Novel phylogenetic clade of avian Haemoproteus parasites (Haemosporida, Haemoproteidae) from Accipitridae raptors, with description of a new Haemoproteus species Author Harl, Josef Institute of Pathology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria Author Fauchois, Anaïs Département Adaptations du Vivant (AVIV), Molécules de Communication et Adaptation des Microorganismes (MCAM, UMR 7245 CNRS), Muséum National d’Histoire Naturelle, CNRS, CP 52, 57 rue Cuvier, 75231 Cedex 05, Paris, France Author Puech, Marie-Pierre Hôpital de la faune sauvage des Garrigues et Cévennes - Goupil Connexion, 34190 Brissac, France Author Gey, Delphine Département Adaptations du Vivant (AVIV), Molécules de Communication et Adaptation des Microorganismes (MCAM, UMR 7245 CNRS), Muséum National d’Histoire Naturelle, CNRS, CP 52, 57 rue Cuvier, 75231 Cedex 05, Paris, France Author Ariey, Frédéric Université de Paris, INSERM 1016, Institut Cochin, Service de Parasitologie-Mycologie Hôpital Cochin, Paris, France Author Izac, Brigitte Université de Paris, INSERM 1016, Institut Cochin, Service de Parasitologie-Mycologie Hôpital Cochin, Paris, France Author Weissenböck, Herbert Institute of Pathology, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria Author Chakarov, Nayden Department of Animal Behaviour, Bielefeld University, Konsequenz 45, 33615 Bielefeld, Germany Author Iezhova, Tatjana Nature Research Centre, Akademijos 2, 08412 Vilnius, Lithuania Author Valkiunas, Gediminas Nature Research Centre, Akademijos 2, 08412 Vilnius, Lithuania Author Duval, Linda Département Adaptations du Vivant (AVIV), Molécules de Communication et Adaptation des Microorganismes (MCAM, UMR 7245 CNRS), Muséum National d’Histoire Naturelle, CNRS, CP 52, 57 rue Cuvier, 75231 Cedex 05, Paris, France text Parasite 2024 Paris, France 2024-02-08 31 5 1 18 http://dx.doi.org/10.1051/parasite/2023066 journal article 299947 10.1051/parasite/2023066 4e1a1e4f-1882-46ea-9d98-b0d75ff02ba4 1776-1042 PMC10854483 38334685 12524609 24FA8E88-C024-4154-88B9-19EA4429AA87 Morphological characteristics of Haemoproteus parasites Among French accipitriform raptor samples, 77/413 (18.6%) were positive for Haemoproteus parasites following microscopic examination of blood films, 53 Buteo buteo , 22 Accipiter nisus , and 2 Circus aeruginosus . These samples were therefore chosen for molecular screenings. In the case of the samples from Austria , the blood films were only available from a subset of individuals kept at the clinic. The samples from Germany originated from 832 nestlings sampled between 2019 and 2022. Blood smears and DNA were available for all samples. Among the German nestling samples, 69 were PCR-positive for hBUBT1 and therefore targeted for close microscopic examination. The examination of all blood films revealed the presence of two distinct Haemoproteus morphospecies based on morphological characteristics of the gametocytes. The microscopypositive samples that were confirmed positive for Haemoproteus spp. by PCR were then used for the molecular characterisation of the found parasite species. The Haemoproteus parasites seen in A. nisus (lineage hACCNIS06) belonged to H. nisi . Haemoproteus parasites observed in blood films of C. aeruginosus (lineage hCIAE08) were morphologically similar to H. nisi but several minor morphological differences were visible; this parasite was considered a variant of H. nisi . Haemoproteus parasites observed in blood films of all PCR-positive and microscopically examined B. buteo from Germany , Austria , and France were morphologically similar and described as Haemoproteus multivacuolatus n. sp. Descriptions of the parasites found are given below. Morphological characterisation of H. nisi Peirce and Marquiss, 1983 , the parasite lineage hACCNIS06 found in the type host A. nisus ( Figs. 1a–1h , Table 1 ) The main diagnostic characters of H. nisi from A. nisus coincide with former descriptions [ 44 , 53 ]. Molecular characterisation of this pathogen was developed for the first time, linking H. nisi to the cytb lineage hACCNIS06. The main features of the blood stages of this lineage are as follows. Macrogametocytes and microgametocytes grow around the nucleus of infected erythrocytes, they markedly enclose the nucleus with their ends but do not displace or only slightly displace it laterally ( Figs. 1a–1h ). Young and growing gametocytes tend to not adhere to the erythrocytes’ nuclei ( Figs. 1b, 1e ). The central part of the advanced growing gametocytes is closely appressed to the erythrocyte’ s envelope, but the ends of the gametocytes usually do not ( Figs. 1b, 1c, 1g ). Advanced growing gametocytes often do not adhere to the erythrocyte’ s nucleus ( Figs. 1b, 1e ), but forms adhering to the nucleus are also common ( Figs. 1c, 1f, 1g ). Fully grown gametocytes are appressed to the nucleus and envelop of infected erythrocytes ( Figs. 1d, 1h ); they are circumnuclear and often completely encircle the nucleus ( Figs. 1d, 1h ) and can occasionally even occupy the entire cytoplasmic space in infected erythrocytes. The cytoplasm of the macrogametocytes is granular in appearance and contains a few small vacuoles ( Figs. 1b, 1h ). Volutin granules are abundant and clumped; they obscure pigment granules ( Figs. 1b–1d, 1f–1h ). The outline of the gametocytes varies markedly; it can be even, slightly wavy, or ameboid. The macrogametocytes’ nuclei are compact, variable in form, and median or sub-median in position ( Figs. 1a, 1b ). The pigment granules are predominantly oval, sometimes roundish, of medium size (0.5–1 µm), randomly scattered throughout the cytoplasm. The configuration of microgametocytes ( Figs. 1e–1h ) resembles that of the macrogametocytes with the usual sexual dimorphic characters, which are the paler stained cytoplasm, the diffuse centrally located nucleus and the predominant gathering of pigment granules close to the ends of the gametocytes ( Figs. 1f–1h ). Voucher blood preparation of H. nisi lineage hACCNIS06 (accession no. MNHN-IR- 2023-01 , 12 September 2013 , HFS Goupil connexion, Hérault , France , collected by Dr. DVM Marie-Pierre Puech ) was deposited in the Muséum National d’ Histoire Naturelle , Paris. Representative DNA sequences: Mitochondrial cytb lineage hACCNIS06 ( GenBank accession OR078933 and OR078931). Table 1. Morphometry of host cells and mature gametocytes of Haemoproteus nisi from Accipiter nisus (lineage hACCNIS06), Circus aeruginosus (hCIAE08), and H. multivacuolatus n. sp. (hBUBT1) from Buteo buteo .

Feature	Measurements (lm)1
H. nisi group		H. multivacuolatus n. sp.
Lineage hACCNIS06	Lineage hCIAE08	Lineage hBUBT1
Uninfected erythrocyte
Length	13.0–16.0 (14.6 ± 0.7)	12.6–15.8 (14.3 ± 05)	9.8–13.5 (11.4 ± 0.9)
Width	6.3–7.9 (6.7 ± 0.4)	6.0–7.7 (6.6 ± 0.3)	6.2–7.8 (6.8 ± 0.4)
Area	61.1–76.0 (69.6 ± 4.2)	59.8–79.0 (67.0 ± 5.0)	50.0–70.4 (62.0 ± 6.0)
Uninfected erythrocyte nucleus
Length	6.4–8.4 (7.5 ± 0.5)	6.0–7.7 (6.5 ± 0.4)	4.5–5.7 (5.1 ± 0.3)
Width	2.1–2.7 (2.4 ± 0.2)	2.1–3.4 (3.1 ± 0.3)	3.0–4.3 (3.7 ± 0.3)
Area	10.5–15.2 (12.1 ± 1.4)	11.8–15.7 (13.3 ± 1.1)	13.0–16.8 (14.8 ± 0.9)
Macrogametocyte
Infected erythrocyte
Length	14.4–17.3 (15.6 ± 0.8)	12.9–16.9 (15.2 ± 0.7)	12.2–15.8 (13.8 ± 0.9)
Width	5.8–7.7 (6.9 ± 0.5)	6.0–7.5 (6.7 ± 0.5)	6.9–8.4 (7.8 ± 0.5)
Area	65.3–91.4 (77.8 ± 7.1)	64.7–94.6 (76.0 ± 6.8)	77.4–100.9 (86.2 ± 6.1)
Infected erythrocyte nucleus
Length	5.9–7.5 (6.7 ± 0.5)	6.0–7.0 (6.3 ± 0.4)	3.7–5.8 (4.6 ± 0.5)
Width	2.1–2.7 (2.3 ± 0.2)	2.1–3.4 (3.0 ± 0.3)	2.9–3.9 (3.4 ± 0.3)
Area	8.4–13.1 (10.1 ± 1.0)	10.1–14.8 (10.0 ± 1.0)	11.1–15.1 (12.8 ± 1.2)
Gametocyte
Length	18.8–31.5 (22.8 ± 3.2)	17.7– 28.7 (22.0 ± 2.2)	16.2–21.2 (18.8 ± 1.3)
Width	1.9–2.7 (2.4 ± 0.3)	2.2–2.8 (2.4 ± 0.2)	2.0–3.7 (2.8 ± 0.4)
Area	42.4–61.8 (48.4 ± 4.3)	39.6–69.7 (52.0 ± 4.8)	44.8– 63.6 (52.6 ± 5.4)
Gametocyte nucleus
Length	2.0–3.7 (2.8 ± 0.4)	2.0–3.9 (2.9 ± 0.4)	–
Width	1.2–2.8 (2.0 ± 0.3)	1.9–3.6 (2.5 ± 0.4)	–
Area	3.1–5.3 (4.2 ± 0.6)	3.6–6.1 (4.9 ± 0.7)	–
Pigment granules	6.0–14.0 (9.4 ± 2.4)	7–18 (12 ± 2.7)	11.0–19.0 (14.5 ± 1.7)
NDR 2	0.7–1.1 (0.9 ± 0.1)	0.8–1.1 (0.9 ± 0.1)	0.4–0.9 (0.6 ± 0.2)
Microgametocyte
Infected erythrocyte
Length	13.1–16.7 (15.1 ± 0.8)	13.9–16.6 (15.0 ± 0.6)	12.2–14.4 (13.4 ± 0.5)
Width	6.0–7.8 (6.9 ± 0.4)	6.0–7.9 (6.8 ± 0.4)	6.5–8.8 (7.7 ± 0.5)
Area	62.7–95.1 (81.7 ± 8.8)	63.8–93.6 (75.5 ± 6.6)	65.8–93.6 (82.4 ± 6.5)
Infected erythrocyte nucleus
Length	5.6–7.7 (6.6 ± 0.6)	6.0–7.7 (6.1 ± 0.4)	4.2–5.8 (4.9 ± 0.4)
Width	1.9–2.8 (2.3 ± 0.2)	2.0–3.6 (3.5 ± 0.2)	2.8–3.7 (3.3 ± 0.2)
Area	8.9–12.7 (10.5 ± 1.0)	10.0–13.8 (11.0 ± 1.2)	10.1–15.1 (12.5 ± 1.4)
Gametocyte
Length	19.0–28.7 (23.9 ± 3.2)	18.8– 28.0 (21.0 ± 2.1)	17.9–24.0 (20.9 ± 1.5)
Width	1.9–3.4 (2.5 ± 0.3)	1.62–2.8 (2.4 ± 0.2)	1.6–3.6 (2.6 ± 0.5)
Area	42.3–61.5 (52.4 ± 5.4)	38.6–68.7 (50.0 ± 5.1)	38.2–66.6 (50.9 ± 6.7)
Gametocyte nucleus
Length	5.8–8.1 (7.0 ± 0.7)	– 3	–
Width	1.8–2.6 (2.1 ± 0.2)	–	–
Area	9.6–21.9 (15.1 ± 3.1)	–	–
Pigment granules	7–19 (13.4 ± 3.1.) 4	8–21 (14 ± 3.2)	13.0–25.0 (18.8 ± 3.1)
NDR	0.7–1.2 (0.8 ± 0.1)	0.8–1.1 (0.9 ± 0.1)	0.4–1.1 (0.7 ± 0.2)

1 All measurements ( n = 21) are given in micrometres. Minimum and maximum values are provided, followed in parentheses by the arithmetic mean and standard deviation. 2 NDR = nucleus displacement ratio according to Bennett and Campbell (1972). 3 Microgametocyte nuclei were markedly diffuse and difficult to measure. 4 Pigment granules were difficult to calculate because they were masked by densely stained volutin. Morphological characterisation of H. nisi Peirce and Marquiss, 1983 , the parasite lineage hCIAE08 found in an additional (non-type) host C. aeruginosus (lineage hCIAE08, Figs. 2a–2l , Table 1 ). The main diagnostic characters of H. nisi from C. aeruginosus coincide with former descriptions [ 44 , 53 ]. Molecular characterisation of the parasite lineage hCIAE08 was developed for the first time. The main features of hCIAE08 blood stages are as follows. Macrogametocytes and microgametocytes grow around the nucleus of infected erythrocytes, they markedly enclose the nucleus with their ends but do not displace or only slightly displace it laterally ( Figs. 2a–2l ). The central part of the growing gametocytes predominantly adheres to the erythrocyte envelope but the ends of the gametocyte usually do not ( Figs. 2c–2f, 2i–2k ); the advanced growing gametocytes tend to adhere to nuclei of infected erythrocytes ( Figs. 2b, 2d ), but gametocytes not adhering to the nuclei were also seen ( Figs. 2c, 2e, 2i ). Fully grown gametocytes are circumnuclear; they often nearly completely or completely encircle the nuclei ( Figs. 2f–2h ) and can occasionally occupy the entire cytoplasmic space in infected erythrocytes. The cytoplasm of the macrogametocyte is heterogeneous in appearance and contains a few small vacuoles ( Figs. 2d, 2g , 2l ); volutin is present and seen as densely stained clamps of variable shape ( Figs. 2d, 2e, 2g , 2h, 2i ); the volutin might obscure the pigment granules, but not noticeably ( Fig. 2h ). The outline of gametocytes varies markedly; it can be even, slightly wavy or ameboid. The nuclei of the macrogametocyte are compact, variable in form, and median or sub-median in position ( Figs. 2c, 2d, 2h ). The pigment granules are roundish or oval, of medium size (0.5–1 µm), and randomly scattered throughout the cytoplasm ( Figs. 2d, 2f, 2i, 2k ). The configuration of the microgametocytes ( Figs. 2i–2l ) resembles that of the macrogametocytes with the usual sexual dimorphic characters, which are the paler stained cytoplasm, diffuse centrally located nuclei ( Figs. 2i, 2l ) and the predominant gathering of pigment granules close to ends of the gametocytes ( Figs. 2i, 2j, 2l ). Volutin is less evident in microgametocytes compared to macrogametocytes (compare Figs. 2e, 2g , 2h and 2i ). Voucher blood preparation of H. nisi lineage hCIAE08 (accession no. MNHN-IR- 2023-02 , 4 April 2019 , HFS Goupil connexion, Hérault , France , collected by Dr DVM Marie-Pierre Puech ) was deposited in the Muséum National d’ Histoire Naturelle , Paris. Representative DNA sequences: Mitochondrial cytb lineage hCIAE08 ( GenBank accession OR078934). Haemoproteus gametocytes observed in blood smears of the two C. aeruginosus infected with the parasite lineage hCIAE08 were morphologically similar to H. nisi hACCNIS06, but some differences were seen, mainly regarding the morphology of the volutin, which was more granular in appearance and more densely stained in parasites of the lineage hACCNIS06 (compare Fig. 1 and Fig. 2 ).