Immune Recruitment and Bactericidal Activity of Neutrophils in Milk of Cows Vaccinated with Staphylococcal α-Toxin

Abstract

The ability of Staphylococcus aureus alpha-toxin to recruit neutrophils in the milk of vaccinated cows and the bactericidal efficiency of these neutrophils were evaluated. Six lactating Holstein cows that were free of intramammary infection received systemic immunization by subcutaneous injection of Freund's incomplete adjuvant with alpha-toxin (n = 2), alpha-toxin mixed with type 5 capsular polysaccharide (n = 2), or a conjugate of these two antigens (n = 2). Controls (n = 4) and vaccinated cows (n = 6) received intramammary infusions of alpha-toxin. No increase in somatic cell count was recorded in quarter milk samples from unimmunized cows; however, 10 micrograms of alpha-toxin induced a local inflammatory reaction in vaccinated cows that was characterized by early and massive cellular recruitment into the mammary gland. More than 90% of the recruited cells were neutrophils. The speed and magnitude of the cellular recruitment were dose-dependent; the threshold dose was 0.6 microgram. Milk samples showed significant bactericidal activity against the type 5 S. aureus strain, regardless of the vaccine used, and showed a decrease in bacterial count of about 2 log10 from the initial inoculum. The best efficiency was recorded during the early phase of cellular recruitment with concomitant activation of blood-derived neutrophils. This study demonstrates that a bacterial virulence factor, alpha-toxin, is able to induce immune recruitment of neutrophils for efficient bactericidal activity in milk when cows are immunized with alpha-toxin that is used either as a nonconjugate vaccinate or as a carrier protein in a conjugate vaccine. The study also suggests that neutrophils that are recruited from blood are activated during inflammation in response to specific antigens.

Mathematical Model of the Acute Inflammatory Response to Escherichia coli in Intramammary Challenge

“…This is precisely the density of blood-derived PMN necessary to exert optimal bactericidal (Staph. aureus) activity in milk, as reported by Herbelin et al (1997), but higher than 6 × 10 5 cells/ mL, as reported by Postle et al (1978). It is also close to the SCC value (1,151,000 cells/mL) reported in the meta-analysis of Djabri et al (2002).…”

Effects of Adjuvants on the Immune Response of Staphylococcal Alpha Toxin and Capsular Polysaccharide(CPS) in Rabbit.

“…Therefore, when multicomponent antigens were used in mastitis vaccine, combined adjuvants may be more effective and useful than one adjuvant in immune response. This results of the study aggrees with earlier studies [5,10,13,14,16].…”

“…The crude alpha-toxin was confirmed by 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot [11]. The hemolytic assay is a serial two-fold dilution of 50% hemolysis end point assay described by Herbelin et al [10] and Harshman et al [9].…”

Effects of Amoxicillin, Gentamicin, and Moxifloxacin on the Hemolytic Activity of Staphylococcus aureus In Vitro and In Vivo

“…The investigators concluded that under circumstances of chronic inflammation, in which the growth of bacteria and the consequent elaboration of ␣-toxin are controlled, toxigenicity may not contribute significantly to virulence. Interestingly, ␣-toxin itself triggers the recruitment of neutrophils to a site of infection (16). The importance of neutrophils in controlling S. aureus growth and toxin production is also evident from studies in which depletion of neutrophils resulted in a conversion of a subclinical infection with toxigenic staphylococci into acute mastitis (29).…”