Lineage-specific tissue distribution and high prevalence of haemosporidian parasites in hooded crows (Corvus cornix) from northwestern Italy

Abstract

Haemosporidian parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon are widespread vector-borne pathogens of birds, yet their tissue distribution and lineage diversity remain poorly understood in many host species. We investigated haemosporidian infections in hooded crows (Corvus cornix) from Northwestern Italy using a multi-organ molecular approach. Forty-seven individuals collected between 2010 and 2011 were screened by nested PCR targeting the mitochondrial cytochrome b gene, followed by sequencing. Overall infection prevalence was 97.9%, with Leucocytozoon spp. detected in all infected individuals, followed by Plasmodium spp. (52.2%) and Haemoproteus spp. (17.4%). Five Leucocytozoon lineages were identified, with lineage COCOR09 being dominant and widely distributed across organs (heart, lung, liver, kidney, spleen skeletal muscle and brain), while other lineages showed more restricted tissue patterns. Sequencing also revealed the Haemoproteus lineage CXPIP27 and three globally distributed Plasmodium lineages (LINN1, GRW06, and SGS1) detected for the first time in this host species. Mixed infections were frequent, occurring in over two-thirds of infected birds. These findings demonstrate extensive tissue dissemination, lineage-specific organ tropism, and high infection complexity in hooded crows, underscoring the importance of multi-organ, lineage-level approaches for understanding the ecology and epidemiology of avian malaria parasites.

1. Introduction

Haemosporidian parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon are vector-borne blood parasites responsible for avian malaria. These parasites exhibit complex heteroxenous life cycles involving avian vertebrate hosts and dipteran insect vectors, and vary markedly in their developmental strategies, vector specificity, and pathogenic potential (1–5). Such biological differences strongly influence host–parasite interactions, transmission dynamics, parasite detection, and disease outcomes.

Plasmodium spp., transmitted by mosquitoes (Culicidae), undergo both exoerythrocytic and erythrocytic merogony in the avian host (6). After an initial phase of tissue replication, merozoites invade erythrocytes, where repeated schizogonic cycles occur and are often associated with clinical disease (7). In contrast, Haemoproteus and Leucocytozoon spp. lack erythrocytic schizogony; their asexual replication is restricted to tissue stages, while only gametocytes circulate in peripheral blood (3, 8, 9). Haemoproteus spp. are transmitted by biting midges or louse flies, whereas Leucocytozoon spp., transmitted mainly by black flies, are characterized by extensive exoerythrocytic development with megaloschizont formation in internal organs such as the liver, spleen, and kidneys (3, 10, 11).

These biological differences have important implications for parasite detection and classification and underpin the widespread use of molecular approaches for haemosporidian lineage identification (8, 12–17).

Avian malaria can severely affect host fitness, causing organ hypertrophy, weight loss, reduced reproductive success, and mortality (18, 19). Clinically, infected birds may exhibit anemia, lethargy, reduced body condition, impaired flight performance, and immunosuppression, while severe infections can lead to multi-organ dysfunction and death, particularly in immunologically naïve hosts (3–5, 20). The disease has also been implicated in population declines, particularly in island ecosystems, with one of the most dramatic examples documented in Hawaii, where the introduction of Plasmodium relictum contributed to the extinction of at least seven species of honeycreepers and to severe range contractions in the remaining endemic forest birds (21–24).

Haemosporidian parasites occur worldwide, except in Antarctica, and exhibit high genetic diversity, with infection prevalence varying widely among avian taxa (3, 25–28). This variation likely reflects differences in host immune responses (29, 30) as well as ecological and behavioral factors such as habitat preference, life-history traits, and vector exposure (31–34).

The bird family Corvidae (Passeriformes, Aves), is a cosmopolitan groupcomprising more than 120 species, including crows (Corvus spp.), jays (e.g., Aphelocoma, Calocitta, Cyanocitta, Garrulus spp.), and magpies (Pica, Cyanopica spp.). Molecular investigations have revealed a high diversity of haemosporidian lineages in this family, with over 150 lineages recorded to date, including approximately 90 in the genus Corvus (12, 14–17, 20, 35–37). While most Leucocytozoon species show host specificity at the family or order level (3), Leucocytozoon berestneffi and L. sakharoffi are considered the most relevant haemosporidian parasites infecting corvids, whereas no Plasmodium species appears to be exclusive to the Corvidae family.

The hooded crow (Corvus cornix Linnaeus, 1758) is a highly adaptable omnivorous species widely distributed across Italy, inhabiting a range of natural and anthropogenic environments. Its increasing sedentariness over recent decades, particularly in anthropogenic environments (38), may enhance exposure to local vectors and haemosporidian parasites, as reduced movement increases the time available for contact with local vector populations and locally circulating haemoparasites. In contrast, migratory birds, owing to their extensive movements, may experience less continuous exposure to any single vector community, despite encountering more heterogeneous parasite assemblages (39).

Previous studies have demonstrated that avian malaria parasites are not confined to peripheral blood but may be widely distributed across host organs, emphasizing the importance of multi-organ sampling for accurate parasite detection (40). In corvids, post-mortem investigations combining molecular and histopathological approaches have revealed haemosporidian DNA in multiple tissues within the same individual (20), suggesting systemic dissemination beyond peripheral blood. Similar molecular studies conducted in corvids from other European populations have demonstrated a high diversity of Plasmodium, Haemoproteus, and Leucocytozoon lineages, as well as frequent co-infections (35, 37). The application of nested PCR protocols targeting mitochondrial cytochrome b has significantly improved parasite detection sensitivity compared with traditional microscopy (13), enabling lineage discrimination and phylogenetic inference (41). However, in the study by Scaglione et al. (20), lineage-level identification was not performed, limiting comparisons with the growing body of molecular epidemiological data available for corvid hosts. The aim of the present study is therefore to characterize haemosporidian infections in hooded crows by identifying the specific parasite lineages present in multiple organs. By applying molecular sequencing approaches, we seek to clarify whether infections involve single or multiple lineages within individual hosts, thereby improving our understanding of parasite diversity, tissue distribution, and infection patterns in this widespread corvid species.

2. Materials and methods

2.1. Study area and sample collection

A total of 47 hooded crows (Corvus cornix; 27 males and 20 females) were collected between 2010 and 2011 within a regional wildlife control program in the Province of Turin, Northwestern Italy (D.G.R. no. 74-6702 [08/03/2007] and subsequent amendments). Sampling was conducted in three districts (ATC TO1, ATC TO4, and AFV DUCA) (Figure 1), and carcasses were submitted to the Pathology Section of the Department of Veterinary Sciences, University of Turin.

Figure 1.

Map of Turin province (Piedmont, Italy), indicating the districts enrolled in the study. Reproduced with permission from (20).

Birds were captured using Larsen cage traps and euthanized with CO₂ by trained personnel in accordance with national legislation and ethical guidelines (DL no. 157, 02/11/1992). A complete necropsy was performed on each individual, and samples of heart, lung, liver, kidney, spleen, skeletal muscle, and brain were collected and stored at −20 °C until molecular analyses.

2.2. Parasite detection and sequencing

Genomic DNA was extracted from sampled organs using a commercial DNA isolation kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. Haemosporidian parasites (Plasmodium, Haemoproteus, and Leucocytozoon spp.) were detected using a nested polymerase chain reaction (nested PCR) protocol targeting a fragment of the mitochondrial cytochrome b gene, following the method described by Hellgren et al. (13), modified as already reported by (20, 42, 43).

In detail: the first PCR amplification was performed using the primers HaemNFl (5′-CATATATTAAGAGAAITATGGAG-3′) and HaemNR3 (5′-ATAGAAAGATAAGAAATACCATTC-3′), which amplify mtDNA from Plasmodium, Haemoproteus, and Leucocytozoon spp. Reactions were carried out in a final volume of 25 μL containing 2 μL of DNA template, 1.5 μM of each primer, and 12.5 μL of HotStarTaq® Master Mix (Qiagen, Hilden, Germany). Amplification was performed after an initial hot-start activation at 95 °C for 15 min, followed by 20 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s, with a final extension step at 72 °C for 10 min.

The second PCR step was conducted using 4 μL of the first-round product as template. For Plasmodium/Haemoproteus spp., primers HaemF (5′-TGGTGCTTTCGATATATGCATG-3′) and HaemR2 (5′-GCATTATCTGGATGTGATAATGGT-3′) were used, whereas Leucocytozoon spp. were amplified using primers HaemFL (5′-ATGGT GTTTTAGATACTTACATT-3′) and HaemR2L (5′-CATTATCTGG ATGAGATAATGGIGC-3′). The thermal profile was identical to that of the first PCR step, except for an increased number of cycles (35 cycles). Positive controls consisted of DNA from individuals with known haemosporidian infections, and negative controls were included in each PCR run.

Amplification products were visualized by agarose gel electrophoresis. PCR-positive samples were sequenced on both strands by Macrogen Europe (Amsterdam, Netherlands) using second-round PCR primers. Sequences were manually edited using BioEdit v7.0.5.3 (44) and compared with reference sequences in GenBank using BLAST. Mixed infections were identified based on the presence of double peaks in electropherograms (45). Novel haplotypes were named according to the MalAvi database nomenclature (12) and deposited in GenBank in March 2014 (KJ128987, KJ128988, KJ128989, KJ128990, and KJ128991).

2.3. Statistical analysis

Statistical analyses were performed using GraphPad InStat (v3.05). Associations between parasite lineages and organ-specific detection were evaluated using Fisher’s exact test or the Chi-squared (χ²) test. Statistical significance was set at p < 0.05.

3. Results

3.1. Infection prevalence and parasite genera distribution

Overall, 46 out of 47 hooded crows (97.9%) tested positive for haemosporidian infection, while only one individual (2.1%) was negative. Leucocytozoon spp. was the most frequently detected genus, occurring in 100% of PCR-positive birds (n = 46), followed by Plasmodium spp. (52.2%, n = 24) and Haemoproteus spp. (17.4%, n = 8). All PCR-positive organs were sequenced to discriminate between Haemoproteus and Plasmodium lineages; however, in a subset of samples, sequencing results remained ambiguous despite repeated attempts. Consequently, lineage-level statistical analyses were restricted to individuals for which complete sequence data were available from all PCR-positive organs, corresponding to 33 of 46 birds infected with Leucocytozoon spp. and 24 of 28 birds infected with Haemoproteus/Plasmodium spp., calculating the detection rate as the ratio between the number of positive organs/the total number of organs.

3.2. Leucocytozoon lineage diversity, organ distribution, and phylogeny

Five Leucocytozoon lineages—COCOR03, COCOR09, COCOR11, COCOR12, and COCOR13—were identified (Table 1). Marked differences in organ-specific prevalence were observed among lineages (Figure 2, Supplementary File 1). Lineage COCOR09 was detected in 100% of infected hooded crows and showed consistently high prevalence across all examined organs, ranging from 91.5 to 96.6%. Intermediate prevalence values were recorded for COCOR03, COCOR12, and COCOR11, with organ-dependent detection rates between 58 and 70%. In contrast, COCOR13 was rare and detected exclusively in the heart, indicating a highly restricted tissue distribution.

Table 1.

GenBank no., Linage, GenBank sequence, percentage of match and references for the five new different lineages of Leucocytozoon identified in the analyzed Hooded crows.

GenBank no.	Lineage	GenBank sequence	% match	Reference
KJ128987	COCOR09	Cc_L2 (MF189963)	100	(37)
		Corvus corone, Germany
		Corvus corone 6 (AB741497)	100
KJ128989	COCOR12	Cc_L5 (MF189966)	99.79	(37)
		Corvus corone, Germany
		Cc_L7 (MF189968)	99.79	(37)
		Corvus corone, Germany
KJ128990	COCOR03	Cc_L3 (MF189964)	100	(37)
		Corvus corone, Germany
KJ128991	COCOR13	Cc_L1 (MF189962)	100	(37)
		Corvus corone, Germany
KJ128988	COCOR11	Cc_L2 (MF189963)	100	(37)
		Corvus corone, Germany

Figure 2.

Organ-specific prevalence of Leucocytozoon lineages detected in hooded crows (Corvus cornix). The figure shows the percentage of examined organs testing positive for each Leucocytozoon lineage, as determined by nested PCR. Each bar represents the proportion of organs positive for a given lineage relative to the total number of organs analyzed for that lineage. Lineage COCOR09 was detected in all infected individuals and exhibited consistently high prevalence across all organs, whereas other lineages showed lower and more variable organ-specific detection rates. Lineage COCOR13 was detected exclusively in heart tissue, indicating a highly restricted tissue distribution. Percentages refer to organ-level prevalence, not individual-level infection status.

Significant associations among Leucocytozoon lineages and positivity in crows were detected (p < 0.0001), as well as a strong association between lineage identity and organ tropism (p < 0.0001). In addition, the number of lineages infecting an individual was positively correlated with the number of parasitized organs (p < 0.05) (Table 2). Single-lineage infections were most common in heart and kidney, whereas double-lineage infections predominated in lung, liver, and spleen. Absence of infection and triple-lineage infections were comparatively rare across all organs.

Table 2.

Distribution of the number of Leucocytozoon lineages detected per organ.

Number of lineage of Leucocytozoon per organ	Heart	Lung	Liver	Kidney	Spleen	Skeletal muscle	Brain
0	2	1	2	3	4	3	3
1	20	13	11	20	3	15	16
2	10	19	19	10	25	14	14
3	1	0	1	0	1	1	0

The table shows the frequency of samples with 0, 1, 2, or 3 Leucocytozoon lineages identified in each examined organ.

3.3. Haemoproteus and Plasmodium lineages

Sequencing of Haemoproteus/Plasmodium-positive samples revealed the presence of multiple lineages. During the 2010–2011 sampling period, we identified the Haemoproteus lineage CXPIP27 (GenBank accession KJ128983), reported in 2014 from northwestern Iberia (Table 3). In addition, three Plasmodium lineages—LINN1 (KJ128984), GRW06 (KJ128985), and SGS1 (KJ128986)—were detected in hooded crows, representing the first records of these MalAvi lineages in this host species (Table 3).

Table 3.

GenBank no., Linage, GenBank sequence, percentage of match and references for the five new different lineages of Haemoproteus and Plasmodium identified in the analyzed Hooded crows.

GenBank no.	Lineage	GenBank sequence	% match	Reference
KJ128983	CXPIP27	H360 (KJ488905)	100	(49)
		Corvus corone, Spain
KJ128985	GRW06	GRW06 (DQ368381)	100	(52)
		Acrocephalus arundinaceus
		GRW06, (DQ368381)	100	(35)
		Corvus corax, Bulgaria
		H012 (KJ488566)	100	(49)
		Cettia cetti, Transcaucasia
KJ128984	LNN1	LINN1 (MF817787)	100	(55)
		Turdus migratorius, Michigan (United States)
		Plasmodium matutinum isolate 9/16R (KY287235)	100	(53)
		Luscinia luscinia, Lithuania
		WWT002 (KX867108)	100	(51)
		Baeolophus bicolor, Mississippi, (United States)
		H144 (KJ488698)	100	(49)
		Cyanistes caeruleus, western Greater Caucasus
		Turdidae, northwest Iberia, western Greater Caucasus and Transcaucasia
KJ128986	SGS1	Plasmodium relictum strain Cc_Pp_P (MF189958)	100	(37)
		Corvus corone, Germany
		Plasmodium relictum strain H011 (KJ488565)	100	(49)
		Garrulus glandarius, western Greater Caucasus and Transcaucasia
		Cyanopica cooki, northwest Iberia

Similar to Leucocytozoon, significant associations among Plasmodium lineages and crows (p = 0.001) were observed. Haemoproteus/Plasmodium spp. was detected in a limited number of organs, with the spleen being the most frequently positive organ. Single-lineage infections of Haemoproteus (1 lineage) were detected only sporadically and at low frequency, occurring in a small number of samples in the heart (5), liver (4), skeletal muscle (4), kidney (2), lung (1), spleen (1), and brain (1). No cases of multiple infections (2 or 3 lineages) were observed in any organ (Table 4, Supplementary File 2).

Table 4.

Distribution of the number of Haemoproteus lineages detected per organ.

Number of lineage of Haemoproteus per organ	Heart	Lung	Liver	Kidney	Spleen	Skeletal muscle	Brain
0	28	32	29	31	32	29	32
1	5	1	4	2	1	4	1
2	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0

The table shows the frequency of samples with 0, 1, 2, or 3 Hemoproteus lineages identified in each examined organ.

The distribution of Plasmodium lineages per organ shows that most samples were negative (0 lineages), particularly in the heart, liver, and kidney (29 negative samples each). Single-lineage infections (1 lineage) were more frequently detected in certain organs. The spleen (13 cases), lung (12 cases), and brain (12 cases) exhibited the highest number of single-lineage infections, suggesting a relatively greater presence of Plasmodium in these tissues. Skeletal muscle showed intermediate values (6 cases), whereas heart, liver, and kidney had fewer positive samples (4 cases each). No multiple infections (2 or 3 lineages) were observed in any organ. Overall, the results indicate that Plasmodium infections were generally low in frequency and restricted to single-lineage detections, with a higher occurrence in spleen, lung, and brain compared to other organs (Table 5, Supplementary File 3).

Table 5.

Distribution of the number of Plasmodium lineages detected per organ.

Number of lineage of Plasmodium per organ	Heart	Lung	Liver	Kidney	Spleen	Skeletal muscle	Brain
0	29	21	29	29	20	27	21
1	4	12	4	4	13	6	12
2	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0

The table shows the frequency of samples with 0, 1, 2, or 3 Plasmodium lineages identified in each examined organ.

3.4. Mixed infections and organ-level patterns

Mixed infections involving two or more haemosporidian lineages were detected in 31 of 46 infected hooded crows (67.4%) (Table 6). Co-infections involving multiple parasite genera were observed in 18 individuals (58.1%). Intra-generic co-infections predominated (93.5%), most frequently involving Leucocytozoon lineages, followed by Plasmodium lineages. Among intergeneric infections, the most common combination was Leucocytozoon + Plasmodium (n = 13), whereas coinfections involving Haemoproteus were rare. Triple infections involving all three genera were detected in three individuals.

Table 6.

Distribution of haemosporidian parasite genera detected across examined organs in hooded crows (n = 31).

Organs	Number of haemoparasites genera				Total
	3	2	1	0
Heart	1	2	1	0
Lung	0	10	19	2
Liver	0	6	23	2
Kidney	0	5	22	4
Spleen	1	10	16	4
Skeletal muscle	0	8	20	3
CNS	0	8	21	2
Total	2	53	142	20	217
Percentage	0.9%	24.4%	65.4%	9.2%

More than half of the birds infected with Leucocytozoon (53.3%) harbored at least one additional lineage. Notably, 41.9% of co-infected individuals carried four distinct lineages, and the most heavily parasitized bird harbored up to seven lineages, with all three genera detected in both heart and spleen. When data from all organs and genera were pooled, 9.2% of organs were negative for haemosporidians, whereas only 0.9% were simultaneously positive for all three genera. Most organs (65.4%) were positive for a single parasite genus.

To clarify the interpretation of organ-level patterns, Table 6 summarizes the number of haemosporidian genera detected per individual organ, rather than per host, thereby illustrating the distribution of single- and multi-genus detections across tissues.

4. Discussion

This study provides a lineage-level characterization of haemosporidian infections in hooded crows (Corvus cornix) from Northwestern Italy, revealing a very high prevalence of infection and substantial parasite diversity. The overall infection rate observed is consistent with previous molecular studies in corvids and confirms that this taxonomic group represents a highly permissive host assemblage for haemosporidian parasites (14–17, 35, 37). In particular, the dominance of Leucocytozoon spp., detected in all infected individuals, reinforces the strong association between corvids and this parasite genus.

The predominance of Leucocytozoon infections likely reflects vector-related factors, as black flies (Simuliidae) may exhibit relatively narrow host preferences and high transmission efficiency within specific avian groups (3). In contrast, mosquitoes transmitting Plasmodium spp. are generally more opportunistic feeders, potentially reducing transmission efficiency in corvids. These differences in vector ecology may explain the markedly higher prevalence of Leucocytozoon compared to other haemosporidian genera in this and other corvid populations (46).

The multi-organ sampling approach adopted here proved critical for capturing the full extent of infection. Haemosporidian parasites were widely distributed across host tissues, confirming that infections are systemic rather than restricted to peripheral blood (3, 40). Moreover, the strong association observed between lineage identity and organ tropism suggests that tissue distribution is not random but reflects lineage-specific biological traits, potentially linked to differences in parasite development or host immune interactions (47).

Among Leucocytozoon lineages, COCOR09 was clearly dominant, being detected in all infected individuals and across nearly all organs. Its widespread occurrence and high organ-level prevalence suggest a well-established host–parasite relationship, since the lack of pathological signs may indicate a stable interaction and host tolerance. Furthermore, such high prevalence values imply that the parasite is widely circulating within the host population (3, 54)). In contrast, other lineages showed more restricted distributions, particularly COCOR13, which was detected only in heart tissue. Such heterogeneity supports the idea that different Leucocytozoon lineages occupy distinct ecological niches within the host, facilitating their coexistence (48).

Lineages COCOR09, COCOR03, COCOR13, and COCOR11 showed 100% sequence identity with lineages previously reported from carrion crows (Corvus corone) in Germany, and COCOR09 was also identical to a sequence detected in C. corone from Japan (Table 1). No sequence in GenBank or the MalAvi database matched COCOR12 with complete identity, although this lineage showed high similarity to sequences isolated from carrion crows in Germany (37).

The detection of the Haemoproteus lineage CXPIP27 prior to its first formal description (49) highlights the value of retrospective molecular analyses in assessing whether a parasite was already present in geographic areas that had not yet been investigated. Similarly, the identification of the Plasmodium lineages LINN1, GRW06, and SGS1—all known generalists—in hooded crows supports the notion that Plasmodium parasites frequently cross host-species boundaries via shared vectors (50). Moreover, the LINN1 lineage has been reported in passerine hosts and showed 100% identity with a sequence obtained from Luscinia luscinia in Lithuania (51). The GRW06 and SGS1 lineages, corresponding to widely distributed MalAvi lineages, have been frequently reported across multiple avian taxa worldwide.

5. Conclusion

In conclusion, hooded crows from Northwestern Italy host a diverse and complex assemblage of haemosporidian parasites characterized by extensive tissue dissemination, lineage-specific organ tropism, and frequent mixed infections. The predominance of intra-generic co-infections, especially among Leucocytozoon lineages, suggests repeated exposure to infective vectors and the ability of multiple lineages to persist within the same host. Such complex infection patterns may influence parasite competition, host immune responses, and transmission dynamics, although the direction and magnitude of these effects remain to be clarified. These findings highlight the potential role of corvids as important reservoirs for avian malaria parasites and underscore the value of multi-organ, lineage-level approaches for understanding the ecology and epidemiology of haemosporidian infections in natural bird populations.

Funding Statement

The author(s) declared that financial support was not received for this work and/or its publication.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Ethics statement

The animal study was approved by Commissione Etica e Benessere Animale, Department of Veterinary Science, University of Turin. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

LS: Writing – original draft, Investigation. PP: Data curation, Formal analysis, Writing – review & editing. AP-R: Investigation, Software, Writing – review & editing. EB: Writing – review & editing, Conceptualization. FS: Writing – original draft, Supervision, Investigation.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that Generative AI was used in the creation of this manuscript. AI was partially used for Figure 2.

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Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2026.1724903/full#supplementary-material