Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection

Abstract

Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1beta (IL-1beta), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.

FIG. 1.

Intramammary growth of E. coli and S. aureus following experimental challenge. Following intramammary infusion of eight quarters with 72 CFU of E. coli or eight quarters with 74 CFU of S. aureus, sterile milk samples were collected from all infused quarters at various times and plated. (A) Number of quarters in which viable bacteria were recovered. (B) Means (± standard error) of log₁₀ CFU per milliliter in those quarters where bacteria were recovered.

FIG. 2.

Effects of intramammary infection with E. coli or S. aureus on daily milk weights and temperatures. (A) Total milk weight (sum of morning and evening outputs) data were collected 1 day prior to infection (−1), the day of infection (0), and for 5 days following infection. The vertical bars represent the means (plus standard error) of milk weights. * and #, significantly decreased compared to prechallenge levels (day −1) in cows challenged with E. coli or S. aureus, respectively (P < 0.05). (B) Rectal temperatures were measured immediately prior to and at various times following intramammary infection as an indicator of the systemic response. Mean (± standard error) temperatures are shown. * and #, significantly increased compared to time zero in cows challenged with E. coli or S. aureus, respectively (P < 0.05).

FIG. 3.

Effects of intramammary infection with E. coli or S. aureus on circulating neutrophils and milk SCC. (A) Total differential neutrophil counts were determined in blood samples collected immediately prior to and at various times following intramammary infection. Mean (± standard error) cell counts are shown. * and #, significantly decreased compared to time zero in cows challenged with E. coli or S. aureus, respectively (P < 0.05). (B) Milk SCC were quantified in milk samples collected from both infected and PBS-infused quarters throughout the study. Mean (± standard error) milk SCC are shown. * and #, significantly increased in E. coli- or S. aureus-infected quarters, respectively, relative to time zero (P < 0.05).

FIG. 4.

Intramammary infection with E. coli or S. aureus increases concentrations of BSA in milk. As a marker of mammary vascular permeability, BSA levels were assayed by ELISA in milk samples obtained from quarters immediately prior to and at various times following intramammary infusion with saline, E. coli, or S. aureus. Mean (± standard error) BSA levels are shown. * and #, significantly increased in E. coli- or S. aureus-infected quarters, respectively, relative to time zero (P < 0.05).

FIG. 5.

Effects of intramammary infection with E. coli or S. aureus on concentrations of IL-8 and C5a in milk. Levels of IL-8 (A) and C5a (B) in milk following intramammary bacterial or saline challenge were determined by ELISA. Mean (± standard error) IL-8 and C5a concentrations are shown. * and #, significantly increased in E. coli- or S. aureus-infected quarters, respectively, relative to time zero (P < 0.05).

FIG. 6.

Effects of E. coli and S. aureus infection on levels of TNF-α and IL-1β in milk. An ELISA was used to measure the concentrations of TNF-α (A) and IL-1β (B) in milk obtained from quarters infused with saline, E. coli, or S. aureus. Mean (± standard error) TNF-α and IL-1β concentrations are shown. * and #, significantly increased in E. coli- or S. aureus-infected quarters, respectively, relative to time zero (P < 0.05).

FIG. 7.

Intramammary infection with E. coli or S. aureus elicits an increase in levels of IL-12, IFN-γ, and IL-10 in milk. ELISAs were used to measure the levels of IL-12 (A), IFN-γ (B), and IL-10 (C) in milk obtained from quarters infused with saline, E. coli, or S. aureus. Mean (± standard error) IL-12, IL-10, and IFN-γ concentrations are shown. * and #, significantly increased in E. coli- or S. aureus-infected quarters, respectively, relative to time zero (P < 0.05).

FIG. 8.

Effects of intramammary infection with E. coli or S. aureus on levels of sCD14 and LBP. Concentrations of sCD14 (A) and LBP (B) in milk obtained from quarters infused with either saline, E. coli, or S. aureus, as well as levels of LBP in plasma (C) obtained from these infected cows, were assayed by ELISA. Mean (± standard error) concentrations of both proteins are shown. * and #, significantly increased in milk (A and B) or plasma (C) obtained from E. coli- or S. aureus-infected cows, respectively, relative to time zero (P < 0.05).