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Effects of frozen storage on the structure of sarcocysts in pig muscle and implications in taxonomic studies
Abstract
Unlabelled:
The morphology of the cyst wall of Sarcocystis has unique characteristics that can be used in species identification. To find a suitable way to preserve Sarcocystis cyst samples for species identification, by light microscopy and electron microscopy, we recorded the morphological changes in the cysts of Sarcocystis suihominis and Sarcocystis miescheriana from pig muscle, induced by storage at -20 degrees C. Comparisons were made between fresh cysts and those subjected to frozen storage for periods of 3 days, 20 days and 30 days.
Results:
cyst wall of the two Sarcocystis species appeared unaffected by storage. There was no obvious change in the length, nor in the width of the protrusions after storage (P>0.05), but the structure of the bradyzoite in the sarcocyst was in many cases disintegrated at -20 degrees C in 20 days for S. miescheriana and 30 days for S. suihominis. To our knowledge this is the first report that Sarcocystis cyst in muscle can be stored at -20 degrees C before and remain suitable for ultrastructural morphological study. Consequently, this paper proposes freezing as a convenient storage method for samples used in taxonomic studies of Sarcocystis.
... Those authors assessed infectivity by feeding cats with cysts and monitoring the presence of oocysts or sporocysts in their faeces; moreover, they did not evaluate viability in the period between 60 and 516 days. Chen et al. (2007) [48] examined the effects of −20 • C freezing over extended periods on Sarcocystis structure. They found that bradyzoites degenerate and dissolve with freezing, resulting in a probable loss of bradyzoite activity and suggesting that cysts lose their infective capacity. ...
... Those authors assessed infectivity by feeding cats with cysts and monitoring the presence of oocysts or sporocysts in their faeces; moreover, they did not evaluate viability in the period between 60 and 516 days. Chen et al. (2007) [48] examined the effects of −20 • C freezing over extended periods on Sarcocystis structure. They found that bradyzoites degenerate and dissolve with freezing, resulting in a probable loss of bradyzoite activity and suggesting that cysts lose their infective capacity. ...
... In domestic freezing, the effect rate is slow. Freezing leads to the formation of large, pointy ice crystals capable of disrupting cell membranes; the concomitant loss of membrane integrity results in parasite death [48]. In theory, longer freezing times lead to the growth of more crystals and, thereby, to a higher percentage of cells with damaged membranes. ...
Sarcocystis spp. are complex apicomplexan parasites that cause a substantial economic impact on livestock used for meat production. These parasites are present worldwide. Our study aimed to identify Sarcocystis species affecting sheep meat in southern–central Spain and to evaluate the effectiveness of freezing for parasite inactivation. A total of 210 condemned samples of sheep meat were thoroughly assessed grossly and microscopically; the presence of macro- and microcysts was confirmed. The samples were then frozen at −20 °C for various time intervals (24, 48, 72, 96, 120, and 144 h) and compared with untreated samples. Bradyzoites were isolated through pepsin digestion for subsequent molecular analysis and viability assessment, employing trypan blue and double fluorescence staining techniques. Our measurements confirmed the presence of S. tenella, S. gigantea, and S. medusiformis in Spanish domestic sheep. Freezing for 96 to 144 h resulted in a significant reduction in parasite viability, with a robust correlation observed between the two staining methods. Both stains effectively measured the viability of Sarcocystis, thereby promising future advances in meat safety.
... Sarcocysts of S. suihominis, on the other hand, have longer and thinner (about 13 μm by 0.8 μm), almost hair-like protrusions, which usually run in an oblique or slanting direction along the cyst surface, and hence, their cyst wall may appear thinner than that in sarcocysts of S. miescheriana, particularly when viewed in histological sections (Saito et al. 1998). By light microscopy, the difference between the two species with respect to their cyst wall protrusions is therefore best seen in wet mounts of fresh cysts removed from their host cells (Erber 1977;Heydorn 1977a;Boch et al. 1978;Hinaidy and Supperer 1979;Chen et al. 2007). However, these differences are also evident by transmission electron microscopy (TEM) Kinder 1974, 1976;Claveria et al. 2001;Chen et al. 2007;Yan et al. 2013) and, to some extent, also by scanning electron microscopy (SEM) (Saito et al. 1996(Saito et al. , 1998. ...
... By light microscopy, the difference between the two species with respect to their cyst wall protrusions is therefore best seen in wet mounts of fresh cysts removed from their host cells (Erber 1977;Heydorn 1977a;Boch et al. 1978;Hinaidy and Supperer 1979;Chen et al. 2007). However, these differences are also evident by transmission electron microscopy (TEM) Kinder 1974, 1976;Claveria et al. 2001;Chen et al. 2007;Yan et al. 2013) and, to some extent, also by scanning electron microscopy (SEM) (Saito et al. 1996(Saito et al. , 1998. The sarcocysts described by TEM under the name S. miescheriana by Bergmann and Kinder (1974) were actually sarcocysts of S. suihominis, but when that study was published, only a single species, known as S. miescheriana, was believed to infect pigs. ...
... By contrast, S. suihominis was found to be quite common in domestic pigs in Germany (60.7% of 219 slaughter pigs; Boch et al. 1978) and Austria (7.5% of 348 sows; Hinaidy and Supperer 1979) in the 1970s. More recently, S. suihominis has been recorded in a few domestic pigs in Japan (Saito et al. 1998) and Spain (González et al. 2006) and seems to be quite common in domestic pigs in China (Yang et al. 2001;Chen et al. 2007) and India (Kaur et al. 2016). ...
A sample of the diaphragm was collected from each of 100 wild boars legally hunted in the Val Grande National Park in north-western Italy and examined for the presence of Sarcocystis infection by histological and molecular methods. In histological sections, thick-walled sarcocysts consistent with those of Sarcocystis miescheriana were detected in 32 wild boars. Genomic DNA extracted from diaphragm samples was initially subjected to PCR amplification of the internal transcribed spacer 1 (ITS1) region, and 97 wild boars were found to harbour a Sarcocystis infection at this screening. Selected DNA samples were then subjected to PCR amplification and sequencing of the ITS1 region and the 18S and 28S ribosomal RNA (rRNA) genes of the nuclear ribosomal DNA unit, while all positive samples were subjected to PCR amplification of the mitochondrial cytochrome c oxidase subunit I (cox1) gene. S. miescheriana was identified in 97 wild boars (97%), while the zoonotic Sarcocystis suihominis was identified in one wild boar (1%), which also harboured S. miescheriana. Intra-specific sequence variation was found in all four DNA regions of S. miescheriana examined and in the 18S rRNA gene and ITS1 region of S. suihominis. The partial cox1 gene was amplified and sequenced from 72 isolates of S. miescheriana, yielding 43 haplotypes with pairwise sequence identities of 97.6–99.9%. These haplotypes were 79.1–79.8% identical with the cox1 sequence of S. suihominis. Phylogeny based on cox1 sequences placed S. miescheriana and S. suihominis as sister species within a clade comprising mainly Sarcocystis spp. of ruminants with felids as known or presumed definitive hosts. The same was true for the phylogeny based on 18S rRNA gene sequences.
... Lane 2, negative control. & Ohsumi, 1999;Chen et al, 2007;Radwan et al. 2015). ...
... This may be due to the low intensity of Sarcocystis in each sample (200 g). This result agrees with Chen et al. (2007) who showed that as frozen storage time's increase, bradyzoite structure becomes indistinct; their arrangement becomes disordered and only electron dense bodies and amylopectin can be seen. In addition, the bounds of the plasma lemma become indistinct, the inner membrane complex, microneme and nucleus of the bradyzoites degenerate and the bradyzoites themselves even dissolve. ...
... In the present study, we found that molecular methods can be applied for the diagnosis of Sarcocystis spp. in frozen tissues and this agrees with Chen et al (2007) who proposed that freezing is a convenient storage method for samples used in taxonomic studies of Sarcocystis. Moreover, Sarcocystis cyst in muscle could be stored at -20 °C and remain suitable for ultrastructural morphological study . ...
The present study was carried out to investigate the prevalence of microscopic Sarcocystis species in the imported frozen Indian and Brazilian beef in Alexandria province, Egypt. In addition, this study aimed to identify the most common types of Sarcocystis spp. using different diagnostic methods as peptic digestion, histopathological examination and PCR in order to compare their results and detect which of them are more accurate and effective in diagnosing Sarcocystosis. Moreover, differential molecular studies were applied between the native strains of Sarcocystis spp. and that in the imported frozen beef. A total number of 384 samples of imported frozen beef (280 Indian buffalo and 104 Brazilian cattle meat) from different butcheries, supermarkets and retail meat shops in Alexandria province, Egypt. Overall examination revealed low prevalence (10.15%) of Sarcocystis spp. infection among the imported frozen beef (39/384). However, about 71% of the infected buffalo samples were microscopic cysts while only 3.9% were macroscopic Sarcocystis with no other gross lesions. A newly designed primer sets were used in molecular identification of four Sarcocystis spp. in both buffalo and cattle (S.fusiformis, S.buffalonis, S.hirsuta & S.hominis) using the primer BLAST tool on the NCBI of the Genbank (http://www.ncbi.nlm.nih.gov/tools/primerblast/ primertool.cgi). This primer yielded positive PCR results with about 46.1% of the infected samples (18/39). Three positive PCR products were sent for DNA sequencing in one direction (Forward). Each sample revealed high identity percent with three Sarcocystis species; S. fusiformis, S. buffalonis and S. hominis depending up on the BLAST comparative analysis. Finally, these three samples were subjected to PCR with newly designed primer sets specific for each species to confirm its identification. The result was positive with each of the three samples.
... This fact may suggest that the chosen non-molecular diagnostic method is convenient and specific procedure, which can apply in studies, as first step, aiming Sarcocystis species identification. Even if various authors (Chen et al., 2007;Dahlgren et al., 2007) demonstrated that freezing have a little or no effect on the morphology of the Sarcocystis affected tissues, the frequently observed ruptured cysts with ambiguous morphology, and the presence of the morphologically similar muscle fragments, can hamper the quantification of microscopically positive results. ...
Domestic and wild pigs, as intermediate hosts, can harbor tissue cysts of three Sarcocystis species namely S. miescheriana, S. suihominis and S. porcifelis. Out of them, S. suihominis is zoonotic. Romania is a country with high consumption of raw and/or undercooked traditional pork products. This fact may greatly favor the acquiring of the zoonotic Sarcocystis infections by humans, as definitive host. Based on this consideration and in order to investigate the occurrence and public health significance of Sarcocystis spp. in two western counties (Caraş-Severin and Timiş) of Romania, a total of 165 heart samples from hunted wild boars (Sus scrofa, n=101) and home slaughtered domestic pigs (Sus scrofa domesticus, n=64) were screened using microscopic fresh examination and molecular methods. Microscopic examination revealed the presence of sarcocysts in 60.4% of wild boars, and 23.4% of domestic pigs. Genetic characterization of isolates through the PCR-RFLP procedure, targeting the 18S rRNA gene, was successfully achieved for all microscopically positive samples, indicating the presence of a single species, S. miescheriana, in both hosts. The identity of 13 selected S. miescheriana isolates was also confirmed through sequencing. The tested hosts older than 27 months were found to be significantly higher infected (p<0.05) with Sarcocystis than the 6 to ≤27months age group. Although the human infective S. suihominis has not been registered, for a more reliable epidemiological picture, further molecular studies enrolling a larger number of animals and diagnosis on human intestinal Sarcocystis infections are still necessary.
The range of vertebrates that serve as intermediate hosts for parasites in the genus Sarcocystis remains incompletely defined. Here, we provide the first report of infections in treeshrews, describe the morphology of encysted parasites using light and transmission electron microscopy, and place this agent within a phylogenetic context by sequencing and comparing its 18S ribosomal DNA to that of related parasites. Muscle infections were diagnosed in four of 45 wild treeshrews captured in the vicinity of Kunming, Yunnan Province, Mainland China. Thread-like cysts (10.773+/-2.411mm in length, 0.106+/-0.009mm in width) had walls (0.538-0.746microm thick) that lacked perpendicular protrusions. The interior of the cyst was packed full with cyst merozoites, the shape of which was typical of Sarcocystis. The primary cyst wall consisted of a thin membrane supported by osmiophilic material, 31-60nm in thickness. The ground substance was about 105-526nm thickness. Cysts conformed to typical of 'type 1' sarcocysts. Freshly examined and frozen specimens did not differ in their cyst wall structure, however, the appearance of bradyzoites did differ: the conoid, rhoptries and micronemes were all visible in fresh bradyzoites; in stored bradyzoites, by contrast, the rhoptries appeared smaller, and although the conoid was visible, the micronemes were not. 18S rRNA gene was distinct from any previously reported sequence in GenBank. Their genetic and morphological uniformity suggest that these parasites, derived from treeshrews, represent a single biological species, Sarcocystis tupaia, sp. nov.
Six Sarcocystis species have previously been described from reindeer in Norway based on sarcocyst morphology and DNA sequencing. The aim of this study was to determine whether reindeer in Iceland, which descend from reindeer imported from Norway in 1787, also were infected with Sarcocystis, and to identify and genetically characterise any species present. Muscle tissue from the heart, diaphragm and/or oesophagus was collected from 36 reindeer in Iceland. Pieces of all tissue samples were examined histologically. Frozen/thawed samples of cardiac muscle, oesophagus and/or diaphragm from 11 of the 36 reindeer were also examined under a stereoscopic microscope and sarcocysts present were identified to species either in situ or under a light microscope. Two cysts of each species, originating from two different reindeer were randomly selected for DNA analyses. The complete ssu rRNA gene was amplified by the polymerase chain reaction (PCR) and sequenced. In addition, two sarcocysts that could not be classified by microscopic examination were selected for partial ssu rRNA gene sequence analysis. By histology, sarcocysts were found in the diaphragm and/or oesophagus of 8 of 36 (22.2%) animals. By examination of fresh tissue, sarcocysts of Sarcocystis rangi, S. tarandivulpes and S. hardangeri were found in the oesophagus of seven of nine (77.8%) animals, suggesting a high prevalence of Sarcocystis in the Icelandic reindeer population. Cyst morphology and the ssu rRNA gene sequence of each of the three species were identical to isolates of the same species from Norwegian reindeer. DNA sequencing was useful in order to identify cysts with an ambiguous morphology. This is the first record of these Sarcocystis species in reindeer outside Norway.
DNA templates were extracted from isolates of Sarcocystis hominis-like cysts collected from cattle and water buffalo, as well as from Sarcocystis fusiformis cysts and Sarcocystis suihominis cysts. The 18S rRNA genes were amplified using DNA from a single cyst as the templates. Approximately 1,367-1,440 bp sequences were obtained. The sequence difference in isolates of Sarcocystis hominis-like cysts from water buffaloes, and isolates of S. hominis cysts from cattle were very low, only about 0.1%, much lower than the lowest value (1.7%) among different species. Combined with their morphological structure, these sequence data indicate that the 4 isolates from cattle and water buffalo might be the same species, i.e., S. hominis, suggesting that both cattle and water buffalo may serve as the intermediate hosts for this parasite. Apparently, this is the first report using a single cyst to do such work and is a useful way to distinguish the Sarcocystis cyst in an intermediate host that may be simultaneously infected by several different Sarcocystis species.
Sarcocystis miescheriana sarcocysts were identified in skeletal muscles of 9 (27%) of 33 swine slaughtered for human consumption. Sarcocysts were 144-180 µm x 20-38 µm in size. Ultrastructurally, the cyst wall resembled the type 10 sarcocyst wall. The villar protrusions (VP) were 3-4.5 µm long and 0.6-1.2 µm wide and had prominent longitudinally arranged microtubules extending from the VP tips to the granular layer (=ground substance). The parasitophorous vacuolar membrane with its underlying electron-dense layer (EDL) measured 25 nm in thickness. The base of the VP exhibited minute (0.42-0.87 µm) bulblike inpocketings. Each VP had 80-90 microtubules situated underneath the EDL. The granular layer was 0.5-1.2 µm thick, and contained hairlike microtubules continuous with those of the VP core. This is the first report of S. miescheriana in Philippine domestic pigs Sus scrofa.
The first detection of Sarcocystis nesbitti Mandour, 1969 in the Chinese mainland is reported and the morphology of the sarcocyst is described in detail. The parasite was detected in the monkey, Macaca fascicularis, maintained on a monkey farm in Yunnan Province; the infection may have occurred via faecal contamination from local rats, mice and/or birds. S. nesbitti was characterized as follows: a macroscopic sarcocyst, length of the cyst up to 2 mm; cyst wall smooth, thin and no perpendicular protrusion is seen under the light microscope; border of cyst wall wavy, primary cyst wall thin (38–65 nm) and invaginated; ground substance about 0.5–0.76 μm thick with electron-dense granules and concentric spherical bodies. The cyst wall is described as type 1 by electron microscopy. It is suspected that S. nesbitti may utilize Macaca mulatta, M. fascicularis, Cercocebus atys, and Papio papionis, as well as human as intermediate hosts. The taxonomy of S. nesbitti is re-appraised in the light of a consideration of possible experimental artefacts and examination of the past literature. Evidence is presented that S. nesbitti may be one of the species infecting humans in South Asia and that the monkey may be a potential reservoir host.
Between 1981 and 1990, 22 intestinal specimens surgically resected due to segmental enterocolitis were collected and examined. Grossly, the specimens were classified into 3 groups 1) Acute inflammation with hemorrhage and necrosis 2) Constrictive lesion 3) False diverticulum with perforation. Mostly, there was unisegmental involvement, distributed in jejunum, ileum and ileocolon. Microscopically, small parasitic structures, interpreted to be unconventional excystation stage of Sarcocystis hominis, (Railliet and Lucet, 1891) Dubey 1976, were present on the luminal border and within the crypt-lining epithelial cells. At the ulcerated area, tissue invasion by Gram-positive bacteria were always seen and considered as second pathogen. Source of the parasite was likely from cyst-containing beef available in markets, (Bos indicus and Bubalus bubalis) along with consumption of undercooked beef. Antismooth muscle antibody, IgG class, with the titer ranging from 1:16-1:256 were detected in 45 per cent of the patients. This is considered as autoimmunity against intestinal smooth muscle damaged previously from subclinical inflammatory condition. Present information suggests a long-standing existence of Sarcocystis in the patients' intestine, associated with Gram-positive bacterial infection, as the mechanism producing segmental enterocolitis found in the Central region.
Using in vitro excystation as a measure of viability, it was found that at 4 degrees C Sarcocystis gigantea sporocysts survived considerably better in tap water (85% excystation after 174 days) than in either 2.5% potassium dichromate (15% excystation after 174 days) or 2% sulphuric acid (0% excystation after 5 days). Although they were able to resist 48 h suspension at room temperature in most laboratory reagents and disinfectants tested, six (sulphuric acid, ammonia, methanol, ethanol, potassium hydroxide, sodium hydroxide, Medol) had substantial sporocysticidal properties. Further investigation with three of these showed that sporocyst excystation was reduced from 65% to less than 10% following contact with 2.5% sulphuric acid for 1 h or with 2% ammonia or 4% Medol for 4 h. Sporocysts were either killed or had their ability to excyst severely impaired by heating to 60 degrees C and 55 degrees C for 5 and 60 min, respectively, by exposure to ultraviolet radiation at a dose of 4000 ET, or by prolonged storage in water at 24 degrees C. Sporocysts exposed to either constant or intermittent freezing at -18 degrees C suffered a comparatively slow decline in excystation rate with time, as did those subjected to desiccation. The duration of survival of desiccated sporocysts was inversely related to relative humidity and after 245 days at 33% relative humidity and temperatures of 15 degrees C or 24 degrees C, 60% of such sporocysts excysted.
The present paper reports on the results of an experimental study on man-cattle-man infection of Sarcocystis hominis, found in Yunnan Province. About ten thousand sporocysts collected from the feces of persons naturally infected with Sarcocystis hominis were fed to a calf, which was dissected 150 days later. Numerous cysts of Sarcocystis hominis were found in the cardiac and skeletal muscles. By light microscopy, the cyst wall of fresh preparation showed numerous thick, finger-like projections, with maximum length of 7.9 microns. By electron microscopy, the cyst had a regularly folded, with primary wall forming palisade-like protrusions. Numerous sharp invaginations found in the protrusions were sawtooth-shaped, covering the whole surface of the protrusions. No fine fibrils were observed within the zone of ground substance beneath the primary cyst wall. Two rhesus monkeys were fed with beef infected with Sarcocystis hominis and sporocysts and oocysts were found in their feces 29 and 31 days later, the patent period of sporocyst excretion being 5 and 7 days, respectively. The senior author had taken voluntarily 60 g beef of the experimentally infected calf, and presented clinical symptoms such as anaemia, abdominal pain, diarrhoea, fatigue and dizziness on d3 post infection with sporocysts and oocysts found in the feces on d8. The patent period of sporocyst excretion was more than 42 days. The mean size of 50 sporocysts was 11.90 +/- 0.04 x 15.88 +/- 0.03 micron and that of 50 oocysts, 15.56 +/- 0.05 x 19.76 +/- 0.04 micron. On d50 he took acetylspiramycin tablets, the initial dose being 0.4 g, followed by 0.2g qid. for 15 days.(ABSTRACT TRUNCATED AT 250 WORDS)
A survey in Kansas compared the prevalence of gastrointestinal helminths and protozoa in 2 raccoon (Procyon lotor) populations; a population in a typical rural setting and a relatively undisturbed population on a military reservation. Gastrointestinal tracts of 128 raccoons were examined. Freeze storage of alimentary tracts prevented collection of data on trematode prevalence. Helminth infections other than trematodes included 1 acanthocephalan, 2 cestodes, and 3 nematodes. Helminths were found in all raccoons from the population on the military installation and 96% of those from the rural population. Prevalence of helminths was generally greater in raccoons from the population living in the rural setting. Eimeria spp. and Sarcocystis sp. were also found, whereas Giardia spp. and Cryptosporidium parvum were not.
Dogs fed buffalo heart muscle containing sarcocysts of Sarcosystis levinei and heated at 65-75 degrees C did not shed sporocysts, whereas other dogs fed infected heart muscle heated between 40 and 60 degrees C shed sporocysts. Dogs fed infected heart muscle stored at -4 degrees C for 48 h did not shed sporocysts, but those fed similar infected tissues stored at -2 degrees C for 24 h shed sporocysts. The results indicate that sarcocysts of S. levinei are rendered noninfective by heating to 65 degrees C or by freezing at -4 degrees C.
Survival of Sarcocystis gigantea macrocysts was studied usingan oxygen electrode and by feeding to cats. Macrocysts were viable after 10 minutes at 52.5°C but not after 20 minutes at 55°C or 10 minutes at 60°C. Macrocysts survived 60 days at — 14°C and infected cats. After storage at 10°C for 13 days and 4°C for 20 days macrocysts still metabolised vigorously. Treatment of sheep meat at 60°C for at least 20 minutes should render it non-infective for cats.
The genus Sarcocystis is composed of about 130 species of heteroxenous cyst-forming coccidia with differences in life cycle and pathogenicity. Pathogenic Sarcocystis spp. can cause disease in their intermediate hosts, in particular in ruminants. Research on Sarcocystis infections has been impeded by several facets of the parasites. Intermediate as well as definitive hosts can be parasitized by several different species with similarities in biology and morphology. Antigen preparations derived from pathogenic Sarcocystis spp. are highly cross-reactive with antibodies directed against non-pathogenic species. As a consequence, none of the currently available immunological tests is species-specific and can differentiate between pathogenic and non-pathogenic Sarcocystis spp. Over the last decade, new techniques in immunology, protein chemistry and molecular biology have facilitated more advanced studies on the molecular composition and molecular biology of Sarcocystis spp. in various laboratories. The development of species-specific monoclonal antibodies and analyses of the molecular composition of some life-cycle stages of Sarcocystis spp. of cattle and sheep showed that species-specific proteins and antigens exist in these species, although they are not highly abundant. In addition, comparisons of rRNA genes of different Sarcocystis spp. identified unique sequences in the rRNA of pathogenic Sarcocystis spp. that are suitable targets for species-specific identification. Thus, tools have become available that facilitate the development of methods for species-specific identification and differentiation of Sarcocystis spp. as well as the identification and study of molecules that are associated with pathogenicity of some of these parasites.
The effect of some chemical and anatomical factors on the excystability and infectivity in cell culture of sporocysts of Sarcocystis cruzi was investigated. A significantly (P < 0.001) higher excystation rate (ER) occurred when sodium bicarbonate was added to the excysting fluid at concentrations between 0.075 and 0.3 M. Sporocysts collected from different dogs had different ER. In contrast, although not uniformly distributed along the intestinal lumen, the sporocysts collected from the different tracts of the intestine showed similar ER. The period of storage did not affect the excystability of the sporocysts; however, it influenced the pattern of growth of the sporozoites in cell culture. Sporozoites excysted from sporocysts stored for approximately 2 years grew slower and produced significantly fewer merozoites compared to those excysted from sporocysts stored for 5-7 months.
Sarcocystis miescheriana sarcocysts were identified in skeletal muscles of 9 (27%) of 33 swine slaughtered for human consumption. Sarcocysts were 144-180 microm x 20-38 microm in size. Ultrastructurally, the cyst wall resembled the type 10 sarcocyst wall. The villar protrusions (VP) were 3-4.5 microm long and 0.6-1.2 microm wide and had prominent longitudinally arranged microtubules extending from the VP tips to the granular layer (=ground substance). The parasitophorous vacuolar membrane with its underlying electron-dense layer (EDL) measured 25 nm in thickness. The base of the VP exhibited minute (0.42-0.87 microm) bulblike inpocketings. Each VP had 80-90 microtubules situated underneath the EDL. The granular layer was 0.5-1.2 microm thick, and contained hairlike microtubules continuous with those of the VP core. This is the first report of S. miescheriana in Philippine domestic pigs Sus scrofa.
Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by infection of the central nervous system with the protozoan parasite Sarcocystis neurona. A herd-level analysis of a cross-sectional study of serum antibodies to S. neurona in Michigan equids was conducted, using data collected in 1997 for study that included 1121 equids from 98 Michigan horse farms. Our objective was to identify specific herd-level risk factors associated with seropositivity. We tested associations between herd seroprevalence and various farm-management practices (including feed-storage methods and wildlife control). Multivariable models were developed for three strata based on relative opossum abundance (opossum districts). Herd seroprevalence ranged from 0 to 100% (median=57%). No risk factor was significantly associated with herd seroprevalence at P< or = 0.05 in all opossum districts. Our results suggest that equids living in areas with large opossum populations might be infected with S. neurona from multiple sources.
The effect of long-term storage on the viability and infectivity of Sarcocystis neurona sporocysts was investigated. S. neurona sporocysts were harvested from the small intestine of Virginia opossums from 1996 to 2002 and stored at 4 degrees C. Viability of sporocysts was assessed by propidium iodide (PI) exclusion assay, in vitro excystation and development in tissue cultures, and bioassay in gamma-interferon gene knockout (gamma-IFN-KO) mice. The rate of excystation was apparently unaffected by long-term storage; sporocysts retained their ability to excyst after 7 years of storage at 4 degrees C. However, the ability of sporocysts to exclude PI stain, to invade and proliferate in cells in vitro, and to cause disease and lesions in gamma-IFN-KO mice appeared to decline as sporocysts age. The results demonstrated that sporocysts of S. neurona were able to survive and maintain moderate to high viability for up to 7 years when stored in phosphate buffered saline and Hank's balanced salt solution containing antibiotic-antimycotic mixture at 4 degrees C.
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