Clinical, pathological and real time rt-pcr diagnosis of canine distemper virus infection in dogs

Abstract

It was aimed to compare the Real Time RT-PCR based on nucleic acid detection and the histopathological method for viral nucleic acid detection by using the Real Time RT-PCR method for rapid diagnosis of the disease in both swab samples and postmortem tissue samples in dogs with canine distemper virus (CDV) infection. The material of study was composed of brain, cerebellum, liver, lung, spleen, kidney, sole pad, blood and swab taken from tonsil, nose and eye samples of dogs with clinical signs of fever, tear discharge and respiratory, digestive and nervous system infections, who started treatment with a clinical diagnosis of CDV infection, but died without responding to treatment, but were diagnosed with CDV according to histopathological findings. When the presence of viral nucleic acid was examined in swab samples taken during necropsy from dogs with intracytoplasmic and intranuclear inclusion bodies that are considered characteristic for CDV infection in histopathological examinations, it was observed that CDV nucleic acid copies were detected in all swabs taken from tonsils, in two of samples taken from the nose and conjunctiva, and viral nucleic acid copies were not detected in blood samples. Although clinical findings in dogs suggest the disease, virological and histopathological examinations used for definitive diagnosis take a long time. This situation brings PCR methods to the fore based on the reproduction of viral nucleic acids, and increases the importance of Real Time RT-PCR applications that minimize operator error, give fast results and can predict the number of viruses among PCR methods.

Keywords

• Canine distemper virus (CDV)
• histopathology
• real time rt-pcr

Köpeklerde canine distemper virüs enfeksiyonunun klinik, patolojik ve real-time RT-PCR ile teşhisi

Abstract

Klinik olarak canine distemper virüs (CDV) teşhisi konulan köpeklerde enfeksiyonun hem sürüntü örneklerinde ve hem de postmortem alınan doku örneklerinde hızlı teşhisine yönelik real-time RT-PCR metodu çalışılarak, viral nükleik asit tespitine dayalı real-time RT-PCR metodu ile histopatolojik yöntemin karşılaştırması amaçlanmıştır. Çalışmanın materyalini, ateş, gözyaşı akıntısı ile solunum, sindirim ve sinir sistemi enfeksiyonlarına ait klinik bulgulara sahip, CDV enfeksiyonu klinik tanısı ile tedaviye başlanmış, ancak tedaviye yanıt vermeden ölen köpeklerden histopatolojik bulgulara göre CDV teşhisi konulmuş köpeklere ait beyin, beyincik, karaciğer, akciğer, dalak, böbrek, taban yastığı ve kan ile tonsil, burun ve gözden alınan sürüntü örnekleri oluşturdu. Histopatolojik incelemelerde CDV enfeksiyonu için karakteristik sayılan intrasitoplazmik ve intranükleer inklüzyon cisimciği belirlenen köpeklerden nekropsi sırasında alınmış sürüntü örneklerinde viral nükleik asit varlığı incelendiğinde tonsillerden alınan sürüntülerin tamamında, burun ve konjunktivadan alınan örneklerin iki tanesinde CDV nükleik asit kopyalarının belirlendiği, kan örneklerinde ise viral nükleik asit belirlenemediği görüldü. Köpeklerde görülen klinik bulgular hastalığı düşündürse de kesin teşhis için kullanılan virolojik ve histopatolojik incelemeler uzun zaman almaktadır. Bu durum viral nükleik asitlerin çoğaltılması esasına dayalı PCR yöntemlerini ön plana çıkarmakta, PCR yöntemleri içerisinde de uygulayıcı hatasını minimize eden, hızlı sonuç veren ve virüs sayısının tahmin edilebildiği real-time RT-PCR uygulamalarının önemini artırmaktadır.

Keywords

• : Canine distemper virüs (CDV)
• histopatoloji
• real-time RT-PCR

References

1. Amude, AM., Carvalho, GA., Alfieri, AA. (2007). Virus isolation and molecular characterization of canine distemper virus by RT-PCR from a matur dog with multifocal encephalomyelit. Braz J Microbiol, 38: 1-6.
2. Appel, MJG. (1987). Canine distemper virus. In: Horzineck MC, ed. Virus infections of carnivores. Virus Infection of Vertebrates,Amsterdam, Elsevier, vol.1, 133-59.
3. Beineke, A., Puff, C., Seehusen, F., Baumgärtner, W. (2009). Pathogenesis and immunopathology of systemic and nervous canine distemper. Veterinary immunology and immunopathology, 127, (1-2), 1-18.
4. Carvalho, OV., Botelho, CV., Ferreira, CGT., Scherer, PO., Soares-Martins, JA., Almeida, MR., Silva Junior, AS. (2012). Immunopathogenic and neurological mechanisms of canine distemper virus. Advances in virology, 4: 1-10.
5. De Almeida, DE., Roveratti, C., Brito, FL., Godoy, GS., Duque, JC., Bechara, GH., Laus, JL. (2009). Conjunctival effects of canine distemper virus-induced keratoconjunctivitis sicca. Vet Ophthalmol, 12: 211-5.
6. Deem, SL., Spelman, LH., Yates, RA., Montali, RJ. (2000). Canine distemper in terrestrial carnivores: a review. Journal of Zoo and Wildlife medicine, 31, (4), 441-51.
7. Demeter, Z., Palade, EA., Rusvai, M. (2009). Canine distemper: still a major concern in Central Europe. Lucrari Stiintifice Universitatea de Stiinte Agricole a Banatului Timisoara, Medicina Veterinara, 42, (1), 136-50.
8. Engelhardt, P., Wyder, M., Zurbriggen, A., Gröne, A. (2005). Canine distemper virus associated proliferation of canine footpad keratinocytes in vitro. Veterinary microbiology, 107, (1-2), 1-12.

9. Goldstein, T., Mazet, JA., Gill, VA., Doroff, AM., Burek, KA., Hammond JA. (2009). Phocine distemper virus in northern sea otters in the Pacific Ocean, Alaska, USA. Emerging infectious diseases, 15, (6), 925.
10. Guo, A., Lu, C. (2000). Canine distemper virus causes apoptosis of Vero cells. Journal of Veterinary Medicine, Series B, 47, (3), 183-90.
11. Higgins, R., Krakowka, S., Metzler, A., Koestner, A. (1982). Primary demyelination in experimental canine distemper virus induced encephalomyelitis in gnotobiotic dogs. Acta neuropathologica, 58, (1), 1-8.
12. Hoskins, JD. (2010). Canine Viral Disease. In: Ettinger SJ, Feldman EC, editors. Textbook of Veterinary İnternal Medicine: Disease of the Dog and Cat. 7th ed. Canada, Elsevier; 961-2.
13. Jones, TC., Hunt, RD., Kıng, NW. (1997). Veterinary Pathology. 6th ed. United States, Williams & Wilkins; 257-61.
14. Kubo, T., Kagawa, Y., Taniyama, H., Hasegawa, A. (2007). Distribution of inclusion bodies in tissues from 100 dogs infected with canine distemper virus. Journal of veterinary medical science, 69, (5), 527-9.
15. Leisewitz, A., Carter, A., Van Vuuren, M., Van Blerk, L. (2001). Canine distemper infections, with special reference to South Africa, with a review of the literature. Journal of the South African Veterinary Association, 72, (3), 127-36.
16. Maclachlan, NJ., Dubovi, EJ. (2011). Fenner’s Veterinary Virology, 4th Ed. USA, Academic Pres ; 299-320.
17. Maxie, G. (2015). Jubb, Kennedy & Palmer's Pathology of Domestic Animals, Volume: 3 volume set No.Ed.6 pp.2456 pp.
18. Moro. L., Martins, A., Alves, C., Santos, F., Del Puerto, HL., Vasconcelos, AC. (2003). Apoptosis in the cerebellum of dogs with distemper. Journal of Veterinary Medicine, Series B, 50, (5), 221-5.
19. Mutinelli. F., Vandevelde. M., Griot, C., Richard, A. (1989). Astrocytic infection in canine distemper virus-induced demyelination. Acta neuropathologica, 77, (3), 333-5. Murphy, FA., Gibbs, EPJ., Horzinek, MC., Studdert, MJ. (1999). Veterinary Virology, 3rd Edition, USA, Acedemic Pres; 411-25.
20. Myers, DL., Zurbriggen, A., Lutz, H., Pospischil, A. (1997). Distemper: not a new disease in lions and tigers. Clinical and diagnostic laboratory immunology, 4, (2), 180-4.
21. Pardo, ID., Johnson, GC., Kleiboeker, SB. (2005). Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America. Journal of clinical microbiology, 43, (10), 5009-17.
22. Sato, H., Masuda, M., Miura, R., Yoneda, M., Kai, C. (2006). Morbillivirus nucleoprotein possesses a novel nuclear localization signal and a CRM1-independent nuclear export signal. Virology, 352, (1), 121-30.
23. Scagliarini, A., Battilani, M., Ciulli, S., Prosperi, S., Morganti, L. (2003). Molecular analysis of the NP gene of Italian CDV isolates. Vet. Res. Commun, 27, 355-357.
24. Sumummers, BA., Appel, MJ. (1987). Demyelination in canine distemper encephalomyelitis:an ultrastructural analysis. Journal of neurocytology, 16, (6), 871-81.
25. Vural, SA., Alcigir, ME. (2010). Distemper virus-induced apoptotic changes in cerebellum. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 57, (2), 83-6.