The Detection of Brucella sp. and Leptospira sp. in Cattle by Multiplex Polymerase Chain Reaction mPCR

Öz

In this study, blood samples obtained from 200 cattle in Aydın Region were used as material. Following DNA extractions, identification of the specific genes related with Brucella and Leptospira was carried out by using multiplex PCR method. Results obtained from this study revealed that Brucella sp. and/or Leptospira sp. were detected from 77 38.5% samples investigated. Bands related with both Brucella sp. and Leptospira sp were detected 24 31.2% out of 77 mPCR positive samples. From the remaining 53 68.8% samples, 33 samples % 62.3 showed positive bands only related with Brucella sp. and 20 samples 37.7% showed positive bands only related with Leptospira sp. The main conclusion of this study was the use of multiplex PCR as a reliable, sensitive and fast technique could prevent the hazards originated from contamination and false results caused by conventional and serum analysis methods in detection of Brucella and Leptospira.

Anahtar Kelimeler

• Brucella sp.,Leptospira sp.,Multipleks PCR

Sığırlarda Brucella ve Leptospira Türlerinin Multiplex Polimeraz Zincir Reaksiyonu mPCR ile Tanımlanması

Öz

Bu çalışmada Aydın ilindeki sığır sürülerinden 200 adet kan örneği materyal olarak toplandı. Bu kan örneklerinden DNA ekstraksiyonları yapıldıktan sonra Brucella ve Leptospira bakterilerine ait spesifik genlerin multipleks PCR yöntemi ile identifikasyonu yapıldı. Yapılan multipleks PCR sonucunda 200 örneğin 77 %38.5 ’sinde Brucella sp. ve Leptospira sp. tespit edilmiştir. mPCR pozitif toplam 77 örneğin 24 %31.2 ’ünde hem Brucella sp.’ne hem de Leptospira sp.’ne ait bantlar ortak olarak görülmüştür. Kalan 53 %68.8 örneğin 33 %62.3 ’ü sadece Brucella sp. ve 20 % 37.7 ’si ise sadece Leptospira sp. için pozitif bulunmuştur. Sonuç olarak çalışmada konvansiyonel mikrobiyolojik kültür yada serum analiz teknikleri ile Brucella ve Leptospira türlerinin tayini konusunda meydana gelen hatalı sonuçların ve kontaminasyon tehlikesinin hassas ve hızlı bir teknik olan multiplex PCR yöntemi ile elimine edilmiştir. Ayrıca her iki bakteri türünün identifikasyonunda da zaman kaybetmeden güvenilir sonuç alınabilecektir.

Anahtar Kelimeler

• Brucella sp.,Leptospira sp.,Multipleks PCR

Kaynakça

1. Ana M, Hugo A. (1997). Barrera-Saldaina Molecular Medicine, Volume 3, Number 11, November 1997 734-739.
2. Arda M, Minbay A, Leloğlu N, Aydın N, Kahraman M, Akay Ö, Ilgaz A, İzgür M, Diker KS (1997). Özel Mikrobiyoloji Gram Negatif Küçük Çomaklar Brusella İnfeksiyonları, Medisan Yayın Serisi No:26, 4. Baskı 110-124.
3. Bricker BJ, Ewalt DR, MacMillan AP, Foster G, Brew S (2000). Molecular characterization of Brucella strains isolated from marine mammals. Journal of Clinical Microbiology, 38, 1258-1262.
4. Cetinkaya B, Ongor H, Muz A, Ertas HB, Kalender H, Ergogan HM (1999). Detection of Brucella species DNA in the stomach content of aborted sheep fetuses by PCR. Veterinary Record, 144, 239–240.
5. Dieffenbach CW, Dveksler GS (1995). PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, p. 714.
6. Fekete A, Bantle AJ, Halling SM (1990). Preliminary development of a diagnostic test for Brucella using polymerase chain reaction. Journal of Applied Bacteriology, 69, 216–227.
7. Ficht TA, Husseinen HS, Derr J, Bearden SW (1996). Species-specifıc sequences at the omp2 locus of Brucella type strains. International Journal of Systematic Bacteriology, 46, 329-331.
8. Heinemann MB, Garcia JF, Nunes CM, Gregori F, Higa ZM, Vasconcellos SA, Richtzenhain LJ (2000). Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism. Veterinary Microbiology, 73, 261–267.

9. Jones LM, Montgomery V, Wilson JB (1965). Characteristics of carbon dioxide-independent cultures of Brucella abortus isolated from cattle vaccinated with strain 19. Journal of Infectious Diseases, 115:312–320.
10. Levett PN (2005). Leptospirosis Principles and Practice of Infectious Diseases 6th edition. New York, Churchill Livingstone 2005;2789- 2794.
11. Me´rien F, Amouriaux P, Perolat P, Baranton G, Saint Girons I (1992). Polymerase chain reaction for detection of Leptospira spp. in clinical samples. Journal Clinical Microbiology, 30, 2219–2224.
12. Moriyo´n I, Berman DT (1982). Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope. Journal of Bacteriology, 152, 822–828.
13. Nicoletti P (1980). The epidemiology of bovine Brucellosis. Advances in Veterinary Science & Comparative Medicine, 24, 69-98.
14. Rijpens NP, James G, Van Asbroeck M, Rossau R, Herman L (1996). Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. Applied and Environmental Microbiology, 62,1683-1688.
15. Romero C, Gamzo C, Pardo M, Lo´pez-Goni I (1995a). Specific detection of Brucella DNA by PCR. Journal Clinical Microbiology, 33, 615–617.
16. Romero C, Pardo M, Grillo MJ, Diaz R, Blasco JM, Lo´pez-Goni I (1995b). Evaluation of PCR and indirect enzyme-links immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle. Journal Clinical Microbiology, 33, 3198–3200.
17. Saad A, Phuong T, Pamela S, Howard CJ (1997). A Polymerase Chain Reaction Assay for the Detection of Leptospira spp. in Bovine Semen. Journal of Veterinary Research, 61, 15-20.
18. Sangari FJ, Garcı´a-Lobo JM, Aguero J (1994). The Brucella abortus vaccine strain B19 carries a deletion in the erythritol catabolic genes. FEMS Microbiology Letters, 121,337-342.
19. Savio ML, Rossi C, Fusi P, Tagliabue S, Pacciarini ML (1994). Detection and identification of Leptospira interrogans serovars by PCR coupled with restriction endonuclease analysis of amplified DNA. Journal Clinical Microbiology, 32, 935–941.
20. Smith LD, Ficht TA (1990) Patogenesis of Brucella. Critical Reviews in Microbiology, 17, 209.
21. Van Eys GJJM, Gravekamp C, Gerritsen MJ, Quint W, Cornelissen MTE, Ter Schegget J, Terpstra WJ (1989). Detection of leptospires in urine by polymerase chain reaction. Journal Clinical Microbiology, 27, 2258–2262.
22. Verger JM, Grimont F, Grimont PAD, Grayson M (1985). Brucella, a monospecific genus as shown by deoxyribonucleic acid hyhridization. International Journal of Systematic Bacteriology, 35, 292-295.
23. Woodward MJ, Sullivan J, Palmer NMA, Woolley JC, Redstone JS (1991). Development of a PCR test specific for Leptospira hardjo genotype bovis. Veterinary Record, 128, 282-283.
24. Woodward MJ, Redstone JS (1993). Differentiation of Leptospira serovars by the polymerase chain reaction and restriction fragment length polymorphism. Veterinary Record, 132, 325-326