Characterisation of extracellular products produced by Mycobacterium spp. and their effects on the fish immune system

Abstract

Mycobacterium spp. isolated from food and ornamental fish in Thailand (strains TB 1, TB40, TB267, TB268), and the type strains Mycobacterium marinum (NCIMB 1298), Mycobacterium fortuitum (NCIMB 1294), and Mycobacterium chelonae (NClMB 1474) were cultured in Long's medium, Eagle's minimum essential medium, Sauton's medium and modified Sauton's medium. The latter enabled excellent growth and production of extracellular products (ECP) from TB40, TB267, TB268 and M marinum in particular, whereas growth and production of ECP for all strains was limited in Long's medium. SDS-PAGE protein profiles of ECPs from 14 day culture supernatants showed major bands at 65, and <14 kDa. After 2 days culture at the higher temperature of 37°C (heat shock), the production of ECP from all mycobacteria strains except M marinum averaged approximately 4 to 10 fold higher than from strains cultured for 14 days at 28°C. The major fibronectin binding proteins from ECP of Mycobacterium spp. isolated from infected fish were identified at 21-25 kDa. Cross reactivity was detected between ECP from Mycobacterium spp. and MAb anti-heat shock protein (60 kDa) and MAb anti-M Tuberculosis. The 65 kDa antigen of TB267 is a strongly immunogenic protein eliciting antibodies in fish, rabbits and mice. Cross-reactivity was found between rabbit anti-65 kDa antibody and sonicated proteins from many other bacterial species. Therefore, the 65 kDa protein from Mycobacterium sp. isolated from snakehead fish may be a common protein in fish bacterial pathogens. Eighteen MAbs to TB 267 and M chelonae were produced. The epitopes to which the MAbs are against located on molecules susceptible to protease treatment. All MAbs recognized the 65 kDa protein. It is one of major proteins in the ECP, whole cell sonicates and lysates from Jv1ycobacterium spp. and is located in the peri plasmic space or cell wall, and is secreted in the medium during culture. A pnmary intraperitoneal (IP) immunisation of extracellular products (ECP) from Jv1ycobacterium spp., (strains TB40, TB267 or M marinum) mixed with Freund's incomplete adjuvant (FIA), followed by a secondary IP injection at 8 wks, resulted in the elevation of both the non-specific immune response (by measuring nitroblue tetrazolium, lysozyme and phagocytosis activity) and the specific immune responses of rainbow trout, Oncorhynchus mykiss (by measuring specific antibody levels). Nile tilapia were immunised by injecting extracellular products (ECP) of Jv1ycobacterium spp. (strain TB40, TB267 or the type strain M marinum) into their swimbladders and this resulted in the elevation of the non specific immune response. The cytological response of rainbow trout head kidney macrophages to ingested Mycobacterium spp was examined in vitro. The bacteria had previously been opsonised with either fresh rainbow trout serum (FS), or serum which had been heat-inactivated (IDS), or rainbow trout antiserum against the extracellular products (ECP) of Mycobacterium strains TB267 or M marinum. MAbs against the ECP were also used as opsonins. Opsonisation of the mycobacteria was found to greatly enhance the phagocytic and killing activity of the rainbow trout macrophage.