The in vivo excystation of Sarcocystis gigantea and S. tenella sporocysts

In vivo studies on the excystation of Sarcocystis gigantea and S. tenella sporocysts indicated that this process was, as in vitro, a diphasic one involving both pretreatment and treatment phases. The studies also tended to support in vitro observations that the requirements for the excystation of these two species are quite different. The results suggested that for neither species was the pretreatment stimulus likely to be provided by conditions in the rumen alone. However, exposure to abomasal conditions only induced moderate levels of excystation in both when they were subsequently treated with trypsin and bile. For S. gigantea, 0.25 to 4 h abomasal exposure was most effective; for S. tenella, 24 hours. The stimuli necessary to complete the excystation process could, apparently, be provided by 1 h placement in the duodenum for S. gigantea but not for S. tenella.     

Recovery of Sarcocystis gigantea sporocysts from cat faeces

A method for the mass recovery of S. gigantea sporocysts from cat faeces involving homogenisation and centrifugation in water, passage through 250- and 53-microns sieves and floatation in 1.2 SG NaCl solution, is described. An examination of the various processes involved in this procedure showed that the greatest yields were obtained when a proportion of faeces to floatation medium of 1:20 and centrifugation at 6000 X g for at least 5 min was used. Ninety-six per cent of the sporocysts recovered were obtained from the first centrifugation in aqueous NaCl solution (SG 1.2). Although neither sieving nor additional washing of homogenised samples prior to floatation significantly affected sporocyst recovery, both reduced the amount of debris present. A considerable reduction in the amount of debris resulted from feeding infected cats on tinned fish rather than tinned meat. The addition of CCl4 to the NaCl solution also improved sporocyst purity but with a marked reduction in the numbers recovered.     

Evaluation of a concentration method for counting Sarcocystis gigantea sporocysts in cat faeces

A technique for determining the numbers of S. gigantea sporocysts in cat faeces using a concentration procedure and haemocytometer was evaluated. The results showed that it was more accurate than a modified McMaster method and had a mean recovery rate of 73% at four levels of infection ranging from about 2000 to over 20,000 sporocysts per gram of faeces.     

The survival of Sarcocystis gigantea sporocysts following exposure to various chemical and physical agents.

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.     

Morphology of Sarcocystis gigantea in experimentally-infected sheep

The development of the parasite and lesions was studied in 32 sheep killed 10 days to 47 months after inoculation with Sarcocystis gigantea sporocysts from cats. At 21-42 days post-inoculation (d.p.i.), there was a mild encephalitis, but organisms were not seen in the brain. Immature sarcocysts were detected from 40-84 d.p.i. The cyst wall was not measurable by light microscopy at 40 d.p.i., but was 1.5-2 microns thick at 84 d.p.i. At 119 d.p.i. both immature cysts containing only metrocytes, and mature cysts containing both metrocytes and merozoites, were present. These mature cysts did not have a secondary cyst wall. A mature cyst, 350 microns in length, was found in a sheep killed at 8 1/2 months p.i. At 10 m.p.i. cysts were up to 0.5 mm long and a secondary cyst wall was present. At 47 m.p.i. cysts were 2-5 X 4.5-7.5 mm, and were found only in the muscles of tongue, oesophagus, pharynx and flank.     

In vitro and in vivo interaction between sporocysts of Sarcocystis muris and mouse peritoneal macrophages

The interaction between the sporocysts of Sarcocystis muris and mouse peritoneal macrophages was studied both in vitro and in vivo in an attempt to determine whether or not resident peritoneal macrophages might effect the excystation of S. muris sporozoites from sporocysts injected intraperitoneally. Sporocysts of S. muris were phagocytosed by peritoneal macrophages both in vitro and in vivo. The addition of either unheated mouse serum or fetal calf serum did not significantly alter the level of phagocytosis. The percentage of phagocytosis in vivo and by thioglycolate-, proteose peptone- and BCG-elicited macrophages in vitro was greater than that shown by unstimulated macrophages in vitro. After 8 h incubation in vivo and in vitro a small proportion of sporocysts (less than 5%) was seen to have collapsed walls and up to 5% to have stained sporozoites, suggesting increased permeability of the sporocyst wall. The significance of increased permeability of the cyst wall in the process of sporozoite excystation is discussed.     

巨型住肉孢子虫烯醇酶基因克隆与表达

巨型住肉孢子虫(Sarcocystis gigantea)是羊体内一种常见寄生虫,多寄生于羊横纹肌内形成包囊,导致羊肉大量废弃,给畜牧业带来巨大经济损失。本研究尝试筛选能有效诊断住肉孢子虫感染的抗原。通过免疫印迹及质谱分析筛选到巨型住肉孢子虫候选诊断抗原烯醇酶,利用染色体步移技术扩增两侧翼未知序列并表达重组蛋白。应用生物信息学分析和免疫印迹对该蛋白诊断价值进行评价。结果筛选到的候选蛋白为巨型住肉孢子虫烯醇酶(SgENO),克隆得到1 181 bp的基因并获得重组蛋白rSgENO。对rSgENO应用进行初步评价,发现其与其他顶复亚门原虫的烯醇酶氨基酸相似性均较高(71%～92.1%),且能被弓形虫和新孢子虫阳性血清所识别,具有交叉反应性。本研究克隆并表达了巨型住肉孢子虫烯醇酶,后期评价发现该重组蛋白与新孢子虫和弓形虫有较强的交叉反应性,不能用于羊住肉孢子虫的血清学诊断,但有成为疫苗候选分子的潜力。     

Viability of Sarcocystis neurona sporocysts after long-term storage

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.     

High prevalence of Sarcocystis calchasi sporocysts in European Accipiter hawks

The emerging Sarcocystis calchasi induces a severe and lethal central nervous disease in its intermediate host, the domestic pigeon (Columba livia f. domestica). Experimental studies have identified the Northern goshawk (Accipiter g. gentilis) as final host. Phylogenetically closely related European sparrowhawks (Accipiter n. nisus) and wood pigeons (Columba palumbus) have been found to harbor genetically closely related Sarcocystis spp. However, data on the prevalence and potential interspecies occurrence of these parasites are lacking. Here, we report that European Accipiter hawks (Accipitrinae) are highly infected with S. calchasi, S. columbae and Sarcocystis sp. ex A. nisus in their small intestine. Thirty-one of 50 (62%) Northern goshawks necropsied during 1997-2008 were positive for S. calchasi in a newly established species-specific semi-nested PCR assay based on the first internal transcribed spacer region. Unexpectedly, 14 of 20 (71.4%) European sparrowhawks tested also positive. In addition, birds of both species were found to be infested with S. columbae and an, as yet, unnamed Sarcocystis sp. recently isolated from European sparrowhawks. These findings raise new questions about the host specificity of S. calchasi and its high virulence in domestic pigeons, since sparrowhawks only rarely prey on pigeons. Notably, isolated sporocysts from both infected Accipiter spp. measured 8 μm × 11.9 μm, precluding a preliminary identification of S. calchasi in feces of Accipiter hawks based on morphology alone. Importantly, three of four Northern goshawks used in falconry tested positive for S. calchasi. In conclusion, the results indicate that both European Accipter spp. in Germany serve as natural final hosts of S. calchasi and suggest that falconry and pigeon sport may serve as risk factors for the spread of this pathogen in domestic pigeons.     

Clinical sarcocystosis in calves fed Sarcocystis hirsuta sporocysts from cats

Lesions of sarcocystosis were studied in 14 calves necropsied between seven and 110 days after inoculation with 5000 to 25 million sporocysts of Sarcocystis hirsuta from cats. Calves developed fever, anemia, and diarrhea between 11 and 30 days after inoculation. The development of first generation meronts in arteries of small intestine, mesentery, and mesenteric lymph nodes seven to 25 days after inoculation was associated with vascular occlusion and necrosis of associated tissues. The development of second generation meronts in capillaries of striated muscles 15 to 23 days after inoculation was associated with necrosis, edema, and nonsuppurative myositis in heart and other muscles. Sixty-two days after inoculation lesions were reduced to focal areas of granulomatous inflammation around degenerating sarcocysts in striated muscles, but not in the heart.     

Lesions in sheep inoculated with Sarcocystis tenella sporocysts from canine feces

Sarcocystosis was studied in 37 sheep after oral inoculation with 10(4)-5 x 10(7) sporocysts of Sarcocystis tenella from canine feces. Two sheep inoculated with 2.5 x 10(7) and 5 x 10(7) sporocysts became moribund 16 and 19 days post-inoculation (DPI), respectively, due to occlusion of arteries of gut and mesentery by first generation meronts. Sheep inoculated with 10(7) sporocysts remained clinically normal until 21 DPI and those inoculated with 10(5)-10(6) became ill 24-28 DPI due to anemia coincident with maturation of second generation meronts. Inflammation, hepatitis and myocarditis were the main lesions of acute and subacute ovine sarcocystosis. Inflammation began to subside by the time (75 DPI) sarcocysts matured. Sarcocystis-induced encephalitis was distinguished from naturally occurring myelomalacia in sheep caused by an unidentified sporozoan.     

Parasitemia in an immunocompetent horse experimentally challenged with Sarcocystis neurona sporocysts

Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in Americans. Most cases are attributed to infection of the central nervous system with Sarcocystis neurona. Parasitemia has not been demonstrated in immunocompetent horses, but has been documented in one immunocompromised foal. The objective of this study was to isolate viable S. neurona from the blood of immunocompetent horses. Horses used in this study received orally administered S. neurona sporocysts (strain SN 37-R) daily for 112 days at the following doses: 100/day for 28 days, followed by 500/day for 28 days, followed by 1000/day for 56 days. On day 98 of the study, six yearling colts were selected for attempted culture of S. neurona from blood, two testing positive, two testing suspect and two testing negative for antibodies against S. neurona on day 84 of the study. Two 10 ml tubes with EDTA were filled from each horse by jugular venipuncture and the plasma fraction rich in mononuclear cells was pipetted onto confluent equine dermal cell cultures. The cultures were monitored weekly for parasite growth for 12 weeks. Merozoites grown from cultures were harvested and tested using S. neurona-specific PCR with RFLP to confirm species identity. PCR products were sequenced and compared to known strains of S. neurona. After 38 days of in vitro incubation, one cell culture from a horse testing positive for antibodies against S. neurona was positive for parasite growth while the five remaining cultures remained negative for parasite growth for all 12 weeks. The Sarcocystis isolate recovered from cell culture was confirmed to be S. neurona by PCR with RFLP. Gene sequence analysis revealed that the isolate was identical to the challenge strain SN-37R and differed from two known strains UCD1 and MIH1. To our knowledge this is the first report of parasitemia with S. neurona in an immunocompetent horse.     

Demonstration of schizogonous stages of Sarcocystis gigantea in experimentally infected sheep

Sarcocystis-free lambs were orally dosed with 1 X 10(6) sporocysts of Sarcocystis gigantea. Schizonts were found in endothelial cells of capillaries and arterioles of the brain, lung and kidney of lambs 7 and 14 days post-inoculation (d.p.i.). Between 21 and 35 d.p.i. there was extensive multi-focal encephalitis; however no organisms were detected in association with these lesions.     

Demonstration of viable Sarcocystis sporocysts in the faeces of a lamb dosed orally

Faecal flotations prepared after dosing a lamb with 3.5 X 10(6) dog-derived Sarcocystis sporocysts proved infective for other lambs for at least 7 days post-dosing. This confirms observations suggesting faecal transfer of sporocysts from treated to control lambs.     

Viability of Sarcocystis neurona sporocysts and dose titration in gamma-interferon knockout mice

Gamma-interferon knockout mice have become the model animal used for studies on Sarcocystis neurona. In order to determine the viability of S. neurona sporocysts and to evaluate the course of the disease in these mice, sporocysts were collected from opossums (Didelphis virginiana), processed, and stored for varying periods of time. Gamma-interferon knockout mice were then inoculated orally with different isolates at different doses. These animals were observed daily for clinical signs until they died or it appeared necessary to humanely euthanize them. 15 of 17 (88%) mice died or showed clinical signs consistent with neurologic disease. The clinical neurologic symptoms observed in these mice appeared to be similar to those observed in horses. 15 of 17 (88%) mice were euthanized or dead by day 35 and organisms were observed in the brains of 13 of 17 (77%) mice. Dose appeared not to effect clinical signs, but did effect the amount of time in which the course of disease was completed with some isolates. The minimum effective dose in this study was 500 orally inoculated sporocysts. Efforts to titrate to smaller doses were not attempted. Direct correlation can be made between molecularly characterized S. neurona sporocysts and their ability to cause neurologic disease in gamma-interferon knockout mice.     

Prevalence, transmission, and pathogenicity of Sarcocystis gigantea of sheep

Between March and May 1983, tongues and esophagi of 355 adult ewes from Colorado and Idaho were examined for grossly visible sarcocysts. Sarcocysts of Sarcocystis gigantea were found in 35 sheep. Cats fed sarcocysts from these naturally infected sheep shed sporocysts in their feces. Two adult ewes and 12 lambs inoculated with 1,000 to 1,000,000 sporocysts were euthanatized at postinoculation days (PID) 146, 230, 265, 391, 721, and 882, and their tissues were fed to Sarcocystis-free cats. All inoculated sheep remained clinically normal except for mild pyrexia between PID 12 and 18. Sarcocysts first became grossly visible at PID 391 and sarcocysts from sheep first became infectious for cats at PID 230.     

Abortion and death in goats inoculated with Sarcocystis sporocysts from coyote feces

Ten 75- to 105-day-pregnant does each were inoculated orally within 1 million (2 does), 10,000 (4 does), or 1,000 (4 does) sporocysts of Sarcocystis from coyote feces. Two does not inoculated with sporocysts served as controls. The 2 does inoculated with 1 million sporocysts died from acute sarcocystosis 21 and 22 days after inoculation (DAI), and each had 2 dead fetuses. The 4 does inoculated with 10,000 sporocysts were ill 19 to 33 DAI but survived; 1 aborted at 33 DAI, 1 had a live kid that died within 2 hours of birth 31 DAI, 1 aborted 2 dead fetuses 23 DAI, and 1 had a normal kid 56 DAI. The 4 does inoculated with 1,000 sporocysts and the 2 control does remained clinically normal and had normal kids. Does and their offspring were killed within 24 hours of parturition, and their tissues were examined histologically and microbiologically. Meronts of Sarcocystis were found in the maternal placenta of does inoculated with 1 million sporocysts. Sarcocystis was not found in the placenta, fetuses, or tissues of kids from does inoculated with 10,000 or 1,000 sporocysts, or from control does. Other abortifacient agents were not found in the placenta, fetuses, or kids from any does.     

Development of sarcocysts of Sarcocystis levinei in water buffalo infected with sporocysts from dogs

Half a million sporocysts of Sarcocystis levinei obtained from experimentally infected dogs were fed to a buffalo calf, and sarcocysts of this species were recovered from its oesophageal muscles when the animal was killed on the 62nd day of inoculation, thus establishing a buffalo-dog-buffalo cycle.