Abstract
BACKGROUND: Haemosporidians (Haemosporida, Apicomplexa), which include malaria parasites, are found in nearly all terrestrial ecosystems. Avian haemosporidians have been extensively studied; however, there is limited information on parasites in owls (order Strigiformes). Here, haemosporidians infecting North American owls were characterized using an integrative methodology. METHODS: Taking advantage of injured/dead owls from rehabilitation centres in the central and northeastern USA, 53 individuals of Bubo virginianus, Strix varia, Megascops asio, Bubo scandiacus, Aegolius acadicus, and Tyto furcata were screened using polymerase chain reaction for all samples and microscopy for those with available blood smears. Parasite mitochondrial genomes were obtained using a long-read sequencing method (PacBio HiFi), which efficiently detects multiple infections in a single host. The relationships between parasite lineages were estimated using phylogenetic and haplotype network methods. RESULTS: In total, 21 individuals from three species were positive by PCR: B. virginianus (14/17, 82.4%), M. asio (3/5, 60%), and S. varia (4/8, 50%). Two Plasmodium, three Haemoproteus, and four Leucocytozoon lineages were identified infecting these hosts, with one Haemoproteus and one Leucocytozoon being new to science. All positive individuals were infected with Haemoproteus parasites, and two B. virginianus had a mixed infection with Leucocytozoon and Haemoproteus species. The hSTVAR01 cytochrome b (cytb) lineage common in North America is linked for the first time to Haemoproteus syrnii. Haemoproteus syrnii was found in all 14 positive B. virginianus and two S. varia. Notably, all the cytb lineages from previously identified H. syrnii, based on erythrocytic stages, were not monophyletic, indicating the existence of an undescribed species. The pPADOM11 cytb lineage was recognized as an allele of Plasmodium elongatum. CONCLUSION: Long reads enabled the detection of mixed/co-infections. The link between genetic data and morphospecies was established in two cases. Several Leucocytozoon clades were observed; however, only one morphospecies, Leucocytozoo danilewskyi, has been described in owls. Thus, there is a need for a detailed analysis of blood stages to determine whether different owl Haemoproteus and Leucocytozoon parasites exhibit morphological differences or represent cryptic species. Overall, this study underscores the importance of high-quality molecular data in characterizing the biodiversity of haemosporidian parasites.