Indirect fluorescent antibody testing of cerebrospinal fluid for diagnosis of equine protozoal myeloencephalitis

OBJECTIVE: To assess the use of CSF testing with an indirect fluorescent antibody test (IFAT) for diagnosis of equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona. SAMPLE POPULATION: Test results of 428 serum and 355 CSF samples from 182 naturally exposed, experimentally infected, or vaccinated horses. PROCEDURE: EPM was diagnosed on the basis of histologic examination of the CNS. Probability distributions were fitted to serum IFAT results in the EPM+ and EPM-horses, and correlation between serum and CSF results was modeled. Pairs of serum-CSF titers were generated by simulation, and titer-specific likelihood ratios and post-test probabilities of EPM at various pretest probability values were estimated. Post-test probabilities were compared for use of a serum-CSF test combination, a serum test only, and a CSF test only. RESULTS: Post-test probabilities of EPM increased as IFAT serum and CSF titers increased. Post-test probability differences for use of a serum-CSF combination and a serum test only were < or = 19% in 95% of simulations. The largest increases occurred when serum titers were from 40 to 160 and pre-test probabilities were from 5% to 60%. In all simulations, the difference between pre- and post-test probabilities was greater for a CSF test only, compared with a serum test only. CONCLUSIONS AND CLINICAL RELEVANCE: CSF testing after a serum test has limited usefulness in the diagnosis of EPM. A CSF test alone might be used when CSF is required for other procedures. Ruling out other causes of neurologic disease reduces the necessity of additional EPM testing.     

Transforming growth factor beta concentrations and interferon gamma responses in cerebrospinal fluid of horses with equine protozoal myeloencephalitis.

The following experiment was performed to test the hypothesis that transforming growth factor beta (TGF-beta) concentration varies in the cerebrospinal fluid and serum of horses with EPM and to determine if cerebrospinal fluid (CSF) alters the interferon-gamma (IFN-gamma) rersponse of equine peripheral blood mononuclear cells (PBMCs). The concentration of transforming growth factor-beta (TGF-beta2) was investigated in the serum and cerebrospinal fluid (CSF) of 18 horses (9 normal, 9 affected with equine protozoal myeloencephalitis [EPM]). The TGF-beta2 assay was validated in a group of 6 normal horses. Intra-assay variability was 4.7%, and interassay variability was 10.7%. The slope of the curve of the unknown samples of various volumes demonstrated parallelism with a curve developed using equal volumes of assay kit standard. Assay of normal and EPM-affected horses found that TGF-beta2 was present in both the serum and CSF of all animals. However, the concentration of TGF-beta2 in the CSF was less (P = 0.03) in EPM-affected horses (144 pg/ml) than in normal horses (256 pg/ml). In addition, the effect of CSF from normal and EPM-affected horses on the production of interferon-gamma (IFN-gamma) by PHA-P stimulated PBMCs from normal horses was investigated using a bioassay. It was found that CSF from normal and EPM-affected horses enhanced IFN-gamma activity from PHA-P stimulated peripheral blood mononuclear cells (P < or = 0.05); however, the response to CSF from EPM-affected horses was no different than the response to CSF from normal horses. Treatment of cells with anti-TGF-beta2 monoclonal antibodies slightly increased the response when co-incubated with CSF from normal horses, and slightly decreased it when co-incubated with CSF from EPM-affected horses. These differences, however, did not achieve statistical significance (P > 0.05). Results of this study indicated that production of TGF-beta2 is altered in horses with EPM, and that CSF appears to contain substances which alter the inflammatory reaction to plant lectins. These findings confirm the immunomodulatory properties of CSF and suggest new techniques for future research regarding the pathophysiology of EPM.     

Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis.

A serum indirect fluorescent antibody test (IFAT) was compared with a Western blot (WB) and a modified Western blot (mWB) for diagnosis of equine protozoal myeloencephalitis (EPM). Using receiver-operating characteristic (ROC) analysis, the area under the curve of the IFAT was greater than the areaunder the curves of the WB and the mWB (P = 0.025 and P = 0.044, respectively). There was no statistically significant difference between the areas under the curves of the WBs (P > 0.05). On the basis of an arbitrarily chosen cut-off titer for a positive test result of 1:80 for the IFAT and interpreting weak positive WB results as positive test results, the sensitivities and 95% confidence intervals (CI) of all 3 tests were identical and equal to 88.9% (51.8-99.7%). The specificities and 95% CIs of the IFAT, WB, and mWB test were 100% (91-100%), 87.2% (72.6-95.7%), and 69.2% (52.4-83%), respectively. The overall accuracy of the IFAT was shown to be better than that of the WBs and, therefore, the test has potential for use in the diagnosis of EPM caused by Sarcocystis neurona.     

Antibody coefficients for the diagnosis of equine protozoal myeloencephalitis

Background: Diagnosis of equine protozoal myeloencephalitis (EPM) remains a challenge for equine practitioners. Current utilized methods have inadequate sensitivity and specificity, because of a high number of false positive results. Hypothesis/Objective: Evaluation of antibody indices to Sarcocystis neurona should provide high sensitivity and specificity for diagnosis of EPM. Animals: Archived samples from 29 clinical patients. Methods: Archived serum and cerebrospinal fluid (CSF) samples from clinical patients with either EPM (14) or cervical vertebral compressive myelopathy (CVM) (15) were examined and tested for anti‐S. neurona antibodies by the SnSAG2 ELISA. The results were used to calculate the antibody index (AI) and C‐value. Sensitivity and specificity were calculated, and the AI, C‐value, immunoglobulin G (IgG) concentrations, and anti‐S. neurona titers compared. In addition, negative CSF was spiked in varying concentrations with blood from a horse with a high anti‐S. neurona titer, and the tests repeated. Results: Results demonstrated that the IgG concentration, anti‐S. neurona titer, AI, and C‐value were significantly higher (P < .05) in horses with EPM than in those with CVM. Sensitivity and specificity of the AI was 71 and 100%, respectively, and that of the C‐value was 86 and 100%, respectively. In addition, the AI and C‐value from the samples spiked with S. neurona positive blood remained below 1 (eg, negative) in CSF with a red blood cell (RBC) count up to 10⁵ RBC/μL. Conclusions/Clinical Importance: Results of the study demonstrate the value of calculating the AI and C‐value in the diagnosis of EPM in horses. In addition, the test is robust in the presence of blood contamination.     

Urinary incontinence and incoordination in three horses associated with equine protozoal myeloencephalitis

Urinary incontinence, weakness and ataxia associated with equine protozoal myeloencephalitis (EPM) was diagnosed in three horses. Rectal examination of all horses revealed distention of the urinary bladder. Urine was expressed when manual pressure was applied to the bladder of each horse during rectal examination. The anal reflex and tone of the anus and tail were normal in all horses. Two horses had bacterial cystitis associated with Enterococcus sp. All horses were treated with pyrimethamine and a sulfonamide for EPM, but there was a variable response to treatment.     

Analysis of risk factors for the development of equine protozoal myeloencephalitis in horses

OBJECTIVE: To investigate risk factors for development of equine protozoal myeloencephalitis (EPM) in horses. DESIGN: Case-control study. ANIMALS: 251 horses admitted to The Ohio State University Veterinary Teaching Hospital from 1992 to 1995. PROCEDURE: On the basis of clinical signs of neurologic disease and detection of antibody to Sarcocystis neurona or S neurona DNA in cerebrospinal fluid, a diagnosis of EPM was made for 251 horses. Two contemporaneous series of control horses were selected from horses admitted to the hospital. One control series (n = 225) consisted of horses with diseases of the neurologic system other than EPM (neurologic control horses), and the other consisted of 251 horses admitted for reasons other than nervous system diseases (nonneurologic control horses). Data were obtained from hospital records and telephone conversations. Risk factors associated with disease status were analyzed, using multivariable logistic regression. RESULTS: Horses ranged from 1 day to 30 years old (mean +/- SD, 5.7 +/- 5.2 years). Risk factors associated with an increased risk of developing EPM included age, season of admission, prior diagnosis of EPM on the premises, opossums on premises, health events prior to admission, and racing or showing as a primary use. Factors associated with a reduced risk of developing EPM included protection of feed from wildlife and proximity of a creek or river to the premises where the horse resided. CONCLUSIONS AND CLINICAL RELEVANCE: Development of EPM was associated with a number of management-related factors that can be altered to decrease the risk for the disease.     

Immune response to Sarcocystis neurona infection in naturally infected horses with equine protozoal myeloencephalitis

Equine protozoal myeloencephalitis (EPM) is one of the most common neurologic diseases of horses in the United States. The primary etiologic agent is Sarcocystis neurona. Currently, there is limited knowledge regarding the protective or pathophysiologic immune response to S. neurona infection or the subsequent development of EPM. The objectives of this study were to determine whether S. neurona infected horses with clinical signs of EPM had altered or suppressed immune responses compared to neurologically normal horses and if blood sample storage would influence these findings. Twenty clinically normal horses and 22 horses with EPM, diagnosed by the presence of S. neurona specific antibodies in the serum and/or cerebrospinal (CSF) and clinical signs, were evaluated for differences in the immune cell subsets and function. Our results demonstrated that naturally infected horses had significantly (P<0.05) higher percentages of CD4 T-lymphocytes and neutrophils (PMN) in separated peripheral blood leukocytes than clinically normal horses. Leukocytes from naturally infected EPM horses had significantly lower proliferation responses, as measured by thymidine incorporation, to a non-antigen specific mitogen than did clinically normal horses (P<0.05). Currently, studies are in progress to determine the role of CD4 T cells in disease and protection against S. neurona in horses, as well as to determine the mechanism associated with suppressed in vitro proliferation responses. Finally, overnight storage of blood samples appears to alter T lymphocyte phenotypes and viability among leukocytes.     

Evaluation of risk factors associated with clinical improvement and survival of horses with equine protozoal myeloencephalitis

OBJECTIVE: To investigate risk factors for use in predicting clinical improvement and survival of horses with equine protozoal myeloencephalitis (EPM). DESIGN: Longitudinal epidemiologic study. ANIMALS: 251 horses with EPM. PROCEDURE: Between 1992 and 1995, 251 horses with EPM were admitted to our facility. A diagnosis of EPM was made on the basis of neurologic abnormalities and detection of antibody to Sarcocystis neurona or S neurona DNA in CSF. Data were obtained from hospital records and through telephone follow-up interviews. Factors associated with clinical improvement and survival were analyzed, using multivariable logistic regression. RESULTS: The likelihood of clinical improvement after diagnosis of EPM was lower in horses used for breeding and pleasure activities. Treatment for EPM increased the probability that a horse would have clinical improvement. The likelihood of survival among horses with EPM was lower among horses with more severe clinical signs and higher among horses that improved after EPM was diagnosed. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of horses with EPM is indicated in most situations; however, severity of clinical signs should be taken into consideration when making treatment decisions. Response to treatment is an important indicator of survival.     

New therapeutic approaches for equine protozoal myeloencephalitis: pharmacokinetics of diclazuril sodium salts in horses

Diclazuril is a triazine-based antiprotozoal agent which may have clinical application in the treatment of equine protozoal myeloencephalomyelitis (EPM). In this study, the use of the sodium salt diclazuril to increase the apparent bioavailability of diclazuril for the treatment and prophylaxis of EPM and various other Apicomplexan mediated diseases is described. In this study, diclazuril sodium salt was synthesized and administered to horses as diclazuril sodium salt formulations. The absorption, distribution, and clearance of diclazuril sodium salt in the horse are described. Diclazuril was rapidly absorbed, with peak plasma concentrations occurring at 8-24 hours following an oral mucosal administration of diclazuril sodium salt. The mean oral bioavailability of diclazuril as Clinacox was 9.5% relative to oral mucosal administration of diclazuril sodium salt. Additionally, diclazuril in DMSO administered orally was 50% less bioavailable than diclazuril sodium salt following an oral mucosal administration. It was also shown that diclazuril sodium salt has the potential to be used as a feed additive for the treatment and prophylaxis of EPM and various other Apicomplexan mediated diseases.     

Utilization of stress in the development of an equine model for equine protozoal myeloencephalitis

Neurologic disease in horses caused by Sarcocystis neurona is difficult to diagnose, treat, or prevent, due to the lack of knowledge about the pathogenesis of the disease. This in turn is confounded by the lack of a reliable equine model of equine protozoal myeloencephalitis (EPM). Epidemiologic studies have implicated stress as a risk factor for this disease, thus, the role of transport stress was evaluated for incorporation into an equine model for EPM. Sporocysts from feral opossums were bioassayed in interferon-gamma gene knockout (KO) mice to determine minimum number of viable S. neurona sporocysts in the inoculum. A minimum of 80,000 viable S. neurona sporocysts were fed to each of the nine horses. A total of 12 S. neurona antibody negative horses were divided into four groups (1-4). Three horses (group 1) were fed sporocysts on the day of arrival at the study site, three horses were fed sporocysts 14 days after acclimatization (group 2), three horses were given sporocysts and dexamethasone 14 days after acclimatization (group 3) and three horses were controls (group 4). All horses fed sporocysts in the study developed antibodies to S. neurona in serum and cerebrospinal fluid (CSF) and developed clinical signs of neurologic disease. The most severe clinical signs were in horses in group 1 subjected to transport stress. The least severe neurologic signs were in horses treated with dexamethasone (group 3). Clinical signs improved in four horses from two treatment groups by the time of euthanasia (group 1, day 44; group 3, day 47). Post-mortem examinations, and tissues that were collected for light microscopy, immunohistochemistry, tissue cultures, and bioassay in KO mice, revealed no direct evidence of S. neurona infection. However, there were lesions compatible with S. neurona infection in horses. The results of this investigation suggest that stress can play a role in the pathogenesis of EPM. There is also evidence to suggest that horses in nature may clear the organism routinely, which may explain the relatively high number of normal horses with CSF antibodies to S. neurona compared to the prevalence of EPM.     

Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (P<0.001, Fisher's exact test). The S. cruzi antibody-blocked Western blot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.     

Risk factors for owner-reported occurrence of equine protozoal myeloencephalitis in the US equine population

BACKGROUND: Equine protozoal myeloencephalitis (EPM) is a serious and often fatal neurologic disease of horses, but few studies have investigated risk factors. OBJECTIVES: To evaluate operation- and individual-level factors associated with likelihood of the occurrence of EPM. ANIMALS: Data were collected as part of a study of the US equine industry from 1,178 operations representing 83.9% of horses and 51.6% of operations with > or =3 horses in 28 states. METHODS: Probability-based sampling was used to enroll representative operations in a cross-sectional study. Interviews were conducted to collect information regarding health and management of horses. A nested case-control study was used to investigate risk factors among individual horses. Interview data were combined with climate data, human population density, and opossum regional ecology categories. Data were analyzed using logistic regression to identify risk factors for the occurrence of EPM. RESULTS: Owners reported that 95% of EPM cases included in this study were diagnosed by veterinarians. Variables associated with EPM occurrence on premises included opossum regional ecology, reported exposure to small wildlife, climate, terrain, housing, choice of bedding material, method of storing feeds, equine stocking density, and primary use of horses. Among individual horses, age was most strongly associated with disease risk. Associations also were identified with sex, breed, primary use, and participation in competitions. CONCLUSIONS AND CLINICAL IMPORTANCE: Because the risk of EPM occurrence on operations is closely tied to factors that impact exposure to opossums, their feces, and their environment, controlling these exposures may be important in preventing the occurrence of EPM.     

A western blot test for the serological diagnosis of equine infectious anemia

After electrophoretic separation in SDS-PAGE structural proteins of the virus of Equine Infectious Anemia (EIA) were easily blotted by the semi-dry-blotting method onto nitrocellulose filters. Strips of these filters were used for antibody demonstration, and positive reactions thereof were intensified by a biotin-avidin-peroxidase system. Sensitivity of this system was so high as to allow readable interpretation of bands up to the dilution of 1:6,400 of a strongly positive serum. Frequently this procedure allowed to make a firm diagnostic Western-Blot diagnosis on far weaker equine sera. Interpretation of results proved, however, difficult with sera, which formed one single band only. This observation, although of weak grade, had to be made in some 5% of sera stemming from horses with a certainly negative history of EIA. Consequently, we conclude, that a policy followed in the serodiagnostic Western-Blot of human AIDS should also be adopted for the interpretation of the EIA Western-Blot, namely to declare as positive merely horse sera which evidence more than one single band, whereof at least one band should represent a viral glycoprotein.     

Assessing the agreement of Western blot test results for paired serum and cerebrospinal fluid samples from horses tested for antibodies to Sarcocystis neuronaf

Equine protozoal myeloencephalitis (EPM) is a neurological disease of equids that is caused by infection of the central nervous system with Sarcocystis neurona. Veterinarians diagnose EPM by performing a neurological examination and by ordering Western blot tests for antibodies to S. neurona in the blood and/or cerebrospinal fluid (CSF). The negative predictive value of the Western blot test is generally accepted to be high for both serum and CSF. If the agreement between serum and CSF test results is strong, serum tests could be used to substitute for CSF tests in some cases. The purpose of this study was to assess the agreement of the results of 181 paired serum and CSF Western blot antibody tests on equine samples submitted to the Michigan State University Animal Health Diagnostic Laboratory. The agreement of the paired serum and CSF results was assessed for three possible test outcomes--negative, positive or suspect. An additional analysis was performed in which samples reported as suspect were reclassified as negative. The kappa statistic for negative, positive and suspect samples was 0.469. The kappa statistic for the analysis in which the suspect results were reclassified as negative was 0.474. In addition, 29% (33/112) CSF samples from seropositive horses were negative. Our results demonstrate that the level of agreement is only moderate in diagnostic samples. This supports the practice of testing CSF of seropositive horses suspected of having EPM.     

Neurologic abnormalities and cerebrospinal fluid changes in horses administered fumonisin B1 intravenously

The objective of this experiment was to characterize a dose-dependent toxic effect of fumonisin B1 (FB1) and to document initial neurologic signs, clinical progression, and terminal cerebrospinal fluid (CSF) changes in horses administered FB1 IV. Seventeen healthy horses were administered 0.00 (n = 4), 0.01 (n = 3), 0.05 (n = 3), 0.10 (n = 3), or 0.20 mg (n = 4) of purified FB1 IV q24h. When neurologic abnormalities observed by a masked observer became severe, atlanto-occipital CSF taps were performed and CSF pressure, cell count, cytology, protein, albumin and glucose concentrations, and creatine kinase activity were measured. Changes in CSF and number of days to 1st observation of neurologic abnormalities were compared between doses by ANOVA, with the level of significance set at P < .05. Control horses and low-dose horses (0.01 mg/kg) remained neurologically normal. In higher dose FB1-treated horses (n = 10), initial clinical signs (days 4-10) included hindlimb ataxia, delayed forelimb placing, and decreased tongue tone and movement. Hindlimb and trunkal ataxia, depression, hyperesthesia, and intermittent dementia gradually became apparent. When data from all horses with neurologic abnormalities were pooled (0.05-0.20 mg/kg FB1), mild clinical signs (mean day 6.3) occurred significantly earlier than did more severe (mean day 8.9) clinical signs (P = .009). Neurologic horses had high CSF protein, albumin, and IgG concentrations and increased albumin quotients (P < .05). It was concluded that FB1-induced neurologic and CSF changes in a dose-dependent manner, with a no-observable-limit of 0.01 mg FB1/kg IV q24h for 28 days. The neurologic and CSF changes were consistent with vasogenic cerebral edema.     

Evaluation of nephelometry for albumin measurement in serum and cerebrospinal fluid: experiences with an indwelling subarachnoidal catheter system for repetitive cerebrospinal fluid collection in horses.

The measurement of albumin concentrations in cerebrospinal fluid (CSF) and serum for albumin quotient (AQ) calculations in normal horses was performed by 2 methods: 1) total protein measurement, followed by electrophoresis of the samples to obtain an albumin percentage; and 2) albumin immunoprecipitation quantitated by nephelometry. The results of both methods correlated well, and nephelometry was chosen to determine the albumin concentrations in CSF samples obtained from an indwelling subarachnoidal catheter for daily sampling. Because the use of an indwelling catheter to collect repetitive CSF samples is a novel technique, routine cytological CSF analysis was performed along with daily clinical evaluation to ascertain the well-being of the horses. The catheters were placed in 2 horses for periods of 14 and 17 days. One horse exhibited pleocytosis on cytological evaluation of CSF on 2 occasions for a 1-2-day duration; however, the AQ showed a significant increase on only 1 occasion. The other horse had a normal cell count in CSF but showed 2 sudden changes in the AQ value; however, these values remained within the 95% confidence interval for AQ in horses. Albumin quotient values of the second horse were consistently below the lower range of the confidence interval. Results from this study indicate that nephelometry can be used for albumin determination in serum and CSF samples from horses. Furthermore, an indwelling subarachnoidal catheter system can provide serial CSF samples in horses, thus obviating the need for repetitive centesis for serial CSF sampling.