HAYDAR ÖZPINAR, İSMAİL HAKKI TEKİNER, Olcay KARAMAN, Yunus KURT

Investigation of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal and bulk milk samples from dairy farms in Thrace region of Turkey

Trakya Türkiyenin Avrupa topraklarında olup, Güney Bulgaristan ve Kuzeydoğu Yunanistan sınırlarının keşiştiği bir bölgedir. FAO 1999 yılında bu bölgeyi zoonozları erken önlemek bakımından stratejik önemi olan Avrupa aşılama tampon bölgesi olarak duyurmuştur. Mycobacterium avium subsp. paratuberculosis (MAP) varlığı Trakyada net şekilde anlaşılamamıştır. Bu çalışmada zoonozlardan ari olarak kabul edilen Trakyada MAP varlığını F57 Real time PCR assay ve IS900 nested-PCR yöntemlerini uygulayarak incelemek amaçlandı. Araştırmada, rastgele seçilen 30 adet süt işletmesinde 2 yaş ve üzeri sığırlardan 270 adet fekal örnek ile bu işletmelerin ve bulundukları köylerin süt toplama tanklarından 45 adet çiğ süt örnekleri toplandı. Bir süt işletmesini temsilen 9 adet fekal örnek tek havuz örnek olacak şekilde karıştırıldı. Örneklerden DNA izolatları elde edildi. İzolatlara F57-Real time PCR assay uygulandı. Elde edilen sonuçların IS900 nested-PCR yöntemi kullanılarak doğrulaması yapıldı. Sonuç olarak, havuz fekal örnekleri ve çiğ süt örneklerinde MAP genomu tespit edilmedi. Trakya bölgesinde MAP varlığının F57 -Real time PCR yönteminin tespit sınırının altında kaldığı sonucuna varıldı ve asemptomatik hayvanlar ve süt örneklerinde yalnızca PCR yöntemi ile MAP tespitinin zorluğu doğrulanmış oldu.

Türkiye Trakya Bölgesindeki süt işletmelerinden toplanan fekal ve çiğ süt örneklerinde Mycobacterium avium subsp. paratuberculosis (MAP) incelemesi

The Thrace, which is Turkeys European part as well as the adjoining parts of Southern Bulgaria and north-east Greece has a strategic importance for being a vaccination bufer zone for Europe as declared by FAO in 1999. The objective of the study was to better understand the occurence of MAP in this animal disease free area of Turkey by applying F57 Real time PCR assay and IS900 nested-PCR. In this study, 270 feces samples from the dairy cattles over 2 years old in 30 randomly selected dairy farms, 45 raw milk samples from each of the bulk tanks belonging to these dairy farms and the villages located in Thrace were collected. Nine fecal samples were used to create the pooled fecal sample for a dairy farm before performing DNA extraction. All the samples were initially subjected to a F57 -Real time PCR analysis, and subsequently an insertion sequence IS900 nested-PCR was performed to verify the results. However, the results revealed that MAP genom could not be detected in any pooled fecal and milk samples. In conclusion, the occurrence of MAP in this part of Turkey may likely be very lower than the capability of the detection limit of the used Real time PCR assay. Furthermore, the results once more confirmed the difficulty of MAP detection in asymptomatic animals and milk samples by performing PCR technique only.

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1. Berger ST, Griffin FT: A comparison of ovine monocyte-derived macrophage function following infection with Mycobacterium avium ssp. avium and Mycobacterium avium ssp. Paratuberculosis . Immunol Cell Biol, 84, 349-356, 2006. DOI : 10.1111/j.1440-1711.2006.01431.x
2. Klee W: Paratuberkulose (Johnesche Krankheit). In, Dirksen G, Grunder HD, Stober M (Eds): Innere Medizin und Chirurgie des Rindes. 586-591, Parey Verlag 5, 2012.
3. Çetinkaya B, Erdoğan HM, Morgan KL: Relationships between the presence of Johnes Disease and farm and management factors in dairy cattle in England. Prev Vet Med, 32, 253-266, 1997. DOI : 10.1016/ S0167-5877(97)00028-7
4. Clarke CJ: The pathology and pathogenesis of paratuberculosis in ruminants and other species. J Comp Pathol, 116, 217-261, 1997. DOI : 10.1016/S0021-9975(97)80001-1
5. Manning EJ, Collins MT: Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis. Rev Sci Tech, 20, 133-150, 2001.
6. Nacy C, Buckley M: Mycobacterium avium paratuberculosis: Infrequent Human Pathogen or Public Health Threat? A Report from American Academy of Microbiology. 1752 N Street, NW Washington DC, 2008.
7. Eltholth MM, Marsh VR, Van Winden S, Guitian FJ: Contamination of food products with Mycobacterium avium paratuberculosis: A systematic review. J Appl Microbiol, 107 (4): 1061-1071, 2009. DOI: 10.1111/j.1365- 2672.2009.04286.x
8. Economou M, Pappas G: New global map of Crohns Disease: Genetic, environmental, and socioeconomic correlations. Infamm Bowel Dis, 14 (5): 709-720, 2008. DOI: 10.1002/ibd.20352
9. Dağlı Ü: Turkish Infammatory Bowel Disease Society recommendations on selected topics of Crohns Disease. Turk J Gastroenterol, 21 (1): 78- 79, 2010. DOI : 10.4318/tjg.2010.0059
10. Behr MA: Paratuberculosis: Organism, Disease, Control. In, Collins DM (Ed): Paratuberculosis: Organism, Disease, Control. AgResearch, New Zealand, ISBN: 978 1 84593 613 6, 2010.
11. NAC: Assessment of food as a source of exposure to Mycobacterium avium subspecies paratuberculosis (MAP). J Food Prot, 73 (7): 1357-1397, 2010.
12. Mihajlovic B, Klassen M, Springthorpe S, Couture H, Farber J: Assessment of sources of exposure for Mycobacterium avium subsp. paratuberculosis in food and water. Int Food Risk Anal J, 1 (2): 1-22, 2011.
13. Gill CO, Saucier L, Meadus WJ: Mycobacterium avium subsp. paratuberculosis in dairy products, meat, and drinking water. J Food Prot, 74 (3): 480-499, 2011. DOI: 10.4315/0362-028X.JFP-10-301
14. Khol JL, Baumgartner W: Examples and suggestions for the control of paratuberculosis in European cattle. Jpn J Vet Res, 60 (Suppl.): S1-S7, 2012.
15. Alibaşoğlu M, Ertürk E, Yücel N: Türkiyede rastlanan ilk keçi paratüberküloz olayları üzerinde patolojik incelemeler. Ankara Üniv Vet Fak Derg, 20 (1): 43-63, 1973.
16. Karadaş E, Metün, N, Yaman Ü: Elazığda bir keçide gözlenen ilk paratüberküloz olgusunda patolojik bulgular. Turk J Vet Anim Sci, 24, 317- 323, 2000.
17. Çetinkaya B, Muz A, Ertaş HB, Öngör H, Sezen İY, Gülcü HB: Süt ineklerinde Paratüberküloz prevalansının polimeraz zincir reaksiyonu (PZR) ile saptanması. Turk J Vet Anim Sci, 24, 371-379, 2000.
18. Yıldırım D, Civelek T: Prevalence of subclinical Paratuberculosis in dairy cattle in Uşak region. Kafkas Univ Vet Fak Derg, 19 (1): 121-126, 2012.
19. Makav M, Gökçe E: Seroprevalence of subclinical Paratuberculosisin cattle in Kars region. Kafkas Univ Vet Fak Derg, 19 (5): 913-916, 2013. DOI: 10.9775/kvfd.2013.8529
20. Ayele WY, Svastova P, Roubal P, Bartos M, Pavlik I: Mycobacterium avium subsp. paratuberculosis cultured from locally and commercially pasteurized cows milk in the Czech Republic. Appl Environm Microbiol, 71, 1210-1214, 2005. DOI : 10.1128/AEM.71.3.1210-1214.2005
21. Fang Y, Wu WH, Pepper JL, Larsen JL, Marras, SAE, Nelson EA, Epperson WB, Christopher-Hennings J: Comparison of real-time, quantitative PCR with molecular beacons to nested PCR and culture methods for detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples. J Clin Microbiol, 40 (1): 287-291, 2002. DOI : 10.1128/JCM.40.1.287-291.2002
22. Johnson-Ifearulundu YJ, Kaneene JB: Management-related risk factors for M. paratuberculosis infection in Michigan, USA, dairy herds. Prev Vet Med, 37: (1-4), 41-54, 1998. DOI : 10.1016/S0167-5877(98)00110-X
23. Naing L, Winn T, Rusli BN: Practical issues in calculating the sample size for prevalaence studies. Arch Orofac Sci, 1, 9-14, 2006.
24. Bull TJ, McMinn EJ, Sidi-Boumedine K, Skull A, Durkin D, Neild P, Rhodes G, Pickup R, Hermon-Taylor J: Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohns disease. J Clin Microbiol, 41, 2915-2923, 2003. DOI: 10.1128/JCM.41.7.2915- 2923.2003
25. Donaghy JA, Johnston J, Rowe MT: Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays. J Appl Microbio, 110 (2): 479-489, 2011. DOI: 10.1111/j.1365-2672.2010.04905.x
26. FAO: Sero surveillance for Foot-and-Mouth Disease in Bulgaria. Session of the Research Group of the Standing Technical Committee of EuFMD. Appendix 10, 1999.
27. Ikiz S, Bagcigil AF, Seyyal AK, Ozgur NY, Lgaz A: Paratuberculosis in cattle in Turkey detected by PCR. Med Weter, 61, 881-883, 2005.
28. OIE - World Organisation for Animal Health: Word Animal Health in 2006. pp.7, Paris, France, 2006.
29. Mpatziou R, Ntafis V, Liandris E, Ikonomopoulos J, Xylouri E: Serological ınvestigation of IBR and MAP in dairy cows in Greece. The 61 st Annual Meeting of The European Association for Animal Production, 23-27 August, Heraklion-Greece, pp.235, 2010.
30. Humphry RW, Cameron A, Gunn GJ: A practical approach to calculate sample size for herd prevalence surveys. Prev Vet Med, 65, 173- 188, 2004. DOI : 10.1016/j.prevetmed.2004.07.003
31. Çetinkaya B, Erdoğan HM, Morgan KL: Risk factors for bovine Paratuberculosis. II. The multiple analysis of risk ractors for bovine Paratuberculosis. Turk J Vet Anim Sci, 21, 303-306, 1997.
32. Raizman EA, Wells SJ, Muñoz-Zanzi CA, Tavornpanich S: Estimated within-herd prevalence (WHP) of Mycobacterium avium subsp. paratuberculosis in a sample of Minnesota dairy herds using bacterial culture of pooled fecal samples. Can J Vet Res, 75 (2): 112-116, 2011.
33. van Schaik G, Pradenas FM, Mella NA, Kruze VJ: Diagnostic validity and costs of pooled fecal samples and individual blood or fecal samples to determine the cow- and herd-status for Mycobacterium avium subsp. paratuberculosis. Prev Vet Med, 82, 159-165, 2007. DOI : 10.1016/j. prevetmed.2007.05.018
34. Sohal JS, Sing SV, Subhodh S, Singh AV, Singh PK, Sheoran N, Sandhu K, Narayansamy K, Maitra A: Mycobacterium avium subspecies paratuberculosis diagnosis and strain typing - Present status and future developments. Indian J Exp Biol, 45, 843-852, 2007.
35. Özpınar H, Tezmen G, Gökçe İ, Tekiner İH: Detection of animal species in some meat and meat products by comparatively using DNA microarray and real time PCR methods. Kafkas Univ Vet Fak Derg , 19 (2): 245- 252, 2013. DOI: 10.9775/kvfd.2012.7616
36. Özpınar H, Turan B, Tekiner İH, Tezmen G, Gökçe İ, Akıneden Ö: Evaluation of pathogenic Escherichia coli occurrence in vegetable s amples from district bazaars of İstanbul using Real Time PCR. Lett Appl Microbiol, 57 (4): 362-367, 2013. DOI : 10.1111/lam.12122
37. Christopher-Hennings J, Dammen MA, Weeks SR, Epperson WB, Singh SN, Steinlicht GL, Fang Y, Skaare JL, Larsen JL, Payeur JB, Nelson EA: Comparison of two DNA extractions and nested PCR, real- time PCR, a new commercial PCR assay, and bacterial culture for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces. J Vet Diagn Invest, 15 (2): 87-93, 2003.
38. Leite FL, Stokes KD, Robbe-Austerman S, Stabel JR: Comparison of fecal DNA extraction kits for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction. J Vet Diagn Invest, 25 (1): 27-34, 2013. DOI : 10.1177/1040638712466395
39. Englund S, Bolske G, Johansson KE: An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis. FEMS Microbiol Lett, 209, 267-271, 2002. DOI: 10.1111/ j.1574-6968.2002.tb11142.x
40. Akineden Ö, Fernández-Silva JA, Weirich S, Abdulmawjood A, Bülte M: Comparison of two decontamination procedures, three culture media, and real time-PCR assay for the detection of Mycobacteriumavium subsp. paratuberculosis (MAP) from artificially contaminated raw sausage. Archiv fur Lebensmittelhygiene, 62,150-156, 2011.
41. Tasara T, Stephan R: Development of an F57 sequence-based Real-Time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk. Appl Environ Microbio, 71 (10): 5957-5968, 2005. DOI: 10.1128/AEM.71.10.5957-5968.2005
42. Herthnek D, Bölske G: New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis. BMC Microbiol, 6, 87, 2006. DOI : 10.1186/1471-2180-6-87
43. Slana I, Paolicchi F, Janstova B, Navratilova P, Pavlik I: Detection method s for Mycobacterium avium subsp. paratuberculosis in milk and milk products: A review. Vet Med Czech, 53 (6): 283-306, 200 8.
44. Schönenbrücher H, Abdulmawjoo A, Failing K, Bülte M: New triplex Real-Time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces. Appl Environ Microbiol, 74 (9): 2751-2758, 2008. DOI : 10.1128/AEM.02534-07
45. Ricchi M, Manini F, Cammi G, Donaghy J, Arrigoni N: Comparison of four diferent PCR methods for the detection of Mycobacterium avium subsp. paratuberculosis in milk. Proceedings of the 10th International Colloquium on Paratuberculosis, August 9-14, Minneapolis, Minnesota- USA. pp.56-59, 2009.
46. Diéguez FJ, González AM, Menéndez S, Vilar MJ, Sanjuán ML, Yus E, Arnaiz I: Evaluation of four commercial serum ELISAs for detection of Mycobacterium avium subspecies paratuberculosis infection in dairy cows. Vet J, 180, 231-235, 2009. DOI : 10.1016/j.tvjl.2007.11.004
47. Aly SS, Anderson RJ, Whitlock RH, Fyock TL, McAdams SC, Byrem TM, Jiang J, Adaska JM, Gardner IA: Cost-efectiveness of diagnostic strategies to identify Mycobacterium avium subspecies paratuberculosis su per-shedder cows in a large dairy herd using antibody enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, and bacterial culture. J Vet Diagn Invest, 24 (5): 821-832, 2012. DOI : 10.1177/1040638712452107
48. Herthnek D, Nielsen SS, Lindberg A, Bölske G: A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk. J Microbiol Methods, 75 (2): 335-340, 2008. DOI : 10.1016/j.mimet.2008.07.009
49. Dzieciol M, Volgger P, Khol J, Baumgartner W, Wagner M, Hein I: A novel real-time PCR assay for specific detection and quantification of Mycobacterium avium subsp. paratuberculosis in milk with the inherent possibility of diferentiation between viable and dead cells. BMC Res Notes, 3, 251, 2010. DOI : 10.1186/1756-0500-3-251
50. Kralik P, Slana I, Kralova A, Babak V, Whitlock RH, Pavlik I: Development of a predictive model for detection of Mycobacterium avium subsp. paratuberculosis in faeces by quantitative real time PCR. Vet Microbiol, 149 (1-2): 133-138, 2011. DOI : 10.1016/j.vetmic.2010.10.009