DIAGN MICROBIOLINFECTDIS 1991;14:11-15
11
Identification of Toxigenic Clostridium botulinum Type E by Enzyme Immunoassay Manoucher Dezfulian and John G. Bartlett
A sensitive double-sandwich enzyme immunoassay for identification of toxigenic Clostridium botulinum type E was developed with a type-E-specific antitoxin. The antitoxin was produced in a rabbit, following inoculation of minute quantities of type-E toxoid. The toxoid was prepared from a commercially available toxin by using a detoxification method. Cross-reactivity of the antitoxin with C. botu-
linum types A and B was avoided by absorption of the serum with alum-precipitated toxoids A and B. The immunoassay proved to be specific for C. botulinum type E because it did not react with culture filtrates of various Clostridium species, including nontoxigenenic "E-like" organisms and Clostridium sporogenes.
INTRODUCTION
MATERIALS A N D METHODS
We recently developed an enzyme-linked immunosorbent assay (ELISA) for detection of botulinum toxin, and identification of Clostridium botulinum types A and B, in clinical specimens (Dezfulian and Bartlett, 1984 and 1985a and b; Dezfulian et al., 1984). This assay has provided a useful alternative to mouse bioassay (MBA) in cases of infant and food-borne botulism. One limitation of the ELISA, however, is its lack of capability for detecting C. botulinum type E, which, together with types A and B, is the major cause of food-borne botulism (Smith, 1977). In this communication, we describe a simple immunological procedure for identification of toxigenic C. botulinum type E.
Animal A male New Zealand white rabbit weighing - 3 kg was used. Fur was shaved from the back of the rabbit before immunization.
Toxoids Alum-precipitated toxoids, types A and B, were kindly provided by Charles Hatheway of the Centers for Disease Control (Atlanta, GA). Clostridium botulinum type-E toxoid was prepared from a commercially available toxin (Sigma Chemical Co., St. Louis, MO) by detoxification with 0.4% formaldehyde.
Method of Immunization
From the Department of MedicalLaboratorySciences (M.D.), Florida InternationalUniversity, Miami, Florida; and InfectiousDisease Division, Department of Medicine (J.G.B.), Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland. Address reprint requests to: ManoucherDezfulian, Department of Medical LaboratorySciences, Florida InternationalUniversity, Miami, FL 33199. Received February27, 1990; revised and acceptedJune 1, 1990. © 1991 ElsevierScience PublishingCo., Inc. 655 Avenue of the Americas, New York, New York 10010. 0732-8893/91/$3.50
The multiple intradermal inoculation procedure of Vaitukaitis et al. (1971), with some modifications, was used. A stable toxoid-adjuvant emulsion was prepared by mixing equal volumes of the type-E botulinat toxoid and incomplete Freund's adjuvant in a double syringe connected with stainless-steel tubing. A 4-ml emulsion containing 10 ~g toxoid was then injected intradermally into 40 sites on the back of the rabbit. Booster injection consisted of 15 ~g toxoid emulsion in incomplete Freund's adjuvant, which was given in four subcutaneous injections 8 weeks later. The rabbit was bled from the ear veins
12
prior to immunization, 6 and 8 weeks after primary immunization and 2, 4, 5, 7, and 8 weeks after the booster injection. The serum samples were aliquoted in small quantities and maintained at -70°C until used.
Organisms The organisms included 13 strains of C. botulinum types A, B, C, D, E, and F (Table 1), 2 nontoxigenic variants of type B, and 18 strains representing 10 Clostridium species, including nontoxigenic "E-like" organisms and Clostridium sporogenes. All strains originated from the stock collection of the Centers for Disease Control (Dezfulian and Dowell, 1980), our own collection, or from American Type Culture Collection.
Culture Media Media used included chopped meat glucose (CMG) broth (Scott Laboratories, Fiskeville, RI) and egg yolk agar (EYA), which was prepared in our laboratory (Dezfulian and Bartlett, 1985b).
ELISA The assay was performed as described for botulinal type-A toxin (Dezfulian and Bartlett, 1984), except type-A antitoxin was replaced by antitoxin of type E. The specimens tested by the ELISA were the culture filtrates of 4-day-old CMG broth culture of test organisms (Dezfulian and Bartlett, 1984). Samples of 25 ~1 of diluted or undiluted test specimens were added to microtitration wells containing 75 ~1 of phosphate-buffer/Tween/gel diluent (Dezfulian and Bartlett, 1984). The diagnostic limits for positive ELISA readings were determined as described previously (Dezfulian et al., 1984). The limit for C. botulinum type E was equal to 0.05.
Serum Absorptions Equal volumes of alum-precipitated type-A and -B botulinal toxoids were mixed with various dilutions of the rabbit type-E antitoxin. The mixture was kept at 37°C for 60 min and at room temperature for 120 min. Following overnight refrigeration, the absorbed serum was separated from the sediment by centrifugation and kept frozen at - 70°C until used.
RESULTS Selection of Rabbit Serum In a preliminary experiment, the immune response of the rabbit was monitored by comparing the reac-
M. Dezfulian and J.G. Bartlett
tivity of serially collected rabbit serum samples, using the ELISA. On the basis of this experiment, the serum sample derived from the fourth bleeding (drawn 2 weeks after booster injection) was selected for further study. The selected serum sample exhibited very low background in the assay and produced nearly the highest absorbance when tested against type-E culture filtrates (data not shown).
Reactivity of the ELISA The assay was highly reactive with culture filtrates of C. botulinum type E strains (Table 1). Except for types A and B, no cross-reaction was observed with other toxigenic C. botulinum tested (Table 1). Likewise, no cross-reaction was demonstrable with the test samples of nontoxigenic "E-like" organisms or C. sporogenes. The assay was also negative with all other clostridial species tested (Table 1).
Avoidance of Cross-Reaction by Serum Absorption The undiluted type-A and type-B toxoid mixed with equal volume of 1:100 dilution of the selected rabbit serum provided the best results in the absorption test. The ELISA values obtained with the absorbed and nonabsorbed sera vs. C. botulinum (types A, B, and E) and C. sporogenes strains are shown in Table 2. Although a slight reduction in sensitivity of the assay was apparent with the absorbed serum, crossreaction with type-A and -B strains was completely avoided.
DISCUSSION The double-sandwich ELISA for C. botulinum typeE toxin has been previously described by other investigators (Notermans et al., 1979 and 1982). The type-E-specific antitoxin has been produced in rabbits by subcutaneous injections of 0.4 mg of purified and detoxified botulinal neurotoxin (Notermans et al., 1979 and 1982). Purification of a highly lethal and relatively unstable protein is obviously a major undertaking. In this study we utilized a small quantity (equivalent to 25 ~g neurotoxin) of commercially available type-E toxin for antitoxin preparation. Successful immunization with small amounts of immunogen saves a great deal of time and expense. Furthermore, by limiting the amount of immunizing toxin to several micrograms, one can greatly reduce or even eliminate any minor impurities that may accompany the toxin. The cross-reaction of our type-E antitoxin with C. botulinum types A and B may indicate that the type-E toxin preparation used for immunization was
C. botulinum Type-E Assays
TABLE 1.
13
M o u s e L e t h a l D o s e a n d E L I S A A b s o r b a n c e of C u l t u r e F i l t r a t e s of V a r i o u s S t r a i n s of Clostridium botulinum a n d O t h e r Clostridial Species Log10 M i n i mu m Lethal Dose per Milliliter~
Bacterial Strain
ELISA Value +- SD b
C. botulinum type A 1696
4.0
0.129 + 0.021
3.0
0.241 -+ 0.035
ND
0.013
ND
0.023
3.0 3.5 3.5 3.9 4.0 4.0
1.004 1.240 1.158 1.530 1.619 1.570
1.0
0.029
0.0 0.0 0.0
- 0.020 - 0.002 - 0.018
0.0 0.0
- 0.005 - 0.006
0.0 0.0 ND
- 0.011 0.018 0.000
C. botulinum type B JHIB1
C. botulinum type C ATCC 17850
C. botulinum type D ATCC 27517
C. botulinum type E ATCC 9564 ATCC 17786 ATCC 17852 ATCC 17854 CDC 2112E E-43 C. botulinum type F F8 E-like organisms ELK-6D ELK-93 ELK-S9 Nontoxigenic variants of C. botulinum type B B27 B30
-+ 0.178 -+ 0.055 + 0.078 +_ 0.091 + 0.045 + 0.071
C. sporogenes 17616 A1068 Other clostridial species c
aND, not determined. bNet absorbance (three replications) at 405 nm _+ SD: values/> 0.05 were considered positive. cSpecies included one strain each of Clostridium haemolyticum (6021), Clostridium paraputrificum (14861), Clostridium septicum (A1252), Clostridium sordellii (19739), and Clostridium novyi A (1872); two strains each of Clostridium histoliticum (A1096 and 1097) and Clostridium subterminalis (18041 and 18183); and three strains of Clostridium novyi B (A741, 6387, and 6388).
T A B L E 2.
A v o i d i n g C r o s s - R e a c t i o n b y M e a n s of S e r u m A b s o r p t i o n
Bacterial Strains
Toxigenic Types
Absorbed Serum
Nonabsorbed Serum
ATCC 17786 2112-E 207-A A28 JHIB1 $7 (C. sporogenes)
E E A A B
0.582 a 0.951 0.004 0.006 0.007 0.010
0.798 1.141 0.141 0.345 0.077 0.008
aNet absorbance (duplicate samples) at 405 nm.
14
not free from botulinal nontoxic components. The toxic and nontoxic components constitute the native or progenitor botulinal toxin, which is normally released by the bacterial cells into the culture medium (Sakaguchi et al., 1981). Although the toxic components (neurotoxin or derivative toxin) of types A, B, and E are antigenically distinct, the nontoxic components are immunologically related (Sakaguchi et al., 1974 and 1981). The antibodies directed against type-E nontoxic components therefore appear to be responsible for cross-reaction of type-E antitoxin with C. botulinum types A and B in our study. It is of note, however, that cross-reactivity does not extend to other toxigenic C. botulinum or other clostridial species used in this investigation. Although a strain of C. botulinum type F was shown to be nonreactive in our ELISA for type-E organisms, some of the more toxigenic strains may show reactivity. This assumption is based on close phenotypic relationship between C. botulinum type-F (proteolytic) and type-A and -B (proteolytic) organisms (Smith, 1977). Several investigators have advocated the use of purified neurotoxin for immunological and diagnostic studies of C. botulinum and botulinal toxin (Sugiyama, 1980; Notermans et al., 1982; DasGupta, 1983). Any immunological assay involving quantitation of or monitoring the activity of botulinal toxin requires utilization of purified neurotoxin; however, in a qualitative diagnostic assay, use of a more stable and readily available progenitor toxin (Sakaguchi et al., 1981) for production of antitoxin seems to be justified. The antiserum to progenitor toxin contains type-specific antibodies directed against neurotoxin and cross-reacting botulinal antibodies directed against botulinal-specific nontoxic components. As
M. Dezfulian and J.G. Bartlett
we have shown previously (Dezfulian and Bartlett, 1984), presence of the latter antibodies actually increases the sensitivity of the immunoassay. Our preliminary data also suggest that this may be true for the ELISA involving C. botulinum type E. In this study the cross-reacting antibodies were conveniently removed from botulinal type-E antitoxin by absorption of the serum with alum-precipitated type-A and -B toxoids. Our previously prepared type-A or -B botulinal antitoxins, which do not react with type E toxin, could also be used in parallel with type-E antitoxin, for distinction of C. botulinum type E from other types. Based on this and other studies (Notermans et al., 1979 and 1982; Dezfulian and Bartlett, 1984 and 1985b; Dezfulian et al., 1984), ELISA provides a useful alternative test to conventional MBA. Advantages are that this assay can be carried out in most diagnostic laboratories, it can readily be adapted to large-scale serological screening of food and clinical specimens, and results are available considerably sooner. Type-E botulinal toxin is a major cause of foodborne botulism in some areas (Smith, 1977). A recent report by McCroskey et al. (1986) describes an unusual case of infant botulism caused by a strain of C. butyricum that produces botulinal type-E toxin. This finding makes further development of ELISA technology for direct analysis of specimens highly desirable.
This work was supported by a research grant from the Center for Alternatives to Animal Testing, the Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD.
REFERENCES DasGupta BR (1983) Microbial food toxicants: Clostridium botulinum toxins. In CRC Handbook of Foodborne Diseases of Biological Origin. Ed, M Recheigl Jr. Boca Raton: CRC Press, pp 25-55. Dezfulian M, Dowell VR Jr (1980) Cultural and physiological characteristics and antimicrobial susceptibility of Clostridium botulinum isolates from foodborne and infant botulism cases. J Clin Microbiol 11:604-609. Dezfulian M, Bartlett JG (1984) Detection of Clostridium botulinum type A toxin by enzyme-linked immunosorbent assay with antibodies produced in immunologically tolerant animals. J Clin Microbiol 19:645648. Dezfulian M, Hatheway CL, Yolken RH, Bartlett JG (1984) Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type A and type B toxins in stool samples of infants with botulism. J Clin Microbiol 10:379383. Dezfulian M, Bartlett JG (1985a) Detection of Clostridium
botulinum type B toxin in the presence of a lethal substance interfering with toxin neutralization. Diagn Microbiol Infect Dis 3:113-118.
Dezfulian M, Bartlett JG (1985b) Selective isolation and rapid identification of Clostridium botulinum types A and B by toxin detection. J Clin Microbiol 21:231233. McCroskey LM, Hatheway CL, Penicia L, Pasolini B, Aureli P (1986) Characterization of an organism that produces type E botulinal toxin but which resembles Clostridium butyricum from the feces of an infant with type E botulism. J Clin Microbiol 23:201-208. Notermans S, Fufrenne J, Kozaki S (1979) Enzyme-linked immunoabsorbent assay for detection of Clostridium botulinum type E toxin. Appl Environ Microbiol 37:11731175. Notermans S, Hagenaars AM, Kozaki S (1982) The enzyme-linked immunoabsorbent assay (ELISA) for the detection and determination of Clostridium
C. botulinum Type-E Assays
botulinum toxins A, B, and E. Methods Enzymol 84:223239. Sakaguchi G, Sakaguchi S, Kozaki S, Sugii S, Ohishi I (1974) Cross-reaction in reversed passive hemagglutination between Clostridium botulinum type A and B toxins and its avoidance by the use of antitoxin component immunoglobulin isolated by affinity chromatography. Jpn J Med Sci Biol 27:161-172. Sakaguchi G, Ohishi I, Kozaki S (1981) Purification and oral toxicities of Clostridium botulinum progenitor toxins,
15
In Biomedical Aspects of Botulism. Ed, GE Lewis. New York: Academic Press, pp 21-34. Smith LD (1977) Botulism: the Organism, Its Toxins, the Disease. Springfield, IL: Charles C Thomas. Sugiyama H (1980) Clostridium botulinum neurotoxin. Microbiol Rev 44:419-448. Vaitukaitis J, Robbins JB, Nieschlag E, Ross GT (1971) A method for producing specific antisera with small doses of immunogen. J Clin Endocrinol 33:988-991.
11
Identification of Toxigenic Clostridium botulinum Type E by Enzyme Immunoassay Manoucher Dezfulian and John G. Bartlett
A sensitive double-sandwich enzyme immunoassay for identification of toxigenic Clostridium botulinum type E was developed with a type-E-specific antitoxin. The antitoxin was produced in a rabbit, following inoculation of minute quantities of type-E toxoid. The toxoid was prepared from a commercially available toxin by using a detoxification method. Cross-reactivity of the antitoxin with C. botu-
linum types A and B was avoided by absorption of the serum with alum-precipitated toxoids A and B. The immunoassay proved to be specific for C. botulinum type E because it did not react with culture filtrates of various Clostridium species, including nontoxigenenic "E-like" organisms and Clostridium sporogenes.
INTRODUCTION
MATERIALS A N D METHODS
We recently developed an enzyme-linked immunosorbent assay (ELISA) for detection of botulinum toxin, and identification of Clostridium botulinum types A and B, in clinical specimens (Dezfulian and Bartlett, 1984 and 1985a and b; Dezfulian et al., 1984). This assay has provided a useful alternative to mouse bioassay (MBA) in cases of infant and food-borne botulism. One limitation of the ELISA, however, is its lack of capability for detecting C. botulinum type E, which, together with types A and B, is the major cause of food-borne botulism (Smith, 1977). In this communication, we describe a simple immunological procedure for identification of toxigenic C. botulinum type E.
Animal A male New Zealand white rabbit weighing - 3 kg was used. Fur was shaved from the back of the rabbit before immunization.
Toxoids Alum-precipitated toxoids, types A and B, were kindly provided by Charles Hatheway of the Centers for Disease Control (Atlanta, GA). Clostridium botulinum type-E toxoid was prepared from a commercially available toxin (Sigma Chemical Co., St. Louis, MO) by detoxification with 0.4% formaldehyde.
Method of Immunization
From the Department of MedicalLaboratorySciences (M.D.), Florida InternationalUniversity, Miami, Florida; and InfectiousDisease Division, Department of Medicine (J.G.B.), Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland. Address reprint requests to: ManoucherDezfulian, Department of Medical LaboratorySciences, Florida InternationalUniversity, Miami, FL 33199. Received February27, 1990; revised and acceptedJune 1, 1990. © 1991 ElsevierScience PublishingCo., Inc. 655 Avenue of the Americas, New York, New York 10010. 0732-8893/91/$3.50
The multiple intradermal inoculation procedure of Vaitukaitis et al. (1971), with some modifications, was used. A stable toxoid-adjuvant emulsion was prepared by mixing equal volumes of the type-E botulinat toxoid and incomplete Freund's adjuvant in a double syringe connected with stainless-steel tubing. A 4-ml emulsion containing 10 ~g toxoid was then injected intradermally into 40 sites on the back of the rabbit. Booster injection consisted of 15 ~g toxoid emulsion in incomplete Freund's adjuvant, which was given in four subcutaneous injections 8 weeks later. The rabbit was bled from the ear veins
12
prior to immunization, 6 and 8 weeks after primary immunization and 2, 4, 5, 7, and 8 weeks after the booster injection. The serum samples were aliquoted in small quantities and maintained at -70°C until used.
Organisms The organisms included 13 strains of C. botulinum types A, B, C, D, E, and F (Table 1), 2 nontoxigenic variants of type B, and 18 strains representing 10 Clostridium species, including nontoxigenic "E-like" organisms and Clostridium sporogenes. All strains originated from the stock collection of the Centers for Disease Control (Dezfulian and Dowell, 1980), our own collection, or from American Type Culture Collection.
Culture Media Media used included chopped meat glucose (CMG) broth (Scott Laboratories, Fiskeville, RI) and egg yolk agar (EYA), which was prepared in our laboratory (Dezfulian and Bartlett, 1985b).
ELISA The assay was performed as described for botulinal type-A toxin (Dezfulian and Bartlett, 1984), except type-A antitoxin was replaced by antitoxin of type E. The specimens tested by the ELISA were the culture filtrates of 4-day-old CMG broth culture of test organisms (Dezfulian and Bartlett, 1984). Samples of 25 ~1 of diluted or undiluted test specimens were added to microtitration wells containing 75 ~1 of phosphate-buffer/Tween/gel diluent (Dezfulian and Bartlett, 1984). The diagnostic limits for positive ELISA readings were determined as described previously (Dezfulian et al., 1984). The limit for C. botulinum type E was equal to 0.05.
Serum Absorptions Equal volumes of alum-precipitated type-A and -B botulinal toxoids were mixed with various dilutions of the rabbit type-E antitoxin. The mixture was kept at 37°C for 60 min and at room temperature for 120 min. Following overnight refrigeration, the absorbed serum was separated from the sediment by centrifugation and kept frozen at - 70°C until used.
RESULTS Selection of Rabbit Serum In a preliminary experiment, the immune response of the rabbit was monitored by comparing the reac-
M. Dezfulian and J.G. Bartlett
tivity of serially collected rabbit serum samples, using the ELISA. On the basis of this experiment, the serum sample derived from the fourth bleeding (drawn 2 weeks after booster injection) was selected for further study. The selected serum sample exhibited very low background in the assay and produced nearly the highest absorbance when tested against type-E culture filtrates (data not shown).
Reactivity of the ELISA The assay was highly reactive with culture filtrates of C. botulinum type E strains (Table 1). Except for types A and B, no cross-reaction was observed with other toxigenic C. botulinum tested (Table 1). Likewise, no cross-reaction was demonstrable with the test samples of nontoxigenic "E-like" organisms or C. sporogenes. The assay was also negative with all other clostridial species tested (Table 1).
Avoidance of Cross-Reaction by Serum Absorption The undiluted type-A and type-B toxoid mixed with equal volume of 1:100 dilution of the selected rabbit serum provided the best results in the absorption test. The ELISA values obtained with the absorbed and nonabsorbed sera vs. C. botulinum (types A, B, and E) and C. sporogenes strains are shown in Table 2. Although a slight reduction in sensitivity of the assay was apparent with the absorbed serum, crossreaction with type-A and -B strains was completely avoided.
DISCUSSION The double-sandwich ELISA for C. botulinum typeE toxin has been previously described by other investigators (Notermans et al., 1979 and 1982). The type-E-specific antitoxin has been produced in rabbits by subcutaneous injections of 0.4 mg of purified and detoxified botulinal neurotoxin (Notermans et al., 1979 and 1982). Purification of a highly lethal and relatively unstable protein is obviously a major undertaking. In this study we utilized a small quantity (equivalent to 25 ~g neurotoxin) of commercially available type-E toxin for antitoxin preparation. Successful immunization with small amounts of immunogen saves a great deal of time and expense. Furthermore, by limiting the amount of immunizing toxin to several micrograms, one can greatly reduce or even eliminate any minor impurities that may accompany the toxin. The cross-reaction of our type-E antitoxin with C. botulinum types A and B may indicate that the type-E toxin preparation used for immunization was
C. botulinum Type-E Assays
TABLE 1.
13
M o u s e L e t h a l D o s e a n d E L I S A A b s o r b a n c e of C u l t u r e F i l t r a t e s of V a r i o u s S t r a i n s of Clostridium botulinum a n d O t h e r Clostridial Species Log10 M i n i mu m Lethal Dose per Milliliter~
Bacterial Strain
ELISA Value +- SD b
C. botulinum type A 1696
4.0
0.129 + 0.021
3.0
0.241 -+ 0.035
ND
0.013
ND
0.023
3.0 3.5 3.5 3.9 4.0 4.0
1.004 1.240 1.158 1.530 1.619 1.570
1.0
0.029
0.0 0.0 0.0
- 0.020 - 0.002 - 0.018
0.0 0.0
- 0.005 - 0.006
0.0 0.0 ND
- 0.011 0.018 0.000
C. botulinum type B JHIB1
C. botulinum type C ATCC 17850
C. botulinum type D ATCC 27517
C. botulinum type E ATCC 9564 ATCC 17786 ATCC 17852 ATCC 17854 CDC 2112E E-43 C. botulinum type F F8 E-like organisms ELK-6D ELK-93 ELK-S9 Nontoxigenic variants of C. botulinum type B B27 B30
-+ 0.178 -+ 0.055 + 0.078 +_ 0.091 + 0.045 + 0.071
C. sporogenes 17616 A1068 Other clostridial species c
aND, not determined. bNet absorbance (three replications) at 405 nm _+ SD: values/> 0.05 were considered positive. cSpecies included one strain each of Clostridium haemolyticum (6021), Clostridium paraputrificum (14861), Clostridium septicum (A1252), Clostridium sordellii (19739), and Clostridium novyi A (1872); two strains each of Clostridium histoliticum (A1096 and 1097) and Clostridium subterminalis (18041 and 18183); and three strains of Clostridium novyi B (A741, 6387, and 6388).
T A B L E 2.
A v o i d i n g C r o s s - R e a c t i o n b y M e a n s of S e r u m A b s o r p t i o n
Bacterial Strains
Toxigenic Types
Absorbed Serum
Nonabsorbed Serum
ATCC 17786 2112-E 207-A A28 JHIB1 $7 (C. sporogenes)
E E A A B
0.582 a 0.951 0.004 0.006 0.007 0.010
0.798 1.141 0.141 0.345 0.077 0.008
aNet absorbance (duplicate samples) at 405 nm.
14
not free from botulinal nontoxic components. The toxic and nontoxic components constitute the native or progenitor botulinal toxin, which is normally released by the bacterial cells into the culture medium (Sakaguchi et al., 1981). Although the toxic components (neurotoxin or derivative toxin) of types A, B, and E are antigenically distinct, the nontoxic components are immunologically related (Sakaguchi et al., 1974 and 1981). The antibodies directed against type-E nontoxic components therefore appear to be responsible for cross-reaction of type-E antitoxin with C. botulinum types A and B in our study. It is of note, however, that cross-reactivity does not extend to other toxigenic C. botulinum or other clostridial species used in this investigation. Although a strain of C. botulinum type F was shown to be nonreactive in our ELISA for type-E organisms, some of the more toxigenic strains may show reactivity. This assumption is based on close phenotypic relationship between C. botulinum type-F (proteolytic) and type-A and -B (proteolytic) organisms (Smith, 1977). Several investigators have advocated the use of purified neurotoxin for immunological and diagnostic studies of C. botulinum and botulinal toxin (Sugiyama, 1980; Notermans et al., 1982; DasGupta, 1983). Any immunological assay involving quantitation of or monitoring the activity of botulinal toxin requires utilization of purified neurotoxin; however, in a qualitative diagnostic assay, use of a more stable and readily available progenitor toxin (Sakaguchi et al., 1981) for production of antitoxin seems to be justified. The antiserum to progenitor toxin contains type-specific antibodies directed against neurotoxin and cross-reacting botulinal antibodies directed against botulinal-specific nontoxic components. As
M. Dezfulian and J.G. Bartlett
we have shown previously (Dezfulian and Bartlett, 1984), presence of the latter antibodies actually increases the sensitivity of the immunoassay. Our preliminary data also suggest that this may be true for the ELISA involving C. botulinum type E. In this study the cross-reacting antibodies were conveniently removed from botulinal type-E antitoxin by absorption of the serum with alum-precipitated type-A and -B toxoids. Our previously prepared type-A or -B botulinal antitoxins, which do not react with type E toxin, could also be used in parallel with type-E antitoxin, for distinction of C. botulinum type E from other types. Based on this and other studies (Notermans et al., 1979 and 1982; Dezfulian and Bartlett, 1984 and 1985b; Dezfulian et al., 1984), ELISA provides a useful alternative test to conventional MBA. Advantages are that this assay can be carried out in most diagnostic laboratories, it can readily be adapted to large-scale serological screening of food and clinical specimens, and results are available considerably sooner. Type-E botulinal toxin is a major cause of foodborne botulism in some areas (Smith, 1977). A recent report by McCroskey et al. (1986) describes an unusual case of infant botulism caused by a strain of C. butyricum that produces botulinal type-E toxin. This finding makes further development of ELISA technology for direct analysis of specimens highly desirable.
This work was supported by a research grant from the Center for Alternatives to Animal Testing, the Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD.
REFERENCES DasGupta BR (1983) Microbial food toxicants: Clostridium botulinum toxins. In CRC Handbook of Foodborne Diseases of Biological Origin. Ed, M Recheigl Jr. Boca Raton: CRC Press, pp 25-55. Dezfulian M, Dowell VR Jr (1980) Cultural and physiological characteristics and antimicrobial susceptibility of Clostridium botulinum isolates from foodborne and infant botulism cases. J Clin Microbiol 11:604-609. Dezfulian M, Bartlett JG (1984) Detection of Clostridium botulinum type A toxin by enzyme-linked immunosorbent assay with antibodies produced in immunologically tolerant animals. J Clin Microbiol 19:645648. Dezfulian M, Hatheway CL, Yolken RH, Bartlett JG (1984) Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type A and type B toxins in stool samples of infants with botulism. J Clin Microbiol 10:379383. Dezfulian M, Bartlett JG (1985a) Detection of Clostridium
botulinum type B toxin in the presence of a lethal substance interfering with toxin neutralization. Diagn Microbiol Infect Dis 3:113-118.
Dezfulian M, Bartlett JG (1985b) Selective isolation and rapid identification of Clostridium botulinum types A and B by toxin detection. J Clin Microbiol 21:231233. McCroskey LM, Hatheway CL, Penicia L, Pasolini B, Aureli P (1986) Characterization of an organism that produces type E botulinal toxin but which resembles Clostridium butyricum from the feces of an infant with type E botulism. J Clin Microbiol 23:201-208. Notermans S, Fufrenne J, Kozaki S (1979) Enzyme-linked immunoabsorbent assay for detection of Clostridium botulinum type E toxin. Appl Environ Microbiol 37:11731175. Notermans S, Hagenaars AM, Kozaki S (1982) The enzyme-linked immunoabsorbent assay (ELISA) for the detection and determination of Clostridium
C. botulinum Type-E Assays
botulinum toxins A, B, and E. Methods Enzymol 84:223239. Sakaguchi G, Sakaguchi S, Kozaki S, Sugii S, Ohishi I (1974) Cross-reaction in reversed passive hemagglutination between Clostridium botulinum type A and B toxins and its avoidance by the use of antitoxin component immunoglobulin isolated by affinity chromatography. Jpn J Med Sci Biol 27:161-172. Sakaguchi G, Ohishi I, Kozaki S (1981) Purification and oral toxicities of Clostridium botulinum progenitor toxins,
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