柱状黄杆菌强毒株和弱毒株胞外蛋白中差异蛋白的初步鉴定

柱状黄杆菌(Flavobacterium columnare)是一种世界范围的水产动物致病菌,是中国重要养殖鱼类草鱼(Ctenopharyngodon idellus)、鳜(Siniperca chuatsi)等烂鳃病的病原。本研究以1972年从患"烂鳃病"草鱼上分离的两株冻干柱状黄杆菌G4和G18菌株为研究对象,并将G4株再次分离纯化得纯化菌株,命名为G4R3。对草鱼鱼苗浸泡攻毒结果显示,G4R3的LD50至少比G18的高3个数量级,因此G4R3为"强毒株",G18为"弱毒株"。利用蛋白质组学方法分析柱状黄杆菌强毒株G4R3和弱毒株G18的胞外蛋白,经过双向电泳并结合图像分析,共发现了34个点是差异表达的蛋白。胶内酶解、肽质量指纹图谱和串联质谱分析后,鉴定出其中的7个蛋白点,代表滑动蛋白K、腺酐甲硫氨酸合成酶和一种可能的膜蛋白等3种蛋白,它们可能是柱状黄杆菌的毒力因子。     

Cell membrane glycoconjugates on virulent and avirulent strains of the haemoflagellate Cryptobia salmositica (Kinetoplastida)

The glycoconjugates on the cell membranes of Cryptobia salmositica (virulent and avirulent strains) were analysed using 13 highly purified lectins. The virulent strain of C. salmositica was agglutinated by two of these lectins (Concanavalin A (Con A) Canavalia ensiformis , specific for α-mannose and α- D -glucosyl residues; Pisum sativau agglutinin (PSA), specific for N-acetyl α- D -galactosaminyl) but the avirulent strain of C. salmositica was agglutinated by 11 lectins. No agglutination of C. salmositica (virulent and avirulent strains) was observed with lectin Tetragonolobus purpureas agglutinin (TPA, specific for α- L -fucose). Glycoprotein analysis with digoxigenin or biotin labelled lectins showed strain specific staining patterns for C. salmositica; with the avirulent strain showing stronger reactions than the virulent strain. These results indicate that the surface carbohydrate residues changed with attenuation of the pathogen and this change may be related to its loss of virulence.     

Adherence, haemagglutination and cell surface characteristics of motile aeromonads virulent for fish

Abstract. Motile Aeromonas spp. virulent for fish were studied with regard to their adhesion profile. Electron microscopy demonstrated the presence of fimbriae (pili) on Aeromonas cells regardless of virulence potential. The results show no significant correlations between ability to haemagglutinate, yeast cell co-agglutination and virulence. All strains expressed mannose-sensitive haemagglutinin activity against guinea pig erythrocytes. Using four types of red blood cells no characteristic haemagglutinin pattern related to virulence could be discerned. Expression of surface haemagglutin(s) on Aeromonas hydrophila appears to be medium dependent; strains grown in liquid media demonstrated enhanced haemagglutination activity. Both virulent and avirulent strains had in vitro epithelial cell adhesive capabilities. Cell surface characteristics measured by agglutination in acriflavine and stability after boiling indicated that most virulent strains agglutinated in the presence of acriflavine, but not all sedimented after boiling. The ability of 10 selected strains of A hydrophila to grow in normal pooled catfish serum was determined. Only 17% of the virulence variations can be explained by their sensitivity to serum.     

Occurrence and characterization of Aerococcus viridans from lobsters, Homarus gammarus L., dying in captivity

Abstract Dead lobsters from five lobster parks have been examined over a period of 5 months for the occurrence of Aerococcus viridans. Isolated strains of the bacterium were characterized biochemically and by means of intracellular protein patterns using thin layer isoelectric focusing in polyacrylamide gel. In a lobster park where a high mortality occurred, A. viridans was isolated from 29 of 33 dead lobsters. The bacterium was also isolated from 7 of 10 dead lobsters in a lobster park with low mortality. Probably, infection with A. viridans occurs more frequently in Norwegian lobster parks than hitherto suspected. Gram-positive cocci sharing many characteristics with A. viridans were isolated from some dead lobsters. These bacteria did not form tetrads, they were catalase positive and had different protein patterns from A. viridans.     

Interaction of the fish pathogen Aeromonas hydrophila with tilapia, Oreochromis aureus (Steindachner), phagocytes

Abstract. Interaction of Aeromonas hydrophila and tilapia, Oreochromis aureus (Steindachner), phagocytes was studied in vitro. All virulent and avirulent strains of A. hydrophila tested could multiply in non-activated and Freund's complete adjuvant activated phagocytes. Activated phagocytes increased the uptake of bacteria into cells, and the rates of intracellular replication for these bacteria were faster than in non-activated phagocytes. Among the A. hydrophila strains examined, virulent strain PPD134/91 replicated at the fastest rate inside phagocytic cells and produced cytopathic effect in the phagocytes in the shortest incubation time. Opsonized avirulent A. hydrophila were sensitive to phagocyte-mediated killing or unable to grow in phagocytes. Serum components and phagocytes may together prevent the growth of avirulent A. hydrophila in fish. The release of extracellular oxygen radicals during phagocytosis was examined using chemiluminescence assay (CL). Virulent strains induced CL responses but avirulent strains did not. This suggests that the virulent strains interacted with the phagocytes somewhat differently from the avirulent strains.     

Comparative analysis of the phenotypic characteristics of high- and low-virulent strains of Edwardsiella tarda

Edwardsiella tarda is a causative agent of edwardsiellosis in freshwater and marine fish. Extracellular enzymic, haemolytic, hydrophobic and serum resistance activities, haemagglutination, autoagglutination and siderophores of high‐ and low‐ virulent E. tarda strains were examined. The results revealed different haemagglutination, autoagglutination, haemolytic, hydrophobic and serum resistance activities in different strains. Analysis of extracellular proteins (ECPs) and outer membrane proteins (OMPs) demonstrated several major, low molecular weight, virulent‐strain‐specific proteins, which could be virulence‐related. Based on the database search with MALDI‐TOF MS data, the closest homologies of the three protein bands Ed1, Ed2 and Ed3 were phosphotransferase enzyme family protein, nitrite reductase [NAD(P)H], large subunit and ATP‐dependent Lon protease, respectively. A comparison of pathogenicity of purified lipopolysaccharide (LPS) and lipid A from virulent and avirulent strains demonstrated that LPS was one of the virulence factors of the E. tarda isolates, and lipid A was a biologically active determinant of LPS.     

Comparative in vitro studies on virulent and avirulent strains of Cryptobia salmositica (Sarcomastigophora: Kinetoplastida)

Abstract. The optimum temperature for in vitro multiplication of Cryptobia salmositica was 10°C. The avirulent strain multiplied more rapidly than the virulent strain. The haemolytic components, lytic component (LC) and immune complex-forming component (ICC), were secreted by the two strains into the culture medium and were detectable from one week post-inoculation. The haemolytic activity in the supernatant increased with increasing parasite numbers in both strains. Although cultures of the avirulent strain had higher parasite numbers than those of the virulent strain, the haemolytic activity was significantly lower than that of the virulent strain. Antiserum against ICC was produced in rabbit by immunization with ICC-coated rainbow trout red blood cells.     

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non‐virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE‐214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non‐virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin‐D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non‐virulent strains were generally serum sensitive.     

Aeromonas hydrophila B-haemolysin: purification and examination of its role in virulence in 0-group channel catfish, Ictalurus punctatus (Rafinesque)

Abstract. A single step procedure for purification of B-haemolysin from Aeromonas hydrophila is described which recovered 94% of the haemolytic activity with a sixfold increase in specific activity. Toxicity of haemolysin in 0-group channel catfish was 0–6 mg of purified protein per gram offish. Of six strains of A. hydrophila evaluated for differential toxin production and virulence in 0-group channel catfish, three B-haemolysin-deficient strains were virulent while three B-haemolysin-producing strains were avirulent.     

鱼源气单胞菌的毒力基因检测、分型及致病力

为调查鱼源气单胞菌毒力基因与其致病力的相关性,以2009--2018年从不同患病鱼分离的173株气单胞菌为研究对象,通过检测毒力相关基因、测定溶血活性、腹腔注射感染异育银鲫等方法开展评价.通过管家基因gyrB分子鉴定结果显示,173株气单胞菌中维氏气单胞菌(119/173,68.9％)和嗜水气单胞菌(50/173,28...     

Virulence of Flavobacterium branchiophilum in experimentally infected salmonids

Abstract. The ability of selected strains of Flavobacterium branchiophilum to attach to and colonize the gills of five species of teleosts (four salmonid and one cyprinid) and cause mortality was investigated. In virulence studies with F. branchiophilum strain LAB4a, cumulative mortality was dose-dependent in rainbow trout, Oncorhynchus mykiss (Walbaum), and ranged from 0 to 75%. However, regardless of dose, the relative amount of gill-associated F. branchiophilum antigen increased 1 h after challenge. The gill-associated F. branchiophilum antigen in fish which survived (moribund fish) increased by four to six times compared to that detected on the gills 1 h after challenge. The gill-associated antigen on moribund fish was not significantly different from that on the gills of dead fish. Flavobacterium branchiophilum strain LAB4a also attached to the gills of brook trout, Salvelinus fontinalis (Mitchell), rainbow trout, chinook salmon, Oncorhynchus tshawytscha (Walbaum), Arctic charr, Salvelinus alpinus (L.), and common shiners, Luxilus cornutus (Mitchell), following a 1-h bath exposure, and caused mortality in all species. The virulence of eight strains of F. branchiophilum for rainbow trout was examined. Both virulent and avirulent strains adhered to the gills following bath exposure (fimbriae were observed on all strains), but only virulent strains had the capacity to further colonize the gills and cause mortality.     

Development and characterization of rifampicin-resistant mutants from high virulent strains of Flavobacterium columnare

Flavobacterium columnare is divided into three genetic groups or genomovars, genomovar II being highly virulent for channel catfish. A modified live vaccine is currently available to prevent columnaris disease under the licensed name Aquavac‐Col^®. The strain of F. columnare used to generate the avirulent rifampicin‐resistant mutant used in Aquavac‐Col^® belonged to genomovar I, the less virulent group towards channel catfish. In this study, we describe the generation and characterization of rifampicin‐resistant mutants from genomovar II strains. A total of 13 new mutants were obtained, and eight of them (two from each parent strain) were genetically and phenotypically characterized. Highly conserved regions within the ribosomal operons were identical between parent and mutant strains. Genetic differences between mutants and their parent strains were revealed by amplified fragment length polymorphism (AFLP). Genetic changes were distinctive among different mutants. Analysis of the lipopolysaccharide (LPS) showed that while some mutants lacked a few molecular bands of the LPS, some exhibited the same LPS profiles as their parent strains. Comparison between immunogenic proteins from mutants and parents was carried out by immunoblot analysis and further confirmed the uniqueness of individual mutants. A complete set of rifampicin‐resistant mutants with different genetic and immunogenic properties from the highly virulent genomovar II has been created. These mutants may have the potential of becoming vaccine candidates against columnaris disease.     

Comparative genome sequencing to assess the genetic diversity and virulence attributes of 15 Vibrio anguillarum isolates

Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. However, the diversity and virulence mechanisms of this pathogen are still insufficiently known. In this study, we aimed to increase our understanding of V. anguillarum diversity and virulence through comparative genome analysis of 15 V. anguillarum strains, obtained from different hosts or non‐host niches and geographical regions, among which 10 and 5 strains were found to be virulent and avirulent, respectively, against sea bass larvae. First, the 15 draft genomes were annotated and screened for putative virulence factors, including genes encoding iron uptake systems, transport systems and non‐ribosomal peptide synthetases. Second, comparative genome analysis was performed, focusing on single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). Five V. anguillarum strains showed a remarkably high nucleotide identity. However, these strains comprise both virulent and avirulent strains towards sea bass larvae, suggesting that differences in virulence may be caused by subtle nucleotide variations. Clearly, the draft genome sequence of these 15 strains represents a starting point for further genetic research of this economically important fish pathogen.     

Immunogenicity of vaccines from a virulent and an a virulent strain of Aeromonas salmonicida

Abstract. Injected vaccines consisting of formalin-killed cells and extracellular antigens prepared from a virulent and an avirulent strain of Aeromonas salmonicida were tested for their efficacy in protecting juvenile coho salmon. Oncorhychus kisutch (Walbaum). Against experimental furunculosis following active and passive immunization. Sera used in the passive immunization experiments were raised in subadult coho salmon and in rabbits. Results indicated that the avirulent strain was inferior to the virulent strain in its immunogenicity for coho salmon. Thus, even though avirulent cells possessed at least one immunogen–an immunogen that was show in passive immunization experiments to be well recognized by rabbits–the immunogen was only inefficiently protection when vaccinated with avirulent cells. Furher, extracellular antigens of the avirulent strain were not protective for coho salmon even though they elicited the production of anti- A. salmonicida agglutinins. In contrast, the killed cells and extracellular antigens of the virulent strain were both immunogenic in coho salmon. By passively immunizing coho salmon with rabbit sera raised against the virulent and the virulent strain produced at least two immunogens, only one of which was shared in common with the avirulent strain. The extra immunogen possessed by the virulent strain is presumably responsible for its superior immunogenicity in coho salmon; the immunogen had the properties of a protein and is believed to be the A-protein.     

Pathogenicity of Edwardsiella tarda to olive flounder, Paralichthys olivaceus (Temminck & Schlegel)

The LD50 and cytotoxic and enzymatic activities of both cells and extracellular products (ECPs) of eight Edwardsiella tarda strains were determined and their bacterial superoxide dismutase gene (sodB) and catalase gene (katB) were sequenced. Strains were also examined for their ability to resist the immune responses of olive flounder, Paralichthys olivaceus. LD50 values of strains (FSW910410, KE1, 2, 3, 4, 5 and 6) in olive flounder ranged between 10(2.5) and 10(5.3) cfu (colony forming units) per fish. Unlike the avirulent strain SU100 (LD50>or=10(7)), all pathogenic strains were able to survive in flounder serum and head kidney leucocytes (except for KE2). The virulent strains possessed type I sodB and katB, whereas SU100 had type II sodB but not katB. However, there was no difference between avirulent and virulent strains in haemolytic and cytotoxic activities. The results of this study demonstrated that the ability of E. tarda to resist complement activity and phagocytosis is conferred by its superoxide dismutase and catalase, which thus play an essential role in the pathogenicity of this bacterium. In addition genotyping of sodB and kat B proved to be a very useful tool to distinguish virulent from avirulent strains.     

Characterization and growth conditions of Vibrio parahaemolyticus strains with different virulence degrees that cause acute hepatopancreatic necrosis disease in Litopenaeus vannamei

The phenotypic characteristics and growth kinetics at several temperatures, salinities, and pH values of three Vibrio parahaemolyticus (Vp) strains with different virulence and one nonpathogenic strain were evaluated. Independent of the virulence of the strain, a high metabolic diversity was found, which yielded different colored phenotypes on the CHROMagar? Vibrio. All strains were resistant to ampicillin and carbenicillin, and Vp AHPND+ organisms were the most sensitive to enrofloxacin. The exponential growth of Vp strains started at 1–2 hr of incubation, although no relationship was observed between the bacterial density and degree of virulence. Moreover, the growth of the most virulent strain was independent of the nutrients in the incubation media during the initial hour postinoculation. No strain grew at 4°C in 0% NaCl and pH 4, but only Vp AHPND+ grew at 44°C. For all strains, the lag phase was proportional to the NaCl concentration, and the growth was better at pH 8–9. However, the Vp AHPND? strain displayed a greater variability, was more sensitive to extreme conditions, and showed a lag phase of 9 hr independent of the pH.     

cel-EIIB蛋白调控罗非鱼无乳链球菌强毒、弱毒株毒力相关基因的表达模式

罗非鱼无乳链球菌纤维二糖-磷酸转移酶系统（cel-PTS）的EIIB蛋白对强毒株毒力影响有限，但对弱毒株毒力似乎存在潜在的影响，但具体机制仍不清晰，有必要弄清该蛋白调控强、弱毒株的毒力相关基因表达模式。在前期研究中，通过同源重组技术，构建了无乳链球菌强毒株cel-EIIB基因缺失株，本研究通过类似的方法，获得弱毒株该基因的缺失株。用无乳链球菌强毒、弱毒株及它们的cel-EIIB缺失株分别感染斑马鱼，结果显示，cel-EIIB缺失后，导致弱毒株毒力明显返强，而强毒株毒力则轻微减弱。qPCR检测发现，cel-EIIB缺失可致cel-PTS系统的cel-EIIA、双组分信号转导系统（TCS）的DltR和CiaH以及毒力基因sodA、cpsD和cpsG在强、弱毒株中呈现相反的表达模式；此外，TCS系统的RgfC、DltS和CsrR以及毒力基因cspA和pavA在强毒突变株中表达未受影响，但在弱毒突变株中的表达却显著上调。研究揭示，EIIB蛋白可能通过调控上述毒力相关基因表达而负调控弱毒菌株的毒力。     

利用mini-Tn10转座子文库筛选鳗弧菌M3表型发生变化的基因

为了寻找影响鳗弧菌(Vibrio anguillarum)表型变化的基因,本研究使用转座子mini-Tn10 (pLOF/Kana)构建了鳗弧菌M3突变株文库,筛选影响表型变化的菌株及相关基因,证明这些表型变化的突变子与毒力存在一定的相关性。对M3突变文库的1152突变子进行筛选,获得泳动能力改变的突变子1个(编号为6G_1),酪蛋白酶活性发生改变的突变子3个(编号为5A_11、7B_12和7E_12),明胶酶活性发生改变的突变子1个(编号为7H_1),以及菌膜形成能力发生显著变化的突变子3个(编号为5E_2、6A_2和6E_12)。对转座子插入位点进一步分析显示,一个磷酸二酯酶相关基因突变引起泳动能力增强(P<0.05),leuD、rseB和thiQ突变引起酪蛋白酶活性显著减弱(P<0.05),potD突变引起明胶蛋白酶活性显著减弱(P<0.05),leuO、ilvH和grpB的突变引起菌膜形成能力明显减弱(P<0.05)。对这些表型变化的突变子进行毒力感染,发现野生型M3是6G_1突变子的半数致死剂量(Lethal dose 50%,LD50)的2.04倍,该突变子毒力相对增强。5A_11、7B_12和7E_12的突变子LD50分别为野生型M3的2.96、3.25和3.36倍。7H_1的LD50是野生型M3的1.25倍,5E_2、6A_2和6E_12的LD50分别为野生型M3的3.34、4.08和1.84倍,这些突变子毒力相对减弱。本研究结果为进一步阐明鳗弧菌的发病机制提供了理论基础。     

Effects of temperature on phagocytosis in Homarus americanus

Phagocytosis constitutes the primary defense mechanism in decapod crustaceans. In this study, monolayers of hemocytes of the American lobster, Homarus americanus, were allowed to phagocytize a strain of Aerococcus viridans. This system was used to investigate the effects of temperature on in vitro phagocytosis. The results indicated a marked decrease in percent phagocytosis at 22°C and above. As this primary response was rendered less efficient at higher temperatures, culture of Homarus americanus at elevated temperatures was cautioned.     

Immunodiagnosis of virulent strains of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) using a monoclonal antibody

Abstract. A virulent strain of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) was used to produce monoclonal antibodies that identified virulent strains of A. hydrophila. Antibodies from a clone, designated as F26P5C8, were found to identify the A. hydrophila serotype I isolates associated with EUS fish, and which were found to be virulent after subsequent inoculation studies. Immunodiagnosis of a large number of A. hydrophila from Australia and Japan showed some additional isolates to be identified by F26P5C8, but the status of their virulence is presently unknown.