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)
Case Description—An 8-year-old Labrador Retriever with diabetes mellitus in which bilateral phacoemulsification had been performed 3 weeks earlier was evaluated for acute onset of blepharospasm, and a 7-year-old Miniature Schnauzer with chronic immune-mediated thrombocytopenia was reevaluated for keratoconjunctivitis sicca that had been diagnosed 4 weeks earlier.
Clinical Findings—Dendritic corneal ulcerations were detected in both dogs. Canine herpesvirus-1 (CHV-1) was isolated from corneal swab specimens obtained during the initial evaluation of each dog and during recheck examinations performed until the ulcerations were healed. Canine herpesvirus-1 serum neutralization titers were detected in both dogs. Results of virus isolation from oropharyngeal and genital swab specimens were negative for both dogs. The isolated viruses were identified as CHV-1 via immunofluorescence, transmission electron microscopy, PCR assay, and gene sequencing. Negative controls for PCR assay and virus isolation included conjunctival swab specimens from 50 dogs without extraocular disease and corneal swab specimens from 50 dogs with corneal ulcers, respectively.
Treatment and Outcome—Lesions resolved in both dogs after topical administration of idoxuridine or trifluridine and discontinuation of topically administered immunosuppressive medications.
Clinical Relevance—To the authors' knowledge, this is the first report of corneal ulcerations associated with naturally occurring CHV-1 infection and may represent local ocular recrudescence of latent CHV-1 infection. The viruses isolated were identified as CHV-1, and the morphology, antigenicity, and genotype were similar to those for CHV-1 isolates obtained from a puppy that died from systemic CHV-1 infection.