J Vet Sci.  2018 Mar;19(2):232-241. 10.4142/jvs.2018.19.2.232.

Recombinant-attenuated Salmonella Pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus (NDV) protects chickens against NDV and Salmonella Pullorum challenge

• Ding K
• Shang K
• Yu ZH
• Yu C
• Jia YY
• He L
• Liao CS
• Li J
• Zhang CJ
• Li YJ
• Wu TC
• Cheng XC

• KMID: 2407622
• DOI: http://doi.org/10.4142/jvs.2018.19.2.232

Abstract

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.

Keyword

HN protein; Newcastle disease virus; immune protective response; recombinant-attenuated Salmonella Pullorum; vaccines

MeSH Terms

Animals
Antibodies
Bacteria
Chickens*
Hemagglutination
HN Protein
Homologous Recombination
Immunoglobulin A, Secretory
Immunoglobulin G
Lymphocytes
Methods
Newcastle disease virus*
Newcastle Disease*
Plasmids
Poultry
Salmonella*
Suicide
Vaccines
Antibodies
HN Protein
Immunoglobulin A, Secretory
Immunoglobulin G
Vaccines

Figure

• 

  Fig. 1 Identification of the hemagglutinin-neuraminidase (HN) protein by western blot assay. (A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis results for the protein expressed from C79-13ΔcrpΔasd (pYA-HN). M, protein molecular weight marker; Lanes 1–3, mycoprotein of recombined bacterium C79-13ΔcrpΔasd (pYA-HN); Lane 4, mycoprotein of recombined bacterium C79-13ΔcrpΔasd (pYA). (B) Western blot analysis of the expressed protein from C79-13ΔcrpΔasd (pYA-HN). M, protein molecular weight marker; Lanes 1 and 2, mycoprotein of recombined bacterium C79-13ΔcrpΔasd (pYA-HN); Lane 3, mycoprotein of recombined bacterium C79-13ΔcrpΔasd (pYA).

• 

  Fig. 2 Comparisons of body weights (A) and spleen- (B), thymus- (C), and bursa- (D) body weight ratios of study groups immunized with C79-13ΔcrpΔasd (pYA-HN), C79-13ΔcrpΔasd (pYA), LaSota, or phosphate buffered saline. For each group, the body, spleen, bursa, and thymus weights of three randomly selected birds were measured at different time after immunization. The asterisks indicate significant differences (*p < 0.05) from C79-13ΔcrpΔasd (pYA-HN) and LaSota.

• 

  Fig. 3 Antibody titers analyzed by using a hemagglutination inhibition (HI) assay. Chicken serum samples were collected on days 0, 7, 14, 21, 28, and 35 post-immunization, and the HI titers were assessed by HI assay. Data are presented as mean ± SD; n = 6 chickens per group. PBS, phosphate buffered saline.

• 

  Fig. 4 Serum immunoglobulin G (IgG) antibody and intestinal secretory IgA (sIgA) were detected by performing enzyme-linked immunosorbent assay. Chicken serum and intestinal lavage samples were collected on days 7, 14, 21, 28, and 35 post-immunization. (A) IgG antibody. (B) Intestinal sIgA. Data are presented as mean ± SD; n = 6 chickens per group. PBS, phosphate buffered saline; OD450, optical density at 450 nm.

• 

  Fig. 5 The Newcastle disease virus specific cell-mediated immune response was determined by performing peripheral blood lymphocyte proliferation assay. Data are presented as mean ± SD; n = 6 chickens per group. *p < 0.05, compared with chickens immunized with phosphate buffered saline (PBS).

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