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
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. (Am J Vet Res 2003;64:1507–1513)