The case of botulinum toxin in milk: experimental data.
ABSTRACT Botulinum neurotoxin (BoNT) is the most toxic substance known to man and the causative agent of botulism. Due to its high toxicity and the availability of the producing organism Clostridium botulinum, BoNT is regarded as a potential biological warfare agent. Because of the mild pasteurization process, as well as rapid product distribution and consumption, the milk supply chain has long been considered a potential target of a bioterrorist attack. Since, to our knowledge, no empirical data on the inactivation of BoNT in milk during pasteurization are available at this time, we investigated the activities of BoNT type A (BoNT/A) and BoNT/B, as well as their respective complexes, during a laboratory-scale pasteurization process. When we monitored milk alkaline phosphatase activity, which is an industry-accepted parameter of successfully completed pasteurization, our method proved comparable to the industrial process. After heating raw milk spiked with a set amount of BoNT/A or BoNT/B or one of their respective complexes, the structural integrity of the toxin was determined by enzyme-linked immunosorbent assay (ELISA) and its functional activity by mouse bioassay. We demonstrated that standard pasteurization at 72 degrees C for 15 s inactivates at least 99.99% of BoNT/A and BoNT/B and at least 99.5% of their respective complexes. Our results suggest that if BoNTs or their complexes were deliberately released into the milk supply chain, standard pasteurization conditions would reduce their activity much more dramatically than originally anticipated and thus lower the threat level of the widely discussed "BoNT in milk" scenario.
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ABSTRACT: Botulinum neurotoxins (BoNTs), which are highly toxic proteins responsible for botulism, are produced by different strains of Clostridium botulinum. These various strains of bacteria produce seven distinct serotypes, labeled A-G. Once inside cells, the zinc-dependent proteolytic light chain (LC) degrades specific proteins involved in acetylcholine release at neuromuscular junctions causing flaccid paralysis, specifically SNAP-25 for BoNT/A. BoNT endopeptidase assays using short substrate homologues have been widely used and developed because of their ease of synthesis, detection limits and cost. SNAPtide, a 13-amino acid FRET peptide, was used in this study as a SNAP-25 homologue for the endopeptidase kinetics study of BoNT/A LC. SNAPtide uses a FITC/DABCYL FRET pair to produce a signal upon substrate cleavage. Signal quenching can become an issue after cleavage since quencher molecules can quench cleaved fluorophore molecules in close proximity, reducing the apparent signal. This reduction in apparent signal provides an inherent error as SNAPtide concentrations are increased. In this study, fluorescence internal quenching (FIQ) correction factors were derived using an unquenched SNAPtide peptide to quantify the signal quenching over a range of SNAPtide concentrations and temperatures. The BoNT/A LC endopeptidase kinetics at the optimally active temperature (37oC) using SNAPtide were studied and used to demonstrate the FIQ Correction Factors in this study. The FIQ Correction Factors developed provide a convenient method to allow for improved accuracy in determining and comparing BoNT/A LC activity and kinetics using SNAPtide over a broad range of concentrations and temperatures.Analytical Chemistry 11/2012; · 5.82 Impact Factor
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ABSTRACT: Using commercially available antibodies and toxoids as templates, an ELISA, immuno-quantitative PCR (iqPCR), and multiplex immuno-PCR (iPCR) were developed for detection of Clostridium botulinum neurotoxins A and B. The obtained sensitivities for ELISA ranged from 1 ng/ml in PBS + 1% BSA to 15 and 10 ng/ml in skimmed milk for botulinum neurotoxins (BoNT)/A and BoNT/B, respectively. In semi-fat milk, the limit of detection (LOD) for both toxoids was 30 ng/ml. Quantitative immuno-PCR (iqPCR) had an LOD of 4.5–9 pg/reaction for BoNT/A in both PBS and semi-fat milk, while this was 18.5–37 pg/reaction for BoNT/B in PBS + 1% BSA and semi-fat milk, respectively. The sensitivities of ELISA and iqPCR were improved to 0.5 ng/ml and 3.75 pg/ml (0.2 pg/reaction) in semi-fat milk, respectively, when toxoid of BoNT/A was substituted with actual toxin. Multiplex iPCR with both toxoids run in the same reaction was able to distinguish presence/absence of tested BoNT/A and BoNT/B at 25 pg/reaction. The tested system offers a realistic alternative with much better sensitivity to the standard mouse assay.Food Analytical Methods 01/2012; 5(3). · 1.97 Impact Factor
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ABSTRACT: Botulinum neurotoxins (BoNTs) are used in a wide variety of medical applications, but there is limited pharmacokinetic data on active BoNT. Monitoring BoNT activity in the circulation is challenging because BoNTs are highly toxic and are rapidly taken up by neurons and removed from the bloodstream. Previously we reported a sensitive BoNT "Assay with a Large Immunosorbent Surface Area" that uses conversion of fluorogenic peptide substrates to measure the intrinsic endopeptidase activity of bead-captured BoNT. However, in complex biological samples, protease contaminants can also cleave the substrates, reducing sensitivity and specificity of the assay. Here, we present a novel set of fluorogenic peptides that serve as BoNT-specific substrates and protease-sensitive controls. BoNT-cleavable substrates contain a C-terminal norleucine, while BoNT-non-cleavable controls contain its isomer 6-aminohexanoic acid. The substrates are cleaved by BoNT subtypes A1-A3 and A5. Substrates and control peptides can be cleaved by non-BoNT proteases (e.g., trypsin, proteinase K, and thermolysin) while obeying Michaelis-Menten kinetics. Using this novel substrate/control set, we studied BoNT/A1 activity in two mouse models of botulism. We detected BoNT/A serum activities ranging from ~3600 to 10 attomol/L in blood of mice that had been intravenously injected one hour prior with BoNT/A1 complex (100 to 4 pg/mouse). We also detected the endopeptidase activity of orally administered BoNT/A1 complex (1 µg) in blood 5 h after administration; activity was greatest 7 h after administration. Redistribution and elevation rates for active toxin were measured and are comparable to those reported for inactive toxin.Analytical Chemistry 05/2013; · 5.82 Impact Factor
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2010, p. 3293–3300
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 10
The Case of Botulinum Toxin in Milk: Experimental Data?†
Oliver G. Weingart,1,2§ Tanja Schreiber,3§ Conny Mascher,3Diana Pauly,3Martin B. Dorner,3
Thomas F. H. Berger,4Charlotte Egger,4Frank Gessler,5Martin J. Loessner,2
Marc-Andre Avondet,1§ and Brigitte G. Dorner3§*
Toxinology Group, SPIEZ LABORATORY, 3700 Spiez, Switzerland1; Institute of Food, Nutrition and Health (IFNH), ETH Zurich,
Schmelzbergstr. 7, 8092 Zurich, Switzerland2; Center for Biological Safety, Microbial Toxins (ZBS3), Robert Koch-Institut,
Nordufer 20, 13353 Berlin, Germany3; Agroscope Liebefeld-Posieux Research Station, Schwarzenburgstr. 161,
3003 Bern, Switzerland4; and miprolab GmbH, Marie-Curie-Str. 7, 37079 Go ¨ttingen, Germany5
Received 4 December 2009/Accepted 22 March 2010
Botulinum neurotoxin (BoNT) is the most toxic substance known to man and the causative agent of botulism.
Due to its high toxicity and the availability of the producing organism Clostridium botulinum, BoNT is regarded
as a potential biological warfare agent. Because of the mild pasteurization process, as well as rapid product
distribution and consumption, the milk supply chain has long been considered a potential target of a
bioterrorist attack. Since, to our knowledge, no empirical data on the inactivation of BoNT in milk during
pasteurization are available at this time, we investigated the activities of BoNT type A (BoNT/A) and BoNT/B,
as well as their respective complexes, during a laboratory-scale pasteurization process. When we monitored
milk alkaline phosphatase activity, which is an industry-accepted parameter of successfully completed pas-
teurization, our method proved comparable to the industrial process. After heating raw milk spiked with a set
amount of BoNT/A or BoNT/B or one of their respective complexes, the structural integrity of the toxin was
determined by enzyme-linked immunosorbent assay (ELISA) and its functional activity by mouse bioassay. We
demonstrated that standard pasteurization at 72°C for 15 s inactivates at least 99.99% of BoNT/A and BoNT/B
and at least 99.5% of their respective complexes. Our results suggest that if BoNTs or their complexes were
deliberately released into the milk supply chain, standard pasteurization conditions would reduce their activity
much more dramatically than originally anticipated and thus lower the threat level of the widely discussed
“BoNT in milk” scenario.
Botulinum neurotoxin (BoNT) is mainly produced by the
rod-shaped anaerobic bacterium Clostridium botulinum but
can also be produced by unique strains of Clostridium baratii
and Clostridium butyricum (15). The toxin is a dichain protein
with a molecular mass of 150 kDa, consisting of a heavy chain
of 100 kDa and a light chain of 50 kDa which are linked by a
disulfide bond. All known BoNT types are secreted bound to
nontoxic neurotoxin-associated proteins (NAPs) and assemble
into large complexes with molecular masses of 300, 600, or 900
kDa, depending on the toxin type (20, 23, 39). There are seven
known antigenically distinct BoNT serotypes, designated types
A through G (11, 32). In recent years, the BoNT serotypes
have been further grouped into subtypes, e.g., A1 to A5, dif-
ferentiated on the basis of the variability of the BoNT genes,
their deduced protein sequences, and their immunological
properties (18, 24, 28, 40). BoNT types A (BoNT/A), /B, /E,
and /F are the causative agents of food-borne botulism in
humans, a serious paralytic illness which is the result of con-
suming improperly preserved food contaminated with C. bot-
ulinum spores and/or BoNT (16, 26, 31). After oral ingestion,
BoNT reaches the intestinal tract, where the NAPs associated
with BoNT in the complex are believed to protect the toxin
against digestive enzymes during its passage through low-pH
gastric juice (25). After crossing the intestinal mucosa, BoNT is
circulated through the blood, ultimately reaching the neuro-
muscular nerve endings (22, 29). Specific binding of the heavy
chain to receptors on the nerve cell surface triggers the trans-
location of BoNT into the lumen of the cell (3, 14, 34). Once
BoNT reaches the cytosol of the nerve cell, the release of
acetylcholine is inhibited by the endopeptidase activity of the
BoNT light chain, leading to symmetric descending, flaccid
paralysis (38). There is no reliable data on the exact oral
toxicity of BoNT for humans. However, from animal studies
using nonhuman primates and from cases of human botulism,
it is estimated that the lethal oral dose of BoNT is between 10
ng and 1 ?g kg?1body weight (17, 27). Generally, the lethal
toxicity depends on the BoNT serotype and the route of expo-
sure (2, 8), and it may vary among individuals.
Given its extreme toxicity, the Centers for Disease Control
and Prevention (CDC, Atlanta, GA) lists BoNT as a Category
A bioterrorism agent: these are high-priority agents and or-
ganisms that pose a high risk to public health and national
security (12). Consequently, there were serious concerns that
terrorists could attack the population by contaminating food
staples with BoNT or a similar agent. The milk supply, in
particular, was considered a likely target since the many trans-
portation and processing steps between cow and consumer
represent critical and vulnerable points at which bioterrorism
agents could be deliberately released. In addition, milk and
other dairy products are distributed rapidly after packaging
* Corresponding author. Mailing address: Center for Biological
Safety, Microbial Toxins (ZBS3), Robert Koch-Institut, Nordufer 20,
13353 Berlin, Germany. Phone: 49 30 18754 2500. Fax: 49 30 18754
2501. E-mail: DornerB@rki.de.
§ These authors contributed equally to the work.
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 2 April 2010.
and, as one of the most widely consumed foodstuffs, reach
large parts of the population.
In 2005, Wein and Liu described a theoretical scenario of a
bioterror attack on the milk supply chain based on a series of
mathematical calculations. It garnered considerable interest
among the scientific community, politicians, and intelligence
organizations (41). Referring to experiments in which the toxic
activity of BoNT in different foodstuffs, but not milk specifi-
cally, was determined after heat treatment (42), they based
their calculation on roughly 70% thermal inactivation of BoNT
during pasteurization, leading to ?104fatalities after the re-
lease of 1 g BoNT into raw milk prior to industrial pasteuriza-
tion. As the scenario was based on theoretical assumptions, it
seemed crucial to us to quantify the actual inactivation rate of
BoNT in raw milk in a pasteurization process similar to the
standard process in the industry, as this would allow us to
calculate a reliable scenario.
The heat treatment commonly used by the dairy industry is
high-temperature short-time (HTST) pasteurization, a contin-
uous process where a plate heat exchanger is used to rapidly
bring the milk up to the required temperature of 72°C and hold
it steady at this temperature for at least 15 s. Subsequently, the
milk is cooled to 4°C and packaged for consumption. The
pasteurization process is controlled by a standard method
which measures the activity of milk alkaline phosphatase
(ALP) as an intrinsic time-temperature integrator (1, 13, 21,
33). If HTST pasteurization is successful, the activity of ALP
falls below 350 mU liter?1, the threshold set by the Interna-
tional Dairy Federation and the International Organization for
In the current work, we describe the thermal inactivation of
BoNT/A, BoNT/A complex, BoNT/B, and BoNT/B complex in
cow’s milk in a pasteurization process similar to the industry
standard. As an internal reference for the experimental setup,
we correlate the activity of the toxins with the enzymatic ac-
tivity of ALP, thereby mimicking the industrial process where
the loss of ALP activity is used to indicate correct pasteuriza-
tion. Unexpectedly, our data show that under industrial pas-
teurization conditions, the BoNTs and BoNT complexes we
analyzed are inactivated by 99.5% or more.
MATERIALS AND METHODS
Milk. A batch of fresh, full-fat, raw bovine milk (pH 6.6) was purchased from
a local retail store, divided into 10-ml aliquots, and stored frozen at ?20°C.
Toxins and antibodies. The study was performed using purified 150-kDa
BoNT/A1 (Hall A) and BoNT/B1 (Okra B) and the corresponding BoNT com-
plexes, all of which were purchased from Metabiologics, Inc. (Madison, WI).
Mouse monoclonal antibody A1688 [IgG1(?)] was used to capture BoNT/A and
BoNT/A complex, and mouse monoclonal antibody B279 [IgG2a(?)] to capture
BoNT/B and BoNT/B complex in the sandwich ELISA (30). Antibodies were
purified from hybridoma supernatants using HiTrap protein G Sepharose col-
umns according to the manufacturer’s instructions (GE Healthcare Bio-Sciences
AB, Uppsala, Sweden). Purity was determined by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis, and protein concentration via absorbance at 280
nm using a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE).
For subsequent studies, IgG antibodies were stored in phosphate-buffered saline
(PBS; pH 7.3) at 1 mg ml?1. For the detection of BoNT and BoNT complexes,
biotinylated polyclonal equine anti-BoNT (anti-BoNT/A, /B, and /E [BoNT/A/
B/E]; Novartis Behring, Marburg, Germany) was used. The antibody was coupled
to biotin according to the manufacturer’s instructions (EZ-Link Sulfo-NHS-LC-
biotin; Pierce, Rockford, IL). Biotinylated antibodies were stored in phosphate-
buffered saline with 0.2% (wt/vol) bovine serum albumin and 0.05% (wt/vol)
Laboratory-scale pasteurization and measurement of ALP activity. Raw milk
was thawed and kept at 4°C before and after thermal treatment. To mimic
industrial milk pasteurization, the raw milk was dispensed at volumes of 50 ?l
into thin-walled 0.2-ml PCR vials (VWR, West Chester, PA) and subjected to
heat treatment in an MJ MiniOpticon cycler (Bio-Rad, Hercules, CA) with a lid
temperature of 99.9°C. All samples were heated to 15°C at a rate of 2.5 K s?1,
maintained there for 30 s, and then heated to 72°C with a slope of 0.5 K s?1and
held at 72°C for 1, 5, 10, 15, 30, 60, 120, or 180 s, respectively. Subsequently, the
samples were cooled to 15°C with a slope of 2.5 K s?1and then to 4°C. Similarly
heated samples were pooled for subsequent analysis. Since the accuracy of the
heat treatment delivered is a crucial point of the experimental setup, the thermal
cycler (Bio-Rad, Hercules, CA) was independently validated using a multichan-
nel temperature acquisition system (Cyclertest, Landgraaf, Netherlands). The
results of the validation are shown in Fig. S1 in the supplemental material:
exemplarily shown for 70°C, a temperature variation of 69.6°C ? 0.2°C was
ALP was used as an intrinsic time-temperature integrator for the heat treat-
ment of milk. The ISO standard (21) requires that pasteurization protocols lead
to the inactivation of ALP activity below the 350 mU liter?1threshold. For
standardized ALP measurement (standard ALP assay) the Fluorophos method
(Advanced Instruments, Inc., Norwood, MA) was used. Following the standard
protocol, Fluorophos substrate [2?-(2-benzothiazolyl)-6?-hydroxybenzothiazole
phosphate] in diethanolamine (DEA) buffer solution at pH 10 was used to detect
the dephosphorylating activity of alkaline phosphatase. Two milliliters of Flu-
orophos was heated to 38°C in a glass cuvette. After 75 ?l of milk was added, the
sample was vortexed and allowed to reach 38°C in the Fluorophos reader.
Measurement of fluorescence was performed at an excitation wavelength of 440
nm and an emission wavelength of 560 nm. Fluorescence was measured every
30 s over a total of 120 s. Calibration of the system and calculation of the
enzymatic activities of ALP were performed as described elsewhere (21).
In the miniaturized ALP measurement system (mini ALP assay), 10-?l
amounts of milk samples were pipetted in triplicate into the bottom of Fluoro-
Nunc F96 MicroWell plates (Nunc, Langenselbold, Germany) which were pre-
heated to 38°C. Amounts of 100 ?l of Fluorophos substrate, also heated to 38°C,
were added to each sample cavity and immediately mixed with the sample by
gentle pipetting. The fluorescence was measured at 38°C with an InfiniTE M200
monochromator (Tecan, Crailsheim, Germany) at an excitation wavelength of
440 nm and an emission wavelength of 560 nm. Fluorescence was measured every
30 s over a total of 120 s. The enzymatic activity of ALP was calculated with the
mean fluorescence min?1of the triplicates, multiplied by the amount of Flu-
oroyellow in calibration solution B, and divided by the calibration ratio and the
sample volume. Due to device-specific limitations, the InfiniTE M200 from
Tecan was only able to show a maximum of 100,000 mU liter?1in the fluores-
cence measurement of the miniaturized Fluorophos method.
Toxin spiking. For the measurement of BoNT inactivation, 1 mg ml?1of each
toxin was diluted in raw milk to give a final concentration of 500 ng ml?1. One
mouse lethal dose (MLD) was defined as the lowest total amount of toxin that
kills 100% of all mice in an experiment. According to this definition, one MLD
was determined by mouse bioassay to be 5 pg for purified BoNT/A and BoNT/B
and 15 pg for BoNT/A complex and BoNT/B complex. These experiments have
been performed with 5 mice for each of the four toxin preparations and have
been highly reproducible (data not shown). Accordingly, 500 ng ml?1comprised
100,000 MLD ml?1for purified BoNT/A and BoNT/B and 33,333 MLD ml?1for
BoNT/A complex and BoNT/B complex.
At each of the different time points indicated in the text, a total volume of
1,000 ?l of milk with toxin at 500 ng ml?1was divided into 50-?l aliquots, heated
in a thermal cycler as described above, and pooled again to 1,000 ?l. From this
volume, (i) 3 ? 10 ?l was used for the ALP assay, (ii) 2 ? 50 ?l was used for the
ELISA, and (iii) either 100 ?l (in the case of the purified neurotoxins) or 300 ?l
(in the case of the neurotoxin complexes) was used for the mouse bioassay,
corresponding to a total amount of 10,000 MLD each.
Enzyme-linked immunosorbent assay. MaxiSorp microtiter plates (F96; Nunc,
Langenselbold, Germany) were coated with monoclonal antibody A1688/2 (anti-
BoNT/A) or B279/5 (anti-BoNT/B) at 10 or 8.7 ?g ml?1, respectively, in phos-
phate-buffered saline (pH 7.2) at 4°C overnight (30). After 60 min of blocking,
heated and unheated samples with BoNTs, as well as negative controls, were
applied in duplicate and incubated at 25°C for 120 min. Sample cavities were
washed and incubated for 60 min with biotinylated polyclonal equine anti-BoNT/
A/B/E antiserum (Novartis Behring, Marburg, Germany) at 60 ?g ml?1, followed
by incubation with streptavidin-coupled horseradish peroxidase (Dianova, Ham-
burg, Germany) for 30 min at a dilution of 1:2,500. TMB (3, 3?, 5, 5?-tetrameth-
ylbenzidine; Sigma-Aldrich, Seelze, Germany) was used as a substrate, and the
3294 WEINGART ET AL.APPL. ENVIRON. MICROBIOL.
average absorbance was measured at 450 nm minus absorbance of impurities at
a 620-nm wavelength.
Biological activity of BoNT measured by mouse bioassay. A mouse bioassay
was used to estimate the biological potencies of unheated and heat-treated raw
milk spiked with BoNT. The assay was performed with female BALB/c mice
weighing between 15 and 21 g (35). The BALB/c mice were raised under specific-
pathogen-free conditions at the German Federal Institute for Risk Assessment
(Berlin, Germany) and were between 6 and 8 weeks old. For the experiments,
mice were maintained under barrier conditions at the Robert Koch-Institut
(Berlin, Germany). All animal experiments were performed in accordance with
the German Animal Protection Law and were approved by the regional authority
for health and social affairs (LAGeSo, Berlin, Germany). Considering that
100,000 MLD ml?1purified BoNTs and 33,333 MLD ml?1BoNT complexes
were spiked into raw milk before the thermal treatment, a total amount of 10,000
MLD was injected into mice intraperitoneally (corresponding to 100 ?l in the
case of the purified neurotoxins, adjusted to 300 ?l with similarly pasteurized
milk, or 300 ?l in the case of the neurotoxin complexes, respectively). Negative-
control mice received raw milk without BoNT, and positive controls received
unheated raw milk with BoNT. Samples and controls at each time point were
tested in duplicate. For dilutions used to investigate the activities of BoNT
complexes after heating, similarly pasteurized milk was again used. For the
critical dilutions (1:10- and 1:50-diluted samples [holding times of 1 and 15 s] and
1:10-diluted samples [holding time of 180 s]), five mice were used per group.
Injected mice were observed for typical botulism symptoms for up to 96 h. When
a wasp-like narrowed waist and immobility due to severe paralysis were observed,
the mouse was sacrificed. The absence of botulism symptoms indicated that no
toxic activity remained. If mice that received samples with purified BoNT or
BoNT complex showed no symptoms, the remaining MLD was considered to be
less than one. In all other cases, dilution factors allowed for the approximation
of the remaining MLD.
Establishment of laboratory-scale pasteurization process
similar to the industry standard. To set up a laboratory-scale
pasteurization process similar to the industry standard, raw
milk was subjected to thermal treatment at 72°C for 15 s. The
efficacy of the thermal treatment was monitored by analyzing
the milk alkaline phosphatase (ALP) activity as an intrinsic
parameter of successfully completed pasteurization. To moni-
tor the pasteurization process, the ALP activity was measured
using a standardized ALP assay (standard ALP assay) which is
regularly used in the dairy industry. According to internation-
ally accepted standards, the activity of ALP after HTST pas-
teurization at 72°C for 15 s must drop below 350 mU liter?1
(21). In order to find the right laboratory parameters and
dimensions which would mimic the industrial process, we
tested two different experimental setups: a fixed volume of 50
?l raw milk was heated either in a thermo mixer or in a thermal
cycler. Using the thermo mixer, samples were subjected to
72°C for different times and then rapidly cooled using liquid
nitrogen. Using the thermal cycler, samples were subjected to
a heating curve up to 72°C, held at that temperature for dif-
ferent times, and then brought down to 15°C using the in-built
cooling system. As shown in Fig. 1, the ALP activities from
samples in the thermo mixer reached values below 350 mU
liter?1only after several minutes at 72°C, whereas the thermal
cycler already showed values below the threshold after a 15-s
holding time at 72°C (Fig. 1 and Fig. 2A). In accordance with
these results, while monitoring the activity of ALP in the
heated milk, we observed enhanced heat transfer using the
thermal cycler. Also, the thermal cycler allowed for better
process control with respect to the heating profile, thereby
allowing us to compare the efficacies of the heating process and
the industrial HTST pasteurization process directly. Conse-
quently, the thermal cycler was used to heat the milk samples
in all further experiments.
To measure ALP activity in milk spiked with BoNT in our
small-scale laboratory setup, the industrial standard ALP assay
was downscaled from 2,000 ?l to a miniaturized ALP assay
format (mini ALP assay) using a 100-?l volume (see Materials
and Methods). The two assays were compared using unspiked
milk heated with the thermal cycler (Fig. 2A). The standard
FIG. 1. Comparison of results from two different experimental set-
ups for laboratory-scale pasteurization. A small volume (50 ?l) of raw
milk was heated either in a conventional thermo mixer or in a thermal
cycler. For both setups, the reduction of ALP activity in milk held
continuously at 72°C for up to 300 s was monitored using the standard
FIG. 2. Reduction of ALP activity during heating of raw milk in a laboratory-scale pasteurization process using a thermal cycler. (A) ALP
activities in raw milk and heated milk (each without toxin) were measured using the standard ALP assay and compared to the activities measured
with the mini ALP assay. (B) ALP activities in unspiked raw and heated milk were measured with the mini ALP assay and compared to ALP activity
in milk spiked with BoNT/A, BoNT/A complex, BoNT/B, or BoNT/B complex. Data are shown for holding times of 0 (unheated), 1, 15, and 180 s
at 72°C. The threshold for successful pasteurization is 350 mU liter?1. Each line represents the means of the results of two independent
experiments. Error bars show standard deviations.
VOL. 76, 2010BOTULINUM TOXIN IN MILK3295
ALP assay showed ALP activities typically found in raw milk,
ranging from 200,000 to 500,000 mU liter?1(33; C. Egger,
personal communication), slightly more than those found with
the miniaturized method. After a 15-s holding time at 72°C, the
ALP activity in milk was reduced to values below the 350 mU
liter?1threshold. As shown in Fig. 2A, the two methods led to
almost identical reductions of ALP activity below the indicated
threshold at the same holding times. The miniaturized ALP
assay, therefore, was used to monitor the loss of ALP activity
during thermal treatment in all further experiments.
To exclude the possibility that milk pasteurization is altered
by the presence of BoNT, raw milk and raw milk spiked with
100,000 mouse lethal doses (MLD) per ml purified BoNT/A or
BoNT/B or 33,333 MLD ml?1BoNT/A complex or BoNT/B
complex were heated in parallel in a thermal cycler and mea-
sured with the mini ALP assay. The results presented in Fig.
2B show that the ALP activities in heated milk were consistent,
regardless of whether the toxin was present in the milk or not.
Based on the measurement of the intrinsic milk parameter
ALP by both the standardized and the miniaturized ALP assay,
the results show that heating for 15 s at 72°C in the thermal
cycler is sufficient to obtain pasteurized milk. Thus, according
to internationally accepted standards, our laboratory-scale pas-
teurization process can be considered similar to the industry
standard, allowing for direct comparison of heat transfer and
Thermal inactivation of BoNT/A, BoNT/B, and BoNT com-
plexes as determined by ELISA. To monitor the thermal inac-
tivation of purified BoNT/A and BoNT/B and the correspond-
ing complexes in milk, we spiked defined amounts of the toxins
into raw milk. From the same samples, we subsequently deter-
mined (i) the ALP activity using the mini ALP assay as indi-
cated above, (ii) the structural integrity of the toxins by sand-
wich ELISAs specific for BoNT/A and BoNT/B, and (iii) the
functional activity of the toxins using the mouse bioassay.
The results of the two sandwich ELISAs used in this study
are indicated in Fig. S2 in the supplemental material and in
Table 1. Both ELISAs were based on the combination of a
monoclonal capture antibody (clone A1688/2 for BoNT/A and
clone B279/5 for BoNT/B) (30) with an equine anti-BoNT/A/
B/E antiserum resulting in detection limits between 47 and 136
pg ml?1for BoNT/A and BoNT/B and 558 and 698 pg ml?1for
the corresponding complexes, both in buffer and in raw milk
(Table 1; see Fig. S2A and B in the supplemental material). As
shown in Fig. S2 in the supplemental material, both ELISAs
were able to discriminate between active and inactive BoNT
(where “inactive BoNT” is equivalent to toxin that has been
heated for 15 s at 72°C) over a range of about three orders of
magnitude of concentration (see Fig. S2C to F in the supple-
mental material), therefore allowing us to reduce the number
of animals used in this study.
After spiking the milk with 500 ng ml?1purified BoNT/A or
BoNT/B or one of the corresponding complexes (equivalent to
100,000 MLD ml?1for the purified neurotoxins or 33,333
MLD ml?1for the toxin complexes, respectively), we per-
formed the laboratory-scale pasteurization as indicated above
and measured the presence of the toxins after appropriate
dilution of the milk samples. As shown in Fig. 3, BoNT/A and
BoNT/B, as well as the respective complexes, could be clearly
detected in the spiked, unheated raw milk samples and were
not present in the unspiked samples. Applying our heating
protocol for different times (1, 5, 15, and up to 180 s at 72°C)
resulted in a dramatic reduction in the toxin-specific signal
detected via sandwich ELISA, both for the purified neurotox-
ins and the neurotoxin complexes. In the case of the purified
neurotoxins, a holding time of only 1 s at 72°C led to a com-
plete loss of the ELISA signal (Fig. 3). For the neurotoxin
FIG. 3. Structural integrity of BoNT/A, BoNT/B, and the corresponding BoNT complexes in milk during laboratory-scale pasteurization.
Purified BoNT/A or BoNT/B or one of the corresponding complexes was spiked into raw milk as indicated in the text and subjected to our
laboratory-scale pasteurization using a thermal cycler. After holding the samples at 72°C for the indicated times, sandwich ELISAs were used to
analyze them specifically for BoNT/A and BoNT/A complex (A) or for BoNT/B and BoNT/B complex (B). Results for purified neurotoxins
(BoNT/A or /B) are depicted by open triangles, and those for BoNT complexes as open squares. Results for unspiked milk samples (negative
control) are shown by filled circles. Each line represents the means of the results of two independent experiments. Error bars show standard
deviations. A, absorbance.
TABLE 1. Sensitivity of sandwich ELISAs for the detection of
BoNT/A, BoNT/B, and the corresponding complexes
in buffer and raw milk
LODa(pg ml?1) in:
53 ? 12
650 ? 221
102 ? 26
653 ? 184
47 ? 9
558 ? 222
136 ? 51
698 ? 245
aLimit of detection (LOD) was calculated on the basis of the arithmetic mean
and the 3-fold standard deviation of the results for 12 to 16 blank samples for
three interassay standard curves.
bPBS containing 0.1% bovine serum albumin.
3296 WEINGART ET AL.APPL. ENVIRON. MICROBIOL.
complexes, the ELISA signal fell sharply even after a holding
time of only 1 s at 72°C. However, it was still detectable at
approximately 20% of the signal intensity of the spiked, un-
heated milk (Fig. 3). For both BoNT complexes, a holding time
of 180 s at 72°C was needed before the signal decreased to 1 to
4% of the original value. Even though the ELISA suggested a
dramatic loss of BoNT integrity within the first few seconds of
heating, a residual functional activity could not be excluded.
Thermal inactivation of BoNT/A, BoNT/B, and BoNT com-
plexes as determined by mouse bioassay. We performed the
mouse bioassay to quantify the residual toxic activities of pu-
rified BoNTs and BoNT complexes spiked into milk after lab-
100,000 MLD ml?1purified BoNTs and 33,333 MLD ml?1
BoNT complexes were spiked into raw milk before thermal
treatment, a total amount of 10,000 MLD was injected into
mice intraperitoneally. The application of unheated raw milk
containing 10,000 MLD BoNT/A, BoNT/A complex, BoNT/B,
or BoNT/B complex to BALB/c mice led to typical symptoms
of botulism, such as a wasp-like narrowed waist and subse-
Interestingly, all mice injected with milk spiked with purified
BoNT/A or BoNT/B held at 72°C for 1 s or longer survived
without showing any symptoms of botulism. Therefore, this
experiment showed that the 10,000 MLD of BoNT originally
spiked into milk were reduced to less than 1 MLD, indicating
that the heat treatment reduced the toxic activity of purified
BoNT/A and BoNT/B by more than 99.99% (Table 2).
Yet, when we applied thermally treated milk spiked with
BoNT/A complex or BoNT/B complex instead of one of the
purified neurotoxins, all mice suffered from typical symptoms
of botulism. Stepwise dilutions of the heated milk originally
containing 10,000 MLD were used to determine the residual
toxicity. A 1:50 dilution, corresponding to 200 MLD of the
original spiked milk held at 72°C for 15 s, was no longer toxic
when injected into mice, demonstrating that less than 1 MLD
was left from the 200 MLD injected. Therefore, the heat treat-
ment reduced the toxicity by at least 99.5%. A similar calcu-
lation leads us to conclude that heat treatment at 72°C for 180 s
reduces the toxicity of both BoNT complexes by more than
99.9% (Table 2).
Taking the results together, heating under standard milk
pasteurization conditions (72°C for 15 s) was sufficient to re-
duce the toxic activity of purified BoNT/A and BoNT/B by
TABLE 2. Toxic activities of BoNT/A, BoNT/B, and the corresponding BoNT complexes in milk after pasteurization
Time sample held
at 72°C (s)
No. of mice surviving/
no. of mice tested
MLD before heating/
MLD after heating
Reduction in toxic
a—, no dilution.
VOL. 76, 2010BOTULINUM TOXIN IN MILK 3297
more than 99.99% and that of the corresponding BoNT com-
plexes by more than 99.5%.
The results presented in this paper show that current con-
ditions of industrial HTST pasteurization are effective in re-
ducing the toxic activity of both BoNT/A and BoNT/B by more
than 99.99% and of the corresponding BoNT complexes by
99.5%. Consequently, these results could provide a significant
contribution to scenarios modeling BoNT as a potential bio-
Milk and milk-derived products are among the most widely
consumed food products worldwide. The per capita milk con-
sumption in 2007 was approximately 80 to 90 liters in the
United States and Western European countries (19). For more
than 100 years, the thermal treatment of milk has been used
effectively to inactivate pathogens that may be present in milk
(10). Common heating processes include high temperature
short time (HTST; 72°C for 15 to 16 s), extended shelf life
(ESL; 80°C and 130°C for 1 to 5 s), and ultra-high temperature
(UHT; 135 to 150°C for 1 to 10 s).
Heat-treated milk has proven to be a safe food, and no cases
of “natural” food-borne botulism resulting from industrially
processed milk have been described. Nevertheless, even when
milk production on farms meets modern standards for food
quality and hygiene, the production process cannot be com-
pletely secured, making the supply chain vulnerable to bioter-
ror attacks. One scenario that could be considered is the de-
liberate release of the agent into raw milk prior to
pasteurization on the farm or while in transit to the dairy
For the current study, it was important to consider the food
matrix used for the experiments, in this case raw milk, since it
has a major impact on the heat inactivation rate of BoNT (37).
Early work by Scott and Stewart (published in 1950) demon-
strated that vegetable juice increased the heat stability of
BoNT/A and BoNT/B due to their being protected by bivalent
cations and organic acid anions present in the juice (36). Later,
Bradshaw et al. (in 1979) showed that BoNT/A and BoNT/B
were more heat stable in beef and mushroom patties than in a
phosphate buffer at the same pH (5). Woodburn et al. (in
1979) also observed increased heat stability of BoNT/A when
1% gelatin was added to a phosphate buffer (42). Recently, it
has been shown that the molten-globule-like character of
BoNT and its interaction with NAPs are responsible for vari-
ations in physical stability at different pH values (6, 7). The
data showed a stabilizing effect of NAPs on the purified neu-
rotoxins. It is worth considering that if the NAPs were dam-
aged, this could influence the stability of the whole complex
and the oral toxicity. Based on the available data, it seemed
conceivable that the actual stability of BoNT in milk during the
pasteurization process cannot be extrapolated directly from its
stability in other food matrices which were analyzed earlier.
Rather, measuring the actual stability of the toxins in milk
appears critical for the generation of reliable numbers.
Our comparisons of different experimental setups for the
thermal treatment of milk highlighted a second critical param-
eter, namely, the importance of finding experimental condi-
tions for heat transfer that mimicked the industrial process as
closely as possible. Our data from comparing the heating of
milk with a thermo mixer and with a thermal cycler suggested
that strict compliance with pasteurization parameters is neces-
sary to guarantee the degree of inactivation described above.
While using a thermo mixer involves heating with an isother-
mal heat source for a defined time, the thermal cycler allows
for temperature-controlled heating with defined holding times,
an approach which complies with the industrial HTST pasteur-
ization process. Compared to ESL and UHT processing, HTST
pasteurization applies less thermal load and is also the most
commonly used and mildest thermal treatment of milk; hence
our decision to apply this process to our experiments. Unlike
the industrial process, which uses forced convective heat trans-
fer, heating in a thermal cycler is achieved by free heat trans-
fer. Nevertheless, ALP, used as an intrinsic time-temperature
integrator for the heat treatment of milk, allowed us to show
that laboratory-scale pasteurization in a thermal cycler met
dairy industry requirements for successful HTST pasteuriza-
tion (i.e., inactivation of ALP within 15 s at 72°C to below a
threshold of 350 mU/liter) (9). These results are also in accor-
dance with the findings of an earlier study that applied the
same methodology of thermal treatment and measuring ALP
activity when investigating the effect of pasteurization on her-
pes virus infectivity in milk (4). Retrospectively, it is not clear
whether the classical work cited above (5, 36, 42) is as well
defined as our current work with respect to the heat transfer
applied, the proven comparability to an industrial process, and
the toxin preparations used (neurotoxin or toxin complexes
and purity of the material).
Due to their high bioavailability, BoNT complexes present
the most toxic form of BoNT if ingested (8, 25), and the
deliberate release of this form would be a worst-case scenario.
For this reason, we spiked purified BoNT or BoNT complex
into raw milk prior to pasteurization. Subsequently, loss of
structural integrity and toxic activity were determined by sand-
wich ELISA and mouse bioassay, respectively. Both the immu-
nological and the functional detection of BoNTs generated
similar results, indicating a dramatic loss of protein structure
and function. In contrast to the 68.4% heat inactivation of
BoNT estimated by Wein and Liu (41), we were able to show
in the mouse bioassay that even after only a 1-s holding time at
pasteurization temperature, the toxic activities of purified
BoNT/A and BoNT/B fell by more than 99.99%. Similarly, we
were able to show that BoNT/A complex and BoNT/B complex
were inactivated by more than 99.5% under common pasteur-
ization conditions. As observed before, the BoNT complexes
showed a slightly higher degree of stability; this was probably
due to the stabilizing effect of associated NAPs (6, 7). In the
case of the complex proteins, the results of the sandwich
ELISAs did not completely reflect those of the functional
assay; this might indicate that distinct epitopes of the BoNT
complexes detected by ELISA were protected somewhat
against thermal inactivation by the accompanying complex pro-
teins and/or by the interaction of the BoNT complex with milk
components. However, considering the results of the mouse
bioassay, the detected epitopes seem not to be directly linked
to the toxic activity of the toxin. Although the ELISA results
provide no information on the remaining toxic activity in the
heated samples, they reflect the overall stability of the toxin
structure under distinct heating conditions.
3298 WEINGART ET AL.APPL. ENVIRON. MICROBIOL.
In the bioterror scenario described by Wein and Liu (41), 1 g
of BoNT released into approximately 230,000 liters (50,000
gallons) of raw milk would lead to 3.2 ? 104possible victims,
based on 68.4% inactivation of the toxin by heat treatment.
However, our experimental results based on a pasteurization
process similar to the industry standard showed 99.5% inacti-
vation of BoNT complexes and 99.99% inactivation of purified
BoNTs. Assuming a scenario in which 1 g of BoNT complex is
deliberately released into raw milk and at least 99.5% of the
toxin is inactivated by HTST pasteurization, the amount of
biologically active toxin would be reduced to 5 mg in total.
Applying our experimental data and assuming a linear rela-
tionship, this would mean that following dilution in a 230,000-
liter bulk milk tank, an average daily serving of 0.5 liter would
contain approximately 11 ng of toxin at most. Considering the
estimates for the human lethal oral dose of BoNT, this amount
dramatically lowers the threat level of the widely discussed
“BoNT in milk” scenario.
Our experimental data show that current conditions of
HTST pasteurization are effective in reducing the toxic activ-
ities of BoNT/A and BoNT/B and the corresponding BoNT
complexes by more than 99.99% and 99.5%, respectively.
Therefore, the HTST pasteurization process in the dairy in-
dustry dramatically reduces the risk of consumer harm even if
larger amounts of BoNT were deliberately released into the
milk supply chain. However, other complex food matrices or
slight changes to the heating parameters might have a different
effect on the stability of BoNT. As a result, we are unable to
draw any general conclusions for other matrices and heating
parameters. These will have to be the subject of further exper-
We thank Reto Zbinden for his thought-provoking contributions to
our discussions and Andreas Spahni for his standardized ALP mea-
This work was supported by a grant from the Swiss Federal Office for
Civil Protection to M.-A.A. and a grant from the German Federal
Ministry of Health to B.G.D.
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