Sarcocystis Species Research Articles

First Molecular Identification and Prevalence of Sarcocystis spp. in Sheep Intended for Human Consumption in Shanxi Province, China.

Sarcocystis species are intracellular coccidian protozoans that can infect a range of animals and humans and cause public health problems and economically significant losses. Sarcocystosis in sheep (Ovis aries) can cause abortion, neurological symptoms, and even death and results in significant economic losses to the livestock industry. To date, however, it is yet unknown whether sheep in Shanxi Province, north China, are infected with Sarcocystis spp. The purpose of this study was to investigate the prevalence of Sarcocystis spp. in sheep in Shanxi Province. Thus, 582 muscle samples of sheep were purchased from farmers' markets from ten representative counties in Shanxi Province, north China, and examined for the presence and prevalence of Sarcocystis spp. by PCR amplification of the mitochondrial cytochrome c oxidase subunit I (cox1) gene. Of the examined 582 mutton samples, 197 samples (33.85%) were Sarcocystis-positive and were sequenced. Of the obtained 197 cox1 sequences, 196 sequences showed nucleotide similarity of 98.56-99.81% with those of S. tenella, and the remaining one cox1 sequence showed nucleotide similarity of 99.71% with that of S. arieticanis. Two representative cox1 sequences of S. tenella (accession nos. PQ189447 and PQ189448) have 99.52% and 99.61% identity with S. tenalla (KC209725) and S. tenalla (MK419984), respectively. The sequence of S. arieticanis (accession no. PQ165949) obtained in this study has 99.71% identity with S. arieticanis (MK419975). This present study documents the occurrence and prevalence of Sarcocystis spp. in sheep in Shanxi Province for the first time, which enriches the data on the distribution of Sarcocystis spp. in sheep in China and has implications for the control of sheep sarcocystosis.

Gross lesions associated with Sarcocystis miescheriana in a wild boar hunted for human consumption: the importance of trained hunters to ensure animal health surveillance and food safety

Sarcocystis is a genus of protozoa with a worldwide distribution infecting a wide range of animals, including humans. Wild boars can harbor at least two species of Sarcocystis, that is, the zoonotic Sarcocystis suihominis, using humans as definitive hosts, and Sarcocystis miescheriana, for which wild and domestic canids serve as definitive hosts. In Portugal, hunting holds significant economic and social importance, and wild boars are among the most appreciated hunted species. As the consumption of wild boar meat can expose humans to several foodborne pathogens, the presence of trained hunters can make a difference in ensuring animal health surveillance and food safety. Herein, we report the detection of macroscopic cystic lesions associated with S. miescheriana in a wild boar hunted for human consumption, resulting in carcass condemnation. To the best of the authors’ knowledge, the presence of S. miescheriana in wild boar tissues has never been associated with macroscopic pathological alterations before. Although S. miescheriana cannot infect humans, carcasses affected by grossly visible pathological changes must be declared unfit for consumption. Therefore, our finding points out the potential economic damage associated with carcass rejection due to the presence of gross lesions associated with generalized sarcocystosis. Nonetheless, further studies are required to investigate these alterations that currently appear to be occasional findings.

Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep.

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.

MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF SARCOCYSTIS INFECTIONS IN THE MUSCLES OF GRAY WOLVES (CANIS LUPUS) FROM MINNESOTA SUGGEST THEY MAY SERVE AS RESERVOIRS FOR INFECTION IN DOMESTICATED DOGS.

Sarcocystis infections were found for the first time in the muscles of 3 of 3 gray wolves (Canis lupus) from Minnesota. Two kinds (thin-walled and thick-walled) of sarcocysts were detected, based on the appearance of the sarcocyst wall. In wolf 1, sarcocysts were thin-walled (<0.5 μm), and without any visible protrusions. Ultrastructurally, the sarcocyst wall was type 1a and identical to Sarcocystis svanai of the domestic dog (Canis familiaris). The second kind of sarcocyst, with a relatively thicker (>1 μm) sarcocyst wall, was detected in wolves 2 and 3. Ultrastructurally, the sarcocyst wall had undulating, pleomorphic villar protrusion of type 9c; these sarcocysts were identical to Sarcocystis caninum from the domestic dog. Molecularly, the 2 Sarcocystis species were characterized using 18S, 28S, COI, ITS-1, and rpoB genetic markers. All these markers showed 100% identity to either of the 2 species previously described from the domestic dog. The thick-walled sarococyst corresponded to Sarcocystis caninum, whereas the thin-walled sarcocyst corresponded to Sarcocystis svanai.

The Genetic Identification of Numerous Apicomplexan Sarcocystis Species in Intestines of Common Buzzard (Buteo buteo).

The common Buzzard (Buteo buteo) was previously shown to transmit two Sarcocystis species (S. glareoli and S. microti) forming cysts in the brains of rodents. Due to a lack of research, the richness of Sarcocystis species spread by these birds of prey is expected to be much higher. A total of 30 samples of the small intestine of the Common Buzzard were collected in Lithuania and subjected to Sarcocystis species identification based on nested PCR of 28S rRNA and ITS1, following the sequencing of amplified DNA fragments. Six known Sarcocystis spp., S. cornixi, S. glareoli, S. halieti, S. kutkienae, S. turdusi, and S. wobeseri, along with three genetically distinct species (Sarcocystis sp. Rod3, Sarcocystis sp. Rod4, and Sarcocystis sp. Rod5), were identified. Phylogenetically, these three potentially new species clustered with Sarcocystis spp. characterised by a rodents-birds life cycle. Sarcocystis spp. employing rodents and birds as their intermediate hosts were detected in 66.7% and 50.0% of samples, respectively. These findings are consistent with the diet preferences of Common Buzzards. Notably, co-infections with two or more species were observed in a half of the samples. Altogether, the obtained results indicate that the Common Buzzard could serve as definitive host of various Sarcocystis species.

Equine Sarcocystosis in the Northern Region of the Republic of Kazakhstan.

A total of 396 samples were taken from the hearts, oesophagi, and diaphragms of 132 horses slaughtered at slaughterhouses in 2023 for subsequent examination. The histological method revealed pathomorphological changes in the muscle tissue. The molecular method identified the pathogen species. Histological examination revealed thick-walled cysts with internal septa and numerous bradyzoites, and mononuclear inflammatory cells with pericyst infiltrates. Microcyst samples were amplified by polymerase chain reaction. Molecular genetic analysis allowed for the identification of 18 sarcocysts. Phylogenetic analysis of Sarcocystis isolates revealed three separate clades of Sarcocystis bertrami and two separate clades of Sarcocystis fayeri. Comparison and phylogenetic analysis revealed a very close relationship between the identified Sarcocystis species and other equine Sarcocystis DNA sequences from China and Japan. Based on the results obtained, the epizootic situation and the parasitic level of sarcocystosis contamination of horses in the northern Kazakhstan were determined. This is the first histological and molecular study to identify Sarcocystis spp. isolated from microscopic forms of equine sarcocysts in the northern Kazakhstan. This research will contribute to the fight against the spread of sarcocystosis in the Republic of Kazakhstan and will allow us to develop proposals for improving the mechanisms of sarcocystosis control.

Sporocysts of Sarcocystis bertrami (syn. Sarcocystis fayeri) shed by dogs: Molecular analysis, morphometry and pattern of excretion

Sarcocystis bertrami (synonym: Sarcocystis fayeri) is a coccidian parasite that infects horses and donkeys in several countries. Dogs are known as definitive hosts of the parasite, however, the patent period is not well defined, and S. bertrami shed by dogs has never been confirmed by molecular methods. Here we investigated the shedding of S. bertrami by experimentally infected dogs and examined the excreted parasites by morphological and molecular tools. Three dogs of small breeds (one Yorkshire terrier and two miniature Pinschers) were acquired with ages of 30 and 60 days and were exclusively fed commercial dog food. Two dogs consumed equine muscle tissues containing cysts of S. bertrami. The third dog served as negative control and was simultaneously fed commercial dog food. The two animals that received equine tissues shed sporocysts and/or oocysts in their feces after prepatent periods of 13 and 23 days. The patent periods were 47 and 14 days. Sporocysts showed average dimensions of 14.19 µm (± 0.53) x 10.06 µm (± 0.44). The control dog did not shed sporocysts or oocysts of the parasite. Interestingly, patent periods had never been reported, and for one dog, the patent period (47 days) was longer than that reported for other Sarcocystidae parasites. PCRs to the gene 18S and to the internal transcribed spacer 1 (ITS1) of the rDNA were successfully performed with DNA extracted from sporocysts. ITS1 sequences were also obtained from the equine tissue cysts used to infect the dogs. Nucleotide sequences of cloned fragments of 18S from sporocysts, and ITS1 from both stages (tissue cysts and sporocysts) matched with S. bertrami (18S: 97.50–99.88 %; ITS1: 88.76–95.21 %), although high molecular diversity was observed with data from these loci. PCR to cox1 using sporocysts’ DNA failed to amplify any product. The possibility of the existence of an additional and undescribed Sarcocystis species in the excreted sporocysts, besides S. bertrami, cannot be excluded from this experiment. To our knowledge, this is the first molecular confirmation of S. bertrami in canine feces. Sporocyst dimensions and prepatent periods observed in this study were similar to those previously described for S. bertrami and S. fayeri. In conclusion, the molecular, morphological and biological data generated here fit in previous descriptions for both S. bertrami and S. fayeri.

Red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) as potential spreaders of Sarcocystis species.

Sarcocystis includes a global group of apicomplexan parasites with two-host life cycle frequently circulating in wildlife and domestic hosts, including humans. Two of the most important wild terrestrial carnivores acting as definitive hosts are the red fox and raccoon dog, due to their wide distribution in Europe and usage of wild and farmed animals as prey. This study was conducted to determine the prevalence of Sarcocystis in hunted red foxes and raccoon dogs from nine regions of the Czech Republic and to identify isolated sporocysts by molecular techniques. Approximately 5 g of the contents of large intestine from 200 animals (197 red foxes and three raccoon dogs) were examined by flotation centrifugation coprological method. Only samples of 50 red foxes and one raccoon dog positive to Sarcocystis spp. were used for the nested PCR (nPCR) method to amplify a fragment or partial sequence on the cox1 gene. Ten species-specific primer pairs for detection of Sarcocystis spp. using farm animals as intermediate hosts were utilized. In total, 38.1% of the red foxes and 66.7% of the raccoon dogs were positive to Sarcocystis by light microscopy. The molecular characterization resulted in the identification of five species in the red fox: S. arieticanis, S. capracanis, S. cruzi, S. miescheriana, and S. tenella, while the PCR was negative for the sole raccoon dog. The highest intraspecific variation was found for S. miescheriana, while S. tenella was the most prevalent. Co-infections occurred in the large intestine of the red fox. No zoonotic species were found in our samples. This is the first study where the potential role of the red fox and raccoon dogs as spreaders of Sarcocystis to farm animals in the Czech Republic is shown. The use of species-specific primers provides a fast and easy method for screening multiple samples for a particular Sarcocystis species.

First findings of Sarcocystis species in game deer and feral pigs in Australia

Several wild game meat species, including deer and feral pigs are hunted and consumed in Australia. Feral pigs and deer are not indigenous to Australia, but they have proliferated extensively and established their presence in every state and territory. Following the report of a sambar deer displaying Sarcocystis like white cysts in its rump muscles, the present study was conducted to explore the prevalence of Sarcocystis infections in wild deer and feral pigs in the southeastern regions of Australia. Oesophagus, diaphragm, and heart tissue from 90 deer and eight feral pigs were examined visually for sarcocysts. All results were negative. PCR testing of randomly selected deer and feral pigs yielded positive results, which were subsequently supported by histopathology. This is the first study to report the presence of Sarcocystis spp. in deer and feral pigs in Australia. As no visual cysts were found on the heart or oesophagus that came back positive with PCR, infected animals, particularly those reared free-range, could be passing through meat quality checks unidentified. If people consume this meat without cooking it properly, it may lead to a human infection of Sarcocystis. However, a more targeted study focused on determining the parasite's prevalence and assessing its risks is necessary to determine if it constitutes a food safety issue. As this species has been found not only in feral pigs but also in domestic pigs, the potential for infection spreading between feral pigs and pigs in free-range livestock systems is high, potentially posing a large problem for the Australian pork industry, particularly with the increased emphasis on free-range pig husbandry. Future studies should concentrate on determining the species of Sarcocystis in feral animals commonly consumed as game meat to determine potential zoonotic risks. This could also include a more in-depth look at the prevalence of Sarcocystis infections in other game animals. Identifying where these parasites are present and to what extent, are important areas for future studies.

Visual and molecular serologic diagnosis of sarcocystosis in cattle

The purpose of the research is to provide a comparative assessment of efficacy of different diagnostic methods for sarcocystosis in cattle.Materials and methods. Seventy-eight bovine carcasses were examined using methods and regulations of veterinary and sanitary examination. Muscles of the esophagus and other parts of the carcass as well as the spleen, lungs, the liver, and kidneys were examined. The compressor research method was used. A total of 156 compressoria from 78 bovine carcasses were analyzed for tissue sarcocystosis. For life-time diagnostics of bovine sarcocystosis, a molecular serologic method (ELISA) was used that was developed based on antibody detection principles.Results and discussion. Two diagnostic methods, visual inspection and compressor microscopy, identified 13 out of 78 carcasses with Sarcocystis species, which was 16.6%. The number of sarcocystosis animals increased by another 23 cases when studying blood sera from the cattle using ELISA. Thus, we found 36 animals suffering from sarcocystosis out of 78 examined, which was 46%, with tests based on different principles.

Diversity of Sarcocystis parasites in southeastern Baltic Sea catchment ecosystems.

Currently, research on apicomplexan Sarcocystis parasites is mainly carried out by analyzing animal carcasses. However, environmental studies would not only allow faster detection of possible sources of infection but also avoid the use of animals for investigations. Therefore, in the current study, we aimed to identify tested Sarcocystis species in sediment collected from water bodies located in the southeastern Baltic countries. A total of 99 sediment samples were collected during the summer from different types of water bodies in Estonia, Latvia, Lithuania, and Poland. Species-specific nested PCR targeting cox1 gene was used for the detection of selected Sarcocystis species (S. cruzi, S. bovifelis, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. miescheriana, and S. bertrami) infecting livestock. The results showed a statistically lower (p < 0.05) occurrence of Sarcocystis parasites in Estonia (50%) compared to three countries, where the detection rate of Sarcocystis spp. DNA was remarkably higher, ranging from 88 to 100%. Among Sarcocystis species tested, S. cruzi (83.8%) and S. arieticanis (55.6%) using cattle and sheep as their intermediate hosts were most commonly identified. The detection rates of some of the analyzed Sarcocystis species were significantly different in southeastern Baltic countries. It is discussed that the detection rates of certain Sarcocystis species depend not only on the number of animals per 1 km2 but also on various ecological factors and farming practices that differ in the amount of contact domestic animals have with predators and the potential for animals to become infected through natural water or food sources.

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

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.

Molecular identification of Sarcocystis species in wild boar (Sus scrofa) and pigs (Sus scrofa domesticus) in Brazil

Sarcocystis spp. are protozoan parasites that form cysts in the organs and musculature of various animal species. The species Sarcocystis miescheriana and Sarcocystis suihominis are pathogenic to pigs and wild boars (Sus scrofa), acting as intermediate hosts, while humans are the definitive host for S. suihominis. To date, there have been no reports of the identification of these coccidian species in Sus scrofa in Brazil. Therefore, in this study, we conducted the first molecular identification of Sarcocystis species using PCR-RFLP and sequencing. A total of 210 samples were analyzed, of this total, 67 tested positive for Sarcocystis spp., representing 31.9% of the total samples assessed. Out of the total positive samples, 55 (82.1%) were identified as S. miescheriana and 8 (11.9%) as S. suihominis, a zoonotic species. Additionally, other species related to bovines, such as S. cruzi and zoonotic S. hominis, were detected in 3.0% of the samples, serving as contaminants in the pork products. The presence of S. suihominis in swine and wild boar samples is concerning due to the zoonotic risk and potential environmental contamination, as humans act as definitive hosts, also for the presence of S. hominis as a bovine contaminant in pork sausages. Furthermore, we confirmed the efficacy of the PCR-RFLP technique as a reliable tool for the identification of Sarcocystis species, demonstrating its potential use in laboratories for molecular diagnosis and rapid identification of these parasites, aiming to protect public health and ensure food safety.

First report of Sarcocystis falcatula in naturally infected Razorbill auks (Alca torda) collected in Tunisian Mediterranean Sea shores.

Sarcocystis spp. are apicomplexan cyst-forming parasites that can infect numerous vertebrates, including birds. Sarcosporidiosis infection was investigated in three muscles (breast, right and left thigh muscle) and one organ (heart) of four Razorbill auks (Alca torda) stranded between November and December 2022 on the shores of the Mediterranean Sea in Nabeul and Bizerte governorates, Northern Tunisia. Two of the four tested A. torda were PCR positive for 18S rRNA Sarcocystis spp. gene. Among the examined 16 muscles/organs, only one breast and one right thigh were Sarcocystis spp. PCR-positive (12.5% ± 8.3, 2/16). Our results showed a relatively high molecular prevalence of Sarcocystis spp. in Razorbill auks (A. torda). Sarcocystis spp. sequence described in the present study (GenBank number: OR516818) showed 99.56-100% identity to Sarcocystis falcatula. In conclusion, our results confirmed the infection of Razorbill auks (A. torda) by S. falcatula. Further research is needed on different migratory seabirds' species in order to identify other Sarcocystis species.

A cyst-forming coccidian with large geographical range infecting forest and commensal rodents: Sarcocystis muricoelognathis sp. nov.

BackgroundThe geographic distribution and host-parasite interaction networks of Sarcocystis spp. in small mammals in eastern Asia remain incompletely known.MethodsExperimental infections, morphological and molecular characterizations were used for discrimination of a new Sarcocystis species isolated from colubrid snakes and small mammals collected in Thailand, Borneo and China.ResultsWe identified a new species, Sarcocystis muricoelognathis sp. nov., that features a relatively wide geographic distribution and infects both commensal and forest-inhabiting intermediate hosts. Sarcocystis sporocysts collected from rat snakes (Coelognathus radiatus, C. flavolineatus) in Thailand induced development of sarcocysts in experimental SD rats showing a type 10a cyst wall ultrastructure that was identical with those found in Rattus norvegicus from China and the forest rat Maxomys whiteheadi in Borneo. Its cystozoites had equal sizes in all intermediate hosts and locations, while sporocysts and cystozoites were distinct from other Sarcocystis species. Partial 28S rRNA sequences of S. muricoelognathis from M. whiteheadi were largely identical to those from R. norvegicus in China but distinct from newly sequenced Sarcocystiszuoi. The phylogeny of the nuclear 18S rRNA gene placed S. muricoelognathis within the so-called S. zuoi complex, including Sarcocystisattenuati, S. kani, S. scandentiborneensis and S. zuoi, while the latter clustered with the new species. However, the phylogeny of the ITS1-region confirmed the distinction between S. muricoelognathis and S. zuoi. Moreover, all three gene trees suggested that an isolate previously addressed as S. zuoi from Thailand (KU341120) is conspecific with S. muricoelognathis. Partial mitochondrial cox1 sequences of S. muricoelognathis were almost identical with those from other members of the group suggesting a shared, recent ancestry. Additionally, we isolated two partial 28S rRNA Sarcocystis sequences from Low’s squirrel Sundasciurus lowii that clustered with those of S. scandentiborneensis from treeshews.ConclusionsOur results provide strong evidence of broad geographic distributions of rodent-associated Sarcocystis and host shifts between commensal and forest small mammal species, even if the known host associations remain likely only snapshots of the true associations.Graphical

First molecular identification and phylogenetic illustration of Sarcocystis species infection in Red Sea shortfin mako shark (Isurus oxyrinchus Rafinesque, 1810)

Backgroundmembers of the genus Sarcocystis are intracellular obligate protozoan parasites classified within the phylum Apicomplexa and have an obligate heteroxenous life cycle involving two hosts. A more comprehensive understanding of the prevalence and geographic range of different Sarcocystis species in marine ecosystems is needed globally and nationally. Hence, the objective of this study was to document the incidence of Sarcocystis infection in sharks within the aquarium ecosystem of Egypt and to identify the species through the characterization of the SSU rDNA gene.MethodsAll organs of the mako shark specimen underwent macroscopic screening to detect the existence of a Sarcocystis cyst. Ten cysts were collected from the intestine and processed separately to extract the genomic DNA. The polymerase chain reaction (PCR) was accomplished by amplifying a specific 18S ribosomal RNA (rRNA) gene fragment. Subsequently, the resulting amplicons were subjected to purification and sequencing processes.ResultsMacroscopic examination of the mako shark intestinal wall sample revealed the presence of Sarcocystis cysts of various sizes and shapes, and sequencing of the amplicons from Sarcocystis DNA revealed a 100% nucleotide identity with the sequence of Sarcocystis tenella recorded from sheep in Iran; The mako shark sequence has been deposited in the GeneBank with the accession number OQ721979. This study presents the first scientific evidence demonstrating the presence of the Sarcocystis parasite in sharks, thereby documenting this specific marine species as a novel intermediate host in the Sarcocystis life cycle.ConclusionsThis is the first identification of Sarcocystis infection in sharks, and we anticipate it will be an essential study for future screenings and establishing effective management measures for this disease in aquatic ecosystems.

Detection of Sarcocystis albifronsi, Eimeria alpacae, and Cystoisospora felis in Eurasian lynx (Lynx lynx) in northwestern China

Eurasian lynx (Lynx lynx) is widely distributed in various habitats in Asia and Europe, and it may harbor multiple pathogens. Currently, the information on protozoan infection in Eurasian lynx is scarce. In this study, we performed nested polymerase chain reaction (nPCR) analysis to detect intestinal protozoan infection in three dead Eurasian lynxes, in northwestern China. Three dead Eurasian lynxes, an adult female (#1), an adult male (#2), and a cub male (#3), were sampled in West Junggar Mountain, the northwestern region of Xinjiang Uyghur Autonomous Region. The intestine samples were analyzed using nPCR. We used primers targeting the cytochrome C oxidase subunit I gene (COI) for detection of Sarcocystis and Eimeria species and targeting the small subunit 18 S ribosomal RNA gene (18S rRNA) for detection of Cystoisospora species. The nPCR-positive products were sequenced, aligned, and phylogenetically analyzed. Three intestinal protozoa, Sarcocystis albifronsi, Eimeria alpacae, and Cystoisospora felis, were found in three Eurasian lynxes. The intestine sample of Eurasian lynx #2 was detected with S. albifronsi and E. alpacae. In addition, C. felis was only found in the intestine sample of Eurasian lynx #3. To the best of our knowledge, S. albifronsi and E. alpacae were detected in Eurasian lynx for the first time. In addition, C. felis was firstly found in Eurasian lynx in China. These findings extend our knowledge of the geographical distribution and host range of intestinal protozoa.

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.

The Distribution of Sarcocsytis Species Described by Ungulates-Canids Life Cycle in Intestines of Small Predators of the Family Mustelidae.

Using molecular techniques, we have previously shown that carnivorous mammals of the family Mustelidae might be common definitive hosts for various protozoan Sarcocystis species. In the present study we aimed to unravel whether Sarcocystis species using ungulates as intermediate hosts and canids or felids as definitive hosts can be found in intestine of mustelids. Small intestine samples of 93 individual mustelids of five different species from Lithuania were examined. Sarcocystis species were identified based on species-specific PCR and subsequent cox1 sequencing. Six Sarcocystis species (S. arieticanis, S. bertrami, S. capracanis, S. capreolicanis, S. linearis and S. morae) defined by ungulate-canid life cycle were detected for the first time in small intestines of mustelids. By contrast, the prevalence of Sarcocystis characterised by ungulate-felid life cycle was low (3.2%). Overall, 76% of the examined animals were positive for at least one of the studied Sarcocystis species. Four species, S. arieticanis, S. bertrami, S. capracanis and S. morae were most commonly found, with the detection rate of about 40%. The current finding, in addition to our previous studies, suggests that mustelids play an important role in the spread of various Sarcocystis species.

Identification of Sarcocystis spp. in Slaughtered Sheep from Spain and Evaluation of Bradyzoite Viability after Freezing.

Sarcocystis spp. are complex apicomplexan parasites that cause a substantial economic impact on livestock used for meat production. These parasites are present worldwide. Our study aimed to identify Sarcocystis species affecting sheep meat in southern-central Spain and to evaluate the effectiveness of freezing for parasite inactivation. A total of 210 condemned samples of sheep meat were thoroughly assessed grossly and microscopically; the presence of macro- and microcysts was confirmed. The samples were then frozen at -20 °C for various time intervals (24, 48, 72, 96, 120, and 144 h) and compared with untreated samples. Bradyzoites were isolated through pepsin digestion for subsequent molecular analysis and viability assessment, employing trypan blue and double fluorescence staining techniques. Our measurements confirmed the presence of S. tenella, S. gigantea, and S. medusiformis in Spanish domestic sheep. Freezing for 96 to 144 h resulted in a significant reduction in parasite viability, with a robust correlation observed between the two staining methods. Both stains effectively measured the viability of Sarcocystis, thereby promising future advances in meat safety.