Molecular Identification of Sarcocystis rileyi and Sarcocystis sp. (Closely Related to Sarcocystis wenzeli) in Intestines of Mustelids from Lithuania

Simple Summary Protozoan parasites of the genus Sarcocystis are characterised by a two-host prey–predator life cycle. To date, more than 25 Sarcocystis species have been confirmed to form sarcocysts in muscles and CNS of birds. Avian Sarcocystis species are transmitted via predatory birds, placental mammals, and opossums. The objective of the study was to examine the role of predatory mammals of the family Mustelidae in the transmission of avian Sarcocystis spp. by means of molecular methods. In total, 115 small intestine samples of mustelids collected in Lithuania were tested for the presence of Sarcocystis spp. using anseriforms and domestic fowl (Gallus domesticus) as their intermediate hosts. Based on the DNA sequence analysis, S. rileyi known as forming macrocysts in muscles of ducks was detected in 11.3% of examined small intestine samples and Sarcocystis sp. was identified in two samples. The latter species was most closely related to Sarcocystis spp. isolates infecting chickens and causing encephalitis. This is the first report of avian Sarcocystis identified by molecular methods in the small intestines of mustelids, indicating the significance of these small predators for the spreading of Sarcocystis spp. using birds as intermediate hosts. Based on current knowledge, canids and mustelids are most likely the definitive hosts of S. rileyi in Europe. Abstract The genus Sarcocystis is a group of numerous protozoan parasites having a two-host life cycle. Based on laboratory experiments and/or phylogenetic analysis results it was shown that seven Sarcocystis spp. producing sarcocsyts in bird tissues are transmitted via predatory placental mammals. To date the role of small mammals of the family Mustelidae in the distribution of avian Sarcocystis spp. have not been studied. During the current investigation, intestinal mucosa scrapings of 115 mustelids belonging to five species were tested for S. albifronsi, S. anasi, S. rileyi, and S. wenzeli infecting anseriforms and chickens. Microscopically, free sporocysts, sporulating oocysts, and loose oocysts were found in 61 samples (53.0%). Using nested PCR targeting the ITS1 region and sequencing, S. rileyi was confirmed in eight American minks, two European polecats and single European badger. Sarcocystis sp. was identified in one American mink and one European pine marten. Based on the partial ITS1 region this parasite showed that 100% identity to pathogenic Sarcocystis sp. caused a fatal infection in backyard chickens from Brazil. Phylogenetically, the Sarcocystis sp. identified in our study was most closely related to S. wenzeli parasitising domestic fowl (Gallus domesticus).


Introduction
Members of the genus Sarcocystis (Apicomplexa: Sarcocystidae) are protozoan parasites distributed worldwide. The genus Sarcocystis has a broad host spectrum encompassing mammals, birds and reptiles. These parasites are distinguished by an obligatory prey-predator two-host life cycle [1]. Sarcocysts are found mainly in muscles or CNS of intermediate hosts, while endogenous sporulation of oocysts take place in the intestine of

Data Analysis
Sterne's exact method [33] was used to compute 95% confidence interval (CI) for the prevalence of Sarcocystis spp. in host species and in animals hunted during different months. Differences in the detection of Sarcocystis species in the examined mustelid species were evaluated using a Chi-squared test. The unconditional exact test was used to compare S. rileyi prevalence in animals collected in different months [34]. Statistical tests were carried out using the Quantitative Parasitology 3.0 software [35].

Microscopical Examination of Sarcocystis spp. Oocysts/Sporocysts
Sarcocystis spp. sporocysts and/or oocysts were noticed in the intestinal epithelium of the small intestines of all five species of mustelids analysed in the current study ( Figure 1). In some samples, only a few (one-five) sporocysts and/or oocysts were detected in the area of the 24 × 24 mm coverslip, while in other samples, numerous parasites of different stages were found and it was even difficult to count the exact number of oocysts/sporocysts.
We observed seasonal changes in the abundance of S. rileyi in our sample ( Figure 2). S. rileyi was determined by molecular methods in one animal hunted in October, in two animals each hunted in September, November, and December, and in four animals hunted in January. The unconditional exact test showed that the detection of S. rileyi in September-January (15.5%, 95% CI = 8.5%-25.9%) was significantly higher (p = 0.0034) than in the February-April period (0%, 95% CI = 0%-8.5%). Meanwhile, Sarcocystis sp. LT-2022 was established in two animals collected in March and April.
brain, pectoral muscle, lung, and heart of native village chickens in Malaysia.
We observed seasonal changes in the abundance of S. rileyi in our sample ( Figure 2). S. rileyi was determined by molecular methods in one animal hunted in October, in two animals each hunted in September, November, and December, and in four animals hunted in January. The unconditional exact test showed that the detection of S. rileyi in September-January (15.5%, 95% CI = 8.5%-25.9%) was significantly higher (p = 0.0034) than in the February-April period (0%, 95% CI = 0%-8.5%). Meanwhile, Sarcocystis sp. LT-2022 was established in two animals collected in March and April.

Phylogenetic Relationships of Identified Sarcocystis Species
Comparing the ITS1 fragments established in the current work, sequences of Sarcocystis sp. were longer at the 5' end and the sequences of S. rileyi were longer at the 3' end. Thus, after multiple alignment and sequence truncation, 547 bp-long sequences of S. rileyi and 554 bp-long sequences of Sarcocystis sp. were used for phylogenetic analysis. In the phylogenetic tree, S. rileyi obtained from mustelids grouped with other S. rileyi isolates obtained from various intermediate hosts ( Figure 3). Based on ITS1, S. rileyi was the sister taxon to S. atraii and these two species formed separate clusters in the phylogram.
Based on the analysed ITS fragment, Sarcocystis sp. LT-2022 isolated from two representatives of two mustelid species were identical to Sarcocystis sp. Chicken-2016-DF-BR isolated from chickens in Brazil and they were placed in one well-supported cluster together with S. wenzeli and Sarcocystis sp. from chickens from Malaysia. Whereas, S. cristata described in the muscles of the great blue turaco (Corythaeola cristata) was the sister taxon to the Sarcocystis isolates established in Brazil, Malaysia, and Lithuania, and S. wenzeli. The remaining Sarcocystis spp., S. albifornsi, S. anasi from anseriforms, and S. chloropusae from the common moorhen (Gallinula chloropus) made a separate cluster in the phylogenetic tree.

Phylogenetic Relationships of Identified Sarcocystis Species
Comparing the ITS1 fragments established in the current work, sequences of Sarcocystis sp. were longer at the 5' end and the sequences of S. rileyi were longer at the 3' end. Thus, after multiple alignment and sequence truncation, 547 bp-long sequences of S. rileyi and 554 bp-long sequences of Sarcocystis sp. were used for phylogenetic analysis. In the phylogenetic tree, S. rileyi obtained from mustelids grouped with other S. rileyi isolates obtained from various intermediate hosts (Figure 3). Based on ITS1, S. rileyi was the sister taxon to S. atraii and these two species formed separate clusters in the phylogram.

The Role of Mustelids in Distribution of Sarcocystis Species
In the present study, free sporocysts, sporulating oocysts, and loose oocysts were Based on the analysed ITS fragment, Sarcocystis sp. LT-2022 isolated from two representatives of two mustelid species were identical to Sarcocystis sp. Chicken-2016-DF-BR isolated from chickens in Brazil and they were placed in one well-supported cluster together with S. wenzeli and Sarcocystis sp. from chickens from Malaysia. Whereas, S. cristata described in the muscles of the great blue turaco (Corythaeola cristata) was the sister taxon to the Sarcocystis isolates established in Brazil, Malaysia, and Lithuania, and S. wenzeli. The remaining Sarcocystis spp., S. albifornsi, S. anasi from anseriforms, and S. chloropusae from the common moorhen (Gallinula chloropus) made a separate cluster in the phylogenetic tree.

The Role of Mustelids in Distribution of Sarcocystis Species
In the present study, free sporocysts, sporulating oocysts, and loose oocysts were found in the intestinal mucosa of the five examined species, American mink, European pine marten, European polecat, European badger, and beech marten ( Figure 1 and Table 1). The morphometric sizes of parasite stages detected in five hosts overlapped. Thus, it was impossible to determine whether the studied host species were infected with the same or different Sarcocystis species. Furthermore, it is known that predators can be simultaneously infected with sporocysts of several Sarcocystis species [16,24,38]. Therefore, the identification of Sarcocystis species was performed using molecular methods.
Based on the nested PCR and subsequent BLAST analyses of the obtained DNA sequences, two Sarcocystis species using birds as intermediate hosts were confirmed. The prevalence of Sarcocystis spp. defined by means of molecular examination was relatively low, reaching 11.3% (13/115). By microscopical analysis, sporocysts and/or oocysts of Sarcocystis spp. were noticed in more than half (53.0%, 61/115) of the investigated samples. Thus, the obtained results of the present work indicate that the examined mustelids spread considerably more than Sarcocystis species, employing mammals rather than birds as their definitive hosts. Our previous research on the small intestine samples of mustelids by species-specific PCR revealed a high prevalence (89.3%, 75/84) of Sarcocystis species using cattle as their intermediate hosts [24]. Furthermore, 32 of the 40 (80.0%) examined small intestine samples of American mink tested positive for S. elongata, S. entzerothi, S. japonica, S. silva, and S. truncata by molecular methods, producing sarcocysts in muscles of ungulates of the family Cervidae [16]. It was also confirmed by experimental infection that possible definitive hosts of S. campestris, S. citellivulpes, S. muris, S. putorii, and S. undulati are members of family Mustelidae [39]. Research carried out until now implies that mustelids play a significant role for the transmission of various Sarcocystis species using hosts that belong to different taxonomic groups.

Mustelids as Possible Definitive Hosts of S. rileyi
Eleven ITS1 sequences obtained in the current study demonstrated 99.18%-100% similarity to the sequences of S. rileyi available in GenBank and showed less than 93% similarity with any other known species of Sarcocystis. Hence, S. rileyi was confirmed in the intestinal mucosa of eight American minks, two European polecats and single European badger ( Table 2). The overall prevalence of S. rileyi in the analysed samples of mustelids was 9.6% (95% CI = 5.1%-16.4%). In the present study, the identified S. rileyi is a wellknown Sarcocystis species forming macroscopic sarcocysts resembling grains of rice in the muscles of ducks. This species was described in the late nineteenth century [40] and redescribed in 2003, providing detailed morphological characterisation [41]. For a long time, macrocysts in numerous duck species were recorded only in North America [42][43][44][45][46][47][48]. Based on light microscopy, transmission electron microscopy, and molecular characterisation at three genetic loci (18S rDNA, 28S rDNA, and ITS1), S. rileyi was identified in Lithuania in 2011 [12]. According to the current data, the distribution of S. rileyi covers the eastern, northern, and central parts of Europe [12,13,29,[49][50][51][52][53]. In this continent, S. rileyi was mostly recorded in mallard [12,13,49,52] and with much less frequency in several other duck species [29,53]. The mallard is the most abundant species of ducks and also one of most important bird game species in Europe [54][55][56]. Sarcocystis rileyi cause economic losses, since hunted duck meat contaminated with macrocysts is not suitable for human consumption [13]. Additionally, severe infection of S. rileyi may result in weakness of hosts, reduced flying capacity, and infected birds may be more easily caught by predators [57]. The stripped skunk (Mephitis mephitis) of the family Mephistidae is an experimentally proved definitive hosts of S. rileyi in North America [58,59]. This small predatory animal lives only in captivity in Europe [60]. Therefore, for a long time it was unclear which predators are responsible for the spread of S. rileyi in Europe. Based on molecular analysis, red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) of the family Canidae were identified as definitive hosts of S. rileyi in Lithuania and Germany [38,50]. Hence, based on the findings of previous and current investigations, S. rileyi is transmitted in Europe by predators of family Canidae and Mustelidae. It should be noted that Mephistidae and Mustelidae families are closely related and together with Ailuridae and Procyonidae compose a superfamily, Musteloidea [61]. Thus, the current findings indicate the coevolution of S. rileyi with their definitive hosts. A co-evolution of Sarcocystis spp. with their definitive host rather than the intermediate host has been shown in the phylogenetic investigations of various groups of Sarcocystis species [15,62,63]. The raccoon (Procyon lotor) of the family Procyonidae which is native to North America is now spreading in Lithuania through the western part of the country [64]. Taking into account the close relationship of raccoon with mustelids and mephistids, this invasive predator should be screened for the distribution of S. rileyi.
In general, Sarcocystis species are more host-specific for their intermediate hosts than for their definitive hosts. For instance, S. cruzi, the most common species of Sarcocystis of cattle worldwide, is transmissible via dogs, coyotes, foxes, and wolves [1]. With the exception of S. wenzeli, Sarcocystis species transmitted by canids cannot be transmitted by felids and vice versa [65]. However, laboratory experiments evidenced that some Sarcocystis spp. transmitted via canids or felids can be spread via mustelids [39,66]. Furthermore, on the basis of molecular investigations of small intestine samples it was shown that S. cruzi can be spread not only by canids, but also by mustelids [24]. The current study on the basis of ITS1 sequence analysis also indicates that S. rileyi can be transmitted in Europe by the members of two families, Canidae and Mustelidae.
It should be emphasised that in the present study S. rileyi was identified in mustelids, which were hunted from September to January (15.5%, 11/71). This Sarcocystis species was mostly confirmed in American mink, while S. rileyi was not detected in 44 animals which were collected during February-April ( Figure 2). In Lithuania, during September, large flocks of ducks and waders concentrate in partly drained fishponds and these birds form a large part of the diet of invasive American mink [67]. In summer-early autumn, birds are also an important food component for other mustelid species in various regions of Lithuania [18]. Whereas, in late autumn and winter mustelids only occasionally hunt ducks [18][19][20]28,[68][69][70]. Oocysts/sporocysts of Sarcocystis spp. are found in the faeces of definitive hosts 7-14 days post infection and excretion of infective parasite stages mostly lasts several months [1]. Thus, the observed variations in the identification of S. rileyi during different months are congruent with the diet of mustelids and the life cycle peculiarities of Sarcocystis parasites. The results of the abundance of S. rileyi depending on the season mustelids were hunted are in congruent with the investigation of S. rileyi in canids. During a previous study conducted in Lithuania, S. rileyi was identified in the small intestines of red foxes and raccoon dogs hunted in November and December, but the parasite was not detected in animals hunted in February and March [50]. Thus, future research on the prevalence of Sarcocystis in predatory mammals through different seasons is needed.

Detection of Sarcocystis sp. Closely Related to S. wenzeli in Small Intestine of Mustelids
Based on the obtained ITS1 sequence analysis, Sarcocystis sp. was identified in a single American mink and in a single American pine marten (Table 2). Two 817 bp-long ITS1 sequences were 100% identical to the sequence of Sarcocystis sp. Chicken-2016-DF-BR Animals 2023, 13, 467 9 of 13 obtained from the brains of two chickens in the midwest of Brazil [36]. This parasite caused fatal outcomes in backyard chickens. The infected chickens suffered from anorexia, weight loss, incoordination, ataxia, and opisthotonos. The histopathological analysis showed necrotizing granulomatous and meningoencephalitis with intralesional Sarcocystis-like schizonts and merozoites. Infected chickens remained free during the day and were kept in the coop at night [36]. It should be noted that severe myositis and encephalitis associated with Sarcocystis parasites has been reported several times in domestic fowl in different geographical regions [71,72].
The ITS1 region is highly variable for Sarcocystis spp. [8]. Due to the large number of indels (insertions/deletions) it is hard to align ITS1 sequences of Sarcocystis spp. sharing relatively low similarity. Therefore, the ITS1 region is not a good choice for the discriminating phylogenetic relationships of genetically remote group of Sarcocystis. However, this genetic locus is suitable for phylogenetic analysis of closely related Sarcocystis species [63,73]. Several studies have demonstrated that ITS1 is an appropriate genetic marker inferring phylogenetic relationships of Sarcocystis spp. using birds as intermediate hosts [5,[9][10][11][73][74][75][76][77][78]. Furthermore, for this group of Sarcocystis species, ITS1 and 28S rDNA give congruous topology [73,79]. Based on the ITS1 sequence analysis conducted in the current study, the topology of the examined Sarcocystis species using birds-predatory mammals in their intermediate-definitive host life cycle ( Figure 3) in general corresponded to that determined in the latest phylogenetic studies [5,36]. Sarcocystis sp. LT-2022 obtained in the present work and Sarcocystis sp. Chicken-2016-DF-BR were placed in one phylogenetic cluster together with S. wenzeli infecting chickens and Sarcocystis sp. isolated from pooled various tissue samples of native village chickens in Malaysia ( Figure 3). Thus, the obtained sequences were grouped with those of Sarcocystis spp. parasitising chickens. Further molecular studies are needed to clarify the number of species that represent S. wenzeli, Sarcocystis sp. Chicken-2016-DF-BR, Sarcocystis sp. from Malaysian chickens, and Sarcocystis sp. LT-2022 identified in the present work.
There is ongoing debate on the classification of Sarcocystis species in chickens [39]. In the latest taxonomic review of the genus Sarcocystis, two species infecting chickens, S. horvathi and S. wenzeli, were distinguished [1]. Sarcocystis wenzeli is characterised morphologically in detail and based on transmission experiments dogs and cats are confirmed as definitive hosts of this species [65], whereas the definitive hosts of S. horvathi are unknown [1].
The data of the current work indicate that mustelids might be involved in the transmission of Sarcocystis species infecting domestic gallinaceous fowl. However, laboratory infection experiments are definitely necessary to test the results obtained. Additionally, here we present the first identification of Sarcocystis sp. in Lithuania, closely related to Sarcocystis parasitizing chickens. Despite extensive studies of Sarcocystis conducted in Lithuania in various groups of wild birds [7,8,12,73,[75][76][77][78][79], these parasites have not been studied in poultry so far. In Lithuania, chickens are mainly raised in poultry farms [80]. However, in rural regions small numbers of domestic fowl are kept free. The main predators of backyard chickens in Lithuania are mustelids and red fox [18]. Mustelids can potentially cause the transmission of highly pathogenic Sarcocystis species in poultry farming.

Conclusions
Based on the nested PCR and sequencing of the ITS1 region, S. rileyi producing macroscopic sarcocysts in muscles of ducks was for the first time confirmed in small intestine scrapings of three mustelid species collected in Lithuania. The prevalence of S. rileyi in the examined mustelids was 9.6%. According to the current data obtained by molecular investigations, canids and mustelids are responsible for the spread of S. rileyi in Europe.
Undescribed Sarcocystis sp. LT-2022 showed 100% similarity within the 817 bp-long ITS1 fragment with Sarcocystis sp. Chicken-2016-DF-BR, which caused a fatal infection in two backyard chickens in Brazil. The detected Sarcocystis parasite was most closely related to S. wenzeli and Sarcocystis sp. using chickens as their intermediate hosts. Thus, this is the first report of Sarcocystis sp. associated with possible infection in gallinaceous birds in Lithuania.