شناسایی مایکوباکتریوم آویوم زیرگونه پاراتوبرکلوزیس در نمونه‌های شیر خام و پاستوریزه با استفاده از روش‌های کشت، IS900 PCR و IS900 nested PCR
محورهای موضوعی :
بهداشت مواد غذایی
مهدی سلطانی
1
(بخش تحقیقات علوم دامی، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی استان خراسان رضوی، سازمان تحقیقات، آموزش و ترویج کشاورزی، مشهد، ایران)
تاریخ دریافت : 1400/07/15
تاریخ پذیرش : 1400/09/14
تاریخ انتشار : 1400/08/01
کلید واژه:
تشخیص,
آلودگی,
شیر,
پاستوریزاسیون,
مایکوباکتریوم پاراتوبرکلوزیس,
چکیده مقاله :
مایکوباکتریوم آویوم زیرگونه پاراتوبرکلوزیس (Mycobacterium avium subspecies paratuberculosis: MAP) یک باکتری بسیار کند رشد است که به‌عنوان عامل بیماری یون، هزینه‌های سنگینی را به صنایع مرتبط با دامداری تحمیل می‌کند. با توجه به علائم بالینی و آسیب‌شناسی مشابه در بیماری‌های یون و کرون، احتمال می‌رود MAP در بروز و پیشرفت کرون نقش داشته باشد. از این‌رو امکان انتقال MAP از طریق شیر موجب ایجاد نگرانی‌های زیادی شده است. برای بررسی وضعیت آلودگی به MAP در نمونه‌های شیر خام و پاستوریزه در استان کرمان از سه روش تشخیص شامل کشت، IS900 PCR و IS900 nested PCR استفاده شد. نتایج نشان داد آلودگی به MAP در شیر خام تولیدی در گله‌های مورد مطالعه نسبتاً زیاد بود؛ به‌نحوی که میزان موارد مثبت در روش کشت، PCR و nested PCR به‌ترتیب 38/4، 16/9 و 55/13 درصد برآورد شد. میزان آلودگی در نمونه‌های شیر پاستوریزه نیز در روش کشت، PCR و nested PCR به‌ترتیب 12/1، 93/3 و 18/6 درصد به‌دست آمد.. مقایسه روش‌های مورد استفاده در مطالعه حاضر توانایی بیشتر nested PCR را برای تشخیص آلودگی MAP نشان داد. بالا بودن سطح آلودگی در شیر خام از یک‌سو و مقاومت نسبتاً بالای این باکتری به تیمارهای حرارتی از سوی دیگر در کنار ویژگی درون‌سلولی بودن آن، موجب بقای بیشتر این باکتری به‌ویژه در جریان پاستوریزه کردن شیر می‌گردد. لذا ضروری است محققین بهداشت مواد غذایی نسبت به آلودگی شیر به این باکتری توجه بیشتری داشته باشند.
چکیده انگلیسی:
Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease, is a very slow-growing bacterium that imposes heavy costs on livestock-related industries. Due to the similar clinical and pathological symptoms in Johne’s and Crohn’s diseases, MAP is likely to play a role in the development and progression of Crohn’s disease. Hence, the possibility of transferring MAP through milk has created many concerns. To investigate the status of MAP contamination in raw and pasteurized milk samples in Kerman province, three diagnostic methods, including culture, IS900 PCR, and IS900 nested PCR was used. The results showed that the level of contamination with MAP in raw milk was relatively high in the studied herds. Based on culture, PCR, and nested PCR assays, 4.38%, 9.16%, and 13.55% were found positive, respectively. In pasteurized milk samples, 1.12%, 3.93%, and 6.18% were found positive for MAP by culture, PCR, and nested PCR, respectively. Comparing the methods used in this study demonstrated the best capability of nested PCR to detect MAP contamination. High levels of MAP contamination in raw milk on the one hand and relatively high resistance to thermal treatments, along with its intracellular characteristics, cause more survival of this bacterium, especially in the milk pasteurization process. Therefore, food hygiene researchers should pay more attention to the public health hazard caused by this bacterium.
منابع و مأخذ:
Alajmi, A., Klein, G., Grabowski, N.T., Fohler, S., Akineden, Ö. and Abdulmawjood, A. (2016). Evaluation of a Commercial Real-Time PCR Kit for the Detection of Mycobacterium avium subspecies paratuberculosis in Milk. Current Microbiology, 73(5):668-675.
Amita, J., Vandana, T., Guleria, R. and Verma, R. (2002). Qualitative evaluation of mycobacterial DNA extraction protocols for polymerase chain reaction. Molecular Biology Today, 3(2):43-49.
Anzabi, Y. (2014). Comparison of culture and PCR methods for detection of Mycobacterium avium paratuberculosis in raw milk of apparently healthy cattle. Food Hygiene, 4(1):1-12. [In Persian]
Anzabi, Y. and Hanifian, S. (2012). Detection of Mycobacterium avium subspecies paratuberculosis in pasteurized milk by IS900 PCR and culture method. African Journal of Microbiology Research, 6(7):1453-1456.
Anzabi, Y. and Khaki, A. (2017). Detection of Mycobacterium avium paratuberculosis in pasteurized milk by PCR according to the search of IS900 gene sequence. Medical Journal of Tabriz University of Medical Sciences and Health Services, 39(3):7-14. [In Persian]
Anzabi, Y., Tabatabayi, A. and Asgharzade, M. (2006). A survey of the infection status of Mycobacterium avium paratuberculosis in dairy cattle using PCR and culture of Tabriz region. Iranian Journal of Veterinary Science, 2(4):297-310. [In Persian]
Beran, V., Havelkova, M., Kaustova, J., Dvorska, L. and Pavlik, I. (2006). Cell wall deficient forms of mycobacteria: a review. Veterinarni Medicina, 51(7): 365-389.
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 Microbiology, 2(1):1-7.
Cousins, D., Whittington, R., Marsh, I., Masters, A., Evans, R. and Kluver, P. (1999). Mycobacteria distinct from Mycobacterium avium paratuberculosis isolated from the faeces of ruminants possess IS 900-like sequences detectable by IS 900 polymerase chain reaction: implications for diagnosis. Molecular and Cellular Probes, 13(6): 431-442.
Eftekhari, M. and Mosavari, N. (2016). Isolation and molecular identification of Mycobacterium from commercially available pasteurized milk and raw milk samples collected from two infected cattle farms in Alborz Province, Iran. International Journal of Mycobacteriology, 5: S222–S223.
Ellingson, J.L., Anderson, J.L., Koziczkowski, J.J., Radcliff, R.P., Sloan, S.J. et al. (2005). Detection of viable Mycobacterium avium paratuberculosis in retail pasteurized whole milk by two culture methods and PCR. Journal of Food Protection, 68(5):966-972.
Fathi, R., Sarkarati, F., Eslami, M., Rezavand, B. and Nourizadeh, A. (2011). Detection of Mycobacterium avium paratuberculosis in cow milk using culture and PCR methods. Archives of Razi Institute, 66(2): 95-100.
Garg, R., Patil, P.K., Singh, S.V., Sharma, S., Gandham, R.K., Singh, A.V. et al., (2015). Comparative evaluation of different test combinations for diagnosis of Mycobacterium avium subspecies paratuberculosis infecting dairy herds in India. BioMed Research International, 1-6.
Grant, I.R., Ball, H.J. and Rowe, M.T. (2002a). Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurized cows' milk from approved dairy processing establishments in the United Kingdom. Applied and Environmental Microbiology, 68(5): 2428-2435.
Grant, I.R., Hitchings, E.I., McCartney, A., Ferguson, F. and Rowe, M.T. (2002b). Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows' milk. Applied and Environmental Microbiology, 68(2):602-607.
Grant, I.R., Williams, A.G., Rowe, M.T. and Muir, D.D. (2005). Efficacy of various pasteurization time-temperature conditions in combination with homogenization on inactivation of Mycobacterium avium paratuberculosis in milk. Applied and Environmental Microbiology, 71(6): 2853-2861.
Hanifian, S. and Khani. S. (2016). Tracking of Mycobacterium avium paratuberculosis Load in Milk Production Chain: A Real‐Time qPCR and Culture Assay. Journal of Food Safety, 36(1): 136-141.
Hanifian, S., Khani, S., Barzegari, A. and Shayegh, J. (2013). Quantitative real-time PCR and culture examination of Mycobacterium avium paratuberculosis at farm level. Veterinary Microbiology, 162(1): 160-165.
Jayarao, B., Pillai, S., Wolfgang, D., Griswold, D., Rossiter, C., Tewari, D. et al., (2004). Evaluation of IS900-PCR assay for detection of Mycobacterium avium subspecies paratuberculosis infection in cattle using quarter milk and bulk tank milk samples. Foodborne Pathogens and Disease, 1(1): 17-26.
Millar, D., Ford, J., Sanderson, J., Withey, S., Tizard, M., Doran, T. et al. (1996). IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales. Applied and Environmental Microbiology, 62(9): 3446-3452.
Nasiri, A., Anzabi, Y. and Mashayekhi, M.R. (2018). Comparing qualitative and quantitative results of some DNA extraction methods from genome of pathogenic and saprophytic mycobacteria. Veterinary Clinical Pathology, 12(1): 79-95. [In Persian]
Nassiri, M., Jahandar, M., Soltani, M., Mahdavi, M. and Doosti, M. (2012). Identification and strain determination of paratuberculosis (MAP) by PCR and REA methods based on IS900 and IS1311 insertion segments. Agricultural Biotechnology 4(1): 83-96. [In Persian]
Patel, A. and Shah, N. (2011). Mycobacterium avium subsp paratuberculosis - Incidences in milk and milk products, their isolation, enumeration, characterization, and role in human health. Journal of Microbiology, Immunology and Infection, 44(6): 473-479.
Piras, C., Soggiu, A., Bonizzi, L., Greco, V., Ricchi, M., Arrigoni, N. et al., (2015). Identification of immunoreactive proteins of Mycobacterium avium paratuberculosis. Proteomics, 15(4): 813-823.
Sadati, R., Jafarpour, M., Mirinargesi, M., Nazemi, A. and Barghi, A. (2012). Prevalence of Mycobacterium avium paratuberculosis in dairy cattle bred in northern Iran by nested PCR. Global Veterinaria, 8(3): 259-263. [In Persian]
Sadeghi, N., Jamshidi, A. and Seyyedin, M. (2020). Detection of Mycobacterium avium paratuberculosis in pasteurized milk samples in northeast of Iran by culture, direct nested PCR and PCR Methods. Iranian Journal of Chemistry and Chemical Engineering, 39(6): 251-258.
Sharafati-chaleshtori, R., Shakerian, A., Momtaz, H. and Sharafati-chaleshtori, F. (2009). Detection of Mycobacterium paratuberculosis using polymerase chain reaction (PCR) in cow raw milk samples in shahre-kord. Medical Laboratory Journal, 3(1):15-19. [In Persian]
Slana, I., Kralik, P., Kralova, A. and Pavlik, I. (2008a). On-farm spread of Mycobacterium avium paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. International Journal of Food Microbiology, 128(2): 250-257.
Slana, I., Paolicchi, F., Janstova, B., Navratilova, P. and Pavlik, I. (2008b). Detection methods for Mycobacterium avium paratuberculosis in milk and milk products: a review. Veterinarni Medicina, 53(6): 283-306.
Soltani, M. (2018). Detection of Mycobacterium avium paratuberculosis in Kerman Province’s Dairy Cows using Microbial Culture, PCR and Nested PCR Methods. Iranian Journal of Animal Science Research, 10(2): 263-273. [In Persian]
Soltani, M., Nassiry, M.R., Shahroudi, F.E. and Bassami, M.R. (2008). Detection of Mycobacterium paratuberculosis in feces and milk samples from Holstein dairy cows by PCR. Biotechnology, 7(3): 582-585.
Taghipour Bazargani, T., Charkhkar, S., Sadeghi, F., Khalaj, M., Rashtibaf, M., Bagheri, M. et al., (2007). Protective effect of Johne’s disease attenuated vaccine in an intensive non-tuberculosis free dairy. Iranian Journal of Veterinary Research, 8(2): 161-165.
Tohidi Moghadam, M., Sarv, S., Moosakhani, F. and Badiie, A. (2010). Detection of Mycobacterium avium Subspecies paratuberculosis in milk and fecal samples in dairy cattle by PCR and nested-PCR. Journal of Animal and Veterinary Advances, 9(24): 3055-3061.
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Alajmi, A., Klein, G., Grabowski, N.T., Fohler, S., Akineden, Ö. and Abdulmawjood, A. (2016). Evaluation of a Commercial Real-Time PCR Kit for the Detection of Mycobacterium avium subspecies paratuberculosis in Milk. Current Microbiology, 73(5):668-675.
Amita, J., Vandana, T., Guleria, R. and Verma, R. (2002). Qualitative evaluation of mycobacterial DNA extraction protocols for polymerase chain reaction. Molecular Biology Today, 3(2):43-49.
Anzabi, Y. (2014). Comparison of culture and PCR methods for detection of Mycobacterium avium paratuberculosis in raw milk of apparently healthy cattle. Food Hygiene, 4(1):1-12. [In Persian]
Anzabi, Y. and Hanifian, S. (2012). Detection of Mycobacterium avium subspecies paratuberculosis in pasteurized milk by IS900 PCR and culture method. African Journal of Microbiology Research, 6(7):1453-1456.
Anzabi, Y. and Khaki, A. (2017). Detection of Mycobacterium avium paratuberculosis in pasteurized milk by PCR according to the search of IS900 gene sequence. Medical Journal of Tabriz University of Medical Sciences and Health Services, 39(3):7-14. [In Persian]
Anzabi, Y., Tabatabayi, A. and Asgharzade, M. (2006). A survey of the infection status of Mycobacterium avium paratuberculosis in dairy cattle using PCR and culture of Tabriz region. Iranian Journal of Veterinary Science, 2(4):297-310. [In Persian]
Beran, V., Havelkova, M., Kaustova, J., Dvorska, L. and Pavlik, I. (2006). Cell wall deficient forms of mycobacteria: a review. Veterinarni Medicina, 51(7): 365-389.
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 Microbiology, 2(1):1-7.
Cousins, D., Whittington, R., Marsh, I., Masters, A., Evans, R. and Kluver, P. (1999). Mycobacteria distinct from Mycobacterium avium paratuberculosis isolated from the faeces of ruminants possess IS 900-like sequences detectable by IS 900 polymerase chain reaction: implications for diagnosis. Molecular and Cellular Probes, 13(6): 431-442.
Eftekhari, M. and Mosavari, N. (2016). Isolation and molecular identification of Mycobacterium from commercially available pasteurized milk and raw milk samples collected from two infected cattle farms in Alborz Province, Iran. International Journal of Mycobacteriology, 5: S222–S223.
Ellingson, J.L., Anderson, J.L., Koziczkowski, J.J., Radcliff, R.P., Sloan, S.J. et al. (2005). Detection of viable Mycobacterium avium paratuberculosis in retail pasteurized whole milk by two culture methods and PCR. Journal of Food Protection, 68(5):966-972.
Fathi, R., Sarkarati, F., Eslami, M., Rezavand, B. and Nourizadeh, A. (2011). Detection of Mycobacterium avium paratuberculosis in cow milk using culture and PCR methods. Archives of Razi Institute, 66(2): 95-100.
Garg, R., Patil, P.K., Singh, S.V., Sharma, S., Gandham, R.K., Singh, A.V. et al., (2015). Comparative evaluation of different test combinations for diagnosis of Mycobacterium avium subspecies paratuberculosis infecting dairy herds in India. BioMed Research International, 1-6.
Grant, I.R., Ball, H.J. and Rowe, M.T. (2002a). Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurized cows' milk from approved dairy processing establishments in the United Kingdom. Applied and Environmental Microbiology, 68(5): 2428-2435.
Grant, I.R., Hitchings, E.I., McCartney, A., Ferguson, F. and Rowe, M.T. (2002b). Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows' milk. Applied and Environmental Microbiology, 68(2):602-607.
Grant, I.R., Williams, A.G., Rowe, M.T. and Muir, D.D. (2005). Efficacy of various pasteurization time-temperature conditions in combination with homogenization on inactivation of Mycobacterium avium paratuberculosis in milk. Applied and Environmental Microbiology, 71(6): 2853-2861.
Hanifian, S. and Khani. S. (2016). Tracking of Mycobacterium avium paratuberculosis Load in Milk Production Chain: A Real‐Time qPCR and Culture Assay. Journal of Food Safety, 36(1): 136-141.
Hanifian, S., Khani, S., Barzegari, A. and Shayegh, J. (2013). Quantitative real-time PCR and culture examination of Mycobacterium avium paratuberculosis at farm level. Veterinary Microbiology, 162(1): 160-165.
Jayarao, B., Pillai, S., Wolfgang, D., Griswold, D., Rossiter, C., Tewari, D. et al., (2004). Evaluation of IS900-PCR assay for detection of Mycobacterium avium subspecies paratuberculosis infection in cattle using quarter milk and bulk tank milk samples. Foodborne Pathogens and Disease, 1(1): 17-26.
Millar, D., Ford, J., Sanderson, J., Withey, S., Tizard, M., Doran, T. et al. (1996). IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales. Applied and Environmental Microbiology, 62(9): 3446-3452.
Nasiri, A., Anzabi, Y. and Mashayekhi, M.R. (2018). Comparing qualitative and quantitative results of some DNA extraction methods from genome of pathogenic and saprophytic mycobacteria. Veterinary Clinical Pathology, 12(1): 79-95. [In Persian]
Nassiri, M., Jahandar, M., Soltani, M., Mahdavi, M. and Doosti, M. (2012). Identification and strain determination of paratuberculosis (MAP) by PCR and REA methods based on IS900 and IS1311 insertion segments. Agricultural Biotechnology 4(1): 83-96. [In Persian]
Patel, A. and Shah, N. (2011). Mycobacterium avium subsp paratuberculosis - Incidences in milk and milk products, their isolation, enumeration, characterization, and role in human health. Journal of Microbiology, Immunology and Infection, 44(6): 473-479.
Piras, C., Soggiu, A., Bonizzi, L., Greco, V., Ricchi, M., Arrigoni, N. et al., (2015). Identification of immunoreactive proteins of Mycobacterium avium paratuberculosis. Proteomics, 15(4): 813-823.
Sadati, R., Jafarpour, M., Mirinargesi, M., Nazemi, A. and Barghi, A. (2012). Prevalence of Mycobacterium avium paratuberculosis in dairy cattle bred in northern Iran by nested PCR. Global Veterinaria, 8(3): 259-263. [In Persian]
Sadeghi, N., Jamshidi, A. and Seyyedin, M. (2020). Detection of Mycobacterium avium paratuberculosis in pasteurized milk samples in northeast of Iran by culture, direct nested PCR and PCR Methods. Iranian Journal of Chemistry and Chemical Engineering, 39(6): 251-258.
Sharafati-chaleshtori, R., Shakerian, A., Momtaz, H. and Sharafati-chaleshtori, F. (2009). Detection of Mycobacterium paratuberculosis using polymerase chain reaction (PCR) in cow raw milk samples in shahre-kord. Medical Laboratory Journal, 3(1):15-19. [In Persian]
Slana, I., Kralik, P., Kralova, A. and Pavlik, I. (2008a). On-farm spread of Mycobacterium avium paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. International Journal of Food Microbiology, 128(2): 250-257.
Slana, I., Paolicchi, F., Janstova, B., Navratilova, P. and Pavlik, I. (2008b). Detection methods for Mycobacterium avium paratuberculosis in milk and milk products: a review. Veterinarni Medicina, 53(6): 283-306.
Soltani, M. (2018). Detection of Mycobacterium avium paratuberculosis in Kerman Province’s Dairy Cows using Microbial Culture, PCR and Nested PCR Methods. Iranian Journal of Animal Science Research, 10(2): 263-273. [In Persian]
Soltani, M., Nassiry, M.R., Shahroudi, F.E. and Bassami, M.R. (2008). Detection of Mycobacterium paratuberculosis in feces and milk samples from Holstein dairy cows by PCR. Biotechnology, 7(3): 582-585.
Taghipour Bazargani, T., Charkhkar, S., Sadeghi, F., Khalaj, M., Rashtibaf, M., Bagheri, M. et al., (2007). Protective effect of Johne’s disease attenuated vaccine in an intensive non-tuberculosis free dairy. Iranian Journal of Veterinary Research, 8(2): 161-165.
Tohidi Moghadam, M., Sarv, S., Moosakhani, F. and Badiie, A. (2010). Detection of Mycobacterium avium Subspecies paratuberculosis in milk and fecal samples in dairy cattle by PCR and nested-PCR. Journal of Animal and Veterinary Advances, 9(24): 3055-3061.
