Molecular Identication of Sarcocystis Halieti in Birds of Prey From Spain

Background: Members of the genus Sarcocystis are protozoan parasites characterized by a prey-predator two-host life cycle. Sarcocysts are formed in muscles or CNS of the intermediate host (IH), while sporocysts develop in the small intestine of the denitive host (DH). Various birds of prey were conrmed to be DH for Sarcocystis spp. By contrast, only two species, S. wobeseri and S. falcatula were identied in the muscles of birds of prey. The latter species is pathogenic and can cause encephalitis in various birds. The aim of the present study was to identify Sarcocystis species in the muscles of birds of prey from Spain. Methods: In the period between 2019 and 2020, muscle tissues of 59 birds collected from Spain were examined for the presence of Sarcocystis spp. Sarcocysts in fresh squashed samples were morphologically characterised under a light microscope (LM). Sarcocystis species were identied by means of 28S rRNA and ITS1 sequence analysis. Results: With the help of methylene blue-staining microscopic sarcocysts were detected in 3/59 (5.1%) birds of prey from Spain. Under LM, one type of sarcocysts was observed. Sarcocysts were thread-like (1050–2160 × 130–158 μm), had a thin (0.7–1.4 μm) and smooth cyst wall. Septa divided the cysts into compartments lled with banana-shaped (5.9 × 1.7 μm) bradyzoites. On the basis of DNA sequence results, S. halieti was identied in the western marsh harrier (Circus aeruginosus) and the black kite (Milvus migrans) for the rst time. Sarcocysts of S. halieti detected in the black kite and the western marsh harrier were shorter and wider as compared to those observed in the great cormorant (Phalacrocorax carbo) and the herring gull (Larus argentatus). Hence, S. halieti might infect birds belonging to three different orders, Suliformes, Charadriiformes

bald eagle (Haliaeetus leucocephalus) [7]. Likewise, an undescribed Sarcocystis species causing encephalitis has been detected in an immature northern goshawk (Accipiter gentilis atricapillus) from Minnesota [4]. Recently, S. wobeseri was identi ed in pectoral and cardiac muscles of the white-tailed sea eagle (Haliaeetus albicilla) [10]. Thus, sarcocysts of two Sarcocystis species, S. falcatula and S. wobeseri, were recorded in the tissues of birds of prey [7,10]. Three morphological types of sarcocysts were detected in the Eurasian buzzard (Buteo buteo) and the long-eared owl (Asio otus), and the third type of sarcocyst distinguished in the owl was recognised as S. otus [11]. However, this species is considered to be invalid [1].
The present paper describes a molecular identi cation of S. halieti in the muscles of birds of prey from Spain.

Methods
In the period between 2019 and 2020, leg muscles of 59 birds of prey (Accipitriformes, Falconiformes and Strigiformes) from Navarra (Spain) were examined for Sarcocystis ( Table 1). The analysed samples come from the birds admitted to the Wildlife Recovery Centre of Ilundain (Navarra). The samples were taken by the Centre's veterinary staff, during the routine diagnostic protocol of the cause of death of the specimens that enter the Centre dead or die there. This Centre belongs to the Government of Navarra and is managed by public company GAN-NIK. Muscle samples were kept frozen (-20ºC) until a morphological detection of sarcocysts. The prevalence and infection intensity of Sarcocystis were evaluated in methylene-blue stained muscle samples as previously described [12]. Genomic DNA was isolated from individual sarcocysts using the GeneJET Genomic DNA Puri cation Kit (Thermo Fisher Scienti c Baltics, Vilnius, Lithuania). Partial 28S rDNA was ampli ed with the help of the KL-P1F/KL-P2R primer pair [13] and the complete ITS1 (internal transcribed spacer 1) region was ampli ed using the SU1F/5.8SR2 primer pair [14]. The PCRs were conducted as described previously [15].
Visualisation, puri cation, and sequencing of PCR products were carried out using the previously described protocol [16]. The sequences obtained in this study were compared with those of various Sarcocystis spp. using the nucleotide BLAST program (megablast option) [17]. The multiple alignment was conducted using the MUSCLE algorithm loaded in MEGA7 software [18]. Selection of a nucleotide substitution model and phylogenetic analysis under Bayesian inference were carried out using TOPALi v2.5 [19].
In leg muscles of one of the black kites (Milvus migrans) sarcocysts were detected in methylene-blue stained muscle samples; however, they were not observed in fresh-squashed samples. Therefore, the muscle sample of this bird was digested with pepsin according to the modi ed protocol of Dubey et al. [1]. Five grams of leg muscles were cut into small pieces and suspended in 15 ml of saline solution (0.9%). The entire substance was homogenized in a commercial blender at top speed for 2 min with breaks. The homogenate was transferred into a 150 ml ask and 15 ml of digestion solution was added to it (pepsin, 0.26 g; NaCl 0.5 g; water up to 15 ml and 37% HCl to pH 1.1). The entire substance was incubated at 37°C for 2 hours and the suspension was used for DNA extraction. Genomic DNA was extracted as described above. External PCR primers were SU1F/5.8SR2 [14], meanwhile internal primers GsShalF1 (5′-GATAATTGACTTTACGCGCCATTAC-3′) and GsShalR1 (5′ GTGCACATCCATATATGCTCATTCT-3′) were designed in the present study. The rst run of a nested PCR assay was conducted as described by [15]. The second run of a nested PCR was carried out in the nal 12.5 µl volume consisting of 6.3 µl of DreamTaq PCR Master Mix, 0.5 µM of each primer, 1 µl from the rst run of PCR, and nuclease-free water. similarity to Sarcocystis sp. from the Cooper's hawk (Accipiter cooperii) (KY348755), and 92.3-92.5% similarity to S. columbae from the herring gull (MN450338-MN450339) and from the woodpigeon (Columba palumbus) (GU253885, HM125052). In ITS1 phylogenetic tree, the obtained sequences of Sarcocystis from the black kites and the western marsh harrier were placed in one cluster together with S. halieti and Sarcocystis sp. from the Chilean skuas (Fig. 1). It should be noted that the sequence of Sarcocystis from the black kite (MmEs1) formed a sister branch to other S. halieti sequences. The 1488 bp 28S rRNA sequence of Sarcocystis from the black kite (MmEs1) differed in 1-2 SNP from those of S. halieti (JQ733512, MF946610, MH130210) and in 7 SNP from those of S. columbae (HM125053), while 1508 bp sequence of Sarcocystis from the western marsh harrier (CaEs1) demonstrated 99.3-100% identity with S. halieti. Thus, on the basis of a molecular examination, S. halieti was identi ed in two black kites and a single western marsh harrier.

Discussion
The present study revealed new IH record for S. halieti. This Sarcocystis species was identi ed in the black kite and the western marsh harrier for the rst time. Thus far, S. halieti have not been observed in the muscles of birds of prey. Previously, S. halieti was detected in the great cormorant [20] and the herring gull [21]. The results of the present study extend the knowledge of S. halieti speci city for the IH and indicate that this species could form sarcocysts in the birds belonging to at least three different orders, Accipitriformes (present study), Charadriiformes [21] and Suliformes [20]. More avian Sarcocystis species, S. calchasi, S. columbae, S. falcatula, S. wobeseri can form sarcocysts in IH belonging to different orders [1,10,21,22]. The development of molecular research and expansion of the diversity of the examined host species leads to the detection of the known Sarcocystis species in different bird orders [22]. Such investigations are particularly important in terms of pathogenic species. It should be noted that highly pathogenic Sarcocystis species, such as, S. neurona, S. canis, S. felis, S. calchasi, S. falcatula are multihost speci c [1]. Up to date, it is not known whether S. halieti is pathogenic. Therefore, extensive histopathological studies of this species are recommended.
Sarcocysts of S. halieti detected in muscles of birds of prey seemingly differed morphologically from those previously described in other IH. For comparative purposes, sarcocysts of S. halieti from the great cormorant were very long, up to 6.5 × 0.1 mm [20], whereas sarcocysts from the herring gull were from 3960 µm to 7930 µm in length and from 43 µm to 128 µm in width [21]. Sarcocysts identi ed from the black kites and the western marsh harrier were shorter and wider (1050-2160 × 130-158 µm). Different shapes of S. halieti sarcocysts may be associated with a diverse type of the anatomical structure of a host. The distribution of muscle forces of accipitrids, falconids and strigiforms tend to possess greater proportions of distally inserted digital exor musculature (53-64%, on average) [23].
Due to a lack of a comprehensive microscopical examination it is di cult to compare morphologically the sarcocysts of S. halieti identi ed in the present work with those observed in other birds of prey. Two types of sarcocysts have been reported in the bald eagles from the USA [24]. The rst type of sarcocyst was microscopic, had a thin cyst wall with spines and contained bradyzoites 5 × 1 µm in size. Type II microscopic sarcocysts were immature and had a 2 µm thick striated cyst wall [24]. These sarcocysts were not similar to those observed in our study. By contrast, type II sarcocysts detected in the Eurasian buzzard [11] measured 694-1850 × 42-235 µm, had a seemingly smooth cyst wall and resembled S. halieti. Also, histologically thin walled (0.5 µm) sarcocysts having a smooth surface with no visible protrusions were found in the cardiac muscle of the white-tailed sea eagle [25]. The length of the sarcocysts was not determined, however, the diameter of the largest cross-sectioned cyst was 40 µm.
Subsequently, S. wobeseri was identi ed in the muscles of the white-tailed sea eagle from the UK [10]. Based on the current knowledge, sarcocsyts of S. halieti and S. wobeseri are morphologically indistinguishable [21]. Lastly, thin-walled (≤ 1 µm) and thick-walled (2-4 µm) sarcocysts were detected in the muscles of raptors from the south-eastern USA, however, no detailed microscopical examination was performed [8]. The most recent studies on Sarcocystis from birds of prey focused on diagnosis of this apicomplexan genus using muscle digestion and subsequent nested PCR [9]

Conclusions
In the present study, S. halieti was identi ed in the black kite and the western marsh harrier from Navarra (Spain) by means of 28S rDNA and ITS1 sequence analysis. This is a third Sarcocystis species detected in the muscles of birds of prey. Studies on Sarcocystis spp. from birds of prey are fragmentary. Therefore, further complex morphological, histopathological and molecular methods should be employed to provide a comprehensive description of Sarcocystis found in birds of prey. Declarations Conceptualization, P.P., A.B., S.Š and D.B.; formal analysis, P.P. and S.Š.; investigation, E.J.N., A.B., P.P. and D.B.; resources D.V. and I.O.; writing-original draft preparation, P.P., A.B., E.J.N., S.Š. and D.B.; writingreview and editing, P.P., A.B., E.J.N., S.Š. and D.B.; visualization P.P. and E.J.N.; supervision, P.P. and D.B.; project administration, P.P. and S.Š.; funding acquisition, A.B. P.P. and S.Š. All authors have read and agreed to the published version of the manuscript.

Funding
Not applicable.

Availability of data and materials
Data supporting the conclusions of this article are included within the article. The 28S rRNA and ITS1 sequences generated in the present study were submitted to the GenBank database under accession numbers MW926916-MW926917 and MW929599-MW929601, respectively.

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