Kuduz Hastalığının Teşhisinde Polimeraz Zincir Reaksiyonu

Bu çalışmada kuduz hastalığının teşhisinde Polimeraz Zincir Reaksiyonu (PCR) tekniğinin kullanım olanakların araştırılması amaçlandı . Toplanan materyallerin Floresan Antikor Tekniği (FAT) ile kontrolü sonuçları heminested RT-PCR teslinden elde edilen sonuçlar ile karşılaştırıldı. Ayrıca kokuşmuş materyallerden heminested RT-PCR ile kuduz hastalığının teşhisi olanaği araştırıIdi. Çalişmada 83 adedi FAT ile değerlendirilebifen ve 30 adedi de kokuşmuş olmaları nedeni ile FAT ile değerlendirilemeyen toplam 113 adet değişik hayvan türüne ait beyin örneği kullanıIdi. FAT ile değerlendirilebilen 83 materyalden 80 adedi kuduz yönünden müspet. 3 adedi ise kuduz yönünden menfi idi. Bu materyaller Tarım ve Köyişleri Bakanfığma bağlı kuduz teşhis lahoratuvarından temin edildi. FAT ile müspel değerlendirilen 80 materyalden 49 (% 61,25) adedinde RT-PCR ile viral nükleik asit tespit edilirken, heminested RT-PCR ile tamamında (% 1 00) viral nükleik asit saptandı. FAT ile menfi olan 3 adet materyal de RT-PCR ve heminested RT-PCR ile de viral nükleik asit tespit edilmedi. 30 adet kokuşmuş materyalden 4 adedinde RT-PCR ile viral nükleik asit (%13.33) ve 6 adedinde de heminested RT-PCR .ile viral nükleik asit (% 20) tespit edildi. Heminested RT-PCR ile viral nükleik asit tespit edilen bu 6 materyalden 3 adedi istanbul Pendik Veteriner Kontrol ve Araştırma Enstitüsünden temin edilen, diğer 3 adedi de Etlik Merkez Veteriner Kontrol ve Araştırma Enstitüsünde aşı üretimi ve deneysel çalişmalarda kullanılan hayvanlara ait beyinlerdi. Deneysel amoçlarla kullanılanlar ve kuduz virusu ile enfekte edilmiş olan hayvanlar (3 adet) hariç tutulacak olursa kokuşmuş materyallerdeki RT-PCR ve heminested RT-PCR pozitiflik oranları sırası ile % 3. 7 ( 1/2 7) ve% 11.11 (3/2 7) olarak belirlendi.

Anahtar Kelimeler:

Teşhis, Kuduz, RT-PCR, Heminested RT-PCR

Polymerase C hain Reaction in the Diagnosis of Rabies

In this study the probability of the use of the PCR technique for the diagnosis of rabies was investigated. The colected specimens were examined using the FAT and the results were compared with the ones thal were obtained from the heminested RT-PCR test. Furthermore. it was investigated the diagnosis of rabies in the decomposed specimens using the heminesled RT-PCR. In this study. 83 brain specimens to evaluate using FAT and 30 decomposed materials not to evatluate using FAT belonging to various animal species totally 113 brain pecimens were used. 80 of these specimens were found to be positive using FAT while 3 of them being negative using FAT All of these specimens were obtained from the rabies diagnosis laboratories belonging to the Ministry of Agriculture and Rural Affairs. Among the 80 specimens that were found to be positive using FAT, 49 of them were determined to be positive using RT-PCR (61,25%) while all of these 80 specimens were found to be pasitive using heminesled RT-PCR (100 %). The 3 specimens that were found to be negative using FAT all of them were found to be negative using RT-PCR and heminested RT-PCR. 4 of the 30 decomposed brain specimens were determined to be positive (13,33%) us ing RT-PCR and 6 of them found to be positive using heminested RT-PCR. If the results of the brain specimens that have been used for experimental purposes and animals that have been injected with rabies virus (3 animals) are excfuded, these rates are 3. 7% and 11, 11I% respeclively in the decomposed materials. Among the 6 decomposed specimens were found to be positive using heminested RT-PCR, 3 of them were obtained. from the İstanbul Pendik Veterinary Control and Research Institute, the other 3 of them were usedfor experimental pwposes and vaccine production in Etlik Central Veterinary Control and Research Institute.

Keywords:

Rabies RT-PCR, Heminested RT-PCR, Diagnosis., Diagnosis,

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ARAl, Y.T., YAMADA, K., KAMEOKA, Y., HORIMOTO, T., YAMAMOTO, K., YABE, S., NAKAYAMA, M., TASHIRO, M. (1997). Nucleoprotein gene analysis of fıxed and street rabies virus variants using RT-PCR. Arch. Viral. 142 : 1787-1796.
BAER, G.M. ( 1 990). Rab i es virus. In: Virus !rifections of Rzmıinantes . Ed. : Z. Din ter, B. Morein, Amsterdam: Elsevier, p. : 393-404.
BAER, G.M., BELLINI, W.J., FISHBEIN, D.B. ( 1 990). Rhabdovinıses In: Fields Virology. Ed.: B.N. Fields, D.M. Knipe, New York : Raven Press, p.: 883-930.
BLANCOU, J. ( 1 988). Epizootiology of rab i es : Eurasia and Africa. In : Rabies, Ed. : J.B. Campbell, K.M. Charlton, London : Kluwer Academic Publishers, p.: 243-299.
BLOOD, D.C., RADOSTITS, O.M. (1990). Veterinary Medicine. 7th Ed. London : Bailliere Tindall, p.: 919-925.
BOURHY, H., ROLLIN, P.E., VINCENT, J., SUREA U, P. ( 1 989). Conıparative field evaluation of the fluorescent antibody test, virus isolation from tissue culture and enzyıne immunodiagnosis for rapid laboratoı·y diagnosis of rabies. J. C/in. Microbiol. 27: 519-523 .
BUXTON, A., FRASER, G. ( 1977). Animal Microbiology. Edinburglı : Blackwell Scientific Publication, p.: 553-567.
DAVID, D., RUPPRECHT, C.E., SMITH, J ., SAMINA, 1., PERL, S., STRAM, Y. ( 1999). Human rabies in lsrael. Emerging li?f'ectious Disease.\·. 5 (2) : 306-308.
DA VIS, B. D., DULBECCO, R., E ISEN, H.N., GINSBERG, H.S. ( 1 990). Microbiology. 4tlı . Ed. Philadelphia: J .B. Lippincott Conıp. p.: 103 5-1043 . DEAN, D.J., ABELSETH, M.K., ATANASI U, P. ( 1 996). The tluorescent antibody test. In: Laboratvorı • Techniques in Rabies. Ed.: F.-X. MESLIN. M.M. Kaplan, H. Koprowski. 4th. Ed. Geneva : WHO. p.: 88-95 .
ERMINE, A., TORDO, N., TS JANG, H. ( 1 988). Rapid diagnosis of rabies infection by nıean s of a dot hybridization assay. Mo/. Ce//. Probes. 2 : 75 - 82.
FENNER, E., BACHMANN, P.A., GIBBS, E.P.J., MURPHY, F.A., STUDDERT, M.S., WHITE, D.O. (1987). Veterinary Virology. Ith. Ed. Orlanclo, Florida : Academic Press. p.: 534-541 .
FU, Z.F. ( 1 997). Rab i es and rab i es research : pa sı. present and future. Vaccine. 15 : 20 - 24.
HEATON, P.R., JOHNSTONE, P., McELHINNEY, L.M., COWLEY, R., O'SULLIVAN, E., WHITBY, J.E. ( 1 997). Heıninested PCR assay for detection of six genotypes of rabies and rabies related vinıses. Journal of Clinical 1\1/icrobiology. 35 (I I) : 2762-2766.
KAMOLVARIN, N., TIREWATNPONG, T., RATTANASIWAMOKE, R., TIREWATNPONG, S., PANPANICH, T., HEMACHUDHA, T. ( 1 993). Diagnosis of rabies by polynıerase chain reaction with nested prinı e rs. Journal of !nfectious diseases. I 67 : 207-21 O.
KING, A.A., TURNER, G.S. ( 1993). Rabies : A Review. J. Comp. Path. 108: 1-39.
KULONEN, K., FEKADU, M., WHITFIELD, S. , WARNER, C.K. (1999). An evaluatioıı of iıııınunofluorescence and PCR nıethod s detection of rabies in archival carııoy-fixed , paraftin eıııbedded brain tissue. Journal Vet. Med. B. 46 : 151-155.
McCOLL, K.A., GOULD, A.R., SELLECK, P.W., HOOPER, P.T., WESTBURY, H.A., SMITH, J.S. ( 1 993). Polynıerase chain reac tioıı and other l a boratoı·y techniques in the diagnos is of long ineabation rab i es in Austral ia. A ustralian Ve !erimttT Journal. 70 (3) : 84-89.
NAOIN-OA VIS, S.A. ( 1998). Polyınerase chain reaction protocols for rabies virus discriınination . Journal of Virologyca/ Methods. 75 : 1-8.
PERRIN, P., SUREAU, P. ( 1988). A new immunoenzymatic test for the rapid laboratoı·y diagnosis of rabies on field speciınens (RREID) In: Virus Diseases in Asia. Ed. : P. Thongcharoen, E. Kurstak. Bangkok : Mahidol University. p.: 345-348.
SACRAMENTO, 0., BOURHY, H., TOROO, N. ( 1991 ). PCR technique as an altenative method for diagnosis and malecular epidemiology of rabies virus. Mo/ecu/ar and Ce/lu/ar Probe.\·. 5 : 229-240.
T.C. SACLIK BAKANLIGI. (1998). Dünya Sağlık Raponı. Çeviri Ed. B. Metin, A. Akm, i. Güngör. Ankara : Dış İlişkiler Dairesi Başkanlığı Yayınları. s.: 53-54.
TSIANG, H. (1995). Current concepts in Rabies. Bul/. lns. Pasteur. p.: 2-4.
WEBSTER, W.A., CASEY, G.A. (1988). Diagnosis of rabies infection. In: Rabies. Ed. : J.B. Camp- beli, K.M. Charlton. London Kimver Acadenıic Publ ishers. p.: 201-221.
WHITBY, J.E., HEATON, P.R., WHITBY, 1-I.E., O'SULLIVAN, E., JOHNSTüN E, P. ( 1997a). Rapid detection of rabies and rabies related viruses by using RT-PCR and enzyme-linked imımınodorbent assay . .Journal of Virologica/ Methods . 69 : 63- 72.
WHITBY, J.E., JOHNSTONE, P. , SILLEROZUBIRI, C. ( 1997b). Rabies virus in the elecanıposed b ra in of an Ethiopian Wolf detec tecl by Nes ı ed RT-PCR. Journal of Wi/d/ife Disease.\. 33 (.f ) .· 912- 915 .
WHITBY, J.E., HEATON, P.R., BLACK, E.IVI., WOOLORIOGE, M., IVIcELHINNEY, L.IVI. , JOHNSTONE, P. (2000). First isolation of a rabie srelated virus from a Daubentoıı ' s bat in the United Kingdoın. Veterinarv Record. 147: 385-388.
WORLO HEALTH ORGANIZATION. (1992) W.H.O. Expert Coınmittee on Rabies, 8'ıı report . Geneva : W. H. O. Technica/ Series. No. 824 .