An ELISA for detecting anti-plasmodium spp. antibodies in African black- footed penguins (Spheniscus demersus)

An enzyme-linked immunosorbent assay (ELISA) utilizing 3 Plasmodium falciparum antigens, R32tet(v2), P.F.R27, and crude red blood cell extract, was developed for the detection of circulating anti-Plasmodium relictum or anti-Plasmodium elongatum antibodies in sera from naturally infected adult African black-footed penguins (Spheniscus demersus) at The Baltimore Zoo, Maryland. A concentration of 2.0 μg/ml of each antigen was optimal in terms of specificity, sensitivity, and test speed. It was possible to detect anti- Plasmodium spp. antibodies at a dilution of 10 ⁴¹¹. Low absorbance values (less than 0.050) of nonspecific background were observed. The binding efficacy of anti-penguin IgG coupled to alkaline phosphatase to antibodies in the penguin sera was significantly higher than the binding efficacy of anti- chicken IgG. All penguins, bled in the winter time, in controlled mosquito- free conditions had anti-Plasmodium spp. antibodies reactive with P. falciparum antigens. The penguins showed age-dependent variation in antibody levels. There was a decrease in antibody titration units that was significantly correlated with the number of outdoor exposure years experienced by the birds, despite the season-comparable epizootiologic conditions in their summer open-air habitat. We concluded that the decrease of anti-malarial antibodies could be explained by an antibody-mediated equilibrium of immunity in naturally immunized birds harboring endothelial- stage parasites. The ELISA described is sensitive, and it requires a minimal amount of equipment to collect the blood samples. The assay can be used for detecting and monitoring levels of anti-Plasmodium spp. antibodies in selected groups of penguins.