M. Özgür ÖZYİĞİT, M. Müfit KAHRAMAN, Ezgi AKDEŞİR

Sığırlarda Mycobacterium bovis’in moleküler ve sito-histopatolojik tanı yöntemleri ile gösterilmesi ve sonuçlarının karşılaştırılması

Bu çalışmada tüberkülin deri testi (PPD) pozitif 30 baş sığırda Mycobacterium bovis’in varlığının klasik ve moleküler teknikler ile ortaya konması ve sonuçların birbirleri ile karşılaştırılması amaçlanmıştır. Klasik yöntemler organların makroskobik muayenesi, örneklenen dokuların Hematoksilen-Eozin (HE), Ziehl Nilseen (ZN) ve immunohistokimya (İHK) ile mikroskobik, dokuda polimeraz zincir reaksiyonu (PCRdoku) ile moleküler; toplanan kan örneklerinin buffy coat’ta immunositokimya (İSKbc) ile mikroskobik, buffy coat’ta polimeraz zincir reaksiyonu (PCRbc) ile moleküler incelemelerinden oluşmuştur. Makroskopide 26 tipik tüberkül gözlenirken, mikroskopide HE ile 27, ZN ile 20 doku tüberkülozla uyumlu bulunmuştur. Akciğer, mediastinal lenf düğümü ve karaciğerden yapılan İHK’da 26,PCRdoku’da 18;İSKbc’de 3, PCRbc’de ise 14 pozitif sonuç elde edilmiştir. İHK pozitif boyanmış etkenlere, makrofaj, dev hücresi, lenfositler ve fibrositlerin sitoplazmalarında rastlanmıştır; İSKbc pozitif reaksiyon ise mononükleer lökositlerin sitoplazmalarında görülmüştür. ZN boyamanın her zaman etkeni göstermediği; İHK’ya kıyasla spesifik olmadığı ve doğrulamak için diğer yöntemlere ihtiyaç olduğu görülmüştür. Mycobacterium bovis’e ait moleküllerin varlığı dokuda (PCRdoku) gösterilmiş ancak, bu molekülleri saptamaya yönelik yöntemlerin sığır tüberkülozunun tanısında kullanılma duyarlılığı, İHK yöntemine kıyasla, düşük bulunmuştur. Yöntemler arasında İHK’nın, PCRbc’ye ve İSKbc’ya kıyasla duyarlılığının (Mc Nemar; sırasıyla p

Demonstration of Mycobacterium bovis in cattle via molecular and cytohistopathological diagnostic tecniques and comparison of the results

In this study the detection of Mycobacterium bovis with classical and molecular techniques and comparisons of the test results in the thirty slaughtered cattle positive to intradermal tuberculin test (PPD) was aimed. The classical methods include macroscopic examination of the organs, microscopic examination of tissues and blood samples that were stained by HE, ZN, immunohistochemistry and immunocytochemistry and PCR as molecular investigation. Characteristic tubercle development was observed in 26 animals’ macroscopic examination yet 27 and 20 cases were found compatible with tuberculosis in HE and ZN respectively. Tuberculosis positive results were also obtained from lungs, mediastinal lymph nodes and liver via immunohistochemistry (IHC), PCRtissue, immunocytochemistry of the buffy coat (ICCbc) and PCR of the buffy coat (PCRbc) in 26, 18, 3 and 14 cows respectively. The agents were observed extracytoplasmically and intracytoplasmically in macrophages, giant cells, lymphocytes and fibrocytes in IHC. ICCbc positivity was observed within the cytoplasm of mononuclear leukocytes. Furthermore, the agent was not always detected via ZN staining, which verifies the necessity of ancillary tests. Among these methods IHC has revealed significant sensitivity compared to those of PCRbc and ICCbc (Mc nemar; p

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1. Ayele WL, Neill SD, Zinsstag J, et al.(2004): Bovine tuberculosis: An old disease but a new threat to Africa. Int J Tuberc Lung Dis, 8, 924-937.
2. Boros DL(2003): Granulomatous infections and inflammations, cellular and molecular mechanisms. 1-21. American Society for Microbiology Press, Washington DC.
3. Brosch R, Gordon SV, Marmıesse M, et al. (2002): A new evolutionary scenario for the mycobacterium tuberculosis complex. Proc Natl Acad Sci, 99, 3684-3689.
4. Buchwalow IB, Böcker W (2010):Immunohistochemistry: Basics and methods. 1-66. Springer Verlag, Heidelberg.
5. Caminiti A, Pelone F, LaTorre G, et al.(2016): Control and eradication of tuberculosis in cattle: a systematic review of economic evidence. Vet Rec, 179, 70-75
6. Cartun RW. (2010): Immunohistochemistry of infectious diseases. J Histotechnol, 3,195-202.
7. Cassidy JP, Bryson DG, Pollock JM, et al.(1999):Lesions in cattle exposed to mycobacterium bovis-inoculated calves. J Comp Pathol, 121, 321–337.
8. Caswell Jeff L, Williams Kurt J(2016): Respiratory System.465-590. In: KVF Jubb, PC Kennedy, NC Palmer(Ed), Pathology of Domestic Animals, 6th edition, Elsevier Saunders, St. Louis, Missouri.
9. CondosR, McClune A, Rom WN, et al. (1996): Peripheral blood based PCR assay to identify patients with active pulmonary tuberculosis.The Lancet, 347, 1082-1085.
10. Divangahi M(2013): The new paradigm of immunity to tuberculosis. 238-239. Springer Science Business Media, New York.
11. Gutierrez Cancela MM,Garcia Marin JF(1993): Comparison of Ziehl-Neelsen Staining and Immunohistochemistry for the Detection of Mycobacterium bovis in Bovine and Caprine Tuberculous Lesions. J Comp Path, Vol. 109,361-370.
12. Hermans PW, Schuitema AR, Van Soolıngen D, et al. (1990): Specific Detection of Mycobacterium tuberculosis Complex Strains by Polymerase Chain Reaction. J Clin Microbiol, 28, 1204-1213.
13. Hernandez-Pando R, Jeyanathan M, Mengistu G, et al.(2000): Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection. The Lancet, 356, 2133-2138.
14. Hirsch DC, Zee YC (1999): Veterinary Microbiology,158-164. Blackwell Science, Massachusetts.
15. Huard RC, Lazzarini LC, Butler WR, et al. (2003): PCR based method to differentiate the subspecies of the mycobacterium tuberculosis complex on the basis of genomic deletions. J Clin Microbiol, 41, 1637–1650.
16. Hunter RL, Ja gannath C, Actor JK (2007):Pathology of postprimary tuberculosis in humans and mice: contradiction of long-held beliefs. Tuberculosis, 87, 267-278.
17. Jabbar A, Khan J, Ullah A, et al. (2015): Detection of mycobacterium tuberculosis and mycobacterium bovis from human sputum samples through multiplex PCR. J Pak Pharm Sci, 28,1275-1280.
18. Kamerbeek J, Schouls L, Kolk A, et al. (1997):Simultaneous detection and strain differentiation of mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol, 35, 907–914.
19. Kaufman S, Medzhitov R, Gordon S (2004):The innate immune response to infection. 444-448. ASM Press, Washington.
20. Kaufmann S, Walker BD (2009):AIDS and Tuberculosis. 34-39. Infection Biology Handbook Series, Wiley-VCH Verlag, Weinheim.
21. Kaya A, İçen H, Tuzcu N, et al. (2015): Comparison of tuberculin skin test, IFN-γ assay, Real Time PCR and Lateral Flow Rapid Test in diagnosis of field outbreaks of bovine tuberculosis.Kafkas Univ Vet Fak Derg, 21,739-743.
22. Kumararatne DS (1997): Tuberculosis and immunodeficiency-of mice and men. Clin Exp Immunol, 107, 11-14.
23. Luna LG(1968):Manual of histologic staining methods of the Armed Forces Institute of Pathology.32-46. Blakiston Division, McGraw-Hill; 3rd edition, New York.
24. Mazurek GH, Lobue PA, Daley CL, et al. (2001):Comparison of a whole-blood interferon γ assay with tuberculin skin testing for detecting latent mycobacterium tuberculosis ınfection.JAMA, 286, 1740-1747.
25. Mirza S, Restrepo BI, McCormik JB, et al. (2003):Diagnosis of tuberculosis lymphadenitis using a polymerase chain reaction on peripheral blood mononuclear cells. Am J Trop Med Hyg, 69, 461-465.
26. Moter A, Göbel UB (2000):Fluorescence in situ hybridisation for direct visualization of microorganism. J Microbiol Methods, 41,85-112.
27. O’ Reilly LM, Daborn CJ (1995): The epidemiology of Mycobacterium bovis infections in animals and man: a review. Tuber Lung Dis, 76, 1-46.
28. O’tole D, Li H, Miller D, Williams WR, et al. (1997):Chronic and recovered cases of sheep associated malignant catarrhal fever in cattle.Vet Rec, 140, 519-524.
29. Parsons LM, Brosch R, Cole ST, et al.(2002): Rapid and simple approach for identification of mycobacterium tuberculosis complex isolates by pcr-based genomic deletion analysis. J Clin Microbiol, 40, 2339–2345.
30. Pritchard DG (1988):A century of bovine tuberculosis 1888-1988: conquest and controversy. J Comp Pathol, 99, 357-399.
31. Purohit MR, Tehmina M, Wiker HG, et al. (2007). Immunohistochemical diagnosis of abdominal lymph node tuberculosis by detecting Mycobacterium tuberculosis complex specific antigen MPT64. Diagn Pathol, 40, 782-791.
32. Russel DG(2007): Who puts the tubercle in tuberculosis.Nat Rev Microbiol, 5, 39-47.
33. Ryan TJ, Buddle BM, De Lisle GW(2000). An evaluation of the gamma interferon test for detecting bovine tuberculosis in cattle 8 to 28 days after tuberculin skin testing. Res Vet Sci,69, 57-61.
34. Sachse K, Frey J(2002): PCR detection of microbial pathogens: Methods and protocols.201-221. Humana Pres, New Jersey.
35. Schiller I, Oesch B, Vordermeier HM, et al. (2010): Bovine Tuberculosis: A Review of Current and Emerging Diagnostic Techniques in View of their Relevance for Disease Control and Eradication. Transbound Emerg Dis, 57, 205–220.
36. Seiler P, Ulrichs T, Bandermann S, et al. (2003): Cell wall alterations as an attribute ofmycobacterium tuberculosis in latent infection. J Infect Dis, 188, 1326-1331.
37. Sheridan MP, Browne JA, Doyle MB, et al. (2017):IL-10 suppression of IFN-γ responses in tuberculin-stimulated whole blood from Mycobacteriumbovis infected cattle. Vet Immunol Immunopathol, 189, 36-42.
38. Smith NH, Gordon SV, Rua-Domenech R, et al. (2006):Bottlenecks and broomsticks: the molecular evolution of mycobacterium bovis. Nature, 4, 670-681.
39. Stender H, Lund K, Petersen KH, et al. (1999): Fluorescence in situ hybridization assay using peptide nucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of mycobacterium cultures. Clin Microbiol Infect, 37, 2760-2765.
40. Sumi S, Radhakrishnan VV (2009):Evaluation of immunohistochemistry with a panel of antibodies against recombinant mycobacterial antigens for the diagnosis of tuberculous lymphadenitis. Int. J. Med. Med. Sci, 1, 215-219.
41. Tehmina M, Wiker HG, Mfinanga SGM, et al. (2006): Immunohistochemistry using a Mycobacterium tuberculosis complex specific antibody for improved diagnosis of tuberculous lymphadenitis.Mod Pathol, 19, 1606-1614.
42. Thierry D, Cave MD, Eisenach KD, et al. (1990): IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res, 18,188.
43. Tüzün-İnce A, Güneş P¸ Şenateş E, et al. (2011):Can an immunohistochemistry method differentiate intestinal tuberculosis from crohn’s disease in biopsy specimens?Dig Dis Sci, 56, 1165– 1170.
44. Whelan C, Shuralev E, O’keeffe G, et al. (2008):Multiplex immunoassay for serological diagnosis of mycobacterium bovis infection in cattle. Clin Vaccine Immunol, 12, 1834–1838.
45. vanCrevel R, Ottenhoff TH, van der Meer JWM(2002): Innate immunity to mycobacterium tuberculosis. Clin Microbiol Rev, 15, 294-309.
46. Vitale F, Capra G, Maxia L, et al.(1998): Detection of Mycobacterium tuberculosis complex in cattle by pcr using milk, lymph node aspirates, and nasal swabs. J Clin Microbiol, 36,1050–1055.
47. Zumarraga MJ, Meikle V, Bernardelli A, et al. (2005): Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection. J Vet Diagn Invest, 17, 232–238.