Efficacy of chitosan-based nanoparticle vaccine administered to broiler birds challenged with Salmonella

Abstract

Two experiments were conducted to evaluate the immune response of broilers vaccinated with Salmonella chitosan-nanoparticle (CNP) vaccine and challenged with Salmonella. The Salmonella CNP vaccine was synthesized with Salmonella enterica outer membrane proteins (OMPs) and flagellin proteins. In Experiment I, birds were orally gavaged with PBS or 500, 1000, or 2000μg of CNP vaccine 1 and 7d-of-age. At 14d-of-age, birds were orally challenged with 1 X 105 CFU/bird of live S. Enteritidis (SE). Macrophage-nitrite production 11d-post-challenge was higher (P<0.05) in the 500μg group when compared to the control. At d14 (8h-post-challenge), broilers vaccinated with 1000μg CNP had higher (P<0.05) serum anti-OMPs IgG and IgA and cloacal anti-OMP IgA amounts. At 11d-post-challenge, birds vaccinated with 1000μg CNP vaccine had greater (P<0.05) bile anti-OMP and anti-flagellin IgA amounts. At 11d-post-challenge, birds administered 1000μg CNP vaccine has increased (P<0.05) IL-1β and IL-10 mRNA in cecal tonsils. In Experiment II, birds were orally gavaged with PBS or 1000μg CNP or a live commercial vaccine at 1 and 7d-of-age. At 14d-of-age, birds were orally challenged with 1 X 105 CFU/bird of live SE or S. Heidelberg (SH). Birds vaccinated with CNP showed higher (P<0.05) serum anti-OMPs IgG amounts at 8h-post-challenge. At 4d-post-SH challenge, birds vaccinated with CNP had higher (P<0.05) bile anti-flagellin IgA amounts. CNP decreased (P<0.05) anti-OMPs IgG levels in serum at 2d-post-SE challenge and 4d-post-SH or SE challenge. Salmonella Enteritidis loads in cecal content at 2d-post-challenge was decreased (P<0.05) by 65.9% in birds vaccinated with CNP, when compared to the control. Chitosan-nanovaccine had no adverse effects on bird's production performance. In conclusion, 1000μg CNP vaccine can induce a specific immune response against Salmonella and has the potential to mitigate SE cecal colonization in broiler birds.

Fig 1. Effect of CNP–Salmonella vaccine on macrophage-nitrite production post-S. Enteritidis challenge.

At 1d of age, chickens were orally vaccinated with either PBS (control-challenged) or different doses of outer membrane + flagellin proteins: 500μg, 1000 μg, or 2000 μg, loaded into CNP. The same route of delivery and doses was repeated at d7 of age. At d14 of age, birds were challenged with live S. Enteritidis (5.4 x 10⁵ CFU/bird). Splenic macrophages were isolated from broilers at d25 (11d-post-challenge) of age and cells were stimulated with 1 μg/mL S. Enteritidis LPS. The nitrite content of the supernatant was determined using Griess assay. Results were reported as average optical density (OD) values. Bars (+SE) with no common superscript differ (P<0.05). n = 3 (Exp 1).

Fig 2. Effect of CNP-S. Enteritidis vaccine on anti-Salmonella IgG and IgA antibody levels.

At 1d of age, chickens were orally vaccinated with either PBS (control-challenged) or different doses of outer membrane + flagellin proteins: 500μg, 1000 μg, or 2000 μg, loaded into CNP. The same route of delivery and doses was repeated at d7 of age. At d14 of age, birds were challenged with live S. Enteritidis (5.4 x 10⁵ CFU/bird). Blood, bile and cloacal swab samples were collected pre- and post-challenge and analyzed for anti-Salmonella antigen-specific IgG and IgA levels using ELISA. Results were reported as average optical density (OD) values. A–OMPs IgG; B–OMPs IgA; C–Flagellin IgA. Bars (+ SE) with no common superscript differ (P<0.05). n = 3 to 5.

Fig 3. Effect of CNP-Salmonella vaccine on anti-S. Enteritidis or Heidelberg IgG and IgA antibody levels.

At 1d of age, chickens were orally vaccinated with either PBS (NV), 1000 μg chitosan nanoparticle vaccine (CNP) or a commercial vaccine (CV). The same route of delivery and doses was repeated at 7d of age. At 14d of age, birds were challenged with 1 X 10⁵ CFU/bird of either S. Enteritidis or S. Heidelberg. Blood, bile and cloacal swab samples were collected pre- and post-challenge and analyzed for anti-Salmonella antigen-specific IgG and IgA levels using ELISA. Results were reported as average optical density (OD) values. A–OMPs IgG; B–OMPs IgA; C–Flagellin IgA. Bars (+ SE) with no common superscript differ (P<0.05). n = 6.

Fig 4. Quantification of S. Enteritidis on cecal content colonization at 2d- post-challenge.

At 1d of age, chickens were orally vaccinated with either PBS (NV), 1000 μg chitosan nanoparticle vaccine (CNP) or a commercial vaccine (CV). The same route of delivery and doses was repeated at 7d of age. At 14d of age, birds were challenged with 1 X 10⁵ CFU/bird of either S. Enteritidis or S. Heidelberg. At 4d-post-challenge S. Enteritidis loads in the cecal content was determined by real-time PCR. The copy numbers of S. Enteritidis was expressed in log units. Bars (+SE) with no common superscript differ (P<0.05). No Vaccine (NV); Chitosan Nanoparticle Vaccine (CNP); Commercial Vaccine (CV). n = 6.

Fig 5. Effect of CNP vaccine on cytokine gene expression by qPCR.

At 1d of age, chickens were orally vaccinated with either PBS (control-challenged) or different doses of outer membrane + flagellin proteins: 500μg, 1000 μg, or 2000 μg, loaded into CNP. The same route of delivery and doses was repeated at d7 of age. At d14 of age, birds were challenged with live S. Enteritidis (5.4 x 10⁵ CFU/bird). Cecal tonsil, liver and spleen samples were collected at 11d-post-challenge and analyzed for cytokine mRNA amounts. A–IL-1β mRNA; B–IL-10 mRNA; C–IL-4 mRNA; D–IFNγ mRNA. Bars (+SEM) with no common superscript differ (P<0.05). n = 6.