Immunopathological studies on Renibacterium salmoninarum the causative agent of bacterial kidney disease ( BKD )

Abstract

Renibacterium salmoninarum isolates collected from the United States, Chile, Japan and Scotland were characterised using API ZYM, multilocus enzyme electrophoresis (MLEE), polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs). The API ZYM profiles of bacteria cultured in kidney disease medium 2 (KDM-2) without foetal calf serum (FCS) were identical in all isolates tested with only slight differences in the intensity of the reaction. However, bacteria cultured in KDM-charcoal showed a notable reduction in the production of butyrate esterase, caprylate esterase and a-glucosidase, while bacteria grown in Muller Hinton Cysteine (MH-C) failed to produce acid phosphatase. The MLEE analysis showed positive reactions for four out of the seven enzymes tested (glucose 6-phosphate, 6-phosphogluconic acid, mannose phosphate isomerase and aldose). Each of these enzymes showed the same electrophoretic type in all the isolates tested. Polyclonal antibodies anti-cell wall and anti-57 kDa protein (p57) plus seven monoclonal antibodies were produced. Three MAbs recognised peptidoglycan epitopes (14C2, 9F5, and 12B7) and three recognised p57 (5A11, 1C7/1E1 and 1C7/3D1). MAb 11D11 recognised both p57 and peptidoglycan. All MAbs were able to recognise the collection of R. salmoninarum isolates using ELISA, immunohistochemistry or Western blot. The crossreactivity of the PAbs and MAbs to 31 different bacterial pathogens was determined. No crossreaction was observed using MAbs C7/3D1 and 1C7/1E1. MAb 11D11 and 12B7 showed crossreaction only with Micrococcus luteus at very low level. PAbs showed crossreaction with several Gram positive species, particularly M. luteus and Arthrobacter sp.

The ELISA analysis of the 18 renibacterial isolates using a library of PAbs and MAbs showed no significant differences between isolates except isolate B88151 which showed a significant reduction in p57. In addition, the production of renibacterial antigens was, however, shown to be dependent on the age, isolate and culture media used. The humoral immune response of rainbow trout to R. salmoninarum was investigated by injecting fish with a variety of purified antigens. The cell wall induced a strong immune response, especially when it was administered with Freund’s complete adjuvant adjuvant (FCA). Immunisation of trout with iron restricted extracellular products (ECP), normal ECP, live or heat killed bacterial cells also induced specific antibodies. Passive immunisation using some of these antibodies, followed by experimental challenge of fish, showed no evidence of protection, however there was a delay in the onset of mortality in some of the groups. By the time that 100 % mortality was reached in most of the experimental groups (day 34), only 57% mortality was observed in the group injected with MAb 1C7/1E1 and 60 % by the one injected with PAb anti cell wall. The antibody probes were also utilised to screen a R. salmoninarum gene bank and identity a gene coding for an unknown cell wall antigen The gene encoding for the major soluble antigen of R. salmoninarum (p57) was isolated and amplified from genomic renibacterial DNA using polymerase chain reaction (PCR). The gene was then inserted into the expression vector pBTtrp2 and the p57 protein synthethised by activating the tryptophan promoter.