METİN GÜRÇAY, Ayşe PARMAKSIZ, Ayşe KILIÇ

Doğal olarak enfekte olmuş farklı tür hayvanlardan, post-mortem olarak elde edilen beyin numunelerinde, klasik kuduzun rutin laboratuvar teşhisinde real time RT-PCR testinin değerlendirilmesi

Bu çalışmada, doğal olarak enfekte olmuş farklı hayvan türlerine ait beyin numunelerinde klasik kuduzun teşhisi için real-time RT-PCR testi değerlendirildi. Çalışma materyalini Elazığ Veteriner Kontrol Enstitüsü‘ne 2011 yılı boyunca Türkiye’nin Doğu ve Güneydoğu Anadolu Bölgelerinde bulunan Elazığ, Malatya, Diyarbakır, Mardin, Tunceli, Muş, Bingöl, Batman, Şırnak, Hakkâri ve Bitlis illerinden getirilen 36 köpek, 13 sığır, 6 koyun, 6 eşek, 4 at, 4 tilki, 3 kurt, 2 kedi ve 1 keçiye ait toplam 75 adet kuduz hastalığı şüpheli beyin numunesi oluşturmuştur. Farklı hayvan türlerine ait beyin numunelerinden kuduz virüsü genomunu tespit etmek amacıyla klasik kuduz virüsüne özgü primer ve TaqMan prob seti kullanıldı. Bu primer ve prob seti, beyin numunelerine real-time RT-PCR testi optimize edilerek uygulandı. Laboratuvara ulaşan beyin numunelerine floresan antikor tekniği (FAT) ve deneme hayvanı inokulasyonu (MIT) testleri ile birlikte real time RT-PCR testi de yapıldı. Real-time RT-PCR testi ile 75 beyin numunesinin 53’ünde kuduz virusuna ait spesifik viral RNA tespit edildi. Otoliz olmuş 3 adet köpek beyni numunesine FAT ve MIT uygulanamazken, real-time RT-PCR testi sonucunda bu numunelerin birinde kuduz virusuna ait spesifik viral RNA tespit edildi. Taze (n=72) beyin dokularından etçillerde C. ammonis, otçullarda Cerebellum’dan elde edilen homojenatlara direkt FAT testi uygulandı, 72 beyin numunesinin 51’inde viral antijenler tespit edildi. Aynı homojenatlardan MIT yöntemiyle 72 beyin numunesinin 52’inde virüs izolasyonu sağlandı. Taze beyin numunelerinden elde edilen MIT testi sonuçlarının, real-time RT- PCR testi sonuçları ile tam uyum gösterdiği (%100) görüldü. Sonuç olarak, klasik kuduz teşhisinde direkt beyin dokusuna uygulanan real-time RT-PCR testinin, hem FAT ve MIT uygulanamayan otoliz olmuş numunelerde güvenle kullanılabileceği ve hem de alt yapısı sağlanmış laboratuvarlarda invitro şartlarda FAT için doğrulayıcı test olarak da kullanılabileceği tespit edilmiştir.

Evaluation of real-time RT-PCR assay for routine laboratory diagnosis of classical rabies in post-mortem brain samples from naturally infected different species of animals

In this study; the real-time RT- PCR assay was evaluated for laboratory diagnosis of classical rabies in post mortem brain samples from naturally infected different animals species. The material consisted of 75 suspected brain samples brought from 11 different provinces, belonging Eastern and Southeastern Anatolia region, including Elazig, Malatya, Diyarbakir, Mardin, Sirnak, Batman, Tunceli, Muş, Bingol, Hakkari and Bitlis in year of 2011. These samples were from 36 dogs (33 normal, 3 autolytic), 13 cattle, 6 sheep, 6 donkeys, 4 horses, foxes, 3 wolves, 2 cats , and one goat. The samples were autolytic in 3 of 75 (4%), whereas 72 (96%) of 75 samples were freshly arrived to the laboratory. Due to badly autolysis of 3 samples, they could not be examined by fluorescent antibody test (FAT) and mouse inoculation test method (MIT). Seventy two of 75 samples were tested with both fluorescent antibody test (FAT) and mouse inoculation test method (MIT), whereas all the samples (75 of 75) were examined by optimizing real-time RT- PCR assay in which the primary for classical rabies virus and TaqMan prob set were used. Fresh brain homogenates from Cornu ammonis in carnivores and Cerebellum in herbivores were used for FAT and MIT. As a result, 51 of 72 samples were positive with FAT, 52 of 72 samples with MIT, whereas 52 of 72 were positive in real-time RT-PCR. In fresh brain samples, with MIT results in mouse brain, real time RT- PCR test results showed complete relation each other. As both fluorescent antibody test (FAT) and mouse inoculation test method (MIT) could not be applied to the autolytic samples, the real time RT-PCR assay was the only choice. Consequently, real-time RT-PCR assay could be utilized in autolytic samples conveniently as well as confirmatory to FAT in in vitro conditions.

PDF

___

1. Wakeley PR, Johnson N, McElhinney LM, Marston D, Sawyer J, Fooks AR: Development of a real-time, Taqman reverse transcriptase- PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6. J Clin Microbiol, 43, 2786-2792, 2005.
2. Singh CK, Sandhu BS: Rabies in South Asia: Epidemiological investigation and clinical perspective. Dev Biol, 131, 133-136, 2008.
3. Luiz FPV, Sílvia RFG, Pereira AC, Galante JG, Castilho RN, Oliveira PEB, Ivanete K: Detection of rabies virus nucleoprotein-RNA in several organs outside the Central Nervous System in naturally-infected vampire bats. Pesq Vet Bras, 31 (10): 922-925, 2011.
4. Ün H, Alkan F: Kuduz hastalığının teşhisinde polimeraz zincir reaksiyonu. J Etlik Vet Microbiol, 15 ( 1-2): 1-13, 2004.
5. The World Organisation for Animal Health (OIE): Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 6th ed., 304-322, OIE, Paris, 2008.
6. Mani RS, Madhusudana SN: Laboratory diagnosis of human rabies: Recent advances. Sci World J, 11, 2013. DOI: 10.1155/2013/569712
7. Dean DJ, Abelseth MK, Atanasiu P: The fluorescent antibody test. In, Meslin FX, Kaplan MM, Koprowski H (Eds): Laboratory Techniques in Rabies. 88-95, World Health Organization, Geneva, 1996.
8. Dopoza CP, Barja JL: Diagnosis and identification of IPNV in salmonids by molecular methods. In, Cunningham CO (Ed): Molecular Diagnosis of Salmonid Disease. 23-48, Kluwer Academic Publishers, Netherlands, 2002.
9. Hoffmann B, Freuling CM, Wakeley PR, Rasmussen TB, Leech S, Fooks AR, Beer M, Müller T: Improved safety for molecular diagnosis of classical rabies viruses by use of a TaqMan real-time reverse transcription- PCR “double check” strategy. J Clin Microbiol, 48 (11): 3970-3978, 2010.
10. Gürçay M: Doğu Anadolu Bölgesinde bazı illerde (Elazığ, Malatya, Tunceli, Bingöl, Muş) 1996-1999 yıllarında görülen kuduz olguları. Kafkas Univ Vet Fak Derg, 8 (2): 153-156, 2002.
11. Un H, Johnson N, Vos A, Muller T, Fooks AR, Aylan O: Genetic analysis of four human rabies cases reported in Turkey between 2002 and 2006. Clin Microbiol Infect, 15 (12): 1185-1189, 2009.
12. Heaton PR, Johnstone P, McElhinney LM, Cowley R, O’Sullivan E, Whitby JE: Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses. J Clin Microbiol, 35 (11): 2762-2766, 1997.
13. Dantas JV, Kimura LMS, Ferreira MSR, Fialho AM, Almeida MMS, Grégio CRV, Romijn PC, Leite JPG: Reverse transcription-polymerase chain reaction assay for rabies virus detection. Arq Bras Med Vet Zootec, 56 (3): 398-400, 2004.
14. Chhabra M, Mittal V, Jaiswal R, Malik S, Gupta M, Lal S: Development and evaluation of an in vitro isolation of street rabies virus in mouse neuroblastoma cells as compared to conventional tests used for diagnosis of rabies. Indian J Med Microbiol, 25, 263-266, 2007.
15. Shankar BP: Advances in Diagnosis of Rabies. Vet World J, 2 (2): 74-78, 2009.
16. Anonim: Başbakanlık Hayvan Deneyleri Etik Kurullarının Çalışma Usul ve Esaslarına Ait Yönetmelik. Resmi Gazete 26220, Tarih: 06.07.2006.
17. Panning M, Baumgarte S, Pfefferle S, Maier T, Martens A, Drosten C: Comparative analysis of rabies virus reverse transcription-PCR and virus isolation using samples from a patient infected with rabies virus. J Clin Microbiol, 48 (8): 2960-2962, 2010.
18. Aravindh Babu RP, Manoharan S, Ramadass P, Chandran NDJ: Evaluation of RT-PCR assay for routine laboratory diagnosis of rabies in post mortem brain samples from different species of animals. Indian J Virol, 23 (3): 392-396, 2012.
19. Dandale M, Singh CK, Ramneek V, Deka D, Bansal K, Sood NK: Sensitivity comparison of nested RT-PCR and TaqMan real time PCR for intravitam diagnosis of rabies in animals from urine samples. Vet World, 6 (4): 189-192, 2013.
20. Fischer M, Wernike K, Freuling CM, Müller T, Aylan O, Brochier B, Cliquet F, Vázquez-Morón S, Hostnik P, Huovilainen A, Isaksson M, Kooi EA, Mooney J, Turcitu M, Rasmussen TB, Revilla-Fernández S, Smreczak M, Fooks AR, Marston DA, Beer M, Hoffmann B: A step forward in molecular diagnostics of Lyssaviruses - Results of a ring trial among European laboratories. PLOS One, 8 (3): 2013. DOI: 10.1371/ journal.pone.0058372