type C isolates remain an important cause of disease in domestic animals, as indicated by the widespread type C vaccination of domestic animals (24
). Type C isolates also are the only non-type A isolates documented to cause human disease (15
). Type C-associated human disease is most closely associated with Papua New Guinea, where it was once the second leading cause of death in children who were more than 1 year old, which led to a childhood vaccine program that helped to reduce this problem (22
). In addition, type C human necrotic enteritis is now seen in developed countries, typically in patients with pancreatic disease (10
Despite the obvious importance of type C isolates for both human and animal disease, the virulence of these isolates is poorly understood. While these bacteria must produce at least CPB and PLC in order to be classified as type C isolates, our results indicate that most type C isolates also produce a third lethal toxin (PFO) and that ~40% of these isolates produce a fourth lethal toxin (CPB2) during late-log-phase growth. This complex lethal toxin repertoire raises the possibility that type C-induced disease results from the combined activities of several toxins. Consistent with this possibility, animal model studies have shown that CPB alone cannot elicit the symptoms of a type C infection (34
). Furthermore, the current type C veterinary vaccines are based on crude toxoids or bacterin/toxoid (killed bacteria plus inactivated supernatants) (35
). In addition, the human vaccine previously used in New Guinea was not prepared with a pure CPB toxoid but instead was prepared with ammonium sulfate-precipitated supernatants from type C vegetative cultures that were formalin inactivated (40
; Gregor Lawrence, personal communication). Therefore, the protective immune response elicited by these type C toxoid vaccines against type C infections could involve protective antibodies not only against CPB (which is the only component for which type C vaccines are validated [19
]) but also against any other lethal toxins produced by the type C isolates used for producing the toxoid vaccine. To our knowledge, type C strains used for vaccine preparation do not have a well-characterized toxin repertoire.
As a first step in determining which toxins play a role in type C-induced disease, we determined the roles of individual type C toxins in the mouse i.v. supernatant injection lethality model. In this research we initially determined the toxin genotypes and phenotypes of a large number of putative type C isolates, which confirmed that all isolates previously genotyped by multiplex PCR were genotype C isolates. However, a number of isolates assigned to type C many years ago using the classical guinea pig skin test were instead classified as type A isolates by multiplex PCR. The explanations for the difference in classification include the loss of the cpb-containing plasmid after toxin neutralization typing and, perhaps, the relatively crude nature of historical guinea pig skin neutralization testing.
Interestingly, our genotypic analyses showed that ~40% of the genotype C isolates surveyed carry the cpb2
gene, which is a considerably higher percentage than the 18% cpb2
-positive isolates identified in a recent survey of type D isolates (28
). The percentage of cpb2
-positive genotype C isolates in our collection is lower than the percentage determined by Bueschel et al., who reported that 64% of their genotype C isolates (n
178) were cpb2
). The cpb2
carriage rate for type C isolates is also less than the 80% cpb2
carriage rate determined for type A isolates carrying a plasmid-borne cpe
). The very strong association between cpb2
and type A isolates carrying a cpe
plasmid is partially attributable to the fact that many, although not all, of the type A isolates carry cpb2
on the same plasmid. In this regard, it is interesting that we identified only a single cpe
-positive type C isolate in the current survey. Studies are now under way to determine whether cpb
reside on the same plasmid in our genotype C isolates.
Phenotypic analyses showed that under our assay conditions, genotype C isolates carrying the cpb2
gene typically produce lower levels of both CPB and PLC than the cpb2
-negative genotype C isolates produce. Since the regulation of PLC or CPB in type C isolates has not been studied yet, we have no explanation for these observations. However, the explanation might involve differences in the VirR/VirS two-component system, which positively regulates both cpb2
expression in type A isolates via its ability to regulate expression of VR-RNA regulatory RNA (2
). Another possibility is that genes present on the putative cpb2
plasmid might specifically down-regulate cpb
Phenotypic analyses also revealed that five of the six cpe-positive genotype C isolates surveyed produced no detectable PFO activity. Two PCR analyses indicated that the pfoA gene was not present in these isolates. In contrast, similar PCR analyses of the pfoA gene in cpe-positive C. perfringens type A isolates demonstrated that the pfoA gene is present in most cpe plasmid isolates but is not present in most chromosomal cpe-positive type A isolates (unpublished observations). Studies should be performed to address the cpe gene location (chromosomal versus plasmid) in cpe-positive genotype C isolates, which would reveal whether the genotypes of these isolates resemble the pfoA-negative genotype of most chromosomal cpe type A isolates.
Sequencing of the pfoA
gene from the three cpe
-negative genotype C isolates that failed to produce PFO yet were PCR positive for pfoA
revealed no nucleotide substitutions in the promoter region that could explain the lack of PFO activity for these isolates (5
). Nor could the lack of PFO activity for these isolates be attributed to consistent nucleotide changes in the pfoA
ORF. However, compared to the pfoA
gene of type A strain 13, there were consistent nucleotide changes in the pfoA
ORF of the type C isolates sequenced that resulted in two different amino acid substitutions. The A71T substitution was identified in all four type C isolates sequenced, including a PFO-positive isolate, while an A215V substitution was present in a single PFO-negative isolate. Based on these sequencing and PCR results, it appears that the lack of detectable PFO activity for some type C isolates results either from an inability to make PFO (for most or all cpe-
negative, type C isolates that were PCR positive for pfoA
), implying that there is a regulatory gene mutation, or from the actual absence of the pfoA
gene (for the cpe
-positive type C isolates negative in both pfoA
PCR assays). However, our results do not rule out the possibility that some pfoA
-positive, PFO-negative isolates could produce PFO under culture conditions different than those tested or that the A215V substitution encoded by the pfoA
gene of one pfoA
-positive PFO-negative isolate eliminates its PFO activity.
Finally, the results of our toxin phenotype analyses of genotype C isolates allowed us to compare the toxin production abilities of these isolates with those of genotype D and A isolates. No statistically significant differences were found between the average amounts of PLC or PFO produced when we compared genotype C isolates to genotype A or D isolates (28
; this study). However, it should be noted that not all of the genotype A isolates used to measure PFO and PLC levels in this study were isolated from gas gangrene cases, which are believed to be associated with genotype A isolates that produce high levels of PFO and PLC. The average levels of CPE (which is produced only during sporulation) in culture supernatants from sporulating genotype C isolates, genotype A isolates (6
), and genotype D isolates (28
; this study) were also not significantly different. It is notable that while some genotype D isolates did not produce detectable levels of their typing toxin (ETX) (28
) even when their supernatants were concentrated, all genotype C isolates tested produced detectable amounts of CPB toxin, although detection sometimes required concentration of the culture supernatants. It is important to note that these findings are specific for the media and test conditions used in this study; i.e., isolates could make more or less PLC (or other toxins) in brain heart infusion media in the late log phase.
The timing of CPB toxin production has not been well documented. Therefore, in this study we tried to determine when this toxin is produced during in vitro growth, because quantifying CPB toxin levels in vegetative culture supernatants is important both for our mouse i.v. lethality studies and for preparing type C vaccines. The results of these analyses indicated that CPB toxin production reaches peak levels during late-log-phase growth in TGY medium. The timing of CPB production agrees with the results of a previous study in which the workers measured levels of CPB activity in culture supernatants from a single type C isolate using a guinea pig skin test animal model (26
). Interestingly, Sakurai and Duncan also found that fermentable sugars can increase CPB production under non-pH-controlled conditions (26
). In the current study, the levels of CPB production were consistently higher in TGY medium (which contained 2% glucose/liter) than in FTG (0.55% glucose/liter), brain heart infusion broth (0.2% glucose/liter), or differential reinforced clostridial broth (0.1% glucose/liter). These analyses also revealed that CPB production varies substantially (>40-fold) among type C isolates, suggesting that there are isolate-dependent variations in cpb
gene regulation. Furthermore, CPB toxin levels were typically found to decrease during the stationary phase, possibly due to decreased CPB production or protease activity present in stationary-phase cultures. Together, the variability in CPB toxin production observed among type C isolates and the reductions in CPB toxin levels detected during longer growth periods highlight the importance of selecting the proper strain and growth conditions for preparing type C toxoid vaccines. To our knowledge, the identities of genotype C strains and the growth conditions used to prepare type C vaccines have not been clearly reported in refereed publications.
To gain further understanding regarding which toxin(s) present in type C supernatants is responsible for virulence (and therefore must be neutralized for a vaccine to elicit protective immunity), sterile late-log-phase supernatants from approximately one-half of our type C isolates were injected i.v. into mice. When the LD50/ml values obtained for these vegetative culture supernatants were correlated with the levels of lethal toxins produced by the isolates, no positive correlation was observed between LD50/ml and PFO levels. In contrast, the levels of three other lethal toxins (CPB, CPB2, and PLC) showed at least some positive correlation with supernatant LD50/ml values, and the CPB levels exhibited a much higher LD50/ml correlation (R2 >0.7) than either the PLC or CPB2 levels (R2, 0.38 and 0.18, respectively). These correlation results suggest that CPB plays a major role in the lethality of type C supernatants in the mouse i.v. injection model.
The importance of CPB for type C supernatant lethality in the mouse i.v injection model was then conclusively demonstrated by toxin neutralization experiments using neutralizing monoclonal antibodies specific for CPB, PLC, or CPE. In these neutralization studies, the lethal properties of late-log-phase type C supernatants were consistently neutralized by anti-CPB monoclonal antibodies, but neutralization was not observed using either anti-PLC or anti-CPE monoclonal antibodies. Interestingly, the time until death for mice treated with supernatants incubated with anti-PLC antibodies was longer than the time until death for mice treated with untreated supernatants. This observation along with the CPB neutralization data may indicate that when our experimental conditions are used, PLC is not the primary mediator of lethality (since the mice still died), but it does make a minor contribution to i.v. lethality, possibly explaining the small positive correlation between supernatant LD50/mls and PLC concentration (R2 = 0.38) (Fig. ).
Previous oral challenge and intestinal loop studies suggested that CPB cannot cause the full pathology of a natural type C infection (34
). This finding along with the current i.v. challenge results suggests that CPB is required for the systemic lethality of type C supernatants, but a secondary factor(s) may facilitate the intestinal absorption of CPB into the circulation during natural disease. This possibility cannot be conclusively addressed by the mouse i.v. challenge model or monoclonal antibody neutralization approaches, which do not address factors such as the breaching of the intestinal permeability barrier or possible synergistic toxin interactions. There is a precedent for such synergistic interactions since previous studies have shown that PLC and PFO act synergistically in the pathogenesis of histotoxic type A infections in mice (1
). While the current results provide important insights into type C pathogenesis, further research is clearly needed to determine which toxin(s) (or the bacteria themselves) contributes to the early stages of type C-induced enteric disease. To address this issue, efforts are being made to develop a small-animal oral challenge model that better mimics the natural type C disease. When available, this oral challenge model should be useful for virulence testing of type C toxin knockout mutants in order to dissect the specific roles of various toxins at each stage of type C disease.