Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens

Abstract

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

FIG. 1.

Parallel Western blot analyses of (A) whole reduced S. neurona lysate (5 × 10⁶ merozoites) and (B) 0.8 μg rSnSAG2 protein showed good correlation between CSF antibody recognition of the rSnSAG2 and native parasite antigen. The immunodominant antigens representing SnSAG1, SnSAG4, and SnSAG2 are indicated, as described previously (20). With the Western blot conditions used, native SnSAG3 was not visible. CSF samples were run at 1:25. +, confirmed EPM positive; −, confirmed EPM negative.

FIG. 2.

Reciprocal antibody titers determined by ELISA analysis of serum samples from 26 EPM-confirmed horses revealed that 24, 24, and 25 sera had detectable antibody titers to rSnSAG2, rSnSAG3, and rSnSAG4, respectively. An antibody titer to rSnSAG1 was detected in only 18 of the 26 sera. All samples were tested in duplicate in serial twofold dilutions ranging from 1:250 to 1:8,000. The end point titer was the final dilution at which the PP was greater than the optimal cutoff defined for each SnSAG ELISA. *, titer of 1:8,000.

FIG. 3.

Reciprocal antibody titers determined by ELISA analysis of CSF from 22 EPM-confirmed horses and four seronegative horses demonstrated that 18, 18, 20, and 21 of the 22 samples had detectable CSF antibodies against rSnSAG1, rSnSAG2, rSnSAG3, and rSnSAG4, respectively. Samples were tested in duplicate in serial dilutions ranging from 1:2 to 1:200. The reciprocal CSF antibody titer was determined by the lowest dilution at which the PP was greater than or equal to the defined cutoff for each rSnSAG ELISA. CSF sample numbering is the same as for the serum samples in Fig. 4 with the addition of horse 27, for which no paired serum was available. Samples n1, n2, n3, and n4 are from the four seronegative horses.

FIG. 4.

Serum antibody analysis of horses challenged with S. fayeri (38) demonstrated that anti-S. fayeri sera at 1:250 dilution do not react with the four rSnSAGs. (A) A representative Western blot of S. neurona merozoites probed with serum samples collected over time from an S. fayeri-challenged horse showed increasing cross-reactivity with numerous undefined parasite antigens. +, positive control serum; * indicates the antigens used as the basis for S. neurona serology (EBI/IDEXX). (B) Mean ELISA PP values (± standard errors of the mean) for the three S. fayeri challenge horses revealed no consistent rise in antibody reactivity over the course of 13 weeks, thus indicating that anti-S. fayeri sera do not cross-react with the four rSnSAGs. The dotted lines indicate PPs of 10 and 15. ⋄, rSnSAG1; □, rSnSAG2; ▵, rSnSAG3; ×, rSnSAG4.