Background
Every year billions of European breeding birds migrate to their wintering ground [
1]. The distances the individuals cover along their routes can reach thousands of kilometres and inevitably these movements involve birds as reservoirs in transporting and potentially spreading other organisms to the new territories [
2,
3]. One group of parasites whose spreading can be enforced by migrating birds causes avian malaria. These parasites belong to Plasmodiidae (order: Haemosporida), are distributed worldwide and are diverse [
4]. More than 100 years of studies on
Plasmodium infecting birds show that some species are virulent to their vertebrate hosts and may cause severe disease [
4‐
10]. Extensive molecular screening of juvenile and adult birds as well as migrant and non-migrating species of birds in Europe reveals which avian malarial parasites are transmitted within Europe and which are exotic species present only in birds after their return from wintering quarters [
11]. Among the latter are
Plasmodium delichoni (genetic lineage pCOLL6),
Plasmodium homonucleophilum (pSW2),
Plasmodium homocircumflexum (pCOLL4),
Plasmodium ashfordi (pGRW2), and some others [
12‐
15]. This is only a small fraction of all recorded
Plasmodium genetic lineages, which are linked to morphologically described species and contains information about their development and virulence for a vertebrate host. In most of the cases, natural vectors are still unknown.
Transmission of the largest number of potentially invasive avian
Plasmodium lineages found in Europe, occurs in Africa as this is the main wintering ground for the European long-distance migrant birds [
4]. Instead of Africa, several bird species migrate to South Asia and SE Asia, these include Common rosefinch (
Ca. erythrinus), Rosy starling (
Pastor roseus), Little bunting (
Emberiza pussila), Read-breasted flycatcher (
Ficedula parva), Blyths reed warbler (
Acrocephalus dumetorum) and a few others [
16]. According to the MalAvi database [
17], there are more than 20 genetic lineages of avian malarial parasites found in breeding European birds which migrate to South Asia and SE Asia, but only few morphologically described lineages of
Plasmodium have been identified, e.g.
Plasmodium circumflexum, pTURDUS1 [
18,
19], and
Plasmodium relictum, pSGS1 [
20] and pGRW4 [
21]. At present, there are no described malarial parasite lineages linked to
Plasmodium species, which are transmitted only in South Asia or SE Asia and annually brought to Europe with migrating birds. However, these parasites should be of prime interest as they may become the main threat to local bird populations in the near future. According to some calculations, the prevalence of avian malaria will increase by two-threefold due to global warming [
22]. Ecological changes and invasive mosquito species may play the main role in the appearance of new interactions between invasive mosquito species and exotic
Plasmodium parasites causing the transmission of these parasites on local, non-migrating or within Europe migrating birds. For instance, the invasive Asian tiger mosquito (
Aedes albopictus), originated from SE Asia, is spreading in some parts of Europe already [
23,
24]. The presence of
Plasmodium vaughani (genetic lineage pSYAT05) DNA was recorded in these mosquitoes collected in Italy [
24] and this mosquito could also be a potential vector for other avian malaria parasites, especially those originated from SE Asia [
25].
The development and virulence in a vertebrate host and insect vector vary between different
Plasmodium species and, therefore, identification of the parasite species and knowledge about their biology is crucial to better understand the epizootiology and potential spread of avian malaria. In recent years, the description of newly found avian malaria parasites includes both morphological and phylogenetic information obtained from the molecular examination [
13,
15,
26,
27]. Some studies go further and provide information about the development patterns in a vertebrate host, erythrocytic and exoerythrocytic stages, the virulence and information about potential vectors [
9,
10,
12].
In the present study, a new species of malaria parasite obtained from a naturally infected long-distance migrant Common rosefinch (wintering in South Asia) in Northern Europe was described. Using morphological and molecular methods the detailed description of blood stages and the phylogenetic relationships of this lineage with other previously described avian malaria parasites is provided. The development in the red blood cells (RBCs) and various tissues together with the caused virulence to the vertebrate hosts was studied on common bird species Eurasian siskins Cr. spinus, which migrate within Europe. Sporogonic development of the newly described species was studied in blood sucking mosquitoes Cx. pipiens form molestus and Cx. quinquefasciatus. The obtained data can help understanding new parasite-host associations and impact on the host health in regard to parasite spread, brought on by global climate change and newly formed ecological conditions.
Discussion
pFANTAIL01 lineage from the Common rosefinch was identified as
Plasmodium (
Novyella)
collidatum n. sp. The morphological analysis of the erythrocytic stages of the parasite showed that
P. collidatum has typical features associated with
Novyella subgenus: elongated gametocytes, small meronts and parasitizing only in mature red blood cells [
4]. Based on several unique morphological features of blood stages of pFANTAIL01 lineage it is possible to distinguish it from other
Novyella parasites and to define this parasite as a separate species (see "
Taxonomic summary").
Analysis of accumulated molecular data shows that
P. collidatum has been recorded predominantly in Oceania and SE Asia (Table
1). It was recorded not only in migrant, but also in resident bird species. The transmission, apparently, takes place in these regions. Several records of pFANTAIL01 were reported in bird species breeding in Europe, Common rosefinch [
18], Rosy starling [
50] and in one of
Milvus sp. [
45]. All above mentioned passerines were adults after their spring migration returning from South Asia, therefore, it is highly probable that transmission of this parasite does not occur in Europe.
Interestingly, there is one case of
P.
collidatum in Spain, from a bird of
Milvus genus [
45]. There might be two species of
Milvus in Europe, one is
M. milvus and the second one,
M. migrans but European populations of both these species do not migrate to South or SE Asia [
45]. However, as this is a single record in a raptor bird, it is difficult to make a definite conclusion where and how the bird was infected [
58].
According to the MalAvi and GenBank databases, pFANTAIL01 lineage has been reported in wide range of birds of 17 species, 14 families and 7 orders prevailing, herewith, in Passeriformes (Table
1). Apparently,
P. colllidatum is of low specificity for the vertebrate host and, therefore, can be considered as a generalist species. Although, it is unclear if
P. collidatum develops gametocytes in all of the mentioned species as the presence of infection in blood was confirmed only by molecular methods and the possibility of abortive parasite development could not be excluded [
9,
59].
The damage caused by malaria parasites can be by the tissue stages of exoerythrocytic merogony or by the pathogenic effect due to severe anaemia caused by erythrocytic stages of the parasite [
10,
60]. The pathogenic effect of the exoerythrocytic meronts was described in bird species of different families and orders [
4,
6,
60] but development and pathogenicity of tissue stages in genera
Novyella is poorly studied [
12,
61]. Phanerozoites were described in
P. nucleophilum toucani with huge infestation of internal organs and caused mortality in experimentally infected canaries [
62]. Phanerozoites were also seen in different internal organs of birds infected by
P. vaughani [
4],
P. paranucleophilum [
57] and
Plasmodium bertii [
4].
The pathogenic effect of the exoerythrocytic stages of
P. collidatum was reported only in two species of cockatoo, a Yellow-tailed black cockatoo
Calyptorhynchus funereus and Glossy black cockatoo
Calyptorhynchus lathami [
53]. Both birds were infected in captivity and died despite of the medical treatment. The histological analysis showed a large infestation of schizonts in their liver, spleen, lungs and intestines together with hemorrhage and necrosis of other tissues [
53]. Birds died, apparently, due to the extensive damage of their internal organs by the tissue stages of
P. collidatum. According to the authors, these Australian species live in habitats where the presence of potential vectors is restricted. Both housing cockatoo birds were kept not in their species-specific conditions thus they most likely were exposed to parasite vectors. In the present experiment, numerous phanerozoites were observed in liver, lungs, spleen and kidney tissue of infected siskins. Two of eight birds died at the end of study after decreasing of parasitaemia.
Two mutually non-exclusive factors, the depleted immune system and pathologies caused by phanerozoites could trigger the death of the host. Similar cases have been reported by Ilgūnas et al. [
63] in experimentally infected crossbill (
Loxia curvirostra), siskin and starling (
Sturnus vulgaris) which were inoculated with a highly virulent parasite
P. (
Giovannolaia)
homocircumflexum (lineage pCOLL4) and mortalities of birds were observed after the peak of parasitaemia.
The negative impact of erythrocytic stages of
Plasmodium is most noticeable when the parasite damages a big number of blood cells [
28,
64‐
66]. The limited information about the development of
Novyella parasites is obtained up to now comparing to some
Haemamoeba or
Giovannolaia species. It was considered that
Novyella parasites are mainly of low virulence to birds [
67]. However, the studies with infection of tropical origin,
P. ashfordi (pGRW2) and
P. delichoni (pCOLL6) showed that these
Novyella parasites develop high intensities of parasitaemia in experimental birds [
12,
15,
68,
69]. According to the present study, siskins are susceptible to
P. collidatum. The prepatent period varied between individuals but was relatively long in all experimental birds (Fig.
4). This data is in consistent manner with the information about the development of other species of
Novyella e.g.
P. vaughani where the prepatent period lasts from 1 to 6 weeks [
4,
70], in
P. ashfordi—2–4 weeks [
15], in
P. delichoni—2–3 weeks [
12]. The dynamic of parasitaemia varied among individuals reaching peak values up to 0.42–80% (Fig.
4), but was rather extended in time comparing to other species from the most studied parasites from subgenus
Haemamoeba which characterized by rapid increase of parasitaemia and rapid decrease to chronic values within 36 dpi [
28,
71].
During the study, the impact of
P. collidatum on body mass and haematocrit level of infected birds was measured (Fig.
6a, b). The negative effect on body mass of the infected siskins was not detected (Fig.
6a). This data agrees with former experimental studies where even severe malaria infection did not affect the body mass of infected individuals, probably, because birds kept in laboratory conditions were receiving food
ad libitum and were able to compensate the energy loss [
10,
28,
72]. Haematocrit level slightly decreased in infected birds on 32 dpi, but the difference between infected and control birds was insignificant (Fig.
6b). This is not a typical case for parasites from other subgenera, because the increase of parasitaemia usually causes the decrease in the number of RBCs [
6,
28,
73]. During the infection with
Haemamoeba parasite
P. relictum (pSGS1), the quick raise of parasitaemia is immediately followed by a sharp drop of haematocrit value [
28]. On the other hand, Palinauskas et al. [
9] showed that single infection with low parasitaemia (less than 1%) by
Huffia subgenus parasite
P. elongatum (pERIRUB01) dramatically decreased the haematocrit value in experimentally infected siskins. The similar situation was observed in canaries infected with
Novyella species
P. paranucleophilum, the bone marrow of infected birds had heavy invasion at low parasitaemia but anemia was clearly manifested by the decrease in haematocrit values [
57]. Apparently, a huge infestation of bone marrow by phanerozoites reduces erythropoiesis and, therefore, decrease haematocrit values. At the present study, the exoerythrocytic stages in the bone marrow of the infected siskins were not detected. Probably, the erythropoietic system was compensating the loss of erythrocytes until its depletion on 32 dpi when slight decrease of haematocrit in infected birds but further experimental studies are needed to clarify this point.
The annual migration of birds is an important factor for a possible invasion of new haemosporidian species [
4,
74]. However, to complete the life cycle on new territories parasites need a competent vector (Culicidae mosquitoes) and suitable environmental conditions. At the present study,
Cx. pipiens form
molestus and
Cx. quinquefasciatus were used for the experimental investigations.
Culex pipiens form
molestus is the common mosquito species distributed around the world and was confirmed as a natural and potential vector for a number of
Plasmodium species [
9,
24,
30,
75,
76].
Culex quinquefasciatus is more distributed in subtropical and tropical regions and is known to transmit avian malaria parasites as well [
77,
78]. However, mosquitoes of both species experimentally exposed to infection of
P. collidatum were not susceptible to this parasite. Neither ookinetes, oocysts or sporozoites were detected in any of the exposed insects. Also, zygotes were not seen in blood smears from engorged mosquitoes. Apparently, sporogonic development was aborted on the stage of forming gametes. Further studies are needed to identify a competent vector species for this parasite.
Knowledge about natural vectors of pathogens causing lethal diseases is a cornerstone for the basic understanding of epizootiology of any disease and possible threats in the future. The introduction of competent vector species could lead to the establishment of the tropical P. collidatum (pFANTAIL01) in Europe and that could further lead to an outbreak of new malarial infection in local birds which did not co-evolve with the introduced parasite. In the present study, it was experimentally demonstrated that susceptible avian species which could enhance the transmission of tropical pathogen exist in Northern Palearctic.
Despite the fact, that
P. collidatum did not develop in
Cx. pipiens form
molestus, there are other mosquitoes, especially invasive species, which potentially, could serve as a vectors of this parasite. The anthropogenic activity and the global warming are the main factors contributing to the increased numbers of invasive species of mosquitoes and other vectors coming from southern regions [
23,
24,
79,
80]. Since recent decades, there are 6 species of mosquitoes and 1 species of biting midges of tropical origin colonizing different parts of Europe. Most of these species are involved in the transmission of various human and animal diseases and could be responsible for the introduction of some of these infections in Europe. For example, the tropical biting midges
Culicoides imicula introduced the bluetongue virus of ruminants widely throughout Europe [
81]. Introduced new mosquito species could serve as competent vectors both for locally already transmitting
Plasmodium spp. and for exotic blood parasite species carried by long-distance avian migrants. Several field and experimental studies indicated that for instance
Culex sasai and
Culex pipiens pallens are competent vectors for some genetic lineages of
Plasmodium in Asian regions [
82,
83]. The introduction of these or other mosquito species from Asia or SE Asia could contribute to transmission of some avian
Plasmodium parasites, including those which are at present not transmitted in Europe, like
P. collidatum. However, precise experimental and field studies are needed to determine the possibility of such assumptions.
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