Invasion of fish epithelial cells by Photobacterium damselae subsp. piscicida: evidence for receptor specificity, and effect of capsule and serum

Abstract

Photobacterium damselae subsp. piscicida is a fish pathogen which causes serious disease in commercial warmwater fish species. Because information on the initial stages of the infection is scarce, an investigation of the invasion ability of this pathogen was undertaken utilizing a fish epithelial cell line (epithelioma papillosum carpio, EPC), a virulent capsulated strain of P. damselae (MT1415), an avirulent non-capsulated strain of P. damselae (EPOY-8803-II) and Escherichia coli HB101 as a non-invasive control. P. damselae was found to be able to adhere to and invade fish epithelial cells and remain inside them for 6-9 h. There were no significant differences in invasiveness between the capsulated and non-capsulated strains. A kinetics study demonstrated that P. damselae invasiveness was more efficient at low m.o.i., reaching saturation at higher m.o.i., suggesting internalization may be receptor-mediated. Invasion efficiency (IE) was significantly higher than in the control E. coli HB101. Engulfment of bacteria was possibly by an endocytic process and was unaffected by killing the bacteria with UV light. However, heat-killed bacteria had significantly reduced invasion capability. Ultrastructural studies showed that inside the epithelial cells, the bacteria remained within large vacuoles for a few hours and no evidence of intracellular replication was found, by either fluorescence or electron microscopic studies. Normal sea bass serum slightly reduced the invasion capability of the MT1415 strain, but heat-inactivated normal serum had no effect. On the other hand, heat-inactivated fish antiserum raised against the same strain reduced the percentage of invaded epithelial cells by 50 %. As for other pathogens, an intracellular phase of P. damselae may be a mechanism to delay or avoid phagocytosis and host immune responses, favouring the spread of infection.

Ecology of oceanic coccolithophores. I. Nutritional preferences of the two stages in the life cycle of Coccolithus braarudii and Calcidiscus leptoporus

“…The bacteria were then labeled by incubation with 0.1 mg FITC (Isomer I, Sigma) ml -1 , 0.1 M NaHCO 3 , pH 9.5, at 4°C in the dark for 1 h. Bacteria were pelleted at 1500 × g for 5 min and washed free of unbound FITC with phosphate-buffered saline (5 to 10 times). After washing, the bacterial concentration was determined in a counting chamber and adjusted to 10 8 -10 9 cells ml -1 (protocol modified from López-Dóriga et al 2000). A culture of the motile haploid phase of C. braarudii was incubated with 2 ml of labeled bacteria and stored at 17°C in the dark.…”

Two Cathelicidin Genes Are Present in both Rainbow Trout ( Oncorhynchus mykiss ) and Atlantic Salmon ( Salmo salar )

“…Photobacterium damselae subsp. piscicida MT1415 was a virulent capsulated strain isolated from sea bass (Dicentrarchus labrax), and EPOY8803II was an avirulent noncapsulated strain isolated from red grouper (Epinephelly akaara) (15). Vibrio anguillarum serogroup O1 MT1741 (4), serogroup O2 MT1742 (3), and Yersinia ruckeri MT252 were isolated from rainbow trout.…”

Identification of a Novel Cathelicidin Gene in the Rainbow Trout,Oncorhynchus mykiss

“…Photobacterium damselae subsp. piscicida MT1415 was a virulent capsulated strain isolated from sea bass Dicentrarchus labrax, and EPOY8803II was an avirulent noncapsulated strain isolated from red grouper Epinephelly akaara (26). Vibrio anguillarum serogroup O1 MT1741 (3) and serogroup O2 MT1742 (4) and Yersinia ruckeri MT252 were isolated from rainbow trout.…”