Immune responses associated with homologous protection conferred by commercial vaccines for control of avian pathogenic Escherichia coli in turkeys

Abstract

Avian pathogenic Escherichia coli (APEC) infections are a serious impediment to sustainable poultry production worldwide. Licensed vaccines are available, but the immunological basis of protection is ill-defined and a need exists to extend cross-serotype efficacy. Here, we analysed innate and adaptive responses induced by commercial vaccines in turkeys. Both a live-attenuated APEC O78 ΔaroA vaccine (Poulvac® E. coli) and a formalin-inactivated APEC O78 bacterin conferred significant protection against homologous intra-airsac challenge in a model of acute colibacillosis. Analysis of expression levels of signature cytokine mRNAs indicated that both vaccines induced a predominantly Th2 response in the spleen. Both vaccines resulted in increased levels of serum O78-specific IgY detected by ELISA and significant splenocyte recall responses to soluble APEC antigens at post-vaccination and post-challenge periods. Supplementing a non-adjuvanted inactivated vaccine with Th2-biasing (Titermax® Gold or aluminium hydroxide) or Th1-biasing (CASAC or CpG motifs) adjuvants, suggested that Th2-biasing adjuvants may give more protection. However, all adjuvants tested augmented humoral responses and protection relative to controls. Our data highlight the importance of both cell-mediated and antibody responses in APEC vaccine-mediated protection toward the control of a key avian endemic disease.

Figure 1

Recoveries of strain EC34195nal ^R from organs of vaccinated turkeys at 24 h post-challenge. Turkey poults were given sterile saline (mock) as a coarse spray (open bars), a formalin-inactivated vaccine of the parent strain EC34195 formulated in aluminium hydroxide adjuvant via the subcutaneous route (black bars) or Poulvac® E. coli as a coarse spray (checked black and white bars) starting at one day-old as indicated in the Materials and methods. All turkeys were challenged via the intra-airsac route at day 41 post-primary vaccination with EC34195nal^R and the indicated tissues recovered 24 h later. The data are expressed as mean log₁₀ CFU/g tissue ± standard error of the mean. The limit of detection by direct plating was 1.79 and limit of detection by enrichment was 1 log₁₀ CFU/g tissue; + denotes values after enrichment of tissue homogenates. *denotes P ≤ 0.05 relative to mock-vaccinated controls.

Figure 2

Levels of cytokine transcripts in the spleens of vaccinated turkeys as measured by real-time qRT-PCR. Results are expressed as corrected ΔCt values ± standard error of the mean after normalization with the Ct value of 28S rRNA product of each sample. Turkey poults were given sterile saline (mock) as a coarse spray (open bars), a formalin-inactivated vaccine based on the parent strain EC34195 formulated in aluminium hydroxide adjuvant via the subcutaneous route (black bars) or Poulvac® E. coli as a coarse spray (checked black and white bars) starting at one day-old. Five birds from each group were sampled per time-point. *denotes P ≤ 0.05 relative to mock-vaccinated controls.

Figure 3

Levels of cytokine transcripts in the livers of vaccinated turkeys as measured by real-time qRT-PCR. Details are as in the legend to Figure 2.

Figure 4

Levels of cytokine transcripts in the lungs of vaccinated turkeys as measured by real-time qRT-PCR. Details are as in the legend to Figure 2.

Figure 5

APEC-specific antibody responses in vaccinated turkeys before and after challenge. Turkey poults were given sterile saline (mock) as a coarse spray (open bars), a formalin-inactivated vaccine based on the parent strain EC34195 formulated in aluminium hydroxide adjuvant via the subcutaneous route (black bars) or Poulvac® E. coli as a coarse spray (checked black and white bars) starting at one day-old. Five birds from each group were sampled at day 28 post-primary vaccination and 10 birds per group at day 42 post-primary vaccination (24 h after intra-airsac challenge with EC34195nal^R) and serum IgY (A) and bile IgA (B) levels determined by an APEC-specific ELISA. *denotes P ≤ 0.05 relative to mock-vaccinated controls.

Figure 6

Turkey splenocyte proliferation responses to Concanavalin A and EC34195 soluble lysate in vaccinated turkeys before and after challenge. Turkey poults were given sterile saline (mock) as a coarse spray (open bars), a formalin-inactivated vaccine based on the parent strain EC34195 formulated in aluminium hydroxide adjuvant via the subcutaneous route (black bars) or Poulvac® E. coli as a coarse spray (checked black and white bars) starting at one day-old. Five birds were sampled from each group at each time and splenocytes re-stimulated with ConA (A) or EC43195 soluble lysate (B). The data are presented as mean counts per minute ± standard error of the mean. *denotes P ≤ 0.05 relative to mock-vaccinated controls.

Figure 7

Recoveries of strain EC34195nal ^R from organs of turkeys vaccinated with a formalin-inactivated bacterin formulated with different adjuvants at 24 h post-challenge. Turkey poults were given sterile saline (negative control), an EC34915-based inactivated bacterin without adjuvant, or formulated with adjuvants that promote a Th1 response (CASAC or CpG-oligonucleotides (A)), or Th2 response (Titermax® Gold or aluminium hydroxide (B)). All vaccines were given by the subcutaneous route at 1 day-old and boosted at day 14. All the birds were challenged at day 41 post-primary vaccination via the intra-airsac route with EC34195nal^R and sampled after 24 h. The data are presented as mean log₁₀ CFU/g tissue ± standard error of the mean. APEC-specific serum IgY levels for corresponding birds at post-mortem examination were measured by ELISA compared to mock- and bacterin-vaccinated groups, Th1- (C) and Th2-stimulating adjuvants (D). + denotes value of EC34195nal^R after enrichment. “a” denotes P ≤ 0.05 relative to turkey poults that received sterile saline (mock-vaccinated group), “b” denotes P ≤ 0.05 relative to bacterin alone, and “ab” is significance relative to both mock- and bacterin-vaccinated groups.