Background
Avian haemosporidians of the genera
Plasmodium,
Haemoproteus, and
Leucocytozoon are arthropod-transmitted blood parasites with almost global distribution, infecting birds of diverse phylogenetic orders [
1]. Counting more than 250 morphospecies [
1,
2] and over 4500 genetic lineages in the MalAvi database (
http://130.235.244.92/Malavi/ [
3]), these protozoans vary considerably in their host specificity and pathogenicity [
4,
5]. While generally considered benign in most birds, avian haemosporidia can cause severe clinical diseases and mortality in susceptible hosts [
1,
6]. Birds without previous parasite exposure are particularly vulnerable and may experience severe parasitaemia during acute infections, resulting in haemolysis, anaemia, and death [
7‐
9]. Birds may also die without premonitory signs, and fatalities are often linked to deleterious effects of exo-erythrocytic parasite stages in internal organs [
10,
11].
Reports of clinical haemosporidioses frequently refer to domesticated or exotic bird species kept outside of their geographic origin such as in zoos or aviaries. In zoological settings, captive birds often live in close contact to the indigenous avifauna, which serves as a reservoir for haemosporidian parasites [
9]. Transmission of the parasites to immunologically naïve bird hosts can result in increased virulence of otherwise non-pathogenic lineages, sometimes leading to disease outbreaks with high mortality rates, as, for example, regularly observed in
Plasmodium-infected penguins [
12] or
Haemoproteus-infected parakeets [
13]. In natural bird populations, haemosporidian disease outbreaks have been associated with the introduction of new lineages to naive host communities [
14]. The probably best known example for the negative impact of haemosporidian infection on hosts without co-evolutionary adaption are Hawaiian honeycreepers, which have suffered severe population declines due to avian malaria after the introduction of both
Plasmodium relictum GRW4 and its mosquito vector
Culex quinquefasciatus to the islands around 1900 [
14‐
16]. Similarly, in New Zealand, the introduction or translocation of avian hosts, together with the spread of invasive mosquito species, has led to multiple mortality events caused by avian malaria in several endemic bird species [
17].
Apart from these well-documented cases of fatal haemosporidioses in naïve bird populations, little is known about Haemosporida-associated diseases and pathogenicity in wild birds inhabiting regions with local haemosporidian transmission. Field studies show that free-ranging birds often appear as asymptomatic carriers with low parasitaemia, suggesting subtle health effects of chronic infections in adapted hosts. Some studies revealed correlations of infections with increased physiological stress [
18], reduced survival [
19] and shorter lifespans [
20], however, other studies yielded more ambiguous results [
21,
22]. Besides consequences for host fitness, fatal haemosporidian infections have been described in a range of wild bird species, including gallinaceous birds [
23], buzzards [
24], falcons [
25], different passerines [
26‐
31], frogmouths [
28], owls [
32], woodpeckers [
33], pigeons [
34], and penguins [
35,
36]. In addition to these mostly incidental reports in naturally infected birds, lethal haemosporidian infections were demonstrated in several experimentally infected passerines, providing indisputable evidence for the pathogenicity of certain lineages in wild birds [
37].
It is important to note that in haemosporidian field studies, techniques for collecting free-ranging birds, such as the use of mist nets, may favour the capture of subclinically infected individuals, as sick birds might exhibit reduced mobility [
38,
39]. In contrast, the detection of diseased or dead birds in the natural environment is problematic due to their rapid decomposition or removal by predators [
40,
41], potentially resulting in biased sampling towards healthy birds. Considering these detection difficulties, it is conceivable that severe haemosporidioses, particularly acute infections, are underreported in wild birds.
Wildlife disease monitoring strategies such as routine pathological investigations of
ad hoc submitted carcasses are fundamental to detecting wildlife morbidity and mortality, particularly when clinical manifestations are less obvious. While these investigations provide important epidemiological data by determining the presence or absence of particular pathogens, and identifying causes of death, they are constrained by the submission of carcasses. Citizen science, the participation of the public community in scientific research, offers a suitable approach to wildlife disease monitoring and has been used for the surveillance of different avian pathogens, including highly pathogenic avian influenza virus [
42], West Nile virus [
41,
43], Usutu virus [
44],
Salmonella typhimurium [
45,
46],
Mycoplasma gallisepticum [
47], or
Trichomonas gallinae [
48]. By involving volunteer participants (“citizen scientists”) in wildlife morbidity and mortality reporting, citizen science can facilitate the detection and recovery of carcasses of target species in both space and time, enabling large-scale epidemiological surveys and the collection of tissue samples [
49].
Exploiting such archives of tissue samples, previous retrospective studies in Austria revealed an association of blackbird mortalities with the presence of widespread haemosporidian lineages [
30,
50]. Notably, severe parasite loads were linked to infections with
Plasmodium matutinum LINN1, pointing towards its pathogenic role in these birds’ mortalities. Single cases of virulent
Plasmodium infections were also recorded in sparrows, tits, chaffinches, and woodpeckers [
30], however, due to low sample sizes, it remains unclear to what extent these and other passerine species are affected by pathogenic lineages present in Austria. To address this question, the present study aimed to collect carcasses of passeriform and piciform birds by means of citizen science, and examine them for haemosporidian parasites.
Discussion
This study reports on haemosporidian infections in wild passerine birds from Austria collected via dead bird monitoring using a citizen science approach. This method of sampling was used for the first time in avian haemosporidian research. Based on previously reported virulent
Plasmodium infections in Common blackbirds [
50], the present study aimed to explore whether and to what extent other native bird species are affected by severe parasite burdens of pathogenic haemosporidian lineages, contributing to avian mortality.
The study showed that citizen science can be beneficial for obtaining carcasses of naturally infected birds within a reasonable time period. In contrast to exploring haemosporidian infections in living birds, the investigation of bird carcasses offers the possibility to address questions related to the exo-erythrocytic development of the parasites, which remains underinvestigated in wild birds. Particularly for
Haemoproteus and
Leucocytozoon species, current knowledge about their exo-erythrocytic life cycle remains fragmentary and requires additional research [
51]. The examination of organ samples of infected birds not only allows the detection and morphological description of developing tissue stages but also histologic assessment of affected organs. This is particularly important for determining infection severity and pathogenicity of haemosporidian species or lineages in different host species and better understanding the pathogenesis of Haemosporida-associated diseases. While citizen science provides an ideal framework to collect samples for such investigations, the sampling outcome in this study was constrained by several factors related to organization of the survey. First, participation of citizen scientists greatly depended on the publicity of the survey. For example, the response quote of citizen scientists was great after the first announcement at the beginning of the survey, but decreased over the following weeks, demanding further advertisement to raise public awareness and motivate citizens to report bird mortalities. Second, not all reported bird carcasses were suitable for collection due to their advanced decomposition or the lack of possibilities by the citizen scientists to store the collected birds adequately. Although haemosporidian parasites can be detected by CISH even in less-well preserved tissues samples, advanced autolysis prevents their morphological assessment in histological sections. Third, for logistic reasons, dead bird collection was geographically restricted, limiting overall bird sample size. Thus, to maximize sampling outcome, good publicity and communication with the citizen scientists before and during the survey, as well as logistical aspects concerning adequate storage, handling and transportation of collected carcasses need to be considered in future studies applying citizen science for the purpose of bird carcass collection.
Another important aspect to consider when collecting dead birds for investigating haemosporidian infections is sampling bias towards certain bird hosts. This is particularly critical with respect to comparing infection rates or apparent prevalences of haemosporidian parasites in bird carcasses to studies investigating living birds, and drawing conclusions about their pathogenicity. In the present study, dead bird sampling was strongly biased towards fringillid species. While species abundance certainly played a role here, this sampling bias can be explained by high mortality rates due to finch trichomonosis, an emerging infectious disease with high seasonality in mid-late summer. This protozoal disease primarily affects green finches, but also other finch species, albeit much less frequently [
58], and has spread through Europe within the last decade, including Austria [
59]. For this reason, the collection of green finch carcasses was restricted to fatalities not indicative for trichomonosis based on the citizen scientists’ reports (e.g. reported lethargy, ruffled plumage in combination with dysphagia). Yet, the pathological examinations revealed, that most of the collected finches had macroscopic lesions consistent with
T. gallinae infection, indicating that these birds probably suffered from trichomonosis.
Keeping in mind its limitations and potential biases regarding bird collection, citizen science could present a complementary tool to study haemosporidian parasites in birds. Apart from generating scientific data, the involvement of citizen scientists in bird carcass collection also increases public awareness about avian diseases and bird health, which can be beneficial for engagement in bird conservation projects and general greater interest in science.
Including 81 birds of 24 passerine and non-passerine species, this investigation showed haemosporidian parasites in 31% of the birds, however, the proportion of infected birds varied considerably across host species. With finches and tits dominating in numbers, the majority of the infected birds presented low parasite burdens in both blood and tissues. Particularly tissue stages of
Haemoproteus and
Leucocytozoon parasites have been reported to cause haemorrhages and necroses or provoke inflammatory processes [
7,
13,
50,
60], however, in none of the cases investigated here, tissue reactions were associated with the detected meronts or megalomeronts, suggesting an overall minor role of these parasites for the bird fatalities. Instead, most bird deaths probably resulted from other causes, most of all traumatic injuries and fatal infections with
Trichomonas parasites (Additional file
1). However, two great tits clearly showed signs of avian malaria, indicating detrimental effects of the involved
Plasmodium parasites. One of the two birds exhibited marked parasitaemia as demonstrated by abundant CISH signals in the blood, whereas the other had numerous parasite stages in the spleen, associated with severe splenic enlargement. Although it remains questionable whether avian malaria was more than a contributory factor for death of the birds, these two cases are particularly interesting, as both featured infections with
P. elongatum GRW06, a generalist parasite rarely documented in species of the Paridae so far. This parasite infects numerous avian species of different families worldwide (MalAvi database, [
2]) and can be highly virulent for non-adapted hosts [
61‐
63]. However, with only two reports of GRW06 in a great tit [
57] and blue tit [
64] from Austria, records from the family Paridae are virtually absent. Notably, in the present study, GRW06 was found in four of ten great tits and one of three blue tits. The positive CISH results with the
P. elongatum-specific probe detecting parasite stages in the tissues suggest that this parasite is able to develop in Paridae species. However, assuming that tits are susceptible to
P. elongatum GRW06, it remains difficult to explain why
P. elongatum GRW06 has been absent in free-living conspecifics sampled in other European localities, despite its prevalence in different sympatric passerines [
65‐
67]. An experimental study showed that certain songbirds, specifically common starlings, are resistant to GRW06 infection [
68]. Similar to starlings, innate resistance of tits would explain the absence of GRW06 in these birds. However, given the detected parasite stages in the tits from this study, innate resistance could hardly explain the lack of GRW06 records in Paridae. It has to be noted, that there is a slight chance, that the CISH signals detected in the
P. elongatum positive tits from this study could also represent sporozoites, the presence of which does not necessarily imply successful infection and thus susceptibility of the birds. In order to confirm the findings and to prove susceptibility of tits to
P. elongatum GRW06, experimental studies are needed.
Aside from tits, the results showed high infection rates in finches, consistent with findings from other studies investigating living birds [
69,
70]. Accordingly, most lineages detected in the finches here were previously recorded in this host family, except for
Plasmodium vaughani SYAT05 and
Leucocytozoon sp. BT2, found in a goldfinch and a bullfinch, respectively.
Plasmodium vaughani SYAT05 typically infects species of Turdidae, most of all blackbirds [
1,
3,
50], however, it has also been recorded in non-Turdidae hosts, including common chaffinches [
71,
72]. While infections of finches with SYAT05 seem to be the exception, the parasite stages detected in the blood of the goldfinch suggest complete parasite development in this host species. The same cannot be concluded definitely for the
Leucocyotzoon lineage BT2 detected in the bullfinch, as this bird was co-infected with another
Leuocytozoon lineage, hampering attribution of the observed parasite stages to either one of the two lineages. However, although BT2 was primarily found in species of Muscicapidae, Phylloscopidae and Sylvidae, it was also recorded in other Fringillidae species [
73,
74], providing support for the occurrence in this bird family.
In addition to blood stages, CISH revealed parasite tissue stages in some birds, contributing new data on the exo-erythrocytic development of
Haemoproteus and
Leucocytozoon lineages in passerines. In hawfinches,
Leucocytozoon meronts were exclusively found in the kidneys, suggesting this organ to be a common site of exo-erythrocytic development of the detected lineages, consistent with descriptions of renal merogony in other passeriform-specific
Leucocytozoon [
51,
75‐
77]. In the hawfinch co-infected with two
Leucocytozoon lineages (HAWF7 and COCCO01), multiple meronts were distributed across the renal parenchyma and seemed to develop primarily in epithelial cells of tubules, sometimes leading to expansion of the infected cells, occupying most of the tubular lumen. While meronts found in neighbouring tubular cells equalled in size and maturity, meronts located in distant renal tubules showed varying stages of maturity, suggesting overall asynchronous renal merogony of the parasites. However, as the observed tissue stages in this bird could not be assigned to one of the two detected
Leucocytozoon lineages, lineage-dependent developmental differences cannot be excluded.
As the results showed, findings of exo-erythrocytic merogony of
Haemoproteus parasites were restricted to single megalomeronts detected in the gizzards of a chaffinch infected with
Haemoproteus sp. CCF23 and a bullfinch infected with
Haemoproteus sp. EMSPO03. Both lineages were previously recorded in finches [
58,
69,
70,
78], but have not been morphologically described yet. Megalomeronts have only been documented in nine
Haemoproteus species so far, and knowledge about the patterns of
Haemoproteus exo-erythrocytic development within different host species remains fragmentary [
51,
79]. In several reports, the formation of megalomeronts were associated with abortive infections in aberrant hosts [
8,
10,
13,
51,
60,
80]. However, recent studies demonstrated
Haemoproteus megalomeronts in naturally infected passerines, proposing that they are regular stages during exo-erythrocytic development [
79,
81]. The findings of the present study support this suggestion, as megalomeronts were found along with erythrocytic stages of the parasites. Notably, the megalomeront found in the bird infected with
Haemoproteus sp. EMSPO03 strongly resembled megalomeronts of three closely related lineages of
H. majoris (PHSIB1, PARUS1, PHYBOR04), sharing the same unique morphological characters such as irregularly formed cytomeres and a thick wall [
79,
81]. Based on the morphological similarities, this study suggest that EMSPO03 also belongs to the
H. majoris species complex, supporting previous suggestions, that closely related lineages have similarly developing megalomeronts [
81]. While EMSPO03 still requires morphological characterization, phylogenetic analyses corroborate its close relationship to other
H. majoris lineages [
79,
82]. Interestingly, in all three other lineages (PHSIB1, PARUS1, PHYBOR04), megalomeronts were particularly numerous in the kidneys of the infected birds, whereas in the finch infected with EMSPO03, only a single megalomeront was found in the gizzard, indicating differences concerning the site of development, which might be a function of the host-lineage combination. Further investigations are needed to elucidate patterns of exo-erythrocytic merogony of
H. majoris lineages.