title: 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.
abstract: 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.
  
  
  METHODS
  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
  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.
authors.affiliation: Department of Medicine, The Bobst Hospital, Animal Medical Center, New York, NY 10021, USA.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Schatzberg
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Haley
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Barr
authors.forename: Alexander
authors.initials: A
authors.lastname: deLahunta
authors.forename: Natasha
authors.initials: N
authors.lastname: Olby
authors.forename: Karen
authors.initials: K
authors.lastname: Munana
authors.forename: Nicholas J H
authors.initials: NJ
authors.lastname: Sharp
mesh_terms: D000818:Animals; D002415:Cats; D002490:Central Nervous System / Q000469:parasitology; D003048:Coccidiosis / Q000175:diagnosis* / Q000662:veterinary*; D017931:DNA Primers; D004285:Dogs; D004587:Electrophoresis, Agar Gel; D018688:Neospora / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014122:Toxoplasma / Q000235:genetics*; D014124:Toxoplasmosis, Animal / Q000175:diagnosis*
chemical_list: D017931:DNA Primers