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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.)

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 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.)

The occurrence of Sarcocystis species was investigated in synanthropic (Muridae) and wild (Cricetidae) rodents from Argentina. Nine species were captured (n = 356). Sarcocysts were detected in muscles of 8.7% (31/356) and 3.7% (4/106) of the rodents by histopathology and direct microscopic observation, respectively. PCR-sequencing targeting the 18S rRNA, cox1, and ITS1 regions was performed on samples with positive histopathology. Four different 18S rRNA sequences or sequence groups with high intra-group identities (99.6-100%) were detected in Mus musculus, Oxymycterus rufus, Akodon azarae, and Necromys lasiurus. Eight sequences showed 99.5-99.7% identity with S. dispersa. Thirteen sequences showed low identity (95.3-96.4%) with other Sarcocystis spp. The obtained coxI sequences (n = 9) were almost identical to each other and showed a high similarity with S. strixi (99.2-99.5%) and S. lutrae (99.1%), despite the 18S rRNA sequences from the same samples suggested the occurrence of at least two species. This suggests that coxI may not show high variability in Sarcocystis spp. that use rodents as intermediate hosts. Six ITS1 sequences were obtained, showing high identity but low coverage with several Sarcocystis spp. Multilocus sequence typing and BLAST analysis did not lead to an accurate species identification. Possible reasons are the detection of new species or the limited molecular information available from previously described Sarcocystis spp. Phylogeny suggests that the detected Sarcocystis spp. may use raptor birds or snakes as definitive hosts. This study represents the first molecular identification of Sarcocystis spp. in naturally infected rodents of the Cricetidae and Muridae families in South America., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Sarcocystis spp. are intracellular protozoan parasites with an obligatory heteroxenous life cycle. The objective of this study is to identify Sarcocystis spp. in wild boar muscles from Argentina by light and transmission electron microscopy and molecular characterization. Muscle samples from diaphragm, tongue, masseter, intercostals, heart, and forelimbs of 240 wild boars were analyzed. Of the animals, 48.3% (116/240) were positive for sarcocysts by light microscopy, whereas 45.8% (110/240) were positive for Sarcocystis spp. by PCR targeting 18S rRNA fragment. These samples were subjected to a specific PCR for S. suihominis coxI gene, 3.6% (4/110) of which were weak positives. Unfortunately, sequence analysis was inconclusive. This could be related to a potentially low S. suihominis cyst load in the samples, or to an incomplete primer matching with the South American S. suihominis sequences. Seventeen individual sarcocysts were positive by PCR for the 18S rRNA fragment, whose sequences showed 99.75-100% identity with each other and with previously reported S. miescheriana sequences. A total of 21 cysts collected from 11 muscle samples and analyzed by TEM presented a cyst wall type compatible with S. miescheriana, and one cyst presented an ultrastructure compatible with S. suihominis. The latter came from a sample that also contained S. miescheriana cysts, indicating that the animal was co-infected. This is the first study that provides infection rates and describes and identifies morphological and molecular features of Sarcocystis spp. cysts in wild boars from South America., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

There is considerable confusion concerning the relationships among species of Sarcocystis found in donkeys and horses. Here, we describe a Sarcocystis species in Chinese donkeys (Equus asinus). Sarcocysts were found in 12 of 32 (37.5%) adult donkeys. By light microscopy, they were divided into two types, thin-walled and thick-walled. The thin-walled were macroscopic (up to 320 μm wide) and had short club-like protrusions (up to 2.7 μm long); the thick-walled were microscopic (up to 135 μm wide) and had villar protrusions (up to 5.4 μm long). Ultrastructures of the two types exhibited similar morphological characteristics, including bundled microtubules in the core of the villar protrusions penetrating diagonally into the ground substance, similar to wall type 11c. Three genetic markers, 18S rDNA, 28S rDNA, and mitochondrial cox1, obtained from the two morphotypes were sequenced and analyzed. The sequences of the three loci in the two morphotypes presented high intraspecific similarities of 97.2-99.5%, 97.8-99.6% and 99.0 - 99.9%, respectively. The most similar sequences in GenBank to the newly obtained 18S rDNA, 28S rDNA and cox1 sequences were those of Sarcocystis spp. in horses, with similarities of 90.0 - 97.5%, 94.7 - 95.1%, and 82.6 - 84.5%, respectively. Phylogenetic analysis using the three genetic markers indicated that the Sarcocystis sp. in donkeys formed an individual clade most closely related to a clade encompassing Sarcocystis spp. in horses. Further studies are needed for taxonomic identification of sarcocysts in donkeys because the Sarcocystis species in donkeys and horses are not successfully cross transmissible despite morphological similarities., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Sika deer (Cervus nippon), which are native to the Japanese islands and the adjacent mainland of eastern Asia, have been introduced into Europe and established free-ranging populations in several countries. Various Sarcocystis species have been identified recently from farmed "mainland sika" deer in Lithuania and native "Japanese sika" deer in Japan. To study the distribution, prevalence and intensity of Sarcocystis infection in free-ranging sika deer outside of their natural range heart and/or diaphragm samples of 311 animals from nine populations in Germany and Austria were examined by histology.Overall, sarcocysts were detected in either heart or diaphragm of 107/311 deer (34.4%) with prevalence ranging roughly from 5 to 50% among the populations. Considering the 263 animals with both heart and diaphragm available, prevalence varied significantly (p < 0.0001) among calves (20.2%), yearlings (40.3%), and adult deer (49.1%) but did not differ between male and female deer (48.3% vs. 43.7%; p = 0.6483). Occurrence of sarcocysts in heart vs. diaphragm indicated a marginal difference (27.8% vs. 20.9%; p = 0.0839). Intensity of infection in all but one heart positive and all diaphragm positive animals was low (< 10 sarcocysts per square centimeter muscle cut). While heart sarcocyst counts of yearlings and adult deer were higher than those of calves and were higher in male compared to female sika deer, diaphragm sarcocyst counts did not differ significantly between age groups and sexes. Sarcocystis infection was demonstrated at variable prevalence in sika deer in all populations but intensity is apparently low. Further studies are needed to identify the species of Sarcocystis infecting sika deer naturalized outside their natural range., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Although three species of Sarcocystis, S. cornixi, S. corvusi and S. kutkienae, have been described in corvids, molecular studies of sarcocysts isolated from these birds are incomplete. Leg muscles of 83 corvids, 35 hooded crows (Corvus cornix), 21 western jackdaws (Coloeus monedula), 11 rooks (Corvus frugilegus), 9 common ravens (Corvus corax), 4 common magpies (Pica pica) and 3 Eurasian jays (Garrulus glandarius), from Lithuania were examined for the presence of Sarcocystis spp. in the present study. In methylene blue-stained squashed samples, sarcocysts were detected in 26 birds (31.0%). Under a light microscope, two morphological types of sarcocysts were distinguished (type A and type B). Sarcocysts of type A had a smooth and thin (about 1 μm) cyst wall, while cysts of type B were characterised by a thicker (1.4-2.5 μm) cyst wall. Based on ITS1 sequence comparison, sarcocysts of type A were identified as S. halieti and Sarcocystis sp. ex Corvus corax, whereas cysts of type B belonged to S. kutkienae and S. cornixi. Furthermore, it was demonstrated that a single bird could host two different Sarcocystis spp. Sarcocystis halieti was detected in corvids for the first time in the common raven and the hooded crow. Also, this study presents the first evidence of S. kutkienae in the hooded crow and the common magpie, and S. cornixi in the western jackdaw. Sarcocystis sp. ex Corvus corax was genetically characterised using almost complete 18S rDNA, partial 28S rDNA and complete ITS1 sequences. Sarcocystis sp. ex Corvus corax clustered together with S. columbae, S. corvusi and S. halieti in phylogenetic trees reconstructed using 28S rDNA and ITS1 sequences., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cattle and buffaloes, popular protein sources worldwide, are intermediate hosts for several Sarcocystis species. These coccidian protozoans cause sarcocystosis resulting in subclinical and chronic infections in striated muscles by forming macrocysts or microcysts. In Thailand, Lao People's Democratic Republic, and Cambodia, Sarcocystis species have been reported, but molecular identification has been lacking. This study investigates the prevalence of infection, histo-morphology, and molecular identification of Sarcocystis species in hearts of cattle and buffalo sold in local markets. A phylogenetic tree inferred from a portion of the 18S ribosomal (r) RNA gene was used to identify the genus and species of Sarcocystis. The mitochondrial cytochrome c oxidase subunit 1 (cox-1 gene) was sequenced to confirm the species of host tissue. In Thailand, Sarcocystis was detected in 66.7% (14/21) of samples. In Lao People's Democratic Republic, 90% (9/10) of samples were infected and in Cambodia 100% (8/8). For the first time from these countries, we report Sarcocystis cruzi, Sarcocystis heydorni, and Sarcocystis levinei found in taurine cattle (Bos taurus) and water buffalo (Bubalus bubalis). Zoonotic protozoan transmission needs to be controlled by inspection activities by local health inspectors, and appropriate action is required at all points in the food chain by competent authorities to protect consumer health and prevent sarcocystosis in cattle and water buffaloes., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Protozoa of the genus Sarcocystis are obligatory heterogenous parasites with both definitive and intermediate hosts. Opossums (Didelphis aurita) can shed multiple species of Sarcocystis with birds as the intermediate host. The pathologies of Sarcocystis species in birds have not been thoroughly elucidated. Therefore, the aim of the present study to determine the main lesions that can occur in acute and chronic infections in intermediate hosts, when they ingest infective sporocysts that are shed in the opossum's feces, using budgerigars as a model. To this end, 12 budgerigars, Melopsittacus undulatus, were divided into two groups that received an inoculum with 60 and 120 sporocysts. Birds that died or were euthanized were necropsied, and the lung, tongue, liver, brain, heart, and skeletal striated muscles were collected and fixed in 10% formalin for histopathological analysis. The infectivity varied according to the sample and infective dose. Acute histopathological lesions were characterized by evidence of slightly degenerated hepatocyte cords that permeated the region of the blood vessel and hepatic sinusoids. Pulmonary tissue lesions were also observed in the parabronchial region with the presence of inflammatory infiltrates associated with areas of edema and atelectasis. In chronic infections, few mature cysts were observed in the chest, and many mature cysts in the thigh and tongue muscles. Thus, it was possible to conclude that lesions are highly characteristic in acute infection and, in chronic infections, cysts were present but without major lesions. In this case, the preferred organs of parasitism were the thigh and the tongue.

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.

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.

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.

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.

A sample of the diaphragm was collected from each of 100 wild boars legally hunted in the Val Grande National Park in north-western Italy and examined for the presence of Sarcocystis infection by histological and molecular methods. In histological sections, thick-walled sarcocysts consistent with those of Sarcocystis miescheriana were detected in 32 wild boars. Genomic DNA extracted from diaphragm samples was initially subjected to PCR amplification of the internal transcribed spacer 1 (ITS1) region, and 97 wild boars were found to harbour a Sarcocystis infection at this screening. Selected DNA samples were then subjected to PCR amplification and sequencing of the ITS1 region and the 18S and 28S ribosomal RNA (rRNA) genes of the nuclear ribosomal DNA unit, while all positive samples were subjected to PCR amplification of the mitochondrial cytochrome c oxidase subunit I (cox1) gene. S. miescheriana was identified in 97 wild boars (97%), while the zoonotic Sarcocystis suihominis was identified in one wild boar (1%), which also harboured S. miescheriana. Intra-specific sequence variation was found in all four DNA regions of S. miescheriana examined and in the 18S rRNA gene and ITS1 region of S. suihominis. The partial cox1 gene was amplified and sequenced from 72 isolates of S. miescheriana, yielding 43 haplotypes with pairwise sequence identities of 97.6-99.9%. These haplotypes were 79.1-79.8% identical with the cox1 sequence of S. suihominis. Phylogeny based on cox1 sequences placed S. miescheriana and S. suihominis as sister species within a clade comprising mainly Sarcocystis spp. of ruminants with felids as known or presumed definitive hosts. The same was true for the phylogeny based on 18S rRNA gene sequences.

This study was conducted to identify the Sarcocystis species that infect the opossum Didelphis aurita in order to determine which sporocysts they are excreating in to the environment and help determine the role of D. aurita in the epidemiology of Sarcocystis. Sporocysts were obtained from intestinal tracts of 8 of 13 D. aurita trapped in Rio de Janeiro state, Brazil, and were orally inoculated into Melopsittacus undulatus and Balb/c nude Mus musculus. Portions of organs and muscles were processed for histology, immunohistochemistry, transmission electron microscopy (TEM), and PCR using primers JNB 33/54, and ITS. Amplification products were subjected to RFLP using DraI and HinfI. Some birds were euthanized 6, 7, 13, 16, and 24 days after inoculation (DAI). All other birds and all mice were euthanized 60 DAI. Schizonts were observed in the lungs using histology and immunostaining in birds examined prior to 60 DAI. Sarcocysts with a ~ 1.5-μm-thick wall were found in the breast, thigh, and tongue of some birds. Sarcocystis asexual stages were isolated in cell cultures inoculated with sporozoites. Parasite DNA isolated from bird tissues and cell cultures demonstrated that S. falcatula-like parasites were present in all samples derived from positive opossums. Asexual stages molecularly characterized as S. lindsayi-like were isolated in cell culture from one opossum with an apparent multiple infection. This study demonstrated that D. aurita is a definitive host for S. falcatula-like parasites and indicates that S. lindsayi-like parasites can be found in coinfections of this opossum species.

The muscles of herbivores commonly harbor sarcocysts of parasites belonging to species in the genus Sarcocystis, but such muscle parasites are rare in carnivores. Here, we report Sarcocystis arctica-like sarcocysts in muscles of Arctic foxes (Vulpes lagopus) from Alaska, USA, for the first time. The tongues of 56 foxes were examined for Sarcocystis infection using several methods. Sarcocystis bradyzoites were detected in pepsin digests of 13 (23.2%), and sarcocysts were found in histological sections stained with hematoxylin and eosin (HE) of 9 (16.0%). By light microscopy, sarcocysts were up to 4 mm long and up to 245 μm wide. In HE-stained sections, the sarcocyst wall appeared smooth and up to 1.5 μm thick without visible protrusions. By transmission electron microscopy, the sarcocyst wall had a wavy parasitophorous vacuolar membrane (pvm) folded as pleomorphic villar protrusions (vp), sometimes with anastomoses of villar tips. The vp and the ground substance (gs) layer were smooth and without microtubules. The gs was up to 2.0 μm thick. The total width of the wall including vp and the gs was up to 4.0 μm. The vp were up to 3.0 μm long and most closely resembled "type 9c." All sarcocysts were mature and contained numerous 8.1 × 2.1 μm sized bradyzoites. Molecular characterization (at 18S rDNA, 28S rDNA, ITS-1, and cox1) showed the highest affinity for S. arctica of the Arctic fox (V. lagopus) from Norway. In the present investigation, we provide evidence that sarcocysts are common in tongues of Alaskan Arctic foxes suggesting that these carnivores are serving as intermediate hosts, and we also provide ultrastructure of S. arctica from the Arctic fox for the first time.

Human sarcocystosis is a rare infection caused by the genus Sarcocystis who human serve as definitive (intestinal form of infection) host or intermediate (extraintestinal form) host for some various Sarcocystis species. The detection of Sarcocystis oocysts/sporocysts in the feces usually incidentally and is achieved by microscopic examination of the fresh or preserved specimens. To rule out any parasitological etiology among 23,875 (aged 2 months to 95 years) apparently immunocompetent Iranian individuals (from October of 2010 to June of 2016) with abdominal discomforts referred to several teaching hospitals and local clinical laboratories in Fars Province, Iran, their fecal samples were examined using light microscopy. Most pathogenic parasite-positive and doubtful samples were sent to the Intestinal Protozoology Laboratories of Fasa and Shiraz Universities of Medical Sciences to further examination to detect probable co-infection with other underdiagnose parasitoses. In addition to the common protozoal and helminthic infections, during the course of examining stool specimens using direct smear mixed with saline or iodine mounts and by formalin-ethyl acetate techniques, four cases of intestinal Sarcocystis infection as only or concurrently infected with other parasites were found. The present paper presents cases of human intestinal Sarcocystis infection in Iran. Since Sarcocystis are small in size and usually rare in stool, they often go unnoticed. It should be noted that stool smears must be examined with great care to avoid misinterpretation of Sarcocystis infections in microscopic examinations. To the best of our knowledge, co-infection of intestinal sarcocystosis and other principal parasitoses in stool investigations has not been reported earlier.

Despite the importance of worldwide goat production, little is known about the prevalence of Sarcocystis spp. in domestic goats (Capra hircus) in China. The aims of the present study were to determine prevalence of Sarcocystis spp. in domestic goats in Kunming, China, as well as to identify parasite species based on morphological characteristics and DNA sequence analysis. Only microscopic sarcocysts of Sarcocystis spp. were detected in 174 of 225 goats (77.3 %). By light and transmission electron microscopy, two species, i.e., Sarcocystis capracanis and Sarcocystis hircicanis, were identified. Two sarcocysts from each of the two species were randomly selected for DNA extraction; the 18S rRNA gene (18S rRNA), the 28S rRNA gene (28S rRNA), and the mitochondrial cytochrome c oxidase subunit 1 (cox1) were amplified by the polymerase chain reaction (PCR) and subsequently sequenced. The results were compared with other previously sequenced Sarcocystis species retrieved from GenBank. There was little sequence variation between two isolates of the same species. S. capracanis was most closely related with Sarcocystis tenella; 18S rRNA, 28S rRNA, and mitochondrial cox1 sequences shared identities of 95.7-99.1, 95.3, and 92.3-93.2 % with those of S. tenella, respectively. Thus, mitochondrial cox1 sequences seem to perform better than 18S rRNA sequences or 28S rRNA sequences for identification of the two species. S. hircicanis was most closely related to Sarcocystis arieticanis, i.e., 18S rRNA and 28S rRNA sequences of the former species shared 97.2-97.4 and 95.6-96.1 % identities with those of latter, respectively. Phylogenetic analysis inferred from the three genetic markers yielded similar results and indicated the two species were within a group of Sarcocystis species with canines as known, or presumed, definitive hosts.

Sarcocystis spp. are protozoan parasites with a heteroxenous life cycle, which produce cysts in the muscle of herbivorous animals. In these animal species, sarcocystosis is frequently asymptomatic, although it may occur with high prevalence. Seven Sarcocystis spp. have been described in red deer (Cervus elephus). The aim of this study was to determine the prevalence of sarcocystosis, and to perform the morphological and molecular characterization of Sarcocystis spp. found in wild red deer of the Nahuel Huapi National Park (NHNP), Patagonia, Argentina. Full necropsies of 62 red deer killed by hunters in the NHNP and neighboring areas were performed. Samples of heart and skeletal muscle were examined histologically and selected samples were also examined by transmission electron microscopy (TEM), PCR and sequencing. Sarcocystis spp. thin walled cysts were detected in 62 % (38/62) of heart, and in 22 % (3/14) of skeletal muscle samples examined histologically. TEM revealed a smooth and thin cyst wall (≤1 μm), with scarce and separated ribbon-like protrusions. A total of three partial and one full 18S ribosomal RNA (rRNA) gene sequences were obtained, and showed the highest identity (≥99 %) with Sarcocystis taeniata, a species described in moose (Alces alces). The morphological and molecular results indicate that red deer in Argentina are frequently infected with S. taeniata, a species for which the definitive host is unknown. The present results also confirm that Sarcocystis spp. using cervids as intermediate host are not host-specific. Further studies are needed to improve the epidemiological knowledge of Sarcocystosis in red deer.

Numerous species of Sarcocystis have been reported from wild ruminants, but none has been from Williamson's mouse deer (Tuagulus williamsoni). Here, we describe a new species, Sarcocystis tuagulusi, infecting five of 12 Williamson's mouse deer from southwest China. Mature sarcocysts are microscopic, up to 2280-μm long. The sarcocyst wall had 8.2-μm long villar protrusions, type 24. Using transmission electron microscopy, the protrusions on sarcocysts appeared as elongated, lancet- or leaf-like shapes in longitudinal sections; in contrast, the cross-sections revealed mushroom-shaped protrusions. In the core of the protrusion, a bundle of microtubules penetrated diagonally into a ground substance along the longitudinal axis. A phyogenetic analysis based on 18S rRNA and cox1 sequences suggested S. tuagulusi is closely related to Sarcocystis species from ruminants that employ felids as definitive hosts.

A total of 33 macroscopically visible (3-11 × 1-5 mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3-99.9 %. In comparison, the sequence identity was 98.8-99.0 % among eight complete 18S rRNA gene sequences (1,873-1,879-bp long), 98.1-100 % among 28 complete ITS1 sequences (853-864-bp long) and 97.4-99.6 % among five partial 28S rRNA gene sequences (1,607-1,622 bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93 % sequence identity at cox1 versus interleaved sequences and 98.7-99.1 % sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8-10 days post-infection (dpi) and were euthanized 15 dpi. Sporocysts isolated from the intestinal mucosa of both cats were identified molecularly as belonging to S. fusiformis through PCR amplification and sequencing of the partial cox1. The two sporocyst-derived cox1 sequences were identical with the most common sarcocyst-derived cox1 haplotype.

Sarcocystis nesbitti, using snakes as the definitive host, is a causative agent of acute human muscular sarcocystosis in Malaysia. Therefore, it is important to explore the distribution and prevalence of S. nesbitti in snakes. Nevertheless, epizootiological information of S. nesbitti in snakes remains insufficient because few surveys have assessed Sarcocystis infection in snakes in endemic countries. In Japan, snakes are popular exotic pet animals that are imported from overseas, but the degree of Sarcocystis infection in them remains unclear. The possibility exists that muscular sarcocystosis by S. nesbitti occurs in contact with captive snakes in non-endemic countries. For a total of 125 snake faecal samples from 67 snake species collected at animal hospitals, pet shops and a zoo, this study investigated the presence of Sarcocystis using polymerase chain reaction (PCR) for the 18S ribosomal RNA gene (18S rDNA). Four (3.2%) faecal samples were positive by PCR. Phylogenetic analysis of the 18S rDNA sequences obtained from four amplification products revealed one isolate from a beauty snake (Elaphe taeniura), Sarcocystis zuoi, which uses rat snakes as the definitive host. The isolate from a Macklot's python (Liasis mackloti) was closely related with unidentified Sarcocystis sp. from reticulated pythons in Malaysia. The remaining two isolates from tree boas (Corallus spp.) were closely related with Sarcocystis lacertae, Sarcocystis gallotiae and unidentified Sarcocystis sp. from smooth snakes, Tenerife lizards and European shrews, respectively. This report is the first of a study examining the distribution of Sarcocystis species in captive snakes in Japan.

Sarcocysts were detected in routinely processed histological sections of skeletal muscle, but not cardiac muscle, of two adult male otters (Lutra lutra; Mustelidae) from northern Norway following their post-mortem examination in 1999 and 2000. The sarcocysts were slender, spindle-shaped, up to 970 μm long and 35-70 μm in greatest diameter. The sarcocyst wall was thin (∼ 0.5 μm) and smooth with no visible protrusions. Portions of unfixed diaphragm of both animals were collected at the autopsies and kept frozen for about 14 years pending further examination. When the study was resumed in 2013, the thawed muscle samples were examined for sarcocysts under a stereo microscope, but none could be found. Genomic DNA was therefore extracted from a total of 36 small pieces of the diaphragm from both otters, and samples found to contain Sarcocystidae DNA were used selectively for PCR amplification and sequencing of the nuclear 18S and 28S ribosomal (r) RNA genes and internal transcribed spacer 1 (ITS1) region, as well as the mitochondrial cytochrome b (cytb) and cytochrome c oxidase subunit 1 (cox1) genes. Sequence comparisons revealed that both otters were infected by the same Sarcocystis sp. and that there was no genetic variation (100 % identity) among sequenced isolates at the 18S and 28S rRNA genes (six identical isolates at both loci) or at cox1 (13 identical isolates). PCR products comprising the ITS1 region, on the other hand, had to be cloned before sequencing due to intraspecific sequence variation. A total of 33 clones were sequenced, and the identities between them were 97.9-99.9 %. These sequences were most similar (93.7-96.0 % identity) to a sequence of Sarcocystis kalvikus from the wolverine in Canada, but the phylogenetic analyses placed all of them as a monophyletic sister group to S. kalvikus. Hence, they were considered to represent a novel species, which was named Sarcocystis lutrae. Sequence comparisons and phylogenetic analyses based on sequences of the 18S and 28S rRNA genes and cox1, for which little or no sequence data were available for S. kalvikus, revealed that S. lutrae otherwise was most closely related to various Sarcocystis spp. using birds or carnivores as intermediate hosts. The cox1 sequences of S. lutrae from the otters were identical to two sequences from an arctic fox, which in a previous study had been assigned to Sarcocystis arctica due to a high identity (99.4 %) with the latter species at this gene and a complete identity with S. arctica at three other loci when using the same DNA samples as templates for PCR reactions. Additional PCR amplifications and sequencing of cox1 (ten sequences) and the ITS1 region (four sequences) using four DNA samples from this fox as templates again generated cox1 sequences exclusively of S. lutrae, but ITS1 sequences of S. arctica, and thus confirmed that this arctic fox had acted as intermediate host for both S. arctica and S. lutrae. Based on the phylogenetic placement of S. lutrae, the geographical location of infected animals (otters, arctic fox) and the distribution of carnivores/raptors which may have interacted with them, the white-tailed eagle (Haliaeetus albicilla) seems to be a possible definitive host of S. lutrae. Some of the muscle samples from both otters were shown to harbour stages of Toxoplasma gondii through PCR amplification and sequencing of the entire ITS1 region (five isolates) and/or the partial cytb (eight isolates) and cox1 (one isolate). These sequences were identical to several previous sequences of T. gondii in GenBank. Thus, both otters had a dual infection with S. lutrae and T. gondii.

Sarcocystis and Hammondia are two obligatory protozoan parasites. These genera belong to cyst-forming coccidia group of the phylum Apicomplexa. They both need two different hosts to complete their life cycles. Felids and canids can act as definitive hosts, while herbivores, such as sheep and cattle, are the most important intermediate hosts. Reports verify that no important disease has been caused by Hammondia spp.; on the other hand, Sarcocystis spp. can cause some severe infectious disease in livestock industry such as abortion. Economic losses are another concern due to carcass condemnation during meat inspection in abattoirs and decrease in the quality and quantity of milk and wool production. Due to the Sarcocystis and Hammondia tissue cysts being similar, the distinction between these different genera is so important. In this study, the prevalence of Sarcocystis and Hammondia in the esophagus tissue of sheep and cattle slaughtered in one of the industrial abattoir in Iran was reported and an easy and rapid method for accurate diagnosing of Sarcocystis and Hammondia bradyzoites was explained.

The breast and leg muscles of a common eider (Somateria mollissima; Anatidae: Anseriformes) from northern Norway contained numerous grossly visible cigar-shaped sarcocysts measuring about 5 × 1 mm. Light microscopic examination of isolated sarcocysts revealed that they were encapsulated by a thin fibrous layer, underneath which there was a thin and fairly smooth cyst wall with no visible protrusions. The cystozoites were straight, spindle-shaped and about 13 μm long. Genomic DNA was extracted from 12 excised sarcocysts and each DNA isolate was subjected to PCR amplification of one to four loci: the 18S and 28S ribosomal RNA genes (four isolates), the internal transcribed spacer 1 (ITS1) region (six isolates) and the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (12 isolates). At the three nuclear loci, the new sequences showed 99.9-100% sequence identity with previous sequences of Sarcocystis rileyi from the mallard duck in Lithuania and USA, and they could therefore be assigned to this species. At cox1, the new sequences of S. rileyi were most similar to Sarcocystis arctica and Sarcocystis neurona, but the most closely related Sarcocystis spp. in birds have not been sequenced at this locus. There was no sequence variation at any locus between the 4-12 examined isolates of S. rileyi. This is the first genetically verified record of S. rileyi in the common eider, as well as in any bird species in Norway. The phylogenetic placement of S. rileyi was inferred separately from 28S rRNA gene and cox1 sequences, and similar results were obtained in both analyses.

Bovines are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta, and Sarcocystis hominis, which use canids, felids, or primates as definitive hosts, respectively. Cattle represent also intermediate hosts of Sarcocystis sinensis, but the definitive hosts of this parasite are not yet known. Sarcocystosis in cattle is frequently asymptomatic. The infection is characterized by the presence of thin-walled (S. cruzi) or thick-walled muscle cysts or sarcocysts (S. hominis, S. sinensis, and S. hirsuta). Recent reports suggest high prevalence of the zoonotic S. hominis in beef in Europe. We therefore aimed at differentiating Sarcocystis spp. in beef offered to consumers in Germany using molecular and microscopical methods, focusing on those species producing thick-walled sarcocysts. A total of 257 beef samples were obtained from different butcheries and supermarkets in Germany and processed by conventional and multiplex real-time PCR. In addition, 130 of these samples were processed by light microscopy and in 24.6% thick-walled cysts were detected. Transmission electron microscopical analysis of six of these samples revealed an ultrastructural cyst wall pattern compatible with S. sinensis in five samples and with S. hominis in one sample. PCR-amplified 18S ribosomal DNA (rDNA) fragments of 28 individual thick-walled cysts were sequenced, and sequence identities of ≥98% with S. sinensis (n = 22), S. hominis (n = 5) and S. hirsuta (n = 1) were observed. Moreover, nine Sarcocystis sp. 18S rDNA full length gene sequences were obtained, five of S. sinensis, three of S. hominis, and one of S. hirsuta. Out of all samples (n = 257), 174 (67.7%) tested positive by conventional PCR and 179 (69.6%) by multiplex real-time PCR for Sarcocystis spp. Regarding individual species, 134 (52%), 95 (37%), 17 (6.6%), and 16 (6.2%) were positive for S. cruzi, S. sinensis, S. hirsuta, and S. hominis, respectively. In conclusion, S. sinensis is the most prevalent thick-walled Sarcocystis species in beef offered for consumption in Germany. Further studies are needed to identify the final host of S. sinensis as well as the potential role of this protozoan as a differential diagnosis to the zoonotic species S. hominis.

In the present investigation, macroscopic sarcocysts of Sarcocystis acanthocolubri were observed in muscles of 42 (4.3%) out of 975 Acanthodactylus sp. lizards collected from different geographical areas in Egypt. The infection rate was 6.4% in Acanthodactylus boskianus, 2.1% in Acanthodactylus sculentus, and 5% in Acanthodactylus paradalis. The highest infection rate was recorded in the lizards captured from Baltem (10% in A. boskianus and 8% in A. paradalis). The infection rate was usually higher in females (7.4%) than in males (3.8%). Moreover, the highest infection rate was recorded in summer (7.53%), autumn (3.57%), and spring (3.11%), and the lowest was recorded in winter (0.91%). Also, old animals had higher infection rates (10.8%) than young ones (0-2.7%). Macrocysts measured 0.95 × 10.12 mm. Both macroscopic and microscopic sarcocysts were enclosed only by a primary cyst wall, which had many finger-like, stalkless, and non-branched protrusions giving it a striated appearance. The primary cyst wall measured 3.9 μm. A dark granulated ground substance was found directly underneath the protrusions and is extended interiorly dividing the cyst cavity into many compartments containing the parasites (metrocytes and merozoites). Metrocytes were found directly under the ground substance and usually multiply asexually by endodyogeny producing two merozoites from each metrocyte. Both metrocytes and merozoites had the apical complex structures characteristic to the genus Sarcocystis. Transmission experiments with three snake species indicated that the snake Spalerosophis diadema is the proper final host belonging to the family Colubridae. The prepatent period was 16 days, while the patent period was 35 days. The results obtained from the present investigation revealed that this is a new species which was named Sarcocystis acanthocolubri.

Four Sarcocystis species, i.e., Sarcocystis fusiformis and Sarcocystis buffalonis with cats as definitive hosts, Sarcocystis levinei with dogs as definitive host, and Sarcocystis dubeyi with unknown definitive host, have previously been described from water buffalo (Bubalus bubalis). The aim of the present study was genetic characterization of the causative agent(s) of water buffalo sarcocystosis in Khuzestan Province, western Iran. RFLP-PCR and partial sequence analysis of 18S rDNA gene were used for the genetic characterization of the specimens directly obtained from water buffalo. In RFLP-PCR, four restriction enzymes (Dra1, Ssp1, Fok1 and Bsl1) were used for species discrimination of Sarcocystis spp. in this host. Comparison of the molecular sequencing results and RFLP-PCR pattern of the samples obtained in the present study with those previously reported for different Sarcocystis spp. revealed that all positive Sarcocystis samples represented S. fusiformis. To our knowledge, this is the first demonstration of the existence of S. fusiformis in the Iranian water buffalo population by a genetic approach. In addition, comparison between the alignments between the Iranian 18S rDNA sequences (HQ703791), made in this study, and those previously reported for S. fusiformis in different geographical location (accession nos. AF176927, AF176926, and U03071) showed the occurrence of local genetic polymorphisms and heterogeneity in this ribosomal locus. Despite the occurrence of some genetic variations in the hypervariable regions of the 18S rDNA in S. fusiformis, Dra I restriction site was conserved among all sequences available. According to the present study, it seems that cats have a more significant epidemiological role than dogs in transmission of sarcocystosis agent to water buffalo in Iran.

The aim of this study was to determine the prevalence of sarcosporidiosis in semi-intensively bred cattle in northwestern Italy. A diagnostic protocol was setup in which infected animals were identified by rapid histological examination of the esophagus, diaphragm, and heart and the detected Sarcocystis spp. were subsequently typed using conventional electron microscopy in combination with molecular techniques. Sarcosporidia cysts were detected in 78.1% of the animals and were seen most often in the esophagus. The cattle is intermediate host for Sarcocystis hominis (final host, humans and some primates), Sarcocystis cruzi (final host, domestic and wild canids), and Sarcocystis hirsuta (final host, wild and domestic cats).All these three species of Sarcocystis were identified, variously associated, with the following prevalence: S. cruzi (74.2%), S. hirsuta (1.8%), and S. hominis (42.7%). Furthermore, a new S. hominis-like (prevalence 18.5%), characterized by hook-like structures of villar protrusion and a different sequence of the 18S rRNA gene, was identified. The cattle sheds testing positive for zoonotic Sarcocystis were assessed for risk factors contributing to the maintenance of the parasite's life cycle. Significant associations emerged between consumption of raw meat by the farm owner, mountain pasturing, and absence of a sewerage system on the farm and cattle breed. Our study demonstrates that sarcosporidiosis may constitute a public health problem in Italy and indicates several issues to be addressed when planning surveillance and prevention actions. The applied diagnostic approach revealed that cattle can harbor a further type of Sarcocystis, of which life cycle and zoonotic potential should be investigated.

A total of 65 free-living carnivores collected on Honshu Island, Japan were examined for muscular Sarcocystis species infections. Among them, 12 Japanese raccoon dogs (Nyctereutes procyonoides viverrinus), one Japanese red fox (Vulpes vulpes japonica), three Japanese martens (Martes melampus melampus), and two Japanese badgers (Meles meles anakuma) were found to have sarcocysts in their muscles. No inflammatory reactions associated with sarcocysts were observed. Ultrastructurally, the sarcocysts detected in the Japanese raccoon dogs, Japanese red fox, and Japanese martens were similar to each other, with the sarcocyst wall being thin and exhibiting minute undulations. On the other hand, the sarcocysts detected in the Japanese badgers had a thick cyst wall with numerous finger-like protrusions which contained microtubules. The species of Sarcocystis in Japanese carnivores remain to be determined. This is the first published report on muscular sarcocystosis in Japanese carnivores.

A total of 206 Virginia opossums ( Didelphis virginiana) collected from the mid-Michigan region, United States, during a period extending from 1996 to 2002 were sampled for the presence of Sarcocystis spp sporocysts. All isolates were phenotypically identified as Sarcocystis spp and genotyped to the species level by PCR-based techniques. The overall prevalence of Sarcocystis spp in opossums was 18% (37/206). The prevalence of Sarcocystis spp differed significantly with age ( P<0.001) and adult opossums were more commonly infected (14.6%; 30/206) than juveniles (3.4%; 7/206). No significant difference in the prevalence of Sarcocystis spp infection was observed between male and female ( P<0.15). The highest prevalence was recorded during summer (9.2%; 19/206). PCR-RFLP analyses demonstrated the majority of Sarcocystis isolates to be S. neurona, with some animals co-infected with sporocysts of S. falcatula. Out of the 37 Sarcocystis-infected opossums, 23 (62%) had sporocysts of S. neurona only, four (11%) had sporocysts of S. falcatula only, and eight (22%) had a mixture of S. neurona and S. falcatula sporocysts. These findings indicate that mixed Sarcocystis infections in opossums are common. The propensity for Sarcocystis spp to co-exist in the opossum gut enhances dissemination and environmental contamination with these coccidia. Additionally, this increases the chance for sexual recombination between Sarcocystis spp, given the proclivity of these species to reproduce sexually at high numbers in the intestinal cells of their definitive host.