In vitro cultivation of meronts of Sarcocystis cruzi

Several established cell lines were tested for their ability to support in vitro development of meronts of Sarcocystis cruzi. Sporozoites penetrated bovine monocytes (BM), bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney cells and mouse macrophages, but developed to meronts in BM and CPA only. Sporozoites developed to large meronts that contained approximately 180-350 merozoites, whereas merozoites formed small meronts with 50-100 merozoites. Mature large meronts were present at 18-86 days after inoculation (DAI) in BM and at 16-72 DAI in CPA. Small meronts were present at 23-115 and 23-91 DAI in BM and CPA. Considerably more merozoites developed in CPA than in BM. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that merozoites harvested at 36 and 48 DAI each had 1 unique protein as well as numerous common proteins.     

Production of Sarcocystis cruzi sporocysts by dogs fed experimentally infected and naturally infected beef.

Twenty-one coccidia-free dogs were fed either experimentally infected or naturally infected beef in 4 experiments. The pretreatment period ranged from days 9 to 33; 7 dogs shed their first sporocysts on day 12. The full length of the patent period was not determined because most dogs were still shedding sporocysts when experiments ended on days 40 and 60. Although the patent period was long, sporocysts were not shed continuously; the total number of days sporocysts were actually shed ranged from 3 to 40. The average number of sporocysts shed by dogs fed 454 to 908 g of beef ranged from 861,000 to over 20 million. Most sporocysts were shed from days 15 to 30; peak numbers were shed on days 23 and 24. Average peak numbers ranged from 145,000 to 408,400 sporocysts. Such data can be applied to facilitate collection of sporocysts in the laboratory or to estimate the potential for transmission of infective organisms under field conditions.     

Early migration of Sarcocystis neurona in ponies fed sporocysts

Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM), a neurologic disease of the horse. In the present work, the kinetics of S. neurona invasion is determined in the equine model. Six ponies were orally inoculated with 250 x 10(6) S. neurona sporocysts via nasogastric intubation and killed on days 1, 2, 3, 5, 7, and 9 postinoculation (PI). At necropsy, tissue samples were examined for S. neurona infection. The parasite was isolated from the mesenteric lymph nodes at 1, 2, and 7 days PI; the liver at 2, 5, and 7 days PI; and the lungs at 5, 7, and 9 days PI by bioassays in interferon gamma gene knock out mice (KO) and from cell culture. Microscopic lesions consistent with an EPM infection were observed in brain and spinal cord of ponies killed 7 and 9 days PI. Results suggest that S. neurona disseminates quickly in tissue of naive ponies.     

Parasitemia and early tissue localization of Sarcocystis neurona in interferon gamma gene knockout mice fed sporocysts.

Early localization and parasitemia of Sarcocystis neurona were studied in gamma interferon gene knockout (KO) mice fed S. neurona sporocysts. Mice were examined for S. neurona infection histologically and immunohistochemically and by bioassay in KO mice. For bioassay, blood and tissue homogenates were inoculated subcutaneously into KO mice. Parasitemia was demonstrated by bioassay in KO mice 1-8 days after feeding sporocysts (DAFS). Sporozoites were seen in histologic sections of all regions of the small intestine and in cells in Peyer's patches of a mouse killed 6 hr after feeding sporocysts. At 1 DAFS, organisms were present in all regions of the small intestine and were also seen in mesenteric lymph nodes. At 3 DAFS, organisms had begun to invade extraintestinal tissues. Sarcocystis neurona was demonstrated histologically in mouse brain as early as 4 DAFS.     

Sarcocystis neurona: parasitemia in a severe combined immunodeficient (SCID) horse fed sporocysts

Sarcocystis neurona was isolated from the blood of a 5-month-old Arabian foal with severe combined immunodeficiency. The foal had been inoculated approximately 3 weeks previously with 5 x 10(5) sporocysts that were isolated from the intestines of an opossum and identified by restriction enzyme analysis of PCR products as S. neurona. The isolate obtained from the blood of this foal was characterized by genetic, serologic, and morphologic methods and identified as S. neurona (WSU1). This represents the first time that S. neurona has been isolated from any tissue after experimental infection of a horse. This is also the first time a parasitemia has been detected during either natural or experimental infection. The severe combined immunodeficiency foal model provides a unique opportunity to study the pathogenesis of S. neurona infection in horses and to determine the role of the immune system in the control of infection with and development of neurologic disease.     

Development of Ox-Coyote Cycle of Sarcocystis cruzi1

The ox-coyote cycle of Sarcocystis cruzi was studied by killing 38 calves between 4 and 153 days postinoculation (DPI) with 55 × 10³-5 × 10⁸ sporocysts from the intestines of coyotes. At 4 DPI, a zoite was found within the lumen of a mesenteric lymph node artery. At 7 DPI, zoites were found in mononuclear cells and in endothelial cells in mesenteric arteries. First generation meronts (41.0 × 17.5 μm in diameter) occurred 7–26 DPI in mesenteric lymph nodes. At 19–46 DPI, second generation meronts occurred in kidneys, muscles, and other tissues: renal meronts were 19.6 × 11.0 μm, and intramuscular meronts were 25.0 × 11.1 μm. Merozoites were found in the peripheral blood 17 DPI and later at 24–46 DPI. They divided by endodyogeny in mononuclear cells. Sarcocysts were seen first in the heart at 45 DPI and contained one or two metrocytes. At 55 DPI, sarcocysts containing only metrocytes were found in striated muscles, heart, and in smooth muscles of the urinary bladder, rumen, omasum, abomasum, and small intestine. At 67, 87, 112, and 153 DPI, sarcocysts were found only in striated muscles and in the heart. At 67 DPI, sarcocysts were up to 360 μm long. They contained only metrocytes and were not infective to the dog. At 86 DPI, sarcocysts contained mostly bradyzoites, a few metrocytes, and were infective to a coyote. The thin-walled sarcocysts grew to a maximum length of 800 μm and contained bradyzoites that were 10.9 × 3.0 μm. At 90 DPI, two mature sarcocysts were found in 2 of 73 sections of brain and spinal cord; hundreds of sarcocysts were present in sections of tongue and heart of this calf. Gametogony occurred in the small intestine of the coyote. Macro-and microgamonts were found in goblet cells of the small intestines of coyotes 6 h after the ingestion of infected meat. Microgamonts were few and contained 3–11 slender gametes. Oocysts were seen at 12 h and sporulation was completed 9 DPI. The prepatent period in the coyote was 8 days. The ox-coyote cycle is compared with ox-dog cycle.     

Experimental induction of the two-host life cycle of Sarcocystis cruzi between dogs and Korean native calves

Eight dogs were experimentally infected with Sarcocystis by oral inoculation of cardiac muscle from naturally infected cattle. The infected dogs commenced discharging of sporocysts in the feces after 10 to 12 days of inoculation, and continued until 20 and 35 days after inoculation. Three dogs were reinfected with cardiac muscle from the naturally infected cattle. Sporocysts reappeared in the feces on 12 to 13 days after reinfection. Sarcocystis sporocysts collected from the experimentally infected dogs were fed to each of the two 30-day-old Korean native calves. The infected calves remained clinically normal, except for the high fever (> or = 40 degrees C) and decreased hematocrit values on day 30 to 40 post inoculation. Muscular cysts of Sarcocystis were found from infected calves on day 40 post inoculation. Proliferative forms of Sarcocystis were also observed in the muscle of infected calves. These results suggest that the Sarcocystis cruzi found in Korean native cattle has a 2-host life cycle with dogs as the definitive host and Korean native calves as the intermediate host.     

Development and Ultrastructure of First-Generation Meronts of Sarcocystis cruzi in Calves Fed Sporocysts from Coyote Feces*

SYNOPSIS. The development of Sarcocystis cruzi Hasselmann (syn. S. fusiformis Railliet) meronts was studied in seven 7- to 10-day-old calves killed 4, 7, 11, 15, 22, 25 and 28 days postinoculation (DPI) with 5 × 10⁷ sporocysts from feces of coyotes. No meronts were found 4 and 7 DPI. Young and intermediate meronts with 1–16 nuclei were found in endothelial cells of arteries in mesenteric lymph nodes, but not in kidneys 11 DPI. Mature meronts were noted in endothelial cells of arteries, arterioles, or capillaries of many organs of calves killed 15 to 25 DPI. No first-generation meronts were found 28 DPI. By electron microscopy, all stages of the first-generation merogony were found free within the host cell cytoplasm and not within a parasitophorous vacuole. The appearance of intranuclear spindles preceded the formation of merozoites by endopolygeny. Mature meronts measured 41.0 × 17.5 (34–50 × 15–24) μm, contained ～ 100–350 merozoites, and had 2 to 4 relatively small residual bodies, 2.8 μm in diameter. Merozoites measured 6.3 × 1.5 (5.5–7 × 1 μm) and contained most of the organelles characteristically found in coccidian merozoites. Micropores were observed in merozoites, but not in young and intermediate meronts. Merozoites were seen free in the lumen of blood vessels, in intracellular areas, and free within the host cell cytoplasm.     

Continuous in vitro cultivation of Sarcocystis cruzi

Sporozoites of Sarcocystis cruzi were inoculated onto monolayer cultures of bovine pulmonary artery endothelial (CPA) cells. Sporozoites entered the cells, formed large and small multinucleate schizonts, and produced large numbers of merozoites. Continuous cultivation from the original sporozoite inoculum has been maintained for more than 1,320 days by subinoculating merozoites onto new cultures of CPA cells. During this time, the capacity to produce both types of schizonts was preserved, and schizogony was the only form of reproduction that was observed.     

In vitro excystation of Sarcocystis capracanis, Sarcocystis cruzi and Sarcocystis tenella (Apicomplexa)

Improved rates of in vitro excystation of sporozoites from sporocysts of Sarcocystis capracanis, Sarcocystis cruzi, and Sarcocystis tenella were obtained by pretreating sporocysts with an aqueous sodium hypochlorite (NaOCl) solution followed by incubation in excysting fluid (EF). After pretreatment with NaOCl, sporocysts were washed 4 times in Hanks' balanced salt solution and then incubated in various EF (pH 7.4) at 38.5 C in 5% CO2-95% air. Maximum rates of excystation (free sporozoites/(sporozoites in sporocysts + free sporozoites) X 100) for all 3 species of Sarcocystis occurred at 4 hr after incubation in EF. These rates were 17% for S. capracanis after incubation in EF containing 2% trypsin + 10% caprine bile; 90% for S. cruzi in 2% trypsin + 10% bovine bile; and 20% for S. tenella in 2% trypsin + 10% caprine bile. Only a 40% excystation rate occurred in sporocysts of S. cruzi that had been stored previously for 14 days in aqueous potassium dichromate. Excysted sporozoites of S. capracanis, S. cruzi, and S. tenella penetrated and developed to mature meronts in bovine pulmonary artery endothelial cells or bovine monocytes.     

Experimental transmission of Sarcocystis from icterid birds to sparrows and canaries by sporocysts from the opossum.

Cowbirds (Molothrus ater) and grackles (Cassidix mexicanus) infected with muscle cysts of Sarcocystis were fed to opposums (Didelphis virginiana) and fecal sporocysts from the latter were given to sparrows (Passer domesticus, Family Ploceidae), canaries (Serinus canarius, Family Fringillidae) and ducks (Anas platyrhynchos, Family Anatidae). Asexual parasites were found in the endothelium of sparrows and canaries but not in ducks. When birds were kept 10 weeks or more after infection, muscle cysts were found grossly and microscopically in the majority of sparrows, and in 1 canary, but not in ducks. Muscle zoites were found in digests of all sparrows and canaries but not in that of ducks. Metrocytes and forms dividing by endodyogeny also were found in the digest. Thus, avian Sarcocystis was transmitted experimentally from 2 genera of 1 family (Icteridae) to 2 different families of passerine intermediate hosts by sporocysts from the definitive host. This is the broadest intermediate host spectrum known for a species of Sarcocystis.     

Effects of high temperature and disinfectants on the viability of Sarcocystis neurona sporocysts

The effect of moist heat and several disinfectants on Sarcocystis neurona sporocysts was investigated. Sporocysts (4 million) were suspended in water and heated to 50, 55, 60, 65, and 70 C for various times and were then bioassayed in interferon gamma gene knockout (KO) mice. Sporocysts heated to 50 C for 60 min and 55 C for 5 min were infective to KO mice, whereas sporocysts heated to 55 C for 15 min and 60 C or more for 1 min were rendered noninfective to mice. Treatment with bleach (10, 20, and 100%), 2% chlorhexidine, 1% betadine, 5% o-benzyl-p-chlorophenol, 12.56% phenol, 6% benzyl ammonium chloride, and 10% formalin was not effective in killing sporocysts. Treatment with undiluted ammonium hydroxide (29.5% ammonia) for 1 hr killed sporocysts, but treatment with a 10-fold dilution (2.95% ammonia) for 6 hr did not kill sporocysts. These data indicate that heat treatment is the most effective means of killing S. neurona sporocysts in the horse feed or in the environment.     

In vitro excystation and structure of Sarcocystis suicanis Erber, 1977, sporocysts

Methods previously described for artificially excysting coccidia were applied to oocysts and sporocysts of Sarcocystis suicanis recovered from the intestinal mucosa of experimentally infected dogs. Observations with a scanning electron microscope showed that sporocysts consisted of 4 plates, each with a series of accessory veinations; plate margins joined at obtuse angles; plates separated at their margins and curled inward, allowing the sporozoites to escape. Transmission electron microscopy suggested that the sporocyst wall is ultrastructurally similar to that of other coccidia.     

Experimental infections of Sarcocystis cruzi, Sarcocystis tenella, Sarcocystis capracanis and Toxoplasma gondii in red foxes (Vulpes vulpes)

Four littermate 6-wk-old red foxes (Nos. 1-4) were fed Toxoplasma gondii, Sarcocystis cruzi, S. tenella and S. capracanis. One littermate fox (No. 5) served as the control. Two foxes (Nos. 1, 2) were fed tissue cysts of T. gondii and two foxes (Nos. 3, 4) were fed oocysts of T. gondii. Twenty-one to 42 days later, the same five foxes were used to test the infectivity of meat of goat, sheep, and ox experimentally inoculated with Sarcocystis. Fox 2 was fed goat meat and shed S. capracanis-like sporocysts 10 days later. Foxes 3 and 4 were fed beef, and they shed S. cruzi-like sporocysts 9 days later. Fox 5 was fed sheep meat and shed S. tenella-like sporocysts 8 days later. Foxes were killed between 36 and 55 days of the experiment and their tissues were inoculated into mice to recover T. gondii. All foxes remained clinically normal and T. gondii was recovered from all inoculated foxes and not from the control. Sarcocystis sporocysts from foxes induced lethal infections in goats, sheep, and ox. The sporocysts, meronts, merozoites, and sarcocysts of fox-derived parasites were similar to those derived from coyotes or dogs. It was concluded that the red fox can act as a final host for the three pathogenic species of Sarcocystis in cattle, sheep, and goats.     

Oligosaccharides from feces of preterm infants fed on breast milk

Nine neutral and five acidic oligosaccharides were isolated from feces of a preterm (30th postmenstrual week) blood group A nonsecretor infant fed on pooled breast milk. Structural analyses were carried out using sugar and methylation analyses, fast atom bombardment mass spectrometry, and 1H NMR. The acidic oligosaccharides are well-known components of human milk. The neutral oligosaccharides are characteristic of nonsecretor milk. Surprisingly, no secretor gene-dependent oligosaccharides were present in the feces. Another preterm (27th postmenstrual week) blood group A, secretor infant fed on pooled breast milk showed the same fecal oligosaccharide pattern as above during the first week after birth, despite being a secretor individual. Also notable was the absence of blood group A-active oligosaccharides in this sample. Another sample of feces collected 8 weeks later from the latter infant contained the expected blood group A-active oligosaccharides. Furthermore, free sialic acid was present at the cost of the sialyl oligosaccharides seen earlier. Thus, infants born prematurely do not show the same degree of development of oligosaccharide metabolism as their more mature counterparts.     

Early Developmental Stages of Sarcocystis cruzi in Calf Fed Sporocysts from Coyote Feces1

ABSTRACT. The development of Sarcocystis cruzi was studied in an 11-day-old calf killed seven days postinoculation with 5 × 10⁸ sporocysts from feces of coyotes. Uninucleate zoites were found in arteries of mesenteric lymph nodes but not in other organs. Zoites measured 4.9 × 3.0 (3.5–7.0 × 2.1–3.5) μm. Of the 36 zoites studied, 31 were in endothelial cells, four were in macrophages in the lumen of arteries, and one was free in the lumen of an artery. Infected endothelial cells were two to three times larger than uninfected cells. Zoites appeared structurally similar to sporozoites. The occurrence of zoites in macrophages suggests that sporozoites of Sarcocystis might use such cells to reach the site of their first merogony.     

Studies of Sarcocystis in Malaysia. II. Comparative ultrastructure of the cyst wall and zoites of Sarcocystis levinei and Sarcocystis fusiformis from the water buffalo, Bubalus bubalis.

The two species of Sarcocystis--S. levinei and S. fusiformis from the water buffalo, Bubalus bubalis, show some ultrastructural similarities in their cyst wall and zoites. The zoites of both species are of about the same size, banana-shaped and have 22 subpellicular microtubules, numerous micronemes, eight rhoptries, a micropore in the region of the micronemes, an elongated mitochondrion, and a nucleus. S. levinei has 200--300 micronemes and S. fusiformis has about 400. The sarcocysts of both species are trabeculated and their cyst walls have cytophaneres containing annulated fibrils and coarse, electron dense granules. The cytophaneres of S. levinei are sloping, with irregular, wavy outlines, whereas S. fusiformis has the cauliflower-type of cytophaneres. This difference in the appearance of the cytophaneres, together with the difference in size of the sarcocysts and their definitive hosts, further confirms that S. levinei and S. fusiformis are two distinct species in the water buffalo.