Immunization of Broiler Chickens With a Killed Chitosan Nanoparticle Salmonella Vaccine Decreases Salmonella Enterica Serovar Enteritidis Load

Abstract

There is a critical need for an oral-killed Salmonella vaccine for broilers. Chitosan nanoparticle (CNP) vaccines can be used to deliver Salmonella antigens orally. We investigated the efficacy of a killed Salmonella CNP vaccine on broilers. CNP vaccine was synthesized using Salmonella enterica serovar Enteritidis (S. Enteritidis) outer membrane and flagella proteins. CNP was stable at acidic conditions by releasing 14% of proteins at pH 5.5. At 17 h post-incubation, the cumulative protein release for CNP was 75% at pH 7.4. Two hundred microliters of PBS with chicken red blood cells incubated with 20 μg/ml CNP released 0% hemoglobin. Three hundred chicks were allocated into 1) Control, 2) Challenge, 3) Vaccine + Challenge. At d1 of age, chicks were spray-vaccinated with PBS or 40 mg CNP. At d7 of age, chicks were orally-vaccinated with PBS or 20 μg CNP/bird. At d14 of age, birds were orally-challenged with PBS or 1 × 10⁷ CFU/bird of S. Enteritidis. The CNP-vaccinated birds had higher antigen-specific IgY/IgA and lymphocyte-proliferation against flagellin (p < 0.05). At 14 days post-infection, CNP-vaccinated birds reversed the loss in gut permeability by 13% (p < 0.05). At 21 days post-infection, the CNP-vaccinated birds decreased S. Enteritidis in the ceca and spleen by 2 Log₁₀ CFU/g, and in the small intestine by 0.6 Log₁₀ CFU/g (p < 0.05). We conclude that the CNP vaccine is a viable alternative to conventional Salmonella poultry vaccines.

Keywords: Enteritidis; Salmonella; broilers; nanoparticles; vaccines.

FIGURE 1

Ex-vivo recall-response of spleenocytes of vaccinated birds. At d12 of age, splenocytes PBMCs were stimulated with either 20 μg/ml OMP, 20 μg/ml Flagellin or 20 μg/ml proteins of different S. Enterica serovars HKA for 3 days. As a negative control splenocytes were stimulated with 0.0 μg/ml of antigen. As a positive control splenocytes were stimulated with 20 μg/ml Con A. (A) OMP and Flagellin. Bars (+SE) with no common superscript differ (p < 0.05); (B) HKA from S. Enterica serovars. Means + SE Bars. n = 6. “*” signifies p < 0.05.

FIGURE 2

Salmonella loads in the ceca, spleen, and small intestines of vaccinated birds. Organ samples were collected from 1 bird per pen (n = 6) at 21 dpi. Samples were analyzed for S. Enteritidis loads by plating. Samples that were double negative for S. Enteritidis presence after selective enrichment were considered negative for Salmonella colonization. Data were recorded as CFU/g of organ and transformed to Log 10 CFU/g of organ for statistical analysis. (A) Ceca; (B) Spleen; (C) Small intestine. Bars (+SE) with no common superscript differ (p < 0.05).

FIGURE 3

FITC-d concentration in the serum of vaccinated birds. At d1 of age birds were allocated into treatment groups: 1) Control; 2) Challenge; or 3) Vaccine + Challenge. At d1 and d7 of age birds in the negative and positive control groups were mock vaccinated with PBS and birds in the treatment group were vaccinated with CNP. At d14 of age birds in the negative control group were given a mock challenge of 0.5 ml PBS/bird and birds in the positive control and the treatment group were orally challenged with 1 × 10⁷ CFU/bird of S. Enteritidis. At 14 dpi, one bird per pen was given 2.2 mg FITC-d by oral gavage. After 2 h blood samples were collected. The Optical Density (OD) was measured at 485 nm. Bars (+SE) with no common superscript differ (p < 0.05).

FIGURE 4

The Effects of Salmonella CNP Vaccine on Gene Expression in the Cecal Tonsils of Vaccinated Birds at 12 h post-vaccination. At d1 of age birds were allocated into treatment groups: 1) Control; 2) Challenge; or 3) Vaccine + Challenge. At d1 and d7 of age birds in the negative and positive control groups were mock vaccinated with PBS and birds in the treatment group were vaccinated with CNP. At d14 of age birds in the negative control group were given a mock challenge of 0.5 ml PBS/bird and birds in the positive control and the treatment group were orally challenged with 1 × 10⁷ CFU/bird of S. Enteritidis. Cecal tonsils were collected from one bird/pen (n = 6) at 12 h post-vaccination. Data represented as fold change compared to control. (A) TNF-α mRNA; (B) TLR 5 mRNA; (C) IL-6 mRNA. Bars (+SE) with no common superscript differ (p < 0.05).

FIGURE 5

The Effects of Salmonella CNP Vaccine on Gene Expression in the Cecal Tonsils of Vaccinated Birds at 12 h post-challenge. At d1 of age birds were allocated into treatment groups: 1) Control; 2) Challenge; or 3) Vaccine + Challenge. At d1 and d7 of age birds in the negative and positive control groups were mock vaccinated with PBS and birds in the treatment group were vaccinated with CNP. At d14 of age birds in the negative control group were given a mock challenge of 0.5 ml PBS/bird and birds in the positive control and the treatment group were orally challenged with 1 × 10⁷ CFU/bird of S. Enteritidis. Cecal tonsils were collected from one bird/pen (n = 6) at 12 h post-challenge. Data represented as fold change compared to control. (A) TNF-α mRNA; (B) IL-17 mRNA; (C) IL-6 mRNA. Bars (+SE) with no common superscript differ (p < 0.05).