Equine Protozoal Myeloencephalitis

EMERGING INFECTIOUS DISEASES

0749-0739 I oo $15.00

+

.oo

EQUINE PROTOZOAL MYELOENCEPHALITIS Robert J. MacKay, BVSc, PhD, David E. Granstrom, DVM, PhD, William J. Saville, DVM, PhD, and Stephen M . Reed, DVM

An unusual neurologic condition of horses termed segmental myelitis was first observed by Rooney et al 85 in Kentucky in 1964. Rooney renamed the syndrome "focal encephalitis-myelitis" because of brain involvement, and Prickett83 reported on 44 cases at the annual meeting of the American Association of Equine Practitioners in 1968. Rooney et al 85 reported on 52 cases in 1970. Affected horses ranged in age from 1 to 24 years, with the highest incidence seen in Standardbreds. It was suggested that Standardbreds might be predisposed to the disease, which seemed to be seasonal, with a greater number of cases observed during the summer months. On the basis of the increased frequency of occurrence in the summer, Rooney further suggested that the condition might be caused by a fungal toxin. In 1974, protozoa were first seen in association with characteristic lesions,2' and the disease was given its current name, equine protozoal myeloencephalitis (EPM), by Mayhew and colleagues,73 who reported on 45 cases at the American Association of Equine Practitioners meeting in 1976. Despite considerable efforts in the ensuing years, the cause and epidemiology of EPM have remained poorly understood. LIFE CYCLE OF CAUSATIVE AGENTS

Sarcocystis neurona and Neospora caninum (N. hughesi) are the currently known causative agents of EPM. Most cases of the disease are believed to be caused by infection with S. neurona. All Sarcocystis organisms alternate serially between definitive and intermediate hosts. 80 The definitive host eats sarcocyst-infested tissue from an intermediate From the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida (RJM); Education and Research Division, American Veterinary Medical Association, Schaumburg, Illinois (DEG); Department of Veterinary Preventive Medicine (WJS), and Department of Clinical Sciences (SMR), College of Veterinary Medicine, The Ohio State University, Columbus, Ohio

VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTICE VOLUME 16 • NUMBER 3 • DECEMBER 2000

405

406

MACKAY et al

host. 32 Cyst protozoa undergo sexual replication in the small intestine and, within 3 weeks, form oocysts, each of which contains two sporocysts. Sporocysts are passed in the feces and are immediately infective. After ingestion by the intermediate host, sporocysts excyst, and liberated sporozoites invade the small intestinal epithelium and gain access to the lymph atic or blood vascular syste m of the host. After several cycles of asexual reproduction (schizogony), usually in endothelial cells or monocytes, the organism organizes into sarcocysts in skeletal muscle. Because the sarcocyst is the only stage of the life cycle of the parasite with species-specific morphology,8° it has often been difficult to find definitive and intermediate hosts for a newly discovered Sarcocystis species. Only 86 of the 189 (45%) known species have had definitive and intermediate hosts identified."" This problem was particularly frustrating in the early years of EPM research, when only schizonts and merozoites (the forms involved in asexual replication) of 5. neurona had b een seen in the central nervous system (CNS) of diseased horses. Progress was achieved when a small section of the nuclear genome, the 18S ribosomal RNA (rRNA) gene, was amplified by polymerase ch ain reaction (PCR) from S. neurona merozoites and sporocysts collected from a variety of candidate hosts.43 When the amplified sequences from sporocysts and merozoites were compared, the sequence from the opossum (Didelphis uirgin iana ) was virtually identical to that of S. neurona. On the basis of this findin g, the opossum was proposed as the definitive host. This is supported by the fact that the geographic distribution of D. virginiana46 correlates closely with the distribution of EPM cases in North America. In South America, D. marsupialis and D. albiventris presumably act as definitive hosts of the parasite."" Additionally, experimental infection of foals w ith sporocysts induced the development of neurologic signs and antibodies against S. neurona in blood and cerebrospinal Auid (CSF).42 In a comparable study, the finding of sequence identity over the " hypervariable" part of the 18S rRNA gene led to the proposal that 5. ji1/rntula, a wellstudied parasite of opossums, a nd 5. neurona w e re the same organism.2 '' Had this been true, it would have implicated numerous species of birds as potential intermediate hosts for S. neurona. Subsequent evidence has confirmed that these two parasites are in fact distinct species; this is based on biologic behavior, DNA sequence, and morphologic characteristics in culture. 22· 3 " · H 9 2 The likely explanation for the initial finding is that the gene chosen for comparison, the 18S rRNA, has evolved slowly over time and so is unlikely to differ significantl y among closely related species.3 " · ·' '· 64· ""· 92 Subsequent analyses of additional nuclear genomic DNA sequ ences ha ve consistently shown tha t 5. neurcma and 5. falcatula are closely related and that opossums are the host to both parasites.''7· " 2 Of 434 trapped or road-killed opossums collected in Florida in 1997 and 1998, 4.5% were infected w ith S. neurona and 3.0°/4, were infected with S. falrntu la.21 Nothing is know n about the life cycle of the Neospom sp that has caused several reported cases of EPM. 17, ·' "· 63• 70• " 2 Although the organism reacts with antibodies against N . rnninum, there is sufficient difference in the amino acid sequence of two immunodominant surface antigens that the organism found in horses is thought to constitute a separate species that has been named N. hughesi.68 Although dogs have recently been shown to be a definitive h ost to N. caninum,74 a definitive host for N. hughesi has not yet been identified.

EPIDEMIOLOGY The first national epidemiologic survey of El'M3" used postmortem data gathered retrospectively from 10 diagnostic centers throughout the United States

EQUINE PROTOZOAL MYELOENCEPHALITIS

407

and Canada. The study included 364 histologically confirmed cases from Pennsylvania, Kentucky, New York, Illinois, Ohio, Oklahoma, Missouri, Texas, New Jersey, Florida, California, Canada, and Brazil, together with some horses that had been exported from the Americas. Most horses (61.8%) were 4 years of age or less, and 19.8% were 8 years of age or older. Although Thoroughbred s, Standardbreds, and Quarter Horses (in that order) were the m ost commonly affected breed s, it was not possible to establish any breed, gender, or seasonal bias. No control group was available for study. In a smalle r retrospective study conducted at the University of Pennsylvania New Bolton Center, 82 horses with histologic lesions compatible w ith EPM were reviewed. 12 Disease risk was highest among m ale Standardbred horses compared with the gender and breed distributions of the attendant hospital population. The mean age of affected horses was 3.6 ± 2.8 years, which was similar to the findin gs of Fayer et al. 37 Again, no matched control population was included for comparison. Recent studies have examined the rates of exposure of ho rses to S. neurona and to N. caninurn/N. l111ghesi. The prevalence of S. neurona-specific serum antibodies (as determined by immunoblot) among Thoroughbreds in an eastern Pennsylvania county was 45.3°/.,, and the percentage increased w ith age. 6 The overall exposure rate to S. 11eurona among randomly sampled horses in Oregon also was 45%,8 with the highest rate (65%) recorded in h orses from the coastal region and the lowest (22%) among horses in the arid eastern quadrant. Although seroconversion against S. neuro11a increased with age, it did not seem to be influenced by breed, gender, or the extent to which the animals were housed. In Ohio, a somewhat higher exposure rate to S. neurona (53.6%) was re ported, and this also increased with age.'7 The highest exposure was found in southwestern Ohio, and the lowest was found in northeastern Ohio. This effect of location on seroprevalence correlated strongly with the average annual number of days below freezing, a factor that may adversely affect parasite transmission. A more recent study from northern Colorado de monstrated age, breed, and seasonal effects on the prevalence of antibodies to S. neurona.''3 Although the exposure rate (33.6%) found in Colorado was lowe r than that found in any other survey, the suggestion of a possible climactic effect is consistent with the findings of the Ohio and Oregon studies. Recently, seroprevalences of 35.6% and 35.5% were reported in horses in Brazil and Argentina, respectively.''· 34 In two surveys conducted in the United States, 23.3%33 and 11.5%'7 of horses were found to have antibodies reactive with N. caninum; however, no seropositive horses were identified in studies carried out in Argentina 34 and Brazil.3' This is in contrast to eviden ce of N. caninurn infection of cattle, which is worldwide. 74 Apparently, about 50% of horses in several regions of the United States have been exposed to S. neurona, and somewhat less than 25% have been exposed to N. caninurn/N. hughesi. Despite relatively low sample numbers, these studies illustrate the w idespread distribution of these two organisms. On the basis of postmortem findings from cases submitted to diagnostic laboratories at the University of Kentucky and elsewhere in the United States, it has been estimated that 1% or less of horses have EPM.47 A recent survey conducted by the National Animal Health Monitoring System (NAHMS) collected data from more than 2900 farms in 28 states for the NAHMS Equine '98 study (March 1, 1997-February 28, 1998).7" Based on this stud y, the average annual incidence of EPM in horses 6 months of age or older was 14 ± 6 cases per 10,000 horses. The annual incidence per 10,000 horses was 1 ± 1 in farm or ranch horses, 6 ± 5 in pleasure horses, 17 ± 12 in breeding horses, 38 ± 16 in

408

MACKAY et al

racing horses, and 51 ± 39 in show and competition horses. Horses kept at racetracks were excluded. During the year before the survey interview, 1% of operations reported at least 1 case of EPM. 78 The role of N. caninum!N. lrughesi in these cases is completely unknown. Seven cases of equine neosporosis have so far been reported in the United States. 17· 25 · "' 50· 5 4 · 70 Of these horses, five exhibited neurologic signs, one was an aborted fetus, and the other had a visceral infection. Many clinical reports of EPM suggest that the disease occurs sporadically and seldom involves more than one horse on an operation.h". 73 Clusters of cases can occur, however. 41 • 48 Case reports have provided meager information on trends associated with the development of EPM. Recently, however, a retrospective study was conducted at The Ohio State University. Data from horses diagnosed with EPM were compared with those of nonneurologic patients and with those of horses admitted with neurologic diseases other than EPM. 88 When compared with the nonneurologic control group, young horses (1-5 years old) and older horses (> 13 years old) had a higher risk of developing EPM than did other horses. The number of cases was lowest in the winter, but increased with rising ambient temperature. Compared with the winter months, the risk was three times higher in spring and summer months and six times higher in the fall months. One possible reason for this seasonal effect may be the influence of days below freezing"7 on rate of exposure to 5. ne11ro11a. Another may be the variable effect of transport stress as a result of the timing of competition events. Other factors associated with increased risk of EPM on a given premises were presence of opossums (2.5-fold), previous diagnosis of EPM (2.5-fold), and presence of woods (twofold). In contrast, the likelihood of EPM was reduced by one third by prevention of wildlife access to feed and one half by the presence of a creek or river. The results of this study emphasize the importance of opossums and their access to horse feed as important risk factors in the occurrence of clinical disease. Stress or advanced age may predispose to development of EPM by immune suppression. 89 A strong dose-response relation was found between various "stressful" events (e.g., heavy exercise, transport, injury, surgery, parturition) and the risk for EPM. 88 After such an event, the risk of manifesting the disease increased with time. Racehorses and show horses had a higher risk of developing EPM compared with breeding and pleasure horses. Not surprisingly, horses with EPM that were treated were lO times more likely to improve than were untreated horses."" Those with moderate to severe clinical signs were less likely to survive than were horses with mild signs. If there was improvement, horses were 50 times more likely to survive than if there was none. Possible risk factors also were investigated as part of the NAHMS Equine '98 study. 78 EPM was more likely to occur on premises where opossums were often or sometimes identified. This risk was sixfold higher for occurrence of EPM in the past year and 12-fold higher for the entire history of the operation. Interestingly, the presence of mice or rats also was associated with increased risk: fourfold for the previous year and fivefold for the operation's history. It is worth noting that rats and mice act as intermediate hosts or transport vectors for other Sarcocystis spp. 1"· 11 Compared with homegrown grain, feedi ng of purchased grain was associated with an increased risk for EPM. 78 This relation must be interpreted cautiously, however, because grain distribution around the country could provide a possible means of dissemination of the parasite. High population density also increased the risk of developing EPM. Horses

EQUINE PROTOZOA L MYELOENCEPHAUTIS

409

in areas with more than 25 people per square mile had a 37-fold (previous year) and a fourfold (operation's history) greater risk of developing EPM compared with horses in areas with less than 25 people per square mile. This finding may be related to the encroachment of human beings on opossum habitats. Interestingly, the presence of woods within 5 miles of an operation was associated with a lower risk for EPM. If surface water was used as the primary water source on operations, the risk for EPM was much lower than was the case when it was made available in buckets or troughs or through the use of other delivery systems. A high stocking density (20 or more horses per operation) resulted in a sevenfold greater chance of EPM. This finding is difficult to explain, because the horse is believed to be a dead-end host for the parasite. Perhaps other risk factors such as increased purchase of feed may be involved. Horses that were housed on wood shavings or chips also were found to have a higher risk for EPM. This is also difficult to explain; it is likely related to management differences among operations. The risk of EPM was seasonal, with most cases occurring in the summer and fall months. CLINICAL SIGNS

EPM is often a progressive debilitating disease affecting the CNS of horses. Clinical signs vary from acute to chronic, with insidious onset of focal or multifocal signs of neurologic disease involving the brain, brain stem, or spinal cord. 66· 84 Affected horses may initially exhibit unusual signs such as evidence of abnormal upper airway function, unusual or atypical lameness, or even seizures.1o Severely affected horses may have difficulty in standing, walking, or swallowing, and the disease may progress rapidly. In other cases, the clinical condition seems to stabilize, only to relapse days or weeks later. The variability of clinical signs is a result of the organism's ability to attack white and gray matter randomly at multiple sites along the entire CNS. Signs of gray matter involvement include focal muscle atrophy and severe muscle weakness, while damage to white matter frequently results in ataxia and weakness in limbs caudal to the site of damage. The early signs of the disease, such as stumbling and frequent interference between limbs, can easily be confused with lameness. Horses affected with EPM commonly experience a gradual progression in the severity and range of clinical signs including ataxia. In some cases, however, a gradual onset may give way to a sudden exacerbation in the severity of clinical illness, resulting in recumbency. The vital signs in affected horses are usually normal, and animals seem bright and alert. Some horses with EPM may look thin and mildly depressed. Neurologic examination often reveals asymmetric ataxia, weakness, and spasticity involving all four limbs. Areas of hyporeflexia, hypalgesia, or complete sensory loss are frequently present. The most commonly observed signs of brain or brain stem disease include depression, head tilt, facial paralysis, and difficulty in swallowing. Signs are not necessa rily limited to these areas. 84 Gait abnormalities most often result from lesions in the spinal cord and may be variable in severity depending on the location and extent of tissue damage. EXPERIMENTAL INFECTIONS

An initial study aimed at reproducing EPM used as a challenge inoculum a mixture of uncharacterized sporocysts collected from 10 feral opossums. 42 Five

410

MACKAY et al

foals negative for antibodies to S. neurona were each given at least 2 X 106 sporocysts over one to three doses. All foals seroconverted 19 to 42 days after the first dose, and antibodies were first present in the CSF at 28 to 42 days after challenge. Each of the CSF-positive foals developed signs of spinal cord disease beginning 28 to 42 days after the challenge; signs ranged from mild toe dragging and weakness in the hind limbs to severe spasticity, hypermetria, and ataxia in all four limbs. At necropsy, no gross lesions were found in the CNS. On histologic examination, multifocal perivascular mononuclear infiltrates were seen in the brain stem or spinal cord of three foals; this was associated with minimal neuronal necrosis. Although no protozoa were detected, these changes were consistent with those seen in some cases of EPM. When molecular markers were identified that were able to differentiate the various Sarcocystis spp sporocysts found in opossums,9 2 it became possible to prepare challenge inocula containing known quantities of S. neurona sporocysts. A number of such experiments have since been carried out. Although the findings from these studies have not yet been analyzed completely, some generalizations can be made. Most of the studies employed single or multiple challenges of 105 to 107 S. neurona sporocysts given intragastrically to seronegative horses. The horses developed serum antibodies to S. neuro11a 2 to 6 weeks after the initial challenge dose of the parasite and converted in the CSF up to 3 weeks later. Mild to moderately severe neurologic signs were observed in some but not all challenged horses. In many cases, signs were progressive initially and then stabilized or even improved. The concentration of antibody against an immunodominant 17-kd S. neurona protein (reported as "Relative Quantity CSF" by Neogen, Lexington, KY) in CSF followed a similar pattern. It began to increase about 6 weeks after challenge and continued to rise until about 9 weeks, at which point it leveled off or began to decrease by the end of the experiment. Evidence of mild to moderate subacute to chronic multifocal changes was generally found in the CNS; these were characterized principally by multifocal neuronophagia and gliosis. Protozoa were not observed in blood or CNS tissue, nor were they detected by immunohistologic staining, PCR, culture, or mouse inoculation. Immunosuppression resulting from dexamethasone administration either reduced the time to the appearance of antibodies in the CSF, suggesting that immunosuppression facilitated invasion of the CNS, or had no effect. Clinical signs and histologic lesions were equivalent to or even milder than those observed in the challenged control horses. The collective results from the various experimental horse infection studies indicate that the horse is relatively resistant to even large challenge doses of S. neurona. In most cases, it was evident that the horses could effectively clear the parasite from the CNS. This type of response to infection with S. neurona may be the norm in young horses after natural exposure to the parasite. Infection of the CNS and the appearance of antibodies in the CSF may be transient phenomena in such horses and may go undetected unless CSF samples are collected for immunoblot analysis. The role of the immune response in the progression of infection to EPM would seem to be complicated. Whereas severe immunosuppression may promote CNS invasion by S. neurona, some element of the immune response may actually be required to permit more widespread infection of the CNS by the parasite with the development of signs of EPM. In rodent models of infection with Toxoplasma gondii or N. caninum, resistance to infection is not only related to strain but is also dependent on generation of a normal Trn cellular immune response. '·" Cytokines such as interferon--y and interleukin-12 are important mediators of this protective response. Similar results

EQUINE PROTOZOAL MYELOENCEPHAUTIS

411

have been seen in limited studies with S. neurona. Protozoa! encephalitis was induced in athymic "nude" or interferon--y "knockout" mice by subcutaneous or intraperitoneal injection of cultured merozoites or by gavage with sporocysts. 29· 67 Immunocompetent mice (C57BL / 6) were apparently resistant to infection even after corticosteroid treatment. Paradoxically, ICR SCID mice (n = 2), who completely lack adaptive immune systems but have a population of natural killer cells, also were resistant to infection. PATHOLOGY

On necropsy examination of cases of EPM, lesions may be grossly visible on cut surfaces of the CNS; these may vary from clearly demarcated discoloration (usually of gray matter) to massive lesions that destroy large portions of the brain or multiple segments of the spinal cord. 72 Histologically, organisms are seen in less than 50'1/o of cases. When present, they can be difficult to detect. They are strongly basophilic, periodic acid-Schiff-negative, and agyrophobic. Merozoites are seen singly or in groups (schizonts) free in tissue or within cells such as macrophages, glia, eosinophils, and neurons. There may be several to more than 100 merozoites per group. Schizonts measure about 20 µm, and the organisms frequently are arranged in stellate or rosette patterns. Histologic lesions are remarkably consistent regardless of the presence of associated organisms. Typically, there is cuffing of blood vessels by mononuclear cells, necrosis of parenchyma with phagocytosis and gitter cell formation, astrocyte proliferation, and gemistocyte formation. Eosinophils are seen commonly, as are multinucleated cells, which may be giant in size. Lesions may extend to produce a nonsuppurative meningitis. Lesions may vary from peracute to chronic with prominent lymphoid vascular cuffing and minimal tissue destruction in the former case and marked tissue loss, prominent astrocyte proliferation, and minimal inflammatory response in chronic cases. The amount of fiber degeneration in ascending and descending pathways below and above the lesions is dependent on the chronicity of the condition. DIAGNOSIS

EPM should always be considered in any horse exhibiting signs of CNS disease. Horses displaying such signs should be subjected to a thorough neurologic examination, and appropriate laboratory tests should be undertaken to support a diagnosis of EPM and to exclude other likely diagnoses. Laboratory testing should be considered ancillary to and not a substitute for an in-depth clinical examination. In many cases of EPM, there is asymmetry of gait and focal muscle atrophy. This combination of signs has proved to be the most useful distinguishing feature of the disease and is helpful in clinically differentiating EPM from similar neurologic conditions affecting the horse. Antemortem diagnosis of mild or atypical cases of EPM can present considerable difficulties for the clinician. Differential Diagnoses

Virtually any neurologic disease of horses can produce clinical signs that mimic those associated with cases of EPM. Cervical vertebral malformation

412

MACKAY et al

(CVM) is a frequently encountered disease that results from compression of the cervical spinal cord. The condition usually is caused by stenosis of the cervical vertebral canal and may be accompanied by intervertebral instability. CVM occurs commonly in horses 1 to 3 years of age.79 Male horses are affected more often than female horses. Signs usually are symmetric and the pelvic limbs are typically more severely affected than the thoracic limbs. Focal muscle atrophy is not a clinical feature of CVM. Trauma also can cause spinal cord damage at any level, potentially causing abnormal neurologic signs in from one to all limbs. Equine herpesvirus-1 myeloencephalopathy can affect any breed. A history of respiratory disease or an outbreak of abortion is a common prelude to the occurrence of equine herpesvirus-1-associated neurologic disease. This disease can occur as single cases or in outbreaks. Affected horses may be febrile at the onset of neurologic signs. Neurologic signs most commonly are symmetric, with primary pelvic limb weakness and ataxia, bladder distention (usually without incontinence), and, more rarely, perinea! hypalgesia, tail paralysis, and fecal retention. Signs of brain involvement are seen rarely. Affected horses may become paraplegic or quadriplegic, recumbent, and unable to rise.56• 8 1 Equine motor neuron disease also produces signs that initially may be confused with those observed in horses affected with EPM. Severe limb weakness with muscle fasciculations and tremors are typical early signs of equine motor neuron disease. With chronicity, there is widespread and profound muscle atrophy. Other causes of spinal cord disease that may result in clinical signs similar to those seen in cases of EPM include extradural and spinal cord tumors, epidural abscess, migrating metazoan parasites, rabies, West Nile viral encephalomyelitis, equine degenerative myeloencephalopathy, vascular malformations, and discospondylopathies. Any of the many equine diseases of the brain or cranial nerves must be considered as potential rule-outs in cases of EPM showing signs attribu table to dysfunction of the brain or cranial nerves. This list includes viral encephalomyelitides, neoplasia, head trauma, brain abscess, migrating parasites, temporohyoid osteoarthropathy, polyneuritis equi, cholesterol granuloma, metabolic derangement, and hepatoencephalopathy. Postmortem Diagnosis

Confirmation of a diagnosis of EPM on postmortem examination is based on the demonstration of protozoa in CNS lesions. The diagnosis frequently is made presumptively even when the organism is not detected if the characteristic inflammatory changes are found. In two reported series, organisms were seen in hematoxylin and eosin sections of CNS tissue in 10% to 36% of suspected cases. 12•53 Sensitivity was increased from 20% to 51 % by immunostaining with S. neurona antibody. 53 The likelihood of finding organisms is reduced by prior treatment with antiprotozoal drugs and may be increased by treatment with corticosteroids. Blood and Cerebrospinal Fluid Analyses

Complete and differential blood cell counts and serum chemistry values do not show consistent changes in cases of EPM.65 Similarly, the results of cytologic and chemical analyses of CSF often are normal. Examination of CSF may be

EQUINE PROTOZOAL MYELOENCEPHALIT1S

413

more useful in differentiating other neurologic diseases from EPM . It should be noted, however, that even slight blood contamination during CSF collection may preclude meaningful analysis. In such cases, another CSF tap can be obtained 1 to 2 weeks later. CSF total albumin concentration and albumin quotient (AQ) may provide useful information about the quality of a CSF sample. 2· 3 Because CSF albumin originates from blood, comparison of these values with established normal ranges can help to evaluate the integrity of the blood-brain barrier. If the values are elevated, the blood-brain barrier permeability has increased or the sample may ha ve been contaminated with blood. The ratio of total IgG concentration in CSF to that in serum can be used in conjunction with the AQ to calculate the IgG index. 2• 3 This index originally was used to detect intrathecal IgG production. A number of CNS diseases, including EPM, may result in an increased IgG index; however, it recently has been established that small amounts of blood may falsely elevate the IgG index even in the presence of a normal AQ, thereby confounding interpretation of the results of this test. 76 Notwithstanding the sensitivity of the IgG index to blood contamination, extremely low-level blood contamination may cause a negative CSF sample to test positive by immunoblot analysis, even when the AQ and IgG index are normal. 71' Horses with the highest circulating serum antibody concentrations present the greatest potential for undetected contamination. Although AQ and IgG index are useful diagnostic aids, results that fall within normal ranges must nonetheless be interpreted with caution. lmmunoblot Testing

Immunoblot analysis detects the presence of S. neurona-specific antibodies in serum or CSF. 49 The test originally was developed in 1991 by examining the reactivity of various sera against lysates of cultured S. neurona merozoites. These test immunoblots included sera from horses with EPM, a pony with experimental S. fayeri infection, and polyclonal rabbit antisera against S. neurona, S. cruzi, and S. muris. On the basis of the reaction of these sera with S. neurona proteins, eight specific proteins were identified. Only serum from horses with EPM or antisera from horses or rabbits injected with S. neurona reacted with these proteins. Several of the proteins form the basis of the immunoblot test used as an EPM diagnostic aid. Although a number of modifications to the test have been introduced over the years, a current commercial immunoblot test for S. neurona antibodies (Equine Biodiagnostics, Lexington, KY) is similar to the original published technique. In a recent report, a purported refinement in test methodology is reported to boost test sensitivity and specificity to nearly 100%.86 The test modification involves preincubation of blots in pooled bovine serum to eliminate crossreactivity to S. cruzi, a parasite of cattle and canids found worldwide. It is worth noting, however, that the original test developed in 1991 was based on excluding any proteins that cross-reacted with S. cruzi, S. muris, or S. fayeri antisera. Most significantly, the reported claims of enhanced sensitivity and specificity in the modified immunoblot test were not substantiated when samples were split between two laboratories in a double-blind study (W.J. Saville, DVM, PhD, unpublished data). S. cruzi apparently does not infect the horse, and horses are not known to produce antibodies to the parasite. 32 Furthermore, sporocysts of S. Jalcatu la, a species closely related to S. neurona obtained from an opossum, failed to stimulate a detectable antibody response after oral administration to horses. 22

414

MACKAY et al

Although S. cruzi and S. fayeri share many common antigens, horses with sarcocystosis (i.e., presumed S. fayeri infection) have routinely tested seronegative in the standard immunoblot test for 5. neurona (T. Cutler, MS, BVM, MRCVS, personal communication, 1999). 95 Because the negative control equine sera used to validate the modified test were obtained from horses native to India and Germany, it is p ossible that these sera may have contained antibodies to equine Sarcocystis spp unique to the Eastern Hemisphere. This may help to explain divergence in the outcomes of the two test procedures. The original immunoblot test was evaluated in 1995 using sera from 300 wild horses from Utah, a region with canids but not opossums. Although anti-Sarcocystis antibodies were detected frequently, only one sample tested positive for antibodies to S. neurona (D. Granstrom, DVM, PhD, unpublished data). In the modified immunoblo t test,"" a sample is considered positive if it is reactive to 30- and 16-kd parasite proteins. A negative test may include reactivity to one protein or the other but not to both. The 30- and 16-kd proteins used in interpretation of the modified immunoblot test are in fact not S. neurona- specific. Both proteins are immunodominant and routinely cross-react with serum from horses infected with S. fayeri. A total of 95% of the negative control equine sera used to validate the modified assay reacted to the 30-kd protein, and 81 °/4, reacted to the 16-kd protein when bovine serum blocking agent was not used. Some 19% of the samples still reacted to the 30-kd protein and 11 % reacted to the 16-kd protein even after incubation of the blots with bovine serum. Several recent studies have attempted to establish the reliability of immunoblot testing using specimens of CSF obtained at postmortem examination. 24,77•90 The risk of blood contamination of CSF and the chronology of disease related to the time of CSF collection (chronically affected or permanently impaired horses often test negative) must be taken into consideration when evalu ating studies of this type. Because the blood-brain barrier deteriorates rapidly after death, CSF samples must be collected with minimal delay, preferably using the atlantooccipital approach. Finally, consistent use of an appropriate sampling plan for histopathologic diagnosis must be adhered to. The results of immunoblot tests on serum and CSF from 295 horses euthanatized because of neurologic disease were compared with the postmortem diagnoses.47 Data were collected on horses from 22 states between 1991 and 1996. Samples available for testing included sera from 191 horses, CSF samples from 254 horses, and sera and CSF samples from 88 ho rses. Any CSF specimen with grossly visible blood contamination was not tested. A postmortem diagnosis of EPM was made in 123 horses. Other diagnoses included cervical vertebral malformation (70 horses), hepatoencephalopathy, viral encephalomyelitis, equine degenerative myeloencephalopathy, leukoencephalomalacia, CNS abscess, epilepsy, neuritis of the cauda equina, neurotoxicity, lymphosarcoma, botulism, guttural pouch mycosis, and aberrant parasite migration. The sensitivity and specificity of immunoblot analysis of the CSF samples were both approximately 89%. Test sensitivity was defined as the percentage of horses diagnosed with EPM that had immunoblot-positive CSF, and specificity was defined as the percentage of horses found not to have EPM that tested negative. Three horses that gave false-negative test results had clinical signs for less than 2 weeks before CFS collection. Although the incubation period of EPM seems to be long enough to allow for the production of detectable amounts of specific antibody in most cases, false-negative CSF test results can occasionally

EQUINE PROTOZOAL MYELOENCEPHALITIS

415

occur in acute cases. Jn addition, some horses affected with EPM simply fail to produce a detectable antibody response. False-positive CSF test results were found in six cases of CVM and in cases of viral encephalomyelitis, trauma, equine degenerative myeloencephaloppahy, leukoencephalomalacia, and CNS abscess. These reactions may have been a result of antibodies from blood leaking into the CSF, pe rhaps from breakdown of the blood-CNS barrier. The most common cause of false-positive CSF test results, however, is thought to be blood contamination of the CSF specimen at the time of collection. The positive predictive value (PPV) for the CSF test results was 85%. The PPV is the percentage of horses that test positive that are truly diseased. Similarly, the negative predictive value (NPV) is the percentage of h orses that test negative by immunoblot analysis that do not h ave EPM. The NPV among the population examined was 92%. The sensitivity and specificity of a diagnostic test are independent of the population sampled; h owever, PPV and N PV vary significantly depending on the prevalence of the disease in the test population. The PPV is most influenced when a test is used on a population w ith low prevalence of a particular disease, (i.e., a normal horse population). Any test with a sensitivity and specificity of 89% would have an 8% PPV in a population with a disease prevalence of 1'.1/o (i.e., mo re or less the population defined by the epidemiologic studies outlined previously). The NPV would be high in the same population. Because the PPV for the immunoblot test on CSF was calculated using samples collected from horses euthanatized because of neurologic disease, it does not provide a reliable assessment of the value of the test when used on CSF fro'll a normal population of horses. The likelihood of clinical disease among normal animals with specific antibodies in the CSF is unknown. Most may remain clinically normal and may not develop signs of EPM. We recommend that CSF samples not be tested from cl inically normal h orses. In the event that a positive result is obtained in CSF from a normal horse, it would seem prudent to instigate a sin gle course of treatment (see below). Seroprevalence studies among normal horse populations have provided ample evidence that positive immunoblot test results on serum merely confirm exposure to the parasite and are not diagnostic per se of EPM. 6• 8• 87 The sensitivity of the immunoblot test on serum collected in the postmortem study w as 89%, whereas the specificity was only 71%. The PPV for the serum test was only 72%, although the NPV was 88%. In a recent report of a horse euthanatized because of N. caninum/N. hughesi myeloencephalitis,7° the authors suggested that antibodies produced against N. caninum cross-react w ith S. neurona-specific proteins. Exposure of this animal to S. neurona and to N. caninum/N. hughcsi may be a more logical explanation for the immunoblot test finding. In support of this notion is the fact that the authors were unable to demonstrate cross-reacti vity w ith S. neurona- specific proteins using rabbit antiserum prepared against N . caninum. Polymerase Chain Reaction Assay

PCR testing of equine CSF for parasite-specific DNA may also confirm the presence of S. neurona in the CNS. 44 Unfortunately, the sensitivity of PCR testing seems to be much lower than originally projected. This may be a result of the fact that the parasite DNA is rapidly destroyed by enzymatic action in the CSF; it may also mean that the parasite DNA is rarely present in the CSF.1,.~

416

MACKAY et al

Notwithstanding the aforementioned limitations, the ability to detect parasite DNA in CSF samples that have tested negative using the immunoblot procedure makes the PCR assay a useful aid in the diagnosis of selected cases of EPM.

TREATMENT Folate-lnhibiting Drugs

The treatment of EPM using folate inhibitors has been reviewed previously.40· 65 Currently, it is recommended that horses be treated w ith a combination of sulfadiazine and pyrimethamine (SDZ / PYR). These drugs block successive steps in protozoa! folate synthesis.'" A dosage regimen that is commonly used is PYR, 1 mg/ kg, and SDZ, 20 mg / kg, administered once daily for at least 6 months. This is referred to hereafter as the " standard" treatment for EPM. Because dietary folate can interfere with the uptake of diaminopyrimidine drugs" like PYR, hay should not be fed for 2 hours before or after treatment. These drugs are usually compounded in a paste or suspension formulation. It is important to emphasize that none of the component drugs or compounded products are currently approved by the US Food and Drug Administration. Recent in vitro studies have shown that the combination of 0.1 µ g/ mL of PYR with 5 µg / mL of either SDZ or sulfamethoxazole completely inhibits the growth of S. neurona in cultured bovine turbinate cells. 5" PYR given orally to horses at 1 mg/kg/d achieves a concentration of approximately 0.02 to 0.10 µg / mL in the CSF 4 to 6 hours after administration. 19 Interestingly, these experimental horses were allowed free access to prairie hay, potentially reducing the bioavailablity of the drug." Additionally, because PYR is concentrated in CNS tissue relative to plasma, 16 the concentration at the desired site of action may be greater than 0.1 µg / mL. Mean peak CSF concentrations of sulfonam ide after single or multiple dosing (22-44 mg/kg) have been rep orted to be approximately 2 to 8 µg/mL. 14·51 These pharmacokinetic studies need to be repeated with compounded drugs to ensure that the bioavailablity of each component drug is comparable to that of its US Food and Drug Administration-approved counterpart. There is little objective information on the usefulness of sulfonamide/PYR for the treatment of EPM. A large multicenter trial involving 105 horses was recently completed by Phoenix Scientific (St Joseph, MO). Affected horses were treated with standard- or double-dose SDZ / PYR given once dail y for 90 to 210 days. Success was defined as two grades or more of improvement on an ataxia score (scale of 0-4) on completion of treatment or reversion to immunoblotnegative status for antibodies to S. 11euro11a in CSF. Based on these criteria, approximately 66% of horses responded favorably to treatment, including 22% with antibody-negative results in the CSF (D.F. Erichsen, DVM, MS, personal communication, 2000). Criteria for discontinuing treatme nt are relatively empiric and arbitrary. Horses that become immunoblot-negative after a standard 6-month course are unlikely to relapse and require no further treatment. In one study, only 1 of 32 horses that were CSF immunoblot-negative relapsed compared with 16 of 17 CSF immunoblot-positive horses. 31 Unfortunately, most horses remain immunoblotpositive. Several options can be considered in the case of such individ uals: (1) continue therapy with SDZ/ PYR (at either standard or higher doses) until, hopefully, the CSF becomes immunoblot-negative; (2) maintain animals o n intermittent therapy (e.g., 2 d ays per week or 1 week per month) indefinitely; (3)

EQUINE l'ROTOZOAL MYELOENCEPHAUTIS

417

switch treatment to a different type of drug (see below); or (4) discontinue therapy and watch closely for evidence of relapse. The toxic effects of these drugs relate to the inhibition of folate synthesis and, fortunately, are rarely serious even when the drug is given at twice the standard dose. Typically, there is progressive mild anemia (packed cell volume in the low 20s) over a 6-month treatment period. 3ii In two different studies in which horses were given SDZ/PYR at the standard or double dose, neutropenia (< 2500 per microliter) was seen in 7.7% and 50°/4,,38 and anemia, leukopenia, or neutropenia occurred in 25% and 50°/4, of animals, respectively (D.F. Erichsen, DVM, MS, personal communication, 2000). Low white blood cell counts usually resolve within 2 weeks if the medication is temporarily discontinued. Although SDZ/PYR treatment apparently had no effect on the semen characteristics of healthy stallions, four of six treated horses exhibited abnormalities in mounting, thrusting, or ejaculatory mechanics from 1 to 3 months after the commencement of treatment. 5 PYR is considered te ratogenic and causes abortions and birth of malformed pups in treated rats. 18 There is widespread belief, without any supportive data, that SDZ/PYR can cause abortion in mares in middle and late gestation. In one study, no detrimental effect from SDZ/PYR treatment was observed during the first 25 days of gestation.13 At least four cases of a fatal syndrome have been observed in neonatal foals born to mares that were given these drugs during the latter stages of pregnancy (R. MacKay, BVSc, PhD, unpublished observations, 1980).'"' Abnormalities included bone marrow hypoplasia, nephrosis, and skin lesions. Three of the mares had been supplemented with fo lic acid. Evidence from other species would suggest that supplementation with folic acid (a synthetic nonreduced form of folate) either does not prevent PYR-induced toxicity" or may even exacerbate it. 1" In light of these observations, the supplemental use of folic acid in SDZ / PYR-treated horses cannot be justified. Triazines

Over the past several years, two members of this group of compounds, diclazuril and toltrazuril, have been used in the United States and Canada to treat horses with EPM. These drugs have been shown to have broad-spectrum anticoccidial activity in many avian and mammalian species. Diclazuril has been evaluated for the treatment of encephalitic toxoplasmosis, cryptosporidiosis, and isosporiasis in acquired immunodeficiency syndrome patients.45· 5~· 75 These drugs are thought to target a protozoa! organelle (the "plastid" body) of members of the apicomplexan group that apparently is of cyanobacterial or algal origin. 52 The anti-S. ncurona activity of diclazuril has been established in a merozoite proliferation assay."" Approximately 95% of merozoite proliferation was inhibited by a concentration of 1 to 100 ng/mL of culture medium. Single oral doses of toltrazuril (10 mg/kg) and diclazuril (5.6 mg / kg) provided mean peak plasma concentrations of 4.5 µg/mL" and 1.1 µg / mL/·27 respectively, approximately 24 hours after administration. The estimated plasma half-lives were 55 hours for toltrazuril and 43 hours for diclaz uril. Daily administration was expected to provide steady-state concentrations of either drug after 10 days. A recent study reported steady-state plasma concentrations for diclazuril of 7 to 9 µg / mL and CSF concentrations of lOO to 250 ng / mL. 27 Serum concentrations at day 10 in horses given toltrazuril at a dosage of either 5 or 10 mg / kg were 20 or 26 µg / mL, with concentrations in CSF of 190 or 387 ng / mL, respectively (M. Furr, DVM, personal communication, December 1999). The formulations of these drugs adapted for clinical u se in horses are

418

MACKAY et al

Clinacox (0.5% diclazuril; Pharmacia-Upjohn, Orangeville, Ontario, Canada), a powdered feed additive used for control of coccidia in chickens, and Baycox (5% toltrazuril; Bayer, Toronto, Ontario, Canada), a suspension used as an anticoccidial agent in swine. Although these drugs are not approved for use in the United States, the US Food and Drug Administration's Center for Veterinary Medicine has allowed the importation of small amounts from Canada.''" The therapeutic efficacy of Clinacox was investigated in a nonmasked trial involving 74 EPM-affected horses. 7 Most of these horses had failed to respond to or had relapsed after standard sulfonamide / PYR therapy. Horses were given 500 g of Clinacox for 21 to 28 days. Of 63 horses that completed this course of treatment, 48 (75%) showed clinical improvement. Three horses relapsed after treatment, and 1 of 5 horses tested had become CSF immunoblot-negative. Baycox usually is used at a rate of either 5 or 10 mg of toltrazuril per kilogram per day for a 1- to 2-month period. Some veterinarians give a 250-mL bottle by stomach tube on two to five occasions at varying intervals (i.e., approximately 25 mg/kg per dose for a 500-kg horse). Although clinical efficacy of the drug has not been established for any of the dosage regimens used so far, based on clinical impressions, the response to treatment seems to be at least as good as that obtained with SDZ/PYR. When 5 horses were given Baycox at a dosage of 50 mg/kg/ d for 10 days, there was mild depression and anorexia, slight colic in 1 horse, but no alteration in a range of blood parameters (M. Furr, DVM, personal communication, January 2000). The efficacy of ponazuril, the sulfone metabolite of toltrazuril, has been evaluated in a multicenter study. This study was prompted by the fact that concentrations of 0.1 to 1.0 µg/ mL of this drug completely protected cell monolayers from destruction by S. neurona. 6 ' A total of 101 horses with presumptive EPM were involved in the study. Each animal was treated with ponaz uril for 28 days at either 5 or 10 mg/kg (T. Kennedy, PhD, personal communication, October 1999). Response to treatment was based on improvement of one grade or more in the ataxia score or reversion to immunoblot-negative CSF by 90 days after treatment. Some 71% of horses in the study showed a successful response with no significant effect of dose. In a parallel study, the mean CSF concentration of ponazuril in horses on 5 mg/kg/d was 150 to 180 ng/mL between days 7 and 28, falling to 20 ng / mL 7 days after discontinuing treatment (M. Furr, DVM, personal communication, February 2000). Nitazoxanide

Nitazoxanide (NTZ) is a 5-nitrothiazole benzamide compound w ith broad activity against protozoa, nematodes, cestodes, trematodes, and bacteria, including Helicobacter pylori. Several studies have been carried out to test its efficacy for the treatment of EPM in horses. After a single oral dose of 50 mg/ kg, maximal concentration (0.97 µg/mL) of the drug was attained w ithin 2.25 hours. 82 NTZ was not detected in CSF samples collected 4 hours after the first and seventh doses. In toxicity studies, anorexia, depression, and diarrhea with some deaths were observed in horses given two to eight times the treatment dosage. Necropsy findings were not remarkable. In view of the depression and anorexia that developed in some of the horses during a pilot study in which 50 mg/kg of NTZ had been given for 28 days, the dosage was halved for the first 5 days of a large multicenter study in which 95 horses were enrolled and 63 completed the study. By 85 days after the start of treatment, the clinical condition of 44 (70%) horses had improved, 7 (11'1/o) had made total recoveries, and

EQUINE PROTOZOAL MYELOENCEPHALITIS

419

8 (13%) had become CSF immunoblot-negative. Transient fever, depression, inappetance, swelling of the limbs, and diarrhea were mild side effects of treatment. Combinations of Antiprotozoal Drugs

Diclazuril and PYR used in combination to treat mice with experimentally induced toxoplasmosis had a synergistic effect on survival. 60 Whether or not such drug combinations would have a similar effect in horses is unknown; howevet; one of the authors (R.J.M.) routinely uses SDZ/PYR (6 months total treatment) and toltrazuril (2 months treatment) concurrently to treat cases of EPM. Data on the efficacy of this treatment approach are not currently available. Anti-Inflammatory Therapy

The use of anti-inflammatory drugs for the treatment of EPM has been reviewed previously.""· "5 In summary, nonsteroidal anti-inflammatory drugs such as flunixin meglumine are frequently given to moderately or severely affected horses during the first 3 to 7 days of antiprotozoal therapy. In the case of horses that are in danger of falling down or exhibit signs of brain involvement, the additional use of corticosteroids (0.1 mg/ kg of dexamethasone twice daily) and dimethyl sulfoxide (1 g/kg as a 10% solution intravenously or by nasogastric tube twice daily) for the first several days may control the inflammatory response and associated clinical signs and provide time for the antiprotozoal drugs to begin to work. Because the damaged CNS is susceptible to oxidant injury, it has become common practice to use pharmacologic doses of the antioxidant vitamin E (e.g., 20 IU/kg daily per os) throughout the period that horses are treated for EPM. Although vitamin E therapy may not significantly alter the course of recovery, it is considered unlikely to do any harm. Biologic Response Modifiers

Based on the assumption that horses that develop EPM may be immune compromised in some respect, immunomodulators have been included by some in treatment of the disease. The drugs used include levamisole (1 mg/ kg orally twice daily for the first 2 weeks of antiprotozoal therapy and for the first week of each month thereafter), killed Propionibacterium acnes (Eqstim; Neogen, Lansing, Ml), and mycobacterial wall extract (Equimune IV; Vetrepharm, London, Ontario, Canada). No study has been performed to date to evaluate the efficacy of any of these adjuvant treatments. PREVENTION

The prevention of EPM is difficult because of the apparent widespread distribution of the causative agents in many parts of the United States and the frequency of movement of horses within the country. Methods for effective control of this disease have not been delineated; however, it is prudent to attempt to eliminate known risk factors. Access of opossums and other wildlife or pests such as mice and rats to

420

MACKAY et al

feed and water should be eliminated. Cereal grains should be kept in rodentproof containers, and forages should also be protected from wildlife access by the use of enclosed facilities. Although it is not known whether birds play a role in the pathogenesis of EPM, it is probably advisable to restrict birds from gaining access to feed storage facilities or horse accommodations. It has been demonstrated that sporocysts ingested by birds may pass through the intestinal tract intact and remain viable. 10• 11 Insects such as flies and cockroaches also may transport S. neurona. Fatal pulmonary disease developed in psittacine birds fed cockroaches maintained on opossum feces that contained S. falcatula. 20 Although this finding suggests that insects may play a role in the transmission of S. neurona, further investigation is needed to determine whether insects may actually be involved in the life cycle of this parasite. Controlling the insect populations on farms may help to reduce the incidence of EPM. The case histories of EPM-affected horses frequently indicate recent adverse health events.88 Close monitoring is warranted of pregnant mares close to term and of horses that have recently experienced a major illness or injury or have been transported a considerable distance or under arduous conditions. Efforts are currently underway by Fort Dodge Animal Health (Fort Dodge, IA) to bring to market a crude vaccine made from lysates of whole cultured S. neurona merozoites. The difficulties experienced in developing other protozoa! vaccines28• 36• 57 raise concerns about the likely effectiveness of this product. Perhaps of more immediate value would be the use of anti-protozoa! drugs given according to a protocol that would allow initial infection and short-term immunity (metaphylaxis) but prevent spread to the CNS. Any of the therapeutic drugs that are thought to kill S. neurona (i.e., the triazines and NTZ) would be logical candidates for this intermittent preventative approach. SUMMARY

Recent advances in the understanding of the parasite life cycle, epidemiology, clinical signs, diagnosis, treatment, and prevention of EPM are reviewed. The NAHMS Equine '98 study and a controlled retrospective study from The Ohio State University College of Veterinary Medicine identified a number of risk factors associated with development of the disease. The national annual incidence of EPM was 1% or less depending on the primary use of the animals. Increased disease risk was associated with age (1-5 and > 13 years of age), season (lowest in winter months and increasing with ambient temperature), previous stressful events, the presence of opossums, the use of nonsurface water drinking systems, and failure to restrict wildlife access to feed. Horses that received treatment were 10 times more likely to improve, and those that improved were 50 times more likely to survive. A number of recent studies confirmed that horses can be experimentally infected with S. neurona; however, large numbers of sporocysts are apparently necessary to achieve infection, and clinical signs and abnormal CNS histology are only seen inconsistently. Results suggest that CNS infection and positive CSF immunoblot findings may be transient phenomena among naturally infected horses. Although immunosuppression may be involved in the development of EPM, some element of the immune response seems to be necessary for the development of clinical signs. Use of the standard immunoblot test for the detection of anti-S, neurona antibodies in CSF continues to provide the most useful adjunct to a detailed neurologic examination for the diagnosis of EPM. Test sensitivity and specificity were 89% in 295 horses euthanatized because of neurologic disease, of which 123 were

EQUINE PROTOZOAL MYELOENCEPHALITIS

421

confirmed cases of EPM. The PPV was 85%, and the NVP was 92°/c,. A number of promising new EPM treatments are under investigation. In addition to standard SDZ/PYR therapy, toltrazuril, ponazuril, diclazuril, and NTZ have shown promise as possible alternatives.

References 1. Alexander J, Scharton-Kersten TM, Yap G, et al: Mechanisms of innate resistance to

Toxop/as111a gondii infection. Philos Trans R Soc Lond B Biol Sci 352:1355- 1359, 1997 2. Andrews FM, Grans trom DE, Provenza M: Differentiation of neurologic d iseases in the horse by the use of albumin quotient and lgG index determinations. In Proceedings of the 41st Annual Convention of the American Association of Equine Practitioners, Lexing ton, 1995, pp 215-217 3. Andrews FM, Maddux JM, Faulk D: Total protein, albumin quotient, IgG, IgG index determinations for horse cerebrospinal fluid. Prog Vet Neurol 1:197- 204, 1990 4. Baszler TV, Long MT, McElwain TF, et al: Interferon-gamma and interleukin-12 mediate protection to acute Neospora ca11inu111 infection in BALB / c mice. Int J Parasitol 29:16351646, 1999 5. Bedford SJ, McDonnell SM: Measurements of reproductive function in stallions treated with trimethoprim-sulfamethoxazole and pyrimethamine. JAVMA 215:1317-1319, 1999 6. Bentz BG, Granstrom DE, Stamper S: Seroprevalence of antibodies to Sarcocystis neurona in horses residing in a county of southeaste rn Pennsylvania. JAVMA 210:517- 518, 1997 7. Bentz B, G ranstrom D, Tobin T, et al: Pre liminary repo rt on diclazuril and equine protozoa! myeloencephalitis. In Proceedings of the Eighth International Conference on Equine Infectious Diseases, Dubai, 1998, p 457 8. Blythe LL, Granstrom DE, Hansen DE, et al: Seroprevalence of antibodies to Sarcocystis neurona in horses residing in Oregon. JAVMA 210:525-527, 1997 9. Bogan JA, Galbraith A, Baxter P, et al: Effect of feeding on the fate of orally administered phenylbutazone, trimethoprim and sulfadiazine in the horse. Vet Rec 115:599600, 1984 10. Box E: Recovery of Sarcocystis sporocysts from feces after oral administration. /11 Proceedings of the Helminthological Society of Washing ton 50:350- 356, 1983 11. Box ED, Smith JH: The intermediate host spectrum in a Sarcocystis species of birds. J Parasitol 68:668-673, 1982 12. Boy MG, Galligan DT, Divers TJ: Protozoa! encephalomyelitis in horses: 82 cases (1972-1986). JAVMA 196:632-634, 1990 13. Brendemuehl JP, Waldridge BM, Bridges ER: Effects of sulfadiazine and pyrimethamine and concurrent folic acid supplementation on p regnancy and early emb ryonic loss rates in mares. In Proceedings of the 44th Annual Convention of the American Association of Equine Practitioners, Phoen ix, 1998, pp 142-143 14. Brown MP, Gronwall RR, Houston AE: Pharmacokinetics and body flu id and endometrial concentrations of ormetoprim-sulfadimethoxine in mares. Can J Vet Res 53:1216, 1989 15. Castles TR, Kintner LD, Lee CC: The effects of folic or folinic acid on the toxicitv of pyrimethamine in dogs. Toxicol Appl Pharmacol 20:447-459, 1971 " 16. Cavallito JC, N ichol CA, Brenckman WDJ, et al: Lipid-soluble inhibitors of dihydrofolate reductase. I. Kinetics, tissue distribution, and extent of me tabolism of pyrimethamine, metoprine, and etoprine in the rat, d og, and man. Drug Metab Dispos 6:329337, 1978 17. Cheadle MA, Lindsay DS, Rowe S, et al: Prevalence of antibodies to Neospora ca11i11um in dogs. Int J Parasitol 29:1537- 1543, 1999 18. Chung MK, Han SS, Roh JK: Synergistic embryotoxicity of combi nation pyrimethamine and folic acid in rats. Reprod Toxicol 7:463-468, 1993 19. Clarke CR, MacAllister CG, Burrows GE, et al: Pharmacokinetics, penetration into cerebrospinal fluid, and hematologic effects after multiple oral administrations of pyrimethamine to horses. Am J Ve t Res 53:2296-2299, 1992

422

MACKAY et al

20. Clubb SL, Frenkel JK: Sarcocystis falca/11111 of opossums: Transmission by cockroaches with fatal pulmonary disease in psittacine birds. J Parasitol 78:11 6-124, 1992 21. Cusick PK, Sells DM, Hamilton DP, e t al: Toxoplasmosis in two horses. JAVMA 164:77-80, 1974 22. Cutler TJ, MacKay RJ, Ginn PE, ct al: Arc Snrcocystis 11e11ro1111 and Sarcocystis fnlcn/11/11 synonymous? A ho rse infecti on cha llenge. J Parasitol 85:301- 305, 1999 23. Cutler TJ, Tanhauser SM, MacKay RJ, c t al: Molecul ar characterization of Sarcocystis spp. sporocysts shed by wild opossums [abstract]. J Vet Intern Med 13:276, 1999 24. Daft B, Ardans A, Barr B, et al: A compa rison of ante mortem and post mortem immunoblot testing for Sarcocystis 11eiiro1111 with post mortem examina tion of neurologically normal and abnormal California horses [abstract]. /11 Abstracts of the 40th Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians, Louisville, 2000, p 86 25. Daft BM, Barr BC, Collins N, et al: Neosporn e ncephalomyelitis and po lyradiculoneuritis in an aged mare w ith Cushing's d isease. Equ ine Vet J 29:240- 243, 1997 26. Dame JB, MacKay RJ, Yowell CA, et al: Snrcocystis falca/11/11 from passerine and psittacine birds: Synonym y with Sarcocystis 11e11nm11, agent of equine protozoa I mycloenccphalitis. J Parasitol 81:930-935, 1995 27. Dirikolu L, Lehner F, Nattrass C, et al: Diclaz uril in the horse: Its identification and detection and preliminary pharmacokinetics. J Vet Pharmacol Thcr 22:374-379, 1999 28. Dubey J: Strategies to reduce transmission of Toxvplas111t1 go11dii to animals and humans. Vet Parasitol 64:65-70, 1996 29. Dubey JE Lindsay DS: Isolation in immunodeficien t mice of S11rcocystis 11e1no1111 from opossum (Oide/phis virginiann) faeces, and its d iffe rentia tion from Sarcocystis falcatula. Int J Parasitol 28:1823- 1828, 1998 30. Dubey JP, Porterfield ML: Neospora cn11i1111111 (Apicomplexa) in an aborted equine fetus. J Parasitol 76:732-734, 1990 31. Dubey JP, Kerber CE, Granstrom DE: Serologi c prevalence o f Sarcon;stis ne11ro11a, Toxoplasma gondii, and Neospora cn11i1111111 in horses in Brazil. JAVMA 215:970- 972, 1999 32. Dubey JP, Speer CA, Fayer R: General biology. In Sarcocystosis of An ima ls a nd Man. Boca Raton, CRC Press, 1989, pp 1- 91 33. Dubey JP, Romand S, Thulliez P, et al: Preva lence of antibod ies to Neosporn c1mi1111111 in horses in North Ame rica. J Parasitol 85:968- 969, 1999 34. Dubey JP, Venturini MC, Venturini L, e t al: Preva lence o f antibod ies to Sarcocystis neurona, Toxoplnsma gondii and Neosporn cn11i1111111 in horses from Argentina. Vet Parasitol 86:59-62, 1999 35. Dunigan CE, Oglesbee MJ, Podell M, et al: Seizure activity associated w ith equine protozoa] m yeloencephalitis. Prog Vet Neurol 6:50-54, 1995 36. Emery DL: Vaccination against worm parasites of animals. Vet Parasitol 64:31--45, 1996 37. Fayer R, Mayhew JG, Baird JD, et al: Epidemio logy of equine protozoa! m yeloencephalitis in North America based on histologicall y confirmed cases: A report. J Vet Inte rn Med 4:54-57, 1990 38. Fenger CK: Equine protozoa! mye loencepha litis: Effi cacy and safe ty of treatment with a pyrimethamine and sulphadiazinc combination. In Proceedings o f the Eigh th International Conference on Equine Infectious Diseases, Duba i, 1998, pp 458--459 39. Fenger CK: Py rimetharnine-sulfonamide dosage and treatment durati on recommendations for equine protozoa! mycloencephalitis: Findings from a re trospective study. In Proceedings of the 17th Annual Veterinary Med ical Forum of the Am erican College of Veterinary Internal Medicine, Lake Buena Vista, 1997, pp 484--485 40. Fenger CK: Treatment of equ ine protozoa! myelocncephalitis. Compe nd Conlin Educ Pract Vet 20:1154-1157, 1998 41. Fenger CK, Granstrom DE, Langeme ier JL, e t al: Epizootic of equine protozoa! myeloencephalitis on a farm. JAVMA 210:923- 927, 1997 42. Fenger CK, Granstrom DE, Gajadhar AA, et al: Experimenta l induction o f equine protozoa] m yeloencephalitis in horses using Sarcocystis sp. sporocysts from the opossum (Didelphis virgininna). Vet Parasitol 68:199-213, 1997 43. Fenger CK, Granstrom DE, Langemeier JL, et al: Identification of opossu ms (Oiddphis

EQUINE PROTOZOAL MYELOENCEPHAL!T!S

423

virginiana) as the putative definitive host of Sarcocystis neurona. J Parasitol 81:916-919, 1995 44. Fenger CK, Granstrom DE, Langemeier JL, et al: Phylogenetic relationship of Sarcocystis neuro1rn to other members of the family Sarcocystidae based on small subunit ribosomal RNA gene sequence. J Parasitol 80:96h- 975, 1994 45. Fung HB, Kirschenbaum HL: Trea tment regimens for patients with toxoplasmic encephalitis. Clin Ther 18:1037- 1056, 1996 46. Gardener AL: Virginia opossum Didelphis virginiana. In Chapman JA, Feldhamer GA (eds): Wild Mammals of North America: Biology, Management, Economics. Baltimore, The Johns Hopkins University Press, 1982, pp 3- 36 47. Granstrom DE: Equine protozoa! m yeloencephalitis: Parasite biology, experimental disease, and laboratory diagnosis. In Proceed ings of the International Equine Neurology Conference, Ithaca, 1997, pp 4- 6 48. Granstrom DE, Alvarez OJ, Dubey JP, et al: Equine protozoa! myelitis in Panamanian horses and isolation of Sarcocystis neurona. J Parasitol 78:909-912, 1992 49. Granstrom DE, Dubey JP, Davis SW, et al: Equine protozoa! myeloencephalitis: Antigen analysis of cultured Snrcocystis ne11ro1111 merozoites. J Vet Diagn In vest 5:88- 90, 1993 50. Gray ML, Harmon BG, Sales L, et al: Visceral neosporosis in a 10-year old horse. J Vet Diagn Invest 8:130-133, 1996 51. Green SL, Mayhew IC, Brown MP, et al: Concentrations of trirnethoprim and sulfamethoxazole in cerebrospinal fluid and serum in mares with and without a dimethyl sulfoxide pretreatment. Can J Vet Res 54:215-222, 1990 52. Hackstein JH, Mackenstedt U, Mehlhorn H, et al: Parasitic apicomplexans harbor a chl orophyll a-D1 complex, the potential target for therapeutic triazines. Parasitol Res 81 :207-216, 1995 53. Hamir AN, Moser C, Galligan OT, et al: lmmunohistochemical study to demonstrate Sarcocystis neurmw in equine protozoa] myeloencephalitis. J Vet Diagn Invest 5:418422, 1993 54. Harnir AN, Tornquist SJ, Cerros TC, et al: Neos pora ca11inum-associated equine protozoa! myeloencephalitis. Vet Parasitol 79:269-274, 1998 55. Hillis OM, Dixon MT: Ribosomal DNA: Molecu lar evolution and phylogenetic inference. Q Rev Biol 66:412-452, 1991 56. Jackson T, Kendrick JW: Paralysis of horses associated with equ ine herpesvirus 1 infection. JAVMA 158:1 351- 1357, 1971 57. Kane M: Malaria, where now? Lancet 348:695-696, 1996 58. Limson-Pobre RN, Merrick S, Gruen D, et al: Use of diclazuril for the treatment of isosporiasis in patients with AIDS [letter]. Clin Infect Dis 20:201-202, 1995 59. Lindsay OS, Dubey JP: Determination of the activity of pyrimethamine, trimethoprirn, sulfonamides, and combinations of pyrimethamine and sulfonamides against Sarcoc1;stis neurona in ceJJ cultures. Vet Parasitol 82:205-210, 1999 60. Lindsay OS, Dubey JP: Determination of the activity of diclazuril against Sarcocystis 11eurona and Sarcocystis /11/catula in cell cultures. J Parasitol 86:164- 166, 2000 61. Lindsay DS, Dubey JP, Kennedy TJ: Activity of Ponaz uril against Sarcocystis neurona and other apicomplexans [abstract]. In Proceedings of the Annual Meeting of the American Association of Veterinary Parasitologists, New Orleans, 1999, p 47 h2. Lindsay OS, Rippey NS, Blagburn BL: Treatment of acute Toxoplas111a gondii infections in mice with diclazuril or a combination of diclazuril and p yrimethamine. J Parasitol 81:315-318, 1995 63. Lindsay OS, Steinberg H, Dubielzig RR, et al: Central nervous system neosporosis in a foal. J Vet Diagn Invest 8:507-510, 1996 64. Lindsay OS, Dubey JP, Horton KM, et a l: Development of Sarcocystis falcatula in cell cultures demonstrates that it is differen t from Sarcocystis neurona. Parasitology 118:227- 233, 1999 65. MacKay RJ: Equine protozoa] myeloencephalitis. Vet Clin North Arn Equine Pract 13:79-96, 1997 66. MacKay RJ, Davis SW, Dubey JP: Equine protozoa] myeloencep halitis. Compend Contin Educ Pract Vet 14:1359-1366, 1992

424

M ACKAY et al

67. Marsh AE, Barr BC, Tell L, et al: Comparison of the inte rnal transcribed spacer, ITS-1, from Sarcocystis falcatula isolates and Sarcocyslis 11 e11ro1w . J Pa rasitol 85:750-757, 1999 68. Marsh AE, Howe DK, Wang C, et al: Differentiation of Neospom lwghesi from Neospora caninu m based on their immunodominan t s urface antigen, SAGI and SRS2. Int J Parasitol 29:1 575- 1582, 1999 69. Marsh AE, Barr BC, Lakritz J, et al: Experi mental in fection of nude mice as a mod e l for Sarcocystis neurona-associated ence phalitis. Parasitol Res 83:706-711, 1997 70. Marsh AE, Barr BC, Madigan J, et al: Neosporosis as a cause of equine p rotozoa) myeloencephalitis. JAVMA 209:1907- 1913, 1996 71. Marsh AE, Barr BC, Madi gan J, et al: Sequence analysis and polym erase chain reaction amplification of sm all subunit ribosomal DNA from Sarcocystis neuronn. Am J Vet Res 57:975- 981, 1996 72. Mayhew, JG, d e Lahunta A: Neuropatho logy. Cornell Vet 68(suppl 6):106- 147, 1978 73. Mayhew IC, de Lahunta A, Whitlock RH, et al: Equine p rotozoa! m yeloencephalitis. /11 Proceed ings of the 22nd Annu al Convention of the American Association o f Equine Practitione rs, Dallas, 1976, pp 107-114 74. McAllister MM, Dubey JP, Lindsay OS, et al: Dogs are de finiti ve hosts of Neospom cani11u111. Int J Parasitol 28:1473-1478, 1998 75. Meniche tti F, Moretti MV, Marroni M, e t a l: Diclazuril for cryptosporidiosis in AIDS [letter]. Am J Med 90:271- 272, 1991 76. Miller MM, Sweeney CR, Russell GE, e t al: Effects of blood contamination of cerebrospinal fluid on Western blot analysis for d etection of antibodies against Sarcocystis neurona and on albumin quo tien t and im mu noglobulin C index in horses. JAVMA 215:67-71, 1999 77. Mohammed HO, Swaney JR: Facto rs associated w ith the ris k o f equine p ro tozoa] rnyeloencephalitis. 1,i Proceedings of the Eighth International Conference on Equine Infectious Diseases, Du bai, 1998, pp 448-449 78. National Animal Health Monitoring System Equine '98: Equine protozoa! myeloencephalitis in the US. Fort Collins, Veterinary Services, United States Department of Agriculture Animal and Plant Health Inspection Service, in press 79. Nixon AJ, Stashak TS, Ingram JT: Diagnosis of cervical vertebral malformation in the horse. In Proceedings of the 28th Annu al Conven tion of the American Association o f Equine Practitioners, Atl anta, 1982, pp 253-256 80. Odening K: The present state o f s pecies-systemati cs in S11rcocystis Lankester, 1882 (Protista, Sporozoa, Coccidie ). Systematic Parasitology 41:209-233, 1998 81. Ostlund EN: The equine herpesviruses. Vet Clin North Am Equ ine Pract 9:283-294, 1993 82. Palma K: Effi cacy of nitazoxanide for the treatment of equine protozoa! m yeloencephalitis. In Convention Notes from the 136th Annual Convention of the American Veterinary Med ical Associati on, New Orleans, 1999, pp 197- 198 83. Prickett ME: Equine spinal a taxia. In Proceedi ngs of the 14th Annu al Convention of the American Association of Eq uine Practiti oners, Philad elphia, 1968, pp 147-148 84. Reed SM, Granstrom DE: Equine p rotozoa! enccpha lom yelitis. In P roceedings of the 13th Annual Veterinary Medical Forum of the American College of Veteri nary Internal Medicine, San Francisco, 1993, pp 591 -592 85. Rooney JR, Prickett ME, Delaney FM, et al: Focal myelitis-encep halitis in horses. Cornell Vet 60:494-501, 1970 86. Rossano MG, M ansfield LS, Kaneene AJ, et al: Improvement of Western blot tes t specificity for detecting equine serum antibodies to S11rcocystis neuronn. J Vet Diagn Invest 12:28- 32, 2000 87. Saville WJ, Reed SM, G ranstrom DE, et al: Serop revale nce of a ntibod ies to Sarcocystis neurona in horses residing in Ohio. JAVMA 210:519-524, 1997 88. Saville WJA, Reed SM, Morley PS, et al: Analysis of risk factors for the development of equine protozoa) myeloencephalitis in horses. JAVMA 217:1174-1180, 2000 89. Saville WJA, Morley PS, Reed SM, et al: Evaluation of risk factors associated with improvement and survival of horses with equine protozoa[ m yeloencephalitis. JAVMA 217:1181-1185, 2000 90. Seilrnan ES, Sweeney CR, Habecker P: Correlation o f ante-mortem Sarcocystis neurona

EQUINE PROTOZO A!" MYELOENCEl'HALITIS

91. 92. 93. 94.

95. 96.

425

testing with post-mortem findings. /11 Proceedings of the 17th Annual Veterin ,1ry Medical Forum of the American College of Veterinary Internal Medicine, Lake Buena Vista, 1997, p 652 Shane B: Fola te chem istry and me tabolism. /11 Bailey LB (ed): Fola te in Health and Disease. New York, Marcel Dekker, pp 1-22, 1995 Tanhauser SM, Yowell CA, C utle r TJ, et al: Multiple DNA markers differentiate Snrcocystis 11e11ru1rn and Si11n1cystis Jiilrn/11/n. J Pa rasitol 85:221- 228, 1999 Tillotson K, McCue l'M, Cranstrom DE, et al: Seropreva lence of antibodies to Sarcoc11stis 11e11ro1111 in horses residing in northe rn Colorado. J Equine Vet Sci 19:122-126, 1999 Toribio RE, Bain FT, Mrad DR, et a l: Congenita l defects in newborn foals of ma res trea ted for equine protozoa ] m yeloe nccphalitis during pregnancy. JAVMA 212:697701 , 1998 Traub-Darga tz JL, Schlipf JWJ, G r<1nstrom DE, et a l: Multi foca l myositis associated with Sarcosystis sp in a horse. JAVMA 205:1 574- 1576, 1994 Woerner TD: Personal import policy. Compend Contin Educ Prnct Vet 20:1265, 1998

Address repri11t request,; to Robert J. MacKay, BVSc, PhD 2015 SW 16th Aven ue, Room VH-136 PO Box 100136 Large Anima l Cl inical Sciences College of Veterinary Med ici ne University of Florida Gainesvi lle, FL 32610-0136