Iraqi Journal of Veterinary Sciences, Vol. 35, Supplement III, 2021 (121-126) Proceedings of the 13th (2nd International) Scientific Conference, College of Veterinary Medicine, University of Baghdad 121 Iraqi Journal of Veterinary Sciences www.vetmedmosul.com Molecular and phylogenetic analysis of methicillin resistant Staphylococcus aureus isolated from subclinical mastitis in lactating ewes Z.A. Ahmed and A.A. Yousif Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq Article information Abstract Article history: Received October 11, 2021 Accepted November 24, 2021 Available online December 16, 2021 This study was conducted for estimation of subclinical mastitis prevalence caused by Staphylococcus aureus (S. aureus) in apparent normal lactating ewes in the areas around Baghdad (Al-Rasheed, Al-Yousifya, Al-Latifya). 400 milk samples from 200 ewes were collected and examined by California mastitis test (CMT), Bacteriological examination was done by culturing on Blood agar, Mannitol salt agar, Staph-110 agar and Chrome agar then bacteria examined by Gram stain and biochemical test. Antibiotic sensitivity test and molecular assay by PCR were done to detect methicillin- resistant strains with the phylogenetic study. The results showed that 49/400 samples gave positive to CMT in the field and positive to S. aureus isolation. The percentage of subclinical mastitis in ewes was 12.25% in this study. All isolated bacteria were resistant to methicillin 100%. The result of PCR revealed that 16.32 % (8/49) of the MRSA were possess mecA gene, while no isolate carried the mecC gene, PCR-product for 8 positive samples of mecA target gene were sequenced, analyzed, and reported in Genbank-NCBI and obtained accession number and became a reference to Iraq and the Middle East and the world. Seven samples that match the global results by 100%, and one sample contains a mutation at the site 1539, Nucleotide. Our conclusion revealed that detection of subclinical mastitis in sheep very important caused by S. aureus and all S. aureus was resistant to methicillin and some strains possess mecA gene. However, absence of mec A in other MRSA isolates requires detecting the alternative genetic possibilities related to the resistance profile and fragment of gene used. Keywords: Subclinical Mastitis Staphylococcus aureus mec A California mastitis test Correspondence: A.A. Yousif afaf.a@covm.uobaghdad.edu.iq DOI: 10.33899/ijvs.2021.131854.2013, ©Authors, 2021, College of Veterinary Medicine, University of Mosul. This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/). Introduction Mastitis defined as a mammary gland infection characterized by physical, chemical, and usually bacteriological changes in milk and glandular tissue abnormalities (1). It is an important disease economically and its subclinical mastitis is difficult in the diagnosis, which makes it mitigation more challenging (2,3). Staphylococcus aureus is Gram-positive, non-spore-forming, facultative anaerobes that grow by aerobic or by fermentation, non- motile, arranged as irregular “grape-like clusters cocci, pairs or tetrads. It is characterized by production of golden pigment. Typical biochemical identification test includes positive catalase to differentiate from Streptococci and Enterococci. Coagulase test is a guide to differentiate between Staphylococcus aureus from coagulase- negative Staphylococcus spp and ferment Mannitol (4). There are numerous studies in Iraq on mastitis in sheep, a survey study by (5) revealed the prevalence of Staphylococcus subclinical ovine mastitis at a percentage 26.9%. Hassan and Yousif (6) found the 12.22% of subclinical mastitis in ewes in Al Anbar Province. Many articles showed that S. aureus is one of the most important bacteria causing mastitis in dairy animals including sheep (7,8). California mastitis test used as a quick field assay to determine subclinical mastitis of ewes and other animals, its