Humoral and cellular immune responses in cattle upon Clostridium chauvoei vaccination and challenge

Abstract

Introduction: Clostridium chauvoei is the causative agent of blackleg, a severe disease in cattle. Vaccination reduces disease incidence but the immune mechanisms that underlie vaccine-induced protection remain poorly understood, particularly the role of cellular immunity. In this study we characterized the humoral and cellular immune responses induced by a polyclostridial vaccine and assessed their correlation with protection against a C. chauvoei challenge.

Methods: Eleven six month old Hereford calves, seronegative for anti C. chauvoei antibodies, were randomized into vaccinated (n=8) and control (n=3) groups. Vaccinated animals received two doses of the vaccine at days 0 and 42-days. All animals were intramuscularly challenged with C. chauvoei spores (8,000 LD50) at day 69 post vaccination and monitored for clinical outcomes. Blood samples were collected at pre-vaccination and pre- and post-challenge. Humoral responses were quantified by specific in-house developed ELISA. Cytokine gene expression was measured in whole-blood (RT-qPCR for IFN γ, TNF α, TGF β1, IL 4, IL 17A, IL 12B, IL 10) upon antigenic stimulation.

Results: While vaccination protected cattle upon challenge and all animals survived, unvaccinated controls developed severe disease and died. Vaccination induced a strong specific antibody response although with inter-individual variation as well as a specific cytokine profile characterized by increased expression of IFN-γ, TGF-β1, and IL-4. Post-challenge, IFN-γ and IL-12B expression declined in vaccinated animals, but TGF-β1 persisted. High pre-challenge IgG, IFN-γ, and TGF-β1 were associated with protection, whereas increased IL-12B post-challenge was associated with disease severity.

Discussion: These findings demonstrate a coordinated interplay between humoral and cellular immune responses in vaccine-induced protection, with IFN-γ emerging as a potential biomarker in conjunction with antibody titre. The study provides a deeper further understanding on the immune mechanisms underlying vaccine-mediated protection against C. chauvoei and shall be relevant for the development of more effective vaccines.

Keywords: C. chauvoei; IFN-γ; cattle; cellular immunity; humoral immunity; polyclostridial vaccine; vaccine efficacy.

Figure 1

Anti-C. chauvoei IgG levels of vaccinated and control cattle at pre-vaccination and pre- and post-challenge. Bars indicate mean ± SEM and the dots represent individual animals. (A) Antibody levels of vaccinated (n = 8) or control (n = 3) cattle at all timepoints. Stratification of vaccinated animals into high and low responders based on their antibody levels pre- (B) and post-challenge (C). Temporal comparisons within each group were assessed using nonparametric paired tests Friedman test, followed by Dunn’s post hoc test. Intergroup comparisons (vaccinated vs. unvaccinated) were performed using the Mann-Whitney test. (*p < 0.05; **p < 0.01).

Figure 2

Cytokine gene expression profile in vaccinated and control cattle. Relative mRNA levels of cytokines (A) IFN-γ, (B) TNF-α, (C) TGF-β1, (D) IL-4, (E) IL-17, (F) IL-12B, (G) IL-1α and (H) IL-10 were quantified at different time points (pre-vaccination, pre- and post-challenge) in whole blood of animals from the vaccinated and control groups stimulated with C. chauvoei antigen. For each cytokine, relative mRNA levels were expressed relative to the average of the unstimulated condition (PBS) of all cattle (vaccinated and unvaccinated) at 0 dpv (calibration condition). Results show mean ± SEM for vaccinated (blue bar) and control (white bar) groups. Temporal comparisons within each group were assessed using nonparametric paired tests Friedman test, followed by Dunn’s post hoc test. Intergroup comparisons (vaccinated vs. unvaccinated) were performed using the Mann-Whitney test. (*p < 0.05; **p < 0.01).

Figure 3

Pre- and post-challenge ratios of immunological parameters in protected vaccinated and no-protected unvaccinated cattle. All ratios (antibody levels or cytokine mRNA expression levels) were normalized to baseline values (0 dpv) for individual animals. (A) α-C. chauvoei IgG levels, (B) IFN-γ, (C) TGF-β1, and (D) IL-4, and (E) IL-12B mRNA expression levels. Statistical analysis was performed using the Mann-Whitney test (*p < 0.05).

Figure 4

Correlation among immunological parameters at pre- and post-challenge. The graphs depict the correlations among the antibody levels ratio and cytokine gene expression ratio, at (A–C) pre-challenge (69 dpv/0 dpv) and at (D–F) post-challenge (70 dpv/0 dpv). Spearman linear correlation coefficient (r) and the significance of the correlation (p-value) are shown in each graph. A p-value < 0.05 was considered to indicate statistical significance in all cases.