Emulsion-Based Postbiotic Formulation Is Comparable to Viable Cells in Eliciting a Localized Immune Response in Dairy Cows With Chronic Mastitis

Abstract

Bovine mastitis is a disease with a multi-etiological nature, defined as an infection and inflammation of the udder. Mastitis represents a significant ongoing concern in the dairy industry, leading to substantial losses in profits and revenue for farmers worldwide. The predominant causes of bovine mastitis include the pathogens Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis, and Escherichia coli. Antibiotic administration is currently the main treatment option for mastitis. However, there is a pressing need for alternative therapies to treat and prevent the disease, especially with the emergence of antibiotic-resistant, mastitis-causing pathogens, resulting in antibiotic treatment failure. One such example is live bio-therapeutics (also known as probiotics), such as Lactococcus lactis DPC3147. The efficacy of this live bio-therapeutic has been demonstrated in several previous trials by our group. The most recent of these trials showed that an emulsion-based formulation of this strain was as effective as a commercial antibiotic formulation in treating sub-clinical and clinical cases of bovine mastitis. Here, we report the results of a follow-up field trial, in which we sought to gain insight into the mechanism of action of such live bio-therapeutics, focussing on chronic mastitis cases. We treated 28 cows with chronic mastitis with two separate emulsion-based formulations containing either viable L. lactis DPC3147 cells (15 cows) or heat-killed L. lactis DPC3147 cells (13 cows). We then evaluated the efficacies of the two formulations (two treatment groups) in terms of stimulating a localized immune response (quantified by measuring IL-8 concentrations in milk collected from udders affected by mastitis) and efficacies in terms of cure rates (quantified by reductions in somatic cell counts and absence of pathogens). We demonstrate that the presence of heat-inactivated bacteria (a postbiotic) was as effective as the live bio-therapeutic in eliciting a localized immune response in cows with chronic mastitis. The response to heat-killed cells (postbiotic) reported herein could have beneficial implications for farmers with regard to prolonging the shelf life of such emulsion-based formulations containing heat-killed cells of L. lactis DPC3147 for curing cows with mastitis.

Keywords: emulsion; lacticin 3147; live bio-therapeutic; mastitis; postbiotic; probiotics; somatic cell counts.

Figure 1

A non-parametric Wilcoxon test was used to compare the IL-8 titres elicited by viable Lactococcus lactis DPC3147 cells versus heat-killed DPC3147 cells. The figure shows that there was no significant difference in IL-8 responses in cows treated with either viable DPC3147 cells or heat-killed DPC3147 cells at any of the time points (p > 0.05). This indicates that heat-killed cells can elicit an equally potent IL-8 response as viable DPC3147 cells.

Figure 2

A spline-based statistical tool was used to compare the value of p of IL-8 titres in the whole time series (R package splinectomeR; Shields-Cutler et al., 2018). The differences between the two treatment groups are not significant for IL-8 (p = 0.93), further highlighting that heat-killed cells have the ability to elicit an equally potent localized IL-8 response as do viable DPC3147 cells.

Figure 3

A non-parametric Wilcoxon test was used to compare the SCC values in cows treated with viable DPC3147 cells versus heat-killed DPC3147 cells. There were no statistically significant differences in the SCC values between the two treatment groups at the designated time points (p > 0.05 in all cases), with the exception of time point Day 0.25 (6 h post-infusion), where there was a statistically significant difference in the SCC values between viable and heat-killed cells (p < 0.05), depicted by an asterisk at Day 0.25. Due to the lack of statistically significant differences at each of the time points except Day 0.25, the data indicates that heat-killed cells may be as efficacious as viable DPC3147 cells in terms of decreasing SCC count in mastitic cows.

Figure 4

A spline-based statistical tool was to compare the value of p of SCC values in the whole time series (R package splinectomeR; Shields-Cutler et al., 2018). Overall, the differences are not statistically significant for SCC between the two groups (p = 0.38). However, this spline-based model predicts that the comparison is statistically significant at Day 0.25 (as shown previously in Figure 2) and also statistically significant between the two treatment groups on Day 5 and Day 7 (p < 0.01). Overall, this spline-based analysis also indicates that heat-killed DPC3147 cells can be as efficacious as viable DPC3147 cells in terms of decreasing SCC count in mastitic cows.

Figure 5

A Wilcoxon comparison in SCC values between cured cows versus non-cured cows was conducted. The Wilcoxon test showed that there was a statistically significant difference in SCC values (p < 0.05) between cured cows versus non-cured cows at Day 5 post-infusion, indicating that it may take up to 5 days post-infusion to see clinical differences in the cows in terms of defining a clinical cure.

Figure 6

Spearman correlations were conducted between IL-8 values and SCC values. When compared overall, SCC and IL-8 are positively and significantly correlated (R = 0.22, p < 0.01).

Figure 7

Spearman correlations between IL-8 values and SCC values in cows which were cured showing that when compared overall, SCC and IL-8 are positively and significantly correlated in cured cows (R = 0.44, p = 0.0012). This suggests that increased IL-8 values in response to infection (and therefore high SCC values) result in the recruitment of PMNs to the site of infection, which likely combat the offending pathogen, eventually resulting in a clinical cure.

Figure 8

Spearman correlations between IL-8 values and SCC values in cows which were not cured. When compared overall for cows which were not cured, SCC and IL-8 are no longer positively correlated (R = 0.035, p = 0.73). This suggests that these cows were likely unable to mount a sufficient localized IL-8 response, leading to inadequate recruitment of PMNs to the site of infection. This insufficient localized IL-8 response may explain why such cows were unable to combat the infection and thereby remained uncured.

Figure 9

A Wilcoxon comparison was conducted for IL-8 values in cows where the etiological agent of mastitis was identified to be Staphylococcus, Streptococcus, Staphylococcus, and Streptococcus together (co-infection) or pathogen negative (where there was no etiological agent detected but the cows still presented mastitis). Overall, the statistical tests show that there were no significant differences in IL-8 values detected in milk samples from the cows infected by each of the above-mentioned pathogens (p > 0.05). However, there were statistical differences in IL-8 values detected in the milk samples in cows infected with Streptococcus versus cows where there was no pathogen detected (but still suffered from mastitis; p < 0.05).