Source: UNIVERSITY OF FLORIDA submitted to NRP
PATHOPHYSIOLOGY, DIAGNOSIS, TREATMENT, AND PREVENTION OF EQUINE PROTOZOAL MYELOENCEPHALITIS (EPM) AND ENDOTOXEMIA OF HORSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0188771
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jun 1, 2001
Project End Date
Jun 1, 2007
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
COLLEGE OF VETERINARY MEDICINE
Non Technical Summary
Equine protozoal myeloencephalitis (EPM) and endotoxemia are 2 serious causes of equine deaths and loss of athletic potential. Currently there is poor understanding of the behavior in nature of the causative organism of EPM. Current methods of diagnosis, treatment and prevention or EPM and endotoxemia are inadequate. The purpose of the proposed work is to fully define the life cycle of the causative agent of EPM. In addition, methods for diagnosis, treatment and prevention of EPM and endotoxemia will be improved.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113810104020%
3114050110350%
3153810116030%
Knowledge Area
311 - Animal Diseases; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
4050 - Protozoa; 3810 - Horses, ponies, and mules;

Field Of Science
1040 - Molecular biology; 1103 - Other microbiology; 1160 - Pathology;
Goals / Objectives
I - Acquire a better understanding of the life cycle of Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis (EPM). II - Refine the ability to reproduce EPM in horses in an experimental setting. III - Investigate ways in which to prevent EPM. IV - Investigate ways in which to treat EPM. V - Investigate ways in which to improve the diagnostic efficacy of tests for EPM. VI - Acquire a better understanding of the pathophysiology of endotoxemia in horses. VII - Investigate ways in which to treat equine endotoxemia.
Project Methods
I - Acquire a better understanding of the life cycle of Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis (EPM). A. Animals of various taxa that are candidate intermediate hosts for S neurona will be acquired as SPF animals and challenged with S neurona sporocysts. Approximately 2 months later, challenged animals will be euthanized and examined for the presence of sarcocysts. Sarcocysts will be characterized as S neurona by both molecular and biological techniques. B. Sarcocyst-infested muscle from naturally-infected animals (candidate intermediate hosts) will be fed to SPF opossums, then opossum feces will be examined for the presence of sporocysts. Sporocysts produced by challenged opossums will be characterized as S neurona by both biological and molecular techniques. C. The possible relevance of the domestic cat (Felis domesticus) will be examined. Cats euthanized by the Alachua county Animal Services Dept. will be examined histologically for the presence of sarcocysts. If sarcocysts are found, they will be characterized by morphologic, immunologic, molecular, and biological techniques to determine their identities. II - Refine the ability to reproduce EPM in an experimental setting. III - Investigate ways in which to prevent EPM. A. Antiprotozoal drugs with the potential to prevent EPM (e.g., ponazuril, nitazoxanide) will be evaluated for their ability to protect sporocyst challenged mice and horses from S neurona CNS infection. Gamma-IFN-knockout mice will be challnged intragastrically with S neurona sporocysts and then given anti-protozoal drugs at intervals thereafter in order to determine whether intermittent therapy can prevent early systemic infection from progressing to neural infection. Similar studies will be done in immunocompetent horses, in which the objective will be to prevent immunoconversion against S neurona in the CSF, and in gamma-IFN-deficient horses with a view to preventing signs of CNS disease. B. A commercially produced EPM vaccine will be evaluated for efficacy in both field and experimental settings. As part of a multi-centered case-control study of equine patients (designed in conjunction with workers at Texas A&M University), vaccine efficacy will be evaluated by comparing the prevalence of vaccination among horses with EPM, horses with other neurologic diseases, and horses without neurologic disease. It is estimated that at least 3 years of data accumulation will be necessary to confer the necessary power upon the study. The vaccine will be evaluated in an experimental setting in which SPF cats will be challenged with S neurona sporocysts. Vaccine efficacy will be determined as the ability to prevent development of S neurona sarcocysts.

Progress 06/01/01 to 06/01/07

Outputs
OUTPUTS: (1) The striped skunk (Mephitis mephitis) was identified as a natural and experimental host for Sarcocystis neurona, the cause of EPM (2) The armadillo is an intermediate host for S neurona. (3) Circulating merozoites were found in immunodeficient SCID foals but not in normal foals. (4) Because domestic cats had been indentified as an experimental host, we investigated 100 feral cats in Florida. No cysts were identified, although 4% of cats were immunoblot positive for S neurona antibodies. We found 5% of cats with sarcocysts of Sarcocystis felis. (5) Seroconversion and immunoconversion in CSF against S neurona was produced in healthy horses inoculated intragastrically with S neurona sporocysts. However, no consistent neurologic or neuropathologic abnormalities were found. (6) We documented stability of sporocysts in various storage media by assay in susceptible gamma interferon knockout mice. (7) Dexamethasone had no significant effect on development of clinical signs or seroconversion rates in experimentally challenged horses. (8) It was shown in susceptible mice that single-dose ponazuril after the time of primary schizogony (4 d after challenge) but before CNS invasion (10 d post-infection) significantly ameliorated the effects of challenge. (9) Single-dose ponazuril given to horses 4 d after challenge and then every 7 d, significantly delayed or suppressed immunoconversion to S neurona, but had not effect on seroconversion. (10) As part of a multi-centered field study, we evaluated the effect of a killed vaccine against S neurona on subsequent development of EPM. There was no significant effect of vaccination. (11) As part of a multi-center multi-dose study of ponazuril, we determined that ponazuril has good clinical efficacy with minimal side-effects. At daily oral dosage of either 5 or 10 mg/kg, given for 28 successive d, approximately 60% of treated horses improved . (12) In collaboration with investigators at the University of California, Davis, we evaluated the accuracy of an IFAT test of CSF and serum of necropsy-confirmed "gold standard" horses positive or negative for EPM. This test had excellent specificity and sensitivity compared with the commonly used western blot assay of CSF. As an important advance it appeared that blood testing with this assay was at least as accurate as CSF immunoblot testing and was robust even in the presence of blood contamination. (13) A specific index was developed which quantified antibody production against S neurona specifically within the CSF, effectively screening out the contaminating effects of systemically produced antibody. (14) Pharmacokinetic data and rational dosing were developed for circulating anti-endotoxic activity following single dose or multiple dosing with polymyxin B. PARTICIPANTS: ROBERT MACKAY, BVSc, PhD, DACVIM, PI, is a full professor at the Department of Large Animal Clinical Sciences in the College of Veterinary Medicine and has been at the University of Florida since 1987. KAREN GILLIS MS was a laboratory technologist for Dr. MacKay and used funding from the Morris Animal to complete an MS before leaving for a position on campus. SUSAN TANHAUSER, DVM, PhD served as a postdoctoral associate after completing her DVM curriculum and was responsible for the molecular studies of S neurona. TARGET AUDIENCES: Target audiences included equine veterinarians, the horse-owning public, and veterinary parasitologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Taken together, the findings reported in the previous section (i.e., that skunks and armadillos were infected with S neurona sarcocysts) identified 2 of the 3 important intermediate hosts thus far found for S neurona, finally solving the complete life cycle after 20 years of searching. These results were published as experimental papers and as reviews. The finding that parasitemia is found only in SCID foals suggests that a competent immune response is capable of suppressing or eliminating circulating infective life cycle stages. The failure to find S neurona sarcocysts in feral domestic cats suggests that cats are not an important intermediate host for S neurona in Florida and as such probably are not involved in the natural history of EPM in this state. The results of horse infection studies reported above suggest that healthy horses are very resistant to CNS invasion by infective sporocysts. We thus concluded that this form of attempted induction of EPM does not replicate well the situation found in naturally infected horses and that alternative models of EPM induction are needed. The finding that dexamethasone had no significant effect on induction of S neurona CNS infection called into question the notion that steroid-induced immunosuppression is a risk factor in S neurona-infected horses for the development of EPM. The results of intermittent dosing of mice and horses given in the previous section under this objective strongly suggest that intermittent dosing with ponazuril may be used as metaphylaxis, preventing infection of the CNS while allowing extraneural schizogony and immune responses. The negative findings of a field study of the preventive efficacy of a killed S neurona vaccine unfortunately fulfill most predictions that protozoal vaccines prepared this way (i.e., killing, mixed antigens) are very unlikely to be effective. Killed vaccines in general induce strong humoral (antibody) responses; however, specific cell-mediated immunity is invariably responsible for protection against protozoa. Future studies with live modified (attenuated) or subunit vaccines will be more relevant. The field study of ponazuril efficacy led to FDA approval of a safe and effective drug for the treatment of EPM. The work on the S neurona IFAT diagnostic test suggested that this test had excellent specificity and sensitivity compared with the commonly used western blot assay of CSF. As an important advance it appeared that blood testing with this assay was at least as accurate as CSF immunoblot testing and was robust even in the presence of blood contamination. Although our published techniques and reagents for specific antibody index testing were developed specifically for a research application, it was clear that the techniques had great application to commercial testing and likely will form the basis of a new generation of diagnostic tests for this disease. The published polymyxin data provide a rational dosing schedule for for use of this commonly used anti-endotoxic treatment and is an important forward step in development of treatment strategies for this vexing and serious condition of horses.

Publications

  • Duarte, P.C., Daft, B.M., Conrad, P.A., Packham, A.E., Saville, W.J., MacKay, R.J., Barr, B.C., Wilson, W.D., Ng, T., Reed, S.M., Gardner, I.A. Evaluation and comparison of an indirect fluorescent antibody test for detection of antibodies to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and experimentally infected, and vaccinated horses. J. Parasitol. 90:379-386, 2004.
  • Ernst, N.S., Hernandez, J.A., MacKay, R.J., Brown, M.P., Gaskin, J.M., Nguyen, A.D., Giguere, S., Colahan, P.T., Troedsson, M.R., Haines, G.R., Addison, I.R., Miller, B.J. Risk factors associated with fecal Salmonella shedding among hospitalized horses with signs of gastrointestinal tract disease. J. Am. Vet. Med. Assoc. 225:275-281, 2004.
  • MacKay, R.J. Equine protozoal myeloencephalitis: Treatment, prognosis, and prevention. Clin. Tech. Equine Pract. 5:9-16, 2006.
  • Ernst, N.S., Freeman, D.E., MacKay, R.J. Progression of mycosis of the auditory tube diverticulum (guttural pouch) after arterial occlusion in a horse with contralateral temporohyoid osteoarthropathy. J. Am. Vet. Med. Assoc. 229:1945-1948, 2006.
  • MacKay, R.J. Brain injury after head trauma: pathophysiology, diagnosis, and treatment. Vet. Clin. North Am. Equine Pract. 20:199-216, 2004.
  • MacKay, R.J. Neurologic disorders of neonatal foals. Vet. Clin. North Am. Equine Pract. 21:387-406, vii, 2005.
  • Christensen, B.W., Troedsson, M.H., Murchie, T.A., Pozor, M.A., Macpherson, M.L., Estrada, A.H., Carrillo, N.A., MacKay, R.J., Roberts, G.D., Langlois, J. Management of hydrops amnion in a mare resulting in birth of a live foal. J. Am. Vet. Med. Assoc. 228:1228-1233, 2006.
  • Morresey, P.R., MacKay, R.J. Endotoxin-neutralizing activity of polymyxin B in blood after IV administration in horses. Am. J. Vet. Res. 67:642-647, 2006.
  • Heskett, K.A., MacKay, R.J. Antibody index and specific antibody quotient in horses after intragastric administration of Sarcocystis neurona sporocysts. Am. J. Vet. Res. 2008;69:403-409.
  • Cohen, N.D., MacKay, R.J., Toby E, Andrews, F.M., Barr, B.S., Beech, J., Bernard, W.V., Clark, C.K., Divers, T.J., Furr, M.O., Kohn, C.W., Levy, M., Reed, S.M., Seahorn, T.L., Slovis, N.M. A multicenter case-control study of risk factors for equine protozoal myeloencephalitis. J. Am. Vet. Med. Assoc. 2007;237:1857-1863.
  • Javsicas, L.H., Watson, E., MacKay, R.J. What is your neurologic diagnosis Equine protozoal myeloencephalitis. J. Am. Vet. Med. Assoc. 2008;232:201-204.
  • MacKay, R.J. Equine protozoal myeloencephalitis: Managing relapses. Comp. Cont. Ed. Pract. Vet. Equine 2008;3:24-27.
  • MacKay, R.J., Tanhauser, S.T., Gillis, K.D., et al. Effect of intermittent oral administration of ponazuril on experimental Sarcocystis neurona infection of horses. Am. J. Vet. Res. 2008;69:396-402.
  • Cheadle, M.A., Tanhauser, S.M., Scase, T.J., Dame, J.B., MacKay, R.J., Ginn, P.E., Greiner, E.C. Viability of Sarcocystis neurona sporocysts and dose titration in gamma-interferon knockout mice. Vet. Parasitol. 95:223-231, 2001.
  • Cheadle, M.A., Yowell, C.A., Sellon, D.C., Hines, M., Ginn, P.E., Marsh, A.E., MacKay, R.J., Dame, J.B., Greiner, E.C. The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona. Int. J. Parasitol. 31:843-849, 2001.
  • Cheadle, M.A., Tanhauser, S.M., Dame, J.B., Sellon, D.C., Hines, M., Ginn, P.E., MacKay, R.J., Greiner, E.C. The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona. Int. J. Parasitol. 31:330-335, 2001.
  • Cutler, T.J., MacKay, R.J., Ginn, P.E., Gillis, K., Tanhauser, S.M., LeRay, E.V., Dame, J.B., Greiner, E.C. Immunoconversion against Sarcocystis neurona in normal and dexamethasone-treated horses challenged with S. neurona sporocysts. Vet. Parasitol. 95:197-210, 2001.
  • Luznar, S.L., Avery, M.L., Dame, J.B., MacKay, R.J., Greiner, E.C. Development of Sarcocystis falcatula in its intermediate host, the Brown-headed Cowbird (Molothrus ater). Vet. Parasitol. 95:327-334, 2001.
  • MacKay, R.J. Update on equine therapeutics: Equine neonatal clostridiosis: Treatment and prevention. Comp. Cont. Ed. Pract. Vet. 23:280-285, 2001.
  • MacKay, R.J. Life cycle of Sarcocystis neurona: An update. Comp. Cont. Ed. Pract. Vet. 23:3-9, 2001.
  • MacKay, R.J. Case notes and commentary: On the true definition of dysphagia. Comp. Cont. Ed. Pract. Vet. 23:1024-1028, 2001.
  • Tanhauser, S.M., Cheadle, M.A., Massey, E.T., Mayer, B.A., Schroedter, D.E., Dame, J.B., Greiner, E.C., MacKay, R.J. The nine-banded armadillo (Dasypus novemcinctus) is naturally infected with Sarcocystis neurona. Int. J. Parasitol. 31:325-329, 2001.
  • Furr, M., MacKay, R.J., Granstrom, D., Schott II, H.C., Andrews, F. Clinical diagnosis of equine protozoal myeloencephalitis (EPM). J. Vet. Intern. Med. 16:618-621, 2002.
  • Furr, M., Kennedy, T.J., MacKay, R., Reed, S., Andrews, F., Bernard, B., Bain, F., Byars, D. Efficacy of ponazuril 15% oral paste as a treatment for equine protozoal myeloencephalitis. Vet. Therapeutics 2:215-222, 2002.
  • Giguere, S., Viel, L., Lee, E., MacKay, R.J., Hernandez, J., Franchini, M. Cytokine induction in pulmonary airways of horses with heaves and effect of therapy with inhaled fluticasone propionate. Vet. Immunol. Immunopathol. 85:147-158, 2002.
  • Long, M.T., Mines, M.T., Knowles, D.P., Tanhauser, S.M., Dame, J.B., Cutler, T.J., MacKay, R.J., Sellon, D.C. Sarcocystis neurona: parasitemia in a severe combined immunodeficient (SCID) horse fed sporocysts. Exp. Parasitol. 100:150-154, 2002.
  • Franklin, R.P., MacKay, R.J., Gillis, K.D., Tanhauser, S.M., Ginn, P.E., Kennedy, T.J. Effect of a single dose of ponazuril on neural infection and clinical disease in Sarcocystis neurona-challenged interferon-gamma knockout mice. Vet. Parasitol. 114:123-130, 2003.
  • Gillis, K.D., MacKay, R.J., Yowell, C.A., Levy, J.K., Greiner, E.C., Dame, J.B., Cheadle, M.A., Hernandez, J., Massey, E.T. Naturally occurring Sarcocystis infection in domestic cats (Felis catus). Int. J. Parasitol. 33:877-883, 2003.
  • Moore, B.D., Balasuriya, U.B., Watson, J.L., Bosio, C.M., MacKay, R.J., MacLachlan, N.J. Virulent and avirulent strains of equine arteritis virus induce different quantities of TNF-alpha and other proinflammatory cytokines in alveolar and blood-derived equine macrophages. Virology 314:662-670, 2003.
  • Porter, M.B., Long, M.T., Getman, L.M., Giguere, S., MacKay, R.J., Lester, G.D., Alleman, A.R., Wamsley, H.L., Franklin, R.P., Jacks, S., Buergelt, C.D., Detrisac, C.J. West Nile virus encephalomyelitis in horses: 46 cases (2001). J. Am. Vet. Med. Assoc. 222:1241-1247, 2003.
  • Porter, M.B., MacKay, R.J., Uhl, E., Platt, S.R., de Lahunta, A. Neurologic disease putatively associated with ingestion of Solanum viarum in goats. J. Am. Vet. Med. Assoc. 223:501-4, 456, 2003.


Progress 03/24/05 to 03/24/06

Outputs
1. Equine protozoal myeloencephalitis. This disease continues to be significant cause of loss to the equine industries. My work during this period has 1. contributed to the evaluation of a commercial equine vaccine for the disease; 2. explored the role of domestic cats in the biology of the causative agent, Sarcocystis neurona, 3. led to the development of an assay system that can distinguish subclinical (inapparent) infections of horses from those that result in damage to the central nervous system, 4. showed that a single dose of the anti-EPM drug ponazuril, if strategically timed, is effective in inhibiting the proliferation of S. neurona in a mouse model of EPM and 5. shown that intermittent "metaphylactic" doses of ponazuril in the horse have great potential to prevent CNS infections with S. neurona while allowing normal immune responses against this organism to go forward. 2. Equine endotoxemia. I previously developed and exhaustively evaluated a new treatment of equine endotoxemia: polymyxin B. During the current reporting period, I have 1. worked out the behavior of this drug when given intravenously to horses, identifying both the enti-endotoxin effect and the duration of effective activity in the blood of treated horses and 2. begun evaluation of a new marker of the effects of endotoxin (HMGB1) and a new treatment for equine endotoxemia (ethyl pyruvate).

Impacts
My work with EPM projects will lead to 1. objective assessment of the possible efficacy of Fort Dodge's EPM vaccine, 2. has shown that cats are rarely if ever naturally infected with the agent of EPM, Sarcocystis neurona, 3. will lead to the adoption of a test that can resolve between extra-neural and CNS infections with S. neurona, 4. has demonstrated both in mice and horses that intermittent administration with ponazuril has the potential to prevent CNS infections with S. neurona and 5. a recent review by the author of all data on treatment of EPM will greatly clarify the incomplete and confusing information currently in the literature. My work in the field of endotoxemia previously has demonstrated the potential utility of polymyxin B in treatment. The impact of the current work is that now the dosing interval for this drug and dose has been worked out such that veterinarians can now give the drug repeatedly knowing that they will maintain continuous therapeutic effect without risk of toxicity and 2. the ongoing work with HMGB1 and ethyl pyruvate will have tremendous impact both on evaluation of and treatment of the endotoxemic horse. Ethyl pyruvate is cheap, non-toxic and acts long after the initial stimulus, making this approach highly attractive.

Publications

  • MacKay RJ. Brain injury: Settings and treatments. 6th Annual Resort symposium, AAEP. In Lecture Workbook, 2004, pp 1-12
  • MacKay RJ. Broken necks, backs, and butts. 6th Annual Resort symposium, AAEP. In Lecture Workbook, 2004, pp 13-16.
  • Long, MT, Hines, MT, Knowles, DP, Tanhauser, SM, Dame, JB, Cutler, TJ, MacKay, RJ, Sellon, DC: Sarcocystis neurona: parasitemia in a severe combined immunodeficient (SCID) horse fed sporocysts, Exp. Parasitol., 100(3):150-154, 2002
  • MacKay RJ. Acute infectious neurologic diseases of the horse. 6th Annual Resort symposium, AAEP. In Lecture Workbook, 2004, pp 27-32
  • MacKay RJ. What, if anything, is new in the field of equine sepsis. 9th Annual Conference for Veterinarians. In Lecture Workbook, 2004, pp 33-38
  • MacKay RJ. EPM: An oldie but a goodie. 9th Annual Conference for Veterinarians, North Carolina Veterinary Conference. In Proceedings 2004, pp 37-41
  • MacKay RJ. Salmonellosis: Clinical update and infection control in the hospital setting. 9th Annual Conference for Veterinarians, North Carolina Veterinary Conference. In Proceedings 2004, pp 151-155
  • MacKay RJ. Intestinal clostridiosis: Under-recognized but fatal.. 9th Annual Conference for Veterinarians, North Carolina Veterinary Conference. In Proceedings 2004, pp 156-159
  • MacKay, R.J.: Equine protozoal myeloencephalitis: In: Robinson, N.E. (ed.): Current Therapy in Equine Medicine, ed. 5. St. Louis, Saunders, 2002, pp 74-77.
  • Furr, M, Mackay, RJ, Granstrom, Schott II, DH, Andrews, F: Clinical diagnosis of equine protozoal myeloencephalitis (EPM). J.Vet. Int.Med. 16:618-621, 2002.
  • MacKay, R.J.: Equine protozoal myeloencephalitis: In: Robinson, N.E. (ed.): Brainstem and cranial nerve diseases, ed. 5. St. Louis, Saunders, 2002, pp 772-778. Also, section editor for Neurologic diseases, pp 735-778.
  • MacKay, RJ:. Update on treatment and management of equine protozoal myeloencephalitis (EPM). In Volume 16, Proceedings of The North American Veterinary Conference (Large Animal Edition), Orlando, FL, January 12-16, 2002, pp. 80-82.
  • MacKay, RJ:. Update on life cycle of S. neurona and pathogenesis of equine protozoal myeloencephalitis (EPM). In Volume 16, Proceedings of The North American Veterinary Conference (Large Animal Edition), Orlando, FL, January 12-16, 2002, pp. 83-85.
  • MacKay, RJ:. Update on treatment and management of equine protozoal myeloencephalitis (EPM). In Volume 16, Proceedings of The North American Veterinary Conference (Large Animal Edition), Orlando, FL, January 12-16, 2002, pp. 80-82.
  • MacKay, RJ:. Update on life cycle of S. neurona and pathogenesis of equine protozoal myeloencephalitis (EPM). In Volume 16, Proceedings of The North American Veterinary Conference (Large Animal Edition), Orlando, FL, January 12-16, 2002, pp. 83-85.
  • MacKay, RJ: Equine protozoal myeloencephalitis: New information about life cycle, pathogenesis, and diagnosis. In Proceedings of the Michigan Veterinary Conference, Lansing, MI, January 24-27, 2002, pp 546-550
  • MacKay, RJ: You think EPM is over-diagnosed? Here are some alternatives. In Proceedings of the Michigan Veterinary Conference, Lansing, MI, January 24-27, 2002, pp 560-564
  • MacKay, RJ: Seizing and other falling down horses. A video tour. In Proceedings of the Michigan Veterinary Conference, Lansing, MI, January 24-27, 2002, pp 556-559
  • MacKay, RJ:. Flat foals, daft foals. In Proceedings of the Michigan Veterinary Conference, Lansing, MI, January 24-27, 2002, pp 551-555
  • Gillis KD, MacKay RJ, Yowell CA, Levy JK, Greiner EC, Dame JB, Cheadle MA, Hernandez J, Massey ET. Naturally occurring Sarcocystis infection in domestic cats (Felis catus). Int J Parasitol. 2003 Jul 30;33(8):877-83.
  • Franklin RP, MacKay RJ, Gillis KD, Tanhauser SM, Ginn PE, Kennedy TJ. Effect of a single dose of ponazuril on neural infection and clinical disease in Sarcocystis neurona-challenged interferon-gamma knockout mice. Vet Parasitol. 2003 May 30;114(2):123-30.
  • RJ MacKay. CNS lesions: Examination and localization. Ninth International Veterinary Emergency & Critical Care symposium. In International VECCS Proceedings 2003, pp 625-628.
  • RJ MacKay. CNS trauma. Ninth International Veterinary Emergency & Critical Care symposium, In International VECCS Proceedings 2003, pp 634-638.
  • RJ MacKay. Acute infectious neurologic diseases of the horse. Ninth International Veterinary Emergency & Critical Care symposium. In International VECCS Proceedings 2003, pp 625-628.
  • RJ MacKay. Vascular Endothelial Changes Leading to SIRS and MODS. Eighth World Congress of Veterinary Anesthesia. In WCVA Proceedings 2003, pp 345-349
  • Duarte PC, Daft BM, Conrad PA, Packham AE, Saville WJ, MacKay RJ, Barr BC, Wilson WD, Ng T, Reed SM, Gardner IA. Evaluation and comparison of an indirect fluorescent antibody test for detection of antibodies to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and experimentally infected, and vaccinated horses. J Parasitol 90: 379-386, 2004.
  • MacKay RJ. Brain injury after head trauma: pathophysiology, diagnosis, and treatment. Vet Clin North Am Equine Pract 20: 199-216, 2004.
  • MacKay RJ. Sepsis in newborn foals: Aseptic management of these patients is even more important than you think. 9th Annual Conference for Veterinarians, North Carolina Veterinary Conference. In Proceedings 2004, pp 228-251
  • MacKay RJ. Diseases that make horses tremble, shiver, shake, or twitch. 6th Annual Resort symposium, AAEP. In Lecture Workbook, 2004, pp 17-26
  • MacKay RJ. Lawsonia, cryptosporidiosis, rotavirus, and other causes of diarrhea in horses. 9th Annual Conference for Veterinarians, North Carolina Veterinary Conference. In Proceedings 2004, pp 160-167
  • MacKay. RJ Cracked heads: Flipping over backwards and running into things. Ocala Equine Conference. In Proceedings 2004, pp 1-8
  • MacKay RJ. Equine protozoal myeloencephalitis. In The Merck Veterinary Manual, 9th Ed, Kahn CM (ed), Whitehouse Station, Merck & Co, 2004, pp 1031-1032.
  • MacKay RJ. Neurologic disorders of neonatal foals. Vet Clin North Am Equine Pract. 21:387-406, 2005.
  • MacKay RJ. Anatomy and physiology of the nervous system . In: In Auer JA, Stick JA (eds): Textbook of Equine Surgery. 3rd ed. WB Saunders Co.Philadelphia, 2005, pp 630-641
  • MacKay RJ. Diagnostic procedures . In: In Auer JA, Stick JA (eds): Textbook of Equine Surgery. 3rd ed. WB Saunders Co.Philadelphia, 2005, pp 642-658.
  • MacKay RJ. Peripheral nerve injury . In: In Auer JA, Stick JA (eds): Textbook of Equine Surgery. 3rd ed. WB Saunders Co.Philadelphia, 2005, pp 684-691.
  • MacKay RJ. Basic neurologic examinations of the horse. Hagyard Bluegrass Equine Symposium. In Proceedings 2005, pp103-109.
  • MacKay RJ. Neurologic examination of the horse and basic: Case examples and lesion localization. 94th WSVMA Conference, 2005 Annual Conference. In Proceedings
  • MacKay RJ. EPM: An oldie but a goodie.. 94th WSVMA Conference, 2005 Annual Conference. In Proceedings
  • MacKay RJ. Cracked heads: Skull trauma and brain injury. 94th WSVMA Conference, 2005 Annual Conference. In Proceedings
  • MacKay RJ. Diagnosis and treatment of spinal cord injuries. Alamo Pintado Equine Medical Center Symposium 2005. In Proceedings 2005, pp 30-33.
  • MacKay RJ. Diagnosis and treatment of spinal cord injuries. Alamo Pintado Equine Medical Center Symposium 2005. In Proceedings 2005, pp 30-33.
  • MacKay RJ. Diagnosis and treatment of head injuries. Alamo Pintado Equine Medical Center Symposium 2005. In Proceedings 2005, pp 34-45.


Progress 10/01/01 to 10/01/02

Outputs
1. Acquire a better understanding of the life cycle of Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis (EPM). Roles for the 9-banded armadillo and striped skunk as intermediate hosts were established. The domestic cat was investigated for its possible role as an intermediate host in North Central Florida. Fifty cats were obtained from the Alachua county Animal Services Dept. and examined histologically for the presence of sarcocysts. Sarcocysts were found by histologic examination in 5 of these. They were characterized by morphologic, immunologic, molecular, and biological techniques and found to be Sarcocystis felis, a species not closely related to S. neurona. DNA sequence from the ITS-1 region of the rRNA gene cluster was obtained and published at GenBank. This was the first identification of S. felis in domestic cats. In addition 5 of 100 cats were found to have S. neurona antibodies by immunoblot. The significance of this observation is uncertain because no cysts suggestive of S. neurona were found. 2 - Refine the ability to reproduce EPM in an experimental setting. No work was done in this area. 3 - Investigate ways in which to prevent EPM. S. neurona sporocysts were given intragastrically to gamma-IFN-knockout mice which were then given ponazuril at 2 different doses at various intervals after challenge. It was found that there was a dose-dependent protective effect of ponazuril therapy. This effect was maximal 7 days after challenge and diminished either side of that. These results indicated that ponazuril could not kill the non-replicative invasive sporozoite form of the protozoan and was ineffective after CNS infection already had occurred. As part of a multi-centered case-control study of equine patients (designed in conjunction with workers at Texas A&M University), vaccine efficacy is being evaluated by comparing the prevalence of vaccination among horses with EPM, horses with other neurologic diseases, and horses without neurologic disease. It is estimated that at least 3 years of data accumulation will be necessary to confer the necessary power upon the study. The efficacy of intermittent ponazuril treatment on prevention of CNS infection by S. neurona was studied. Horses (5/group) were given 5 x 105 sporocysts, then 20 mg ponazuril/kg every 7 or 14 d for 84 d. Control horses (5/group) were either not treated (+) or not challenged (-).There was significant (P <0.05) effect of treatment on immunoconversion (detected by immunoblot) in CSF but not in serum. All challenged horses became serum-positive within 56 d. Only 2/5 horses treated every 7 d converted in CSF; all other S neurona-challenged horses became CSF-positive within 84 d. Magnitude of the anti-17-kD antibody response in CSF ("relative quantity CSF") was significantly reduced by 7-d but not by 14-d treatments. 4. Establish pharmacokinetic/pharmacodynamic parameters for the anti-endotoxic drug polymyxin B. This has been done in 6 horses and the results are being prepared for publication.

Impacts
Clearly the causative organism of EPM, S. neurona, is widespread in the environment; thus, it is essential to determine the natural life cycle of the organism. We have determined that the 9-banded armadillo and the striped skunk are two likely relevant natural intermediate hosts. This observation will be useful in devising strategies to control the life cycle. We also have shown that the cat is unlikely to be a very important intermediate host. Preliminary experiments in mice and horses have shown the potential for ponazuril has intermittent therapy for the prevention of EPM. Finally, appropriate dose rates for polymyxin B have been determined, which should enable appropriate treatment of horses with endotoxemia in clinical situations.

Publications

  • Furr, M., Kennedy, T., Mackay, R., Reed, S., Andrews, F., Bernard, B., Bain, F., Byars, D. Efficacy of ponazuril 15
  • Costa, L.R.R., Eades, S.C., Tulley, R.T., Koch, C.E., Polkes, A., MacKay, R.J., Moore, R.N. Plasma magnesium concentrations in neonatal foals admitted to the intensive care unit. Advances in magnesium research: Nutrition and health. Yves Rayssiguier, Andrzej Mazur and Jean Durlach (eds.) 2001 John Libbey & Company Ltd, pp. 487-490.
  • Long, M.T., Hines, M.T., Knowles, D.P., Tanhauser, S.M., Dame, J.B., Cutler, T.J., MacKay, R.J., Sellon, D.C. Sarcocystis neurona: parasitemia in a severe combined immunodeficient (SCID) horse fed sporocysts. Exp Parasitol 100 (2002) 150-154.


Progress 10/01/00 to 10/01/01

Outputs
1. Acquire a better understanding of the life cycle of Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis (EPM). A role for the 9-banded armadillo as a natural host for S. neurona in North Central Florida was confirmed by biological and molecular techniques outlined in the original CRIS proposal. The life cycle for the parasite also was completed in the laboratory in the striped skunk (Mephitis mephitis). It remains to be seen whether this will prove to be an important natural host for the parasite. The domestic cat was investigated for its possible role as an intermediate host in North Central Florida. Fifty cats were obtained from the Alachua county Animal Services Dept. and examined histologically for the presence of sarcocysts. Sarcocysts were found by histologic examination in 5 of these. They were characterized by morphologic, immunologic, molecular, and biological techniques and found to be Sarcocystis felis, a species not closely related to S. neurona. In addition 5 of 100 cats were found to have S. neurona antibodies by immunoblot. The significance of this observation is uncertain because no cysts suggestive of S. neurona were found. 2 - Refine the ability to reproduce EPM in an experimental setting. No work was done in this area although projects are planned. 3 - Investigate ways in which to prevent EPM. S. neurona sporocysts were given intragastrically to gamma-IFN-knockout mice which were then given ponazuril at 2 different doses at various intervals after challenge. It was found that there was a dose-dependent protective effect of ponazuril therapy. This effect was maximal 7 days after challenge and diminished either side of that. These results indicated that ponazuril could not kill the non-replicative invasive sporozoite form of the protozoan and was ineffective after CNS infection already had occurred. As part of a multi-centered case-control study of equine patients (designed in conjunction with workers at Texas A&M University), vaccine efficacy is being evaluated by comparing the prevalence of vaccination among horses with EPM, horses with other neurologic diseases, and horses without neurologic disease. It is estimated that at least 3 years of data accumulation will be necessary to confer the necessary power upon the study. 4. Establish pharmacokinetic/pharmacodynamic parameters for the anti-endotoxic drug polymyxin B. This has been done in 6 horses and the results are being prepared for publication.

Impacts
Clearly the causative organism of EPM, S. neurona, is widespread in the environment; thus, it is essential to determine the natural life cycle of the organism. We have determined that the 9-banded armadillo and the striped skunk are two likely relevant natural intermediate hosts. This observation will be useful in devising strategies to control the life cycle. We also have shown that the cat is unlikely to be a very important intermediate host. Preliminary experiments in mice have shown the potential for ponazuril has intermittent therapy for the prevention of EPM. Finally, appropriate dose rates for polymyxin B have been determined, which should enable appropriate treatment of horses with endotoxemia in clinical situations.

Publications

  • Cheadle, M.A., Tanhauser, S.M., Scase, T.J., Dame, J.B., MacKay, R.J., Ginn, P.E. and Greiner, E.C. 2001. Viability of Sarcocystis neurona sporocysts and dose titration in gamma-interferon knockout mice. Vet. Parasitol. 95:223-231.
  • Luznar, S.L., Avery, M.L., Dame, J.B., MacKay, R.J. and Greiner, E.C. 2001. Development of Sarcocystis falcatula in its intermediate host, the Brown-headed Cowbird (Molothrus ater). Vet. Parasitol. 95:327-334.
  • Tanhauser, S.M., Cheadle, M.A., Massey, E.T., Mayer, B.A., Schroedter, D.E., Dame, J.B., Greiner, E.C. and MacKay, R.J. 2001. The nine-banded armadillo (Dasypus novemcinctus) is naturally infected with Sarcocystis neurona. Int. J. Parasitol. 31:325-329.
  • Cheadle, M.A., Tanhauser, S.M., Dame, J.B., Sellon, D.C., Hines, M., Ginn, P.E., MacKay, R.J. and Greiner, E.C. 2001 The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona. Int. J. Parasitol. 31:325-329.
  • MacKay, R.J. 2001. Update on equine therapeutics: Equine neonatal clostridiosis: Treatment and prevention. Comp. Cont. Ed. Pract. Vet. 23:280-285.
  • MacKay, R.J. 2001. Case notes and commentary: On the true definition of dysphagia. Comp. Cont. Ed. Pract. Vet. 23:1024-1028.
  • Cheadle, M.A., Yowell, C.A., Sellon, D.C., Hines, M., Ginn, P.E., Marsh, A.E., MacKay, R.J., Dame, J.B. and E. C. Greiner. 2001 Corrigendum to "The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona" Int. J. Parasitol. 31:843-849.