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All issues Volume 32 (2025) Parasite, 32 (2025) 13 Full HTML
Open Access
Issue
Parasite
Volume 32, 2025
Article Number 13
Number of page(s) 12
DOI https://doi.org/10.1051/parasite/2025007
Published online 21 February 2025

© G.A. Gutiérrez-Liberato et al., published by EDP Sciences, 2025

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Parasites from the order Haemosporida comprise a wide range of species that infect several vertebrate hosts. These vector-borne blood parasites are of medical and veterinary importance as etiological agents of human malaria, and several other diseases in poultry and wild birds [1, 6, 10, 14, 37, 43, 49, 55]. Some parasites, such as Plasmodium spp., can be found in a high diversity of vertebrate hosts, while other haemosporidians are found exclusively in birds, which is the case for those belonging to the genus Leucocytozoon [49].

Haemosporidian parasites have a worldwide distribution, and infections have been reported from almost all zoogeographic regions, except Antarctica [50]. Currently, there are approximately 50 species of Leucocytozoon, which were described based mainly on a combination of their morphological features, ecological traits, and development patterns in their vertebrate hosts [48–50]. However, there is a marked discrepancy between the number of morphospecies and available molecular information, with more than 1,500 cytochrome b (cytb) gene lineages reported in MalAvi (MalAvi database, http://130.235.244.92/Malavi/ accessed on February 22, 2024) [4] and only a few of them (24) are linked to a morphospecies [4, 50], with 12.2% (3) of them reported in Paridae birds, also known as tits and chickadees [4].

Morphological description of blood stages of Leucocytozoon spp. is complicated even for experienced parasitologists [50]. Their unique morphological features (absence of hemozoin pigments, no merogony in the peripherical blood, and the conspicuous deformation on the infected host cell) form a parasite-host cell complex, making them easy to differentiate from other closely related parasites, such as those from the genera Haemoproteus and Plasmodium [32, 49], but not within the Leucocytozoon genus. Due to the lack of structures, the identification and differentiation of Leucocytozoon species has mainly been done relating to the bird order and the influence of the parasites on the infected host cell [49]. For instance, nine Leucocytozoon morphospecies have been described to infect Passeriformes: five with only roundish parasite-host cell complex (Leucocytozoon majoris, L. sakharoffi, L. berestneffi, L. fringillinarum, and L. dubreuili), two with only fusiform parasite-host cell complex (L. grallarie and L. neotropicalis), and two with both parasite-host cell complexes (L. balmorali and L. maccluri) [50]. The most common parasite-host cell complex found in passerine birds is the roundish one, but some species are found only in birds belonging to the Corvidae family, such as L. sakharoffi and L. berestneffi [49]. The other Leucocytozoon spp. forming roundish parasite-host cell complexes are differentiated based on the shape assumed by the nucleus of the infected cell. A host cell nucleus with a band-like shape is characteristic of L. majoris (Laveran, 1902), a dumbbell-like shape of L. dubreuili, and a cap-like shape of L. fringillinarum Woodcock, 1910 [49].

Concerning knowledge about the exo-erythrocytic stages, the information available for Leucocytozoon spp. is scarce and fragmentary, especially in Passeriformes [50]. Research on tissue stages development has focused on L. caulleryi infecting chickens [10, 36, 37], L. smithi infecting turkeys [55], and L. simondi infecting ducks [26], mainly due to their economic importance, with a few studies in wild birds [2, 9, 27, 30]. Investigations of the tissue development of Leucocytozoon sp. were mainly done by experimental infection several decades ago [15, 18, 49, 51, 56] and currently, few studies have focused on studying the development of these stages in natural infections in wild birds [9, 24, 27]. Additionally, the microscopic study of histological sections is a time-consuming methodology that requires expertise to correctly recognize a parasite stage from other possible structures with which it could be confused, such as cellular debris or tissue necrosis [24]. The application of chromogenic in situ hybridization (CISH) has allowed a more accurate diagnosis of different haemosporidians in microscopic analyses of histological sections [9, 16, 17, 24, 25, 27].

The Paridae birds comprise a diverse group of small passerine birds with 13 genera and 63 species and are known for a high prevalence of Leucocytozoon spp. infections [33], as well as co-infections with other closely related haemosporidian parasites [38]. With that in mind, this study aimed (i) to investigate exo-erythrocytic development of Leucocytozoon spp. in Paridae birds; (ii) to identify genetic lineages involved in these infections and link them with the different Leucocytozoon sp. morphotypes; and (iii) to check the phylogenetic relationships between Leucocytozoon spp. lineages infecting Paridae birds.

Materials and methods

Ethics

Specimens were collected under collection permits approved by the Lithuania Environmental Protection Agency, Vilnius (2020 04 08 Nr. A4E-2892; 2021 05 05 Nr. SR-96, and 2023 03 15 Nr. SR-197).

Study site and sampling

Individuals belonging to the Paridae family were sampled at the Ventės Ragas ornithological station (55°20′38.93″ N, 21°11′34.05″ E), Lithuania during the spring (May) 2021, autumn (September and October) seasons of 2020 and 2021, and autumn (October) 2023. The birds were caught using a large stationary trap and mist nets. Blood samples were obtained from the brachial vein by puncturing with a needle. A few drops of blood were used to prepare thin blood smears from each individual, and the remaining blood was stored in SET-buffer (0.05 M tris, 0.15 M NaCl, 0.5 M EDTA, pH 8.0) for subsequent molecular analyses. The thin blood smears were dried at room temperature with a battery-operated fan and fixed with absolute methanol (~1 s). One slide per individual was stained with a 30% Giemsa solution for 15 min [7]. Microscopic examination was immediately conducted to detect the presence of Leucocytozoon sp. gametocytes in the field, following the available protocols [52]. The remaining slides were stained with 10% Giemsa solution for 1 h [52], examined for at least 30 min to identify the parasite morphotype, and to calculate the parasitemia. The intensity of parasitemia was determined as a percentage of parasites per 10,000 red blood cells [20]. Representative preparations of blood stages (49759NS–49764NS) were deposited at the Nature Research Centre, Vilnius, Lithuania.

Eight infected individuals were selected and euthanized by decapitation according to available permits (see Ethics). The internal organs were collected and stored for histological examination (described below). Furthermore, 33 individuals that were found dead at the study site and in its surrounding areas, during the autumn season (September, October, and November) 2022, and frozen until dissections in May 2023, were included in this study (Table 1). Blood cloths were collected from the heart and stored in SET-buffer for molecular analyses, and internal organs were processed as for the other birds. No blood smears were prepared for these birds.

Table 1

Examined Paridae birds, with the results on thin blood smears microscopy, parasite cytochrome b gene (cytb) lineages, and histology using hematoxylin-eosin (H&E) staining and chromogenic in situ hybridization (CISH).

DNA extraction, PCR, and sequencing

DNA was extracted from the blood samples stored in SET-buffer using an ammonium acetate protocol [40]. A standard nested PCR protocol was performed targeting a ~ 480 bp fragment of the parasite cytb gene. The set of primer HaemNFI/HaemNR3 was used in the first run, and the second PCR was done with two sets of primers: HaemFL/HaemR2L which amplifies Leucocytozoon spp. DNA, and HaemF/HaemR2 which amplifies Haemoproteus spp. and Plasmodium spp. DNA. The thermal cycle profile was the same as described in the original protocols [23, 54]. On each PCR reaction, microscopically positive samples for Leucocytozoon spp., Haemoproteus spp./Plasmodium spp. and ultrapure water were used as positive and negative controls, respectively. PCR products were visualized in a 2% agarose gel and positive amplifications were purified using differential precipitation with ammonium acetate protocol [40]. Positive samples were sequenced from both ends with the respective primers, using Big Dye Terminator V3.1 Cycle Sequencing Kit and ABI PRISMTM 3100 capillary sequencing robot (Applied Biosystems, Foster City, CA, USA). The resulting sequences were analyzed using Geneious Prime 2023.2.1 (https://www.geneious.com). Both strands were aligned to form a contig sequence and analyzed for the presence of double peaks and quality. Co-infection with other lineages was considered if double peaks were found. These sequences were not included in the phylogenetic analysis.

Obtained lineages were compared with other sequences deposited in the MalAvi [4] and GenBank (https://www.ncbi.nlm.nih.gov/ accessed on February 22, 2024) databases using BLAST. Lineage identities were considered when the obtained contig had 100% similarity with existing lineages deposited in the MalAvi database. If the obtained sequences had at least one base pair of difference, they were considered new lineages and named according to the MalAvi protocol [4]. All obtained sequences were deposited in both the GenBank (PP988073–PP988098) and MalAvi databases.

Phylogenetic analysis

A total of 92 Leucocytozoon spp. lineage sequences reported in the MalAvi database from Paridae birds, as well as the ones linked to a morphospecies, were used for the phylogenetic analysis (see https://www.parasite-journal.org/10.1051/parasite/2025007/olm). The alignment was constructed using MEGA 11 V11.0.13 [47]. A Bayesian Inference (BI) analysis was carried out in CIPRES Science Gateway V3.3 [35]. Sequences from other haemosporidian parasites (Leucocytozoon (Akiba) sp., and Haemoproteus sp.) were used as outgroups. This analysis was performed under the general time-reversal model (GTR + I + G) suggested by jModelTest 2.1.1 [12]. For the BI, independent Markov Chain Monte Carlo (MCMC) simulations were run simultaneously with six chains; using 1 × 107 generations sampled every 500 generations. After discarding 25% of the trees as “burn-in”, the remaining trees were used to build the majority rule consensus tree, which was visualized and edited using FigTree version 1.4.3 [39] and MEGA 11 V11.0.13 [47]. Genetic distances within and between groups were estimated using a Kimura two-parameter model of substitution, implemented in MEGA 11 V11.0.13 [47].

Histological examination and chromogenic in situ hybridization (CISH)

The organs collected (brain, heart, trachea, lungs, kidneys, liver, spleen, pancreas, esophagus, proventriculus, gizzard, intestine, reproductive organs, pectoral, and leg muscles) were fixed in 10% neutral formalin for 24 h, then washed in distilled water for 1 h, and stored in ethanol 70% until processing [46]. Later, tissue samples were dehydrated with increasing ethanol concentrations (from 70 to 100%), clarified in xylene, and embedded in paraffin wax-blocks [46]. Histological sections of 3 μm were prepared with a microtome from all the paraffin blocks containing the collected material. At least four sections per organ were prepared and stained with hematoxylin and eosin-staining procedures, hereafter H&E, and one section per organ was used for application of the CISH method. Histological preparations were mounted on glass slides for H&E and on Fisherbrand™ Superfrost™ Plus Microscope Slides (Thermo Fisher Scientific, Waltham, MA, USA) for CISH. The latter was applied using the genus-specific oligonucleotide probe (Leuco18S), following the original protocol [24].

H&E preparations were used to investigate exo-erythrocytic stages of Leucocytozoon spp. using 100x, 200x, and 400x magnifications with an Olympus CX23 or BX51 microscope (Olympus, Tokyo, Japan). Positive preparations were photographed using a DP12 camera with the software DP-SOFT (Olympus) [24]. Representative preparations (49765NS–49772NS) were deposited at the Nature Research Centre, Vilnius, Lithuania.

Results

Microscopy findings

In total, blood samples from eight individuals, two Eurasian Blue tits Cyanistes caeruleus and six Great tits Parus major, were microscopically positive for Leucocytozoon spp. (Table 1). Microscopic analyses of thin blood smears revealed the presence of two Leucocytozoon sp. morphotypes: L. majoris (present in six individuals, two Blue tits and four Great tits; Table 1, Figs. 1A–1B) and L. fringillinarum (present in two Great tits; Table 1 and Figs. 1C–1D). Parasitemia intensity for the L. majoris morphotype ranged from 0.002% to 0.09% and for the L. fringillinarum morphotype from 0.004% to 0.01% (Table 1). Co-infections with other haemosporidian parasites of the genera Plasmodium and Haemoproteus were also detected in three individuals, all Great tits (Table 1). For the individuals that were found dead, parasitemia was not determined since clotted blood is not suitable for the preparation of thin blood smears and microscopic analysis (Table 1).

thumbnail Figure 1

Gametocytes of Leucocytozoon spp. with roundish parasite-host cell complexes from Great tit Parus major blood samples. Two morphotypes were found during microscopic examination: Leucocytozoon majoris (A, B) in the individual H10/20R (lPARUS4) and Leucocytozoon fringillinarum (C, D) in the individual H83/21R (lPARUS22). Microgametocytes (A, C). Macrogametocytes (B, D). Short simple black arrow: parasite nucleus. Black arrowhead: nucleus of infected host cell. Methanol-fixed and Giemsa-stained. Scale bar 10 μm.

Parasite molecular diversity and phylogenetic analysis

Using PCR methods, Leucocytozoon spp. infections were confirmed in all the samples that were positive by microscopy. Single Leucocytozoon sp. infections were confirmed in five individuals and with four different lineages (Table 1). One individual had co-infection with different Leucocytozoon spp. lineages (individual H44/21R); one individual with Plasmodium circumflexum (cytb lineage pTURDUS1) (individual H84/21R), and one individual with Haemoproteus majoris (hPARUS1) (individual H81/21R).

Among the opportunistically sampled individuals, PCR-based methods detected single infections by Leucocytozoon sp. in eight individuals, and three individuals had co-infection with different Leucocytozoon spp. lineages (Table 1). Four individuals had co-infection with P. circumflexum pTURDUS1. No co-infections with Haemoproteus sp. were detected by molecular methods. Single infections with Plasmodium sp. were detected in eight individuals, three with Plasmodium sp. pBT7 and five with P. circumflexum pTURDUS1 (Table 1).

Seven of the eight Leucocytozoon spp. lineages recovered in the analyzed birds were previously reported in Paridae birds, and one new lineage was found in a Great tit (Leucocytozoon sp. lPARUS95). The most prevalent lineage in this study was Leucocytozoon sp. lPARUS4 (Table 1). Furthermore, the prevalence of infection with P. circumflexum pTURDUS1 was high, being reported in ten individuals.

In the phylogenetic analysis, all Leucocytozoon lineages that presented L. majoris morphotype (lPARUS4, lSYBOR07, lPARUS16 and lPARUS19) were placed in the same clade, together with the other lineages obtained from the opportunistically sampled birds (lPARUS14, lPARUS95, and lPARUS25) (Fig. 2A). The estimated genetic divergence within clade A was 0.027. Both samples that presented gametocytes with the L. fringillinarum morphotype had the same parasite lineage Leucocytozoon sp. lPARUS22 that was placed in a separate clade (Fig. 2B). The estimated genetic divergence within clade B was 0.030. Additionally, the estimated genetic divergence between clades A and B was 0.079.

thumbnail Figure 2

Bayesian inference tree of partial cytochrome b gene sequences of 92 Leucocytozoon lineages and sequences linked to a morphospecies. Two lineages (Haemoproteus columbae hHAECOL01 and Leucocytozoon (Akiba) caullery lGALLUS05) were used as the outgroup. Parasite lineages were presented as they appear in the MalAvi database. Two clades were highlighted according to the morphotypes some of the lineages were linked to, being L. majoris (A) and L. fringillinarum (B). Bold font indicates the lineages obtained in this study. The new lineage is indicated with an asterisk. All the posterior probabilities ≥ 0.7 were provided with a black dot. GenBank accession numbers of the sequences used in the phylogeny are given in Supplementary Table S1.

Histological findings

Leucocytozoon spp. meronts were seen in the histological preparations stained with H&E of two Great tits (Figs. 3B, 3C, 3E, 3F), and one megalomeront was observed in a third individual (Table 1, Figs. 3H, 3I). No tissue stages were found in the Eurasian Blue tits or in the Coat tits. A high number of blood stages (present in the H&E preparations and CISH-treated samples) were found in different organs, including the oviducts (Figs. 3A, 3D). Blood stages were not evident in the brain, pectoral muscle, and leg muscle.

thumbnail Figure 3

Leucocytozoon spp. exo-erythrocytic and blood stages in naturally infected Great tits Parus major. Individual H44/21R with Leucocytozoon spp. (molecular co-infection) (A–C) presented blood stages in oviduct (A) and meront development found in the kidneys (B, C). Tissue stages visualized in H&E-stained preparations were confirmed by chromogenic in situ hybridization (CISH) using a Leucocytozoon genus-specific probe (Leuco18S) indicated by a purple signal in the inserts of panels. Individual H58/21R infected with Leucocytozoon sp. (cytochrome b lineage lPARUS16) (D–F) had blood stages in the liver vessels (D) as well as meronts in the liver (E) and kidneys (F). All meronts seemed to be growing, as merozoites were not recognizable. Individual H83/21R infected with Leucocytozoon sp. (lPARUS22) (G–I) had blood stages in the spleen (G) and a megalomeront developing in the heart (H, I). The megalomeront, covered by a thick eosinophilic wall, showed growing cytomeres. Black arrowheads: gametocytes. Black triangle arrowheads: meronts. Wide black arrow: enlarged host cell nucleus. Simple black arrow: megalomeront-wall. Scale bars: A, D, G, I 20 μm. B–C, E–F, H 50 μm. (insert I, 50 μm. All remaining inserts, 20 μm).

Two meronts were found in the individual H44/21R (undetermined lineage) in the kidneys (Figs. 3B–3C), one presenting an elongated shape and darker coloration (note the strong purple staining of the structure) of approximately 50 μm in length and no evidence of cytomere formation, a feature of early growing meronts (Fig. 3B); while the other one had an oval shape, ≤40 μm in width and marked segmentation that is associated with cytomere formation (Fig. 3C). These results were confirmed by CISH-treated histological sections (Fig. 3 inserts). No signs of necrosis or inflammation were found near these meronts.

In the individual H58/21R (lPARUS16), the meronts were found in the liver (Fig. 3E) and kidneys (Fig. 3F). At least five meronts were seen in the liver, and four in the kidneys. These meronts had differences in length and shape, depending on the organ they were infecting. In the kidneys, the meronts were irregularly shaped, without cytomere differentiation, length not exceeding 50 μm, and present mainly in the spaces of the renal tubules (Fig. 3F). In the liver, developing meronts were seen presenting round forms, and with lengths no longer than 30 μm, they were mainly seen in the hepatic sinusoids (Fig. 3E). Of note, a high number of blood stages was seen in the liver; these could be evidenced both in the H&E slides and in the histological preparations treated with CISH (Fig. 3D). No signs of necrosis or inflammation were seen in any of the organs of this individual. Additionally, the brain was the only organ where blood stages were not seen.

A megalomeront was found in the heart of the individual H83/21R (lPARUS22) (Figs. 3H, 3I). The length of this structure was approximately 60 μm width, and it was covered by a conspicuous capsule-shaped wall (Fig. 3I). The large number of unstained areas that look like empty spaces and the strong signal obtained with the Leuco18S probe during CISH suggest that it is a growing stage (Fig. 3I). No other tissue stages were seen in this individual. However, blood stages were observed in histological sections of almost all organs (Fig. 3G). No signs of inflammation or necrosis were observed.

Discussion

The presence of Leucocytozoon spp. exo-erythrocytic development in naturally infected Great tits was confirmed in this study. Meronts were seen in the liver and kidneys, and a megalomeront in the cardiac muscle (Fig. 3). The presence of co-infections was common in the studied individuals (eight out of 29 PCR-positive individuals; Table 1). Morphological identification of Leucocytozoon species was not possible; however, parasites that resemble two Leucocytozoon sp. morphotypes were found, L. majoris and L. fringillinarum (Fig. 1, Table 1). Phylogenetic analysis placed all the lineages found in samples presenting L. majoris morphotype in the same clade and separated from the one with L. fringillinarum morphotype (Fig. 2).

The presence of co-infections with other haemosporidian parasites (Haemoproteus sp. and Plasmodium sp.) was evident by microscopy. Except one, all other microscopically detected co-infections were confirmed by PCR (Table 1). It is to be noted that most of the co-infections were detected from the dead birds opportunistically sampled during the study. The presence of co-infections between different Leucocytozoon sp. lineages in the same individual were evidenced in some of the analyzed samples (Table 1). The high prevalence of co-infections was not a surprising result, since Paridae birds are known for having co-infections between different haemosporidian genera, and within the same parasite genus [41, 53].

The difficulties of morphological characterization of Leucocytozoon spp. have been discussed previously [50]. In the present study, we could not confirm the Leucocytozoon sp. involved in the infections as this requires us to investigate the type-host in the type locality, which has not yet been done. While the blood stages do not provide enough information to delimit Leucocytozoon species [50], underestimating parasite diversity, their cytb genetic diversity can overestimate its diversity [19, 22]. Nonetheless, including other morphological features, type of host cell infected by the parasite, parasite biology, and molecular information is essential to try to untangle the species characterization of Leucocytozoon spp.

Approximately 130 different lineages of Leucocytozoon spp. have been reported to infect Paridae birds, with 64 of them reported in Great tits, 33 in Blue tits, and 13 in Coal tits (MalAvi database, http://130.235.244.92/Malavi/, accessed on February 22, 2024). In the present study, we could confirm the presence of eight of them. Among the isolated Leucocytozoon spp. lineages, lPARUS4 was the most common one, present in five individuals (Table 1). This Leucocytozoon sp. lineage was previously reported to develop in lymphocytes and was associated with the L. majoris morphotype [8]. During this study, we could associate the lineages lSYBOR07 and lPARUS16 to the L. majoris morphotype; however, due to the absence of early stages of Leucocytozoon spp., we could not confirm on which type of host cell the parasite develops. The Leucocytozoon spp. lineages lPARUS4 and lPARUS19 have frequently been reported in the three bird species mentioned above [4]; in this study, the presence of both lineages was confirmed in two of the three species (Table 1).

No Leucocytozoon sp. infections were found in the analyzed Coal tits, which were infected only with Plasmodium sp. pBT7 (Table 1). This could be due to the low number of individuals analyzed in this study. Additionally, when compared to the other Paridae species, the prevalence of haemosporidians in Coal tits is lower. Numerous studies reporting new Leucocytozoon spp. lineages in wild birds are based only on molecular methodologies. Some authors point out that reports based on PCR alone may be the result of DNA amplification of sporozoites persisting in the bird after injection by the vector. However, some of these sporozoites may be unable to continue development in non-adapted hosts [13, 50], which does not allow us to know whether the infection is real or abortive [28, 34, 42, 44, 45]. Therefore, more studies targeting Coal tits should be encouraged to understand whether the prevalence of haemosporidian infections in this species is low.

Microscopic analysis of thin blood smears of birds infected with Leucocytozoon sp. lPARUS22 revealed the presence of L. fringillinarum morphotype (Figs. 1C–1D). The phylogenetic analyses placed this parasite lineage in a separate clade than the ones with L. majoris morphotype (Fig. 2). To date, associating the morphospecies L. majoris with its molecular information is challenging, resulting in difficulties in determining the phylogenetic relationships of the species within the phylogeny of Leucocytozoon spp. [8, 51]. It is noteworthy that Leucocytozoon sp. lPARUS22 clustered with other parasite lineages that have similar morphological features (e.g., the nucleus of the infected cell assumes a cap-like form, which is one of the main features of L. fringillinarum) [21, 49]. Leucocytozoon fringillinarum was described in Eurasian chaffinch Fringilla coelebs in England [49], but to date, no L. fringillinarum lineage has been associated with the type species and type locality [50, 51]. Based on microscopic analyses, it was reported that parasites displaying the L. fringillinarum morphotype possibly develop in thrombocytes, supporting the importance of this character in phylogenetic relationships within Leucocytozoon spp. Two other Leucocytozoon lineages, lTFUS04 and lZOLEU02, have been pointed out as L. fringillinarum; however, they are placed in different and separated branches in the phylogenetic analysis (Fig. 2) [8, 44]. Based on morphological analyses, parasites with this morphotype have a wide distribution and the only zoogeographic region where L. fringillinarum has not been reported is Antarctica [49]. This may be because lZOLEU02 was isolated from a White-crowned sparrow Zonotrichia leucophrys and lTFSU04 from a Great thrush Turdus fuscarter, in the United States and Colombia, respectively. More studies using an integrative approach, including a combination of molecular and morphological techniques, are necessary to better understand the relationships within the genus Leucocytozoon and its relationship with other Haemosporida.

The genetic distance estimate between clades A and B, exceeding 7.9%, indicates that these two groups are likely distinct taxonomic entities. However, analysis of the genetic distances within each clade – 2.7% for clade A and 3% for clade B – suggests that the observed divergence may result from the clustering of lineages into multiple subclades within each group. This implies that the genetic diversity within each clade is higher than expected. Further investigation is required to explore this phenomenon in more detail.

It should be noted that none of the lineages obtained in this study have previously been associated with any described Leucocytozoon morphospecies. Additionally, several investigations reporting Leucocytozoon sp. infections are conducted mainly using molecular analysis [19]. The main reason for this is the lack of experts in parasitology for morphological investigations and species identification [5]. Morphological analysis of thin blood smears should be planned and encouraged in future studies, as well as training in species identification of avian blood parasites, since some characters can be of great value for better understanding phylogenetic analyses and for description of new parasites species.

Research focused on exo-erythrocytic stages has been carried out for some Leucocytozoon species, but these studies were mainly based on experimental infections [11, 14, 18, 26, 29–31, 36, 43, 56], with only a few reports on natural infections [1, 9, 27, 50]. The Leucocytozoon spp. life cycle begins when sporozoites are injected into the avian host by the infected Simuliidae insect during the blood meal, and these sporozoites can be persistent for up to 11 days, the longest persistence time of sporozoites in birds [14]. The role of this feature is still unknown and needs to be studied further [50]. After that, sporozoites invade the parenchymal cells in the liver, where the first generation of merogony occurs [49]. First-generation meronts produce individual merozoites and clusters of nuclei within the cytoplasm, known as “syncytia”. Subsequent generations of meronts, which stem from merozoites created by hepatic meronts, are able to mature within various fixed tissue cells, such as hepatocytes, renal epithelial cells, endothelial cells, and macrophages throughout multiple organs. These subsequent generations are especially common in the spleen and lymph nodes [50]. In the current study, it was not possible to confirm which generations of merogony were present in these infections. The development of meronts was evidenced in two individuals of Great tits (Figs. 3B–3F). The differences in the lengths of meronts found in both the liver (no larger than 30 μm) and the kidneys (no larger than 50 μm), and the high agglomeration of blood stages in capillary vessels suggest asynchronous development of these parasites in their avian hosts. It is also necessary to consider that the merogonial cycles of Leucocytozoon spp. can differ depending on the host species. In experimental infections, the number of generations of tissue stages of this genus is still unknown [51].

The complete exo-erythrocytic development of L. majoris has not been reported in the literature, and the tissue development of this parasite in their avian hosts is completely unknown. Although Leucocytozoon spp. parasite seems to be widely distributed in passerines, given the numerous lineages reported (MalAvi database, http://130.235.244.92/Malavi/ accessed on February 22, 2024), studies directed at understanding the merogonial development of these parasites need to be developed. However, it is known that some Leucocytozoon species, such as L. fringillinarum, can initiate merogony in the kidneys and liver [29, 30]. An experimental study investigated the exo-erythrocytic development of L. fringillinarum obtained from Zonotrichia leucophrys, and injected in Quiscalus quiscula [29, 30]. Meronts found in the kidneys were usually larger than those found in the liver, and they took longer to complete their development. However, in this experiment the authors reported that only the liver and kidneys were analyzed, so it is not possible to confirm whether the tested parasite can develop tissue stages in other organs [29, 30]. It is worth mentioning that the development of irregular meronts in the kidneys and the formation of cytomeres during merozoites development seems to be a general pattern in the maturation of Leucocytozoon spp. parasites [30, 50].

In our study, meront stages were found only in two individuals: H44/21R, with Leucocytozoon spp. unknown lineage (due to co-infection of lineages), and H58/21R Leucocytozoon sp. lPARUS16. The gametocytes found in both individuals were of L. majoris morphotype, and meronts were found only in the liver and kidneys. The megalomeront found in the heart of one analyzed Great tit (with lineage lPARUS22 which clusters with other lineages that resemble the L. fringillinarum morphotype) presented a markedly enlarged and deformed host cell nucleus and a thick capsule-like structure surrounding it (Figs. 3H, 3I). It can be inferred that this was a growing megalomeront, given the absence of individual distinguishable merozoites within the structure (Figs. 3H, 3I). The wall surrounding it is quite thick (≥ 1 μm), and deformed cells and fibers are present around it (Fig. 3I), which is part of the formation of the capsular wall [49, 51]. Furthermore, studies of other haemosporidian parasites of the genus Haemoproteus reported that the intensity of the signal produced by CISH may be different between mature (where the signal is weak) and growing (where the signal is more intense) megalomeronts. This could be because of the difference in ribosomal content of the mature stages of the parasite [16, 17, 24]. The same pattern could also be applied to the Leucocytozoon sp. megalomeront found in this study where an intense signal in the CISH-labelled structure was observed (Figs. 3H–3I). No meronts in any organ were found in this individual, suggesting that the infection by this parasite already had at least one generation of tissue stage, given that experimental infections have shown that the formation of megalomeronts is associated with the second or later generations of development in Leucocytozoon spp. [49–51]. This suggests that the stage found in this individual may be associated with a persistent stage of the parasite in the host, responsible for relapse of the infection. It is important to note that to have a more general overview of the parasite’s life cycle, we should have data from July to August, the period of active transmission at our study site [49]. Unfortunately, in our dataset, this period is not represented, which may or may not be related to the low prevalence of stages in organs. The developmental patterns of the exo-erythrocytic stages of Leucocytozoon spp. in most of the naturally infected passerines are still unknown, which shows that more research should be done in this field to better understand the presence of these stages. While general conclusions on the patterns of exo-erythrocytic development of Leucocytozoon spp. in Paridae birds cannot be drawn based on three positive individuals, the findings indicate possible directions for future investigations.

Conclusion

Leucocytozoon spp. develop different types of exo-erythrocytic stages in natural infections in Great tits. Leucocytozoon sp. meronts were mainly found in the kidneys, although they can also be found in the liver, reinforcing the presence of asynchronous development. One growing megalomeront was found in the heart, confirmed by the CISH signal intensity, indicating that some Leucocytozoon sp. lineages can also infect other organs than those mentioned above, which could represent a serious threat to bird health. The development of certain parasite stages, such as megalomeronts, could be associated with the parasite lineage, as it was found in a single infection with a parasite resembling the morphospecies of L. fringillinarum. Co-infections (Haemoproteus sp., Plasmodium sp., and Leucocytozoon sp.) were reported in several analyzed individuals. Parasites with morphological features resembling L. majoris and L. fringillinarum morphotypes clustered in different clades in the phylogenetic tree. Phylogenies based on partial cytb genes seem to indicate the morphology of parasite-host cell complexes in Leucocytozoon sp. Further studies should be carried out to understand the dynamics, pathogenicity, and life stages of Leucocytozoon spp. in wild birds.

Acknowledgments

We would like to acknowledge the Nature Research Centre for supporting this study and Gediminas Valkiūnas and Tatjana Iezhova for their assistance during fieldwork.

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

Supplementary Table S1: GenBank accession numbers for the sequences used in the phylogenetic analysis. Access here

References

  1. Akiba K. 1958. Studies on Leucocytozoon of chickens in Japan. I. Natural infection of L. caulleryi. Bulletin of the National Institute of Animal Health, 34, 163–180. [Google Scholar]
  2. Allison FR, Desser SS, Whitte LK. 1978. Further observations on the life cycle and vectors of the haemosporidian Leucocytozoon tawaki and its transmission to the Fiordland crested penguin. New Zealand Journal of Zoology, 5, 371–374. [CrossRef] [Google Scholar]
  3. Bennett GF, Laird M, Khan RA, Herman CM. 1975. Remarks on the status of the genus Leucocytozoon Sambon, 1908, Journal of Protozoology, 22, 24–30. [CrossRef] [PubMed] [Google Scholar]
  4. Bensch S, Hellgren O, Pérez‐Tris J. 2009. MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages. Molecular Ecology Resources, 9, 1353–1358. [CrossRef] [PubMed] [Google Scholar]
  5. Bradbury RS, Sapp SGH, Potters I, Mathison BA, Frean J, Mewara A, Sheorey H, Tamarozzi F, Couturier MR, Chiodini P, Pritt B. 2022. Where have all the diagnostic morphological parasitologists gone? Journal of Clinical Microbiology, 60, e00986–22. [CrossRef] [PubMed] [Google Scholar]
  6. Cavalier-Smith T. 2014. Gregarine site-heterogeneous 18S rDNA trees, revision of gregarine higher classification, and the evolutionary diversification of Sporozoa. European Journal of Protistology, 50, 472–495. [PubMed] [Google Scholar]
  7. Chagas CR, Binkienė R, Ilgūnas M, Iezhova T, Valkiūnas G. 2020. The buffy coat method: a tool for detection of blood parasites without staining procedures. Parasites & Vectors, 13, 104. [CrossRef] [PubMed] [Google Scholar]
  8. Chagas CRF, Duc M, Gutiérrez-Liberato GA, Valkiūnas G. 2023. Host cells of Leucocytozoon (haemosporida, leucocytozoidae) gametocytes, with remarks on the phylogenetic importance of this character. Pathogens, 12, 712. [CrossRef] [PubMed] [Google Scholar]
  9. Chagas CRF, Duc M, Himmel T, Eigirdas V, Weissenböck H, Valkiūnas G. 2023. Exo-erythrocytic development of Leucocytozoon parasites (Haemosporida, Leucocytozoidae) in song thrushes Turdus philomelos. International Journal for Parasitology: Parasites and Wildlife, 22, 60–68. [CrossRef] [Google Scholar]
  10. Chew M. 1968. Megaloschizonts of Leucocytozoon in the eyes and sciatic nerves of the domestic fowl. Veterinary Record, 83, 518–519. [CrossRef] [PubMed] [Google Scholar]
  11. Clark GW. 1965. Schizogony and gametocyte development of Leucocytozoon berestneffi in the Yellow‐Billed Magpie, Pica nuttalli. Journal of Protozoology, 12, 584–589. [CrossRef] [Google Scholar]
  12. Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest 2: more models, new heuristics and high-performance computing. Nature Methods, 9, 772. [CrossRef] [Google Scholar]
  13. De Amaral F, Wilson RE, Sonsthagen SA, Sehgal R. 2023. Diversity, distribution, and methodological considerations of haemosporidian infections among Galliformes in Alaska. International Journal for Parasitology: Parasites and Wildlife, 20, 122–132. [CrossRef] [Google Scholar]
  14. Desser SS. 1967. Schizogony and gametogony of Leucocytozoon simondi and associated reactions in the avian host. Journal of Protozoology, 14, 244–254. [CrossRef] [PubMed] [Google Scholar]
  15. Desser SS, Ryckman AK. 1976. The development and pathogenesis of Leucocytozoon simondi in Canada and domestic geese in Algonquin Park, Ontario. Canadian Journal of Zoology, 54, 634–643. [CrossRef] [PubMed] [Google Scholar]
  16. Duc M, Himmel T, Harl J, Iezhova T, Nedorost N, Matt J, Ilgūnas M, Weissenböck H, Valkiūnas G. 2023. Comparative analysis of the exo-erythrocytic development of five lineages of Haemoproteus majoris, a common haemosporidian parasite of European passeriform birds. Pathogens, 12, 898. [CrossRef] [PubMed] [Google Scholar]
  17. Duc M, Himmel T, Ilgūnas M, Eigirdas V, Weissenböck H, Valkiūnas G. 2023. Exo-erythrocytic development of two Haemoproteus species (Haemosporida, Haemoproteidae), with description of Haemoproteus dumbbellus, a new blood parasite of bunting birds (Emberizidae). International Journal for Parasitology, 53, 531–543. [CrossRef] [PubMed] [Google Scholar]
  18. Eide A, Fallis AM. 1972. Experimental studies of the life cycle of Leucocytozoon simondi in ducks in Norway. Journal of Protozoology, 19, 414–416. [CrossRef] [PubMed] [Google Scholar]
  19. Galen S, Nunes R, Sweet P, Perkins S. 2018. Integrating coalescent species delimitation with analysis of host specificity reveals extensive cryptic diversity despite minimal mitochondrial divergence in the malaria parasite genus Leucocytozoon. BMC Evolutionary Biology, 18, 128. [CrossRef] [PubMed] [Google Scholar]
  20. Godfrey Jr RD, Fedynich AM, Pence DB. 1987. Quantification of hematozoa in blood smears. Journal of Wildlife Diseases, 23, 558–565. [CrossRef] [PubMed] [Google Scholar]
  21. Groff TC, Lorenz TJ, Iezhova TA, Valkiūnas G, Sehgal RNM. 2022. Description and molecular characterization of novel Leucocytozoon parasite (Apicomplexa: Haemosporida: Leucocytozoidae), Leucocytozoon polynuclearis n. sp. found in North American woodpeckers. Systematic Parasitology, 99, 103–114. [CrossRef] [PubMed] [Google Scholar]
  22. Harl J, Himmel T, Valkiunas G, Ilgūnas M, Bakonyi T, Weissenböck H. 2020. Geographic and host distribution of haemosporidian parasite lineages from birds of the family Turdidae. Malaria Journal, 19, 335. [CrossRef] [PubMed] [Google Scholar]
  23. Hellgren O, Waldenström J, Bensch S. 2004. A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. Journal of Parasitology, 90, 797–802. [CrossRef] [PubMed] [Google Scholar]
  24. Himmel T, Harl J, Kübber-Heiss A, Konicek C, Fernández N, Juan-Sallés C, Ilgūnas M, Valkiūnas G, Weissenböck H. 2019. Molecular probes for the identification of avian Haemoproteus and Leucocytozoon parasites in tissue sections by chromogenic in situ hybridization. Parasites & Vectors, 12, 282. [CrossRef] [PubMed] [Google Scholar]
  25. Himmel T, Harl J, Matt J, Nedorost N, Lunardi M, Ilgūnas M, Iezhova T, Valkiūnas G, Weissenböck H. 2024. Co-infecting Haemoproteus species (Haemosporida, Apicomplexa) show different host tissue tropism during exo-erythrocytic development in Fringilla coelebs (Fringillidae). International Journal for Parasitology, 54, 1–22. [CrossRef] [PubMed] [Google Scholar]
  26. Huff CG. 1942. Schizogony and gametocyte development in Leucocytozoon simondi, and comparisons with Plasmodium and Haemoproteus. Journal of Infectious Diseases, 71, 18–32. [CrossRef] [Google Scholar]
  27. Ilgūnas M, Himmel T, Harl J, Dagys M, Valkiūnas G, Weissenböck H. 2022. Exo-erythrocytic development of avian haemosporidian parasites in european owls. Animals, 12, 2212. [CrossRef] [PubMed] [Google Scholar]
  28. Imura T, Suzuki Y, Ejiri H, Sato Y, Ishida K, Sumiyama D, Murata K, Yukawa M. 2012. Prevalence of avian haematozoa in wild birds in a high-altitude forest in Japan. Veterinary Parasitology, 183, 244–248. [CrossRef] [PubMed] [Google Scholar]
  29. Khan RA, Fallis AM. 1969. Endogenous stages of Parahaemoproteus fringillae (Labbe, 1894) and Leucocytozoon fringillinarum (Woodcock, 1910). Canadian Journal of Zoology, 47, 37–39. [CrossRef] [Google Scholar]
  30. Khan RA, Fallis AM. 1970. Life cycles of Leucocytozoon dubreuili Mathis and Leger, 1911 and L. fringillinarum woodcock, 1910 (Haemosporidia: Leucocytozoidae). Journal of Protozoology, 17, 642–658. [CrossRef] [Google Scholar]
  31. Lee HR, Koo B-S, Jeon E-O, Han M-S, Min K-C, Lee SB, Bae Y, Mo I-P. 2016. Pathology and molecular characterization of recent Leucocytozoon caulleryi cases in layer flocks. Journal of Biomedical Research, 30, 517. [CrossRef] [PubMed] [Google Scholar]
  32. Lotta IA, Pacheco MA, Escalante AA, González AD, Mantilla JS, Moncada LI, Adler PH, Matta NE. 2016. Leucocytozoon diversity and possible vectors in the neotropical highlands of Colombia. Protist, 167, 185–204. [CrossRef] [PubMed] [Google Scholar]
  33. Lynton-Jenkins JG, Bründl AC, Cauchoix M, Lejeune LA, Sallé L, Thiney AC, Russell AF, Chaine AS, Bonneaud C. 2020. Contrasting the seasonal and elevational prevalence of generalist avian haemosporidia in co-occurring host species. Ecology and Evolution, 10, 6097–6111. [CrossRef] [PubMed] [Google Scholar]
  34. Mata VA, da Silva LP, Lopes RJ, Drovetski SV. 2015. The Strait of Gibraltar poses an effective barrier to host-specialised but not to host-generalised lineages of avian Haemosporidia. International Journal for Parasitology, 45, 711–719. [CrossRef] [PubMed] [Google Scholar]
  35. Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees, in: 2010 Gateway Computing Environments Workshop (GCE) on November 14, 2010 at New Orleans, LA, USA, pp. 1–8. [Google Scholar]
  36. Morii T, Fukuda M. 1992. Observations on first‐generation schizogony of Leucocytozoon caulleryi in chickens. Journal of Protozoology, 39, 281–287. [CrossRef] [Google Scholar]
  37. Morii T. 1992. A review of Leucocytozoon caulleryi infection in chickens. Journal of Protozoology Research, 2, 128–133. [Google Scholar]
  38. Pigeault R, Cozzarolo C-S, Choquet R, Strehler M, Jenkins T, Delhaye J, Bovet L, Wassef J, Glaizot O, Christe P. 2018. Haemosporidian infection and co-infection affect host survival and reproduction in wild populations of great tits. International Journal for Parasitology, 48, 1079–1087. [CrossRef] [PubMed] [Google Scholar]
  39. Rambaut A. 2009. FigTree. Tree figure drawing tool. http://tree.bio.ed.ac.uk/software/Figtree/. [Google Scholar]
  40. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual. New York, NY, USA: Cold Spring Harbor Laboratory Press. [Google Scholar]
  41. Schumm YR, Wecker C, Marek C, Wassmuth M, Bentele A, Willems H, Reiner G, Quillfeldt P. 2019. Blood parasites in Passeriformes in central Germany: Prevalence and lineage diversity of Haemosporida (Haemoproteus, Plasmodium and Leucocytozoon) in six common songbirds. PeerJ, 6, e6259. [CrossRef] [PubMed] [Google Scholar]
  42. Stanković D, Jönsson J, Rakovic M. Diversity of avian blood parasites in wild passerines in Serbia with special reference to two new lineages. Journal of Ornithology, 160, 545–555. [Google Scholar]
  43. Steele EJ, Noblet GP. 1992. Schizogonic development of Leucocytozoon smithi. Journal of Protozoology, 39, 530–536. [CrossRef] [PubMed] [Google Scholar]
  44. Strehmann F, Becker M, Lindner K, Masello J, Quillfeldt P, Schumm Y, Farwig N, Schabo D, Rösner S. 2023. Half of a forest bird community infected with haemosporidian parasites. Frontiers in Ecology and Evolution, 11, 1107736. [CrossRef] [Google Scholar]
  45. Šujanová A, Spitalská E, Václav R. 2021. Seasonal dynamics and diversity of haemosporidians in a natural woodland bird community in Slovakia. Diversity, 13, 439. [CrossRef] [Google Scholar]
  46. Suvarna KS, Layton C, Bancroft JD. 2018. Bancroft’s theory and practice of histological techniques. Elsevier Health Sciences. [Google Scholar]
  47. Tamura K, Stecher G, Kumar S. 2021. MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38, 3022–3027. [CrossRef] [PubMed] [Google Scholar]
  48. Valkiūnas G. 2011. Haemosporidian vector research: marriage of molecular and microscopical approaches is essential. Molecular Ecology, 20, 3084–3086. [CrossRef] [PubMed] [Google Scholar]
  49. Valkiūnas G. 2005. Avian malaria parasites and other haemosporidia. Boca Raton, FL, USA: CRC Press, Taylor & Francis Group. [Google Scholar]
  50. Valkiūnas G, Iezhova TA. 2023. Insights into the biology of Leucocytozoon species (Haemosporida, Leucocytozoidae): Why is there slow research progress on agents of leucocytozoonosis? Microorganisms, 11, 1251. [CrossRef] [PubMed] [Google Scholar]
  51. Valkiunas G, Iezhova TA. 2017. Exo-erythrocytic development of avian malaria and related haemosporidian parasites. Malaria Journal, 16, 101. [CrossRef] [PubMed] [Google Scholar]
  52. Valkiūnas G, Iezhova TA, Križanauskienė A, Palinauskas V, Sehgal RN, Bensch S. 2008. A comparative analysis of microscopy and PCR-based detection methods for blood parasites. Journal of Parasitology, 94, 1395–1401. [CrossRef] [PubMed] [Google Scholar]
  53. Van Rooyen J, Lalubin F, Glaizot O, Christe P. 2013. Avian haemosporidian persistence and co-infection in great tits at the individual level. Malaria Journal, 12, 40. [CrossRef] [PubMed] [Google Scholar]
  54. Waldenström J, Bensch S, Hasselquist D, Ostman O. 2004. A new nested polymerase chain reaction method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood. Journal of Parasitology, 90, 191–194. [CrossRef] [PubMed] [Google Scholar]
  55. Wehr EE. 1962. Studies on leucocytozoonosis of turkeys, with notes on schizogony, transmission, and control of Leucocytozoon smithi. Avian Diseases, 6, 195–210. [CrossRef] [Google Scholar]
  56. Yang YJ, Desser SS, Fallis AM. 1971. Elongate and round gametocytes of Leucocytozoon simondi (Mathis & Léger) in ducks inoculated with megaloschizonts. Journal of Protozoology, 18, 553–556. [CrossRef] [PubMed] [Google Scholar]

Cite this article as: Gutiérrez-Liberato GA, Duc M, Eigirdas V & Chagas CRF. 2025. Leucocytozoon infections in tits (Aves, Paridae): blood and tissue stages investigated using an integrative approach. Parasite 32, 13. https://doi.org/10.1051/parasite/2025007.

All Tables

Table 1

Examined Paridae birds, with the results on thin blood smears microscopy, parasite cytochrome b gene (cytb) lineages, and histology using hematoxylin-eosin (H&E) staining and chromogenic in situ hybridization (CISH).

In the text

All Figures

thumbnail Figure 1

Gametocytes of Leucocytozoon spp. with roundish parasite-host cell complexes from Great tit Parus major blood samples. Two morphotypes were found during microscopic examination: Leucocytozoon majoris (A, B) in the individual H10/20R (lPARUS4) and Leucocytozoon fringillinarum (C, D) in the individual H83/21R (lPARUS22). Microgametocytes (A, C). Macrogametocytes (B, D). Short simple black arrow: parasite nucleus. Black arrowhead: nucleus of infected host cell. Methanol-fixed and Giemsa-stained. Scale bar 10 μm.
In the text
thumbnail Figure 2

Bayesian inference tree of partial cytochrome b gene sequences of 92 Leucocytozoon lineages and sequences linked to a morphospecies. Two lineages (Haemoproteus columbae hHAECOL01 and Leucocytozoon (Akiba) caullery lGALLUS05) were used as the outgroup. Parasite lineages were presented as they appear in the MalAvi database. Two clades were highlighted according to the morphotypes some of the lineages were linked to, being L. majoris (A) and L. fringillinarum (B). Bold font indicates the lineages obtained in this study. The new lineage is indicated with an asterisk. All the posterior probabilities ≥ 0.7 were provided with a black dot. GenBank accession numbers of the sequences used in the phylogeny are given in Supplementary Table S1.
In the text
thumbnail Figure 3

Leucocytozoon spp. exo-erythrocytic and blood stages in naturally infected Great tits Parus major. Individual H44/21R with Leucocytozoon spp. (molecular co-infection) (A–C) presented blood stages in oviduct (A) and meront development found in the kidneys (B, C). Tissue stages visualized in H&E-stained preparations were confirmed by chromogenic in situ hybridization (CISH) using a Leucocytozoon genus-specific probe (Leuco18S) indicated by a purple signal in the inserts of panels. Individual H58/21R infected with Leucocytozoon sp. (cytochrome b lineage lPARUS16) (D–F) had blood stages in the liver vessels (D) as well as meronts in the liver (E) and kidneys (F). All meronts seemed to be growing, as merozoites were not recognizable. Individual H83/21R infected with Leucocytozoon sp. (lPARUS22) (G–I) had blood stages in the spleen (G) and a megalomeront developing in the heart (H, I). The megalomeront, covered by a thick eosinophilic wall, showed growing cytomeres. Black arrowheads: gametocytes. Black triangle arrowheads: meronts. Wide black arrow: enlarged host cell nucleus. Simple black arrow: megalomeront-wall. Scale bars: A, D, G, I 20 μm. B–C, E–F, H 50 μm. (insert I, 50 μm. All remaining inserts, 20 μm).
In the text

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