Furunculosis In Fish Research Articles

Furunculosis-inducing ability of microcosms seeded with Aeromonas salmonicida correlates with colony-forming ability but not with PCR and ELISA data

Two laboratory microcosms, constructed of sterilised sediment and water taken from different locations, were seeded with Aeromonas salmonicida. A non-quantitative PCR, a heterogeneous, non-competitive, sandwich ELISA and colony formation were employed to generate survival data. In one microcosm, all three analytical methods generated positive results over the full 279 days of the incubation of the system at 18 °C. In the second microcosm, constructed with humic acid-rich material, colony formation was undetectable after 1 day but the PCR and ELISA methods generated positive results for up to 269 days. Direct injection into fish of material from the two microcosms both seeded with a passaged strain of A. salmonicida was used to determine the disease-producing potential of the bacteria in these systems. A direct correlation was observed between the presence of colony-forming units and the ability to produce furunculosis in test fish. None of the samples that generated positive PCR and ELISA signals but in which colony formation was not detected proved capable of inducing disease in fish. The significance of these data for the application of proxy methods in epizootiological studies is discussed.

The Effect of Depuration on Transmission ofAeromonas salmonicidabetween the Freshwater BivalveAmblema plicataand Arctic Char

Abstract A model system was used to study bacterial fish pathogen transmission between the freshwater bivalve Amblema plicata and two strains (Nauyuk and Labrador) of Arctic char Salvelinus alpinus. Aeromonas salmonicida, the cause of fish furunculosis, was readily transmitted from Arctic char to A. plicata and vice versa via simple cohabitation. Clinical furunculosis was artificially established in Nauyuk Arctic char via horizontal exposure to Labrador Arctic char that received intraperitoneal injections of A. salmonicida. After the Nauyuk Arctic char began to die, A. plicata were placed in the tank with the fish. After 33 d of cohabitation, a group of 10 A. plicata was cultured, and A. salmonicida was isolated from all 10. The remaining A. plicata were transferred to other tanks being supplied with specific-pathogen-free water. At 1, 5, 15, and 30 d after transfer, 60 uninfected Labrador Arctic char were cohabitated with the A. plicata. Transmission of A. salmonicida from A. plicata to the Arctic char w...

The postponement of non‐culturability in Aeromonas salmonicida

Abstract 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.

Recombinant infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout (Oncorhynchus mykiss).

Fragments of the glycoprotein genes of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) were cloned into a bacterial broad-host-range expression vector under the control of the plac promoter. Western blot (immunoblot) analysis with monoclonal antibodies specific to the glycoproteins demonstrated the inducible expression of the fusion proteins in Escherichia coli. Aeromonas salmonicida is the causative agent of furunculosis in salmonid fish. It was confirmed that an avirulent strain of A. salmonicida, A440, which contains a deletion in the structural gene for the paracrystalline surface protein array, will provide protective immunity against furunculosis when used as a live attenuated vaccine. The plasmid-encoded viral epitopes were then mobilized into A440 for use as a shuttle system for the expression of fragments of the glycoprotein genes of IHNV and VHSV. Vaccination of rainbow trout with A440 containing the viral epitopes resulted in the development of protective immunity against both VHSV and IHNV. This indicates that the use of cloned fragments of the glycoproteins and the use of A. salmonicida as a shuttle system constitute a feasible approach to fish vaccine development.

Phenotypic and molecular characteristics of Aeromonas salmonicida subsp. salmonicida isolated in Southern and Northern Finland

Aeromonas salmonicida subsp. salmonicida causes outbreaks of furunculosis in salmonid fish. Furunculosis was first detected in Finland in 1986 on fish farms located on the Finnish coast of the Bothnian Bay. Molecular methods, SDS-PAGE, ribotyping, randomly amplified polymorphic DNA (RAPD) and plasmid profile analysis as well as phenotypic characteristics (biochemical characteristics, maximum growth temperature, pigment and elastase production) were used both for typing the strains and to study the possible routes of transmission of the organism to Finland and the spread of infection within Finland from 1986 to 1993. Ribopattern analysis of chromosomal DNA digested with SmaI, BglI, PstI and ClaI of 28 Finnish strains and eight foreign strains (Denmark, Sweden, Norway or Canada) showed that all Finnish strains and the Swedish strain originating from the Swedish coast of the Bothnian Bay had identical ribopatterns. All other foreign strains had distinct, unique ribotypes except for the second Swedish strain studied, the ribotype of which was identical with that of one Danish strain. RAPD typing, based on the results of two arbitrary primers, yielded 15 types for the Finnish strains. Except for both Danish strains, which had the RAPD type which was identical with that of one Finnish strain, the foreign strains had RAPD patterns differing from those of the Finnish strains. Plasmid profile typing and RAPD profile typing did not correlate. Ribotyping with four different enzymes proved to be the most sensitive method for studying genetically homogeneous Aer. salmonicida subsp. salmonicida. Ribopattern analysis showed that the infection which first started in 1984/1985 on the Swedish coast of the Bothnian Bay may have been transmitted to Finland where the first outbreaks occurred in 1986. The strains infecting Finnish fish farms were very homogeneous, with most differences seen, for example, in maximum growth temperature, plasmid profiles and the RAPD profiles of the strains.

Acute effects of chlorinated resin acid exposure on juvenile rainbow trout,Oncorhynchus mykiss

AbstractThe effects of an acute exposure to either 14–monochlorodehydroabietic acid (MCDHAA) or 12,14–dichlorodehydro‐abietic acid (DCDHAA) were examined in juvenile rainbow trout,Oncorhynchus mykiss.The experimentally determined 96–h LC50 values (and their 95% confidence limits) were 1.03 (0.72, 1.48) and 0.91 (0.70, 1.21) mg/L, for MCDHAA and DCDHAA, respectively. To measure effects on several biochemical parameters, swimming performance, and disease resistance, juvenile trout were exposed for 24 h to sublethal concentrations of one or the other resin acid in an intermittent‐flow respirometer. Hematocrit, plasma lactate, and liver protein were significantly affected by exposure to the highest dose (80% of the 96‐h LC50 value) of either of the resin acids. Plasma cortisol levels were 14‐and 3‐fold higher than were controls. Resistance to infection byAeromonas salmonicidawas significantly reduced; the cumulative percent mortalities due to furunculosis in fish exposed to MCDHAA or DCDHAA reached 20 and 26%, respectively. Swimming performance, measured as critical swimming speed (mean values 6.32 ± 0.20 and 5.93 ± 0.15 body lengths per second for MCDHAA and DCDHAA, respectively), was not significantly affected by resin acid exposure.

Detection of Aeromonas salmonicida, causal agent of furunculosis in salmonid fish, from the tank effluent of hatchery-reared Atlantic salmon smolts

The fish pathogen, Aeromonas salmonicida, could be detected only by bacteriological culture from the kidney of dead or moribund fish in one tank in a hatchery rearing Atlantic salmon (Salmo salar L.) smolts. However, by using a DNA probe specific for this species, allied to a PCR assay, the pathogen could be detected in water, feces and effluent samples taken from this fish tank. Also, the presence of the pathogen was found in effluent samples from two fish tanks containing apparently healthy fish. Subsequently, the presence of pathogen in these tanks was confirmed by an increase in the daily mortality rate and by a plate culture from moribund fish.

An aromatic-dependent mutant of the fish pathogen Aeromonas salmonicida is attenuated in fish and is effective as a live vaccine against the salmonid disease furunculosis

Aeromonas salmonicida is the etiological agent of furunculosis in salmonid fish. The disease is responsible for severe economic losses in intensively cultured salmon and trout. Bacterin vaccines provide inadequate protection against infection. We have constructed an aromatic-dependent mutant of A. salmonicida in order to investigate the possibility of an effective live-attenuated vaccine. The aroA gene of A. salmonicida was cloned in Escherichia coli, and the nucleotide sequence was determined. The codon usage pattern of aroA was found to be quite distinct from that of the vapA gene coding for the surface array protein layer (A layer). The aroA gene was inactivated by inserting a fragment expressing kanamycin resistance within the coding sequence. The aroA::Kar mutation was introduced into the chromosome of virulent A. salmonicida 644Rb and 640V2 by allele replacement by using a suicide plasmid delivery system. The aroA mutation did not revert at a detectable frequency (< 10(-11). The mutation resulted in attenuation when bacteria were injected intramuscularly into Atlantic salmon (Salmo salar L.). Introduction of the wild-type aroA gene into the A. salmonicida mutants on a broad-host-range plasmid restored virulence. A. salmonicida mutant 644Rb aroA::Kar persisted in the kidney of brown trout (Salmo trutta L.) for 12 days at 10 degrees C. Vaccination of brown trout with 10(7) CFU of A. salmonicida 644Rb aroA by intraperitoneal injection resulted in a 253-fold increase in the 50% lethal dose (LD50) compared with unvaccinated controls challenged with a virulent clinical isolate 9 weeks later. A second vaccination after 6 weeks increased the LD50 by a further 16-fold.

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

Abstract.Cultures ofAeromonas salmonicida, subspeciessalmonicidaandachromogenes, 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. Subspeciessalmonicidaalso 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 subspeciessalmonicidacould be increased considerably by moderate heating of the material. The effect of inhibitors produced by other relevant Gram‐negative bacteria on the proteases of theA. salmonicidasubspecies included was more limited. Considerable quantities of inhibitors against proteases of the subspeciessalmonicidaandachromogeneswere demonstrated in cell‐free filtrates of two commercially available vaccines against furunculosis in fish.

Immersion vaccination for control of fish furunculosis

immersion vaccination for control of fish furunculosis

Lack of relationship between virulence of Aeromonas salmonicida and the putative virulence factors: A‐layer, extracellular proteases and extracellular haemolysins

Abstract The role of A‐layer (A), protease (P) and haemolysin (H) as virulence factors of Aeromonas salmonicida, the aetiological agent of fish furunculosis, was a investigated using three strains of the bacterium. Strain MT004 lacked the A‐Iayer (A−) and produced extracellular caseinase and gelatinase (P+) and haemolysin (T‐lysin; H+). Strain MT028 was A−, P− and H−, and strain MT048 was A+, P+ and H−. The pathology and LD50 produced in rainbow trout by cells or extracellular products (ECP) of each strain were determined. The ECP was produced by two different methods where the protease and haemolytic activities differed in relative levels, or when the protease of MT004 ECP was inhibited by the serine protease inhibitor PMSF. The results indicate that the presence of A‐layer is not essential, at least for a moderate degree of virulence; that in vitro production of extracellular proteases is not a requisite of virulent strains; that presence of protease and haemolysin in ECP can be correlated with the development of certain lesions and a rapid time to death but cannot be correlated with the lethal toxicity of the ECP. The authors conclude that an as‐yet unrecognized component of ECP is responsible for killing fish.

28] Preparation of hemolysin from Aeromonas

Publisher Summary This chapter describes the preparation of Hemolysin from Aeromonas hydrophila. A. hydrophila, a gram-negative bacterium of the family Vibrionaceae is ubiquitously found in nature and commonly isolated from cold- and warm-blooded animals, various waters and sewage, and may act as a primary human pathogen. It causes furunculosis and septicemia in fish. In contrast to most gram-negative organisms A. hydrophila produces several extracellular enzymes and toxins during growth—namely, hemolysins, enterotoxin, proteases, phospholipases, lipase, elastase, phosphatase, aminopeptidases, and ribonuclease. Hemolysin is produced during growth in various media, and released into the culture medium during the logarithmic growth phase. Unless otherway stated all operations were carried out at 4°. A precursor of hemolysin is detected in culture fluid during the logarithmic phase of growth. It is converted to mature toxin by proteolytic enzymes, hence very little precursor is present in the stationary phase. It is about 250 times less hemolytic than hemolysin and possibly nonhemolytic.

Inhibition of the Aeromonas salmonicida extracellular protease by α 2-macroglobulin in the serum of rainbow trout

One of the major pathogenic factors of Aeromonas salmonicida, the bacterium causing furunculosis in fish, is considered to be a protease secreted in the extracellular products. This protease can be inhibited by normal trout and rabbit serum but the factor responsible has not hitherto been identified. The present results demonstrate that the A. salmonicida protease is inhibited by the α 2-macroglobulin of rainbow trout serum. This inhibitor accounts for about 9% of the total trypsin inhibiting capacity of trout serum, is α-migrating in electrophoresis, is moderately stable on storage at −20 °C, is destroyed by heating at 45 °C, has no requirement for cations and is inactivated by methylamine. The A. salmonicida protease is resistant to inhibition by 91.9% of the total trout serum trypsin inhibitors, without inactivating them, and to human α 1-antiproteinase. It is proposed that trout α 2M may have a defensive function against furunculosis but the resistance of the bacterial protease to inhibition by the majority of the serum protease inhibitors may represent pathogenic adaptation.

Electrostatic mechanism of survival of virulent Aeromonas salmonicida strains in river water.

The ecological mechanism of survival of Aeromonas salmonicida, the bacterial pathogen of fish furunculosis, in river water was investigated by laboratory-based experiments with two virulent strains (which were autoagglutinating) and two virulent strains (which were nonagglutinating). A difference in net electrical charge of A. salmonicida cells was detected by electrophoresis; cells of the virulent strains were negative, whereas cells of the avirulent strains were positive. Despite the loss of viable cells within a week in distilled water and physiological saline (0.85% sodium chloride), the cells of the virulent strains survived for more than 15 weeks in the presence of diluted humic acid (10 micrograms/ml), tryptone (10 micrograms/ml), and cleaned river sand (100 g/100 ml of medium), but loss of viable cells occurred within 5 weeks in the absence of sand. The cells of the avirulent strains lost viability within 2 weeks with no relation to the presence of sand. Using ion-exchange columns, humic acid and the amino acids of tryptone were found to be anionic and cationic in water (pH 7.0), respectively. Sand particles had a high capacity to adsorb humic acid alone and amino acid-humic acid complexes. Thirty to fifty times the environmental concentration of amino acids (10 micrograms/ml) were accumulated on the surface of sand particles, thereby permitting only bacterial cells carrying net negative electrical charges (virulent cells) to survive for a long period on the surface of the sand particles. These electrostatic interrelationships among river sand, humic acid, and bacterial cells are closely implicated in the mechanism of long-term survival of virulent A. salmonicida in river sediments.

Susceptibility of Salmonids to Furunculosis: Differences between Serum and Mucus Responses against Aeromonas salmonicida

Abstract At the Reeds Creek (West Virginia) state hatchery, mortality from enzootic furunculosis is most severe among brown trout Salmo trutta, intermediate in brook trout Salvelinus fontinalis, and virtually nonexistent in rainbow trout Salmo gairdneri. In each species, survivors of epizootics had developed similar levels of protective antibodies in their sera as demonstrated by microtiter agglutination and passive immunization experiments. We concluded that serum antibodies were not related to graded differences in resistance to furunculosis. These same fishes, however, produced a mucus precipitin that reacted with cell-free bacterial extracts in single-radial immunodiffusion assays. The mucus precipitin activity correlated with the resistance of the three species to furunculosis. Breeding studies with brown trout also showed that fish selected for a high level of mucus precipitin activity against Aeromonas salmonicida, the cause of fish furunculosis, produced progeny that were more resistant to furuncu...

Significance of Extracellular Protease for Growth of a Heterotrophic Bacterium, Aeromonas salmonicida

The role of protease produced by a heterotrophic bacterium during growth was investigated with Aeromonas salmonicida, the pathogen of fish furunculosis, strain A-7301 and its protease-deficient mutant NTG-1 induced by mutagenesis. Strain A-7301 produced extracellular protease in a mixed amino acid medium (composed of Gly, Ala, Val, Ile, Leu, Thr, Ser, Cys, Met, Phe, Tyr, Lys, Arg, Pro, His, Try, Asp, Asn, Glu, and Gln at equal concentrations of 0.1 g/liter). Its multiplication rate was limited by the amounts of amino acids present, whereas strain NTG-1 showed no protease production despite considerable growth similar to that of A-7301. There was no difference between A-7301 and NTG-1 in amino acid requirements for growth, and seven amino acids (Gly, Ala, Val, Thr, Cys, Met, and His) were found to be indispensable. A defined level of the mixed amino acids (0.4 to 0.5 g/liter) was needed for A-7301 to initiate a large production of protease. Neither of the strains grew well in a casein medium, to which no amino acids were added. However, when a protease fraction obtained from extracellular products of A-7301 by DEAE-cellulose column chromatography was added, NTG-1 successfully reproduced in the casein medium. These results indicate that the extracellular protease plays an important role in supplying A. salmonicida cells with available amino acids as nutrients and that higher growth is closely associated with protease production which stimulates further reproduction.

Loss of virulence in a protease-deficient mutant of Aeromonas salmonicida.

The importance of extracellular protease production by Aeromonas salmonicida, the bacterial pathogen of fish furunculosis, was investigated with four virulent strains (which were autoagglutinative, hemagglutinative, resistant to fish serum, adhesive to fish tissue culture, protease positive, hemolysin positive, and leukocytolysin positive) and three avirulent strains (which were nonagglutinative, nonhemagglutinative, sensitive to serum, nonadhesive, protease positive, hemolysin positive, and leukocytolysin positive). A protease-deficient mutant (NTG-1) was induced by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine from virulent strain A-7301, showing protease production, which was common to the two strain groups. Strain NTG-1 showed loss of virulence in determinations of 50% lethal doses, although it remained autoagglutinative, hemagglutinative, serum resistant, adhesive, hemolysin positive, and leukocytolysin positive. A protease fraction separated from extracellular products of strain A-7301 by DEAE-cellulose column chromatography had the capacity to produce skin lesions (furuncles) and high mortality in sockeye salmon. A comparable protein fraction from extracellular products of NTG-1 resulted in no protease activity and no pathological effects on the fish. The avirulent strains were eliminated from rainbow trout in a short time, whereas the virulent strains (including A-7301) were highly infective and proliferated in hosts. NTG-1 preserved its infectivity, but fish showed no signs of disease and no mortality. These findings indicate that extracellular protease is a major virulence factor and that protease production in the host is closely implicated in the pathogenesis of fish furunculosis.

Relationship between agglutinative properties of Aeromonas salmonicida strains isolated from fish in Japan and their resistance to mechanisms of host defense.

Phenotypic variation in spontaneous agglutination of Aeromonas salmonicida was investigated using 114 strains isolated from naturally epizootics of furunculosis in salmonid fish in Japan from 1967 through 1982. These strains were categorized into three different types: an agglutinating (AG) type which produced cell flocks resulting in precipitation and loss of turbidity in cell suspensions; a nonagglutinating (NAG) type, representing no loss in turbidity; and an intermediate (IM), weakly autoagglutinating type. Based on the occurrence of the AG, NAG and IM types determined in 1978 and again 1982, it was shown that a transition occurs progressing from the AG to the NAG via the IM type. The strains involved in the AG and NAG types were virulent and avirulent, respectively, based on LD50 determination in rainbow trout (Salmo gairdneri). The NAG type was much less susceptible than the AG to the bactericidal activity of fresh normal rainbow trout serum, to immune bacteriolysis and to phagocytosis by cells from pertioneal exudate. When growth properties of AG and NAG types were compared under mixed culture conditions, the NAG type predominated in the culture broth 2-5 days after inoculation. In contrast, when rainbow trout were inoculated intraperitoneally with cells of both types, the NAG-type cells were selectively eliminated from the kidneys within a period of a few days. The AG-type cells, however, greatly proliferated in the kidneys and brought about clinical furunculosis. These results indicate that the AG type possesses a capacity to escape the defense mechanisms of the host by resisting the bactericidal activity of serum and phagocytosis by leucocytes.

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.

Quantitative and Qualitative Studies of Aeromonas salmonicida Bacteriophage

A comprehensive bacteriophage typing scheme for Aeromonas salmonicida, the causal agent of furunculosis in fish, is described. It distinguishes 27 bacterial groups based on sensitivity patterns to 18 bacteriophage isolates. In addition, the sensitivity patterns are shown to reflect the morphological characteristics of the host bacterium. The ‘rough’,‘smooth’ and ‘G-phase’ forms possess different quantities of lipopolysaccharide in the cell wall and this influences bacteriophage attachment. The problems related to these morphological variations are discussed.