A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain

Abstract

Lactococcus lactis is one of the most important microorganisms in the dairy industry and has "generally recognized as safe" (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were also found in L. lactis-inoculated glands. The above findings seem to suggest that food-grade L. lactis at a high-inoculum dose such as an overnight culture may elicit a suppurative inflammatory response in the mammary gland, thus becoming a potential mastitis-causing pathogen. Because of the unpredictable potential of L. lactis in acting as a potential mastitis pathogen, this organism cannot be considered a safe treatment for bovine mastitis.

Fig 1. L. lactis, S. chromogenes and S. aureus counts.

All inoculated bacteria were counted in mammary glands at 24 h p.i. in treated (LL, S.CH, S.CH-LL, S.AU and PBS) and untreated (UNT) groups. Results are expressed as mean values with standard deviation of log₁₀ CFU/g values. The initial bacterial inoculum size is shown. Significant differences are indicated. * P ≤ 0.05, ** P ≤ 0.01.

Fig 2. Histological changes in the murine mammary glands.

Panels A: UNT group, score 0, no infiltration and undamaged tissue; B: LL group, score 1, mild PMN infiltration in isolated areas of tissue sections; C: LL group, score 2, moderate PMN alveolar and interstitial infiltration; D: LL group, score 3, severe PMN infiltration and focal areas of tissue damage; E: S.AU group, score 4, severe PMN infiltration and extensive necrotic areas. Magnifications 20X, bar = 100 μm.