Corynebacterium bovis is one of the most commonly isolated bacteria from aseptically collected bovine milk samples. The objective of the current study was to characterize the bovine innate immune response by evaluating milk polymorphonuclear neutrophilic leukocytes (PMNL) in mammary glands infected with C. bovis. Twenty quarters infected with C. bovis and 28 culture-negative quarters (with milk somatic cell count <1×10(5) cells/mL) were used. The percentages of milk PMNL and the PMNL expression of L-selectin (CD62L), β2-integrin (CD11b), and one of the endothelial-selectin ligands (CD44), as well as the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus, were evaluated by flow cytometry. The apoptosis and necrosis rates of the PMNL were quantified using dual-color flow cytometry with fluorescein-labeled annexin and propidium iodide. The present study revealed a higher percentage of PMNL in the milk from C. bovis-infected quarters, although no significant differences were found in levels of CD44, CD62L, or CD11b expression among the PMNL. A lower percentage of apoptotic PMNL was observed in C. bovis-infected quarters, as well as higher percentages of viable PMNL and of PMNL that produced intracellular ROS. However, no alterations were observed in phagocytosis of Staph. aureus by the PMNL or in intensity of intracellular ROS production by PMNL. Thus, results from this investigation of the PMNL function support, at least in part, the fact that intramammary infections by C. bovis may offer protection against intramammary infections by other bacteria.
The effects of bovine leukemia virus (BLV) on the immune response have been extensively investigated; however, its effects on mammary gland immunity are only speculative. Although BLV has a tropism for B cells, it can affect both adaptive and innate immunities because these systems share many effector mechanisms. This scenario is the basis of this investigation of the effects of BLV on mammary gland immunity, which is largely dependent upon neutrophilic functions. Thus, the present study sought to examine neutrophilic functions and the lymphocyte profile in the milk of naturally BLV-infected cows. The viability of the milk neutrophils and the percentage of milk neutrophils that produced reactive oxygen species (ROS) or phagocytosed Staphylococcus aureus were similar between BLV-infected and BLV-uninfected dairy cows. Furthermore, the expression of CD62L and CD11b by the milk neutrophils and the percentage of milk neutrophils (CH138+ cells) that were obtained from the udder quarters of the BLV-infected cows were not altered. Conversely, the median fluorescence intensity (MFI) representing intracellular ROS production and the phagocytosis of S. aureus, the expression of CD44 by the milk neutrophils and the percentage of apoptotic B cells were lower in the milk cells from BLV-infected dairy cows, particularly those from animals with persistent lymphocytosis (PL). The lymphocyte subsets were not different among the groups, with the exception of the percentage of CD5−/CD11b− B cells, which was higher in the milk cells from BLV-infected cows, particularly those with PL. Thus, the present study provides novel insight into the implications of BLV infection for mammary gland immunity.
The influences of age in calves' immune system are described in their first phase of life. We hypothesized that variations that occur in the main mechanisms of lung innate response can help to identify periods of greater susceptibility to the respiratory diseases that affect calves in the first stage of their life. This study aimed to evaluate the innate immune system. Nine healthy calves were monitored for 3 mo and 8 immunologic evaluations were performed. Bronchoalveolar lavage samples were recovered by bronchoscopy. The alveolar macrophages in samples were identified by protein expression of cluster of differentiation 14 (CD14) and underwent functional evaluation of phagocytosis (Staphylococcus aureus stained with propidium iodide and Escherichia coli). Data was assessed by one-way ANOVA (unstacked and parametric) and the Mann-Whitney test (nonparametric). Functional alterations in CD14-positive phagocytes were observed, with punctual higher intensity of phagocytosis in the third week and its decrease starting at 45 d of life. A gradual increase in phagocytosis rate was observed starting at this date. It is concluded that from 45 d of life on, alveolar macrophages have less phagocytic capacity but more cells perform this function. We suggest that this occurs because lung macrophages of calves start to maintain their immune response without passive immunity influence. Until 90 d of life, calves did not achieve the stability to conclude the maturation of local innate immune response.
We analyzed a large number of immune response parameters from quarter milk samples with distinct bacteriological and quarter somatic cell count (qSCC) statuses. Furthermore, we sought to explore and identify displayed immune response patterns in milk samples from mammary glands with nonspecific mastitis. Thus, 92 quarter milk samples from 28 cows were stratified into 4 groups, as follows: (1) 49 culture-negative control quarters with a low qSCC (<1 × 10 5 cells/mL) from 19 dairy cows (so-called healthy quarters); (2) 15 culture-negative quarters with high qSCC (>2 × 10 5 cells/mL; so-called quarters with nonspecific mastitis) from 10 dairy cows; (3) 8 culture-positive quarters with low qSCC (noninflammatory quarters with low qSCC) from 5 dairy cows; and (4) 20 culture-positive quarters with high qSCC (so-called truly infected quarters) from 8 dairy cows. Using flow cytometry, we evaluated the percentage of milk neutrophils and their viability, intracellular reactive oxygen species production, phagocytosis, and the expression of CD62L, CD11b, and CD44 for each of the 4 quarter strata. Furthermore, the percentage of monocyte/macrophages, B cells, and T lymphocyte subsets were evaluated by flow cytometry. Milk samples from bacteriologically negative quarters (both with a low and elevated qSCC) had a lower qSCC than those with bacteriologically positive outcomes (both with a low and elevated qSCC). As expected, the healthy quarters showed the lowest percentage of neutrophils and also showed a higher percentage of milk monocytes/macrophages and lower percentage of T lymphocytes than truly infected quarters. The most prominent result of the present study is that quarters with nonspecific mastitis showed the highest percentage of milk CD4 + T lymphocytes. The healthy quarters had a lower percentage of apoptotic neutrophils than noninflammatory and truly infected quarters, although it did not differ from those from the quarters with nonspecific mastitis. Our study supports the role of differential cell counting in the diagnosis of mastitis, as the milk leukocyte populations markedly fluctuate under healthy and inflammatory conditions. Furthermore, an increase in milk CD4 + T cells was associated with nonspecific mastitis, suggesting an increase in this leukocyte subpopulation is correlated with low bacterial shedding. Our study allows us to go further in our understanding of mammary gland immunity, providing further insights on potential protective mammary gland immunity, which we hypothesize can open new avenues for the development of novel targets that can promote bovine udder health.
Streptococcus dysgalactiae is a bacterium that accounts for a notable proportion of both clinical and subclinical intramammary infections (IMIs). Thus, the present study explores the function of milk neutrophils and the lymphocyte profile in mammary glands naturally infected with Streptococcus dysgalactiae. Here, we used 32 culture-negative control quarters from eight clinically healthy dairy cows with low somatic cell counts and 13 S. dysgalactiae-infected quarters from six dairy cows. Using flow cytometry, we evaluated the percentage of milk monocytes/macrophages and neutrophils, expression of CD62L, CD11b and CD44 by milk neutrophils, the levels of intracellular reactive oxygen species (ROS) production and phagocytosis of Staphylococcus aureus by milk neutrophils, and neutrophil viability. Furthermore, the percentages of B cell (CD21(+)) and T lymphocyte subsets (CD3(+)/CD4(+)/CD8(-); CD3(+)/CD8(+)/CD4(-); and CD3(+)/CD8(-)/CD4(-)), and the expression of CD25 by T milk lymphocytes (CD3(+)) and T CD4(+) milk cells were also assessed by flow cytometry using monoclonal antibodies. The present study showed a higher SCC and percentage of milk neutrophils, and a decrease in the percentage of milk monocytes/macrophages from S. dysgalactiae-infected quarters when compared to uninfected ones. We also observed a higher expression of CD11b by milk neutrophils and a tendency toward a decrease in neutrophil apoptosis rate in S. dysgalactiae-infected quarters. In addition, the S. dysgalactiae-infected quarters had higher percentages of milk T cells (CD3(+)) and their subset CD3(+)CD8(+)CD4(-) cells. Overall, the present study provided new insights into S. dysgalactiae IMIs, including distinct lymphocyte profiles, and a tendency toward an inhibition of apoptosis in milk neutrophils.
The immune system of newborn calves is immature and must mature gradually. Understanding how this immunity is established may define different profiles. Twelve healthy calves were monitored during 8 time periods to assess the innate immune system during the first 90d. Blood samples were collected, and the blood phagocytes, identified by the expression of CD14 and CH138 surface molecules, were evaluated for phagocytic functionality (Staphylococcus aureus and Escherichia coli stained with propidium iodide) and the intracellular production of reactive oxygen species (2,7'-dichlorofluorescein diacetate oxidation). Functional changes in the CD14+ and CH138+ cells occurred at 40d of age, with sporadic increases in phagocytosis intensity and reactive oxygen species production, and decreased phagocytosis occurred at 60d of age. Therefore, fewer phagocytes were active from 40d of age, although those that were active performed their roles with greater efficacy. That change presumably occurred because the calf phagocytes began to support the immune response without the influence of passive immunity. The animals failed to reach the stability needed to complete the maturation of the innate immune response by 90d of age. These data are applicable for healthy calves only.
An important question about intramammary infections that is still debated in the literature is the independence or interdependence of the quarters of dairy cows. The present study sought to explore milk neutrophil function and the milk lymphocyte profile of uninfected quarters from uninfected and infected (one infected quarter per cow) udders to evaluate interdependence of the quarters. Thus, 32 (8 cows) and 18 (6 cows) uninfected quarters from uninfected and infected udders were used, respectively. Using flow cytometry, we evaluated the percentage of milk neutrophils and their expression of adhesion molecules L-selectin (CD62L), β2-integrin (CD11b), and an endothelial-selectin ligand (CD44); levels of intracellular reactive oxygen species (ROS); phagocytosis of Staphylococcus aureus by milk neutrophils; and neutrophil viability. Furthermore, we assessed the percentage of B-cell (CD21(+)) and T-lymphocyte subsets (CD3(+)/CD4(+)/CD8(-), CD3(+)/CD8(+)/CD4(-), CD3(+)/CD4(+)/CD25(-), CD3(+)/CD4(+)/CD25(+), and CD3(+)/CD4(-)/CD25(-)) using flow cytometry with monoclonal antibodies. The infected quarter did not affect somatic cell count or the percentage of neutrophils in the neighboring uninfected quarters. Furthermore, the infected quarter did not influence neutrophil viability, intracellular reactive oxygen species production, or phagocytosis of S. aureus by milk neutrophils. Conversely, the expression of adhesion molecules CD11b, CD62L, and CD44 by milk neutrophils differed between uninfected quarters from infected versus uninfected udders. The lymphocyte subsets did not differ between groups, except for a higher percentage of B cells in uninfected quarters from infected udders than in those from uninfected udders. Thus, our study strongly supports the hypothesis of interdependence of quarters based on the influence of infection on both the percentage of B cells and the expression of adhesion molecules by milk neutrophils in the neighboring uninfected quarters.
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