Comparative Analysis of the Exo-Erythrocytic Development of Five Lineages of Haemoproteus majoris, a Common Haemosporidian Parasite of European Passeriform Birds

Abstract

Haemoproteus parasites (Apicomplexa, Haemosporida) are widespread pathogens of birds, with a rich genetic (about 1900 lineages) and morphospecies (178 species) diversity. Nonetheless, their life cycles are poorly understood. The exo-erythrocytic stages of three Haemoproteus majoris (widespread generalist parasite) lineages have been previously reported, each in a different bird species. We aimed to further study and compare the development of five H. majoris lineages—hCCF5, hCWT4, hPARUS1, hPHSIB1, and hWW2—in a wider selection of natural avian hosts. A total of 42 individuals belonging to 14 bird species were sampled. Morphospecies and parasitemia were determined by microscopy of blood films, lineages by DNA-barcoding a 478 bp section of the cytochrome b gene, and exo-erythrocytic stages by histology and chromogenic in situ hybridization. The lineage hCWT4 was morphologically characterized as H. majoris for the first time. All lineage infections exclusively featured megalomeronts. The exo-erythrocytic stages found in all examined bird species were similar, particularly for the lineages hCCF5, hPARUS1, and hPHSIB1. Megalomeronts of the lineages hWW2 and hCWT4 were more similar to each other than to the former three lineages. The kidneys and gizzard were most often affected, followed by lungs and intestines; the site of development showed variation depending on the lineage.

Unexpected absence of exo-erythrocytic merogony during high gametocytaemia in two species of Haemoproteus (Haemosporida: Haemoproteidae), including description of Haemoproteus angustus n. sp. (lineage hCWT7) and a report of previously unknown residual bodies during in vitro gametogenesis

“…CISH was performed following a previously established protocol using a Haemoproteus- specific oligonucleotide probe (Haemo18S, sequence: 5′-GCTAACCGTAGT TATAGTCGCCATCTC-3′) targeting the 18S ribosomal RNA of parasites of the subgenus Parahaemoproteus ( Himmel et al, 2019 ). It was proved to be sensitive in the diagnostics of tissue stages of the parasites in several recent studies ( Duc et al, 2023a , 2023b ; Himmel et al, 2024 ). Briefly, FFPE tissue sections were deparaffinized and pre-treated with proteinase K (Roche, Basel, Switzerland) 3 μg/ml for 40 min at 37 °C.…”

“…life cycle is obvious, but details of the development remain insufficiently investigated in most described parasite species. For example, it remains unclear if megalomeronts (huge-sized tissue stages) develop in all Haemoproteus species and what is their relationship with meronts (small-sized tissue stages) during development in birds ( Duc et al, 2023a , 2023b ; Himmel et al, 2024 ). These issues are important for better understanding the mechanisms of pathologies during avian haemoproteosis.…”

“…Consequently, these findings were questioned and assumed to relate to other apicomplexan protists ( Bennett et al, 1993 ). Recent molecular studies, and particularly the application of chromogenic in situ hybridization (CISH) diagnostic methods, which use genus- and species-specific probes targeting the parasites’ RNA, have proved that tissue stages of Haemoproteus parasites are remarkably diverse and are responsible for the damage of various internal organs ( Ilgūnas et al, 2019 ; Himmel et al, 2019 , Himmel et al, 2024 ; Duc et al, 2023a , 2023b ). Marked damage of lungs, heart, brain, and many other organs by Haemoproteus spp.…”

“…Marked damage of lungs, heart, brain, and many other organs by Haemoproteus spp. exo-erythrocytic stages hardly can be considered as benign in affected hosts ( Ortiz-Catedral et al, 2019 ; Hernández-Lara et al, 2021 ; Duc et al, 2023a , 2023b ; Himmel et al, 2024 ; Cococcetta et al, 2023 ). Nevertheless, the virulence and patterns of exo-erythrocytic development remain only fragmentally investigated in haemoproteids and remain unassessed in most described species ( Valkiūnas and Iezhova, 2017 ; Duc et al, 2023a , 2023b ; Himmel et al, 2024 ).…”

“…exo-erythrocytic stages hardly can be considered as benign in affected hosts ( Ortiz-Catedral et al, 2019 ; Hernández-Lara et al, 2021 ; Duc et al, 2023a , 2023b ; Himmel et al, 2024 ; Cococcetta et al, 2023 ). Nevertheless, the virulence and patterns of exo-erythrocytic development remain only fragmentally investigated in haemoproteids and remain unassessed in most described species ( Valkiūnas and Iezhova, 2017 ; Duc et al, 2023a , 2023b ; Himmel et al, 2024 ). These are obstacles to better understand the general patterns of exo-erythrocytic development in haemoproteids, particularly for unravelling the question if the development of both meronts and megalomeronts is obligatory for each Haemoproteus species or if these different tissue stages occur independently in some parasite species.…”