The present study assessed the effects of intramammary infusion of Bifidobacterium breve (B. breve) on mastitis‐causing pathogens and on the somatic cell counts (SCC) in lactating cows with chronic subclinical mastitis. The bacteriological cure rates of 42 quarters from 42 cows infected with Staphylococcus aureus, Corynebacterium bovis, coagulase‐negative staphylococci, and environmental streptococci were 18.2% (2/11), 14.3% (1/7), 58.8% (10/17), and 28.6% (2/7), respectively, on day 14 after B. breve infusion. In a second trial, B. breve was infused into 18 quarters from 18 cows with chronic subclinical mastitis from which pathogens had not been isolated; the rates of quarters showing SCC > 50 × 10⁴ cells/ml prior to B. breve infusion that decreased to < 30 × 10⁴ cells/ml after infusion were significantly (p < .01) increased to 61.1% (11/18) on day 14 compared to that prior to infusion (0/18). The intramammary infusion of B. breve appears to be a non‐antibiotic approach for elimination of minor pathogens and decreasing SCC in quarters with chronic subclinical mastitis in dairy cows.

Mycoplasma pneumonia, caused by Mycoplasma bovis (Mycoplasmopsis bovis; M. bovis), is linked with severe inflammatory reactions in the lungs and can be challenging to treat with antibiotics. Biofilms play a significant role in bacterial persistence and contribute to the development of chronic lesions. A recent study has shown that polymicrobial interactions between species are an important factor in biofilm formation, yet the precise mechanism of biofilm formation in M. bovis remains unknown. By assuming multiple pathogen infections in the bovine respiratory disease complex (BRDC), this study examined the characterisation of the polymicrobial relationship between M. bovis and Trueperella pyogenes (T. pyogenes) during biofilm formation. Autopsies were performed on four Holstein calves (two chronic Mycoplasma pneumonia calves and two control calves). Bacterium-like aggregation structures (> 10 μm), which were assumed to be biofilms of M. bovis in vivo, were observed adhering to the cilia in calves with Mycoplasma pneumonia. M. bovis released an extracellular matrix to connect with neighbouring bacteria and form a mature biofilm on the plate. Biofilm formation in the co-culture of M. bovis and T. pyogenes (strain T1: 1 × 10⁵ and 1 × 10⁶ CFU/well) significantly increased (p < 0.05 and p < 0.01; 64.1% and 64.8% increase) compared to that in a single culture of these bacteria. Furthermore, some large aggregates (> 40 μm), composed of M. bovis and T. pyogenes, were observed. The morphological characteristics of this biofilm were similar to those observed in vivo compared to a single culture. In conclusion, the polymicrobial interaction between M. bovis and T. pyogenes induces biofilm formation, which is associated with increased resistance to antimicrobial agents, and this exacerbates the progression of chronic Mycoplasma pneumonia.

Mycoplasma bovis mastitisis highly contagious and disrupts lactation, posing a significant threat to the dairy industry. While the mammary gland’s defence mechanism involves epithelial cells and mononuclear cells (MNC), their interaction with M. bovis remains incompletely understood. In this study, we assessed the immunological reactivity of bovine mammary epithelial cells (bMEC) to M. bovis through co-culture with MNC. Upon co-culture with MNC, the mRNA expression levels of interleukin (IL)-1β,IL-6, IL-8 and tumor necrosis factor (TNF)-α in bMEC stimulated by M. bovis showed a significant increase compared to monoculture. Additionally, when stimulated with M. bovis, the culture supernatant exhibited significantly higher concentrations of IL-6 and interferon (IFN)-γ, while IL-1β concentration tended to be higher in co-culture with MNC than in monoculture. Furthermore, the mRNA expression levels of toll-like receptor (TLR) 2 in bMEC stimulated with M. bovis tended to increase, and TLR4 significantly increased when co-cultured with MNC compared to monocultures. However, the surface expression levels in bMEC did not exhibit significant changes between co-culture and monoculture. Overall, our research indicates that the inflammatory response of bMEC is increased during co-culture with MNC, suggesting that the interaction between bMEC and MNC in the mammary gland amplifies the immune response to M. bovis in cows affected by M. bovis mastitis.