Nested Cytochrome B Polymerase Chain Reaction Diagnostics Detect Sporozoites of Hemosporidian Parasites in Peripheral Blood of Naturally Infected Birds

Abstract

Some discrepancies between microscopy and PCR-based methods have been recently recorded in the diagnosis of Leucocytozoon spp. infection in naturally infected birds. To clarify this issue, blood samples from 109 yellow-whiskered greenbuls Andropadus latirostris were investigated using both the microscopic examination of blood films and a nested mitochondrial cytochrome b PCR. The overall prevalence of Leucocytozoon spp. infection was 4% after the standard microscopic examination and 17% using the PCR diagnostics. Samples from 9 randomly chosen birds that were microscopy negative, but PCR positive, were then examined microscopically by screening 2 entire blood films from each individual bird. Sporozoites of Leucocytozoon spp. were observed in 4 birds, and 1 gametocyte of the parasite was seen in each of 2 birds. We conclude that sensitive PCR-based diagnostics are able to detect extremely light parasitemias of circulating sporozoites and gametocytes of hemosporidian parasites. Because of the PCR detection of sporozoites of unknown fate in the peripheral circulation, conclusions regarding the distribution of hemosporidian lineages in wildlife should be made with caution. To be accepted as the lineages of successfully developing species of hemosporidians, such PCR-based information should be supported with the detection of blood stages of the parasites. The present study emphasizes the crucial need for a synthesis of information provided by the tools of traditional parasitology and molecular biology, particularly in field studies of blood parasites.