Analysis of exotoxins produced by atypical isolates of Aeromonas salmonicida,by enzymatic and serological methods

In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion.     

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.     

Clinical, microbiological and epidemiological findings in recent outbreaks of goldfish ulcer disease due to atypical Aeromonas salmonicida in south-eastern Australia

Abstract. The spread of goldfish ulcer disease (GUD) from Victoria to New South Wales, Australia, and the first isolation of Aeromonas salmonicida from wild goldfish are reported. Cultural, biochemical and protein SDS-PAGE characteristics of these recent isolates are compared with those of existing Australian isolates, with strains recovered from goldfish in Italy and the USA (atypical strains) and with strain ATCC 14174 (typical strain). The Australian isolates were identical and closely resembled the exotic atypical strains. Although there were several biochemical differences between the atypical isolates and the typical ATCC 14174 strain, the results of SDS-PAGE confirmed that these strains were closely related. The homology of the Australian and overseas strains recovered from goldfish supports the common view that A. salmonicida was introduced first into Australia with diseased goldfish in 1974. The three widely separated outbreaks of GUD reported here confirm that an atypical strain of A. salmonicida is now endemic in Australia.     

Evaluation of an experimental Aeromonas salmonicida epidemic in chinook salmon, Oncorhynchus tshawytscha (Walbaum)

To determine the dynamics of the transmission of Aeromonas salmonicida ssp. salmonicida infection, chinook salmon, Oncorhynchus tshawytscha, were exposed to bacteria by cohabitation. The latent period (time between exposure and infectivity) was determined by exposing a group of chinook salmonid fingerlings to A. salmonicida by bath, then, at daily intervals, by holding five exposed (donor) fish with approximately 50 naive fish for 24 h. The latent period was 3 days post-infection and the time period between the initial exposure to bacteria and the beginning of bacterial shedding was 4.5 days for the same animals. The prevalence and intensity of infection in the donor fish, to which recipient fish were exposed, i.e. the level of exposure, was highly correlated with the development of disease in recipient (susceptible) chinook salmon (r2 = 0.57). An experiment was conducted to determine the daily progress of infection and development of a furunculosis epidemic among recipient fish by cohabiting a single exposed fish with 43 unexposed salmon. At daily intervals, all fish (in seven treatment tanks and one control tank daily) were sacrificed and tested for the presence of A. salmonicida in the kidney (n = 3520). Over 10 days, mean prevalence among recipient fish reached 75% and disease related mortality exceeded 50%. Bacterial concentrations in the water continued to increase over the duration of the experiment in concert with the number of infected animals present in the population.     

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.     

Transmission of protease genes into a protease-deficient mutant of Aeromonas salmonicida in river sediments

Abstract. The transformation of Aeromonas salmonicida with DNA fragments from bacterial cell-free sonicates was investigated with intraspecific, interspecific band intergeneric fish pathogenic bacteria including Aeromonas salmonicida, Aeromonas hydrophila, Pseiidomonas fluorescens and Vibrio anguillarum strains as donor bacteria. A phenotypic marker for transformation was extracellular protease production since a protease-deficient mutant NTG-1 induced from pathogenic A. salmonicida strain A-7301 by mutagenesis was used as a recipient. This mutant was non-pathogenic to rainbow trout. The mutant was incubated with each sonicate at 20°C for 20 days with a nutrient-poor medium containing a trace (5 μg/ml each) of both humic acid and tryptone in the presence of clean river sand (100 g/100 ml medium) corresponding with an environment of rivers. During the incubation, the survival of mutant NTG-1 cells was observed and protease positive NTG-1 cells were isolated from each culture. The protease production of the isolates was due to the transmission of protease genes of the donor strains. The activity of proteases produced by the transformants extra-cellularly was determined. These transformants induced with the sonicates of the parent strain, intraspecific strain and with the sonicates of the interspecific A. hydrophila strain were pathogenic to rainbow trout, whereas the transformants derived with the sonicates of the intergeneric strains P. fluorescens and V. anguiUarum showed non-pathogenicity, although all the donor strains, with the exception of the P. fluorescens strain, were pathogenic. These findings are interesting since they demonstrate that trausformation in A. salmonicida occurs with considerable ease even intergenencally and interspecifically, as well as intraspecifically in river environments, and that there is a large difference in the lethal toxicity of extracellular protease produced by these bacteria.     

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.     

Autoagglutination, growth on tryptone-soy-Coomassieagar, outer membrane protein patterns and virulence of Aeromonas salmonicida strains

Abstract. Twelve Aeromonas salmonicida isolates were examined for autoagglutination, colony colour on tryptone soy agar supplemented with Coomassie brilliant blue (TSA-C), and outer membrane protein pattern by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoagglutination did not correlate with colony colour on TSA-C, nor with the presence of the additional 49-kDa protein (A-protein) in all strains tested. However, there was a correlation between colour on TSA-C and A-protein. Two weakly autoagglutinating isolates with no A-protein detectable in SDS-PAGE and with negative staining behaviour on TSA-C shared another additional protein, designated X-protein. One of these isolates was virulent for juvenile rainbow trout, the other one avirulent. Thus, for the strains examined, neither autoagglutination nor X-protein were reliable indicators of virulence, and absence of A-protein did not necessarily mean avirulence.     

Infectious load of Aeromonas salmonicida subsp. salmonicida during the initial phase of a cohabitant infection experiment with Atlantic salmon, Salmo salar L.

Abstract The abundances of Aeromonas salmonicida subsp. salmonicida in the water and in the surface microlayer was studied during the initial phase of a cohabitant infection experiment with Atlantic salmon, Salmo salar L., smolt. Aeromonas salmonicida was detected in the water samples only until the intraperitoneally infected smolt were dead and had been removed. In the lipid rich surface microlayer, A. salmonicida was detected in high concentrations from the day of the first fish mortality and throughout the rest of the experiment. The significance of the high cell surface hydrophobicity is discussed as a possible reason for enrichment of A. salmonicida at the air-water interface.     

Atypical Aeromonas salmonicida infection in the black rockfish, Sebastes schlegeli Hilgendorf, in Korea

Cultured black rockfish, Sebastes schlegeli, suffered mass mortalities during winter 2008 and spring 2009 in Korea, showing clinical signs of ulcer lesions and haemorrhages over their body surface. The aetiological agent was identified as Aeromonas salmonicida (strains RFAS-1, -2 and -3), which is a non-pigmented, slow-growing bacterium. Phenotypes of RFAS strains showed variation, while 16S rRNA, gyrB, rpoD, dnaJ and recA gene sequences of all the strains were affiliated to A. salmonicida. In particular, vapA gene sequences of the strains were most closely related to one of the five subspecies of A. salmonicida subsp. masoucida (=KCCM 40239(T) ). LD(50) values of RFAS-1 for intraperitoneal and intramuscular injection were 1.5 × 10(5.25) and 1.5 × 10(6.4) cfu/rockfish, respectively. However, A. salmonicida strains KCCM 40239(T) and SAS-1, which originate from masou and chum salmon, respectively, were not pathogenic to black rockfish. RFAS strains, possessing A-layer protein on their surface, exhibited β-haemolytic activity against rockfish erythrocytes and capability to survive in rockfish serum, which seem to be associated with virulence.     

In vitro activities of oxolonic acid, ciprofloxacin and norfloxacin against Aeromonas salmonicida

Abstract. Oxolinic acid and two new fluoroquinolones, ciprofloxacin and norfloxacin, were evaluated for in vitro antibacterial activity against Aeromonas salmonicida. Although oxolinic acid was as active as ciprofloxacin in terms of minimum inhibitory concentration (MIC), fluoroquinolones were significantly more active in terms of their ability to kill both oxolinic acid sensitive and resistant strains of A. salmonicida. Furthermore, the fluorinated drugs were active against non-dividing A. salmonicida. It would appear worthwhile to carry out further investigations with fluoroquinolones as they may be more effective in treating A. salmonicida infections than the current regime of oxolinic acid.     

Aetiology of an ulcerative disease in goldfish, Carassius auratus (L.): characterization of the causative agent

Abstract. A bacterium resembling Aeromonas salmonicida and determined to be the aetiologic agent of a cutaneous ulcerative disease in goldfish Carassius auratus (L.) was further characterized in this study.
Forty-five isolates of the bacterium (43 from the United States and one each position (moles % guanine plus cytosine) and DNA homology. The bacteriological atypical A. salmonicida previously described. Several important biochemical characteristics distinguished the goldfish isolates from typical A. salmanicida , but the DNA binding experiments indicated a high degree of relatedness between the goldfish isolates and typical A. salmonicida strains.     

Aeromonas salmonicida subsp. salmonicida: correlation of protein patterns, antibiotic resistance, exoprotease activity, haemolysis and pathological lesions produced in vivo

Abstract. A collection of 130 strains of the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida isolated from diseased salmonids in Denmark, Norway, North America and Scotland has been characterized with regard to protein patterns, antibiotic resistance and exoprotease activity. Whole cell and outer membrane protein profiling could distinguish three different profiles in A. salmonicida. Eight outer membrane proteins were demonstrated (49, 40, 38, 37, 33, 31, 30 and 29 kDa). One protein profile was deficient in a 38 kDa outer membrane protein and instead contained an outer membrane protein of 37 kDa which was not detectable among the other protein profiles. Strains with the 37 kDa outer membrane protein showed multiple low-level antibiotic resistance towards cephalothin, penicillin, chloramp-henicol, tetracycline and quinolones. In addition, these strains were exoprotease deficient. Strains with the 37 kDa protein were unable to degrade cattle and trout serum proteins and displayed a delayed degradation of casein. Haemolysis on cattle blood agar plates was similarly delayed. In vivo examination of extracellular products from a normal protein profile strain and one with the 37 kDa outer membrane protein demonstrated major differences in pathological effects in rainbow trout. The strain possessing the 37 kDa outer membrane protein produced almost no pathological effects while the normal protein profile strain produced typical furuncles.     

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.     

Efficacy studies of passive immunization against Aeromonas salmonicida infection in brook trout, Salvelinus fontinalis (Mitchill)

Abstract. Aeromonas salmonicida , the aetiologic agent of furunculosis, causes high mortality in cultured salmonids. Experiments were conducted to determine the therapeutic and prophylactic efficacy of passive immunization against furunculosis in brook trout, Salvelinus fontinalis (Mitchill), infected by immersion. Rabbit hyperimmune serum was produced against a virulent strain of A. salmonicida and an aliquot of this serum was absorbed with cells of a non-virulent strain of A. salmonicida. Immunoglobulins from aliquots of the absorbed and non-absorbed serum were purified using affinity chromatography. Each serum or immunoglobulin preparation was tested in passive immunization experiments. Brook trout were infected by immersion in a suspension of virulent A. salmonicida , and passively immunized by intraperitoneal injection at the time of experimental infection, or at various periods after experimental infection. Passive immunization of brook trout against furunculosis was therapeutically efficacious when effected either at zero, 24 or 48h post-infection, but not at 72 or 96h. Purified rabbit immunoglobulins specific to virulent A. salmonicida were as protective as the initial rabbit hyperimmune serum in protecting brook trout against furunculosis. To determine the prophylactic efficacy of this treatment, the groups of fish passively immunized at the time of the experimental infection were challenged a second time at either 14, 35, 41 or 56 days after passive immunization. Brook trout were protected against a second experimental bath challenge with virulent A. salmonicida for a period of 35–41 days.     

Effect of three dietary oils on disease susceptibility in Arctic charr (Salvelinus alpinus L.) during cohabitant challenge with Aeromonas salmonicida ssp. salmonicida

Arctic charr ( Salvelinus alpinus L.) were fed diets based on a commercial recipe supplemented with either linseed, soybean or marine oil prior to cohabitant challenge with Aeromonas salmonicida ssp. s almonicida . Mortality varied significantly between the three dietary groups. Highest mortality (48%) was observed in fish fed the marine oil and the lowest mortality (20%) was in the group fed soybean oil. Transmission electron microscopy (TEM) examination of the digestive tract of uninfected fish demonstrated substantial numbers of bacterial cells between microvilli. However, only a few bacteria were recovered that were associated with the microvilli of infected fish. Immunocytochemical staining/labelling investigations using TEM and an immunogold method were performed on mid-gut segments of fish fed the marine oil diet and showed augmentation of goblet cells and the presence of A. salmonicida ssp. salmonicida in the gastrointestinal tract of diseased fish after challenge with the pathogen. It is suggested that the gastrointestinal tract could be an infection route of A. salmonicida ssp. salmonicida. The greater prevalence of goblet cells supports the suggestion that sloughing off mucus is a protective response against bacterial infections. These results make an important contribution to our understanding of how nutrition can affect the disease resistance of fish.     

Identification of cultured Aeromonas salmonicida by an indirect dot blot immunoassay

Abstract. A method is described which improves both the specificity and paracticability of immune identification of Aeromonas salmonicida. The modified assay employs antisera raised against outer membrane proteins (OMP) of A. salmonicida cells and is carried out as a dot blot test on nitrocellulose membranes. Performance of the test with 55 non- A. salmonicida bacterial isolates from fish and water revealed weak cross reactivity in five cases. However, these cross reactive only occur at very high antigen concentrations and can be overcome by adequate dilution.     

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.     

Antibiotic resistance of Aeromonas salmonicida isolated from Atlantic salmon, Salmo salar L., in Scotland

Abstract. Antibiotic sensitivity patterns of 304 isolates of Aeromonas salmonicida from 229 outbreaks of furunculosis among salmon in Scotland between 1988 and 1990 were investigated. Fifty-five per cent were resistant to oxytetracycline and 37% resistant to oxolinic acid. Multiple resistance was common (52%) and 18 out of 19 antibiograms which were found in the first year recurred in the succeeding year. More than a quarter of the outbreaks were associated with two or more A. salmonicida variants distinguishable by their antibiotic sensitivity patterns. The implications of these findings in the control of furunculosis are considered.     

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.