Immunogenicity of live phoP gene deletion strain of Riemerella anatipestifer serotype 1

Abstract

Duck infectious serositis is an acute and infectious disease caused by Riemerella anatipestifer (R. anatipestifer) that leads to perihepatitis, pericarditis, meningitis, and airbag inflammation in ducks, which causes serious economic losses to the global duck industry. The phoP/phoR is a novel 2-component signal transduction system first reported in gram-negative bacteria, of which phoP acts as a global regulator and virulence factor. In this study, the phoP gene from the R. anatipestifer YM strain was knocked out using homologous recombination technology and replaced with the spectinomycin resistance gene (Spec). The virulence of the R. anatipestifer YMΔphoP strain was reduced by approximately 47,000 times compared to that of the wild-type R. anatipestifer YM strain. Ducks were immunized with live R. anatipestifer YMΔphoP strain by subcutaneous inoculation at a dose of 10⁶ to 10⁷ CFU (0.2 mL per duck) and challenged with the wild-type R. anatipestifer YM strain 14 days later. The protection rate in the immunized group was 100%. The growth characteristics of ducks in the immunized and negative control groups were normal, and the research demonstrated R. anatipestifer YMΔphoP strain have suitable immunogenicity and protective effects. Thus, the study findings suggest that the novel R. anatipestifer YMΔphoP strain may provide a candidate for the development of a gene deletion activated vaccine against duck infectious serositis.

Keywords: Immunity; R. anatipestifer YMΔphoP; Riemerella anatipestifer.

Figure 1

Construction of the R. anatipestifer YMΔphoP strain. (A) Diagram of the designed R. anatipestifer YMΔphoP mutant strain. (B) PCR amplification of the left and right homologous arms of the phoP and Spec genes. Lanes 1–3: PCR products of the left homologous arm. Lanes 4–6: PCR products of the right homologous arm. Lanes 7–9: PCR products of the Spec gene. (C) Overlap extension PCR amplification of the left and right homologous arms of the phoP and Spec genes. Lanes 1 and 2: PCR amplification using the left arm, right arm, and Spec gene as templates. (D) Identification of the R. anatipestifer YMΔphoP strain. Lane 1: Identification of deletion strain with phoP primers. Lane 2: Identification of deletion strain with Spec primers. Lane 3: Identification of deletion strain with phoP primers. Lane 4: Identification of deletion strain with Spec primers. RA, Riemerella anatipestifer.

Figure 2

Protection and antibody levels for different groups. IgY antibody levels in ducks on days 7 and 14. All data are expressed as the mean ± SEM of 3 independent experiments. ^⁎⁎⁎⁎P < 0.0001.

Figure 3

Determination of serum cytokine levels in different groups, as measured by ELISA. (A) IFN-γ, (B): IL-2, (C): IL-4, (D): IL-10. All data are expressed as the mean ± SEM of 3 independent experiments. ^⁎⁎⁎⁎P < 0.0001; *P < 0.05.

Figure 4

Staining results for histopathological sections from the ducks. (A, B) Left to right, Observations from tissue sections from the immunized and negative control groups. (C) Abnormal myocardial structure with large amounts of viscous material in the epicardial membrane. Abnormal liver tissue structures, with extensive steatosis of the liver cells. Some spleen cells were necrotic, and a small amount of red homogeneous material was detected in the splenic nodules. Some neurons were pyknotic and hyperchromatic, showing neurotropic effects. The tissues were stained with hematoxylin and eosin (200 ×).