BACKGROUND
the ubiquitous, spore forming, gram-positive bacterium clostridium perfringens is considered to be the most widespread pathogenic bacterium in the world .
it can cause a wide range of diseases including, amongst others, gas gangrene in man and necrohemorrhagic enteritis in suckling and veal calves .
most of these diseases follow a very rapid, often fatal course.
therefore, curative treatment is difficult and control must rely on preventive measures, including vaccination.
virulence properties of different c. perfringens strains are largely determined by their ability to secrete a variety of proteinaceous toxins and enzymes, which can cause different forms of tissue damage .
alpha toxin and perfringolysin o have been identified as the principal toxins involved in the pathogenesis of both c. perfringens-induced gas gangrene and bovine necrohemorrhagic enteritis  <cit> .
these toxins exert different effects in both diseases.
bovine necrohemorrhagic enteritis is characterized by congestion of the capillaries, hemorrhages and inflammation.
this is in contrast to gas gangrene, where these toxins lead to tissue necrosis, thrombosis and lack of leukocyte infiltration at the site of infection .
it is well known that humoral antibodies against secreted proteinaceous virulence factors of c. perfringens can be protective, as shown in different animal models.
as the enzymes and toxins of c. perfringens are highly destructive to tissues, vaccines against a variety of clostridial diseases have been developed using the denatured proteins .
despite the usefulness of formaldehyde toxoids for other c. perfringens-associated diseases, there is controversy about the efficacy of such vaccines for gas gangrene, as opposed to crude toxin preparations .
in addition, multivalent clostridial vaccines based on formaldehyde inactivated exotoxins derived from culture supernatant are commercially available for domestic livestock, including bovines, but no studies on their efficacy for necrohemorrhagic enteritis in calves are available.
the objective of the present study was to evaluate whether antibodies against c. perfringens toxins could protect against the development of necrotic lesions in the intestine.
therefore, calves were immunized with native c. perfringens toxins.
to evaluate whether we could eliminate the undesired toxin activity, but conserve the immune-protective potential, a previously described, modified formaldehyde treatment was also tested  <cit> .
also a commercial formaldehyde inactivated multivalent clostridial vaccine was used.
as necrohemorrhagic enteritis in veal calves is an unpredictable event and experimental reproduction of the disease is difficult, the neutralizing activity of the antibodies was evaluated in a previously developed intestinal loop model  <cit> .
to further unravel the mechanism of protection, the inhibitory effect of the evoked antibodies on c. perfringens-induced cytotoxicity to bovine endothelial cells was evaluated and the toxin-neutralizing capacity against alpha toxin and perfringolysin o was analyzed.
RESULTS
western blot analysis
the proteins in the c. perfringens toxin preparation were visualized by sds-page .
in the vaccinated calves, the production of circulating antibodies against c. perfringens supernatant and the c. perfringens toxin preparation was analyzed by western blot in three separate experiments .
no immune reaction was detected in the sera before immunization .
sera obtained from calves six weeks after initial vaccination with either native toxins or the l-lysine protected, formaldehyde inactivated toxins, revealed immunoreactivity towards two proteins.
immune sera from calves vaccinated with the commercial formaldehyde inactivated clostridial vaccine showed immunoreactivity towards more proteins.
the two proteins that were immunoreactive with antisera raised against all vaccine preparations were further identified as alpha toxin and perfringolysin o by maldi analysis.fig.
<dig> western blot analysis of the immune sera.
a sds-page of the c. perfringens toxin preparation after coomassie staining.
b-d representative western blots showing the immunoreactivity towards crude c. perfringens supernatants  and the c. perfringens toxin preparation .
the immune sera from calves vaccinated with native toxins  and formaldehyde inactivated, l-lysine protected c. perfringens toxins  detect only two proteins, whereas the commercial formaldehyde inactivated multivalent clostridial vaccine  reacts with multiple proteins.
the blots shown are representative pictures of one out of three experiments
elisa
in the vaccinated calves, the production of circulating antibodies directed against alpha toxin and perfringolysin o was also monitored by elisa.
no antibodies against alpha toxin or perfringolysin o were detected in the sera before immunization.
in all calves a strong antibody response against both alpha toxin and perfringolysin o was detected 6 weeks after initial immunization.
calves vaccinated with the native c. perfringens toxins showed the highest antibody titers, whereas vaccination with formaldehyde inactivated toxins  resulted in a more variable immune response .table  <dig> calves were immunized with either a c. perfringens toxin preparation , l-lysine protected, formaldehyde inactivated c. perfringens toxins  or a commercial multivalent formaldehyde inactivated clostridial vaccine
the anti-alpha toxin and perfringolysin o response was measured by elisa.
the data represent antibody titers , six weeks after initial immunization
protective effect of antisera against c. perfringens-induced necrosis in an intestinal loop model
the potential of the antisera, derived after vaccination of calves with the respective vaccines, to inhibit c. perfringens-induced necrosis, was evaluated in an intestinal loop assay.
all positive control loops inoculated with c. perfringens developed necrosis.
injection of loops with c. perfringens together with sera from naive calves  also resulted in a high percentage of necrotic loops.
injection of c. perfringens together with antisera raised against native toxins resulted in significantly fewer necrotic loops as compared to the positive control loops  and the loops injected with the pre-immune sera .
antisera raised against formaldehyde inactivated toxoid  were unable to significantly neutralize the necrosis-inducing activity of c. perfringens .fig.
<dig> neutralization of the lesion-inducing potential of c. perfringens.
the graph represents the percentage of ligated intestinal loops in which necrotic lesions were present after 5 h of incubation with sterile culture medium , c. perfringens alone  or c. perfringens in combination with naive sera , antiserum to c. perfringens toxins , formaldehyde inactivated, l-lysine protected c. perfringens toxins  and commercial formaldehyde inactivated multivalent clostridial vaccine .
**  <dig>  ≤ p <  <dig>  or *** p <  <dig>
fig.
<dig>
c. perfringens-induced necrosis in experimentally infected intestinal loops in calves.
a representative histological section from an intestinal loop without necrotic lesions.
this loop was injected with c. perfringens in combination with antiserum to native c. perfringens toxins.
b representative section from an intestinal loop from the same calf, showing hemorrhage and extensive necrosis of the villi.
this loops was injected with c. perfringens in combination with naïve immune serum
neutralization of alpha toxin and perfringolysin o activity in vitro
the inhibitory capacity of the sera towards alpha toxin and perfringolysin o activities was further examined using recombinant toxins.
all antisera decreased the activity of alpha toxin in vitro .
up to a final dilution of  <dig>  the antisera against the native toxins neutralized 50 % of the alpha toxin activity.
to the contrary, in order to obtain the same inhibition of alpha toxin activity, antisera against l-lysine protected, formaldehyde inactivated toxoid or against the commercial formaldehyde inactivated clostridial vaccine could only be diluted up to final dilutions of  <dig>  or  <dig> , respectively.table  <dig> in vitro neutralization of biological activities of alpha toxin and perfringolysin o. calves were immunized with either a c. perfringens toxin preparation , l-lysine protected, formaldehyde inactivated c. perfringens toxins  or a commercial multivalent formaldehyde inactivated clostridial vaccine
aneutralization of 10 μg/ml alpha toxin.
the inhibitory capacity of the antiserum is expressed as the dilution that gives 50 % inhibition of the alpha toxin activity
bneutralization of 2 μg/ml perfringolysin o. the inhibitory capacity of the antiserum is expressed as the highest dilution that inhibited perfringolysin o-induced hemolysis
alpha toxin activity was determined by measuring its lecithinase activity on egg yolk lipoproteins.
perfringolysin o  activity was determined by measuring the hemolysis of horse erythrocytes
the hemolytic activity of perfringolysin o towards equine erythrocytes in vitro was decreased by all antisera .
up to a final dilution of  <dig> either antiserum inhibited the perfringolysin o activity completely.
this neutralizing ability of the sera was observed up to a final dilution of  <dig> for the anti-native toxins antisera, a final dilution of  <dig> for the anti-l-lysine protected, formaldehyde inactivated toxoid or a final dilution of  <dig> when the antiserum obtained after vaccination with the commercial vaccine was used.
the pre-immune sera had no effect on the alpha toxin or perfringolysin o activity in vitro.
neutralization of c. perfringens cytotoxicity to bovine endothelial cells
to determine whether the cytotoxic activity of c. perfringens could be inhibited by the antisera to the vaccines, c. perfringens supernatants were incubated with serial dilutions of the antisera.
exposure of the endothelial cells to untreated c. perfringens supernatant resulted in 100 % cell death.
more than 80 % cell viability could be measured by pre-incubation of the c. perfringens supernatant with a 32-fold dilution of the native toxins antiserum.
at this concentration, neither the antisera raised against l-lysine protected, formaldehyde inactivated toxins nor the antisera raised against the commercial formaldehyde inactivated clostridial vaccine had an effect on the cytotoxicity .
the pre-immune sera had no effect on the c. perfringens cytotoxicity.table  <dig> in vitro neutralization of c. perfringens cytotoxicity.
calves were immunized with either a c. perfringens toxin preparation , l-lysine protected, formaldehyde inactivated c. perfringens toxins  or a commercial multivalent formaldehyde inactivated clostridial vaccine
the cytotoxicity of c. perfringens supernatant to primary bovine endothelial cells was measured using a neutral red uptake  assay.
the inhibitory capacity of the antiserum is expressed as the highest dilution that yields 80 % cell viability
discussion
necrohemorrhagic enteritis caused by c. perfringens in suckling and veal calves is characterized by sudden death.
due to the very rapid course of the disease, curative treatment is not possible and therefore, protection by vaccination would be of high value.
the virulence of c. perfringens is due to the many extracellular toxins it produces.
in this study we showed that toxin neutralizing antibodies protect against c. perfringens-induced necrotic lesions in an intestinal loop assay and are able to prevent endothelial damage.
western blot analysis revealed antibodies towards alpha toxin and perfringolysin o as the most abundant antibodies in the immune sera from calves vaccinated against c. perfringens toxins.
we previously reported congestion and leakage of the capillaries as an early event in the pathogenesis of necrohemorrhagic enteritis as shown in an intestinal loop assay  <cit> .
furthermore we showed that alpha toxin and perfringolysin o may exert their effect by directly targeting the endothelial cells  <cit> .
this points towards endothelial damage as a key event in the pathogenesis of bovine necrohemorrhagic enteritis.
indeed, in the present study antisera which protected against c. perfringens-induced cytotoxicity to bovine endothelial cells also offered protection against c. perfringens-associated necrosis in an intestinal loop assay.
moreover, the protective antisera were shown to inhibit the activity of alpha toxin and perfringolysin o, which further underscores the roles of these toxins in the pathogenesis of bovine necrohemorrhagic enteritis.
it can, however, not be ruled out that antibodies induced against other substances present in the vaccines also played a role in the protection observed in the intestinal loop model.
formaldehyde inactivation of c. perfringens toxins diminished their capacity to induce protective antibodies.
antisera raised against l-lysine protected, formaldehyde inactivated c. perfringens toxins were also not protective in the intestinal loop model.
this result is in disagreement with previous studies showing high antigenicity, low toxicity, and protection in mice that were immunized with l-lysine protected, formaldehyde inactivated toxoid and subsequently challenged with lethal doses of c. perfringens  <cit> .
in the present study we demonstrated that vaccinations with c. perfringens toxins, either in their native forms or as formaldehyde inactivated toxoids, all resulted in high antibody responses as detected by elisa.
however, only serum derived from animals immunized with the native toxins offered protection against necrosis in an intestinal loop assay.
there is thus a discrepancy between the antibody titers against formaldehyde inactivated c. perfringens toxins measured by elisa and the protective capacity of these antibodies in the intestinal loop model.
nevertheless, the value of vaccines based on formaldehyde inactivated c. perfringens toxins has been demonstrated for diseases associated with toxins other than alpha toxin and perfringolysin o .
this suggests that the protective immunogenicity of other c. perfringens toxins, such as, amongst others, netb and epsilon toxin, is not affected by formaldehyde inactivation.
although the use of c. perfringens native toxins represents an efficient strategy for vaccine development, active toxins cannot be regarded as safe.
therefore methods for the development of toxoids other than formaldehyde inactivation are needed.
possible strategies include the use of genetically modified toxoids based on site-directed mutants with reduced toxic activity or the use of immunologically active fragments of the essential toxins.
immunization with the carboxy-terminal domain of alpha toxin has previously been shown to provide protection in a mouse model against c. perfringens gas gangrene and may be a good candidate for development of a vaccine against bovine necrohemorrhagic enteritis  <cit> .
the identification of the structural elements responsible for membrane interaction of perfringolysin o provides opportunities for the development of non-toxic site-directed mutants as alternatives for native perfringolysin o  <cit> .
in order to obtain the ultimate evidence that vaccination against c. perfringens toxins protects against bovine necrohemorrhagic enteritis, field trials need to be performed.
however, since necrohemorrhagic enteritis is a low incidence disease, this would be a huge cost and more evidence concerning the immune-protective potential of the antisera is needed before considering this type of trial.
unfortunately, no in vivo model to validate the protective immune-potential of the candidate vaccines against bovine necrohemorrhagic enteritis is available.
niilo and colleagues were able to induce a mild diarrhea in cattle inoculated intraduodenally or per os with c. perfringens type a cultures, but no necrohemorrhagic enteritis was established  <cit> .
also we were unable to develop a reliable model of bovine necrohemorrhagic enteritis after per os or intraduodenal administration of c. perfringens type a cultures .
CONCLUSIONS
this study showed that toxin-neutralizing antibodies protect against c. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.
immunization of calves with either native or formaldehyde inactivated toxins resulted in a strong immune response against alpha toxin and perfringolysin o, but only antibodies raised against native toxins were protective in the intestinal loop model.
therefore it seems that, at least for alpha toxin mediated diseases, antibody titers detected by elisa are not a guarantee for protection, even if protection against the disease is antibody mediated.

 the ubiquitous, spore forming, gram-positive bacterium clostridium perfringens is considered to be the most widespread pathogenic bacterium in the world. it can cause a wide range of diseases including, amongst others, gas gangrene in man and necrohemorrhagic enteritis in suckling and veal calves. most of these diseases follow a very rapid, often fatal course. therefore, curative treatment is difficult and control must rely on preventive measures, including vaccination. virulence properties of different c. perfringens strains are largely determined by their ability to secrete a variety of proteinaceous toxins and enzymes, which can cause different forms of tissue damage. alpha toxin and perfringolysin o have been identified as the principal toxins involved in the pathogenesis of both c. perfringens-induced gas gangrene and bovine necrohemorrhagic enteritis. these toxins exert different effects in both diseases. bovine necrohemorrhagic enteritis is characterized by congestion of the capillaries, hemorrhages and inflammation. this is in contrast to gas gangrene, where these toxins lead to tissue necrosis, thrombosis and lack of leukocyte infiltration at the site of infection. it is well known that humoral antibodies against secreted proteinaceous virulence factors of c. perfringens can be protective, as shown in different animal models. as the enzymes and toxins of c. perfringens are highly destructive to tissues, vaccines against a variety of clostridial diseases have been developed using the denatured proteins. despite the usefulness of formaldehyde toxoids for other c. perfringens-associated diseases, there is controversy about the efficacy of such vaccines for gas gangrene, as opposed to crude toxin preparations. in addition, multivalent clostridial vaccines based on formaldehyde inactivated exotoxins derived from culture supernatant are commercially available for domestic livestock, including bovines, but no studies on their efficacy for necrohemorrhagic enteritis in calves are available. the objective of the present study was to evaluate whether antibodies against c. perfringens toxins could protect against the development of necrotic lesions in the intestine. therefore, calves were immunized with native c. perfringens toxins. to evaluate whether we could eliminate the undesired toxin activity, but conserve the immune-protective potential, a previously described, modified formaldehyde treatment was also tested. also a commercial formaldehyde inactivated multivalent clostridial vaccine was used. as necrohemorrhagic enteritis in veal calves is an unpredictable event and experimental reproduction of the disease is difficult, the neutralizing activity of the antibodies was evaluated in a previously developed intestinal loop model. to further unravel the mechanism of protection, the inhibitory effect of the evoked antibodies on c. perfringens-induced cytotoxicity to bovine endothelial cells was evaluated and the toxin-neutralizing capacity against alpha toxin and perfringolysin o was analyzed.  western blot analysis the proteins in the c. perfringens toxin preparation were visualized by sds-page. in the vaccinated calves, the production of circulating antibodies against c. perfringens supernatant and the c. perfringens toxin preparation was analyzed by western blot in three separate experiments. no immune reaction was detected in the sera before immunization. sera obtained from calves six weeks after initial vaccination with either native toxins or the l-lysine protected, formaldehyde inactivated toxins, revealed immunoreactivity towards two proteins. immune sera from calves vaccinated with the commercial formaldehyde inactivated clostridial vaccine showed immunoreactivity towards more proteins. the two proteins that were immunoreactive with antisera raised against all vaccine preparations were further identified as alpha toxin and perfringolysin o by maldi analysis.fig. western blot analysis of the immune sera. a sds-page of the c. perfringens toxin preparation after coomassie staining. b-d representative western blots showing the immunoreactivity towards crude c. perfringens supernatants and the c. perfringens toxin preparation. the immune sera from calves vaccinated with native toxins and formaldehyde inactivated, l-lysine protected c. perfringens toxins detect only two proteins, whereas the commercial formaldehyde inactivated multivalent clostridial vaccine reacts with multiple proteins. the blots shown are representative pictures of one out of three experiments elisa in the vaccinated calves, the production of circulating antibodies directed against alpha toxin and perfringolysin o was also monitored by elisa. no antibodies against alpha toxin or perfringolysin o were detected in the sera before immunization. in all calves a strong antibody response against both alpha toxin and perfringolysin o was detected 6 weeks after initial immunization. calves vaccinated with the native c. perfringens toxins showed the highest antibody titers, whereas vaccination with formaldehyde inactivated toxins resulted in a more variable immune response.table calves were immunized with either a c. perfringens toxin preparation, l-lysine protected, formaldehyde inactivated c. perfringens toxins or a commercial multivalent formaldehyde inactivated clostridial vaccine the anti-alpha toxin and perfringolysin o response was measured by elisa. the data represent antibody titers, six weeks after initial immunization protective effect of antisera against c. perfringens-induced necrosis in an intestinal loop model the potential of the antisera, derived after vaccination of calves with the respective vaccines, to inhibit c. perfringens-induced necrosis, was evaluated in an intestinal loop assay. all positive control loops inoculated with c. perfringens developed necrosis. injection of loops with c. perfringens together with sera from naive calves also resulted in a high percentage of necrotic loops. injection of c. perfringens together with antisera raised against native toxins resulted in significantly fewer necrotic loops as compared to the positive control loops and the loops injected with the pre-immune sera. antisera raised against formaldehyde inactivated toxoid were unable to significantly neutralize the necrosis-inducing activity of c. perfringens.fig. neutralization of the lesion-inducing potential of c. perfringens. the graph represents the percentage of ligated intestinal loops in which necrotic lesions were present after 5 h of incubation with sterile culture medium, c. perfringens alone or c. perfringens in combination with naive sera, antiserum to c. perfringens toxins, formaldehyde inactivated, l-lysine protected c. perfringens toxins and commercial formaldehyde inactivated multivalent clostridial vaccine. **  ≤ p <  or *** p <  fig. c. perfringens-induced necrosis in experimentally infected intestinal loops in calves. a representative histological section from an intestinal loop without necrotic lesions. this loop was injected with c. perfringens in combination with antiserum to native c. perfringens toxins. b representative section from an intestinal loop from the same calf, showing hemorrhage and extensive necrosis of the villi. this loops was injected with c. perfringens in combination with naïve immune serum neutralization of alpha toxin and perfringolysin o activity in vitro the inhibitory capacity of the sera towards alpha toxin and perfringolysin o activities was further examined using recombinant toxins. all antisera decreased the activity of alpha toxin in vitro. up to a final dilution of the antisera against the native toxins neutralized 50 % of the alpha toxin activity. to the contrary, in order to obtain the same inhibition of alpha toxin activity, antisera against l-lysine protected, formaldehyde inactivated toxoid or against the commercial formaldehyde inactivated clostridial vaccine could only be diluted up to final dilutions of or, respectively.table in vitro neutralization of biological activities of alpha toxin and perfringolysin o. calves were immunized with either a c. perfringens toxin preparation, l-lysine protected, formaldehyde inactivated c. perfringens toxins or a commercial multivalent formaldehyde inactivated clostridial vaccine aneutralization of 10 μg/ml alpha toxin. the inhibitory capacity of the antiserum is expressed as the dilution that gives 50 % inhibition of the alpha toxin activity bneutralization of 2 μg/ml perfringolysin o. the inhibitory capacity of the antiserum is expressed as the highest dilution that inhibited perfringolysin o-induced hemolysis alpha toxin activity was determined by measuring its lecithinase activity on egg yolk lipoproteins. perfringolysin o activity was determined by measuring the hemolysis of horse erythrocytes the hemolytic activity of perfringolysin o towards equine erythrocytes in vitro was decreased by all antisera. up to a final dilution of either antiserum inhibited the perfringolysin o activity completely. this neutralizing ability of the sera was observed up to a final dilution of for the anti-native toxins antisera, a final dilution of for the anti-l-lysine protected, formaldehyde inactivated toxoid or a final dilution of when the antiserum obtained after vaccination with the commercial vaccine was used. the pre-immune sera had no effect on the alpha toxin or perfringolysin o activity in vitro. neutralization of c. perfringens cytotoxicity to bovine endothelial cells to determine whether the cytotoxic activity of c. perfringens could be inhibited by the antisera to the vaccines, c. perfringens supernatants were incubated with serial dilutions of the antisera. exposure of the endothelial cells to untreated c. perfringens supernatant resulted in 100 % cell death. more than 80 % cell viability could be measured by pre-incubation of the c. perfringens supernatant with a 32-fold dilution of the native toxins antiserum. at this concentration, neither the antisera raised against l-lysine protected, formaldehyde inactivated toxins nor the antisera raised against the commercial formaldehyde inactivated clostridial vaccine had an effect on the cytotoxicity. the pre-immune sera had no effect on the c. perfringens cytotoxicity.table in vitro neutralization of c. perfringens cytotoxicity. calves were immunized with either a c. perfringens toxin preparation, l-lysine protected, formaldehyde inactivated c. perfringens toxins or a commercial multivalent formaldehyde inactivated clostridial vaccine the cytotoxicity of c. perfringens supernatant to primary bovine endothelial cells was measured using a neutral red uptake assay. the inhibitory capacity of the antiserum is expressed as the highest dilution that yields 80 % cell viability discussion necrohemorrhagic enteritis caused by c. perfringens in suckling and veal calves is characterized by sudden death. due to the very rapid course of the disease, curative treatment is not possible and therefore, protection by vaccination would be of high value. the virulence of c. perfringens is due to the many extracellular toxins it produces. in this study we showed that toxin neutralizing antibodies protect against c. perfringens-induced necrotic lesions in an intestinal loop assay and are able to prevent endothelial damage. western blot analysis revealed antibodies towards alpha toxin and perfringolysin o as the most abundant antibodies in the immune sera from calves vaccinated against c. perfringens toxins. we previously reported congestion and leakage of the capillaries as an early event in the pathogenesis of necrohemorrhagic enteritis as shown in an intestinal loop assay. furthermore we showed that alpha toxin and perfringolysin o may exert their effect by directly targeting the endothelial cells. this points towards endothelial damage as a key event in the pathogenesis of bovine necrohemorrhagic enteritis. indeed, in the present study antisera which protected against c. perfringens-induced cytotoxicity to bovine endothelial cells also offered protection against c. perfringens-associated necrosis in an intestinal loop assay. moreover, the protective antisera were shown to inhibit the activity of alpha toxin and perfringolysin o, which further underscores the roles of these toxins in the pathogenesis of bovine necrohemorrhagic enteritis. it can, however, not be ruled out that antibodies induced against other substances present in the vaccines also played a role in the protection observed in the intestinal loop model. formaldehyde inactivation of c. perfringens toxins diminished their capacity to induce protective antibodies. antisera raised against l-lysine protected, formaldehyde inactivated c. perfringens toxins were also not protective in the intestinal loop model. this result is in disagreement with previous studies showing high antigenicity, low toxicity, and protection in mice that were immunized with l-lysine protected, formaldehyde inactivated toxoid and subsequently challenged with lethal doses of c. perfringens. in the present study we demonstrated that vaccinations with c. perfringens toxins, either in their native forms or as formaldehyde inactivated toxoids, all resulted in high antibody responses as detected by elisa. however, only serum derived from animals immunized with the native toxins offered protection against necrosis in an intestinal loop assay. there is thus a discrepancy between the antibody titers against formaldehyde inactivated c. perfringens toxins measured by elisa and the protective capacity of these antibodies in the intestinal loop model. nevertheless, the value of vaccines based on formaldehyde inactivated c. perfringens toxins has been demonstrated for diseases associated with toxins other than alpha toxin and perfringolysin o. this suggests that the protective immunogenicity of other c. perfringens toxins, such as, amongst others, netb and epsilon toxin, is not affected by formaldehyde inactivation. although the use of c. perfringens native toxins represents an efficient strategy for vaccine development, active toxins cannot be regarded as safe. therefore methods for the development of toxoids other than formaldehyde inactivation are needed. possible strategies include the use of genetically modified toxoids based on site-directed mutants with reduced toxic activity or the use of immunologically active fragments of the essential toxins. immunization with the carboxy-terminal domain of alpha toxin has previously been shown to provide protection in a mouse model against c. perfringens gas gangrene and may be a good candidate for development of a vaccine against bovine necrohemorrhagic enteritis. the identification of the structural elements responsible for membrane interaction of perfringolysin o provides opportunities for the development of non-toxic site-directed mutants as alternatives for native perfringolysin o. in order to obtain the ultimate evidence that vaccination against c. perfringens toxins protects against bovine necrohemorrhagic enteritis, field trials need to be performed. however, since necrohemorrhagic enteritis is a low incidence disease, this would be a huge cost and more evidence concerning the immune-protective potential of the antisera is needed before considering this type of trial. unfortunately, no in vivo model to validate the protective immune-potential of the candidate vaccines against bovine necrohemorrhagic enteritis is available. niilo and colleagues were able to induce a mild diarrhea in cattle inoculated intraduodenally or per os with c. perfringens type a cultures, but no necrohemorrhagic enteritis was established. also we were unable to develop a reliable model of bovine necrohemorrhagic enteritis after per os or intraduodenal administration of c. perfringens type a cultures.  this study showed that toxin-neutralizing antibodies protect against c. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis. immunization of calves with either native or formaldehyde inactivated toxins resulted in a strong immune response against alpha toxin and perfringolysin o, but only antibodies raised against native toxins were protective in the intestinal loop model. therefore it seems that, at least for alpha toxin mediated diseases, antibody titers detected by elisa are not a guarantee for protection, even if protection against the disease is antibody mediated. 
alpha toxin and perfringolysin o were identified as the most immunogenic proteins in the vaccine preparations. all vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin o, as detected by elisa. all antibodies were able to inhibit the activity of alpha toxin and perfringolysin o in vitro. although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin o, only antibodies raised against native toxins protect against c. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis. bovine necrohemorrhagic enteritisclostridium perfringensneutralizing antibodiesalpha toxinperfringolysin ohttp://dx.doi.org/http://dx.doi.org/ /501100003132agentschap voor innovatie door wetenschap en technologie090910issue-copyright-statement© the author 2016 in this study, c. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. however, the antibodies raised against the native toxins were more inhibitory to the c. perfringens-induced cytotoxicity and only these antibodies protected against c. perfringens challenge in the intestinal loop model. bovine necrohemorrhagic enteritis is caused by clostridium perfringens type a. due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. we determined whether antisera raised in calves against these toxins were able to protect against c. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.
 bovine necrohemorrhagic enteritis is caused by clostridium perfringens type a. due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. in this study, c. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. we determined whether antisera raised in calves against these toxins were able to protect against c. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.  alpha toxin and perfringolysin o were identified as the most immunogenic proteins in the vaccine preparations. all vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin o, as detected by elisa. all antibodies were able to inhibit the activity of alpha toxin and perfringolysin o in vitro. however, the antibodies raised against the native toxins were more inhibitory to the c. perfringens-induced cytotoxicity and only these antibodies protected against c. perfringens challenge in the intestinal loop model.  although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin o, only antibodies raised against native toxins protect against c. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis. keywords bovine necrohemorrhagic enteritisclostridium perfringensneutralizing antibodiesalpha toxinperfringolysin ohttp://dx.doi.org/http://dx.doi.org/ /501100003132agentschap voor innovatie door wetenschap en technologie090910issue-copyright-statement© the author 2016 