Optimizing <i>Lankesterella</i> Detection in Avian Hosts: A Comparative Analysis of Microscopic and Molecular Techniques

Lankesterella Labbe, 1899 is a genus of blood parasites found in reptiles, amphibians, and birds that has been poorly understood. In birds, it was originally classified as Hepatozoon Miller, 1908 or Atoxoplasma Garnham, 1905, but recent studies have revealed the diversity, prevalence, and host specificity of Lankesterella using integrative approaches, particularly the 18S rDNA gene. The introduction of new diagnostic protocols focusing on mitochondrial genes requires assessing the comparability of different diagnostic methods. This study aims to compare microscopy and molecular detection methods for Lankesterella spp. infections in wild birds, while also exploring the phylogenetic relationships inferred from different genetic markers. A total of 99 samples from nine passerine species were analyzed in a double-blind study using microscopy and PCR protocols targeting 18S rDNA, CytB, and COI. The overall Lankesterella sp. prevalence was 33%, with 17% detected by microscopy and 23% by molecular methods. In our study, the highest prevalences were found in European robins Erithacus rubecula (8.08%) and wrens Troglodytes troglodytes (9.09%). CytB detected the highest number of infections among the three used protocols, followed by COI, whereas 18S rDNA yielded the lowest detection rate. Only four samples were positive for all three genes and by microscopy. CytB and microscopy combined detected the highest number of infections, despite the low correspondence. Phylogenetic analysis confirmed high host specificity, with the best phylogenetic tree resolutions seen in COI and CytB. The study emphasizes the importance of using multiple methods, especially CytB and microscopy, to determine infection prevalence.