| Summary: | <i>Lactococcus garvieae</i> (<i>L. garvieae</i>) is a pathogenic gram-positive, catalase-negative (GPCN) bacterium that causes bovine mastitis. A total of 49 <i>L. garvieae</i> isolates were identified from 1441 clinical mastitis (CM) samples. The pathogenic effects of <i>L. garvieae</i> were studied with two infection models: bovine mammary epithelial cells cultured in vitro and murine mammary infections in vivo. The overall farm prevalence was 15.5% (13/84 farms in 9/19 provinces) and sample prevalence was 3.40% (49/1441). Post-treatment somatic cell count (SCC) post <i>L. garvieae</i> infection was significantly higher than the other GPCN pathogens isolated, and the bacteriological cure fraction was 41.94% (13/31) after intramammary antibiotic treatment. All <i>L. garvieae</i> isolates were resistant to rifaximin, 12.24% of isolates were resistant to cephalexin, and 10.20% (5/49) were multidrug-resistant (MDR). The most prevalent virulence genes were Hemolysin 1 (hly1)(100%), Hemolysin 2 (hly2) (97.96%), NADH oxidase (NADHO) (100%), Superoxide dismutase (SOD) (100%), Adhesin Pav (Pav) (100%), Adhesin PsaA (PsaA) (100%), Enolase (eno) (100%), Adhesin cluster 1(AC1) (100%), Adhesin cluster 2 (AC2) (100%), and several exopolysaccharides. <i>L. garvieae</i> rapidly adhered to bovine mammary epithelial cells, resulting in an elevated lactate dehydrogenase release. Edema and congestion were observed in challenged murine mammary glands and bacteria were consistently isolated at 12, 24, 48, 72, and 120 h after infection. We concluded that <i>L. garvieae</i> had good adaptive ability in the bovine and murine mammary cells and tissue. Given the resistance profile, penicillin and ampicillin are potential treatments for CM cases caused by <i>L. garvieae</i>.
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