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

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

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

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

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

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.

Molecular Epidemiology, Species Distribution, and Zoonotic Importance of the Neglected Meat-Borne Pathogen Sarcocystis spp. in Cattle (Bos taurus): A Global Systematic Review and Meta-analysis.

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.

Infection of the Asian gray shrew Crocidura attenuata (Insectivora: Soricidae) with Sarcocystis attenuati n. sp. (Apicomplexa: Sarcocystidae) in China.

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.

Molecular identification of Sarcocystis halieti in the muscles of two species of birds of prey from Spain.

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.

Molecular identification of Sarcocystis species in diaphragm muscle tissue of European mouflon (Ovis gmelini musimon) from Austria.

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.

Sarcocystis species in bovine carcasses from a Belgian abattoir: a cross-sectional study.

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.

Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis.

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.

Sarcocystis cristata sp. nov. (Apicomplexa, Sarcocystidae) in the imported great blue turaco Corythaeola cristata (Aves, Musophagidae).

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.

Morphologic and molecular identification of three macroscopic Sarcocystis species infecting domestic sheep (Ovis aries) and cattle (Bos taurus) in Egypt.

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.

Molecular identification of four Sarcocystis species in cattle from Lithuania, including S. hominis, and development of a rapid molecular detection method.

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.

Molecular and morphological description of Sarcocystis kutkienae sp. nov. from the common raven (Corvus corax).

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.

Morphological and molecular characterization of Sarcocystis wenzeli in chickens (Gallus gallus) in China.

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.

Sarcocystis spp. infection in South American deer huemul (Hippocamelus bisulcus) and pudu (Pudu puda) from Patagonian National Parks, Argentina.

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.

Redescription and molecular characterization of sarcocysts of Sarcocystis cymruensis from Norway rats (Rattus norvegicus) and Sarcocystis ratti from black rats (R. rattus) in China.

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.

First molecular characterization of Sarcocystis miescheriana in wild boars (Sus scrofa) from Latvia.

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.

Genetic characterization and phylogenetic analysis of Sarcocystis suihominis infecting domestic pigs (Sus scrofa) in India.

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.

Presence of Sarcocystis sybillensis in Japanese sika deer (Cervus nippon) captured in its native territory and its phylogenetic relationship with Sarcocystis nipponi.

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.

Molecular phylogeny of Sarcocystis fayeri (Apicomplexa: Sarcocystidae) from the domestic horse Equus caballus based on 18S rRNA gene sequences and its prevalence.

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.