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REVIEW ARTICLE
BOTULISM: A FREQUENTLY FORGOTTEN OLD MALADY
Teguh Thajeb1,2, Yi-Min Chen3, Dao-Fu Dai4 , Daniel Daile Thajeb5, Peterus Thajeb3,6,7*
1
Department of Internal Medicine, Chang-Gung Memorial Hospital, Lin-Kou, 2Department of Internal Medicine,
Li-Shin Hospital, Ping-Jen City, Taoyuan, 3Department of Neurology and Medical Research,
Mackay Memorial Hospital, Taipei, Taiwan, 4Department of Pathology, School of Medicine,
University of Washington, Seattle, USA, 5School of Biotechnology, Asia University, Taichung,
6
School of Medicine, National Yang-Ming University, and 7School of Medicine,
Taipei Medical University, Taipei, Taiwan.
SUMMARY
A frequently forgotten old malady called botulism has been recognized for more than a century. This ailment
occurs worldwide, afflicts human of all age groups from infants to elderly and affects Oriental people more often
in several regions of China. Occurrence in Taiwan is uncommon, and therefore, it is often overlooked. The outbreaks of human botulism in various regions of the world, the clinical types, the molecular mechanisms, and the
electrophysiologic findings will be highlighted. [International Journal of Gerontology 2007; 1(3): 118–124]
Key Words: Botox, botulinum neurotoxin, botulism, Clostridium botulinum, paralysis
Introduction
Botulism, a Latin name for sausage, was first reported
by van Ermengem in 18971. He eloquently observed
that in a group of musicians who developed a unique
clinical syndrome of autonomic dysfunction with ocular and bulbar weakness, the rapid and subsequent
descending paralysis led to death within 16 to 60
hours after ingestion of ham. He also noted that unlike myasthenia gravis, the pupillary constriction of
eyes was impaired in this syndrome. A century later,
botulism was redefined as a neuroparalytic illness
caused by the action of an exotoxin, the botulinum
neurotoxins (Botox) that inhibit the release of acetylcholine at the peripheral nerve endings, subsequently
blocking the presynaptic neuromuscular transmission2–5. Thus, the paralysis of muscles, which involves
the ocular, bulbar and skeletal muscles and eventually
*Correspondence to: Dr Peterus Thajeb, School of Medicine,
National Yang-Ming University, P.O. Box Nei-Hu 6-30, Taipei City
11499, Taiwan.
E-mail: [email protected]
Accepted: March 15, 2007
118
the respiratory muscles, may be widespread, leading
to death or moribund outcome. Until three decades
ago, the chemical and molecular structures of botulinum neurotoxin and the mechanisms of action were
identified6–8.
Botox is a mixture of several protoplasmic proteins6,7,9–17 that are released from cells after autolysis18.
The toxins are resistant to low pH and are readily
destroyed by alkali, such as 0.1 N sodium hydroxide. The
neurotoxin is a unique protein produced by an obligate
anaerobic, spore-forming, Gram-positive, rod-shaped
bacterium called Clostridium botulinum (C. botulinum).
C. botulinum has a widespread distribution in the terrestrial and marine environments throughout the world.
To date, there are more than eight types of botulinum
neurotoxins recognized, based on their distinct antigenicity. They are types A, B, C1, C2, D, E, F, and G10–21.
Most colonies of C. botulinum in culture produce only
one single type of neurotoxin. The exceptions are types
C and D, and types A and F, that can be produced by
the same colony. Different cultural characteristics of
C. botulinum has been identified. The germination of
the spores, the outgrowth, and toxin production by
C. botulinum do not occur in food with pH 4.5 or less.
International Journal of Gerontology | September 2007 | Vol 1 | No 3
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Botulism: A Frequently Forgotten Old Malady
Molecular Properties of Botox
The chemical structures and molecular properties of
Botox have been described in detail in a number of
articles6–21. By using polyacrylamide gel electrophoresis with sodium dodecyl sulfate, all types of Botox,
except type E, showed a similar disulfide-linked dichain molecular structure. The heavy chain (H-chain)
is the larger subunit with molecular weight of about
twice as heavy as that of the complementary light
chain subunit. H-chain can be cleaved by trypsin or
chymotrypsin into H-1 and H-2 fragments. All of these
subunits are antigenically distinctive. The maximal
toxicity of all Botox depends upon the integrity of the
disulfide-linked di-chain configuration.
Six other physical and molecular properties of
Botox are listed as follows:
1. The neurotoxic crystals following protein purification
from type A culture yields a homogeneous entity of
90,000 daltons (90 kDa) protein molecule.
2. The molecular moiety of the crystal has a hemagglutination activity that is being absorbed by red
blood cells without affecting the toxic titer. This
specific hemagglutination effect was first reported
by Lamanna et al in 19718.
3. Two protein peaks have been recognized from the
ion-exchange chromatography by using diethylaminoethyl Sephadex column. The first peak is
the alpha fraction (20% of the applied protein) that
shows high toxicity, approximately three- to fivefold
more toxic than the crystals per se, and it reveals
little hemagglutinating activity. The second peak is
the beta fraction that has the reverse properties.
4. Botox molecules show a core strand within a coil
of a hemagglutinin helix on electron microscopic
examination.
5. On ultracentrifugation of type A culture, Botox shows
toxic peaks at 7.2S, M-complex, L-complex, and 19S
components.
6. On gel filtration, the neurotoxic peak reveals a homogeneous molecule of 150,000 daltons (150 kDa).
Relative Toxicities of Botulinum
Neurotoxin Complexes
The toxicity of botulinum neurotoxin complexes depends upon several factors, some of which are intrinsic factors and others extrinsic. The relative toxicity
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potencies of the components of type A culture, 7S,
M-complex, L-complex, and 19S, are 1, 12, 20, and 360,
respectively. For type B culture, the potency of 7S/
M-complex/L-complex is 1:20:16,000. By oral route of
intoxication, the larger complexes of Botox have the
greater potency of toxicity, probably due to the associated protein on the surface of the molecules which protects the neurotoxins per se from inactivation in the gut
and gives them a greater opportunity to be absorbed
before being inactivated. Further, pure components of
Botox are more easily detoxified than the complexes by
pepsin, pancreatin, and gastric and intestinal juices22.
The estimated LD50 of Botox for humans is 5 to
50 ng/kg of body weight23. Once the toxin and cell
binding occurs, the axon terminals are permanently
destroyed24, and partial recovery is only possible when
the sprouting of axonal terminals makes new neuronal
connections.
How does Botox affect the nervous system? Three
steps of action on the axon terminals and the neuromuscular transmission have been recognized.
1. Botox irreversibly binds to the axon terminal. It is
calcium independent and minimally temperature
dependent25. At the earliest stage, the toxin does not
interfere with the neuromuscular transmission. Use
of antitoxin in early phase of botulism is still effective, because the antitoxin reacts with the toxin and
neutralizes its neurotoxicity.
2. Translocation step. Botox is internalized into the cell
and exerts its effect in the axoplasm, and thus, use
of antitoxin becomes ineffective.
3. Lytic step. The toxins interfere with the release of
acetylcholine from the axon terminals to the neuromuscular junctions. The lytic step is calcium milieu
dependent and temperature sensitive.
Clinical Forms of Human Botulism
Botulinum toxins affect humans by several routes, and
thus cause various clinical forms.
Food-borne botulism is caused by ingestion of preformed neurotoxin in the contaminated food. It is intriguing that clinical botulism will not develop in all
people who ingest the toxin26. The diversity of vulnerability of every individual to this neurotoxin may dictate
different genetic backgrounds of the susceptibility. It
has been reported that 31% of 36 patients died of food
poisoning caused by C. botulinum toxin type A in 14
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T. Thajeb et al
different areas of Japan27. Most of these victims had
eaten a special local food product used for gift in
Kumamoto. The commercially available food product
was made of fried lotus-rhizome solid mustard.
In vivo Botox production in human body has two
common routes. In infant botulism, Botox are produced
in vivo after multiplication of C. botulinum in the infant
intestinal tract. Type A and B Botox are the most common cause of infant botulism. It has been reported that
as low as 10 out of 280 cases of sudden infant death
syndrome (SIDS) were caused by infantile botulism28.
In an elegant pathologic study of 70 necropsies of infants
died of SIDS, as high as 15% of all cases of SIDS were
shown to bear Botox and C. botulinum of different types
(A, B, C, F, and G) in the contents of the ileojejunum or
colon29. The second route of human in vivo botulism
is well recognized as “wound botulism”. The Botox are
produced in the infected wound and circulated to the
body by hematogenous spread.
In some cases of human botulism, neither vehicle
nor toxin can be identified. We call this “unclassified
human botulism”. Some other form of unidentified
new strain may play a role. In an autopsied series of
95 patients with unexplained death, four adults and an
18-month-old infant had C. botulinum type G isolated
from their intestines30.
Diagnosis of Human Botulism
The diagnosis of botulism can only be reached if the
clinician keeps the possibility of botulism in mind.
The clues to rapid diagnosis are as follows.
Clinical signs of acute descending neuroparalytic
syndromes start from ocular and bulbar weakness,
followed by more generalized limb and respiratory
paralysis. Consciousness remains clear prior to the respiratory failure and the subsequent complication of
brain hypoxia. It is important to exclude many other
diseases that resemble botulism, such as Guillain–Barré
syndrome and myasthenia gravis.
Demonstration of the source of Botox intoxication is
utmost important. Necessary survey and work-up has to
be done for confirming the food-borne botulism or the
wound botulism, the endemic occurrence, or the sporadic unclassified human botulism. Different cultural
backgrounds and common habits for processing foodstuff may explain the high incidence of human botulism
in several geographic regions of the world. The higher
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incidence of human botulism in certain parts of China
is shown in Figure 1. Taiwan is an area of low incidence.
Shih and Chao31 have reported 986 outbreaks of botulism during the period from 1958 to 1983. The outbreaks affected 4,377 individuals and resulted in 548
deaths in China31. The incidence was highest in Xinjiang
province. Type A botulinum toxin was the most common in the northwest region, whereas type B in the
north, and type E in the northeast of China. The most
frequent offending food was homemade strong smelling
preserved bean curd (74%). We speculate that although
this type of bean curd is very popular in Taiwan, botulism is uncommon in Taiwan probably because of the
habit of Taiwanese using high-temperature cooking
process before eating bean curd and good public sanitation. An eloquent ecologic study on C. botulinum in
the soils of Taiwan has been reported32.
Identification of the strains of the invading microorganisms, C. botulinum, and the types of Botox they
produce may be rewarding. In the 12-year period of
a survey among 336 infants with suspected infantile
botulism, 113 out of 336 patients had positive stool
culture for group I (proteolytic) C. botulinum. Among
them, 69 showed type B botulinum neurotoxin, and 28
patients revealed type A toxin. Type E, type F, and two
or more different strains of organism were also found
in a few cases. The neurotoxins could be identified in
the feces of 98 out of 111 culture-positive victims,
whereas it could only be seen in the serum of 9 out of
67 stool culture-positive infants33. Therefore, early detection of the neurotoxins is more sensitive in the stool
specimens than in the serum. In affected Chinese adults,
types A and B are the most common toxins encountered. In other parts of the world, such as the northern
Quebec of Canada, type E neurotoxin is more common
(52 of 61 outbreaks)34. The outbreaks were attributed
to eating raw, parboiled or fermented meats (59%)
from marine mammals, fermented salmon eggs, or
fish (23%). Similarly, an outbreak of type E botulism
has been reported in the Middle East country Iran35. In
contrast, the largest outbreak of food-borne botulism
in the United Kingdom in June 1989 was attributed to
type B botulinum intoxication that came from improperly processed hazelnut yoghurt contaminated with
C. botulinum spores36. Different strains of food-borne
botulism were reported in the United States37.
Electrodiagnosis of human botulism include objective evidence showing a failure of neuromuscular transmission38, such as a decrementing response following
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∗
Nei Monggol
Botulism: A Frequently Forgotten Old Malady
∗
Heilongjiang
Northeast
Xinjiang
∗
l
rea
Qinghai
Xizang (Tebet)
∗
∗
Liaoning
∗
∗
∗Henan
∗
ng
do
an
Sh
Jiangsu
Anhui
Hubei
Sichuan
Jianxi
Hunan
Zhejiang
Fujian
∗
Taiwan
ai
Guandong
∗
H
Guanxi
n
Yunnan
Area line
Na
Province line
Se
a
Guizhou
∗
Hebei
a
h are
Nort
st a
Shaanxi
∗
Mo
Shanxi
∗
we
gxia
Nin
Gansu
rth
i
Ne
Jilin
∗
n
No
∗
o
gg
area
Provinces with botulism
Hainan Island
Islands of the Nan Hai Sea
Figure 1. Map of mainland China and Taiwan shows areas of high incidence (mustard colour) and low incidence (light yellow).
Botulism-free areas are in white.
repetitive stimulation of nerves (Figure 2). These decrementing responses simulate myasthenia gravis. However, botulism differs from myasthenia gravis in that
the former causes decrease in acetylcholine quantal
release from the autonomic and motor nerve endings39,
whereas the latter reveals an immune-mediated defective function of the postsynaptic acetylcholine receptors. Further, nerve conduction studies reveal a pattern
of axonal change with reduced amplitude of the compound muscle action potentials and nerve action
potential in case of botulism, but not in the patients
with myasthenia gravis. The nerve conduction velocities are relatively preserved in botulism (Figure 3). The
characteristic features on electrophysiologic tests may
also distinguish botulism from another common acute
motor paralytic syndrome, called the classic form of
Guillain–Barré syndrome. Rarely, a patient with botulism
can be associated with Guillain–Barré syndrome40.
A pathologic study of biopsied muscles taken from
the biceps brachii of three patients with chronic type A
botulism revealed not only the ultrastructural alterations at the presynaptic region, but also the postsynaptic structures such as denuded nerve terminals at
the motor end-plate and hypertrophy of the junctional
folds. These findings were not seen in patients with
International Journal of Gerontology | September 2007 | Vol 1 | No 3
Figure 2. Repetitive stimulation of the median nerve at 3-Hz,
5-Hz and 10-Hz stimulation frequencies showed a 39% decrement response, suggesting a defective transmission at the
neuromuscular junction. The initial incremental response
in the case of botulism makes it different from myasthenia
gravis.
Eaton–Lambert myasthenic syndrome41. Finally and
most importantly, it should be emphasized that the
autonomic dysfunction is an early sign of botulism. The
control of heart rate and blood pressure responsiveness is remarkably impaired in the early stage of botulism. Loss of heart rate variability and blood pressure
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variability can be seen in the first 48 hours of onset of
botulism (Figure 4). At recovery, the sympathetic functions normalized earlier than the parasympathetic functions, and the neuromuscular transmission recovers
earlier than the autonomic function42. In contrast, the
autonomic dysfunctions are not early signs of myasthenia gravis.
Regarding the bioassay, the intraperitoneal injection of Botox, prepared from the patients’ specimen, into
a group of 18–20 g female Swiss-Webster mice yields
toxic effect that causes a certain number of mouse
lethality/mortality. The dose that produces 50% of
mouse lethality (LD50, lethal dose 50) is defined as one
mouse unit. Commercially available Botox for therapeutic usage contains 50 ng of freezed and dried toxin
hemagglutinin in one-vial preparation, which is equal
A1
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T. Thajeb et al
to 2,000 mouse units. Therefore, one mouse unit is
approximately equal to 0.4 ng of the protein toxin.
Treatment of Human Botulism
The keystone to the success of treatment of human
botulism depends on the early detection of the disorder.
Use of botulism antitoxin is effective only in the early
stage of the disease. There is lack of evidence-based
data to support use of plasma exchange in botulism,
although it has been tried on some occasions. Supportive
treatment, especially respiratory care, is utmost important. Recovery of the upper airway muscles may take as
short as 12 weeks to as long as 12 months following
appropriate ventilation support and exercise performance43. Nutritional support and extensive rehabilitation
may be helpful. Guanidine hydrochloride (50 mg/kg)
may be useful to reverse in part the motor paralysis5.
However, the neurologic recovery from the axonal
nerve injury is often slow and incomplete.
L1
Acknowledgments
This work was supported in part by grant number
MMH-9565.
L1
B1
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Figure 3. Nerve conduction studies showed axonal change
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