Post-milking application of a Lacticaseibacillus paracasei strain impacts bovine teat microbiota while preserving the mammary gland physiology and immunity.

Bovine mastitis (BM) is a major disease in dairy industry. The current approaches - mainly antibiotic treatments - are not entirely effective and may contribute to antimicrobial resistance dissemination, rising the need for alternative treatment. The present study aims to evaluate the impact of post-milking application of Lacticaseibacillus paracasei CIRM BIA 1542 (Lp1542) on the teat skin (TS) of 20 Holstein cows in mid lactation, in order to reinforce the barrier effect of the microbiota naturally present on the teat. Treatment (Lp1542, iodine or no treatment) was applied post-milking twice a day on the 4 teats of healthy animals for 15 days. Blood and milk samples, and TS swabs were collected at day (D)1, D8, D15 and D26 before morning milking and at D15 before evening milking (D15E) to evaluate Lp1542 impact at the microbial, immune and physiological levels. Lp1542 treatment resulted in a higher lactic acid bacteria and total microbial populations on TS and in foremilk (FM) at D15(E) compared with iodine treatment. Metabarcoding analysis revealed changes in the composition of TS and FM microbiota, beyond a higher Lacticaseibacillus abundance. This included a higher abundance of Actinobacteriota, including Bifidobacterium, and a lower abundance of Pseudomonadota on TS of Lp1542 compared with iodine-treated quarters. In addition, Lp1542 treatment did not trigger any major inflammatory response in the mammary gland, except interleukin 8 production and expression which tended to be slightly higher in Lp1542-treated cows compared with the others. Finally, Lp1542 treatment had no impact on the mammary epithelium functionality (milk yield and composition) and integrity (epithelial cell exfoliation into milk and milk Na+/K+ ratio). Altogether, these results indicate that a topical treatment with Lp1542 is safe with regard to mammary gland physiology and immune system, while impacting its microbiota, inviting us to further explore its effectiveness for mastitis prevention.

Heat inactivation partially preserved barrier and immunomodulatory effects of Lactobacillus gasseri LA806 in an in vitro model of bovine mastitis.

Probiotics could help combat infections and reduce antibiotic use. As use of live bacteria is limited in some cases by safety or regulatory concerns, the potential of inactivated bacteria is worth investigating. We evaluated the potential of live and heat-inactivated Lactobacillus gasseri LA806 to counteract Staphylococcus aureus and Escherichia coli infection cycles in an in vitro model of bovine mastitis. We assessed the ability of live and inactivated LA806 to impair pathogen colonisation of bovine mammary epithelial cells (bMECs) and to modulate cytokine expression by pathogen-stimulated bMECs. Live LA806 induced a five-fold decrease in S. aureus adhesion and internalisation (while not affecting E. coli colonisation) and decreased pro-inflammatory cytokine expression by S. aureus-stimulated bMECs (without interfering with the immune response to E. coli). The ability of inactivated LA806 ability to diminish S. aureus colonisation was two-fold lower than that of the live strain, but its anti-inflammatory properties were barely impacted. Even though LA806 effects were impaired after inactivation, both live and inactivated LA806 have barrier and immunomodulatory properties that could be useful to counteract S. aureus colonisation in the bovine mammary gland. As S. aureus is involved in various types of infection, LA806 potential would worth exploring in other contexts.

Lactobacillus casei BL23 modulates the innate immune response in Staphylococcus aureus-stimulated bovine mammary epithelial cells.

Probiotics have been adopted to treat and prevent various diseases in humans and animals. They were notably shown to be a promising alternative to prevent mastitis in dairy cattle. This inflammation of the mammary gland is generally of infectious origin and generates extensive economic losses worldwide. In a previous study, we found that Lactobacillus casei BL23 was able to inhibit the internalisation of Staphylococcus aureus, one of the major pathogens involved in mastitis, into bovine mammary epithelial cells (bMEC). In this study, we further explored the capacity of this strain to modulate the innate immune response of bovine mammary epithelial cells during S. aureus infection. L. casei BL23 was able to decrease the expression of several pro-inflammatory cytokines, including interleukins 6, 8, 1α and 1ß and tumour necrosis factor alpha, in S. aureus-stimulated bMEC, 8 h post-infection. On the other hand, L. casei did not impair the induction of defensins, such as lingual antimicrobial peptide and defensin ß1 in the presence of S. aureus, and even slightly increased the induction of tracheal antimicrobial peptide during S. aureus infection. Finally, this strain did not alter the expression of the pattern recognition receptor nucleotide-binding oligomerisation domain proteins (NOD2). This study demonstrates that L. casei BL23 displayed anti-inflammatory properties on S. aureus-stimulated bMEC. These results open the way to further characterisation of the BL23 probiotic potential in a bovine mammary gland context and to a better understanding of how all these beneficial properties combine in vivo to combat mastitis pathogens.