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Detection of Preformed Type A Botulinal Toxin in Hash Brown Potatoes by Using

the Mouse Bioasssay and a Modified ELISA Test

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1460 FERREIRA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001
FOOD CHEMICAL CONTAMINANTS
Detection of Preformed Type A Botulinal Toxin in Hash Brown
Potatoes by Using the Mouse Bioasssay and a Modified ELISA
Test
JOSEPH L. FERREIRA and STACEY J. ELIASBERG
U.S. Food and Drug Administration, 60 8th St, Atlanta, GA 30309
MARK A. HARRISON
University of Georgia, Department of Food Science and Technology, Athens, GA 30602-7610
PAUL EDMONDS
Georgia Institute of Technology, School of Biology, Atlanta, GA 30332
A foodborne illness caused by type A
toxin-producing Clostridium botulinum was inves-
tigated by using the standard mouse bioassay and
a rapid invitro test for toxin detection. The patient,
who consumed improperly stored hash brown po-
tatoes that contained the preformed toxin, was di-
agnosed with type A botulism. C. botulinum type A
toxin was detected in the hash brown potatoes as
well as in the tryptone–peptone–glucose–yeast ex-
tract (TPGY) medium subcultures of this food us-
ing the mouse bioassay and an amplified ELISA
technique. The mouse bioassay revealed pre-
formed toxin at 10 000 minimum lethal dose
(MLD)/g uncooked product and the amplified
ELISA an equivalent 50 000 MLD/g. The cultural
toxin from the uncooked product killed mice at the
106 dilution and a modification of the ELISA proce-
dure was positive at the 103 dilution. Cooked food
obtained from the consumer’s waste can contained
100 MLD/g and the ELISA was also positive at the
same dilution of the product. The culture of the
cooked product obtained from the waste can was
lethal for mice at the 107 dilution and positive using
the modified ELISA at the 104 dilution. The unmodi-
fied amplified ELISA method indicated a toxin level
of approximately 1 ng/mL (equivalent to 5 ´
105 MLD/mL) in diluted culture fluid from the un-
cooked food and the culture of cooked food ob-
tained from the waste can. The hash brown pota-
toes were negative for types B, E, and F preformed
and cultural botulinal toxins using both assays.
here are 7 antigenically distinct botulinal types (A–G)
T but only types A, B, E, and F have been implicated in
human botulism (1). Botulism has been associated with
many food types, including vegetable, dairy, fish, meat, and
Received January 26, 2001. Accepted by AP April 3, 2001.
honey products (1, 2). Potato products such as baked potatoes,
potato salad, and a potato dip (skordalia) have been involved
in foodborne botulism outbreaks (3–6). Recently, a frozen po-
tato product (hash brown potatoes) that was stored improperly
at room temperature rather than refrigerator temperature prior
to preparation for consumption was the cause of a botulism
outbreak.
The classical method for the detection of botulinal toxins is
the mouse bioassay (7, 8). Several rapid in vitro methods have
been developed that approximate the sensitivity of the in vivo
method and possess greater specificity (2, 9, 10). In this study,
an amplified ELISA method (AE) previously used for the
qualitative detection of cultural botulinal toxins (2) was modi-
fied to detect and estimate the amount of preformed type A
botulinal toxin in a hash brown potato sample. Cultural toxin
formed in anaerobic media inoculated with the food samples
was tested using the unmodified amp-ELISA. The
amp-ELISA and the mouse bioassay were also used to test
samples for types B, E, and F botulinal toxins.
Experimental
Samples
The samples consisted of previously cooked hash browns
collected from the kitchen waste container and uncooked hash
browns from the kitchen cupboard of the consumer hospital-
ized with botulism.
Reagents
(a) Amp-ELISA IgG reagents.—FDA Southeast Regional
Laboratory (SRL), Atlanta, GA.
(b) Type A standard neurotoxin.—50 minimum lethal
dose (MLD) ng; B.R. DasGupta, Food Research Institute,
University of Wisconsin, Madison, WI.
(c) ELISA amplification substrate.—GibCo-BRL (Grand
Island, NY).
(d) Neutravidin-alkaline phosphatase (NAP) conju-
gate.—Pierce Chemical Co., Rockford, IL.
 FERREIRA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001 1461

Figure 1. Estimation of Clostridium botulinum type A neurotoxin level in hash brown potato samples by using the
modified amp-ELISA method. Uncooked sample absorbance value obtained from the 103 dilution of the food. The
cooked sample absorbance value was from the 101 dilution of food.

Food Sample Preparation for the Modified
amp-ELISA (MAE)
Equal amounts of solid food samples (w/v) and casein
buffer (0.05M Tris, 0.085M NaCl–0.5% casein, pH 7.6) were
mixed and ground with a mortar and pestle into a slurry (1:2
dilution) that was centrifuged at 7000 × g for 30 min at 4°C.
The 1:2 and 10-fold dilutions (up to 105) of the supernatant
were made in casein buffer and tested in duplicate by 2 differ-
ent analysts for botulinal toxin using the MAE. The sensitivity
of this modification of the AE was determined by using
10-fold dilutions (100 to 0.01 ng/mL casein buffer) of type A
purified neurotoxin and plotting a standard curve. This stan-
dard curve was then used to estimate the level of toxin in the
food samples.
MAE
The method of Ferreira and Crawford (2) was modified for
the detection of the preformed C. botulinum toxins in the im-
plicated food samples. Briefly, type A antitoxin (IgG) was di-
luted to 1 µg/mL in 0.05M carbonate–bicarbonate buffer,
pH 9.6, and 100 µL added to each well of an Immulon ll
(Dynex, Chantilly, VA) microtiter plate. The plate was stored
at 35°C for 2 h (instead of overnight at 4°C for the original
AE), then the plate was washed 3× in TBS-T (0.05M Tris,
0.15M NaCl, pH 7.5 with 0.05% Tween 20) wash buffer. The
plate was blocked in casein buffer for 30 min. The samples,
biotinylated IgG, and NAP were all diluted in casein buffer
and incubated at room temperature on a microtiter plate
shaker. The casein buffer was then discarded and 100 µL sam-
ples of the diluted food samples and dilutions of type A toxin
standard were added to the microtiter plate. A known type A
C. botulinum culture obtained from the SRL culture collection
and negative controls were also examined. The samples were
incubated 2 h and then washed as above. Biotin-labeled anti-
body (ca 2.5 µg/mL casein buffer) was added and incubated
for 1 h. The plates were washed as above and 100 µL NAP
(1:2500 dilution in casein buffer) was added to the plate wells.
The conjugate was incubated for 1 h and the plate was washed
7× in TBS-T. The GibCo-BRL ELISA amplification system
was used to detect the presence of bound conjugate. The
absorbance was read at 490 nm with 410 nm subtraction.
In an attempt to increase the sensitivity of the MAE, plates
were coated with 10 µg type A IgG/mL bicarbonate buffer and
incubated for 2 h at 35°C. The remainder of the revised proto-
1462 FERREIRA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001
Table 1. Detection of preformed and cultural botulinal toxin from hash brown potatoes
Preformed toxin
Sample Identification Bioassaya
Hash browns Cooked, from waste can 102
Hash browns Uncooked, from cupboard 104
d
Positive control Type A NA
Negative control – –
Toxin standard Type A NA
a
MLD/g.
b
Endpoint dilution positive.
c
MLD/mL.
d
Not analyzed.
col was the same as for the modified protocol except for the
following: the biotinylated IgG concentration was reduced to
1.25 µg/mL, plates were incubated at 35°C instead of room
temperature with no shaking, and plates were washed 5× be-
tween each step instead of 3×. The final wash was stored for
10 min at room temperature before removal of the wash
buffer.
Cultural Toxin Preparation for the Original AE
One to 2 g of the food samples were cultured in TPGY me-
dium and incubated at 26°C for 5 days. Ten-fold dilutions of
culture fluid were made in casein buffer and 100 µL volumes
of the dilutions tested by the AE procedure.
Mouse Bioassay
The procedures described in the AOAC INTERNA-
TIONAL Official Methods of Analysis (7) or the FDA Bacte-
riological Analytical Manual (8) were used to determine
botulinal toxin levels in foods and culture media. Briefly, for
preformed botulinal toxin analysis, an equal weight of gel
phosphate buffer was added to the food samples and ground
with a mortar and pestle into a slurry that was centrifuged at
7000 × g for 30 min at 4°C. The supernatant was removed and
the pH adjusted to 6.2 prior to making higher 10-fold dilutions
and injecting mice intraperitoneally; 0.5 mL/mouse,
2 mice/dilution. The highest dilution that killed both mice
(MLD) was established and the toxin type determined using a
neutralization assay with homologous antitoxin. Cultural
toxin endpoints were determined similarly by injecting mice
as above with 10-fold gel phosphate buffer dilutions of clari-
fied culture supernatants. The mouse bioassay data was gener-
ated by one analyst and the assay was not replicated.
Results
The MAE results showed that the cooked hash brown sam-
ple collected from the waste can was positive for type A toxin
at up to the 102 dilution (Table 1) and contained approxi-
mately 2.2 ng type A neurotoxin/mL at the 101 dilution (Fig-
Cultural toxin
ELISAb Bioassayc ELISAb
102 107 >103
103 106 >104
+ NA +
– –
1 ng/mL NA 1 ng/mL
ure 1). Therefore, the original food sample contained approxi-
mately 22 ng type A toxin/g food based on the toxin standard
curve using the MAE.
The uncooked potato sample obtained from the room tem-
perature cupboard (Figure 1) contained 1.8 ng type A
toxin/mL at the 103 dilution using the MAE. The original food
contained approximately 1800 ng type A toxin/g.
When cooked and uncooked samples were cultured in
TPGY, the MAE detected type A toxin at 103 and 104 dilution,
respectively (Table 1). The toxin concentration of TPGY cul-
tures from the cooked and uncooked foods was approximately
1 ng/mL at the 105 dilution of the cultures (approximately
500 000 MLD/mL) using the AE method (data not shown).
Endpoint ELISAs for isolates from cultures of the product
ranged from 104L–105/mL of TPGY using the original
amp-ELISA method. No other botulinal toxins (types B, E, or
F) were detected from culture using the AE.
The standard curve (Figure 1) shows that the MAE sensi-
tivity was approximately 1 ng toxin protein/mL. When revi-
sions were made to the MAE method in an attempt to increase
the sensitivity, type A neurotoxin was detected at approxi-
mately 0.2 ng neurotoxin protein/mL casein buffer (Figure 2).
The mouse bioassay results (Table 1) revealed preformed
type A toxin was present at the 102 dilution (100 MLD/g) in
the cooked sample and at 10 000 MLD/g in the uncooked po-
tato sample. The TPGY culture of the uncooked potato prod-
uct killed mice at the 106 dilution and the cooked (waste can)
culture was toxic at the 107 dilution. No other botulinal toxins
(types B, E, or F) were detected using the mouse bioassay.
Discussion
The MAE sensitivity at the endpoint dilution was approxi-
mately 10-fold less than the mouse bioassay for the detection
of preformed type A botulinal toxin in the uncooked food.
These assays were equally sensitive for the detection of type A
toxin in the cooked food sample. Sensitivity of preformed
toxin detection was adequate, especially at the lower dilutions
of the product. The absorbance values measured at the 10–1 di-
 FERREIRA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001 1463
Figure 2. Standard curve for Clostridium botulinum type A neurotoxin obtained by using the revised modified
amp-ELISA method.
lution of food samples were 2.2 and 0.37 for the cooked and
the uncooked samples, respectively. In TPGY cultures, toxin
detection sensitivity was greater using the mouse bioassay
compared with the MAE. Previous data using the overnight
coating of microtiter plates showed that the original AE
method detected from 1–10 MLD/mL (2). For detection of
cultural toxins it is not necessary to coat the plates the same
day as the analysis because the media are incubated for 5 days
prior to analysis and plates can be coated the day before analy-
sis. It also is not necessary to obtain an endpoint titer using the
AE for qualitative detection and typing. Undiluted cultures or
very low dilutions of foods can be tested for the qualitative
identification of the toxin. An endpoint titer is needed for
toxin typing with the mouse bioassay because the typing anti-
toxin can neutralize only a limited amount of toxin.
The MAE results were positive for type A cultural toxin
between the 103 and 104 dilutions, with a toxin concentration
of approximately 1 ng/mL (equivalent to 50 000 and
500 000 MLD/mL culture).
It is possible that other foods may contain substances that
inhibit the sensitivity or cause false positive reactions with the
MAE. There are also substances in some foods that cause non-
specific deaths in the mouse bioassay.
We can not explain the unusually high toxin levels observed
using the mouse bioassay for the TPGY cultures of the foods.
The mouse bioassay results can vary depending on the tech-
nique of the analyst and the condition of the animals. There is
also no clear reason for the culture of the heated food product
(107 MLD/mL) to be more toxic than the unheated food culture
(106 MLD/mL). Organisms isolated from these cultures and
transferred to fresh TPGY were positive at the 105 dilution us-
ing the original AE but were not tested using the mouse
bioassay (data not shown). Because the AE is about 10-fold less
sensitive than the mouse bioassay, we would expect the
bioassay to have about 106 MLD/mL for these isolates.
The revised MAE used a higher concentration of coating IgG
and detected lower levels of type A standard neurotoxin com-
pared with the MAE but the revised MAE method was not used
to test the original food samples for preformed type A toxin.
The MAE method was useful for the rapid detection of pre-
formed type A botulinal toxin in hash brown potatoes. The ap-
proximate toxin concentration and the toxin type in the food sam-
ples using this method was determined in almost 8 h. Based on the
increased sensitivity obtained using the revised MAE, toxin detec-
tion in foods may also be enhanced utilizing this method. To deter-
mine its usefulness in other food matrixes, this method should also
be tested for the detection of type A toxin in other food types and
with other botulinal types. In outbreaks of botulism, this procedure
could be used for a rapid 8 h screening of samples while regula-
tory decisions are made with respect to legal actions.
1464 FERREIRA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 5, 2001
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