Protection against carp erythrodermatitis following bath or subcutaneous exposure to sublethal numbers of virulent Aeromonas salmonicida subsp. nova

Abstract. A bath challenge system was used to infect carp. Cyprinus carpio L., with Aeromonas salmonicida subsp. nova , the causative agent of carp cruthrodermatitis. Bath-challenged fish became infected with the bacterium exihibitinng typical signs of the disease, Carp that were sublethally bath exposed became infected and exhibited some skin lesions, but after one week, these quickly healed and the animals fully recovered from the infection, Naive fish that had not been previously exposed to the bacterium had mortalities of 100% when infected by the subcutaneous route and 40–60% by the bath route of infection. Carp that received sublethal infections were able to withstand subsequent lethal infection and recover regardless of the route of infection. Sublethally bath-exposed carp were protected from subsequently lethal challenges of A. salmonicida subsp. nova for at least 5 months.     

Aeromonas salmonicida: determination of an antigen associated with protective immunity and evaluation of an experimental bacterin

Abstract. Protection against Aeromonas salmonicida was determined by passive immunization and with various bacterin preparations. Rabbit antiserum was prepared against a rough, virulent strain of A. salmonicida (AS-1R), the same strain boiled (AS-1R, boiled), and an avirulent, smooth strain of this same isolate (AS-1S). Cross-absorption, cross-passive protection and analysis by counter immunoelectrophoresis of various extraction methods were studied. It was shown that AS-1R cells contained an additional antigen not present in AS-1R (boiled) and AS-1S cells. Antiserum to the AS-1R antigen passively protected sockeye salmon, Oncorhynchus nerka (Walbaum), against a virulent challenge, and antisera to AS-1R (boiled) and AS-1S were not protective. The antigen was not destroyed by formalin or heat at 5°C for 60 min, but it appeared to be partially inactivated with proteolytic enzymes. The antigen was produced in casein yeast beef (CYB) broth up to 32 h but not thereafter, and low yields were obtained in tryptic soy or brain heart infusion (BHI) broth. It was extracted from cells with ethylenediamine tetraacetic acid (EDTA) and especially alkaline hydrolysis, but not with proteolytic enzymes or detergents. The detergents appeared to destroy the antigen. We concluded that the antigen was protein and is most likely the external A-protein (AP) reported for rough, virulent strains of A, salmonicida. Various methods of preparing A. salmonicida bacterins were evaluated by determining the level of protective immunity induced in intraperitoneally (i.p.) vaccinated fish. Growth of cells in CYB or BHI broth resulted in production of only rough (autoagglutinated in saline) variants of A. salmonicida. Although only rough variants were associated with protective immunity, one strain was not protective, it was avirulent by bath challenge. Bacterins prepared in CYB were more efficacious than those grown in BHI, but inactivation with formalin, iodine, or glutaraldehyde worked equally well. However, boiling the bacterin or filtering the cells from the bacterin removed its efficacy. Methods of releasing the AP were evaluated by sonification, pH-lysis, disaggregation and treatment with EDTA, and all treatments worked equally well. Also, precipitation on to aluminium or use of Freund's complete adjuvant did not significantly improve the protection. In parenterally vaccinated fish, protection was demonstrated by challenging the fish at various levels by bath, injection or cohabitation with infected fish. The best protection was demonstrated using the cohabitation challenge method. The potency and field efficacy of an A. salmonicida bacterin prepared in CYB broth and extracted with 5 mM EDTA was evaluated. Fish were vaccinated by i.p, injection and potency was determined in the laboratory by experimental challenge and in the field by natural challenge. Chinook salmon, O.ishawytschu (Walbaum), developed immunity within seven days at 10°C. The bacterin could be diluted up to 1:2000 without loss of potency. The field tests results were equivocal; however, (he prevalence of infection was lower in vaccinated fish.     

Potential for immersion vaccination against Aeromonas salmonicida

Abstract. The efficacy of the immersion method of vaccination was evaluated using Aeromonas salmonicida bacterin. In general a 2 min immersion vaccination was not as effective as vaccination by intraperitoneal injection; however, the level of immunity was improved by giving multiple vaccinations several days apart. A primary immersion vaccination with bacterin diluted 1:4 followed by a secondary vaccination diluted 1:2 gave good results. The most concentrated secondary booster was more effective than boosting with more dilute bacterin.     

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.     

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.     

Fluoroquinolones display rapid bactericidal activity and low mutation frequencies against Aeromonas salmonicida

Abstract. Oxolinic acid and five new fluoroquinolones, sarafloxacin, enrofloxacin, PD127, 391, PD117, 596 and C1934, were evaluated for in vitro activity against Aeromonas salmonicida. Sarafloxacin, enrofloxacin, PD127, 391 and PD117, 596 were significantly more active than cither oxolinic acid or C1934 in terms of their bactericidal activity against both oxolinic-acid-resistant and sensitive strains of A. salmonicida , killing 99% of the bacteria after 1h exposure. Resistant mutants developed at lower frequency to the fluoroquinolones than to oxolinic acid or oxytctracycline at 5, 10 and 20 times the MIC. However, mutants selected with one quinolone were cross-resistant to all the other quinolones tested.     

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.     

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.     

Cell-mediated protection in carp, Cyprinus carpio L., against Aeromonas hydrophila

Abstract. The mechanism of protection against Aeromonas hydrophila infection in carp was studied. Recipient fish, into which pronephric cells from carp previously immunized by dipping in bacterial crude lipopolysaccharide (LPS) at 25°C for 2h were transferred, demonstrated almost the same level of protective ability as an immunized control group. The protective ability was transmitted by non-adherent (to nylon fibre) immune pronephric cells. These non-adherent cells were damaged by anti-carp thymocyte serum and were, thus, considered to be T-like cells. The protective ability was depressed in immunized carp treated with anti-carp thymocyte serum in vivo and was also remarkably reduced in immunized carp whose macrophage function was impaired by Dextran sulphate-500 treatment. These results indicate that the protection shown by carp immunized by dipping in crude LPS is dependent on cellular immunity regulated by a T-like cell-macrophage system.     

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.     

Tissue localization of Aeromonas salmonicida in Atlantic salmon, Salmo salar L., following experimental challenge

Three groups of Atlantic salmon, Salmo salar L., were exposed to live, colony-forming, radiolabelled Aeromonas salmonicida bacteria in a bath challenge: (1) fish with artificial wounds; (2) fish with a reduced epidermal mucus layer caused by removal of the mucus layer on two occasions by a swabbing procedure; and (3) a control group of untreated fish. Fish were killed 2, 6 and 24 h after challenge, and radioactivity (cpm g^–1) was measured in the blood, mucus, skin, wound area, gills, anterior kidney, posterior kidney, spleen, midgut and hindgut. The highest levels of radioactivity were measured in the wound areas and in the gills. There was a significant positive correlation between the levels of radioactivity in the gills and blood, and between the mucus and skin at 2 h post-challenge. Two hours after the bath challenge, live A. salmonicida bacteria were found in the blood of fish in the 'swabbed' and 'artificial wound' groups, and not in the control group. Twenty-four hours after the bath challenge, the kidney of fish from all groups contained viable bacteria, whereas the blood was negative.     

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.     

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.     

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.     

Enhancement of protective immunity in blue gourami,Trichogaster trichopterus (Pallas), against Aeromonas hydrophila and Vibrioanguillarum by A. hydrophila major adhesin

Blue gourami, Trichogaster trichopterus (Pallas), were intraperitoneally immunized with major adhesin, a 43 kDa OMP protein isolated from fish Aeromonas hydrophila, in the presence of Freund's complete adjuvant (FCA). Three weeks later, a booster injection of adhesin without FCA was administered. Control group fish were similarly treated with phosphate‐buffered saline (PBS) and FCA. Results showed that anti‐adhesin serum obtained from fish after booster immunization exhibited very strong ability in agglutinating bacterial cells. Although this antiserum had no bactericidal effect, it could significantly inhibit serologically different strains of A. hydrophila from invading EPC (Epithelioma papillosum of carp) cells in vitro. In addition, the proliferative response of head kidney leucocytes of these immune fish was significantly increased as compared to that of the control. The results also showed that the major adhesin could provide significant protective immunity to fish against the challenge by homologous and heterologous strains of A. hydrophila and one virulent strain of Vibrio anguillarum.     

Immunological responses in Atlantic salmon, Salmo salar L., against purified serine protease and haemolysins from Aeromonas salmonicida

Abstract. A method for purification of the 70-KDa extracellular serine protease of Aeromonas salmonicida by hydrophobic chromatography and ion exchange is described. The purified protease, adsorbed onto mineral particles, was used for immunization of salmon. Other groups of salmon were immunized with particles coated with purified glycerophospholipid:cholesterol acyltransferase (GCAT) or with the acyltransferase complexed with LPS (GCAT-LPS). Humoral immune responses assayed after 6 weeks by ELISA, showed relatively good responses against GCAT-LPS, while titres of antisera against the purified protease and GCAT were scarcely above those of control sera. However, the antibody responses to each toxin were shown by Western blotting to be specific and qualitatively similar to responses seen in rabbits. The toxin preparations were also used (in combination with whole bacteria) for vaccination of salmon. On challenge 3 months later, only GCAT-LPS elicited significant immunological protection. However, a more convincing protection was seen when total extracellular product was present in the vaccine.     

The role of Aeromonas salmonicida extracellular products in the pathology of furunculosis

Abstract. The extracellular products (ECP) of Aeromonas salmonicida , prepared by the cellophane overlay method, are lethal to rainbow trout when administered parenterally. Sublethal doses when injected i.p. or i.m. are shown to reproduce all the lesions that have been described in the literature as being associated with furunculosis. In addition, meningitis may be an important feature of furunculosi s and is reproduced by injection of ECP. A serum factor, probably an α-globulin, present in normal serum of rainbow trout, is capable of neutralizing the toxic effects of ECP. The pathology is discussed with reference to the proteolytic and leucocidal properties of the ECP and its effects upon the eosinophilic granular cells which are caused to disperse and degranulate with the possible release of histamine.     

Aeromonas salmonicida isolated from wild and farmed fish and invertebrates in Oman

International Aquatic Research - Aeromonas salmonicida was isolated from red spot emperor, king soldier bream, white-spotted rabbit fish and tilapia, and an invertebrate (abalone) in Oman during...