The present study aimed to the isolation and identification of Penicillium chrysogenum from subclinical bovine mastitis as well as the evaluation of their potential to produce the main virulence factors by assessing proteinase production, urease production, growth rate at 37 ̊C, and hemolytic activity on Blood agar. One hundred milk samples were assembled from the White Gold village and surrounded outlying farms of Abu-Ghraib, Baghdad province, during the period from November 2018 to March 2019. Each milk sample was tested for California Mastitis (CMT). The results indicated that 85% of the samples gave positive (+ve) results for CMT. Sixty six mycotic isolates were detected, including 31 isolates of Penicillium spp. (46.9%) and 23 isolates of P. chrysogenum (34.8%). All of P. chrysogenum isolates were identified by culturing on Sabouraud Dextrose Agar and Czapek Doxes Agar at 25 ºC for 5-7 days. P. chrysogenum was diagnosed by polymerase chain reaction (PCR) based on the internal transcribed spacer (ITS) region of fungal ribosomal DNA (rDNA). The results of genetic identities showed that this fungus had 94% matching with the reference strain. Also, this study indicated that P. chrysogenum has several virulence factors with the ability of this fungus to degrade both proteins (albumin and casein), hydrolyse urea, and grow ate 37 ̊C, but not to confer hemolytic activity on Blood Agar.
