Effect of Piscirickettsia salmonis inoculation on the ASK continuous cell line.

Piscirickettsia salmonis (P. salmonis) is a Gram-negative aquatic pathogen that causes piscirickettsiosis, a contagious systemic disease which was first described in coho salmon Oncorhynchus kisutch (Walbaum) cultured in sea net pens (Fryer et al. 1990). Since then, this condition has been detected in a variety of teleost fish from distant geographical regions in the world, but so far it has been consistently more severe in salmonid species reared in the South Pacific Ocean in Chile (Arkush & Bartholomew 2011; Rojas et al. 2013). First isolation of P. salmonis was in the chinook salmon, O. tshawytscha (Walbaum), embryo (CHSE-214) cell line (Fryer et al. 1990), and these cells were since then the most extensively used substrate for this bacterium propagation (Arkush & Bartholomew 2011; Rojas et al. 2013). For some years, it was thought that P. salmonis could replicate in vitro only in cell cultures, but rather recently, it was found that it can also be grown in some enriched artificial media (Mauel, Ware & Smith 2008; Mikalsen et al. 2008), and therefore at present, it is characterized as a facultative, instead of an obligated, intracellular organism. Although several fish cell lines in which P. salmonis replicates have been described, no attempts have been made to know whether the Atlantic salmon, Salmo salar L., kidney (ASK) cell line developed by Devold et al. (2000) is permissive to this bacterium. The objective of this work was to determine the effect of P. salmonis inoculation on ASK cells through the cytopathic effect (CPE) description and bacterial titration after cell exposure to this fish pathogen. Monolayers of the ASK (ATCC CRL 2797) and the CHSE-214 (ATCC CRL 1681) cell lines, both cultured at 18 °C in absence of antimicrobials, were used. The ASK cells were cultured with L-15 Leivobitz medium with L-glutamine (2.05 mM) and supplemented with b mercaptoethanol (38.5 lM) and foetal calf serum (FCS) at 10% (all from Gibco, Life Technologies, Carlsbad, CA). The CHSE-214 cells were grown in Eagle0s minimal essential medium with Earle0s salts (Automod Sigma-Aldrich, Lenexa, KS), L-glutamine (2 mM) and FCS (10%). An isolate of P. salmonis obtained from an Atlantic salmon suffering clinical piscirickettsiosis, sampled at a sea-site of the south of Chile, was used. It was cultured in CHSE-214 cells. Infectious supernatant was harvested when CPE reached 100%, and it was titred by end-point dilution assay using 96-well microplates containing monolayers of CHSE-214 cells, with 6 wells per dilution, employing the method of Reed and M€ uench (1938) to estimate the tissue culture infectious dose 50% per mL (TCID50 mL ). Finally, the inoculum used to expose the cells in the infectivity assays explained below was a bacterial suspension having 10 TCID50 contained in 0.2 mL. Correspondence P A Smith, Department of Animal Pathology, Faculty of Veterinary Sciences, University of Chile, Santa Rosa 11735, Santiago, Chile (e-mail: [email protected])