Threats of bioterrorism have renewed interest in learning more about all aspects of anthrax, particularly its deadly spores. In 1878, Louis Pasteur conducted a famous experiment on the virulence of anthrax bacilli. In so doing, he provided a sound experimental basis for the current day concept of how the environment of humans and animals can contribute to the development of infectious disease. Louis Pasteur, one of the fathers of the germ theory of disease, was confronted with a dilemma in 1878.
He conducted several experiments with disease in chickens, sheep and cattle. He could easily infect and cause disease and death in all of these species with blood from animals that had died of cholera. These experimentally infected animals apparently contained bacteria that we now know as Vibrio cholera. Similarly, when sheep and cattle were inoculated with a different pathogenic microbe, Bacillus anthracis, they all died of anthrax. But chickens responded differently.
Pasteur was perplexed because chickens did not suffer an untimely demise after being exposed to B. anthracis. Instead, they remained quite healthy. So, if germs cause disease, as postulated by Robert Koch and himself, why did chickens not succumb to the putative germs that lead to symptoms and consequences of anthrax?
Sixteen years earlier, Pasteur had conducted an experiment that ultimately put to rest a prevalent theory of the time. Spontaneous generation embraced the idea that non-living substances could evolve into living organisms. That is, organisms do not need to have parents because any biological organism could develop from non-living material. By sterilizing a meat-broth-containing medium and leaving the flask exposed to open air, Pasteur discovered that bacteria appeared and multiplied in the broth.
However, if he prevented aerial dust from entering the flask and contacting the broth, which he did by crafting an “S” shape at the end of the flask that resembled a swan (hence the name “col de cygne”), bacteria did not appear in the broth – for years! As a result of this experiment, Louis Pasteur was credited with the discovery that microorganisms beget other microorganisms, thus discrediting the theory of spontaneous generation.
This conclusion complemented his finding in 1859 that food spoilage was caused by microorganisms, work that ultimately led to the discovery of the biological basis of fermentation. The process of pasteurization to preserve foods such as milk and wine was successful because it greatly reduced viable microbes in these products. Today, nearly all dairy products sold in the United States are required to be pasteurized.
As Pasteur expanded the germ theory of disease, he was confronted with a distraction. He was studying the disease known as “la maladie charbonneuse,” now more commonly known as anthrax, the etiologic agent being B. anthracis, discovered by Robert Koch in 1877.
These bacilli are large, aerobic, Gram-positive, spore forming, rod-shaped bacteria. The cutaneous form of the disease causes black necrotic skin lesions, hence the derivation from the French noun for coal, “charbon.” However, the systemic form of anthrax results in malaise, coughing, septicemia, fever, diarrhea, dehydration, hypotension, shock, respiratory failure and rapid death. It is an awful way to die.
Systemic anthrax was exacting a costly economic toll on French farmers, resulting in the death of their cattle and sheep within hours after signs first appeared. Pasteur wanted to do something about it. Most shepherds knew that pastures that were used to graze sheep that had died of the disease were somehow contaminated because sheep permitted access to the same pasture the following year subsequently died of anthrax. We now know the reason for this phenomenon was due to the presence of spores of B. anthracis.
These spores can survive for years in soil because they are very resistant to weather extremes. Inhalation of just a few spores of B. anthracis is very deadly. This property of B. anthracis has been often exploited as a bioterrorist threat, as highlighted in 2001 by the mailing of letters through the U.S. Postal Service that contained spores of B. anthracis. As a result of that threat, biohazard detection systems have been installed by the U.S. Postal Service to scan for anthrax at central distribution centers.
To Pasteur’s surprise, in early experiments, cattle injected with B. anthracis survived. Edward Jenner had shown 80 years earlier that cowpox could be used to provide protection for humans from smallpox. Pasteur reasoned that the cattle that he used had already been exposed to anthrax and somehow had acquired resistance to the bacteria.
After initial successful laboratory experiments with vaccines using guinea pigs and cattle, Pasteur designed another one of his famous experiments. However, in this case, he was challenged to do so by a prestigious French veterinarian by the name of Rossignol, an editor of the journal, La Presse Vétérinaire. Rossignol and a colleague, Professor Gabriel Collin, goaded Pasteur into testing the hypothesis that pre-exposure of sheep to heat-treated avirulent B. anthracis would protect sheep against death when subsequently injected with virulent B. anthracis.
A humorous description of their plan to publically challenge Pasteur was created by the Association for Science Education (ASE) in the United Kingdom. One of the techniques used by this organization to teach the history of vaccines was the application of theater. Although the veracity of this drama could not be confirmed, the following lines were taken verbatim from scene 5. This conversation between three men revolved around Pasteur’s prior discoveries on anthrax:
SCENE 5 Inside the office of the vet in the town of Melun, near Paris, in 1881. The vet, Rossignol, is talking to Dr Colin, a scientist from Paris, and to Monsieur Dubois, a local farmer.
ROSSIGNOL – (sneering) I hear the great Pasteur has announced a new wonder vaccine for anthrax. Is it any good, Colin?
COLIN – I don’t suppose so. Most of his ideas are nonsense, but it’s no good telling Pasteur that. If you try to criticise him, he soon shows what he thinks of you. He called me an idiot at a public meeting last month.
ROSSIGNOL – That’s no way for him to treat a fellow scientist like yourself. But I hear he has suggested using his vaccine on a large scale. Suppose we forced him to do it in public – it would only take a few deaths of vaccinated sheep to ruin him forever.
COLIN – Excellent idea! It’s time Monsieur Pasteur learned what it is like to be called an idiot in front of his fellow scientists.”
Pasteur was far from being shy, actually being pretty infatuated with himself. He readily accepted the challenge and injected virulent bacteria into 25 sheep that had been pre-treated with an avirulent B. anthracis vaccine and 25 non-vaccinated control animals. The experiment was subsequently expanded to include six vaccinated and 4 control cows. Two goats were even substituted for two sheep!
The results were astounding: All of the control animals died with clinical signs of anthrax, whereas all of the animals that had been previously exposed to the heat-treated B. anthracis vaccine survived. Pasteur had therefore developed an effective vaccine for anthrax, and the results of this experiment were greeted with great fanfare throughout France.
Over a century later, it was revealed that Pasteur had used a killed rather than an attenuated anthrax vaccine. This issue remains highlighted in biomedical research not only because of the tremendous importance of vaccines in medicine, but also because of the potential use of anthrax spores as a deadly biological weapon. A number of vaccines have been developed against the bacillus and its toxins, although these anthrax vaccines are more effective for prevention of disease in domestic animals than in humans.
As might have been expected in view of what has come to be known as the Semmelweis effect in scientific culture, many scientists were quite skeptical of Pasteur’s results with experimental vaccinations. They just seemed too good to be true. The scientist mentioned above, Professor Colin, conducted research at the School of Veterinary Medicine in Maisons Alfort in Paris. He was one of Pasteur’s most inimical adversaries.
The School of Veterinary Medicine at Maisons Alfort is a famous institute that was established a century earlier in 1776 and remains the second oldest veterinary school in the world (the oldest veterinary school was also established in France in Lyon fifteen years earlier in 1761). The enmity of Professor Colin for Pasteur really began at least three years before the anthrax-vaccination trials. The relationship between Colin and Pasteur turns out to have been the impetus for the first well-publicized study of stress and immunity. This is how so.
First, a bit of background. Pasteur’s earlier experiments had consistently shown that it was not difficult to infect mammals with B. anthracis, but chickens seemed to be particularly resistant to the infectious agent. Colin chided Pasteur by claiming that he could easily reproduce anthrax in chickens. Pasteur agreed to give Professor Colin a culture of B. anthracis that readily caused disease in mammals, and Colin agreed to provide Pasteur with a chicken suffering from anthrax within a week. Pasteur left Paris on vacation, and upon returning to his laboratory, asked Professor Colin for a hen that died of anthrax. Colin eventually admitted that his experiments had been unsuccessful. He even claimed that wild dogs broke into the cage where his infected chickens had been kept, which ruined his experiment.
Figure 1. Pasteur’s experiment using cold water stress to induce susceptibility to Bacillus anthracis in chickens. Pasteur’s earlier experiments with chickens revealed that they did not die of anthrax following an infection with the putative germs that cause this disease, now known as B. anthracis. This was a dilemma because the data did not support the concept of the germ theory of disease. Chickens have a normal body temperature that is three Celsius degrees higher than mammals. Pasteur hypothesized this is why avian species do not contract anthrax. To test this hypothesis, he made chickens hypothermic by restraining chickens with either gray or white feathers in a bucket and exposing the bottom third of their body to ice water. The chicken with white feathers was inoculated with B. anthracis and subsequently died. The other chicken with gray feathers that was subjected to hypothermia and not inoculated remained healthy. The chicken with black feathers that was inoculated with B. anthracis but not subjected to hypothermia remained healthy. This experiment confirmed Pasteur’s earlier results by demonstrating that, at least in chickens, B. anthracis alone is not sufficient to cause anthrax. Furthermore, hypothermia alone did not cause the disease, although this finding certainly did not refute the germ theory. Instead, both a cold water stress and pathogenic bacteria were needed to cause anthrax in chickens. This experiment convinced Pasteur that B. anthracis is the true cause of anthrax in chickens. These data established an experimental basis for disease by environment interactions.
The story continues. Pasteur had already discovered that B. anthracis germs did not survive well at elevated temperatures, and this finding set the stage for another one of his famous experiments. Humans maintain a core body temperature of roughly 37°C, whereas the core temperature of birds is higher, around 40°C. Pasteur reasoned that the higher body temperature of chickens might be responsible for their resistance to B. anthracis. To test this idea, the feet and legs of two chickens were fastened to the bottom of a bucket and roughly a third of the hens’ bodies were submerged in ice cold water: a gray one was subjected to only the cold and a chicken with white feathers was inoculated with B. anthracis (Fig. 1).
Another chicken with black feathers received twice the dose of bacteria but it was not placed in the bucket of ice water, instead remaining in a comfortable thermal environment. Only the chicken with white feathers died of anthrax; whereas the gray and black hens remained healthy. Pasteur exclaimed, “On ne peut donc douter que la mort de la poule blanche soit due uniquement à l’inoculation charbonneuse.” On that date, March 19, 1878, in the prestigious Académie de Médecine de Paris, Pasteur and his fellow scientists were convinced that B. anthracis is the sole cause of anthrax. Pasteur and his predecessor in this discovery, Robert Koch, had been correct. Voila! – Pasteur had solved the dilemma!
Incidentally, Pasteur may have considered other experiments in which the core body temperature of sheep or cattle was artificially elevated to determine if hyperthermic mammals resisted the pathogenic properties of B. anthracis, but we have no documentation of this possibility.
Ten years later, a Russian zoologist, Élie Metchnikoff, came to the Pasteur Institute after conducting research in Italy. He had discovered that some cells of starfish larvae engulfed dyes and even splinters, and he called these cells phagocytes (Greek for “devouring cells”). He extended his results to the blood of animals, finding that some types of leukocytes are capable of phagocytosis. This discovery formed his theory of cellular immunity, for which he shared with Paul Ehrlich the 1908 Nobel Prize in Physiology or Medicine.
Hypothermia, either in vitro or in vivo, is now known to impair phagocytosis by neutrophils (known as heterophils in birds). Therefore, René Dubos, a French-American microbiologist and a humanist philosopher, offered a different interpretation than the one originally proposed by Pasteur. Dubos proffered that it was unlikely that hypothermia actually promoted the proliferation of B. anthracis in the cold-stressed chicken with white feathers.
Nearly a century later, in 1973, Dubos wrote the following: “Je ne m”attarderai pas sur la fameuse expérience par laquelle il rendit une poule, normalement résistante au charbon, malade de cette infection en la plaçant dans un bain d”eau froide. Il est probable que l”effet de l”abaissement de la température ne s”exerçait pas, comme il le croyait, directement sur le microbe en facilitant sa multiplication, mais plutôt qu”il consistait en un affaiblissement des défenses cellulaires de la poule. On pourrait imaginer une analyse de ce problème par les méthodes d”observation que Metchnikoff introduisait à cette même époque pour établir le rôle protecteur des phagocytes.”
In other words, a previously unrecognized protective immunologic mechanism had been impaired in the hens that were cold-stressed in Pasteur’s experiment: Host defenses such as phagocytosis were compromised in both of the restrained, cold-stressed chickens that were held in ice water. However, the chicken with gray feathers survived whereas the one with white feathers died because the white hen was the only one that was exposed to B. anthracis.
To the best of our knowledge, this is the first documented scientific report of a disease-environment interaction. So it is that Pasteur contributed to modern research in brain, behavior and immunity. Only the hen that was both infected with B. anthracis and cold-stressed succumbed to the anthrax. The hen that was only cold-stressed survived. The hen that was inoculated with a hyperdose B. anthracis but kept in a comfortable thermal environment also survived.
A digression: To help students grasp the meaning of interaction between main effects, we ask them, “Did cold stress cause anthrax in Pasteur’s chickens?” If the student answers “No,” we note that s/he is wrong: although cold exposure per se did not have a clinical result, it was required for the chicken with white feathers to become infected with and die of anthrax.
Alternatively, if the student says “Yes,” we point out that the chicken with gray feathers was cold-stressed but remained healthy.
The correct answer is “It depends.” In Pasteur’s chickens, B. anthracis alone did not induce anthrax. Neither did cold stress alone cause anthrax. Instead, both cold stress and B. anthracis were required to induce the disease.
Importantly, during the past century, the concept of disease-environment interaction has been extended to other pathogenic bacteria and viruses, with the effect on morbidity and mortality due to the infectious disease depending upon dose, virulence and degree of environmental stress.
The study of stress, immunity and disease is one of the long-standing cornerstones in brain, behavior and immunity research. Although Pasteur’s experiment can be criticized, it was a useful one. The take-home lesson is that well-designed experiments, conducted appropriately, last forever.
They are what good science is made of. What can change is interpretation of the results, which depends very much on the current state of knowledge. It is incumbent upon us as scientists to accurately and honestly report what we have done. Although other scientists before Louis Pasteur may have performed similar experiments, it was Pasteur who shared his results with scientists and laymen alike. Consequently, we owe another debt of gratitude to the father of the germ theory of disease, Louis Pasteur. His insightful experiments demonstrated that interactions between pathogenic microbes, host and host environment can be crucial in the etiology of disease.
Supported by grants from the National Institutes of Health to KWK (MH 51569 and AG 029573) and RD (MH 079829 and MH 71349).
K Kelley – Laboratories of Integrative Immunophysiology, Integrative Immunology and Behavior Program, Departments of Animal Sciences and Medical Pathology, University of Illinois, Urbana, IL 61801
S Curtis – Departments of Animal Sciences, University of Illinois, Urbana, IL 61801
R Dantzer – Laboratories of Integrative Immunophysiology, Integrative Immunology and Behavior Program, Departments of Animal Sciences and Medical Pathology, University of Illinois, Urbana, IL 61801
Information for this article was derived from numerous articles that have been written on the discoveries of Louis Pasteur. Some of the articles that provided very useful information are cited below.
Akriotis, V. and W.D. Biggar. The effects of hypothermia on neutrophil function in vitro. J Leukocyte Biol 1985; 37:51-61.
Decker, Janet. Deadly Diseases and Epidemics, Anthrax. Chelsea House Publishers, ISBN 0-7910-7302-5 2003; 27–28.