مطالعه کارآیی روش تشخیصی آزمایش مستقیم میکروبی، PCR900IS و کشت میکروبی در تشخیص باکتری مایکوباکتریوم اویوم تحت گونه پاراتوبرکلوزیس در مدفوع گاوهای به ظاهر سالم

محورهای موضوعی : آسیب شناسی درمانگاهی دامپزشکی

یونس انزابی ¹ , صمد فراشی بناب ² , غلامعلی مقدم ³

1 - گروه پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه آزاد اسلامی واحد تبریز، تبریز، ایران
2 - گروه میکروبیولوژی و ایمونولوژی، دانشکده دامپزشکی، دانشگاه تهران، تهران، ایران
3 - گروه علوم درمانگاهی، دانشکده دامپزشکی، دانشگاه تبریز، تبریز، ایران

تاریخ دریافت : 1387/10/29 تاریخ پذیرش : 1388/01/31 تاریخ انتشار : 1387/12/01

کلید واژه: PCR, یماری یون, آزمایش مستقیم میکروبی, کشت مدفوع, مایکوباکتریوم اویوم تحت‌گونه پاراتوبرکلوزیس,

چکیده مقاله :

بیماری یون یا پاراتوبرکلوزیس نوعی آنتریت گرانولوماتوزی مزمن در نشخوارکنندگان با عامل مسبب مایکوباکتریوم اویوم تحت گونه پاراتوبرکلوزیس (MAP) می باشد و خسارات اقتصادی فراوانی به صنعت دامپروری به‌خصوص گاوداری شیری در سراسر دنیا وارد می کند. تشخیص MAP در نمونه های بالینی با روش کشت میکروبی به عنوان روش استاندارد طلایی به 16-6 هفته زمان نیاز دارد. از طرف دیگر شناسایی سریع و دقیق گاوهای دفع کننده میکروب فوق در مدفوع برای کنترل موفق بیماری در گله لازم است. در این تحقیق، بر روی مدفوع 100 رأس گاو به ظاهر سالم از گاوداری های صنعتی تبریز با سابقه بیماری یون، آزمایش مستقیم میکروبی با رنگ آمیزی ذیل نلسن، کشت میکروبی در محیط زرده تخم مرغ هرولد و دو روش direct PCRبر پایه عنصر 900IS انجام شد. در آزمایش مستقیم میکروسکوپی 7 نمونه (7 درصد)، در کشت میکروبی 14 نمونه (14 درصد)، در PCR با جفت پرایمر 91F/90F 15 نمونه (15 درصد) و در PCR با جفت پرایمر 26FP/25FP 25 نمونه (25 درصد) مثبت ثبت شدند. این نتایج نشان داد روش PCR تعداد موارد مثبت بیشتری را شناسایی می کند، بنابراین می توان از آن برای شناسایی سریع و در عین حال دقیق تر گاوهای دفع کننده MAP در مدفوع استفاده کرد. هم‌چنین نوع پرایمر در حساسیت تست PCR نقش مهمی ایفا می کند.

چکیده انگلیسی:

     Johne’s disease or paratuberculosis is a chronic granulomatous enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease is responsible for significant economic losses in dairy industry worldwide.  Microbial culture as a golden standard test for detection of MAP in faecal specimens requires 6-16 weeks to complete, whereas accurate and rapid identification of cattle shedding MAP in their feces is essential for successful control of the disease in herds. In the present study, direct microbial diagnosis by Ziehl-Neelsen acid-fast staining, microbial culture on Herrolds’ egg yolk media and two IS900 direct PCR assays were carried out on 100 fecal specimens of apparently healthy cattle collected from dairy herds of Tabriz with a history of Johne’s disease. The number of positive specimens identified by the direct microbial diagnosis, microbial culture and PCR with F90/F91 and FP25/FP26 primes were 7 (7%), 14 (14%), 15(15%) and 25(25%) respectively. These results indicated that PCR detected more positive cases therefore it can be employed for rapid and accurate diagnosis of cattle shedding MAP it their feces and the type of primer used has a significant role in the sensitivity of this test. Direct microbial diagnosis by Ziehl-Neelsen acid-fast staining identified 7 (7%) specimens, two IS900 direct PCR assays identified 15 (15%) and 25 (25%) specimens, respectively, and microbial culture identified 7 (7%) specimens as positive. Collectively, these data indicate that PCR detection of MAP was more sensitive than direct microbial diagnosis by Ziehl-Neelsen acid-fast staining or faecal culture, especially if appropriate primers were used.

منابع و مأخذ:

1.    1.   طباطبائی، ع.ح. و فیروزی، ر. (1380): بیماری­های باکتریائی دام. انتشارات دانشگاه تهران- صفحات: 423- 414.


2. Bhide, M., Chakurkar, E., Tkacikova, L., Barbuddhe, S., Novak, M., and Mikula, I. (2006). IS900 PCR-based detection          and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep. Veterinary Microbiology. 112: 33–41.


3. Clarke, C.J. (1997). The pathology and pathogenesis of paratuberculosis in ruminants and other species. J. Comp. Pathol. 116: 217–261.


4.  Clark, D.L.Jr., Koziczkowski, J.J., Radcliff, R.P., Carlson, R.A., and Ellingson, J.L.E. (2008). Detection of Mycobacterium avium Subspecies paratuberculosis: Comparing Fecal Culture Versus Serum Enzyme-Linked Immunosorbent Assay and Direct Fecal Polymerase Chain Reaction. J. Dairy Sci. 91: 2620-2627.


5. Cocito, C., Gilot, P., Coene, M., de Kesel, M., Poupart, P., and Vannuffel, P. (1994). Paratuberculosis. Clin. Microbiol. Rev. 7: 328–345.


6. Corti, S., and Stephan, R. (2002). Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland. BMC Microbiol. 2: 15.


7. Crossley, B.M., Zagmutt-Vergara, F.J.,  Fyock, T.L., Whitlock, R.H., and Gardner, I.A. (2005): Fecal shedding of Mycobacterium avium subsp. paratuberculosis by dairy cows. Veterinary Microbiology. 107: 257–263.


8. Dieguez, F.G., Gonzalez, A.M., Menendez, S., Vilar, M.J., Sanjuan, M.L., Yus, E., and Arnaiz, I. (2007). Evaluation of four commercial serum ELISAs for detection of Mycobacterium avium subsp. paratuberculosis infection in dairy cows. The Veterinary Journal. 56: 243-252.


9. Englund, S., Bolske, G., Ballagi-Pordany, A., and Johansson, K.E. (2001). Detection of Mycobacterium avium subsp. paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene. Veterinary Microbiology. 81:  257–271.


10. Englund, S., Bolske, G., and Johansson, K.E. (2002). An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis. FEMS Microbiology Letters. 209: 267-271.           


11. Garrido, J.M., Cortabarria, N., Oguiza, J.A., Aduriz, G., and Juste, R.A. (2000). Use of a PCR method on fecal samples for diagnosis of sheep paratuberculosis. Veterinary Microbiology. 77: 379-386.


12. Giese, S.B., and Ahrens, P. (2000). Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture. Veterinary Microbiology. 77: 291-297.


13. Glanemann, B. (2003). Detection of Mycobacterium avium subspecies paratuberculosis in Swiss Dairy Cattle by Culture and Serology. internete, xxxxx, 1-20.


14. Greenstein, R.J. (2003). Is Crohn’s disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne’s disease. THE LANCET Infectious Diseases. 3: 507-514.


15. Greenstein, R.J.; Collins, M.T. (2004). Emerging pathogens: is Mycobacterium avium subspecies paratuberculosis zoonotic? Lancet. 31 (364): 396–397.


16. Harris, N.B. and Barletta, R.G. (2001). Mycobacterium avium subsp. paratuberculosis in veterinary medicine. Clin. Microbiol. Rev. 14: 489-512.


17. Huntley, J.F.J., Whitlock, R.H., Bannantine, J.P., and Stabel, J.R. (2005): Comparison of Diagnostic Detection Methods for Mycobacterium avium subsp. paratuberculosis in North American Bison. Vet Pathol. 42: 42–51.


18. Irenge, L.M., Walravens, K., Govaerts, M., Godfroid, J., Rosseels, V., Huygen, K., and Gala, J. (2008): Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples. Veterinary Microbiology. xxx: xxx–xxx.


19. Klausen, J., Huda, A., Ekeroth , L., and Ahrens, P. (2003). Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle. Preventive Veterinary Medicine. 58: 171–178.


20. Kudahl, A.B., Sorensen, J.T., Nielsen, S.S., and Ostergaard, O. (2007). Simulated economic effects of improving the sensitivity of a diagnostic test in paratuberculosis control. Preventive Veterinary Medicine. 78: 118–129.


21. Lilenbaum, W., Marassi, C.D., and Oelemann, W.M.R. (2007). Paratuberculosis: an update. Braz. J. Microbiol. 38: 580-590.


22. Nielsen, S.S., and Toft, N. (2008). Ante mortem diagnosis of paratuberculosis: A review of accuracies of ELISA, interferon-γ assay and faecal culture techniques. Veterinary Microbiology. 129:  217–235.


23. Manning, E.J.B., and Collins, M.T. (2001). Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis. Rev. Sci. Tech. Off. Int. Epiz. 20: 133–150.


24. Millar, D., Ford, J., Sanderson, J., Withey, S., Tizard, M., Doran, T., and Hermon-Taylor, J. (1996). IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cow' milk in England and Wales. Appl. Environ. Microbiol. 62: 3446-3452.


25. Pillai, S.R., and Jayarao, B.M. (2002). Application of IS900 PCR for Detection of Mycobacterium avium subsp. paratuberculosis Directly from Raw Milk. J. Dairy Sci. 85: 1052–1057.


26. Pinedo, P.J., Rae, D.O., Williams, JE., Donovan, G.A., Melendez, P.  and Buergelt, CD. (2008). Association among Results of Serum ELISA, Faecal Culture and Nested PCR on Milk, Blood and Faeces for the Detection of Paratuberculosis in Dairy Cows. Transboundary and Emerging Diseases. 55 (2): 125–133.


27. Rajeev, S., Zhang, Y., Sreevatsan, S., Motiwala, A.S., and Byrum, B. (2005). Evaluation of multiple genomic targets for identification and confirmation of Mycobacterium avium subsp. paratuberculosis isolates using real-time PCR. Veterinary Microbiology. 105: 215–221.


28. Reddacliff, L.A., Vadali, A., and Whittington, R.J. (2003). The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated from tissues and faeces. Vet. Microbiol. 95: 271-282.


29. Romero, C., Hamdi, A., Valentine, J.F. and Naser, S.A. (2005). Evaluation of surgical tissue from patients with Crohn’s disease for the presence of Mycobacterium avium subspecies paratuberculosis DNA by in situ hybridization and nested polymerase chain reaction. Inflamm. Bowel. Dis. 11: 116–125.


30. Shamoradi, A.H., Arefpajohi, R., Tadayon, K., and Mosavari, N. (2008). Paratuberculosis in Holstein-Friesian cattle farms in Central Iran. Trop. Anim. Health Prod. 40: 169–17.


31. Sharma, G., Singh, S.V., Sevilla, I., Singh, A.V., Whittington, R.G., Juste, R.A., et al. (2008). Evaluation of indigenous milk ELISA with m-culture and m-PCR for the diagnosis of Bovine Johne’s disease (BJD) in lactating Indian dairy cattle. Research in Veterinary Science. 84: 30–37.


32. Singh, A.V., Singh, S.V., Makharia, G.K., Singh, P.K., and Sohal, J.S. (2007). Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn’s disease and in the healthy human population in India. Int. J. Infect. Dis. 18: 254-258.


33. Singha, S.V., Singha, A.V. Singhb, R., Sandhub, K.S., Singha, P.K.,  Sohala, J.S., et al. (2007). Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne’s disease (BJD) in India. Comparative Immunology, Microbiology & Infectious Diseases. 30: 175–186.


34. Sola, C., Filliol, I., Legrand, E., Lesjean, S., Locht, C., Supply, P., Rastogi, N. (2003). Genotyping of the Mycobacterium tuberculosis complex using MIRUs: association with VNTR and spoligotyping for molecular epidemiology and evolutionary genetics. Infection, genetics and evolution. 3: 125-133.


35. Stabel, J.R., Bosworth, T.L., Kirkbride, T.A., Forde, R.L., and Whitlock, R.H. (2004). A simple, rapid, and effective method for the extraction of Mycobacterium paratuberculosis DNA from fecal samples for polymerase chain reaction. J Vet Diagn Invest. 16: 22–30.


36. Taddei, S., Robbi, C., Cesena, C., Rossi, I., Schiano, E., Arrigoni, N., Vicenzoni, G. and Cavirani, S. (2004). Detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples: comparison of three polymerase chain reaction–based diagnostic tests with a conventional culture method. J Vet Diagn Invest. 16: 503–508.


37. Wells, S.J., Collins, M.T., Faaberg, K.S., Wees, K., Tavornpanich, S., Petrini, K.R., Collins, J.E., Cernicchiaro, N., and Whitlock, R.H. (2006). Evaluation of a Rapid Fecal PCR Test for Detection of Mycobacterium avium subsp. Paratuberculosis in Dairy Cattle. Clinical and Vaccine Immunology. 13 (10): 1125–1130.


38. Zimmer, K., Drager, K., Klawonn, W., and Hess, R. (1996). Contribution to the diagnosis of Johne’s disease in cattle. Comparative studies on the validity of Ziehl– Neelsen staining, faecal culture and a commercially available DNA-probe test in detecting Mycobacterium paratuberculosis in faeces from cattle. Zentralbl. Veterinarmed. [B]. 46 (2): 137–140.