Evaluation of a restriction fragment length enzyme assay for differentiation of Haemoproteus and Plasmodium across a standard region of the mitochondrial genome.

Avian hemosporidian parasites are a genetically diverse group of parasites with a near cosmopolitan distribution. Over the past 2 decades, several PCR protocols have been designed to detect these parasites. The majority of these protocols amplify part of or the entire mitochondrial cytochrome b gene. However, many of these protocols co-amplify 2 genera (Haemoproteus and Plasmodium), making it impossible to determine which genus is amplified without post-PCR analysis. A uniform database (MalAvi), containing sequences amplified with the primers HAEMF and HAEMR2, has been developed to increase comparability across studies. We analyzed sequences from the MalAvi database and new sequences and found that digestion with EcoRV could be used to distinguish Haemoproteus from the majority of Plasmodium sequences. In addition, we tested 220 wild birds from Costa Rica and the United States for avian hemosporidians and assessed the ability of EcoRV to distinguish these 2 genera. Thirty-six positive samples were sequenced to confirm the restriction profiles, and we also analyzed 63 new hemosporidian sequences from ongoing studies in the United States for the restriction site. Among these new samples, all of the 85 Haemoproteus (subgenus Parahaemoproteus) and 14 Plasmodium were distinguishable. Overall, 887 of 898 (98.8%) sequences from our studies and the MalAvi database were assigned to the correct genus. Of these samples, all Haemoproteus samples were correctly identified and all but 11 Plasmodium samples were correctly identified by the EcoRV assay. Overall, this restriction enzyme protocol is able to quickly and efficiently classify these 2 genera of avian malarial parasites and would be useful for researchers interested in identifying parasites to genus-level, studies focused on sequence analysis of only a single genus, or for detecting co-infections that would need cloning prior to sequence analysis.