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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Vicuñas (Vicugna vicugna) are wild South American camelids (SACs) protected by law in Argentina, and information on pathogens that infect them is scarce. In this study, an adult vicuña found dead in the province of Salta was examined, and evidence of infection by Sarcocystis sp. protozoans was sought. Infection of skeletal muscles by S. aucheniae, with the production of macroscopic sarcocysts, a disease known as SAC sarcocystosis, has been described in the other three SACs - llamas, alpacas, and guanacos - but its occurrence in vicuñas has so far remained unknown. In the analyzed individual, many macroscopic cysts compatible with S. aucheniae were found upon necropsy in the muscular tissue of the neck and diaphragm. Analysis of 18 S rRNA and cytochrome c oxidase subunit 1 (cox-1) gene sequences by BLAST searches and construction of phylogenetic trees demonstrated that the etiological agent was S. aucheniae. Our results show for the first time that vicuñas act as intermediate hosts in the life cycle of this parasite. In addition, this study provides the first cox-1 sequences for S. aucheniae isolates from the four SAC species acting as intermediate hosts and suggests that this marker could be useful for genotypification of this parasite species. The impact of SAC sarcocystosis on the health, well-being, and fitness of vicuñas, and the relevance of vicuña infections in the epidemiology of S. auchaniae, remain to be elucidated., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Sarcocystis spp. are cyst-forming coccidia characterized by a two-host predator-prey life cycle. Sarcocysts are formed in muscles or nervous system of the intermediate host, while sporocysts develop in the small intestine of the definitive host. The intermediate hosts of Sarcocystis falcatula are wild birds. Colombia is one of the countries with the greatest biodiversity of birds, however, there are few studies related to this parasite in wild birds. This study presents the morphological and molecular detection of Sarcocystis falcatula collected from the emerald toucanet (Aulacorhynchus albivitta), a wild bird species endemic to South America. Pectoral muscle samples were obtained, and microscopic and molecular detection was performed by light microscopy, transmission electron microscopy, and amplifying of the first internal transcribed spacer (ITS-1) and surface antigen-encoding genes (SAGs). Sarcocystis measured an average of 161  × 42 μm, with a cyst wall ∼0.4 μm thick. Ultrastructurally, the sarcocyst wall type 11b-like consisted of numerous villar protrusions of 850 nm wide on average. The ITS-1 sequence showed 97.0-99.7% identity to S. falcatula previously described from birds in the United States and Brazil, respectively. Concatenated phylogenetic analysis based on SAG2, SAG3 and SAG4 confirmed that the new isolate is grouped with other sequences of Sarcocystis from South America, but divergent from those isolates obtained in North America. The results of this study demonstrate for the first time the presence of S. falcatula in a wild bird from Colombia., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 × 10 5 copies/L for S. tenella and 6 × 10 4 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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Abstract: 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., (© American Society of Parasitologists 2024.)

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., Competing Interests: Declaration of competing interest The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript., (Copyright © 2024. Published by Elsevier B.V.)

Muscles of 25 bobcats (Lynx rufus) from remote areas of Mississippi in 2017 were tested for parasites. Testing for Sarcocystis infections included microscopic examination of fresh unstained muscle squashes, pepsin digestion of hearts and tongues, and histological sections of paraffin-embedded tissues. Sarcocystis spp. infections were detected in the muscles of 21 (84%) by a combination of methods. Sarcocysts were detected in the unstained tongue squashes of 2 bobcats. Sarcocystis sp. bradyzoites were detected in the pepsin digests of 3 of 19 hearts, and 12 of 19 tongues. In paraffin-embedded histological sections, sarcocysts were detected in 7 of 25 hearts, 17 of 25 tongues, and 5 of 23 limb muscles. Based on the character of the cyst wall, at least 3 morphologic types of sarcocysts were detected: those with small spikes on the cyst wall, corresponding to Sarcocystis felis, those with long villar protrusions, corresponding to Sarcocystis neurona, and those lacking visible cyst wall protrusions, representing an unidentified type of sarcocyst. Myositis associated with sarcocysts was seen in the tongues of 3, and in the limb muscles of 1 bobcat. Multilocus genotyping of the DNA extracted from paraffin-embedded sections from 2 bobcats, employing 18S, 28S, COI, ITS-1, and 5.8S and rpoB genes, diagnosed Sarcocystis caninum, S. felis, Sarcocystis lutrae, and S. neurona. An encapsulated species of Trichinella was identified in the tongue of 1; it represents the first documented occurrences in bobcats from Mississippi. Taken together, these observations suggest intensive exposure of these wild carnivores to Trichinella tissue cysts, implies predation or scavenging on these tissues promotes parasite transmission, and raises caution concerning zoonotic risk when such meat is rendered for human consumption., (© American Society of Parasitologists 2024.)

The genus Sarcocystis includes protozoan parasites with an indirect life cycle. Sarcocystis spp. can infect various animal species and humans, causing sarcocystosis, a parasitosis of economic importance and zoonotic concern. Wild boars can act as intermediate hosts for Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis that infects humans by consumption of raw or undercooked infected swine meat. In the present study, the diaphragmatic muscle tissue of 123 wild boars hunted in Greece was examined to determine the frequency of Sarcocystis spp. The samples were examined by tissue compression and molecular techniques. Under light microscopy, 34 out of 123 (27.6%) wild boars tested positive for Sarcocystis spp., while a higher infection prevalence (75%) was revealed by multiplex PCR performed in 100 of the samples. The partial mtDNA cox1 gene (~ 1100 bp) of 20 samples tested positive for S. miescheriana by multiplex PCR was amplified and sequenced. Sarcocystis miescheriana was identified as the only species involved in these infections. This is the first study on the prevalence of Sarcocystis spp. in wild animals in Greece. Further, large-scale surveys are needed to assess the prevalence and species of this parasite in Greece and to design efficient control and preventive measures in a One Health perspective., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In the present study, tissue samples (tongue, esophagus and heart) were investigated from dromedary camels of India for identification and characterization of Sarcocystis spp. using histopathology, PCR and gene sequencing. Genomic DNA extracted from these tissue samples was used for PCR amplification of the cytochrome c oxidase subunit I gene (cox1) of Sarcocystis spp. and the partial sequence of small subunit ribosomal RNA (18S rRNA) gene of the S. cameli. The PCR products were purified, sequenced and analyzed using bioinformatics tools. Based on phylogenetic analysis of the cox1 gene, the sequences of the present study clustered with those of S. cameli, hosted by dromedary camels of Iraq and a close association was observed with S. masoni hosted by dogs and alpacas of China. Until now, there are no 18S rRNA sequences of S. cameli available in GenBank and this is the first study recording 18S rRNA sequences of S. cameli which were grouped with S. masoni from alpaca of China and guanaco and llama of Argentina in phylogenetic analysis. These findings could be useful for further studies on the characterization through molecular epidemiology, genetic diversity and host specificity of S. cameli., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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 km 2 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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: 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., Methods: 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., Results: 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%., Conclusions: The current finding, in addition to our previous studies, suggests that mustelids play an important role in the spread of various Sarcocystis species., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Sarcocystis species are diverse apicomplexan parasites, though only two zoonotic species (S. hominis and S. heydorni) circulate between cattle and humans. Due to the importance of cattle in the human food chain and to prevent the consequences of parasitosis in humans, the first global systematic review and meta-analysis on molecular epidemiology, species distribution, and zoonotic significance of Sarcocystis infection in cattle was performed., Methods: For this aim, four international English databases (PubMed, Scopus, Google Scholar, and Web of Science) were systematically searched till 20th September 2021, and random-effect models were drawn to calculate total estimates and their 95% confidence intervals (CIs)., Results: Finally, 44 papers from 21 countries were qualified for this review which examined 8526 cattle regarding Sarcocystis infection, rendering a total prevalence of 62.7% (95% CI 53-71.5%). Globally, 12 Sarcocystis spp. have been reported from cattle, including S. cruzi, S. hominis, S. hirsuta, S. rommeli, S. heydorni, S. bovifelis, S. bovini, S. sinensis, S. gigantea, S. fusiformis, S. hjorti and S. tenella. Among them, S. cruzi (37 studies), S. hominis (22 studies) and S. hirsuta (19 studies) were the 3 most common species, with 76.4% (95% CI 64.8-85%), 30.2% (95% CI 19.3-44%) and 8.7% (95% CI 3.8-18.6%), respectively. However, molecular identification was not performed in 48.4% (95% CI 27.3-70.1%) of the positive samples., Conclusion: Despite the zoonotic significance of Sarcocystis spp., particularly S. hominis, the epidemiology and distribution of Sarcocystis infection in cattle remains unclear and demands more extensive researches around the world., (© 2022. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Background: Data on the genus Sarcocystis in insectivores are limited. The Asian gray shrew Crocidura attenuata is one of the most common species of the insectivore family Soricidae in South Asia and Southeast Asia. To our knowledge, species of Sarcocystis have never been recorded previously in this host., Methods: Tissues were obtained from 42 Asian gray shrews caught in 2017 and 2018 in China. Sarcocysts were observed using light microscopy (LM) and transmission electron microscopy (TEM). To describe the parasite life cycle, muscle tissues of the host infected with sarcocysts were force-fed to two beauty rat snakes Elaphe taeniura. Individual sarcocysts from different Asian gray shrews, and oocysts/sporocysts isolated from the small intestines and feces of the experimental snakes, were selected for DNA extraction, and seven genetic markers, namely, two nuclear loci [18S ribosomal DNA (18S rDNA) and internal transcribed spacer region 1 (ITS1)], three mitochondrial genes [cytochrome oxidase subunit 1 (cox1), cox3 and cytochrome b], and two apicoplast genes (RNA polymerase beta subunit and caseinolytic protease C), were amplified, sequenced and analyzed., Results: Sarcocysts were found in 17 of the 42 (40.5%) Asian gray shrews. Under LM, the microscopic sarcocysts showed saw- or tooth-like protrusions measuring 3.3-4.5 μm. Ultrastructurally, the sarcocyst wall contained numerous lancet- or leaf-like villous protrusions, similar to those described for type 9h of the common cyst wall classification. The experimental beauty rat snakes shed oocysts/sporocysts measuring 11.9-16.7 × 9.2-10.6 μm with a prepatent period of 10-11 days. Comparison of the newly obtained sequences with those previously deposited in GenBank revealed that those of 18S rDNA and cox1 were most similar to those of Sarcocystis scandentiborneensis recorded in the tree shrews Tupaia minor and Tupaia tana (i.e., 97.6-98.3% and 100% identity, respectively). Phylogenetic analysis based on 18S rDNA or ITS1 sequences placed this parasite close to Sarcocystis spp. that utilize small animals as intermediate hosts and snakes as the known or presumed definitive host. On the basis of morphological and molecular characteristics and host specificity, the parasite was proposed as a new species, named Sarcocystis attenuati., Conclusions: Sarcocysts were recorded in Asian gray shrews, to our knowledge for the first time. Based on morphological and molecular characterization, a new species of parasite is proposed: Sarcocystis attenuati. According to the LM and TEM results, S. attenuati sarcocysts are distinct from those of Sarcocystis spp. in other insectivores and those of S. scandentiborneensis in tree shrews. The 18S rDNA or cox1 sequences of Sarcocystis attenuati shared high similarity with those of Sarcocystis scandentiborneensis, Sarcocystis zuoi, Sarcocystis cf. zuoi in the Malayan field rat, and Sarcocystis sp. in the greater white-toothed shrew. Therefore, we suggest that more research on the relationships of these closely related taxa should be undertaken in the future., (© 2021. The Author(s).)

Background: Members of the genus Sarcocystis are protozoan parasites characterized by a prey-predator two-host life-cycle. Sarcocysts are formed in the muscles or central nervous system of the intermediate host (IH), while sporocysts develop in the small intestine of the definitive host (DH). Various birds of prey have been confirmed to be DH for Sarcocystis spp. Three Sarcocystis species, S. wobeseri, S. halieti and S. falcatula, have been identified in the muscles of birds of prey, of which the latter are known to be pathogenic and can cause encephalitis in various birds. The aim of this study was to identify Sarcocystis spp. in the muscles of birds of prey from Spain., Methods: Between 2019 and 2020, muscle tissue samples taken from 59 birds of prey admitted to the Wildlife Recovery Centre in Ilundain (Navarra, Spain) were examined for the presence of Sarcocystis spp. Sarcocysts in fresh squashed samples were morphologically characterized under the light microscope (LM). Sarcocystis spp. were identified by means of 28S ribosomal RNA and internal transcribed spacer 1 sequence analysis., Results: Microscopic examination of squashed tissue samples stained with methylene blue revealed the presence of sarcocysts in three of the 59 (5.1%) birds examined. Only one sarcocyst type was observed under the LM. Sarcocysts were thread-like (1050-2160 × 130-158 μm) and had a thin (0.7-1.4 μm) and smooth cyst wall. Septa divided the cysts into compartments filled with banana-shaped (5.9 × 1.7 μm) bradyzoites. On the basis of DNA sequence results, S. halieti was identified in the western marsh harrier (Circus aeruginosus) and the black kite (Milvus migrans) for the first time. Sarcocysts of S. halieti were shorter and wider compared to those observed in the great cormorant (Phalacrocorax carbo) and the herring gull (Larus argentatus). According to current knowledge, S. halieti may infect birds belonging to four different orders: Suliformes, Charadriiformes, Strigiformes and Accipitriformes., Conclusions: This is the first report of S. halieti in the western marsh harrier and the black kite as IH. So far, little research has been conducted on birds of prey as IH for Sarcocystis spp. These results indicate that further studies combining morphological, histopathological, and molecular methods are required., (© 2021. The Author(s).)

Previous morphological studies suggested that mouflon may have sarcocysts similar to those of sheep. However, to date, no molecular-based studies of the species of Sarcocystis infecting mouflon have been done. The present study identified Sarcocystis species in diaphragm muscle samples from 20 European mouflon (Ovis gmelini musimon). Molecular identification using the cox1 sequence analysis was performed on sarcocysts excised from muscle tissue and on DNA from digested muscle samples. Both frequency and intensity of infection in mouflon were high with 19 of 20 animals testing Sarcocystis positive and > 50 cysts per gram of tissue recovered from 10 of the 19 Sarcocystis positive animals. Molecular analysis revealed dominant Sarcocystis tenella (18/19 animals) and Sarcocystis arieticanis (1/19 animals), whose known intermediate hosts are sheep. In addition, Sarcocystis capracanis, which is known to form sarcocysts in goats, was detected in two animals. The results of this study demonstrated the digestion method to be superior over the direct isolation of sarcocysts for the molecular identification of Sarcocystis species in a certain host. Future research of Sarcocystis diversity in wild ovine and caprine species is needed.

Background: Sarcocystis species are obligatorily heteroxenous parasites, of which some are zoonotic, representing a public health and economic impact. This study investigated the occurrence of Sarcocystis spp. in cattle sampled from a Belgian slaughterhouse., Methods: A total of 200 carcasses were included in the study, sampled during 10 sampling days. The sedimentation method was applied to isolate the sarcocysts from both heart and diaphragm muscles collected from each carcass. Multiplex PCR, PCR-RFLP as well as cox1 gene sequencing techniques were applied serially on collected sarcocysts for species identification., Results: Sarcocystis spp. were detected in 64% (128/200; 95% CI 57-71%) of the sampled carcasses. Female dairy cattle presented the highest Sarcocystis occurrence rate (91%) as well as the highest Sarcocystis species diversity compared to female beef and male beef. Sarcocystis spp. were detected more often in the heart muscles than in the diaphragm among female beef (p < 0.001) and dairy carcasses (p = 0.001), while in male carcasses no significant difference was observed (p = 0.763). The effect of age was not significant in male carcasses (p = 0.872), while the odds of finding sarcocysts significantly increased with age (p = 0.003) within both types of female carcasses. S. cruzi was the most prevalent species and was found in 56.5% (113/200) of the carcasses, followed by S. hominis (21.0%, 42/200), S. bovifelis (12.5%, 25/200), S. bovini (2.0%, 4/200), S. hirsuta (1.5%, 3/200) and S. heydorni (0.5%, 1/200). Six different species were detected in the diaphragm, while only two species were recovered from the heart. S. cruzi was the most prevalent species in heart, while in the diaphragm, this was S. hominis., Conclusions: The detection of S. hominis in 21% of the sampled carcasses presents a potential food safety issue, and further research is warranted into controlling this infection.

Background: Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis., Methods: To reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification., Results: Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses., Conclusions: Our study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis.

In a survey study on the macroscopic species of Sarcocystis infecting domestic sheep (Ovis aries) and cattle (Bos taurus) in Egypt, the macrosarcocysts of Sarcocystis gigantea and Sarcocystis medusiformis were detected in the carcasses of 33 domestic sheep out of a total of 250 (13.20%), whereas Sarcocystis hirsuta macrosarcocysts were found in 17 out of 150 cattle (11.33%) slaughtered at the municipal abattoirs of two different provinces in Egypt. The sarcocysts of each species were thoroughly described morphologically through gross inspection, histopathologic and transmission electron microscopic (TEM) examination. By TEM, S. gigantea primary cyst wall was 6-7.5 μm thick and had irregular highly branched cauliflower-like villar protrusions (VP).The VP contained microtubules (mt) and multiple electron dense granules (edg) that were dispersed inside the cores of the branched VP. Besides, the parasitophorous vacuolar membrane (PVM) had minute blister-like invaginations all over the entire surface of the sarcocyst. S. medusiformis cyst had a thin sarcocyst wall (~2 μm thick) as compared to that of S. gigantea. The cyst wall had trapezoidal or nearly pyramidal VP that were surrounded by thick PVM in addition to a ground substance GS that contained electron-dense fine particles. S. hirsuta sarcocyst wall was 7-9 μm thick and possessed rhomboid-shaped VP that contained microtubules (mt) and electron-dense granules (edg) of variable sizes. The edg were arranged in rows and running parallel to the longitudinal axis of the protrusions. The VP had characteristic narrow neck-like constrictions at their bases, dilated middle portions, and tapered distal ends. The detected macrosarcocysts were eventually confirmed by molecular characterization on the levels of 18S rRNA, 28S rRNA, and Cox1 sequences. Phylogenetic analyses based on the sequences of the 18S rRNA and Cox1 genetic markers gave rise to robust associations of the currently identified isolates of S. gigantea, S. medusiformis, and S. hirsuta within a major clade of Sarcocystis species with felines as presumed or known definitive hosts.

Background: Species of Sarcocystis are parasitic protozoa in poikilothermic and homeothermic animals. Out of the 26 valid species in birds as intermediate hosts, none has been reported in those of the order Musophagiformes, such as the great blue turaco Corythaeola cristata (Vieillot, 1816), which is a bird endemic to Central and Western Africa. The examination of great blue turacos imported from the Central Africa Republic to Czech Republic allowed the morphological and molecular characterization of a new species of Sarcocystis., Methods: Four turacos imported from the Central Africa Republic to a private breeder (Czech Republic) underwent parasitological examination for the presence of sarcocysts through wet mounts of breast, heart and leg muscles. Found parasites were molecularly and histologically studied by four loci (18S rRNA, 28S rRNA, ITS1 and cox1) and haematoxylin and eosin staining, respectively., Results: Three out of four examined birds harboured numerous sarcocysts in the breast and leg muscles. No macroscopic lesions where observed. Sarcocysts were microscopic, elongate and ribbon-shaped with a wall characterised by the presence of finger-shaped villar protrusions and filled with numerous elongate, banana-shaped bradyzoites, 11.87-14.84 × 2.05-2.92 µm in size. The new species was most closely related to Sarcocystis albifronsi, Sarcocystis anasi, Sarcocystis atraii, Sarcocystis chloropusae, Sarcocystis rileyi, Sarcocystis wenzeli and Sarcocystis sp. isolate from chicken in the four loci., Conclusions: To our knowledge, this is the first species of Sarcocystis found in a musophagiform bird worldwide. Genetically, S. cristata sp. nov. represents a distinct species. Phylogenetic analyses are useful for predicting potential definitive hosts of the new Sarcocystis species.

Background: Six Sarcocystis species are known to use cattle (Bos taurus) as the intermediate host, two of which, S. hominis and S. heydorni, are zoonotic. There is a need for a method that will enable rapid identification of the Sarcocystis species in cattle., Methods: The diaphragm muscles of 102 cattle from Lithuania were examined for the presence of Sarcocystis spp., using two different methods for species identification. Individual sarcocysts were isolated from squash preparations of the diaphragm muscle under the light microscope, followed by genetic characterisation of excised cysts using sequence analysis of the 18S rRNA (18S rRNA) and cytochrome c oxidase subunit I (cox1) genes. The same cattle muscle samples were digested and species-specific PCR analyses targeting cox1 were developed to identify the Sarcocystis isolates to the species level., Results: Under the light microscope, sarcocysts were detected in 87.3% of animals, and Sarcocystis infection was verified in all digested samples. Three species, namely S. cruzi (n = 20), S. bovifelis (n = 23) and S. hirsuta (n = 6), were identified by DNA sequence analysis of isolated sarcocysts. Based on sequence analysis of cox1, the level of genetic variability depended on Sarcocystis species and geographical location. Four Sarcocystis species, S. cruzi (96.1%), S. bovifelis (71.6%), S. hirsuta (30.4%) and S. hominis (13.7%), were confirmed in the digested samples. In individual samples, the most common finding was two species of Sarcocystis (44.1%), followed by three species (26.5%), a single species (24.5%) and four species (4.9%)., Conclusions: Although examination of tissue preparations under the light microscrope did not detect any sarcocysts belonging to S. hominis, this species was identified in the digested samples subjected to a cox1-specific PCR analysis. These results demonstrate the need for effective molecular diagnosis techniques to detect Sarcocystis spp., which may be present at a lower prevalence and not detectable among the limited number of sarcocysts identified individually under the light microscope.

Until now, two Sarcocystis species, S. cornixi and S. corvusi, were known to employ members of the family Corvidae as intermediate hosts. Between 2013 and 2019, having examined leg muscles of 23 common ravens in Lithuania, sarcocysts were detected in 18 birds (78.3%). Using light microscopy, transmission electron microscopy (TEM), and molecular analysis (three genetic loci, 18S rDNA, 28S rDNA, and ITS1), sarcocysts found in the common raven were described as a new species S. kutkienae. Under a light microscope, the observed sarcocysts were ribbon-shaped (1500-8147 × 53-79 μm) and had a wavy striated cyst wall that reached up to 1.5 μm. Lancet-shaped bradyzoites were 7.7 × 2.2 μm (6.1-9.0 × 1.2-3.0 μm) in size. Ultrastructurally, the sarcocyst wall was 1.5-1.8 μm in thickness and had conical-like protrusions with minute invaginations of a parasitophorous vacuolar membrane. The cyst wall was type 1e-like. Limited genetic variability was observed between the 18S rDNA and 28S rDNA sequences of S. kutkienae and other Sarcocystis spp. using birds as intermediate hosts. In contrast, S. kutkienae could be clearly identified by comparing sequences. At this locus, sequences of S. kutkienae shared the highest similarity (89.5-89.7%) with those of S. cornixi. Phylogenetic analysis showed that S. kutkienae was most closely related to Sarcocystis spp. that employs birds as intermediate and definitive hosts. The issue relating to which species might serve as definitive hosts of S. kutkienae in Lithuania is addressed.

In the present study, sarcocysts of Sarcocystis cymruensis were found in four of 42 (9.5%) Norway rats and those of S. ratti were observed in six of 60 (10%) black rats in China. With light microscopy, the sarcocysts of the two parasites were microscopic, and had smooth, thin cyst walls (≤ 1 μm). Ultrastructurally, the sarcocysts of S. cymruensis had small, osmiophilic, bleb-like protrusions, similar to type 1c; those of S. ratti had a cyst wall with regular, short, conical protrusions, similar to type 1 g. Three loci, i.e., 18S rDNA, the mitochondrial cox1 gene (Cox1), and the mitochondrial Cytb gene (Cytb), of the two parasites were sequenced and analyzed, and the Cytb sequences of the two parasites constituted the first records of this marker in GenBank. A comparison of the newly obtained sequences of the three loci between the two parasites revealed that the interspecific similarities of 18S rDNA, Cox1, and Cytb were 96.4-97.2%, 96.5%, and 93.7%, respectively. Therefore, the two species could be better discriminated with Cytb than with 18S rDNA and Cox1. Phylogenetic analysis based on 18S rDNA sequences and Cox1 sequences indicated that the two parasites had a close relationship with Sarcocystis in nonruminant animals, especially birds and canids.

Various muscle samples of wild boar (Sus scrofa) from Latvia were studied for the presence of Sarcocystis infection by means of morphological and molecular methods. Sarcocysts were detected in 122 out of 140 (87.1%) wild boar examined. According to the morphological appearance of sarcocysts, the observed cysts belonged to one morphological type and resembled Sarcocystis miescheriana. Twenty-three sarcocysts isolated from the muscles of Latvian wild boars were molecularly characterized at 18S rRNA, ITS1 and cox1. Additionally, eight sarcocysts obtained from Lithuanian wild boars were subjected to molecular analysis in order to compare intraspecific genetic variability. The amplified 18S rRNA region using newly designed primers is sufficiently variable to separate S. miecheriana from S. suihominis. All Latvian and Lithuanian isolates were confirmed belonging to S. miescheriana. No genetic variation was detected within 18S rRNA and ITS1. By contrast, the high intraspecific genetic variability of S. miescheriana was observed within cox1 since each newly obtained sequence represented a unique haplotype. The comparison made using S. miescheriana isolates from Italian and Japanese wild boar and Chinese domestic pig revealed the genetic similarity of the samples depending on their geographical distances. The current study provides the first detection of Sarcocystis infection in wild boars from Latvia and molecular characterization of S. miescheriana.

Sarcocystis spp. are intracellular protozoan parasites with heteroxenous life cycles. This study described Sarcocystis spp. infection in adult South American native deer huemul (Hippocamelus bisulcus) and pudu (Pudu puda). Heart, diaphragm, tongue, and skeletal muscle samples were collected from 5 huemuls and 2 pudus, found dead in National Parks. Direct microscopic examination, transmission electron microscopy, PCR, and sequencing were performed. Sarcocystis spp. microscopic thin-walled cysts were identified in 3 huemuls and 1 pudu. Several cysts from 1 huemul and 1 pudu were observed by TEM; ultrastructure was similar to previously reported as cyst wall type 17 and types 2 and 8, respectively. Fragments of the 18S rRNA and cytochrome c oxidase subunit I (cox1) genes were amplified and sequenced from 3 individual cysts from 2 huemuls and 2 cysts from the pudu. The sequences from huemuls showed a high identity among them (> 99%) at both amplified targets. The highest identities were > 99.7% at 18S rRNA and 93% at cox1 with S. tarandivulpes sequences. The 18S rRNA gene sequences from pudus showed an identity > 99.7% with Sarcocystis sp., S. taeniata, and S. linearis sequences, while the cox1 sequences were different, one showing 99.42% identity with S. venatoria and the other 98.22% with S. linearis. A single species, similar to S. tarandivulpes, was identified in all huemul samples while 2 molecularly different Sarcocystis spp. were found in 1 pudu with high similarities to either S. venatoria or to S. linearis, S. taeniata-like, and S. morae. Based on the cox1 sequence identities, at least the Sarcocystis sp. in huemuls might represent a new species, primarily occurring in this host. Additional sarcocyst isolates from both hosts need to be examined molecularly in order to firmly establish whether these species are indeed native to huemuls and/or pudus or are derived from introduced deer species.

Background: There has been considerable confusion concerning the number and classification of Sarcocystis spp. in chickens. Scarce nucleotide data of Sarcocystis spp. from chickens are provided in GenBank. The study aimed to investigate the morphological and molecular characteristics of Sarcocystis spp. found in chickens in China., Methods: Tissues from 33 chickens were collected in 2019. Sarcocysts were observed using light (LM) and transmission electron microscopy (TEM). Individual sarcocysts from different chickens were selected for DNA extraction, and five loci, 18S rDNA, 28S rDNA, ITS1 region, the mitochondrial cox1 gene and the apicoplastic rpoB gene, were amplified from each sarcocyst, sequenced and analyzed., Results: Only S. wenzeli was found in 14 of 33 (42.4%) chickens. Under LM, the sarcocysts were microscopic and exhibited palisade-like villar protrusions measuring 1.5-2.8 μm. Ultrastructurally, the sarcocyst wall contained numerous stubby hill-like villar protrusions. The protrusions included scattered microtubules, which extended from the tips of the protrusions into the ground substance. The five loci were successfully sequenced and the sequences deposited in GenBank. At 18S rDNA, ITS1 and cox1, the most similar sequences in GenBank were those of Sarcocystis sp. obtained from the brains of chickens, i.e. 99.9-100%, 98.1-98.5% and 99.3% identity, respectively. The five loci (18S rDNA, 28S rDNA, ITS1, cox1 and rpoB) showed different levels of interspecific sequence similarity with other closely related species of Sarcocystis (e.g. 99.8%, 99.0-99.2%, 89.3-89.7%, 98.5%, and 97.5%, respectively, with S. anasi). Phylogenetic analysis based on four of the loci (18S rDNA, cox1, rpoB and ITS1) revealed that S. wenzeli formed an independent clade with Sarcocystis spp. that utilize geese or ducks as intermediate hosts and canines as the known or presumed definitive host., Conclusions: To our knowledge, the sequences of 28S rDNA and rpoB reported here constitute the first records of genetic markers of Sarcocystis spp. in chickens. Based on molecular analysis, S. wenzeli might be responsible for the neurological disease in chickens, and ITS1 and rpoB are more suitable for discriminating it from closely related Sarcocystis spp. Phylogenetic analysis revealed that S. wenzeli presents a close relationship with Sarcocystis spp. in geese or ducks.

A total of 57 tissue samples of domestic pigs (Sus scrofa) were collected from the meat outlets of five north Indian states and examined for sarcocystosis by histological and molecular methods. The genomic DNA extracted from five representative positive isolates was subjected to PCR amplification of the partial 18S rRNA gene followed by cloning and sequencing. Sequence analysis of the newly generated Indian isolates recorded 96.9-100.0% identity with published sequences of Sarcocystis suihominis. Two new haplotypes that have not been previously described manifested 99.5-100.0% nucleotide homology within themselves. In the phylogenetic analysis, Indian isolates of S. suihominis grouped together with S. suihominis originating from Italy, and they collectively formed a sister clade with Sarcocystis miescheriana within a clade containing various Sarcocystis spp. of ruminants having felids as final hosts. At the same time, this clade separated from a sister clade containing Sarcocystis spp. of bovid or cervid ruminants using canids as known or surmised definitive host. The current study established the phylogenetic relationship of Indian isolates of S. suihominis with various Sarcocystis spp. as well as with other taxa of Sarcocystidae family based on 18S rRNA gene for the first time.

Sarcocystosis is a parasitic disease caused by an intracellular protozoan parasite Sarcocystis belonging to the phylum Apicomplexa. These parasites have a requisite two-host life cycle. Recently, there are many Sarcocystis species that identified morphologically. In the present study, diaphragmatic muscle samples from the domestic horse (Equus caballus) were examined for Sarcocystis infection. The natural infection with sarcocysts was recorded to be 62·5% for only microcysts in the infected muscles. Molecular analysis using the 18S rRNA gene was conducted to swiftly and accurately identify the recovered species. Studies on the expression of the 18S rRNA gene have confirmed that the present parasite isolates belong to the Sarcocystis genus. The sequence data showed significant identities (>80%) with archived gene sequences from species within the Sarcocystidae family, and a dendrogram showing the phylogenetic relationship was constructed. The most closely related species were the previously described Sarcocystis fayeri and Sarcocystis bertrami. The current data showed that the present species was identified as S. fayeri and deposited in GenBank (accession number MF614956.1). This study highlights the importance of the genetic data in the exact taxonomy within sarcocystid species., (© 2020 The Society for Applied Microbiology.)

The first study reporting the morphological characterization of Sarcocystis sybillensis was performed in 1983; however, without any molecular analysis. Sarcocystis nipponi has been recently described as a species synonymic to S. sybillensis. We reconfirmed the presence of S. sybillensis in Japanese sika deer (Cervus nippon) captured in its native territory; and performed its molecular and phylogenetic characterization. The morphological characteristics of the sarcocysts were consistent with those of S. nipponi and S. sybillensis described in the first report. However, the nucleotide sequence of 18S rRNA gene of S. sybillensis showed only 91.9% identity to that of S. nipponi, suggesting low homology among the concerned Sarcocystis spp. Accordingly, S. sybillensis was found to occupy a clade distinct from that of S. nipponi in a phylogenetic tree of Sarcocystis. Therefore, the present study provides essential information on 18S rRNA-based molecular characterization of S. sybillensis and disproves the existing notion of morphology-based species synonymity of S. sibillensis and S. nipponi. These results also suggest that S. sybillensis belongs to type 2 Sarcocystis.

Purpose: Sarcocystis spp. are protozoan parasites of livestock which also infect birds, lower vertebrates and mammals, including man. Wild and domestic ruminants such as red deer, roe deer, fallow deer, cattle, sheep and goats may act as intermediate hosts for many Sarcocystis species, some of which are significant pathogens causing sarcocystosis in livestock and humans. The purpose of the present study was to determine the prevalence of Sarcocystis species in fallow deer farmed in an open pasture system., Methods: Samples of heart and oesophagus tissue taken from five fallow deer were examined by light microscope for the presence of sarcocysts. Genomic DNA was extracted from individual sarcocysts. ssu rRNA was successfully amplified using their DNA as templates., Results: Analysis of the ssu rRNA identified the presence of two S. morae sarcocysts in the heart tissue; similarly, S. gracilis sarcocysts were identified in the heart and oesophagus, and Sarcocystis sp. most closely related to S. linearis and S. taeniata were detected in oseophagus., Conclusions: These findings confirm the presence of Sarcocystis spp. in farmed fallow deer in Poland; however, more molecular studies are needed.

Background: Apicomplexan parasites of the genus Sarcocystis have an obligate two-host life-cycle and comprise about 200 species, which infect different cold- and warm-blooded hosts, including humans. Recently, morphological and molecular studies of sarcocysts in broadly spread carnivore hosts have been on the rise. The description of muscular tissues infection by Sarcocystis in the raccoon dog and the common raccoon from the Czech Republic is herein presented., Methods: During January-August 2019, 15 raccoon dogs and 1 common raccoon were examined from 5 districts (Česká Lípa, Liberec, Mladá Boleslav, Trutnov and Ústí nad Labem) of the Czech Republic. Muscle parts (diaphragm, forearm, hind-limb, tongue and heart) were examined in wet preparations under compression by light microscopy. After finding Sarcocystis sp., morphological characteristics and molecular analyses of 18S rRNA, 28S rRNA, ITS1 and cox1 loci were used to identify the species., Results: Sarcocysts were detected and identified in 1 out of 15 raccoon dogs and in the single common raccoon. Preferential infection sites were diaphragm and tongue, followed by forearm and hind limb. To our knowledge, this is the first identification of microscopic sarcocysts by multi-locus genetic analysis from both host species. Molecular analyses revealed 100% similarity at 18S rRNA, 28S rRNA, and cox1 genes with S. lutrae for both hosts and 98-100% identity at the ITS1 region of the isolate from the common raccoon., Conclusions: Both widely distributed non-indigenous wild carnivores represent new intermediate host records for S. lutrae and the first report of this parasite in a member of the family Procyonidae, but still with an unknown natural definitive host. Molecular data revealed that this parasite species appears more closely related to the Sarcocystis spp. using raptorial birds as definitive hosts. Therefore, further studies aimed at its identification, including the complete life-cycle, remain necessary.

The roe deer (Capreolus capreolus) has been identified as an intermediate host for six known Sarcocystis species, S. capreolicanis, S. entzerothi, S. gracilis, S. linearis, S. oviformis, and S. silva. In this study, we identified Sarcocystis species in the diaphragm and tongue muscles from the Lithuanian and Spanish roe deer, respectively, on the basis of a microscopic examination and DNA analysis. A total of 43 and 27 sarcocysts were isolated and characterized from the Lithuanian and Spanish roe deer, respectively. Overall six Sarcocystis species were identified in roe deer from Lithuania, and only three of them, S. gracilis, S. linearis, and S. silva were found to have infecting animals from Spain. The current paper represents first molecular results of Sarcocystis species in the Spanish roe deer. Furthermore, transmission electron microscopy examination revealed specific wall structure of sarcocysts studied, S. linearis was characterized by ribbon-like villar protrusions (vp) (type 8a), and S. oviformis was distinguished by elongated vp resembling spades or mushroom-like structures (type 39). Based on 18S rDNA and cox1 sequences, Sarcocystis species from the roe deer showed considerable intraspecific genetic variability. However, similar values of intraspecific genetic variation were estimated at both genes analysed. The highest variability was observed for S. capreolicanis and S. linearis in both genes and for S. silva at cox1. Consequently, the level of genetic variability of Sarcocystis from the roe deer varied depending on species rather than on gene analysed or geographical area.

Sarcocystosis is a protozoal disease affecting a wide range of animals. The aims of this study were to characterize the following in sheep: (1) the muscle pathology in Sarcocystis infection, (2) the inflammatory infiltrate and its relationship to severity of infection, and (3) immune markers expressed by parasitized muscle fibers and parasitic cysts. Skeletal muscle samples from 78 sheep slaughtered in southern Italy were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Polymerase chain reaction (PCR) and sequencing were used for Sarcocystis species identification. All 40 muscle samples tested were PCR-positive for Sarcocystis tenella. Histologically, cysts were identified in 76/78 cases (97%), associated with an endomysial infiltrate of lymphocytes and plasma cells. The T cells were predominantly CD8+, with fewer CD4+ or CD79α+ cells. Eosinophils were absent. Notably, sarcolemmal immunopositivity for major histocompatibility complex (MHC) I and II was found in 76/78 cases (97%) and 75/78 cases (96%), respectively, both in samples with and in those without evident inflammatory infiltrate. The number of cysts was positively correlated with inflammation. In addition, MHC I was detected in 55/78 cyst walls (72%), and occasionally co-localized with the membrane-associated protein dystrophin. The findings suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II. The possible role of MHC I and II in the inflammatory response and on the cyst wall is also discussed.

Background: Reports from UK hunters of 'rice grains' in muscles of shot wildfowl (Anatidae) coincided temporally with the finding of sarcocystosis in a number of ducks found as part of the Wildfowl & Wetlands Trust long-term general surveillance of found dead waterbirds. Sarcocystis rileyi has also been relatively recently confirmed in wildfowl in north-eastern Europe., Methods: This study uses four approaches to investigate UK wildfowl sarcocystosis: first, through a hunter questionnaire that captured historical case data; secondly, through an online reporting system; thirdly, DNA sequencing to characterise UK cases; and fourthly, histological myopathy assessment of infected pectoral muscle., Results: Our questionnaire results suggest Sarcocystis infection is widely distributed throughout the UK and observed in 10 Anatidae species, reported cases increased since the 2010/2011 shooting season, with the online reporting system reflecting this increase. DNA sequencing (18S rRNA gene and internal transcribed spacer-1 region) of UK isolates confirmed S rileyi in the five dabbling duck host species tested and the associated histopathological myopathy is described., Conclusion: This work highlights an emerging issue to European wildfowl species and provides much opportunity for further research, including the impacts of S rileyi and the described myopathy on host health, fitness and survival., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Birds of the family Laridae have not been intensively examined for infections with Sarcocystis spp. To date, sarcocysts of two species, S. lari and S. wobeseri, have been identified in the muscles of gulls. The aim of the present study was to evaluate the species richness of Sarcocystis in the herring gull, Larus argentatus, from Lithuania., Methods: In the period between 2013 and 2019, leg muscles of 35 herring gulls were examined for sarcocysts of Sarcocystis spp. Sarcocystis spp. were characterised morphologically based on a light microscopy study. Four sarcocysts isolated from the muscles of each infected bird were subjected to further molecular examination. Sarcocystis species were identified by means of ITS1 sequence analysis., Results: Sarcocysts were detected in 9/35 herring gulls (25.7%). Using light microscopy, one morphological type of sarcocysts was observed. Sarcocysts were microscopic, thread-like, had a smooth and thin (about 1 µm) cyst wall and were filled with banana-shaped bradyzoites. On the basis of ITS1 sequences, four Sarcocystis species, S. columbae, S. halieti, S. lari and S. wobeseri, were identified. Furthermore, it was demonstrated that a single infected herring gull could host two Sarcocystis species indistinguishable under light microscopy., Conclusions: Larus argentatus is the first bird species found to act as intermediate host of four Sarcocystis spp. According to current knowledge, five species, S. falcatula, S. calchasi, S. wobeseri, S. columbae and S. halieti can use birds belonging to different orders as intermediate hosts.

The major aim of the present study was to determine by molecular methods whether the wide and narrow types of macroscopic sarcocysts in Spanish sheep belonged to different species, that is, Sarcocystis gigantea and Sarcocystis medusiformis, respectively. Additionally, we wanted to identify and characterize molecularly the species forming microscopic sarcocysts and determine the phylogenetic placement of all species found. Portions of the oesophagus, diaphragm and hind legs containing macroscopic sarcocysts were collected from slaughtered culled ewes at an abattoir in the Province of Madrid, Central Spain, but both macroscopic and microscopic sarcocysts were isolated for molecular examination. Genomic DNA from 63 sarcocysts (21 macroscopic, 42 microscopic) were examined at the cytochrome c oxidase subunit I gene (cox1), while selected isolates of each species found were further examined at the 18S and 28S ribosomal RNA (rRNA) genes. The 63 sarcocysts comprised five cox1 sequence types, each corresponding to a particular sarcocyst type, and thus represented five Sarcocystis spp. The slender fusiform and thick macrocysts belonged to S. medusiformis and S. gigantea, respectively. The microscopic sarcocysts belonged to Sarcocystis arieticanis, Sarcocystis tenella and a Sarcocystis mihoensis-like species with slanting thorn-like cyst wall protrusions, which was characterised molecularly for the first time. Based on its phylogenetic position, the S. mihoensis-like species probably uses corvids as definitive hosts.

Three cyst-forming Sarcocystis species have been identified in horsemeat; however, there exists considerable confusion concerning their relationships. Here, 74% (34/46) of the examined tissue samples from horses contained sarcocysts based on examination by light microscopy (LM), and the organism was identified as Sarcocystis bertrami based on cyst structure. The S. bertrami cysts were microscopic (up to 6750 μm in length) and exhibited a striated wall with 2.0-5.1 μm villar protrusions (vps) under LM. Transmission electron microscopy (TEM) observations showed that the vps were tightly packed, similar to "type 11c". Four genetic markers (18S, 28S, ITS1 and the mitochondrial cox1 gene) of S. bertrami were sequenced and analyzed. The 28S and ITS1 sequences are the first records for Sarcocystis in horses. The newly obtained sequences of the 18S and cox1 genes both shared the highest similarities with those of S. bertrami and S. fayeri obtained from horses. Phylogenetic analysis based on the 18S, 28S and cox1 sequences revealed that S. bertrami and S. fayeri formed an independent clade within a group comprising Sarcocystis spp. from ruminants and pigs. Therefore, S. bertrami and S. fayeri are considered to represent the same species of Sarcocystis in horses, and S. fayeri is a junior synonym of Sarcocystis bertrami., (© C.-L. Ma et al., published by EDP Sciences, 2020.)

Objective: Sarcocystosis is an important zoonotic protozoal disease with worldwide distribution and wide range of hosts. The aim of the present study was to determine the intensity of Sarcocystis spp. infection and to show histopathological features of their cystic lesions in slaughtered cattle of Zabol- Iran., Methods: From April to September 2018, 500 tissue samples from esophagus, heart, diaphragm, tongue and masticatory muscles were prepared from 100 slaughtered cattle. All samples were fixed in 10% neutral buffered formalin and routine tissue processing protocol was performed., Results: The microscopic results showed that 92.2% of specimens had thin-walled cysts of S. cruzi and 14% had thick-walled Sarcocystis ( S. hirsuta and S. hominis ) but macrocyst was only observed in one cattle. The positivity rate of thin walled cysts was 58.8% for heart, 13.9% for masticatory muscles, 10.2% for tongue, 9.3% for esophagus and 7.8% for diaphragm. The positivity rate of thick walled cysts was 32.8% for esophagus, 28.6% for tongue, 22.9% for heart, 15.7% for masticatory muscles and 0% for diaphragm, which could represent either S. hominis or S. hirsuta . The most infected tissue was heart and the least infected tissue was diaphragm. Thin walled cysts ( S. cruzi ) were mostly found in heart and were less found in diaphragm. However, thick-walled cysts ( S. hirsuta and S. hominis ) were mostly detected in esophagus. No thick-walled cysts were found in diaphragm muscle., Conclusion: A high positivity rate of sarcocystosis in slaughtered cattle in Zabol abattoir revealed heavily environmental contamination of Sistan region by this important parasitic disease.

In this study, 36.8% (28/76) of tissue samples collected from domestic pigs (Sus scrofa) contained sarcocysts, as determined by light microscopy. The organisms were identified as Sarcocystis miescheriana and Sarcocystis suihominis based on their morphological and molecular characteristics. Four genetic markers, i.e., 18S rDNA, 28S rDNA, ITS-1 region (ITS-1), and the mitochondrial COX1 gene (COX1), of the two parasites were sequenced and analyzed, and the 28S rDNA and ITS-1 of S. suihominis obtained from pigs constituted the first records of these markers in GenBank. The sequences of the four loci (18S rDNA, 28S rDNA, ITS-1, and COX1) of S. miescheriana shared high identities with those of S. miescheriana obtained from domestic and/or wild pigs in GenBank, with similarities of 99.6%, 99.6%, 95.9%, and 95.4%, respectively. The 18S rDNA sequences of S. suihominis exhibited 99.4% identity with those of S. suihominis from domestic and wild pigs. The comparison of the newly obtained sequences of the four genetic markers between the two parasites revealed that the interspecific similarities of 18S rDNA, 28S rDNA, ITS-1, and COX1 were 97.7%, 96.6%, 80.3%, and 81.2%, respectively. Therefore, the two species could be better discriminated with ITS-1 and mitochondrial COX1 compared with 18S rDNA or 28S rDNA. The phylogenetic analysis using 28S rDNA indicated that the two Sarcocystis species in domestic pigs had a close relationship.

Diaphragm samples from 65 hunted sika deer (Cervus nippon yesoensis) from Hokkaido, Japan were examined for the presence of sarcocysts based on histological sections. Morphologically, the detected sarcocysts grouped into three types: (Type 1) 108.0-305.0 μm in width, thick-walled (4.3-7.0 μm) with tombstone-like protrusions; (Type 2) 25.0-69.5 μm in width, thick-walled (3.8-8.0 μm) with finger-like protrusions; and (Type 3) 22.5-55.0 μm in width, thin-walled (under 1 μm) with no visible protrusions under light microscopy. All samples contained at least one sarcocyst type, and multiparasitism was apparent in 58 samples. Morphologically, Type 1 sarcocysts were found in 19 (29.2%) samples, Type 2 in 62 (95.4%) samples, and Type 3 in 60 (92.3%) samples. The sarcocysts were collected using laser microdissection, the DNA extracted from them was PCR-amplified, and their 18S ribosomal RNA and cytochrome c oxidase subunit 1 genes were sequenced. Phylogenetic analysis showed that, for both genes, each morphological sarcocyst type (Types 1, 2, and 3) aligned most closely with S. silva/S. truncata, S. tarandi/S. elongata, and S. pilosa, respectively. Based on the sequence identities between taxa and the molecular information for sarcocysts in C. nippon centralis, the sarcocyst types were presumed to be S. truncata-like (Type 1), S. tarandi-like (Type 2), and S. pilosa (Type 3). The phylogenetic analyses based on the present comprehensive molecular characterization of three Sarcocystis spp. from C. nippon yesoensis in Hokkaido suggest that canids (e.g., wild foxes) may be the definitive hosts for S. pilosa, and felids (or unknown species) the definitive hosts for the other two species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The reported prevalence of Sarcocystis infection in cattle in Europe ranges between 66 and 94%. Although in the Netherlands a prevalence of 100% was reported in 1993, this study aimed to develop a method for sensitive and specific molecular detection and species identification of Sarcocystis spp., in order to provide more recent data on the prevalence and identification of these protozoa in cattle meat intended for human consumption in the Netherlands. For this purpose, 104 cattle samples were obtained from Dutch slaughterhouses. Genomic DNA was extracted, and analysed by 18S and cox1 PCR. Magnetic capture was used to extract and amplify 18S-specific DNA. Sarcocystis DNA was detected in 82.7% of the samples. PCR amplicons of both targets were sequenced, and sequence identities of ≥97% were observed for Sarcocystis cruzi (65.4%), Sarcocystis hominis (12.5%), Sarcocystis bovifelis (8.7%), Sarcocystis hirsuta and Sarcocystis heydorni (both 1.0%). Mixed infections were observed in 17.3% of the samples. The magnetic capture was not significantly more sensitive compared with standard DNA extraction, but magnetic capture did add to the overall sensitivity. Using cox1 sequencing, all species are clearly distinguished, whereas for 18S the variation between species is limited, which particularly hampers reliable identification of thick walled Sarcocystis spp. Furthermore, the detection of 12.5% S. hominis and 1% S. heydorni points towards an established transmission route between cattle and humans in the Netherlands. The availability of four additional well-identified and well-referenced S. hominis cox1 sequences in public databases enables development of species-specific diagnostic PCRs targeting cox1, which in combination with magnetic capture could provide the means to determine the prevalence of human sarcocystosis., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Members of the genus Sarcocystis are frequently found infecting members of the family Cervidae. Although Sarcocystis species are generally host specific for their intermediate hosts, species in cervids appear to be less host specific. Here, we report fallow deer ( Dama dama ) as a new host for Sarcocystis morae , originally described from the red deer ( Cervus elaphus ). Tongues of 69 legally hunted animals in Spain were tested for sarcocysts, and the species were characterized by light microscopy, ultrastructurally and molecularly. Sarcocysts were identified in 66.7% of D. dama . Sarcocysts had thin (<2 μm thick) cyst wall with hair-like villar protrusions bifurcated at their tips resembling type 8a. Genetic sequences obtained for 18S rRNA and COI reached 99.6-100% and 97.9-98.7% similarity, respectively, to those of S. morae from the red deer. The present study provides new data concerning lower level of host specificity within Sarcocystis genus for cervids.

Background: In spite of the global economic significance of sheep production, little is known about the prevalence of various Sarcocystis spp. infecting the domestic sheep (Ovis aries) in Egypt., Materials and Methods: Muscle samples were collected from 175 sheep (> 2 years) slaughtered at El-Mahalla El-Kubra abattoir, Gharbia governorate, Egypt. Samples were initially examined by naked eye for the existence of macrosarcocysts. The microscopic sarcocysts were detected and identified using the light microscopy and the Transmission electron microscopy (TEM). Different microscopic species of ovine Sarcocystis were molecularly confirmed by PCR, sequence analyses and phylogeny., Results: Preliminary light microscopic inspection of the muscle specimens revealed the existence of only the microscopic sarcocysts of Sarcocystis tenella and Sarcocystis arieticanis in 152 (86.8%) out of the175 examined animals. Sarcoysts of S.tenella had striated thick cyst wall that amounted from 3.5-5.5 μm in thickness whereas, S.arieticanis sarcocysts had a thin cyst wall that ranged from 1-3 μm in thickness. S.tenella sarcocysts were detected in 115 sheep (65.7%), and were more prevalent than those of S.arieticanis, observed only in 68 sheep (38.8%). No macroscopic sarcocysts were observed in any of the examined carcasses. Transmission electron microscopy (TEM) of the cyst wall of S.tenella revealed the existence of the short stubby villar protrusions (VP) with the characteristic disk-like structures at the tips of the (VP). While, TEM of S.arieticanis showed that the cyst wall had elongated tubular protrusions that measured approximately 5-7 μm in length. Each (VP) consisted of a dome-shaped base (0.3-0.9 μm in diameter), a relatively thick middle portion (0.1-0.3 μm) in width, and a thin hair-like distal portion that measured about (0.03 x 1-4.5 μm)., Conclusion: Comparative analyses of the sequences of the four genetic markers (18S rRNA, 28S rRNA, mitochondrial cox1 and ITS-1) for S.tenella and S.arieticanis isolates detected herein, revealed genetic variations of 95% and 95- 96% among the different isolates on the level of the 18S rRNA and 28S rRNA, respectively. Whereas, the cox1 and ITS-1 shared sequence identities of 76-78% and 70-73%, respectively. S.tenella was strongly related to S.capracanis infecting goats (Capra hircus). Sequence identity of 98% on the level of 18S rRNA, 28S rRNA genes was observed between the currently identified isolates of S.tenella and the formerly GenBank deposited isolates of S.capracanis. While, cox1 sequences shared identities of 92-93%. Furthermore, S.arieticanis isolates identified here were closely related to the formerly published sequences of S.hircicanis. The 18S rRNA and 28S rRNA sequences of S.arieticanis shared 98% and 94-95% identities with those of S.hircicanis, respectively. However, 87-88% homologies were observed between the cox1 sequences of S.arieticanis and S.hircicanis. Consequently, cox1 and ITS-1 gene sequences act as better genetic markers than 18S rRNA and 28S rRNA sequences for the characterization of ovine Sarcocystis spp. Maximum parsimony analyses based on the sequences of three genetic markers, (18S rRNA, 28S rRNA and mitochondrial cox1), yielded the same placement of the currently identified isolates of the two taxa (S.tenella and S.arieticanis) within a clade of Sarcocystis species with carnivorous animals as known, or assumed, final hosts.

Rodents have been widely studied as intermediate hosts of Sarcocystis; however, only a few reports on these parasites in the black rat (Rattus rattus) are known. Having examined 13 black rats captured in Latvia, sarcocysts were found in skeletal muscles of two mammals and were described as Sarcocystis ratti n. sp. Under a light microscope, sarcocysts were ribbon-shaped, 0.9-1.3 × 0.09-0.14 mm in size and had a thin (0.8-1.3 μm) and smooth cyst wall. The lancet-shaped bradyzoites were 8.3 × 4.3 (7.5-9.3 × 3.9-4.8) μm. Under a transmission electron microscope, the cyst wall was up to 1.3 μm thick, wavy, the ground substance appeared smooth, type 1a-like. Morphologically, sarcocysts of S. ratti were somewhat similar to those of S. cymruensis, S. rodentifelis, and S. dispersa-like previously identified in the brown rat (Rattus norvegicus). On the basis of 18S rDNA, 28S rDNA, and cox1, significant genetic differences (at least 2.3, 4.5, and 5.8%, respectively) were observed when comparing S. ratti with other Sarcocystis species using rodents as intermediate hosts. While ITS1 sequences of S. ratti were highly distinct from other Sarcocystis species available in GenBank. Phylogenetic and ecological data suggest that predatory mammals living near households are definitive hosts of S. ratti.

Several surveys of Sarcocystis infection in sika deer in Japan have shown a high prevalence, but the identification has been unclear because molecular data have been lacking or have been limited to 18S ribosomal RNA gene sequences. Thus, in our previous study based on such sequences, some Sarcocystis isolates from sika deer were not clearly separated from other species in the phylogenetic analysis. In the present study, we therefore characterized sarcocyst isolates from sika deer (Cervus nippon centralis) at the mitochondrial cytochrome c oxidase subunit I gene (cox1). Moreover, we developed a multiplex PCR based on cox1 sequences of all species found, so that we could rapidly identify sarcocysts of these species. Twenty-one sarcocysts from nine sika deer were examined. Five distinct cox1 sequence types, each with a high sequence identity (> 99%), were found, and the sarcocysts could thus be classified into five species. Based on the sequence comparisons and the phylogeny, Sarcocystis spp. of types 1, 3, and 5 are considered to represent three new species, which were most closely related to Sarcocystis silva/Sarcocystis truncata, Sarcocystis entzerothi, and Sarcocystis iberica/Sarcocystis venatoria, respectively. There was a slight uncertainly whether Sarcocystis sp. with type 2 sequences represented a new species or was identical to Sarcocystis tarandi. Type 4 sequences showed 99% identity with those of Sarcocystis pilosa from sika deer in Lithuania and have therefore been assigned to this species. In the multiplex PCR, type-specific fragments were successfully amplified for all five Sarcocystis spp., indicating that this assay may be useful for a rapid identification of sarcocysts of these species.

Various muscle tissue samples from 60 moose (Alces alces) in the Baltic region were examined for Sarcocystis species. Sarcocysts were detected in 49 out of 60 (81.7%) moose investigated. Six species, Sarcocystis alces, Sarcocystis hjorti, Sarcocystis linearis, Sarcocystis silva, Sarcocystis ovalis, and Sarcocystis sp., were identified using light microscopy (LM), and DNA sequence analysis (cox1 and 18S rDNA). Sarcocysts of S. alces, S. ovalis, and S. hjorti were studied using transmission electron microscopy (TEM); sarcocyst walls of S. alces, S. ovalis, and S. hjorti were type 25, type 24, and type 7a, respectively. Sarcocystis linearis previously found in roe deer and red deer was also shown to use moose as an intermediate host for the first time. The unknown Sarcocystis sp. was rare and might employ another main intermediate host. Phylogenetic results demonstrated that Sarcocystis sp. was most closely related to Sarcocystis tarandivulpes, using canids as definitive hosts.

Sarcocystis tenella is a common tissue coccidian parasite of sheep. It is reported worldwide with high prevalence rate ranging from 9 to 100%. However, there are very limited reports of this parasite from the Indian context and those reports are totally based on the morphology alone. When it comes to molecular characterization, such studies are absent from India. The present communication reports the first characterization study of S. tenella from India. 18S rRNA ribosomal gene and mitochondrial cytochrome c oxidase subunit I (cox1) genes were used for molecular characterization and phylogenetic analysis alongside standard histopathology of sarcocysts. Five Indian isolates were characterized for each gene, and respective sequences were submitted in the NCBI. Two haplotypes were noticed, both for the 18S rRNA and cox1 gene showing 99.8-100.0% and 99.7-100.0% nucleotide homologies within themselves, respectively. When compared with other sequences of S. tenella across the globe, the present isolates showed 93.3-99.9% nucleotide homology based on 18S rRNA gene and 95.2-99.8% nucleotide homology based on cox1 gene, respectively. In both the 18S and cox1 phylogenetic trees, respective sequences of S. tenella were placed with monophyletic cluster which was sister to a cluster comprising of sequences of S. gracilis and S. alces.