title: Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections.
abstract: Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections.
authors.affiliation: Nature Research Centre, Akademijos 2, Vilnius 2100, LT-08412, Lithuania. Electronic address: rasab@ekoi.lt.
authors.forename: Rasa
authors.initials: R
authors.lastname: Bernotienė
authors.affiliation: Nature Research Centre, Akademijos 2, Vilnius 2100, LT-08412, Lithuania. Electronic address: vaidas@ekoi.lt.
authors.forename: Vaidas
authors.initials: V
authors.lastname: Palinauskas
authors.affiliation: Nature Research Centre, Akademijos 2, Vilnius 2100, LT-08412, Lithuania. Electronic address: tatjana@ekoi.lt.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Iezhova
authors.affiliation: Nature Research Centre, Akademijos 2, Vilnius 2100, LT-08412, Lithuania. Electronic address: d.murauskaitei@gmail.com.
authors.forename: Dovilė
authors.initials: D
authors.lastname: Murauskaitė
authors.affiliation: Nature Research Centre, Akademijos 2, Vilnius 2100, LT-08412, Lithuania. Electronic address: gedvalk@ekoi.lt.
authors.forename: Gediminas
authors.initials: G
authors.lastname: Valkiūnas
mesh_terms: D000818:Animals; D001715:Bird Diseases / Q000175:diagnosis* / Q000469:parasitology; D001717:Birds; D016054:DNA, Protozoan / Q000097:blood / Q000737:chemistry; D016787:Haemosporida / Q000235:genetics / Q000302:isolation & purification*; D008289:Malaria, Avian / Q000175:diagnosis / Q000469:parasitology; D010961:Plasmodium / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011529:Protozoan Infections, Animal / Q000175:diagnosis* / Q000469:parasitology; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan
keywords: Avian malaria; Detectability; Haemoproteus; Mixed infection; Plasmodium; Polymerase chain reaction