شناسایی مایکوباکتریوم آویوم زیرگونه پاراتوبرکلوزیس (Map) در گاوهای شیری استان کرمان با استفاده از روش‌های کشت میکروبی، PCR و nested PCR

نوع مقاله : علمی پژوهشی- ژنتیک و اصلاح دام و طیور

نویسنده

تحصیلات تکمیلی صنعتی و فناوری پیشرفته کرمان

چکیده

مایکوباکتریوم آویوم زیرگونه پاراتوبرکلوزیس (Map) عامل بیماری یون یا پاراتوبرکلوزیس است که با اسهال غیرقابل درمان، کاهش وزن و کاهش تولید مشخص می‌شود. به سبب شیوع بالا در گله‌های گاو شیری، Map خسارات زیادی را به صنایع مرتبط با دامپروری وارد می‌کند. احتمال دارد Map در برخی از بیماری‌ها مانند بیماری کرون در انسان نیز نقش داشته باشد. اولین گام در کنترل بیماری یون، شناسایی حیوانات بیمار تحت بالینی است. برای ارزیابی وضعیت آلودگی Map در استان کرمان، از سه روش کشت، واکنش PCR و nested PCR روی نمونه‌های مدفوع استفاده شد. سویه جدایه‌های شناسایی شده Map نیز تعیین گردید. این مطالعه اولین گزارش در مورد شناسایی Map در گله‌های گاو شیری در جنوب شرق ایران است. مقایسه روش‌های استفاده شده در این مطالعه نشان داد روش Nested PCR برای شناسایی Map توان بالاتری دارد. تمامی جدایه‌های شناسایی شده در این مطالعه مربوط به سویه گاوی بودند. با توجه به امکان انتقال Map از طریق شیر حیوانات آلوده به انسان، بایستی معیارهای بسیار دقیق برای کنترل بیماری یون در نظر گرفته شود تا احتمال انتقال آلودگی به انسان به حداقل برسد. بر خلاف سل گاوی، به بیماری یون توجه کمتری شده است، بنابراین در نظر گرفتن استانداردهای شدیدتر برای مقابله با مشکلات احتمالی اقتصادی و بهداشتی حاصل از این بیماری ضروری به نظر می‌رسد. از نتایج مطالعه حاضر می‌توان برای طرح‌ریزی تحقیقات آینده و اتخاذ تصمیمات مدیریتی مناسب در موضوع Map استفاده کرد.

کلیدواژه‌ها


عنوان مقاله [English]

Detection of Mycobacterium avium subsp. paratuberculosis in Kerman Province’s Dairy Cows using Microbial Culture, PCR and Nested PCR Methods

نویسنده [English]

  • Mahdi Soltani
Graduate University of Advanced Technology, Kerman
چکیده [English]

Introduction Mycobacterium avium subsp. paratuberculosis (Map) is a very slow growing bacterium that solely infect the digestive tract and leads to Johne’s disease (JD) that is characterized by immedicable diarrhea. Considering innate resistance and different transmission modes of Map and its possible role in development or progression of some human diseases (mainly Crohn’s disease), strong zoonotic potential has been proposed for Map. Diagnosis of JD depends on isolation of viable Map (culture), tracking of its consequences on host immune system (ELISA) or amplification of Map genome (PCR). Fecal culture considered as gold standard for Map detection but obstacles like very slow growth rate of Map, decontamination issues and its high expenses led to development of alternatives like PCR based approaches to gain PCR advantages like high sensitivity, specificity and rapidity for diagnosis of Map. Since no published report was found on Map infection status in Kerman, the current study aimed to investigate the presence of Map in bovine feces samples using 3 different detection methods including culture, IS900 PCR and IS900 nested PCR. Also IS1311 PCR-REA was used for strain typing of Map isolates.
Materials and Methods 212 fecal samples were taken from 4 dairy cattle farms located in Kerman province, southeast Iran. The fecal fractions for culture and PCR were prepared separately to inhibit potential cross-contaminations. Per each sample, 300 µl of prepared inoculum was cultured on slope of Herrold’s egg yolk medium (HEYM) alone, and two HEYM + 2.0 mg/L of mycobactin J slopes. Inoculated slopes were incubated at 37 °C for 4 months and monitored at biweekly intervals. After extraction of DNA from fecal samples, PCR reaction performed in 25 µl volume and products were analyzed by electrophoresis on 2% agarose gels. The samples were considered as positive if 413 bp amplified band was present. To avoid false positive results, a REA approach using AlwI restriction enzyme was adopted. For IS900 nested PCR reaction, 3 ml from IS900 PCR products were used as template. Resultant products were screened as before. To evaluate the limit of detection of PCR reactions, the DNA extracted from confirmed MAP field strain culture was 10 fold serially diluted (1 µg – 1 fg) and the minimum detection level of PCR and nested PCR were observed. Strain typing of Map isolates was based on IS1311 PCR-REA.
Results and Discussion It takes 3-4 months of incubation for appearance of MAP positive colonies. Map was detected in 32/212 (15.1%) of cultured samples. All colonies were verified as MAP using IS900 PCR and REA with AlwI. 44 positive samples were detected by IS900 PCR (20.7%) while nested PCR was able to find 52 infected samples (24.5%). All isolates detected by PCR based methods were also verified as MAP by IS900 PCR-REA. According to expectations, nPCR offered higher sensitivity than conventional PCR. However, comparing the methods used in the present study showed that as a simple and cheap assay, nested PCR is inherently successful in amplification even with rare amount of starting template and has the better ability to detect the Map infection in fecal samples and can be used as a routine method in diagnostic processes. Although this study was the first attempt to access the Map infection status in Kerman province, as summarized in table 3, many studies were conducted all around Iran that can be compared with. Altogether, Map incidence in other central Iran provinces (Isfahan, Fars and Chaharmahal and Bakhtiari) were between 14.1 to 31.8 percent of tested populations based on examination of fecal samples. Considering that most of reported values came from clinically infected or suspected animals, the estimates obtained in this study showed the possibility that the Map incidence in Kerman province may be higher than other parts of central Iran, but it more investigations needed to create a realistic scheme of Map infection status in Kerman province. Remembering that a copy of genome was considered as equivalent to 5 fg of DNA, sensitivity analysis using serially diluted DNA preparations revealed that the minimum detection level were 1 pg (200 genomes) and 10 fg (2 genomes) for PCR and nested PCR, respectively. According to results, compared to nested PCR, conventional PCR is not sensitive enough for diagnostic tests on field samples. Analysis of fragments produced after IS1311 REA showed the presence of explicit patterns of four bands for all obtained isolates that is the indication of MAP C type. Obtained DNA sequences from the amplified IS1311 locus was exactly consistent with previously published sequence for MAP C type. S type was not detected in current study that was consistent with results of researches performed in Razavi Khorasan province. Failure to find S Map strain was not surprising, since its global prevalence in cow population is very low and hard to find. Also, there are few papers dealing with Map strain typing in Iran, so it is conceivable to find S Map type in Iran’s cattle population if more research conducted in this respect.
Conclusion Although paratuberculosis is a global neglected disease, the situation is worse in developing countries like Iran. Contrary to bovine tuberculosis, due to absence of a national program to control paratuberculosis infection in Iran’s dairy herds, no reliable data is available for arrangement of tools for efficient restriction of disease which leads to inadequate attention to JD and eventually receive the least priority to control. Unfortunately, paratuberculosis as an overlooked disease, is a severe hazard for cows’ health and also for economic activities related to dairy cattle industry. On the other hand, Map is still considered as an important suspicious zoonotic agent which cause serious health problems for humans. Meanwhile, considering aforementioned warnings, enactment of crucial health standards is vital to minimize the chance of infection transmission to non-infected populations and alleviating health problems and economic losses due to Map infection.

Materials and Methods 212 fecal samples were taken from 4 dairy cattle farms located in Kerman province, southeast Iran. The fecal fractions for culture and PCR were prepared separately to inhibit potential cross-contaminations. Per each sample, 300 µl of prepared inoculum was cultured on slope of Herrold’s egg yolk medium (HEYM) alone, and two HEYM + 2.0 mg/L of mycobactin J slopes. Inoculated slopes were incubated at 37 °C for 4 months and monitored at biweekly intervals. After extraction of DNA from fecal samples, PCR reaction performed in 25 µl volume and products were analyzed by electrophoresis on 2% agarose gels. The samples were considered as positive if 413 bp amplified band was present. To avoid false positive results, a REA approach using AlwI restriction enzyme was adopted. For IS900 nested PCR reaction, 3 ml from IS900 PCR products were used as template. Resultant products were screened as before. To evaluate the limit of detection of PCR reactions, the DNA extracted from confirmed MAP field strain culture was 10 fold serially diluted (1 µg – 1 fg) and the minimum detection level of PCR and nested PCR were observed. Strain typing of Map isolates was based on IS1311 PCR-REA.
Results and Discussion It takes 3-4 months of incubation for appearance of MAP positive colonies. Map was detected in 32/212 (15.1%) of cultured samples. All colonies were verified as MAP using IS900 PCR and REA with AlwI. 44 positive samples were detected by IS900 PCR (20.7%) while nested PCR was able to find 52 infected samples (24.5%). All isolates detected by PCR based methods were also verified as MAP by IS900 PCR-REA. According to expectations, nPCR offered higher sensitivity than conventional PCR. However, comparing the methods used in the present study showed that as a simple and cheap assay, nested PCR is inherently successful in amplification even with rare amount of starting template and has the better ability to detect the Map infection in fecal samples and can be used as a routine method in diagnostic processes. Although this study was the first attempt to access the Map infection status in Kerman province, as summarized in table 3, many studies were conducted all around Iran that can be compared with. Altogether, Map incidence in other central Iran provinces like Isfahan, Fars and Chaharmahal and Bakhtiari were between 14.1 to 31.8 percent of tested populations based on examination of fecal samples. Considering that most of reported values came from clinically infected or suspected animals, the estimates obtained in this study showed the possibility that the Map incidence in Kerman province may be higher than other parts of central Iran, but it more investigations needed to create a realistic scheme of Map infection status in Kerman province. Remembering that a copy of genome was considered as equivalent to 5 fg of DNA, sensitivity analysis using serially diluted DNA preparations revealed that the minimum detection level were 1 pg (200 genomes) and 10 fg (2 genomes) for PCR and nested PCR, respectively. According to results, compared to nested PCR, conventional PCR is not sensitive enough for diagnostic tests on field samples. Analysis of fragments produced after IS1311 REA showed the presence of explicit patterns of four bands for all obtained isolates that is the indication of MAP C type. Obtained DNA sequences from the amplified IS1311 locus was exactly consistent with previously published sequence for MAP C type. S type was not detected in current study that was consistent with results of researches performed in Razavi Khorasan province. Failure to find S Map strain was not surprising, since its global prevalence in cow population is very low and hard to find. Also, there are few papers dealing with Map strain typing in Iran, so it is conceivable to find S Map type in Iran’s cattle population if more research conducted in this respect.
Conclusion Although paratuberculosis is a global neglected disease, the situation is worse in developing countries like Iran. Contrary to bovine tuberculosis, due to absence of a national program to control paratuberculosis infection in Iran’s dairy herds, no reliable data is available for arrangement of tools for efficient restriction of disease which leads to inadequate attention to JD and eventually receive the least priority to control. Unfortunately, paratuberculosis as an overlooked disease, is a severe hazard for cows’ health and also for economic activities related to dairy cattle industry. On the other hand, Map is still considered as an important suspicious zoonotic agent which cause serious health problems for humans. Meanwhile, considering aforementioned warnings, enactment of crucial health standards is vital to minimize the chance of infection transmission to non-infected populations and alleviating health problems and economic losses due to Map infection.

کلیدواژه‌ها [English]

  • Culture
  • Detection
  • Johne’s disease
  • Mycobacterium avium subsp. Paratuberculosis
  • Nested PCR
  • PCR
1. Ansari-Lari, M., M. Haghkhah, A. Bahramy, and A. M. N. Baheran. 2009. Risk factors for Mycobacterium avium subspecies paratuberculosis in Fars province (Southern Iran) dairy herds. Tropical Animal Health and Production, 41(4):553-557.
2. Corti, S. and R. Stephan. 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.
3. Cousins, D., R. Whittington, I. Marsh, A. Masters, R. Evans, and P. Kluver. 1999. Mycobacteria distinct from Mycobacterium avium subsp. 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.
4. Englund, S., A. Ballagi-Pordany, G. Bölske, and K.-E. Johansson. 1999. Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis. Diagnostic Microbiology and Infectious Disease, 33(3):163-171.
5. Grant, I. and M. Rowe. 2004. Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat‐treated milk. Letters in Applied Microbiology, 38(4):283-288.
6. Hanifian, S. and S. Khani. 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.
7. Hanifian, S., S. Khani, A. Barzegari, and J. Shayegh. 2013. Quantitative real-time PCR and culture examination of Mycobacterium avium subsp. paratuberculosis at farm level. Veterinary Microbiology, 162(1):160-165.
8. Ikonomopoulos, J., M. Gazouli, I. Pavlik, M. Bartos, P. Zacharatos, E. Xylouri, E. Papalambros, and V. Gorgoulis. 2004. Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis. Journal of Microbiological Methods, 56(3):315-321.
9. Jayarao, B., S. Pillai, D. Wolfgang, D. Griswold, C. Rossiter, D. Tewari, C. Burns, and L. Hutchinson. 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 & Disease, 1(1):17-26.
10. Kralik, P., I. Slana, A. Kralova, V. Babak, R. H. Whitlock, and I. Pavlik. 2011. Development of a predictive model for detection of Mycobacterium avium subsp. paratuberculosis in faeces by quantitative real time PCR. Veterinary Microbiology, 149(1):133-138.
11. Liapi, M., G. Botsaris, I. Slana, M. Moravkova, V. Babak, M. Avraam, A. Di Provvido, S. Georgiadou, and I. Pavlik. 2015. Mycobacterium avium subsp. paratuberculosis sheep strains isolated from Cyprus sheep and goats. Transboundary and emerging diseases, 62(2):223-227.
12. Marsh, I., R. Whittington, and D. Cousins. 1999. PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311. Molecular and Cellular Probes, 13(2):115-126.
13. Millar, D., J. Ford, J. Sanderson, S. Withey, M. Tizard, T. Doran, and J. Hermon-Taylor. 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.
14. Nassiri, M., M. Jahandar, M. Soltani, M. Mahdavi, and M. Doosti. 2012. Identification and strain determination of M. paratuberculosis (MAP) by PCR and REA methods based on IS900 and IS1311 insertion segments. Agricultural Biotechnology, 4(1):83-96. (In Persian).
15. Nebbia, P., P. Robino, S. Zoppi, and D. De Meneghi. 2006. Detection and excretion pattern of Mycobacterium avium subspecies paratuberculosis in milk of asymptomatic sheep and goats by Nested-PCR. Small Ruminant Research, 66(1):116-120.
16. Rapp, D. 2010. DNA extraction from bovine faeces: current status and future trends. Journal of Applied Microbiology, 108(5):1485-1493.
17. Ronai, Z., Á. Csivincsik, M. Gyuranecz, Z. Kreizinger, Á. Dan, and S. Janosi. 2015. Molecular analysis and MIRU‐VNTR typing of Mycobacterium avium subsp. paratuberculosis strains from various sources. Journal of Applied Microbiology, 118(2):275-283.
18. Sanderson, J., M. Moss, M. Tizard, and J. Hermon-Taylor. 1992. Mycobacterium paratuberculosis DNA in Crohn's disease tissue. Gut, 33(7):890-896.
19. Sevilla, I. A., J. M. Garrido, E. Molina, M. V. Geijo, N. Elguezabal, P. Vazquez, and R. A. Juste. 2014. Development and evaluation of a novel multicopy-element-targeting Triplex PCR for detection of Mycobacterium avium subsp. paratuberculosis in feces. Applied and Environmental Microbiology, 80(12):3757-3768.
20. Shahmoradi, A. H., R. Arefpajohi, K. Tadayon, and N. Mosavari. 2008. Paratuberculosis in Holstein-Friesian cattle farms in central Iran. Tropical Animal Health and Production, 40(3):169-173.
21. Shariati, S. H., A. Alaei, R. Keshavarz, N. Mosavari, A. Rabbani, M. Niegowska, L. A. Sechi, and M. M. Feizabadi. 2016. Detection of Mycobacterium avium subsp. paratuberculosis in Iranian patients with type 1 diabetes mellitus by PCR and ELISA. The Journal of Infection in Developing Countries, 10(08):857-862.
22. Soltani, M., M. R. Nassiry, F. Shahroudi, and B. Sadeghi. 2010. PCR-restriction endonuclease analysis for strain typing of Mycobacterium avium subsp. paratuberculosis based on polymorphisms in IS 1311. Middle East Journal of Scientific Research, 5(4):311-315.
23. Soltani, M., M. R. Nassiry, F. E. Shahroudi, and M. R. Bassami. 2008. Detection of Mycobacterium paratuberculosis in feces and milk samples from holstein dairy cows by PCR. Biotechnology, 7(3):582-585.
24. Stevenson, K. 2015. Genetic diversity of Mycobacterium avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review. Veterinary Research, 46(1):1.
25. Sting, R., M. Hrubenja, J. Mandl, G. Seemann, A. Salditt, and S. Waibel. 2014. Detection of Mycobacterium avium subsp. paratuberculosis in faeces using different procedures of pre-treatment for real-time PCR in comparison to culture. The Veterinary Journal, 199(1):138-142.
26. Tohidi Moghadam, M., S. Sarv, F. Moosakhani, and A. Badiie. 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.
27. Whittington, R., A. Hope, D. Marshall, C. Taragel, and I. Marsh. 2000. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia. Journal of Clinical Microbiology, 38(9):3240-3248.
28. Whittington, R., I. Marsh, S. McAllister, M. Turner, D. Marshall, and C. Fraser. 1999. Evaluation of Modified BACTEC 12B Radiometric Medium and Solid Media for Culture of Mycobacterium aviumsubsp. paratuberculosis from Sheep. Journal of Clinical Microbiology, 37(4):1077-1083.
29. Whittington, R., I. Marsh, and R. Whitlock. 2001. Typing of IS 1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock. Molecular and Cellular Probes, 15(3):139-145.
30. Whittington, R. J. 2009. Factors affecting isolation and identification of Mycobacterium avium subsp. paratuberculosis from fecal and tissue samples in a liquid culture system. Journal of Clinical Microbiology, 47(3):614-622.