Molecular Confirmation of Accipiter Birds of Prey as Definitive Hosts of Numerous Sarcocystis Species, including Sarcocystis sp., Closely Related to Pathogenic S. calchasi

The present study aimed to test intestinal scrapings of the Northern Goshawk (Accipiter gentilis) and the Eurasian Sparrowhawk (Accipiter nisus) from Lithuania for S. calchasi and other Sarcocystis species characterised by bird–bird life cycles. The protozoan parasite Sarcocystis calchasi can cause respiratory and neurological diseases in a variety of birds; however, the distribution of this parasite is not well-examined. Sarcocystis species were identified with nested PCR and sequencing of the partial ITS1 region. Sporocysts and/or sporulated oocysts of Sarcocystis spp. were observed in 16 (100%) Northern Goshawks and 9 (56.3%) Eurasian Sparrowhawks. Four species, S. columbae, S. halieti, S. turdusi, and S. wobeseri, were confirmed in the Eurasian Sparrowhawk. Apart from the latter four species, S. calchasi, S. cornixi, S. kutkienae, and S. lari were established in the Northern Goshawk. A higher prevalence of Sarcocystis spp. and species richness in Northern Goshawks is associated with the differences in the diet of two examined Accipiter species. This study is the first report of S. calchasi in Lithuania. Furthermore, the genetically distinct species Sarcocystis spp. 23LTAcc, which is most closely related to S. calchasi, was found in three Northern Goshawks.


Introduction
Protozoans of the genus Sarcocystis (Apicomplexa: Sarcocystidae) are abundant and worldwide-distributed parasites that might infect reptiles, birds, and mammals, including man [1]. They are characterised by an obligatory two-hosts life cycle. Sarcocysts mainly develop in the muscles and CNS of the intermediate host (prey), and sporulation of oocysts occurs in the small intestine of the definitive host (predator, scavenger) [2]. The morphology of sarcocysts is the main phenotypic feature used for the differentiation of Sarcocystis species, whereas parasite species cannot be discriminated by the morphometric parameters of oocysts and sporocysts [1,2].
Definitive hosts of S. calchasi and S. halieti are hawks, mainly of the genera Accipiter [4,5,18,46,[50][51][52]. These birds of prey become infected by ingesting infected tissues (usually muscle) of various birds, which are intermediate hosts that harbour the asexual stages of the parasite [1,[50][51][52]. In Lithuania, S. halieti was detected in the muscles of the Great Cormorant (Phalacrocorax carbo) [53], the Herring Gull (Larus argentatus) [54], the Hooded Crow (Corvus cornix), and the Common Raven (Corvus corax) [55]. Two species of hawks, the Northern Goshawk (Accipiter gentilis) and the Eurasian Sparrowhawk (Accipiter nisus), which may act as definitive hosts of pathogenic avian Sarcocystis species, are widespread and common in Lithuania [56,57]. The estimated breeding population of the Northern Goshawk (further in the text: Goshawk) is 500-800 pairs, while that of the Eurasian Sparrowhawk (further in the text: Sparrowhawk) is 2000-4000 pairs [57]. Based on geographical reports of the parasite and the distribution of intermediate and definitive hosts, it could be expected that S. calchasi might be prevalent in Lithuania. However, this Sarcocystis species has not been recorded in Lithuania yet.
There is a lack of knowledge about which species of Sarcocystis are spread by birds of prey belonging to the genus Accipiter. Previously, based on molecular methods, Sarcocystis spp. have been identified in the intestinal tract of Accipiter hawks in Germany [50] and the USA [18]. Therefore, the aim of the present study was to examine intestinal samples of Goshawks and Sparrowhawks collected in Lithuania for the presence of S. calchasi and other Sarcocystis species employing birds as their intermediate and definitive hosts. Sarcocystis species were identified using species-specific PCR and sequencing of amplified fragments.

Sample Collection
A total of 32 birds (16 Goshawks and 16 Sparrowhawks) were collected between 2016 and 2022. All birds were found dead (as a result of collisions with motor vehicles, power lines, buildings, etc.) and obtained from the Kaunas T. Ivanauskas Zoology Museum, the Lithuanian national authority responsible for monitoring dead birds. Birds were kept frozen at −20 • C until the animals were dissected.

Isolation of Oocysts/Sporocysts
Sarcocystis spp. were extracted from the entire intestine of each Accipiter bird using a modified Verma et al. [68]. technique. This technique allows isolation of different development stages of Sarcocystis spp., i.e., sporocysts, sporulated oocysts, and unsporulated oocysts, in definitive hosts. The small intestine was removed from the bird, cut lengthwise, and spread over the dissection table with the luminal side up. The intestinal epithelium was lightly scraped with the help of glass slides and suspended in 50 mL of distilled water (dH 2 O). The suspended scrapings in dH 2 O were homogenized in a commercial blender at top speed for 1-2 min or more, with breaks to prevent frothing. The homogenate was Pathogens 2023, 12, 752 3 of 13 centrifuged for 6 min at 1600 rpm at 20 • C in 50 mL centrifuge tubes. The supernatant was discarded, sediments were re-suspended in 50 mL of water, and the homogenate was repeatedly homogenized in a commercial blender and centrifuged at the same conditions. The centrifugation and decantation were repeated until most sporulated oocysts and sporocysts were released from the host tissue. At this stage, the obtained sediments were examined for oocysts/sporocysts under a light microscope using at ×400 magnification. The 400 µL of re-suspended sediments were taken from each sample and used for DNA extraction. The isolation of DNA was performed on all bird samples, regardless of whether Sarcocystis spp. oocysts/sporocysts were found in them.
The purification of the genomic DNA from the sediments of intestinal scrapings was performed using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the manufacturer's instructions. The DNA samples were kept frozen at −20 • C for further molecular analysis.
The identification of Sarcocystis species was carried out using partial sequences of the ITS1 region, situated between the 18S rRNA and 5.8S rRNA genes. To amplify DNA fragments of the surveyed parasite species, nested PCR was used. In the first step, a forward SU1F and reverse 5.8SR primer pair was used [73]. These primers are suitable for the amplification of Sarcocystis spp. isolated from various hosts. In the second step of nested PCR, species-specific primers developed in our previous study were applied [65]. Additionally, for the diagnosis of S. calchasi, the newly designed primers GsScalF2 (5 -CCTTTTGTAAGGTTGGGGACATA-3 )/GsScalR2 (5 -GCCTCCCTCCCTCTTTTTG-3 ) were used. The following primers were chosen using the Primers 3 Plus program [74]. A negative control (nuclease-free water instead of target DNA) and a positive control (DNA of each Sarcocystis spp., except of S. calchasi, extracted from single sarcocysts) were used in each round of PCR. Overall, three negative controls were used: (i) from the first amplification step (ii), from the second amplification step, and (iii) two µL of solution obtained from the negative control of the first nested-PCR step was transferred to the negative control of the second amplification step.
PCR reactions were carried out using DreamTaq PCR Master Mix (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the manufacturer's instructions. The PCR cycling conditions were as follows: initial denaturation for 5 min at 95 • C; 35 cycles of 45 s at 94 • C; 45 s at 55-65 • C, depending on the primer pair; 60 s at 72 • C; and final extension for 10 min at 72 • C. All positive PCR samples were sequenced, and the DNA fragments obtained were used for the confirmation of Sarcocystis species. The visualization, purification, and sequencing of amplified products were carried out using a previously described protocol [8]. The obtained ITS1 sequences of Sarcocystis spp. were deposited in GenBank with accession numbers OQ848675-OQ848736.

Analysis of DNA Sequence and Statistical Data
The obtained chromatograms were manually analysed in Chromas 2.6.5 for ambiguously placed nucleotides. The resulting sequences were truncated to exclude nucleotide binding sites. The sequences were then compared with each other and with the sequences of various Sarcocystis species available in GenBank by nucleotide BLAST (e.g., http://blast.ncbi.nlm.nih.gov/ (accessed on 6 April 2023)). Within-group mean genetic distances, as well as between-group mean genetic distances, were calculated using MEGA7 [75]. The Tamura 3-parameter [76] was set for the estimation of genetic distances. Phylogenetic analysis was also performed with MEGA7. Sequences were aligned with the Pathogens 2023, 12, 752 4 of 13 MUSCLE algorithm [75]. The general time reversible evolutionary model [77] with gamma distribution (GTR + G) was determined to be the most suitable for the analysed alignment. For the evaluation of the robustness of the implied phylogeny, a bootstrap test with 1000 replicates was performed.
The prevalence of distinct Sarcocystis species established in two birds of prey, the Goshawk and Sparrowhawk, was compared using the unconditional exact test, which is a good choice when comparing small samples [78]. Statistical analysis was conducted with Quantitative Parasitology 3.0 software [79]. http://blast.ncbi.nlm.nih.gov/ (accessed on 6 April 2023)). Within-group mean genetic distances, as well as between-group mean genetic distances, were calculated using MEGA7 [75]. The Tamura 3-parameter [76] was set for the estimation of genetic distances. Phylogenetic analysis was also performed with MEGA7. Sequences were aligned with the MUS-CLE algorithm [75]. The general time reversible evolutionary model [77] with gamma distribution (GTR + G) was determined to be the most suitable for the analysed alignment. For the evaluation of the robustness of the implied phylogeny, a bootstrap test with 1000 replicates was performed.

Microscopical Examination of Sarcocystis spp. Oocysts/Sporocysts Isolated from Accipiter Hawks
The prevalence of distinct Sarcocystis species established in two birds of prey, the Goshawk and Sparrowhawk, was compared using the unconditional exact test, which is a good choice when comparing small samples [78]. Statistical analysis was conducted with Quantitative Parasitology 3.0 software [79].

Molecular Identification and Phylogenetic Analysis of Sarcocystis spp. Closely Related to S. calchasi
Four samples isolated from Goshawks were amplified using the GsScalF/GsScalR primer pair, which was previously created to identify S. calchasi [65]. However, the comparison of the resulted sequences showed a match with S. calchasi only in one analysed isolate (AgLT7), whereas three other isolates (AgLT3, AgLT6, and AgLT9) were attributed to Sarcocystis spp. 23LTAcc. In the present study, designed GsScalF2/GsScalR2 primers were used to amplify a single AgLT7 isolate, and the resulted sequence (OQ848675) displayed 100% identity to sequences of S. calchasi (FJ232948) from the racing pigeon (Columba livia domestica).

Molecular Identification and Phylogenetic Analysis of Sarcocystis spp. Closely Related to S. calchasi
Four samples isolated from Goshawks were amplified using the GsScalF/GsScalR primer pair, which was previously created to identify S. calchasi [65]. However, the comparison of the resulted sequences showed a match with S. calchasi only in one analysed isolate (AgLT7), whereas three other isolates (AgLT3, AgLT6, and AgLT9) were attributed to Sarcocystis spp. 23LTAcc. In the present study, designed GsScalF2/GsScalR2 primers were used to amplify a single AgLT7 isolate, and the resulted sequence (OQ848675) displayed 100% identity to sequences of S. calchasi (FJ232948) from the racing pigeon (Columba livia domestica).
Three 427 bp-long ITS1 sequences of Sarcocystis spp. 23LTAcc (OQ848676-OQ848678) showed 100% identity between each other and the greatest similarity with S. calchasi and S. wobeseri (Table 1). It is noteworthy that the interspecific and intraspecific genetic similarity of the compared Sarcocystis spp. did not overlap. Additionally, the genetic differences of Sarcocystis spp. 23LTAcc as compared with other Sarcocystis spp. were more than 4% within ITS1. Therefore, it is very likely that Sarcocystis spp. 23LTAcc represent a genetically new non-described Sarcocystis species. In the phylogenetic tree, Sarcocystis spp. 23LTAcc was placed together with a dozen Sarcocystis spp. employing birds as their intermediate and definitive hosts (Figure 2). The analysed Sarcocystis species were represented by a range from 1 to 100 sequences. Isolates of S. halieti, S. columbae, S. calchasi, S. wobeseri, S. turdusi, S. cornixi, and S. kutkienae, which were represented by more than one sequence, formed monophyletic clades with high bootstrap support (99-100). Based on the partial ITS1, Sarcocystis spp. 23LTAcc was a sister species to S. calchasi, and these two taxa made a sister clade to S. wobeseri. The grouping of the following three species into one cluster was supported by a high bootstrap value (93). In addition, the placement of several Sarcocystis spp., S. halieti, Sarcocystis spp. from Chilean Skua (Stercorarius chilensis) (GenBank: MW160469), Sarcocystis spp. from Common Raven (GenBank: MZ707151), S. corvusi, Sarcocystis spp. from Cooper's Hawk (Accipiter cooperi) (GenBank: KY348755), and S. columbae into one cluster was supported by the close-to-themaximum bootstrap value. By contrast, the grouping of the four remaining Sarcocystis spp., S. fulicae, S. turdusi, S. cornixi, and S. kutkienae, was not well-supported (59 bootstrap values).
showed 100% identity between each other and the greatest similarity with S. calchasi and S. wobeseri (Table 1). It is noteworthy that the interspecific and intraspecific genetic simi larity of the compared Sarcocystis spp. did not overlap. Additionally, the genetic differ ences of Sarcocystis spp. 23LTAcc as compared with other Sarcocystis spp. were more than 4% within ITS1. Therefore, it is very likely that Sarcocystis spp. 23LTAcc represent a genet ically new non-described Sarcocystis species.  (Figure 2). Th analysed Sarcocystis species were represented by a range from 1 to 100 sequences. Isolate of S. halieti, S. columbae, S. calchasi, S. wobeseri, S. turdusi, S. cornixi, and S. kutkienae, which were represented by more than one sequence, formed monophyletic clades with high bootstrap support (99-100). Based on the partial ITS1, Sarcocystis spp. 23LTAcc was a siste species to S. calchasi, and these two taxa made a sister clade to S. wobeseri. The grouping of the following three species into one cluster was supported by a high bootstrap valu (93). In addition, the placement of several Sarcocystis spp., S. halieti, Sarcocystis spp. from Chilean Skua (Stercorarius chilensis) (GenBank: MW160469), Sarcocystis spp. from Com mon Raven (GenBank: MZ707151), S. corvusi, Sarcocystis spp. from Cooper's Hawk (Accip iter cooperi) (GenBank: KY348755), and S. columbae into one cluster was supported by th close-to-the-maximum bootstrap value. By contrast, the grouping of the four remaining Sarcocystis spp., S. fulicae, S. turdusi, S. cornixi, and S. kutkienae, was not well-supported (5 bootstrap values).

The Identification and Distribution of Sarcocystis spp. in Intestines of Accipiter spp.
By using nested PCR targeting the ITS1 region and subsequent Sanger sequencing, eight previously known species (S. calchasi, S. columbae, S. cornixi, S. halieti, S. kutkienae, S. lari, S. turdusi, and S. wobeseri) and Sarcocystis spp. 23LTAcc were confirmed in the small intestines of two Accipiter species (Figure 3). Interspecific genetic differences of analysed Sarcocystis spp. were higher than 4%. Three species, S. cornixi, S. kutkienae, and S. lari, were genetically most distant from other species within ITS1 (possessing more than 9% interspecific genetic differences). By contrast, the lower interspecific genetic differences were calculated comparing S. columbae, S. halieti, and Sarcocystis spp. 23LTAcc. In general, for all examined Sarcocystis spp., the difference between the highest interspecific and intraspecific differences was more than 2%. Apart from Sarcocystis spp. 23LTAcc, other tested Sarcocystis spp. were characterised by up to 1.8% intraspecific genetic variation. Even so mean values of intraspecific genetic differences were small and ranged between 0.1-0.6%. Thus, in the present study, the detected species can be reliably distinguished from other Sarcocystis spp. with the suggested molecular method.

The Identification and Distribution of Sarcocystis spp. in Intestines of Accipiter spp.
By using nested PCR targeting the ITS1 region and subsequent Sanger sequencing, eight previously known species (S. calchasi, S. columbae, S. cornixi, S. halieti, S. kutkienae, S. lari, S. turdusi, and S. wobeseri) and Sarcocystis spp. 23LTAcc were confirmed in the small intestines of two Accipiter species (Figure 3). Interspecific genetic differences of analysed Sarcocystis spp. were higher than 4%. Three species, S. cornixi, S. kutkienae, and S. lari, were genetically most distant from other species within ITS1 (possessing more than 9% interspecific genetic differences). By contrast, the lower interspecific genetic differences were calculated comparing S. columbae, S. halieti, and Sarcocystis spp. 23LTAcc. In general, for all examined Sarcocystis spp., the difference between the highest interspecific and intraspecific differences was more than 2%. Apart from Sarcocystis spp. 23LTAcc, other tested Sarcocystis spp. were characterised by up to 1.8% intraspecific genetic variation. Even so mean values of intraspecific genetic differences were small and ranged between 0.1-0.6%. Thus, in the present study, the detected species can be reliably distinguished from other Sarcocystis spp. with the suggested molecular method. Overall, 48 and 14 sequences of Sarcocystis spp. were obtained by examining the Goshawk and Sparrowhawk, respectively. However, differences in the proportion of infected birds by means of DNA analysis were small compared to Goshawk 12/16 (75.0%) and Sparrowhawk 11/16 (68.8%). Two Accipiter hawks differed in the detected parasite species richness and infection rates of Sarcocystis spp. (Figure 4). Only four species were identified in the Sparrowhawk, as compared to nine Sarcocystis spp. determined in the Goshawk. The prevalence of certain species detected varied from 6.25% (1/16) to 62.5% (10/16). A higher infection rate of all identified Sarcocystis spp. was established in Goshawk. Nevertheless, statistically significant differences were determined only when comparing the prevalence of S. cornixi and S. kutkienae among two Accipiter species, while marginal differences were estimated in the case of S. columbae and S. wobeseri. Overall, 48 and 14 sequences of Sarcocystis spp. were obtained by examining the Goshawk and Sparrowhawk, respectively. However, differences in the proportion of infected birds by means of DNA analysis were small compared to Goshawk 12/16 (75.0%) and Sparrowhawk 11/16 (68.8%). Two Accipiter hawks differed in the detected parasite species richness and infection rates of Sarcocystis spp. (Figure 4). Only four species were identified in the Sparrowhawk, as compared to nine Sarcocystis spp. determined in the Goshawk. The prevalence of certain species detected varied from 6.25% (1/16) to 62.5% (10/16). A higher infection rate of all identified Sarcocystis spp. was established in Goshawk. Nevertheless, statistically significant differences were determined only when comparing the prevalence of S. cornixi and S. kutkienae among two Accipiter species, while marginal differences were estimated in the case of S. columbae and S. wobeseri.

Determination of the Definitive Hosts of Sarcocystis spp.
In the current study, sporulated oocysts and free sporocysts were detected in the intestinal mucosa of Goshawk and Sparrowhawk (Figure 1). The morphometric sizes of parasite sexual stages detected in Accipiter hawks overlapped. Thus, morphologically, it was impossible to determine how many different Sarcocystis species were present in the inves-

Determination of the Definitive Hosts of Sarcocystis spp.
In the current study, sporulated oocysts and free sporocysts were detected in the intestinal mucosa of Goshawk and Sparrowhawk (Figure 1). The morphometric sizes of parasite sexual stages detected in Accipiter hawks overlapped. Thus, morphologically, it was impossible to determine how many different Sarcocystis species were present in the investigated samples. It is well known that birds of prey can be simultaneously infected with sporocysts of several Sarcocystis spp. [18,50,51]. In general, the transmission experiments reveal the definitive host of Sarcocystis species [1,[58][59][60][61]. The need for an infection experiment raises ethical questions regarding the non-pathogenicity or low pathogenicity of the investigated Sarcocystis species, whereas molecular methods can be advantageous for the identification of definitive hosts of Sarcocystis spp. It is important to mention that the molecular identification of Sarcocystis species in intestinal samples does not conclusively prove that the analysed predator or scavenger is the definitive host of the parasite [51]. Furthermore, the obtained molecular analysis data on the definitive hosts of certain Sarcocystis species should be indirectly combined with ecological data [65][66][67].

Distribution of Detected Sarcocystis spp. in the Definitive Hosts
Sporulated oocysts and free sporocysts of Sarcocystis spp. were observed in both investigated Accipiter species. Based on the comparison of the obtained ITS1 sequences, nine Sarcocystis species (S. calchasi, S. columbae, S. cornixi, S. halieti, S. kutkienae, S. lari, S. turdusi, S. wobeseri, and Sarcocystis spp. 23LTAcc) were identified in the intestinal samples of birds of prey in Lithuania for the first time (Table 2). Furthermore, this is the first confirmation of S. wobeseri and S. kutkienae in predatory birds. The prevalence of Sarcocystis spp. in the Goshawk obtained using morphologic methods (100%) was higher than detected by DNA analysis (75.0%). Such findings can be explained by the fact that, unlike the Sparrowhawk, the Goshawk is a generalist predator, and small mammals comprise a part of its diet. In southern Finland, small mammals (mostly squirrels, hares, and voles) formed 10% of all prey items for Goshawk [80].
Notably, the distribution of the identified Sarcocystis species in the current work was unequal in two Accipiter hawks (Figure 4). Two species, S. cornixi and S. kutkienae, were found only in the Goshawk (p < 0.05). Birds of the family Corvidae serve as intermediate hosts for both Sarcocystis species [62,69]. In Europe, corvids are among the few preferred prey groups of the Goshawk [81]. In southern Finland, corvids, mostly the Hooded Crow and the Magpie (Pica pica), formed more than 30% of the total diet of the Goshawk, particularly in sub-urban habitats [80]. On the contrary, Sparrowhawks do not prey on corvid birds [56].
Marginal differences (p = 0.0502-0.0579) were observed when comparing the higher prevalence of S. columbae and S. wobeseri in Goshawk than in Sparrowhawk. Previously, S. columbae was detected in the muscles of bird families Columbidae and Laridae [5,54,82], while up to date, anatids, larids, and accipitrids are known intermediate hosts of S. wobeseri [54,70,83]. Columbids (pigeons and doves) comprise the bulk of the Goshawk diet during the breeding season [84]. A rapid increase in Feral Pigeons (Columba livia domestica) was recently recorded as prey for Goshawk in urban and sub-urban habitats of Europe [80]. Columbids are the preferred prey of the Sparrowhawk [85]. In the United Kingdom, the Wood Pigeon (Columba palumbus), Eurasian Collared Dove (Streptopelia decaocto), and Feral Pigeon were identified as the key diet items of the species [86]. Larids and waterfowl species comprise a small part of the diet of tested Accipiter hawks [56,84]. In southern Finland, waterfowl and shorebird species formed about 3% of the total diet of the Goshawk [80].
Goshawks breeding in the eastern Baltic region are partly migratory, with most ringed individuals recorded in winter being up to 400 km from their nesting sites [56]. Birds breeding in Latvia, Estonia, and Finland were found in Lithuania in October-April. During the migration period, domestic poultry (mostly hens) form a small part of the diet of the Goshawk, while in winter, the diet includes hares and the Grey Partridge (Perdix perdix) [56,80]. Sparrowhawks In this work, S. halieti and S. turdusi were the most commonly identified species in two Accipiter species. Notably, S. halieti is multi-host adapted, employing birds of several different orders as intermediate hosts [22,49,[51][52][53][54][55]87], while S. turdusi was found in the muscles of small passeriform birds of the families Turdidae [71] and Muscicapidae. Thrushes are the preferred prey items for the Sparrowhawk [85]. In Poland, the Song Thrush (Turdus philomelos) comprised 29% of the total diet of the Sparrowhawk [88]. Remarkably, as in this work, S. fulicae and S. corvusi were not found in the small intestine samples of corvids. It is assumed that S. corvusi is rare in the area under examination [55]. Whereas, in Lithuania, other birds of prey specialized in the predation of waterbirds, such as the Western Marsh Harrier (Circus aeruginosus) and the White-tailed Eagle (Haliaetus albicilla), prey on the Eurasian Coot (Fulica atra), which acts as an intermediate host for S. fulicae [56,57].
In conclusion, the composition and distribution of identified Sarcocystis species in the analysed birds of prey are consistent with the diet of the Goshawk and the Sparrowhawk.
Moreover, it should be clarified whether S. wobeseri, which is closely related to S. calchasi, might be pathogenic for intermediate hosts [83]. This species was detected in the leg muscles of anseriforms [70] and Herring Gulls [54] and in the pectoral and cardiac muscles of the White-tailed Eagle [83]. However, a comprehensive histopathological examination of S. wobeseri is lacking [54,83].

Identification of S. calchasi in Lithuania
Sarcocystis calchasi infection can lead to various clinical outcomes, depending on the bird species and the severity of the infection [4,12,[15][16][17]19,20,23,[41][42][43][44][45][46][47][48]. In pigeons, S. calchasi infection can cause neurological symptoms such as ataxia, head tilt, and torticollis [47], as well as severe diffuse, fibrinous perihepatitis [42]. In psittacines, the infection can cause neurological signs such as seizures and paralysis [89]. In Brandt's Cormorants (Phalacrocorax penicillatus), the clinical signs of S. calchasi infection are not well-characterised, but it is known that the parasite can cause encephalitis and lead to neurological dysfunction in these birds [17]. The pathogenicity of S. calchasi for Accipiter hawks is not fully understood, but recent studies suggest that these birds of prey are a natural reservoir for this parasite species [50][51][52]. Epidemiological studies have shown that the prevalence of S. calchasi in wild bird populations can vary depending on the region and the bird species. A country-wide study in Germany demonstrated that the prevalence of S. calchasi in free-ranging Accipiter hawks was 7.3% (95% CI = 4.9-10.5%), with a significantly higher infection rate in juveniles (13.7%; CI = 7.7-22.0%) than in adult birds (5.8%; CI = 2.7-9.3%). Additionally, a slightly higher prevalence of S. calchasi infection was established in Goshawk (10.1%; 95% CI = 5.3-17.0%) than in Sparrowhawk (6.0%; 95% CI = 3.4-9.7%). However, the difference between these two species was not statistically significant (p = 0.17) [46]. In the present research, the S. calchasi species was discovered for the first time in Lithuania. However, the infection was found to be low, as only one Goshawk was confirmed to be positive for this pathogenic Sarcocystis species (Figure 4). Therefore, it can be assumed that S. calchasi is not widespread in Lithuania.

Conclusions
Using species-specific nested PCR targeting the partial ITS1 region and Sanger sequencing, eight known Sarcocystis species, S. calchasi, S. columbae, S. cornixi, S. halieti, S. kutkienae, S. lari, S. turdusi and S. wobeseri, were identified in the small intestines of Goshawk and Sparrowhawk from Lithuania. Higher Sarcocystis species richness and detection rates of examined parasites were established in the Goshawk. The distribution of the detected Sarcocystis species was related to the diet of two Accipiter hawk species.
The study represents the first evidence that Accipiter hawks play a significant role in the distribution of S. wobeseri and S. kutkienae, producing sarcocysts in the muscles of the birds of the family Corvidae. In addition, our research provides the first report of pathogenic S. calchasi in Lithuania. Furthermore, a genetically new species, Sarcocystis spp. 23LTAcc, closely related to S. calchasi, was detected in three Goshawks.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The ITS1 sequences of Sarcocystis spp. from intestinal mucosa scrapings of Goshawk and Sparrowhawk were submitted in GenBank database with accession numbers OQ848675-OQ848736.