Prevention of mastitis in multiparous dairy cows with a previous history of mastitis by oral feeding with probiotic Bacillus subtilis

Abstract

Mastitis is a very common inflammatory disease of the mammary gland of dairy cows, resulting in a reduction of milk production and quality. Probiotics may serve as an alternative to antibiotics to prevent mastitis, and the use of probiotics in this way may lessen the risk of antibiotic resistant bacteria developing. We investigated the effect of oral feeding of probiotic Bacillus subtilis (BS) C-3102 strain on the onset of mastitis in dairy cows with a previous history of mastitis. BS feeding significantly decreased the incidence of mastitis, the average number of medication days and the average number of days when milk was discarded, and maintained the mean SCC in milk at a level substantially lower than the control group. BS feeding was associated with lower levels of cortisol and TBARS and increased the proportion of CD4⁺ T cells and CD11c⁺ CD172a^high dendritic cells in the blood by flow cytometry analysis. Parturition increased the migrating frequency of granulocytes toward a milk chemoattractant cyclophilin A in the control cows, however, this was reduced by BS feeding, possibly indicating a decreased sensitivity of peripheral granulocytes to cyclophilin A. These results reveal that B. subtilis C-3102 has potential as a probiotic and has preventative capacity against mastitis in dairy cows.

Keywords: Bacillus subtilis C-3102 strain; bovine mastitis; dairy cattle; probiotics; prophylactic effect.

FIGURE 1

Prophylactic effect of Bacillus subtilis (BS) feeding on mastitis in dairy cows. All dairy cows had a history of mastitis. The control group (n = 10, calving number = 4.1 ± 1.7 [mean ± S. D.]) was fed a normal diet. The experimental group (n = 6, calving number = 4.5 ± 1.0) was fed a BS C‐3102 in their diet between 1 month before parturition and the end of the lactation period. Blue or red bars represent the mean of each group with standard errors. *P < 0.05.

FIGURE 2

Effect of Bacillus subtilis (BS) feeding on somatic cell count (SCC) in milk of dairy cows. Cows with a history of mastitis were divided into the control group (n = 6, blue circle) and the experimental group (n = 6, red circle) (a). Heifers were separated into the control group (n = 11, blue circle) and the experimental group (n = 12, red circle) (b). The control group was fed a normal diet, and the experimental group was fed the BS diet from 1 month before parturition. Milk samples were collected for 100 days after parturition, and their SCC was determined. These data were divided into four periods of 25 days each over the 100 days. The values below the lower limit (1000 cells/ml) are not shown in the figures. Blue or red bars represent the mean of SCC with standard errors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 3

Effect of Bacillus subtilis (BS) feeding on the concentrations of plasma metabolites. The control group (n = 10) was fed a normal diet, and the experimental group (n = 10) was fed a BS diet between 1 month before parturition and the end of the lactation. Blood samples were obtained on 12 occasions, once a month from the dairy cows. The concentrations of glucose, urea nitrogen (BUN), non‐esterified fatty acid (NEFA), total cholesterol (T‐chol), cortisol and thiobarbituric acid reactive substances (TBARS) in plasma were measured (a). The comparison analysis shows the differences in the averages of plasma metabolites between the control group and the BS‐feeding group in the total lactation period, the early and the mid and late stages of lactation (b). *P < 0.05, **P < 0.01.

FIGURE 4

Effect of Bacillus subtilis (BS) feeding on the proportion of peripheral leucocytes during lactation. The control group (n = 10) was fed a normal diet, and the experimental group (n = 10) was fed a BS diet between 1 month before parturition and the end of the lactation. Peripheral blood was obtained on 12 occasions, once a month from the dairy cows. The percentages of granulocytes, monocytes, T cells, and B cells were analyzed in peripheral blood by flow cytometry (a). The comparison analysis shows the differences in the averages of peripheral leucocytes between the control group and the BS‐feeding group in the total lactation period, the early and the mid and late stages of lactation (b). *P < 0.05, **P < 0.01.

FIGURE 5

Effects Bacillus subtilis (BS) feeding on the proportion of peripheral CD3⁺ T cells during lactation. During the present lactation, the control group (n = 10) was fed a normal diet, and the experimental group (n = 10) was fed a BS diet between 1 month before parturition and the end of the lactation. Peripheral blood was obtained from the dairy cows on 12 occasions from just before parturition to the end of lactation. The percentages of CD4⁺ T cells, CD8⁺ T cells, γδ⁺ T cells, WC1⁺ γδ⁺ T cells and CD8⁺ γδ⁺ T cells were analyzed in CD3⁺ T cells by flow cytometry (a). The comparison analysis shows the differences in CD3⁺ T cells between the control group and the BS‐feeding group in the total lactation period, the early and the late stages of lactation, respectively (b). *P < 0.05, **P < 0.01.

FIGURE 6

Effect of Bacillus subtilis (BS) feeding on peripheral dendritic cells during lactation. During the present lactation period, the control group (n = 10) was fed a normal diet, and the experimental group (n = 10) was fed a BS diet between 1 month before parturition and the end of the lactation. Peripheral blood was obtained from dairy cows on 12 occasions from just before parturition to the end of lactation. T cells, B cells, and monocytes were removed from peripheral blood mononuclear cell (PBMC) by the negative selection using magnetic‐activated cell sorting (MACS) with anti‐CD3 (MM1A), anti‐B cells (BAQ44A), and anti‐CD14 (CAM36A) antibodies. After the negative selection, the expression of the surface molecules CD172a and CD11c was analyzed on the negative‐selected cells by flow cytometry (a). The percentages of CD11c⁺ CD172a⁺, CD11c⁺ CD172a^high and CD11c⁺ CD172a^dim dendritic cells were counted during the lactation (b). The comparison analysis shows the differences in peripheral dendritic cells between the control group and the BS‐feeding group in the total lactation period, the early and the late stages of lactation, respectively (c). *P < 0.05.

FIGURE 7

Effect of Bacillus subtilis (BS) feeding on chemotactic activity of peripheral granulocytes. Blood samples were obtained from dairy cows at 1 month before parturition. Then, the control group (n = 5) was fed a normal diet, and the experimental group (n = 10) was fed a BS diet from 1 month before parturition. At 1 month after parturition, second blood samples were obtained from dairy cows. Granulocytes were purified by centrifugation with Lympholyte‐H. Chemotactic analysis with 100 ng/ml of rbCyPA was performed using Transwell® inserts. After incubations for 90 min, the membranes were stained with Giemsa reagent. The migrating granulocytes were counted using a microscope. The ratio of the cell number of migrating granulocytes with rbCyPA to that without it is indicated. Results are reported as mean ± SEM. *P < 0.05.