The effect of in vivo growth on the cellular and extracellular components of the marine bacterial pathogen Photobacterium damsela subsp. piscicida

Abstract

Photobacterium damsela subsp. piscicida, the causative agent of fish pasteurellosis, was grown in vivo. Bacterial cells and extracellular products (ECPs) were analysed via electrophoresis and immunoblot analysis, using specific sea bass antisera. Growth in vivo induced the synthesis of unique bacterial cell proteins at > 206, 206, 21.3, 18, 7.6 and < 7.6 kDa. Sea bass serum raised against live bacterial cells of the pathogen and especially a sea bass serum raised against formalin-inactivated bacterial cells grown in a specific novel medium recognized the novel antigens at > 206 (associated with iron sequestration), 21.3, 7.6 and < 7.6 kDa, suggesting that the latter medium conserves the synthesis of natural bacterial cell proteins in vitro. In vivo growth of the pathogen induced the synthesis of more toxic ECPs in comparison with in vitro growth and an inverse correlation between total protein concentration in the ECPs and toxicity per unit of protein was observed. Substrate-polyacrylamide electrophoresis revealed the presence of in vivo synthesized ECPs of the pathogen (proteases) at 175, 132, < 79 and 48.3 kDa. Histological examination of tissues isolated from fish injected with these ECPs revealed inflammatory and necrotic lesions in the spleen, liver, head kidney, intestine and heart as soon as 48 h post-introduction of the ECPs.

In vivomorphological and antigenic characteristics ofPhotobacterium damselaesubsp.piscicida

“…piscicida recognised antigens of differing molecular weights depending on whether the bacteria had been cultured in vitro or in vivo . The absence of low molecular weight antigens in bacteria cultured in vivo agrees with previous observations [3,4]. Bakopoulos et al [4] identified bands at 52, 44, and 21.3 kDa using sea bass sera raised against live bacteria, and bands at 52, 44, 39.5, 34.7, and 21.3 kDa with sea bass sera raised against formalin inactivated cells cultured in a modified yeast based medium.…”

“…subsp. piscicida was examined following bacterial growth in dialysis tubing implanted in the peritoneal cavity of sea bass [4]. These authors identified a number of novel antigens associated with the bacterium and ECP, and demonstrated that antigens induced under iron-restriction were conserved on bacteria grown in vivo .…”

Prevention of fish photobacteriosis. Comparison of the efficacy of intraperitoneally administered commercial and experimental vaccines

“…The differences between the two experiments were that in this study a higher cumulative mortality was achieved, the entire bacterial culture was used as the vaccine without prior isolation of bacterial cells and ECPs, and the challenge was performed with Phdp grown in BHIB 2% NaCl. Despite the fact that the culture medium plays a very important role in the antigens that a pathogen synthesizes and virulence (Bakopoulos et al, 2003a;Bakopoulos et al, 2004), the differences in challenge severity and the modification of the preparation method applied for the experimental vaccine, may account for the contradictory results obtained in this study. Unfortunately, protection of vaccinated sea bass against photobacteriosis under such extreme conditions cannot be compared with previous studies.…”

Variation in the molecular weight of Photobacterium damselae subsp. piscicida antigens when cultured under different conditions in vitro

“…d. subsp. piscicida have been identified [1,3,4,21-23,26,28]. The influence of culture conditions on the expression of virulence factors of Ph.…”

“…d. subsp. piscicida , which are recognized by the immune responses of fish [1,3-5,26]. In the present study, differences in the antigenicity of the bacterium were examined when the bacterium was cultured in vitro in tryptone soya broth (TSB) or under "near in vivo " conditions in TSB + IR - , GRM, or GRM+ IR - .…”