Aeromonas hydrophila induces apoptosis in Carassius auratus lymphocytes in vitro

Aeromonas hydrophila is a Gram-negative opportunistic pathogen that can cause bacterial haemorrhagic septicemia and epizootic ulcerative syndromes in numerous freshwater fish species. In this study, we investigated the in vitro effect of A. hydrophila on lymphocytes of Carassius auratus. We found that lymphocytes infected with A. hydrophila at a multiplicity of infection (MOI) of 1:1 to 10:1 for 12 h exhibited typical characteristic features of cells undergoing apoptosis as shown by changes in morphology and DNA fragmentation. The morphological changes, including condensation of nuclear chromatin, formation of apoptotic bodies, maintenance of organelles structure, cytoplasmic vacuolization and blebbing of cell membrane, were measured by fluorescent and transmission electron microscope, and DNA fragmentation was detected by TUNEL reaction and agarose gel electrophoresis. To understand the role of A. hydrophila-induced apoptosis, we examined the changes of apoptotic lymphocytes in response to A. hydrophila at different doses for varying time periods. The result showed that A. hydrophila induced lymphocyte apoptosis in a dose- and time-dependent manner. Furthermore, we used exotoxins isolated from culture supernates of A. hydrophila to induce apoptosis in lymphocytes, and found that exotoxins were also able to trigger apoptosis in lymphocytes. So we drew a conclusion that exotoxins produced by A. hydrophila could be involved in apoptosis of C. auratus lymphocytes in vitro. Our study provides the first evidence that pathogenic bacteria induce apoptosis in fish immune cells. The result indicates that the existence of a novel anti-host strategy may be used by bacteria based on the elimination of host immune cells through apoptosis.