Early eradication of persistent Salmonella infection primes antibody-mediated protective immunity to recurrent infection

Abstract

Typhoid fever is a systemic, persistent infection caused by host-specific strains of Salmonella. Although the use of antibiotics has reduced the complications associated with primary infection, recurrent infection remains an important cause of ongoing human morbidity and mortality. Herein, we investigated the impacts of antibiotic eradication of primary infection on protection against secondary recurrent infection. Using a murine model of persistent Salmonella infection, we demonstrate protection against recurrent infection is sustained despite early eradication of primary infection. In this model, protection is not mediated by CD4(+) or CD8(+) T cells because depletion of these cells either alone or in combination prior to rechallenge does not abrogate protection. Instead, infection followed by antibiotic-mediated clearance primes robust levels of Salmonella-specific antibody that can adoptively transfer protection to naïve mice. Thus, eradication of persistent Salmonella infection primes antibody-mediated protective immunity to recurrent infection.

Fig. 1

Protection against secondary Salmonella infection despite early eradication of primary infection. A. Number of recoverable Salmonella CFUs in the spleen and liver day 5 post-secondary challenge (10⁴ CFUs) in B6.129F1 mice treated with antibiotics beginning day 20 after primary infection (10⁴ CFUs) or control mice treated with antibiotics in parallel without primary infection. B. Number of recoverable Salmonella CFUs in the spleen and liver day 5 post- secondary challenge (10⁴ CFUs) in B6.129F1 mice treated with antibiotics beginning either day 5 or day 20 after primary infection (10⁴ CFUs), or control mice without primary infection. These results are representative of two independent experiments each containing 3–5 mice per group. Bar, one standard error.

Fig. 2

Enrofloxacin eradicates persistent S. typhimurium infection in B6.129F1 mice. Number of recoverable Salmonella CFUs in the spleen and liver at the indicated time points after infection for mice with (open squares) or without (filled squares) enrofloxacin (2mg/ml) supplementation in the drinking water beginning day 5 post-infection. These results are representative of two independent experiments each containing 3–5 mice per group per time point. Dotted line, limit of detection.

Fig. 3

Protection against secondary challenge with virulent S. typhimurium despite early eradication of primary infection. Percent survival following secondary challenge with virulent S. typhimurium (10⁶ CFUs) in mice treated with antibiotics beginning 5 days post-primary infection (10⁴ CFUs) (open squares), mice treated with the same dose of heat-killed Salmonella (filled circles), or naïve mice given antibiotics in parallel (open circles). The number of mice in each group is indicated and combined from two to three independent experiments.

Fig. 4

T cell depletion does not impact protection against secondary Salmonella infection conferred by antibiotic eradication of primary infection. A. Number of recoverable Salmonella CFUs in the spleen and liver day 5 post-challenge for mice with CD4⁺ and CD8⁺ T cells depleted beginning one day prior to secondary Salmonella challenge (10⁴ CFUs). B. Representative FACS plots demonstrating the efficiency of in vivo CD4⁺ and CD8⁺ T cell depletions. The numbers in each plot indicate the percent cells in each gate. These results are combined from two independent experiments containing 7–10 mice per experimental group.

Fig. 5

Salmonella-specific IgG is sustained despite early eradication of primary Salmonella infection. Antibody titers of Salmonella-specific IgG, IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 in the serum of mice 45 days post-infection (10⁴ CFUs) treated with enrofloxacin beginning day 5 (open squares) or without antibiotic treatment (filled squares), mice treated the same dose of heat-killed Salmonella (filled circles), or naïve mice (open circles). These results represent four independent experiments containing 4–6 mice per group.

Fig. 6

Adoptively transferred serum from mice eradicated of primary Salmonella infection confers protection to naïve recipients. A. Number of recoverable Salmonella CFUs in the spleen and liver day 5 after infection with virulent S. typhimurium (10⁴ CFUs) for mice transferred serum from mice eradicated of primary Salmonella or no transfer control mice (top). Number of recoverable Salmonella CFUs in the spleen and liver day 5 post-challenge for mice transferred serum from naïve antibiotic-treated mice or no transfer control mice (bottom). B. Percent survival after infection with virulent S. typhimurium (10⁶ CFUs) in mice transferred serum from mice eradicated of primary Salmonella (open squares) or control mice without primary infection (filled squares). These results are representative of two independent experiments containing 8 – 12 mice per group. Bar, one standard error.