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. 2020 Mar 13;44(2):462–466. doi: 10.1007/s12639-020-01211-x

Haemoparasites in red-legged partridge (Alectoris rufa): first record of Haemoproteus sp. in Italy?

Paolo Tizzani 1,✉,#, Angela Fanelli 1,#, Ennio Negri 2, Fabrizio Silvano 2, Arianna Menzano 3, Annarita Molinar Min 1, Pier Giuseppe Meneguz 1
PMCID: PMC7244661  PMID: 32508424

Abstract

During a health survey in the Province of Alessandria (NW Italy) 267 free ranging red-legged partridge (Alectoris rufa; 137 males and 130 females) were captured in 2009 (n = 101), 2010 (n = 130) and 2011 (n = 36). After biometric data and blood samples collection, animals were released on site. Blood samples were used for the preparation of blood smears. A total of 1.5% of smears presented parasites with a light blue finely granular cytoplasm in red blood cells. The parasites were identified, on the basis of their morphology (no molecular identification was possible), as being Haemoproteus sp. juvenile forms. The infestation prevalence was 0.8% in 2010 and 8.3% in 2011. No parasite was observed in 2009. The size of the infested erythrocytes was not altered (P > 0.05) and the nuclear displacement ratio was 0.8 ± 0.2. All the birds were apparently in good health status. To our knowledge this is the first description of Haemoproteus sp. in red-legged partridge, and more in general in birds belonging to Galliformes Order in Italy.

Keywords: Haemoproteus, Galliformes, Alectoris rufa, North-Western Italy

Introduction

Haemoproteus sp. is an obligate heteroxenous blood protozoan parasite of birds transmitted by blood-sucking insects like mosquitos, of the Families Ceratopogonidae (Culicoides) and Hippoboscidae (Hippoboscidae) (Valkiūnas 2005; Taylor et al. 2007). Asexual development of this parasite occurs in the peripheral blood of the host and sexual development in the vector louse fly (Sá 2011). The infestations are characterized by schyzogony in visceral endothelial cells, gametocyte development in circulating erythrocytes and presence of pigment in granules in infested erythrocytes (Springer 1997). Normally Haemoproteus is considered nonpathogenic in most avian species (Bennett et al. 1982) or in adapted natural hosts (Bennett et al. 1993), but it has been described to cause severe and lethal disease in naive bird species (Donovan et al. 2008; Olias et al. 2011; Ortiz-Catedral et al. 2019; Valkiūnas and Iezhova 2017; Valkiūnas et al. 2017). Moreover some species of Haemoproteus have been described as highly pathogenic, causing severe myositis in avian hosts (Cardona et al. 2002; Olias et al. 2011).

The red-legged partridge (Alectoris rufa) is a medium-sized galliform whose natural breeding range includes Spain, Portugal, Andorra, Italy and France, as well as Corsica, Elba and Balearic islands. Large introduced populations are also reported in the UK, Ireland and Greece (BirdLife International 2012). Despite its wide geographic range, few parasitological investigations are currently available on this species (Millan 2009). Specifically, in Italy, only three scientific articles are available on the pathology of Alectoris rufa and none of these deal with the sanitary status of wild population (Millan 2009).

The occurrence of Haemoproteus sp. in red-legged partridges was reported for the first time in Spain by Millan et al. (2002) at low prevalence (4 infested animals out of 40). The aim of this study was to investigate the occurrence and prevalence of this parasite in a free roaming population of red-legged partridge in Italy.

Materials and methods

In January 2009, 2010 and 2011 267 free ranging red-legged partridges (137 males and 130 females) were captured in the province of Alessandria (NW Italy), in a riparian habitat along the Scrivia river between Villalvernia and Novi Ligure (44° 49′ N, 8° 50′ E).: 101 in 2009, 130 in 2010, and 36 in 2011. The study area is characterized by pastures and mowed meadows, woods, scrublands, and crops of corn, vines and wheat (altitude range 100–200 m a.s.l.). All birds were ringed and sexed by morphologic characteristics (Speek et al. 2001). Blood samples for the preparation of blood smears were obtained from the brachial vein. Following the collection of data, animals were released on site. Blood smears were prepared immediately after withdrawal of the blood, air-dried and subsequently fixed in absolute methanol (1 h) and stained with Giemsa’s solution (45 min, 12% Giemsa, pH 7.4). (Melhorn et al. 1992). The slides were examined by screening the entire film with conventional light microscopy under low magnification (40×). Then, smears were examined under oil immersion (1000×) for at least 15 min for parasite detection. Using a calibrate ocular micrometer, we measured the same number of parasitized and unparasitized red cells (Bennett and Campbell 1972). Parasite identification was made basing on morphologic characteristics. To calculate nuclear displacement ratio (NDR), the formula 2X/(X + Y) was used, where Y is the distance between the periphery of the cell and the periphery of the host cell nucleus on the side the parasite occupies. X represents the distance between the host cell membrane and host cell nucleus on the other side of the erythrocyte; NDR = 1 means no nuclear displacement (Savage et al. 2004).

Holotype and paratype of the haemosporida has been deposited in the Department of Veterinary Sciences, Grugliasco, Turin (Piedmont Region), Italy.

The Mann–Whitney non-parametric test was used to compare the size of parasitized and not-parasitized red blood cells. Data are reported as average ± SD; statistical significance was set at P < 0.05. Statistical analyses were performed using R-2.15.2 software (R Core Team 2012).

Results

Prevalence of Haemoproteus sp. was 1.5% (4/101) in 2010 and 8.3% (3/36) in 2011. The observed parasites are elongate and sausage-shaped with a light blue finely granular cytoplasm; the ends of the parasite extend only partially around the host nucleus and the parasite border is generally well defined. The cytoplasm contains diffusely scattered blue to black pigment granules. Parasites at different development stages (trophozoites and gametocytes) are reported in Fig. 1a, b.

Fig. 1.

Fig. 1

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a, bHaemoproteus sp. parasitizing red blood cells

It is often difficult to count individual granules. The parasite is closed to the host cell nucleus, usually occupies the entire cytoplasm on one side, but rarely displaces the host cell nucleus (mean NDR = 0.8 ± 0.2). The dimensions of the parasitized red blood cells were not altered (P > 0.05). Measurements of parasitized and not-parasitized red blood cells are presented in Table 1.

Table 1.

Morphometric parameters of infested/not infested red blood cells examined and Haemoproteus sp. (mean ± 1SD)

Infected birds (ID) Average
364 365 N28206 N28268
Uninfected erythrocytes (N) 2 11 10 3
Cell length (µm) 13 ± 0 12.6 ± 0.7 14.9 ± 0.7 13.7 ± 0.6 13.6 ± 1.2
Cell width (µm) 7 ± 0 6.7 ± 0.5 7.5 ± 0.5 7.3 ± 0.6 7.1 ± 0.6
Nucleus length (µm) 6 ± 1.4 5.4 ± 0.7 5.6 ± 0.5 6.0 ± 1.0 5.6 ± 0.7
Nucleus width (µm) 2.5 ± 0.7 2.4 ± 0.5 3.3 ± 0.5 2.3 ± 0.6 2.7 ± 0.7
Infected erythrocytes (N) 4 27 10 4 45
Cell length (µm) 13.1 ± 1.2 11.6 ± 0.8 15.2 ± 1.9 13.2 ± 1.7 12.7 ± 1.9
Cell width (µm) 7.2 ± 1.5 6.9 ± 1.0 7.1 ± 0.9 7.5 ± 1.0 7.0 ± 1.0
Nucleus length (µm) 5 ± 1.2 4.8 ± 0.6 5.7 ± 0.5 5.5 ± 1.3 5.1 ± 0.8
Nucleus width (µm) 2 ± 0 2.5 ± 0.5 3.0 ± 0.8 2.0 ± 0 2.5 ± 0.6
Parasite length (µm) 7.7 ± 2.2 5.0 ± 1.7 4.9 ± 2.7 4.75 ± 3.4 5.2 ± 2.2
Parasite width (µm) 3.2 ± 0.5 2.6 ± 0.8 2.4 ± 0.6 2.62 ± 1.8 2.6 ± 0.9
NDR 0.7 ± 0.3 0.9 ± 0.2 0.8 ± 0.2 0.7 ± 0.5 0.8 ± 0.2
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NDR nuclear displacement ratio

Discussion

Despite the importance of red-legged partridge for biodiversity conservation and as economic resource, to date, limited sanitary surveys are available on this species (Millan 2009). Currently, Haemoproteus sp. has been described in A. rufa only in Spain (Millan et al. 2002). Considering the Alectoris genus, Haemoproteus sp. was also described in Chukar Partridges (A. chukar) in Nevada (Christensen 1970) and in Rock Partridge (A. graeca) in Iraq (Mohammad et al. 2001). The only Haemoproteus species described in Europe is H. mansoni, parasite of the Red Grouse (Lagopus l. scoticus) in Scotland (White and Bennett 1979), but Fallis and Bennett (1960) demonstrated that it could not be transmitted to members of other subfamilies of Phasianidae. Therefore, as suggested by Millan et al. (2002), the haemoproteid described in A. rufa could be a different species. We performed parasites identification basing on morphologic features, but to obtain species classification a molecular approach is needed (Fallon and Ricklefs 2008).

The parasited birds were apparently in good health condition, but splenomegaly, previously described by Millan et al. (2002) in the same species or in other bird species (Cordero del Campillo and Rojo 1999; Shivaprasad 2002) is not visible without a necroscopy.

The prevalence of infestation and the NDR we found were in accordance with Millan et al. (2002), but lower than the prevalence found in Phasianidae (P = 23.8%) by Bennett et al. (1982), and Mohammad et al. (P = 16.1%) (2001). This could be probably due not to a different species affinity for the parasite but to the period of investigation. Both the study of Millan et al. (2002) and ours were in fact carried on in winter when, as reported by Valkiunas (1996), gametocytes could disappear from the blood. The same author indicates May–July as the best period to increase the possibility to found blood parasites. Measurements of Haemoproteus collected in this study are bigger than the ones provided by Millan et al. (2002), may be due to a different development stadium of the parasites (Peirce 1981). In Italy the parasite has been previously described in other wild species belonging to the Orders Charadriformes, Falconiformes, Gruiformes, Passeriformes, Pelecaniformes and Strigiformes (Sacchi and Prigioni 1984, 1986, 1989; Sacchi et al. 1992; Zehtindjiev et al. 2012). To our knowledge this is the first report of Haemoproteus sp. in A. rufa and in general in Galliformes order in Italy.

The main limitation of our study is the absence of molecular confirmation of the species reported. Confirmation was not possible due to the reduced availability of blood for molecular analysis. Due to the absence of molecular confirmation, it is still possible that the parasite identified could be a plasmodium. However, the morphological evaluation of the parasites is in line with the description of Mayer (1911), referring to an “oval or ring-like shape”, as characteristic of the species. The same shape of the parasite as characteristic of the species is reported by Karadjian et al. (2013). Moreover, in plasmodium merogony is observed in blood cells, while in Haemoproteus the erythrocytic stages produce only gametocytes (Remple 2004). Considering that merogony had not been observed in any of the infested birds, this can be considered an indirect confirmation of the correct identification of the parasite as Haemoproteus.

The data presented in this work are important, considering that are the only available for the Italian population and can be used as a basis to improve the knowledge on the sanitary status of this species.

Acknowledgements

We would like to thank ATF (Ambiente Territorio Formazione) for financial support; Renzo Ceria, Benito Raschia, Mario Traverso, Franco Volpara (manager directors of Alessandria restocking areas) for field work and logistic support.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with ethical standards

Conflict of interest

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.

Ethical approval

Approval on the capture and manipulation of animals for scientific purposes was provided by the Department of Environment, Province of Alessandria (Italy), with authorization number 20090031830.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Paolo Tizzani and Angela Fanelli have contributed equally to this work.

Contributor Information

Paolo Tizzani, Email: paolo.tizzani@unito.it.

Angela Fanelli, Email: angela.fanelli@unito.it.

Ennio Negri, Email: ennio.negri@gmail.com.

Fabrizio Silvano, Email: fabriziosilvano@virgilio.it.

Arianna Menzano, Email: arianna.menzano@gmail.com.

Annarita Molinar Min, Email: annarita.molinar@unito.it.

Pier Giuseppe Meneguz, Email: piergiuseppe.meneguz@unito.it.

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