Both epsilon-toxin and beta-toxin are important for the lethal properties of Clostridium perfringens type B isolates in the mouse intravenous injection model

Abstract

Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the pathogenesis of C. perfringens type B infections in domestic animals.

FIG. 1.

Growth versus production of CPB and ETX. (A) TGY was inoculated with an overnight starter culture and incubated at 37°C. Supernatant samples were collected at the indicated time intervals and measured for turbidity (optical density at 600 nm [OD_600nm]). Samples were then electrophoresed on a 12% SDS-polyacrylamide gel and then Western blotted with MAb-CPB (B) or MAb-ETX (C). The times (in h) of samples used for Western blotting are listed underneath the blots in panels B and C. The locations of protein molecular mass standards are shown on the left of each blot (B and C). Purified CPB or ETX was run on each gel as a positive control for Western blotting.

FIG. 2.

Western blots for ETX, CPB2, and CPB production by representative type B isolates. Isolates were grown in TGY medium to late log phase, and supernatants were collected and run on 12% SDS-PAGE gels. Detection was performed with MAb-ETX, polyclonal Ab-CPB2, or MAb-CPB. The migration of protein standards are shown to the left of each blot, and isolates are indicated at the top. Purified toxin was run on the far left of each blot as a positive control, and the genotype A cpb2-negative strain ATCC3624 was used as a negative (neg) control for ETX, CPB, and CPB2. MAb-ETX and MAb-CPB blots were visualized with film, whereas the anti-CPB2 blot was visualized with a Bio-Rad imager.

FIG. 3.

Toxin levels produced by genotype B isolates. Toxin levels in late-log-phase culture supernatants were quantified as described in Materials and Methods. Charts shown are for (A) ETX, CPB, and CPB2 (all quantified via Western blots) and for (B) PFO and (C) CPA (quantified via toxin activity assays). PLC, phospholipase C.

FIG. 4.

Comparative analysis of toxin production by different genotypes of C. perfringens isolates in late-log-phase culture supernatants. Results shown are for CPB (A), ETX (B), CPA (C), and PFO (D) as relative percentages of surveyed isolates found in each range of toxin levels, quantified as described in Materials and Methods. For all panels, ND indicates that toxin could not be detected in culture supernatants. Total numbers of isolates were 10 (type A), 19 (type B), 34 (type C), and 39 (type D).

FIG. 5.

Lethalities of non-trypsin-treated (non-tryp) and trypsin-treated C. perfringens genotype B isolate culture supernatants expressed as LD₅₀/ml values. Pairs of mice were injected i.v. through the tail vein with dilutions of culture supernatants, and mice were observed for up to 48 h for the development of clinical signs of distress.

FIG. 6.

Correlation analyses of genotype B toxin levels versus mouse i.v. LD₅₀/ml values for non-trypsin-treated culture supernatants. LD₅₀/ml values were correlated with the amount of CPA (A), CPB (B), CPB2 (C), ETX (D), and PFO (E) present in each vegetative culture supernatant. A linear equation was then used to draw a best-fit line based on the data points, and the R² value for this line is reported for each graph.

FIG. 7.

Correlation analyses of genotype B toxin levels versus mouse i.v. LD₅₀/ml values for trypsin-treated culture supernatants. LD₅₀/ml values were correlated with the amounts of CPA (A), CPB (B), CPB2 (C), ETX (D), and PFO (E) present in the vegetative culture supernatants prior to trypsin treatment. A linear equation was then used to draw a best-fit line based on the data points, and the R² value for this line is reported for each graph.