Detection of Mycobacterium avium Sub sp. paratuberculosis in Pasteurized Milk Samples in Northeast of Iran by Culture, Direct Nested PCR and PCR Methods

Document Type : Research Article

Authors

1 Department of Food Hygiene, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, I.R. IRAN

2 Razi Vaccine and Serum Research Institute, Mashhad, I.R. IRAN

Abstract

Mycobacterium avium Subsp. paratuberculosis (MAP) is a gram-positive, small, acid-fast bacillus with high environmental resistance. In animals, especially ruminants, it leads to Paratuberculosis (PTB) or Johne's disease, which is chronic granulomatous enteritis. This bacterium as the main causative agent of Crohn's disease can be a serious threat to human health. This study aimed to detect MAP in pasteurized milk samples produced in Khorasan Razavi province, Iran, using Direct Nested PCR, PCR, and culture methods. In this study, 544 milk samples from Pasteurized Milk Production Companies were selected randomly during the 3-month period. DNA was extracted from milk fat after centrifugation. In order to identify the bacteria, Direct Nested PCR and PCR tests were applied using IS900 and f57, respectively. Furthermore, to detect viable MAP, positive samples resulted from Direct Nested PCR assays were cultured on Herrold's egg medium. For identification of mycobacterial isolates, all colonies were processed by PCR based on f57. A total of 544 pasteurized milk samples were assayed, and Mycobacterium paratuberculosis was detected in 39% of them by IS900 Nested PCR, and only 4.9% of samples were positive in the culture method. All the colonies were positive for the f57using PCR. The results of this study indirectly indicated a high level of contamination of pasteurized milk to Mycobacterium paratuberculosis which is due to the large number of affected animals in livestock farms in Khorasan Razavi province. However, in comparison with the other researches, the low percentage of viable bacteria in pasteurized milk can be due to changes in temperature and time in pasteurizing systems of milk production companies in Khorasan Razavi province, Northeast of Iran.

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[1] Abubakar I., Myhill D.J., Hart A.R., Lake I., A Case-Control Study of Drinking Water and Dairy Products in Crohn's Disease Further Investigation of the Possible Role of Mycobacterium avium paratuberculosis, American Journal of Epidemiology., 165(7): 776-783 (2007).
[3] Colombani A.G.F.F.B.T.G., Detection of Mycobacterium Avium Subsp. Paratuberculosis in a Sheep Flock in Tuscany, Trop Anim Health Prod., 47: 1567–1571 (2015)
[4] Cocito C., Gilot P., Coene M., Kesel M.d., Poupart P., Vannuffel P., Paratuberculosis, Clin Microbiol Rev., 7: 328-345 (1994)
[5] Feller M., Huwiler K., Stephan R., Altpeter E., Shang A., Furrer H., Pfyffer G.E., Jemmi T., Baumagartner A., Egger M., Mycobacterium Avium Subspecies Paratuberculosis and Chron’s Disease: a Systematic Review and Meta-Analysis, Lancet Infect Dis., 7: 607-613 (2007)
[7] Alexejeff-Goloff NA., A Possible Case of Congenital Johne's Disease, Comparative Pathology., 48:81 (1935)
[8] Taylor T.K. WCR, McQueen D.S., Isolation of Mycobacterium Paratuberculosis from the Milk of a Cow with Johne's Disease, The Veterinary Record., 109: 532-533 (1981)
[11] Grant I.P. C., O'Riordan L., Ball H., Rowe M., Improved Detection of Mycobacterium Avium Subsp. Paratuberculosis in Milk by Immunomagnetic PCR, Vet. Microbiol., 77: 369-378 (2000).
[12] Castellanos E., de Juan L., Dominguez L., Aranaz A., Progress in Molecular Typing of Mycobacterium Avium Subspecies Paratuberculosis, Res Vet Sci., 92: 169-179 (2012)
[13] Millar D., Ford J., Sanderson J., Withey S., Tizard M., Doran T., Hermon-Taylor J., IS900 PCR to Detect Mycobacterium Paratuberculosis in Retail Supplies of Whole Pasteurized Cows' Milk in England and Wales, Appl. Environ. Microbiol., 62: 3446-3452 (1996)
[14] Vary P.H., Andersen P.R., Green E., Hermon-Taylor J., McFadden J.J., Use of Highly Specific DNA Probes and the Polymerase Chain Reaction to Detect Mycobacterium Paratuberculosis in Johne’s Disease, J Clin Microbiol., 28: 933-937 (1990)
[16] Green G.T.M., Thompson J., Winterbourme D., Mc Fadden J., Hermon-Taylor J., Sequence and Characteristic of IS900, and Insertion Sequence Identified in Human Crohn's Disease Isolate of Mycobacterium Avium Subsp. Paratuberculosis, Nucleic Acid Res., 17: 9063-9073 (1989)
[18] Ellingson J.A.J., Koziczkowski J., Radcliff R., Sloan S., Allen S., Sullivan N., Detection of Viable Mycobacterium Avium Subsp. Paratuberculosis in Retail Pasteurized Whole Milk by Two Culture Methods and PCR, J. Food Prot., 68: 966-972 (2005)
[19] Taddei S., Robbi C., Cesena C., Rossi I., Schiano E., Arrigoni N., Vicenzoni G., Cavirani S., Detection of Mycobacterium Avium subsp. Paratuberculosis in Bovine Fecal Samples: Comparison of Three Polymerase Chain Reaction-Based Diagnostic Tests with a Conventional Culture Method, Journal of Veterinary Diagnostic Investigation, 16: 503-8 (2004).
[20] Jain Amita, R.S.G. Verma R.K., Qualitative Evaluation of Mycobacterial DNA Extraction Protocols for Polymerase Chain Reaction, Molecular Biology Today., 3: 43-50 (2002)
[21] Pirondini A., Bonas U., Maestri E., Visioli G., Marmiroli M., Marmiroli N., Yield and Amplificability of Different DNA Extraction Procedures for Traceability in the Dairy Food Chain, Food Control., 21:661-668 (2010).
 [22] Dundee L. GIR, Ball H.J., Rowe M.T., Comparative Evaluation of Four Decontamination Protocols for the Isolation of Mycobacterium Avium Subsp.Paratuberculosis from Milk, Letters in Applied Microbiology., 33:173-177 (2001).
[23] Grant I.R. KRB, Hitchings E., Rowe M.T., Comparative Evaluation of the MGIT and BACTEC Culture Systems for the Recovery of Mycobacterium Avium Subsp. Paratuberculosis from Milk, Journal of Applied Microbiology., 95: 196-201 (2003)
[24] Hammer P., Kiesner C., Walte H.G., Knappstein K., Teufel P., Heat Resistance of Mycobacterium Avium Subsp. Paratuberculosis in Raw Milk Tested in Pilot Plant Pasteuriser, Kieler Milchwirtschaftlich., 275: 303-354 (2002)
[25] Fernando Paolicchi K.C., Claudia  Morsella1; Andrea Gioffré., First Isolation of Mycobacterium Avium Subsp Paratuberculosis from Commercial Pasteurized Milk in Argentina, Brazilian Journal of Microbiology., 1034-1037(2012)
[26] JR, S., On-Farm Batch Pasteurization Destroys Mycobacterium Paratuberculosis in Waste Milk, Journal Dairy Science., 84: 524–527 (2001)
[27] Slana, I.P., F.; Janstova, B.; Navratilova, P.; Pavlík, I., Detection Methods for Mycobacterium Avium Subsp. Paratuberculosis in Milk and Milk Products, Vet. Med. Czech., 53: 286-306 (2008)
[30] Anzabi, Y. and K. A., Detection of Mycobacterium Avium Paratuberculosis in Pasteurized Milk by PCR According to the Search of IS900 Gene Sequence, Med J Tabriz Uni Med Sciences Health Services., 39(3):7-14 (2016).
[31] Ikonomopoulos J. Pavlik I., Bartos M., Svastova P., Ayele W.Y., Roubal P., Lukas J., Cook N., Gazouli M., Detection of Mycobacterium Avium Subsp. paratuberculosis in Retail Cheeses from Greece and the Czech Republic, Applied and Environmental Microbiology., 71:8934-8936 (2005).
[32] Gao A M.L., Raymond M., Odumeru J., Improved Template DNA Preparation Procedure for Detection of Mycobacterium Avium Subsp. Paratuberculosis in Milk by PCR, Journal of Microbiological Methods., 69:417-420 (2007).
[33] McDonald W.L., O'Riley K.J., Schroen C.J., Condron R.J., Heat Inactivation of Mycobacterium Avium Subsp. Paratuberculosis in Milk, Appl Environ Microbiol., 71(4):1785–1789 (2005).