همسانی ژنتیکی سویه های واکسن مایکوپلاسما آگالاکتیه جدا شده از طالقان، لرستان و شیراز در ایران
محورهای موضوعی : میکروب شناسی مولکولیخاطره کبیری 1 , کیوان تدین 2 , سید علی پوربخش 3 , جمیله نوروزی 4
1 - دانشجوی دکتری، گروه میکروبیولوژی، دانشکده علوم زیستی، دانشگاه آزاد اسلامی واحد تهران شمال، تهران
2 - دانشیار، سازمان تحقیقات آموزش و ترویج کشاورزی، موسسه تحقیقات واکسن و سرم سازی رازی
3 - استاد، سازمان تحقیقات آموزش و ترویج کشاورزی، موسسه تحقیقات واکسن و سرم سازی رازی
4 - استاد، گروه میکروبیولوژی، دانشکده علوم زیستی، دانشگاه آزاد اسلامی واحد تهران شمال، تهران
کلید واژه: مایکوپلاسما آگالاکتیه, MLVA, دسته بندی ژنتیکی, سویه های واکسن, آگالاکسی,
چکیده مقاله :
سابقه و هدف: ایران یکی از اصلی ترین کانون های فعالیت مایکوپلاسما آگالاکتیه در دام های نشخوارکننده در جهان شناخته است. سه سویه بومی طالقان، لرستان و شیراز برای تولید تنها نمونه واکسن کشته علیه آگالاکسی در ایران مورد استفاده قرار می گیرند. این مطالعه با هدف آگاهی از ارتباط ژنتیکی میان این سویه ها انجام شد.مواد و روش ها: از هر سه سویه بومی طالقان، لرستان و شیراز کشت تازه در محیط آبکوشت PPLO تهیه و ماده ژنتیکی با روش جوشاندن استخراج گردید. چهار واکنش مستقل PCR بر مبنای 4 لوکوس VNTR5، VNTR9، VNTR17 و VNTR19 تنظیم و در مورد هر سه سویه اجرا گردید. توالی نوکلئوتیدهای تمامی محصولات PCR تعیین گردید.یافته ها: نتایج به دست آمده بر مبنای توالی نوکلئوتیدها در هر 4 لوکوس وجود همسانی کامل در میان ژنوم سه سویه را نشان داد. از طرف دیگر به جز لوکوس VNTR19 سویه های سه گانه ایرانی در هر سه لوکوس دیگر با سویه شاخص PG2 مایکوپلاسما آگالاکتیه همسان مشاهده شدند. در لوکوس VNTR19 تنها تفاوت مشهود اضافه شدن 3 نوکلوتید به طول این لوکوس در سویه های ایرانی بود.نتیجه گیری: همسانی ژنتیکی در میان سه سویه ایرانی می تواند نشانه فعالیت یک یا چند کلون های بومی اجدادی باکتری در جغرافیای ایران باشد که در طول تاریخ دامپروری این کشور پیدایش و تکامل یافته اند. در نتیجه ضعف در اعمال سیاست های کنترل بیماری به صورت هموژن و غالب منتشر شده است. در توضیح چگونگی شباهت میان سه سویه ایرانی و سویه PG2 می توان آنرا به تشابه تصادفی در فرآیند تکاملی (حالت هموپلازی) و یا متاثر از مداخلات انسانی از راه فعالیت های دامپروری مانند واردات دام از خارج مربوط دانست. اعمال روش های استاندارد ژنوتایپینگ بر روی تعداد بیشتر جدایه های ایرانی می تواند به درک صحیح این موضوع کمک نماید.
Background & Objectives: Iran continues to hold one of the most currently active world foci of agalaxy in ruminants. The three local strains of Taliqan, Lorestan, and Shiraz have been used for many years in preparation of the only commercially available killed vaccine against agalaxy in the Iranian market. This study was conducted to determine the genetic link between these strains. Materials & Methods: All three strains of Taleghan, Lorestan, and Shiraz were cultivated freshly on PPLO agar media. The genetic material was extracted by boiling method. In order to investigate the genomic relations between these strains, a Multi-Locus Variable Number Tandem Repeat Analysis (MLVA) strategy concentrating on 4 VNTR loci of 5, 9, 17, and 19 was employed. The nucleotide sequences of all PCR products were determined, as well. Results: Nucleotide sequence analysis showed an identical MLVA pattern by all the three strains. When compared to the Mycoplasma agalactiae PG2 laboratory strain, the only VNTR19 locus was different between Iranians and the PG2 strain, with respect to a 3bps addition at this locus in Iranian strain. Conclusion: The identical genetic pattern of the three Iranian strains is likely an indication of the activity of one or more indigenous ancestral clone in the geography of Iran that has been appeared and evolved throughout its livestock history and became homogeneous and predominant due to the absence of efficient disease control policies. The similarity between three Iranian and the PG2 strains might be due to homoplasy or human intervention through animal husbandry activities such as livestock importation. Applying standard genotyping methods to a larger number of local isolates help to better assess this observation.
Rosengarten R, Citti C. Molecular typing of Mycoplasma agalactiae: tracing European-wide
genetic diversity and an endemic clonal population. Comp Immunol Microbiol Infect Dis. 2012;
35(5): 487-496.
2. Noaman V. Identification of Mycoplasma agalactiae by conventional and molecular methods on
small ruminants in central zone of Iran. Comp Clin Path. 2015; 24(3): 653-657.
3. Khezri M, Pourbakhsh SA. A survey of Mycoplasma agalactiae in small ruminants with
contagious agalactiae syndrome in Iran. Bangladesh J Vet Med. 2014; 12(1): 67-72.
4. Mohammadpour SH, Pourbakhsh SA, Kheirkhah B. Isolation and identification of Mycoplasma
agalactiae by polymerase chain reaction (PCR) in suspected sheep samples in Kerman Province,
Iran. Afr J Microbiol Res. 2013; 7(10): 885-889.
5. Khezri M, Pourbakhsh SA, Ashtari A, Rokhzad B, Khanbabaie H. Isolation and prevalence of
Mycoplasma agalactiae in Kurdish sheep in Kurdistan, Iran. Vet World. 2012; 5(12): 727-731.
6. Bidhendi SM, Khaki PLangroudi RP. Isolation and identification of Mycoplasma agalactiae by
culture and polymerase chain reaction in sheep and goat milk samples in Kordestan province,
Iran. Arch Razi Inst. 2011; 66(1): 11-16.
7. Amir Momen H, Roshdi Maleki M. Identification of Mycoplasma agalactiae in milk by culture
and PCR methods in Hamedan of Iran. Ind J Fundament Appl Life Sci. 2014; 4 (S4): 2175-2180.
8. Sotoodehnia A, Moazenijula A, Rad AN, Jabbari A. Preparation of agalactia vaccine in
fermentor. Arch Razi Inst. 2007; 62(1): 45-48.
9. Tola S, Idini G, Manunta D, Casciano I, Rocchigiani AM, Angioi ALeori G. Comparison of
Mycoplasma agalactiae isolates by pulsed field gel electrophoresis, SDS-PAGE and
immunoblotting. FEMS Microbiol Lett. 1996; 143(2-3): 259-65.
10. De la Fe C, Amores J, Tardy F, Sagne E, Nouvel LX, Citti C. Unexpected genetic diversity of
Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of
Spain. BMC Vet Res. 2012; 8: 146.
11. McAuliffe L, Gosney F, Hlusek M, de Garnica ML, Spergser J, Kargl M, Rosengarten R,
Ayling RD, Nicholas RA, Ellis RJ. Multilocus sequence typing of Mycoplasma agalactiae. J
Med Microbiol. 2011; 60(6): 803-811.
12. McAuliffe L, Churchward CP, Lawes JR, Loria G, Ayling RD, Nicholas RA. VNTR analysis
reveals unexpected genetic diversity within Mycoplasma agalactiae, the main causative agent of
contagious agalactia. BMC Microbiol. 2008; 8: 193.
13. Ataee RA, Golmohammadi R, Alishiri GH, Mirnejad R, Najafi A, Esmaeili D, Jonaidi-Jafari N.
Simultaneous detection of Mycoplasma pneumoniae, Mycoplasma hominis and Mycoplasma
arthritidis in synovial fluid of patients with rheumatoid arthritis by Multiplex PCR. Arch Iran
Med. 2015; 18(6): 345-350.
14. Subramaniam S, Bergonier D, Poumarat F, Capaul S, Schlatter Y, Nicolet J, Frey J. Species
identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by
PCR. Mol Cell Probes. 1998; 12(3): 161-169.
15. Prats-van der Ham M, Tatay-Dualde J, de la Fe C, Paterna A, Sanchez A, Corrales JC,
Contreras A, Gomez-Martin A. Detecting asymptomatic rams infected with Mycoplasma
agalactiae in ovine artificial insemination centers. Theriogenol. 2017; 89: 324-328.
16. Notifiable diseases: Contagious agalactia pathogen confirmed in Wales. Vet Rec. 2014; 175
(19): 468.
17. Ruiz-Fons F, Gonzalez-Barrio D, Aguilar-Rios F, Soler AJ, Garde JJ, Gortazar C,
Fernandez-Santos Mdel R. Infectious pathogens potentially transmitted by semen of the black
variety of the Manchega sheep breed: Health constraints for conservation purposes. Anim
Reprod Sci. 2014; 149(3-4): 152-157.
18. do Nascimento NC, Santos AP, Guimaraes AM, Sanmiguel PJ, Messick JB. Mycoplasma
haemocanis- the canine hemoplasma and its feline counterpart in the genomic era. Vet Res.
2012; 43: 66.
19. Dos Santos LF, Clavijo MJ, Sreevatsan S, Rovira A, Moreira MA, Pieters M. Genotyping of
Mycoplasma hyorhinis using multiple-locus variable number tandem repeat analysis. J
Microbiol Methods. 2015; 111: 87-92.
20. Pantoja LG, Pettit K, Dos Santos LF, Tubbs R, Pieters M. Mycoplasma hyopneumoniae
genetic variability within a swine operation. J Vet Diagn Invest. 2016; 28(2): 175-179.
21. Pinho L, Thompson G, Rosenbusch R, Carvalheira J. Genotyping of Mycoplasma bovis
isolates using multiple-locus variable-number tandem-repeat analysis. J Microbiol Methods.
2012; 88(3): 377-385.
22. Hata E, Suzuki K, Hanyu H, Itoh M, Higuchi H, Kobayashi H. Molecular epidemiology of
cases of Mycoplasma californicum infection in Japan. Appl Environ Microbiol. 2014; 80(24):
7717-7724.
23. Ferandon C, Peuchant O, Renaudin H, Bebear C. Diversity of Mycoplasma hominis clinical
isolates from Bordeaux, France, as assessed by multiple-locus variable-number tandem repeat
analysis. BMC Microbiol. 2013; 13: 120.
24. Cazanave C, Charron A, Renaudin H, Bebear C. Method comparison for molecular typing of
French and Tunisian Mycoplasma genitalium-positive specimens. J Med Microbiol. 2012; 61(Pt
4): 500-506.
25. Nwankpa ND, Manso-silvan L, Lorenzon S, Yaya A, Lombin LH, Thiaucourt F. Variable
Number Tandem Repeat (VNTR) analysis reveals genetic diversity within Mycoplasma
mycoides mycoides small colony isolates from Nigeria. Vet Microbiol. 2010; 146(3-4):354-355.
26. Mahdavi S, Salehi TZ, Madani R, Keyvanfar H. Comparative study of homology of
cytoplasmic membrane protein 40 KDa of Mycoplasma agalactiae in isolated strains in Iran.
Afr J Microbiol Res. 2009; 3(9): 528-532.
27. Candela MG, Serrano E, Sevila J, Leon L, Caro MR, Verheyden H. Pathogens of zoonotic and
biological importance in roe deer (Capreolus capreolus): Seroprevalence in an agro-system
population in France. Res Vet Sci. 2014; 96(2): 254-259.
28. Murai K, Lehenbauer TW, Champagne JD, Glenn K, Aly SS. Cost-effectiveness of diagnostic
strategies using quantitative real-time PCR and bacterial culture to identify contagious mastitis
cases in large dairy herds. Prev Vet Med. 2014; 113(4): 522-535.
29. Sanna G, Lecca V, Foddai A, Tola S. Development of a specific immunomagnetic
capture-PCR for rapid detection of viable Mycoplasma agalactiae in sheep milk samples. J
Appl Microbiol. 2014; 117(6): 1585-1591.
30. Gomez-Martin A, Uc N, Vieira LA, Gadea J, Cadenas J, Sanchez A, De la Fe C. Survival
capacity of Mycoplasma agalactiae and Mycoplasma mycoides subsp capri in the diluted semen
of goat bucks and their effects on sperm quality. Theriogenol. 2015; 83(5): 911-919
_||_
Rosengarten R, Citti C. Molecular typing of Mycoplasma agalactiae: tracing European-wide
genetic diversity and an endemic clonal population. Comp Immunol Microbiol Infect Dis. 2012;
35(5): 487-496.
2. Noaman V. Identification of Mycoplasma agalactiae by conventional and molecular methods on
small ruminants in central zone of Iran. Comp Clin Path. 2015; 24(3): 653-657.
3. Khezri M, Pourbakhsh SA. A survey of Mycoplasma agalactiae in small ruminants with
contagious agalactiae syndrome in Iran. Bangladesh J Vet Med. 2014; 12(1): 67-72.
4. Mohammadpour SH, Pourbakhsh SA, Kheirkhah B. Isolation and identification of Mycoplasma
agalactiae by polymerase chain reaction (PCR) in suspected sheep samples in Kerman Province,
Iran. Afr J Microbiol Res. 2013; 7(10): 885-889.
5. Khezri M, Pourbakhsh SA, Ashtari A, Rokhzad B, Khanbabaie H. Isolation and prevalence of
Mycoplasma agalactiae in Kurdish sheep in Kurdistan, Iran. Vet World. 2012; 5(12): 727-731.
6. Bidhendi SM, Khaki PLangroudi RP. Isolation and identification of Mycoplasma agalactiae by
culture and polymerase chain reaction in sheep and goat milk samples in Kordestan province,
Iran. Arch Razi Inst. 2011; 66(1): 11-16.
7. Amir Momen H, Roshdi Maleki M. Identification of Mycoplasma agalactiae in milk by culture
and PCR methods in Hamedan of Iran. Ind J Fundament Appl Life Sci. 2014; 4 (S4): 2175-2180.
8. Sotoodehnia A, Moazenijula A, Rad AN, Jabbari A. Preparation of agalactia vaccine in
fermentor. Arch Razi Inst. 2007; 62(1): 45-48.
9. Tola S, Idini G, Manunta D, Casciano I, Rocchigiani AM, Angioi ALeori G. Comparison of
Mycoplasma agalactiae isolates by pulsed field gel electrophoresis, SDS-PAGE and
immunoblotting. FEMS Microbiol Lett. 1996; 143(2-3): 259-65.
10. De la Fe C, Amores J, Tardy F, Sagne E, Nouvel LX, Citti C. Unexpected genetic diversity of
Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of
Spain. BMC Vet Res. 2012; 8: 146.
11. McAuliffe L, Gosney F, Hlusek M, de Garnica ML, Spergser J, Kargl M, Rosengarten R,
Ayling RD, Nicholas RA, Ellis RJ. Multilocus sequence typing of Mycoplasma agalactiae. J
Med Microbiol. 2011; 60(6): 803-811.
12. McAuliffe L, Churchward CP, Lawes JR, Loria G, Ayling RD, Nicholas RA. VNTR analysis
reveals unexpected genetic diversity within Mycoplasma agalactiae, the main causative agent of
contagious agalactia. BMC Microbiol. 2008; 8: 193.
13. Ataee RA, Golmohammadi R, Alishiri GH, Mirnejad R, Najafi A, Esmaeili D, Jonaidi-Jafari N.
Simultaneous detection of Mycoplasma pneumoniae, Mycoplasma hominis and Mycoplasma
arthritidis in synovial fluid of patients with rheumatoid arthritis by Multiplex PCR. Arch Iran
Med. 2015; 18(6): 345-350.
14. Subramaniam S, Bergonier D, Poumarat F, Capaul S, Schlatter Y, Nicolet J, Frey J. Species
identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by
PCR. Mol Cell Probes. 1998; 12(3): 161-169.
15. Prats-van der Ham M, Tatay-Dualde J, de la Fe C, Paterna A, Sanchez A, Corrales JC,
Contreras A, Gomez-Martin A. Detecting asymptomatic rams infected with Mycoplasma
agalactiae in ovine artificial insemination centers. Theriogenol. 2017; 89: 324-328.
16. Notifiable diseases: Contagious agalactia pathogen confirmed in Wales. Vet Rec. 2014; 175
(19): 468.
17. Ruiz-Fons F, Gonzalez-Barrio D, Aguilar-Rios F, Soler AJ, Garde JJ, Gortazar C,
Fernandez-Santos Mdel R. Infectious pathogens potentially transmitted by semen of the black
variety of the Manchega sheep breed: Health constraints for conservation purposes. Anim
Reprod Sci. 2014; 149(3-4): 152-157.
18. do Nascimento NC, Santos AP, Guimaraes AM, Sanmiguel PJ, Messick JB. Mycoplasma
haemocanis- the canine hemoplasma and its feline counterpart in the genomic era. Vet Res.
2012; 43: 66.
19. Dos Santos LF, Clavijo MJ, Sreevatsan S, Rovira A, Moreira MA, Pieters M. Genotyping of
Mycoplasma hyorhinis using multiple-locus variable number tandem repeat analysis. J
Microbiol Methods. 2015; 111: 87-92.
20. Pantoja LG, Pettit K, Dos Santos LF, Tubbs R, Pieters M. Mycoplasma hyopneumoniae
genetic variability within a swine operation. J Vet Diagn Invest. 2016; 28(2): 175-179.
21. Pinho L, Thompson G, Rosenbusch R, Carvalheira J. Genotyping of Mycoplasma bovis
isolates using multiple-locus variable-number tandem-repeat analysis. J Microbiol Methods.
2012; 88(3): 377-385.
22. Hata E, Suzuki K, Hanyu H, Itoh M, Higuchi H, Kobayashi H. Molecular epidemiology of
cases of Mycoplasma californicum infection in Japan. Appl Environ Microbiol. 2014; 80(24):
7717-7724.
23. Ferandon C, Peuchant O, Renaudin H, Bebear C. Diversity of Mycoplasma hominis clinical
isolates from Bordeaux, France, as assessed by multiple-locus variable-number tandem repeat
analysis. BMC Microbiol. 2013; 13: 120.
24. Cazanave C, Charron A, Renaudin H, Bebear C. Method comparison for molecular typing of
French and Tunisian Mycoplasma genitalium-positive specimens. J Med Microbiol. 2012; 61(Pt
4): 500-506.
25. Nwankpa ND, Manso-silvan L, Lorenzon S, Yaya A, Lombin LH, Thiaucourt F. Variable
Number Tandem Repeat (VNTR) analysis reveals genetic diversity within Mycoplasma
mycoides mycoides small colony isolates from Nigeria. Vet Microbiol. 2010; 146(3-4):354-355.
26. Mahdavi S, Salehi TZ, Madani R, Keyvanfar H. Comparative study of homology of
cytoplasmic membrane protein 40 KDa of Mycoplasma agalactiae in isolated strains in Iran.
Afr J Microbiol Res. 2009; 3(9): 528-532.
27. Candela MG, Serrano E, Sevila J, Leon L, Caro MR, Verheyden H. Pathogens of zoonotic and
biological importance in roe deer (Capreolus capreolus): Seroprevalence in an agro-system
population in France. Res Vet Sci. 2014; 96(2): 254-259.
28. Murai K, Lehenbauer TW, Champagne JD, Glenn K, Aly SS. Cost-effectiveness of diagnostic
strategies using quantitative real-time PCR and bacterial culture to identify contagious mastitis
cases in large dairy herds. Prev Vet Med. 2014; 113(4): 522-535.
29. Sanna G, Lecca V, Foddai A, Tola S. Development of a specific immunomagnetic
capture-PCR for rapid detection of viable Mycoplasma agalactiae in sheep milk samples. J
Appl Microbiol. 2014; 117(6): 1585-1591.
30. Gomez-Martin A, Uc N, Vieira LA, Gadea J, Cadenas J, Sanchez A, De la Fe C. Survival
capacity of Mycoplasma agalactiae and Mycoplasma mycoides subsp capri in the diluted semen
of goat bucks and their effects on sperm quality. Theriogenol. 2015; 83(5): 911-919