Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non-salmonid fish.

Michigan's fisheries rely primarily upon the hatchery propagation of salmonid fish for release in public waters. One limitation on the success of these efforts is the presence of bacterial pathogens, including Aeromonas salmonicida, the causative agent of furunculosis. This study was undertaken to determine the prevalence of A. salmonicida in Michigan fish, as well as to determine whether biochemical or gene sequence variability exists among Michigan isolates. A total of 2202 wild, feral and hatchery-propagated fish from Michigan were examined for the presence of A. salmonicida. The examined fish included Chinook salmon, Oncorhynchus tshawytscha (Walbaum), coho salmon, O. kisutcha (Walbaum), steelhead trout, O. mykiss (Walbaum), Atlantic salmon, Salmo salar L., brook trout, Salvelinus fontinalis (Mitchill), and yellow perch, Perca flavescens (Mitchill). Among these, 234 fish yielded a brown pigment-producing bacterium that was presumptively identified as A. salmonicida. Further phenotypic and phylogenetic analyses identified representative isolates as Aeromonas salmonicida subsp. salmonicida and revealed some genetic and biochemical variability. Logistic regression analyses showed that infection prevalence varied according to fish species/strain, year and gender, whereby Chinook salmon and females had the highest infection prevalence. Moreover, this pathogen was found in six fish species from eight sites, demonstrating its widespread nature within Michigan.

Association of Cytophaga psychrophila with mortality among eyed eggs of Atlantic salmon (Salmo salar).

Although Pseudomonas fluorescens was the predominant bacterium associated with Atlantic salmon (Salmo salar) eggs incubated at the White River National Fish Hatchery (Bethel, Vermont) during January 1992, the fish pathogen Cytophaga psychrophila was isolated only from specific lots of eggs that displayed poor survival (35% eye-up).

Relationship between resistance of salmonids to furunculosis and recovery of Aeromonas salmonicida from external mucus.

Fish were sampled at the Ed Weed State Fish Hatchery (South Hero, Vermont, USA) in September 1992. Aeromonas salmonicida was common, with concentrations as high as 10(5) to 10(7) colony-forming units per gram of mucus, and readily recovered from most mucus samples obtained from furunculosis-sensitive populations of brook trout (Salvelinus fontinalis), lake trout (Salvelinus namaycush), and Atlantic salmon (Salmo salar). The pathogen was the predominant microorganism and accounted for greater than 85% of the total number of bacteria isolated from the mucus of these fish. By comparison, A. salmonicida was recovered only from two rainbow trout (Oncorhynchus mykiss), and bacterial frequencies did not exceed 10(3) colony-forming units per gram of mucus. The pathogen was not recovered from the mucus of steelhead (O. mykiss) or Rome brown trout (Salmo trutta) selectively bred for resistance to furunculosis, even though there was widespread contagion throughout the hatchery and fish were cultured on a common, unprotected water supply.

Isolation of Aeromonas salmonicida from paddlefish, Polyodon spathula.

Aeromonas salmonicida was isolated from paddlefish (Polyodon spathula) mortalities collected during an epizootic of furunculosis at the Spring River State Hatchery, Arkansas (USA), in 1992. Isolates of the bacterium were obtained from culture of gill and kidney tissue. This is the first epizootic of bacterial etiology to be reported in paddlefish.

Molecular and genetic characterization of cytochrome oxidase-negative Aeromonas salmonicida isolated from coho salmon (Oncorhynchus kisutch).

Cytochrome oxidase variants of the bacterial fish pathogen, Aeromonas salmonicida, were characterized for genetic and molecular homology with cytochrome oxidase-positive isolates that typically induce furunculosis in salmonids. Protein and lipopolysaccharide moieties of the cytochrome oxidase-negative variants were similar to their typical counterparts, based on sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. Pathogenicity of aberrant isolates to brook trout (Salvelinus fontinalis) was similar to typical cytochrome oxidase-positive isolates. Colorimetric deoxyribonucleic acid (DNA) hybridization in 96-well microplates yielded homology values greater than 82.5% for typical aberrant A. salmonicida isolates when photobiotinylated DNA for reference A. salmonicida 3.101 was used as a probe. The only variation of these isolates from typical A. salmonicida was a negative cytochrome oxidase reaction.

Isolation and phenotypic characterization of an oxidase-negative Aeromonas salmonicida causing furunculosis in coho salmon (Oncorhynchus kisutch).

An oxidase-negative Aeromonas salmonicida was isolated from coho salmon (Oncorhynchus) kisutch) suffering from an epizootic of furunculosis at the state hatchery near Belfair, Washington. Typical, oxidase-positive A. salmonicida was isolated concurrently from the same population of fish. Mortality was controlled with medicated feed treatments. Evidence supporting the identification of the two types of A. salmonicida is presented. Methods for the proper identification of oxidase-negative A. salmonicida isolates are evaluated.

Serological investigation of the fish pathogen Edwardsiella ictaluri, cause of enteric septicemia of catfish.

The serological relationships among 32 isolates of Edwardsiella ictaluri obtained from fish were studied. The strains were extremely homogeneous in protein and lipopolysaccharide preparations as observed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. Only minor variations were observed in the structural O-side chain subunits in three isolates; however, such variation did not preclude antigenic recognition by two E. ictaluri antisera in either microagglutination or Western blot immunoassays. The antigenic homogeneity of E. ictaluri was further demonstrated by microagglutination assays with both formalin-killed and heat inactivated cellular antigens. The minimal degree of antigenic variability observed suggested that most isolates of E. ictaluri compose a single antigenic serotype.

Selection for virulence in the fish pathogen Aeromonas salmonicida, using Coomassie Brilliant Blue agar.

Coomassie Brilliant Blue Agar was used to quantify the frequency of the A-layer phenotype in different isolates of Aeromonas salmonicida. Hydrophilic, non-clumping isolates of A. salmonicida consisted predominantly of the A-layer minus phenotype. These bacteria were avirulent by intraperitoneal injection into susceptible brook trout (Salvelinus fontinalis) and could not be reisolated from infected fish. By contrast, hydrophobic, clumping isolates were predominantly of the A-layer positive phenotype, highly virulent in brook trout, and easily recovered from dead or moribund fish. A-layer positive and negative clones of A. salmonicida were derived by plating bacteria on Coomassie Blue Agar. The plating showed clearly that Coomassie Blue Agar could be used as a highly selective in vitro screening method to reclaim the virulence of certain isolates of A. salmonicida having a relatively low percentage of A-layer positive phenotypes.

Immunization of salmonids against Yersinia ruckeri: significance of humoral immunity and cross protection between serotypes.

Brook trout (Salvelinus fontinalis) were immunized with bacterins containing either Serotype 1 or 2 isolates of Yersinia ruckeri to determine the relative degree of cross-protection afforded when the fish were challenged with the homologous or heterologous serotype. While fish immunized with pH-lysed bacterins produced highly specific agglutinins that did not cross-react with antigens derived from a heterologous serotype of Y. ruckeri all fish were protected against experimental challenge, regardless of which serotype was used for bacterin production and experimental challenge. Other experiments indicated that brook trout injected intraperitoneally with highly specific antibodies could not be passively immunized against experimental challenge.

An epizootic in chinook salmon (Oncorhynchus tshawytscha) caused by a sorbitol-positive serovar 2 strain of Yersinia ruckeri.

Enteric redmouth disease is described in chinook salmon (Oncorhynchus tshawytscha) at a state hatchery in Sand Ridge, Illinois. Biochemical, isoenzyme, and serological data indicated that the epizootic was caused by a sorbitol-fermenting Serovar 2 strain of Yersinia ruckeri. In laboratory experiments the isolate was pathogenic for both brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar).

Detection of Vibrio anguillarum antigen by the dot blot assay.

The dot blot assay, modified and adapted for detection of antigens from Vibrio anguillarum in fish tissues, was specific for V. anguillarum and did not react with antigens of V. ordalii, Pseudomonas sp., or Yersinia ruckeri. The blot assay enabled detection of as little as 2.3 ng of a mixture of protein antigens obtained from cell-free extracts of V. anguillarum; it was about 100 times more sensitive than either the indirect fluorescent antibody technique or bacterial isolation for detecting V. anguillarum in fish tissues.

Comparative sensitivities of diagnostic procedures used to detect bacterial kidney disease in salmonid fishes.

Kidney and spleen homogenates from each of 60 coho salmon (Oncorhynchus kisutch) and steelhead trout (Salmo gairdneri) were examined for detection of Renibacterium salmoninarum. The proportions of positives differed widely with the detection procedures used: in coho salmon, 5% were positive by the Gram-stain procedure, 10% by the direct fluorescent antibody test, 48% by bacteriological isolation, 65% by staphylococcal coagglutination, and 73% by counterimmunoelectrophoresis; in steelhead trout, 3% were positive by Gram-stain, 8.3% by fluorescent antibody, 17% by bacteriological isolation, and 67% by counterimmunoelectrophoresis. Renibacterium salmoninarum was not detected in either coho salmon or steelhead trout by immunodiffusion analysis.