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

Equine protozoal myeloencephalitis (EPM) is a neurological disease of horse s and ponies caused by the apicomplexan protozoan parasite Sarcocystis neur ona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and speci ficity 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 minimi ze false-positive results in the western blot test for S. neurona. Sarcocys tis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyac rylamide gel electrophoresis in a 12-20% linear gradient gel. Separated pro teins were electrophoretically transferred to polyvinylidene fluoride membr anes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered sa line. Serum samples from 6 horses with S, neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from t he Eastern Hemisphere, where S. neurona does not exist) were tested by West ern blot. Horses from bath 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 s amples. After this modification of the Western blot test, positive infectio n 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 Weste rn 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 b locking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.