The present work was designed to make sequencing of M. synoviae isolates for identification of nucleotide differences in the M. synoviae vlhA gene which isolated from the respiratory system and Joint from chickens. A total of 224 chicken samples (109 respiratory samples, 115 joint samples) collected from birds from different flocks showing respiratory manifestation and arthritis were examined. Identification of M. synoviae by PCR Using primers corresponding to the single copy preserved 5´ end for vlhA genetic factor, amplicons for 350~400 bp then, generated which 6 and 26 out of 224 samples were positive samples for M. synoviae from culture and tissue by PCR respectively. High-resolution melting curve analysis (HRM) for amplicons using SYBR green fluorescent dye of 9 selected isolates (5 respiratory isolates and 4 arthirtic isolates) showed that different Melting temperature curves were ranged between 84.6-85.1°C for isolates MS from the respiratory samples and other melting temperature curves ranged between 73.6-74.6°Cfor isolates Ms from the arthirtic samples. The results proved that, there was a broad concordance among nucleotide sequence of all isolates which isolated from the respiratory system except in one sample which observed the point mutations and the frame-shift mutation in some nucleotide differ than other respiratory isolates in addition there is complete concordance between nucleotide sequence of all isolates which isolated from joint. However, in comparison the Respiratory M. synoviae isolates with M. synoviae joint isolates, there was clear variation in nucleotide sequence which was confirm the result of HRM. Compared to vaccine MS-H strain arrangement, all isolates show clear variation from (MsH) vaccine strain. This study proved a difference between M. synoviae isolated from the respiratory system and Joint and live commercial vaccine (MsH) strain. Also, these informations showed that variations in the vlhA gene sequence could be introduced into the expressed vlhA gene amino acid codons and effective in pathogenesis rate in herds.
Mycoplasma synoviae, VlhA gene, PCR, RHM with high dissociation curve, Sequence analysis.
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