract. Monoclonal antibodies were made against the reference strain of Edwardsiella ictaluri (ATCC 3320). Antibody produced by one of seven anti- E. ictaluri hybridomas reacted positively by the immunofluorescent antibody technique against 17 other E. ictaluri isolates. All hybridoma antibodies failed to react with six other bacterial species pathogenic to fish including E. tarda . Ouchterlony tests indicated that four anti- E. ictaluri clones produced only one kind of immunoglobulin. Electrophoresis of 14 different E. ictaluri isolates indicated identical protein bands at 36 and 60 kilodaltons (KD) in all isolates except an isolate from Thailand. Using the immunoblot method, channel catfish anti- E. ictaluri serum reacted with protein bands at 34 and 60 KD, which indicates that this molecular weight protein in the bacterium may be the dominant immunoprotein.     

Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm‐raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive‐sequence‐mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep‐PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep‐PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.     

Edwardsiella ictaluri infection in Pangasius catfish imported from West Bengal into the Southern Caribbean

In response to a mortality event, seven Pangasius catfish (Pangasianodon hypophthalmus) were submitted to the University of the West Indies, School of Veterinary Medicine, Trinidad and Tobago, for diagnostic evaluation. These fish were part of a consignment that arrived from Kolkata two weeks earlier. Fish presented with perianal haemorrhage and blister‐like swellings on the skin which ruptured to leave ulcers. Edwardsiella ictaluri was consistently recovered from the brain and skin. Repetitive sequence‐mediated PCR analysis revealed genetic fingerprints consistent with E. ictaluri isolates from farm‐raised channel catfish in Mississippi, USA. Plasmid analysis of the case isolates identified two unique plasmids that differ slightly in conformation and content from the pEI1 and pEI2 plasmids described for E. ictaluri from other fish hosts. The case isolates were also PCR negative for several E. ictaluri virulence factors. The biological implications of these genetic differences are unclear and warrant further study. This is the first report and documentation of E. ictaluri infection in Trinidad and Tobago, suggesting the pathogen may have been introduced concurrently with the importation of fish. This report emphasizes the importance of adequate health screenings of imported lots to minimize the threat of introducing E. ictaluri to non‐endemic areas.