Systemic dissemination of Clostridium difficile toxins A and B is associated with severe, fatal disease in animal models

Abstract

Background: Clostridium difficile infection (CDI) can cause a wide range of disease, from mild diarrhea to fulminant systemic disease. The incidence of systemic CDI with fatal consequence has increased rapidly in recent years.

Methods: Using an ultrasensitive cytotoxicity assay, we measured C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB) in sera and body fluids of piglets and mice exposed to C. difficile to investigate the relationship between the presence of toxins in body fluids and systemic manifestations of CDI.

Results: We found that both TcdA and TcdB disseminate systemically, with toxins present in the sera and body fluids of infected animals, and toxemia is significantly correlated with the development of systemic CDI. The systemic administration of neutralizing antibodies against both toxins blocked the development of systemic disease in mice. We measured cytokine concentrations in the sera of mice and piglets with systemic and nonsystemic CDI and found that proinflammatory mediators were considerably elevated in animals with systemic CDI.

Conclusion: Our study demonstrates the existence of a strong correlation between toxemia and the occurrence of systemic disease, supporting the hypothesis that systemic CDI is most likely due to the toxicity of TcdA and TcdB and the induction of proinflammatory cytokines by the toxins.

Figure 1.

Cell rounding caused by piglet and mouse serum samples. A, 10 ng/mL of Clostridium difficile toxin A (TcdA); B, Serum from a piglet with severe C. difficile infection (CDI); C, Piglet serum in (B) plus anti-TcdA and C. difficile toxin B (TcdB) polysera; D, Piglet preinoculation serum; E, Serum from a mouse with severe CDI; F, Mouse serum in (E) plus anti-TcdA and anti-TcdB polysera.

Figure 2.

Toxins in piglet and mouse sera. A, Serum from 1 representative piglet with systemic Clostridium difficile infection (CDI) was assessed for the presence of toxemia, using antibodies against C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB) separately to demonstrate the presence of each toxin. B, Sera from 1 representative mouse with systemic CDI and 1 mouse with nonsystemic CDI were assessed for the presence of toxemia. Control = cells only; anti AB = goat anti-TcdA and anti-TcdB polysera; anti A = alpaca anti-TcdA polysera; anti B = alpaca anti-TcdB polysera.

Figure 3.

Glucosyltransferase activity in mouse and pig sera. The mRG1-1 cells were exposed to mouse (A) or pig (B) sera before being harvested for detection of Rac1 glucosylation by immunoblotting as described in Materials and Methods. A, Lane 1: cells only; lane 2: A1H3 antibody only; lane 3: 10 ng/mL Clostridium difficile toxin A (TcdA) + A1H3; lane 4: 1 ng/mL TcdA + A1H3; lane 5: mouse preinoculation serum (1:10) + A1H3; lane 6: mouse serum (1:20) 1 d postinoculation (PI) + A1H3; lane 7: mouse serum (1:20) 2 d PI + A1H3; lane 8: mouse serum (1:50) 2 d PI + A1H3; lane 9: mouse serum (1:200) 2 d PI + A1H3; lane 10: mouse serum (1:20) 2 d PI + A1H3 + anti-TcdA and anti-TcdB polysera (1:1000); lane 11: mouse serum (1:20) 2 d PI + A1H3 + anti-TcdA polysera (1:1000); lane 12: mouse serum (1:20) + A1H3 + anti-TcdB polysera (1:1000); B, Lane 1: cells-only control; lane 2: A1H3 antibody control; lane 3: 10 ng/mL TcdA + A1H3; lane 4: piglet serum (1:10) 5 d PI + A1H3; lane 5: piglet serum (1:50) 7 d PI + A1H3; lane 6: piglet serum (1:50) + A1H3 + anti-TcdA and anti-TcdB polysera.

Figure 4.

Necropsy and histopathologic images from piglets and mice with Clostridium difficile infection (CDI). A, Necropsy image of the thorax of a piglet with systemic CDI showing pleural effusion. B, Necropsy image of the thorax of a mouse with systemic CDI showing pleural effusion; C, Necropsy image of the abdomen of a mouse with systemic CDI showing ascites; D, Section of lung from a normal piglet for comparison; E, Section of lung from a piglet with systemic CDI showing regional atelectasis and interstitial thickening with no bacteria or neutrophilic inflammation present.

Figure 5.

Survival and body-weight changes in mice treated with systemic polyclonal anti–Clostridium difficile toxin A and anti–C. difficile toxin B antibodies. A, Kaplan-Meier survival curves of antitoxin antibody-treated mice compared with those of mice treated with nonimmune sera (CRT) (P = .007); B, Relative body-weight change in mice after inoculation with C. difficile. The data shown are pooled from 2 independent experiments (n = 15). Significant differences were determined using the t test. *P < .001.

Figure 6.

Piglet and mouse serum cytokine concentrations. A, Serum from piglets (n = 69) was grouped according to disease severity, as systemic (black bar, n = 29) or nonsystemic (open bar, n = 40) Clostridium difficile infection (CDI); B, Serum samples from 7 mice with systemic CDI (black bar), 7 mice with nonsystemic CDI (open bar), or 5 mice treated with anti–Clostridium difficile toxin A and anti–C. difficile toxin B polysera (anti AB, gray bar) were run separately in cytokine assays. Columns represent the mean cytokine concentration with standard error bars for each cytokine. Statistical differences were determined by the t test. *P < .05; ** P < .01. Abbreviations: IFN-γ, interferon γ; IL, interleukin; TGF-β, transforming growth factor β; TNF-α, tumor necrosis factor α.