Erythrocytes infected with the Nigerian strain of P. ovale were concentrated from chimpanzee blood using Percoll gradients (4). The greatest concentration and separation from white blood cells was obtained when the buffy coat was removed before centrifugation of the Percoll gradients. Band 1 of the gradient contained 99% infected erythrocytes with less than 1% white blood cells. Monoclonal antibodies were subsequently produced against the asexual stages of P. ovale. Four distinct patterns were observed using the indirect fluorescent antibody assay, a spotted fluorescence pattern within the infected erythrocyte, fluorescence of the parasite itself, a diffuse pattern of fluorescence over the entire infected erythrocyte, and a diffuse pattern over the entire cell plus the parasite itself. Three monoclonal antibodies produced against P. ovale reacted only with P. ovale, whereas others reacted either with all four human malaria parasites or with P. falciparum, P. vivax, and P. ovale.
Antisporozoite monoclonal antibody 110-54.3 was used to characterize the circumsporozoite protein of P. ovale (83). In Western blot analysis with P. ovale sporozoites, three distinct species-specific polypeptides were recognized. A single-antibody, two-site enzyme-linked immunosorbent assay demonstrated the presence of a repeating epitope. However, the sequence of the repeating epitope has yet to be determined. This antibody was used to demonstrate the circumsporozoite protein in midgut oocysts by immunoelectron microscopy (77). The monoclonal antibody bound primarily to the plasma membrane of sporoblasts that contained budding sporozoites. Gold particles were not found in immature, nonvacuolated oocysts. An enzyme-linked immunosorbent assay has been developed for the identification of mosquitoes infected with P. ovale. This test has been used successfully to identify mosquitoes infected with P. ovale in Kenyan field studies (9).
Analyses have indicated that there are two types of P. ovale based on nucleotide deletions and substitutions in the 18S rRNA gene, and these parasites have been found to coexist in Vietnam, Thailand, and Myanmar (60, 102, 98). Two types of P. ovale were shown to have distinct sequences for ookinete surface proteins that suggested that there may be two subspecies of the parasites (94).