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

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.

Diagnostic performance of the equine IgM capture ELISA for serodiagnosis of West Nile virus infection.

The objectives of these studies were to assess the diagnostic performance (sensitivity and specificity) of the IgM capture enzyme-linked immunosorbent assay (ELISA; MAC) for diagnosis of West Nile (WN) virus in horses and to examine the performance of this test by using different criteria for seropositivity. A total of 36 horses classified as WN virus infected (group 1) and 383 horses from 4 subpopulations of hoses classified as noninfected (groups 2, 3, 4, and 5) were used in the study. The sensitivity (proportion of infected horses that tested positive for WN virus IgM antibodies) and specificity (proportion of noninfected horses that tested negative) were calculated at different cutoff points by using receiver operating curve (ROC) analysis. Using a selected cutoff point = 2.0, the sensitivity and specificity of the MAC were 91.7 and 99.2%, respectively. The area under the ROC curve = 0.95 (95% confidence interval [CI], 0.89 to 1.0), suggesting that the MAC is a useful tool for diagnosis of recent WN virus exposure in horses. In fulfillment of the 2nd objective, 2 other indices were developed and these indices approached 1.0 for the AUC with smaller 95% CIs. These indices were then used to test 602 additional diagnostic samples submitted from suspect horses between 2002 and 2004. Using the standard cutoff, 194 (32%) of the horses were interpreted as positive. Utilizing newly predicted cutoff criteria from each index, additional horses were identified as positive. In conclusion, the MAC as used for identification of WN virus-diseased horses undergoing recent exposure performs reliably at the standard cutoff for seropositivity. A negative test might not completely rule out WN virus disease, but horses that test negative were most likely not exposed to WNV. Performance of the test can be further improved by investigation of other indexes of seropositivity.

Depletion of natural killer cells does not result in neurologic disease due to Sarcocystis neurona in mice with severe combined immunodeficiency.

Sarcocystis neurona is an apicomplexan parasite that is the primary etiologic agent of equine protozoal myeloencephalitis in horses. Protective immune responses in horses have not been determined, but interferon-gamma (IFN-gamma) is considered critical for protection from neurologic disease in mice. The role of adaptive and innate immune responses in control of parasites was explored by infecting BALB/c, IFN-gamma knockout (GKO), and severe combined immune deficient (SCID) mice with S. neurona (10(4) sporocysts/mouse). Immune competent BALB/c mice eliminated parasites within 30 days, with no sign of neurologic disease, whereas GKO mice developed fulminant neurologic disease. In contrast, SCID mice remained healthy throughout the experimental period despite the persistence of parasite at low levels in some mice. Treatment with anti-IFN-gamma antibody resulted in neurologic disease in infected SCID mice. Although SCID mice lack adaptive immune responses, they have natural killer (NK) cells capable of producing significant quantities of IFN-gamma. Therefore, SCID mice were infected with sporocysts of S. neurona and treated with anti-asialo GM1. Depletion of NK cells, confirmed by flow cytometry, did not result in neurologic disease in SCID mice. These results indicate that IFN-gamma mediates protection from neurologic disease in SCID mice. Protective levels of IFN-gamma may originate from a low number of nondepleted NK cells or from a non-T cell, non-NK cell population.

Effect of a single dose of ponazuril on neural infection and clinical disease in Sarcocystis neurona-challenged interferon-gamma knockout mice.

Interferon gamma-knockout mice were challenged with 5000 Sarcocystis neurona sporocysts acquired from a naturally infected opossum. Ponazuril was administered once, by gavage, at day 1, 3, 7, 10, or 14 post-infection (pi). Ponazuril was given at either 20 or 200mg/kg. Mice that survived to day 30 pi were euthanized. Severity of CNS infection was quantified as schizont density in the cerebellum. Unchallenged mice in treatment and non-treatment groups remained free of disease and gained weight throughout the experiment. All challenged mice, regardless of treatment, developed histologic evidence of CNS infection even though clinical signs were prevented in some groups. The greatest treatment benefits were seen in mice given 200mg/kg ponazuril between days 4 and 14 pi. Weight gain over the course of the experiment occurred only in mice that were given 200mg/kg ponazuril on day 7 or 10 pi. With the exception of groups given 200mg/kg ponazuril on day 7 or 14 pi, mice in groups that got sporocysts developed abnormal neurologic signs. No deaths before day 30 pi occurred in mice given ponazuril at 20mg/kg on day 7 pi or 200mg/kg on day 1, 7, 10, or 14 pi. This effect was not significant. Mice given 200mg/kg on day 7 pi had significantly fewer cerebellar schizonts than did those of the control group that was not given ponazuril. These results indicate that single-dose administration of ponazuril for prevention of CNS infection is partially protective when given between days 4 and 14 pi.