Abstract

An enzyme-linked immunosorbent assay (ELISA) has been developed to quantitate levels of antibody specific for Erysipelothrix rhusiopathiae in the cetacean species Tursiops truncatus, Lagenorhynchus obliquidens, and Delphinapterus leucas.² This assay utilizes as capture antigen a mixture of E. rhusiopathiae surface antigens extracted by the method described by Frasch.¹ In order to develop a more sensitive ELISA, crude E. rhusiopathiae antigen extracts have been studied in an attempt to identify the component(s) most important in eliciting a humoral immune response in cetaceans. The antigens from an isolate of E. rhusiopathiae (kindly provided by SPAWARSYSCEN), known to be lethal in cetaceans, were extracted using one the following protocols: 1) suspension of the culture in a 0.2M lithium-chloride and 0.1M sodium acetate extraction buffer (pH 5.8) and incubation at 50°C for two hours,¹ or suspension of the culture in a 1mM Tris buffer (pH 8.0) containing either 2) 10mM SDS, 3) 1mM EDTA, or 4) 0.25% Triton X-100 and incubation for thirty minutes at 37°C.³ The antigens were then separated by SDS-PAGE using 4-15% Tris-glycine polyacrylamide gels in a Bio-Rad Mini Protean® II system (60 minutes at 160 V), blotted to nitrocellulose membranes with the Bio-Rad Trans-Blot® system (60 minutes at 100 V) and detected with either Colloidal Gold (total protein stain) or by immunostaining.

Before immunostaining, the nitrocellulose membranes were blocked with 3% bovine serum albumin (BSA). In preparation for immunostaining, the serum test samples were processed to remove serum proteins other than immunoglobulins that may bind nonspecifically to the membrane. Processing of the sera included centrifugation for 15 minutes at 10,000 rpm in an Eppendorf microcentrifuge, syringe filtration (0.2µ), and centrifugation through a Centricon® Plus-20 device with a molecular weight cutoff membrane of 100,000 daltons. The sera were then diluted to contain a protein concentration of 10 mg/ml using Tween 20·Tris buffered saline (TTBS) containing 1% BSA as diluent; the diluted sera were then used to probe the membranes. Several types of sera were evaluated by immunostaining: 1) rabbit anti-E. rhusiopathiae antiserum; 2) T. truncatus serum with a high ELISA titer (1:100,000); 3) T. truncatus serum with low a ELISA titer (< 1:100); 4) L. obliquidens serum with a high ELISA titer (1:1000); 5) L. obliquidens serum with a low ELISA titer (< 1:100); 6) D. leucas serum with a high ELISA titer (1:10,000); 7) D. leucas serum with a low ELISA titer (< 1:100); 8) normal rabbit serum; 9) T. truncatus serum with anti-E. rhusiopathiae antibodies removed (by adsorption); 10) L. obliquidens serum with anti-E. rhusiopathiae antibodies removed; and 11) D. leucas serum with anti-E. rhusiopathiae antibodies removed. Following the probing, an indicator system consisting of biotin-labeled rabbit anti-cetacean specific IgG antiserum, ExtrAvidin® conjugated alkaline phosphatase, and BCIP/NBT (chromogenic substrate) was used to visualize the bound immunoglobulins. The immunostained extracts were then analyzed for the presence of antigens, which were immunogenic in the three cetacean species, and for the extraction technique which liberated the highest amount of those antigens.

Acknowledgements

This research was supported by a DEPSCoR grant through the Office of Naval Research. The E. rhusiopathiae isolates were graciously provided by Dr. William Van Bonn, SPAWARSYSCEN.

References

1.  Frasch CE. 1979. Noncapsular surface antigens of Neisseria meningitidis. In: Weinstein, L. and B.N. Fields (eds.) Seminars of Infectious Diseases, Volume 2. Stratton Intercontinental Medical Book Company., New York, Pp.304-337.

2.  Jones JC, RA Patterson, BL Middlebrooks. 1995. A biotin-avidin amplified ELISA for the detection of antibodies against Erysipelothrix rhusiopathiae in captive dolphins. Proceedings of the 26th Annual International Association of Aquatic Animal Medicine Conference. P 52.

3.  Lachmann PG, H Deicher. 1986. Solubilization and characterization of surface antigenic components of Erysipelothrix rhusiopathiae T28. Infection and Immunity 52: 818-822.