Accurate Antemortem Diagnosis of Equine Protozoal Myeloencephalitis (EPM) Based on Detecting Intrathecal Antibodies againstSarcocystis neuronaUsing the SnSAG2 and SnSAG4/3ELISAs

Abstract

Background: Recent work demonstrated the value of antigen-specific antibody indices (AI and C-value) to detect intrathecal antibody production against Sarcocystis neurona for antemortem diagnosis of equine protozoal myeloencephalitis (EPM).Objectives: The study was conducted to assess whether the antigen-specific antibody indices can be reduced to a simple serum : cerebrospinal fluid (CSF) titer ratio to achieve accurate EPM diagnosis.Animals: Paired serum and CSF samples from 128 horses diagnosed by postmortem examination. The sample set included 44 EPM cases, 35 cervical-vertebral malformation (CVM) cases, 39 neurologic cases other than EPM or CVM, and 10 non-neurologic cases.Methods: Antibodies against S. neurona were measured in serum and CSF pairs using the SnSAG2 and SnSAG4/3 (SnSAG2, 4/3) ELISAs, and the ratio of each respective serum titer to CSF titer was determined. Likelihood ratios and diagnostic sensitivity and specificity were calculated based on serum titers, CSF titers, and serum : CSF titer ratios.Results: Excellent diagnostic sensitivity and specificity was obtained from the SnSAG2, 4/3 serum : CSF titer ratio. Sensitivity and specificity of 93.2 and 81.1%, respectively, were achieved using a ratio cutoff of ≤100, whereas sensitivity and specificity were 86.4 and 95.9%, respectively, if a more rigorous cutoff of ≤50 was used. Antibody titers in CSF also provided good diagnostic accuracy. Serum antibody titers alone yielded much lower sensitivity and specificity.Conclusions and Clinical Importance: The study confirms the value of detecting intrathecal antibody production for antemortem diagnosis of EPM, and they further show that the antigen-specific antibody indices can be reduced in practice to a simple serum : CSF titer ratio.

Utility of C‐reactive protein and serum amyloid A in the diagnosis of equine protozoal myeloencephalitis

“…Additionally, there is widespread exposure of horses to the primary causative agent Sarcocystis neurona ; seroprevalence in the United States ranges from 33% to 89% depending on the region 1. However, annual incidence of clinical disease is still <1%2 and is thought to occur when parasites invade and propagate in the CNS.…”

Equine Protozoal Myeloencephalitis: An Updated Consensus Statement with a Focus on Parasite Biology, Diagnosis, Treatment, and Prevention

“…This study also showed that minor blood contamination of the CSF sample (ie, up to 10,000 red cells per μL) will not confound the assay results. Subsequently, 2 additional studies examining a more extensive collection of horses with neurologic disease showed that a simple serum:CSF antibody titer ratio was sufficient in many cases for an accurate diagnosis of EPM caused by S. neurona 71, 72. Although use of a serum:CSF titer ratio should be equally effective for diagnosis of EPM caused by N. hughesi , an optimal serum:CSF titer ratio cut‐off needs to be established.…”

“…Descriptions of testing options and reported test performance are shown in Table 1 71, 72, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93. All tests can be performed on serum or CSF, and none is considered a gold standard.…”

“…The IFAT is a quantitative (end‐point titer) test for antibodies against culture‐derived whole merozoites. Although serum titers obtained with the IFAT have been used to predict the likelihood of EPM, with higher titers suggesting greater probability of disease, studies that have used diverse collections of neurologic disease cases have shown that a serum titer alone is a poor predictor of EPM 71, 72. As a quantitative test, the IFAT can be used to calculate a serum:CSF titer ratio.…”

“…These molecules have proven to be good serologic targets in the assays because of their high level of expression in the parasite and their immunogenicity in infected horses 94, 95, 96. The SnSAG2 ELISA and the SnSAG4/3 ELISA accurately detect antibodies against S. neurona in equine serum and CSF samples88, 91 and were used to demonstrate the value of detecting intrathecal antibody production for EPM diagnosis 71, 72. An ELISA based on the SnSAG1 surface protein has been described 86.…”