Different Immune Control of Gram-Positive and Gram-Negative Mammary Infections in Dairy Cows

Abstract

In the dairy industry, bovine mastitis represents a major concern due to substantial production losses and costs related to therapies and early culling. The mechanisms of susceptibility and effective response to intra-mammary infections are still poorly understood. Therefore, we investigated innate immunity in acellular bovine skim milk through cytofluorimetric analyses of bacterial killing activity against both Gram-positive and Gram-negative pathogens. Freshly cultured E. coli and S. aureus strains were incubated with colostrum and milk samples at different lactation time points from two groups of cows, purportedly representing mastitis-resistant and mastitis-susceptible breeds; bacterial cells were analyzed for vitality by flow cytometry following incorporation of vital dyes. N-acetyl-β-D-glucosaminidase (NAGase) activity was also investigated in milk and colostrum samples. Our findings revealed that colostrum and milk bacterial killing activity was greater against S. aureus compared to E. coli., with this activity correlated with milk NAGase levels. Furthermore, both killing of S. aureus and NAGase activity were negatively correlated to the elapsed time of lactation. Interestingly, samples from the allegedly mastitis-resistant breed displayed higher bacterial killing and NAGase activities. Our study suggests that diverse control mechanisms are exerted against Gram-positive and Gram-negative pathogens in the mammary glands of cows, probably beyond those already described in the literature.

Keywords: NAGase; bacterial killing activity; dairy cow; innate immune response; mammary gland; mastitis.

Figure 1

Milk production data. Daily milk production and the content of proteins and fat in milk were registered for each animal under study: (A) total milk production during the analyzed lactation (kg); (B) daily milk yield average (kg); (C) milk protein content (%); (D) milk fat content (%). The number of asterisks (*, **, ***) indicates the level of statistical significance.

Figure 2

Quarters with negative bacterial growth. Bacteriological examination was conducted on all milk quarter samples. After cultural analysis, the milk samples that did not show bacterial growth were defined as “no growth”, whereas those with bacterial colonies were defined as “positive” for bacterial growth (Fisher’s test; n HF = 72 samples, n MOD = 96 samples).

Figure 3

S. aureus killing activity. (A) The killing activity was measured by flow cytometry as a cumulative percentage of dead and subvital bacteria after incubation of log-phase S. aureus in skim acellular milk (1:2 in BHI medium) compared to the same bacteria incubated only in BHI medium (control). (B) Defining 6% as the cut-off of S. aureus killing ability, we compared the % of quarters in both animal groups (HF and MOD) with a killing activity higher or lower compared to the cut-off. The number of asterisks (*) indicates the level of statistical significance.

Figure 4

Milk S. aureus and E. coli killing activity at different time points. The killing activity was measured by flow cytometry as cumulative percentage of dead and subvital bacteria after incubation of log-phase S. aureus or E. coli in skim acellular milk (1:2 in BHI medium) compared to the same bacteria incubated only in BHI medium (control) at different time points: T1 (dry-off), T2 (1 day postpartum), T3 (7–10 days postpartum), and T4 (30 days postpartum). The number of asterisks (*, **, ***, ****) indicates the level of statistical significance.

Figure 5

NAGase activity of milk at different time points. NAGase activity was measured using a fluorescence-based procedure on quarter milk samples of HF and MOD cows. NAGase activity of HF and MOD cows is shown at different time points (T1 to T4). The last two groups depict all the milk samples of the whole study. The number of asterisks (* to **) indicates the level of statistical significance.

Figure 6

NAGase activity of milk. NAGase activity was measured using a fluorescence-based procedure on quarter milk samples. (A) NAGase activity was analyzed in samples with negative bacterial growth and with a SCC < 200,000 compared to negative bacterial growth samples with SCC > 200,000. (B) NAGase activity was evaluated in quarter samples divided into negative, inflamed, latent, and subclinical. Inflamed quarters were not analyzed because of their paucity. The number of asterisks (*, **, ***) indicates the level of statistical significance.