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1.
Sarcocystis species sporocysts were found in intestinal scrapings from 24 of 72 opossums (Didelphis virginiana) from rural Mississippi. The number of sporocysts in each opossum varied from a few ( < 100000) to 187 million. Sporocysts from 24 opossums were bioassayed for Sarcocystis neurona infections by feeding to gamma-interferon knockout (KO) mice. S. neurona was detected in the brains of KO mice fed sporocysts from 19 opossums by immunohistochemical staining with anti-S. neurona specific polyclonal rabbit serum, and by in vitro culture from the brains of KO mice fed sporocysts. The isolates of S. neurona from opossums were designated SN16-OP to SN34-OP. Merozoites from 17 of 19 isolates tested at the 25/396 locus were identical to previously described S. neurona isolates from horses. The high prevalence of S. neurona sparocysts in D. virginiana suggests that this opossum constitutes an ample reservoir of infection in the southern United States.  相似文献   

2.
Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM) in horse in the Americas. The only known definitive host for this parasite in the United States is the opossum (Didelphis virginiana); however, despite the importance of the disease, the epidemiology of the parasite in the definitive host is poorly understood. To begin addressing these data gaps, potential risk factors were evaluated for their association with the presence of sporocysts of S. neurona in opossums live-trapped in March 1999 and November 1999 to May 2000. Sporocysts of S. neurona were found in 19 of the 72 animals examined. Potential risk factors evaluated were locality, trap date, age, gender, the presence of young in the pouch of females, and body condition score. Variables that were associated with the presence of S. neurona sporocysts were used in logistic regression analysis. Of the factors examined, season and body condition score were associated with increased odds of an animal harboring sporocysts.  相似文献   

3.
The Virginia opossum (Didelphis virginiana) is a definitive host for multiple Sarcocystis species including Sarcocystis neurona, one of the causative agents of equine protozoal myeloencephalitis (EPM), a severe, neuromuscular disease of horses. Size and morphologic characteristics of isolates of Sarcocystis shed by the opossum were examined to determine if differences were useful in discriminating between the isolates and/or species. Collections of sporocysts from 17 opossums were molecularly characterized and measured using an ocular micrometer. The mean sporocyst size of isolates of S. neurona was 10.7 microm x 7.0 microm, Sarcocystis falcatula 11.0 microm x 7.1 microm, Sarcocystis speeri 12.2 microm x 8.8 microm, 1085-like isolate 10.9 microm x 6.8 microm, and 3344-like isolate 19.4 microm x 10.5 microm. The length and width of S. speeri were statistically different (p < 0.05) from the sporocysts of other types. The length of S. neurona and S. falcatula sporocysts were statistically different (p < 0.05) from each other and the width of S. falcatula and 1085 differed (p < 0.05). The fifth sporocyst type (3344) was observed, but due to pronounced morphological characteristics, statistical analysis was not performed. There was no consistent difference between the taxa based on internal structure of the sporocyst.  相似文献   

4.
Fifteen gamma-interferon gene knockout mice were each orally inoculated with 5 x 10(3) Sarcocystis sporocysts derived from Virginia opossums (Didelphis virginiana) fed nine-banded armadillo (Dasypus novemcinctus) muscle containing sarcocysts. Three mice were inoculated with similarly obtained homogenates, but in which no sporocysts were detected. Mouse M8 was pregnant when inoculated and gave birth during the trial. Fifteen of 15 (100%) mice inoculated with sporocysts developed neurologic signs and/or died by day 30 d.p.i. One of 3 (33.3%) mice inoculated with homogenates in which no sporocysts were detected developed clinical signs and died at 34 d.p.i. All young of mouse M8 had maternally acquired antibodies to Sarcocystis neurona, but none developed clinical neurologic signs or had protozoal parasites in their tissues. All brains from mice that developed clinical signs contained merozoites that reacted positively to S. neurona antibodies using immunohistochemical techniques. Evidence from this study further supports the nine-banded armadillo being an intermediate host of S. neurona.  相似文献   

5.
We tested the hypothesis that brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona, the agent of equine protozoal myeloencephalitis (EPM), and act as intermediate hosts for this parasite. In summer 1999, wild caught brown-headed cowbirds were collected and necropsied to determine infection rate with Sarcocystis spp. by macroscopic inspection. Seven of 381 (1.8%) birds had grossly visible sarcocysts in leg muscles with none in breast muscles. Histopathology revealed two classes of sarcocysts in leg muscles, thin-walled and thick-walled suggesting two species. Electron microscopy showed that thick-walled cysts had characteristics of S. falcatula and thin-walled cysts had characteristics of S. neurona. Thereafter, several experiments were conducted to confirm that cowbirds had viable S. neurona that could be transmitted to an intermediate host and cause disease. Specific-pathogen-free opossums fed cowbird leg muscle that was enriched for muscle either with or without visible sarcocysts all shed high numbers of sporocysts by 4 weeks after infection, while the control opossum fed cowbird breast muscle was negative. These sporocysts were apparently of two size classes, 11.4+/-0.7 microm by 7.6+/-0.4 microm (n=25) and 12.6+/-0.6 microm by 8.0+/-0 microm (n=25). When these sporocysts were excysted and introduced into equine dermal cell tissue culture, schizogony occurred, most merozoites survived and replicated long term and merozoites sampled from the cultures with long-term growth were indistinguishable from known S. neurona isolates. A cowbird Sarcocystis isolate, Michigan Cowbird 1 (MICB1), derived from thin-walled sarcocysts from cowbirds that was passaged in SPF opossums and tissue culture went on to produce neurological disease in IFNgamma knockout mice indistinguishable from that of the positive control inoculated with S. neurona. This, together with the knowledge that S. falcatula does not cause lesions in IFNgamma knockout mice, showed that cowbird leg muscles had a Sarcocystis that fulfills the first aim of Koch's postulates to produce disease similar to S. neurona. Two molecular assays provided further support that both S. neurona and S. falcatula were present in cowbird leg muscles. In a blinded study, PCR-RFLP of RAPD-derived DNA designed to discriminate between S. neurona and S. falcatula showed that fresh sporocysts from the opossum feeding trial had both Sarcocystis species. Visible, thick-walled sarcocysts from cowbird leg muscle were positive for S. falcatula but not S. neurona; thin-walled sarcocysts typed as S. neurona. In 1999, DNA was extracted from leg muscles of 100 wild caught cowbirds and subjected to a PCR targeting an S. neurona specific sequence of the small subunit ribosomal RNA (SSU rRNA) gene. In control spiking experiments, this assay detected DNA from 10 S. neurona merozoites in 0.5g of muscle. In the 1999 experiment, 23 of 79 (29.1%) individual cowbird leg muscle samples were positive by this S. neurona-specific PCR. Finally, in June of 2000, 265 cowbird leg muscle samples were tested by histopathology for the presence of thick- and thin-walled sarcocysts. Seven percent (18/265) had only thick-walled sarcocysts, 0.8% (2/265) had only thin-walled sarcocysts and 1.9% (5/265) had both. The other half of these leg muscles when tested by PCR-RFLP of RAPD-derived DNA and SSU rRNA PCR showed a good correlation with histopathological results and the two molecular typing methods concurred; 9.8% (26/265) of cowbirds had sarcocysts in muscle, 7.9% (21/265) had S. falcatula sarcocysts, 1.1% (3/265) had S. neurona sarcocysts, and 0.8% (2/265) had both. These results show that some cowbirds have S. neurona as well as S. falcatula in their leg muscles and can act as intermediate hosts for both parasites.  相似文献   

6.
Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease in the horse most commonly caused by Sarcocystis neurona. The domestic cat (Felis domesticus) is an intermediate host for S. neurona. In the present study, nine farms, known to have prior clinically diagnosed cases of EPM and a resident cat population were identified and sampled accordingly. In addition to the farm cats sampled, samples were also collected from a mobile spay and neuter clinic. Overall, serum samples were collected in 2001 from 310 cats, with samples including barn, feral and inside/outside cats. Of these 310 samples, 35 were from nine horse farms. Horse serum samples were also collected and traps were set for opossums at each of the farms. The S. neurona direct agglutination test (SAT) was used for both the horse and cat serum samples (1:25 dilution). Fourteen of 35 (40%) cats sampled from horse farms had circulating S. neurona agglutinating antibodies. Twenty-seven of the 275 (10%) cats from the spay/neuter clinic also had detectable S. neurona antibodies. Overall, 115 of 123 (93%) horses tested positive for anti-S. neurona antibodies, with each farm having greater than a 75% exposure rate among sampled horses. Twenty-one opossums were trapped on seven of the nine farms. Eleven opossums had Sarcocystis sp. sporocysts, six of them were identified as S. neurona sporocysts based on bioassays in gamma-interferon gene knockout mice with each opossum representing a different farm. Demonstration of S. neurona agglutinating antibodies in domestic and feral cats corroborates previous research demonstrating feral cats to be naturally infected, and also suggests that cats can be frequently infected with S. neurona and serve as one of several natural intermediate hosts for S. neurona.  相似文献   

7.
Opossums (Didelphis virginiana) are exposed to a wide range of coccidia through feeding on a variety of foods, including, but not limited to, carrion, insects, and nestling birds. Abundant D. virginiana populations in urban and suburban areas can be important reservoirs of parasitic infection because of their profuse and prolonged excretion of the sporocysts of several species of Sarcocystis, their omnivorous diet, and their relatively long life span. This report describes 2 adult female opossums found to be simultaneously infected with the tissue cysts of Besnoitia darlingi, sarcocysts of Sarcocystis inghami, as well as with the intestinal sporocysts of S. neurona. Cysts typical of B. darlingi based on gross, histological, and ultrastructural characteristics were disseminated throughout the visceral organs, musculature, ears, and skin. The S. neurona and B. darlingi infections were confirmed by comparative sequence analysis of polymerase chain reaction-amplified diagnostic genetic loci. Sarcocysts of S. inghami are also described. Such examples of multiple parasitic infections show that concurrent infections occur naturally. The propensity for species to coexist should be considered in the differential diagnosis of tissue cyst-forming coccidian protozoa and may have important epidemiological and evolutionary implications.  相似文献   

8.
Sarcocystis neurona is the parasite most commonly associated with equine protozoal myeloencephalitis (EPM). Recently, cats (Felis domesticus) have been demonstrated to be an experimental intermediate host in the life cycle of S. neurona. This study was performed to determine if cats experimentally inoculated with culture-derived S. neurona merozoites develop tissue sarcocysts infectious to opossums (Didelphis virginiana), the definitive host of S. neurona. Four cats were inoculated with S. neurona or S. neurona-like merozoites and all developed antibodies reacting to S. neurona merozoite antigens, but tissue sarcocysts were detected in only two cats. Muscle tissues from the experimentally inoculated cats with and without detectable sarcocysts were fed to laboratory-reared opossums. Sporocysts were detected in gastrointestinal (GI) scrapings of one opossum fed experimentally infected feline tissues. The study results suggest that cats can develop tissue cysts following inoculation with culture-derived Sarcocystis sp. merozoites in which the particular isolate was originally derived from a naturally infected cat with tissue sarcocysts. This is in contrast to cats which did not develop tissue cysts when inoculated with S. neurona merozoites originally derived from a horse with EPM. These results indicate present biological differences between the culture-derived merozoites of two Sarcocystis isolates, Sn-UCD 1 and Sn-Mucat 2.  相似文献   

9.
Sarcocystis neurona was isolated from sporocysts from two of eight South American opossums, Didelphis albiventris, from Brazil. Interferon gamma gene knock out (KO) mice fed sporocysts from two opossums developed neurologic sarcocystosis. S. neurona was demonstrated in the brains of infected KO mice by immunohistochemical staining with anti-S. neurona antibody. The parasite was cultivated in cell culture and S. neurona DNA was isolated from cultured merozoites. This is the first report of isolation of S. neurona from Brazil and the first report from its new host, D. albiventris.  相似文献   

10.
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.  相似文献   

11.
Equine protozoal myeloencephalitis is a common neurologic disease of horses in the Americas usually caused by Sarcocystis neurona. To date, the disease has not been induced in horses using characterized sporocysts from Didelphis virginiana, the definitive host. S. neurona sporocysts from 15 naturally infected opossums were fed to horses seronegative for antibodies against S. neurona. Eight horses were given 5x10(5) sporocysts daily for 7 days. Horses were examined for abnormal clinical signs, and blood and cerebrospinal fluid were harvested at intervals for 90 days after the first day of challenge and analyzed both qualitatively (western blot) and quantitatively (anti-17kDa) for anti-S. neurona IgG. Four of the challenged horses were given dexamethasone (0.1mg/kg orally once daily) for the duration of the experiment. All challenged horses immunoconverted against S. neurona in blood within 32 days of challenge and in CSF within 61 days. There was a trend (P = 0.057) for horses given dexamethasone to immunoconvert earlier than horses that were not immunosuppressed. Anti-17kDa was detected in the CSF of all challenged horses by day 61. This response was statistically greater at day 32 in horses given dexamethasone. Control horses remained seronegative throughout the period in which all challenged horses converted. One control horse immunoconverted in blood at day 75 and in CSF at day 89. Signs of neurologic disease were mild to equivocal in challenged horses. Horses given dexamethasone had more severe signs of limb weakness than did horses not given dexamethasone; however, we could not determine whether these signs were due to spinal cord disease or to effects of systemic illness. At necropsy, mild-moderate multifocal gliosis and neurophagia were found histologically in the spinal cords of 7/8 challenged horses. No organisms were seen either in routinely processed sections or by immunohistochemistry. Although neurologic disease comparable to naturally occurring equine protozoal myeloencephalitis (EPM) was not produced, we had clear evidence of an immune response to challenge both systemically and in the CNS. Broad immunosuppression with dexamethasone did not increase the severity of histologic changes in the CNS of challenged horses. Future work must focus on defining the factors that govern progression of inapparent S. neurona infection to EPM.  相似文献   

12.
At least three species of Sarcocystis (S. neurona, S. falcatula, S. speeri) have recently been shown to use opossums of the genus Didelphis as their definitive host. In order to evaluate the evolutionary relationships among Sarcocystis spp. isolates from the Americas, and to determine whether organisms representing the same parasite lineages are transmitted north and south of the Panamanian isthmus, we inferred the phylogenetic relationships from nucleotide sequence variation in parasites isolated from three opossum species (D. virginiana, D. albiventris, D. marsupialis). In particular, we used variation in the 25/396 marker to compare several isolates from Brazil, Argentina, and the United States to each other and to cloned S. neurona and S. falcatula whose morphology and host affinities have been defined in the laboratory. S. neurona was identified from a Brazilian D. albiventris, as well as from North American D. virginiana. Parasites resembling the Cornell isolate of S. falcatula are transmitted both south and north of the Panamanian isthmus by D. albiventris and D. virginiana, respectively. Distinct attributes at two genetic loci differentiated a Brazilian isolate of S. falcatula from all other known parasite lineages. We confirm S. neurona as the causative agent of recently reported neurologic disease in Southern sea otters, Enhydra lutris nereis. And we found that S. speeri could not be compared to the other opossum-derived Sarcocystis isolates on the basis of nucleotide variation at the 25/396 locus. The widespread distribution of certain species of Sarcocystis may derive from their ability to parasitize migratory bird hosts in their intermediate stage.  相似文献   

13.
Neurologic disease in horses caused by Sarcocystis neurona is difficult to diagnose, treat, or prevent, due to the lack of knowledge about the pathogenesis of the disease. This in turn is confounded by the lack of a reliable equine model of equine protozoal myeloencephalitis (EPM). Epidemiologic studies have implicated stress as a risk factor for this disease, thus, the role of transport stress was evaluated for incorporation into an equine model for EPM. Sporocysts from feral opossums were bioassayed in interferon-gamma gene knockout (KO) mice to determine minimum number of viable S. neurona sporocysts in the inoculum. A minimum of 80,000 viable S. neurona sporocysts were fed to each of the nine horses. A total of 12 S. neurona antibody negative horses were divided into four groups (1-4). Three horses (group 1) were fed sporocysts on the day of arrival at the study site, three horses were fed sporocysts 14 days after acclimatization (group 2), three horses were given sporocysts and dexamethasone 14 days after acclimatization (group 3) and three horses were controls (group 4). All horses fed sporocysts in the study developed antibodies to S. neurona in serum and cerebrospinal fluid (CSF) and developed clinical signs of neurologic disease. The most severe clinical signs were in horses in group 1 subjected to transport stress. The least severe neurologic signs were in horses treated with dexamethasone (group 3). Clinical signs improved in four horses from two treatment groups by the time of euthanasia (group 1, day 44; group 3, day 47). Post-mortem examinations, and tissues that were collected for light microscopy, immunohistochemistry, tissue cultures, and bioassay in KO mice, revealed no direct evidence of S. neurona infection. However, there were lesions compatible with S. neurona infection in horses. The results of this investigation suggest that stress can play a role in the pathogenesis of EPM. There is also evidence to suggest that horses in nature may clear the organism routinely, which may explain the relatively high number of normal horses with CSF antibodies to S. neurona compared to the prevalence of EPM.  相似文献   

14.
Sarcocystis neurona is an important cause of neurological disease in horses (equine protozoal myeloencephalitis, EPM) and sea otters in the United States. In addition, EPM-like disease has been diagnosed in several other land and marine mammals. Opossums are its only definitive hosts. Little genetic diversity among isolates of S. neurona from different hosts has been reported. Here, we used 11 microsatellites to characterize S. neurona DNA isolated from natural infections in 22 sea otters (Enhydra lutris) from California and Washington and in 11 raccoons (Procyon lotor) and 1 striped skunk (Mephitis mephitis) from Wisconsin. By jointly analyzing these 34 isolates with 26 isolates previously reported, we determined that geographic barriers may limit S. neurona dispersal and that only a limited subset of possible parasite genotypes may have been introduced to recently established opossum populations. Moreover, our study confirms that diverse intermediate hosts share a common infection source, the opossum (Didelphis virginiana).  相似文献   

15.
Sarcocystis neurona is the most important cause of a neurologic disease of horses, equine protozoal myeloencephalitis (EPM). Cats and other carnivores can act as its intermediate hosts and horses are aberrant hosts. Little is known of the sero-epidemiology of S. neurona infections in cats. In the present study, antibodies to S. neurona were evaluated by the S. neurona agglutination test (SAT). Cats fed sporocysts from the feces of naturally infected opossums or inoculated intramuscularly with S. neurona merozoites developed high levels (> or =1:4000) of SAT antibodies. Antibodies to S. neurona were not found in a cat inoculated with merozoites of the closely related parasite, Sarcocystis falcatula. These results should be useful in studying sero-epidemiology of S. neurona infections in cats.  相似文献   

16.
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.  相似文献   

17.
Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in the Americas. The apicomplexan protozoan most commonly associated with EPM is Sarcocystis neurona. A direct agglutination test (SAT) was developed to detect antibodies to S. neurona in experimentally infected animals. Merozoites of the SN6 strain of S. neurona collected from cell culture were used as antigen and 2-mercaptoethanol was added to the antigen suspension to destroy IgM antibodies when mixed with test sera. Mice fed sporocysts of S. speeri or S. falcatula-like sporocysts from opossums did not seroconvert in the SAT. The sensitivity of the SAT was 100% and the specificity was 90% in mice.  相似文献   

18.
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19.
Gamma interferon knockout (KO) mice (n=74) were fed a lethal dose of approximately 1000 sporocysts of the SN15-OP isolate of Sarcocystis neurona. Groups of mice were given pelleted rodent feed containing 50ppm of diclazuril at different times before and after feeding sporocysts. All mice were examined at necropsy and their tissues were examined immunohistochemically for S. neurona infection. Twenty mice were fed sporocysts and given diclazuril starting 5 days before feeding sporocysts and continuing 30-39 days post-infection (p.i.). One mouse died of causes unrelated to S. neurona with no demonstrable parasites; the remaining 19 mice remained clinically normal and S. neurona organisms were not found in their tissues. Sarcocystis neurona organisms were not demonstrable by bioassay of the brains of these 19 mice in uninfected KO mice. Sarcocystis neurona organisms were not found in tissues of five mice treated with diclazuril, starting 7 days after feeding sporocysts and continuing up to 39 days p.i. Therapy was less efficient when diclazuril was given 10 days p.i. Sarcocystis neurona organisms were found in two of 19 mice treated with diclazuril starting 10 days after feeding sporocysts, in two of five mice starting therapy 12 days p.i., and in 10 of 10 mice when treatment was delayed until 15 days p.i. All 15 mice fed S. neurona, but not given diclazuril, developed neural sarcocystosis and were euthanized 22-30 days after feeding sporocysts. Six mice not fed S. neurona, but given diclazuril for 44 days, remained clinically normal. Results indicate that diclazuril can kill the early stages of S. neurona.  相似文献   

20.
Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses caused primarily by the protozoal parasite Sarcocystis neurona. Currently available antemortem diagnostic testing has low specificity. The hypothesis of this study was that serum and cerebrospinal fluid (CSF) of horses experimentally challenged with S neurona would have an increased S neurona-specific IgM (Sn-IgM) concentration after infection, as determined by an IgM capture enzyme linked immunoassay (ELISA). The ELISA was based on the S neurona low molecular weight protein SNUCD-1 antigen and the monoclonal antibody 2G5 labeled with horseradish peroxidase. The test was evaluated using serum and CSF from 12 horses experimentally infected with 1.5 million S neurona sporocysts and 16 horses experimentally infected with varying doses (100 to 100,000) of S neurona sporocysts, for which results of histopathologic examination of the central nervous system were available. For horses challenged with 1.5 million sporocysts, there was a significant increase in serum Sn-IgM concentrations compared with values before infection at weeks 2-6 after inoculation (P < .0001). For horses inoculated with lower doses of S neurona, there were significant increases in serum Sn-IgM concentration at various points in time after inoculation, depending on the challenge dose (P < .01). In addition, there was a significant increase between the CSF Sn-IgM concentrations before and after inoculation (P < .0001). These results support further evaluation of the assay as a diagnostic test during the acute phase of EPM.  相似文献   

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