Evaluation of the Efficacy of a Cholera-Toxin-Based Staphylococcus aureus Vaccine against Bovine Intramammary Challenge

Abstract

Staphylococcus aureus (S. aureus) is a primary agent of bovine mastitis and a source of significant economic loss for the dairy industry. We previously reported antigen-specific immune induction in the milk and serum of dairy cows following vaccination with a cholera toxin A₂ and B subunit (CTA₂/B) based vaccine containing the iron-regulated surface determinant A (IsdA) and clumping factor A (ClfA) antigens of S. aureus (IsdA + ClfA-CTA₂/B). The goal of the current study was to assess the efficacy of this vaccine to protect against S. aureus infection after intramammary challenge. Six mid-lactation heifers were randomized to vaccinated and control groups. On days 1 and 14 animals were inoculated intranasally with vaccine or vehicle control, and on day 20 animals were challenged with S. aureus. Clinical outcome, milk quality, bacterial shedding, and somatic cell count (SCC) were followed for ten days post-challenge. Vaccinated animals did not show signs of clinical S. aureus mastitis and had lower SCCs compared to control animals during the challenge period. Reductions in bacterial shedding were observed but were not significant between groups. Antibody analysis of milk and serum indicated that, upon challenge, vaccinated animals produced enhanced IsdA- and ClfA-CTA₂/B specific immunoglobulin G (IgG) responses, while responses to CTA₂/B alone were not different between groups. Responses after challenge were largely IgG1 against the IsdA antigen and mixed IgG1/IgG2 against the ClfA antigen. In addition, there was a significant increase in interferon gamma (IFN-γ) expression from blood cells in vaccinated animals on day 20. While preliminary, these findings support evidence of the induction of active immunity by IsdA + ClfA-CTA₂/B, and further assessment of this vaccine is warranted.

Keywords: Staphylococcus aureus; bovine; mastitis; vaccine.

Figure 1

S. aureus cholera toxin A₂/B (CTA₂/B) chimeric mucosal vaccines. (A) pLR001 for expression of IsdA-CTA₂/B, and pLR003 for expression of ClfA-CTA₂/B, and (B) SDS-PAGE of purified IsdA-CTA₂/B (1, IsdA-CTA₂~38 kD, CTB~11 kD) and ClfA-CTA₂/B (2, ClfA-CTA₂~37 kD, CTB~11 kD).

Figure 2

Trial design summary. Animals (n = 3 per group, #) were vaccinated intranasally on day 1 and boosted on day 14 with 5 mL of either phosphate-buffered saline (PBS) + 5% glycerol vehicle control or 600 µg IsdA + ClfA-CTA₂/B vaccine (orange arrows). On day 20 animals were challenged once with 400 colony-forming units (CFU) of S. aureus Newbould 305 in two quarters (yellow arrow) and on day 30, animals were treated (end of challenge period, green arrow). Samples of blood were taken on days 1, 14, 20, and 30 (X). Samples of milk were taken on days 1 and 14 (X), and every day for ten days over the challenge period (days 20–30, X→X).

Figure 3

Vaccination outcomes during the trial period. (A) Quantification of bacterial shedding by cows during the challenge period. Log10 of CFU/mL of Staphylococcus aureus on mannitol salt agar (MSA). Mean ± standard error, n = 3 per group, and analyzed using repeated measures analysis of variance (ANOVA). No significance after false discovery rate (FDR) adjustment for multiple comparisons. (B) Somatic cell count (SCC) (×1000 cells/mL) by cow. Mean ± standard error, n = 3 per group, and analyzed using repeated measures ANOVA. During the challenge period, control cows uniformly had higher SCC than vaccinated cows (main model effect p = 0.002). (C) Rectal temperature in degrees Fahrenheit (°F). Mean ± standard error, n = 3 per group, and analyzed using repeated measures ANOVA showing no significance between groups. Orange arrows indicate day of booster vaccination (14), yellow arrows indicate day of bacterial challenge (20) and green arrows indicates the last day of challenge (30).

Figure 4

Immunoglobulin G (IgG) antibody responses in serum and milk as determined by enzyme-linked immunosorbent assay (ELISA). Anti-IsdA-CTA₂/B IgG responses in (A) serum and (B) milk, anti-ClfA-CTA₂/B IgG responses in (C) serum and (D) milk, and anti-CTA₂/B IgG responses in (E) serum and (F) milk. Serum was analyzed on days 14, 20, and 30 and milk on days 14, 20, 22, 24, 26, 28, and 30 during the trial period. Results are reported as ELISA ratios of day X/day 1 at O.D. 370 at serum dilutions of 1:1600 and milk dilutions of 1:160. Shown are mean and standard error by treatment with control (gray) and vaccinated (blue) (n = 3 per group). Significant differences between groups are represented as p ≤ 0.05 (*). The log10 of the values were analyzed using repeated measures analysis of variance (ANOVA) with a compound symmetric covariance structure for cows across days. Model-based estimates were compared between groups within days and adjusted for multiple comparisons.

Figure 5

Serum IgG1, IgG2, and cytokine expression analysis. (A) Anti-IsdA-CTA₂/B IgG1, (B) anti-IsdA-CTA₂/B IgG2, (C) anti-ClfA-CTA₂/B IgG1, and (D) anti-ClfA-CTA₂/B IgG2 responses in serum on days 14, 20, and 30. Results are reported as ELISA ratios of day X/day 1 at O.D. 370 at serum dilutions of 1:1600. Shown are mean and standard error by treatment with control (gray) and vaccinated (blue) (n = 3 per group). The log10 of the values were analyzed using analysis of variance (ANOVA), with a compound symmetric covariance structure for cows across days. Model-based estimates were compared between groups within days and adjusted for multiple comparisons. (E) IL-10, IL-6, and IFN-γ expression as determined by quantitative RT-PCR of peripheral blood mononuclear cells (PBMCs) isolated from whole blood after boost on day 20. Results are shown as relative gene expression to GAPDH (2^−ΔΔCt). Data are presented as mean and standard error of control (gray) and vaccinated (blue) showing median and range (n = 3 per group). Data were analyzed using a two-group t-test between vaccinated and control. Significant differences between groups are represented as p ≤ 0.05 (*).