The key results of this study comprise (i) the first report and description of exo-erythrocytic development of the P. matutinum lineage pLINN1 during single infection, (ii) the first report of this infection in the fieldfare, and (iii) the first report of phanerozoite development in ocular structures, suggesting that this infection contributes to avian road kills due to impaired vision.
Plasmodium matutinum (pLINN1) is common in palearctic birds with a broad geographic distribution. Molecular characterization was developed using blood stages of the parasite isolated from the Thrush nightingale (Luscinia luscinia) [14]. This lineage is particularly common in species of the genus Turdus in Europe with the Common blackbird (Turdus merula) being the most common host [13]. This study expands the range of natural avian hosts for this malaria infection by the fieldfare, a migratory bird species with broad palearctic distribution [33].
Blood stages of P. matutinum can be readily distinguished due to marked vacuolization of the cytoplasm in trophozoites and growing meronts (Fig. 1a–e). Additionally, erythrocytic merogony is markedly synchronized, with meront maturation peaking in the morning and the cycle of the merogony being close to 24 h [5, 6, 34]. Vectors of the lineage pLINN1 are currently unknown, but likely to be Culex mosquitoes, as they were shown to be competent vectors of unidentified lineages of P. matutinum in America and Europe [11, 35, 36]. This parasite lineage was found in naturally infected Culex mosquitoes in Italy [37], however it remains to be proved that this parasite lineage completes sporogony and develops sporozoites in these mosquitoes.
To date, a primary exo-erythrocytic development has not been described for any strain of P. matutinum [5, 6, 14]. This paper describes for the first time a secondary exo-erythrocytic cycle with characteristic development of phanerozoites after a natural infection in a wild bird for the lineage pLINN1 during single natural infection. The observed morphology and location of the exo-erythrocytic meronts were indistinguishable from those seen in canaries (Serinus canaria) experimentally infected with unknown lineages of P. matutinum isolated from the Common blackbird and redwing in Italy [5, 6, 10] and the Common blackbird in Switzerland [11]. Previous studies reported the presence of phanerozoites in reticulo-endothelial cells of brain, liver, spleen, kidneys, lungs, heart muscle and bone marrow. According to our case report this list must be complemented with ocular structures and skeletal muscle. Furthermore, massive infection of circulating leucocytes and tissue macrophages was seen for the first time during this infection.
Characteristic features of the phanerozoite development of P. matutinum include the development of roundish phanerozoites in large mononuclear cells and occasionally granulocytes, the presence of elongate phanerozoites in endothelial cells of capillaries in various organs with particular prominence in brain and lung; the presence of conspicuous vacuolization in the cytoplasm of immature phanerozoites, the development of usually > 100 merozoites in mature phanerozoites with up to > 300 merozoites in the largest phanerozoites. The latter feature together with the presence of phanerozoites in brain tissue distinguishes P. matutinum from the morphologically similar malaria parasite Plasmodium giovannolai, which also parasitizes species of the genus Turdus in Europe [5, 6, 38, 39] but remains non-characterized molecularly [13]. It is worth to mention that numerous elongate phanerozoites were seen in blood films (Fig. 1u). This suggests that mature large phanerozoites might be washed out from fixed tissues into the circulation during intense P. matutinum infection, but this observation remains speculative due to possible mechanical impact. However, G. Valkiūnas (unpublished, pers. obs.) has observed occasionally similar structures in blood films of Common blackbirds, whose were naturally infected with Plasmodium sp. in Europe, indicating that such process might occur naturally and may be worth more attention of researchers. The biological meaning of this phenomenon remains unclear.
Conspicuous circular vacuoles were described in phanerozoites and/or erythrocytic meronts of P. matutinum, P. giovannolai, Plasmodium griffithsi, Plasmodium lutzi and Plasmodium tejerai, which belong to the subgenus Haemamoeba [5, 6]. Similar vacuoles have been reported in zygotes, ookinetes, early oocysts and gametocytes of many species of haemosporidian parasites belonging to the families Plasmodiidae, Haemoproteidae, Leucocytozoidae and Garniidae [6, 14, 40, 41]. The origin and function of such vacuoles, however, remains insufficiently understood. It is believed that they contain material, which plays a role in energy metabolism and is involved in the lipid metabolism of actively growing parasites. Similar to other fatty structures, this material might be washed out during alcohol fixation leaving a vacuole-like space in stained samples [6, 7, 14, 42]. Further studies are needed to elucidate the true nature of these structures. Absence of vacuoles in mature phanerozoites in P. matutinum suggests that vacuolization is a feature of immature, growing phanerozoites.
Himmel et al. [43] investigated the occurrence of various haemosporidian infections in a large sample of Eurasian blackbirds and song thrushes (Turdus philomelos) whose were found dead in Austria. Co-infections of various haemosporidians predominated in these samples, and numerous new lineages of Plasmodium parasites were found. It was shown that P. matutinum (pLINN1) often caused high exo-erythrocytic meront intensities in various organs. With the presence of cytomeres in maturing exo-erythrocytic meronts and the absence of conspicuous vacuolization in most of the illustrated exo-erythrocytic meronts in P. matutinum (pLINN1) two unusual characters of the exo-erythrocytic stages were reported. These features were not observed (cytomeres) or not characteristic (absence of vacuoles in growing meronts) in our study, which was based on a single P. matutinum (pLINN1) infection. These observations were in accordance with former studies dealing with this parasite morphospecies [5, 6, 10, 11, 35]. It is difficult to rule out that some of the described exo-erythrocytic stages, which were attributed to pLINN1 [43] might belong to other Plasmodium lineages, which could occur in co-infection. These observations raise questions for future research on exo-erythrocytic development of avian Plasmodium species, particularly in regard of the presence of cytomeres in developing exo-erythrocytic meronts of these pathogens. So far, cytomeres were not observed in tissue stages of avian malaria parasites [1, 5,6,7, 43].
Different strains of P. matutinum showed differences in virulence when inoculated to domestic canaries [5, 6, 36, 44]. High virulence and mortality were described in canaries after experimental exposure to Italian and Swiss strains [11, 38], whereas American strains seemed to be less aggressive with frequent recovery of the birds [5, 36]. Experimental inoculation with infected blood showed that domestic canaries were susceptible to a strain of pLINN1 isolated from the Thrush nightingale (Luscinia luscinia), but parasitaemia was low and mortality was not observed [14]. The virulence of this parasite in wild birds remains insufficiently understood. Corradetti et al. [10] reported the death of one redwing (Turdus iliacus) after experimental infection with an unknown lineage of P. matutinum and speculated that the stress of prolonged captivity keeping might have impaired the host parasite balance. This study shows that natural infection of the lineage pLINN1 is virulent and pathogenic in the free-living fieldfares without relationship to captivity stress. As the parasites matured and produced merozoites in immune cells (Fig. 1j), immune evasion from cellular immunity was suspected. The prominent presence of phanerozoite stages in several organs in comparison to the rather low parasitaemia further suggests that the examined fieldfare was not adapted to P. matutinum (pLINN1). The finding of large, nearly mature phanerozoites in the peripheral blood may represent an artificial contamination of the samples, as the blood films were taken from traumatically ruptured heart and lung tissue. Phagocytosis of such phanerozoites by monocytic cells, however, indicate an immune reaction intra vitam against phanerozoite laden endothelial cells, which are washed out from capillaries into the circulation during massive infections.
Multiple phanerozoites in samples of brain, skeletal muscle, and eye tissue, in combination with signs of vascular blockage and thrombus formation raise suspicion of an impaired vision and neuromuscular responsiveness as cause of the unexpected collision with a slow driving car. Impairment of vascular perfusion of the Pecten oculi due to blockage by endothelial phanerozoites may have a direct impact on the eye function itself. The Pecten oculi is a unique structure of the avian eye composed of multiple capillaries and larger blood vessels surrounded by pigment cells. It is assumed to serve as a nutritive organ for the avascular retina and to balance the intraocular microenvironment by regulation of pressure, pH, and physical stability of the vitreous body [45,46,47,48,49,50,51]. To prove this hypothesis, however, ophthalmologic examinations intra vitam would have been necessary.
Plasmodium matutinum (pLINN1) lineage is common in wild birds in Austria [4, 13] and pathogenic for local endemic birds in New Zealand [12, 52], where it was probably introduced together with their Turdus host species. The same lineage was recently reported to cause lethal malaria in captive African penguins Spheniscus demersus and Lovebirds Agapornis roseicolli in Italy [37, 53], and Atlantic puffins Fratercula arctica in Switzerland [54]. These and other exotic to Europe bird species likely are non-adapted to pLINN1 infection. It is worth to note that – similar to this case in the fieldfare—parasitaemia was low during most reported mortalities, which raises suspicion of tissue damage by exo-erythrocytic stages as cause of death. Exo-erythrocytic merogony in various organs is reported to be the most striking histologic lesion in pet and aviary birds and is particularly prominent in non-adapted hosts [55]. Furthermore, it is considered to be key pathogenic stage in experimental infections of naïve poultry flocks with Plasmodium durae, Plasmodium gallinaceum, and Plasmodium octamerium [56]. Mortality in most of these cases was caused by cerebral dysfunction due to the early and prominent development of exo-erythrocytic phanerozoites in endothelial cells. The brain capillaries were occluded by the swollen endothelial cells, preventing normal blood flow, and causing anoxic conditions resulting in clinical symptoms resembling cerebral stroke. Likewise, the foci of degeneration and necrosis of single fibres seen in the cardiac and skeletal muscle in close proximity to blocked and deteriorated vessels are considered a sequela of local ischaemia.
Pulmonary oedema is one of the key findings of avian malaria in captive birds and could also be confirmed for the fieldfare. Right ventricular hypertrophy (RVH) due to hypoxic pulmonary arterial hypertension is a well-documented sequela of Plasmodium species and Aegyptianella pullorum infections in poultry. It is caused by hypoxic pulmonary arterial vasoconstriction as a response to anaemia [56, 57]. The moderate left shift of the erythroid line seen in the heart blood and the bone marrow of the fieldfare indicates an increased erythropoietic activity, whose key trigger is peripheral tissue oxygen deficiency. Although the values for the PCV were not measured, these findings strongly indicate hypoxia most likely caused by anaemia and/or tissue malperfusion due to the diffuse blockage of capillary beds by phanerozoites.
Liver and spleen were only slightly enlarged, inflammatory infiltrates were mild to moderate, and parasitic stages and pigment granules were few to absent. Consequently, the organs also did not show a blackish pigmentation frequently reported in literature. This discoloration results from haemozoin accumulation in macrophages. Haemozoin is birefringent and negative on Prussian blue stain for iron, while haemosiderin is golden-brown and stains positively with Prussian blue [58]. Both pigments were present in low numbers.
Mild gastrointestinal worm infections are a common finding in necropsies of wild birds with usually little impact on the general body condition and health. Compared to the prominent lesions in brain, heart, and lung related to the Plasmodium infection, the low-grade helminthiasis seen in the intestine and the lung was considered of subordinate importance for the fatal roadkill. The concurrent seromucous diarrhoea, however, speaks for clinically manifest gastrointestinal disease. It may have been caused by the intestinal nematodiasis with or without an undetected gastrointestinal microbial co-infection and was possibly facilitated by the malarial infection [59].
Clarification of a possible concurrent microbial disease with special emphasis on West Nile Virus, Usutu Virus, and Chlamydiaceae was of particular interest, as the inflammatory patterns and organ distribution of lesions seen corresponded to patterns of these diseases described in literature [55].
Testing for microbial co-infections was negative in the extraintestinal compartment, and NGS was negative for both the extraintestinal and the intestinal compartment. This shows that the moderate to prominent inflammatory infiltrates observed in several organs outside the gastrointestinal tract were related to the malaria infection.
Like many parasites of the phylum Apicomplexa, infections with Plasmodium species trigger a predominantly mononuclear inflammatory reaction consisting of various portions of lymphocytes, plasma cells, and macrophages/histiocytes. Depending on the host immunity and the pathogenicity of the lineages the inflammatory lesions vary from mild to dramatic. Severe reactions may be mistaken for lymphoid neoplasia [55]. The findings in the SSS and PWP of the spleen suggest a prominent reaction of the highly phagocytic reticular ellipsoid associated cells and an increased reactivity of the B-cells in the surrounding peri ellipsoid lymphocytic sheath. Interpretation of the eosinophilia seen in the heart blood is limited as the nature of this cell type differs from mammals and remains not fully understood in avian species [60,61,62,63]. Prominent exo-erythrocytic development, phagocytosis of phanerozoites seen in circulating monocytes and concurrent successful maturation of phanerozoites within leukocytes raises suspicion of both an active antiparasitic immune response and immunoevasive mechanisms playing a role in the pathogenesis of this malaria case. As this report is based on a single natural infection of unknown history with a co-infection with helminths, these considerations remain speculative. Targeted immunological studies under experimental conditions would be of interest to clarify a true correlation of these findings to an infection with P. matutinum, to characterize the anti-parasite response profile of the host immune system as reported for other apicomplexan pathogens [64], and to elucidate possible immunoevasive strategies which allowed the parasite to escape from cellular immunity.
This study shows that P. matutinum (pLINN1) is an aggressive malaria parasite, which can develop even in immune cells and is dangerous for non-adapted wild birds. This study supports formerly fragmental observations that avian Haemamoeba malaria parasites can develop and produce merozoites in monocytes and macrophages [5, 6]. The true role of this infection as a potential threat for wildlife bird populations remains to be investigated.
Millions of birds are killed on roads due to collisions with vehicles each year [65]. This study shows that severe malaria infections likely contribute to such mortalities. Interestingly, examination of roadkill juvenile chaffinches Fringilla coelebs revealed exceptionally high (up to 7%) parasitaemia of Haemoproteus species in comparison to the same age bird species, which were mist-netted at the same area [6]. This indicates possible involvement of avian haemoproteosis in road mortalities. This is not unexpected due to recent findings of megalomeronts of Haemoproteus parasites in brain of naturally infected birds [3]. This limited available information suggests that avian haemosporidian infections are worth more attention as agents of avian diseases. The described case of severe P. matutinum (pLINN1) malaria in the fieldfare emphasizes the importance of further studies on exo-erythrocytic stages of haemosporidian parasites as potential underestimated cause of fatal disease in wild bird populations in general, and in roadkill in particular.