Molecular identification of Mycoplasma agalactiae in Iran based on P30 gene

Document Type: Original Articles

Authors

1 Departmen of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Mycoplasma Reference Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

3 Central Laboratory Department, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

Abstract

Mycoplasma agalactiae (M. agalactiae) is known as the main etiological agent of contagious agalactia (CA) which is a disease affecting dairy sheep and goats and its main characteristics include keratoconjunctivitis, arthritis and mastitis. This pathogen results in milk production reduction and suppression and hence leads to serious economic loss. In the present research, 125 sheep and goat samples were collected from the 15 provinces of Iran. Cultural and molecular methods were used for sample characterization. After extracting genomic DNAs using phenol/chloroform method, PCR technique was employed to detect Mycoplasma genus in 163bp fragment of 16S rRNA gene (M-PCR) and M. agalactiae in 800bp fragment of conserve and specific P30 lipoprotein gene (P30-PCR) in Cultural and also clinical samples. Finally, to validate the experimental approach, a 375 bp amplicon of P80 lipoprotein was amplified (MA-PCR). Fourty three cases of the 125 samples under investigation were positive, as Mycoplasma colonies were observed in the PPLO (pleuropneumonia-like organisms) agar culture. Based on the results of M-PCR method, 61 specimens (out of 125 samples) were scored positive for Mycoplasma presence. Twenty samples were positive according to P30-PCR data. We also performed MA-PCR based on P80 gene on 125 total samples to further verify our results for M.agalactiae detection. Based on the obtained data, P30 and P80 genes were presented and amplified in all Iranian M. agalactiae isolates (Twenty samples). Our results indicated that P30 gene is conserve and specific in all Iranian M. agalactiae isolates and this new P30-PCR method (as an MA-PCR technique) may be useful in the detection of this pathogen.

Keywords