VENOM
ANTIVENOM
and
IMMUNITY
With an illustrated program in the deliberate induction of an
Immune response against the venom of the Eastern Coral Snake
By Norman Benoit
Table of Contents
3
Introduction
5
Snake Bite
8
The history of vaccines
15
Antivenom
34
So what’s the problem?
36
The immune system
39
From Horse to Human
42
Basic premise- Simple over view of this program
43
Materials
63
Serial Dilution
68
Actual case study
This compilation is intended to be distributed free of charge
Introduction
This is a presentation of a program to make a man immune to the
venom of the Eastern Coral snake. It is one of the first fully documented
cases of its kind, complete with step by step account for the purpose of
clarity and completeness. It is the intention that this information be
included in the body of available materials regarding this subject, and a
basis for further discovery. We may be at the beginning of modern
investigation into venoms and their usefulness to mankind. Besides the
obvious research into more effective antivenom and the production of a
snake bite vaccine, there remains much to be learned in how venoms can
be used in man’s quest to cure or mitigate some common human
degenerative conditions.
William Haast is the much celebrated founder of Miami Serpentarium
Labs. He was the American pioneer in immunizing himself to the deadly
venomous snakes he worked with in the course of performing daily
venom extractions. He has been quoted as saying that since he began
injecting himself with venoms he has never been sick a day in his life,
“Never even had a common cold.” At the time of this writing he has
survived 174 venomous snake bites and is ninety eight years old. The
fact that transfusions of his immunized blood have been used to save the
lives of snake bite victims, he is the true inspiration for this work, and
the author’s life-long interest in venomous reptiles.
Snake Bite
“Don’t Tread On Me” the slogan of the Gadsden Flag. The rattlesnake
was seen as a symbol of the American colonies and patriotism when Ben
Franklin wrote his famous anonymous letter to the Pennsylvania Gazette
in 1751. He was proposing the rattlesnake as a national symbol of the
United States, and was also known to oppose using the bald eagle which
he described as a bird “of bad moral character".
"She never begins an attack, nor, when once engaged, ever surrenders:
She is therefore an emblem of magnanimity and true courage. ... she
never wounds 'till she has generously given notice, even to her enemy,
and cautioned him against the danger of treading on her."
It is a rough estimate that about 125,000 people die each year from
snake bite. This is out of the estimated five million bites. Fatalities occur
in regions where people walk bare footed and are usually located in
remote regions that are some distance from life saving antivenom. It is
true that some people die even with treatment, but a great many lives
have been saved with the availability of the serums to counter act the
poisons.
In the United States, about eight thousand venomous snake bites
occur each year, and about nine people will die. It is interesting that in
the United States most bites are on the hands from people gardening,
moving brush or deliberately handling a snake, where in other countries
most bites are to the feet or lower extremities. This is again due to
people not wearing any protection and walking in tropical regions at
night or places where venomous snakes are more common.
Unlike the rattlesnake, that gives fair warning to those about to
“tread” on her, many snakes, including the subject of this presentation,
are secretive, shy and deadly silent. Some rely on camouflage to remain
undetected and silently allow people to pass within striking distance with
untold greater occurrence than the bite statistics. Some are reluctant to
bite at all unless quite provoked. Certainly stepping on a snake will
usually cause the snake to lash out to protect itself. You could say that
any bite that is not intended for capturing a meal is a defensive strike,
not at all different from any harmless snake or creature that is protecting
itself out of fear and survival instinct.
The never ending expansion of civilization into rural areas does create
more encounters with creatures of every remaining kind, and venomous
snakes are no exception. It is a common reaction to grab the nearest
stick and beat any snake to death with just a bare presumption the
creature might be a venomous kind. In some instances that matters not,
really. Children, in particular, are sometimes attracted out of curiosity, a
lack of learned fear, or in the case of the coral snakes and a few others,
the remarkable pretty coloration. With the coral snake in particular, they
are almost candy-cane like in coloration. They do not buzz their tails, coil
and strike, and in every way seem very inoffensive. They are quite often
mistaken as diminutive harmless potential pets and play things. It is
often reported that bites have occurred after the snake has been handled
for an hour or more by children (and some adults). Coral snakes are
usually fairly secretive by nature. They spend much of their lives on the
cool ground below the grass and leaf litter. They can be fairly common in
residential areas of suitable habitat and thrive unnoticed among the
flower beds and landscaping of encroaching suburbia. This will
occasionally be sensationalized by media reports of coral snakes invading
neighborhoods and attacking dogs and children when an incident does
occur. It is always true that “the snake was there first” but the creature
is typically portrayed as the relative of the cobra and the most venomous
snake in North America.
According to published reports from the Miami Dade venom rescue
unit, there are about eighty coral snake bites reported every year in
Florida. Many are treated at hospitals that stock antivenin and go
unreported, so the actual number might be as high as 150 bites. Despite
the fact that the venom is actually extremely potent, many of these
might be from extremely small snakes that are actually incapable of
injecting a fatal dose of venom in anything weighing over sixty pounds or
so.
Even these smaller snakes, with limited venom production and delivery
capability, do pose a great risk to children and most dogs. Certainly an
average adult coral snake of twenty three inches or so is quite capable of
delivering a fatal dose of venom with a single bite to an adult.
Fortunately the venom is a slow acting neurotoxin, which usually will
provide ample time to get to a medical facility and begin treatment with
the life saving serum. Until the death of 29 year old Fernando Hernandez
in Bonita Springs, Florida from a coral snake on June 10, 2006, there had
not been a reported death by a coral snake since the introduction of
Wyeth's Micrurus fulvius Antivenin in 1967.
The history of vaccines
Man’s understanding of the power of snake venoms must be very
ancient. One can hardly think of Egyptians without the cobra, which is
prominently depicted in so many of their carvings, paintings and
artifacts. One of the surviving Egyptian papyrus scrolls in the collection
of the Brooklyn Museum of Art, dating from Dynasty XXX (380-343 B.C.),
is a snake charmer’s field guide. It contains descriptions of over thirty
snakes by color, name, lengths attained, and for each a section on
treating the bites, remedies, treatment and the chances for the victims
recovery.
Venoms have held special fascination, no doubt, as one of the most
potent of natural potions to ancient shaman and medicine men from
prehistoric times. There is a Sumerian vase from about 2600 BC that
depicts a snake wrapped around a staff, which is today the familiar
medical symbol. Venom is derived from the word Venus and originally
meant love potion.
The concept of immunizing oneself against a poison by taking small
doses and building up a resistance is called mithridatization. It got the
name from King Mithridates (132–63 BC) who did this to protect himself
from poisoning by his enemies.
The concept was probably passed to Mirithridates by his physician, the
herbalist Krateuas. His writings were included in the early “De Materia
Medica”, a five volume book written by the ancient Greek pharmacologist
Pedanius Dioscorides. Dioscorides traveled throughout the Greek world
collecting herbal remidies and medicinal substances and compiled the
volumes that would become the most influential pharmacopeia in history.
Snake venoms have been used by healers since ancient Chinese
practice. It is thought that human immunity to snake venom is one of
the oldest forms of vaccinology known.
The Marsi people (150 BC) inhabiting the Abruzzi Mountain region of
Italy reputed to have immunity to snake venoms. The Marsi were great
snake hunters, charmers and druggists.
The Psylli Tribe in Africa practiced human immunity to snake venoms in
what is now Lybia in AD 60. They were an isolated people, who were
very helpful to outsiders such as the Romans. They helped clear the way
of dangerous snakes and were able to treat snakebite. They were known
to treat the wounds by sucking the venom out with their mouths. The
Romans wrote that the Psylli used their magic towards saving other
peoples lives. They describe the neurotoxic effects of envenomation
when noting, “the course of the wound does not allow one to catch his
breath”. A fitting description of a neurotoxic venom that is known to kill
by suffocation, paralyzing the diaphragm.
Vaccination is the most effective manipulation of the human immune
system ever discovered. The Chinese learned to innoculate against
smallpox by having patients snort the dried powdered scabs of smallpox
victims. This small dose exposure would provide protection to a more
serious exposure. These were the first known innoculations. The method
was known in America as early as 1706.
The practice of smallpox innoculation was being performed in Turkey
in 1716. Lady Mary Ward Montagu was the wife of a British Ambassador
who is credited for bringing the practice back to England. She had lost a
brother to smallpox, and bore the facial scars of a bout herself.
Upon learning this innoculation technique in
Constantinople, which used smallpox, not cowpox, she
had her own children innoculated. Upon returning to
England, she promoted and popularized the procedure
among the socially connected. The royal family had their
children innoculated, despite resistance to the technique
from the medical establishment of the day, who regarded
it a crude “Oriental” procedure.
When a smallpox epidemic broke out in Boston in 1721, it was an
African slave named Onesimus who informed his master of an ancient
practice of scratchng with a thorn into the skin of an affected person and
scratching the skin of an unaffected person. Thus exposed, the person
suffers a mild form or nothing at all, but is then immune.
An English farmer named Benjamin Jesty noticed that milkmaids
exposed to cowpox (a much milder form of smallpox) were not only then
never reinfected with cowpox, but also never contracted the frequently
deadly smallpox. He deliberately vaccinated his wife and children with
cowpox as an immunization to smallpox in 1774.
In 1794 a more prominent English medical
doctor named Edward Jenner became the first
doctor to write about the practice of inducing
immunity in the manner that had been
performed at least twenty years before by
Benjamin Jesty, and probably centuries before
by others. He is still usually credited with this
discovery, despite the timeline and his
incorrect theory that the disease started in
horses, which he named “the grease”, was
transferred by humans from horses to cows,
and then became cowpox. He most certainly
did publish a report of his finding in twenty
three cases to the Royal Society. The
“discovery”, and it’s acceptance by the
medical establishment led to revolutionary
work in infectious diseases.
The practice of canning was invented in the late eighteenth century
after Napolean Bonapart offered a reward for anyone who could come up
with a practicle way for him to feed his army. Wine maker Nicholas
Appert took the prize in 1810 when he discovered that foods heated and
preserved in airtight containers would not spoil. It was then easy to store
and transport enough food to feed, well, an army.
Pasteurization consists of, basically, the idea that heating liquids like
milk and wine to kill most of the germs and bacteria would inhibit
spoilage. Louis Pasteur, for whom the process gets it’s name, is credited
with this discovery in1862. In fact, the first factory for preserving foods,
The House of Appert, became the first commercial cannery in the world
in 1811, over fifty years before Pasteur explained why it worked.
Pasteur was able to demonstrate that microorganisms were
responsible for the contamination seen in foods, infections that occurred
following surgical techniques, infectious diseases in both animals and
humans, and the validity of the “germ theory” of disease. Pasteur also
used the smallpox vaccine idea to develop other vaccines. In one fateful
experiment, an attempt to infect a group of chickens failed because the
source of the disease turned out to be too weak. They showed only mild
symptoms of the disease. Upon trying again to infect the chickens with a
fresh batch of disease, he found he was unable to infect them at all. The
chickens that had been exposed to the weakened disease and showed
only mild infection had developed immunity to even the stronger dose.
His innovation over the ancient smallpox vaccine, which had remained
largely unchanged from the ancient Chinese method of variolation, was
artificially creating weakened versions of these diseases, which he named
“vaccine” ("vacca" means cow in Latin) as a tribute to the earlier
discoveries with cowpox.
A vaccine is a preparation which is used to improve immunity to a
particular disease. The immune system responds to the intruder by
producing an antibody, and remembering how to produce it again. He
was able to develop, among others, the first vaccines for anthrax,
chicken cholera and rabies (1885). The Pasteur Institute, a non-profit
private foundation dedicated to the study of biology, microorganisms,
diseases and vaccines was founded on June 4, 1887.
It would be almost two thousand years after Mithridates began actively
immunizing himself against poison until Lois Pasteur would be credited
with proving the germ theory of disease and developing the first
vaccines, but surely the ideas are very ancient.
Antivenom
American Henry Sewall discovered that by
gradually injecting animals with small
quantities of venom they would build up a
resistance to it, eventually reaching a point
where they could comfortably survive
normally lethal doses. He worked with the
venom of the Massaugua rattlesnake and
pidgeons. His experiments showed that the
prophylactic effect of repeated inoculations
was persistant over the interval of five
months. He published his findings in 1887 in
his paper Experiments on the Preventive
Inoculation of Rattlesnake Venom.
Robert Koch worked with Louis Pasteur
to establish the germ theory of diseases,
for which Koch had a lifelong passion. He
is considered one of the founding fathers
of both microbiology and bacteriology.
He developed a method of growing
cultures in agar in specially made flat
dishes invented by his assistant Julius
Richard Petri. The “Petri” dish is still in
use today. With these innovations he was
able to discover the bacterium causing
tuberculosis. In 1885, he became
professor of hygiene at the University of
Berlin. His pupils were creditied to
discovering the organisms responsible for
diphtheria, typhoid, pneumonia,
gonorrhoea, cerebrospinal meningitis,
leprosy, bubonic plague, tetanus, and syphilis, among others, by using
his methods.
One of his students at the University of Berlin was
Bacteriologist Shibasaburo Kitasato. Born in Japan
in1852, Kitasato began working in the laboratory of
Robert Koch in Berlin shortly after graduating from
the University of Tokyo in 1883.
Emil Adolf von Behring also became an assistant at the Institute of
Hygiene under Robert Koch. He made
a great advancement in the
development of vaccines when he,
along with Shibasaburo Kitasato
discovered that animals injected with
microbes that cause tetanus not only
produced substances that neutralized
the toxins, but that those antigens
could be extracted and injected into
other animals. They had coined the
term “antitoxin”. They demonstrated
that sterilized cultures of tetanus or
diphtheria, injected in small increasing
doses would stimulate the production
of antitoxins that could then be
transferred as passive immunity into
another animal that had contracted
the disease. This discovery led to
what would be modern therapeutic
Immunology. Working together they published their findings on passive
immunity in 1890.
Emile Roux was a co-founder with Louis Pateur of the Pasteur Institute
in Paris. Roux joined Pasteur’s laboratory as a research assistant in 1878.
He became one of the founding fathers working with Pasteur in the
research into the vaccines for avian cholera, rabies, and is credited as
the discoverer of the anti-diphtheria serum, which was based on the
work of Adolf von Behring and Shibasaburo Kitasato published the year
before. Roux was one of the earliest experts in medical microbiology and
immunology. As a professor, he taught a course on bacteriology. One of
his students was Albert Calmette.
Emile Roux and Albert Calmette
Léon Charles Albert Calmette was born in Nice, France in 1863.
Perhaps in part to suffering from typhoid at thirteen, he developed an
interest in fighting disease and became a medical doctor. As an assistant
doctor in the Naval Medical Corps he served a squadron in the China Sea
and Vietnam. He studied malaria while serving in Hong Kong. Later he
served in West Africa, in Gabon and French Congo. Fertile ground for a
young researcher. In 1890 He returned to France and became an
associate of Louis Pasteur. Calmette was an immunologist, bacteriologist
and physisian who became an ardent toxocologist, studying snake and
bee venoms.
In 1891 a branch of the Pasteur Institute was founded in Saigon in the
newly formed French Indochina. Pasteur sent Albert Calmette, an officer
of the Pasteur Institute to found and direct the branch. It was the first
Pasteur Institute established outside of France.
In 1894, after returning to Lille,
France, using the same method that
Emile Roux used in developing the antidiphtheria serum, he developed the first
antivenom against the Indian Cobra
from the sera of immunized horses. It
was called “Calmette’s Antivenomous
Serum”.
The principle of antivenin is that a
hyperimmunized serum is transfused
into the patient. This was the passive
immunity discovered by Emil Adolf von
Behring and Shibasaburo Kitasato from
four years before.
Calmette published his experiments with cobra and other venoms,
including their lethal doses in rabbits, immunization of animals with
venom and modified venoms, and the use of “antivenene” in treating
snakebite in volume 8, Annls Inst. Pasteur, in 1884. The title of his
paper, written in French, was ‘Contribution a L'Etude Du Venin Des
Serpents Immunisation Des Animaux Et Traitement De L'Envenimation’.
As an aside, this paper led to the use of the words commonly used today
as the French “antivenin” and the English translation, “antivenom”. Both
words are acceptable and used interchangeably. Hence the use of the
English “antivenom” throughout this text.
The experiments he documented also included the venom of the
Australian tiger snake and red bellied black snake. The next year, he
published ‘Contribution a L'Etude Des Venins, Des Toxines Et Des Serums
Antitoxiques', Annls Inst. Pasteur (Paris), vol. 9, 1895. This paper
documented experiments with the tiger snake, red bellied black snake,
death adder and broad-headed snake from snakes that had been sent to
him from Sydney, Australia by bacteriologist M’Garvie Smith.
On August 4th 1894 an article appeared in the Sydney Daily Telegraph
titled, “The Cure of Snakebite”. The article described the contribution
M’Garvie Smith had made in providing venoms for research at the
Pasteur Institute. They reprinted a thank you letter from A. Roux, who
noted that one of his “pupils” had carefully studied the venoms and the
results would lead to “a practical treatment of snakebites”. Shortly after,
a small pamphlet including Albert Calmette’s paper arrived detailing the
“venom of serpents and the method of rendering it innocuous”. There
was a brief description of the process of inoculating animals with
frequently repeated small but safe quantities of venom, and the notion of
taking the serum of animals so treated and injecting it into the bite
victim to render the poison innocuous. “The theory on which Dr.
Calmette has proceeded is on the lines of the principle of vaccination”,
they wrote. The full account of the results of experiments made at the
Pasteur Institute was anticipated with much interest, “as the acquisition
of an assured remedy for snakebite is one of first-rate importance to the
colony”.
The article was reprinted, and panned, in the Australasian Medical
Gazette on Sptember 15, 1894. They commented that “the idea and the
supposed antidote are too thin. The practical application of the theory,
even if there is anything in it, which we doubt greatly, would be beset
with much difficulty”. Their article concluded, “But why wait for the
results from the Pasteur Institue? Put on the right track, it should be
easy for Mr M’Garvie Smith to follow it up, and get the credit for the
acquisition of an assured remedy for snakebite”.
A Vaccine Institute was opened in 1847 in Bent Street, Sydney.
Australian George B. Halford had experimented with snake venoms as
early as 1865. He claimed early success by attaching leaches to the
throat of a snakebite victim. Later he adopted a method of intravenous
injection of ammonia to treat snake bite. His method was widely
discounted by 1870.
The first suggested trials of Australian antivenom were proposed by
Charles James Martin in Sidney in 1897. Born in London on January 9,
1866, Sir Charles was knighted in1927. He was a pathologist and
physiologist who only spent twelve years in Australia, but served as
demonstrator in physiology at the University of Sydney, lecturer in
physiology and professor at the University of Melbourne, and did
extensive research on black snake venom.
In 1898 an Australian named Frank Tidswell immunized a former
ambulance horse with Tiger snake venom. Tidswell wrote a book on
Australian venoms in 1906, including the platypus and red-spotted
spider. He experimented with venom and concluded that it would not be
possible to produce a single universal snake antivenom, that a separate
serum was needed for each snake venom. Frank Tidswell became
Director of the NSW Government Bureau of Microbiology in 1908.
Adolpho Lutz was born to Swiss emigrants in
Rio de Janero in 1855. He became a physician,
graduating from the University of Bern in
Switzerland in 1879. He continued his studies in
Germany, and several places in europe,
including Paris, where he studied with Louis
Pasteur. He returned to Brazil for six years and
then went back to Germany. He specialized in
infectious disease and tropical medicine. Before
returning to Brazil in 1892, his studies took him
to Hawaii (not then a part of the United States)
and California in America. He was then made
director of the the Bacteriological Institute in Sao
Paulo. In 1897 Adolfo began work on the
preparation of sera against bubonic plague with another young physician
there named Vital Brazil.
Vital Brazil was a physician known for his clinical activities in Public
Health dedicated to important diseases such as the yellow fever, bubonic
pest, smallpox and cholera, among others. While living in Botucatu, Soa
Paulo he was moved by the number of snake bite cases to begin his
study and experiments with snake venoms. He was taught the
production of anti-snake serum from Drs Adolfo Lutz and Albert
Calmette. He developed his own antivenom by progressively injecting
small amounts of raw venom in dogs and goats.
In 1901 Vital Brazil developed the first polyvalent
anti-ophidic serum by combining the venom from the
Jararaca, rattlesnake and coral snake at the Instituto
Butantan, located in São Paulo, Brazil.
The Bacteriological Institute began production of anti-plague serum in
1899.
Vital Brazil began his pioneering research on snake venoms. On
February 3rd, 1901, Adolpho Lutz and Vital Brazil co-founded a new
Serum Therapy Institute. It was the first research facility dedicated to
the science of venomous animals and the production of vaccines for
tropical diseases. It soon became leading research center in vaccines and
sera of all kinds. Vital was convinced that antisera could be used to treat
envenomations by spider, bee and snake bite. Vital began experimenting
with Calmette’s serum, and was able to prove very quickly that the
serum made to treat the bites of the Indian cobra were ineffective
against the South American snakes. He became the first to produce
monovalent antivenom for the jararaca, the South American rattlesnake,
and the coral snake, the most common snakebites in Brazil. He then
became the first to produce a polyvalent serum that would be effective
against envenomations by the three most common snake bites in Brazil.
Using the same methods, he produced the first antivenom against the
spider and scorpion.
As antivenom became available throughout Brazil, the mortality from
snake bite dropped to less than 2% which ammounted to thousands of
lives saved.
In the early 1900’s the foremost medical research institutes were in
Europe, with the Pasteur Institute most noteworthy. American
laboratories were primarily university chemistry labs and government
labs set up for national defense. The first organization of a world class
research facility began with the founding of the Rockefeller Institute for
Medical Research, created in 1901 through an endowment by John D.
Rockefeller. The first director of the Rockefeller Institute for Medical
Research was Dr. Simon Flexner, professor of pathology at the University
of Pennsylvania.
One of his associates and research
assistants was Japanese physician
Dr.
Hideyo Noguchi, a prominent Japanese
bacteriologist. Noguchi was a herpetologist,
who studied snake venom. Together,
Flexner and Noguchi produced the first
antivenom for rattlesnakes in America in
1903.
The incident of venomous snake bite in North America has never
approached the numbers of bites recorded annually in tropical South
America, India or Africa. Even before the widespread availability of the
serum there were usually slightly more than one hundred fatal bites per
year in North America. This is a significant amount, but pales in
comparison to, for example, the three thousand deaths per year in
Brazil.
In North America, the copperhead snake is regarded as being deadly
in rare cases involving large snakes or small victims. The snakes of the
Appalachians up through Maine spend the winter months in hibernation.
The rattlesnakes give warning to their presence with the famous rattle,
and are less frequently stepped on. In fact, the production of the
antivenomous serum for North American snakes was considered
unprofitable, as the demand would not equal the supply.
In 1904, Vital Brazil
visited Europe. The
“Instituto Serumthérapico,”
gained a reputation around
the world as a first class
institute. During a 1914
South American expedition,
Theodore Roosevelt visited
with Dr. Brazil to obtain
antivenom as “there is
always a certain danger
from snakes.” He wrote of
the visit in Through the
Brazilian Wilderness, and
included “I know of no
institution of similar kind
anywhere.”
The American Society of Ichthyologists and Herpetologists was formed
in 1913 by John Treadwell Nichols.
In 1915 the Serum Therapy Institute was renamed the Butantan
Institute, which is still a major tourist attraction of the same name in Sao
Paulo, Brazil.
On January 27, 1916, Bronk Zoo worker John Toomey was bitten by a
Western diamondback rattlesnake and admitted to German hospital on
Manhattan's Upper East Side. Thanks to a special invitation from the
Carnegie Foundation for the improvement of peace, Vital Brazil and a
group of Brazillian scientists happened to be in New York. Dr. Brazil
made headlines in the United States when four vials of his antivenom
were used to save the man’s life. One of the first palces Dr. Brazil visited
was the Reptile house in the Bronx Zoological Garden, just four days
before.
It was on this visit that Vital met
Raymond L. Ditmars, curator of the
New York Zoological Park (Bronx
Zoo). Ditmars was an avid
herpetologist who had written The
Reptile Book 1907 and Reptiles of
the World in 1910. Raymond Ditmars
provided a number of North
American snakes and a large Gila
Monster for Vital to take back with
him for researching the production of
specific antivenom. In exchange,
Vital and Ditmars developed a safety
protocol for the Reptile House, with
agreement to ship fresh tubes of the
North American antivenoms
developed from Brazil every six
months for the Park’s reptile house.
The friendship of Vital Brazil and Raymond Ditmars was instrumental in
establishing a serum production facility in the United States. Theirs was
such that Ditmars visited the serpentarium in Brazil on zoo expeditions
and brought back several hundred tubes of antivenom from Dr. Brazil’s
Institute, which he liberally distributed for free. Even then, the South
American product could not be distributed if sold in the United States due
to lack of approvals here. Later when the next director of the Butantan
Institute, Afranio do Amaral came to New York, he brought forty South
American snakes and presented them for the collection at Ditmars’ New
York Zoological Park.
The Commonwealth Serum Laboratories was established in Australia
in 1916.
Unlike in the United States, the demand for antivenom in South
America was such that in 1920 Vital Brazil opened a second facility called
the Instituto Vital Brazil.
Formerly known as the Pasteur Institute, the Queen Saowapha
Memorial Institute (or The Snake Farm) in Bankok Thailand is only the
second of this type of farm to be started in the world, was opened on
November 22 1923.
In 1923 the Bangkok Snake Farm was established in Thailand to
produce antivenin.
It was the experiments and research done by Vital Brazil that led to
more understanding of the properties of different venoms. Calmette’s
serum derived from the venom of Indian cobra was not effective on the
venom of bothrops type South American snakes. Research done by
Hideyo Noguchi showed variation in rattlesnake species. The only
consistent North American antivenom was for the water moccasin.
Probably because it was a single species. Venoms collected from
rattlesnakes might be from Timber rattlesnakes, Eastern diamondback
and Canebrake snakes in the east, or Western diamondback, sidewinder
or many other rattlesnake species in the west. Monospecific antivenin
would depend on proper identification and uniformity of venom samples.
Experiments were done to identify the differences in each. Polyvalent
antivenom could be made using a mixture of the types that would
provide the best cross protection. If two or more venoms were found to
be identical, it wouldn’t be necessary to use all in the production of the
antivenom. In Brazil, Vital used the venom of the Jararaca to represent
all bothrops type snakes. The serum proved effective against the Fer De
Lance and Bushmaster. In North America, the water moccasin and
copperhead were so similar that an antivenom for water moccasin would
work on copperhead envenomation. Venom samples were collected from
living specimens in the collections of the New York Zoological Park.
Experiments were done to analyze the venoms from the families of
venomous snakes, to go beyond the Indian cobra used in the first
antivenom. The characteristics unique to bothrops, crotalus, elapidae and
macrurinae began to be recognized and understood.
The commercial production of antivenom in the United States began
due to the demand from the military as troops began training in desert
camps and border areas of the American southwest. Of equal significance
was the expansion of American exploration and development of mining
and agriculture in central and South America. The need for a quality
product manufactured with U.S. approvals for the supply chain became
necessary.
Antivenom produced in America would have to be produced under the
supervision and inspection of the U. S. Public Health Service. With the
passage of the Food and Drug Act by President Theodore Roosevelt in
1906, all vaccines and biologival products had to be listed in the United
States Pharmacopoeia or the National Formulary. This would require
products to meet the standards of strength, purity and quality.
The 1902 Biologics Control Act authorized the Hygenic Laboratory to
inspect firms producing vaccines and grant licenses to those who met
rigorous standards of cleanliness and product purity. Mulford Biological
Laboratories was recognized as a leader in modern sanitary
manufacturing. They formed a division of the company to begin research
and production of antivenom that would meet the requirements of the
United States.
The H. K. Mulford Company was founded in 1891
with a retail pharmacy in Philadelphia. The original
small laboratory was upstairs from a stable in
Western Philadelphia. They became the first U. S.
firm to commercially produce diphtheria antitoxin.
After moving eight miles away to a two hundred acre
facility in Glenolded, Mulford Biological Laboratories
attained a leadership position in the production of
vaccines and serums. At one point they maintained
1,500 horses dedicated to producing diphtheria
antitoxin alone.
Mulford Biological Laboratories made New York Times front page
headlines when their diphtheria vaccine was sent by dogsled over snow
and ice in a January blizzard 650 miles from Nenana, the end of the
railroad in Anchorage, to Nome Alaska. The delivery of the vaccine is
commemorated today by the Iditarod Trail dog sled race.
Due in part to their achievements with the production of their
diphtheria vaccine and impressive clean production facilities, Mulford was
able to become the first manufacturers of approved antivenom in North
America.
In 1924 the Antivenin Institute of America, was formed as a division
of Mulford Laboratories. The Antivenin Institute of America was formed
through a joint effort of the United Fruit Company, the largest employer
in Central America, Mulford Biological Laboratories, and Harvard
University.
The Antivenin Institute of America was formed to supply Mulford
Biological Laboratories with the venoms to be used in production of
antivenom.
The first Director of the Antivenin Institute was Dr. Afranio do Amaral, of
the Institute at Butantan, who brought the techniques from Brazil.
Among those first contributors to the
Antivenom Institute was Dr. Thomas
Barbour. Thomas Barbour was an
American herpetologist and director of
the Museum of Comparative Zoology at
Harvard University in Cambrdge,
Massachusetts. His scientific travels took
him through Africa, Asia, North America,
South America, and Central America,
among other regions. Along with more
than 400 scholarly articles, Barbour
wrote several books.
Col. Martin L. Crimmins, of San Antonio Texas. Crimmins was a
Roosevelt Rough Rider, and friend of President Theodore Roosevelt. He
was a writer of western history and a herpetologist who possessed
immunity to snake bite following several bites. A transfusion of his blood
was used to treat a snake bite victim. He was a herpetologist, snake
wrangler and expert on the rattlesnakes of the American west. He and
Dr. Dudley Jackson conducted many experiments using dogs at the
Robert B. Green hospital in San Antonio.
Dr Herbert C. Clark, Director of the The Gorgas Memorial Institute of
Tropical and Preventive Medicine in Panama. Dr. Clark was a former
employee of United Fruit Company, where he had been Director of
Laboratories and Preventive Medicine in Panama. The Gorgas Memorial
Institute of Tropical and Preventive Medicine, was founded in 1921 in
Philadelphia, Pennsylvania. Its mission was to create a health education
program to train researchers in tropical health, disease, and medicine
and to establish a research institute focusing on tropical and preventative
medicine in Panama.
Raymond L. Ditmars was an avid American herpetologist,
entomologist and naturalist. Best know even today for his books on
reptiles, he could be credited with creating popular interest in the captive
maintenance and understanding of snakes and reptiles in America.
The New York Zoological Society was founded by a number of
prominent New Yorkers, including Theodore Roosevelt. The Society built
New York Zoological Park (the Bronx Zoo) in 1899, where Ditmars
worked as an assistant in charge of the reptiles. He donated his personal
reptile collection to the zoo, and helped it achieve a world-class status.
Raymond Ditmars was placed in charge of the mammals in 1926.
Ditmars made numerous collecting expeditions for the zoo, including
South America where he first visited Vital Brazil at the Serum Institute.
Ditmars helped bring about the antivenom centers in both the United
States and Brazil.
There were others who contributed to the Antivenom Institute. They
formulated a plan to provide a way to supply Mulford with a reliable
supply of venoms, so that the product could be manufactured to all
relevant U.S. approvals. Snakes would be collected and brought to
several “depots”, like the San Diego Zoo. There the snakes would be
identified, and venom extracted by the “milking” method. These samples
would be freeze dried according to a new improved method of vacuum
desiccation invented in 1909 by L. F. Shackel, assistant in Physiology, St.
Louis University. This “freeze dried” or lyophilized venom powder would
be shipped to Mulford for creating the antiserums.
Commercial sale of antivenom produced at Mulford Biological
Laboratories was authorized by U. S. Public Health Service on April 25,
1927. The Mulford Company was absorbed by Sharp and Dohme in 1929
(later Merek) and in 1947 production was transferred to American Home
Products, which is the company we know today as Wyeth.
Coral snake antivenin was produced in rabbits from a coral snake
colony maintained at the University of Florida Medical College starting in
the in the early sixties. This colony was eventually moved to the Miami
Serpentarium, where Bill Haast was able to perform the countless venom
milking extractions to provide the lyophilized powder that would become
(beginning in 1967) the Wyeth (Antielapid) North American coral snake
antivenin.
Bill Haast
Wyeth is one of the world’s largest pharmaceutical companies. In
2006 they had reported revenue of 20 billion dollars. Not bad for a
company started by two brothers back in 1860 as a pharmacy and small
research lab. Things really took off when an employee devised an
improvement to a “rotary tablet machine” that enabled mass production
of medicines with great speed and precise dosages. In 1885, Wyeth
began vaccine production. Four years later, a fire destroyed Frank and
John Wyeth’s original store on Walnut Street in Philadelphia. The
brothers sold the retail business and began focusing on mass-production.
The company became a leading vaccine and medicine producer and grew
through the years by acquiring such well known brands as Chef Boyardee
and producing a multitude of recognized products like Advil, ChapStick,
Preparation H, Dimetapp, Robitussin, and Anacin. The conglomerate we
know today that began as “John Wyeth & Brother” and “American Home
Products”, actually became “Wyeth” on March 11, 2002.
Two primary antivenoms were produced by Wyeth. One was the
polyvalent blend (Antivenin (Crotalidae) Polyvalent by Wyeth
Laboratories), designed to offer cross protection to the venom of
crotalids. This was achieved by using the venoms from the Eastern
diamond rattlesnake, Western diamond rattlesnake, Cascabel, and Ferde-lance in the production of the serum. The venoms are simmilar
enough so that the antibodies produced will work on venoms not used in
the origional mix, including the copperhead, water moccasin,
bushmaster, and other rattlesnakes. This was introduced in 1954.
The other was a monovalent for treating envenomation by North
American coral snakes. These snakes differ from all other North
American venomous snakes in having a primarily neurotoxic venom. The
cotalid venoms used in the production of the polyvalent serum provides
no cross protection to this type of venom.
Both of these antivenoms were produced by immunizing horses with
venom. The blood serums have always been associated with the potential
for a side effect that can range from a mild allergic reaction to the horse,
to full blown anaphalaxis, itself a life threatening emergency. Horses
have been used for large scale production, but it is just as viable to
produce serums in rabbits, goats, chickens, camels, or even people.
The actual package of antivenin includes two vials. One contains a
freeze dried pellet of antivenin and 0.005% thimerosal as a preservative.
This vial has a vacuum and is obviously sealed with a cap that allows a
needle to be inserted. The other vial is sterile water used to reconstitute
the pellet. The water contains the preservative phenylmercuric nitrate at
0.001% concentration. The procedure is to use a hypodermic needle and
syringe to take up the water from one vial, and inject it into the vial with
the pellet. The vacuum will actually draw the water from the syringe into
the vial with the pellet. Once the pellet has dissolved into the water, it is
ready to use. It has been “reconstituted”. This has been the typical
packaging and method of reconstituting vaccines, including common
children’s vaccines, for the last seventy years or so. From a packaging
stand point, often the only difference is the ingredient in the pellet and
the labels from one vaccine to another.
So what’s the problem?
Thimerosal is the most widely used preservative in vaccines.
Thimerosal contains mercury in the form of ethyl mercury. There have
been instances where as many as fifty vials have been used to treat
cases of severe envenomation. It is more common to see fifteen vials
used in a typical treatment. The preservatives used in the vaccine,
thimerosal (50 micrograms per milliliter) and phenylmercuric nitrate (10
micrograms per milliliter) means a person receiving fifteen vials of
antivenin would also be receiving 4.7 milligrams of mercury.
Russia, Denmark, Austria, Japan, Great Britain and all the
Scandinavian countries banned the use of thimerosal as a preservative
as long as twenty years ago due to research showing it caused nervous
system injury, and in some cases, death.
In 1997, the FDA became involved when
concerns were raised that vaccines containing the
preservative Thimerosal might be a cause in the
dramatic spike (1500%) in childhood autism seen
in the early nineties, when the number of
recommended vaccinations for children doubled.
The Food and Drug Administration
Modernization Act of 1997 required the FDA to compile a list of drugs and
foods that contain intentionally introduced mercury compounds. As part
of this review, U.S. vaccine manufacturers responded to a December
1998 and April 1999 FDA Federal Register request to provide more
detailed information about their products which included thimerosal as a
preservative. FDA concluded that reducing or eliminating exposure to
thimerosal in vaccines was merited.
In August 1999, the National Vaccine Advisory Committee did urge, as
a prudent measure, that thimerosal be removed from vaccines.
FDA’s OVRR Office of Vaccine Research and Review has been
encouraging manufacturers to develop new vaccines without thimerosal
as a preservative and to remove or reduce the thimerosal content of
existing, licensed vaccines.
To further encourage the development of thimerosal-free vaccines, the
Center for Biologics Evaluation and Research sent another letter to
vaccine manufacturers on May 31, 2000. CBER requested an update on
progress toward the goal of thimerosal-free vaccines, particularly for
vaccines administered to infants and children.
According to 53 scientists from the CDC, FDA, and the ACIP, who
reviewed the findings of Dr. Thomas Verstraten, an epidemiologist hired
by the Center for Disease Control (CDC) to review data collected from
500,000 children from the North California Kaiser Permanente cohort on
vaccine safety: "A statistically significant connection was found between
thimerosal and tics, verbal delays, ADHD/ADD and autism."
Meanwhile at Therapeutic Antibodies, Inc (now Protherics), a new
antivenom was being developed that was made from sheep instead of
horses. This eliminated the allergic reaction to the horse serum, primarily
from the better processing of the new serum, and they were able to
eliminate the thimerosal preservative. Ironically, there is still a trace of it
in the new product because it is used in the production process, but it is
not used as the preservative.
The ethyl mercury content was limited to not more than 104.5
micrograms ethyl mercury per vial, with a recommended maximum dose
of 18 vials. A patient receiving this product would receive about 1.88
milligrams of mercury, compared to the 4.7 milligrams in fifteen vials of
the old antivenom.
On October 2, 2000 the new crotalidae polyvalent antivenom (the first
one in almost fifty years) was licensed by the FDA. In January 2001
Wyeth quietly announced the discontinuation of the antivenom they had
been producing since 1954. On the last day of that same month, Savage
Laboratories announced the availability of CroFab.
January 2001, Wyeth announced the discontinuation of their antivenom.
In October, 2008, the last of the remaining discontinued product
reached the label’s stated expiration date. This was later extended for
another year.
The immune system
The most basic needs of life are the ability to survive and replicate.
One key to the ability to survive is a properly functioning immune
system. The most basic life forms must have a system of protection from
the constant barrage of bacteria and pathogens in the bloodstream. The
system works by recognizing the DNA sequence of itself. When it detects
a different sequence, it interprets that as “non-self”. These non-self
invaders are what generates a response, hence the name, “antigen”, or
antibody generating.
All cells are composed of the proteins that make up our DNA. These
proteins are a unique combination expressed on the surface of all cells
and help identify “self” from non self. All DNA has a large section called
the major histocompatibility complex. This is the section used by the
immune system to communicate and function. An antigen begins by
entering the body and attatching itself to another cell. It becomes part of
the bloodstream. White blood cells are known as lymphocytes. There are
T cells and B cells. T cells are developed in the Thymus. They attack
virus-infected cells, foreign cells, and cancer cells. B cells are produced in
the bone marrow. B cells make antibodies.
The body makes millions of Both B and T cells that circulate
throughout the blood and lymph systems every day. Their response is
activated by the antigen. When they find an antigen hitching a ride on a
cell, they can decide if it is a cell that is damaged and malfunctioning or
a non-self. They can destroy the cell on the spot, or call for other white
blood cells to help attack it. White blood cells, or plasma cells, produce
immunoglobulins. Each antigen will have a different DNA sequence that
signals it’s difference. The antigen will have several epitopes, the
sequences that makes it unique. The immunoglobulins that will be
produced will clone the epitope that induced the immune response.
Antibodies are Y-shaped proteins that are produced to bind to the
epitopes of the antigen. The tips of the Y contain the sites that bind to
the antigen like a key to a lock. These cells can then recognise and bind
to the antigen. By attaching to the antigen, the invader can no longer
move through cell walls. A large number of antibodies can bind to the
invader and signal that the invader needs to be removed.
When an antibody binds to a toxin, it is called an antitoxin. If the toxin
is a venom, it is called an antivenom.
In addition to attacking, neutralizing and removing antigens, the B
and T cells, when activated, also begin to replicate. Some of the cells
become long-lived memory cells. These clones that have sequenced the
epitopes of the invader are reproduced forever throughout the lifetime of
the system. When ever the same pathogen is encountered, these
memory cells will produce an army of cells that can attack. There are
millions of unique specific immunoglobulins constantly produced ready to
respond circulating in the bloodstream. When a horse, or any mammal,
is injected with venom, the immune system clones the unique markers of
the venom, produces an antibody that binds to the epitopes, neutralizes
it, produces memory cells of the antibody, and eliminates the toxin.
There is a limitation to what the system can respond to, which is the
lethal dose. When the invasion exceeds the ability of the system to
mount the defense, you die.
When injecting a dose to elicit the response without causing damage,
you are manipulating the system to produce an immune response. This is
called vaccination, or immunization. Immunity can be natural or artificial,
passive or active. It can also be innate or aquired. Passive natural is how
a child’s immunity is passed from the mother. Once a child is born, the
contact with infection begins the life-long process of active natural
immunity. The long term gradual immunity provided by immunization is
called active artificial. The immediate temporary immunity provided by
antivenom is called passive artificial. In a snake bite treatment, the
victim is transfused with specific immunoglobulins that bind to the toxins
to provide the temporary passive immunity. The patient is artificially
made hyperimmune to a normally lethal quantity of toxin by an abnormal
level of the antitoxin. The early discovery of the ability to transfer this
hyperimmunity is the basis for the antivenomous serum, which went
virtually unchanged for fifty years.
The first generation of antivenoms for commercial production were
manufactured by harvesting the immunoglobins from horse blood. The
hyperimmune animal’s blood would be rich in polyclonal antibodies
specific to the antigen, which would be whatever venoms were used to
elicit the response. This serum is what we call antiserum for it’s
abundance of antitoxins. Wyeth-Averst’s whole IgG antivenom was the
only approved product in the United States for decades.
One of the adverse effects from the serum was a potential for allergic
reaction to the whole immunoglobulin derived from the horse.
A simple skin test for horse sensitivity had to be performed before using
the antivenom. In a hospital, the antivenom could still be given to people
who had an alergic reaction, but it required appropriate medication. As
many as 23% had anaphylactoid reactions and nearly 50% suffered from
serum sickness. Whole IgG horse serum was the only antivenom
available until the production of Protherics’ CroFab was launched in 2001.
CroFab was the first new snake bite antivenom in 50 years. There are
a couple of important differences in the new antivenom. The new CroFab
was made from Australian sheep (ovine) instead of horses (equine).
CroFab was developed by Dr. Richard Dart, head of the Rocky Mountain
Poison Center in Denver Colorado. Dr. Dart worked tirelessly to develop a
safer serum that caused fewer of the sometimes violent allergic reactions
seen with horse-based product. In 2002 Dr. Dent was honored with the
FDA’s Commissioner’s Citation for his efforts in bringing this product to
market. In addition to the serum being produced from sheep, there was
another innovation that reduced the incidence of allergic reaction. Unlike
the whole IgG immunoglobulin, which used the entire “Y” structure,
CroFab used an enzyme to cleave the Y structure into Fab (antigen
binding) fragments so they could be separated from the whole
immunoglobulin in the final product. This eliminated the Fc fragment,
thought to be responsible for the allergic reactions. There are always
possible reactions to
all animal products,
but the new CroFab
has proven to cause
fewer than the whole
IgG horse serum.
Another potential
sourse for an allergic
reaction is from the
enzyme used to cleave
the immunoglobulin
molecule. Papain is
used to break the
hinge region into the
fragments in the
production process. People treated for snake bite who have allergies to
papain, chymopapain, other papaya extracts, or the pineapple enzyme
bromelain may also be at risk for an allergic reaction to CroFab.
From Horse to Human
Passive immunity can be acquired artificially through a transfusion of
antibody-rich, or hyperimmune serum, and is the basis for the concept of
antivenomous serum.
As early as 430 BC, ancient Greek historian Thucydides noticed that
people who survived the plague could treat the sick without contracting
the sickness a second time. He was considered the father of scientific
history for his method of analysing cause and effect and physical
evidence without referencing intervention by the gods.
Louis Pasteur would later experiment with aquired immunity to develop
the first vaccines. The body has the system that responds to the invader,
and man has learned to manipulate and exploit the capability artificially.
Vaccines represent a very effective manipulation of the immune system.
Transfering that immunity to others is the basis for all commercial
production of antiserums.
To harvest a substantial amount of blood for commercial purposes,
horses became a logical choice. They were fairly easy to maintain, docile
and readily available. The breed of choice for serum production is the
Percheron horse, whose average mature weight is about 1,500 pounds.
The mighty Percheron draft horses originated in the town of La
Perche, France from ancient war horses. These were large muscular
horses, bred for their temperament and ability to pull stage coaches and
heavy wagons. The breed is very alert and intelligent, and very good
around children. It is the horse of choice pulling decorative displays and
wagons down Main Street USA in Disneyland today. John Baughman is
one of the reasons the Percheron horse became, and is still, the
preferred breed to use in equine serum production.
Daniel E. Baughman was the founder of the Fort Dodge Serum
Company. He was born the fifth of twelve children on April 18, 1867, on
the homestead of his parents in Panola Township, Woodford County,
Illinois. His parent’s co founded the Flanagan Mennonite Congregation in
Illinois. In 1892 Daniel Baughman’s father, John Baughman, had
purchased 720 acres near Manson, in Calhoun County, Iowa. He became
a breeder of Percheron draft horses and formed the German American
Horse Company. At that time a Percheron horse had the same value as a
160-acre farm.
One account of the sturdyness of this breed is the story of a farmer
that rode to the home of Dr. Baughman. The farmer pleaded with him to
come take a look at his sick mare. Dr. Baughman could see the concern
of the farmer and agreed to ride back to his farm and have a look at the
horse. When the two finally arrived at the farm Dr. Baughman asked the
farmer where the mare was. The farmer said it was the horse he had just
ridden all the way to the doctors house and back!
Daniel E. Baughman became a Doctor of Veterinary Medicine and
moved his veterinary practice to nearby Ft. Dodge in 1898. He was
primarily a large animal practitioner with emphasis on horses. In 1906
the U.S. government patented the
first anti-hog cholera serum. Dr.
Baughman bought the serum from
the government to treat hogs in
Ames, Iowa. He saw an opportunity
to start a business manufacturing
the serum. In 1912 he formed the
Ames Vaccine Company by hiring
the man that had worked in the
production of the serum for the
federal government (Mr. Hamilton),
and the director of the production of
hog cholera serum with the United
States Department of Agriculture,
Bureau of Animal Industry (Dr. H. P.
Lefler) and a former employee of the
Virus and Serum Division of the
Department of Agriculture (Howard
Shore) who was particularly suited
to obtaining approvals from the
USDA of the serums developed by
their new company. Within a year the business moved to Ft. Dodge
where it was renamed the Fort Dodge Serum Company. It eventually
became the largest industry in Fort Dodge, Iowa, and a leader in animal
serum and vaccine manufacturing.
In 1945, one of the companies acquired by the future Wyeth (still
known as American Home Products at that time) was the Fort Dodge
Serum Company.
In the production of antivenom, the horse is slowly immunized with
minute doses of venom. Gradually the dose is increased until the horse
can receive a normally lethal amount of venom, and more. The horses
immune system has responded to the very first exposure with the
process of producing the antitoxin. The horse can not mount a defense
against a lethal quantity until it has produced enough antitoxin to do so.
The first few injections, or exposures, serve only to manipulate the
immune system into producing enormous ammount of clones of the
antitoxin. These clones will remain in circulation until they are eliminated
from the body. New ones are constantly being produced, but after all
enemies have been neutralized, production slows considerably if the
enemy is no longer detected. In a program of gradually increasing doses,
“booster” shots are spaced out to maximize production for the
antivenom. The horse quickly reaches a hyperimmune state, where there
is much more antitoxin than the toxin that could be delivered by a
normal snake bite. It is at this point and beyond where the blood can be
harvested for the production of a serum.
Horses are used
because they are
generaly calm, gentle,
and easily maintained.
Perhaps most important,
a commercially viable
volume of blood sera can
be harvested repeatedly
without harming the
animal. The same
process that is described
using a horse for the
production of the
antivenom could be
applied to sheep, or any
other suitable animal, (in
Saudi Arabia they use
camels!) or a human
being.
Basic premise- Simple over view of this program.
The presentation of this simple case study is intended to provide
insight into one example of self immunization using raw snake venom.
There is a bit of irony with using the venom of the Eastern coral snake, in
that the process has come full circle. At present there is no FDA
approved antivenom being produced, and here one solution is to revert
to the ancient crude method of the Egyptian snake charmer. The basic
premise of inoculating with gradually increasing doses has been practiced
for centuries and is the basis for all modern pharmaceutical antivenom
production. Here the remedy for a lack of available modern packaged
product is as old as man himself. Human adaptive immunity.
In the example herein, a series of injections is recorded which resulted
in the ability to survive injections of entire venom extractions from a
large adult coral snake. This was achieved with a total of seventeen
injections, beginning with the first injection on 4/08/08 an continuing up
to and past the injection of an entire bite equivalent on 9/16/08, which
was injection number twelve. This is by all means an anecdotal account
of one person’s experience, not an instruction manual. The subject is a
forty-seven to forty-eight year old adult male weighing approxamately
220 lbs throughout the experiment. There were no adverse effects noted
throughout the test period. There were a total of four different snakes
used randomly through the course of extractions, but most were from a
snake that exceeded 42 inches, including the entire extraction injections.
Venom was not weighed, dried, saved or detoxified. All venom used was
collected within moments of use, dispensed from the collection cup into
the dilution cups using the same size pipettes. Measurements were made
by drops. Experiments showed drops from the same pipette were very
consistant in actual weight and liquid volume. It should be noted that
drops from a pipette are not the same as drops from a snake fang.
Consistency comes from the specific gravity of the liquid being
dispensed, and the size of the pipette. Dosages were estimated in
diluting the liquid drop or drops of venom by serial dilution and working
back to the whole drop of raw venom and then multiple drops or entire
extractions. This technique proved simple and effective to elicite the
immune response desired.
Materials
Snakes were milked into a shot glass which was cleaned, sterilized
and covered with a plastic bag held taught across the opening by rubber
bands. This insured that the fangs penetrated the plastic allowing the
venom to collect in the clean glass container. Larger snakes would
obviously require a suitable collecting jar. The snakes mouth or any other
foreign matter was prevented from contact with the inside of the
collection glass. The venom would usually be seen as a slight streak
down the glass from each fang and a drop or two pooled at the bottom
edge. Coral snakes typically deliver a very small amount of venom. In
some cases the venom collected was so small that drops of sterile water
were added to the collection cup to enable transfering the liquid
containing the venom. The venom was sometimes nothing more than the
streaks on the side of the collection glass.
Water used for dilutions was distilled and boiled to make sterile. It
was kept in sterile centrifuge tubes until used. The tubes were unsealed
and used for the injection and discarded. A fresh tube was used with
each injection.
15ml Polystyrene Centrifuge Tubes were purchased from:
Lake Charles Manufacturing
4905 Common St
Lake Charles LA 70607
Toll Free: +1 866 739-4600
International: +1 337 479 2480
Fax: +1 337 479-2481
A single tray of 50
tubes, sterilized,
were $12.00.
The dilution cups were actually Sample Analyzer Cups. They are
.05ml, clear. These were used to perform dilutions. I would typically
dispense nine drops of sterile water from the centrifuge tube into the
dilution cup using the pipette. Then, using the pipette, draw up the
venom from the collection vial (the shot glass) and carefully dispense
one single drop into the dilution cup already containing the nine drops of
sterile water. I was almost
always dealing with ten drops in
the first dilution cup.
Each individual drop in the
dilution cup would represent one
tenth of a drop of venom.
Dividing the contents of the
dilution cup by half, for
example, would represent one
half a drop of venom.
Subsequent dilutions could
easily be made by ten by
repeating the dilution sequence
from each cup into another.
For example, using the pipette to draw up a sample from the dilution
cup, and adding just one drop into a second dilution cup containing nine
drops of sterile water would give you ten drops that would be diluted
1/100 from the first drop of venom added to the first cup. Now by using
the contents of the second cup instead of the first, you have a range of
1/100, one tenth of the second cup, to 1/10 of a drop- the entire
contents of cup two. I will elaborate on serial dilution later.
These cups also came from Lake Charles Manufacturing.
1000 of these cups cost $28.70. These were used once and discarded.
The pipettes used to transfer and
measure all liquids were
Disposable plastic Fine Tip Standard
Transfer Pipettes.
A pack of twenty cost $2.52, also from Lake Charles Manufacturing.
Syringes used were ½ cc Terumo Insulin syringes.
These were purchased from:
TNB
152 Congressional Lane #307
Rockville Md, 20852
Model number Terumo SS05M2813 , 100 per box cost $22.00
This picture shows a simple way to set up for an injection. A small 2x4
wooden platform is drilled to hold a pipe fitting that serves as a holder
for the centrifuge tube with the sterile water. Several additional holes are
drilled to hold the small sample analyzer cups that are used as the
dilution cups. The pipette seen in the picture is a very inexpensive plastic
disposable type. It provides a very consistent drop size when used
throughout the program.
Drops are dispensed very carefully with
the pipette held at the exact angle.
“Units” are the graduations printed on the side of the syringe being
used. These are insulin syringes, and the basic measure of insulin is U100, which is 100 units per mililiter, or cubic centimeter of solution.
These syringes are ½ cc, so will hold 50 units. When I use the dosage
amount of five units, for example, it would be the fifth line (indicated
with the number five in the picture). These micro syringes make it very
easy to inject very small quantity such as three units. If the dosage is a
hair over the line, I might call that a “fat five”.
Usually when drawing the diluted venom and water mixture into the
syringe, it will be about ten units or so. After allowing the air to rise to
the top and squeezing out, there is usually going to be about seven units
available. I tried to work the dilutions to be fairly consistant with
injecting five units every time.
No guts, no glory!
Injections were made in the fatty area of the inner thigh, upper front of
thigh, or calf area. Just because it was fairly painless, easy to observe,
and not as ugly if these big red patches were anywhere else. You’ll see.
All injections were made through the skin, a subcutaneous injection. If
you don’t go deep enough, it is considered an intradermal injection.
Those hurt. It will pop up in the skin like a blister between the colored
layer and the white layer below. You will not do that twice.
Use alchohol pads to sterilize the injection site, just as you have seen
your doctor do it when you get any shot or needle. Get the injection
through the skin and pull back on the plunger to make sure you have not
hit a vein. If you draw blood into the syringe, use another spot. If you do
not draw blood into the syringe, push the plunger and dispense the
desired amount. It is much easier to be accurate by having the exact
amount in the syringe and shooting it all.
Sometimes I would draw a circle around the site, but it quickly becomes
unnecessary as the site turns pink, or worse.
This is a typical reaction that happens within a few minutes. The injection
site will puff up in a circle about the size of a quarter, somewhat like a
massive mosquito bite.
The immediate mosquito bite swell usually fades away within fifteen
minutes or so and becomes a pink spot that continues to spread out. As
it gets bigger, the skin begins to swell with a feeling I call “fat skin”. As if
the skin becomes engorged with fluid in the pink area. It becomes tight,
warm, and might resemble the skin on an orange.
The pink area will sometimes have a very distinct sharp edge betweeen
the swollen “fat skin” area and surrounding normal skin. This will
continue to spread out from the injection site. This would be pretty
typical about an hour after the injection.
Starting to see a nice fat swell here. This could be an hour or two in,
depending on the strength of the booster. At this point it will feel warm,
maybe hot. It does not feel good, but not really painful.
Now the skin is really feeling fat, swollen, and sore. The skin is
usually not going to get any fatter or more swollen from this, but it is
going to spread out. This will be a constant irritation, to say the least.
Hard to ignore, but still not a terrible pain.
This is actually a different injection from the last picture, just happens
to be very close to the same place. The pink spot has a distinct edge in
color and swelling. You can grab it with your hand and it feels like a
handful. It will feel hot. Not a throbbing thing at all, but a constant
irritated sore spot. Feels alot like it looks. This would be about three
hours in. it is a pretty typical sequence. There may be some venoms that
do not produce this type of swelling, and I didn’t expect this from the
neurotoxic venom of the coral snake, but it always swelled in a similar
manner, even after showing resistance later on. It would just run it’s
course a little faster.
This shows the pink area has spread out over a good sized area of the
right upper thigh. You can see the profile of the swelling. The venom, or
the effect, is still spreading further from the injection site. The reddness
and swelling do seem to advance away from the heart rather than
towards it for some reason. An injection like this on the front of the thigh
will also eventually migrate towards the back of the thigh when you
sleep, it seems. At this point, the swelling is not getting worse, as before,
just keeps spreading out.
This picture is from about six
hous in after building up quite a
good resistance. This is not
typical in the beginning. The pink
area is quite spread out, but the
swelling is actually starting to
really fade away at this point. It
takes a couple hours for the
“immunity” to kick in and fight
back to this extent. This is quite
good. In the beginning, this will
not happen for a couple of days.
This is more typical, where the pink area, and maybe you’d call it red,
keeps spreading even after the swelling has stabilized for a few hours.
This is what it will look like after about twelve hours.
It is not unusual for an
injection in the thigh to
spread out from the groin
to the knee.
These two pictures are
from one day to the next.
It keeps spreading until the
immune system can
contain it.
This picture is twelve hours after
an injection and there is an
obvious resistance when you see
the swelling is gone and the pink
is already fading out.
In the early stages, it would take
about four days for the pink to
fade this much!
This was the first injection, a single drop of venom diluted one thousand
times. From this to an entire exraction injection in about five months.
A picture of the second injection. Same dilution rate and amount
injected. Even at this early stage, the immune system already has seen
this invader before and produced an antivenom to neutralize it. It has
produced memory cells that will recognise this second invasion and
signal the production of more antivenom to attack this second exposure.
It hasn’t yet learned to respond to a significant threat, as this is only one
thousanth of one drop. By increasing the venom in each injection, the
immune system will learn to produce greater quantities of the antivenom
very quickly. There will also be more constantly circulating antibodies as
it comes to expect the next invasion. If you were to discontinue the
boosters, eventually the circulating antibodies are eliminated and there
will be nothing to stimulate production of quantities. The memory to
produce the exact antitoxin will never be completely forgotten.
Everything in this presentation
can be found online and it is
hoped that you will use this as
inspiration for more thourough
and detailed inquiry. There is so
much to learn.
This is a basic stick figure
drawing of an immunoglobulin.
The very basics were described
in the section about the immune
system.
This is a more detailed
illustration of the same IgG.
It is meant to illustrate the
complexity, and the extent
that the presentation was
extremely basic and meant to
be a very general introduction.
About the equivalent of the
stick figure illustration above.
Please read more about this
fascinating system.
Serial Dilution
When working with venoms, it is necessary to work with minute
quantities. It may be desirable to work with single drops or quantities as
low as one thousandth of one drop. It would be pretty difficult to try and
divide a single drop into one hundred or one thousand parts. It would be
almost impossible to read a syringe and accurately administer a quantity
so small. There is an easier way.
I use 15ml polystyrene centrifuge tubes for storing the sterile water I will
be using with each injection. These are just convenient screw top
containers that can hold plenty of water for setting up a booster. I boil
the distilled water and fill the tubes myself. This is the water I will use to
dilute the venom and inject. You could use any sterile or bacteriostatic
water. Usually the commercially prepared bacteriostatic water will have
.9% benzyl alchohol added to it. A 30ml vial is usually about $7.00. I feel
comfortable using my own sterilized water. I do not reseal and save the
container. Once I have taken water from it and set up for an injection,
the rest of the water in that tube is discarded and the tube will be
resterilized and refilled for the next use.
I use a plastic standard fine tip transfer pipette to measure all liquids in
exact drops. It is a clear plastic bulb with a tube and a fine tip at the
end, basically like an eye-dropper. I use these one time and throw them
away.
I use 0.5ml sample analyzer cups for dilutions. These are very small,
sturdy plastic cups. They come in a bag of 1000, so I use them once and
throw them away. I put nine drops of sterile water in these and add one
drop so I am always working with ten drops in all my calculations and
dilutions. I draw from one of these cups right into the syringe I am using
for dosages. I also call these dilution cups.
The syringes I use are Terumo Sterile 1/2cc Insulin Syringes with 28g x
1/2" Needle. The syringes are marked in “units”. I keep track of my
dosage in units at certain dilution levels. I may be using, for example, a
one hundredth drop solution and inject five units.
centrifuge tube
analyzer cup
pipette
Typically, I set up my boiled sterile water and two or three analyzer
cups. I have made a wooden platform with holes drilled to support these
items. I use the pipette to transfer the sterile water into the analyzer
cups, very carefully dispensing nine drops in each cup I will be using to
make dilutions. Then I use the pipette to transfer the freshly milked
venom, dispensing one pure drop of raw venom into the first analyzer
cup. That makes a total of ten drops in analyzer cup number one, one
drop of venom in the cup and nine drops of water. At this point it really is
a dilution cup, because I have diluted the drop of venom with nine drops
of water.
Each drop in cup number one now represents one-tenth of a drop of
venom. If we were to draw up all the contents of cup number one into a
syringe and inject half of it, we would be injecting one half a drop of
venom.
Usually I will further dilute the venom from cup number one in a process
known as serial dilution.
If we use our pipette and take up the one in nine mix from dilution cup
one, and carefully drop one single drop into the nine drops of pure water
we put into cup number two, we will have ten total drops in cup number
two. We have now diluted the original drop of raw venom by one
hundred. If we inject only one tenth of cup number two, we would be
getting one hundredth of one drop of the original drop of raw venom.
This can be further diluted with each successive dilution by ten. By taking
one drop from cup number two, and putting it into nine drops of pure
water in cup number three, we have diluted our one drop of venom by
one thousand. One drop from cup number three now represents one
thousandth of a drop of raw venom.
It is always important to remember that the dilution cup contains the
same amount of venom that you added to it. It is the division of the cup
that dilutes the venom. For this example, using the contents of the third
cup, if you were to draw up all ten drops into a syringe, you would inject
one-tenth of the contents of the syringe to get one thousandth of one
drop of venom. You could inject half the amount in the syringe and get
five-hundredth of one drop. If you injected the entire syringe, you would
be getting one hundredth of one drop. That is how much venom you put
in cup number three.
Just be careful with the
dilutions, it is easy to
miscalculate by ten times. Not
good when you are using a
potent venom, or just starting
out. The venom should be
considered potentially diluted,
depending on how you use it.
The consistency comes from using the exact pipette so that the drops are
equal. You can experiment with different droppers. The drops will be
bigger as the opening gets bigger. A fine tip pipette will have different
sized drops than a medium tip. But the drops from the same instrument
are actually quite consistent. The specific gravity and surface tension will
make each drop nearly exactly the same.
By using this method, it is fairly simple to start using one thousandth of
a drop of venom and progressively inject stronger doses until you are
taking all of the contents (all ten drops) and then move up to the
stronger dilution in your second cup and on to the first. By using a very
small syringe, you will have fairly precise control of dispensing even one
drop in an injection.
Actual case study
In this section I will provide all of my actual notes for each of the
injections. These are reproductions of my handwritten notes just as they
were written on the yellow legal pad. This is my account of the program I
followed to achieve virtual immunity to coral snake venom. I prefer the
term resistance, but once you are resistant to a bite equivalent from a
very large adult snake, you can use the word “immune”. Whatever you
call it, these snakes can not hurt me. And this is exactly how I did it.
Number
one
Date
4/18/08
Interval
start
Dilution *
1/1000
Units
four
Location
Back R calf
Notes:
1 drop micrurus fulvius venom into nine drops sterile water.
Into second cup of nine, a single drop for 1/100 dilution
Into third cup, a single drop from the 1/100 mix to make 1/1000 dilution
Injected four units into back of right calf, 4:00 pm.
Notes:
1” red spot around site, with burning sensation.
12 hrs- 2” red spot feeling of swelling, but none observed
redness is down and away from injection site. Actual inj. site is ½” red
spot. Soreness is spread to red area.
24 hrs- 2.5” wheal, very sore.
26 hrs- soreness is fading. Is into muscle depth.
4/21/08 8:00 a.m.
Soreness is gone, slight pink where red was. Wheal around injection site
is same small ½” red spot. Not sore.
* Dilution recorded in the box are really just a general indication if using
from the first, second or third dilution cup, not exactly accurate
Number
two
Date
5/21/08
Interval
33 days
Dilution
1/1000
Units
four
Location
Above R calf
Notes:
Same repeat of dose one.
four units @ 1/1000 Right leg, above calf.
Notes:
No burning- thought there was nothing
Next day- redness. Second day quite a red spot, some soreness. Red
area felt hard, like thicker. Some discomfort, not bad. Redness goes
down and away from injection.
Number
three
Date
5/30/08
Interval
9 days
Dilution
1/1000
Units
eight
Location
Back of L
knee
Notes:
Same dilution as previous, but more units injected.
eight units @ 1/1000
Notes:
No burning- thought there was nothing
Next day- very small (quarter size) slight red spot, some tenderness
inside. Hard spot like before, maybe not so much. Slight swelling. Very
similar to last one.
Number
four
Date
6/13/08
Interval
14 days
Dilution
1/100
Units
five
Location
upper R
thigh
Notes:
Who knows? 2:15 p.m. Right inner thigh
Big female, foaming action of the pipette made getting the usual dosage
a crap shoot.
Put entire extraction into cup one with nine drops sterile water. Made a
lot of bubbles, was hard to calculate how much venom was in there.
Tapped on the cup to try to get liquid to form at bottom to have
something to work with. Was able to get a very small amount of clear
liquid below the bubbles to transfer one drop into cup two. Not the usual
one drop added to cup one, more like a third of the extraction (two thirds
were foaming bubbles in the cup) diluted by ten, but really don’t know.
Able to transfer one good solid drop into cup two. Drew up ten units and
injected a fat five. Could be a fairly strong mix compared to previous?
Would be 10x stronger if it was straight serial dilution of solid drops, so
who knows what this is?
Notes:
Very sore, very big red circle, pretty fast. Three hours later, very sore.
By far the worst injection so far. Puffy too. Might have f’ed up? Guess
we’ll see what happens…
10:00 Red spot about the size of a tennis ball. Very tender and sore.
Really smarts.
7:00 a.m. took two Tylenol, a lot of pain like a deep muscle bruise. Red
area is like a four x four square. Not real red, but very sensitive to touch.
Still really smarts.
6/15/08 10:00 a.m. red area is quite large, but after a rough day
yesterday, seems to be less painful and fading a bit. The area continued
to get bigger, now about eight inches square. The original four inch
square is still pretty red, the rest is quite pinkish red, but not so sore. It
may even continue to get bigger, I hope not…
6/17/08 All is well. Entire affected area is still slightly pink, like a slight
sunburn. Is slowly fading back to normal. Feels normal again.
Number
five
Date
6/27/08
Interval
14 days
Dilution
1/1000
Units
eight
Location
Inner L
thigh
Notes:
Was very careful to not make foam or bubbles with venom!
Went back to previous known mix, 1/1000 and injected eight units.
Notes:
Same as 5/30 so far.
Left inner thigh. 12:00 next afternoon- 8” x 5” rather square area of
thickened skin, somewhat sore and itchy. Just pink, not red. No red spot
around injection. Fairly mild discomfort, but skin is puffed up, hard and
feels thick throughout pink area. Looks like a sun burn. Swelling is quite
pronounced from surrounding normal area, like it has quite an edge to it.
10:30 P.M. pink area is much larger, almost entire thigh from knee to
groin. Is no longer puffy at all, but redder than earlier, and itchy.
Somewhat sore, not terrible. No red spot at injection site, but fairly red
like a sunburn. Hard to believe such a small diluted amount of venom
could be doing this? Maybe it’s an infection or something…
6/29 11:00 p.m.- All swelling was gone in morning, but quite pink and
irritated, itchy all the way around back of thigh. Now it is still pink and
itchy. Not really fading very much, pretty much the same all day. Down
to knee.
6/30 10:00 Still pink like mild sunburn still itchy. Feels good to rub area.
Is complete from knee to groin in area, around back of thigh to half way
around front. Not swollen at all.
Number
six
Date
7/7/08
Interval
10 days
Dilution
1/1000
Units
five
Location
Upper R
thigh
Notes:
Very neat set up and dilution. Did not want to do eight units, I’d rather
mix hotter and do less.
Did the usual dilution to 1/1000 except I added two drops into last cup.
Should be a bit stronger than 1/1000.
Injected fat five units. Upper right thigh.
Notes:
Immediate reaction of red spot, puffed up. Over all, much less stinging.
Same usual 4” x 8” reddish square in a couple of hours. Same feeling of
thickened skin through pink area. Not bad. By 3:00 a.m. swelling was
gone, slight pink area, seemed to be fading already. Thickened skin is
not so noticeable, just very slight pink color about ten by six inches.
7/10 10:00 a.m. Still faintly pink, feels pretty normal. Pink always
migrates around to the back of the leg, as if by gravity from sleeping? Inj
site is clearing up, was never very noticeable from other areas. This
method is always uncomfortable, gives a dull constant ache that gives
you a headache after a day of it, makes you grumpy. Not pleasant. The
boiled water seemed to be a lot less stingy than the saline. The inj site
was really no big deal This was the easiest to do to start, but exactly the
same throughout with the pink, puffy thick skin, spreading area from
knee to groin, around leg inside to back, etc. Takes a few days to run it’s
course every time. I will keep doing this dosage until effects noticeably
diminish.
Number
seven
Date
7/21/08
Interval
14 days
Dilution
1/100
Units
three
Location
Upper L
thigh
Notes:
Same dilution of one drop venom into nine drops of sterile water. Second
cup same for 1/1000 dilution
Drew into syringe from second cup and injected three units. Depending
on how much I actually did in Dose Four, this should be the strongest
dilution so far.
Notes:
Very easy. No pain, no red, no swelling after one hour.
1:30 a.m. Thick skin, very hard about four inch square area. Easiest shot
so far.(?) Really bearable minor pain. No red, slight pink. Not very
swollen, just that thick skin. Slightly raised but pretty mild compared to
the others.
7/22 9:30 p.m. Thick skin area is now about 12 inch by 8 inches, down
to top of knee. Not swollen, not red, just sunburn pink. Is itchy as usual,
but this was really easy. Sore as expected, but not as bad as any other
time. Right now it looks like it is basically over and going to quickly get
back to normal. No red in this, but the pink is pretty much the same
along the area of thick skin. Almost flat, not the pronounced edge
swelling of other doses, just that feeling of thick warm skin. Never was
anything detectable at injection site like a red spot, etc.
Number
eight
Date
8/3/08
Interval
13 days
Dilution
1/100
Units
five
Location
Upper R
thigh
Notes:
Upper right thigh. Got a very small amount from milking, snake just had
no interest in envenomating. Had to use the syringe as a pipette to try to
suck up the one decent drop. Put that into nine drops, and two drop into
another nine. Used five units from that, which should be my strongest
dose yet.
Notes:
Very easy to take. Got quite swollen quickly, obviously a decent dose.
Injection site never did anything of note except immediate puff up after
injection, which was unusually fast and pretty puffed.
8/4 Next day, very swollen tennis ball (diameter, not that big!) sized
lump quite unlike any yet. When getting my keys out of my pocket, could
feel a big lump. Got fairly red, same thick feeling skin. Spot felt hot, in
shower lukewarm water hitting it felt cold. Pink area is about five inch
circle, maybe red even. Quite a distinct edge, from one color to normal,
no gradual area.
8/5 Morning, swelling is completely gone. Skin is light pink about eight
inch square, skin feels a little hard, not the thick feeling when swollen.
Very easy with quick recovery. Symptoms are nearly gone. Definite
quicker return to normal from previous injections. Never migrated
around the sides of the thigh, maybe a bit on the inside about to half
way point. Not itchy this time. Pink area actually did eventually spread
very similar to previous, but seemed milder.
Number
nine
Date
8/11/08
Interval
8 days
Dilution
1/100
Units
five
Location
Upper L
thigh
Notes:
Upper left. Very similar to eight, had hard time getting venom to work
with. Put nine drops right into collection vial, sucked up with syringe as
pipette. Put two into nine in cup two and drew up seven units in syringe.
Injected five units.
Notes:
Had virtually no reaction at all. There was a very burgundy colored spot
about pea sized around injection, unlike any other. Perhaps from using
the needle as a pipette in vial? Slight pink blush spot about three inches
max. Very little of the thick skin thing. Basically nothing.
Number
Date
8/22/08
Notes:
Failed
no venom in mix
Interval
11 days
Dilution
Units
Location
Number
Ten
Date
8/25/08
Interval
14 days
Dilution
1/10
Units
four
Location
Upper R
thigh
Notes:
Entire milking into vial from small coral. Put nine drops into vial, took up
seven units into syringe. Injection four units, upper right thigh. This is
about the same as half a bite from this small snake.
This is really a pure venom injection with water added to measure the
amount injected.
Notes:
Had an allergic reaction. Itchy all over, very red around collar, wrists,
under arm pits, crotch, scalp. Had a spot like bubbles on left thigh in
area of previous injections. Weird. Took two 25mg DIPHENHYDRAMINE,
everything o.k. Injection swelled up and was sore. More so than others,
good bruise feeling. Basically gone in 24 hrs. Just mild pink area 8”
square.
Number
Eleven
Date
9/02/08
Interval
7 days
Dilution
1/10
Units
six
Location
Upper L
thigh
Notes:
Only about two drops in the entire extraction. Added nine drops of water
right into collection glass. Put three drops from this mix into dilution cup
one with nine drops of water. Injected six units from this dilution.
upper left thigh.
Notes:
Kinda painful injection, maybe hit a nerve? Not sure. No allergic reation
at all. Some swelling, very mild pink 8” square. 9/3 Barely anything. Pink
square.
Number
Twelve
Date
9/16/08
Interval
14 days
Dilution
1/10
Units
six
Location
Upper R
thigh
Notes:
This is a challenge dose, an entire extraction from a very large coral
snake.
Put nine drops right into extraction cup, took up all the venom.
Injected fat six units. 10:30 a.m.
Notes:
Almost immediate tingling in lips, face. Stinging all over- not good. Took
two Benadryl right away. Had a very severe and almost immediate
reaction. Became very weary, felt like I was going to pass out. Nauseous,
red, hives, itchy. Dreanching sweat. Could barely keep eyes open for one
hour. 11:30, was basically knocked out for an hour. Cleared a place on
the floor, laid down with a fan on me for another half hour. Hard to keep
eyes open. Allergic reaction. Lips and hands tingling. Waist band red,
getting hives like little bubbles all over. Injection doesn’t look that bad,
all symptoms are allergic, not venom- I think. After half hour, more
mentally together. Took another Benadryl. Arms coated in little bubbles.
Weird. Just want to lay down and sweat it out. No feeling of breathing
problems or anaphylactic shock, just kicked my butt bad. 4:30—injection
site is red, about 5” square with blotching all around the thigh. Thick skin
thing again, typical of some others. Next day morning- nothing there.
Slight swelling, very slight pink. Very tender to touch- really hurts to
press. Like a sting when pressed, but doesn’t look bad. All allergic
symptoms are gone.
Number
Thirteen
Date
10/21/08
Interval
35 days
Dilution
1/100
Units
four
Location
Upper L
Notes:
Entire bite amount was very small, as usual. At this point I am adding
nine drops to extractions, not single drops of venom, although
sometimes an extraction is about the same as a good drop anyway. Put
into nine drops, mixed with pipette. Got foam so I know there was
something there. Put two fat drops into nine, sucked up six units in
syringe and injected four.
Notes:
Very slight red area after one hour.
(This was the entire entry for this injection, must have been pretty
uneventful)
Number
Fourteen
Date
1/26/09
Interval
87 days
Dilution
1/10
Units
two
Location
Upper R
thigh
Notes:
Very little yield again. Used the nine drops of water to collect the venom
that was only slight streaks on the inside of the collection glass. Mixing
with pipette made foam. Drew up mix. Drew up solid five units into
syringe. Pushed out air, had two units left- injected two units.
Notes:
10:49 am left thigh. Some stinging, very slight swell inside, some red.
11:20 no allergic so far. No problems.
Number
Fifteen
Date
2/27/09
Interval
32 days
Dilution
1/100
Units
five
Location
Upper L
thigh
Notes:
Very nice extraction of venom. Able to use fat drop into nine drops, then
one into nine, for a very nice 1/100 dilution. Injected five units in right
thigh.
Notes:
2:45. Some immediate swelling at injection site with red patch about two
and a half inches by one and a half within twenty minutes. Some
stinging. Eleven hours later, thick skin slight pink about two and a half
inches around. Very easy, not sore. No itching or allergic reaction at all.
Number
Sixteen
Date
4/1/09
Interval
33 days
Dilution
1/10
Units
five
Location
Upper R
thigh
Notes:
Very nice extraction again. Put water into extraction cup to suck up all
venom, probably a really nice fat drop. Put all into first cup, took up 15
units and shot a fat five. One third of an entire extraction amount. Into
inside fat part of left thigh.
Notes:
Had some stinging, maybe close to a nerve or something? Got a big pink
spot about 3.5” with a raised white spot at injection like an ant bite. Not
too bad. Pink blush spread slowly from knee to groin over two days then
slowly faded out. Easy. 4/4/09 Can only tell if I press the fingers and
release that it is still slightly pink. Pretty easy dose.
Number
Seventeen
Date
7/8/09
Interval
98 days
Dilution
1/10
Units
four
Location
Upper L
thigh
Notes:
Very nice extraction from new snake @ 30” long female. Into inside
upper left thigh. Fat drop (sorta bubbles) into nine drops of water. Mixed
well by drawing up into pipette and discharging a few times. Put back
into cup one, tapped on cup until there was enough water at bottom to
suck up about seven units. Used four in injection. Basically a pure venom
injection, about one drop.
Notes:
10:30a.m. Had immediate swell around site. In one hour, nice fat pink
spot.
12:30 3” very fat thick skin area with very sharp edge around pink spot.
Very fat puffy swell that is very flat throughout pink area, just a very
sharp distinct edge to the swell. Itchy, but no allergy. It hurts a bit.
10:30 p.m. Pink area is about seven inches, pretty spread out. Not
swollen at all, feels hot. Not itchy. Immunity working to quickly reverse
the effects from earlier. Twelve hours after injection, not bad. Just a big
bright pink area spread out.
7/9 Pink spot is fading away. Very nice, this use to take four days!
Almost completely back to normal.
Presently continuing with this program to maintain a high level of
circulating antibodies.
This venom is an acetylcholinesteraise inhibitor and has a biological
function that is identical to some degenerative neurological diseases.
I hope that a study of a healthy immune system’s resistance could lead
to a method of stimulating the immune system of those people whose
lack of resistance to a similar effect expresses itself as a disorder.
Dedicated to my grandmother