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

• Han JI
• Chang DW
• Na KJ

• KMID: 2344307
• DOI: http://doi.org/10.4142/jvs.2015.16.3.341

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.

Reference

1. Arceneaux KA, Taboada J, Hosgood G. Blastomycosis in dogs: 115 cases (1980-1995). J Am Vet Med Assoc. 1998; 213:658–664.

2. Berthelin CF, Bailey CS, Kass PH, Kegendre AM, Wolf AM. Cryptococcosis of the nervous system in dogs. Part 1: Epidemiologic, clinical, and neuropathologic features. Prog Vet Neurol. 1994; 5:88–97.

3. Bhat N, O'Brien KL, Karron RA, Driscoll AJ, Murdoch DR. Pneumonia Methods Working Group. Use and evaluation of molecular diagnostics for pneumonia etiology studies. Clin Infect Dis. 2012; 54:Suppl 2. S153–S158.

4. Chang YF, Novosel Y, Chang CF, Summers BA, Ma DP, Chiang YW, Acree WM, Chu HJ, Shin S, Lein DH. Experimental induction of chronic borreliosis in adult dogs exposed to Borrelia burgdorferi-infected ticks and treated with dexamethasone. Am J Vet Res. 2001; 62:1104–1112.

5. Cherubini GB, Platt SR, Anderson TJ, Rusbridge C, Lorenzo V, Mantis P, Cappello R. Characteristics of magnetic resonance imags of granulomatous meningoencephalomyelitis in 11 dogs. Vet Rec. 2006; 159:110–115.

6. Cherubini GB, Platt SR, Howson S, Baine E, Brodbelt DC, Dennis R. Comparison of magnetic resonance imaging sequences in dogs with multi-focal intracranial disease. J Small Anim Pract. 2008; 49:634–640.

7. Cho YI, Han JI, Wang C, Copper V, Schwartz K, Engelken T, Yoon KJ. Case-control study of microbiological etiology associated with calf diarrhea. Vet Microbiol. 2013; 166:375–385.

8. Cho YI, Kim WI, Liu S, Kinyon JM, Yoon KJ. Development of a panel of multiplex real-time polymerase chain reaction assays for simultaneous detection of major agents causing calf diarrhea in feces. J Vet Diagn Invest. 2010; 22:509–517.

9. Chou J, Wünschmann A, Hodzic E, Borjesson DL. Detection of Borrelia burgdorferi DNA in tissues from dogs with presumptive Lyme borreliosis. J Am Vet Med Assoc. 2006; 229:1260–1265.

10. Courtney JW, Kostelnik LM, Zeidner NS, Massung RF. Multiplex real-time PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi. J Clin Microbiol. 2004; 42:3164–3168.

11. Diaz MH, Bai Y, Malania L, Winchell JM, Kosoy MY. Development of a novel genus-specific real-time PCR assay for detection and differentiation of Bartonella species and genotypes. J Clin Microbiol. 2012; 50:1645–1649.

12. Doyle CK, Labruna MB, Breitschwerdt EB, Tang YW, Corstvet RE, Hegarty BC, Bloch KC, Li P, Walker DH, McBride JW. Detection of medically important Ehrlichia by quantitative multicolor TaqMan real-time polymerase chain reaction of the dsb gene. J Mol Diagn. 2005; 7:504–510.

13. Elia G, Decaro N, Martella V, Cirone F, Lucente MS, Lorusso E, Di Trani L, Buonavoglia C. Detection of canine distemper virus in dogs by real-time RT-PCR. J Virol Methods. 2006; 136:171–176.

14. Flegel T, Henke D, Boettcher IC, Aupperle H, Oechtering G, Matiasek K. Magnetic resonance imaging findings in histologically confirmed Pug dog encephalitis. Vet Radiol Ultrasound. 2008; 49:419–424.

15. Gerber JE, Johnson JE, Scott MA, Madhusudhan KT. Fatal meningitis and encephalitis due to Bartonella henselae bacteria. J Forensic Sci. 2002; 47:640–644.

16. Ghalmi F, China B, Kaidi R, Daube G, Losson B. Detection of Neospora caninum in dog organs using real time PCR systems. Vet Parasitol. 2008; 155:161–167.

17. Kerl ME. Update on canine and feline fungal diseases. Vet Clin North Am Small Anim Pract. 2003; 33:721–747.

18. Krimer PM, Miller AD, Li Q, Grosenbaugh DA, Susta L, Schatzberg SJ. Molecular and pathological investigations of the central nervous system in Borrelia burgdorferi-infected dogs. J Vet Diagn Invest. 2011; 23:757–763.

19. Lavely J, Lipsitz D. Fungal infections of the central nervous system in the dog and cat. Clin Tech Small Anim Pract. 2005; 20:212–219.

20. Lin MH, Chen TC, Kuo TT, Tseng CC, Tseng CP. Real-time PCR for quantitative detection of Toxoplasma gondii. J Clin Microbiol. 2000; 38:4121–4125.

21. Lowrie M, Smith PM, Garosi L. Meningoencephalitis of unknown origin: investigation of prognostic factors and outcome using a standard treatment protocol. Vet Rec. 2013; 172:527.

22. Menaut P, Landart J, Behr S, Lanore D, Trumel C. Treatment of 11 dogs with meningoencephalomyelitis of unknown origin with a combination of prednisolone and cytosine arabinoside. Vet Rec. 2008; 162:241–245.

23. Panciera RJ, Ewing SA, Confer AW. Ocular histopathology of ehrlichial infections in the dog. Vet Pathol. 2001; 38:43–46.

24. Park KS, Ki CS, Lee NY. Laboratory experience in phenotypic and molecular identification of Blastomyces dermatitidis first isolated in Korea. Korean J Clin Microbiol. 2012; 15:114–116.

25. Rudd PA, Bastien-Hamel LE, von Messling V. Acute canine distemper encephalitis is associated with rapid neuronal loss and local immune activation. J Gen Virol. 2010; 91:980–989.

26. Schatzberg SJ, Haley NJ, Barr SC, de Lahunta A, Sharp NJH. Polymerase chain reaction screening for DNA viruses in paraffin-embedded brains from dogs with necrotizing meningoencephalitis, necrotizing leukoencephalitis, and granulomatous meningoencephalitis. J Vet Intern Med. 2005; 19:553–559.

27. Sidamonidze K, Peck MK, Perez M, Baumgardner D, Smith G, Chaturvedi V, Chaturvedi S. Real-time PCR assay for identification of Blastomyces dermatitidis in culture and in tissue. J Clin Microbiol. 2012; 50:1783–1786.

28. Talarico LR, Schatzberg SJ. Idiopathic granulomatous and necrotising inflammatory disorders of the canine central nervous system: a review and future perspectives. J Small Anim Pract. 2010; 51:138–149.

29. Thonur L, Maley M, Gilray J, Crook T, Laming E, Turnbull D, Nath M, Willoughby K. One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3. BMC Vet Res. 2012; 8:37.

30. Veron V, Simon S, Blanchet D, Aznar C. Real-time polymerase chain reaction detection of Cryptococcus neoformans and Cryptococcus gattii in human samples. Diagn Microbiol Infect Dis. 2009; 65:69–72.

31. Williams JH, Köster LS, Naidoo V, Odendaal L, van Veenhuysen A, de Wit M, van Wilpe E. Review of idiopathic eosinophilic meningitis in dogs and cats, with a detailed description of two recent cases in dogs. J S Afr Vet Assoc. 2008; 79:194–204.