Short communication Evaluation of the internalization process of the fish patho- gen Renibacterium salmoninarum in cultured fish cells M Gonza Âlez 1 , F Sa  nchez 1 , M I Concha 1 , J Figueroa 1 , M I Montecinos 2 and G Leo Ân 1 1 Instituto de Bioquõ Âmica, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile 2 Instituto de Microbiologõ Âa, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile Renibacterium salmoninarum is an obligate fish pathogen responsible for bacterial kidney disease (BKD) in both farmed and wild stocks of salmonid fish. Bacterial kidney disease is difficult to control because its causative agent can be transmitted both from fish to fish and to the progeny through infected eggs (Mitchum & Sherman 1981; Evelyn, Ketcheson & Prosperi-Porta 1986). The ability to adhere to and enter non- phagocytic cells constitutes an important strategy for the survival and multiplication of a number of pathogens, and it also seems to be an important aspect of the pathogenicity of these microorganisms (Bliska, Gala Ân & Falkow 1993). Bacterial uptake usually involves exploitation of normal host cell functions by altering the cytoskeleton architecture, as manifested by a dramatic rearrangement of microtubules or microfilaments (Finlay & Cossart 1997). Regarding R. salmoninarum, it has been suggested that one of its most relevant pathogenic mechanisms is its ability to enter the cells of its host (Evenden, Grayson, Gilpin & Munn 1993; Flan Äo, Lo Âpez-Fierro, Razquin, Kaattari & Villena 1996). Since little is known about R. salmoninarum infection, understanding the interaction of this pathogen with its host, in particular the mechan- isms involved in the internalization of this bacterium into the cells, may be helpful in the development of new control strategies for this disease. Therefore, the aim of the present study was to evaluate different fish cell lines as in vitro models to study R. salmoninarum internalization, and subsequently, to determine, with the help of inhibitors, the participation of cytoskeleton com- ponents in this endocytic process. All the fish cell lines (CHSE-214, RTG-2, EPC, BF-2) were cultured in MEM-NEA-HEPES- EARLE medium containing 10% foetal bovine serum and incubated at 18 °C in a humidified, 5% CO 2 incubator. For the invasion assays, each well of a six-well tissue culture plate was seeded with 9 ´ 10 5 cells, incubated to obtain semiconfluent to confluent monolayers and infected with R. salmo- ninarum at a multiplicity of infection of 50 for 5 h. Renibacterium salmoninarum strain ATCC33209 was cultured on SKDM agar plates (Evelyn 1977) for 3±4 weeks at 15±18 °C. The in vitro invasion assay was evaluated by indirect immunofluorescence (IFAT) and transmission electron microscopy, as previously described (Maule Ân, Morales, Aruti, Figueroa, Concha, Krauskopf & Leo Ân 1996). All the IFAT results shown are representative of experiments performed at least in triplicate. Although the internalization of R. salmoninarum into several non-phagocytic fish cell lines has previously been demonstrated (Maule Ân et al. 1996; McIntosh, Flan Äo, Grayson, Gilpin, Austin & Villena 1997), it is interesting to establish which of the cell lines is the most appropriate for studying the internalization process for this bacterium. The IFAT results, shown in Fig. 1, indicate that the association of the bacterium with the cells varies Journal of Fish Diseases 1999, 22, 231±235 Correspondence Dr G LeoÂn, Instituto BioquõÂmica, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile. E-mail: gleon@uach.cl 231 Ó 1999 Blackwell Science Ltd.