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.     

The protective effect of humic‐rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.)

When challenged with atypical Aeromonas salmonicida subsp. salmonicida, exposure of the common carp (Cyprinus carpio L.) to different humic‐rich compounds resulted in a significant reduction in infection rates. Specifically, in fish exposed to (i) humic‐rich water and sludge from a recirculating system, (ii) a synthetic humic acid, and (iii) a Leonardite‐derived humic‐rich extract, infection rates were reduced to 14.9%, 17.0% and 18.8%, respectively, as compared to a 46.8% infection rate in the control treatment. An additional set of experiments was performed to examine the effect of humic‐rich components on the growth of the bacterial pathogen. Liquid culture medium supplemented with either humic‐rich water from the recirculating system, the synthetic humic acid or the Leonardite humic‐rich extract resulted in a growth reduction of 41.1%, 45.2% and 61.6%, respectively, as compared to the growth of the Aeromonas strain in medium devoid of humic substances. Finally, in a third set of experiments it was found that while the innate immune system of the carps was not affected by their exposure to humic‐rich substances, their acquired immune system was affected. Fish, immunized against bovine serum albumin, displayed elevated antibody titres as compared to immunized carps which were not exposed to the various sources of humic substances.     

Prophylactic effect of β(1,3)-D-glucan (laminaran) against experimental Aeromonas salmonicida and Vibrio salmonicida infections

Immersion and intraperitoneal vaccination are the two most common and successful methods for vaccination of fish (Holm & Júrgensen 1987; Lillehaug 1989). Effective vaccines and the improvement of environmental conditions have dramatically reduced the amount of antibiotics used in aquaculture in Norway from 48.5 t in 1987 to 1.03 t in 1996. Nevertheless, the labour intensive process of vaccination and the often undesirable side-effects of the adjuvants incorporated in injectable vaccines (Anderson 1992; Mulvey, Landolt & Busch 1995; Midtlyng, Reitan, Speilberg 1996), have resulted in a search for alternative approaches to achieve immune stimulation. Hence. there is growing interest in the development of immunomodulators that do not cause or have only minor unwanted effects on the host and preferably can be given as a feed additive. It is widely acknowledged that the beneficial (or in some instances undesirable) effects of immunomodulators such as b-glucans and bacterial products in an infection are mainly a result of their stimulatory effects on macrophages (Johnston 1978; Chung & Secombes 1988; Júrgensen, Lunde & Robertsen 1993).     

Differences in resistance of carp, Cyprinus carpio L., to atypical Aeromonas salmonicida

Abstract. The degree of resistance to an atypical strain of Aeromonas salmonicida , the causative bacterium of carp erythrodermatitis, was examined in two strains of carp, Cyprinus carpio L.: a Polish line. R3, sixth generation of conventional inbreeding (full-sib matings); and a Hungarian line. R8, fifth generation of conventional inbreeding. Comparisons were made between and within the two strains. Results showed a significant difference ( P < 0·001) in the degree of resistance, with the Hungarian carp showing greater resistance than the Polish carp. Differences within each strain were also observed indicating a maternal influence on resistance. Two transferrin genotypes in three genetic combinations were identified (DD, DG, GG) but were not found to influence resistance.     

An experimental model for fish furunculosis caused by Aeromonas salmonicida

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

Antibiotic protection against recrudescence of latent Aeromonas salmonicida during furunculosis vaccination

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

The 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.     

被动转移免疫在鲫感染温和气单胞菌中的作用

实验用鲫平均体重100g，采用免疫物质被动转移试验，探讨体液免疫在鲫感染温和气单胞菌（Aeromonas sobria)中的保护作用。结果表明，无论免疫物质是来自异源还是自源，是由相关的嗜水气单胞菌（A.hydrophia)诱导产生还是由攻毒的温和气单胞直接刺激，均具有良好的免疫保护作用。实验还发现，再次攻毒时，在所有抗体组的鲫存活率为100%时，全血被动转移组的死亡率高达55.6%；免疫前后免疫复合物发生明显变化。说明抗体在保护鲫免疫受温和气单胞菌的致死性感染中发挥决定性的作用。     

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.     

Autoaggregation and extracellular A-layer protein in Aeromonas salmonicida

Fifteen strains of Aeromonas salmonicida were examined for the presence of an extracellular protein A-layer. The presence of an A-layer has been associated with the property of bacterial autoaggregation. However, three of the ten autoaggregating strains examined in this study showed no detectable A-layer subunit protein.     

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.