Molecular Identification of Protozoan Sarcocystis in Different Types of Water Bodies in Lithuania

Representatives of the genus Sarcocystis are unicellular parasites having a two-host life cycle and infecting mammals, birds, and reptiles. Until now, Sarcocystis spp. have been mainly investigated in definitive and intermediate hosts. Only a few studies have been conducted on the detection of Sarcocystis parasites in water samples. The aim of this research was to examine whether the prevalence of Sarcocystis spp. parasitizing farm animals varies in different types of water bodies. Water samples (n = 150) were collected from the entire territory of Lithuania, dividing water bodies into five groups (lakes, rivers, ponds/canals, swamps, and the inshore zone of the territorial Baltic Sea area). One-liter samples were filtered and subsequently analyzed using nested PCR. At least one of the analyzed Sarcocystis spp. (S. arieticanis, S. bertrami, S. bovifelis, S. capracanis, S. cruzi, S. hirsuta, S. miescheriana, and S. tenella) was determined in all examined samples from water bodies. No significant difference in Sarcocystis spp. prevalence between different types of water sources was detected. Our research proved that selecting appropriate primers is important for the accurate identification of parasites in samples collected from water bodies.


Introduction
Unicellular parasites of the genus Sarcocystis are characterized by an obligatory twohost life cycle. These parasites infect mammals, birds, and reptiles. Sarcocysts mostly develop in the muscle tissues of intermediate hosts; meanwhile, sporulation of oocysts occurs in the small intestine of the definitive host [1]. Four of more than 200 species belonging to this genus are zoonotic, S. hominis, S. suihominis, S. heydorni, and S. nesbitti [2]. However, few studies have been carried out to determine the prevalence of these species, as they are often misidentified during morphological or molecular examination [3].
To date, most research has been conducted on Sarcocystis spp. infecting economically important domestic animals. Farm animals can become infected through food or water contaminated with fecal sporocysts of Sarcocystis spp. It has been observed that Sarcocystis species transmitted through canids are more dangerous to farm animals, and acute infections can cause such symptoms as fever, weight loss, anemia, reduction in wool and milk yield, abortion, or even death [4][5][6][7].
Until now, Sarcocystis infection was mainly investigated by performing morphological or molecular analysis of animal carcasses. However, the use of natural environment studies by avoiding animal carcass-based research is increasing. Even so, only a few studies were conducted to test for Sarcocystis spp. in water [8][9][10]. The aim of our previous study was to assess different sample preparation and common PCR methodologies for identifying Sarcocystis species in water. The environmental water samples were collected from ponds (n = 49), lakes (n = 35), rivers (n = 18), canals (n = 10), and lagoons (n = 2). DNA of at least one of eight tested Sarcocystis species (S. arieticanis, S. bertrami, S. bovifelis, S. capracanis, S. cruzi, S. hirsuta, S. miescheriana, and S. tenella) was detected in 111 of 114 (97.4%) water samples using nested PCR targeting cox1 gene [10]. However, Sarcocystis species occurrence capracanis, S. cruzi, S. hirsuta, S. miescheriana, and S. tenella) was detected in 111 of 114 (97.4%) water samples using nested PCR targeting cox1 gene [10]. However, Sarcocystis species occurrence rates in different areas and types of water bodies were not compared. Therefore, the aim of this study was to compare detection rates of different Sarcocystis species in five types of water bodies-lakes, rivers, canals/ponds, swamps, and the inshore zone of the Baltic Sea-using molecular methods.

Sample Collection
Samples (n = 150) were collected from water bodies throughout the territory of Lithuania in the summer of 2021 ( Figure 1). Water bodies were divided into five groups with an equal number of samples each-lakes (stagnant water), rivers (flowing water), canals and ponds (small water bodies that are usually close to pastures), swamps (acidic water) and the inshore zone of the Baltic Sea (saline water). Water samples were collected in sterile containers of 1 L capacity and transported in portable coolers with ice batteries. Until further processing, water samples were stored at +4 °C.

Preparation of Water Samples and Genomic DNA Extraction
First, the water sample was filtered through a metal sieve with 1 mm pores, then through Whatman™ Qualitative Filter Paper Grade 4 and finally filtered using MF-Millipore ® 5 μm pore membranes. Two milliliters of distilled water were used for membrane washing and collection of material, which was stored at +4 °C until further processing.
Genomic DNA (gDNA) was isolated from 200 μL of concentrated water samples using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania), according to the manufacturer's recommendations. The resulting DNA samples were kept frozen at −20 °C until further analysis.

Preparation of Water Samples and Genomic DNA Extraction
First, the water sample was filtered through a metal sieve with 1 mm pores, then through Whatman™ Qualitative Filter Paper Grade 4 and finally filtered using MF-Millipore ® 5 µm pore membranes. Two milliliters of distilled water were used for membrane washing and collection of material, which was stored at +4 • C until further processing.
Genomic DNA (gDNA) was isolated from 200 µL of concentrated water samples using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania), according to the manufacturer's recommendations. The resulting DNA samples were kept frozen at −20 • C until further analysis.

Nested PCR-Based Identification of Sarcocystis Parasites
During this work, samples from environmental water bodies were analyzed to distinguish the prevalence of different Sarcocystis spp. (S. arieticanis, S. bertrami, S. bovifelis, S. capracanis, S. cruzi, S. hirsuta, S. miescheriana, and S. tenella) using farm animals (cattle, sheep, goats, horses, and pigs) as intermediate hosts. Since the cox1 gene is considered the most appropriate for the identification of selected Sarcocystis parasites [11][12][13], primers targeting this gene were selected [10]. It was observed that, having selected adequate PCR primer pairs, detection rates of farm animals infecting Sarcocystis spp. in water samples were associated with those observed in the muscles of the intermediate host [10,[14][15][16][17]. However, in our earlier research, the prevalence of S. bertrami, S. cruzi, S. miescheriana and S. tenella identified in water samples was significantly lower [10]. Thus, 10 new primers ( Table 1; highlighted in bold) were designed for the identification of these species. To detect S. miescheriana and S. bertrami, primers were redesigned to give shorter products, whereas to diagnose S. cruzi and S. tenella, different binding sites of primers were chosen. Reverse ATTGAAGGGCCACCAAACAC PV are primer pairs selected in the previous study [10], PS are primer combinations selected in the present study. Ta is annealing temperature, Ext is extension time, ProS is product size. Primers designed in the present study are in boldface.
During all PCR reactions, both positive (DNA extracted from sarcocysts of the corresponding Sarcocystis species) and negative (distilled water) controls were used. Primers were checked for cross reactions with other Sarcocystis species. The specificity of primer sets was confirmed. To check for possible contamination, distilled water and tap water were examined after the first batch, in the middle of our experiments and after the last batch. Based on the examination of distilled and tap water, PCRs were negative with all primers used in the study.
Preparation of PCR reaction mixtures and cycling conditions were as described previously [10]. The annealing temperatures were modified depending on the primers used (Table 1). Agarose gel electrophoresis was used to visualize PCR amplicons.
The selected PCR products were purified and directly sequenced as described previously [10]. Five positive samples of each species were used for sequencing, except for S. hirsuta, since only three samples were positive for this species. Four positive samples of the species S. tenella, S. cruzi, S. bertrami and S. miescheriana were additionally sequenced with the primers used in the previous study [10].
The editing of resolved sequences was performed manually with subsequent comparative BLAST analysis (http://blast.ncbi.nlm.nih.gov/, accessed on 10 October 2022). Differences in the prevalence of the identified Sarcocystis species were evaluated using the Chi-squared test. The cox1 sequences of Sarcocystis species generated in the present study were deposited in the GenBank under the accession numbers OP681467-OP681524.

Identification of Sarcocystis spp. Using Different PCR Primer Sets
GenBank accession numbers, length, and similarity of the obtained cox1 sequences of S. bovifelis, S. cruzi, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. bertrami and S. miescheriana are presented in Table 2. In no case did the obtained intraspecific and interspecific genetic differences overlap. Therefore, the primer sets used in this study were appropriate for the identification of Sarcocystis species in water samples examined. Four of eight investigated Sarcocystis species, S. bertrami, S. cruzi, S. miescheriana and S. tenella, were identified in the same water samples using two different primer combinations, the primer set chosen in our previous study (21 PV) [10] and the primer set selected in the current work (21 PS). The Sarcocystis parasite occurrence rate for the above-mentioned species was significantly higher (p < 0.05) using the primers selected in this study (21 PS) (Figure 2a). Depending on the primers used, the prevalence of S. bertrami was 16.0% and 26.0% (χ 2 = 4.52, p < 0.05), whereas the prevalence of S. miescheriana accounted for 6.7% and 19.3% (χ 2 = 10.64, p < 0.01). Most significant differences were identified when evaluating the primers tested for the detection of S. cruzi and S. tenella. The prevalence of S. cruzi was 35.3% and 98.7% (χ 2 = 136.02, p < 0.00001) and the occurrence of S. tenella was 38.7% and 82.0% (χ 2 = 58.85, p < 0.00001), using 21 PV and 21 PS, respectively.  [11] and 2021 (present study). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Sarcocystis spp. Occurrence Rates in Different Types of Water Bodies
The detection rate of Sarcocystis species examined was compared in five types of water bodies-lakes, rivers, ponds/canals, swamps, and the inshore zone of the Baltic Sea ( Figure 3a). The comparison showed a significantly higher (χ 2 = 6.65, p < 0.01) detection rate of S. bertrami in lakes (43.3%) than that in rivers (13.3%) and a significantly higher (χ 2 = 5.45, p < 0.05) detection rate of S. bovifelis in swamps (40.0%) than that in lakes (13.3%). In the case of S. tenella, significantly higher detection rates were calculated in lakes (96.7%) than those in swamps (60.0%) (χ 2 = 11.88, p < 0.001) and ponds/canals (73.3%) (χ 2 = 6.41, p < 0.05); moreover, they were higher in the inshore zone of the Baltic Sea (93.3%) than the rates in swamps (χ 2 = 9.32, p < 0.01) and ponds/canals (χ 2 = 4.32, p < 0.05), and finally, they were higher (86.7%) in rivers as compared to those in swamps (χ 2 = 5.45, p < 0.05). The overall frequency of Sarcocystis species (calculated by summing up all PCR-positive samples and dividing them by the total number of samples tested) varied depending on water type: from 102 (42.5%) positive cases in the Baltic Sea to 116 (48.3%) positive cases in lakes (Figure 3b). However, the differences observed in occurrence rates of Sarcocystis spp. in five types of water bodies were insignificant (χ 2 = 1.85, df = 4, p > 0.05). In summary, the overall prevalence of Sarcocystis species did not depend on the type of water body.
Life 2023, 13, x FOR PEER REVIEW 7 of 12 than those in swamps (60.0%) (χ 2 = 11.88, p < 0.001) and ponds/canals (73.3%) (χ 2 = 6.41, p < 0.05); moreover, they were higher in the inshore zone of the Baltic Sea (93.3%) than the rates in swamps (χ 2 = 9.32, p < 0.01) and ponds/canals (χ 2 = 4.32, p < 0.05), and finally, they were higher (86.7%) in rivers as compared to those in swamps (χ 2 = 5.45, p < 0.05). The overall frequency of Sarcocystis species (calculated by summing up all PCR-positive samples and dividing them by the total number of samples tested) varied depending on water type: from 102 (42.5%) positive cases in the Baltic Sea to 116 (48.3%) positive cases in lakes ( Figure 3b). However, the differences observed in occurrence rates of Sarcocystis spp. in five types of water bodies were insignificant (χ 2 = 1.85, df = 4, p > 0.05). In summary, the overall prevalence of Sarcocystis species did not depend on the type of water body.

Distribution of Sarcocystis spp. in Water Samples
Summarizing the results, at least one Sarcocystis species was identified in all 150 examined samples. The number of Sarcocystis species per individual sample was estimated by combining data obtained in the analysis using different primer sets (21 PV and 21 PS). Single species was identified only in three cases (2.0%). The detection of two (18.7%), three (21.3%), four (32.0%) and five (19.3%) Sarcocystis species per sample was more frequent. Finally, six and seven species of Sarcocystis in one water sample were identified in nine

Distribution of Sarcocystis spp. in Water Samples
Summarizing the results, at least one Sarcocystis species was identified in all 150 examined samples. The number of Sarcocystis species per individual sample was estimated by combining data obtained in the analysis using different primer sets (21 PV and 21 PS). Single species was identified only in three cases (2.0%). The detection of two (18.7%), three (21.3%), four (32.0%) and five (19.3%) Sarcocystis species per sample was more frequent. Finally, six and seven species of Sarcocystis in one water sample were identified in nine cases (6.0%) and one (0.7%) case, respectively.

Discussion
Based on nested PCR, we identified eight Sarcocystis species (S. arieticanis, S. bertrami, S. bovifelis, S. capracanis, S. cruzi, S. hirsuta, S. miescheriana, and S. tenella) in different types of water bodies (Table 2, Figure 3). Cattle are intermediate hosts for the first three Sarcocystis species tested, S. arieticanis and S. tenella use sheep as their intermediate hosts, while goats, horses and pigs/wild boar are hosts of S. capracanis, S. bertrami, and S. miescheriana, respectively [5]. Based on current knowledge, the European bison (Bison bonasus) can be an alternative host for Sarcocystis species parasitizing cattle [18]. The sarcocysts of such species as S. arieticanis, S. tenella and S. capracanis can be found in muscle tissues of European mouflon (Ovis aries musimon) [19]. Nevertheless, in the areas under investigation, the mentioned wild animals are rare [20]. Other wild ungulate species that could be intermediate hosts for the tested Sarcocystis species are not free-ranging in Lithuania.
To date, most studies have been conducted on the prevalence of protozoan infection in drinking water treatment facilities [21][22][23][24], whereas only a few studies have investigated the prevalence of parasitic protozoa in environmental water sources, such as rivers [25,26], reservoirs [27], lakes [28] or private wells [29]. Typically, studies cover small regions, basins of a particular river or several nearby villages where an outbreak of parasitic protozoa was identified. The number of studies on other parasitic protozoa is much smaller; for example, only three studies were devoted to the identification of Sarcocystis spp. in water bodies [8][9][10]. The current study is the first attempt to compare the prevalence of eight Sarcocystis species in different types of water bodies. It is noteworthy that equal numbers of samples representing five groups of water bodies (lakes, rivers, ponds/canals, swamps, and the Baltic Sea) were collected throughout the entire territory of the country. The majority of other investigations of parasitic protozoa in water samples were limited to small geographic regions or the location of infection outbreaks [22,[27][28][29]. During the present study, it was found that overall detection of the analyzed Sarcocystis species did not depend on the type of water body (Figure 3). The number of positive cases was very similar in all types of water and varied from 102 to 116 positive cases. As a result, it can be assumed that environmental conditions and the location of water bodies had no effect on the prevalence of Sarcocystis parasites in different water sources. However, insignificant differences were noticed when comparing the distribution of eight species in different types of water bodies. Distribution of individual Sarcocystis species may be determined by the abundance of final hosts in a particular area and different characteristics of water bodies, such as water salinity, acidity, or flow turbidity.
Our current research showed that Sarcocystis spp. DNA detection depended on the primer combinations used (Figure 2). The use of different primer pairs for identification of S. bertrami, S. miescheriana, S. cruzi, and S. tenella revealed statistically significant differences. As compared to the prevalence of these four Sarcocystis species obtained using primers selected in the previous study (21 PV) [10], a significantly higher (p < 0.05) prevalence was determined when the primers selected in this study (21 PS) were applied to PCR. Previous studies also showed that amplification success of cox1 fragments of Sarcocystis spp. using ungulates as their intermediate hosts depended on the chosen PCR primers [30][31][32]. It is assumed that some Sarcocystis species exhibit high interspecific genetic variability [17,33]. However, intraspecific genetic variation of Sarcocystis spp. is poorly studied [34][35][36], and selection of suitable primers has been one of the challenges to the diagnosis of Sarcocystis species thus far.
Due to the large morphological and genetic differences between genera or even species of parasitic protozoa, universal methods for identification of these parasites in various environmental samples have not been developed yet. The concentration of many protozoa species in water samples is relatively low [37]; therefore, detection requires very sensitive techniques, such as molecular methods. While some protozoa are undetectable in water samples under a microscope [38], molecular methods for the identification of parasitic protozoa are not fully developed [27,37]. It should be noted that molecular-based techniques are not standardized for the diagnosis of Sarcocystis and other parasitic protozoa in water samples [10,37]. Therefore, a wide variety of methodologies, e.g., conventional PCR and derivatives, DNA hybridization, loop-mediated isothermal amplification (LAMP), or quantitative PCR, are used [10,27,39,40]. During environmental sample testing, discrimination between DNA from a living cell versus DNA from a dead one might be considered impossible. Consequently, the prevalence of parasites in the samples collected can be overestimated [37]. Oocysts or sporocysts of protozoan Sarcocystis parasites are also known to be resistant to various environmental conditions (freezing, low humidity, high temperatures, etc.) and can remain viable in nature for months [5,41]. However, to amplify DNA only from viable sporocysts, water samples can be treated with dyes that penetrate only membrane-damaged cells. After dye has entered the cell, it is covalently cross-linked to DNA. Consequently, PCR amplification is strongly inhibited [42]. To date, fluorescent dyes, such as ethidium monoazide (EMA), propidium monoazide (PMA), ethidium bromide (EB), and propidium iodide (PI), have been successfully used in protozoan studies [43]. Accordingly, further research of Sarcocystis spp. in water samples could focus on additional testing, such as measuring the sensitivity of sporocyst recovery, a spike-and-recovery experiment (controlled contamination of water samples with purified sporocysts) or adaptation of quantitative PCR. However, during the previous studies on animal carcasses, a high prevalence of Sarcocystis spp. infection was determined in some intermediate hosts [14,15,20]. As much as 100% of cattle and sheep bred in Lithuania were found to be infected with Sarcocystis parasites [14,15]. Meanwhile, the prevalence of infection in horses and pigs accounted for 47.2% and 40.2%, respectively [14]. Since it is known that animals can become infected with these parasites through food and water, there seem to be large amounts of viable and infective sporocysts in the environment.

Conclusions
The present study is the first attempt to compare detection rates of Sarcocystis parasites using farm animals as their intermediate hosts in five different types of water bodies (lakes, rivers, ponds/canals, swamps, and the inshore zone of the Baltic Sea). The prevalence of the analyzed Sarcocystis species did not vary significantly between the examined water source groups. The environmental conditions of water bodies do not affect the prevalence of Sarcocystis parasites. Based on the nested PCR, eight Sarcocystis species were identified-S. hirsuta (2.0%), S. miescheriana (19.3%), S. bovifelis (26.0%), S. bertrami (26.7%), S. capracanis (44.7%), S. arieticanis (61.3%), S. tenella (89.3%) and S. cruzi (99.3%). Further, it was established that the detection frequency of Sarcocystis species in water samples depended on the combinations of selected primers. The present study showed that in general, Sarcocystis parasites were widespread in water bodies and could easily infect livestock.