C500 variants conveying complete mucosal immunity against fatal infections of pigs with Salmonella enterica serovar Choleraesuis C78-1 or F18+ Shiga toxin-producing Escherichia coli

Abstract

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) C500 strain is a live, attenuated vaccine strain that has been used in China for over 40 years to prevent piglet paratyphoid. However, this vaccine is limited by its toxicity and does not offer protection against diseases caused by F18+ Shiga toxin-producing Escherichia coli (STEC), which accounts for substantial economic losses in the swine industry. We recently generated a less toxic derivative of C500 strain with both asd and crp deletion (S. Choleraesuis C520) and assessed its efficacy in mice. In addition, we demonstrate that C520 is also less toxic in pigs and is effective in protecting pigs against S. Choleraesuis when administered orally. To develop a vaccine with a broader range of protection, we prepared a variant of C520 (S. Choleraesuis C522), which expresses rSF, a fusion protein comprised of the fimbriae adhesin domain FedF and the Shiga toxin-producing IIe B domain antigen. For comparison, we also prepared a control vector strain (S. Choleraesuis C521). After oral vaccination of pigs, these strains contributed to persistent colonization of the intestinal mucosa and lymphoid tissues and elicited both cytokine expression and humoral immune responses. Furthermore, oral immunization with C522 elicited both S. Choleraesuis and rSF-specific immunoglobulin G (IgG) and IgA antibodies in the sera and gut mucosa, respectively. To further evaluate the feasibility and efficacy of these strains as mucosal delivery vectors via oral vaccination, we evaluated their protective efficacy against fatal infection with S. Choleraesuis C78-1, as well as the F18+ Shiga toxin-producing Escherichia coli field strain Ee, which elicits acute edema disease. C521 conferred complete protection against fatal infection with C78-1; and C522 conferred complete protection against fatal infection with both C78-1 and Ee. Our results suggest that C520, C521, and C522 are competent to provide complete mucosal immune protection against fatal infection with S. Choleraesuis in swine and that C522 equally qualifies as an oral vaccine vector for protection against F18+ Shiga toxin-producing Escherichia coli.

Keywords: Salmonella enterica serovar Choleraesuis C500; edema disease of swine; host; in vivo; mucosal immunity.

FIGURE 1

Expression of rSF by S. Choleraesuis C520, C522 and construction of vector pYA3493. C522 (pYA-SF; vaccine strain; rSF expression) and C521 (pYA3493; vector control) vaccine strains were cultured in LB broth at 37°C. Total cells (1.2 × 10⁹) and concentrated culture supernatants (750 μl at an OD600 of 0.8) were subjected to SDS-PAGE analysis, and rSF was detected by Coomassie blue staining or immunoblotting with anti-rSF rabbit polyclonal antibody. (A) Immunoblot of concentrated culture supernatants (lane 1) and total cell extracts (lane 2) of the C522 strain detected with anti-rSF rabbit polyclonal antibody. (B) Coomassie brilliant blue gel staining of concentrated culture supernatant from C522 (lane 1) and inclusion bodies from C522 (lane 2). (C) Coomassie brilliant blue-stained gel of total cell extracts from C522 (pYA-SF) (lane 1) and C521 (pYA3493) (lane 2). Molecular markers are indicated to the right. The position corresponding to the predicted MW of rSF protein is indicated by an arrow. (D) Construction of vector pYA3493.

FIGURE 2

(A) Percentage of pigs experiencing diarrhea and fever 1–3 days and more than 14 days (>14) after vaccination C500, C520, C521, and C522. (B) Enumeration of the vaccine strains in lymphoid tissue after vaccination (day 0, day 1, day 14, and day 21). Pigs were orally vaccinated with 2.0 × 10⁹ CFU of C522 (pYA-SF) vaccine strain, C521 (pYA3493) vector control strain or C500 parental vaccine strain. Colonization of each vaccine strain from the Peyer’s patches (Facinelli et al., 2019). Facinelli et al. (2019), mesenteric lymph nodes (MLN) and spleen were measured on day 0, day 1, day 14, and day 21 post-vaccination. (C) Cytokine expression in the spleen from spleens of pigs immunized with C521. Pigs were orally vaccinated with 2.0 × 10⁹ CFU of C521 (pYA3493) vector control strain. Quantitative RT-PCR was performed to measure the level of interferon Y (IFN-Y), tumor necrosis factor-α (TNF-α) and interleukin 4 (IL 4) on day 0, day 1, day 14, and day 21 post-vaccination. Day 0 as a control group means that the pigs have been unvaccinated. The level of each cytokine gene was normalized to the corresponding GAPDH value. Data represent the means ± SE (n = 3). The alphabet indicate the statistically significant differences between CFU of Cytokine at 0, 1, 14, and 21 days post-vaccination. P < 0.05 was considered significant. Error bars indicate standard deviations.

FIGURE 3

ELISA analysis of the anti-S. Choleraesuis immune response after oral vaccination of pigs with C521, C522 (pYA-SF) and C500-vaccinated pigs after oral vaccinations. Pigs were inoculated with the recombinant vaccine C522 (pYA-SF), C521 or parent (vector control) or the parental vaccine C500. Samples from 5 pigs were collected on day 0, day 14, and day 21. Individual pig serum, gut mucus (wash fluid from mucosa of terminal ileum) and MLN samples were tested for total IgG antibody or IgA antibody against whole Salmonella cells by ELISA. (A,B) Anti-Salmonella IgG or IgA titers obtained in serum; (C,D) anti-Salmonella IgG or IgA titers obtained in gut mucus; (E,F) anti-Salmonella IgG or IgA titers obtained in MLN. The data show the mean maximum end-point dilutions from the serum generating an optical density at 630 nm (OD630) two times that of undiluted pre-immune serum from the PBS-treated group (OD630 < 0.1). Statistical differences between groups were analyzed by the T-Test. The asterisk indicates the statistically significant differences (P < 0.05) between lipopolysaccharide (LPS) titers at 14 and 21 days post-vaccination. Error bars indicate standard deviations.

FIGURE 4

ELISA analysis of the anti-rSF immune response for pigs vaccinated orally with C522 (pYA-SF). Pigs were inoculated with the recombinant vaccine C522 (pYA-SF) on day 0, day 14, and day 21. Samples from 5 pigs in each group were collected at each time point. Individual pig serum, gut mucus and MLN samples were tested for total IgG antibody or IgA antibody against rSF by ELISA. (A,B) Anti-rSF IgG or IgA titers obtained in serum; (C,D) anti-rSF IgG or IgA titers obtained in gut mucus; (E,F) anti-rSF IgG or IgA titers obtained in MLN. The titers represent the maximum end-point dilutions from the sample yielding an optical density at 630 nm (OD630) two times that of undiluted pre-immune serum from the PBS-treated group (OD630 < 0.1). Mean values from each group were compared using the T-Test. The asterisk indicates the statistically significant differences (P < 0.05) between rSF titers at 14 and 21 days post-vaccination. Error bars indicate standard deviations.

FIGURE 5

(A) CFU of Ee strain was detected in rectal swabs from pigs orally vaccinated with C522, C520, C500, and PBS from day 0 until day 21 post-challenge. The asterisk indicates the statistically significant differences between CFU of Ee strain at 0, 3, 14, and 21 days post-vaccination. P < 0.05 was considered significant. Error bars indicate standard deviations. (B) Temperatures of pigs during the 21 days post-vaccination. Pigs with temperatures above 41 degrees have an increase in body temperature. (C) ELISA analysis of the anti-rSF immune response in pigs vaccinated orally with C522 (pYA-SF) at 21 days after C78-1 and Ee challenge. Somatic of S. Choleraesuis C500-based IgG or IgA in mesenteric lymph nodes (MLN) from the C522-vaccinated pigs 21 days after C78-1 challenge and rSF-based IgG or IgA in MLN from the C522 vaccinated pigs 21 days after Ee challenge were determined by ELISA.

FIGURE 6

Pathological observation post-challenge with C78-1 and STEC Ee strains. Piglets were vaccinated orally with 1 dose of 2 × 10⁹ CFU of C522 live vaccine, or PBS as blank control. Three weeks later, the piglets were challenged orally with 2.0 × 10¹⁰CFU of virulent Salmonella C78-1 or 25 × 10¹⁰ CFU of STEC Ee field strain. (A1,A2) The PBS control of C78-1 and Ee on the gut, respectively. (A1) Mesenteric lymph nodes are diffusely hyperemic and mildly enlarged. (A2) There is moderate amount of yellowish fluid in the intestine, and the small intestine is hyperemic and bleeding. (A3,A4) The PBS control of C78-1 and Ee on the lung, respectively. (A3) Interstitial pneumonia, alveolar septal hyperemia, edema, inflammatory cell infiltration, septum widening, and alveolar shrinkage were seen in the lungs of piglets that challenged C78-1. (A4) The piglets that challenged Ee had fibrinous exudate on the apical lobes of their lungs, and the entire lung was covered with rubber-like exudate. (A5) The intestinal mucosal wall of piglets that challenged C78-1 is destroyed, the intestinal mucosa is detached, and inflammatory cells are infiltrated. (A6) The small intestinal lymph nodes of piglets that challenged Ee showed hemorrhagic and necrotic pathological manifestations. (A7,A8) The piglets that challenged C78-1 and Ee had hyperemia and thickened capillaries in the alveolar wall, and a large number of neutrophils and serous and fibrinous exudation in the alveolar cavity. (B1,B2) The protective effects of C78-1 and Ee on the gut after vaccination with C522, respectively. (B3,B4) The protective effects of C78-1 and Ee on the lung after vaccination with C522, respectively. (B5) The intestinal tissue of piglets in the C522 immune group had no lesions and the intestinal mucosa was intact. (B6) The lungs of piglets in the C522 immune group had no lesions. Magnification, 10 × 4 (A5,B5), 10 × 40 (others).