Experimental infection of ponies with Sarcocystis fayeri and differentiation from Sarcocystis neurona infections in horses

Sarcocystis neurona and Sarcocystis fayeri infections are common in horses in the Americas. Their antemortem diagnosis is important because the former causes a neurological disorder in horses, whereas the latter is considered nonpathogenic. There is a concern that equine antibodies to S. fayeri might react with S. neurona antigens in diagnostic tests. In this study, 4 ponies without demonstrable serum antibodies to S. neurona by Western immunoblot were used. Three ponies were fed 1 x 10(5) to 1 x 10(7) sporocysts of S. fayeri obtained from dogs that were fed naturally infected horse muscles. All ponies remained asymptomatic until the termination of the experiment, day 79 postinoculation (PI). All serum samples collected were negative for antibodies to S. neurona using the Western blot at the initial screening, just before inoculation with S. fayeri (day 2) and weekly until day 79 PI. Cerebrospinal fluid samples from each pony were negative for S. neurona antibodies. Using the S. neurona agglutination test, antibodies to S. neurona were not detected in 1:25 dilution of sera from any samples, except that from pony no. 4 on day 28; this pony had received 1 X 10(7) sporocysts. Using indirect immunofluorescence antibody tests (IFATs), 7 serum samples were found to be positive for S. neurona antibodies from 1:25 to 1:400 dilutions. Sarcocystis fayeri sarcocysts were found in striated muscles of all inoculated ponies, with heaviest infections in the tongue. All sarcocysts examined histologically appeared to contain only microcytes. Ultrastructurally, S. fayeri sarcocysts could be differentiated from S. neurona sarcocysts by the microtubules (mt) in villar protrusions on sarcocyst walls; in S. fayeri the mt extended from the villar tips to the pellicle of zoites, whereas in S. neurona the mt were restricted to the middle of the cyst wall. Results indicate that horses with S. fayeri infections may be misdiagnosed as being S. neurona infected using IFAT, and further research is needed on the serologic diagnosis of S. neurona infections.     

Prevalence and development of two Sarcocystis spp. in the horse (author's transl)

The prevalence of Sarcocystis spp. in horses was investigated in a survey at the Munich abattoir during 1978/79. Muscle specimens (oesophagus, diaphragm, sublingual muscle, myocardium) were examined using tryptic digestion. Out of 200 horses 31 (15.5%) were found to be carriers of sarcocysts. No parasites were found in the myocardium. In three animals sarcocysts could be isolated and differentiated in fresh preparations. Cysts with 5 to 11 microns by less than 0.5 microns hairlike, unstable protrusions were classified as Sarcocystis equicanis, whereas those with 2.5 to 4.5 microns by 0.8 to 1.0 microns fingerlike, stabile protrusions were assigned to be S. fayeri. Histologically S. equicanis cysts were thin-walled and S. fayeri cysts were thick-walled and often striated. For both species the dog acts as final host. A mixture of sporocysts of both species measured: 12.0--14.4 (13.4 +/- 0.7) X 9.3--10.5 (9.8 +/- 0.4) microns. The prepatent period is 11 to 17 days. Two ponies experimentally infected with 100,000 sporocysts each did not show clinical signs. In fresh preparations and in histopathological examinations of biopsied (111th, 130th, 152th, and 165th day post-infection (p.i.) and postmortem material (167th and 189th day p.i.) different developmental stages of sarcocysts of both species were seen and the following pathological alterations observed: circumscribed non-purulent inflammation, moderate Zenker's degeneration of muscle fibres, and degenerated cysts, of which sometimes only parts of the cyst wall were left. In fresh preparations S. equicanis and S. Fayeri could be differentiated 111 days p.i. The observed disappearance of the sarcocysts is suggested to be a self-cleaning process.     

Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease of the horse. The causative agent. Sarcocystis neurona, has been suggested to be synonymous with Sarcocystis falcatula, implying a role for birds as intermediate hosts. To test this hypothesis, opossums (Didelphis virginiana) were fed muscles containing S. falcatula sarcocysts from naturally infected brown-headed cowbirds (Molothrus ater). Ten horses were tested extensively to ensure no previous exposure to S. neurona and were quarantined for 14 days, and then 5 of the horses were each administered 10(6) S. falcatula sporocysts collected from laboratory opossums. Over a 12-wk period, 4 challenged horses remained clinically normal and all tests for S. neurona antibody and DNA in serum and cerebrospinal fluid were negative. Rechallenge of the 4 seronegative horses had identical results. Although 1 horse developed EPM, presence of S. neurona antibody prior to challenge strongly indicated that infection occurred before sporocyst administration. Viability of sporocysts was confirmed by observing excystation in equine bile in vitro and by successful infection of naive brown-headed cowbirds. These data suggest that S. falcatula and S. neurona are not synonymous. One defining distinction is the apparent inability of S. falcatula to infect horses, in contrast to S. neurona, which was named when cultured from equine spinal cord.     

Sarcocystis arieticanis and other Sarcocystis species in sheep in the United States

Histological sections of tongues, esophagi, and diaphragms from 512 adult ewes from the northwest United States and Texas were examined for Sarcocystis spp. Sarcocysts were found in sections of 82.1% of 504 tongues, 44.4% of 478 esophagi, and 51.7% of 89 diaphragms. Sarcocystis tenella was the predominant species and was found in 430 (84.0%) sheep; S. arieticanis was found in 18 (3.5%) sheep. The mean number of S. tenella sarcocysts in tissue sections was approximately 10 times higher than that of S. arieticanis. The identification of S. arieticanis was confirmed by ultrastructural studies and by transmission to dogs. Macroscopic sarcocysts of S. gigantea were also found but were not quantitated in all sheep; sarcocysts of S. medusiformis were not observed.     

Sarcocystis speeri N. sp. (Protozoa: Sarcocystidae) from the opossum (Didelphis virginiana)

The North American opossum (Didelphis virginiana) is host to at least 3 species of Sarcocystis: Sarcocystisfalcatula, Sarcocystis neurona, and a recently recognized Sarcocystis sp. A new name, Sarcocystis speeri, is proposed for the third unnamed Sarcocystis. Immunodeficient mice are an experimental intermediate host for S. speeri. Sarcocystis speeri sporocysts are 12-15 x 8-10 microm in size, and its schizonts are found in many organs of mice. Sarcocysts of S. speeri are found in skeletal muscles and they are up to 5 mm long and filiform. By light microscopy, the sarcocyst wall is thin (<1 microm thick); ultrastructurally, the cyst wall is up to 1.8 microm thick and has characteristic steeple-shaped villar protrusions surmounted by a spire. Sarcocystis speeri schizonts are morphologically and antigenically distinct from schizonts of S. neurona, and S. speeri sporocysts were not infective to budgerigars (Melopsittacus undulatus).     

Transmission studies with Sarcocystis idahoensis of deer mice (Peromyscus maniculatus) and gopher snakes (Pituophis melanoleucus)

Transmission studies with Sarcocystis idahoensis of deer mice (Peromyscus maniculatus) and gopher snakes (pituophis melanoleucus) were conducted to determine host specificity of various stages of the parasite. Sporocysts were not passed by four dogs or four cats fed infected skeletal muscle from deer mice. Seven white mice (Mus musculus) and 34 white-footed mice (Peromyscus leucopus) were negative for sarcocysts and liver meronts following oral inoculation with S. idahoensis sporocysts; however, excystation of sporocysts occurred in two white-footed mice killed four hours post inoculation (PI). A gopher snake orally inoculated with sporocysts remained negative for coccidia for two months PI. Three deer mice orally inoculated and three intraperitoneally (IP) inoculated with tachyzoites from liver meronts developed sarcocysts in their skeletal muscles similar to those seen in deer mice orally inoculated with sporocysts. Liver meronts were not found. Ten deer mice orally inoculated and 10 deer mice inoculated IP with bradyzoites from S. idahoensis sarcocysts remained negative for sarcocysts and liver meronts at necropsy 17 days PI.     

Development of Sarcocystis fayeri in the equine

Eight ponies and a horse were inoculated orally with sporocysts of Sarcocystis fayeri from dogs. They were examined for clinical signs of infection and killed 10, 15, 20, 25, 30, 50 (horse), 77, 101, and 156 days after inoculation (DAI). Elevated temperature was observed in three ponies 20 and 26 DAI and anemia was observed in three ponies and the horse 15 to 69 DAI. Schizonts were found in or near cells lining capillaries or arteries of the heart, brain, and kidney 10, 20, and 25 DAI. Immature cysts containing only metrocytes were first found in muscles 50 DAI. Mature intramuscular cysts containing metrocytes and zoites or zoites alone were found 77, 101, and 156 DAI and produced patent infections in dogs fed infected meat.     

Experimental induction of equine protozoan myeloencephalitis (EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the development of clinical neurologic disease

The effect of inoculation dose of Sarcocystis neurona sporocysts on the development of clinical neurologic disease in horses was investigated. Twenty-four seronegative weanling horses were subjected to the natural stress of transport and then randomly assigned to 6 treatment groups of 4 horses each. Horses were then immediately inoculated with either 10(2), 10(3), 10(4), 10(5), or 10(6) S. neurona sporocysts or placebo using nasogastric tube and housed indoors. Weekly neurologic examinations were performed by a blinded observer. Blood was collected weekly for antibody determination by Western blot analysis. Cerebrospinal fluid was collected before inoculation and before euthanasia for S. neurona antibody determination.Horses were killed and necropsied between 4 and 5 wk after inoculation. Differences were detected among dose groups based on seroconversion times, severity of clinical neurologic signs, and presence of microscopic lesions. Seroconversion of challenged horses was observed as early as 14 days postinfection in the 10(6) sporocyst dose group. Mild to moderate clinical signs of neurologic disease were produced in challenged horses from all groups, with the most consistent signs seen in the 10(6) sporocyst dose group. Histologic lesions suggestive of S. neurona infection were detected in 4 of the 20 horses fed sporocysts. Parasites were not detected in equine tissues by light microscopy, immunohistochemistry, or bioassay in gamma-interferon gene knockout mice. Control horses remained seronegative for the duration of the study and had no histologic evidence of protozoal infection.     

Simplified technique for isolation, excystation, and culture of Sarcocystis species from opossums

Sarcocystis neurona is a protozoan parasite that causes a neurological disease in horses called equine protozoal myeloencephalitis. The route of transmission is speculated to be by fecal-oral transfer of sporocysts shed from opossums. Controversy exists regarding both the natural life cycle for this parasite as well as the species identity of opossum Sarcocystis. To provide stage-specific material for species comparison, 27 opossums from southern Michigan were screened for Sarcocystis spp. sporocysts. Seven opossums were positive for Sarcocystis sporocysts by fecal flotation. A simplified, effective technique for isolation, excystation, and culture of opossum Sarcocystis sp. from mucosal scrapings was developed. All 7 Sarcocystis sp. isolates were successfully cultured to grow long term in equine dermal cells to the merozoite stage. Merozoites were observed between 5 and 15 days after inoculation. In conclusion, opossums shed Sarcocystis sp. sporocysts that may be manipulated to excyst and grow in vitro in equine dermal cell lines to the merozoite stage using the simplified technique described.     

Experimental infection of dogs with Sarcocystis from wapiti.

Ten domestic dogs became infected with Sarcocystis when fed simple portions of heart, esophagus and diaphragm from a two-year-old female wapiti (Cervus canadensis). The prepatent period was 14 days in all exposed dogs; the patent period ranged from 8 to 20 days. Neither the 10 control dogs, nor two dogs fed sporocysts collected from the infected dogs passed sporocysts within the study period. Sporocysts averaged 16.5 by 11.1 micron in size.     

The gamma interferon knockout mouse model for sarcocystis neurona: comparison of infectivity of sporocysts and merozoites and routes of inoculation.

The dose-related infectivity of Sarcocystis neurona sporocysts and merozoites of 2 recent isolates of S. neurona was compared in gamma interferon knockout (KO) mice. Tenfold dilutions of sporocysts or merozoites were bioassayed in mice, cell culture, or both. All 8 mice, fed 1,000 sporocysts, developed neurological signs with demonstrable S. neurona in their tissues. Of 24 mice fed low numbers of sporocysts (100, 10, 1), 18 became ill by 4 wk postinoculation, and S. neurona was demonstrated in their brains; antibodies (S. neurona agglutination test) to S. neurona and S. neurona parasites were not found in tissues of the 6 mice that were fed sporocysts and survived for >39 days. One thousand culture-derived merozoites of these 2 isolates were pathogenic to all 8 mice inoculated subcutaneously (s.c.). Of the 24 mice inoculated s.c. with merozoites numbering 100, 10, or 1, only 3 mice had demonstrable S. neurona infection; antibodies to S. neurona were not found in the 21 mice that had no demonstrable organisms. As few as 10 merozoites were infective for cell cultures. These results demonstrate that at least 1,000 merozoites are needed to cause disease in KO mice. Sarcocystis neurona sporocysts were infective to mice by the s.c. route.     

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 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.     

Ultrastructural development of the sarcocyst of Sarcocystis rauschorum (Apicomplexa: Sarcocystidae) in the varying lemming Dicrostonyx richardsoni

The development of the sarcocyst of Sarcocystis rauschorum in its intermediate host was studied. Lemmings were orally administered sporocysts of S. rauschorum obtained from snowy owls (Nyctea scandiaca). Beginning at 9 days postinoculation (DPI) and at various intervals to 84 DPI, skeletal muscle tissue taken from the infected lemmings was examined by electron microscopy. At 9 DPI the sarcocysts contained few metrocytes and the cyst wall was flat. The metrocytes underwent endodyogeny, and within a few days the cyst wall of the rapidly growing sarcocyst developed numerous tubulovesicular invaginations into the electron-dense layer, and the wall had a few irregular infoldings. By 21 DPI, banana-shaped bradyzoites appeared, and by 84 DPI the mature cysts were filled with bradyzoites in groups subdivided by septa and by deep infoldings of the cyst wall. The fine structure of the wall remained simple throughout maturation, with no conspicuous invagination or protrusion. The sarcocyst produced in response to S. rauschorum is unlike those from many species of Sarcocystis, which have complex walls that change markedly as the sarcocysts mature; however, its simple appearance is similar to other species that have rodents as intermediate hosts and raptorial birds as definitive hosts.     

Ultrastructure of the cyst and life cycle of Sarcocystis sp. from wild sheep (Ovis musimon)

Sarcocystis sp. (Eimeriina: Sarcocystidae) is described as a heteroxenous coccidian with domestic dogs as an experimental definitive host and wild sheep (Ovis musimon) as natural intermediate hosts. Mature sarcocysts of this Sarcocystis sp. were examined by transmission electron microscopy. Sarcocysts in various muscle tissues were microscopic, had a thin primary cyst wall and septa and measured 81.0 x 30.5 microns. The cysts were located within muscle cells and were limited by a primary cyst wall (PCW). The cyst surface was highly folded forming densely packed projections. Between the PCW projections the surface of the cyst was marked with pit-like invaginations. The ground substance of the cyst formed a layer at the periphery of the cyst, filled the projections and formed septa which divided the cyst into compartments. Sarcocysts contained numerous bradyzoites that were 15.2 x 3 microns and few metrocytes 11.5 x 3.5 microns. Twelve days after ingesting Sarcocystis sp.-infected wild sheep meat, four dogs began passing sporocysts in their feces: two domestic cats did not pass oocysts or sporocysts after ingesting meat from the same animals. Sporocysts measured 14.8 x 9.9 microns.     

Sarcocystis sigmodontis n. sp. from the cotton rat (Sigmodon hispidus)

Sarcocystis sarcocysts were found in 3 of 4 cotton rats (Sigmodon hispidus) from Atlanta, Georgia. Sarcocysts were several centimetres long and were present only in skeletal muscles. The sarcocyst wall appeared thin (less than 1 micron), with minute projections in the light microscope. By transmission electron microscopy, the sarcocyst wall had 0.6-1.0 x 0.21-0.36-micron villar protrusions without microtubules. The metrocytes were 6.5 x 3.8 micron, and the bradyzoites were 8 x 2.7 micron. The sarcocysts were not infectious for dogs and cats. The parasite was named Sarcocystis sigmodontis because it differed from all sarcocysts in rodents.     

Life cycle of Sarcocystis tenella in sheep and dog

For Sarcocystis tenella, the second microscopic sarcocyst in sheep, the dog was shown to act as final host shedding sporocysts measuring 13.75-15.8 (14.8 +/- 0.8) X 9.7-10.8 (10.1 +/- 0.4) micron after a prepatent period of 8-13 days. The clinical signs and the course of experimental infections in sheep were most similar to S. ovicanis. After high doses of sporocysts sheep had temperatures up to 42 degrees C, anaemia, and paresis; they finally died from haemorrhagic diathesis. The development of S. tenella in sheep was studied and it resulted in microscopic cysts in the musculature that measured 300-650 X 20-50 micron. They showed hair-like delicate protrusions of the cyst wall measuring 6-8 X less than 0.5 micron, by which S. tenella could be clearly differentiated from S. ovicanis from day 60 p.i. onwards. The decreasing number of S. tenella through degeneration of cysts is suggested to be a self-cleaning process.     

Sarcocystis Idahoensis Sp. N. In Deer Mice Peromyscus Maniculatus (Wagner) and Geopher Snakes Pituophis Melanoleucus (Daudin)*,†

SYNOPSIS Deer mice Peromyscus maniculatus (Wagner) were trapped near Hammett, Idaho, as a possible source of Besnoitia jellisoni Frenkel and species of Sarcocystis to be used for life cycle studies. Forty-nine deer mice were necropsied; 20 (40.8%) were positive for sarcocysts structurally identical with those of Sarcocystis idahoensis sp. N. the source of S. idahoensis used for life cycle studies was a Great Basin gopher snake Pituophis melanoleucus deserticola Stejneger killed near Hammett, Idaho; 20 sporulated sporocysts measured 11.1 × 13.4 (11-12 × 13-14) μm. Structurally identical sporocysts were found in 7 of 14 Pacific gopher snakes P. m. catenifer (Blainville), and in 6 of 10 San Diego gopher snakes, P. m. annectens Baird & Girard. Totals of 148 deer mice and 17 gopher snakes were necropsied in the course of life cycle studies. Development of the first generation meronts took place within the hepatocytes of deer mice 2-10 days post-inoculation (PI) with sporulated sporocysts. Rosette-shaped meronts (6-8 days PI) contained tachyzoites attached by their posterior poles to a residual body. After release from the residual body, tachyzoites were initially retained in a meront wall and later released from the hot cells Within muscle cells a single tachyzoite-shaped structure was found 11 days PI and PAS-negative metrocyte-containing sarcocysts (2nd generation meronts) 13-34 days PI. PAS-positive material was first seen in sarcocysts 34 days II at which time bradyzoite formation became apparent. At 160 days PI, 10 sarcocysts measured 0.4 × 5.8 (0.2-0.9 × 1.8-9.9) μ and appeared to be mature and structurally identical with those from naturally infected deer mice. After ingestion of S. idahoensis-infected deer mice by gopher snakes, bradyzoites developed directly into microgamonts and macrogametes. These stages were first seen 5 days PI. Microgamonts were generally located above and macrogametes below the epithelial host cell nucleus. Seven to 11 days PI microgamonts were seen with mature microgametes, and oocysts which had not yet begun sporogony were found with oocyst walls. Clinical signs of illness were generally not observed in infected gopher snakes; however, one snake developed anorexia and cachexia, and became moribund after repeated ingestion of heavily infected deer mice. Acute hepatitis associated with developing meronts often was noted in deer mice given over 15,000 sporocysts each. Five to 6 days PI anorexia, weakness, ataxia, and dyspnea were observed: these clinical signs increased in severity until 6-8 days PI, when mice became recumbent and died, or were killed while moribund. Hepatosplenomegaly, petechial hemorrhage o the serosal and cut surfaces of the liver, and icterus were common. Diffuse coagulative necrosis with cellular infiltration (primarily neutrophils) was noted on microscopic examination.     

Sarcocystis Infection in Sheep from South-Western Norway

The occurrence of Sarcocystis infection and pathological changes were recorded in samples of the heart, diaphragm, and oesophagus from 198 healthy sheep representing 3 different age groups, obtained from an abattoir. The infection rate of S. gigantea (syn. S. tenella) was 18.2 %, and the distribution within groups was: ewes 30.0 %, yearlings 11.6 %, lambs nil. The infection rate of S. tenella (syn. S. ovicanis) was 65.1 %, and the corresponding distribution was: ewes 83.5 %, yearlings 74.4 %, and lambs 25.0 %. A third type of Sarcocystis sp. displaying thick wall was found in 3 samples. Focal interstitial infiltrates of mononuclear cells were demonstrated in 47.9 % of the hearts, in 19.6 % of the diaphragms and in 31.3 % of the oesophagi. The occurrence of Sarcocystis and the focal interstitial mononuclear cell infiltrates were positively correlated (P < 0.0001). Morphologically identical sporocysts typical of S. tenella were produced by dogs and foxes fed naturally infected sheep tissues. A cat fed S. gigantea macrocysts produced sporocysts characteristic for the species. Sarcocystis; pathology; life cycle; final hosts; sheep.