First Observations of Buzzards (Buteo) as Definitive Hosts of Sarcocystis Parasites Forming Cysts in the Brain Tissues of Rodents in Lithuania

Simple Summary Some species of Sarcocystis parasites form cysts in the brains of small mammals. These parasites have been shown in laboratory experiments to be transmitted by Buteo buzzards. However, there is a lack of studies identifying these parasites in natural definitive hosts. In the current investigation, we examined brain tissues of small mammals and small intestines of two buzzard species collected in Lithuania for Sarcocystis spp. Species of Sarcocystis were confirmed using DNA sequence analysis. Of the eleven small mammal species inspected, only bank voles were infected with cysts of Sarcocystis glareoli. The prevalence of this parasite in the brain of vole hosts reached 9.1%. Based on genetic examination, half of the investigated common buzzards were positive for S. glareoli. Furthermore, two Sarcocystis species, including a putative new species, were detected in the small intestines of rough-legged buzzards. Our results indicate that Buteo buzzards play an important role in transmitting rarely studied Sarcocystis parasites forming cysts in the tissues of small mammals. Abstract Representatives of the genus Sarcocystis are worldwide distributed apicomplexan parasites characterised by two-host prey-predator relationships. Sarcocystis spp. produce sarcocysts in the muscles and brains of intermediate hosts and develop sporocysts in the intestines of definitive hosts. Two species, Sarcocystis glareoli and Sarcocystis microti, previously assigned to the genus Frenkelia, form cysts in the brains of rodents and are transmitted through the common buzzard (Buteo buteo). In our study, brain samples of 694 small mammals caught in different regions of Lithuania were examined for Sarcocystis spp. Additionally, 10 B. buteo and two rough-legged buzzards (Buteo lagopus) were tested for sporocysts of the analysed parasites. Sarcocystis species were identified based on 28S rRNA sequence comparison. Of the eleven species of small mammals tested, Sarcocystis parasites were observed only in the bank vole (Clethrionomys glareolus). Cysts of S. glareoli were detected in 34 out of 374 C. glareolus (9.1%, 95% CI = 6.4–12.5%). Molecular investigation showed the presence of only S. glareoli in the intestines of 50% of B. buteo. Furthermore, two species, Sarcocystis sp. Rod3 and Sarcocystis sp. Rod4, were confirmed in B. lagopus. Our results demonstrate the need for further studies on Sarcocystis cycling between rodents and birds.


Introduction
The genus Sarcocystis (Apicomplexa: Sarcocystidae) is a widespread group of parasites worldwide, comprising more than 200 species.These parasites infect reptiles, birds and mammals, including humans.Members of the genus Sarcocystis are characterised by a two-host life cycle.Typically, the intermediate host of the parasite is the prey, and the definitive host is the predator.Sarcocysts develop mainly in the muscles and central nervous system (CNS) of the intermediate host, while oocysts sporulate in the small intestine of the definitive host [1,2].Some species of Sarcocystis are pathogenic, and the pathogenicity of the parasite is expressed in intermediate hosts [3].Due to their widespread prevalence, small mammals are fundamental components in food webs, playing important roles in ecosystem functioning.They serve as the base of the food chain for many birds of prey [4,5].Small mammals represent one of the animal groups where the highest number of Sarcocystis species has been identified.To date, over 45 different Sarcocystis species have been reported to use small mammals as intermediate hosts [1], and S. attenuati [6], S. funereus [7], S. kani [8], S. muricoelognathis [9], S. myodes [10], S. ratti [11] and S. scandentiborneensis [12] have been described in recent years.However, as surveys for Sarcocystis spp. in these hosts are fragmentary, the actual number of Sarcocystis species is likely much higher [8,13].
In previous taxonomical reviews, members of the family Sarcocystidae were traditionally categorized into two groups.The genera Sarcocystis and Frenkelia were classified under the subfamily Sarcocystinae, while other genera such as Besnoitia, Hammondia, Neospora, Toxoplasma, and others were attributed to subfamily Toxoplasamatinae.Within the genus Frenkelia, only two species, Frenkelia glareoli and F. microti have been identified that form cysts in the CNS of rodents.The subfamily Sarcocystinae was characterised by obligate development in two hosts, with the sexual sporogony stage occurring in the intestine of the definitive hosts.Traditionally, the Frenkelia and Sarcocystis genera were distinguished based on the localisation and morphology of their asexual stages in the intermediate host.The asexual phase of the life cycle of Frenkelia species occurs in the liver of the intermediate host, with only one generation of merozoites, while cysts are exclusively detected in the CNS [14].Molecular analyses conducted between 1998 and 2000 suggested the synonymisation of these two genera [15][16][17].Finally, the species F. glareoli was reclassified as S. glareoli, F. microti was renamed S. microti and a new name S. jaypeedubeyi was proposed for the former S. microti [18,19].
Sarcocystis microti infections have been documented in small mammals across North America [26], Europe [23,[27][28][29], and Japan [30].Similarly, the presence of S. glareoli has been reported in Europe [23,24,28,29,[31][32][33].Both S. microti and S. glareoli utilize the common buzzard (Buteo buteo) as their definitive host.However, the rough-legged buzzard (Buteo lagopus) is also suspected to serve as a definitive host for S. glareoli [34], while S. microti employs red-tailed hawks (Buteo jamaicensis) [26].Thus, the available data do not resolve the issue of intermediate and definitive host specificity of S. glareoli and S. microti.Since the identification of S. glareoli and S. microti has primarily relied on morphological studies, further molecular investigations are necessary to identify these Sarcocystis species in different hosts.
Both S. glareoli and S. microti were previously confirmed in Lithuania through microscopic analysis of 560 rodents caught across different regions of the country between 1995 and 2001 [28].However, Buteo buzzards in Lithuania have not been examined for Sarcocystis parasites.The objectives of our present study are threefold: (i) to establish the prevalence of Sarcocystis spp. in the brains of small mammals collected in Lithuania, (ii) to identify Sarcocystis species in the brain tissues of these animals using molecular methods, and (iii) to investigate the potential role of Buteo buzzards in the transmission of Sarcocystis spp.forming cysts in the brains of small mammals using molecular analysis.

Trapping of Small Mammals
A standard snap trap line method (25 traps at 5 m intervals) was deployed to capture small mammals at 13 distinct locations within Lithuania during the autumn of the year 2023.In three cases, sites in close proximity were combined into one sample (Figure 1).Sarcocystis spp. in the brains of small mammals collected in Lithuania, (ii) to identify Sarcocystis species in the brain tissues of these animals using molecular methods, and (iii) to investigate the potential role of Buteo buzzards in the transmission of Sarcocystis spp.forming cysts in the brains of small mammals using molecular analysis.

Trapping of Small Mammals
A standard snap trap line method (25 traps at 5 m intervals) was deployed to capture small mammals at 13 distinct locations within Lithuania during the autumn of the year 2023.In three cases, sites in close proximity were combined into one sample (Figure 1).

Morphological Examination of Sarcocystis spp. from Brain Tissues of Small Mammals
To determine the prevalence of Sarcocystis spp. in the brain of small mammals, fragments of brain tissue (~0.2 g) were stained with 0.2% methylene blue solution for 24 h.Then samples were cleared in 1.5% acetic acid solution for 45-50 min.Afterwards, samples were squeezed between glass compressors and studied under a light microscope (LM) at 40× and 100× magnification.The parasite load was evaluated by counting cysts in ~0.2 g of sample.

Morphological Examination of Sarcocystis spp. from Brain Tissues of Small Mammals
To determine the prevalence of Sarcocystis spp. in the brain of small mammals, fragments of brain tissue (~0.2 g) were stained with 0.2% methylene blue solution for 24 h.Then samples were cleared in 1.5% acetic acid solution for 45-50 min.Afterwards, samples were squeezed between glass compressors and studied under a light microscope (LM) at 40× and 100× magnification.The parasite load was evaluated by counting cysts in ~0.2 g of sample.

Genetic Characterization of Sarcocystis spp. Detected in Small Mammals
The DNA isolation from sarcocysts was conducted using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the manufacturer's recommendations.The partial 28S rRNA was amplified using KL-P1F/KL-P1R primer pairs (Table 1) [35].Each PCR reaction was carried out in a 25 µL mixture containing 12.5 µL of DreamTaq PCR Master Mix (Thermo Fisher Scientific Baltics, Vilnius, Lithuania), 4 µL of DNA template, 0.5 µM of both forward and reverse primers and nuclease-free water.The PCR was initiated with the initial hot start at 95 • C for 5 min followed by 35 cycles of 94 • C for 45 s, annealing at 52 • C for 60 s and 72 • C for 80 s, and a final extension at 72 • C for 7 min.The visualisation, purification, and sequencing of PCR products followed the previously described protocol [36].In order to detect essentially similar DNA sequences, and evaluate the interspecific and intraspecific genetic variability of detected Sarcocystis parasites, the 28S rRNA sequences generated in this study were compared with those of various Sarcocystis spp.using the nucleotide BLAST program (http://blast.ncbi.nlm.nih.gov/,accessed on 10 March 2024).

Collection of Buteo Buzzards
A total of 12 birds (10 B. buteo and two B. lagopus) were collected between 2017 and 2020.All birds, obtained from the Kaunas Tadas Ivanauskas Zoology Museum (the Lithuanian national authority responsible for monitoring dead birds), were found dead as a result of collisions with motor vehicles, power lines, buildings, etc., and were kept frozen at −20 • C until they were dissected.

Morphological Examination of Sarcocystis spp. from Intestines of Buteo Buzzards
Sarcocystis spp.oocysts/sporocysts were excreted from the entire intestine of each buzzard using a slightly modified method by Verma et al. [38].Initially, the small intestine was removed from the bird.Then, the faeces from each intestine were squeezed, and the gut was cut lengthwise.The intestinal epithelium was scraped with the help of a scalpel and suspended in 50-100 mL of distilled water.The homogenization was performed in a commercial blender at top speed for 1-2 min with breaks to prevent frothing.The homogenate was centrifuged for 6 min at 1600 rpm, 25 • C in a 50 mL centrifuge tube.The supernatant was discarded, and the sediments were re-suspended in 50 mL water.The above-described process was repeated 5-8 times until most oocysts/sporocysts were released from the host tissue.Thereafter, the sporocyst pellet was suspended in HBSS and filtered through cheesecloth.The homogenate was emulsified in 5.25% sodium hypochlorite (bleach) solution (1:1 ratio) in a cold bath for 30 min.The centrifugation for 6 min at 1600 rpm, 25 • C, and removal of supernatant were repeated until the smell of bleach (chlorine) was gone.Eventually, the oocysts/sporocysts of Sarcocystis spp.were examined under LM at 400× and 1000× magnification.The 400 µL of re-suspended sediments were taken from each sample and used for DNA extraction.

The Genetic Identification of Sarcocystis spp. from Intestines of Buteo Buzzards
The DNA isolation from the mucosal suspension was performed using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania).The DNA samples were then stored frozen at −20 • C until further molecular analysis.
Sarcocystis spp.were identified by nested PCR of partial 28S rDNA sequences.In the first step of nested PCR, forward Sgrau281 and reverse Sgrau282 primers were used (Table 1).Whereas in the second step of nested PCR, two primer pairs, GsSglaF1/GsSglaR1 and GsSmicF1/GsSmicR1 were used.The primers of the second round of nested PCR were designed with the help of Primer3Plus program [39].These primers were designed for amplification of S. glareoli and S. microti.However, due to relatively small variance of Sarcocystis spp.using rodents and birds as their intermediate and definitive hosts, respectively, within rRNA genes [40], it was suspected that GsSglaF1/GsSglaR1 and GsS-micF1/GsSmicR1 might not only be suitable for the amplification of S. glareoli and S. microti.Positive controls (DNA of S. glareoli extracted from single sarcocysts) were used in each set of PCRs.Three negative controls (nuclease free water instead of target DNA) were used: one control for the first amplification step and two controls for the second step of nested PCR.The third negative control was obtained by transferring 1 µL from the negative control of the first amplification step to the negative control of the second amplification step.PCRs were conducted under the conditions described above in Section 2.3 and using annealing temperatures provided in Table 1.For the second PCR assay, 1 µL from the first PCR assay was used.
The visualisation, purification, and sequencing of PCR products were carried out using the previously described protocol [36].Our 28S rRNA sequences were compared with those of various Sarcocystis spp. with the Nucleotide BLAST program (megablast option) (http://blast.ncbi.nlm.nih.gov/,accessed on 10 March 2024).The 28S rDNA sequences of Sarcocystis spp.isolated from intestinal scrapings of Buteo buzzards were made available in GenBank with PP535696-PP535702 accession numbers.

Phylogenetic Analysis
Based on the molecular results, a putative new Sarcocystis species was identified in single sample of intestinal mucosa of B. lagopus, prompting further phylogenetic analysis.Phylogenetic relationships were reconstructed using the Maximum Likelihood (ML) method.Multiple alignments of partial 28S rRNA sequences of 20 Sarcocystis taxa was carried out using the MUSCLE algorithm incorporated into MEGA7 version 7.0.26software [41].The selection of the evolutionary model was conducted using MEGA7, based on the lowest Bayesian Information Criterion values generated.The robustness of the resulted phylogeny was tested using bootstrap test with 1000 replications.
Furthermore, phylogenetic analysis was performed to evaluate the relationships between the species identified in this study and closely related ones (S. glareoli, S. microti, S. jamaicensis and Sarcocystis sp.).A phylogenetic tree was constructed using the Maximum Likelihood algorithm, as described in the paragraph above.Additionally, phylogenetic relationships among the different sequences were inferred through coalescent simulations using a median-joining model implemented in NETWORK v. 10.2.0.0 [42].

Statistical Tests
Confidence intervals (95% CI) for the proportions of infection prevalence were estimated using online software [43].Differences in infection prevalence between study sites were tested using the G-test on an online calculator [44].The number of cysts in the samples was compared between the different study sites using the Student's t-test in STATISTICA for Windows, version 6.0 (StatSoft, Inc., Tulsa, OK, USA).The level of significance was set at p < 0.05.

Prevalence and Parasite Load of Sarcocystis spp. in Small Mammals
Cysts of Sarcocystis were observed exclusively in the brains of a single small mammal species, C. glareolus.The prevalence of infection reached 9.1% (34 infected of 374 tested, 95% CI = 6.4-12.5%).The infection rates varied among these animals across different study areas.Sarcocystis spp.were detected in rodents collected from eight out of the 13 localities, accounting for 61.5% of the sampled sites (Table 2).The highest detection rates of cysts of Sarcocystis were found in Kamasta, Zabarauskai, and Aukštikalniai.In other sampling sites the prevalence of Sarcocystis spp.ranged from 2.1% to 12.5%.The prevalence of Sarcocystis spp.infection was significantly different only between the two study sites, Kamasta and Lukštas (G = 7.4, p < 0.05).The number of cysts in 0.2 g brain samples varied from 1 to 50, with an average of 13.4 ± 1.89 (average number of cysts ± standard error).We compared cyst counts across various locations, focusing on three areas where more than one infected individual was captured.The Kamasta study site exhibited the highest average number of cysts (18.0 ± 4.90), correlating with the highest prevalence of examined parasites.The average number of cysts in Kamasta was higher than in the Bileišiai site (11.3 ± 2.24) and the Utena site (9.6 ± 3.17), though the differences were not statistically significant (t = 1.45, p = 0.16 and t = 1.19, p = 0.26).
(Figure 2b), or irregular round structures measuring 48.2-89.0× 75.7-104.1 µm (average size: 73.6 × 94.0 µm; n = 4) (Figure 2c).Based on molecular analysis, sarcocysts isolated from brain tissues of C. glareolus in Lithuania were identified as S. glareoli.Our analysis of ten 944 bp long 28S rRNA sequences of S. glareoli isolated from C. glareolus demonstrated 100% identity among them.These sequences have been deposited in Gen-Bank under the accession number PP535695.Despite significant variation in the size and shape of the cysts, the 28S rRNA region tested showed no differences among the S. glareoli isolates.
The 944 bp sequences of S. glareoli obtained in this study exhibited 99.9% identity to S. glareoli sequence AF044251.In our sequences, the C insertion was noticed in 537 nucleotide position to be compared (in TAGGTCCCCCG region, insertion is bolded and underlined).Notably, numerous other related Sarcocystis spp.had the C insertion in a homologous nucleotide position indicating highly probable sequencing error in AF044251 GenBank record.

Morphological Characterisation of Sarcocystis spp. Sporocysts/Oocysts Found in Intestines of Buteo Buzzards
Both examined B. lagopus harboured sporocysts and sporulated oocysts of Sarcocystis spp.
The examination of sporocysts and sporulated oocysts found in the intestines of B. lagopus and B. buteo indicated that morphological parameters of Sarcocystis spp.overlapped in size.Our analysis of ten 944 bp long 28S rRNA sequences of S. glareoli isolated from C. glareolus demonstrated 100% identity among them.These sequences have been deposited in GenBank under the accession number PP535695.Despite significant variation in the size and shape of the cysts, the 28S rRNA region tested showed no differences among the S. glareoli isolates.
The 944 bp sequences of S. glareoli obtained in this study exhibited 99.9% identity to S. glareoli sequence AF044251.In our sequences, the C insertion was noticed in 537 nucleotide position to be compared (in TAGGTCCCCCG region, insertion is bolded and underlined).Notably, numerous other related Sarcocystis spp.had the C insertion in a homologous nucleotide position indicating highly probable sequencing error in AF044251 GenBank record.

Molecular Investigation of Sarcocystis spp. Detected in Intestines of Buteo Hawks
When analysing samples of DNA extracted from scrapings of the intestinal mucosa of two hawk species, amplification was unsuccessful using GsSmicF1 and GsSmicR1 primers in the second round of nested PCR.Out of the 10 examined intestinal mucosa samples from B. buteo, five tested positive (50.0%) by nested PCR using GsSglaF1 and GsSglaR1 as internal primers.The resulting five sequences of 28S rRNA without primer binding sites were 515 bp long.These sequences showed 100% identity with each other and to those of S. glareoli from C. glareolus from Lithuania.
Based on BLAST analysis, the 28S rRNA sequences obtained in this study differed from those of S. jamaicensis (KY994650) by two T/G and A/G SNPs (99.6% similarity), and from those of S. microti (AF044252) by four SNPs and single indel (99.0%similarity), and by ≥2.3% from those of other Sarcocystis spp.available in GenBank.Therefore, molecular investigation confirmed the presence of S. glareoli in the intestinal mucosa of five common buzzards.
Sequencing of the 28S rRNA fragment amplified by the GsSglaF1/GsSglaR1 primer pair, both B. lagopus were positive for Sarcocystis parasites.Comparison of the analysed 515 bp long and 522 bp long sequences revealed the presence of two different Sarcocystis species, Sarcocystis sp.Rod3 and Sarcocystis sp.Rod4.
It is worth noting that the experimental intermediate host of S. funereus and S. strixi are laboratory mouse, while owls serve as definitive hosts of these Sarcocystis species [7,46,47].
Our 515 bp long 28S rRNA sequences of S. glareoli and Sarcocystis sp.Rod3 exhibited significant similarity to 299-316 bp long sequences (OK576420, OK576425, OK576428-36, The examination of sporocysts and sporulated oocysts found in the intestines of B. lagopus and B. buteo indicated that morphological parameters of Sarcocystis spp.overlapped in size.

Molecular Investigation of Sarcocystis spp. Detected in Intestines of Buteo Hawks
When analysing samples of DNA extracted from scrapings of the intestinal mucosa of two hawk species, amplification was unsuccessful using GsSmicF1 and GsSmicR1 primers in the second round of nested PCR.Out of the 10 examined intestinal mucosa samples from B. buteo, five tested positive (50.0%) by nested PCR using GsSglaF1 and GsSglaR1 as internal primers.The resulting five sequences of 28S rRNA without primer binding sites were 515 bp long.These sequences showed 100% identity with each other and to those of S. glareoli from C. glareolus from Lithuania.
Based on BLAST analysis, the 28S rRNA sequences obtained in this study differed from those of S. jamaicensis (KY994650) by two T/G and A/G SNPs (99.6% similarity), and from those of S. microti (AF044252) by four SNPs and single indel (99.0%similarity), and by ≥2.3% from those of other Sarcocystis spp.available in GenBank.Therefore, molecular investigation confirmed the presence of S. glareoli in the intestinal mucosa of five common buzzards.
Sequencing of the 28S rRNA fragment amplified by the GsSglaF1/GsSglaR1 primer pair, both B. lagopus were positive for Sarcocystis parasites.Comparison of the analysed 515 bp long and 522 bp long sequences revealed the presence of two different Sarcocystis species, Sarcocystis sp.Rod3 and Sarcocystis sp.Rod4.
It is worth noting that the experimental intermediate host of S. funereus and S. strixi are laboratory mouse, while owls serve as definitive hosts of these Sarcocystis species [7,46,47].

Phylogenetic Relationships of Examined Sarcocystis Species
Based on the 28S rRNA fragment amplified with GsSglaF1/GsSglaR1 primers, the three Sarcocystis species identified in this study were categorized into two clusters, alongside Sarcocystis species utilizing rodents as intermediate hosts and birds of prey as definitive hosts (Figure 4).Our sequences of S. glareoli grouped together with the sequence of S. glareoli taken from the GenBank AF044251.Sarcocystis sp.Rod3 was found to be most closely related to S. jamaicensis and S. microti.However, the grouping of Sarcocystis sp.Rod3 with S. microti was not well supported (68 bootstrap value).Notably, the branch lengths within the clade comprising S. glareoli, S. jamaicensis, Sarcocystis sp.Rod3 and S. microti was shorter compared to that established in another clade consisting of Sarcocystis spp.using rodents and birds of prey as their intermediate and final hosts.The following clade included S. funereus, S. strixi, S. cf.strixi and Sarcocystis sp.Rod4 from B. lagopus.Sarcocystis funereus formed a sister branch to S. strixi and S. cf.strixi, while Sarcocystis sp.Rod4 formed a sister branch to the aforementioned taxa.

Phylogenetic Relationships of Examined Sarcocystis Species
Based on the 28S rRNA fragment amplified with GsSglaF1/GsSglaR1 primers, the three Sarcocystis species identified in this study were categorized into two clusters, alongside Sarcocystis species utilizing rodents as intermediate hosts and birds of prey as definitive hosts (Figure 4).Our sequences of S. glareoli grouped together with the sequence of S. glareoli taken from the GenBank AF044251.Sarcocystis sp.Rod3 was found to be most closely related to S. jamaicensis and S. microti.However, the grouping of Sarcocystis sp.Rod3 with S. microti was not well supported (68 bootstrap value).Notably, the branch lengths within the clade comprising S. glareoli, S. jamaicensis, Sarcocystis sp.Rod3 and S. microti was shorter compared to that established in another clade consisting of Sarcocystis spp.using rodents and birds of prey as their intermediate and final hosts.The following clade included S. funereus, S. strixi, S. cf.strixi and Sarcocystis sp.Rod4 from B. lagopus.Sarcocystis funereus formed a sister branch to S. strixi and S. cf.strixi, while Sarcocystis sp.Rod4 formed a sister branch to the aforementioned taxa.We conducted another phylogenetic analysis by comparing our S. glareoli and Sarcocystis sp.rod3 28S rRNA sequences with those of Sarcocystis sp. from Rogers et al. [48] as well as those of S. glareoli (AF044251), S. microti (AF044252) and S. jamaicensis (KY994650).Out of the 32 analysed sequences, 13 haplotypes were identified, and four haplotypes (sequences: OK576420, OK576425, OK576431, OK576432, OK576433, OK576435, OK576436, OK576440, OK576447, OK576451, OK576459, OK576461, OK576462, OK576464, OK576465) were clustered together with a high support value of 89 (Figure 5a).These four haplotypes were sepa- We conducted another phylogenetic analysis by comparing our S. glareoli and Sarcocystis sp.rod3 28S rRNA sequences with those of Sarcocystis sp. from Rogers et al. [48] as well as those of S. glareoli (AF044251), S. microti (AF044252) and S. jamaicensis (KY994650).Out of the 32 analysed sequences, 13 haplotypes were identified, and four haplotypes (sequences: OK576420, OK576425, OK576431, OK576432, OK576433, OK576435, OK576436, OK576440, OK576447, OK576451, OK576459, OK576461, OK576462, OK576464, OK576465) were clustered together with a high support value of 89 (Figure 5a).These four haplotypes were separated from the remaining ones by at least six mutations (Figure 5b) and presumably belonged to a single Sarcocystis species isolated from B. jamaicensis and B. lineatus.However, clustering of other haplotypes belonging to S. glareoli and Sarcocystis sp.rod3, S. jamaicensis, S. microti and remaining haplotypes of Sarcocystis sp.(OK576428, OK576429, OK576430, OK576434, OK576438, OK576439, OK576441, OK576442, OK576443, OK576448, OK576449, OK576456) was not well defined.Thus, the variability of the examined fragment was not sufficient to reveal how many distinct taxa comprised the nine haplotypes.It also should be pointed out that at 307-308 bp 28S rRNA fragment, Sarcocystis sp.rod3 showed 100% identity to OK576428 and OK576430 sequences of Sarcocystis sp. from two Buteo buzzard species from the USA.
Biology 2024, 13, x FOR PEER REVIEW 10 of 16 of other haplotypes belonging to S. glareoli and Sarcocystis sp.rod3, S. jamaicensis, S. microti and remaining haplotypes of Sarcocystis sp.(OK576428, OK576429, OK576430, OK576434, OK576438, OK576439, OK576441, OK576442, OK576443, OK576448, OK576449, OK576456) was not well defined.Thus, the variability of the examined fragment was not sufficient to reveal how many distinct taxa comprised the nine haplotypes.It also should be pointed out that at 307-308 bp 28S rRNA fragment, Sarcocystis sp.rod3 showed 100% identity to OK576428 and OK576430 sequences of Sarcocystis sp. from two Buteo buzzard species from the USA.

Sarcocystis Parasites in the Brain Tissues of Rodents
A previous study conducted in Lithuania reported that 72 out of 341 (21.1%) of the analysed C. glareolus had cysts of S. glareoli in their brains [28].However, in the present examination, the prevalence of S. glareoli was significantly lower (p < 0.001), as only 9.1% (95% CI = 6.4-12.5) of the studied C. glareolus specimens were infected.
In this study, no cysts of S. microti were detected, despite its previous detection in Lithuania.Formerly, S. microti was identified in two rodent species; however, the prevalence of this parasite was low, with rates of 2.9% (1/35) in A. oeconomus and 4.2% (1/24) in M. agrestis [28].Two comprehensive investigations (n > 1700) carried out in Europe determined a lower prevalence of S. microti compared to S. glareoli [23,29].In the study conducted in France, cysts of S. microti were found in M. agrestis, M. arvalis, and C. glareolus, with prevalence ranging from 1.0% to 9.2% in each species [23].Meanwhile, in the Czech Republic, S. microti was detected in M. arvalis, C. glareolus, and Apodemus sp., with infection rates ranging from 0.6% to 5.0%

Sarcocystis Parasites in the Brain Tissues of Rodents
A previous study conducted in Lithuania reported that 72 out of 341 (21.1%) of the analysed C. glareolus had cysts of S. glareoli in their brains [28].However, in the present examination, the prevalence of S. glareoli was significantly lower (p < 0.001), as only 9.1% (95% CI = 6.4-12.5) of the studied C. glareolus specimens were infected.
There are multiple reports that the prevalence of Sarcocystis spp. in small mammals is affected by seasonal changes.A study on Norwegian lemmings (Lemmus lemmus) concluded that S. microti infestation of intermediate hosts depends on the season, with the highest number of parasites found in summer (9%) and the lowest in autumn (0-4%) [27].Furthermore, research conducted in the Netherlands analysed seasonal dynamics of S. cernae infection in the intermediate host M. arvalis and definitive host the common kestrel (Falco tinnunculus) and concluded that prevalence of S. cernae varies throughout the seasons and was highest in the spring [49].Similar results were obtained in a study in France, where a variety of small mammals belonging to genera Myodes, Arvicola, Microtus, Apodemus and Sorex were investigated [23].In our study, rodents were collected in autumn when lower infection rates of Sarcocystis spp.are observed based on aforementioned research.In the previous study conducted in Lithuania [28] rodents were collected not only in autumn, but also in spring and summer thus probably leading to the detection of higher total prevalence of Sarcocystis spp. in the brains of rodents.

Buteo Buzzards as Definitive Hosts of Sarcocystis spp.
Although detailed studies on S. glareoli and S. microti have been carried out in intermediate hosts [23,28,29], these parasites have been scarcely studied in their natural definitive hosts.Based on morphological examination, "Frenkelia-like" sporocysts were detected in the droppings and intestines of Buteo buzzards in Europe [29,50] and North America [26,51,52].Without molecular analysis, it is not possible to ascertain whether the sporocysts discovered in the above studies belonged to S. glareoli and/or S. microti, which form cysts in the brains of rodents, or to some other Sarcocystis spp.This is because the morphometrical parameters of sporocysts of many different species of Sarcocystis overlap [1,52].
Due to the lack of data, the prevalence of Sarcocystis spp.sporocysts in different Buteo species, geographical regions, seasons, etc. is still generally unknown.A detailed study conducted in the Czech Republic revealed that the prevalence of Sarcocystis spp. in faeces from nests of B. buteo ranged from 28.3% to 88.6%, depending on the season and locality [29].Another study performed in North America examined droppings from red-tailed hawks (Buteo borealis) in a rehabilitation centre and determined that half of them contained Sarcocystis spp.sporocysts [52].In this work, by microscopical examination, 80% of B. buteo and both analysed B. lagopus were infected with oocysts/sporocysts of Sarcocystis spp.Based on 28S rRNA sequence analysis, S. glareoli was confirmed in B. buteo and two Sarcocystis species (Sarcocystis sp.Rod3 and Sarcocystis sp.Rod4) were detected in B. lagopus.Thus, our results indicate that Buteo buzzards in Europe can transmit not only S. glareoli and S. microti, previously assigned to the genus Frenkelia.
The common buzzard nests across nearly the entire European region within the Western Palearctic [53].This migratory species breeds in Lithuania and is commonly found throughout the country, although it rarely stays for the winter season.It nests in woodlands and small clusters of trees near cultivated fields, displaying remarkable adaptability in choosing its breeding sites [54].A wide array of prey is consumed, encompassing small mammals, birds, reptiles, amphibians, larger insects, and earthworms [55].The primary portion (38.3%) of B. buteo diet consists of small mammals, predominantly voles [54,56].Studies conducted in Poland indicate that these raptors mostly catch common voles [57].
Of the five B. buteo in which S. glareoli were identified, only two of them have a known collection date (February and September).Taking into account that definitive hosts excrete sporocysts of Sarcocystis spp. up to several months [1] and based on migratory data, birds collected in February and September were most likely infected with S. glareoli in Lithuania.
Another species, B. lagopus, only winters in Lithuania.This buzzard is distributed in the northern part of the Western Palearctic.Its range stretches eastward, predominantly across the tundra belt, reaching the Pacific Ocean.From there, it tracks along the coastal areas southward to Sakhalin and extends across the northern reaches of North America on the opposite side of the Pacific [53].The rough-legged buzzard predominates in the diet, mainly small rodents, accounting typically for 80-95% of the food, where they occur in large numbers [52,54].In Lithuania, ringed B. lagopus chicks were found in central Europe: Slovakia, Hungary, France, and Italy [54].In autumn and winter in Lithuania, the ringed B. lagopus come from Norway, Finland, and mostly from Sweden [54].Both of B. lagopus examined in current work were collected in November of 2018.Based on ecological data, infected B. lagopus were migrants most likely from Scandinavia [54].It is likely that these buzzards were infected with Sarcocystis sp.Rod3 and Sarcocystis sp.Rod4 before entering Lithuania.

Genetic Identification of Sarcocystis sp. Rod3 and Sarcocystis sp. Rod4
Based on phylogenetic analysis using 28S rRNA, Sarcocystis sp.Rod3 was found to be closely related to three Sarcocystis species, S. glareoli, S. microti and S. jamaicensis (Figures 4 and 5).These Sarcocystis species produce sarcocysts with thin and smooth cyst walls [1,40].The natural intermediate host of S. jamaicensis is unknown.However, laboratory experiments have shown that this species circulates between rodents and B. jamaicensis [40,45].Two IFN-c gene knockout mice orally infected with sporocysts of S. jamaicensis had meningoencephalitis associated with schizonts and merozoites of this parasite, while sarcocysts were detected in the muscles of the animals [45].Thus, the developmental stages of these three species, S. glareoli, S. microti and S. jamaicensis, are found in the brain.The detection of Sarcocystis sp.Rod3 in the intestine of a single B. lagopus collected in Lithuania suggests that it might be another species in Europe, apart from S. glareoli and S. microti, which can infect rodent brains.Also, our work demonstrated that 28S rRNA locus is not sufficiently variable for the reliable discrimination of S. glareoli, S. microti, S. jamaicensis and Sarcocystis sp.Rod3.To date, only sequences of 18S rRNA and 28S rRNA of S. glareoli and S. microti are available in GenBank [15,16], while S. jamaicensis has been characterised within 18S rRNA, 28S rRNA, mitochondrial cytochrome oxidase 1 (cox1) and ITS1.The ITS1 genetic marker has been proven to be the best choice for the separation of Sarcocystis species using birds and predatory mammals as their intermediate hosts [58,59].Although this genetic region has not been commonly applied for the characterization of Sarcocystis spp.from rodents, it has shown more variability compared to cox1 [9,13,59].In contrast, cox1 is relatively conservative considering Sarcocystis spp.parasitizing small mammals [59].Therefore, the highly variable ITS1 can be helpful for the distinguishing the aforementioned Sarcocystis species.
Furthermore, the results of the current study and that described by Rogers et al. [48] indicate that different species of the genus Buteo from North America and Europe can transmit the same Sarcocytis species.Intriguingly, based on short at 307-308 bp 28S rRNA sequences, Sarcocystis sp.Rod3 was identified in three different Buteo species.Nevertheless, further studies are needed to clarify the specificity of Sarcocystis species for Buteo buzzards as their definitive hosts.
Sarcocystis sp.Rod 4, identified in a single B. lagopus, phylogenetically clustered with S. funereus, S. strixi, and S. cf.strixi (Figure 4).Two owl species, the Tengmalm's owl (Aegolius funereus) and the barred owl (Strix varia), are proven definitive hosts of S. funereus [47] and S. strixi [46], respectively.Sarcocysts of these two Sarcocystis species have not yet been discovered in natural intermediate hosts.However, recently the DNA of S. funereus has been detected in blood sample of a single C. glareolus from Lithuania [60].There is a lack of investigations on the role of the Strigiformes in the transmission of Sarcocystis spp.[37,46,47].Even so, the close relationships of Sarcocystis sp.Rod 4 to S. funereus and S. strixi imply that Sarcocystis species using Accipitriformes and Strigiformes as their definitive hosts might not have evolved separately.Further examinations are needed to clarify whether the same Sarcocystis species can employ representatives of both orders Accipitriformes and Strigiformes as their definitive hosts.

Conclusions
After examining brain samples from 694 small mammals belonging to eight Rodentia and three Eulipotyphla species, cysts of S. glareoli were observed in only 9.1% of the C. glareolus investigated.The literature suggests that this molecularly confirmed Sarcocystis species is more common in Europe compared to another species, S. microti, which also forms cysts in the brains of small mammals.The 28S rRNA sequence analysis confirmed the presence of S. glareoli in half of the B. buteo examined, indicating that these predatory birds are natural definitive hosts of S. glareoli in Lithuania.Our results also highlight the necessity of using other genetic markers, such as ITS1, to distinguish between Sarcocystis spp.parasitizing the brains of small mammals.
Based on molecular examination, two species were identified in B. lagopus: Sarcocystis sp.Rod3, which is closely related to S. jamaicensis, S. glareoli and S. microti, and a putative new species, Sarcocystis sp.Rod4.Ecological data suggest that B. lagopus were likely infected outside Lithuania.Overall, our work underscores the significant role of Buteo buzzards in the transmission of Sarcocystis parasites that produce cysts in small mammals.

Figure 4 .
Figure 4. Maximum likelihood phylogenetic tree of Sarcocystis spp.based on 28S rRNA alignment containing 532 nucleotide positions.Sarcocystis myodes was chosen as an outgroup.The HKY + G evolutionary model was used for analysis.GenBank accession numbers of sequences are displayed in parenthesis.Bootstrap values higher than 50 are indicated next to branches.Three Sarcocystis species identified in intestinal mucosa of buzzards from Lithuania are highlighted in red.

Figure 4 .
Figure 4. Maximum likelihood phylogenetic tree of Sarcocystis spp.based on 28S rRNA alignment containing 532 nucleotide positions.Sarcocystis myodes was chosen as an outgroup.The HKY + G evolutionary model was used for analysis.GenBank accession numbers of sequences are displayed in parenthesis.Bootstrap values higher than 50 are indicated next to branches.Three Sarcocystis species identified in intestinal mucosa of buzzards from Lithuania are highlighted in red.

Figure 5 .
Figure 5.The maximum likelihood phylogram (a) and median-joining network (b) of selected Sarcocystis spp.based on 299 bp long fragment of 28S rRNA.Sarcocystis turdusi was used as an outgroup.The Tamura-Nei + G + I evolutionary model was used for ML analysis.GenBank accession numbers are given after species name.The figures next to branches display bootstrap values higher than 50.When multiple sequences were assigned to a single haplotype, only sequences from different hosts are shown in the figure.Hypothetical not determined haplotypes are named mv1-mv4.Dashes indicate mutational steps.Colours respond to Buteo buzzards from which Sarcocystis spp.oocysts/sporocysts were isolated.The S. sp. in our figure correspond to S. jamaicensis in [48].

Figure 5 .
Figure 5.The maximum likelihood phylogram (a) and median-joining network (b) of selected Sarcocystis spp.based on 299 bp long fragment of 28S rRNA.Sarcocystis turdusi was used as an outgroup.The Tamura-Nei + G + I evolutionary model was used for ML analysis.GenBank accession numbers are given after species name.The figures next to branches display bootstrap values higher than 50.When multiple sequences were assigned to a single haplotype, only sequences from different hosts are shown in the figure.Hypothetical not determined haplotypes are named mv1-mv4.Dashes indicate mutational steps.Colours respond to Buteo buzzards from which Sarcocystis spp.oocysts/sporocysts were isolated.The S. sp. in our figure correspond to S. jamaicensis in [48].

Table 1 .
List of oligonucleotides used for the identification of Sarcocystis spp.