Exo-Erythrocytic Development of Avian Haemosporidian Parasites in European Owls

Abstract

Avian haemosporidian parasites (Haemosporida, Apicomplexa) are globally distributed and infect birds of many orders. These pathogens have been much investigated in domestic and wild passeriform birds, in which they are relatively easy to access. In birds belonging to other orders, including owls (order Strigiformes), these parasites have been studied fragmentarily. Particularly little is known about the exo-erythrocytic development of avian haemosporidians. The goal of this study was to gain new knowledge about the parasites infecting owls in Europe and investigate their exo-erythrocytic stages. Tissue samples of 121 deceased owls were collected in Austria and Lithuania, and examined using polymerase chain reactions (PCR), histology, and chromogenic in situ hybridization (CISH). PCR-based diagnostics showed a total prevalence of 73.6%, revealing two previously unreported Haemoproteus and five novel Leucocytozoon lineages. By CISH and histology, meronts of several Leucocytozoon lineages (lASOT06, lSTAL5, lSTAL7) were discovered in the brains, heart muscles, and kidneys of infected birds. Further, megalomeronts of Haemoproteus syrnii (lineage hSTAL2) were discovered. This study contributes new knowledge to a better understanding of the biodiversity of avian haemosporidian parasites infecting owls in Europe, provides information on tissue stages of the parasites, and calls for further research of these under-investigated pathogens relevant to bird health.

Keywords: Strigiformes; birds; exo-erythrocytic development; haemosporidian parasites; tissue stages.

Figure 1

Bayesian Inference tree of 47 partial (474 bp) Haemoproteus cytB lineages. Lineages found in the present study are presented in red bold font. All but one lineage (hARW1) clustered within a clade featuring multiple lineages from owls. The latter (regular letters) were included in the analyses, but approximately 100 lineages from non-strigiform birds clustering in this clade were excluded a priori. For comparison, three lineages closely related to lineage hARW1 were also included. A sequence of Haemoproteus columbae hCOLIV03 was used as outgroup. Lineages found for the first time are marked with an asterisk. Bayesian inference posterior probabilities and Maximum-likelihood bootstrap values are indicated at each node. The scale bars indicate the expected number of substitutions per site according to the model of sequence evolution applied. GenBank accession numbers are provided, followed by lineage names according to MalAvi database (http://130.235.244.92/Malavi/, accessed on 6 July 2022). In case the lineages were linked to morphospecies, the taxon name is indicated.

Figure 2

Bayesian Inference tree of 43 partial (474 bp) Leucocytozoon cytB lineages (474 bp). Lineages found in the present study are provided in red bold letters. Most lineages (excluding lSTAL7, lSTAL9, and lSTAL10) clustered in a clade featuring multiple lineages from owls and other birds. For comparison, all owl parasite lineages clustering in this clade were included but approximately 40 lineages from non-strigiform birds were excluded a priori. A sequence of Leucocytozoon toddi lBUBT2 was used as outgroup. Lineages found for the first time are marked with an asterisk. Bayesian inference posterior probabilities and Maximum-likelihood bootstrap values are indicated at each node. The scale bars indicate the expected number of substitutions per site according to the model of sequence evolution applied. GenBank accession numbers are provided, followed by lineage names according to MalAvi database (http://130.235.244.92/Malavi/, accessed on 6 July 2022). In case the lineages were linked to morphospecies, the taxon name is indicated.

Figure 3

Leucocytozoon sp. (lineage lSTAL5) meronts in the brain of a tawny owl (Strix aluco). Several clusters of meronts were located in different brain areas (a–o) and identified by CISH using a Leucocytozoon genus- and a lSTAL5 lineage-specific probe (see inserts (a,e,h,i)). Each cluster contained numerous young (light basophilic) and mature (dark basophilic) meronts ((b,f,j,n), black arrows) and was covered by a thick eosinophilic wall (white arrowheads), the inside of which was lined by epithelial-like, cuboid host cells, likely of endothelial origin. Sometimes, intermingling erythrocyteys were observed ((f), white arrow). Individual meronts showed the formation of roundish cytomeres (l), giving the meronts a grape-like appearance (see (k,l), yellow line—cytomere outline, blue dotted line—meront outline). Developing meronts comprised multiple roundish cytomeres, which were separated by vacuolic spaces (e.g., (d,l) short black arrows), whereas mature meronts contained numerous round merozoites ((d,l) black arrowheads). Tiny, intracellular early meronts were observed in vacuoles within host cells lining the wall ((h), open arrowheads) and were labeled by the Leucocytozoon probe ((h), insert). Notably, blood capillaries located close to the observed meront clusters also contained small meronts ((j,n,o,p), open arrows). Figure (o) shows a higher magnification of a small meront developing close to the large structure (arrows) with the insert picture demonstrating its intraendothelial location (note the endothelial host cell nucleus— short, white arrow, and the immunohistochemical labeling of actin—brown diaminobenzidine (DAB), a marker for smooth muscle cells and pericytes around microvessels). Protrusions of the wall bordering the meront clusters towards nearby microvessels (as seen in (o,p)) indicate a vascular connection. Long black arrow ()—meront, short black arrow ()—cytomere, black arrowhead ()—merozoite, open arrowhead ()—intracellular young parasite stages, open arrow ()—meront containing capillary, long white arrow ()—erythrocytes, short white arrow ()—host cell nucleus, white arrowhead ()—wall. All scale bars are 25 µm.

Figure 4

Leucocytozoon sp. (lineage STAL5) meronts in the kidney of a tawny owl (Strix aluco). A cluster of meronts morphologically similar to those observed in the brain was located in the renal cortex of the same individual and identified by CISH using a lSTAL5 lineage-specific probe (a, insert). The meront clusters were covered by an entire thick eosinophilic wall-like structure, encompassed by multiple layers of smooth muscle cells ((a,b), asterisks), as demonstrated by immunohistochemical labeling of actin—the smooth muscle cell marker ((c), asterisk—brown DAB staining)—indicating their intravascular location. Numerous erythrocytes were observed in the central lumen of the meront cluster ((a), long, white arrow). Subfigures (d,g,j,m) represent magnifications of the four different areas of the meront cluster indicated by rectangles in (b). The meronts appeared grape-like due to the formation of roundish cytomeres ((e,h), short black arrows). Young meronts were closely associated with the host cells beneath the wall and characterized by slight basophilic staining and loosely distributed large nuclei ((f), open arrowhead). Note the host cell nucleus in a growing meront ((f), short white arrow). Mature meronts were mostly observed in the center of the cluster and contained numerous small, round, and strongly basophilic merozoites ((f,i), black arrowheads). The inside of the eosinophilic wall was lined by epithelial-like squamous to cuboidal cells, likely attributable to endothelium. Many of these cells contained small young parasite stages within a vacuole ((l,o), open arrowheads). Note the transition from squamous, endothelial-like host cells along the wall in areas with little parasite disturbance ((k), bottom left) over to cuboidal cells in areas with extensive parasite involvement ((k), top right). In the middle left area of the entire host-parasite cell complex, a loop-like evagination was observed, likely representing a vascular branch (m,n). High magnification of this area reveals intracellular parasite stages in endothelial cells ((o), open arrowheads). Long black arrow ()—meront, short black arrow ()—cytomere, black arrowhead ()—merozoite, open arrowhead ()—intracellular young parasite stages, long white arrow ()—erythrocytes, short white arrow ()—host cell nucleus, white arrowhead ()—wall, asterisk ()—smooth muscle layer. Scale bars are 25 µm.

Figure 5

Leucocytozoon spp. meronts in the heart muscles of a long-eared owl (Asio otus) and three tawny owls (Strix aluco) infected with Leucocytozoon. Meronts found in HE-stained sections were identified by CISH using a Leucocytozoon-specific probe (inserts (a–i)). (a,b) Meronts in cardiomyocytes of a A. otus infected with lineage lASOT06 (Table 1 individual: AH1539). (c–i) Meronts found in the myocardium of three S. aluco infected with multiple, not further characterized Leucocytozoon lineages (Table 1 individuals: (c)—AH1957, (d,e)—AH0410, (f–i)—ZA21/18). Generally, meronts found in the heart were roundish or oval (a,e–i) but appeared elongate in longitudinal cuts of the myocardium (b,d). Most meronts seemed to be growing as merozoites were not discernable, whereas mature/nearly mature meronts were only exceptionally observed (e), indicating asynchronous development. Occasionally, a thin eosinophilic wall-like structure around the meront was visible ((i), white arrowhead). Long black arrow ()—meront, white arrowhead ()—wall. Scale bars are 25 µm.

Figure 6

Haemoproteus syrnii (lineage hSTAL2) megalomeronts in the skeletal muscle of a Ural owl (Strix uralensis). Multiple growing megalomeronts (long arrows) were detected by HE-staining and CISH using a Haemoproteus-specific probe ((b,e–i), inserts). The megalomeronts were located in muscle fibers and were covered by a thin eosinophilic wall (arrowheads). Maturity of the stages varied among megalomeronts, indicating asynchronous development. Young megalomeronts were characterized by a basophilic more or less uniform content interspersed with vacuolic and cleft-like spaces (g,i). Megalomeronts more advanced in their development showed roundish cytomeres of varying sizes, which contained plenty of cytoplasm and large basophilic nuclei ((a–f), short arrows). Note that the infected muscle fibers, particularly those containing more advanced megalomeronts, were marked by hyaline degeneration with a loss of myofiber striation and hypereosinophilia ((b,d,e,h), asterisks). Long black arrow ()—megalomeront, short black arrow ()—cytomere, white arrowhead ()—wall, asterisk ()—necrosis. Scale bars are 50 µm.