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Yersinia Ruckeri, the Enteric Redmouth Bacterium S

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Yersinia Ruckeri, the Enteric Redmouth Bacterium S INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1988, p. 49-55 Vol. 38, No. 1 0020-7713/88/010049-07$02.0010 Copyright 0 1988, International Union of Microbiological Societies Deoxyribonucleic Acid Relatedness of Serovars of Yersinia ruckeri, the Enteric Redmouth Bacterium S. A. DE GRANDIS, P. J. KRELL, D. E. FLETT, AND R. M. W. STEVENSON* Department of Microbiology, University of Guelph, Guelph, Ontario NI G 2 WI, Canada Received 20 July 1987lAccepted 20 October 1987 The name Yersinia ruckeri was initially applied to a group of serologically and biochemically homogeneous bacteria from diseased salmonid fish. Subsequently, isolates that differed in serology and in their ability to ferment D-sorbitol were also called Y. ruckeri. Strains of serovars 11, 111, V, and VI showed a high degree of relatedness in dot-blot deoxyribonucleic acid hybridization assays on nitrocellulose filters. In reciprocal tests, serovar I and I1 isolates had relative binding ratios (RBRs) of greater than 70%. Serovar V isolates appeared more closely related to serovar I1 (85% RBR) than to serovar I (70% RBR) strains. Serovar I11 strains appeared closely related to both serovar I and I1 strains, with the exception of a single sorbitol-fermenting strain. The single serovar IV isolate was excluded from Y. ruckeri (8% RBR), as were other isolates that ferment L-arabinose, D-XylOSe, and L-rhamnose. Y. ruckeri had low levels of hybridization with Hafnia alvei, Salmonella cholerae-suis subsp. arizonae, and Yersinia enterocolitica in dot blots and colony hybridization. Overall, the description of Y. ruckeri can be broadened to include isolates that differ from previously studied strains in their serological reactions and ability to ferment D-sorbitol. Yersinia ruckeri is the causative agent of enteric redmouth disease bacterium. In this study, DNA hybridization reac- (ERM) disease of salmonid fish, which has produced signif- tions were used to test relationships between the various icant losses of rainbow trout (Salmo gairdneri) in aquacul- serovars and strains, using filter-immobilized dot-blot and ture operations (4). The disease was first a problem in the colony-blot methods (13, 15). These are rapid methods, with Hagerman Valley of Idaho, but later was found in other potential applications in diagnostic screening. We demon- places where salmonids are intensively cultured (2, 3). Ross strate here that the general description of the species Y. et al. (22) first characterized the disease agent as a member ruckeri can be broadened to include D-sorbitol-fermenting of the family Enterobacteriaceae, but it continued to be isolates and several additional serological groups. called the redmouth or ERM bacterium because it was not closely similar to any particular one of the described species and genera. Ewing et al. (10) eventually placed the redmouth MATERIALS AND METHODS bacterium in the genus Yersinia, as Yersinia ruckeri, on the Bacterial strains. The isolates of Y. ruckeri examined are basis of deoxyribonucleic acid (DNA) hybridization and listed in Table 1, with their sources and serovars indicated. guanine-plus-cytosine content. The 29 isolates examined in The serovar designations were based on microtiter aggluti- that work were from disease outbreaks in the western United nation tests with antisera raised against Formalin-killed States and appeared to be homogeneous in their biochemical whole cells and on cross-absorbance tests, as described reactions and serology (10, 22). previously (24, 25). The serovar I strains include Y. ruckeri Since that study, new serological groups of the ERM ATCC 29473T and Y. ruckeri ATCC 29904, the DNA refer- bacterium have been found, notably the sorbitol-fermenting ence strain (10). The serovar I1 strains include the sorbitol- isolates from chinook salmon (Oncorhynchus tshawytscha) fermenting reference strain Big Creek 74 described by described by O'Leary et al. (21), which form serovar 11. O'Leary et al. (20, 21). Yersinia enterocolitica CDC 2383 Bullock et al. (3) suggested that a group of isolates from (RS31) was provided by P. Gemski, Walter Reed Army Australian fish formed a third serological group (serovar 111). Institute of Research, Washington, D.C. Other bacteria used Other Australian isolates, called the salmonid blood spot were obtained from the stock culture collection of the bacterium by Llewellyn (16), partially cross-reacted with Department of Microbiology, University of Guelph, or were serovar I isolates of Y. ruckeri and were designated serovar isolates identified by the Fish Health Laboratory, Depart- I' (23, 24). Three additional serovars, IV, V, and VI, have ment of Microbiology, University of Guelph. In general, also been identified (6, 23, 24). cultures were grown on Trypticase soy agar (BBL Microbi- Isolates belonging to the new serovars were identified as ology Systems, Cockeysville, Md.) or in Trypticase soy Y. ruckeri largely on the basis of biochemical test reactions. broth (BBL Microbiology Systems) at 25°C. Stock cultures Some isolates differed from the strains described by Ewing were maintained both on tightly stoppered slants at 15°C and et al. (10) in one or more tests, particularly D-sorbitol as lyophilized cultures. fermentation. Serovar I strains are the most common iso- Biochemical tests. Isolates were characterized and identi- lates from diseased fish (19), although serovar I1 isolates fied as before (25). Biochemical tests were performed by the have been associated with ERM in chinook salmon (5,21). It conventional methods described by Ewing (9) and with API was important to test the authenticity of the additional 20E test strips (Analytab Products, Montreal, Quebec, Can- serovars, as many fish health protection regulations equate ada), both incubated at 25°C. Fermentation tests were done finding isolates of Y. ruckeri with detection of the ERM in Hugh-Leifson basal medium, with filter-sterilized sugars added to a final concentration of 0.5% (wthol). A Steers * Corresponding author. multipoint replicator apparatus was used to inoculate plates 49 50 DE GRANDIS ET AL. INT. J. SYST.BACTERIOL. TABLE 1. Y. ruckeri isolates used in this study Laboratory Fish sourceb and code Other designation Geographic source Serovaf year isolated RS 1 ONT270 Ontario IV Brown trout, 1981 RS2 ONT258 Ontario I1 Rainbow trout, J981 RS3 BC74 Oregon I1 Chinook salmon, 1974 RS4 11.4 Utah I Rainbow trout, 1968 RS6 ONT193 Ontario I1 Brook trout, 1980 RS7 RO8-85 Ontario I Muskrat, 1978 RS8 ONT288 Ontario I1 Cisco, 1981 RS9 9-4-5 Nova Scotia I Rainbow trout, 1976 RS 10 ONT339 Ontario I1 Brown trout, 1981 RSll ATCC 29473T‘ Idaho I Rainbow trout RS12 ATCC 29908 Washington I Rainbow trout RS13 C75-199 British Columbia I Rainbow trout, 1975 RS14 ONT347 Ontario I1 Rainbow trout, 1981 RS20 0634 Australia I’ Brook trout RS21 0589 Australia I‘ Atlantic salmon RS24 2.87 Colorado V Rainbow trout, 1978 RS26 Path 126-76 Idaho I Rainbow trout, 1977 RS27 2#C-M#8 Idaho I Rainbow trout, 1980 RS28 9-4-1 Unknown I Unknown RS32 9-4-5 Nova Scotia Id Rainbow trout, 1976 RS33 YR12 New Mexico I Rainbow trout, 1978 RS34 Y R20 California I Rainbow trout, 1978 RS35 JW10-76 Unknown I Unknown RS36 JW8-78 Unknown 1 Unknown RS37 JW76-78 Wisconsin I Rainbow trout, 1976 RS38 JW77-46 Indiana I Rainbow trout, 1977 RS39 JW77-71 Ohio I Rainbow trout, 1977 RS40 JW78-74 Wisconsin I Rainbow trout, 1978 RS41 JW79-52 Wisconsin I Rainbow trout, 1979 RS42 JW81-29 Missouri I Rainbow trout, 1981 RS43 JW81-48-4 Ohio I Rainbow trout, 1981 RS44 82.091 British Columbia I1 Rainbow trout, 1982 RS45 82.077 British Columbia I Steelhead trout, 1982 RS46 JW82-A Ohio I Rainbow trout, 1982 RS47 JW82-B Ohio I Rainbow trout, 1982 RS48 OS76 Oregon I Rainbow trout, 1976 RS49 11.54 Virginia I Rainbow trout, 1978 RS50 TH75 Oregon I Coho salmon, 1975 RS51 11.34 Colorado I Rainbow trout, 1978 RS52 11.40 North Carolina I Rainbow trout, 1978 RS53 82-75-3 Colorado I1 Rainbow trout, 1982 RS54 YR-40 British Columbia I’ Dolly Varden trout, 1982 RS55 ONT487 Ontario I Rainbow trout, 1982 RS61 4218 1 England I Rainbow trout, 1981 RS62 83-206-1 British Columbia V Whitefish, 1983 RS63 F.15.1183 West Germany UT Rainbow trout, 1983 RS64 F ,1 1 1/81 West Germany I Rainbow trout, 1981 RS66 ONT518 Ontario I1 Lake trout, 1983 RS68 83- 106-7 British Columbia I Steelhead trout, 1983 RS69 ONT534 Ontario I Lake trout, 1983 RS72 Unknown I Unknown RS74 Manitoba UT Whitefish, 1984 RS76 F979.T43/5 France I Rainbow trout RS77 11.43 Australia I11 Rainbow trout, 1959 RS78 11.44 Australia I11 Rainbow trout, 1959 RS79 11.45 Australia I11 Rainbow trout, 1959 RS80 16A Ontario VI Rainbow trout, 1984 RS8l 84-024#3 British Columbia V Cutthroat trout, 1984 RS82 84-081 British Columbia I1 Chinook salmon, 1984 RS83 025#3 British Columbia I Steelhead trout, 1984 RS85 Saskatchewan I Unknown, 1984 RS86 84-247# 1 British Columbia I Steelhead, 1984 RS88 8405-25 412 Denmark I Rainbow trout, 1984 Serovars are as defined previously (3, 6, 24). Untyped (UT) strains did not react with antisera to any of the other strains. Fish sources are rainbow trout (S. gairdneri); lake trout (Salvelinus namaycush); steelhead trout (S. gairdneri); Dolly Varden trout (Salvelinus malma); .whitefish (Coregonus clupeaformia); cutthroat trout (Salmo clarkii); chinook salmon (0.tshawytscha); brown trout (Salmo trutta); cisco (Coregonus artedii); brook trout (Salvelinus fontinalis); Atlantic salmon (Salmo salmo). Muskrat is Ondatra zibethica. T, Type strain. Isolation source is the same as RS9, but a minor antigenic difference occurs (24). VOL.38, 1988 DNA RELATEDNESS OF YERSZNZA RUCKERZ SEROVARS 51 for testing citrate, malonate, and acetate utilization and strain used as the source of the radioactive DNA probe and reactions on casein, Tween, and blood agars.
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