J Vet Sci.  2015 Sep;16(3):341-347. 10.4142/jvs.2015.16.3.341.

A multiplex quantitative real-time polymerase chain reaction panel for detecting neurologic pathogens in dogs with meningoencephalitis

Affiliations
  • 1Laboratory of Veterinary Laboratory Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea. sigol@cbnu.ac.kr
  • 2Laboratory of Veterinary Radiology, College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea.

Abstract

Meningoencephalitis (ME) is a common inflammatory disorder of the central nervous system in dogs. Clinically, ME has both infectious and non-infectious causes. In the present study, a multiplex quantitative real-time polymerase chain reaction (mqPCR) panel was optimized for the detection of eight canine neurologic pathogens (Blastomyces dermatitidis, Cryptococcus spp., Neospora caninum, Borrelia burgdorferi, Bartonella spp., Toxoplasma gondii, Ehrlichia canis, and canine distemper virus [CDV]). The mqPCR panel was subsequently applied to 53 cerebrospinal fluid (CSF) samples collected from dogs with ME. The analytic sensitivity (i.e., limit of detection, expressed as molecules per 1 microL of recombinant vector) was 3.8 for CDV, 3.7 for Ehrlichia canis, 3.7 for Bartonella spp., 3.8 for Borrelia burgdorferi, 3.7 for Blastomyces dermatitidis, 3.7 for Cryptococcus spp., 38 for Neospora caninum, and 3.7 for Toxoplasma gondii. Among the tested CSF samples, seven (15%) were positive for the following pathogens in decreasing order of frequency: Cryptococcus spp. (3/7), Blastomyces dermatitidis (2/7), and Borrelia burgdorferi (2/7). In summary, use of an mqPCR panel with high analytic sensitivity as an initial screen for infectious agents in dogs with ME could facilitate the selection of early treatment strategies and improve outcomes.

Keyword

canine; meningoencephalitis; multiplex polymerase chain reaction; neurologic pathogen

MeSH Terms

Animals
Bacteria/genetics/*isolation & purification
Dog Diseases/*diagnosis/microbiology/parasitology
Dogs
Meningoencephalitis/diagnosis/microbiology/parasitology/*veterinary
Multiplex Polymerase Chain Reaction/*veterinary
Prevalence
Real-Time Polymerase Chain Reaction/*veterinary
Republic of Korea/epidemiology

Figure

  • Fig. 1 Detection of viral and bacterial meningoencephalitis pathogens in canine cerebrospinal fluid samples by mqPCR. Specific primer and probe sets were used to amplify sequences from (A) canine distemper virus, Ehrlichia (E.) canis, Bartonella spp., Borrelia (B.) burgdorferi, (B) Blastomyces (B.) dermatitidis, Cryptococcus (C.) neoformans, Neospora (N.) caninum, and Toxoplasma (T.) gondii. Known quantities (i.e., copy number per µL) of recombinant vector encoding the target gene from each pathogen were prepared as serial 10-fold dilutions and used as controls. Cycle threshold (CT) values are shown on the Y-axis. Each regression line was obtained from triplicate measurements.


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