Genotyping of Edwardsiella tarda isolated from freshwater fish culture system

The applicability of PCR-RFLP of 16S rDNA and conventional phenotypic methods for differentiation of Edwardsiella tarda associated in freshwater fish culture system was studied. In this study, by conventional biochemical tests and antibiotic resistant patterns 2 and 14 groups were obtained. But these methods failed to discriminate the isolates habitat wise. However, PCR-RFLP of 16S rDNA was found to be specific to detect habitat-specific isolates. All the fish isolates belonging to particular genotypes were found only in fish, not in water or sediment. Some of the genotypes were exclusively present in water and sediment. This study indicates the prevalence of site-specific genotypes in freshwater ecosystems. Molecular method is found to be superior to discriminate the E. tarda habitat wise to conventional typing methods.     

Isolation of Edwardsiella tarda from a sea lion and two alligators

Edwardsiella tarda was isolated from a sea lion and 2 alligators on 3 separate occasions. The 3 isolates were not serologically identical. Specific pathologic effects of E. tarda could not be determined.     

Production of monoclonal antibodies specific to major outer membrane protein of Edwardsiella tarda

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and gastro and extra-intestinal infections in humans. Monoclonal antibodies (MAbs) were produced against outer membrane proteins (OMPs) of E. tarda ET-7, isolated from diseased snakehead (Ophiocephalus punctatus). Two stable hybridoma clones, designated as 3F10 and 2C3 MAbs were found to be potentially specific for E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These MAbs recognized major immunogenic OMP band at 44kDa in Western blotting. Both MAbs belonged to the IgG1 isotype and recognized different epitopes of OMP as seen by competitive ELISA. These MAbs strongly reacted with all 17 isolates of E. tarda used in our study by indirect ELISA and Western blotting. Interestingly, no reaction was observed with the reference strain of E. tarda (MTCC 2400). The sensitivity of 3F10 MAb to detect whole cells of E. tarda was up to a level of 1x10(4)CFU/ml in indirect ELISA. No cross-reactivity of MAbs were seen with Escherichia coli, Salmonella arizonae, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae, Flavobacterium ferrugineum and Mycobacterium tuberculosis. These MAbs could be used for specific detection of E. tarda infection in fish by immunoassays.     

Microbiological comparative study of isolates of Edwardsiella tarda isolated in different countries from fish and humans

It is difficult to use tissue culture assays to investigate adherence and other properties of Edwardsiella tarda because the organism is invasive and produces a potent hemolysin. We therefore relied on polymerase chain reaction (PCR) to determine the occurrence of genes for enterotoxins (LT-I, EAST-1), Shiga toxin (Stx-1, Stx-2), cytotoxic necrotizing factors (CNF-1, CNF-2), aerobactin, invasion plasmid of enteroinvasive Escherichia coli, EPEC adherence factor (EAF), intimin (Eae), enterohemolysin (EntHly) and hemolysin (Hly) in 53 isolates of E. tarda from humans and fish from several countries. All isolates were negative for all genes investigated by PCR. Adhesion to and invasion of HeLa cells were determined by using the unusually short incubation time of 1h or 30 min. All isolates adhered and invaded in these tests. Finally, a random amplified polymorphic DNA (RAPD) test distinguished, with a few exceptions, isolates of human and fish origin.     

Potential of auxotrophic Edwardsiella tarda double-knockout mutant as a delivery vector for DNA vaccine in olive flounder (Paralichthys olivaceus)

To evaluate potential of an auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) as a delivery vehicle for DNA vaccine in fish, olive flounder (Paralichthys olivaceus) were immunized with the E. tarda mutant harboring plasmids (pG02-ASD-CMV-eGFP) for eukaryotic expression of the enhanced green fluorescent protein (eGFP) gene through either intraperitoneal (i.p.) or oral route, and the expression of eGFP in the internal organs and generation of antibody against eGFP in fish were analyzed. In fish i.p. injected with 2×10(7)CFU/fish of Δalr Δasd E. tarda harboring pG02-ASD-CMV-eGFP, expression of eGFP was detected in liver, kidney, and spleen from 1 day to 28 days post-injection. In fish orally administered with 1×10(9)CFU/fish of the bacteria, the eGFP band was detected in liver, kidney, and spleen from 1 day to 14 days post-administration, whereas, in intestine, the band was detected only at 1 day post-administration. Either oral or i.p. immunization of olive flounder with recombinant E. tarda that carried eGFP-expressing eukaryotic plasmids was successful to induce humoral adaptive immunity against not only E. tarda that was used as a delivery vehicle but also eGFP that was used as the reporter protein of DNA vaccine, suggesting attenuated E. tarda-vectored DNA vaccine has a potential to be used as a combined vaccine against infectious diseases in fish.     

豫陕两省14株鸡杆菌的gyrB、16S rRNA和rpoB基因序列比较分析

分析河南、陕西分离的14株鸡杆菌(Gallibacterium)之间的进化关系,及gyrB基因序列比较在该菌进化分析中的作用。PCR扩增鸡杆菌分离株的gyrB、16SrRNA和rpoB3个看家基因,PCR产物纯化后直接测序。将鸡杆菌分离株、国外参考株的3个看家基因序列进行比较分析,用Phylip 3.67软件构建进化树。结果表明,14株鸡杆菌与鸭源鸡杆菌(Gallibacterium anatis)模式株间的相似性为96.3%～98.0%(gyrB)、97.7%～99.6%(16SrRNA)和97.7%～99.0%(rpoB);14株鸡杆菌与鸡杆菌复合群1(Gallibacterium genomosp.1)参考株间的相似性为88.8%～89.9%(gyrB)、96.2%～97.5%(16SrRNA)和92.6%～93.6%(rpoB)。基于3个看家基因序列的进化分析,均显示14株鸡杆菌和鸭源鸡杆菌模式株形成单独的一个群。14株鸡杆菌分离株均属于鸭源鸡杆菌种;在3个看家基因位点,鸡杆菌河南株与陕西株之间、鸡杆菌输卵管炎病鸡分离株与健康鸡分离株之间均无明显遗传上的差异;gyrB基因序列分析可用于鸡杆菌分离株的种类鉴定,且对14株鸡杆菌与复合群1参考株的区别能力优于另外2个看家基因。     

Immuno-response in tilapia Sarotherodon niloticus vaccinated with Edwardsiella tarda by hyperosmotic infiltration method

Sarotherodon niloticus with average weight of 28.42 +/- 1.87 g were immunized with formalin-killed Edwardsiella tarda using the hyperosmotic infiltration method. Test fish maintained in 30 l aquaria were grouped into four treatments. Group 1 and 2 were exposed to a single hyperosmotic treatment on day 0. Group 1 was bled on day 14 and group 2 was bled on day 28. Group 3 was given hyperosmotic treatments twice: on day 0 and day 14 and bled on day 28. Group 4 was an untreated control bled on day 28. All sera were analyzed for agglutinating antibody titer against E. tarda flagellar and somatic antigens. Results showed that flagellar and somatic agglutinin titers in all treatments were not statistically significant. Likewise, infection experiments where test fish were challenged with intraperitoneal injection of the test bacterium showed that the vaccination experiment did not effectively protect the test fish from infection by Edwardsiella tarda.     

鱼类迟钝爱德华菌病诊断与防治研究进展

迟钝爱德华菌(Edwardsiella tarda)是目前水产养殖中危害极大的病原菌,它可引起鱼类产生迟钝爱德华菌病,使鱼类腹部积水肿胀、体表出血、肠内出现黏液,造成鱼类的大量死亡,严重危害了鱼类的养殖,带来了巨大的经济损失.迟钝爱德华菌能够侵染鱼类宿主细胞,抵抗宿主免疫机制,并能分泌毒素使正常细胞发生病变.防治鱼类爱德华菌病的方法主要为化学治疗法、疫苗防治法和微生态制剂防治法.论文对国内外有关迟钝爱德华菌病的发病情况、致病性研究、诊断方法及防治等诸方面的研究概况进行了系统的综述.     

Health status of salmonids in river systems in Natal. III. Isolation and identification of bacteria

Both pathogenic and non-pathogenic bacteria were isolated from fish, both salmonid and non-salmonid, from selected river systems in Natal. Pasteurella pisicida was isolated for the first time from fish in South Africa. The isolation of Yersinia ruckeri, Aeromonas salmonicida, and Edwardsiella tarda were recorded for the first time from fish in Natal. A. hydrophila and Flexibacter columnaris were found to be widespread throughout the river systems in Natal. The Streptococcus species which caused serious disease problems in trout in the Cape Province and Transvaal was not isolated from any of the fish examined in Natal.     

大菱鲆爱德华氏菌病:病例报告

对不同养殖场所发生的3起大菱鲆（Turbot，Scophthalmus maximus L．）暴发病害进行了调查，发病情况、临床表现、病理变化提示为败血感染症。经细菌学检验确定系迟钝爱德华氏菌（Edwardsiella tarda）所引起的爱德华氏菌病（Edwardsiellasis）。通过对分离后纯培养的18株菌的生物学性状测定，确认均为典型的迟钝爱德华氏菌野生型（E．tarda wild type）菌株；血清型检定表明均为同一血清型。人工感染试验更确证了其相应的病原学意义及较强的致病作用。药敏试验结果表明，对头孢唑啉等28种抗菌药敏感、对苯唑青霉素等9种抗菌药耐受。     

迟缓爱德华菌FliC-TolC融合蛋白表达及免疫特性研究

鞭毛蛋白FliC与外膜蛋白TolC均具有免疫保护效果,其中FliC蛋白具有疫苗佐剂的特性,但目前尚未有关迟缓爱德华菌(E.tarda)FliC-TolC融合产物免疫动物的免疫效果的相关报道。为研究E.tarda FliC-TolC融合蛋白的免疫特性,本研究利用融合PCR方法扩增获得fliC-tolC片段,构建重组载体pET-28a-fliC-tolC,并利用E.coli BL21表达系统表达了融合蛋白FliC-TolC。将纯化的FliC-TolC、TolC、FliC蛋白免疫小鼠后,以E.tarda强毒株攻毒评估免疫效果;并利用斑马鱼模型进行了平行试验。结果显示,本研究克隆了E.tarda fliC-tolC基因并表达和纯化了相应重组蛋白FliC-TolC;FliC-TolC蛋白激发小鼠产生的抗体水平优于FliC、TolC蛋白单独免疫组,表明FliC-TolC蛋白具有优良的免疫原性。攻毒试验表明FliC-TolC蛋白组小鼠对强毒株可产生较好的抵抗力,相对保护率为95%;斑马鱼攻毒试验相对保护率为60%。本研究证实了E.tarda重组蛋白FliC-TolC的免疫效力,为进一步研制E.tarda亚单位疫苗提供了参考和借鉴。     

迟缓爱德华菌人工感染斑马鱼的致病性及组织病理学变化

用迟缓爱德华菌真鲷分离株(E.t-CD)对斑马鱼进行人工感染试验,观察其发病、死亡以及相关病理学变化。结果表明,该菌对斑马鱼的半数致死浓度(LD50)为2.69×102 cfu/尾;感染发病鱼呈现岀血、溃疡、腹水、败血等症状;病理组织学检查以肝脏水肿变性,肝细胞萎缩、坏死、脱落;脾脏散在增生性结节、充血、水肿、淋巴细胞大量缺失等病变为主。结果表明,斑马鱼可作为研究迟缓爱德华菌致病性的动物模型。     

Molecular identification of bacteria associated with canine periodontal disease

Hsp90 is a molecular chaperone that is involved in diverse cellular processes including protein folding/repairing and signal transduction. Edwardsiella tarda is a serious fish pathogen that affects fish aquaculture worldwide. The aim of this study was to investigate the potential importance of HtpG, the prokaryotic homologue of Hsp90, in the pathogenesis of E. tarda. E. tarda HtpG is 627-residue in length and contains domain structures that are conserved among Hsp90 family members. Quantitative real time RT-PCR analysis indicated that expression of htpG is induced by heat shock and oxidative stress. Recombinant HtpG (rHtpG) purified from Escherichia coli exhibits apparent ATPase activity, which is optimal at 40°C. Mutation of htpG (i) affects bacterial growth at elevated temperature and renders the cells more sensitive to stress induced by reactive oxygen species, (ii) causes dramatic reduction in blood dissemination and general bacterial virulence, (iii) weakens the ability of E. tarda to block head kidney macrophage activation and to resist against the bactericidal effect of macrophages, and (iv) upregulates the expression of pro-inflammatory cytokines in macrophages. Taken together, these results indicate that HtpG is a biologically active protein that is required for E. tarda to cope with various stress conditions especially that encountered in vivo the host system during infection.     

Immunomodulating potency of lipopolysaccharides (LPS) derived from smooth type of bacterial pathogens in Indian major carp

Lipopolysaccharides (LPS), the outer membrane of the Gram-negative bacteria, are reported to stimulate the immunity of different vertebrates including fish. However, their potency and spectrum of actions often differ among different bacteria. In this study, effect of crude LPS, derived from three species of smooth Gram-negative bacterial fish pathogens viz. Edwardsiella tarda, Escherichia coli, and Pseudomonas fluorescens, on certain innate immune parameters of Indian major carp, Labeo rohita was studied. L. rohita yearlings, when injected intraperitoneally with crude LPS extracted from these bacteria showed little variations in different innate immune parameters. Furthermore, LPS injected fish were protected against a virulent E. tarda challenge. Although, no significant difference (p>0.05) in most of the immune parameters were found with LPS of different bacteria, the E. coli LPS injected fish elucidated high resistivity during challenge study. Hence, there could be some variations in LPS with respect to the bacterial type which needs to be further explored.     

Cardiac tamponade in largemouth bass (Micropterus salmoides)

A public aquarium with a 4-mo history of occasional fish mortalities submitted for necropsy an adult female largemouth bass (Micropterus salmoides) that died unexpectedly. Gross necropsy revealed that the pericardial cavity was markedly distended with partially coagulated blood. Examination of the heart revealed multiple nodular masses in the area of the atrium and two small perforations on the surface of one of the nodular masses. Histopathologic exam of the atrium revealed severe fibrinonecrotic endocarditis and transmural myocarditis with intralesional bacteria. A pure culture of Edwardsiella tarda was obtained from culture of posterior kidney and spleen. An area of stagnant water that may serve as the source of E. tarda was identified, and steps to rectify this problem were taken. Low-level supersaturation was also a significant stressor; the source of the supersaturation was not identified. To our knowledge, this is the first report of cardiac tamponade in a largemouth bass.     

Phenotypic and genetic characterization of Edwardsiella tarda isolated from pond sediments

The extent of genotypic and phenotypic diversity of Edwardsiella tarda isolated from pond sediment was assessed by SDS-PAGE, Plasmid Profiling and ERIC-PCR. SDS-PAGE of whole cell protein extracts reveals 20-23 discrete bands with molecular wt of 14-110 kDa. Several bands with molecular weight range of 38-83 kDa were present in all the isolates. Numerical analysis of protein electrophoregram delineated the isolates into four clusters. Two different types of plasmids having molecular mass of 23 kDa and 29 kDa were obtained by plasmid profiling. About 51% of the isolates carried both the plasmids. ERIC-PCR generates 3-7 bands with molecular mass of 14-1013 bp. Numerical analysis differentiated the ERIC pattern into 5 clusters at 60% similarly level. It was concluded that out of three methods ERIC-PCR was found to be more sensitive for intraspecific typing of E. tarda and can be used as a potential tool for epidemiological studies in the future.     

Intestinal bacterial flora of the household lizard, Gecko gecko

A total of 114 isolates was recovered from the intestines of 43 househould lizards, Gecko gecko. Among the important ones were Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, Proteus mirabilis and Edwardsiella tarda.     

鳗鲡爱德华菌病的诊断和病原学研究

采用病理学技术、细菌培养技术、生化试验和药敏试验对珠海市某养殖场鳗鲡暴发病的病理特点和病原菌的特征进行了研究。在分离到的６株细菌中,４株为迟钝爱德华菌,另２株为继发感染的运动性气单胞菌。综合分析判断,该场鳗鲡暴发病为鳗鲡爱德华菌病。     

Outer membrane protein profiles of Edwardsiella ictaluri from fish.

Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)     

Septicaemia caused by Edwardsiella tarda and Plesiomonas shigelloides in captive penguin chicks

Three cases of fatal septicaemia due to Plesiomonas shigelloides and one due to Edwardsiella tarda were diagnosed in newborn penguins from the Basle Zoo, Switzerland from 2003 to 2007. The affected penguins were of two different species (king penguin, Aptenodytes patagonicus, and African penguin, Spheniscus demersus) and between 2 and 10 days old at the time of death. The causative agents, E. tarda and P. shigelloides are ubiquitous bacteria which are reported to be present in the normal intestinal flora of wild and captive aquatic animals, including penguins. Their occurrence and infectious potential is discussed.