Immunodiagnosis

Immunoblot analysis of serum and cerebrospinal fluid (CSF) provides antemortem information about exposure to S. neurona. The test utilizes cultured merozoites to detect antibodies directed against proteins that are unique to S. neurona. Antibodies produced to other organisms can be differentiated. Immunoblot testing of CSF has demonstrated 89% specificity and sensitivity among approximately 300 neurologic cases that received post mortem examination. Positive CSF indicates that parasites have penetrated the blood-brain barrier and stimulated a local immune response. If the integrity of the blood-brain barrier is compromised, circulating antibodies may leak across and produce a false positive test result. False negative results have been rare, but may occur. Some horses may simply fail to respond to the S. neurona-specific proteins identified. The possible causes of false negative responses are important to consider so that affected horses are not misdiagnosed.

Horses that initially tested positive have become negative after several weeks of treatment and are apparently recovered. Chronically affected horses may test negative and still be infected or the horse may still exhibit neurologic signs. We speculate that this may be due to permanent CNS damage and that parasites are either no longer present or, antibody production is below test sensitivity. The use of PCR testing aids in parasite detection when antibody level is low. Acute cases that test negative should be re-tested in two to three weeks. However, the incubation period appears to be sufficiently long to allow production of detectable amounts of IgG before the onset of clinical signs in most cases. As was discussed before, one exception with a very short incubation period has been observed.

Use of PCR as a diagnostic tool has been developed. Using a random primed polymorphic DNA assay (RAPD), molecular comparisons were made between S. neurona and eight related coccidia, namely two Sarcocystis sp., one Toxoplasma sp. and five Eimeria sp. The DNA fragments from the S. neurona isolates had sequence homology, whereas, there was no sequence homology between the 550 bp of S. neurona and the other species of coccidia tested. It was therefore suggested that this S. neurona DNA fragment could be utilized as a species-specific probe for this parasite which will enable differentiation of S. neurona from other equine coccidia as well as having potential to help solve the life cycle by examining Sarcocystis sp. from possible definitive hosts. Further study was performed to determine the phylogenetic relationship of S. neurona to other members of the family Sarcocytidae. This was done based on the sequence of the small ribosomal subunit gene of S. neurona which was amplified using polymerase chain reaction (PCR).

This study led to development of species-specific amplification primers which could be utilized for wildlife testing to elucidate the life cycle of S. neurona as well as utility in antemortem diagnostics of EPM. Other research examined the specificity and sensitivity of polymerase chain reaction (PCR) for detection of S. neurona (falcatula) DNA in the CSF. The test was both sensitive and specific, however, it appears to be related to the presence of parasite in the CSF. In this study, there was a 40% sensitivity which is not unexpected as in most cases of EPM, the parasite is in the tissues, not free in the CSF. Therefore, it appears that the PCR testing may be useful prior to development of an antibody response in the CSF or in chronic cases where the antibody level has waned below detectable levels.

Other testing modalities have been used to help determine the disease status of neurologic horses. Previously, the CSF cytology, total protein, and CSF enzmes such as creatine kinase (CK) and aspartate aminotransferase (AST) were used to help diagnose neurologic diseases. Research at OSU would suggest that this information is of limited value, but may help determine if the CSF sample has been contaminated during the collection process. Other tests that appear to have more value in determining the true disease status of neurologic horses are the CSF indices.

These are calculations based on serum and CSF concentrations of albumin and immunoglobulin G. The calculations are called albumin quotient (AQ) and the IgG Index. The AQ will help determine the permeability of the blood-brain barrier (BBB) and helps to determine if the CSF sample collected was contaminated with serum or blood. The IgG Index will determine if the antibodies in the CSF were produced in the central nervous system (intrathecal) or whether they are due to leakage from the serum as well. Although the bugs have not been worked out conclusively, these tests may have an important role in augmenting clinicians in the diagnosis of EPM.