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 5 8 15 34 36 39 42 43 63 68

Introduction Snake Bite The history of vaccines Antivenom So whats the problem? The immune system From Horse to Human Basic premise- Simple over view of this program Materials Serial Dilution 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 mans 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 authors life-long interest in venomous reptiles.

Snake Bite Dont 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 Mans 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 charmers 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 (13263 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 its 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 its 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 Pasteurs 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 Lon 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 Calmettes 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 MGarvie Smith. On August 4th 1894 an article appeared in the Sydney Daily Telegraph titled, The Cure of Snakebite. The article described the contribution MGarvie 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 Calmettes 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 MGarvie 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 So 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 Calmettes 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 1900s 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 Serumthrapico, 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 mans 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 Parks 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. Brazils 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. Calmettes 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 wouldnt 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 worlds 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 Wyeths 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 childrens 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 whats 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. FDAs 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 labels 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 its 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 childs 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 animals 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 its abundance of antitoxins. Wyeth-Aversts 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 FDAs Commissioners 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 parents co founded the Flanagan Mennonite Congregation in Illinois. In 1892 Daniel Baughmans 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 persons 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 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 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. Youll see.

All injections were made through the skin, a subcutaneous injection. If you dont 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 didnt 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 its 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 youd 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 hasnt 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 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 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 dont 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 fed up? Guess well 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 its 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, Id 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 its 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

Interval 11 days

Dilution

Units

Location

Notes: Failed no venom in mix

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 doesnt 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. 4:30injection 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 doesnt 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 systems 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