- Email: [email protected]
Evaluation of different fluids for detection of Clostridium perfringens type D epsilon toxin in sheep with experimental enterotoxemia
ARTICLE IN PRESS
Anaerobe 12 (2006) 204–206 www.elsevier.com/locate/anaerobe
Short communication—Veterinary anaerobes and diseases
Evaluation of different fluids for detection of Clostridium perfringens type D epsilon toxin in sheep with experimental enterotoxemia Jorge E. Layanaa, Mariano E. Fernandez Miyakawab, Francisco A. Uzalb, a
The National Institute of Agricultural Technology. CC 277, (8400) Bariloche, Argentina California Animal Health and Food Safety Laboratory, San Bernardino Branch, School of Veterinary Medicine, University of California, Davis. 105 W Central Ave, San Bernardino, CA 92408, USA
b
Received 31 March 2006; received in revised form 18 May 2006; accepted 18 May 2006 Available online 20 July 2006
Abstract Enterotoxemia caused by Clostridium perfringens type D is a highly lethal disease of sheep, goats and other ruminants. The diagnosis of this condition is usually confirmed by detection of epsilon toxin, a major exotoxin produced by C. perfringens types B and D, in the intestinal content of affected animals. It has been suggested that other body fluids can also be used for detection of epsilon toxin. This study was performed to evaluate the usefulness of intestinal content versus other body fluids in detecting epsilon toxin in cases of sheep enterotoxemia. Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 15 sheep with experimentally induced enterotoxemia, were analysed for epsilon toxin using a capture ELISA. Epsilon toxin was detected in 92% of the samples of ileal content, 64% of the samples of duodenal content, 57% of the samples of colon content and in 7% of the samples of pericardial fluid and aqueous humor. No epsilon toxin was found in samples of abdominal fluid or urine from the animals with enterotoxemia or in any samples from six clinically healthy sheep used as negative controls. The results of this study indicate that with the diagnostic capture ELISA used, intestinal content (preferably ileum) should be used for C. perfringens type D epsilon toxin detection in suspected cases of sheep enterotoxemia. r 2006 Elsevier Ltd. All rights reserved. Keywords: Body fluids; Clostridium perfringens; Enterotoxemia; Epsilon toxin; Intestinal content; Sheep
1. Introduction Enterotoxemia is a highly lethal disease of sheep, goats and other animal species, produced by Clostridium perfringens type D [1]. In sheep, the main clinical signs and pathological changes of the disease are produced by epsilon toxin, a major exotoxin produced by the types B and D of C. perfringens [1]. Epsilon toxin is produced in the intestine of sheep in the form of an inactive prototoxin, which becomes active by the action of intestinal trypsin or a metalloproteinase produced by C. perfringens itself [2]. The active toxin is then absorbed into the general circulation and exerts its effects on several organs, principally lungs and brain [3,4].
Corresponding author. Tel.: +909 383 4287; fax: +909 884 5980.
E-mail address: [email protected] (F.A. Uzal). 1075-9964/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2006.05.001
In a suspected case of enterotoxemia, the mere isolation of C. perfringens type D from the intestinal content of sheep is of little diagnostic significance since this microorganism can be part of the normal intestinal flora of sheep and other animal species [5]. Histological examination of the brain for changes (i.e. perivascular proteinaceous edema and/or focal symmetrical degeneration and necrosis) can be of diagnostic importance for C. perfringens enterotoxemia, despite some drawbacks that arise from cases with absence of brain lesions [6] and the need for formalin fixed specimens for examination. Currently, the most widely used method to confirm the diagnosis of enterotoxemia in sheep is the detection of epsilon prototoxin or toxin in the intestinal content of dead animals [7]. In addition to intestinal content, other body fluids (i.e. abdominal, pleural and/or pericardial fluid) are frequently submitted to diagnostic laboratories for epsilon
ARTICLE IN PRESS J.E. Layana et al. / Anaerobe 12 (2006) 204–206
toxin detection when enterotoxemia is suspected, and it has been suggested that any of these fluids can be used for epsilon toxin detection [8,9]. However, to the best of the authors’ knowledge, in cases of enterotoxemia of sheep, no information is available in the literature regarding the presence of epsilon toxin in body fluids other than intestinal contents. In addition, there is no information available about the suitability of contents from different intestinal segments for the diagnosis of sheep enterotoxemia. This study was designed to examine the diagnostic efficacy of different body fluids for detection of epsilon toxin in experimental cases of sheep enterotoxemia by C. perfringens type D. 2. Materials and methods Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 21, 6-month-old Merino lambs from a previous experiment [6], were used for this study. These animals had been weaned at the age of 2 months, reared on natural pastures and had not been vaccinated against clostridial diseases. A week before the experiment they were housed in pens with alfalfa hay and water at libitum. Fifteen of these animals had been challenged with an experimental model of enterotoxemia, as described elsewhere [6]. Briefly, the lambs were subjected to a laparotomy and inoculated intraduodenally with 200 ml of a 20% solution of corn flour in 0.85% saline into the abomasum. Then, 300 ml of whole cultures of C. perfringens type D (enterotoxin and beta2 toxin negative) containing between 200 and 800 mouse lethal doses fifty per ml (MLD50/ml) of epsilon toxin were inoculated into the duodenum. All the animals showed nervous and/or respiratory signs within 26 h of inoculation (range 2–26 h) and they were euthanized as soon as they developed severe clinical signs. The other six lambs (controls) were subjected to the same experimental conditions and treatment with the exception that they did not receive whole cultures intraduodenally. These animals did not show any clinical alteration and they were euthanized 26 h after the surgical procedures. Samples were collected immediately after death from all the animals and stored frozen at 20 1C until processed within 2 months of collection. The detection of epsilon toxin was performed with a commercial capture ELISA, purchased from Bio-X (Brussels, Belgium) following the instructions of the manufacturer. This test had previously been validated for detection of epsilon toxin in several body fluids and compared with the diagnostic efficacy of other three techniques [10], being found to have the highest sensitivity (amongst the techniques compared) to detect epsilon toxin in all fluids examined. The test used 96-well plates sensitized by specific monoclonal antibodies for epsilon toxin. The samples were added to the wells and the plates were incubated for 60 min at room temperature, washed and incubated 60 min with a
205
Table 1 Results of a capture ELISA used for detection of Clostridium perfringens type D epsilon toxin in different body fluids of 15 sheep with enterotoxemia Animal #
Duod
Ileum
Colon
PF
AF
AH
Urine
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
P P P P P P nd P P N P N N N N
nd N P nd P P P P P P P P P P P
nd N P P P N N N P P N P P P N
N N N N P N N N N N N N N N N
N N N N N N N N N N N N N N N
N N P N N N N N N N N N N N N
N nd N N N N N N N N N N N N nd
P, positive; Duod, duodenal content PF, pericardial fluid; N, negative Ileum, ileal content; AF, abdominal fluid; nd, not done Colon, colonic content; AH, aqueous humor.
peroxidase-labelled anti-epsilon toxin polyclonal antibody. Plates were washed again, and a solution of chromogen substrate (hydrogen peroxide and tetramethyl bendizine) was added. The enzymatic reaction was stopped by acidification with phosphoric acid. The optical densities were read using an ELISA reader with a 450 nm filter. Semi-purified epsilon toxin was used in positive control wells, while the toxin was replaced by buffer in negative control wells. Results were calculated according to the manufacturer’s instructions. 3. Results The individual ELISA results of the samples from the 15 animals with experimental enterotoxemia are shown in Table 1. Epsilon toxin was detected in 92%, 64% and 57% of the samples of ileal, duodenal and colon contents, respectively, and in 7% each of the samples of pericardial fluid and aqueous humor. No abdominal fluid or urine sample gave positive result for epsilon toxin. No epsilon toxin was detected in any of the samples from the 6 clinically healthy animals (controls). 4. Discussion In the animals with experimental enterotoxemia, the number of positive results using intestinal content was much higher than with any other body fluid. Out of all positive samples, epsilon toxin was most frequently detected in ileal and duodenal contents. The capture ELISA used in this study was selected because in a previous experiment [10] comparing the diagnostic efficiency of this test with an indirect ELISA
ARTICLE IN PRESS 206
J.E. Layana et al. / Anaerobe 12 (2006) 204–206
technique, mouse neutralization test and counterimmunoelectrophoresis, the capture ELISA showed the highest sensitivity to detect epsilon toxin in different body fluids artificially spiked with that toxin [10]. Possible causes for epsilon toxin not being detected in most samples of body fluids other than intestinal content of animals with experimental enterotoxemia are the relative limited sensitivity of the ELISA technique used and the absence of toxin in a given fluid when the samples were collected. Although the capture ELISA described was chosen because of its relative higher sensitivity when compared with other techniques, still the epsilon toxin detection limit of 0.075 MLD50/ml of intestinal content of this technique [10] could have been above the level of toxin present in the samples evaluated. In this study, epsilon toxin was not detected in any of the intestinal contents from the clinically healthy sheep. Information about the presence of epsilon toxin in intestinal content of healthy sheep is scant and contradictory [10,11]. The results of one study [11] indicate that up to 250 MLD50 of this toxin can be found in the intestinal content of healthy sheep, while another study using a more sensitive diagnostic technique, did not find epsilon toxin in intestinal content of healthy sheep and goats [10]. The differences in results of these studies could be due to several factors, including variations in environmental conditions and in breed, age, and nutritional status of the animals used. More research is needed to clarify this issue. This study was performed using sheep with experimental enterotoxemia. It is possible that in natural cases of the disease the distribution and concentration of epsilon toxin in body fluids may differ from the results presented here. The experiments previously reported [6] from which the samples for the current study originated, were performed using whole cultures and provided a reasonable model of enterotoxemia in sheep, with clinical signs and gross and microscopic findings very similar to those observed in natural cases of the disease. Samples from animals that presented clinical signs before 2 h of inoculation were not included in this study because it was considered likely that they were intoxicated by the toxin present in the inoculum. Furthermore, intestinal samples collected from animals that died shortly after inoculation likely contained epsilon toxin from the inoculum, which would have increased the positivity level in those samples and biased the results against more-distant samples (to which toxin would not have had time to diffuse). Further research is needed to study distribution and detection of epsilon toxin in different body fluids in natural cases of enterotoxemia. Samples were stored at 20 1C before processing. Previous studies have shown that epsilon toxin retains its
full activity in intestinal content for almost a year when stored at 4 1C [12]. Also, in previous experiments (data not shown) we observed that epsilon toxin remains fully active for several years in samples of intestinal content and other body fluids stored at 20 1C. The results presented here show that while the presence of epsilon toxin in intestinal contents might be considered an indicator of enterotoxemia, the absence of this toxin in samples other than small intestinal content cannot be used to rule out a diagnosis of enterotoxemia. Also, the results of this study indicate that small intestinal content (preferably ileum) should be the sample of choice for epsilon toxin detection in suspected cases of enterotoxemia in sheep. Acknowledgements We thank Drs. G. Songer and R. Walker for their valuable suggestions, Ms. S. Fitisemanu for typing this manuscript and Ms. S.J. Uzal for reviewing it. This study was partly funded by Fondo Nacional para la Ciencia y la Tecnologı´ a (PICT-01-3591), Argentina. References [1] Niilo L. Clostridium perfringens in animal disease: a review of current knowledge. Can Vet J 1980;21:141–8. [2] Minami J, Katayama S, Matsuyita O, Okabe A. Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N and C terminal peptides. Microbiol Immunol 1997;41:527–35. [3] Barker IK, Van Dreumel AA, Palmer N. The alimentary system. In: Jubb KVF, Kennedy PC, Palmer N, editors. Pathology of domestic animals. California: Academic Press; 1993. p. 241–4. [4] Nagahama M, Sakurai J. Distribution of labeled Clostridium perfringens epsilon toxin in mice. Toxicon 1991;29:211–7. [5] Uzal FA. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. Anaerobe 2004;2:135–43. [6] Uzal FA, Kelly WR, Morris WE, Bermudez J, Baison M. The pathology of per acute experimental Clostridium perfringens type D enterotoxemia in sheep. J Vet Diagn Invest 2004;6:403–11. [7] Sterne M, Batty I. Pathogenic clostridia. London: Butterworths; 1975. p. 96–145. [8] Carter GR. Diagnostic procedures in veterinary microbiology. Illinois: Thomas; 1984. p. 229–51. [9] Timoney JF, Gillespie JH, Scott FW, Barlough JE. Hagan and Bruner’s microbiology and infectious diseases of domestic animals. New York: Comstock Publishing Associates; 1988. p. 207–12. [10] Uzal FA, Kelly WR, Thomas R, Hornitzky M, Galea F. Comparison of four techniques for the detection of Clostridium perfringens type D epsilon toxin in intestinal contents and other body fluids of sheep and goats. J Vet Diagn Invest 2003;15:94–9. [11] Bullen JJ. The influence of the diet on the pathogenesis of enterotoxaemia of sheep. Bull Internatl Off Epiz 1963;59:1453–61. [12] Niilo L. Bovine enterotoxemia. III. Factors affecting the stability of the toxins of Clostridium perfringens types A, C and D. Can Vet J 1965;6:38–42.
Anaerobe 12 (2006) 204–206 www.elsevier.com/locate/anaerobe
Short communication—Veterinary anaerobes and diseases
Evaluation of different fluids for detection of Clostridium perfringens type D epsilon toxin in sheep with experimental enterotoxemia Jorge E. Layanaa, Mariano E. Fernandez Miyakawab, Francisco A. Uzalb, a
The National Institute of Agricultural Technology. CC 277, (8400) Bariloche, Argentina California Animal Health and Food Safety Laboratory, San Bernardino Branch, School of Veterinary Medicine, University of California, Davis. 105 W Central Ave, San Bernardino, CA 92408, USA
b
Received 31 March 2006; received in revised form 18 May 2006; accepted 18 May 2006 Available online 20 July 2006
Abstract Enterotoxemia caused by Clostridium perfringens type D is a highly lethal disease of sheep, goats and other ruminants. The diagnosis of this condition is usually confirmed by detection of epsilon toxin, a major exotoxin produced by C. perfringens types B and D, in the intestinal content of affected animals. It has been suggested that other body fluids can also be used for detection of epsilon toxin. This study was performed to evaluate the usefulness of intestinal content versus other body fluids in detecting epsilon toxin in cases of sheep enterotoxemia. Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 15 sheep with experimentally induced enterotoxemia, were analysed for epsilon toxin using a capture ELISA. Epsilon toxin was detected in 92% of the samples of ileal content, 64% of the samples of duodenal content, 57% of the samples of colon content and in 7% of the samples of pericardial fluid and aqueous humor. No epsilon toxin was found in samples of abdominal fluid or urine from the animals with enterotoxemia or in any samples from six clinically healthy sheep used as negative controls. The results of this study indicate that with the diagnostic capture ELISA used, intestinal content (preferably ileum) should be used for C. perfringens type D epsilon toxin detection in suspected cases of sheep enterotoxemia. r 2006 Elsevier Ltd. All rights reserved. Keywords: Body fluids; Clostridium perfringens; Enterotoxemia; Epsilon toxin; Intestinal content; Sheep
1. Introduction Enterotoxemia is a highly lethal disease of sheep, goats and other animal species, produced by Clostridium perfringens type D [1]. In sheep, the main clinical signs and pathological changes of the disease are produced by epsilon toxin, a major exotoxin produced by the types B and D of C. perfringens [1]. Epsilon toxin is produced in the intestine of sheep in the form of an inactive prototoxin, which becomes active by the action of intestinal trypsin or a metalloproteinase produced by C. perfringens itself [2]. The active toxin is then absorbed into the general circulation and exerts its effects on several organs, principally lungs and brain [3,4].
Corresponding author. Tel.: +909 383 4287; fax: +909 884 5980.
E-mail address: [email protected] (F.A. Uzal). 1075-9964/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2006.05.001
In a suspected case of enterotoxemia, the mere isolation of C. perfringens type D from the intestinal content of sheep is of little diagnostic significance since this microorganism can be part of the normal intestinal flora of sheep and other animal species [5]. Histological examination of the brain for changes (i.e. perivascular proteinaceous edema and/or focal symmetrical degeneration and necrosis) can be of diagnostic importance for C. perfringens enterotoxemia, despite some drawbacks that arise from cases with absence of brain lesions [6] and the need for formalin fixed specimens for examination. Currently, the most widely used method to confirm the diagnosis of enterotoxemia in sheep is the detection of epsilon prototoxin or toxin in the intestinal content of dead animals [7]. In addition to intestinal content, other body fluids (i.e. abdominal, pleural and/or pericardial fluid) are frequently submitted to diagnostic laboratories for epsilon
ARTICLE IN PRESS J.E. Layana et al. / Anaerobe 12 (2006) 204–206
toxin detection when enterotoxemia is suspected, and it has been suggested that any of these fluids can be used for epsilon toxin detection [8,9]. However, to the best of the authors’ knowledge, in cases of enterotoxemia of sheep, no information is available in the literature regarding the presence of epsilon toxin in body fluids other than intestinal contents. In addition, there is no information available about the suitability of contents from different intestinal segments for the diagnosis of sheep enterotoxemia. This study was designed to examine the diagnostic efficacy of different body fluids for detection of epsilon toxin in experimental cases of sheep enterotoxemia by C. perfringens type D. 2. Materials and methods Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 21, 6-month-old Merino lambs from a previous experiment [6], were used for this study. These animals had been weaned at the age of 2 months, reared on natural pastures and had not been vaccinated against clostridial diseases. A week before the experiment they were housed in pens with alfalfa hay and water at libitum. Fifteen of these animals had been challenged with an experimental model of enterotoxemia, as described elsewhere [6]. Briefly, the lambs were subjected to a laparotomy and inoculated intraduodenally with 200 ml of a 20% solution of corn flour in 0.85% saline into the abomasum. Then, 300 ml of whole cultures of C. perfringens type D (enterotoxin and beta2 toxin negative) containing between 200 and 800 mouse lethal doses fifty per ml (MLD50/ml) of epsilon toxin were inoculated into the duodenum. All the animals showed nervous and/or respiratory signs within 26 h of inoculation (range 2–26 h) and they were euthanized as soon as they developed severe clinical signs. The other six lambs (controls) were subjected to the same experimental conditions and treatment with the exception that they did not receive whole cultures intraduodenally. These animals did not show any clinical alteration and they were euthanized 26 h after the surgical procedures. Samples were collected immediately after death from all the animals and stored frozen at 20 1C until processed within 2 months of collection. The detection of epsilon toxin was performed with a commercial capture ELISA, purchased from Bio-X (Brussels, Belgium) following the instructions of the manufacturer. This test had previously been validated for detection of epsilon toxin in several body fluids and compared with the diagnostic efficacy of other three techniques [10], being found to have the highest sensitivity (amongst the techniques compared) to detect epsilon toxin in all fluids examined. The test used 96-well plates sensitized by specific monoclonal antibodies for epsilon toxin. The samples were added to the wells and the plates were incubated for 60 min at room temperature, washed and incubated 60 min with a
205
Table 1 Results of a capture ELISA used for detection of Clostridium perfringens type D epsilon toxin in different body fluids of 15 sheep with enterotoxemia Animal #
Duod
Ileum
Colon
PF
AF
AH
Urine
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
P P P P P P nd P P N P N N N N
nd N P nd P P P P P P P P P P P
nd N P P P N N N P P N P P P N
N N N N P N N N N N N N N N N
N N N N N N N N N N N N N N N
N N P N N N N N N N N N N N N
N nd N N N N N N N N N N N N nd
P, positive; Duod, duodenal content PF, pericardial fluid; N, negative Ileum, ileal content; AF, abdominal fluid; nd, not done Colon, colonic content; AH, aqueous humor.
peroxidase-labelled anti-epsilon toxin polyclonal antibody. Plates were washed again, and a solution of chromogen substrate (hydrogen peroxide and tetramethyl bendizine) was added. The enzymatic reaction was stopped by acidification with phosphoric acid. The optical densities were read using an ELISA reader with a 450 nm filter. Semi-purified epsilon toxin was used in positive control wells, while the toxin was replaced by buffer in negative control wells. Results were calculated according to the manufacturer’s instructions. 3. Results The individual ELISA results of the samples from the 15 animals with experimental enterotoxemia are shown in Table 1. Epsilon toxin was detected in 92%, 64% and 57% of the samples of ileal, duodenal and colon contents, respectively, and in 7% each of the samples of pericardial fluid and aqueous humor. No abdominal fluid or urine sample gave positive result for epsilon toxin. No epsilon toxin was detected in any of the samples from the 6 clinically healthy animals (controls). 4. Discussion In the animals with experimental enterotoxemia, the number of positive results using intestinal content was much higher than with any other body fluid. Out of all positive samples, epsilon toxin was most frequently detected in ileal and duodenal contents. The capture ELISA used in this study was selected because in a previous experiment [10] comparing the diagnostic efficiency of this test with an indirect ELISA
ARTICLE IN PRESS 206
J.E. Layana et al. / Anaerobe 12 (2006) 204–206
technique, mouse neutralization test and counterimmunoelectrophoresis, the capture ELISA showed the highest sensitivity to detect epsilon toxin in different body fluids artificially spiked with that toxin [10]. Possible causes for epsilon toxin not being detected in most samples of body fluids other than intestinal content of animals with experimental enterotoxemia are the relative limited sensitivity of the ELISA technique used and the absence of toxin in a given fluid when the samples were collected. Although the capture ELISA described was chosen because of its relative higher sensitivity when compared with other techniques, still the epsilon toxin detection limit of 0.075 MLD50/ml of intestinal content of this technique [10] could have been above the level of toxin present in the samples evaluated. In this study, epsilon toxin was not detected in any of the intestinal contents from the clinically healthy sheep. Information about the presence of epsilon toxin in intestinal content of healthy sheep is scant and contradictory [10,11]. The results of one study [11] indicate that up to 250 MLD50 of this toxin can be found in the intestinal content of healthy sheep, while another study using a more sensitive diagnostic technique, did not find epsilon toxin in intestinal content of healthy sheep and goats [10]. The differences in results of these studies could be due to several factors, including variations in environmental conditions and in breed, age, and nutritional status of the animals used. More research is needed to clarify this issue. This study was performed using sheep with experimental enterotoxemia. It is possible that in natural cases of the disease the distribution and concentration of epsilon toxin in body fluids may differ from the results presented here. The experiments previously reported [6] from which the samples for the current study originated, were performed using whole cultures and provided a reasonable model of enterotoxemia in sheep, with clinical signs and gross and microscopic findings very similar to those observed in natural cases of the disease. Samples from animals that presented clinical signs before 2 h of inoculation were not included in this study because it was considered likely that they were intoxicated by the toxin present in the inoculum. Furthermore, intestinal samples collected from animals that died shortly after inoculation likely contained epsilon toxin from the inoculum, which would have increased the positivity level in those samples and biased the results against more-distant samples (to which toxin would not have had time to diffuse). Further research is needed to study distribution and detection of epsilon toxin in different body fluids in natural cases of enterotoxemia. Samples were stored at 20 1C before processing. Previous studies have shown that epsilon toxin retains its
full activity in intestinal content for almost a year when stored at 4 1C [12]. Also, in previous experiments (data not shown) we observed that epsilon toxin remains fully active for several years in samples of intestinal content and other body fluids stored at 20 1C. The results presented here show that while the presence of epsilon toxin in intestinal contents might be considered an indicator of enterotoxemia, the absence of this toxin in samples other than small intestinal content cannot be used to rule out a diagnosis of enterotoxemia. Also, the results of this study indicate that small intestinal content (preferably ileum) should be the sample of choice for epsilon toxin detection in suspected cases of enterotoxemia in sheep. Acknowledgements We thank Drs. G. Songer and R. Walker for their valuable suggestions, Ms. S. Fitisemanu for typing this manuscript and Ms. S.J. Uzal for reviewing it. This study was partly funded by Fondo Nacional para la Ciencia y la Tecnologı´ a (PICT-01-3591), Argentina. References [1] Niilo L. Clostridium perfringens in animal disease: a review of current knowledge. Can Vet J 1980;21:141–8. [2] Minami J, Katayama S, Matsuyita O, Okabe A. Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N and C terminal peptides. Microbiol Immunol 1997;41:527–35. [3] Barker IK, Van Dreumel AA, Palmer N. The alimentary system. In: Jubb KVF, Kennedy PC, Palmer N, editors. Pathology of domestic animals. California: Academic Press; 1993. p. 241–4. [4] Nagahama M, Sakurai J. Distribution of labeled Clostridium perfringens epsilon toxin in mice. Toxicon 1991;29:211–7. [5] Uzal FA. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. Anaerobe 2004;2:135–43. [6] Uzal FA, Kelly WR, Morris WE, Bermudez J, Baison M. The pathology of per acute experimental Clostridium perfringens type D enterotoxemia in sheep. J Vet Diagn Invest 2004;6:403–11. [7] Sterne M, Batty I. Pathogenic clostridia. London: Butterworths; 1975. p. 96–145. [8] Carter GR. Diagnostic procedures in veterinary microbiology. Illinois: Thomas; 1984. p. 229–51. [9] Timoney JF, Gillespie JH, Scott FW, Barlough JE. Hagan and Bruner’s microbiology and infectious diseases of domestic animals. New York: Comstock Publishing Associates; 1988. p. 207–12. [10] Uzal FA, Kelly WR, Thomas R, Hornitzky M, Galea F. Comparison of four techniques for the detection of Clostridium perfringens type D epsilon toxin in intestinal contents and other body fluids of sheep and goats. J Vet Diagn Invest 2003;15:94–9. [11] Bullen JJ. The influence of the diet on the pathogenesis of enterotoxaemia of sheep. Bull Internatl Off Epiz 1963;59:1453–61. [12] Niilo L. Bovine enterotoxemia. III. Factors affecting the stability of the toxins of Clostridium perfringens types A, C and D. Can Vet J 1965;6:38–42.