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 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 four Sarcocystis spp., including Sarcocystis linearis n. sp., from roe deer (Capreolus capreolus) in Italy.

Fresh (frozen/thawed) muscle samples from four 2-12-year-old roe deer (Capreolus capreolus) from the Sondrio province in north-eastern Italy were examined under a dissecting microscope, and about 180 sarcocysts were isolated and identified to morphological type in wet mounts by light microscopy (LM). Seventy-seven of these sarcocysts were subsequently examined by molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of all isolates, as well as PCR amplification, cloning and sequencing of the complete18S ribosomal RNA (rRNA) gene of two isolates of each species found. By LM, three major sarcocyst types were recognised: spindle-shaped sarcocysts, 0.5-3 mm long, either with no clearly recognisable protrusions (thin-walled) or with finger-like protrusions (thick-walled); and slender, thread-like sarcocysts, 2-3 mm long, with hair-like protrusions. Sequencing of cox1 revealed that the sarcocysts belonged to four different species. Those with no visible protrusions either belonged to Sarcocystis gracilis (n = 24) or to a Sarcocystis taeniata-like species (n = 19), whereas those with finger- and hair-like protrusions belonged to Sarcocystis silva (n = 27) and Sarcocystis capreolicanis (n = 7), respectively. The 19 cox1 sequences of the S. taeniata-like species, comprising five haplotypes, differed from each other at 0-16 of 1038 nucleotide positions (98.5-100% identity). They differed from 25 previous cox1 sequences of S. taeniata from moose and sika deer (with 98.0-100% intraspecific identity), at 33-43 nucleotide positions (95.9-96.8% interspecific identity), and there were 20 fixed nucleotide differences between the two populations. In the phylogenetic analysis based on cox1 sequences, the two populations formed two separate monophyletic clusters. The S. taeniata-like species in roe deer was therefore considered to represent a separate species, which was named Sarcocystis linearis n. sp. At the 18S rRNA gene, however, the two species could not be clearly separated from each other. Thus, there was considerable intraspecific sequence variation in the 18S rRNA gene of S. linearis (98.1-99.9% identity between 24 sequences), which was similar both in magnitude and nature to the variation previously found in this gene of S. taeniata. The new 18S rRNA gene sequences of S. linearis shared an identity of 97.9-99.6% with those of S. taeniata (overlap between intra- and interspecific identity), and in the phylogenetic tree, sequences of the two species were interspersed. By scanning electron microscopy (SEM), the sarcocysts of S. linearis were found to possess regularly spaced, thin and narrow ribbon-like cyst wall protrusions (about 2.8-3.2 µm long, 0.3-0.4 µm wide and about 0.02-0.03 µm thick), terminating in a plate-like structure of the same thickness but with an elliptic outline (about 0.3-0.4 µm wide and 0.7-0.9 µm long). The terminal plates were connected in the middle with the band-like portion of the protrusions like the board of a seesaw (tilting board). The terminal plates of adjacent protrusions were neatly arranged in a hexagonal pattern resembling tiles on a roof. Together, they formed an outer roof-like layer facing the surrounding cytoplasm of the host cell and completely covering the band-like proximal portion of the protrusions, which overlapped and were stacked in three to four layers close to the cyst surface. The sarcocyst morphology of S. linearis was consistent with that of an unnamed Sarcocystis sp. in roe deer previously found by transmission electron microscopy in several countries, including Italy. A few sarcocysts of S. gracilis and S. silva were also examined by SEM, confirming the presence of regularly distributed, short knob-like protrusions in S. gracilis (as seen in previous SEM studies) and revealing tightly packed, erect 6-7-µm-long villus-like protrusions having regularly distributed round depressions on their surface in S. silva. The sequencing of cox1 of 7, 24 and 27 new isolates of S. capreolicanis, S. gracilis and S. silva, respectively, recovered 7, 11 and 10 new haplotypes from each of the three species and expanded our knowledge on the intraspecific sequence variation at this marker. Similarly, the study revealed a more extensive intragenomic sequence variation at the 18S rRNA gene of S. capreolicanis and S. silva than known from previous studies and confirmed a near absence of such variation in the 18S rRNA gene of S. gracilis.

Sarcocystis caninum and Sarcocystis svanai n. spp. (Apicomplexa: Sarcocystidae) Associated with Severe Myositis and Hepatitis in the Domestic Dog (Canis familiaris).

There are several reports of Sarcocystis sarcocysts in muscles of dogs, but these species have not been named. Additionally, there are two reports of Sarcocystis neurona in dogs. Here, we propose two new names, Sarcocystis caninum, and Sarcocystis svanai for sarcocysts associated with clinical muscular sarcocystosis in four domestic dogs (Canis familiaris), one each from Montana and Colorado in the USA, and two from British Columbia, Canada. Only the sarcocyst stage was identified. Most of the sarcocysts identified were S. caninum. Sarcocysts were studied using light microscopy, transmission electron microscopy (TEM), and polymerase chain reaction. Based on collective results two new species, S. caninum and S. svanai were designated. Sarcocystis caninum and S. svanai were structurally distinct. Sarcocystis caninum sarcocysts were up to 1.2 mm long and up to 75 µm wide. By light microscopy, the sarcocyst wall was relatively thin and smooth. By TEM, the sarcocyst wall was "type 9", 1-2 µm thick, and contained villar protrusions that lacked microtubules. Bradyzoites in sections were 7-9 µm long. Sarcocysts of S. svanai were few and were identified by TEM. Sarcocystis svanai sarcocysts were "type 1", thin walled (< 0.5 µm), and the wall lacked villar protrusions but had tiny blebs that did not invaginate. DNA was extracted either from infected frozen muscle biopsies or formalin-fixed paraffin-embedded sections. Dogs were either singly infected with S. caninum or multiply co-infected with S. caninum and S. svanai (the result of a mixed infection) based on multilocus DNA sequencing and morphology. BLASTn analysis established that the sarcocysts identified in these dogs were similar to, but not identical to Sarcocystis canis or Sarcocystis arctosi, parasites found to infect polar bears (Ursus maritimus) or brown bears (Ursus arctosi), respectively. However, the S. caninum sequence showed 100% identify over the 18S rRNA region sequenced to that of S. arctica, a parasite known to infect Arctic foxes (Vulpes lagopus).

Sarcocystis clethrionomyelaphis Matuschka, 1986 (Apicomplexa: Sarcocystidae) infecting the large oriental vole Eothenomys miletus (Thomas) (Cricetidae: Microtinae) and its phylogenetic relationships with other species of Sarcocystis Lankester, 1882.

Sarcocystis clethrionomyelaphis Matuschka, 1986 was first identified in skeletal muscles of 47 (75.8%) of 62 large oriental voles Eothenomys miletus (Thomas) captured between March 2012 and May 2014 in Anning Prefecture of Yunnan Province (China). Sarcocyst walls were thick and possessed villous protrusions measuring 3.5-5.5 µm in length. Beauty rat snakes Elaphe taeniura (Cope) fed sarcocysts of the species shed sporulated oöcysts measuring 13-18×9-13 (16×12) µm with a prepatent period of 16 to 17 days. Phylogenetic analysis based on 18S rRNA gene sequences revealed a close relationship between S. clethrionomyelaphis and other colubrid-transmitted species of Sarcocystis Lankester, 1882. This is the first report identifying S. clethrionomyelaphis from its natural intermediate host.

Sarcocystis arieticanis (Apicomplexa: Sarcocystidae) infecting the heart muscles of the domestic sheep, Ovis aries (Artiodactyla: Bovidae), from K. S. A. on the basis of light and electron microscopic data.

In the present study, the heteroxenous life cycle of Sarcocystis species from three strains of the slaughtered sheep at Al-Azizia and Al-Saada abattoirs in Riyadh city, K.S.A., was studied. Muscle samples of the oesophagus, diaphragm, tongue, skeletal and heart muscles were examined. Varied natural infection rates in the muscles of the examined sheep strains were recorded as 83% in Niemy, 81.5% in Najdy and 90% in Sawakny sheep. Muscles of the diaphragm showed the highest infection level above all organs except Najdy sheep in which oesophagus has the highest rate. Also, the heart was the lowest infected organ (40% Niemy, 44% Najdy and 53% Sawakny). Microscopic sarcocysts of Sarcocystis arieticanis are easily identified in sections through the heart muscles of the domestic sheep Ovis aries (Artiodactyla: Bovidae). Cysts measured 38.5-64.4 µm (averaged 42.66 µm) in width and 62.4-173.6 µm (averaged 82.14 µm) in length. The validity of this species was confirmed by means of ultrastructural characteristics of the primary cyst wall (0.1-0.27 µm thick) which revealed the presence of irregularly shaped crowded and hairy-like projections underlined by a thin layer of ground substance. This layer consisted mainly of fine, dense homogenous granules enclosing the developing metrocytes and merozoites that usually contain nearly all the structures of the apical complex and fill the interior cavity of the cyst. Several septa derived from the ground substance divided the cyst into compartments. The merozoites were banana-shaped and measured 12-16 µm in length with centrally or posteriorly located nuclei. Experimental infection of carnivores by feeding heavily infected sheep muscles revealed that the dog, Canis familiaris, is the only final host of the present Sarcocystis species. Gamogony, sporogonic stages and characteristics of sporulated oocysts were also investigated.

Muscular sarcocystosis in two arctic foxes (Vulpes lagopus) due to Sarcocystis arctica n. sp.: sarcocyst morphology, molecular characteristics and phylogeny.

The arctic fox (Vulpes lagopus) is a critically endangered species in Norway, and therefore, the small population is closely monitored, and most foxes found dead are subjected to necropsy. In two deceased foxes, thin-walled muscular sarcocysts were first detected in histological sections, and numerous sarcocysts were later found in frozen and thawed muscle samples from Fox 1. These sarcocysts measured 1-12 × 0.1-0.25 mm and had closely spaced, short, knob-like protrusions, giving the cysts a serrated outline. Genomic DNA was extracted from eight isolated sarcocysts (Fox 1) and two muscle samples (Fox 2) and subjected to polymerase chain reaction amplification at four loci: the nuclear 18S and 28S ribosomal RNA genes and internal transcribed spacer 1 region and the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Both foxes were infected by the same Sarcocystis sp., which displayed little or no genetic variation at the three nuclear loci (99.9-100% identity) and slightly more variation at cox1 (99.4-100% identity). Sequence comparisons and phylogenetic analyses revealed that this species was distinct from other named Sarcocystis spp. but was closely related to various species using avian intermediate hosts and possibly identical to an unnamed species reported from two American dogs. The species described from the two arctic foxes was named Sarcocystis arctica n. sp.

Prevalence of Sarcocystis spp. and Hammondia spp. microcysts in esophagus tissue of sheep and cattle, emphasized on their morphological differences.

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.

Morphological and molecular characterization of Sarcocystis miescheriana from pigs in the central region of China.

Sarcocystosis is an important food-borne parasitosis in humans and various animals. Sarcocystis miescheriana and Sarcocystis suihominis are pathogenic to pigs; S. suihominis is also distinctly pathogenic to humans. Intermediate and final hosts can harbor more than one Sarcocystis species, so the exact identification for Sarcocystis infection in various hosts is essential to control sarcocystosis in humans and important economic animals including pigs. In this study, four isolates of sarcocysts from slaughtered pigs (SmJY1-SmJY4) in the central region of China, in Henan province, were collected and examined by transmission electron microscopy and 18S rRNA sequence analysis to identify the Sarcocystis species in pigs in China. The results showed that cysts in the diaphragm muscles have a thick cyst wall with a number of palisade-like protrusions up to 4.38 µm in length. Inside these protrusions, there were 13-16 fibrils per protrusion. Bradyzoites in cysts showed typical characteristics of Apicomplexa including a conoid, many micronemes, dense bodies, one big nucleus, and a number of amylopectin granules. These ultrastructural results suggest that characteristics of tissue cysts of the isolates SmJY1-SmJY4 were similar to those of S. miescheriana. The sequence similarities of SmJY1-SmJY4 with S. miescheriana were 99-99.5 %, and the sequence similarities of SmJY1-SmJY4 with S. suihominis were much lower. Results of the ultrastructural observation in combination with molecular characterization based on the 18S rRNA sequence represent the first demonstration of S. miescheriana in pigs in China. In addition, results of the histological examination showed that the cysts of S. miescheriana had two types of cyst wall, a palisade-like thick wall and another smoothly thin wall, and could cause obvious atrophy, degeneration, and necrosis of muscle fibers in the diaphragm of naturally infected pigs. These findings will provide an important reference for the examination of Sarcocystis species in the slaughter quarantine of live pigs and in the control of sarcocystosis in pigs.

Sarcocystis acanthocolubri sp. n. infecting three lizard species of the genus Acanthodactylus and the problem of host specificity. Light and electron microscopic study.

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.

Description of Sarcocystis anasi sp. nov. and Sarcocystis albifronsi sp. nov. in birds of the order Anseriformes.

On the basis of the already published morphological, 18S rDNA, 28S rDNA data (Kutkiene et al., Parasitol Res 99:562-565, 2006; Parasitol Res 102:691-696, 2008; Parasitol Res 104:329-336, 2009), and ITS-1 region investigation results of sarcocysts presented in this paper, Sarcocystis albifronsi sp. nov. from the white-fronted goose (Anser albifrons) and Sarcocystis anasi sp. nov. from the mallard duck (Anas platyrhynchos) are described.

Ongoing outbreak of an acute muscular Sarcocystis-like illness among travellers returning from Tioman Island, Malaysia, 2011-2012.

As of 4 November, 2012, 100 patients with an acute muscular Sarcocystis-like illness associated with travel to Tioman Island, Malaysia, have been identified. Thirty-five travelled there mostly during July and August 2011 and 65 mostly during July and August 2012, suggesting an ongoing outbreak. Epidemiological investigations are ongoing. Public health agencies and practicing clinicians should be aware of this rarely-reported disease in humans and consider it as differential diagnosis in travellers returning from Tioman Island.

Infection survey and morphological characteristics of Sarcocystis spp. in naturally infected Tibetan sheep from Qinghai in northwestern China.

Sarcocystosis is a parasitic disease caused by intracellular coccidian protozoans that belong to the genus Sarcocystis. These parasites can cause diseases of the nervous system, abortion and economically significant losses in host animals. Previous studies have reported that Sarcocystis is found in mammals, birds and reptiles, while molecular and morphological studies of infected Tibetan sheep have not been performed in the Qinghai region. The aim of this study was to determine the prevalence of Sarcocystis spp. in Tibetan sheep in Qinghai, northwestern China. The results showed that in 1155 samples, sarcocysts from unspecified species were found in 50% (577/1155) of the sheep tissues by microscopy detection. The positive rates of sarcocysts in the diaphragmatic, esophageal and cardiac muscles were 78.4% (175/223), 29.1% (207/711), and 88.2% (195/221), respectively. Ultrastructural features were exclusively observed in Sarcocystis gigantea in the esophageal tissues. The specific architecture was characterized as a space between the two layers of the original capsule wall, which was filled with fiber bundles and tissue fluid. Cauliflower-like protrusions of the original capsule wall were observed toward the outer surface of the capsule. Prominent protrusions contained fibers and matrix. In addition, the Sarcocystis 18S rRNA genes from 6 esophageal tissue samples were cloned, sequenced, and aligned to related sequences from GenBank. All 5 S. gigantea sequences examined in this study were grouped into the same cluster and belonged to the same genotype. The other 5 Sarcocystis tenella sequences were obtained from cardiac muscle and diaphragm muscle and belonged to the same clade. Overall, this study revealed a high infection rate of Sarcocystis in Tibetan sheep in the region. The results of this study may provide a reference for further research investigating the sarcocystosis epidemic in Qinghai, China.

Corvid birds (Corvidae) act as definitive hosts for Sarcocystis ovalis in moose (Alces alces).

Epidemiological data and a unique phylogenetic position had suggested that Sarcocystis ovalis in moose and red deer might use a definitive host other than canids, felids, or humans. Corvid birds and rats were therefore evaluated as potential definitive hosts for this species in a small pilot study. Four laboratory rats were each inoculated with 10 or 25 sarcocysts of S. ovalis isolated from moose, but no Sarcocystis oocysts were detected in their intestinal mucosa upon euthanasia 2 to 3 weeks later. At a site where large flocks of corvid birds (hooded crows, ravens and European magpies) fed on remnants of moose carcasses during the hunting period in October, fresh bird droppings were collected on the ground and examined microscopically and by molecular methods. By microscopy, a small number of typical Sarcocystis sporocysts, measuring 12.8 × 8.4 µm, were found in the faecal samples. These sporocysts were identified as belonging to S. ovalis by a polymerase chain reaction assay using specific primer pairs targeting the ssu rRNA gene, followed by sequence analysis. The intestinal contents of a crow and two magpies shot near the dumping site were also examined. Sarcocystis oocysts (16.1 × 12.4 µm) and free sporocysts (12.5 × 7.9 µm) were found in the intestinal mucosa/contents of one magpie (Pica pica). These oocysts/sporocysts were also found to belong to S. ovalis by the same molecular assay. This is the first report of corvid birds acting as definitive hosts for a species of Sarcocystis.

The mallard duck (Anas platyrhynchos) as intermediate host for Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis).

Morphometric and DNA investigation results of Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis) and Sarcocystis sp. (cyst type IV) from the mallard duck (Anas platyrhynchos) are presented. No significant morphometric differences between the investigated Sarcocystis species were found. ITS-1, 18S rRNA, and 28S rRNA gene sequences of these species showed 100% identity. The conclusion is drawn that it is one and the same Sarcocystis species in different intermediate hosts.

Sarcocystis calchasi sp. nov. of the domestic pigeon (Columba livia f. domestica) and the Northern goshawk (Accipiter gentilis): light and electron microscopical characteristics.

A novel highly pathogenic Sarcocystis species has been shown to cycle between the Northern goshawk (Accipiter gentilis) as definitive host and the domestic pigeon (Columba livia f. domestica) as intermediate host. However, genetically based characteristics are only available from very few bird-infecting Sarcocystis species. We therefore further characterised morphological properties of this protozoan in both hosts. Using light and electron microscopy, oocysts and sporocysts as well as schizonts and sarcocysts were characterised and compared with available morphological features of previously reported Sarcocystis species of Northern goshawks, Columbidae and genetically closely related species of other avian hosts. Sporocysts shed from day 6 on after experimental infection by the Northern goshawk were of ovoid appearance (11.9 x 7.9 microm). Ultrastructurally, schizonts of all developmental stages were found in the liver, spleen and next to or in endothelial cells of various organs of domestic pigeons 7 to 12 days after experimental infection. The cyst wall surface of slender sarcocysts (1 to 2 mm in length and 20 to 50 microm in width) was smooth and lacked protrusions. Cystozoites were lancet-shaped and measured 7.5 x 1.5 microm in Giemsa stain smears. The morphological findings, when combined with data of experimental infection and genetic studies, convergently indicate that the recently discovered Sarcocystis species represents a new species. We therefore propose to name this parasite Sarcocystis calchasi species nova.

Sarcocystosis among wild captive and zoo animals in Malaysia.

Sarcocystis sp. infection was investigated in 20 necropsied captive wild mammals and 20 birds in 2 petting zoos in Malaysia. The gross post-mortem lesions in mammals showed marbling of the liver with uniform congestion of the intestine, and for birds, there was atrophy of the sternal muscles with hemorrhage and edema of the lungs in 2 birds. Naked eye examination was used for detection of macroscopic sarcocysts, and muscle squash for microscopic type. Only microscopically visible cysts were detected in 8 animals and species identification was not possible. Histological examination of the sections of infected skeletal muscles showed more than 5 sarcocysts in each specimen. No leukocytic infiltration was seen in affected organs. The shape of the cysts was elongated or circular, and the mean size reached 254 x 24.5 µm and the thickness of the wall up to 2.5 µm. Two stages were recognized in the cysts, the peripheral metrocytes and large numbers of crescent shaped merozoites. Out of 40 animals examined, 3 mammals and 5 birds were positive (20%). The infection rate was 15% and 25% in mammals and birds, respectively. Regarding the organs, the infection rate was 50% in the skeletal muscles followed by tongue and heart (37.5%), diaphragm (25%), and esophagus (12.5%). Further ultrastructural studies are required to identify the species of Sarcocystis that infect captive wild animals and their possible role in zoonosis.

Molecular identification of two Sarcocystis species in fallow deer (Dama dama) from Lithuania.

Due to the lack of molecular research conducted, little is known about Sarcocystis species diversity in the fallow deer (Dama dama). Until now, Sarcocystis jorrini and Sarcocystis morae were described to form sarcocysts in the muscles of this host. In the present study diaphragm muscle samples of free-ranging fallow deer from Lithuania were investigated for Sarcocystis species. Sarcocysts were detected in 39 out of 48 (81.3%) fallow deer examined. Under a light microscope two types of sarcocysts having hair-like and finger-like protrusions were observed. Based on DNA sequence analysis of cox1 and 18S rDNA, two species, S. morae and Sarcocystis entzerothi were identified. In prior studies, the latter species was only detected in Lithuanian roe deer (Capreolus capreolus) and in sika deer (Cervus nippon). The haplotype network of S. morae sequences specified close relationships between haplotypes found in the same country. According to current knowledge, the fallow deer is characterised by low Sarcocystis species richness as compared with other cervid species from Europe.

Sarcocystis: development in cultured avian and mammalian cells.

Sarcocystis, a protozoan that parasitizes muscle tissue of reptiles, birds, and mammals, including man, developed in avian and mammalian cell cultlures. Mctile banana-shaped organisms, released from cysts in grackle muscle, entered cells and transformed into enlarged ellipsoid or oblong stages, which developed into either multinucleate or cyst-like stages, or both.

Gametogony of Sarcocystis sp. in cell culture.

Sexual stages and cystlike bodies of Sarcocystis sp., a protozoan parasite found in muscles of reptiles, birds, and mammals, including man, developed in cell culture. Motile organisms, obtained from leg muscles of wild grackles, were inoculated into cell line cultures of embryonic bovine kidney. Mature micro-and macrogametes and the cystlike forms were found 30 and 42 hours after inoculation respectively. These observations indicate that the parasite is probably a coccidium.