Use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs

Objective-To develop a multiplex polymerase chain reaction (PCR) assay for the detection of Toxoplasma gondii and Neospora caninum DNA in canine and feline biological samples. Sample Population-Biological samples from 7 cats with systemic (n = 4) or CNS (3) toxoplasmosis, 6 dogs with neospora- or toxoplasma-associated encephalitis, and 11 animals with nonprotozoal disease. Procedure-Primers for T gondii, N caninum, and the canine ferritin gene (dogs) or feline histone 3.3 gene (cats) were combined in a single PCR assay. The DNA was extracted from paraffin-embedded brain tissue, CSF, or skeletal muscle. The PCR products with positive results were cloned, and sequence identity was confirmed. Results-Of 7 cats and 4 dogs with immunohistochemical or serologic evidence of toxoplasmosis, PCR results were positive for all cats and 3 dogs for T gondii, and positive for T gondii and N caninum for 1 dog. Another dog had negative PCR results for both parasites. Of 2 dogs with immunohistochemical or serologic evidence of neosporosis, PCR results were positive for 1 for N caninum and positive for the other for T gondii. All negative-control samples yielded negative results for T gondii and N caninum on the PCR assay. Conclusions and Clinical Relevance-Standard tests for toxoplasmosis or neosporosis associated with the CNS rely on serologic, histologic, or immunohistochemical analysis and can be difficult to interpret. The multiplex PCR assay with built-in control reactions could be a complementary clinical tool for the antemortem diagnosis of toxoplasmosis or neosporosis associated with the CNS.