筛选用转座子Tn916诱变的具有_省略_疫原性的嗜水气单胞菌蛋白酶缺失株

以携带质粒pAM120(Apr、Tcr/Tn916)的大肠杆菌CG120株为供体菌，与受体菌嗜水气单胞菌J-1株，采用滤膜接合法进行接合转移，在选择平板(含Tc、cfz和脱脂奶)上进行筛选，筛选出6株蛋白酶缺失株(ECPase-)，ECPase-菌株其生长速度，对正常鲫血清的抗性降低，用脱脂奶平板法及底物法检测，蛋白酶产量明显降低。与亲本J-1株相比，突变株致病力降低，对鲫的半数致死量大于108(J-1株对鲫的半数致死量为5×103)。用ECPase-突变株MJ-1株的18h培养物(5×106CFU)接种健康鲫，不产生亲本J-1株引起的病变及死亡。用MJ-I活菌免疫鲫，在第5周产生较高的凝集抗体，且对强毒株J-1的攻击有保护作用。表明蛋白酶是嗜水气单胞菌的一个重要的毒力因子，ECPase-突变株仍具有免疫原性。     

杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种PCR检测方法的建立

杀鲑气单胞菌(Aeromonas salmonicida)是一种重要的鱼类致病菌,可以感染多种海淡水鱼类。杀鲑气单胞菌包括5个亚种,目前常用的生理生化特征和16S rDNA序列分析方法很难实现亚种的快速精确区分。为实现杀鲑气单胞菌亚种的快速鉴定和检测,针对我国常见的杀鲑气单胞菌杀鲑亚种(A. salmonicida subsp. salmonicida)和杀日本鲑亚种(A. salmonicida subsp. masoucida),本研究开发了其特异性的PCR检测方法。根据Gene Bank已公布的杀鲑气单胞菌基因组信息,选择杀鲑亚种phoB基因和杀日本鲑亚种LOC111476736基因作为目标基因,根据其序列设计特异性引物,进一步对PCR反应的退火温度、引物浓度、dNTPs浓度、Mg2+浓度和酶浓度5个方面进行了优化,并测试了该方法的特异性、敏感性和应用效果。结果显示,2对引物分别可以扩增出杀鲑气单胞菌杀鲑亚种522 bp的phoB特异性基因片段和杀日本鲑亚种515 bp的LOC111476736特异性基因片段。杀鲑亚种特异性引物最适退火温度为64 ℃,10 µmol/L引物、2 mmol/L dNTPs、25 mmol/L MgSO4和1 U/µL KOD酶的最适添加量分别为1.5、2、1.5和0.5 µL。杀日本鲑亚种特异性引物最适退火温度为64 ℃,10 µmol/L引物、2 mmol/L dNTPs、25 mmol/L MgSO4和1 U/µL KOD酶的最适添加量分别为0.75、1、1.5和0.5 µL。以鳗弧菌(Vibrio anguillarum)、美人鱼发光杆菌(Photobacterium damselae)、杀鱼爱德华氏菌(Edwardsiella piscicida)、杀鲑气单胞菌其他亚种等14种其他水产病原菌或常见环境菌为模板进行PCR检测,均无特异性条带。该方法对杀鲑气单胞菌杀鲑亚种的检测灵敏度为12.8 CFU/反应(菌体)或17.6 fg/反应(DNA),对杀鲑气单胞菌杀日本鲑亚种的检测灵敏度为23.8 CFU/反应(菌体)或27.2 fg/反应(DNA)。利用杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种分别对大菱鲆(Scophthalmus maximus)进行人工感染实验,感染后取病鱼组织进行PCR检测,结果显示,本方法可以从感染后的大菱鲆中分别检测到相应病原。综上所述,本研究建立了杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种的特异性PCR检     

The occurrence of a 70-kDa serine protease gene in typical and atypical strains of Aeromonas salmonicida

Abstract. The typical strains of Aeromonas salmonicida examined secreted a PMSF-sensitive proteolytic activity, and with few exceptions, gave a positive colony hybridization with a 70-kDa scrinc protease gene probe. By contrast, atypical strains produced a spectrum of responses with various combinations of (1) secreted/did not secrete PMSF-inhibited protease, (2) colonies hybridized/did not hybridize with the gene probe, and (3) produced protease not inhibited by PMSF. It is suggested that certain strains might be reassigned as typical or atypical on the basis of the presence or absence of the protease gene.     

Extracellular glycerophospholipid:cholesterol acyltransferase from Aeromonas salmonicida: activation by serine protease

Abstract. Extracellular hacmolytic activities of Aeromonas salmonicida ssp. salmonicida to salmon red blood cells were shown to be due to different forms of the membrane-active enzyme glyccrophospholipidrcholcstcrol acyltransferase (GCAT). About 10% of the total haemolytic activity was due to a high molecular mass complex of LPS and GCAT (mol. mass >1000kDa), containing 35–50% neutral sugars and 1.5–2.0% protein. Some haemolytic activity (30–40% of total), corresponding to 50–70kDa by gel filtration, also contained GCAT-activity and may represent aggregated forms of GCAT. However, about 50% or more of the haemolytie activity was due to a protein of 26kDa free GCAT. Rabbit antibodies to GCAT neutralized the hacmolytic activity of both GCAT and GCAT-LPS. A transposon-produccd serinc protease negative mutant of the same A. salmonicida strain showed reduced haemolytic activity. The mutant produced a 38-kDa GCAT proform of low hacmolytic activity. The proform was processed by autogenous scrinc protease to a highly hacmolytic 26-kDa molecule with pl 6.3, similar to GCAT of the parent strain. The weakly haemolytic GCAT-LPS analogue of the mutant strain did not contain detectable amounts of the 26-kDa molecule and was not activated by proteases.     

The specificity of the major (70 kDa) protease secreted by Aeromonas salmonicida

Abstract. The specificity of the major protease secreted by Aeromonas salmonicida has been explored using a number of proteins and p-nitroanilides as substrates. The 70kDa protease was found to hydrolyse two p-nitroanilides which have been reported to be specific substrates for thrombin. Kinetic parameters (k_cat, and K_m) were compared for the 70kDa protease and for thrombin as were the effects of a number of inhibitors. The 70kDa protease is able to degrade proteins which have a relatively open structure, for example, caseins or denatured bovine scrum albumin, to small fragments mostly of M_r<2500. However, proteins with a more compact structure are more resistant to the protease. It was concluded that the 70kDa protease shows some of the specificity features of thrombin, although it is less discriminating in its choice of both low and high M_r substrates than thrombin. In preliminary experiments, the 70 kDa protease was found, like thrombin, to decrease the clotting time of rainbow trout blood. The possible physiological significance of these results is discussed.     

The survival of Aeromonas salmonicida subsp. salmonicida in sea water

Abstract. The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sea water under a variety of conditions. Survival in different types of microcosms (glass or dialysis bags); of bacteria grown under both in vivo and in vitro (broth culture) conditions; and in sterile and non-sterile sea water were compared. In all cases, survival was found to be of short duration (<10 days) and did not conflict with the previously stated obligate nature of the pathogen. The spread of furunculosis may depend less on its ability to survive in the environment than on its rate of shedding from infected fish and prevailing hydrographic conditions. Survival was extended and growth occurred in sterile sea water to which nutrients (tryptone soya broth) had been added. However, sea water obtained from beneath a commercial salmon cage, which would have been expected to be nutrient rich, did not prolong the survival of the pathogen. In vivo infectivity studies provided no evidence for the existence of unculturable but infective forms of A. salmonicida subsp. salmonicida which, therefore, validates the use of colony-forming units as a measure of survival.     

Direct analysis of starved Aeromonas salmonicida

Survival of Aeromonas salmonicida was monitored during prolonged incubation in either distilled water or lake water. Culturability was determined from colony forming units enumerated on tryptone soy agar, whilst flow cytometry was used for direct analysis of viable cells after staining with fluorescent dyes which differentially stained bacteria in relation to defined cellular properties. Over time, populations of culturable cells steadily declined and were not detected after 10 days incubation in distilled water or 33 days in lake water. Flow cytometric analysis revealed that cellular properties related to viability were lost shortly after culturable cells became undetectable in distilled water. In contrast, those incubated in lake water showed little change in these properties over a 57-day experimental period. The implications of these differences are discussed, and it is concluded that A. salmonicida is capable of remaining intact and active upon prolonged incubation in lake water, although this does not conclusively prove viability.     

Resistance of Aeromonas salmonicida to amoxicillin

Abstract. Isolates of Aeromonas salmonicida were obtained from farmed Atlantic salmon, salmo salar L., with amoxicillin minimum inhibitory concentrations (MICs) of 2·5–10 μg ml^-1 Several antibiotic sensitivity patterns were found among the isolates including resistance to the four UK-licensed antibacterial drugs. Combination of clavulanic acid with amoxicillin was not effective, but all isolates were sensitive to florfenicol and to a cephalosporin, the latter having very low MICs.     

The postponement of non-culturability in Aeromonas salmonicida

Aeromonas salmonicida, the causative agent of furunculosis in fish, has been shown to become non-culturable but viable after inoculation in fresh water. The onset of non-culturability is entirely predictable, but can be delayed by inoculation at high concentration or amendment with nutrients. This paper reports that non-culturability can be postponed by the addition of both the amino acids methionine and arginine to microcosms inoculated with A. salmonicida. During these experiments, A. salmonicida decreased in cell size and became rounded. This was regardless of whether it received an amino acid supplement or not. We observed that cells receiving both amino acids remained culturable despite their reduction in cell size to less than 1 μm. Therefore, it would appear that the reduction in size and associated morphological change cannot be taken as an indicator of non-culturability, although it may be a significant step in that direction in some cases.     

Serological comparison of selected isolates of Aeromonas salmonicida ssp. salmonicida

Abstract. Eight isolates of Acronionus salmonicida ssp. salmonicida were collected during furunculosis epizootics in North American Pacific coast states and provinces. Both virulent and avirulent forms of each isolate, confirmed by challenge and electron microscopy, were examined. Serological comparisons by cross-absorption agglutination tests revealed no serological differences between isolates. Using the double diffusion precipitin test, a single band was observed when antigen from a sonicated virulent strain was reacted with antiserum against a sonicated, virulent strain absorbed with homologous, avirulent strain. The presence of the single band was eliminated by excess sonication.     

Efficacy of specific antibody against agglutinating Aeromonas salmonicida strains on infectivity and vaccination with inactivated protease

Efficacy of specific antibody on serum resistance and adhesion was investigated using a pathogenic strain of Aeromonas salmonicida A-7301 (which was autoagglutinative, haemagglutinative and protease production positive), a protease-deficient, non-pathogenic mutant NTG-1 induced from A-7301 (autoagglutinative and haemagglutinative), and a non-pathogenic strain GH-7501 (non-agglutinative, non-haemagglutinative and protease positive). A-7301 could grow and produce protease extracellularly in the presence of rainbow trout anti-A-7301 serum, resulting in a considerable reduction of the antibody titre. NTG-1 similarly grew, but the titre scarcely decreased. GH-7501 could not survive in this medium. A-7301 and NTG-1 possessed a high capacity to adhere to the surface of fish monolayer cell cultures, whereas GH-7501 lacked the capacity. The capacity for adhesion was not inhibited by the antibody. Although live NTG-1 cells were ineffective as a live vaccine, sockeye salmon receiving protease fraction (obtained from extracellular products of A-7301 by DEAE-cellulose column chromatography) inactivated with normal serum, suffered only a low mortality when challenged with A-7301. Thus, although the antibody specific to autoagglutinating cells showed no effects on serum resistance and adhesion, which are involved in the infectivity of this pathogen, the possibility of protease as an effective protective antigen was demonstrated.     

Amoxycillin resistance in Scottish isolates of Aeromonas salmonicida

Abstract. Eighty isolates of Aeromonas salmonicida , recovered from separate outbreaks of furunculosis in farmed and wild salmon in Scotland during 1988 and 1989, were examined for susceptibility to the β-lactam antibiotic amoxycillin. Susceptibility was determined in terms of minimum inhibitory concentration (MIC). All of the A. salmonicida subsp. salmonicida isolates investigated were susceptible to amoxycillin, with MICs of 0.30–1.50mg1^-1. All of the A. salmonicida subsp. achromogenes isolates tested were resistant to amoxycillin, with MICs in excess of 500mgl^-1. The A. salmonicida subsp. achromogenes produced a β-lactamase enzyme with a pI of approximately 8.0. The enzyme was inducible and its production was unaffected by plasmid curing with ethidium bromide, suggesting that resistance was chromosomal rather than plasmid mediated.     

An appraisal of the extracellular toxins of Aeromonas salmonicida ssp. salmonicida

Abstract. Aeromonas salmonicida produces many extracellular enzymes, some of which are known to play an important role in pathogenesis and virulence, while the role of others is presently speculative. The latter group includes amylase, aryl-sulphatase, glucosidases, esterases and lysophospholipase. There are two enzymes which are known to be of prime importance in pathogenesis: a 70-kDa protease (caseinase) and a 25-kDa phospholipase (glycerophospholipid: cholesterol acyltransferase, GCAT). The protease causes extensive tissue liquefaction, activates the blood clotting system and is lethal for fish at 2·4 μg/g fish. It is inhibited by α2-macroglobulin but resistant to all the other serum protease inhibitors. Its role in vivo appears to be as a broad spectrum protease providing amino acids for in vivo growth. The GCAT is mainly present in a high molecular weight complex with LPS. The complex is extremely haemolytic for fish (but not mammalian) erythrocytes. It is the most lethal component of the exotoxins (lethal dose 45 ng/g fish). The complex with LPS confers enhanced toxicity to the GCAT and stability to heat and proteolytic degradation. In vitro , this toxin also has high leucocytolytic and cytolytic (RTG-2) activity. On injection into fish, it causes very little histopathology other than a marked degranulation of eosinophilic granular cells (EGCs) in the gills. Its precise mode of pathogenesis is uncertain and appears complex. The protease and the GCAT/LPS have an additive relationship in respect to lethal doses and mixtures of the two produce extensive liquefactive and haemorrhagic lesions typical of furuncles. The possible relationship of the GCAT/LPS to other less well characterized factors (cytotoxin, leucocytolysin, haemolysin, salmolysin) is discussed.     

Utilization of haem compounds by Aeromonas salmonicida

Abstract. The ability of A-layer-positi ve (A⁺) and A-layer-negative (A⁻) strains of Aeromonas salmonicida to utilize haem sources of iron under conditions of iron-restriction was evaluated. In a plate bioassay, only A⁺ strains of A. salmonicida were able to utilize haem from a variety of sources including haem, haemin, myoglobin, haemoglobin, haemoglobin- haptoglobin and haem-albumin complexes. Trypsin-digestion of whole cells abolished haem- binding, indicating that binding was cell-surface associated, involving a protein binding site or receptor. Competitive binding studies indicated that all haem compounds were bound by a common receptor, which was not iron-regulated and was associated with the presence of the 49-kDa A-layer protein. The ability of both typical A⁺ (siderophore-positive) and atypical A⁺ (siderophore-negative) strains to utilize haem indicated that the mechanism of haem utilization was not siderophore-mediated and that A. salmonicida possesses both siderophore-dependent and siderophore-independent mechanisms to overcome iron-restricted conditions encountered in vivo.     

Proteases secreted by strains of Aeromonas salmonicida

Abstract. The proteases secreted by four strains (MT004, 1102, 480 and 480P^-) of Aeromonas salmonicida grown in liquid culture have been studied. Strains MT004, 1102 and 480 all show a similar pattern with two types of proteases produced; one of molecular weight 70 000 which is active against casein and gelatin and one (or more) of lower molecular weight (about 20 000) which is (are) active against gelatin but not casein. Strain 480P^- produces only the latter type of protease(s). The protease of molecular weight 70 000 is classified as a serine-type protease, but further characterization of the features of its active site has not yet proved possible. The results are discussed in terms of the previously published but often contradictory data on the proteases of A. salmonicida.     

Autoaggregation and extracellular A-layer protein in Aeromonas salmonicida

Fifteen strains of Aeromonas salmonicida were examined for the presence of an extracellular protein A-layer. The presence of an A-layer has been associated with the property of bacterial autoaggregation. However, three of the ten autoaggregating strains examined in this study showed no detectable A-layer subunit protein.     

Partial purification and characterization of extracellular metalloproteases from Aeromonas salmonicida ssp. salmonicida

Abstract. Aeromonas salmonicida ssp, salmonicida is shown to produce several extracellular proteins having gelatinolytic activity. Among the six isolates tested, two (NCMB 1102 and 84–14–R) produced both high (89–100 kDa) and low molecular (37 kDa) weight gelatinases, while the other four demonstrated only the 89–100 kDa forms. The low molecular form (metalloprotease 1: MP 1, 37 kDa) was isolated by ammonium sulphate precipitation, hydrophobic, ion exchange and size exclusion chromatography. The isolated enzyme was inhibited by the metal-chelating agents o-phenantroline and EDTA, and by excess Zn ions, and thus was defined as a metalloprotease. Its pH-optimum was 7–5, optimal activity was at 40°C and its pI 5.2. Specificity studies demonstrated cleavage of gelatin and azocoll, but not casein.