Evaluation of Different PCR Systems for the Detection of Mycoplasma gallisepticum in Chicken Trachea

Yıl 2017, Cilt: 28 Sayı: 1, 51 - 54, 15.06.2017

Serpil Kahya Demirbilek

https://doi.org/10.35864/evmd.530104

Öz

 In this work, we detected the MG-serologic condition by rapid plate agglutination tests, used Air Thermal Cycler (ATC PCR) (Idaho Technologies) and LightCycler real-time PCR system (LC PCR) (Roche Diagnostics, Manheim, Germany) for rapid and reliable detection of Mycoplasma gallisepticum (MG) from tracheal swab samples of naturally infected breeder chickens, and determinated MG-DNA detection limit by MG LC PCR from both pure culture and artificially spiked samples. One hundred and seventy seven tracheal swab samples from 16 flocks of 3 different companies were tested by LC PCR. Despite 117 chickens from 10 flocks were diagnosed as MG-seropositive, only 41 (35%) of tracheal swab samples from 3 (%30) flocks were found positive by LC PCR. Sixty (33.8%) of the samples from 6 MG-seronegative flocks were also found to be MG negative by LC PCR. Two hundred twelve MG-seropositive samples from 4 companies (3 of them are same companies tested previously by LC) were tested by ATC PCR and detected only 4 (1.8%) tracheal swab samples MG-positive. The LC PCR gives the results in approximately 6 hours DNA extraction, and is rapid and reliable confirmation and detection test ready to be implemented for screening MG-infected flocks in poultry companies.

Anahtar Kelimeler

Chicken, Mycoplasma gallisepticum, PCR

Kaynakça

• 1. Anonymous (2008). Manual of Diagnostic tests and vaccines for terrestrial animals. Chapter 2.3.5. Avian Mycoplasmosis (Mycoplasma gallisepticum, M. synoviae). http://www. oie.int/eng/normes/manual/2008/pdf/2.03.05_%20avıan_ MYCO.pdf 2. Avakian AP, Kleven, SH, Glisson, JR, (1988). Evaluation of the specificity and sensitivity of two commercial enzymelinked immunosorbent assay kits, the serum plate agglutination test and the hemagglutination-inhibition test for antibodies formed in response to Mycoplasma gallisepticum. Avian Dis. 32, 262-272. 3. Callison SA, Riblet SM, Sun S, Ikuta N, Hilt D, Letting V, Kleven SH, Suarez DL, Garcia M, (2006). Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds. Avian Dis. 50, 527-544. 4. Carli KT, Eyigor A, (2003). Real-time polymerase chain reaction for detection of Mycoplasma gallisepticum in chicken trachea. Avian Dis. 47, 712-717. 5. Garcia M, Jackwood MW, Leviohn S, Kleven SH, (1995). Detection of Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma iowae by multi-species polymerase chain reaction and restriction fragment length polymorphism. Avian Dis. 39, 606-612. 6. Garcia M, Ikuta N, Levisohn S, Kleven SH, (2005). Evaluation and comparison of various PCR methods for detection of Mycoplasma gallisepticum infection in chickens. Avian Dis. 49, 125-132. 7. Glisson JR, Dawe JF, Kleven SH, (1984). The effect of oilemulsion vaccines on the occurance of nonspecific plate agglutination reactions for Mycoplasma gallisepticum and Mycoplasma synoviae. Avian Dis. 28, 397-405. 8. Harasawa R, Mizusawa H, Fuji M, Yamamoto J, Mukai H, Ueomori T, Asada K, Kato I, (2005). Rapid detection and differentiation of the major Mycoplasma contaminants in the cell cultures using Real-Time PCR with SYBR Green I and melting curve analyses. Microbiol İmmunol. 49, 859- 863. 9. Kleven SH, (1998). Mycoplasmas in the etiology of multifactorial respiratory diseases. Poult Sci. 77, 1146-1149. 10. Lauerman LH, (1998). Mycoplasma PCR assays, In. Lauerman LH (Ed) Nucleic Amplification Assays for Diagnosis of Animal Disases, American Association of Veterinary Laboratory Diagnosticians, Auborn, AL, USA, pp. 41-52. 11. Ley DH, Berkhoff JE, McLaren JM, (1996). Mycoplasma gallisepticum isolated from house finches (carpodacus mexicanus) with conjunctivitis. Avian Dis. 40, 480-483. 12. Ley DH, (2003). Mycoplasma gallisepticum infection. In: Saif YM, Barnes HJ, Fadly AM, Glisson JR, McDougald LR & Seayne DE (Eds), Diseases of Poultry. Ames, Iowa State University Press, USA, 11th ed., pp. 122-144. 13. Moscoso H, Thayer SG, Kleven SH, (2004). Materials and methods optimization and application of PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae. Avian Dis. 48, 841-850. 14. Raviv Z, Kleven SH, (2009). The development of diagnostic real-time PCR’s for the four pathogenic avian Mycoplasmas. Avian Dis. 53, 103-107. 15. Silveria RM, Fiorentin I, Marques EK, (1996). Polymerase chain reaction optimization for Mycoplasma gallisepticum and M. synoviae diagnosis. Avian Dis. 40, 218-222. 16. Slavik MF, Wang RF, Cao WW, (1993). Development and evaluation of polymerase chain reaction method for diagnosis of Mycoplasma gallisepticum infections in chickens. Mol Cell Probes. 7, 459-463.