Prevention of Ulcer Disease in Goldfish by Means of Vaccination

Abstract

A vaccine comprising cells of Aeromonas bestiarum grown in tryptic soy broth and atypical A. salmonicida cells produced in iron-limited and iron-supplemented media protected goldfish Carassius auratus when administered by immersion (dosage ≈ 5 × 10⁷ cells/mL for 60 s) followed after 28 d by an oral booster (dosage = 5 × 10⁷ cells/g of feed), which was fed for 7 d so that each fish received about 1 g of vaccine-containing feed. After challenge by intramuscular injection of a virulent culture of atypical A. salmonicida, the relative percent survival (RPS) was more than 90%. The approach was more successful than using a commercial furunculosis vaccine with or without supplementation with A. bestiarum or atypical A. salmonicida cells. Moreover, a smooth derivative of the virulent rough culture of atypical A. salmonicida was less effective as a vaccine candidate, yielding an RPS of only 65%. Low antibody titers of 1:39–1:396 were found in the vaccinated fish. The vaccinated fish had a significantly higher proportion of dead head kidney macrophages (10.9 ± 3.5%; P = 0.0149) than did the controls (6.8 ± 3.1%). However, differences in the number of erythrocytes and leukocytes, the level of phagocytic and lysozyme activities, and the proportion of lymphocytes, monocytes, and polymorphonuclear cells were not statistically significant between the two groups.     

In Vitro Antimicrobial Activity of Sarafloxacin against Clinical Isolates of Bacteria Pathogenic to Fish

Abstract

An in vitro susceptibility assay of sarafloxacin (A-56620), a new 4-quinolone, was performed against five important bacterial species that are pathogenic to fish. A collection of 44 clinical isolates and five corresponding type strains were included in the study. The objectives were to determine the minimal inhibitory concentrations (MICs) of sarafloxacin by a drug microdilution method and to compare the MIC values at two different temperatures, 4 and 15°C. Sarafloxacin was active against all species tested and showed the following mean MIC values at 15 and 4°C, respectively, against the bacterial pathogens investigated: Aeromonas salmonicida subspecies salmonicida, 0.029 and 0.045 μg/mL; atypical A. salmonicida, 0.053 and 0.041 μg/mL; Vibrio anguillarum, 0.085 and 0.054 μg/mL; V. salmonicida, 0.125 and 0.123 μg/mL; and Yersinia ruckeri, 0.023 and 0.031 μg/mL. The MICs ranged from 0.0025 μg/mL (or less) for two strains of A. salmonicida salmonicida to 0.32 μg/mL for one strain of atypical A. salmonicida and one strain of V anguillarum. A decrease in antimicrobial activity was observed as the incubation temperature was lowered from 15 to 4°C; however, no significant statistical difference between the measured values was demonstrated.     

Cell-Surface-Associated Properties of Fish Pathogenic Bacteria

Abstract

Pathogenicity assays showed that 33 of 42 potentially pathogenic strains of bacteria tested were virulent to rainbow trout Oncorhynchus mykiss. Regardless of their degree of virulence to fish, strains of motile Aeromonas, A. salmonicida, and Vibrio anguillarum were moderately hydrophobic. Only 46 and 25°10 of the strains were able to hemagglutinate human and trout erythrocytes, respectively. Hydrophobicity and hemagglutination were practically absent in isolates of Yersinia ruckeri. A notable number of the strains positively adhered to salmonid (51%) and nonsalmonid (55%) fish cells. Whereas the treatment of the bacteria with proteinase K or trypsin did not decrease the hydrophobicity of the isolates, within motile Aeromonas and A. salmonicida species, strains with both protease-sensitive and -resistant hemagglutinating and adhesive abilities occurred. The effects of heat and sugars on hemagglutinating and hydrophobic properties varied within all bacterial groups. Although treatment of strains with D-mannose or L-fucose had distinct effects on adhesiveness according to the bacterial species and the cell system used, none of the heat-treated (80°C for 15 min) bacteria lost their capacity to adhere to cultured fish cells. The results showed that there was no direct relationship between any of the cell surface properties analyzed and the degree of virulence of the strains.     

Effect of Low Dietary Magnesium on Immune Response and Osmoregulation of Atlantic Salmon

Abstract

The influences of dietary magnesium on immune response and on osmoregulation in parr of Atlantic salmon Salmo salar were determined. Groups of fish were fed a casein–gelatin diet unsupplemented (containing about 200 mg Mg/kg) or supplemented with either 300 or 500 mg Mg/kg dry diet (as MgSO₄) for 12 weeks before vaccination to produce fish with different Mg levels, and the feeding regime was continued throughout the study. No differences were observed between the treatment groups in serum-specific antibody levels every second week for 8 weeks after vaccination against Vibrio anguillarum serotypes O1 and O2. Both lysozyme levels and spontaneous hemolytic activities in serum were elevated in vaccinated fish compared with unvaccinated fish. Neither lysozyme activity, complement hemolytic activity, total protein in serum nor blood hemoglobin were affected by dietary Mg. The spontaneous hemolytic activity in serum was lower in fish fed the unsupplemented diet and highest in fish fed the diet supplemented with 500 mg Mg/kg. After 28 weeks on the diets supplemented with graded levels of Mg, a salinity tolerance test (32.5 g/L, 24 h) was performed. High mortality and elevated serum chloride concentrations in all groups after 24 h reflected a general salinity intolerance, but the highest serum chloride level was observed in fish fed the unsupplemented diet. This indicates that low dietary Mg affects the osmoregulation of Atlantic salmon.     

Communications: Protective Immunity Induced by a Commercial Vibrio Vaccine in Hybrid Striped Bass

Abstract

Commercial Vibrio anguiliarum-V. ordalii bacterin was used to vaccinate hybrid striped bass (Morone saxatilis ♀ × M. chrysops ♂) to test the vaccine efficacy against vibriosis. Vaccination by direct immersion of fish in diluted Vibrio vaccine for 20 s resulted in increased protective immunity. The relative percent survival of hybrid striped bass challenged 35 d after vaccination was 66.7% for those challenged by 1-h immersion exposure to 7.03 × 10⁷ V. anguillarum cells/mL, 75.0% for those challenged by injection with 3.51 × 10⁵ cells/fish, and 86.7% for those challenged by injection with 3.51 × 10⁴ cells/fish.     

Occurrence and Distribution of Aeromonas salmonicida Infections on Swedish Fish Farms, 1951–1987

Abstract

The occurrence of typical and atypical Aeromonas salmonicida infections on Swedish fish farms was surveyed, and the methods for controlling the disease are described here. This review is based on data for a period of 36 years from fish health control studies conducted by the National Veterinary Institute and the Salmon Research Institute. The epizootiology and routes of transmission of the pathogens are described, and policy and methods for disease control are discussed. The distribution of atypical A. salmonicida infections was entirely different from that of typical infections. There was also circumstantial evidence of different routes of transmission for typical and atypical A. salmonicida infections. Although subclinical carriers of atypical A. salmonicida occurred among Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss on fish farms, acute disease outbreaks were most frequent among Arctic char Salvelinus alpinus and brown trout Salmo trutta. On the other hand, acute outbreaks of typical A. salmonicida infections were frequent among Atlantic salmon and rainbow trout.     

Nature of Aeromonas salmonicida Carriage on Asymptomatic Rainbow Trout Maintained in a Culture System with Recirculating Water and Fluidized Sand Biofilters

Abstract

An asymptomatic carrier population of rainbow trout Oncorhynchus mykiss was examined for Aeromonas salmonicida by primary dilution counts on Coomassie Brilliant Blue agar and also by streaking on bacteriological media after a 24–48-h pre-enrichment in tryptic soy broth. The pathogen was detected by primary dilution plate counts in 1 spleen, 15 gill, and 19 mucus samples of the 100 trout examined. Aerornonas samonicida was detected only after 48-h preenrichment in the one spleen that had already tested positive via primary dilution counts. The pathogen was not detected in kidney, liver, and intestinal samples. The occurrence of the pathogen in mucus and gills suggests a predominantly external nature of asymptomatic carriage of A. salmonicida within this population of fish. Repeated examination of fluidized biofilters and tank water showed that A. salmonicida did not become established in the recirculation system.     

Effect of Nanophyetus salmincola and Bacterial Co-Infection on Mortality of Juvenile Chinook Salmon

The freshwater trematode Nanophyetus salmincola has been demonstrated to impair salmonid immune function and resistance to the marine pathogen Vibrio anguillarum, potentially resulting in ocean mortality. We examined whether infection by the parasite N. salmincola similarly increases mortality of juvenile Chinook Salmon Oncorhynchus tshawytscha when they are exposed to the freshwater pathogens Flavobacterium columnare or Aeromonas salmonicida, two bacteria that juvenile salmonids might encounter during their migration to the marine environment. We used a two-part experimental design where juvenile Chinook Salmon were first infected with N. salmincola through cohabitation with infected freshwater snails, Juga spp., and then challenged with either F. columnare or A. salmonicida. Cumulative percent mortality from F. columnare infection was higher in N. salmincola-parasitized fish than in nonparasitized fish. In contrast, cumulative percent mortality from A. salmonicida infection did not differ between N. salmincola-parasitized and nonparasitized groups. No mortalities were observed in the N. salmincola-parasitized-only and control groups from either challenge. Our study demonstrates that a relatively high mean intensity (>200 metacercariae per posterior kidney) of encysted N. salmincola metacercariae can alter the outcomes of bacterial infection in juvenile Chinook Salmon, which might have implications for disease in wild fish populations.

Received February 24, 2015; accepted September 7, 2015     

Serological Analysis of Thermolabile Antigens of Vibrio anguillarum

Abstract

Serological analysis of thermolabile antigens of representative strains belonging to the J-O-1, J-O-2, and J-O-3 serotypes of Vibrio anguillarum, which are the major serotypes of thermostable antigens (O-antigens) in Japan, showed that the strains belonging to each O-serotype possessed a common thermolabile antigen designated as k-1. In addition to the k-1 antigen, strains of the J-O-1 serotype of biovar II V. anguillarum possessed another antigen designated as k-2. These findings were confirmed by examining additional strains belonging to each O-serotype. The k-1 antigen was found in all the strains of J-O-4, J-O-5, J-O-6, J-O-7, and J-O-8 serotypes, which are minor serotypes in Japan. The same antigen was also found in some strains of V. metschnikovii, Aeromonas hydrophila, and A. salmonicida.     

Journal of Aquatic Animal Health: Guide for Authors 2014

Abstract

Brook trout Salvelinus fontinalis were treated with single 60-min static baths of 250 mg formalin/L, 3% NaCl, and 15 mg Chloramine-T/L to evaluate the efficacy of these compounds against external infections of Aeromonas salmonicida. Prevalence of A. salmonicida was significantly lower in brook trout treated with Chloramine-T than among those treated with formalin or salt. Further laboratory tests substantiated the therapeutic value of a single treatment of ChloramineT (15 mg/L) against A. salmonicida. In two experiments, viable counts of A. salmonicida in mucus did not vary among replicate groups of treated brook trout, but the counts for treated fish were significantly (P < 0.05) lower than those for untreated controls. In vitro tube dilution assays indicated that mean minimum inhibitory concentrations of Chloramine-T for 10 isolates of A. salmonicida were 9.0 mg/L for 1 h and 2.25 mg/L, for 24 h. In field trials at the White River National Fish Hatchery (Bethel, Vermont), the pathogen was detected principally as an external infection of juvenile Atlantic salmon Salmo solar maintained in two culture ponds. In one pond, the bacterium accounted for 100% of the total distribution of tnicroflora isolated from mucus. Seven days after treatment with Chloramine-T, A. sahnonicida accounted for 11% of the total bacterial counts identified from these fish. In the second pond, A. salmonicida composed 3% of the counts of bacteria isolated from the mucus of fish before treatment but was not isolated after treatment.     

Effect of Vitamin E on the Immune Response of Channel Catfish to Edwardsiella ictaluri

Abstract

Three-month-old fingerling channel catfish Ictalurus punctatus were fed purified diets supplemented with ∝-tocopherol acetate to provide 0, 60, and 2,500 mg vitamin E/kg for 180 d. A 30-s immersion bath and an oral booster were used to deliver a bacterin of formalin-killed Edwardsiella ictaluri to half of the fish from each dietary treatment. Resistance of red blood cells to peroxidation was used as an index of antioxidant status. The susceptibility of red blood cells to oxidative hemolysis decreased with increasing levels of dietary vitamin E. Vaccinated and nonvaccinated fish were evaluated for agglutinating antibody titers and macrophage activity. Humoral antibody titers in response to E. ictaluri were significantly (P ≤ 0.05) higher in vaccinated fish than in nonvaccinated fish; however, no such differences in agglutinating antibody titers were detected among any of the dietary treatment groups. Both vaccination and vitamin E significantly enhanced the ability of macrophages to phagocytize virulent E. ictaluri. Results of this study indicate that elevated levels of dietary vitamin E may affect the ability of channel catfish to respond immunologically to bacterial pathogens.     

Monoclonal Antibody-Based Analysis of the Renibacterium salmoninarum p57 Protein in Spawning Chinook and Coho Salmon

Abstract

Renibacterium salmoninarum, causative agent of bacterial kidney disease of salmonid fish, produces large amounts of soluble proteins during infection and broth culture. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies was developed for the precise quantification of p57, a major component of these proteins. Kidney, spleen, blood, and reproductive fluids of adult Pacific salmon Oncorhynchus spp. were examined by means of the assay. Kidney and spleen harbored the highest concentrations of this antigen. In the populations of returning salmon tested, 5–25% of the fish had p57 concentrations above a baseline level of 3 ng antigenig tissue, and antigen concentrations as high as 200 μg/g tissue were detected in kidneys of individual fish. The ELISA was compared to direct fluorescent antibody analysis, in which rabbit anti-R. salmoninarum antiserum was used to identify infected fish. There was 99% agreement (199 of 201 examined fish) between the two methods. Western blot analysis was used to demonstrate that antigenic proteins present in infected fish were similar to those seen from antigen prepared from broth culture of R. salmoninarum, although less degradation of p57 appears to occur in vivo.     

Demonstration of the Specificity of the Salmonid Hurnoral Response to Renibacterium salmoninarum with a Monoclonal Antibody against Salmonid Immunoglobulin

Abstract

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytscha was compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarum preparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarum antibody.     

Thiaminase Activity of Crucian Carp Carassius carassius Injected with a Bacterial Fish Pathogen,Aeromonas salmonicida subsp. salmonicida

Abstract

Dietary thiaminase I is a cause of thiamine deficiency in animals. The physiological significance of thiaminase in the organisms containing this enzyme is not known, nor are the factors causing variation in their thiaminase activity. Tests were performed to evaluate the effect a pathogen might have on thiaminase activity in fish, when analyzed both with a cosubstrate added (CATA tests) and no cosubstrate added (NCATA tests). Pyridine is known as a cosubstrate specific for thiaminase I activity that does not accelerate thiaminase II activity. Crucian carp Carassius carassius known to harbor thiaminase I activity were injected intramuscularly with live Aeromonas salmonicida, a pathogenic bacterium of fish. For comparison, other groups were injected with formalin-killed bacteria and phosphate-buffered saline, respectively; an untreated group of fish was kept as a control. The bacteria did not contain any thiaminase activity. Significantly higher thiaminase activities (CATA and NCATA) were measured in all tissues (whole blood, injected muscle, uninjected muscle, and whole fish homogenates) of fish injected with live bacteria than in the saline-injected and the uninjected groups. The thiaminase activity of blood and that in the injected, inflamed muscle tissue followed different allocation patterns in fish injected with live A. salmonicida. The amount of thiaminase I enzyme appeared to be elevated in the whole blood of injected fish in the absence of natural cosubstrate(s). The thiaminase activity of the injected, inflamed muscle suggested that both the amount of thiaminase enzyme and some yet-unidentified natural cosubstrate(s) were elevated. This suggests that in addition to the enzyme, some cosubstrate(s) of fish or pathogen origin play a regulatory role in the so-far-unknown physiological significance of thiaminase I activity in vivo. It is suggested that the health of fish should be considered when searching for factor(s) affecting its thiaminase activity.     

Screening for the Fish Disease Agent Aeromonas salmonicida with an Enzyme-Linked Immunosorbent Assay (ELISA)

Abstract

Serological detection of bacterial pathogens in fish tissue is an important tool for surveying epidemiological situations. Whenever antibacterial treatment of fish is recommended, it becomes necessary, however, to culture the pathogen for sensitivity testing. An enzyme-linked immunosorbent assay (ELISA) was developed to identify Aeromonas salmonicida in culture. This serological identification can partly substitute for biochemical characterization of the organism and thus decrease the time between isolation and sensitivity testing by at least 3 d. The ELISA works with only one bacterial colony and yields results within 4 h. During this time, a bacterial suspension can be prepared for the resistance test. The specificity of an antiserum, raised in rabbits, against whole cells of A. salmonicida can be increased by adsorption with strains of cross-reacting species. However, difficulties arise when serologically heterogeneous species (e.g., A. hydrophila) are used as the cross-reacting bacterium. In the present study, severalfold adsorption with four isolates did not totally rule out cross-reactivity against additional strains. Therefore, the strain in question should also be checked for colony morphology, production of pigment, or presence of cytochrome oxidase to validate the serologically obtained result.     

Stress and Humoral Innate Immune Response of Gilthead Seabream Sparus aurata Cultured in Sea Cages

Innate and acquired immune responses of Gilthead Seabream Sparus aurata was studied under normal culture and short-term stressful conditions for 18 months in offshore sea cages in Alghero Bay, Italy. Every 45 d, 50 fish were sampled and divided into two groups: fish in the first group (normal culture conditions) were bled after harvesting; fish in the second group were put into a tank under stressful conditions (crowding and confinement) and bled after 2 h. Innate humoral immunity, such as complement-like, hemagglutination, and lysozyme activities, was determined in the sera of both groups. Pathogen challenge was not performed, but the specific humoral immune response was assessed against the most common pathogens affecting cultured fish in Sardinia. Stressed fish, compared with the control, showed a lower lysozyme activity against Vibrio (Listonella) anguillarum, which was not clearly correlated with temperatures. Complement-like activity differed between the first and second half of the study and, at the end of the trial, a slightly higher activity was recorded in the controls than in the stressed fish. Hemagglutination activity was mainly higher in the stressed fish than in control fish. Confinement, crowding, and cold water temperature caused decreased lysozyme activity in short-term stressed Gilthead Seabream compared with those reared normally. The specific humoral immune response, against V. anguillarum, Tenacibaculum mesophilum, Enterococcus Seriolicida, and Aeromonas sobria, fluctuated during the rearing period, particularly during the first year of culture.

Received October 12, 2015; accepted March 24, 2016 Published online August 2, 2016     

Infections of Edwardsiella tarda among Brook Trout in Quebec

Abstract

Acute bacterial septicemia is commonly diagnosed in brook trout Salvelinus fontinalis of Quebec, Canada. The agents most commonly isolated include Aeromonas salmonicida (furunculosis), Aeromonas hydrophila (motile aeromonad septicemia), and Pseudomonas species. Septicemia in brook trout caused by the gram-negative bacterium Edwardsiella tarda was diagnosed for the first time in the province of Quebec from two different fish farms producing stock for fee fishing establishments. Affected fish displayed nonspecific lesions associated with bacterial septicemia including hemorrhages on the gills and viscera and exophthalmia. Stress-associated immunosuppression due to an increase in summer water temperatures and lack of precipitation were considered as primary causes of these disease outbreaks.     

Host factors associated with the detection of Aeromonas salmonicida and Yersinia ruckeri in Ontario, Canada government fish hatcheries

This paper presents an epidemiological investigation of Ontario Ministry of Natural Resources Fish Health Laboratory data from 1981 to 1997, to determine whether fish species and age were associated with lot-level detection of Aeromonas salmonicida and Yersinia ruckeri in hatchery fish. In stepwise logistic regression, the species brook trout and back-cross (lake trout crossed with the hybrid “splake”) were more likely to test A. salmonicida-positive compared to all other species reared in the hatcheries. Similarly, the species brook trout was significantly more likely to test Y. ruckeri-positive compared to all other species. For both pathogens, the 1–5-month age group was associated significantly with detection. These findings suggest that purposive sampling of higher-risk fish lots could increase the likelihood of detecting both study pathogens.     

Occurrence of Aeromonas salmonicida,Renibacterium salmoninarum,and Infectious Pancreatic Necrosis Virus in Ontario Salmonid Populations

Abstract

The results of samples collected from private and government fish farms and wild and feral fish populations in Ontario from 1981 to 1995 were synthesized to obtain prevalence estimates in salmonids at both the fish and site levels for three pathogens. Renibacterium salmoninarum and Aeromonas salmonicida were both detected on at least one site for every year investigated. Ontario Ministry of Natural Resources (OMNR) culture stations had the highest percentages of sites with infected fish for R. salmoninarum. Natural water bodies had the highest percentages of sites with infected fish for A. salmonicida. Infectious pancreatic necrosis virus (IPNV) was only detected sporadically on some commercial farms and never in OMNR hatcheries or in wild or feral fish. Although screening for any virus that would yield cytopathological effect was carried out during all the years surveyed, no virus other than IPNV was isolated. The low prevalence and “source-specific” presence of IPNV in Ontario demonstrates the necessity of representative sampling for the detection of rare pathogens. It was estimated that, overall, less than 1% of all fish in the sampled populations were infected with each of the three pathogens for almost every year studied. The importance of summarizing pathogen-testing data and the possible implications on disease control policy planning and assessment are discussed.     

Disease Susceptibility of Hatchery Snake River Spring–Summer Chinook Salmon with Different Juvenile Migration Histories in the Columbia River

Abstract

Various methods have been developed to mitigate the effects of dams on juvenile Pacific salmon Oncorhynchus spp. migrating to the Pacific Ocean through the Columbia River basin. In this study, we examined the health of hatchery Snake River spring and summer Chinook salmon relative to two mitigating strategies: dam bypass and transportation (e.g., barging). The health of out-migrants was assessed in terms of the difference in the incidence of mortality among fish, categorically grouped into no-bypass, bypass, and transportation life histories, in response to challenge with the marine pathogen Listonella anguillarum during seawater holding. These three life histories were defined as follows: (1) fish that were not detected at any of the juvenile bypass systems above Bonneville Dam were classified as having a no-bypass life history; (2) fish that were detected at one or more juvenile bypass systems above Bonneville Dam were classified as having a bypass life history; and (3) fish that were barged were classified as having the transportation life history. Barged fish were found to be less susceptible to L. anguillarum than in-river fish—whether bypassed or not—which suggests that transportation may help mitigate the adverse health effects of the hydropower system of the Columbia River basin on Snake River spring–summer Chinook salmon. The findings of this study are not necessarily transferable to other out-migrant stocks in the Columbia River basin, given that only one evolutionarily significant unit, that is, Snake River spring–summer Chinook salmon, was used in this study.