Contradictory data is available on the intermediate host specificity of Sarcocystis spp. in farm animals. Therefore, the current work aimed at molecularly testing samples of sheep and goats reared in Lithuania to identify Sarcocystis species described in other intermediate hosts but suspected to be non-canonical parasites to these small ruminants. For this purpose, muscle samples from 47 domestic sheep and nine goats were examined. Sarcocystis species were identified using direct and nested PCR targeting cox1 and sequencing of positive amplified products. Along with the detection of the canonical Sarcocystis spp. in their respective intermediate hosts, the DNA of S. capracanis and S. morae was detected in sheep, although these species were previously thought to be specific to goats and deer, respectively. In addition, DNA from S. arieticanis and S. tenella was found in goats, even though these two species were believed to be sheep-specific. Notably, under light microscopy, only sarcocysts of S. capracanis specific to goats were observed. Thus, future research on the life cycle and host-specificity of Sarcocystis spp. examined is warranted.

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.

Records of Sarcocystis spp. diversity in rodents are relatively extensive; however, the increasing application of molecular approaches indicates that our current knowledge of these parasites remains incomplete. In the present study, morphological, genetic and phylogenetic data are provided on Sarcocystis arvalis n. sp. from the common vole (Microtus arvalis). Using light microscopy, the observed sarcocysts had a relatively thin (<1 μm) and smooth cyst wall. Via transmission electron microscopy, the sarcocyst wall thickness ranged from 0.7 to 1 μm, and the parasitophorous vacuolar membrane exhibited small knob-like blebs and was slightly wavy, type 1a. Based on 18S rRNA, 28S rRNA, cox1 and rpoB loci, S. arvalis n. sp. showed the highest similarity with Sarcocystis myodes from the bank vole (Clethronomys glareolus). According to the phylogenetic placement, S. arvalis n. sp. is the most closely related to Sarcocystis spp. with a rodent–mammal lifecycle. Morphologically, S. arvalis n. sp. forms sarcocysts that share a lot of similarities with those of S. myodes, Sarcocystis ratti and Sarcocystis cernae and molecular analysis is necessary for accurate species identification. Based on the abundance of the hosts and the proportion of voles in their diet, the most likely definitive hosts of S. arvalis n. sp. are red foxes, stone martens, least weasels, and domestic cats.

Sarcocystis, a diverse and species-rich protist genus infecting reptiles, birds, and mammals, remains poorly understood in terms of true diversity and their lifecycles. Typically, sarcocysts are found in the muscle tissue of the intermediate host (IH), while oocysts undergo sporulation in the intestines of the definitive host (DH). Rodent-associated Sarcocystis species often form cryptic species complexes with strong specificity to their DHs; however, their presence in the intestines of wild carnivores, whose IHs are rodents, is understudied. The aim of this study was to investigate the distribution of rodent-associated Sarcocystis species in the intestines of wild Mustelidae and Canidae from Lithuania using light microscopy (LM) and nested PCR targeting 28S rRNA. LM analysis of intestinal scraping revealed Sarcocystis spp. in 56.3% of canids and mustelids, while DNA sequence analysis identified 41.0% of mustelids and 11.6% of canids as positive. Three Sarcocystis species, S. arvalis, S. myodes, and S. ratti, and the genetic lineage Sarcocystis sp. Rod8, which belong to the same cryptic species complex, were identified in mustelids, while S. arvalis and S. myodes were detected in canids. Thus, mustelids contribute more than canids to the natural transmission of Sarcocystis spp. from rodents in Lithuania.

Toxoplasma gondii, a widespread parasite, poses significant public health concerns. It infects humans and animals, with rodents serving as important intermediate hosts. The present study investigated the prevalence and genetic ITS1 diversity of T. gondii in wild rodents from Lithuania. A total of 469 rodents from eight species were captured across various regions, and DNA from neural and cardiac tissues was analyzed using nested PCR. Overall prevalence of T. gondii was 26.2% (95% CI = 22.3–30.5). The prevalence of infection varied among rodent species (0–50.0%) and across geographic locations. A mere few rodents exhibited concurrent infections in both tissues examined. Toxoplasma gondii was detected more frequently in the brains of Apodemus flavicollis and hearts of Clethrionomys glareolus, and in the males of Microtus arvalis. A total of 19 distinct ITS1 genotypes were identified, including 17 novel ones; Genotype 1 was the most prevalent and widely distributed. Phylogenetic and network analyses revealed a star-like topology centered on Genotype 1 and confirmed the accurate identification of T. gondii in Lithuanian rodents. This study provides the first evidence of T. gondii in wild rodents in Lithuania, highlighting the need for further research on its prevalence and potential impact on public health and wildlife.

Micromammals are abundant in nature and have a significant role as reservoirs for various parasites and pathogens. Very little is currently known about Sarcocystis prevalence and species richness in wild micromammals from Mediterranean Spain. In the present study, a pool of heart, lungs, and brain of 371 individuals representing three species, Algerian mouse ( Mus spretus ), wood mouse ( Apodemus sylvaticus ), and white-toothed shrew ( Crocidura russula ), was screened for Sarcocystis using molecular methods. Animals were sampled across 4 seasons in both natural and agricultural habitats. Prevalence in white-toothed shrew (28%) was significantly higher than in wood and Algerian mouse (17% and 18%, respectively). The lowest prevalence occurred in autumn, whereas no significant differences were detected between habitats. Analysis of 28S rRNA sequences revealed 11 Sarcocystis lineages, with the highest lineage diversity observed in the Algerian mouse. Based on comparative sequence and phylogenetic analysis, most of these lineages are suspected to use snakes as definitive hosts (DHs), three are associated with birds, and the DHs for two lineages are likely mammals. Notably, cox1 sequences could not be used to confidently identify all lineages. Our results reveal substantial diversity of Sarcocystis in Mediterranean micromammals and provide new insights into their transmission cycles.