The lack of a good diagnostic method for avian malaria infections is an important factor in limiting the affectivity of control programs in captive systems. We have tested an ELISA using P. falciparum antigens to help solve this problem.

Plasmodium relictum and P. elongatum infections were separately induced in 2 groups of 3, 2-wk-old ducklings (Peking strain) through blood transfer (1.0 ml/animal) from infected ducks. Animals were closely observed for parasitemia by Giemsa-stained thin film beginning on the 5th postinfection (PI). The thin film method gave positive results for all challenged birds. At day 13th PI, 1 ml of blood was collected by syringe venepuncture and processed for trial as follow: 1. Drop of blood (50 ul) was deposited on filter paper, air-dried and stored at 4 C. Plasma and blood were eluded from the filter by 12 hr incubation in 0.4% of Tween 20 (TW 20) H20 solution, 2. 0.5 ml of blood was centrifuged (2,500 rpm, 10 min), supernatant was collected and stored at -70 C, 3. Whole blood (0.5 ml) was stored at -70 C.

An enzyme-linked immunosorbent assay (ELISA) for circulating IgG antibody to P. relictum and P. elongatum was developed for use with the avian sera. Optimum sensitivity was achieved using Immulon 2 microtitration plate, coated with 100 µ (2 µg/ml) of 3 P. falciparum antigens in phosphate-buffered saline (PBS), blocked with casein/TW 20 blocking buffer and filled with 100 Ml of serum diluted with PBS (1:100). Sera from 3 noninfected ducklings were used as a negative control. After 3 hr incubation 100 µl of anti-chicken IgG coupled to alkaline phosphatase was added for I hr then wells were filled with 100 Ml of 1 mg/ml p-Nitrophenyl Phosphate (p-NPP) substrate in 0.1 M glycine buffer. Reaction was stopped after 45 min by 50 µl of 0.5 mM NaOH and absorbance was read at a MAXline Microplate Reader, in 405 nm wavelength.

All birds infected with P. elongatum were strongly positive for all P. falciparum antigens indicating severe infection. All ducklings infected with P. relictum were positive for P. falciparum gametocyte and crude parasite antigens, 1 bird was negative for circum sporozoite (CS) protein.

The described ELISA method is much more sensitive than examination of thick and/or thin bird blood smear. The proposed method is fast, easy to perform, sensitive, and requires a minimal amount of equipment to perform and to collect the blood samples. The proposed assay can be used for diagnosis of avian malaria in captive birds along with monitoring the level of antibody in selected groups of animals, and may be of value in other serological studies.