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.     

Neurologic disease in gamma-interferon gene knockout mice caused by Sarcocystis neurona sporocysts collected from opossums fed armadillo muscle.

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.     

Determination of the activity of ponazuril against Sarcocystis neurona in cell cultures

The present study examined the efficacy of ponazuril in inhibiting merozoite production of Sarcocystis neurona in cell cultures. Ponazuril inhibited merozoite production by more that 90% in cultures of S. neurona treated with 1.0 microg/ml ponazuril and greater than 95% inhibition of merozoite production was observed when infected cultures were treated with 5.0 microg/ml ponazuril. Ponazuril may have promise as a therapeutic agent in the treatment of S. neurona induced equine protozoal myeloencephalitis (EPM) in horses.     

Effect of daily administration of pyrantel tartrate in preventing infection in horses experimentally challenged with Sarcocystis neurona

OBJECTIVE: To determine whether daily administration of pyrantel tartrate can prevent infection in horses experimentally challenged with Sarcocystis neurona. ANIMALS: 24 mixed-breed specific-pathogen-free weanling horses, 10 adult horses, 1 opossum, and 6 mice. PROCEDURE: Sarcocystis neurona-na?ve weanling horses were randomly allocated to 2 groups. Group A received pyrantel tartrate at the labeled dose, and group B received a nonmedicated pellet. Both groups were orally inoculated with 100 sporocysts/d for 28 days, 500 sporocysts/d for 28 days, and 1000 sporocysts/d for 56 days. Blood samples were collected weekly, and CSF was collected monthly. Ten seronegative adult horses were monitored as untreated, uninfected control animals. All serum and CSF samples were tested by use of western blot tests to detect antibodies against S. neurona. At the end of the study, the number of seropositive and CSF-positive horses in groups A and B were compared by use of the Fisher exact test. Time to seroconversion on the basis of treatment groups and sex of horses was compared in 2 univariable Cox proportional hazards models. RESULTS: After 134 days of sporocyst inoculation, no significant differences were found between groups A and B for results of western blot tests of serum or CSF There were no significant differences in number of days to seroconversion on the basis of treatment groups or sex of horses. The control horses remained seronegative. CONCLUSIONS AND CLINICAL RELEVANCE: Daily administration of pyrantel tartrate at the current labeled dose does not prevent S. neurona infection in horses.     

Diclazuril preventive therapy of gamma interferon knockout mice fed Sarcocystis neurona sporocysts

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.     

Pathology of sarcocystis neurona in interferon-gamma gene knockout mice

Pathologic changes were studied in 27 interferon-gamma gene knockout mice 34-54 days after being fed graded doses of Sarcocystis neurona sporocysts derived from a naturally infected opossum. The target tissue for S. neurona infection was the central nervous system. Characteristic histopathologic changes present in all mice consisted of an inflammatory infiltrate consisting of mostly neutrophils and macrophages, fewer eosinophils, and rare multinucleated giant cells. Intralesional protozoa and scattered subacute perivascular cuffs were present. Where the infiltrates were extensive, neuropil rarefaction was frequent. Pathologic changes were much more frequent and severe in the caudal portion of the brain, especially in the cerebellum, than in the middle and cranial portions. Changes were present in all spinal cords examined (10 of 10). Lesions were equally distributed in white and gray matter of the brain and spinal cord and their meningeal linings.     

Anthelmintic activity of pyrantel tartrate against Ascaridia galli in fowls.

At a dose of 15 mg/kg body weight, pyrantel tartrate was 18-51, 99-63, 100 and 100 per cent effective in chickens treated at 10, 20, 30 and 40 days respectively after infection with Ascaridia galli. Similarly, 25 mg/kg was 14-44, 100, 100 and 99-63 per cent effective.     

Serologic responses of cats against experimental Sarcocystis neurona infections

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.     

Migration and development of Sarcocystis neurona in tissues of interferon gamma knockout mice fed sporocysts from a naturally infected opossum

Migration and development of Sarcocystis neurona was studied in 50 gamma interferon knockout mice fed graded doses of S. neurona sporocysts from the intestine of a naturally infected opossum. Mice were examined at necropsy 1-62 days after feeding sporocysts (DAFS). All tissue sections were reacted with anti-S. neurona-specific polyclonal rabbit serum in an immunohistochemical (IHC) test. Between 1 and 3 DAFS, organisms were seen mainly in intestines. Between 4 and 11 DAFS, organisms were seen in several visceral tissues. Beginning with 13 DAFS, schizonts and merozoites were present in sections of brains of all infected mice. All regions of the brain were parasitized but the hind brain was most severely affected. S. neurona was found in the spinal cord of all 10 mice examined 22-30 DAFS. Of the 28 infected mice examined 20-62 DAFS, S. neurona was found in the brains of all 28, lungs of 14, hearts of 8 and eyes of 3. More organisms were seen in IHC-stained sections than in sections stained with hematoxylin and eosin. Treatment of tissues with glutaraldehyde, Karnovsky fixative, and ethylene diamino tetra acetic acid (EDTA, used for decalcification) did not affect staining of organisms by IHC.     

Immunoconversion against Sarcocystis neurona in normal and dexamethasone-treated horses challenged with S. neurona sporocysts

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.     

Determination of the activity of pyrimethamine, trimethoprim, sulfonamides, and combinations of pyrimethamine and sulfonamides against Sarcocystis neurona in cell cultures.

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. The activities of pyrimethamine, trimethoprim, sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfamethazine, and sulfathiazole were examined against developing S. neurona merozoites in bovine turbinate cell cultures. A microtiter plate host cell lesion based assay was used to determine the effects of agents on developing merozoites. A cell culture flask assay was used to determine if selective concentrations of the agents killed or only inhibited development of S. neurona. Pyrimethamine was coccidiocidal at 1.0 microg/ml and trimethoprim was coccidiocidal at 5.0 microg/ml. None of the sulfonamides had activity when used alone at 50.0 or 100.0 microg/ml. Combinations of sulfonamides (5.0 or 10.0 microg/ml) with 0.1 microg/ml pyrimethamine demonstrated improved activity.     

Detection of antibodies against Sarcocystis neurona in cerebrospinal fluid from clinically normal neonatal foals

OBJECTIVE: To determine whether antibodies against Sarcocystis neurona could be detected in CSF from clinically normal neonatal (2 to 7 days old) and young (2 to 3 months old) foals. DESIGN: Prospective study. ANIMALS: 15 clinically normal neonatal Thoroughbred foals. PROCEDURE: Serum and CSF samples were obtained from foals at 2 to 7 days of age and tested for antibodies against S. neurona by means of western blotting. Serum samples from the mares were also tested for antibodies against S. neurona. Additional CSF and blood samples were obtained from 5 foals between 13 and 41 days after birth and between 62 and 90 days after birth. RESULTS: Antibodies against S. neurona were detected in serum from 13 mares and their foals; antibodies against S. neurona were detected in CSF from 12 of these 13 foals. Degree of immunoreactivity in serum and CSF decreased over time, and antibodies against S. neurona were no longer detected in CSF from 2 foals 83 and 84 days after birth. However, antibodies could still be detected in CSF from the other 3 foals between 62 and 90 days after birth. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that antibodies against S. neurona can be detected in CSF from clinically normal neonatal (2 to 7 days old) foals born to seropositive mares. This suggests that western blotting of CSF cannot be reliably used to diagnose equine protozoal myeloencephalitis in foals < 3 months of age born to seropositive mares.     

Sarcocystis neurona and Sarcocystis falcatula: monitoring of schizogony in cell culture using fluorescent nuclear labeling

The nuclei of merozoites of Sarcocystis neurona and Sarcocystis falcatula were labeled with the fluorescent marker Syto21. It was shown that the marker would label the parasites and that they would retain the marker throughout schizogony. Thus, there was sufficient marker in the daughter merozoites to make them easily visible with fluorescence microscopy. This technique will be helpful in studying the developmental biology of these parasites in vitro.