Methodological Challenges of Detecting Haemoproteus (Haemosporida, Haemoproteidae) Co-infections in Wild Avian Hosts

Accurate detection and characterization of avian haemosporidian infections remain challenging due to frequent co-infections of morphologically similar and genetically diverse parasite lineages. This study aimed to assess the concordance of different diagnostic approaches in detecting Haemoproteus co-infections.

We investigated Haemoproteus infections in the Eurasian blackcap (Sylvia atricapilla) using microscopy, PCR from blood and tissue samples, and histological methods including haematoxylin and eosin staining and chromogenic in situ hybridization (CISH).

All 45 examined birds were microscopically positive for Haemoproteus. The most prevalent parasites belonged to Haemoproteus parabelopolskyi, H. pallidulus, and H. homogeneae, while PCR additionally detected H. majoris (hWW2) and H. attenuatus (hROBIN1). Blood- and tissue-derived PCR results were congruent in only 17 individuals (37.8%), indicating that single-method screening substantially underestimates infection diversity. Tissue stages were histologically confirmed in 14 birds, and parasite DNA was detected by CISH in 12 individuals. Although all tissue samples were PCR-positive, only 37 blood samples yielded positive PCR results. The H. parabelopolskyi group showed high genetic diversity, comprising ten closely related cytochrome b lineages. Kidneys were the most frequently affected organ.

Combining microscopic, molecular, and histological approaches is essential for comprehensive detection of Haemoproteus co-infections and improves our understanding of parasite diversity and host-parasite relationships.