شناسایی مایکوپلاسما بویس در گاوهای مبتلا به ورم پستان بالینی با استفاده از کشت و روش واکنش زنجیره‌ای پلیمراز بر اساس ژن‌های 16SrRNA و uvrC
محورهای موضوعی :
آسیب شناسی درمانگاهی دامپزشکی
محسن ایمان‌‌دار
1
,
سید علی پوربخش
2
,
محمود جمشیدیان
3
,
تقی زهرائی صالحی
4
1 - گروه میکروبیولوژی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
2 - آزمایشگاه رفرانس مایکوپلاسما، مؤسسه تحقیقات واکسن و سرم سازی رازی، کرج، ایران.
3 - گروه میکروبیولوژی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
4 - گروه میکروبیولوژی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
تاریخ دریافت : 1396/06/24
تاریخ پذیرش : 1396/10/04
تاریخ انتشار : 1397/08/01
کلید واژه:
گاو,
PCR,
ورم پستان,
مایکوپلاسما بویس,
ژن uvrC,
چکیده مقاله :
مایکوپلاسما بویس از عوامل اصلی ایجاد پنومونی، ورم پستان و آرتریت مایکوپلاسمائی در گاو می‌باشد. هدف از این مطالعه، شناسایی مایکوپلاسما بویس در گاوهای مبتلا به ورم پستان بالینی با استفاده از کشت و روش زنجیره‌ای پلیمراز بود. این مطالعه روی تعداد 328 نمونه شیر گاو مبتلا به ورم پستان بالینی که در طول یک سال به آزمایشگاه مرجع مایکوپلاسمای مؤسسه تحقیقات واکسن و سرم‌سازی رازی ارجاع می‌شد، اجرا گردید. نمونه‌ها در دمای 4 درجه سلسیوس و در مدت کمتر از 24 ساعت به آزمایشگاه ارسال و به مدت 24-18 ساعت در 37 درجه سلسیوس انکوبه می شدند و پس از فیلتر شدن به محیط اختصاصی PPLO، در انکوباتور Co2دار به مدت 10-7 روز از نظر رشد پایش می شدند. همزمانDNA نمونه‌ها به وسیله روش فنل- کلروفرم استخراج و خالص سازی شده و از روش PCR برای تشخیص جنس مایکوپلاسما بر اساس ژن 16SrRNA و برای تشخیص گونه بویس از جستجوی وجود ژن uvrC استفاده گردید.از تعداد 328 نمونه، 58 مورد (69/17 درصد) در محیط کشت PPLO مثبت شناخته شدند. از نظر حضور ژن مربوط به جنس مایکوپلاسما، تعداد 97 نمونه (57/29 درصد) مثبت شناخته شدند که از این تعداد، 14 نمونه (26/4 درصد) ژن اختصاصی گونه بویس (uvrC) را نشان دادند.نتایج این مطالعه روش PCR را به عنوان یک تست سریع و قابل اعتماد در شناسایی مایکوپلاسما بویس در نمونه‌های شیر معرفی کرده و نشان داد که میزان حضور مایکوپلاسما بویس در گله‌های شیری در ایران بالا است و می‌تواند به عنوان یکی از عوامل اصلی مسبب ورم پستان بالینی در گاو مطرح باشد.
چکیده انگلیسی:
Mycoplasma bovis is one of the main causes of pneumonia, mastitis and arthritis in cattle. The aim of this study was to identify the M. bovis in cows with clinical mastitis using culture and PCR based on 16SrRNA and uvrC genes. A total of 328 milk samples were obtained from cattle with clinical mastitis in industrial dairy herds. Samples were sent to the Mycoplasma reference laboratory, Razi Vaccine and Serum Research Institute at 4°C in less than 24 hr and incubated for 18-24 hr at 37°C. After filtering to specific PPLO medium, they were incubated in the presence of Co2 and were monitored for 7-10 days. Simultaneously, DNA of the samples was extracted and purified by Phenol-Chloroform method and PCR was used to detect mycoplasma genus based on 16SrRNA and detection of M. bovis species based on uvrC genes. Out of 328 samples, 58 samples were positive in PPLO agar media. Ninty-seven samples (29.57%) were positive for mycoplasma gene, of which 14 samples (4.26%) showed specific gene of M. bovis. The results of this study introduced the PCR method as a rapid and reliable test for detection of M. bovis in milk specimens. The results also showed that the presence of M. bovis in dairy herds in Iran is high and it can be considered as one of the main causes of the clinical manifestation of cattle mastitis.
منابع و مأخذ:
Ahmad, Z.S., Babar, F., Abbas, M.A., Awan, M., Shafee, M.M., Tariq, M., et al. (2014). Prevalence of Mycoplasma bovis in respiratory tract of cattle slaughtered in Balochistan, Pakistan. Pakistan Veterinary Journal, 34(1): 46-49.
Arcangioli, M.A., Chazel, M., Sellal, E., Botrel, M.A., Bezille, P., Poumarat, F., et al. (2011). Prevalence of Mycoplasma bovis udder infection in dairy cattle: preliminary field investigation in southeast France. New Zealand Veterinary Journal, 59(2): 75-78.
Baird, S.C., Carman, J., Dinsmore, R.P., Walker, R.L. and Collins, J.K. (1999). Detection and identification of Mycoplasma from bovine mastitis infections using a nested polymerase chain reaction. Journal of Veterinary Diagnosis Investigation, 11: 432-435.
Cai, H.Y., Bell-Rogers, P. and Parker, L. (2005). Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples. Journal of Veterinary Diagnosis Investigation, 17: 537-545.
Cremonesi, P., Vimercati, C., Pisoni, G., Perez, G., Miranda, R., Castiglioni, A.B., et al. (2007). Development of DNA extraction and PCR amplification protocols for detection of Mycoplasma bovis directly from milk samples. Veterinary Research Communications, 31(1): 225-227.
Foddai, A., Idini, G., Fusco, M., Rosa, N., De la Fe, C., Zinellu, S., et al. (2005). Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-PCR and a PCR-RFLP. Molecular and Cellular Probes, 19: 207-212.
Fox, L.K., Kirk, J.H. and Britten, A. (2008). Mycoplasma mastitis: a review of transmission and control. Journal of Veterinary Medical Infectious Disease, Veterinary Public Health, 52: 153-160.
Fu-Rong Z., Xue-Liang Z., Min-Jun X., Si-Yang H., Dong-Hui Z., Hui-Yan X., et al. (2012). First report of Mycoplasma bovis infection in dairy cattle in Guangzhou, subtropical southern China. African Journal of Microbiology Research, 6(27): 5668-5671.
Ghadersohi, A., Coelen, R.J. and Hirst, R.G. (1997). Development of specific DNA probe and PCR for the detection of Mycoplasma bovis. Veterinary Microbiology, 56: 87-98.
Ghazaei, C. (2006). Detection of mycoplasmal mastitis and determination of its prevalence rate in dairy cattle herds in Moghan-Ardabil state, Iran. Journal of Animal and Veterinary Advances, 5(4): 280-283.
Gunning, R.F. and Shepherd, P.A. (1996). Outbreak of bovine Mycoplasma bovis mastitis. Veterinary Records, 139: 23-24.
Hidetoshi, H., Hideto, M., Kazuhiro, K., Takehiro, O. and Tetsu, O. (2010). A simplified PCR assay for fast and mycoplasma mastitis screening in dairy cattle. Journal of Veterinary Science, 12(2): 191-193.
Hirose, K., Kawasaki, H. and Dotani, K. (2001). Detection of Mycoplasma in mastitic milk by PCR analysis and culture method. Journal of Veterinary Medical Science, 63: 691-693.
Justice-Allen, A., Trujillo, J., Goodell, G. and Wilson, D. (2011). Detection of multiple Mycoplasma species in bulk tank milk samples using real-time PCR and conventional culture and comparison of test sensitivities. Journal of Dairy Science, 94: 3411-3419.
Kampa, J., Sukolapong, V., Buttasri, A. and Charoenchai, A. (2009). Prevalence of Mycoplasma bovis and Other Contagious Bovine Mastitis Pathogens in Bulk Tank Milk of Dairy Cattle Herds in Khon Kaen Province, Thailand. Thailand Journal of Veterinary Medicine, 39(3): 275-280.
Karahan, M., Kalin, R., Atil, E. and Çetinkaya B. (2010). Detection of Mycoplasma bovis in cattle with mastitis and respiratory problems in eastern Turkey. Veterinary Record, 166: 827-829.
Kirk, J.H. and Lauerman, L.H. (1994). Mycoplasma mastitis in dairy cows. Compendium of Continious Education Practice Veterinary, 16: 541-551.
Kojima, A., Takahashi, T., Kijima, M., Ogikubo, Y., Nishimura, Y., Nishimura, S., et al. (1997). Detection of mycoplasma in avian live virus vaccines by polymerase chain reaction. Biologicals, 25: 365-71.
McAuliffe, L., Branko, K.R., Ayling R.D. and Nicholas, R.A. (2006). Molecular epidemiological analysis of Mycoplasma bovis isolates from the United Kingdom shows two genetically distinct clusters. Journal of Clinical Microbiology, 42: 4556-4565.
McDonald, W.L., Rawdon, T.G., Fitzmaurice, J., Bolotovski, I., Voges, H., Humphrey, S., et al. (2009). Survey of bulk tank milk in New Zealand for Mycoplasma bovis, using species-specific nested PCR and culture. New Zealand Veterinary Journal, 57(1): 44-49.
Mosaferi, S. and Haghighat Kaghazchian, M. (2003). Mastitis in Farm Animals. 1st ed., Iran: Tabriz, Islamic Azad University Press, pp: 159-170. [In Persian]
Moshkelani, S., Rabiee, S. and Javaheri-Koupaei, M. (2011). Occurrence of Mycoplasma in bovine mastitic milk in Iran. Research Opinions in Animal and Veterinary Sciences, 1(11): 694-696.
Nicholas, R.A.J. and Ayling, R.D. (2003). Mycoplasma bovis: disease, diagnosis and control. Research in Veterinary Science, 74: 105-112.
Osman, K.M., Abd El-Razik, K.A., Barbar, E.E., ELShafey, D.Y.H. and Arafa, A.A. (2008). Molecular typing of mycoplasma species recovered from bovine mastitis. Global Veterinaria, 2(6): 360-368.
Pfützner, H. and Sachse, K. (1996). Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Review Scientific Technical Journal, 15: 1477-1494.
Pinnow, C.C., Butler, J.A., Sachse, K., Hotzel, H., Timms, L.L. and Rosenbusch, R.F. (2001). Detection of Mycoplasma bovis in preservative-treated field milk samples. Journal of Dairy Science, 84: 1640-1645.
Pourbakhsh, S.A., Shokri, G.R., Banani, M., Elhamnia, F. and Ashtari, A. (2010). Detection of Mycoplasma synoviae infection in broiler breeder farms of Tehran province using PCR and culture methods. Archives of Razi Institute, 65(2): 75-81.
Radaelli, E., Castiglioni, V., Losa, M., Benedetti, V., Piccinini, R. and Nicholas, R.A.J. (2011). Outbreak of bovine clinical mastitis caused by Mycoplasma bovis in a North Italian herd. Research in Veterinary Science, 91: 251-253.
Rossetti, B.C., Frey, J. and Pilo, P. (2010). Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR. Molecular and Cellular Probes, 24: 321-323.
Sayin, Z., Aslı, S., Uçkun S.U., Uslu, A., Hadimli, H.H., Aras, Z., et al. (2016). Mycoplasma infections in dairy cattle farms in Turkey. Turkish Journal of Veterinary and Animal Sciences, 40: 1-6.
Stokka, G.L., Lechtenberg, K. and Edwards, T. (2001). Lameness in Feedlot Cattle. Veterinary Clinical North Amer-Food Animal Practice, 17(1): 196-202.
Subramaniam, S., Bergonier, D., Poumarat, F., Capaul, S., Schlatter, Y., Nicolet, J., et al. (1998). Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR. Molecular and Cellular Probes, 12: 161-169.
Taleb Khan Garousi, M., Navid Mehr, J. and Rahimi, R. (2006). Preliminary study on the prevalence of mycoplasma mastitis in a number of herds of dairy cattle around Mashhad. Journal of Veterinary Research, University of Tehran, 61(4): 321-323. [In Persian]
Thomas, A., Dizier, I., Linden, A., Mainil, J., Frey, J. and Vilei, E.M. (2004). Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis. The Veterinary Journal, 168: 100-102.
Wise, K.S., Calcutt, M.J., Foecking, M.F., Roske, K., Madupu, R. and Methe, B.A. (2011). Complete genome sequence of Mycoplasma bovis type strain PG45 (ATCC 25523). Infection and Immunity, 79(2): 982-983.
_||_
Ahmad, Z.S., Babar, F., Abbas, M.A., Awan, M., Shafee, M.M., Tariq, M., et al. (2014). Prevalence of Mycoplasma bovis in respiratory tract of cattle slaughtered in Balochistan, Pakistan. Pakistan Veterinary Journal, 34(1): 46-49.
Arcangioli, M.A., Chazel, M., Sellal, E., Botrel, M.A., Bezille, P., Poumarat, F., et al. (2011). Prevalence of Mycoplasma bovis udder infection in dairy cattle: preliminary field investigation in southeast France. New Zealand Veterinary Journal, 59(2): 75-78.
Baird, S.C., Carman, J., Dinsmore, R.P., Walker, R.L. and Collins, J.K. (1999). Detection and identification of Mycoplasma from bovine mastitis infections using a nested polymerase chain reaction. Journal of Veterinary Diagnosis Investigation, 11: 432-435.
Cai, H.Y., Bell-Rogers, P. and Parker, L. (2005). Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples. Journal of Veterinary Diagnosis Investigation, 17: 537-545.
Cremonesi, P., Vimercati, C., Pisoni, G., Perez, G., Miranda, R., Castiglioni, A.B., et al. (2007). Development of DNA extraction and PCR amplification protocols for detection of Mycoplasma bovis directly from milk samples. Veterinary Research Communications, 31(1): 225-227.
Foddai, A., Idini, G., Fusco, M., Rosa, N., De la Fe, C., Zinellu, S., et al. (2005). Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-PCR and a PCR-RFLP. Molecular and Cellular Probes, 19: 207-212.
Fox, L.K., Kirk, J.H. and Britten, A. (2008). Mycoplasma mastitis: a review of transmission and control. Journal of Veterinary Medical Infectious Disease, Veterinary Public Health, 52: 153-160.
Fu-Rong Z., Xue-Liang Z., Min-Jun X., Si-Yang H., Dong-Hui Z., Hui-Yan X., et al. (2012). First report of Mycoplasma bovis infection in dairy cattle in Guangzhou, subtropical southern China. African Journal of Microbiology Research, 6(27): 5668-5671.
Ghadersohi, A., Coelen, R.J. and Hirst, R.G. (1997). Development of specific DNA probe and PCR for the detection of Mycoplasma bovis. Veterinary Microbiology, 56: 87-98.
Ghazaei, C. (2006). Detection of mycoplasmal mastitis and determination of its prevalence rate in dairy cattle herds in Moghan-Ardabil state, Iran. Journal of Animal and Veterinary Advances, 5(4): 280-283.
Gunning, R.F. and Shepherd, P.A. (1996). Outbreak of bovine Mycoplasma bovis mastitis. Veterinary Records, 139: 23-24.
Hidetoshi, H., Hideto, M., Kazuhiro, K., Takehiro, O. and Tetsu, O. (2010). A simplified PCR assay for fast and mycoplasma mastitis screening in dairy cattle. Journal of Veterinary Science, 12(2): 191-193.
Hirose, K., Kawasaki, H. and Dotani, K. (2001). Detection of Mycoplasma in mastitic milk by PCR analysis and culture method. Journal of Veterinary Medical Science, 63: 691-693.
Justice-Allen, A., Trujillo, J., Goodell, G. and Wilson, D. (2011). Detection of multiple Mycoplasma species in bulk tank milk samples using real-time PCR and conventional culture and comparison of test sensitivities. Journal of Dairy Science, 94: 3411-3419.
Kampa, J., Sukolapong, V., Buttasri, A. and Charoenchai, A. (2009). Prevalence of Mycoplasma bovis and Other Contagious Bovine Mastitis Pathogens in Bulk Tank Milk of Dairy Cattle Herds in Khon Kaen Province, Thailand. Thailand Journal of Veterinary Medicine, 39(3): 275-280.
Karahan, M., Kalin, R., Atil, E. and Çetinkaya B. (2010). Detection of Mycoplasma bovis in cattle with mastitis and respiratory problems in eastern Turkey. Veterinary Record, 166: 827-829.
Kirk, J.H. and Lauerman, L.H. (1994). Mycoplasma mastitis in dairy cows. Compendium of Continious Education Practice Veterinary, 16: 541-551.
Kojima, A., Takahashi, T., Kijima, M., Ogikubo, Y., Nishimura, Y., Nishimura, S., et al. (1997). Detection of mycoplasma in avian live virus vaccines by polymerase chain reaction. Biologicals, 25: 365-71.
McAuliffe, L., Branko, K.R., Ayling R.D. and Nicholas, R.A. (2006). Molecular epidemiological analysis of Mycoplasma bovis isolates from the United Kingdom shows two genetically distinct clusters. Journal of Clinical Microbiology, 42: 4556-4565.
McDonald, W.L., Rawdon, T.G., Fitzmaurice, J., Bolotovski, I., Voges, H., Humphrey, S., et al. (2009). Survey of bulk tank milk in New Zealand for Mycoplasma bovis, using species-specific nested PCR and culture. New Zealand Veterinary Journal, 57(1): 44-49.
Mosaferi, S. and Haghighat Kaghazchian, M. (2003). Mastitis in Farm Animals. 1st ed., Iran: Tabriz, Islamic Azad University Press, pp: 159-170. [In Persian]
Moshkelani, S., Rabiee, S. and Javaheri-Koupaei, M. (2011). Occurrence of Mycoplasma in bovine mastitic milk in Iran. Research Opinions in Animal and Veterinary Sciences, 1(11): 694-696.
Nicholas, R.A.J. and Ayling, R.D. (2003). Mycoplasma bovis: disease, diagnosis and control. Research in Veterinary Science, 74: 105-112.
Osman, K.M., Abd El-Razik, K.A., Barbar, E.E., ELShafey, D.Y.H. and Arafa, A.A. (2008). Molecular typing of mycoplasma species recovered from bovine mastitis. Global Veterinaria, 2(6): 360-368.
Pfützner, H. and Sachse, K. (1996). Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Review Scientific Technical Journal, 15: 1477-1494.
Pinnow, C.C., Butler, J.A., Sachse, K., Hotzel, H., Timms, L.L. and Rosenbusch, R.F. (2001). Detection of Mycoplasma bovis in preservative-treated field milk samples. Journal of Dairy Science, 84: 1640-1645.
Pourbakhsh, S.A., Shokri, G.R., Banani, M., Elhamnia, F. and Ashtari, A. (2010). Detection of Mycoplasma synoviae infection in broiler breeder farms of Tehran province using PCR and culture methods. Archives of Razi Institute, 65(2): 75-81.
Radaelli, E., Castiglioni, V., Losa, M., Benedetti, V., Piccinini, R. and Nicholas, R.A.J. (2011). Outbreak of bovine clinical mastitis caused by Mycoplasma bovis in a North Italian herd. Research in Veterinary Science, 91: 251-253.
Rossetti, B.C., Frey, J. and Pilo, P. (2010). Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR. Molecular and Cellular Probes, 24: 321-323.
Sayin, Z., Aslı, S., Uçkun S.U., Uslu, A., Hadimli, H.H., Aras, Z., et al. (2016). Mycoplasma infections in dairy cattle farms in Turkey. Turkish Journal of Veterinary and Animal Sciences, 40: 1-6.
Stokka, G.L., Lechtenberg, K. and Edwards, T. (2001). Lameness in Feedlot Cattle. Veterinary Clinical North Amer-Food Animal Practice, 17(1): 196-202.
Subramaniam, S., Bergonier, D., Poumarat, F., Capaul, S., Schlatter, Y., Nicolet, J., et al. (1998). Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR. Molecular and Cellular Probes, 12: 161-169.
Taleb Khan Garousi, M., Navid Mehr, J. and Rahimi, R. (2006). Preliminary study on the prevalence of mycoplasma mastitis in a number of herds of dairy cattle around Mashhad. Journal of Veterinary Research, University of Tehran, 61(4): 321-323. [In Persian]
Thomas, A., Dizier, I., Linden, A., Mainil, J., Frey, J. and Vilei, E.M. (2004). Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis. The Veterinary Journal, 168: 100-102.
Wise, K.S., Calcutt, M.J., Foecking, M.F., Roske, K., Madupu, R. and Methe, B.A. (2011). Complete genome sequence of Mycoplasma bovis type strain PG45 (ATCC 25523). Infection and Immunity, 79(2): 982-983.
