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.

Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.

BackgroundHealth of mammary glands is fundamental for milk and dairy products hygiene and quality, with huge impacts on consumers welfare.MethodsThis study aims to investigate the microbial agents (bacteria, fungi and lentiviruses) isolated from 89 macroscopically healthy udders of regularly slaughtered small ruminants (41 sheep, 48 goats), also correlating their presence with the histological findings. Multinomial logistic regression was applied to evaluate the association between lesions and positivity for different microbial isolates, animal age and bacteria.ResultsTwenty-five samples were microbiologically negative; 138 different bacteria were isolated in 64 positive udders. Coagulase-negative staphylococci were the most prevalent bacteria isolated (46.42%), followed by environmental opportunists (34.76%), others (10.14%) and pathogens (8.68%). Most mammary glands showed coinfections (75%). Lentiviruses were detected in 39.3% of samples. Histologically, chronic non-suppurative mastitis was observed in 45/89 glands, followed by chronic mixed mastitis (12/89) and acute suppurative mastitis (4/89). Only 28 udders were normal. Histological lesions were significantly associated with the animal species and lentiviruses and coagulase-negative staphylococci infections. Goats had significantly higher risk to show chronic mixed mastitis compared to sheep. Goats showed a significantly lower risk (OR = 0.26; 95% CI [0.06–0.71]) of being infected by environmental opportunists compared to sheep, but higher risk (OR = 10.87; 95% CI [3.69–37.77]) of being infected with lentiviruses.DiscussionThe results of the present study suggest that macroscopically healthy glands of small ruminants could act as a reservoir of microbial agents for susceptible animals, representing a potential risk factor for the widespread of acute or chronic infection in the flock.

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