Simultaneous Protective Immune Responses of Ducks against Duck Plague and Fowl Cholera by Recombinant Duck Enteritis Virus Vector Expressing Pasteurella multocida OmpH Gene

Abstract

Duck enteritis virus and Pasteurella multocida are major duck pathogens that induce duck plague and fowl cholera, respectively, in ducks and other waterfowl populations, leading to high levels of morbidity and mortality. Immunization with live attenuated DEV vaccine containing P. multocida outer membrane protein H (OmpH) can provide the most effective protection against these two infectious diseases in ducks. We have recently reported the construction of recombinant DEV expressing P. multocida ompH gene using the CRISPR/Cas9 gene editing strategy with the goal of using it as a bivalent vaccine that can simultaneously protect against both infections. Here we describe the findings of our investigation into the systemic immune responses, potency and clinical protection induced by the two recombinant DEV-ompH vaccine constructs, where one copy each of the ompH gene was inserted into the DEV genome at the UL55-LORF11 and UL44-44.5 intergenic regions, respectively. Our study demonstrated that the insertion of the ompH gene exerted no adverse effect on the DEV parental virus. Moreover, ducklings immunized with the rDEV-ompH-UL55 and rDEV-ompH-UL44 vaccines induced promising levels of P. multocida OmpH-specific as well as DEV-specific antibodies and were completely protected from both diseases. Analysis of the humoral and cellular immunity confirmed the immunogenicity of both recombinant vaccines, which provided strong immune responses against DEV and P. multocida. This study not only provides insights into understanding the immune responses of ducks to recombinant DEV-ompH vaccines but also demonstrates the potential for simultaneous prevention of viral and bacterial infections using viral vectors expressing bacterial immunogens.

Keywords: CRISPR/Cas 9; duck plague; fowl cholera; recombinant vaccine; viral vector.

Figure 1

DEV viral load in organs infected with rDEV-OmpH-UL55, rDEV-OmpH-UL44 or DEV-WT. All the ducks in the three groups were humanely euthanized on day 7 post-inoculation, and their organs (liver, spleen and lungs) were obtained to determine virus titers using a one-step real-time PCR assay. (p < 0.05).

Figure 2

Examination of immunogenicity of rDEV-UL55 and rDEV-UL44 compared with DEV-WT, rOmpH and PBS post-immunization in ducks against DEV (A) and P. multocida X-73 (B) using ELISA.

Figure 3

Protective efficacy of the rDEV-UL55 and rDEV-UL44 against virulent DEV (A) and P. multocida X-73 (B) challenge in ducks. Ducks were examined daily for 1 week after challenge.

Figure 4

Cellular response to rDEV-UL55 & rDEV-UL44 vaccination. Serum IL-4 (A) and IFN-γ (B) cytokine levels in ducks were assessed before challenge at 4, 6, and 8 weeks post-immunization. Statistically significant differences are indicated by (***) p  <  0.001, (**) p  <  0.01, and (*) p  <  0.05 compared with the PBS control.

Figure 5

Cellular response to rDEV-UL55 and rDEV-UL44 post-challenge with DEV (A) or P. multocida (B). Serum IFN-γ and IL-4 cytokine levels in surviving ducks were analyzed at 7 days post-challenge. Statistically significant differences are indicated by (***) p  <  0.001 and (*) p  <  0.05 comparing the recombinant DEV-OmpHs with the DEV-WT or rOmpH group.

Figure 6

Peripheral blood T-lymphocyte proliferation assays were evaluated by MTT assay at 8 weeks post-immunization. Data are shown as the SI mean  ±  SD. Statistically significant differences are indicated by (**) p  <  0.01 and (*) p  <  0.05 compared with the PBS control.

Figure 7

Analysis of CD4+ (A) and CD8+ (B) T lymphocytes in peripheral blood using flow cytometry at 4 weeks and 8 weeks post-vaccination. Statistically significant differences are indicated by (***) p  <  0.001, (**) p  <  0.01, and (*) p  <  0.05 compared with the PBS control.

Figure 8

Levels of CD4+ and CD8+ response in survived ducks were analyzed at 7 days post-challenge with lethal DEV (A) and P. multocida X-73 (B) in different groups. Statistically significant differences are indicated by (***) p  <  0.001, (**) p  <  0.01 comparing the recombinant DEV-OmpHs with the DEV-WT or rOmpH group.