Thursday March 20, 2008 - Vol. VII Issue 2[Download PDF for Printing]
Biotoxins: Part 2
The December 2007
AristaTek Newsletter examined the biotoxins, Saxitoxin and Ricin. These were two biotoxins which appeared in a
list of toxins which the U.S. Department of Health considered as a severe
threat to public safety. This list, as
it appeared in the August 23, 2002 Federal Resister, (see also 42 CFR Part 72,
Appendix A) is as follows:
perfringens epsilon toxin
Conceivably, a terrorist
could potentially harvest and purify these biotoxins and use them against an
enemy or the public. Congress passed
the Public Health and Safety Act of 2002 (PL 107-188), and the President signed
it into law on June 12, 2002, which required the U.S. Department of Health to
maintain a list of biotoxins and biological agents that a terrorist might use.
This Newsletter looks at
Abrin, Botulinum neurotoxins, and Clostridium perfringens epsilon toxin.
Abrin is a potent toxin
extracted from the seeds of the Jequirity Pea (Abrus precatorius)
plant. The adult fatal dose for abrin
is about 0.0026 mg if ingested, or about 0.04 micrograms per kilogram
(μg/kg) of body weight. The fatal
dose can vary between individuals. Its
toxicity is roughly 75 times that of ricin.
It is also fatal by inhalation or by absorption through sensitive skin
areas as in the eyes.
Like ricin, the toxin
acts by inhabitation of body protein synthesis. Initial symptoms by ingestion include watery diarrhea at first,
later nausea, vomiting, abdominal cramps, and chills. The vomiting and diarrhea becomes bloody. Severe dehydration may result followed by
low blood pressure. Other symptoms may
include hallucinations, seizures, and blood in the urine. Within days, the person’s liver, spleen, and
kidneys may stop working. Death could
take place within 36 to 72 hours of exposure.
If death has not occurred within 5 days, the person usually recovers but
may suffer long-term organ damage.
There is no specific antidote.
If abrin powder is inhaled, pulmonary edema and hemorrhaging can
result. Abrin dust in air can result in
blindness or at minimum severe eye irritation.
There have also been several reports of people who have ingested the
seeds and slipped into a coma, a condition called “acute demyelinating
encephalitis” [see http://www.ncbi.nlm.nih.gov/pubmed/17357388
and Clin. Toxicol
(Phil, PA) 2007, 45
(1) 77-79 for a case study,
the patient died]
The plant itself goes by
many names, including: Rosary Pea, Jequirity Pea, Jequirity Bean, Crab’s Eye,
Deadly Crab’s Eye, Precatory pea, Precatory bean, Roseary Pea, Abrus seed,
Jumble beans, Ratti seeds, Prayer beads, Tentos de America, Tentos dos mundos,
Jequirite, Aivoeiro, Buddist rosary bead, Ruti, Indian bead, and Wild
liquorice. The plant itself is a vine
which grows up to 10 feet tall and has clusters of rose pink or purplish
flowers about 0.5 to 0.8 inches long.
The flowers produce brown seedpods with shiny red and black seeds about
5 to 8 mm long. The plant itself is
grown in tropical and subtropical areas and has been planted in warmer parts of
the United States. The red and black
seeds are sometimes used in necklaces and other decorative items.
Illustrations of the
plant and seeds are available on the Internet.
Seeds of jequirity pea, about 0.7 cm long
Steve Hurst @ NRCS PLANTS Database
jequirity plant vines
Seeds and seed pod with leaves
Mark Skinner, USDA NRCS NPDC
Leaf detail, each leaflet 1.2 to 1.8 cm long
One seed if chewed can
kill, especially a child. All parts of
the plant are toxic to some degree, but the seed, which contains abrin, is
especially toxic. If the dried seed is
swallowed whole, it is less toxic but some of the toxic elements can still
leach out by the digestive enzymes.
Sucking on the seeds can release some of the toxic contents. Immature seeds are poisonous if ingested
even whole. If holes are drilled in the
seed as in a necklace and the seed is ingested, the toxic contents will leach
out by intestinal secretions.
Five glycoproteins have
been purified from the seeds, four of which (abrin a, b, c, and d) are
extremely toxic. The other glycoprotein
is “abrus agglutinin” which is a powerful hemagglutinator but is relatively
non-toxic to animal cells; its molecular weight is approximately 134,900.
Arbin (a, b, c, and d)
are the toxic parts. The molecular
weight is between 63,000 and 67,000.
Abrin has been given CAS# 1393-62-0.
Purified arbin is a yellowish-white powder. Most of its toxicity is destroyed if heated to 80o
F) for 30 minutes.
The U.S. Department of
Health and Human Services – Centers for Disease Control (CDC) Website http://www.bt.cdc.gov/agent/abrin/erc1393-62-0.asp
provides the following information about first responder response to an Abrin
Table 1 CDC
Emergency Preparedness and Response for Abrin
Irritation (see ingestion for other symptoms)
demand, self-contained breathing apparatus (SCBA) (SCBA CBRN, if available)
is recommended in response to non-routine emergency situations.
Response (pressure demand) HEPA PAPR.
Fresh air, rest, half-upright position. Perform CPR if necessary. Seek medical attention immediately.
Potential for allergic skin reaction; redness, blisters,
May be absorbed
Tychem® BR or Responder® CSM protective clothing. Eyes should be protected when possible.
contaminated clothes. Rinse skin with
plenty of water or shower (and soap if available).
Seek medical attention immediately.
Tearing, swelling of the eyelids, pain, redness, corneal
Goggles with respiratory protection or full face-piece
flush with large amounts of tepid water for at least 15 minutes.
Seek medical attention immediately.
Table 1 continued
- Cardiovascular (heart
and blood circulation) shock from severe dehydration, life-threatening
low blood pressure, fast heart rate (tachycardia) and irregular heart
- Central Nervous
System (brain) - drowsiness, disorientation, hallucinations, seizures,
(stomach and intestines) - burning pain in the mouth, abdominal pain,
nausea, vomiting, diarrhea, bleeding and swelling of the lining of the
GI tract, liver cell damage and death.
(kidneys and urine) - blood in urine, low or no urinary output, kidney
cell damage and death.
- Musculoskeletal -
muscle weakness, tremors, and muscle spasm (tetany)
- Eyes - dilated pupils
and bleeding in the back of the eye (retinal hemorrhage)
Skin - blue skin (cyanosis) and redness (flushing)
Symptoms delayed 1-3 days.
May be fatal.
Do not eat, drink, or smoke during work. Wash hands before eating.
mouth. Do not induce vomiting. Use slurry of activated charcoal. In the event of vomiting, lean patient
forward or place on left side (head-down position, if possible) to maintain
open airway and prevent aspiration.
Early and aggressive IV fluid and electrolyte replacement.
medical attention immediately.
As far as we have been
able to determine from an Internet search, Abrin is not known (to date) to have
been used in any wars or terrorist attacks.
Abrin also has potential medical uses such as in treatment to kill
1. Summary, to Congress, bioterrorism report, 2003
2. EY Laboratories Inc test kit for Abrin and Ricin
One of the most toxic
biotoxins known is botulinum neurotoxin, with an estimated human lethal dose of
0.001 micrograms per kilogram of body weight (μg/kg) by inhalation, or 1
μg/kg if ingested. The poisoning
is called botulism. There are actually
seven types of botulinum neurotoxins recognized, labeled serotypes A through G,
with type A being the most toxic and types B and E causing less severe toxicity
to humans. The botulinum neurotoxins
are produced primarily by a spore-forming bacterium known as Clostridium
and to a lesser extent by spore-forming bacteria Clostridium
and Clostridium butyricum
does not produce the
neurotoxin. These bacteria and its
spores are found worldwide in soil, ponds, costal waters, sediments, the
intestinal tracks of fish and mammals, and in shellfish.
photo of Clostridium botulinum stained with a dye (Gram Stain),
spore pink and rest of bacterium stained purple
of unstained Clostridium botulinum bacterium, length 3 to 8 μm, spore diameter (not
visable in photo) about 0.5 μm., photo from
Human botulism is
primarily caused by types A, B, E, and F neurotoxin. Types C and D most commonly affect fowl, birds, cattle, and
horses and do not affect humans. Type E
typically occurs from eating contaminated fish. Type G, isolated from a soil in Argentina, has not yet been
linked to any outbreak. All of them are
single polypeptide chains (molecular weight about 150,000) that work by
blocking the release of acetylcholine from peripheral cholinergic nerve
endings. What this means is that the
nerve terminals are blocked, and the muscles do not work, usually starting at
the eyes and face, then the throat, chest, and extremities. When the diaphragm and chest muscles become
fully involved, respiration is inhibited and death occurs. There are differences on how the different
types interact with nerve endings and block the release of acetylcholine, which
is the reason why some types are more toxic than others or are more toxic to
bird or animal species or to humans.
The neurotoxin itself is
destroyed if heated to 80o
F) for 10 minutes or
longer. If heated to 100o
F), destruction occurs quicker. However, cooking food containing spores does not destroy the
spores at 100o
When canning food, the spores must be destroyed by moist heat at
temperatures of at least 121o
F). The spores if ingested normally do not
prorogate and produce neurotoxin in adult humans because gut conditions are too
acidic, but the spores can colonize and produce neurotoxin in the intestinal tracks
of infants under 12 months of age.
Four different kinds of
botulism poisoning are recognized:
1. Inhalation botulism. This kind does not occur naturally and could mean that a
terrorist has released the toxin as an aerosol.
2. Food borne botulism. This is usually the result of ingestion of food that has been
inadequately processed or inadequately cooked before being eaten. If food is contaminated with Clostridium
spores, and the bacteria allowed to grow under anaerobic
conditions, the neurotoxin will be produced.
If not cooked properly to destroy the toxin, botulism food poisoning
will occur. The most common source is
home-canned foods. There have been
incidences of poisoning in commercial products. Neurological symptoms usually appear within 6 hours to 8 days of
ingestion of the food, the shorter time associated with more severe poisoning.
3. Infant (intestinal) botulism. This results from ingestion of bacteria
spores which germinate in the intestinal gut and produce the toxin. This can happen as the result of food, soil,
or dust contaminated with the spores.
The infant gut is most susceptible to spore germination, but adult
infections can occur if acidic conditions are not maintained in the gut. This is the most common incidence of botulism
poisoning reported to the CDC in the United States. Pediatricians do not recommend feeding honey to infants as the
honey could contain the spores.
4. Wound botulism.
This happens when wounds become contaminated with dirt containing
botulism spores. This is especially a
problem with chronic injection drug users.
The incubation period is between 4 and 14 days since contamination, the
shorter time corresponding to the more severe infection. Neither the spores nor the neurotoxins are
able to penetrate intact skin.
About 110 cases of U.S.
botulism poisoning are reported to the CDC annually.
Conformation of botulism
poisoning is done by either detection of botulinum toxin or by isolation of Clostridium
in a clinical specimen and
the patient also displays
clinical symptoms of botulism poisoning.
The specimen could be a person’s stool sample in the case of infant or
food borne botulism or a serum sample, or a food sample. The standard test for botulinum toxin
testing is the mouse neutralization test.
A sensitive method for
detection of botulinum neurotoxin in foods has been published recently (see http://aem.org/cgi/content/abstract/72/2/1231
for abstract). The citation is S.K.
Sherma, J. L Ferreira, B.S. Eblen, and R.C. Whiting, “Detection of Type A, B,
E, and F Clostridium botulism
Neurotoxins in Foods by Using an Amplified
Enzyme-linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies”, Applied
and Environmental Microbiology
, 72(2), Feb. 2006, p. 1231-1238. The enzyme-linked immunosorbent assay
(ELISA) test is an alternative test to the mouse neutralization test.
Antitoxin therapy is
available and is administrated to adult patients with food borne or wound or
inhalation botulism. The antitoxin must
be administrated early. It is not
administrated to infants. The CDC must
release and approve its use. The
antitoxin is a horse serum product and may cause serum sickness in
approximately 20% of treated persons. A
human-derived antitoxin product is under evaluation in California (see World
Health Organization Poisons Information Monograph 858) for infants. Also, the decision to administer the
antitoxin often can’t wait for conformation by testing for the toxin or
bacteria, and must be made on the basis of display of symptoms especially if a
suspect food is identified or there is evidence of a wound which may have been
contaminated. The patient’s serum
should be collected before administration of the antitoxin.
The specific antitoxins
available from the CDC are
Anti-AB equine antitoxin, Aventis-Pasteur
IND Univalent Anti-E
equine antitoxin, Aventis-Pasteur
despeciated (Fab’2) equine Anti-ABCDEFG antitoxin, U.S. Army
California Dept. of
Health Services, Infant Botulism Treatment and Prevention Program
Licensed Anti-AB human
antitoxin for infant botulism “Baby-BIG”
(reference: University of Alabama School of Medicine
website at http://www.bioterrorism.uab.edu/CategoryA/Botulinum/summary.asp
The classic symptoms of
botulism poisoning include double vision, blurred vision, drooping eyelids,
slurred speech, difficulty swallowing, dry mouth, and muscle weakness. Infants appear lethargic, feed poorly, are
constipated, have a weak cry, and have a poor muscle tone. If untreated, these symptoms may progress to
cause paralysis of the arms, legs, trunk, and respiratory muscles. (from CDC website, at http://www.cdc.gov/ncidod/dbmd/diseaseinfo/botulism_g.htm
Because of respiratory
failure, the patient may need to be on a breathing machine (ventilator) for
weeks. If severe, the person may
require intensive medical and nursing care for months.
The person affected by
botulism poisoning is not infectious.
Several countries are
reported to have produced botulinum neurotoxins as weapons. The United States once produced botulinum
neurotoxin under military code name X.
The entire stockpile of botulinum neurotoxin was ordered destroyed by
President Nixon (1969) along with other biological agents. The former Soviet Union continued their
biological weapons research program. In
April 1992, President Boris Yeltsin declared that his country had continued a
massive offensive biological warfare buildup which included botulinum warfare
buildup. In 1995, Iraq admitted to the
United Nations Special Commission inspection team that it had produced 19,000
liters of botulinum neurotoxin concentrate for use in specially designed
missiles and sprayers, including approximately 10,000 liters loaded in military
weapons. In 1990, Iraq deployed 13
missiles with a 600 km range containing botulinum toxin. (source:
“The Comprensive Sourcebook of Bacterial Protein Toxins” (book), edited
by J.E. Alouf and M.R. Popoff, Academic Press, 2006).
neurotoxins are unstable in the environment.
Therefore the range of an aerosol attack out in the open is limited. The neurotoxin would be expected to degrade
in the environment within one or two days.
Contaminated surfaces can be cleaned with a 0.1% chlorine bleach
Additional reading: (from Journal of American Medical
The University of Florida
Environmental Health and Safety Manual lists LD50
values for each of
the Botulinin toxins, which can be obtained at http://www.ehs.ufl.edu/Bio/toxin.htm
Table 2 LD50
values for Botulinin Toxins
, [Univ. of Florida, citing D.M.
Gill, Microbiological Reviews 46
: 86-94. (1982) and other
perfrigens epsilon toxin
Botulinin toxin A
Botulinin toxin B
Botulinin toxin C1
Botulinin toxin C2
Botulinin toxin D
Botulinin toxin E
Botulinin toxin F
is the name of the
microorganism and epsilon toxin is one of several biotoxins that it
produces. Gas gangrene results from
wound contamination by this microorganism, which grows in the wound and
produces biotoxins. In addition, Clostridium
can cause food poisoning.
Clinical symptoms resulting from food poisoning include intense
abdominal cramps and diarrhea which begins between 8 to 24 hours after eating
food containing these bacteria. The
toxins are a particular problem with farm animals, in particular, goats, sheep,
young caves, and pigs. Theoretically, a
person could also become poisoned by inhalation of dust containing the
microorganism or its toxin. The toxins
can be transmitted in contaminated food, water, or as an aerosol. Specific information on human poisoning by
the epsilon toxin is minimal, but farm animals are affected.
There are 5 different
strains of Clostridium perfringens
(labeled Types A, B, C, D, and E) and
perhaps at least 20 toxins (labeled alpha, beta, beta2, etc, epsilon, iota,
etc.; some sources cite 12 toxins). The
toxins vary in toxicity and in their effect on animals and people. The epsilon toxin (which is produced by
strains B and D) is singled out as being the most toxic, based primarily on
intervenous injection of the toxin to test animals. The LD50
value (lethal dose resulting in death of 50%
of the test animals) for the epsilon toxin using a mouse as the test animal is
0.78 nanograms (0.1 μg/kg of body weight), (ref: Iowa State University,
Center for Food Security and Public Health website). Goats and lambs injected with the epsilon toxin develop severe
pulmonary edema (fluid in their lungs) and show neurological symptoms. Rats injected (intraperitionally) develop
cerebral edema. Onset of neurological
symptoms by intervenous injection may occur anywhere from 2 minutes to one hour
in test calves to 0.5 hours to 3 hours for goats and lambs. The epsilon toxin works by causing potassium
and fluid leakage from body cells.
Neurological symptoms include stupor (mild poisoning) to coma and death
(more severe poisoning).
Table 3 LD50
values for Clostridium perfringens Toxins
, [Univ. of Florida, citing D.M. Gill, Microbiological Reviews
: 86-94. (1982) and other sources]
13 to 16
Clostridium perfringens gram positive bacterial
stain, anaerobic, rod-shaped
Tissue smear showing Clostridium
photos from Kirksville College of Osteopathic Medicine website, at
About one-third of the
incidences of food poisoning in the United States is caused by Clostridium
mostly from the type A strain producing the alpha toxin, or
enterotoxin. A more serious form is the
type C strain producing the beta2 toxin.
Other strains besides type A produce the alpha toxin. Symptoms (from the alpha toxin) of food
poisoning include diarrhea, nausea, severe abdominal cramps, and bloating but
rarely vomiting or fever. The symptoms
appear 8 to 24 hours after ingestion of contaminated food. Patients generally recover within a day or
two, although the elderly can take longer.
Deaths are rare. However, the
type C strain producing the beta2 toxin can cause sloughing of the mucosa off
the intestines and intestinal perforation, and severe infection, often
resulting in death. Symptoms include a
bloody stool and probably vomiting and fever in addition to type A
symptoms. Intervention including
removal of part of the intestine with prolonged antibiotic treatment and
intravenous fluid replacement may be necessary. This type of food poisoning may be called “necrotizing enteritis”
or “pig-bel disease”. The beta2 toxin
is usually associated with enteritis in pigs, therefore the name “pig-bel
Information on human food
poisoning from the epsilon toxin is sketchy and is complicated by proper
identification of the strain and maybe more than one toxin can be
produced. Fluid replacement including
electrolyte monitoring is part of treatment as potassium loss is a feature of
epsilon toxin poisoning. Limited
information available on the Internet seems to suggest that the symptoms are
similar to that produced by the alpha toxin.
The purified alpha toxin
can be lethal by inhalation as an aerosol, causing acute pulmonary disease,
vascular leak, hemolysis, thrombocytopenia, and liver damage.
The toxins including the
epsilon toxin can be destroyed by heat.
Thorough cooking of food is required.
The vegetative form of the bacteria can be destroyed at 70o
F), but much higher temperatures and prolonged heating is
required to kill the spores. The high
temperatures required to kill all of the spores also compromise the nutritional
value of the product, and sometimes food processors use a combination of gamma
irradiation treatment and heat, or add salt or other preservatives. When cooking meat, it is also important to
cool the meat product down quickly and refrigerate if consumed at a later
time. More details are at a University
of Wisconsin, Food Research Institute
, in a paper published in 2002, © Food Research
Institute, “Survival and Growth of Clostridium perfringens
Cooling Step of Thermal Processing of Meat Products”.
Toxin identification in
humans and animals can be done using the mouse neutralization test (MNT) or
enzyme-linked immunosorbent assay test (ESILA) on clinical samples (e.g. feces,
intestinal fluids). Another test is the
polymerase chain reaction assay test (PCR).
An Internet search failed
to turn up evidence that Chostridium perfringens epsilon toxin has been
weaponized. The South African
biological weapons program between 1981 and 1984 developed the bacteria as a
potentially lethal agent to resemble food poisoning. In 1991, Iraq representatives informed the United Nations Special
Commission inspection team that Iraq had researched the offensive use of Chostridium
as a potentially lethal agent, and had produced 90 gallons of
Presumably the toxin can
be manufactured by fermentation of Clostridium perfringens
likely by another microorganism expressing the cloned gene for the toxin. The microorganism. E. coli
already been cloned to produce the epsilon toxin for the production of a
vaccine for inoculation of farm animals.
The purified toxin or even the crude material could be released as an
aerosol. A person inhaling the material
would be expected to suffer severe pulmonary edema (fluid buildup in the lungs)
in addition to vascular leak, organ damage, and neurological problems including
cerebral edema, as the result of potassium leakage from body cells. The alpha toxin is also a potentially lethal
agent when dispersed as an aerosol.
Additional information on biotoxins may be found at the
under Weapons of Mass Destruction.
There is a good overview article on toxins formed by Clostridium
bacteria published by the University of Wisconsin (2008), Madison
Department of Bacteriology, at http://www.textbookofbacteriology.net/clostridia.html
In searching the Internet, I found some discrepancies between LD50
values cited in different sources, and also which Clostridium sp.
are more toxic. Also there are
discrepancies as to the number of toxins produced by Clostridium
I have not
resolved these differences by searching out the original publications where the
work was done]