Microbiology (1 997), 143,3349-3356 Printed in Great Britain Production of putative virulence factors by Renibacterium salmoninarum grown in cell culture D. Mclntosh,’t E. Flat70,~ T. H. Grayson,2 M. L. Gilpin,2 B. Austin’ and A. J. ViIlena3 Author for correspondence: B. Austin. Tel: +44 131 451 3452. Fax: +44 131 451 3009. e-mail : B.Austin @hw.ac.uk Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, UK Department of Biological Sciences, University of Plymouth, Plymouth PL4 8AA, UK Departamento de Biologla Celular y Anatomla, Facultad de Biologla, Universidad de Le6n, 24071-Le6nf Spain A cell culture system, employing the fish cell line Epithelioma papillosum cyprini (EPC), was developed to study the synthesis of intracellular antigen and the expression of putative virulence factors by Renibacterium salmoninarum. EPC cultures infectedwith R. salmoninarum could be maintained for 7 weeks, during which the pathogen multiplied intracellularly. lmmunohistochemical examination of infected cultures revealed the production of the p57 antigen, haemolysin and cytolysin. The intracellular nature of the infection was confirmed by transmission electron microscopic examination of EPC monolayen. A comparison of the relative virulence of bacterial cells cultured in EPC cells and on agar plates revealed that the former were markedly more virulent in challenge experiments with juvenile rainbow trout (Oncorhynchus mykiss Walbaum). The EPC cell culture model provided a system for the study of R. salmoninarum under more natural conditions than those achieved with plate culture techniques. INTRODUCTION Keywords : cell culture, Renibacterium salmoninarum, in vitro infection, antigen detection Renibacterium salmoninarum is the causative agent of bacterial kidoey disease (BKD), a chronic debilitating disease of salmonids (Fryer & Sanders, 1981). The disease is characterized by a granulomatous inflamma- tory response with marked affinity for kidney tissue (Wolke, 1975). The pathogen is capable of multipli- cation in host cells, particularly macrophages (Young & Chapman, 1978 ; Gutenberger, 1993) and non-profes- sional phagocytic cells (Flaiio et al., 1996b, c), and can evade host immune surveillance with the result that asymptomatic carriers may develop (Evelyn, 1993). This intracellular nature limits the effectiveness of chemo- therapy (Austin & Austin, 1993). R . salmoninarum is a Gram-positive, slow-growing, nutritionally fastidious organism which is difficult to t Present address: Departamento Bioquimica e Biologia Molecular, lnstituto Oswaldo Cruz, FIOCRUZ, Avenida Brad 4365, Rio de Janeiro, 21045 900, Brazil. Abbreviations: EP, extracellular products; EPC cells, Epithelioma papillosum cyprini cells; BKD, bacterial kidney disease. culture in vitro, requiring prolonged incubation on cysteine-containing media. The inability to conveniently culture the organism may be the main reason why so little is known about the virulence or pathogenicity mechanisms of R. salmoninarum (Evenden et al., 1993). Following the identification of the p57 protein (Getchell at al., 1985) and subsequent studies confirming it to be the predominant cell surface antigen and constituent of the extracellular products (EP) in vitro and in vivo (Turaga et al., 1987; Wiens & Kaattari, 1989; Griffiths et al., 1991), workers have sought to define a role for the protein in the pathogenesis of BKD. Properties ascribed to the p57 protein include the ability to bind to salmonid erythrocytes (Kaattari et al., 1986), haemagglutination of mammalian erythrocytes (but not usually those of fish species) (Daly & Stevenson, 1987; Bandin et al., 1989), agglutination of salmon spermatazoa (Daly & Steven- son, 1989) and the leukocytes of various salmonids (Wiens & Kaattari, 1991),and hydrophobicity of the cell surface of R. salmoninarum (Daly & Stevenson, 1990). Also, the p57 protein may be involved in the attachment of R. salmoninarum to phagocytes in the absence of specific antibody, as a consequence of opsonization by 0002-1571 0 1997 SGM 3349