تعیین تیپهای ایزولههای Clostridium perfringens به روش Multiplex PCR در شتر مرغ

Subject Areas : Camel

E.  Zandi ¹ (Department of Animal Science, Shahid Bahonar University of Kerman, Kerman, Iran)
M.R.  Mohammadabadi ² (Department of Animal Science, Shahid Bahonar University of Kerman, Kerman, Iran)
M.  Ezzatkhah ³ (Razi Vaccine and Serum Research Institute, Kerman, Iran)
A.K.  Esmailizadeh ⁴ (Department of Animal Science, Shahid Bahonar University of Kerman, Kerman, Iran)

Received: 2014-01-11 Accepted : 2014-04-15 Published : 2014-12-01

Keywords: Multiplex PCR, کلستریدیوم پرفرینجنس, تست‌های بیوشیمیایی, تعیین تیپ ژنتیکی,

Abstract :

کلستریدیوم پرفرینجنس یک پاتوژن مهم است که بیماریهای گوناگون زیادی را ایجاد میکند. این باکتری دارای 5 تیپ مختلف است که هر تیپ قادر به ایجاد بیماری مشخصی میباشد. روشهای مختلفی برای شناسایی باکتریها وجود دارند که بیشتر آنها مشکل، زمان بر، پرهزینه و دارای حساسیت و اختصاصیت پایینی هستند. هدف این پژوهش تعیین انواع نامشابه کلستریدیوم پرفرینجنس با روش PCR بود. در این مطالعه، نمونه‌های مدفوع 120 شترمرغ به صورت تصادفی از نواحی اطراف شهر کرمان واقع در جنوب شرقی ایران جمع‌آوری شدند. بعد از عمل‌آوری و کشت نمونه‌ها، کلونی‌های تولید شده از نظر مورفولوژیکی مطالعه شدند، تست رنگ آمیزی گرم نیز انجام شد و جنس این باکتری‌ها با استفاده از تست‌های بیوشیمیایی تعیین گردید. DNA استخراج شده از باکتریهای جدا شده برای تعیین ژنوتیپ توسط multiplex PCR با آغازگرهای اختصاصی آزمون شد. بر اساس طول قطعات سنتز شده توسط PCR، تیپ‌های توکسین و استرین‌های باکتریایی شناسایی شدند. تیپ‌های جدا شده کلستریدیوم پرفرینجنس عبارت بودند از: 100 درصد تیپ A، 0 درصد تیپ B، 0 درصد تیپ C و 0 درصد تیپ D. این نکته را می‌توان مورد تأکید قرار داد که تا کنون تیپ A کلستریدیوم پرفرینجنس گزارش نشده است.

References:

Ahsani M.R., Mohammadabadi M.R. and Shamsaldini M. (2010a). Clostridium perfringens isolate typing by multiplex PCR. J. Venom. Anim. Toxin. Incl. Trop. Dis. 16, 573-578.
Ahsani M.R., Mohammadabadi M.R., Shamsaldini M., Ezatkhah M., Hasani M. and Esmailizadeh K.A. (2010b). Using triplex PCR for detecting of A, B and C types of Clostridium perfringens.Iranian J. Anim. Sci. Res. 2, 184-189.
Ahsani M.R., Shamsadini M.B., Esmailizadeh A.K. and Mohammadabadi M.R. (2011). Genotyping of isolates of Clostridium perfringens from vaccinatedand unvaccinated sheep. Small Rumin. Res. 95, 65-69.
Ahsani M.R. (2009). Molecular study of different native type of Clostridium perfringens in Kerman province: MS Thesis. Shahid Bahonar University of Kerman. Kerman, Iran.
Albini S., Brodard I., Jaussi A., Wollschlaeger N., Frey J. and Miserez R. (2008). Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates.Vet. Microbiol. 127, 179-185.
Augustynowicz E., Gzyl A. and Slusarczyk J. (2002). Detection of enterotoxigenic Clostridium perfringens with a duplex PCR. J. Med. Microbiol. 51, 169-172.
Babe T., Jafari B. and Bahmanpour S. (2012). Clostridiumperfringens isolated typing by ELISA in ostrich.African J. Microbiol. Res. 6, 1766-1769.
Baez L.A. and Juneja V.K. (1995). Nonradioactive colony hybridization assay for detection and enumeration of enterotoxigenic Clostridium perfringens in raw beef. Appl. Environ. Microbiol. 61, 807-810.
Baums C.G., Schotte U., Amtsberg G. and Goethe R. (2004). Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet. Microbiol. 100, 11-16.
Cruz  W.P.D., Gozum M.M.A., Lineberry S.F., Stassen S.D., Daughtry M., Stassen N.A., Jones M.S. and Johnson O.L. (2006). Rapid detection of enterotoxigenic Clostridium perfringens by real-time fluorescence resonance energy transfer PCR. J. Food Prot. 69, 1347-1353.
Daube G., China B., Simon P., Hvala K. and Mainil J. (1994). Typing of Clostridium perfringens by in vitro amplification of toxin genes. J. Appl. Bacteriol. 77, 650-655.
Fach P. and Popoff M. (1997). Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test. Appl. Environ. Microbiol. 63, 4232-4236.
Garmory H.S., Chanter N., French N.P., Bueschel D., Songer J.G. and Titball R.W. (2000). Occurrence of Clostridium perfringens β-2-toxin amongst animals, determined using genotyping and subtyping PCR assays. Epidemiol. Infect. 124, 61-67. 
Gkiourtzidis K., Frey J., Bourtzi-Hatzopoulou E., Iliadis N. and Sarris K. (2001). PCR detection and prevalence of alpha-, beta-, beta 2-, epsilon-, iota- and enterotoxin genes in Clostridium perfringens isolated from lambs with clostridial dysentery. Vet. Microbiol. 82, 39-43.
Gokce H.I., Genc O., Sozmen M. and Gokce G. (2007). Determination of Clostridium perfringens toxin-types in sheep with suspected enterotoxemia in Kars Province, Turkey. Turk J. Vet. Anim.Sci. 31, 355-360.
Greco G., Madio A., Buonavoglia D., Totaro M., Corrente M. and Martella V. (2005). Clostridium perfringens toxin-types in lambs and kids affected with gastroenteric pathologies in Italy. Vet. J. 170, 346-350.
Gurjar A.A., Hegde N.V., Love B.C. and Jayarao B.M. (2008). Real-time multiplex PCR assay for rapid detection and toxin typing of Clostridium perfringens toxin producing strains in feces ofdairy cattle. Mol. Cell Prob. 22, 90-95.
Herholz C., Miserez R., Nicolet J., Frey J., Popoff M., Gibert M., Gerber H. and Straub R. (1999). Prevalence of beta 2-toxigenic Clostridium perfringens in horses with intestinal disorders. J. Clin. Microbiol. 37, 358-361.
Juneja V.K., Marks H. and Thippareddi H. (2008). Predictive model for growth of Clostridium perfringens during cooling of cooked uncured beef. Food Microbiol. 25, 42-55.
Kadra B., Guillou J.P., Popoff M. and Bourlioux P. (1999). Typing of sheep clinical isolates and identification of enterotoxigenic Clostridium perfringens strains by classical methods and by polymerase chain reaction (PCR). FEMS. Immunol. Med. Microbiol. 24, 259-266.
Kalender H., Ertas H.B., Cetinkaya B., Muz A., Arslan N. and Kilic A. (2005). Typing of isolates of Clostridium perfringens from healthy and diseased sheep by multiplex PCR. Vet. Med. 50, 439-442.
Kanakaraj R., Harris D.L., Songer J.G. and Bosworth B. (1998). Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed. Vet. Microbiol. 63, 29-38.
Kim S., Labbe R.G. and Ryu S. (2000). Inhibitory Effects of Collagen on the PCR for Detection of Clostridium perfringens. Appl. Environ. Microbiol. 66, 1213-1215.
Kokai-Kun J., Songer J., Czeczulin J., Chen F. and McClane B. (1994). Comparison of western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens. J. Clin. Microbiol. 32, 2533-2539.
Komoriya T., Hashimoto A., Shinozaki A., Inoue M. and Kohno H. (2007). Study on partial purification of α-toxin produced from obligate anaerobe Clostridium perfringens. Research Institute of Industrial Technology, Nihon University. 88, 1-11.
MacFaddin J.F. (2000). Biochemical Tests for Identification of Medical Bacteria. Baltimore: Lippincott Williams and Wilkins.
Meer R.R. and Songer J.G. (1997). Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens. Am. J. Vet. Res. 58, 702-705.
Miwa N., Nishina T., Kubo S. and Atsumi M. (1997). Most probable number method combined with nested polymerase chain reaction for detection and enumeration of enterotoxigenic Clostridium perfringens in intestinal contents of cattle, pig and chicken. J. Vet. Med. Sci. 59, 89-92.
Miwa N., Nis hina T., Kubo S. and Fujikura K. (1996). Nested polymerase chain reaction for detection of low levels of enterotoxigenic Clostridium perfringens in animal feces and meat. J. Vet. Med. Sci. 58, 197-203.
Miyashiro S., Nassar A.F.C., Del Fava C., Cabral A.D. and Silva M. (2007). Clostridium perfringens types Aand D associated with enterotoxemia in an 18-month-old goat. J. Venom. Anim. Toxin. Incl. Trop. Dis. 13, 885-893.
Piatti M., Ikuno A.A. and Baldassi L. (2004). Detection of bovine Clostridium perfringens by polymerase chain reaction. J. Venom. Anim. Toxin. Incl. Trop. Dis. 10, 154-160.
Ridell J., Björkroth J., Eisgruber H., Schalch B., Stolle A. and Korkeala H. (1998). Prevalence of the enterotoxin gene and clonality of Clostridium perfringens strains associated with food-poisoning outbreaks. J. Food Prot. 61, 240-243.
Saito M., Matsumoto M. and Funabashi M. (1992). Detection of Clostridium perfringens enterotoxin gene by the polymerase chain reaction amplification procedure. Int. J. Food Microbiol. 17, 47-55.
Sambrook J. and Russell D.W. (2002). Molecular Cloning a Laboratory Manual. Woodbury: ColdSpring Harbor Laboratory Press.
Sawires Y.S. and Songer J.G. (2006). Clostridium perfringens: insight into virulence evolution and population structure. Anaerobe. 12, 23-43.
Settanni L. and Corsetti A. (2007). The use of multiplex PCR to detect and differentiate food and beverage-associated microorganisms: a review. J. Microbiol. Method. 69, 1-22.
Songer J.G. (2006). Bovine enteritis and enterotoxemia. Pp 43-80 in Genus Clostridium Clostridia in Medical, Veterinary and Food Microbiology: Diagnosis and Typing. J. Mainil, Ed. Luxembourg: European Commission / EU Concerted Action.
Songer J.G. (1996). Clostridial enteric diseases of domestic animals. Clin. Microbial. Rev. 9, 216-234.
Songer J. and Meer R. (1996). Genotyping of Clostridium perfringens by polymerase chain reaction is a useful adjunct to diagnosis of Clostridial enteric disease in animals. Anaerobe. 2, 197-203.
Tansuphasiri U. (2001). Development of duplex PCR assay for rapid detection of enterotoxigenic isolates of Clostridium perfringens. Southeast Asian J. Trop. Med. Public Health. 32, 105-113.
Tansuphasiri U., Wongsuvan G. and Eampokalap B. (2002). PCR detection and prevalence of enterotoxin (cpe) gene in Clostridium perfringens isolated from diarrhea patients. J. Med. Assoc. Thailand. 85, 624-633.
Timoney J.F., Gillespie J.H., Scott F.W. and Barlough J.E. (1988). Hagan and Bruner’s Microbiology and Infectious Diseases of Domestic Animals. Ithaca: Comstock Publishing Associates.
Van Asten A.J.A.M., van der Wiel C.W., Nikolaou G., Houwers D.J. and Grone A.A. (2009). Multiplex PCR for toxin typing of Clostridium perfringens. Vet. Microbiol. 136, 411-412.
Wojdat E., Kwiatek K. and Kozak M. (2006). Occurrence and characterization of some Clostridium species isolated from animal feeding stuffs. Bull Vet. Inst. Pulawy. 50, 63-67.
Yoo H.S., Lee S.U., Park K.Y. and Park Y.H. (1997). Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR.J. Clin. Microbiol. 35, 228-232.