The role of the lethal extracellular cytolysin of Aeromonas salmonicida in the pathology of furunculosis

Abstract. The histopathological effects of intramuscular (i.m.) and intraperitoneal injections of the purified lethal cytolytic toxin of A. salmonicida in Atlantic salmon, Salmo salar L., were restricted to extensive degranulation of eosinophilic granular cells (EGCs) in the gills (both routes) and a limited coagulative necrosis of muscle fibres at the site of i.m. injection. The severity of these lesions did not apparently differ amongst fish that were moribund within 48 h and the surviving fish at 90 h. Previous studies indicate a maximum time to death caused by this toxin of 48 h. Thus, it is difficult to account for death of the fish from the toxin solely on the grounds of the histopathological features. It is suggested that a complex metabolic dysfunction results in death. The mechanism of 'furuncle' formation was demonstrated to result from a combined effect of the cytolysin and the purified 70-kDa protease present in the ECP. When i.m.-injected singly, the purified cytolysin produced a limited coagulative necrosis and the purified protease produced a limited liquefaction and haemorrhaging. In combination, they induced the extensive liquefactive haemorrhagic 'furuncle' characteristic of the whole ECP.     

Toxicity and immunogenicity of Aeromonas salmonicida extracellular products to salmonids

Abstract. Aeromonas salmonicida extracellular products (ECP) were fractionated by gel filtration chromatography. Three protein fractions were found. The first and second fractions showed low and high haemolytic activity, respectively, whereas the third fraction showed protcolytic activity. By intramuscular injection into juvenile rainbow trout, the median lethal doses of the first, second and third fractions were calculated at >669, 152 and <1514μg/g body weight, respectively. Cytopathic effects of the fractionated ECP to coho salmon lymphocytes were observed in vitro. The first and second fractions caused cell deformation, nuclear granulation and cytoplasmic streaming after 3h. No cytologic effects with the third fraction were observed. A mixture of the first and third fractions, a mixture of the second and third fractions, and non-fractionated ECP caused nuclear granulation and cytoplasmic streaming after 3Qmin. Using immunodiffusion analysis, the first and second fractions formed a single precipitating line each against white-spotted char anti-ECP sera, but the third fraction did not formed a precipitating line against the antisera.     

An experimental model for fish furunculosis caused by Aeromonas salmonicida

Abstract. Development of an experimental bath challenge method for fish furunculosis caused by Aeromonas salmonicida is described. The influence of certain important experimental variables was investigated and standardized. Possible application of the model is discussed.     

Antibiotic protection against recrudescence of latent Aeromonas salmonicida during furunculosis vaccination

Problems of temporary immunosuppression following vaccination against Aeromonas salmonicida infection had to be overcome in the development of a furunculosis vaccine. Empirical observations have indicated that immunosuppression persists for some time after vaccination, rendering fish, especially subclinical carriers of A. salmonicida, highly vulnerable to bacterial invasion. The efficacy of simultaneous application of furunculosis vaccine and a long-lasting amoxycillin preparation to a population of Atlantic salmon smolts was evaluated. Control groups were treated with either vaccine alone, amoxycillin alone or were untreated. Moderate stress, simulating smolt transfer with a 5°C temperature rise, resulted in a rapid outbreak in mortalities reaching 100% in the vaccinates. Losses among the untreated controls were more gradual and rose to about 50%. Both amoxycillin-treated groups survived well. Further severe stress resulted in total mortalities among the untreated fish but no further losses in the amoxycillin groups. Four months after vaccination, evidence of a specific immune response was confirmed by ELISA, demonstrating circulating antibodies in the blood of vaccinates. In a severe and in a moderate challenge with A. salmonicida., the relative specific protection was 63 and 86%, respectively. Thus, effective protection against furunculosis was achieved without jeopardizing the stock during the vaccination process and with elimination of the carrier state.     

The development of live vaccines for furunculosis lacking the A-layer and O-antigen of Aeromonas salmonicida

Abstract. Mutants of Aeromonas salmonicida strains lacking either the A-protein, O-antigen or both of these major surface antigens were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), for their suitability as live vaccines (LV). All of these mutants were shown to be attenuated, as fish receiving ∼5 × 10⁷ of the respective strains showed no clinical signs of furunculosis. Immersion vaccination of fish in 5 × 10⁷ cfu ml^-1 of these strains with an identical immersion dose 14 days later resulted in significant protection by all strains from challenge with a heterologous virulent strain of A. salmonicida 5 weeks later. The levels of protection conferred were all greater than or equal to that provided by an injected bacterin using the same vaccination schedule. With one exception, all LV strains that still possessed a functional O-antigen provided protective indices (PI) four- to seven-fold greater than the PI for the fish injected with bacterin. When antibody responses of vaccinated fish were compared, it was found that only vaccination by bacterin gave rise to a measurable agglutinating litre. Western immunoblots using the immune fish sera failed to reveal any major differences in antigen recognition in fish that received any of the vaccines tested. These data suggest that the immune response generated by the use of live vaccine strains is different from that generated by a bacterin, and that these useful mutations may be incorporated into existing furunculosis LVs for further attenuation.     

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.     

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.     

Evaluation of profiles of Aeromonas salmonicida as epidemiological markers of furunculosis infections in fish

Abstract. Strains of Aeromonas salmonicida subsp. salmonicida , the agent of furunculosis disease of salmonid fish, have fairly uniform plasmid patterns. Of 35 strains examined by agarose gel electrophoresis, 28 had a pattern consisting of four small plasmids (4.2, 3.6, 3.5, 3.3 Mda) and a larger plasmid. The larger plasmid was most often 50–56 Mda, but it was larger in some strains. In the remaining seven strains, the same general profile was seen, but one of the small plasmids was missing. An additional plasmid was present in six strains. The pattern seen in 30 strains collected from Ontario fish over an 8-year period did not differ significantly from five reference isolates from other locations. Plasmid profiles of A. salmonicida strains appear too uniform to provide a useful epidemiological tool. The non-pigmented. atypical strains of A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida, A. media , and brown-pigmented strains of A. hydrophila had different plasmid DNA profiles, which were distinct from those of typical isolates of A. salmonicida subsp. salmonicida . Antibiotic susceptibility patterns, determined by the agar dilution method, were uniform for most typical strains. A non-transferable resistance to tetracyclmes was found in two Ontario isolates, but antibiotic resistance was relatively uncommon among the Ontario isolates.     

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.     

Modified extracellular product antigens of Aeromonas salmonicida as potential vaccines for the control of furunculosis in Atlantic salmon, Salmo salar L.

Abstract. Extracellular product (ECP) antigens of Aeromonas salmonicida were modified in an attempt were tested on an antigen-induced proliferation assay, for the ability to induce antibodies as measured by dot blot dot assay and as vaccines in vaccination/challenge trials. Modifications tested included particularization on to polystyrene beads, coating on to sheep red blood cells, mixing with BCG vaccine as adjuvant, and attachment to the T-independent carrier Fieoll. The only modification that resulted in increased protection levels was the particularization on to polystyrene beads.     

Isolation and characterisation of two extracellular metalloproteases from Aeromonas salmonicida ssp. salmonicida

Two extracellular metalloproteases were purified from a culture filtrate derived from Aeromonas salmonicida ssp. salmonicida . One enzyme, leucine aminopeptidase (LAP), which had a molecular mass 37 kDa, hydrolysed aminoterminal l -leucine and l -phenylalanine. The activity was inhibited by 1,10-o-phenanthroline, but not by EDTA. The addition of excess Zn²⁺ to an o-phenanthroline-inhibited enzyme restored most of its activity. The peptidase was temperature stable, and had an optimum temperature and pH of 60 °C and 8, respectively. The other enzyme, metalloprotease 3 (MP3), which had a molecular mass 20 kDa, was an endoprotease, and hydrolysed azocoll and hide powder-azure, but not gelatine. The MP3 enzyme had an optimum temperature and pH of ≈40 °C and 7.5, respectively, and a cationic isoelectrical point.     

杀鲑气单胞菌杀日本鲑亚种胞外产物毒性及免疫原性分析

用玻璃纸平板法提取了杀鲑气单胞菌杀日本鲑亚种Aeromonassal monicida masoucida的胞外产物（ECP）。毒性试验证实,ECP对剑尾鱼Xiphophorus helleri具有致死性,其半致死量（LD50）为4.72μg蛋白/g体重。SDS-PAGE表明,ECP由13条蛋白带组成。利用大鼠抗ECP血清进行的Western-blot印迹显示,组成ECP的13条蛋白带中有7条具有免疫原性,能够引发机体的免疫应答反应产生抗体,其分子量分别为88、70、42、39、36、22和15kDa。     

The humoral immune response of rainbow trout, Salmo gairdneri Richardson, and rabbits to Aeromonas salmonicida extracellular products

Abstract. Controversy exists concerning the efficacy of vaccinating fish against furunculosis. Where success is claimed, there has been little attempt to characterize the protective antigens or confirm their immunogenicity. In this report, the immunogenicity of native extracellular products (ECP) of Aeromonas salmonicida and a formalin-inactivated toxoid of ECP (f-ECP) was studied in rainbow trout and rabbits, with particular attention to the putative bacterial virulence factors protease and haemolysin. Using crossed immunoelectrophoresis and Protein-A absorption, antibodies to seven ECP components were detected in the rabbit following immunization with native ECP; antihaemolysin antibodies were found but antibodies to the protease could not be detected. Antibodies to at least 14 components of ECP, including haemotysin and protease, were detected in the rabbit following immunization with f-ECP. In trout immunized either with native ECP or f-ECP, antibodies to only four ECP antigens were detected and no antibodies to haemolysin or protease were found. The results may explain previous reports that passive immunization with rabbit antisera gave superior protection against furunculosis compared with antisera raised in fish, and indicate that many extracellular antigens of A. salmonicida may require modification in order to improve their immunogenicity in fish.     

Do extracellular products of Aeromonas salmonicida induce thrombosis by entering the fish coagulation system at factor X?

Abstract. Thrombosis of minute vessels is a common feature of acute furunculosis in Atlantic salmon. Crude extracellular products (F.CP) of Aeromans, salmonicida injected into the dorsal aorta of cannulated fish elicited an activation of the coagulations systems of the fish When screened with a number of chromogenic substrates, the ECP protease revealed a factor-X like activity. Thus, it may he hypothesized that the ECP induces thrombosis by entering the fish coagulation systems at factor X. and that thrombus formation is a way of creating a site of infection.     

Bacterial inhibitors affecting proteases produced by Aeromonas salmonicida, and the occurrence of such inhibitors in furunculosis vaccines

Abstract. Cultures of Aeromonas salmonicida , subspecies salmonicida and achromogenes , produced considerable quantities of inhibitors that affected extracellular bacterial proteases (endopeplidases), as well as some animal trypsins (from pig, salmon and trout) included in this study. Electrophoretic separation of these inhibitors revealed a complex of three to four single factors which were similar for the two subspecies. One of the factors only inhibited protease from the homologus subspecies, while another only affected enzyme from the other subspecies (cross-wise inhibition). Both subspecies produced a factor which inhibited animal trypsins only. Subspecies salmonicida also possessed a factor which inhibited most of the examined proteases unspecifically. In young cultures (2–3 days at 15°C), the inhibitors were demonstrable both in the disintegrated cell material and in cell-free culture filtrate. The activity of the factor which only inhibited the protease from subspecies salmonicida could be increased considerably by moderate heating of the material. The effect of inhibitors produced by other relevant Gram-negative bacteria on the proteases of the A. salmonicida subspecies included was more limited. Considerable quantities of inhibitors against proteases of the subspecies salmonicida and achromogenes were demonstrated in cell-free filtrates of two commercially available vaccines against furunculosis in fish.     

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.     

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.     

养殖刺参溃疡病杀鲑气单胞菌的分离、致病性及胞外产物特性分析

从患溃疡病的养殖刺参(Apostichopus japonicus)病灶处分离出1株优势菌H1,以浸浴、创伤浸浴、体腔注射和体壁肌肉注射等方式进行感染实验,证实菌株H1为养殖刺参溃疡病病原菌,并证明该菌通过体表创伤侵入的方式感染刺参,以创伤浸浴和体壁肌肉注射感染的LD50(半数致死量)分别为2.26×107CFU/尾和1.80×107CFU/尾。经形态学观察、生理生化特性分析和mini API系统鉴定,确定菌株H1为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida ma-soucida)。提取菌株H1的胞外产物(ECP)进行致病性实验,结果表明ECP可导致刺参死亡,其对刺参的LD50为5.24μg蛋白/g体质量。H1-ECP具有酪蛋白酶、明胶蛋白酶、几丁质酶和淀粉酶活性,并具有溶血素活性;对底物偶氮酪蛋白(Azocasin)作用的酶比活力可达到674.5活力单位/mg蛋白,最适作用温度为50℃;对热不稳定,70℃作用30 min时,酪蛋白酶活性降到0;100℃作用30 min,ECP对刺参的毒性消失;ECP酶活可被10 mmol/L EDTA完全抑制,可被5 mmol/L PMSF抑制98.8%,Ca2 和Mg2 可使酶活性分别提高约9%和4%。结论认为,该病原菌通过体表创伤侵入方式感染宿主刺参,菌株H1胞外产物是其对刺参致病的因子之一。     

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