Detection of Three Sarcocystis Species (Apicomplexa) in Blood Samples of the Bank Vole and Yellow-Necked Mouse from Lithuania

The genus Sarcocystis is an abundant group of Apicomplexa parasites found in mammals, birds, and reptiles. These parasites are characterised by the formation of sarcocysts in the muscles of intermediate hosts and the development of sporocysts in the intestines of definitive hosts. The identification of Sarcocystis spp. is usually carried out in carcasses of animals, while there is a lack of studies on the detection of Sarcocystis species in blood samples. In the current study, blood samples of 214 yellow-necked mice (Apodemus flavicollis) and 143 bank voles (Clethrionomys glareolus) from Lithuania were examined for Sarcocystis. The molecular identification of Sarcocystis was carried out using nested PCR of cox1 and 28S rRNA and subsequent sequencing. Sarcocystis spp. were statistically (p < 0.01) more frequently detected in the bank vole (6.3%) than in yellow-necked mice (0.9%). The analysed parasites were observed in four different habitats, such as mature deciduous forest, bog, natural meadow, and arable land. Three species, Sarcocystis funereus, Sarcocystis myodes, and Sarcocystis cf. glareoli were confirmed in the bank vole, whereas only Sarcocystis myodes were found in yellow-necked mice. The obtained results are important in the development of molecular identification of Sarcocystis parasites in live animals.


Introduction
Sarcocystis (Apicomplexa: Sarcocystidae) is a genus of intracellular parasites that were first discovered in 1843 by F. Miescher in the muscles of the house mouse (Mus musculus) [1].All Sarcocystis species use two hosts to complete their life cycle.Hosts of Sarcocystis species are usually determined by a prey-predator ecological relationship [2].Intermediate hosts are infected by consuming food or water contaminated with mature sporocysts of Sarcocystis spp.After intake, sporozoites are released from the intestine of the host and enter the bloodstream where the schizogony takes place.Schizogony consists of several stages while the number of generations and the type of host cell varies based on Sarcocystis species [1,3].Asexual reproduction results in the formation of sarcocysts in muscle tissues or CNS [1,4,5].Definitive hosts become infected through the ingestion of tissues containing mature sarcocysts.Upon ingestion, sexual reproduction occurs within the small intestines of the definitive host.After the sporulation of oocysts, sporocysts are released into the environment together with the faecal matter [1,6].
Some Sarcocystis species are pathogenic to intermediate hosts, whereas in most cases they are not hazardous to definitive hosts [1].These parasites are often found in livestock and annually cause losses in the animal husbandry industry [7].Rodents are important for the transmission of various diseases [8].However, limited data exists regarding the pathogenic Sarcocystis species that utilize rodents as intermediate hosts.Sarcocystis glareoli and Sarcocystis microti, which were previously assigned to the genus Frenkelia, form cysts in the brain of various rodent species [9].The latter two Sarcocystis species are transmitted through the common buzzard (Buteo buteo) and potentially other members of the genus Buteo [10].Additionally, Sarcocystis singaporensis has been used as a biological control agent against rats in a variety of agroforestry and agricultural environments [11].
To date, over 40 different Sarcocystis species have been identified in rodents.However, due to the low level of research on different rodent species, it is suggested that the true number of Sarcocystis spp. in these hosts is higher [12,13].In recent years, several new Sarcocystis species have been described in rodents [14][15][16].It should be noted that Sarcocystis spp.are most thoroughly examined in the house mouse (Mus musculus) and brown rat (Rattus norvegicus).However, data on the prevalence and richness of Sarcocystis species infecting wild mice and voles is limited.
Only some species of Sarcocystis produce sarcocysts that are visible to the naked eye, while the cysts of the other species of this genus are microscopic.The use of light or an electron microscope allows the differentiation of Sarcocystis species based on the size, and shape of the sarcocysts, their wall structure, and the morphometric features of bradyzoites that are located inside the cysts [16][17][18].Nonetheless, the microscopical characterization and the isolation of sarcocysts from host tissues require specific competencies [19].Additionally, the detection of morphologically similar Sarcocystis species in tissues of closely related intermediate hosts complicates microscopical analysis [1,20].Therefore, the list of known Sarcocystis species is revised and new species are described by using combined morphological and molecular methods [21].Currently, ribosomal RNA genes (28S rRNA or 18S rRNA), internal spacer region 1 (ITS1), and mitochondrial cytochrome oxidase 1 (cox1) are mostly used for Sarcocystis species identification [12].The selection of the genetic regions for the identification of the parasites depends on its host taxonomic group as it has been shown that Sarcocystis species co-evolved with their hosts [22].
To date, Sarcocystis parasites were mainly examined in animal carcasses by the aforementioned methods [23].Sarcocystis species identification in blood would allow the detection of the parasite in living organisms.Conventional immunological methods are poorly suitable for the separation of Sarcocystis species due to the difficulties in the generation of species-specific antibodies [1,24].Therefore, DNA analysis methods are appropriate for the diagnosis of Sarcocystis spp. in blood samples [25].The first attempt to use molecular methods in blood samples of llamas instead of muscle tissues for the identification of Sarcocystis species was conducted in 2016 in Argentina [26].A year later, the first known attempt at using the blood of rodents to identify Sarcocystis spp. was carried out in Japan.Even though the number of screened samples was small, Sarcocystis species were successfully detected in some of them [27].In the following years, two unrelated studies were carried out in Nigeria and Turkey in which researchers successfully identified the DNA of Sarcocystis species in the blood samples of rodents [28,29].
An increasing amount of data indicates that 18S rRNA is unsuitable for distinguishing sequences of related Sarcocystis species [17,21,30,31].A recent study indicates that amplification of ITS1 sequences from Sarcocystis spp. in rodents proves to be challenging [22].Consequently, 28S rRNA and cox1 genetic regions were used for our study.Low concentrations of Sarcocystis species DNA in the bloodstream limit the use of conventional PCR methods.Thus, the nested PCR approach was applied in the current study to generate enough of the amplified products.
In 2016-2017, research was carried out to determine if rodents in Lithuania are infected with blood parasites such as Babesia spp., Trypanosoma spp., and Hepatozoon spp.[32].Blood samples collected by Baltr ūnait ė et al. [32] and additional samples gathered between 2018 and 2019 were employed in this investigation.For this study, we selected two of the most common and abundant rodent species in Lithuania, the yellow-necked mouse (Apodemus flavicollis) and the bank vole (Clethrionomys glareolus).These rodents dominate in forests but are also frequent in other habitats [33].Thus, the objective of our work was to determine the prevalence of Sarcocystis spp.and to molecularly identify parasite species in the blood samples of A. flavicollis and C. glareolus from Lithuania.

Blood Sample Collection
The study was carried out in Lithuania, Vilnius, and Mol ėtai districts, in 2016-2019 (May-November) (Figure 1).
Life 2024, 14, x FOR PEER REVIEW 3 of 1

Blood Sample Collection
The study was carried out in Lithuania, Vilnius, and Molėtai districts, in 2016-201 (May-November) (Figure 1).A highly fragmented landscape composed of various open and forest habitats wa typical of the study area.The habitats ranged from the ones not disturbed by human ac tivity (e.g., natural meadows, bogs) to the intensively used (e.g., arable land).During th four-year period, a total of 357 blood samples were collected, with 214 of the samples be longing to A. flavicollis and 143 to C. glareolus.Small mammals were trapped in variou habitats, namely mature deciduous forests, and mixed forests, planted young forests bogs, natural and shrubby meadows, and arable land.Sherman live traps were baited with pieces of bread soaked in sunflower oil and bedding material.Traps were set in the even ing and checked early in the morning.Live animals were humanely killed by cervical dis location.Blood from the heart was collected in SET buffer immediately after death and stored in the freezer at −20 °C until further molecular analysis [34].Trapped rodents wer identified to species and weighed.The sex and age (juveniles, sub-adults, adults) wer determined during dissection.The age was based on the atrophy of the thymus gland and the status of reproductive organs [35][36][37].

Molecular Analysis of A. flavicollis and C. glareolus Blood Samples
Total DNA extraction was performed using standard ammonium acetate protoco [38].Nested PCR of 28S rRNA and cox1 was employed to detect the presence of Sarcocysti spp. in the blood samples of A. flavicollis and C. glareolus.External primer pair of SF1/SR and internal primer pair of SgraucoF1/SgraucoR1 were used for the amplification of cox A highly fragmented landscape composed of various open and forest habitats was typical of the study area.The habitats ranged from the ones not disturbed by human activity (e.g., natural meadows, bogs) to the intensively used (e.g., arable land).During the four-year period, a total of 357 blood samples were collected, with 214 of the samples belonging to A. flavicollis and 143 to C. glareolus.Small mammals were trapped in various habitats, namely mature deciduous forests, and mixed forests, planted young forests, bogs, natural and shrubby meadows, and arable land.Sherman live traps were baited with pieces of bread soaked in sunflower oil and bedding material.Traps were set in the evening and checked early in the morning.Live animals were humanely killed by cervical dislocation.Blood from the heart was collected in SET buffer immediately after death and stored in the freezer at −20 • C until further molecular analysis [34].Trapped rodents were identified to species and weighed.The sex and age (juveniles, sub-adults, adults) were determined during dissection.The age was based on the atrophy of the thymus gland and the status of reproductive organs [35][36][37].

Molecular Analysis of A. flavicollis and C. glareolus Blood Samples
Total DNA extraction was performed using standard ammonium acetate protocol [38].Nested PCR of 28S rRNA and cox1 was employed to detect the presence of Sarcocystis spp. in the blood samples of A. flavicollis and C. glareolus.External primer pair of SF1/SR5 and internal primer pair of SgraucoF1/SgraucoR1 were used for the amplification of cox1 sequences, while the Sgrau281/Sgrau282 and Sgrau283/Sgrau284 primer pairs were employed for the 28S rRNA [22].
The first PCR reaction was carried out in 25 µL reaction volume, containing 12.5 µL of Dream Taq PCR Master Mix (Thermo Fisher Scientific Baltics, Vilnius, Lithuania), 7.5 µL of nuclease-free water, 0.5 µM of each primer and 4 µL of the template DNA.For the second step of the PCR, 2 µL of the products obtained from the initial PCR were utilized.The rest of the reaction volume contained 12.5 µL of Dream Taq PCR Master Mix (Thermo Fisher Scientific Baltics, Vilnius, Lithuania), 9.5 µL nuclease-free water, and 0.5 µM of each primer.Water was used as the negative control instead of the DNA template for both steps of the nested PCR.DNA extracted from Sarcocystis myodes cysts [16] served as positive control in this study.
The amplification of the first PCR was conducted using the following program: initial denaturation at 95 • C for 5 min, followed by 35 cycles of 45 s at 95 • C, 55 s at 59-61 • C (depending on the annealing temperature of primers), followed by 65 s at 72 • C and the final extension for 7 min at 72 • C. The second round of the PCR was performed as follows: initial denaturation at 95 • C for 5 min, followed by 35 cycles of 35 s at 95 • C, 45 s at 59 • C, 55 s at 72 • C and ended with the final extension at 72 • C for 7 min.After completion of each PCR step products were visualized on 1% agarose gels using electrophoresis.
All the positive samples were purified with alkaline phosphatase FastAP and exonuclease ExoI (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the instructions of the manufacturer.The purified samples were sequenced with forward and reverse primers used for a second PCR step.Big-Dye ® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Vilnius, Lithuania) and the 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) were employed for Sanger sequencing.All the acquired chromatograms were pure, without double peaks.Nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/,accessed on 27 January 2024) tool was utilized to compare sequences detected in this study with Sarcocystis spp.sequences in the NCBI GenBank database.
Multiple alignments of cox1 and 28S rRNA sequences were created with the help of the MUSCLE algorithm implemented in MEGA7 [39].The alignment of cox1 contained 22 taxa and 619 nucleotide positions without gaps, whilst 28S rRNA alignment consisted of 27 taxa and 803 nucleotide positions including gaps.The selection of evolutionary models and generation of phylogenetic trees was performed while using TOPALi v2.5 [40].The Bayesian method was applied to uncover phylogenetic relationships.The F81 + G and HKY + G nucleotide substitution models were chosen for cox1 and 28S rRNA analysis.Toxoplasma gondii was chosen as the outgroup in the generation of phylogenetic trees.The analyses were carried out in two runs, using one million generations with a sample frequency of 10 and 25% burn-in value.The 28S rRNA and cox1 sequences of the Sarcocystis spp.from the present study are available in GenBank under the accession numbers PP350819-PP350829 and PP358794-PP358804.

Statistical Analysis
Quantitative parasitology 3.0 software was utilized for statistical tests [41].Sterne's exact method was applied to determine a 95% confidence interval (CI) for the detection rates of Sarcocystis spp.Fisher's exact test was utilized to assess the statistical significance of the differences comparing the prevalence of Sarcocystis spp.observed in the samples of A. flavicollis and C. glareolus.

Detection Rates of Sarcocystis spp. in the Blood Samples of A. flavicollis and C. glareolus
For this study, 143 samples of C. glareolus were collected from traps in seven different habitats, such as mature deciduous forests and mixed forests, planted young forests, bogs, natural and shrubby meadows, and arable land (Table 1).The number of specimens caught in each habitat ranged from 8 to 34.Molecular examination of blood samples revealed the presence of Sarcocystis spp. in individuals collected from four different habitats: arable land, bog, mature deciduous forest, and natural meadow.In the latter habitats, the rate of positive samples ranged from 11.11% (95% CI = 2.02-33.02) to 12.50% (95% CI = 0.64-50.00).Differences in detection rates of Sarcocystis spp.were statistically insignificant when comparing the habitats tested (p > 0.05).The overall frequency of Sarcocystis spp. in the investigated blood samples of C. glareolus was 6.29% (95% CI = 3. 19-11.45).Additionally, 214 individuals of A. flavicollis were caught in seven different habitats, including mature deciduous forests and mixed forests, planted young forests, bogs, natural and shrubby meadows, and arable land (Table 2).In all but two habitats, between 29 and 63 individuals were trapped.In the remaining two habitats, planted young forest and bog, only five and eight individuals were collected, respectively.Molecular analysis of blood samples revealed the presence of Sarcocystis spp. in individuals collected only in arable land 3.17% (95% CI = 0.57-10.87).Statistically significant differences in detection rates of Sarcocystis spp.across the analysed habitats were not observed (p > 0.05).The detection frequency was significantly lower in A. flavicollis (0.93%; 95% CI = 0.17-3.40)compared to C. glareolus (p = 0.009).

Molecular Characterisation of Sarcocystis spp. in A. flavicollis and C. glareolus
In this study, 11 isolates were successfully characterized within partial cox1 and 28S rRNA.The length of the analysed cox1 sequences was 619 bp, while 28S rRNA sequences ranged between 721 bp and 735 bp.At the cox1 loci, sequences obtained in this study shared a similarity of 94.67-100% amongst themselves, while at the 28S rRNA loci the similarity was 87.25-100%.Thus, 28S rRNA was more variable compared with the cox1 for the Sarcocystis spp.detected.Based on the molecular analysis, three Sarcocystis species (Sarcocystis funereus, S. myodes and Sarcocystis cf.glareoli) were identified (Table 3).The lengths of the analysed fragment are indicated in parentheses under the name of Sarcocystis species.
According to the analysis of cox1 gene fragments, the sequences of S. myodes demonstrated 99.84-100% similarity compared to the sequences of this species deposited in the NCBI GenBank.Additionally, fragments acquired from the amplification of the 28S rRNA loci demonstrated intraspecific similarity of 99.46-100% in comparison with other S. myodes sequences.Based on 28S rRNA, the sequences of S. myodes showed the highest similarity to those of Sarcocystis ratti and Sarcocystis sp.Rod1 (96.46-97.82%).
The second detected Sarcocystis species (isolates LTKrCgla7A and LTKrCgla25C) exhibited the highest genetic similarity with two Sarcocystis species (S. glareoli and S. microti) previously classified in the genus Frenkelia and Sarcocystis jamaicensis (Table 3).However, cox1 sequences of S. glareoli and S. microti are yet to be established.The analysed Sarcocystis isolate displayed 100% similarity with S. jamaicensis within the cox1.Based on 28S rRNA, this organism differed from S. jamaicensis by two single nucleotide polymorphisms (SNPs) and by a single indel (insertion/deletion) from S. glareoli.Therefore, as nucleotide substitutions have more value for species divergence than indels [42], this Sarcocystis organism was denoted as S. cf.glareoli.
The last found species in this investigation was recently described as S. funereus [43].At 28S rRNA, obtained sequences shared 99.72-100% similarity with those of S. funereus, 95.60% with those of Sarcocystis lari, and 95.25% with those of Sarcocystis strixi.Cox1 sequences of S. funereus have not been identified to date.Thus, based on this gene, sequences of S. funereus obtained in our work demonstrated the highest similarity with those of S. lari, Sarcocystis lutrae, and S. strixi (99.35-99.52%).

Phylogenetic Analysis of Identified Sarcocystis Species
The phylogenetic analyses based on two analysed loci (cox1 and 28S rRNA) resulted in longer branches of phylograms when using 28S rRNA sequences (Figure 2).In the 28S rRNA phylogenetic tree, sequences of three Sarcocystis species detected in this work were grouped with other isolates of the corresponding species, thus confirming the identification of the species studied.In both phylograms, two of the examined species, S. funereus and S. cf.glareoli were placed together with Sarcocystis species employing rodents, birds, and carnivorous mammals as their intermediate hosts and birds of their identified or presumed definitive hosts according to phylogenetic studies.Meanwhile, S. myodes was grouped into several Sarcocystis species using rodents and carnivorous mammals as their defined or proposed definitive hosts.Based on sequences of more variable 28S rRNA, S. cf.glareoli was most closely related to S. jamaicensis and S. microti, whereas S. funereus was sister species to S. strixi and finally S. myodes was grouped with S. ratti and Sarcocystis sp.Rod1.
sister species to S. strixi and finally S. myodes was grouped with S. ratti and Sarcocystis sp.Rod1.
Specifically, the reported prevalence of Sarcocystis parasites in C. glareolus in Lithuania varies from 1.34% to 14.38% [16,22,45,48,49].In this study, DNA of Sarcocystis spp. was detected in 6.29% (95% CI = 3.19-11.45) of C. glareolus blood samples, which is consistent with results from prior studies using muscle samples in Lithuania.Similar detection rates were observed in the Czech Republic and Finland, with Sarcocystis spp.detection in C. glareolus being relatively low, at 1.46% and 6%, respectively [50,51].Nevertheless, studies on other vole species highlight significantly higher Sarcocystis species prevalence.In
Formerly, species producing cysts in the muscle tissues were classified under the genus Sarcocystis [54], while parasite species forming cysts in the brains of small mammals were placed under the genus Frenkelia [55] due to differences in sarcocyst morphology and its location.However, the merging of these genera has been proposed by several authors based on phylogenetic studies [9,56].In Lithuania, it was reported that the prevalence of Frenkelia spp. in C. glareolus was 21.11% [57].By comparison, in the Czech Republic, the prevalence of Frenkelia spp. was 38.5% [51], while in Germany it ranged from 10.34% to 55.93% [58,59], and in France, it varied from 1.02% to 48.14% [60,61].
Investigations on the prevalence of Sarcocystis spp.and Frenkelia spp. in A. flavicollis are notably scarce.In Lithuania, previously reported detection rates of Sarcocystis parasite in muscles ranged from 0% to 0.84%, consistent with the findings of this investigation, which reported a prevalence of 0.93% (95% CI = 0.17-3.40)[22,45,48,49].In studies conducted in the Czech Republic and Spain, no Sarcocystis species were identified in the muscle samples of mice [51,62].Taking into consideration other species of the genus Apodemus, higher infection rates of Sarcocystis spp.were recorded, namely in the large Japanese field mouse (Apodemus speciosus) and the small Japanese field mouse (Apodemus argenteus), the prevalence of Sarcocystis spp. was 18.99% and 21.43%, respectively [52].In a previous Lithuanian study, Frenkelia spp. was absent in all A. flavicollis specimens [57].Meanwhile, the prevalence of Frenkelia spp. in A. flavicollis from the Czech Republic was 2.40% [51], and in Germany, it ranged from 0.45% [59] to 8.54% (95% CI = 4.2-16.6)[58].In summary, studies carried out in Lithuania, Germany, and the Czech Republic indicate that Sarcocystis spp. is more commonly found in C. glareolus than in A. flavicollis (present study, [51, [57][58][59]).

Detection of Sarcocystis spp. in Blood Samples of Intermediate Hosts
During this investigation, blood samples were used to determine the prevalence and Sarcocystis spp.richness in Lithuania for the first time.Additionally, prior documented research on blood samples to identify Sarcocystis spp.has been conducted in Argentina, Japan, Turkey, Nigeria, and Australia, indicating precedence for such studies [25][26][27][28][29]63].Three separate studies utilized rodent blood samples for investigation, successfully detecting Sarcocystis spp. in the blood of grey-sided vole (Myodes rufocanus), wood mouse (Apodemus sylvaticus), black rat and brown rat [27][28][29].Remarkably, the prevalence of Sarcocystis spp. in M. rufocanus from Japan was relatively high, reaching 16.67%; nonetheless, this observation could be attributed to the small sample size (n = 6) that was examined [27].Blood samples collected in Nigeria and Turkey from A. sylvaticus, R. rattus, and R. norvegicus exhibited an overall prevalence of Sarcocystis parasites ranging between 0.19% and 2.1% [28,29], aligning with the low prevalence rates reported in our study.
Regrettably, in this study, it was not possible to compare data from samples collected from blood and muscle tissues of the same individuals.Despite this, a study in Lithuania was carried out recently to identify Sarcocystis spp. in the muscle samples of rodents from commercial orchards [22].Due to the sheer volume of the samples, the authors pooled muscle samples of the rodents that belonged to the same species, digested them, and identified parasite species using nested PCR and sequencing.In the study, the prevalence of Sarcocystis spp. in A. flavicollis was recorded as 0.84% (95% CI = 0.15-2.75),a finding that correlates closely with the results of our investigation, which showed a detection rate of 0.93% (95% CI = 0.17-3.40).Meanwhile, the rate of Sarcocystis infection in the muscles of C. glareolus was 1.34% (95% CI = 0.08-6.43)[22], showing a lower detection rate compared to our study, in which the rate was 6.29% (95% CI = 3. 19-11.45).The variance in the results may be due to the different stages of Sarcocystis spp.infection, distinct sample collection locations, and potential seasonal effects [3,53].Findings from a study conducted on camelids suggest that the DNA of Sarcocystis spp.can be detectable in the blood during the initial phases of the infection but become undetectable once encystment occurs [25].
The identification of three Sarcocystis species in this study highlights the significance of utilizing blood samples from intermediate hosts as a valuable method for examining both Sarcocystis prevalence and species diversity.This is the first report of S. funereus in C. glareolus from Lithuania, although molecular studies of muscle tissues were conducted before [16,22].Although the molecular method used in this investigation is not suitable for the detection of Sarcocystis species coinfections.To identify more than one Sarcocystis species in a single sample, other methods, e.g., cloning, might be used [64].

Characteristics of Sarcocystis spp. in A. flavicollis and C. glareolus
Our molecular analysis showed the presence of three Sarcocystis species in A. flavicollis and C. glareolus from Lithuania.Among these species, S. myodes was only recently identified and characterised in the muscles of a single C. glareolus specimen [16].Subsequently, sarcocysts of S. myodes have been detected in other intermediate hosts, such as A. flavicollis, A. agrarius, and M. arvalis [22].Morphological analysis revealed sarcocysts of S. myodes to be microscopic (600-3000 × 70-220 µm) with a thin (~1 µm), smooth cyst wall lacking visible protrusions [16].The characteristics of S. myodes sarcocysts closely resemble those of S. microti and S. glareoli.However, the latter species are exclusively found in the brains of small mammals [10,65].Molecular analysis data on 18S rRNA and 28S rRNA loci demonstrates reliable differences between the three species [16,47,66].While S. glareoli and S. microti utilise buzzards (Buteo spp.) as their definitive host [16,65], the primary host of S. myodes remains speculative due to insufficient data.Nevertheless, phylogenetic analysis suggests a potential association of S. myodes with predatory mammals [22].
During our study, one of the Sarcocystis organisms was identified tentatively as S. cf.glareoli.The lack of molecular data for the S. glareoli species, which was once placed under the genus Frenkelia, causes a significant challenge in the identification of this species.Currently, only sequences of the 18S rRNA and 28S rRNA genes have been obtained [9,10].Apart from cox1, which was used in numerous studies [12,16,21,47,70,71], additional genetic markers, such as ITS1, mitochondrial cytochrome b (cytb), complete ITS1-5.8SrRNA-ITS2 region, two apicoplast genes-RNA polymerase beta subunit (rpoB) and caseinolytic protease C (clpC) demonstrate promising prospects for improved discrimination of Sarcocystis spp. in small mammals [47,66,72,73].
The last Sarcocystis species identified in our investigation is S. funereus, recently detected in the Tengmalm's owl (Aegolius funereus) population in Finland [43].Formerly identified as Sarcocystis sp.isolate Af1, this species lacked comprehensive data concerning its intermediate host and sarcocyst structure, despite characterisation using four genetic loci (ITS1, cox1, 18S rRNA, and 28S rRNA) [74].However, S. funereus has been differentiated from other Sarcocystis species primarily through the utilization of the 28S rRNA and ITS1 region [43].The latter region demonstrates superior sensitivity in discerning genetic variances among Sarcocystis species with birds and carnivores as intermediate hosts [74][75][76].While the natural intermediate host remains elusive, experimental findings proposed C. glareolus as a potential candidate [77].Notably, A. funereus exhibits a dietary preference for voles, with bank voles comprising more than 40% of its diet [78].Our investigation provides additional evidence supporting the hypothesis that C. glareolus serves as the natural intermediate host for S. funereus.

Conclusions
Our comprehensive study marks a significant advance in the research of Sarcocystis spp.by using blood samples for the investigation of these parasites in two rodent species, A. flavicollis and C. glareolus.The findings revealed varying frequencies of Sarcocystis spp.across habitats, with no statistically significant differences observed in detection rates.Moreover, prior investigations carried out in Lithuania, Germany, and the Czech Republic indicate that C. glareolus tends to have higher rates of Sarcocystis spp.detection compared to A. flavicollis, which is consistent with the prevalence rates observed in the current study.Additionally, molecular analysis revealed three distinct Sarcocystis species-S.myodes, S. cf.glareoli, and S. funereus, with the latter being identified in Lithuania for the first time.Thus, blood samples can be successfully used for the studies of Sarcocystis spp.richness in small mammals.Informed Consent Statement: Not applicable.

Figure 1 .
Figure 1.Small mammal trapping areas in Lithuania during the 2016-2019 period.Sampled district are marked in grey.

Figure 1 .
Figure 1.Small mammal trapping areas in Lithuania during the 2016-2019 period.Sampled districts are marked in grey.

Figure 2 .
Figure 2. The phylogenetic relationships of the Sarcocystis spp.identified in blood samples of C. glareolus and A. flavicollis based on cox1 (a) and 28S rRNA (b) sequences.Phylograms were generated using Bayesian methods, scaled according to the branch length, and rooted on Toxoplasma gondii.The posterior probability support values are indicated next to branches, and GenBank accession numbers are given behind the species name.The sequences of three Sarcocystis species (S. funereus, S. myodes, and S. cf.glareoli) obtained in this work are shown in red.The coloured rectangles and triangles indicate the identified or presumed intermediate hosts and definitive hosts of Sarcocystis species, respectively.

Figure 2 .
Figure 2. The phylogenetic relationships of the Sarcocystis spp.identified in blood samples of C. glareolus and A. flavicollis based on cox1 (a) and 28S rRNA (b) sequences.Phylograms were generated using Bayesian methods, scaled according to the branch length, and rooted on Toxoplasma gondii.The posterior probability support values are indicated next to branches, and GenBank accession numbers are given behind the species name.The sequences of three Sarcocystis species (S. funereus, S. myodes, and S. cf.glareoli) obtained in this work are shown in red.The coloured rectangles and triangles indicate the identified or presumed intermediate hosts and definitive hosts of Sarcocystis species, respectively.

Author Contributions:
Conceptualisation, L.B. and P.P.; methodology, L.B. and P.P.; software, N.G. and P.P.; validation, L.B., N.K. and P.P.; formal analysis, N.G. and D.L.B.; investigation, L.B., N.K., N.G. and D.L.B.; resources, L.B., N.K. and P.P.; writing-original draft preparation, L.B., N.G., D.L.B. and P.P.; writing-review and editing, L.B., N.G., D.L.B., N.K. and P.P.; visualization, P.P.; supervision, P.P.; funding acquisition, L.B. and P.P.All authors have read and agreed to the published version of the manuscript.Funding: This research received no external funding.Institutional Review Board Statement: The research was conducted according to Lithuanian laws.The use of wild mammals for research is regulated by the Republic of Lithuania Law of Wildlife (22 June 2010, No. XI-920) and the Order of the Director of the State Food and Veterinary Service (30 June 2014, No. D1-369/B1-380, as last amended on 15 February 2014, No. D1-119/B1-197).According to the current procedure, no permit was required to perform the investigation in the current study.Investigated species are not classified as protected species in accordance with the Republic of Lithuania Law on protected Animals, Plants and Mushrooms (Order of the Director of the State Food and Veterinary Service, Chapter III, Article 50.2).The study was approved by the Animal Welfare Committee of the Nature Research Centre (protocol No GGT-2, 15 November 2016).

Table 1 .
Detection rates of Sarcocystis spp. in blood samples of C. glareolus.
* Sarcocystis myodes was detected in three samples and S. funereus was detected in a single sample.

Table 2 .
Detection rates of Sarcocystis spp. in blood samples of A. flavicollis.
* Only one species S. myodes was confirmed by molecular methods.

Table 3 .
Sarcocystis species identified in this study, and the percentage of similarity compared with the most closely related species.