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 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.

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.

Identification of opossums Didelphis aurita (Wied-Neuweid, 1826) as a definitive host of Sarcocystis falcatula-like sporocysts.

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.

Detection and Identification of Sarcocystis cruzi (Protozoa: Apicomplexa) by Molecular and Ultrastructural Studies in Naturally Infected Korean Cattle (Bos taurus coreanae) from Daejeon, Korea.

To survey the prevalence of Sarcocystis infections, 210 heart samples were collected from Korean native cattle (Bos taurus coreanae) at an abattoir in Daejeon Metropolitan City, Republic of Korea. Sarcocysts were detected form 31 specimens (14.8%) and identified as Sarcocystis cruzi via transmission electron microscopy. The wall of S. cruzi has flattened protrusions that did not contain fibrils or microfilaments. The protrusions arose irregularly from the base, contained a fine granular substance, lacked internal microfilaments, and measured approximately 0.21-1.25 µm in length and 0.05-0.07 µm in width. Sequence analysis revealed 99.5% homology to S. cruzi. This is the first report on the prevalence of S. cruzi in native cattle from the Republic of Korea.

Sarcocystis dehongensis n. sp. (Apicomplexa: Sarcocystidae) from water buffalo (Bubalus bubalis) in China.

Water buffalo (Bubalus bubalis) is the intermediate host for at least four species of Sarcocystis: S. fusiformis, S. buffalonis, S. levinei, and S. sinensis/S. dubeyi. Here, a new species, Sarcocystis dehongensis, is reported in 51 of 756 (6.7%) water buffaloes in China. By light microscopy, the cysts of S. dehongensis were macroscopic, up to 18.5 mm long and 95 µm in diameter; 4.9-11.9 µm villous protrusions extended beyond the sarcocyst wall. Using transmission electron microscopy, the sarcocyst wall had lancet- or leaf-like protrusions in longitudinal section, but the cross section showed that the protrusions appeared as mushroom-like in shape with a core of tightly packed microtubules, similar to "type 24." BLAST searches revealed that S. dehongensis shared the most similarities with the 18S rDNA sequence of S. hardangeri (92.4%) and mitochondrial cox1 gene sequence of S. ovalis (81.0%), whereas no sequences in GenBank were found to be significantly similar to the ITS-1 region of S. dehongensis. A phylogenetic analysis based on 18S rDNA and mitochondrial cox1 gene sequences suggested that S. dehongensis was closely related to Sarcocystis species from cervids that employ corvids as definitive hosts.

Morphological and molecular characteristics of six Sarcocystis spp. from red deer (Cervus elaphus) in Spain, including Sarcocystis cervicanis and three new species.

Samples of muscle tissue from the diaphragm, oesophagus and/or heart of eight adult red deer (Cervus elaphus hispanicus) from the Quintos de Mora Park in Toledo, Central Spain, were screened for sarcocysts by means of the compression method. From positive samples, individual sarcocysts were excised and examined in wet mounts under a light microscope (LM) in order to study their basic morphology before being preserved for molecular studies. In all red deer examined, only microscopic sarcocysts were found. Those in the diaphragm and oesophagus were spindle-shaped and about 1 × 0.1 mm in size, while those in cardiac muscle were sac-like and 500-800 × 80-180 µm. By LM, the sarcocysts either had densely packed, about 8-µm-long, hair-like protrusions (type 1), sparsely distributed indistinct projections (fuzzy outline; type 2) or no visible protrusions (smooth surface; type 3). In cardiac muscle, only sarcocysts without visible protrusions were found. One of the latter sarcocysts was examined by scanning electron microscopy (SEM) and found to possess thin ribbon-like protrusions. Forty-eight sarcocysts isolated from the diaphragm, oesophagus and heart of one red deer, as well as 55 sarcocysts from the heart of three other red deer, 103 sarcocysts in total, were characterized molecularly through PCR amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of the mitochondrial genome, revealing the presence of six major cox1 sequence types. Each type comprised either a single sequence (three types) or a collection of several identical or nearly identical sequences. From selected isolates possessing each of these cox1 sequence types, the complete 18S ribosomal RNA (rRNA) gene was amplified and sequenced directly and/or after cloning of the 5' end half. Supported by the sequence data from the latter gene, as well as the morphology of the sarcocysts from which the sequences originated, the six cox1 sequence types were considered to represent six separate Sarcocystis spp. Two cox1 sequence types were identified as belonging to the previously characterized species Sarcocystis hjorti (one sequence/sarcocyst) and Sarcocystis linearis (38 sequences/sarcocysts), respectively, whereas four sequence types were new. One of the latter types was assigned to the previously named species Sarcocystis cervicanis from red deer, since this sequence type was obtained from 52 sarcocysts from cardiac muscle, which matched the original morphological description (smooth surface) and habitat of this species. The remaining three sequence types were assigned to the three new species Sarcocystis iberica (one sequence/sarcocyst) Sarcocystis venatoria (10 sequences/sarcocysts) and Sarcocystis morae (one sequence/sarcocyst), respectively. The two species S. iberica and S. venatoria shared the same sarcocyst morphology (type 1) and habitat (diaphragm) and had virtually identical 18S rRNA gene sequences, but differed by 4% at cox1, which was considered sufficient to regard them as separate species. The single sarcocyst of S. morae (from the oesophagus) examined by LM had a smooth wall and this species was therefore believed to have the same type of ribbon-like protrusions (ultrastructurally) as sarcocysts of S. cervicanis and S. linearis, which were also the species most closely related to S. morae at cox1. Thus, there seems to be three species with similar ribbon-like cyst wall protrusions in red deer (S. cervicanis, S. linearis, S. morae), as well as three species with similar hair-like protrusions (S. hjorti, S. iberica, S. venatoria). Sarcocysts of S. cervicanis were only identified in cardiac muscle, whereas sarcocysts of S. linearis were found mainly in the diaphragm and oesophagus and rarely in the heart. The relative number of cox1 haplotypes was greater among sequences/isolates of S. linearis (17/38) than among isolates of S. cervicanis (7/52). Four of the species examined (S. cervicanis, S. linearis, S. iberica, S. venatoria) possessed considerable intra-isolate (intra-genomic) sequence variation (insertions/deletions, substitutions) in the 18S rRNA gene.

Morphological and molecular characterization of Sarcocystis arctica-like sarcocysts from the Arctic fox (Vulpes lagopus) from Alaska, USA.

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.

Sarcocystis menglaensis n. sp. (Apicomplexa: Sarcocystidae) from Williamson's mouse deer Tuagulus williamsoni (Kloss) (Artiodactyla: Tragulidae).

Williamson's mouse deer, Tuagulus williamsoni (Kloss), is one of the smallest ungulates among tragulid species found in northern Thailand, and Yunnan Province, China. Here we describe Sarcocystis menglaensis n. sp., infecting two of 14 (14.3%) Williamson's mouse deer from south-western China. By light microscopy, sarcocysts of S. menglaensis are microscopic, up to 2,170 µm in length, and have a striated sarcocyst wall with 1.5-3.6 µm long palisade-like protrusions. Transmission electron microscopy observations revealed that sarcocyst wall is of "type 10f", and has numerous villar protrusions folded over the cyst wall. The villar protrusions contained microtubules dispersed throughout the protrusions. Phylogenetic analysis based on 18S rDNA and mitochondrial cox1 gene sequences indicated that S. menglaensis shared a close affinity with species of Sarcocystis Lankester, 1982 from ruminants, which utilise felids as definitive hosts.

Sarcocystis rommeli, n. sp. (Apicomplexa: Sarcocystidae) from Cattle (Bos taurus) and its Differentiation from Sarcocystis hominis.

Cattle (Bos taurus) are intermediate hosts for three named species of Sarcocystis, S. cruzi, S. hirsuta, and S. hominis. Recently, a fourth species was identified and named S. sinensis. However, S. sinensis originally named a species of Sarcocystis in water buffalo (Bubalus bubalis) in China. Based on unverifiable evidence, it was suggested that the same parasite infects cattle. In addition, S. sinensis was recently declared as nomen nudum because its naming violated the rules of International Code of Zoological Nomenclature. Thus, the fourth species using cattle as an intermediate host does not have a valid name. Here, we propose a new name, Sarcocystis rommeli for the S. sinensis-like parasite from cattle in Argentina, and differentiate it ultrastructurally from S. hominis sarcocysts from experimentally infected cattle. Sarcocystis rommeli sarcocysts were microscopic with a 5-µm-thick wall with slender villar protrusions (Vp); the Vp were up to 5 µm long, up to 0.5 µm wide, and of uneven thickness, often bent at an angle. The ground substance layer (Gs) was up to 0.8 µm thick and smooth. Vesicular structures were seen at the base of the Vp. The bradyzoites were 10-12 µm long. Sarcocystis hominis sarcocysts had Vp that were often upright, up to 7.5 µm long, and up to 1.8 µm wide; the Gs was up to 2 µm thick and without vesicles. Its sarcocyst wall was up to 5.6 µm thick, the vp were bent at an angle, up to 5.8 µm long, the Gs was up to 2 µm thick, but without vesicles seen in S. rommeli. Beef containing sarcocysts of S. rommeli was not orally infectious for two human volunteers and a red fox (Vulpes vulpes). The Sarcocystis described here is molecularly different from S. cruzi, S. hirsuta, and S. hominis based on 18S rRNA and cox1 gene sequences.

Ultrastructure of Sarcocystis bertrami sarcocysts from a naturally infected donkey (Equus asinus) from Egypt.

There is considerable confusion concerning Sarcocystis species in equids. Little is known of Sarcocystis infections in donkeys (Equus asinus). Here we describe the structure of Sarcocystis bertrami-like from the donkey by light microscopy (LM) and transmission electron microscopy (TEM). Nineteen sarcocysts from the tongue of a donkey from Egypt were studied both by LM and TEM. By LM, all sarcocysts had variably shaped and sized projections on the sarcocyst walls, giving it a thin-walled to thick-walled appearance, depending on individual sarcocyst and plane of section. By TEM, sarcocysts walls had villar protrusions (vp) of type 11. The sarcocyst wall had conical to slender vp, up to 6 µm long and 1 µm wide; the vp were folded over the sarcocyst wall. The total thickness of the sarcocyst wall with ground substance layer (gs) was 1-3 µm. The vp had microtubules (mt) that originated deeper in the gs and continued up to the tip. The apical part of the vp had electron dense granules. The mt were configured into 3 types: a tuft of electron dense mt1 extending the entire length of the vp with a tuft of medium electron dense mt2 appearing in parallel, and fine mt3 present only in the villar tips. The gs was mainly smooth with few indistinct granules. All sarcocysts were mature and contained metrocytes and bradyzoites. Bradyzoites were approximately 11-15 × 2-3 µm in size with typical organelles.

Sarcocystis masoni, n. sp. (Apicomplexa: Sarcocystidae), and redescription of Sarcocystis aucheniae from llama (Lama glama), guanaco (Lama guanicoe) and alpaca (Vicugna pacos).

There is considerable confusion concerning the species of Sarcocystis in South American camelids (SAC). Several species names have been used; however, proper descriptions are lacking. In the present paper, we redescribe the macroscopic sarcocyst forming Sarcocystis aucheniae and describe and propose a new name, Sarcocystis masoni for the microscopic sarcocyst forming species. Muscles samples were obtained from llamas (Lama glama) and guanacos (Lama guanicoe) from Argentina and from alpacas (Vicugna pacos) and llamas from Peru. Individual sarcocysts were processed by optical and electron microscopy, and molecular studies. Microscopic sarcocysts of S. masoni were up to 800 µm long and 35-95 µm wide, the sarcocyst wall was 2·5-3·5 µm thick, and had conical to cylindrical villar protrusions (vp) with several microtubules. Each vp had 11 or more rows of knob-like projections. Seven 18S rRNA gene sequences obtained from sarcocysts revealed 95-96% identity with other Sarcocystis spp. sequences reported in the GenBank. Sarcocysts of S. aucheniae were macroscopic, up to 1·2 cm long and surrounded by a dense and laminar 50 µm thick secondary cyst wall. The sarcocyst wall was up to 10 µm thick, and had branched vp, appearing like cauliflower. Comparison of the 11 sequences obtained from individual macroscopic cysts evidenced a 98-99% of sequence homology with other S. aucheniae sequences. In conclusion, 2 morphologically and molecularly different Sarcocystis species, S. masoni (microscopic cysts) and S. aucheniae (macroscopic cysts), were identified affecting different SAC from Argentina and Peru.

The resurrection of a species: Sarcocystis bovifelis Heydorn et al., 1975 is distinct from the current Sarcocystis hirsuta in cattle and morphologically indistinguishable from Sarcocystis sinensis in water buffaloes.

In the mid-1970s, it was established through transmission experiments and ultrastructural studies of sarcocysts by transmission electron microscopy (TEM) that cattle was the intermediate host of three Sarcocystis spp. using dogs, cats and humans, respectively, as definitive hosts. The cat-transmitted species with microscopic sarcocysts was initially named Sarcocystis bovifelis, but it was soon renamed Sarcocystis hirsuta, since it was considered to be identical with a previously named species. In recent years, an apparently new species has been detected in cattle in several countries by molecular methods and TEM and found by both methods to be indistinguishable from Sarcocystis sinensis in water buffaloes. This species was recently named Sarcocystis rommeli. Beginning in August 2014, a thorough review of papers comprising TEM micrographs of thick-walled sarcocysts in cattle was made in order to determine whether S. sinensis-like sarcocysts had been reported previously under other designations. Surprisingly, the review showed that the species S. bovifelis Heydorn et al., 1975 as described from cattle in Germany was S. sinensis-like and that indistinguishable sarcocysts had also been found in cattle in New Zealand and Canada in the 1980s. However, in the New Zealand study, these small sarcocysts were erroneously thought to represent developmental stages of a species with ultrastructurally similar but macroscopic sarcocysts, since the macroscopic cysts were found to be infective for cats. Thus, in the late 1980s, the cat-transmitted S. bovifelis, after having been renamed S. hirsuta, was erroneously synonymised with a second cat-transmitted species in cattle and then slid into obscurity until recently being rediscovered as a S. sinensis-like species in cattle and then named S. rommeli. Following the erroneous synonymisation, the name S. hirsuta has consistently been used for a taxon with macroscopic sarcocysts, and this usage should be continued. The name S. bovifelis should again be used not only for the species originally described from cattle in Germany but also for morphologically indistinguishable taxa recently reported from cattle under the names S. sinensis and S. rommeli. Because of the morphological similarity between S. bovifelis and S. sinensis, it is likely that cats also act as definitive hosts for S. sinensis. The present paper also gives a thorough review of all research in the 1970s pertaining to S. bovifelis, including its development in cats and cattle; a review of reports of S. bovifelis-like sarcocysts in cattle, water buffaloes and other hosts; and a review of reports of the taxon currently named S. hirsuta in cattle. The usage of the name S. sinensis versus Sarcocystis dubeyi for the S. bovifelis-like taxon in water buffaloes is discussed, and the latter name is found to represent a nomen dubium since the original description concerned a mixture of a S. sinensis- and a Sarcocystis hominis-like species. Based on available transmission (TEM) and scanning electron microscopy (SEM) images, the three-dimensional configuration of the cyst wall protrusions of S. bovifelis/S. sinensis and the current S. hirsuta has been inferred and is described. The protrusions of S. bovifelis/S. sinensis are shaped like soft plastic tubes, having a cylindrical basal portion and a flattened distal portion, making them prone to fold over. The protrusions of the current S. hirsuta are thin, flattened and flexible rectangular structures (like a soft cover note book), which are attached to the cyst surface with a narrow stalk. The appearance of both types of protrusions in ultrathin sections viewed by TEM is highly dependent on how the sarcocysts and the protrusions themselves have been sectioned.

Sarcocystis arctica (Apicomplexa: Sarcocystidae): ultrastructural description and its new host record, the Alaskan wolf (Canis lupus).

Sarcocystis sarcocysts are common in muscles of herbivores but are rare in muscles of carnivores. Here, we report sarcocysts in the muscles of a gray wolf (Canis lupus) from Alaska, USA, for the first time. Sarcocysts extracted from the tongue of the wolf were up to 900 µm long and slender and appeared to have a relatively thin wall by light microscope. By transmission electron microscopy, the sarcocyst wall most closely resembled "type 9c," and had a wavy parasitophorous vacuolar membrane folded as pleomorphic villar protrusions (vp), with anastomoses of tips. The vp and the ground substance (gs) layer were smooth without tubules or granules. The gs was up to 2.0 µm thick. The total width of the wall including vp and the gs was 3.5 µm. The vp were up to 1.5 µm long. Mature sarcocysts contained numerous bradyzoites and few metrocytes. The bradyzoites were 9.5 µm long and 1.5 µm wide, and contained all organelles found in Sarcocystis bradyzoites with at least two rhoptries. Molecular characterization showed the highest identity for 18S rRNA, 28S rRNA, ITS-1, and cox1 sequences of Sarcocystis arctica of the Arctic fox (Vulpes lagopus) from Norway. The ultrastructure of S. arctica from the fox is unknown. Here, we provide ultrastructure of S. arctica from the Alaskan wolf for the first time. The definitive host of S. arctica remains unknown.

Molecular differentiation of Sarcocystis buffalonis and Sarcocystis levinei in water buffaloes (Bubalus bubalis) from Sarcocystis hirsuta and Sarcocystis cruzi in cattle (Bos taurus).

The purpose of the present study was to obtain sarcocysts of Sarcocystis buffalonis and Sarcocystis levinei from water buffaloes and characterize the isolates by molecular methods in order to determine whether the two species were genetically different from Sarcocystis hirsuta and Sarcocystis cruzi, respectively, from cattle, which had been characterized before. About 35 macroscopically visible (3-4 × 1-2 mm) and 20 barely visible (1-3 × 0.2 mm) sarcocysts were excised from the esophagus of 18 naturally infected and freshly slaughtered adult water buffaloes at three slaughterhouses in Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were first characterized at the mitochondrial cytochrome c oxidase subunit I gene (cox1) gene through PCR amplification and direct sequencing. Selected isolates were subsequently further characterized at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit by direct sequencing or cloning. Only six of the isolated macroscopic sarcocysts belonged to S. buffalonis, whereas the others belonged to Sarcocystis fusiformis. Twelve of the smaller cysts belonged to S. levinei and seven to Sarcocystis sinensis. The characterization of the sarcocysts of S. sinensis and some of the sarcocysts of S. fusiformis have been reported before. Fifteen additional sarcocyst isolates of S. fusiformis were characterized at cox1 in the present study and found to be identical or closely similar to previous isolates. At cox1, the sequence identity between the six isolates of S. buffalonis was 99.8-100 % (two haplotypes), whereas the identity between the 12 isolates of S. levinei was 99.0-100 % (10 haplotypes). The identity between cox1 sequences of S. buffalonis and S. hirsuta (n = 56) was 92.9-93.6 % (on average 93.4 %), and the identity between cox1 sequences of S. levinei and S. cruzi (n = 22) was 92.9-94.0 % (on average 93.5 %). The phylogenetic analyses placed with high support the cox1 sequences of S. buffalonis and S. hirsuta into two monophyletic sister groups, and the same was true for the cox1 sequences of S. levinei and S. cruzi. Hence, the study established that S. buffalonis and S. levinei are distinct species different from S. hirsuta and S. cruzi, respectively. Nucleotide sequences of S. buffalonis could be distinguished from those of S. hirsuta also at the 28S rRNA gene (clearly different) and the ITS1 region (small and uncertain difference) but not at the 18S rRNA gene. Sequences of S. levinei could be distinguished from those of S. cruzi both at the 18S and 28S rRNA genes (ITS1 region not examined). However, the cox1 gene was superior to the 18S and 28S rRNA genes as regards the ability to unambiguously delimit the species within each species pair, since at the latter markers, the number of consistent nucleotide differences between the species was low and there was a slight overlap between the intraspecific and interspecific sequence divergence. Comparison of the newly generated 18S rRNA gene sequences of S. levinei from water buffaloes with similar sequences deposited in GenBank suggested that S. levinei and S. cruzi are not strictly intermediate host specific but might occasionally infect cattle and water buffaloes, respectively.

Prevalence, morphology, and molecular characteristics of Sarcocystis spp. in domestic goats (Capra hircus) from Kunming, China.

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.

Redescription of Sarcocystis fusiformis sarcocysts from the water buffalo (Bubalus bubalis).

Four valid species of Sarcocystis have been reported from the water buffalo (Bubalus bubalis): Sarcocystis fusiformis, Sarcocystis buffalonis, Sarcocystis levinei and Sarcocystis dubeyi. Here, we redescribe structure of S. fusiformis sarcocysts by scanning and transmission electron microscopy (SEM, TEM). Twenty-one macroscopic sarcocysts from oesophagus of the water buffalo in Egypt were examined by light microscopy, SEM and TEM. The sarcocyst wall was up to 9 µm thick, depending on the section and the technique. In 5 µm paraffin-embedded sections, the sarcocyst wall was indistinct, 2-5 µm thick and appeared smooth. In 1 µm plastic-embedded sections stained with toluidine blue, the sarcocyst wall was 2.5-5.2 µm thick and had branched villar protrusions (vp)-like branches of a dead tree. By SEM, the sarcocyst wall had a mesh-like structure with irregularly shaped vp that were folded over the sarcocyst wall. On each vp there were uniform papillomatous structures that were 100 nm wide. By TEM, vp were up to 6 µm long and contained filamentous tubular structures, most of which were parallel to the long axis of the projections; granules were absent from these tubules. By TEM, bradyzoites within the same cyst varied from 11.2 to 16.8 µm in length. By TEM, bradyzoites had a very long (10 µm) convoluted mitochondrion, up to 12 dense granules, but only 2 rhoptries. This redescription should help to differentiate the sarcocysts of S. fusiformis from similar sarcocysts in domestic and wild ruminants.

A review of sarcocystosis in camels and redescription of Sarcocystis cameli and Sarcocystis ippeni sarcocysts from the one-humped camel (Camelus dromedarius).

There is considerable confusion concerning Sarcocystis species in camels. Five species: Sarcocystis cameli, Sarcocystis ippeni, Sarcocystis camelicanis, Sarcocystis camelocanis and Sarcocystis miescheri were named with inadequate descriptions and no type specimens. Here, we review literature on sarcocystosis in camels worldwide and redescribe structure of S. cameli and S. ippeni sarcocysts by light- and transmission electron microscopy (LM and TEM). Eight sarcocysts from the oesophagi of two camels (Camelus dromedarius) from Egypt were studied. By LM, all sarcocysts were thin-walled with barely visible projections on the cyst walls. By TEM, two structurally distinct sarcocysts were recognized by unique villar protrusions (vp) not found in sarcocysts from any other host. Sarcocysts of S. cameli had vp of type 9 j. The sarcocyst wall had upright slender vp, up to 3.0 µM long and 0.5 µM wide; the total thickness of the sarcocyst wall with ground substance (gs) layer was 3.5 µM. On each vp, there were rows of knob-like protrusions that appeared to be interconnected. The vp had microtubules that originated at midpoint of the gs and continued up to the tip; microtubules were smooth, without any granules or dense areas. Bradyzoites were approximately 14-15 × 3-4 µM in size with typical organelles. Sarcocystis ippeni sarcocysts had type 32 sarcocyst wall characterized by conical vp with an electron dense knob. The total thickness of the sarcocyst wall (from the base of gs to vp tip) was 2.3-3.0 µM. The vp were up to 1.2 µM wide at the base and 0.25 µM at the tip. Microtubules in vp originated at midpoint of gs and continued up to tip; microtubules were criss-crossed, smooth and without granules or dense areas. Bradyzoites were 12.0-13.5 × 2.0-3.0 µM in size. Sarcocystis camelicanis, S. camelocanis and S. miescheri are considered invalid.

Sarcocystis chloropusae (protozoa: Sarcocystidae) n. sp. from the common moorhen (Gallinula chloropus) from Egypt.

A new name Sarcocystis chloropusae is proposed for a parasite previously found in two of 25 common moorhen (Gallinula chloropus) from Brolos Lake, Egypt. Sarcocysts were microscopic, up to 650 µm long, the cyst wall was up to 4.5 µm thick, and contained villar protrusions that were up to 4 µm long and up to 2 µm wide. The villar protrusions were crowded, contained vesicles but lacked microtubules. The ground substance layer was smooth. The bradyzoites were up to 12 µm long and up to 2 µm wide. Molecular characterization and phylogenetic analysis of the (ITS-1) supported the conclusion that the Sarcocystis in G. chloropus is a distinct species.

Molecular characterization and development of Sarcocystis speeri sarcocysts in gamma interferon gene knockout mice.

The North American opossum (Didelphis virginiana) is the definitive host for at least three named species of Sarcocystis: Sarcocystis falcatula, Sarcocystis neurona and Sarcocystis speeri. The South American opossums (Didelphis albiventris, Didelphis marsupialis and Didelphis aurita) are definitive hosts for S. falcatula and S. lindsayi. The sporocysts of these Sarcocystis species are similar morphologically. They are also not easily distinguished genetically because of the difficulties of DNA extraction from sporocysts and availability of distinguishing genetic markers. Some of these species can be distinguished by bioassay; S. neurona and S. speeri are infective to gamma interferon gene knockout (KO) mice, but not to budgerigars (Melopsittacus undulatus); whereas S. falcatula and S. lindsayi are infective to budgerigars but not to KO mice. The natural intermediate host of S. speeri is unknown. In the present study, development of sarcocysts of S. speeri in the KO mice is described. Sarcocysts were first seen at 12 days post-inoculation (p.i.), and they became macroscopic (up to 4 mm long) by 25 days p.i. The structure of the sarcocyst wall did not change from the time bradyzoites had formed at 50-220 days p.i. Sarcocysts contained unique villar protrusions, 'type 38'. The polymerase chain reaction amplifications and sequences analysis of three nuclear loci (18S rRNA, 28S rRNA and ITS1) and two mitochondrial loci (cox1 and cytb) of S. speeri isolate from an Argentinean opossum (D. albiventris) confirmed its membership among species of Sarcocystis and indicated an especially close relationship to another parasite in this genus that employs opossums as its definitive host, S. neurona. These results should be useful in finding natural intermediate host of S. speeri.