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

Abstract

The antigenicity of Photobacterium damselae (Ph. d.) subsp. piscicida, cultured in four different growth media [tryptone soya broth (TSB), glucose-rich medium (GRM), iron-depleted TSB (TSB + IR(-)), and iron-depleted GRM (GRM + IR(-))] was compared by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using sera obtained from sea bass (Dicentrarchus labrax) raised against live or heat-killed Ph. d. subsp. piscicida. The antigenic expression of Ph. d. subsp. piscicida was found to differ depending on the culture medium used. A significantly higher antibody response was obtained with iron-depleted bacteria by ELISA compared with non-iron depleted bacteria obtained from the sera of sea bass raised against live Ph. d. subsp. piscicida. The sera from sea bass raised against live bacteria showed a band at 22 kDa in bacteria cultured in TSB + IR(-) or GRM+ IR(-) when bacteria that had been freshly isolated from fish were used for the screening, while bands at 24 and 47 kDa were observed with bacteria cultured in TSB or GRM. When bacteria were passaged several times on tryptic soya agar prior to culturing in the four different media, only bands at 24 and 47 kDa were recognized, regardless of the medium used to culture the bacteria. It would appear that the molecular weight of Ph. d. subsp. piscicida antigens change in the presence of iron restriction, and sera from sea bass infected with live bacteria are able to detect epitopes on the antigens after this shift in molecular weight.

Fig. 1

Western blot analysis of sea bass antisera (raised against live Photobacterium damselae (Ph. d.) subsp. piscicida I752) with Ph. d. subsp. piscicida whole cells (I752) grown under different culture conditions. Ph. d. subsp. piscicida used in A~E were recently isolated and cultured in (1) TSB+IR^-, (2) TSB, (3) GRM+IR^-, and (4) GRM. Bacteria in F~H were passaged several times in artificial medium before culturing in the four conditions. Each gel represents the analysis of antisera from an individual fish sampled 3 weeks post-injection. The same antiserum was used in A and F.