Evidence for the facultative intracellular behaviour of the fish pathogen <i>Vibrio ordalii</i>

<jats:title>Abstract</jats:title><jats:p><jats:italic>Vibrio ordalii</jats:italic> is an extracellular, Gram‐negative bacterium that produces vibriosis in salmonids. While pathogenesis is not fully understood, this bacterium has numerous likely genes for adhesion, colonization, invasion factors and, as recently suggested, intracellular behaviour. Therefore, this study aimed to clarify possible intracellular behaviour for <jats:italic>V. ordalii</jats:italic> Vo‐LM‐18 and ATCC 33509<jats:sup>T</jats:sup> in the fish‐cell lines SHK‐1 and CHSE‐214. Confocal microscopy revealed Vo‐LM‐18 and ATCC 33509<jats:sup>T</jats:sup> inside cytoplasm in both fish‐cell lines at 4 hr post‐inoculation (hpi). At 8 and 16 hpi, the proportion of fish cells invaded by both strains increased. Moreover, intracellular <jats:italic>V. ordalii</jats:italic> were observed after 8 hpi inside mouse embryonic fibroblasts (MEF), demonstrating that entry was not due to a cellular phagocytosis process. Flow cytometry confirmed immunocytochemistry results, with both <jats:italic>V. ordalii</jats:italic> evidencing statistically significant differences in the number of infected cells between 8 and 16 hpi. Interestingly, <jats:italic>V. ordalii</jats:italic> infection did not significantly damage fish cells, as determined by LDH liberation. Viable counts at 8 hpi detected, on average for both lines, 176 ± 47 CFU/ml of culturable intracellular Vo‐LM‐18 and ATCC 33509<jats:sup>T</jats:sup> cells. These in vitro findings support the facultative intracellular behaviour of <jats:italic>V. ordalii</jats:italic> and may be of importance for understanding pathogenicity and survival in aquatic environments.</jats:p>