In vitro cytotoxicity and multiplex PCR detection of virulence factors of Yersinia ruckeri isolated from rainbow trout in North West Germany

Abstract

The aim of this study was to investigate differences in presence and expression of virulence factors between biotype 1 and 2 strains of 82 Yersinia (Y.) ruckeri isolates, collected from North West Germany during the period of 2004-2012, and to analyze the cytotoxicity of these strains to different fish cell lines. The common virulence factor genes, such as yhlA and yhlB encoding for hemolysin YhlA, rucC and rupG encoding for ruckerbactin, yrp1 and yrpDEF for ABC exporter protein system, and two flagellar genes, including flgA for flagellar secretion chaperones and flhA for flagellar secretion apparatus, were found present in both biotype 1 and 2 isolates of Y. ruckeri collected from North West Germany using multiplex PCR. mRNA expression of these genes was compared between the two biotypes of Y. ruckeri. There was no significant diversity (p > 0.05) in the expression of these genes between biotype 1 and 2 strains. 27 Y. ruckeri isolates from different typing groups were analysed in cytotoxicity tests to common carp brain (CCB), epithelioma papulosum cyprini (EPC), fathead minnow epithelial (FHM) and rainbow trout gonad-2 (RTG-2) cells, respectively. In vitro cytotoxicity of the isolates to CCB, EPC and FHM was higher than that to RTG-2 (p < 0.05). At 15 degrees C the maximum cytotoxicity to FHM and EPC was higher in non-motile strains than in motile stains after an incubation of 24 h (p < 0.05), however, after 48 h, there was no significant difference (p > 0.05) of cytotoxicity between those two biotypes. Our results suggest that biotype 2 strains from North West Germany are homogenous with biotype 1 strains on the basis of genetic virulence factor genes. At lower temperature non-motile Y. ruckeri isolates were found more active than motile strains, which could explain why in winter non-motile strains were found more often responsible for ERM outbreaks than motile strains.