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.     

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.     

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.     

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.     

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.     

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.     

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.     

The nucleotide sequence of the gene encoding GCAT from Aeromonas salmonicida ssp. salmonicida

The nucleotide sequence of the gene encoding GCAT (glycerophospholipid: cholesterol acetyltransferase) in Aeromonas salmonicida ssp. salmonicida has been determined. The sequence revealed an open reading frame of 1005 bp encoding 335 amino acids, an 18-amino-acid signal peptide and a 317-amino-acid mature polypeptide with a deduced molecular mass of 35 380 Da and an estimated pI of 6.25. Within the encoding region, the homology with the corresponding gene fromAeromonas hydrophila was 92.9% for the nucleotide sequence and 93.7% for the deduced amino acid sequence.     

Glycogen, the preferred energy source for extracellular protein formation and growth of Aeromonas salmonicida

Abstract. A study was made of the effect of supplementing a rich 3% (w/v) tryptone soya broth (TSB) medium and a poorer 1.7% (w/v) tryptone-based medium with glucose, maltose and glycogen, as carbon sources, on growth and exoprotein formation by Aeromonas salmonicida. In TSB, glucose inhibited growth and repressed exoprotein formation whilst maltose and glycogen had little effect, up to 20h, when compared with an unsupplemented control. By contrast, in the poorer medium, over a 24-h incubation period, growth was stimulated three-fold by glycogen, and whilst exoprotein formation was low in comparison with that observed in TSB, the greatest production was observed in the presence of glycogen. Extracellular α-amylase was measured in the tryptone medium in the presence of the three carbon sources and the highest level, produced in the presence of glycogen, was 1.6 times that with added maltose whilst none was detectable with glucose present. This pattern was repeated in the case of the maltose-inducible porin, LamB, of the outer membrane.     

Carrier state in furunculosis: secondary infection of trout with different Aeromonas salmonicida strains results in advantage for the primarily harboured one

Abstract. On the basis of previous observations, in vivo experiments were conducted in rainbow trout, Oncorhynchus mykiss (Walbaum), to detect possible competition between strains of Aeromonas salmonicida differing in their pattern of resistance to antimicrobial drugs. Serial infections, or reinfection of naturally-infected trout, were induced by intramuscular injection. Surviving fish were artificially stressed, and all dying fish were examined using selective media to assess the type of latent infection. Secondary infections with virulent strains always resulted in mortality and reisolation of the secondary strain in pure culture. However, when trout having undergone the two successive infections were stressed, the primary strain was always reisolated. Primary strains seem to be able to survive in unknown locations within cells or tissues, regardless of their virulence or growth potency. Although a protective effect conferred by latent infection to secondary challenge could not be clearly proven, such mechanisms resemble what occurs in premunition and could be of epidemiological interest.     

Evaluation of florfenicol in Atlantic salmon,Salmo salar L.: efficacy against furunculosis due to Aeromonas salmonicida and cold water vibriosis due to Vibrio salmonicida

Two replicated controlled trials were conducted to determine the efficacy of florfenicol against Aeromonas salmonicida and Vibrio salmonicida infections in Atlantic salmon, Salmo salar L., smolts kept in 25‰ salt water. Infection with A. salmonicida was treated with florfenicol, oxolinic acid, oxytetracycline, trimethoprim/sulphadiazine or flumequine, whereas the V. salmonicida infection was treated with florfenicol or oxolinic acid only. A. salmonicida infection was induced by the introduction of cohabitant fish previously inoculated intraperitoneally. Medication started simultaneously in all test tanks on the first day of specific mortality among test fish. V. salmonicida infection was induced by intraperitoneal inoculation of all test fish. Medication started 1 day after infection. Medicated feeds were produced by coating the antibacterials on standard feed pellets, and administered twice daily for 10 consecutive days. With the dose used in the present trials, florfenicol was highly effective in reducing specific mortalities due to both infections. It was slightly more effective than oxolinic acid and trimethoprim/sulphadiazine against A. salmonicida infection. There was no significant difference between florfenicol and oxolinic acid in reducing specific mortalities due to V. salmonicida.     

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.