Expression of immunogenic structural proteins of cyprinid herpesvirus 3 in vitro assessed using immunofluorescence.

Cyprinid herpesvirus 3 (CyHV-3), also called koi herpesvirus (KHV), is the aetiological agent of a fatal disease in carp and koi (Cyprinus carpio L.), referred to as koi herpesvirus disease. The virus contains at least 40 structural proteins, of which few have been characterised with respect to their immunogenicity. Indirect immunofluorescence assays (IFAs) using two epitope-specific monoclonal antibodies (MAbs) were used to examine the expression kinetics of two potentially immunogenic and diagnostically relevant viral antigens, an envelope glycoprotein and a capsid-associated protein. The rate of expression of these antigens was determined following a time-course of infection in two CyHV-3 susceptible cell lines. The results were quantified using an IFA, performed in microtitre plates, and image analysis was used to analyse confocal micrographs, enabling measurement of differential virus-associated fluorescence and nucleus-associated fluorescence from stacks of captured scans. An 8-tenfold increase in capsid-associated protein expression was observed during the first 5 days post-infection compared to a ≤ 2-fold increase in glycoprotein expression. A dominant protein of ~100 kDa reacted with the capsid-associated MAb (20F10) in western blot analysis. This band was also recognised by sera obtained from carp infected with CyHV-3, indicating that this capsid-associated protein is produced in abundance during infection in vitro and is immunogenic to carp. Mass spectrometry carried out on this protein identified it as a previously uncharacterised product of open reading frame 84. This abundantly expressed and immunogenic capsid-associated antigen may be a useful candidate for KHV serological diagnostics.

In vivo morphological and antigenic characteristics of Photobacterium damselae subsp. piscicida.

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacterium's structure and antigenicity when cultured in vivo is discussed.

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

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.