Clostridium Botulism

Clostridium botulinum
I. Organism Information
A. Taxonomy Information
1. Species:
a. Clostridium botulinum(Website 1):
i. GenBank Taxonomy No.: 1491
ii. Description: The threat posed by botulism, classically a food- and waterborne disease with a high
morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum
neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to
contaminate the food or water supply, the Centers for Disease Control and Prevention and the
National Institute of Allergy and Infectious Diseases has classified it as a category A agent
(Kobayashi et al., 2005). Food-borne botulism probably has accompanied mankind since its
beginning. However, we have only few historical sources and documents on food poisoning before
the 19th century. Some ancient dietary laws and taboos may reflect some knowledge about the life-
threatening consumption of poisoned food. One example of such a dietary taboo is the 10th century
edict of Emperor Leo VI of Byzantium in which manufacturing of blood sausages was forbidden.
Some ancient case reports on intoxications with Atropa belladonna probably described patients with
food-borne botulism, because the combination of dilated pupils and fatal muscle paralysis cannot be
attributed to an atropine intoxication. At the end of the 18th century, some well-documented
outbreaks of "sausage poisoning" in Southern Germany, especially in Wurttemberg, prompted early
systematic botulinum toxin research. The German poet and district medical officer J ustinus Kerner
(1786-1862) published the first accurate and complete descriptions of the symptoms of food-borne
botulism between 1817 and 1822. Kerner did not succeed in defining the suspected "biological
poison" which he called "sausage poison" or "fatty poison." However, he developed the idea of a
possible therapeutic use of the toxin. Eighty years after Kerner's work, in 1895, a botulism outbreak
after a funeral dinner with smoked ham in the small Belgian village of Ellezelles led to the discovery
of the pathogen Clostridium botulinum by Emile Pierre van Ermengem, Professor of bacteriology at
the University of Ghent. The bacterium was so called because of its pathological association with
the sausages (Latin word for sausage- "botulus") and not-as it was suggested-because of its shape.
Modern botulinum toxin treatment was pioneered by Alan B. Scott and Edward J . Schantz (Erbguth,
2004). Over 100 years ago, the classic food-borne type was found to be caused by ingesting
contaminated food containing the toxin produced by a bacteria. In the first half of the 20th century a
second form, wound botulism, was discovered. Three additional forms (infant, hidden, and
inadvertent) were first described in the last quarter of the 20th century (Cherington, 2004). Botulism
is today divided into 5 clinical forms: classic or food-borne botulism, infant botulism, wound
botulism, hidden botulism, and inadvertent botulism (Caya et al., 2004). BoNTs are serologically
differentiated according to their neutralization with type-specific antitoxins into seven serotypes,
designated by the letters A through G (Dineen et al., 2003). Based on the toxin type produced, C.
botulinum strains are divided in groups I to IV, with groups I and II being the main human
pathogens. Group I consists of proteolytic types A, B, and F, and group II consists of nonproteolytic
types B, E, and F. The two groups are completely different in their phenotypical characteristics, such
as temperature requirements, biochemical profile, and production of metabolites (Lindstrom et al.,
2001). Types C and D provoke botulism in animal species, including the avian form (Arimitsu et al.,
2004). All eight neurotoxins (BoNT/A to BoNT/G and TeTx (tetanus toxin)) are synthesized as a
single-chain, 150,000 Da molecule which subsequently becomes nicked to the more potent dichain
form, composed of a heavy (H) (approximately 100,000 Da) chain and a light (L) (50,000 Da) chain
polypeptide linked by at least one disulfide bridge (Whelan et al., 1992 (a)).
iii. Variant(s):
Clostridium botulinum A :
GenBank Taxonomy No.: 36826
Description: Types A, B, and F are coded for by genes located on clostridial chromosomal
material (Caya et al., 2004). The complete sequence of botulinum neurotoxin type A
(BoNT/A; 1,296 amino acid residues, Mr 149,425). A comparison of the protein sequence
revealed an overall identity of 33.8% to that of tetanus toxin (TeTx). No significant similarity
to other known proteins including ADP- ribosylating toxins could be detected (Binz et al.,
1990 (b)). Conservation of Cys residues flanking the position at which the toxins are cleaved
to yield the heavy chain and light chain allowed the tentative identification of those residues
which probably form the disulfide bridges linking the two toxin subfragments (Thompson et
al., 1990). From 1990 to 2000, 160 foodborne botulism events affected 263 people in the
United States, an annual incidence of 0.1 per million. Toxin type A caused 50% of all cases.
The implicated foods in 40 (50%) were home-canned products (27 vegetable items, two home-
canned soups, two home-canned tuna items, one each home-canned tomato juice, garlic-in-oil,
and stew). Other implicated home-prepared foods included five events from potato salad or
potatoes, four from homemade soup, two from homemade sausage, and one from each of the
following: roast beef, liver pate, bread pudding, salsa, apple pie, hamburger, and chili. In 18
(23%) events caused by botulinum toxin type A, the specific food vehicle was not identified
(Sobel et al., 2004).
Clostridium botulinum B :
material (Caya et al., 2004). Translation of the nucleotide sequence derived from all three
clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative
alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus
toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which
component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest
degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most
homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were
shown to be composed of highly conserved amino acid domains interceded with amino acid
tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are
absolutely conserved between L chains and 110 of 845 amino acids are conserved between H
chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was
particularly striking. The most divergent region corresponds to the extreme carboxy terminus
of each toxin, which may reflect differences in specificity of binding to neuron acceptor sites
(Whelan et al., 1992 (a)). During the period under study, 15 events were caused by type B
botulinum toxin. The implicated foods in five (33%) of the type B toxin events were home-
canned products (corn, eggs, green beans, olives, and an unspecified vegetable), and one event
was due to each of the following: a commercially manufactured burrito, commercially caught
and sold fish, pasta sauce and meatballs, salsa, turnip greens, and peyote. In four (25%) events,
the food vehicle was not identified (Sobel et al., 2004).
Clostridium botulinum C :
Description: The production of neurotoxins C, D, and E is coded for by genes carried by
bacteriophage (Caya et al., 2004). The complete C I DNA sequence shows a single open
reading frame which begins with an ATG at position 207 and encodes for 1291 amino acids
(Mr 148,698 Da). Cys437 and Cys453 are probably involved in a disulfide bridge linking the
light and heavy chains (Hauser et al., 1990). In Japan, some farmers have used ducks, named
"Aigamo" in J apanese, which are cross strain of J apanese Mallard and Khaki Campbell, for
reducing the chemicals in the rice. A few hundred ducks died of botulism in a certain area of
Ishikawa prefecture. These ducks showed symptoms of leg and wing paralysis and became
weak and listless. C. botulinum type C organisms were isolated from the contents of the gastric
tract of the carcass and environmental materials such as soil, maggots, food, and (or) straw
mats (Arimitsu et al., 2004). Botulism due to Clostridium botulinum type C causes
considerable mortality in gulls in the UK, and refuse disposal sites are suspected as a major
source of toxin (Ortiz and Smith, 1994).
Clostridium botulinum D :
bacteriophage (Caya et al., 2004). The sequence of the BoNT/D gene contains a single open
reading frame beginning at the ATG codon in position 47 and encodes polypeptide of 1276
amino acid residues (Mr 146,872 Da). Cys437 and Cys450 are probably involved in the
disulfide bond between the light and the heavy chain. BoNT/D shares 49.6% amino acid
sequence identity with BoNT/C (46.3% within the light chain, 67% within the N-terminal part
of the heavy chain, and merely 35% within the putative fragment C) (Binz et al., 1990 (a)).
Fifty-two feedlot cattle exhibited clinical signs suggestive of botulism. Clostridium botulinum
type D organisms were recovered from ruminal fluid of 4 of the 5 affected animals tested and
were isolated from bakery waste fed to the cattle. Clostridium botulinum type D has not been
reported previously in Canadian cattle (Heider et al., 2001).
Clostridium botulinum E :
bacteriophage (Caya et al., 2004). Translation of the sequence has shown botulinum
neurotoxin type E (BoNT/E) to consist of 1252 amino acids and, as such, represents the
smallest BoNT characterized to date. The light chain of the toxin exhibits the highest level of
sequence similarity to tetanus toxin (TeTx, 40%). The light chains of BoNT/A and BoNT/D
share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity. In contrast, the
TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT
heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively
(Whelan et al., 1992 (b)). From 1990 to 2000, 160 foodborne botulism events affected 263
people in the United States. During the period under study, 58 botulism events occurred in
Alaska; 103 persons were affected, with a median of 5 events (range 015) and 8 cases (range
020) occurring per year. Forty-nine (84%) events and 91 (88%) cases were caused by toxin
type E, all involving foods from aquatic animals. All identified foods were homemade Alaska
Native foods. Eleven events, affecting 21 persons, were caused by foods consisting of
fermented aquatic mammal tissues, such as mukluk (whale skin and blubber), beaver tail, and
seal flipper. Fourteen events, affecting 20 persons, were caused by seal oil; 14 events, affecting
28 persons, were caused by fish foods such as fermented salmon heads and whitefish; 7 events,
affecting 18 persons, were caused by fermented fish eggs; and 3 events, affecting 5 persons,
were caused by foods with mixed ingredients (Sobel et al., 2004). Clostridium botulinum type
E was found in 81% of sea and 61% of freshwater samples. No other toxinotypes were found.
These findings indicate the possibility of Clostridium botulinum type E multiplication or at
least, suitable conditions for spore survival, in anoxic sediments (Hielm et al., 1998).
Clostridium botulinum F :
material (Caya et al., 2004). Primers designed to conserved regions of botulinum and tetanus
clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium
botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments
were cloned and the complete nucleotide sequence of the gene encoding type F toxin
determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame
encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of
the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein
with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin (East et
al., 1992). Botulism caused by type F botulinum toxin accounts for less than 0.1% of all
human botulism cases and is rarely reported in the literature. The treating physician initially
considered the possibility of paralytic shellfish poisoning due to a report of shellfish ingestion,
which was later determined to be frozen shrimp and a can of tuna, but no gastroenteritis or
paresthesias were present (Richardson et al., 2004).
Clostridium botulinum G :
Description: The gene responsible for type G neurotoxin is present on a plasmid (Caya et al.,
2004). The neurotoxin gene from Clostridium botulinum type G was cloned as a series of
overlapping DNA fragments generated using polymerase chain reaction (PCR) technology and
primers designed to conserved regions of published botulinal toxin (BoNT) sequences.
Translation of the nucleotide sequence derived from the cloned PCR fragments demonstrated
that the gene encodes a protein of 1297 amino acid residues. Comparative alignment of the
determined BoNT/G sequence with those of other clostridial neurotoxins revealed highest
sequence relatedness (approx. 58% amino acid identity) with BoNT/B of proteolytic and non-
proteolytic C. botulinum. Tetanus toxin and other BoNT types revealed lower levels of
relatedness with BoNT/G (approximate range 35-42% amino acid identity) (Campbell et al.,
1993). The last antigenic toxin type to be discovered was type G. Clostridium botulinum
producing neurotoxin type G was originally isolated from soil samples in Argentina, (Bhandari
et al., 1997) now recognized as Clostridium argentinense (Dineen et al., 2003).
B. Lifecycle Information:
No lifecycle information is currently available here.
C. Genome Information:
No genome information is currently available here.
II. Epidemiology Information
A. Outbreak Locations:
1. From 1973 through 1996 in the United States, 724 cases of foodborne botulism (median, 24 cases
annually (range, 8 to 86 cases)), 103 cases of wound botulism (median, 3 cases annually (range, 0 to 25
cases)), 1444 cases of infant botulism (median, 71 cases annually (range, 0 to 99 cases)), and 39 cases of
botulism of undetermined type were reported to the Centers for Disease Control and Prevention (CDC). In
the United States, approximately half of the cases of foodborne botulism are caused by toxin type A; the
remaining foodborne cases are almost equally divided between toxins type E and type B. Among cases of
infant botulism, approximately half are caused by toxin type A and half by toxin type B; among cases of
wound botulism, approximately 80% are caused by toxin type A and 20% by toxin type B. In the United
States, type A botulism is most common west of the Mississippi River, and type B is most common east
of the Mississippi River. Type E outbreaks are most common in Alaska (Shapiro et al., 1998).
B. Transmission Information:
1. From: Clostridium botulinum Environmental Reservoir To: Human(Shapiro et al., 1998):
Mechanism: Foodborne Botulism. Foodborne botulism is caused by ingestion of preformed toxin
produced in food by C. botulinum. The most frequent source is home-canned foods, in which spores that
survive an inadequate cooking and canning process germinate, reproduce, and produce toxin (Shapiro et
al., 1998). Spores of C. botulinum are ubiquitous in the environment, but growth and elaboration of toxin
occur only under particular conditions that include an anaerobic, low-salt, low-acid environment. The
canning and fermentation of foods are particularly conducive to creating anaerobic conditions that allow
C. botulinum spores to germinate. Foodborne botulism, while rare, remains a public health emergency
because of its severity and epidemic potential. Home-canned foods and Alaska Native foods remain the
leading causes in the United States, and restaurant-associated outbreaks continue to account for a
disproportionate number of illnesses (Sobel et al., 2004).
2. From: Clostridium botulinum Environmental Reservoir To: Human(Caya et al., 2004):
Mechanism: Wound Botulism (WB). Since 1988, California has experienced a dramatic increase in
wound botulism associated with injecting "black tar" heroin (BTH), a dark, tarry form of the drug. Among
the 26 patients, the median age was 41.5 years, 15 (58%) were women, 14 (54%) were non-Hispanic
white, 11 (42%) were Hispanic, and none were positive for the human immunodeficiency virus. Nearly all
participants (96% of patients and 97% of controls) injected BTH, and the mean cumulative dose of BTH
used per month was similar for patients and controls (27 g and 31 g, respectively; P~.6). Patients were
more likely than controls to inject drugs subcutaneously or intramuscularly (92% vs 44%, P less .001) and
used this route of drug administration more times per month (mean, 67 vs 24, P less .001), with a greater
cumulative monthly dose of BTH (22.3 g vs 6.3 g, P less .001). A dose-response relationship was
observed between the monthly cumulative dose of BTH injected subcutaneously or intramuscularly and
the development of WB ({chi}2 for linear trend, 26.5; P less .001). In the final regression model,
subcutaneous or intramuscular injection of BTH was the only behavior associated with WB among IDUs
(odds ratio, 13.7; 95% confidence interval, 3.0-63.0). The risk for development of WB was not affected
by cleaning the skin, cleaning injection paraphernalia, or sharing needles. From 1951 through 1995, 68
cases of WB were reported to California Department of Health Services (CDHS). An average of 0.49 WB
cases per year were reported from 1951 through 1987; 2.25 cases per year were reported in 1988 through
1991, 3 cases in 1992, 4 in 1993, 11 in 1994, and 23 in 1995. 18 From 1988 through 1995, only 2 WB
cases among IDUs were reported from outside California, and they occurred in Arizona (Foodborne and
Diarrheal Disease Branch, Centers for Disease Control and Prevention, unpublished data, 1996) (Passaro
et al., 1998). Under conditions of tissue necrosis and anaerobiosis, such as those seen in a subcutaneous
abscess, C. botulinum spores can germinate and produce neurotoxinin vivo, with the same clinical
features as those seen in food-borne botulism, except for a lack of acute gastrointestinal signs and
symptoms (Caya et al., 2004).
Mechanism: Infant botulism, first described in 1976, is now the most frequently reported form
(Cherington, 1998). Since 1980, it has become the most common form of botulism reported in the United
States. Unlike food-borne botulism, the infant form is a combination infection intoxication, including
ingestion of C botulinum spores, germination within the gastrointestinal tract, andin vivo production of
toxin; these events occur in part due to the lack of a protective gastrointestinal bacterial flora and in part
due to the relatively reduced levels of clostridial-inhibiting bile acid as compared to the adult
gastrointestinal tract (Caya et al., 2004). The source of ingestion is unknown in approximately 85% of
cases; in up to 15% of cases, the ingestion of honey is suspected (Shapiro et al., 1998). A significant risk
factor for the development of infant botulism is honey consumption; 15% to 25% of honey products
harbor botulinum spores (especially type B) (Caya et al., 2004). In addition, honey samples across the
United States have tested positive for Clostridium botulinum spores and toxins. Such substantial evidence
led the CDC to recommend that honey not be given to infants younger than 12 months old. It is important
that clinicians be familiar with this risk and should not recommend honey-containing products or
supplements or the use of honey as a flavoring agent for infants in this age group (Tanzi and Gabay,
2002).
Mechanism: Hidden Botulism. Hidden botulism (also known as intestinal colonization botulism) refers to
those cases of botulism in adults for which there is no readily apparent source of botulinum toxin. Many
of these patients may actually represent an adult form of infant botulism (also known as adult form of
intestinal botulism). Clostridium botulinum organisms are present within the gastrointestinal tracts of
these patients, where they proliferate and produce neurotoxinin vivo (Caya et al., 2004). Such patients
often have a history of abdominal surgery, gastrointestinal tract abnormalities, or recent antibiotic
treatment that may disrupt the natural gastrointestinal flora (Shapiro et al., 1998).
Mechanism: Inadvertent botulism. Inadvertent botulism, the most recent type to be recognized by the
medical community, is either an iatrogenic disease (resulting from the therapeutic use of botulinum toxin)
or occurring as an accidental exposure in laboratory workers. Indeed, the literature includes a recent
report of full-blown botulism resulting from therapeutic botulinum toxin use in at least 2 patients. Three
cases of botulism have been reported in laboratory workers who apparently contracted the disease by
inhalation of the toxin; an inhalational route provides a potential basis for the use of botulinum toxin as an
agent of bioterrorism or biocrime warfare (Caya et al., 2004).
C. Environmental Reservoir:
1. Clostridium botulinum Environmental Reservoir :
a. Description: These neurotoxigenic organisms are anaerobic, gram-positive, spore-forming bacilli and
are commonly found in soils throughout the world (Shapiro et al., 1998). Clostridium botulinum spores
are quite widespread throughout the world, inhabiting soil as well as both freshwater and saltwater mud
(Caya et al., 2004). The prevalence of C. botulinum in Swedish cattle was established to be 73% for
non-proteolytic type B and less than 5% for types E and F. Twenty-eight (64%) of the positive faecal
samples had a spore load of less than 4 spores/g. Statistical analysis (ANOVA) showed that seasonal
variation (summer and winter) had a significant effect on the prevalence of C. botulinum type B in
cattle, whereas the effect of geographical location of rearing of the cattle (southern and central Sweden)
was less significant (Dahlenborg et al., 2003).
b. Survival Information: These very resilient spores are capable of surviving for up to 2 hours at 100C.
The botulinum neurotoxin may be inactivated by 0.2 M sodium hydroxide. Clostridium botulinum is
inactivated by a 1:10 dilution of household bleach with a 20-minute contact time. For successful
inactivation of both organism and toxin, both bleach and sodium hydroxide must be applied for a total
of 40 minutes (Caya et al., 2004).
D. Intentional Releases:
1. Intentional Release information :
a. Description: Botulinum neurotoxin is considered the most potent lethal substance known. It is 15 000
to 100 000 times more toxic than sarin, the potent organophosphate nerve agent. Studies in monkeys
indicate that, if aerosolized, botulinum toxin also can be absorbed through the lungs; this could occur in
the case of a terrorist attack (Shapiro et al., 1998). It is sobering to consider that botulinum toxin is a
potentially effective agent for the mass destruction of human life in biological warfare/bioterrorism
contexts and is indeed categorized as a biothreat level A biological warfare agent. Botulinum toxin is
not only highly lethal after ingestion of minute amounts (in water and/or food), but can also cause
disease via the inhalational route and therefore potentially lends itself to biowarfare/bioterrorism
activity. Indeed, unsuccessful attempts to utilize botulinum toxin in the bioterrorism context occurred
in J apan on at least 3 occasions between 1990 and 1995. In 1995. In the same year, the Aum Shinrikyo
cult in J apan prepared botulinum toxin before its attack on the Tokyo subway system, even though it
instead chose to deploy the nerve agent sarin. Although bioterrorism-related botulinum toxin could be
introduced into its target population by contaminating food and water supplies, these vectors are
associated with significant limitations, including logistical difficulties. Therefore, most health care
experts and government officials have focused their strongest bioterrorism-related concerns on the
inhalational delivery of botulinum toxin into a target population. It has been suggested that inhaled,
aerosolized toxin is typically not identifiable in either stool or serum (Caya et al., 2004).
III. Infected osts
A. uman:
1. Taxonomy Information:
a. Species:
i. uman :
Scientific Name: o o i n (Website 4)
Description: It should be emphasized that botulism (in any form, including inhalational) is a
noncontagious disease that cannot be transmitted from affected to nonaffected persons, and that the
botulinum toxin cannot pass through intact skin (Caya et al., 2004).
2. Infection rocess:
a. Infectious Dose: Clostridium botulinum organisms elaborate a very potent neurotoxin that is one of the most
(if not the most) potent toxins known to man; as little as 10 pg is sufficient to kill a mouse, and the estimated
ingested human toxic dose is 1 ng/kg body mass (Caya et al., 2004).
b. Description: Botulism is a neuroparalytic illness caused by a neurotoxin produced from the anaerobic, spore-
forming bacterium Clostridium botulinum (Shapiro et al., 1998). Growth from bacterial spores occurs
through a number of steps that are distinguishable but poorly understood. The first of these steps is
germination. As the safety margin of minimally heat-processed refrigerated foods is small, it is imperative
that spore germination is well understood for non-proteolytic C. botulinum, the pathogen of most concern for
the safety of these foods. When germination occurred, it proceeded rapidly, with little further germination
after 6 h. Heat activation increased the initial germination rate, and increased or had no effect on the final
extent of germination (Plowman and Peck, 2002). FOOD-BORNE BOTULISM.This form occurs after
ingestion of food containing preformed neurotoxin, produced by clostridial organisms that contaminate
inadequately processed food. The archetypical example of food-borne botulism is that resulting from
ingestion of improperly prepared home-canned foodstuffs (Caya et al., 2004).
3. Disease Information:
a. Botulism :
i. athogenesis Mechanism: The botulinum neurotoxin is a metalloproteinase that enters nerve cells and
blocks neurotransmitter release by zinc-dependent cleavage of protein components of the neuroexocytosis
apparatus. Specifically, botulinum toxin types A and E target a protein known as SNAP-25, and toxin
types B, D, F, and G target the protein VAMP/synaptobrevin. Toxin C targets both SNAP-25 and syntaxin.
Target protein cleavage significantly disrupts release of the neurotransmitter acetylcholine. In its native
state, this neurotoxin is bound to nontoxic proteins, which greatly enhances toxin molecular stability.
Botulinum toxin introduced into the body (potentially by a number of mechanisms but most commonly by
absorption from the gastrointestinal tract) is carried throughout the body by the bloodstream. Toxin binds
to nerve-ending receptors, becomes internalized within the neuron, and causes an irreversible blockade of
cholinergic transmission at the following sites: all ganglionic synapses, all postganglionic parasympathetic
synapses, and all neuromuscular junctions. The far-reaching results at these sites include widespread
flaccid paralysis and potentially devastating autonomic nervous system perturbations (Caya et al., 2004).
ii. Incubation eriod: Onset usually occurs 18 to 36 hours after exposure (range, 6 hours to 8 days) (Shapiro
et al., 1998).
iii. rognosis: The prognosis for botulism has improved greatly during the past 50 years, coincident with the
advent of modern supportive care measures provided in the intensive care setting, especially featuring
pulmonary function protection with the mechanical ventilator. Thus, the case fatality rate for food-borne
botulism has decreased from the 50% to 70% range (worse for C botulinum neurotoxin type A) to the 5%
to 20% range seen today. Infant botulism and wound botulism currently have case fatality rates of
approximately 15% and 1%, respectively. It should be stressed that survival of an episode of botulism
provides essentially no immunity and therefore offers no protection from future bouts of this disease (Caya
et al., 2004).
iv. Diagnosis Overvie : CLINICAL FINDINGS. Botulism is underdiagnosed because many clinicians are
unfamiliar with the disease and because symptoms can be mistaken for more common clinical entities,
such as stroke or the Guillain-Barre syndrome. However, the diagnosis of botulism is not difficult in most
cases once it has been considered. Botulism should be suspected in a patient with acute onset of
gastrointestinal, autonomic (such as dry mouth or difficulty focusing eyes), and cranial-nerve (diplopia,
dysarthria, dysphagia) dysfunction. The diagnosis is even more likely if the patient has recently eaten
home-canned foods or if family members or companions who have shared meals are similarly ill.
ANCILLARY TESTING. The diagnosis of botulism is supported by ancillary testing, such as
documentation of a normal result on magnetic resonance imaging or computed tomography of the brain to
rule out stroke syndrome; a normal result on lumbar puncture to differentiate botulism from the Guillain-
Barre syndrome, which typically causes elevated levels of protein in the cerebrospinal fluid (although
protein levels may be normal initially); and a negative edrophonium chloride test result to rule out
myasthenia gravis (although transient responses may occasionally be noted in botulism).
Electromyography usually reveals decreased amplitude of action potentials in affected muscle groups, but
this finding is relatively nonspecific. An incremental increase in amplitude to rapid repetitive
electromyography by using frequencies of 20 to 50 Hz is more helpful and may distinguish botulism from
the Guillain-Barre syndrome or myasthenia gravis but not the Eaton-Lambert syndrome.
Electromyography should be performed by a person experienced in performing rapid repetitive testing. In
most cases, lumbar puncture and brain imaging can be performed within hours of presentation. Negative
results may raise the clinical suspicion for botulism and should prompt close monitoring for respiratory
compromise; rapid repetitive electromyography; and, possibly, edrophonium chloride testing. State or
local health officials should be contacted to discuss potential measures for preventing additional cases; the
possible release of antitoxin by CDC; and the collection of serum and stool samples at the earliest possible
opportunity to confirm the diagnosis of botulism by the detection of toxin if none of the ancillary tests is
pathognomonic. TOXIN - MICROBIOLOGICAL TESTING. In cases of suspected foodborne botulism,
serum and stool specimens and epidemiologically implicated foods should be tested for botulism
neurotoxin. The most reliable method for the detection of toxin is the mouse inoculation test; this can be
performed at the CDC or some state public health laboratories. Botulinum toxin type is determined by
neutralizing the biological activity of toxic samples injected into mice with type-specific botulism anti-
toxin. Symptoms of botulism and death occur in mice injected with unneutralized samples but not in mice
injected with neutralized samples. Toxin is detected in serum or stool specimens in approximately 46% of
clinically diagnosed cases. Stool specimens also should be cultured for C. botulinum because a positive C.
botulinum culture from stool is also considered confirmatory for botulism. Isolation of neurotoxigenic
organisms from stool specimens increased the sensitivity of laboratory testing to 73% in one case series
and 67% in another. Detection of botulinum toxin from epidemiologically implicated food may provide
additional confirmatory evidence for botulism; however, the isolation of C. botulinum organisms from a
food devoid of toxin usually has little significance because spores are ubiquitous in the environment. If
wound botulism is suspected, such specimens as wound exudate, a tissue sample, or a swab sample should
be obtained for anaerobic culture in addition to a serum toxin assay. A stool specimen may be examined to
exclude food or intestinal colonization as sources of toxin. Infant botulism should be suspected in any
infant with constipation, poor feeding, diminished sucking and crying ability, neck and peripheral muscle
weakness, or ventilatory distress. Stool cultures for C. botulinum and testing for the presence of toxin in
stool should be performed in such patients (Shapiro et al., 1998). GENERAL PRINCEPELS. Appropriate
samples must usually be sent to a reference laboratory, typically a state health department laboratory
facility or the CDC. Laboratory evaluation includes anaerobic culture and toxin assays of appropriate
samples. Specimens should be obtained and sent to a reference laboratory after consultation with the state
epidemiologist, the state health department, and the CDC (emergency telephone number for the CDC:
(770) 488-7100. SPECIMEN TYPES. Food should be submitted in its original container. If the original
container is not available, the food should be put into a sterile, unbreakable, leak-proof container. In the
rare event of a suspect commercial food product, the unopened food container should be sent immediately
to the Food and Drug Administration. SPECIMEN TRANSPORT. Clinical specimens should be packaged
in an insulated container with refrigerant and transported to the laboratory as quickly as possible. All
specimens for isolation of C botulinum should be collected in an anaerobic transport medium. If there is an
unavoidable delay in transport, serum or feces may be frozen and shipped on dry ice; freezing clinical
specimens will not interfere with toxin detection but may compromise microbiologic culture detection of
clostridia (Caya et al., 2004).
v. Symptom Information :
General Description: BOTULISM SYMTOMS IN ADULTS:
Fatigue (Arnon et al., 2001):
Observed: 77 % (Arnon et al., 2001)
Di iness (Arnon et al., 2001):
Observed: 51% (Arnon et al., 2001)
Double vision (Arnon et al., 2001):
Blurred vision (Arnon et al., 2001):
Dysphagia (Arnon et al., 2001):
Dry mouth (Arnon et al., 2001):
Dysarthria (Arnon et al., 2001):
Sore throat (Arnon et al., 2001):
Dyspnea (Arnon et al., 2001):
Constipation (Arnon et al., 2001):
Nausea (Arnon et al., 2001):
Vomiting (Arnon et al., 2001):
Abdominal cramps (Arnon et al., 2001):
Diarrhea (Arnon et al., 2001):
Arm eakness (Arnon et al., 2001):
Leg eakness (Arnon et al., 2001):
aresthesia (Arnon et al., 2001):
vi. Treatment Information:
Antitoxin therapy: The administration of antitoxin is the only specific pharmacologic treatment
available for botulism. The currently available licensed antitoxin is an equine product with antibodies to
toxin types A, B, and E. The administration of trivalent equine antitoxin to humans by the intravenous
route neutralizes toxin molecules that are not yet bound to nerve endings. Before 1996, two to four 10-
mL vials were administered to each adult patient suspected of having botulism; however, one vial (7500
IU of type A, 5500 IU of type B, and 8500 IU of type E antitoxins) per patient is now administered, and
it is believed that no additional doses are necessary. Each vial contains an amount of antitoxin that is
more than 100-fold greater than that needed to neutralize the largest amount of circulating antitoxin
ever measured at the CDC. The circulating antitoxins have a half-life of 5 to 8 days, and a
hypersensitivity reaction has been reported for up to 9% of patients. After the change to single-vial
dosing, the incidence of hypersensitivity may be smaller than that previously reported. More than 80%
of patients with adult infectious botulism in the United States are treated with antitoxin. The remaining
20% generally have such a prolonged delay in diagnosis that treatment is considered to be of no benefit;
therefore, antitoxin is not administered. As of J une 1998, this product is available in the United States
solely for the treatment of infant botulism, under a Treatment Investigational New Drug protocol. For
information on obtaining human botulism immune globulin, contact the California Department of
Health Services at 510-540-2646 (24 hours) (Shapiro et al., 1998).
Contraindicator: Equine antitoxin therapy has not been recommended for infant botulism because
of early observations (since disproved) that serum toxin was not detected in such cases and because
of concerns about hypersensitivity reactions to this product (Shapiro et al., 1998)
Complication: Equine-derived antitoxin preparations have a number of possible side effects,
including serum sickness and anaphylaxis, and are not recommended for use in infant botulism but
do have a role in the treatment of food-borne and wound botulism in adults and noninfant children
(Caya et al., 2004).
Success Rate: If it is administered early during the course of neurologic dysfunction, it is effective
in preventing progression of illness and shortening the duration of ventilatory failure in severe cases
of botulism. A retrospective analysis of 134 cases of type A botulism showed an overall mortality
rate of 10% among patients who received early treatment with antitoxin (within 24 hours of
symptom onset) compared with 15% among those who received late treatment (more than 24 hours
after symptom onset) and 46% among those who did not receive antitoxin. In addition, survivors
who received antitoxin early had a median hospital stay of only 10 days compared with 41 days for
those who received antitoxin late and 56 days for those who did not receive antitoxin. The safety and
efficacy of a human-derived antitoxin product (human botulism immune globulin) administered to
infants with botulism are being determined (Shapiro et al., 1998).
Supportive care: Rapid diagnosis and the availability of well-coordinated, multidisciplinary
supportive-care capabilities constitute the most effective measures in the treatment of botulism
(whatever its type). Indeed, the keystone for supportive care is ventilatory support; protection and
control of the airway by intubation and mechanical ventilation may not be necessary in mild cases of
botulism, but they should be available and used whenever appropriate because these measures become
an absolute necessity in cases of severe botulism. Although it is true that botulinum neurotoxins bind
irreversibly to presynaptic endplates and irreparably impair these structures, axons possess a remarkable
regenerative capability by which they can sprout new endplates if the patient can be successfully
shepherded through the acute botulism crisis. Thus, supportive care is generally necessary for several
days to several weeks until the patient is capable of survival without these measures. Antibiotics and
debridement have very limited but nonetheless bona fide roles, principally in botulism of the wound
type, to treat infection and clean up an injury site (Caya et al., 2004).
Contraindicator: Aminoglycosides, clindamycin, and polymyxin B should especially be eschewed
because they have intrinsic neuromuscular blocking properties (Caya et al., 2004).
4. revention:
a. Botulism revention:
Description: C. botulinum spores are ubiquitous. Safe food preservation methods destroy spores or inhibit
their germination and growth. To reduce the risk for botulism when pickling, food items should be washed
and cooked adequately, and utensils, containers, and other surfaces in contact with food, including cutting
boards and hands, should be cleaned thoroughly with soap and warm water. Containers (e.g., jars and lids)
in which pickling will occur should be sterilized (e.g., placed in boiling water for the prescribed period
published in the container instructions). Adequate acidification to a pH less than or equal to 4.6 is
essential. Refrigeration at 39 F (4 C) during pickling is advisable, especially in foods that may be acidified
inadequately such as whole eggs. Once opened, any canned or pickled food should be refrigerated.
Pricking, poking holes, or otherwise handling whole eggs in a manner that might allow spores or bacteria
into the yolk should be avoided. When foodborne botulism is suspected, clinicians and public health
investigators should inquire about the preparation and eating of foods preserved by any home method
(e.g., canning, pickling, curing, and fermenting). Persons seeking advice on home-food preservation
should consult their local county or university cooperative extension service, or contact the U.S.
Department of Agriculture Food Safety Hotline, telephone (800) 535-4555. CDC provides epidemiologic
consultation and laboratory diagnostic services for suspected botulism cases and authorizes release of
botulism antitoxin. Through state health departments, these services are available 24 hours a day from
CDC (CDC report, 2000). Although ingesting honey is a known risk factor, the source of spores for the
majority of infant botulism cases is unknown. Ingestion of Clostridium botulinum spores, which exist
worldwide in the soil and dust, is believed to be the principal route of exposure. Avoid feeding honey to
infants aged less then 12 months. Report all cases to local and state health departments (CDC report,
2003).
5. Model System:
a. uman erythrocytes:
i. Model ost: Binding of the progenitor toxins and recombinant proteins to erythrocytes was determined in
a microassay (Fujinaga, 2000).
ii. Description: Microtiter plates (EIA/RIA Plate, Costar) were coated with the toxins or recombinant
proteins (100 mg/ml, 50 ml per well) at 4C for 16 h. The wells were washed twice with PBS, and free
binding sites were blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 2 h.
Then the wells were incubated with a 1% suspension of human type O erythrocytes in PBS containing 1%
BSA at room temperature for 30 min. The wells were carefully washed five times with PBS, the adherent
erythrocytes were lysed with H2O (50 microliters per well), and an absorbance at 405 nm of the
hemoglobin was taken as a measure of the number of adherent erythrocytes (Fujinaga, 2000).
b. Guinea pigs:
i. Model ost: Cavia. Guinea pigs (Website 9)
ii. Description: Binding experiments of the progenitor toxins and recombinant proteins to paraformaldehyde-
fixed sections of guinea pig upper small intestine (Fujinaga, 2000). Binding experiments were performed
using intestinal tissue sections. Type A HA(hemagglutinin)-positive progenitor toxin (a mixture of 16S
and 19S toxins) showed intense binding to the microvilli, whereas HA-negative progenitor toxin (12S
toxin) did not show any binding, as reported for type C progenitor toxins. Given that type C 16S toxin
exhibits NeuAc (N-acetylneuraminic acid)-dependent binding activity, the ability of type A HA-positive
progenitor toxin to adhere to sialidase-treated intestinal sections was tested. The sialidase treatment
showed no change in type A HA-positive progenitor toxin binding. In order to examine ligand(s) for type
A HA-positive progenitor toxin on microvilli, binding inhibition experiments using three different
saccharides were carried out. Binding was drastically inhibited by 100 mM lactose (Lac) and 100 mM
galactose (Gal), but not by 100 mM glucose (Glc). These results indicate that the HA but not NTNH (non-
toxic non-HA) or NTX (neurotoxin), components of type A HA-positive progenitor toxin exhibit binding
activity on microvilli, as observed for type C. However, unlike type C HA, the Gal moieties on the
microvilli glycoconjugates mediate toxin binding (Fujinaga, 2000).
c. Mice:
i. Model ost: Mus sp. Mice (Website 10)
ii. Description: Mice were injected intragastrically with 100000 spores of a Clostridium botulinum type A
culture. Botulism was not observed, but 80% or more of mice challenged when 8 to 11 days old had
botulinum toxin in the large intestine 3 days later. Mice younger than 7 days or older than 15 days were
resistant to the challenge. Whenin vivo toxin production was started by spores given to 9-day-old mice,
toxin was present in the intestine at 1 through 7 days post-challenge but with greatest consistency between
1 and 4 days. Total toxin in an intestine ranged up to 1,920 50% lethal doses as titrated intraperitoneally in
adult mice. The dose infecting 50% of a group of 9-day-old mice was 700 (95% confidence limits of 170
to 3,000) spores per animal. Toxin was formed in the lumen of the large intestine; it was not associated
with the ileum. Injection of 10(5) spores intraperitoneally into 9-day-old mice resulted in toxin production
in the large intestines of 30% of the test animals (Sugiyama and Mills, 1978).
d. Mice:
i. Model ost: Mus sp. Mice (Website 10)
ii. Description: An animal model of wound botulism was developed in mice using an inoculum of
Clostridium botulinum type A spores. The number of C. botulinum in infected wounds was quantitated by
culturing on egg yolk agar, and the level of C. botulinum toxin in infected wound tissue was measured by
a bioassay in mice and by an enzyme-linked immunosorbent assay. All infected mice receiving no further
treatment developed neuroparalytic symptoms consistent with botulism after an incubation period of ca. 48
h, and all of these animals died. Serotherapy with C. botulinum type A antitoxin initiated 24 h post-
challenge reduced the mortality rate to 5%. Treatment with metronidazole 2 to 24 h post-challenge
resulted in recovery rates of 40 to 91% (Dezfulian and Bartlett, 1985).
e. Rats:
i. Model ost: Rattus sp. Rats (Website 11)
ii. Description: A rat model of localized disuse induced by the Clostridium botulinum toxin (Chappard,
2001). Twelve male Wistar rats, aged between 19 and 20 weeks and weighing 506 57 g were used in
experiments of generalized paralyzing disease caused by the botulinum toxin. Six rats received a 2-U
injection of toxin in the right quadriceps. Six rats were similarly injected with saline and were used as
control. The toxin acts 34 days after injection by blocking the release of acetylcholine to the muscle.
Paralysis of the quadriceps was obtained 45 days after the injection (Chappard, 2001).
f. Rats:
i. Model ost: Rattus sp. Rats (Website 11)
ii. Description: The rat as an animal model for infant botulism (Moberg and Sugiyama, 1980) Only animals
from 7 to less than 13 days old at time of intragastric challenge with 10(5) spores were colonized; 9-
dayold rats were the most susceptible. The colonized infants did not become ill, but C. botulinum toxin
was present in the cecum-colon segment from 1 to 6 days postchallenge. The highest incidence of
positives was obtained when the animals were tested for toxin on day 4 after administration of the
inoculum. The mean infective dose for 9-day-old rats (1,500 spores per animal with 95% confidence limits
of 1,300 to 1,800) was not significantly different from that for infant mice of the most susceptible age (700
spores with 170 to 3,000 confidence limits). Susceptibility to intraintestinal Clostridium botulinum
colonization of conventional infant and germfree adult rats is comparable to that of mice. C. botulinum-
monoassociated rats pass C. botulinum toxin in their milk (Moberg and Sugiyama, 1980).
IV. Lab ork Information
A. Biosafety Information:
1. General biosafety information :
a. Biosafety Level: For Clostridium botulinum and its toxin, the recommended BSL (Biosafety Levels for
Laboratories) for clinical specimen handling is BSL-2. Any procedures resulting in aerosolization of
toxin and/or production of toxin necessitate BSL-3 precautions. Most clinical laboratories function at
BSL-2 and are therefore equipped to handle clinical specimens suspected to contain C. botulinum
organisms and/or toxins. Once the organism is cultured and preliminarily identified, the culture plates
and tube must be transferred immediately to a class II biological safety cabinet and the supervisor
notified of the potential biosafety hazard. For many small to mid-sized laboratories, shipment of the
specimen and culture to a reference laboratory, such as a public health laboratory, would then be
appropriate. The prescribed practice for BSL-3 is that all manipulations for the open culture be carried
out in a biological safety cabinet (Caya et al., 2004).
b. Applicable: Clostridium botulinum Precautions (Caya et al., 2004)
c. recautions:
BSL-2 requirements state that all microbiologic laboratory procedures that may generate aerosols
(such as blending, shaking, or vortexing) should be conducted in a class II biological safety cabinet,
with a laboratory coat, disposable surgical gloves, and a face shield (Caya et al., 2004).
d. Disposal:
The botulinum neurotoxin may be inactivated by 0.2 M sodium hydroxide. Clostridium botulinum is
inactivated by a 1:10 dilution of household bleach with a 20-minute contact time. For successful
inactivation of both organism and toxin, both bleach and sodium hydroxide must be applied for a
total of 40 minutes (Caya et al., 2004).
B. Culturing Information:
1. Costridium botulinum Culturing :
a. Description: Trypticase-Peptone-Glucose-Yeast Extract Broth (TPGY).
b. Medium:
i. Trypticase-Peptone-Glucose-Yeast Extract Broth (TPGY). Trypticase 50 g, Peptone 5 g, Yeast
extract 20 g, Dextrose 4 g, Sodium Thioglycollate 1 g, Distilled water 1 liter. Before sterilization
(121C for 15 min), anaerobic conditions are created by boiling the medium for 10 min and, during
cooling, flushing the medium with nitrogen gas (Dahlenborg et al., 2001).
c. Optimal Temperature: 37C (Dahlenborg et al., 2001).
C. Diagnostic Tests :
1. Organism Detection Tests:
a. Light microscopy:
i. Description: Organisms from the growth are examined by Gram stain or phase-contrast
microscopy. Clostridium botulinum is a gram-positive, straight rod. Spores may be demonstrated by
phase-contrast microscopy of wet mounts, where they appear mature and refractile (Caya et al.,
2004).
2. Immunoassay Tests:
a. amp ELISA:
i. Description: An amplified enzyme-linked immunosorbent assay (amp-ELISA) was compared with
the AOAC Official Method 977.26 for detection of Clostridium botulinum and its toxins in foods.
Eleven laboratories participated and the results of 10 laboratories were used in the study. Two
anaerobic culture media, tryptone-peptone-glucose-yeast extract (TPGY) and cooked meat medium
(CMM) were used to generate toxic samples with types A, B, E, and F strains. The toxicity of each
botulinal culture was determined by the AOAC method, and the cultures were then diluted, if
necessary, to high (about 10,000 minimal lethal dose [MLD]/mL) and low (about 100 MLD/mL)
test samples. The overall sensitivity of detection in TPGY and CMM cultures with the amp-ELISA
was 94.7% at about 100 MLD/mL and 99.6% for samples with more or equal 10,000 MLD/mL
toxicity. The amp-ELISA detection sensitivity for low toxin samples was 92.3% in TPGY and
99.4% in CMM. The amp-ELISA could be used to screen suspect cultures for botulinum toxin
(Ferreira et al., 2003).
ii. False ositive: The false-positive rate ranged from 1.5% for type A to 28.6% for type F in TPGY,
and from 2.4% for type A to 11.4% for type F in CMM. Most of the cross-reactivity was due to
detection of other types, especially in high toxin samples (Ferreira et al., 2003).
b. Immuno CR ELISA:
i. Description: Immuno-PCR, using the specificity of an antibody, and a reporter DNA molecule
amplified by PCR, enabled the development of a sensitive assay to detect BTx-A antigen. This
method combines the amplification power of PCR and a method, similar to enzyme-linked
immunosorbent assay (ELISA), which detects an antigen-antibody reaction; however, instead of an
enzyme being conjugated to an antibody, a reporter DNA was used which could be amplified by
PCR. Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-
linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA
conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte
were demonstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) exceeded the
conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) mol) by a 1000-fold
enhancement in detection sensitivity (Wu et al., 2001).
3. Nucleic Acid Detection Tests: :
a. Multiplex CR:
i. Description: The method employs four new primer pairs with equal melting temperatures, each
being specific to Clostridium botulinum neurotoxin gene type A, B, E, or F, and enables a
simultaneous detection of the four serotypes. Other bacterial species, including Clostridium
sporogenes and the nontoxigenic nonproteolytic Clostridium botulinum-like organisms, do not yield
a PCR product. Sensitivity of the PCR for types A, E, and F is 10(2) cells and for type B is 10 cells
per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples is
from 10(-2) spore/g for types A, B, and F to 10(-1) spore/g of sample material for type E. The assay
is sensitive, specific and provides a marked improvement in the PCR diagnostics of Clostridium
botulinum (Lindstrom et al., 2001).
ii. rimers:
For ard: CBMLA1: AGC TAC GGA GGC AGC TAT GTT (Lindstrom et al., 2001)
Reverse: CBMLA2: CGT ATT TGG AAA GCT GAA AAG G (Lindstrom et al., 2001)
roduct
Si e: 782 bp
roduct source: Clostridium botulinum A
For ard: CBMLB1: CAG GAG AAG TGG AGC GAA AA (Lindstrom et al., 2001)
Reverse: CBMLB2: CTT GCG CCT TTG TTT TCT TG (Lindstrom et al., 2001)
roduct
Si e: 205 bp
roduct source: Clostridium botulinum B
For ard: CBMLE1: CCA AGA TTT TCA TCC GCC TA (Lindstrom et al., 2001)
Reverse: CBMLE2: GCT ATT GAT CCA AAA CGG TGA (Lindstrom et al., 2001)
roduct
Si e: 389 bp
roduct source: Clostridium botulinum E
For ard: CBMLF1: CGG CTT CAT TAG AGA ACG GA (Lindstrom et al., 2001)
Reverse: CBMLF2: TAA CTC CCC TAG CCC CGT AT (Lindstrom et al., 2001)
roduct
Si e: 543 bp
roduct source: Clostridium botulinum F
b. CR Southern hybridi ation:
i. Description: A PCR procedure was developed for the detection of Clostridium botulinum in foods.
PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR
was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of
the method in broth was high (2.1-8.1 cfu/ml) for types A and B, but rather low (10(4) cfu/ml) for
types E and F. However, after enrichment at 37 degrees C for 18 h, it was possible to detect
Clostridium botulinum types A, B, E and F in food samples at initial levels of about 1 cfu/10 g of
food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine
detection of Clostridium botulinum in foods (Aranda et al., 1997).
ii. rimers:
For ard: BoNT1.1: CC(C/A)TAT(A/G)TAGG(A/T)C(T/C)TGCTTTAAATATAGG(A/T)A

(A/T)T (Aranda et al., 1997)
Reverse: BoNT2: TTAGT(T/A)ATAGTTACAAAAATCCA(T/C)(T/C)T(A/G)
TTTATATATA (Aranda et al., 1997)
roduct
Si e: 1.1 kbp
roduct source: Clostridium botulinum
4. Other Types of Diagnostic Tests:
a. The Standard Lab Test for Botulism:
i. Description: Clinical diagnosis of botulism is confirmed by specialized laboratory testing that often
requires days to complete. At present, laboratory diagnostic testing for botulism in the United States
is available only at the CDC and approximately 20 state and municipal public health laboratories.
The laboratory should be consulted prospectively about specimen collection and processing.
Samples used in diagnosis of botulism include serum (more or equal to 30 mL of blood in "tiger"-
top or red-top tubes from adults, less from children), stool, gastric aspirate, and, if available, vomitus
and suspect foods. Serum samples must be obtained before therapy with antitoxin, which nullifies
the diagnostic mouse bioassay. An enema may be required to obtain an adequate fecal sample if the
patient is constipated. Sterile water should be used for this procedure because saline enema solution
can confound the mouse bioassay. Gastric aspirates and, perhaps, stool may be useful for detecting
inhaled aerosolized botulinum toxin released in a bioterrorist attack. A list of the patient's
medications should accompany the diagnostic samples because anticholinesterases, such as
pyridostigmine bromide, and other medicines that are toxic to mice can be dialyzed from samples
before testing. All samples should be kept refrigerated after collection. The standard laboratory
diagnostic test for clinical specimens and foods is the mouse bioassay, in which type-specific
antitoxin protects mice against any botulinum toxin present in the sample. The mouse bioassay can
detect as little as 0.03 ng of botulinum toxin and usually yields results in 1 to 2 days (range, 6-96
hours). Fecal and gastric specimens also are cultured anaerobically, with results typically available
in 7 to 10 days (range, 5-21 days). Toxin production by culture isolates is confirmed by the mouse
bioassay. Foods suspected of being contaminated should be refrigerated until retrieval by public
health personnel. The US Food and Drug Administration and the US Department of Agriculture can
assist other public health laboratories with testing of suspect foods by using methods similar to those
applied to clinical samples (Arnon et al., 2001).
V. References
A. ournal References:
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Krieglstein K, Henschen A, Gill DM, Niemann H. Nucleotide sequence of the gene encoding
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Chappard : Chappard D, Chennebault A, Moreau M, Legrand E, Audran M, Basle MF.
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Dahlenborg et al. : Dahlenborg M, Borch E, Radstrom P. Prevalence of Clostridium
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Dineen et al. : Dineen SS, Bradshaw M, J ohnson EA. Cloning, nucleotide sequence, and
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East et al. : East AK, Richardson PT, Allaway D, Collins MD, Roberts TA, Thompson DE.
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B. Book References:
No book references used.
C. ebsite References:
ebsite : NCBI. Clostridium botulinum
[ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?
mode=Info&id=1491&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
ebsite : NCBI. Clostridium botulinum A genome
[ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=27532928 ].
ebsite : NCBI. Clostridium botulinum plasmid
[ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=9454296 ].
ebsite : NCBI. Homo Sapiens [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?
id=9606 ].
ebsite : Clostridium botulinum neurotoxin type A
[ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=33356571 ].
ebsite : Clostridium botulinum neurotoxin type B
ebsite : Clostridium botulinum neurotoxin type E
ebsite : Clostridium botulinum neurotoxin type F
ebsite : NCBI. Taxonomy. Guinea pigs
[ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?
mode=Info&id=10140&lvl=3&lin=f&keep=1&srchmode=1&unlock ].
ebsite : NCBI. Taxonomy. Mice
[ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=10095 ].
ebsite : NCBI. Taxonomy. Rats
[ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=10118 ].
D. Thesis References:
No thesis or dissertation references used.
VI. Curation Information
Curators: George Abramochkin
Date: . .
Version: .
Note: This is the first complete version of the Botulism background information
Contact information:
Email: georg vbi.vt.edu

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