Rocco C. Cipriano

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.

Intraovum Infection Caused by Flavobacterium psychrophilum among Eggs from Captive Atlantic Salmon Broodfish

Abstract This study indicated that the bacterium Flavobacterium psychrophilum induced an infection within eggs of Atlantic salmon Salmo salar that were held at federal New England restoration facilities. The pathogen, which originated from the Connecticut, Penobscot, Machias, East Machias, Dennys, Narraguagus, and Sheepscot rivers, was obtained from these eggs at concentrations that ranged from 5.0 × 102 to 2.5 × 108 colony-forming units per gram of egg, despite successive treatments with povidone iodine (I2). Treatments consisted of 50 mg/L of water for 30 min, then 100 mg/L for 10 min, followed at the eyed egg stage by 100 mg/L for 60 min. Collectively, 63% of the egg lots (77 of 122) obtained from paired matings of these captive broodfish were infected; 39% of these lots contained 10 or fewer infected eggs (60 eggs sampled per lot), and less than 10% of the lots contained more than 20 positive eggs. Consequently, standard iodophor disinfection procedures were ineffective. Eggs were positive from each o...

Comparative analysis of Edwardsiella isolates from fish in the eastern United States identifies two distinct genetic taxa amongst organisms phenotypically classified as E. tarda.

Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, has been implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated PCR (rep-PCR) using 4 different primer sets (ERIC I & II, ERIC II, BOX, and GTG5) and multi-locus sequence analysis of 16S SSU rDNA, groEl, gyrA, gyrB, pho, pgi, pgm, and rpoA gene fragments identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Isolates that fell into DNA group II demonstrated more similarity to E. ictaluri than DNA group I, which contained the reference E. tarda strain (ATCC #15947). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Fluorometric determination of G+C content demonstrated 56.4% G+C content for DNA group I, 60.2% for DNA group II, and 58.4% for E. ictaluri. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II may represent an unrecognized, genetically distinct taxa of Edwardsiella that is phenotypically indistinguishable from E. tarda.

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.

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.

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.

Reappraisal of the Federal Fish Health Recommendation for Disinfecting Eggs of Atlantic Salmon in Iodophor

Abstract The U.S. Fish and Wildlife Service federal protocol for dual disinfection of fish eggs in 50 mg/L iodine solution for 30 min followed by a secondary disinfection in 100 mg/L iodine for 10 min was investigated during six spawning cycles of Atlantic salmon Salmo salar held at the Richard Cronin National Salmon Station (Sunderland, Massachusetts). This population of salmon had undergone an epizootic of furunculosis, and the surviving fish maintained a persistent infection of Aeromonas salmonicida throughout the course of study. Eggs from 20 individual paired matings of salmon were obtained annually during the first 2 weeks of November in each spawning cycle from 1995 through 2000 except for 1999, when fertilized eggs from 35 pairs of salmon were examined. Aeromonas salmonicida was isolated from 19 of the total 135 groups of fertilized eggs investigated during this study. In those cases, all isolations of the pathogen were made only in fertilized eggs that had not yet undergone disinfection in iodoph...

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).

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).