Abstract
Background:
Staphylococcus aureus (S. aureus) is identified as the foremost cause of inflammation of the milksecreting glands of buffalo and all ruminant animals. This bacterium carries various genes that encode virulence factors.

Aim:
This study aimed to identify the prevalence of S. aureus in milk specimens from buffalo farms, identify specific genes associated with virulence, and compare the genetic similarity of the study isolates with S. aureus isolates from different regions worldwide.

Methods:
Sixty subclinical milk samples were collected from buffaloes from October to December 2024 across various locations in the Nineveh governorate. This study employed standard microbiological procedures, including the use of various types of media and biochemical tests. Additionally, the presence of nuc, mecA, clfA, clfB, sea, seb, and sec genes in the isolates was determined using polymerase chain reaction.

Results:
Staphylococcus aureus was detected in 15% (9/60) of buffalo subclinical mastitis milk samples, with a higher prevalence rate of 30% (3/10) in buffalo milk samples from the Kaneitra district. While nuc, clfA, and clfB were present in all S. aureus isolates, the mecA, sea, seb, and sec genes were not. Using clfA gene analysis, five distinct strain sequences were identified and deposited to GenBank.

Conclusion:
Five S. aureus strains were found to share characteristics with strains from all over the world using phylogenetic analysis; the isolates exhibit distinct characteristics according to strain type and global distribution.

Key words: Genetic tree; S. aureus; Buffalo milk; Virulence factors.