Abstract
Background:
Mycoplasmosis is widespread in farm animals and is a major contributor to respiratory disease caused by Mycoplasma bovis, a genetically diverse organism.

Aim:
To molecularly detect M. bovis and its antimicrobial resistance-associated genes through Polymerase Chain Reaction (PCR) amplification and Sanger sequencing.

Methods:
A total of 60 samples (50 milk and 10 nasal swabs) were collected in Sulaimaniyah province, Iraq, from March to July 2025. Conventional PCR for the detection of M. bovis (targeting 16S rRNA and 16S-23S rRNA IGS genes) was conducted, and positive samples were tested for the resistant gene of fluoroquinolone (gyrA and parC) and macrolides (L4 and L22). Then, gene sequencing was performed using the Sanger method, and the resulting sequences were deposited in GenBank. Finally, a phylogenetic tree was constructed using the Neighbour-joining method and the gene mutations at different positions were detected.

Results:
Among the tested samples, only 11 (18.33%) were positive for M. bovis, including 10 (20%) milk samples and one (10%) nasal swab. Most positive samples exhibited high sequence similarity (>98%) with reference strains. The gyrA gene shows amino acid variations among field strains, with mutations at S111F and K115E, while the parC gene shows amino acid variations with mutations at Q2R and N80D. Also, the L4 gene, with the PG45 reference, exhibits amino acid variations among field strains, including multiple nonsynonymous substitutions and one insertion, whereas the L22 gene shows a Q90H mutation. Three field strains exhibited substitutions (G748A and A534T).

Conclusions:
Gene mutations may be the primary cause of antimicrobial resistance in isolated M. bovis strains.

Key words: Antimicrobial resistance, Mycoplasmosis, molecular detection, Sanger sequencing, mutation