• View in gallery
    Figure 1—

    Proportions of horses that seroconverted against Sarcocystis neurona after inoculation with 612,500 S neurona sporocysts via nasogastric tube (day 0) followed by treatment with ponazuril (20 mg/kg, PO) every 7 days beginning on day 5 (gray bars; n = 5) or every 14 days (striped bars; 5) beginning on day 12 or no treatment (black bars; 5) during a 12-week study period. Five control horses were not inoculated with sporocysts and received no treatment (white bars). Ponazuril was formulated as a paste (0.15 g of toltrazuril sulfone/g). Blood samples were collected before challenge (day – 1) and at 14-day intervals after challenge. *Within a group, value was significantly (P ≤ 0.05) different from value at day – 1. †Value was significantly (P ≤ 0.05) different from the value for challenged, untreated horses at the same time point.

  • View in gallery
    Figure 2—

    Proportions of the horses in Figure 1 that immunoconverted against S neurona as determined by positive results of western blot analysis of CSF samples. See Figure 1 for key.

  • View in gallery
    Figure 3—

    Mean ± SEM relative concentration of antibody against the S neurona 17-kd antigen in CSF samples collected from the horses in Figure 1. See Figure 1 for key.

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Effect of intermittent oral administration of ponazuril on experimental Sarcocystis neurona infection of horses

Robert J. MacKayDepartment of Large Animal Clinical Sciences, University of Florida, Gainesville, FL 32610.

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Susan T. TanhauserDepartment of Large Animal Clinical Sciences, University of Florida, Gainesville, FL 32610.

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Karen D. GillisDepartment of Large Animal Clinical Sciences, University of Florida, Gainesville, FL 32610.

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Ian G. MayhewDepartment of Veterinary Clinical Studies, Easter Bush Veterinary Centre, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland.

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Tom J. KennedyBayer Animal Health, Merriam, KS 66202.

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Abstract

Objective—To evaluate the effect of intermittent oral administration of ponazuril on immunoconversion against Sarcocystis neurona in horses inoculated intragastrically with S neurona sporocysts.

Animals—20 healthy horses that were seronegative for S neurona–specific IgG.

Procedures—5 control horses were neither inoculated with sporocysts nor treated. Other horses (5 horses/group) each received 612,500 S neurona sporocysts via nasogastric tube (day 0) and were not treated or were administered ponazuril (20 mg/kg, PO) every 7 days (beginning on day 5) or every 14 days (beginning on day 12) for 12 weeks. Blood and CSF samples were collected on day – 1 and then every 14 days after challenge for western blot assessment of immunoconversion. Clinical signs of equine protozoal myeloencephalitis (EPM) were monitored, and tissues were examined histologically after euthanasia.

Results—Sera from all challenged horses yielded positive western blot results within 56 days. Immunoconversion in CSF was detected in only 2 of 5 horses that were treated weekly; all other challenged horses immunoconverted within 84 days. Weekly administration of ponazuril significantly reduced the antibody response against the S neurona 17-kd antigen in CSF. Neurologic signs consistent with EPM did not develop in any group; likewise, histologic examination of CNS tissue did not reveal protozoa or consistent degenerative or inflammatory changes.

Conclusions and Clinical Relevance—Administration of ponazuril every 7 days, but not every 14 days, significantly decreased intrathecal anti–S neurona antibody responses in horses inoculated with S neurona sporocysts. Protocols involving intermittent administration of ponazuril may have application in prevention of EPM.

Abstract

Objective—To evaluate the effect of intermittent oral administration of ponazuril on immunoconversion against Sarcocystis neurona in horses inoculated intragastrically with S neurona sporocysts.

Animals—20 healthy horses that were seronegative for S neurona–specific IgG.

Procedures—5 control horses were neither inoculated with sporocysts nor treated. Other horses (5 horses/group) each received 612,500 S neurona sporocysts via nasogastric tube (day 0) and were not treated or were administered ponazuril (20 mg/kg, PO) every 7 days (beginning on day 5) or every 14 days (beginning on day 12) for 12 weeks. Blood and CSF samples were collected on day – 1 and then every 14 days after challenge for western blot assessment of immunoconversion. Clinical signs of equine protozoal myeloencephalitis (EPM) were monitored, and tissues were examined histologically after euthanasia.

Results—Sera from all challenged horses yielded positive western blot results within 56 days. Immunoconversion in CSF was detected in only 2 of 5 horses that were treated weekly; all other challenged horses immunoconverted within 84 days. Weekly administration of ponazuril significantly reduced the antibody response against the S neurona 17-kd antigen in CSF. Neurologic signs consistent with EPM did not develop in any group; likewise, histologic examination of CNS tissue did not reveal protozoa or consistent degenerative or inflammatory changes.

Conclusions and Clinical Relevance—Administration of ponazuril every 7 days, but not every 14 days, significantly decreased intrathecal anti–S neurona antibody responses in horses inoculated with S neurona sporocysts. Protocols involving intermittent administration of ponazuril may have application in prevention of EPM.

Equine protozoal myeloencephalitis is an important neurologic disease that is enzootic among horses in North and South America. Although < 1% of adult horses in the United States develop EPM,1 results of surveys have indicated that 32.6% to 89.2% have serologic evidence of infection with the usual causative agent, Sarcocystis neurona.2,3

The life cycle of S neurona involves reciprocal transmission of the agent between the definitive host (the opossum) and any of several intermediate hosts, including skunks, nine-banded armadillos, raccoons, and domestic cats.4–7 It is likely that horses are infected naturally via ingestion of sporocysts derived from opossums. The fate of ingested sporocysts in horses is not fully understood, but on the basis of findings in mice with experimentally induced S neurona infection,8 it appears that at least 1 cycle of schizogony occurs systemically before the organism infects the CNS. Although it has long been assumed that tissue cysts do not develop in S neurona–infected horses, there is a recent report9 of S neurona sarcocysts in the tongue and skeletal muscle of a foal that died as a result of EPM.

Investigations of experimentally induced S neurona infections in skunks, raccoons, domestic cats, and immunodeficient mice have been performed.7,10,11 Encephalitis associated with S neurona organisms in the CNS has been detected in immunodeficient mice and raccoons inoculated with sporocysts.8,10 In other studies,12–18 administration of sporocysts to horses elicited synthesis of S neurona–specific IgG and IgM in blood and CSF, but protozoa were not detected in the CNS tissues of challenged horses. Neurologic signs and histologic evidence of CNS inflammation have developed inconsistently in horses that were experimentally infected with S neurona13,14,16,19; however, long-distance transportation of horses prior to sporocyst administration has been reported to increase the severity of subsequent infection-related clinical signs.15

Strategies for prevention of EPM have been inferred from epidemiologic investigations of risk factors for the disease,1,20 and a vaccine has been marketed under conditional licensure.21 However, because data to support the efficacy of these approaches are not available, it seems prudent to explore other means of EPM prevention. Ponazuril is a triazinone anticoccidial agent that has been approved by the FDA as a treatment for horses with EPM. Approximately 62% (63/101) of horses with EPM improved neurologically after oral administration of ponazuril for 28 days.22 Daily oral administration of ponazuril to horses at a dosage of 2.5 or 5 mg/kg before and after experimental S neurona challenge reduced the severity of subsequent ataxia and delayed immunoconversion against S neurona in blood and CSF.19

By contrast with daily prophylactic administration, strategically timed intermittent administration of antiprotozoal drugs offers the potential advantage of allowing systemic immunity against extraneural proliferating stages to develop while invasion of the CNS is prevented. A single dose of ponazuril partially protected interferon-γ–knockout mice when administered via gavage at 4 to 13 days after intragastric challenge with S neurona sporocysts.23 This interval of apparent susceptibility to ponazuril corresponds to first-generation schizogony in interferon-γ–knockout mice during the period between sporozoite migration and invasion of the CNS.8 The purpose of the study reported here was to evaluate the effect of intermittent oral administration of ponazuril on immunoconversion against S neurona in horses inoculated intragastrically with S neurona sporocysts. We hypothesized that ponazuril given every 7 days, but not every 14 days, to S neurona–challenged horses would prevent CNS infection without affecting systemic anti–S neurona antibody responses.

Materials and Methods

Experimental animals—Horses were purchased from Prince Edward Island, Canada. Before transportation to Florida, blood samples from all horses were assessed via western blot analysis for S neurona–specific IgG; all results were either negative or nonspecific.13 Among the 20 horses, there were 9 geldings and 11 mares that were 2 to 15 years old (median, 5 years); 17 were Standardbreds, and 3 were mixed breed. Horses were transported to the University of Florida and kept in an enclosure that had been modified to exclude opossums. The paddock was enclosed by a woven-wire fence (4 × 2-inch mesh); there were 2 electric wires on the outside of the fence. A complete pelleted rationa was provided, and horses were given water ad libitum. Routine preventative care was provided to horses throughout the study, including ivermectin administration every 2 weeks and vaccination against eastern and western equine encephalomyelitides and tetanus. At the completion of experiments, horses were euthanized by IV overdose with a product containing pentobarbital sodium and phenytoin sodium.b All procedures were approved by the University of Florida's Institutional Animal Care and Use Committee.

Sporocyst preparation—Sporocysts were scraped from the small intestinal mucosa of 20 road-killed opossums, then characterized via restriction enzyme digestion of the PCR-amplified genomic DNA sequences TGF, 33/54, 63/65, 48/50, and ITS-1, as described previously.24 Sporocyst preparations from 7 opossums were determined by these analyses to be S neurona and were stored at 4°C in saline (0.9% NaCl) solution supplemented with penicillin G sodium (100 U/mL), gentamicin (50 μg/mL), and amphotericin B (1.25 μg/mL). Sarcocystis neurona yields for these opossums ranged from 1.10 × 105 sporocysts/opossum to 1.69 × 106 sporocysts/opossum (median, 1.25 × 106 sporocysts/opossum). These yields were combined into a stock suspension for inoculation of horses. The median storage time of sporocysts before use in the challenge study was 59 days (range, 21 to 105 days).

Sporocyst inoculation—After transportation, horses were allowed to adapt to the experimental environment and pelleted diet for at least 2 weeks. On the day before challenge (day – 1), horses were transferred to individual isolation pens. On day 0, an inoculum of 612,500 sporocysts in 25 mL of Hank's buffered salt solution was administered via nasogastric tube to each horse. Horses were confined in pens for 10 days to allow for intestinal transit of any potentially infective sporocysts. After completion of this isolation period, inoculated horses were returned to the experimental enclosure.

Treatment groups—Horses were assigned randomly to 1 of 4 treatment groups (5 horses/group). In group 1, horses were inoculated with S neurona sporocysts and no antiprotozoal treatment was given. In group 2, horses were inoculated with S neurona sporocysts and treatment with the antiprotozoal drug ponazuril (20 mg/kg, PO) was given every 7 days (beginning on day 5). In group 3, horses were inoculated with S neurona sporocysts and treatment with ponazuril (20 mg/kg, PO) was given every 14 days (beginning on day 12). In group 4, control horses were neither challenged nor treated. Ponazuril was formulated as a paste containing 0.15 g toltrazuril sulfone/g of paste and was administered orally via syringe. Treatments were continued according to those schedules during the entire 12-week experiment. Blood and CSF samples used in the study were obtained immediately prior to sporocyst challenge (day – 1) and at day 84. At the times of sample collection, body weights were estimated by use of a weight tape.

Physical and neurologic examinations—Beginning at acclimation, then every 2 weeks for the remainder of the experiment, all horses underwent complete physical and neurologic examinations by 1 examiner (RJM). Rectal temperature and pulse and respiratory rates were recorded, and neurologic signs (limb and truncal ataxia and weakness) were graded on a scale of 0 to 4 according to the system of Mayhew.25 Grade assignments were as follows: grade 0 = no ataxia or weakness; grade 1 = minimal ataxia or weakness detectable by careful examination including circling; grade 2 = ataxia or weakness easily detectable during neurologic examination; grade 3 = obvious weakness or ataxia that may cause falling during manipulations of the neurologic examination; and grade 4 = severe ataxia or weakness that may result in falling during walking in straight lines. The examiner was unaware of the horses' assigned treatment groups.

Collection of blood and CSF samples—Blood and CSF samples were collected on day – 1 (immediately before sporocyst challenge at the start of the isolation period [day 0]) and then every 14 days thereafter until day 84. Each blood sample was collected via jugular venipuncture for a CBC, plasma biochemical analyses, and serum western blot testing. For collection of CSF samples, horses were administered xylazine (1.1 mg/kg) and ketamine (2.2 mg/kg) IV and anesthetized for a brief period. Cerebrospinal fluid was collected aseptically from the atlantooccipital cistern. Each sample was grossly clear and colorless when collected and underwent western blot analysis. The concentrations of albumin in CSF and serum was measured and used to calculate Qalb as a measure of blood contamination of CSF samples.26 Cell counts were not performed for these CSF samples; however, CSF samples collected under the same conditions in previous studies13,19 performed by our group and other researchers have all had RBC counts < 100 cells/μL.

Western blot analysis—Western blot analysis of serum and CSF samples for S neurona–specific IgG were performed at a commercial laboratory.c At that laboratory, reactivity against a 17-kd band in S neurona lysate was interpreted as a positive result, and reactivity against a 30-kd band was interpreted as a nonspecific positive result. A lack of reactivity against either band in a sample was interpreted as a negative result. In addition, the reactivity of CSF against the 17-kd antigen of S neurona was quantified in western blots, as previously described.13 This analysis of reactivity against the 17-kd antigen yielded a unitless value between 0 and 100, which was designated as the relative anti–17-kd antigen concentration in CSF.

Postmortem examinations—Following euthanasia of the horses, the brains and spinal cords were removed in manageable sections and fixed in neutral-buffered 10% formalin. Tissues were sectioned at approximately 1-cm intervals, and selected portions were prepared routinely with paraffin, sectioned at 7 Mm, glass-mounted, stained with H&E stain, and examined by use of a binocular optical microscope. Sections selected for histologic examination were from the temporal, parietal, and occipital cortices; the basilar region at the level of the putamen, pallidum, and internal capsule; the thalamus at the region of the lateral geniculate nucleus; the midbrain at the level of the oculomotor nucleus; the pons; the medulla at the levels of the vestibular nuclei, facial nuclei, obex, and confluence of the peduncles; and the cerebellar vermis. Three sites in the cervical spinal cord, 3 sites in the thoracic spinal cord, and 3 sites in the lumbosacral spinal cord were selected. In addition, other sections were obtained where there was any suspicion of a gross lesion. The presence of mononuclear inflammatory cells was scored as 0, 1, 2, or 3 when 0, 1 to 10, 11 to 20, or 21 to 30 cells, respectively, were counted at a site. These sites included the meninges, perivascular regions (cuffs), and individual glial scars. The presence of Wallerian-like neuronal fiber degeneration and neuroaxonal dystrophy was scored as 0 to 3 on the basis of the number of degenerating fibers per ascending or descending tract.27 Thus, degeneration and neuroaxonal dystrophy were scored as 0 (absent), 1 (1 to 5 fibers), 2 (6 to 10 fibers), or 3 (11 to 50 fibers). The presence of pigment and mineralized vessels was scored as 0 (absent), 1 (1 to 5 sites), 2 (6 to 10 sites), or 3 (> 10 sites). For analyses, scores for all sites in each horse were combined and recorded as a total score. In addition, overall scores were obtained for inflammation (combination of meningitis, perivascular cuffing, and glial scars) and for degeneration (combination of Wallerian degeneration, neuroaxonal dystrophy, mineralization of vessels, and pigmentation). The examiner (IGM) was unaware of treatment groups.

Statistical analysis—Because data did not meet criteria for normal distribution and equality of variance, nonparametric analyses were performed. Proportional data (ie, proportion of samples from each group that yielded positive results via analysis) were analyzed pairwise for the effects of time and treatment by use of Fisher exact tests. The effects of time on reactivity against the 17-kd antigen, CSF albumin concentration, and Qalb were explored by use of a Friedman ANOVA on ranks, and the effects of treatment were analyzed by use of a Kruskal-Wallis ANOVA. Where significant effect was found, differences between points were explored by use of a Dunn test. The effects of treatment on ataxia and quantifiable histologic findings (ie, inflammatory, degenerative, and total scores) were analyzed by use of a Kruskal-Wallis ANOVA. A commercial statistical software packaged was used to perform analyses. In all analyses, significance was ascribed to a value of P ≤ 0.05.

Results

Clinical signs—Values for rectal temperature and pulse and respiratory rates remained within reference ranges for the duration of the experiments. Overall, horses gained weight during the study (mean ± SD increase, 13.6 ± 7.3 kg), and there was no significant difference in weight gain among the groups. Consistent signs of neurologic disease were not evident among the horses of any group (Table 1). Only 3 of 20 horses had overall ataxia scores ≥ 2; 1 of those horses was in the unchallenged control group (group 4), and there was 1 in each of the challenged ponazuril-treated groups (groups 2 and 3). There was no significant effect of either sporocyst challenge or ponazuril treatment on ataxia score.

Table 1—

Histologic findings (at the end of a 12-week study period) and ataxia grade (assessed at 2-weekly intervals during the study in horses that were inoculated with 612,500 Sarcocystis neurona sporocysts via nasogastric tube (day 0) followed by no treatment (group 1; n = 5) or treatment with ponazuril (20 mg/kg, PO) every 7 days beginning on day 5 (group 2; 5) or every 14 days (group 3; 5) beginning on day 12. Five control horses were not inoculated with sporocysts and received no treatment (group 4). Ponazuril was formulated as a paste (0.15 g of toltrazuril sulfone/g)

GroupMedian histologic scores (range)  Median ataxia grade (range)
 Infammatory*DegenerativeTotal
12 (0–16)1 (0–4)2 (0–20)0 (0–1)
24 (0–14)2 (0–7)4 (0–21)0.5 (0–3)
30 (0–2)1 (0–6)2 (1–6)0 (0–2)
42 (0–4)1 (0–9)4 (0–9)1 (0–2)
Pvalue§0.1980.9550.9550.457

CNS infammation score represents the combined score for perivascular cuffng, meningitis, and glial scarring at a minimum of 9 sites. Scores of 0 to 3 were assigned to designate no changes through severe abnormality.

CNS degeneration score represents the combined score for neuroaxonal dystrophy, Wallerian degeneration, pigment accumulation, and mineralization of vessel walls at a minimum of 9 sites.

Median of highest ataxia grade assigned during experiment. Grades of 0 to 4 represented no limb ataxia or weakness through severe limb ataxia or weakness.

Kruskal-Wallis ANOVA tests of significance of treatment group; a value of P ≤ 0.05 was considered significant.

CSF albumin concentration and Qalb—All Qalb values (median, 1.38; range, 1.05 to 1.96) were less than the published upper limit (2.35) for adult horses.26 Concentrations of albumin in CSF samples ranged from 21.8 to 51.6 mg/dL (median, 32.4 mg/dL). A significant effect of either treatment or time after S neurona challenge on CSF albumin and Qalb was not identified.

Western blot analysis—With the exception of a single positive result in 1 horse at day 14, sera from all unchallenged control horses in group 4 yielded negative results via western blot analysis throughout the study (Figure 1). By day 56 after inoculation, all sporocystchallenged horses had seroconverted against S neurona. This represented a significant increase in the proportion of horses that had positive western blot results in each of the challenged groups, compared with results obtained before challenge. A significant effect of ponazuril treatment on seroconversion was not detected.

Figure 1—
Figure 1—

Proportions of horses that seroconverted against Sarcocystis neurona after inoculation with 612,500 S neurona sporocysts via nasogastric tube (day 0) followed by treatment with ponazuril (20 mg/kg, PO) every 7 days beginning on day 5 (gray bars; n = 5) or every 14 days (striped bars; 5) beginning on day 12 or no treatment (black bars; 5) during a 12-week study period. Five control horses were not inoculated with sporocysts and received no treatment (white bars). Ponazuril was formulated as a paste (0.15 g of toltrazuril sulfone/g). Blood samples were collected before challenge (day – 1) and at 14-day intervals after challenge. *Within a group, value was significantly (P ≤ 0.05) different from value at day – 1. †Value was significantly (P ≤ 0.05) different from the value for challenged, untreated horses at the same time point.

Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.396

The CSF samples from all unchallenged control horses in group 4 yielded negative results via western blot analysis throughout the study. By 42 days after sporocyst inoculation, there was a significant increase in the proportion of western blot–positive horses in the challenged groups that received either no treatment or biweekly ponazuril (groups 1 and 3; Figure 2). All horses in these 2 groups had immunoconverted by day 84. By contrast, the proportion of western blot–positive horses in the group given ponazuril on a weekly basis (group 2) did not increase significantly after challenge, and CSF from only 2 of 5 of these horses yielded positive results via western blot analysis at the end of the study. The proportion of horses with positive western blot results in this group was lower than that in the untreated challenged horses (group 1) at all time points after day 14; this difference was significant at day 56. Similar changes in reactivity against the 17-kd antigen were evident (Figure 3). For horses in the challenged groups that were not treated or that received biweekly treatment (groups 1 and 3), values increased after day 14 to become significantly higher than prechallenge values by day 42. The reactivity against 17-kd antigen in CSF samples from group 2 (horses that were given ponazuril weekly) also increased after day 14, but mean values did not differ significantly from those obtained before challenge. On day 70, reactivity against 17-kd antigen in group 2 was significantly lower than that in the challenged untreated group 1 horses.

Figure 2—
Figure 2—

Proportions of the horses in Figure 1 that immunoconverted against S neurona as determined by positive results of western blot analysis of CSF samples. See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.396

Figure 3—
Figure 3—

Mean ± SEM relative concentration of antibody against the S neurona 17-kd antigen in CSF samples collected from the horses in Figure 1. See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.396

Histologic findings—A range of inflammatory and degenerative changes was evident in some tissue sections from at least 1 horse in each of the study groups. The presence and severity of these changes did not vary significantly according to treatment group (Table 1).

CBC and plasma biochemical findings—With the exception of 2 WBC counts that were greater than the upper reference limit in 1 control horse (day 28) and 1 challenged horse (day 56; 13,800 and 14,110 cells/μL, respectively), CBC and plasma biochemical analysis results were within reported reference ranges28 throughout the 12 weeks of the study.

Discussion

The dose of ponazuril (20 mg/kg) used in the present study was higher than that approved for treatment of EPM (5 mg/kg) in horses. In horses, ponazuril is known to have minimal toxic potential when given at a dosage of 30 mg/kg daily for as long as 56 days.29 We wanted to ensure that each preventative dose would achieve serum concentrations well in excess of 1 μg/mL (the concentration that kills > 90% of S neurona merozoites grown in bovine turbinate cells in vitro) and possibly as high as 5 μg/mL (a dose that kills > 95% of cultured merozoites30). Although serum concentrations in horses following single oral doses of ponazuril have not been reported to our knowledge, ponazuril at daily dosage of 5 mg/kg must be given to horses for > 2 weeks to reach a mean maximal serum concentration of 5.6 μg/mL.31 The objective of the experimental dosage regimen used in the present study was to kill S neurona organisms before they could reach the CNS from systemic sites of replication.

As we expected from our previous experiments of this type,12,13 horses given S neurona sporocysts intragastrically did not develop reliable clinical or histologic evidence of CNS disease. The apparent resistance of horses to experimental infection is consistent with the observation that EPM is relatively rare, even in some areas of the United States where seroconversion against S neurona is common.1,2,32 Saville et al15–17,19 attempted to increase the susceptibility of horses to experimental infection via exposure to 1 or 2 episodes of long-distance transportation at approximately the time of sporocyst challenge. Although this approach reportedly resulted in more reliable induction of neurologic signs, protozoa were not detected histologically in CNS tissues of challenged horses; more specifically, S neurona was not detected in CNS tissues of challenged horses via inoculation of interferon-γ–knockout mouse or via immunohistochemical techniques. In contrast, protozoal stages were detected in the CNS of a horse given autologous blood mononuclear cells that had been cocultured with S neurona merozoites.33 This latter result, which needs confirmation in a larger number of horses, is consistent with the notion that merozoites that are injected IV, unlike sporocysts that are ingested, gain access to the CNS before a protective immune response is elicited.

Although the organisms apparently do not persist in the CNS of healthy horses given S neurona sporocysts experimentally, there is convincing evidence that systemic infection does develop in this setting. Parasitemia was detected in 1 of 6 yearlings that were administered S neurona sporocysts daily for 98 days.34 Almost all horses given sporocysts were reported to seroconvert against S neurona between 12 and 40 days after challenge.12–17 This finding is in accordance with the period of 3 to 5 weeks reported for the appearance of specific IgG in sera of cattle, pigs, sheep, and mice after inoculation with Sarcocystis spp sporocysts.35 In cattle receiving Sarcocystis cruzi sporocysts, both antibody production and immunity likely are dependent on first-generation schizogony or sporozoite migration.35

In experimentally infected horses, anti–S neurona antibody is detected in CSF at or after the time of seroconversion.13,14,17 The finding of intrathecal antibody in sporocyst-challenged horses has been interpreted as evidence of transient CNS infection,13 and there was apparent correlation between ataxia and the presence of anti–S neurona antibody in CSF of horses that underwent sporocyst challenge and transportation stress.19 Such infection presumably is cleared by a competent local immune response. In the present study, weekly treatment with ponazuril beginning 5 days after challenge with sporocysts significantly reduced the intrathecal antibody response against S neurona without affecting the proportion of horses that became seropositive. These differences among treatment groups apparently could not be explained by blood contamination or differences in blood-brain barrier function. Although RBC counts were not performed on CSF samples, previous extensive experience with puncture of the atlantooccipital space by our group and other researchers has shown that this technique results in samples with minimal blood contamination.13,19 Furthermore, comparisons of CSF albumin concentrations and Qalb within and among groups in the present study revealed no effect of time or treatment on either variable. It has been previously argued that Qalb is an insensitive method of detecting blood contamination in individual horses36; however, direct comparison of results should be a relatively sensitive method of detecting treatment differences, even when all results are within published reference ranges.

In a previous study,23 interferon-γ–knockout mice were protected from S neurona–induced weight loss when a single dose of ponazuril was given 7 days after sporocyst challenge; however, there was no protection associated with ponazuril administration 3 or 14 days after challenge. On the basis of the known development of S neurona in sporocyst-challenged interferon-γ–knockout mice,8 it was postulated that the organism was susceptible to ponazuril during first-generation schizogony (7 to 11 days after challenge) but not during sporozoite migration (1 to 3 days) or after invasion of the CNS (beginning at 11 days). In light of the fact that CSF concentrations of ponazuril in horses were < 5% of those found in serum,31 S neurona located in the CNS may be relatively inaccessible to treatment, compared with stages located outside the CNS. In horses in the present study, it is likely that weekly ponazuril treatment removed most infective S neurona stages before they could circulate to the CNS from sites of multiplication, thereby preventing or minimizing intrathecal antibody production. By contrast, there was no effect on intrathecal anti–S neurona antibody when treatment was administered every 2 weeks beginning 12 days after challenge, suggesting that sporozoite migration, systemic schizogony, and invasion of the CNS all could occur within this period. Daily administration of either 2.5 or 5 mg of ponazuril/kg was reported to reduce the proportion of horses that seroconverted within 28 days of sporocyst challenge,19 suggesting that regular treatment protocols may suppress or prevent active systemic antibody responses (and perhaps all specific immune responses) to invading organisms. On the basis of the discrepant effects on seroconversion against S neurona derived from daily versus weekly administration of ponazuril, it is reasonable to hypothesize that weekly, but not daily, treatment may allow normal immune responses against S neurona while still preventing neuroinvasion.

It must be acknowledged that the presence of S neurona–specific IgG in the CSF of challenged horses has not been established as definitive proof of CNS infection, transient or otherwise. The specificity of a positive CSF western blot result for CNS infection with S neurona was reported to be only 44% for horses with and 60% for horses without neurologic signs.37 Inoculation of adult horses with a killed S neurona vaccine results in specific antibody within the CSF, presumably because of transfer of antibody across the blood-brain barrier.38 Similarly, antigen-specific antibody was detected in the CSF of horses after IM administration of ovalbumin.39 Thus, it could be argued that the effect of ponazuril on anti–S neurona antibody in the CSF may actually reflect changes in plasma antibody concentration in treated horses. The data gathered in the present experiment do not permit resolution of the issue of whether CNS infection develops consistently in experimentally infected horses, independent of clinical disease. Support for the suggestion that protozoa do invade and proliferate in the CNS of sporocyst-challenged horses is provided by the finding of mild to moderate brain and spinal cord lesions (including lymphocytic perivascular cuffing and gliosis) 28 to 84 days after sporocyst inoculation.13,14,16,17 Such changes are consistent with prior protozoal infection and are presumed to account for the clinical signs of ataxia and limb weakness in challenged horses. In future work with sporocyst challenge of horses, it will be important to distinguish between intrathecal and systemic production of IgG that is detected in the CSF. Albumin quotients and IgG indices have been used previously to measure the integrity of the blood-brain barrier and intrathecal IgG production, respectively,40 but lack sufficient sensitivity to detect low-level transfer of IgG into the CSF.35 The specific antibody index, which relates the ratio of concentrations of specific and total immunoglobulin in blood or CSF, theoretically identifies a fraction of specific antibody made in the CNS.41 Antibody indices are used to identify CNS involvement in humans infected with agents such as Borrelia burgdorferi,42 Angiostrongylus cantonensis,43 Toxoplasma gondii,44 and Trypanosoma brucei45 and may have application to the diagnosis of EPM in horses. A study46 of antibody index in samples generated during the present study has been completed.

Results of the present study have provided support for the hypothesis that weekly treatment with ponazuril is sufficient to greatly reduce or prevent invasion of the CNS by S neurona in horses. Administration of ponazuril at intervals may have value in horses as metaphylaxis and prophylaxis against EPM.

ABBREVIATIONS

EPM

Equine protozoal myeloencephalitis

Qalb

Albumin quotient

a.

Horse Chow 100, Purina Mills, St Louis, Mo.

b.

Beuthanasia-D Special, Schering-Plough Animal Health, Summitt, NJ.

c.

Neogen Corp, Lexington, Ky.

d.

SPSS, version 12.0 for Windows, SPSS Inc, Chicago, Ill.

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Contributor Notes

Dr. Tanhauser's present address is Newberry Equine Veterinary Service, 1210 SW 218th St, Newberry, FL 32669.

Dr. Gillis' present address is Environmental Health and Safety, University of Florida, Gainesville, FL 32611.

Dr. Mayhew's present address is Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand.

Dr. Kennedy's present address is Farnam Companies, 3851 N 28th St, Phoenix, AZ 85013.

Supported by Bayer Animal Health.

Address correspondence to Dr. MacKay.