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, R32tet32, 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 micrograms/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(-4.11). 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.

Subclinical avian malaria infections in African black-footed penguins (Spheniscus demersus) and induction of parasite recrudescence.

The subclinical and clinical Plasmodium elongatum and Plasmodium relictum infections of captive-reared African black-footed penguins (Spheniscus demersus) were evaluated in nine adult and 29 juvenile penguins in the Baltimore Zoo (Maryland, USA) during summer 1988 and winter 1989. Two diagnostic methods were used: Giemsa-stained thin blood films, and subinoculation of penguin blood into 1-day-old Peking ducklings. Chloroquine and primaquine treatment was applied to all parasitemic juvenile penguins. Twenty-nine parasite-free, juvenile penguins were monitored for parasitemia by Giemsa-stained thin blood films every two weeks for 26 weeks of their first outdoor exposure. Eighteen of 29 penguins experienced naturally acquired malaria; 14 were infected with P. elongatum, three with P. relictum, and one bird had a mixed P. relictum and P. elongatum infection. Eleven of 18 juveniles became parasitemic again after chloroquine and primaquine treatments. Based on Giemsa-stained thin blood smears and subinoculation of penguin blood into 1-day-old ducklings, performed in a mosquito-free environment in winter, nine adult penguins had no evidence of Plasmodium spp. infection. After dexmethasone-induced immunosuppression, four of six of these nonparasitemic adult penguins were found to be infected with P. relictum by the blood inoculation method.