Journal zyxwvutsrqponm of Applied Bacteriology zyxwvutsrqpon 1993, zyxwvutsr 74, 360-366 Culture media for the differentiation of isolates of Yersinia ruckeri, based on detection of a virulence factor M.D. Furones’, M.L. Gilpin zyxwvut and C.B. Munn Department of Biological Sciences zyxwvutsr ~ University of Plymouth, Plymouth, UK 4259/06/92: accepted 19 October 1992 M.D. FURONES, M.L. GILPIN AND C.B.MUNN. 1993. Strains of the bacterial fish pathogen zyx Yersznia ruckeri were identified with the API 20E system and distinguished on the basis of whole cell agglutination with antisera, sorbitol fermentation and polymyxin B sensitivity. Strains which were shown to possess the virulence-associated heat-sensitive factor (HSF) were shown to grow preferentially on culture media containing sodium dodecyl sulphate (SDS) and to produce a creamy deposit around the colonies. By contrast, strains lacking this factor (HSF-) grew poorly and without forming a deposit. Enhancement of the differentiation between the two types was shown by the incorporation of Coomassie brilliant blue dye into agar containing 1% SDS, and the uptake of Coomassie blue and Congo red was shown to be temperature-dependent. Most strains tested were shown to belong to serotype I, and were sensitive to polymyxin and did not ferment sorbitol. With the medium developed most serotype I strains but not those of other serotypes were shown to possess HSF. It is suggested that the medium is used in epidemiological studies of Y. ruckeri. INTRODUCTION scheme for characterization of Y. ruckeri based on the Yersinia ruckeri is the causative agent of Enteric Redmouth (ERM) disease of fish (Austin and Austin 1987). Since its discovery in the USA in 1955 (Rucker 1966), ERM appears to have spread rapidly throughout the world, assuming major economic importance as a limiting factor which can affect the intensive aquaculture of trout and, more recently, salmon (Austin and Austin 1987). The taxonomic position of Y. ruckeri remains uncertain. The general characteristics of the bacterium indicate that it is a member of the Entero- bacteriaceae (Ross et al. 1966) and, on the basis of bio- chemical reactions, guanine plus cytosine ratios and DNA hybridization studies, it was assigned to the new species Y. ruckeri (Ewing et al. 1978). Bercovier and Mollaret (1984), however, expressed their doubts about the inclusion of the ERM bacterium in the genus Yersinia on the basis of DNA relatedness and phenotypic characteristics. Initially, it was thought that Y . ruckeri was a very homogeneous taxon, both biochemically and serologically. Comparison of the serotyping schemes used by different authors, however, leads to the realization that the antigenic characteristics of Y. ruckeri are very complex (Furones 1990). Recently, a Correspondence to : zyxwvu Dr C.B. Munn, Department of Biological Sciences, Unicersity zyxwvutsrqpo of Plymouth, Plymouth PL4 8AA, UK. 07006. Palma de Mallorra, Spam. Present address: Serveis de Apiculture Martna S.A., Carrer Foners 10, analysis of outer membrane profiles has been described (Davies 1990). It has been reported that the ability to ferment sorbitol (O’Leary et al. 1982; Stevenson and Daly 1982), growth at 37°C and resistance to polymyxin B (De Grandis 1987) are related to serological grouping and, poss- ibly, to virulence. In order to study the epidemiology of this important disease simple laboratory methods are needed which enable isolates of the bacterium to be differentiated from other bacteria in samples taken from fish, the environment, or possible vectors. Rodgers (1992) described a ribose- ornithine deoxycholate citrate (ROD) medium which is both selective and differential for Y. ruckeri. The use of such a medium might be extended if characters linked with other intra-species differences, especially virulence, could be detected. Despite a large number of pathogenicity studies little is known about the mechanisms of virulence of Y. ruckeri. Plasmid involvement in virulence has been suggested (Cook and Gemski 1982; Stave et al. 1987), as in the description of a 70 MDa plasmid in the serotype I strain of Y. ruckeri (Cook and Gemski 1982). Similar size plasmids are also present in other representatives of the genus Yersinia (Portnoy and Falkow 1981 ; Portnoy et al. 1984), and a clear correlation between plasmid presence and virulence has been reported in these species (Charnetzky and Shuford