Abstract

Mycoplasma Gallisepticum (MG) is considered as one of the most economically important mycoplasmal pathogens among poultry industry worldwide. This pathogen has various strains, therefore their detection using culture method is not sufficient. From this point of view, this study was designed to develop a novel TaqMan® real-time polymerase chain reaction (TaqMan RT-PCR) assay for direct detection of MG using cytadhesin to encode a surface protein (mgc2) containing a TaqMan FAM-labeled minor groove binder probe that targets this gene and to compare it with conventional polymerase chain reaction and immunological methods. Using serial broth dilution methods against identified MG isolates, minimum inhibitory concentrations of various antimicrobial agents were detected. Throughout Al-Qassim region, Kingdom of Saudi Arabia, 208 specimens were collected from 18 commercial chicken broiler farms where respiratory diseases were present. Furthermore, 180 blood serum samples were investigated for serological diagnosis of MG. Serum plate agglutination and enzyme-linked immunosorbent assay techniques detected positive serological identification in 83 (46.11%) and 97 (53.88%) isolates, respectively. The sensitivity recorded for TaqMan RT-PCR was 10–3 CFU/ml for MG template DNA. Results of TaqMan RT-PCR revealed 169 positive samples (81.25%), while 108 samples (51.92%) were identified as positive through conventional polymerase chain reaction assay. The results of MIC for 11 antimicrobial agents against 60 identified MG isolates indicated that all groups of MG exhibited a higher degree of sensitivity to tiamulin (93.33%) at low level of MIC50 and MIC90 (≤0.032 µg/ml) followed by tylosin (85%) and doxycycline (81.66%) with MIC50 and MIC90 ranged from ≤0.032 to 2 µg/ml; In contrast, gentamycin, tilmicosin, erythromycin, ciprofloxacin, oxytetracylcine and enrofloxacin did not show a higher activity at low concentrations. In conclusion, the developed TaqMan RT-PCR exhibited higher sensitivity and applicable accuracy than other molecular and serological techniques and thus could be highly recommended for the management of MG susceptibility and to facilitate implementation of effective treatment and prophylactic measures.

Key words: Mycoplasma Gallisepticum, TaqMan RT-PCR assay, Antimicrobial resistance