迟缓爱德华菌分子伴侣蛋白GroEL原核表达及免疫特性

GroEL是细菌内分子伴侣蛋白,参与细菌多种生命活动,在布鲁菌、链球菌研究中,GroEL蛋白展示了良好的疫苗潜力。前期利用迟缓爱德华菌抗体Pull-down技术筛选到GroEL蛋白,揭示了该蛋白的潜在应用价值,本试验以迟缓爱德华菌GroEL蛋白作为研究对象,利用大肠杆菌表达系统表达纯化GroEL重组蛋白(rGroEL),进一步通过动物模型评估rGroEL的免疫效果。结果显示,rGroEL能够激发小鼠产生高水平的特异性抗体,具有较好的免疫原性,免疫小鼠对迟缓爱德华菌感染具有较好的抵抗力,相对保护率达95%。斑马鱼感染试验结果表明,rGroEL免疫对迟缓爱德华菌感染具有一定保护效果,保护率为60%。用rGroEL免疫斑马鱼可以产生针对嗜水气单胞菌的免疫保护力,保护率可达45%。本试验初步探索了迟缓爱德华菌重组蛋白rGroEL的免疫特性,为研制迟缓爱德华菌亚单位疫苗和核酸疫苗提供了依据。     

迟缓爱德华菌外膜蛋白FadL原核表达及免疫原性

FadL是革兰阴性细菌编码的外膜通道蛋白,参与长链脂肪酸的转运,沙门菌中该蛋白作为免疫原使用具有优良的保护效果。本试验以迟缓爱德华菌FadL蛋白作为研究对象,原核表达并纯化该蛋白,进一步通过动物试验确定该蛋白的免疫特性和免疫效果。结果显示,成功表达和纯化重组FadL蛋白(rFadL),蛋白大小为50 000。小鼠试验结果表明,rFadL免疫小鼠能产生高水平特异性抗体;斑马鱼感染试验结果表明,rFadL能够对斑马鱼迟缓爱德华菌产生一定免疫保护力,保护率可达55%。本试验揭示了迟缓爱德华菌FadL蛋白的免疫特性,为迟缓爱德华菌疫苗研发提供了参考和借鉴。     

迟缓爱德华菌延伸因子EF-Tu的免疫特性研究

EF-Tu是细菌的延伸因子,参与细菌众多生理进程。前期在布鲁氏菌、鼠伤寒沙门氏菌的研究中,EFTu蛋白的免疫原性已被证实,但在迟缓爱德华菌上关于EF-Tu蛋白的研究还未见报道。为研究迟缓爱德华菌延伸因子EF-Tu的免疫特性,本试验利用原核表达系统表达迟缓爱德华菌EF-Tu蛋白,并利用小鼠模型和斑马鱼模型分别评估该蛋白的免疫原性和免疫保护效果。结果显示,重组蛋白rEF-Tu免疫可以使斑马鱼产生针对迟缓爱德华菌的免疫保护力,保护率为50%。本次研究明确了迟缓爱德华菌EF-Tu蛋白的免疫特性,为迟缓爱德华菌疫苗研发提供了借鉴和参考。     

迟缓爱德华菌鞭毛蛋白FliC原核表达及免疫试验

为了解迟缓爱德华菌鞭毛蛋白FliC的免疫特性,从迟缓爱德华菌基因组中克隆出鞭毛基因fliC,并将其构建到原核表达载体上,用原核表达的方法获得大量重组蛋白,将蛋白纯化后,通过动物模型确定该蛋白的免疫特性。结果表明,迟缓爱德华菌鞭毛蛋白FliC具有较强的免疫原性,对免疫动物应对强毒株感染具有一定保护效果。将FliC与牛血清白蛋白(BSA)混合免疫小鼠能够刺激机体产生较高水平抗BSA抗体,说明其佐剂特性。研究表明,迟缓爱德华菌鞭毛蛋白FliC具有优良的免疫原性和佐剂特性,是迟缓爱德华菌的保护性抗原,具有潜在的应用价值。     

养殖鳜鱼出血病病原的分离与鉴定

2021年上海市一家鳜鱼养殖场暴发出血病，初始典型症状为下颌出血。本研究通过细菌分离、分子鉴定、生化反应以及健康鳜鱼感染试验等，确认鳜鱼发病死亡由维氏气单胞菌(Aeromonas veronii)和迟缓爱德华氏菌(Edwardsiella tarda)共同感染所致。分离的维氏气单胞菌鸟氨酸脱羧酶(ODC)和赖氨酸脱羧酶(LDC)反应均为阴性，表明其既不属于维氏气单胞菌维氏生物群(A. veronii biogroup veronii)，也不属于维氏气单胞菌温和生物群(A. veronii biogroup sobria)，但在遗传上与温和生物群更近；迟缓爱德华氏菌生化反应不产生H₂S。分离菌腹腔注射感染健康鳜鱼幼鱼后，被感染幼鱼均出现了死亡现象，且相同数量级病原回感鳜鱼，维氏气单胞菌感染后，鳜鱼发病更快，死亡率更高，感染量为1×10⁷ cfu/尾时即可导致鳜鱼幼鱼在24 h内100%死亡。两种病菌都对硫酸新霉素和红霉素耐药，而对氟苯尼考都高度敏感。本研究为养殖鳜鱼出血病的治疗和控制提供了参考。     

迟缓爱德华菌menF基因缺失株的构建及生物学特性研究

为研究异分支酸合成酶MenF在迟缓爱德华菌(E.tarda)致病中的作用,本研究利用自杀载体构建了E.tarda menF基因缺失菌株(ET-CLΔmenF),并分析其生物学特性。结果显示,ET-CLΔmenF株比野生菌株生长减缓。体外应激试验结果表明,ET-CLΔmenF菌株对铁饥饿的耐受能力明显下降,应对酸性和过氧化氢处理的能力显著下降。细胞感染实验表明ET-CLΔmenF菌株的胞内存活能力显著下降(p0.01)。对斑马鱼的致病性结果表明,ET-CLΔmenF株毒力下降。本研究为E.tarda致病机制研究以及疫苗的研制提供了参考。     

饲料维生素C含量对半滑舌鳎抵抗迟缓爱德华氏菌感染的影响

本试验旨在研究维生素C对半滑舌鳎在感染迟缓爱德华氏菌后红细胞抗氧化能力及葡萄糖-6-磷酸脱氢酶(G-6-PD)活性的影响。在基础饲料中添加L-抗坏血酸-2-磷酸酯(作为维生素C添加剂),制成维生素C含量分别为0.2(对照)、104.8、209.6、313.8、521.6、837.3 mg/kg的6种试验饲料。将健康的半滑舌鳎[平均体重(99.12±1.58)g]分别喂食上述6种饲料84 d后,感染迟缓爱德华氏菌,试验期为14 d。结果显示:试验鱼的终末体重、特定生长率、增重率均以209.6 mg/kg组最高。随着饲料中维生素C含量的升高,半滑舌鳎血浆维生素C含量及红细胞血红蛋白(Hb)含量和G-6-PD活性均呈上升趋势,且除104.8 mg/kg组红细胞G-6-PD活性外各添加组均显著高于对照组(P0.05)。饲料维生素C含量为209.6 mg/kg时,红细胞超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性显著高于对照组(P0.05),而后随着维生素C含量继续升高,红细胞SOD和CAT活性呈下降趋势。对照组红细胞丙二醛(MDA)含量和红细胞膜一氧化氮(NO)含量显著高于各添加组(P0.05)。以上结果表明,维生素C能有效降低感染迟缓爱德华氏菌半滑舌鳎的红细胞氧化损伤程度;经过折线模型分析,推荐饲料中维生素C含量为257.04~341.36 mg/kg。     

新肝宝对鲶爱德华氏菌感染黄颡鱼的保护作用研究

为了考察新肝宝对鲶爱德华氏菌感染黄颡鱼的保护作用,采用不同粉碎粒度的新肝宝拌饲投喂7 d,随后用鲶爱德华氏菌腹腔注射感染,观察死亡率、酶学指标及组织病理变化。结果显示,新肝宝组死亡率(48.57%和44.28%)低于阴性对照组(54.29%),溶菌酶及超氧化物歧化酶活性显著高于对照组(P<0.05),肝脏、肾脏、脾脏损伤情况均小于阴性对照组,其中600目作用效果优于200目。结果表明,新肝宝能够提高黄颡鱼非特异性免疫力,增强黄颡鱼对鲶爱德华氏菌的抵抗能力,在一定程度上减轻鲶爱德华氏菌感染导致的肝、脾、肾损伤,其中600目作用效果优于200目。     

迟缓爱德华菌鞭毛蛋白FlgJ原核表达及免疫原性

为研究迟缓爱德华菌鞭毛蛋白FlgJ的免疫保护性,本研究利用PCR方法扩增迟缓爱德华菌flgJ基因,构建重组载体pET-32a-flgJ,将其转化大肠杆菌BL21后进行诱导表达,表达产物经SDS-PAGE和Western blot分析显示,重组蛋白大小约54 000;将纯化的重组蛋白免疫小鼠后,以迟缓爱德华菌分离株ET-13进行攻毒,结果显示该重组蛋白对免疫组小鼠具有保护力,保护率为70%。本研究克隆了迟缓爱德华菌外膜蛋白flgJ基因并表达了相应重组蛋白,免疫小鼠后能够提供一定保护,为重组FlgJ蛋白亚单位疫苗的研制奠定基础。     

迟缓爱德华菌外膜蛋白OmpA原核表达及其免疫原性研究

为研究迟缓爱德华菌外膜蛋白OmpA免疫保护性,本研究利用PCR方法扩增迟缓爱德华菌ompA基因,构建重组载体pET-32a-ompA,将其转化大肠杆菌BL21后诱导表达,表达产物经SDS-PAGE和western blot分析显示,重组蛋白大小约58 ku;将纯化的重组蛋白免疫小鼠后,以迟缓爱德华菌强毒株ET-13攻毒,结果显示该重组蛋白对免疫组小鼠具有保护力,保护率为55%。本研究克隆了迟缓爱德华菌ompA基因并表达了相应重组蛋白,免疫小鼠后能够提供一定保护,为重组OmpA蛋白亚单位疫苗的研制奠定基础。     

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。     

Multi-locus Sequence Analysis (MLSA) of Edwardsiella tarda isolates from fish

Edwardsiella tarda is an enteric fish pathogen that has caused significant economic losses in a range of fish species residing in diverse ecological conditions. Several molecular methods relying on DNA fingerprinting (RAPD, RFLP and ERIC-PCR) and the gyrB gene marker have been used to characterize E. tarda isolates. However, all had drawbacks in resolving power and reproducibility. The present study was aimed at developing a novel Multi-locus Sequence Analysis (MLSA) scheme for genetic characterization of E. tarda isolates originating from multiple sources. MLSA has been described as an effective molecular tool with superior discriminatory power and reproducibility for exploring intra-species genetic diversity of several bacterial species. Nucleotide sequence fragments of eight protein coding housekeeping genes (gyrB, mdh, adk, dnaK, phoR, metG, pyrG and aroE2) were obtained from 23 fish pathogenic E. tarda isolates of different geographical origins, one human isolate and 3 reference strains. The phylogenetic relationships between isolates in individual gene analyses were not consistent, although some common patterns were apparent. Phylogenetic analysis based on concatenated sequences of seven gene loci, however, buffered the conflicting phylogenetic signals and resolved isolates according to their geographical origin and/or fish host. The MLSA revealed two major genetically diverging clusters in E. tarda isolates examined, one cluster representing isolates from fish and the other representing (in the main) human isolates, with E. ictaluri cluster situated in between. The results suggest, therefore, that the fish pathogenic E. tarda isolates may have been previously misclassified and probably represent one or more as yet unrecognized taxa within the genus Edwardsiella. The MLSA described here was robust enough in discriminating E. tarda isolates not only with respect to their geographical origins but also within different hosts from the same geographical location, high-lighting its potential application in tracing the source of infection and understand the epidemiological relationships among isolates of environmental, fish, other domestic animals or human origins.     

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.     

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.     

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.     

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.     

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.     

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.     

Enhanced susceptibility of channel catfish to the bacterium Edwardsiella ictaluri after parasitism by Ichthyophthirius multifiliis

Bacterium Edwardsiella ictaluri and parasite Ichthyophthirius multifiliis (Ich) are two common pathogens of cultured fish. The objective of this study was to evaluate the susceptibility of channel catfish Ictalurus punctatus to E. ictaluri and determine bacterial loads in different fish organs after parasitism by Ich. Fish received the following treatments: (1) infected by I. multifiliis at 5000 theronts/fish and exposed to E. ictaluri; (2) infected by I. multifiliis alone; (3) exposed to E. ictaluri alone; and (4) non-infected control. E. ictaluri in fish organs were quantified by quantitative real-time polymerase chain reaction and reported as genome equivalents per mg of tissue (GEs/mg). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (91.7%) when exposed to E. ictaluri than non-parasitized fish (10%). The bacterial loads in fish infected by 5000 theronts/fish ranged from 6497 to 163,898 GEs/mg which was between 40 and 2000 fold higher than non-parasitized fish (49-141 GEs/mg). Ich infection enhanced the susceptibility of channel catfish to bacterial invasion and increased fish mortality.     

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.