The Danish Peace Academy

SCIENCE AND SOCIETY

John Avery
H.C. Ørsted Institute, University of Copenhagen

Chapter 10 VICTORY OVER DISEASE

Jenner

If the Europeans and Americans of the 19th century felt that their scientific civilization had something to offer to humanity as a whole, they may have had in mind not only factories, steamships, railways and telegraphs, but also great victories won against disease. The first of these victories was won against smallpox, a disease which at one time was so common that almost everyone was sure of getting it. In the more severe epidemics, one person out of three who contracted smallpox died of the disease. Those who recovered were often so severely disfigured that their faces were hardly human.

Since smallpox was so common that people scarcely hoped to avoid it entirely, they hoped instead to have a mild case. It had been noticed that anyone who survived an attack of smallpox could never be attacked again. In Turkey and China, people sometimes inoculated themselves with pus taken from the blisters of patients sick with smallpox in a mild form. The Turkish and Chinese custom of inoculation was introduced into Europe in the 18th century, and Diderot, the editor of the Encyclopedia, did much to make this practice popular. However, this type of inoculation was dangerous: It gave protection against future attacks, but often the inoculated person became severly ill or died. It was like “Russian roulette”.

The story of safe immunization against smallpox began when an English physician named Edward Jenner (1749-1823) treated a dairymaid. He suspected that she might have smallpox; but when he told her this, she replied: “I cannot take the smallpox sir, because I have had the cowpox”. She told him that it was common knowledge among the people of her district that anyone who had been ill with cowpox (a mild disease of cattle which sometimes affected farmers and dairymaids), would never be attacked by smallpox.

Jenner realized that if her story were true, it might offer humanity a safe method of immunization against one of its most feared diseases. On May 14, 1796, he found a dairymaid with active cowpox, and taking a little fluid from a blister on her hand, he injected it into a boy. The boy became ill with cowpox, but he recovered quickly, because the disease is always mild.

Jenner then took the dangerous step of inoculating the boy with smallpox. If the boy had died, Jenner would have been a criminal - but he was immune! It took Jenner two years to find the courage and the opportunity to try the experiment again; but when he repeated it in 1798 with the same result, he decided to publish his findings. So great was the terror of smallpox, that Jenner was immediately besieged with requests for immunization by inoculation with cowpox (which he called “vaccination” after vacca, the Latin word for “cow”).

The practice quickly became accepted: The English Royal Family was vaccinated, and Parliament voted Jenner rewards totalling thirty thousand pounds - in those days an enormous sum.

In 1807, Bavaria made vaccination compulsory, and celebrated Jenner’s birthday as a holiday. Russia also enthusiastically adopted vaccination. The first child in Russia to be vaccinated was given the name “Vaccinov”, and was educated at the expense of the state. Thanks to Jenner and the dairymaid, smallpox began to disappear from the earth.

Pasteur

In 1800, when vaccination began to be used against smallpox, no one understood why it worked. No one, in fact, understood what caused infectious diseases. It had been more than a century since Anton van Leewenhoek had studied bacteria with his home-made microscopes and described them in long letters to the Royal Society. However, the great Swedish naturalist, Carolus Linnaeus, left microscopic organisms out of his classification of all living things on the grounds that they were too insignificant and chaotic to be mentioned.

This was the situation when Louis Pasteur was born in 1822, in the Jura region of France, near the Swiss border. His father was a tanner in the small town of Arbois. Pasteur’s parents were not at all rich, but they were very sincere and idealistic, and they hoped that their son would one day become a teacher.

As a boy, Louis Pasteur was considered to be a rather slow student, but he was artistically gifted. Between the ages of 13 and 19, he made many realistic and forceful portraits of the people of his town. His ambition was to become a professor of the fine arts; and with this idea he studied to qualify for the entrance examination of the famous ´Ecole Normale of Paris, supporting himself with a part-time teaching job, and sometimes enduring semi-starvation when the money sent by his father ran out.

The earnest, industrious and artistically gifted boy would certainly have succeeded in becoming an excellent professor of the fine arts if he had not suddenly changed his mind and started on another path. This new path was destined to win Louis Pasteur a place among the greatest benefactors of humanity.

The change came when Pasteur attended some lectures by the famous chemist Jean Baptiste Dumas. Professor Dumas was not only a distinguished researcher; he was also a spellbinding speaker, whose lectures were always attended by six or seven hundred excited students. “I have to go early to get a place”, Pasteur wrote to his parents, “just as in the theatre”. Inspired by these lectures, Pasteur decided to become a chemist. He put away his brushes, and never painted again.

While he was still a student, Pasteur attracted the attention of Antoine Jerome Balard, the discoverer of the element bromine. Instead of being sent to teach at a high-school in the provinces after his graduation, Pasteur became an assistant in the laboratory of Balard, where he had a chance to work on a doctor’s degree, and where he could talk with the best chemists in Paris. Almost every Thursday, he was invited to the home of Professor Dumas, where the conversation was always about science.

Pasteur’s first important discovery came when he was 25. He had been studying the tartarates - a group of salts derived from tartaric acid. There was a mystery connected with these salts because, when polarized light was passed through them, they rotated the direction of polarization. On the other hand, paratartaric acid (now called racemic acid), did not exhibit this effect at all, nor did its salts. This was a mystery, because there seemed to be no chemical difference between tartaric acid and racemic acid.

Studying tiny crystals of paratartaric acid under his microscope, Pasteur noticed that there were two kinds, which seemed to be mirror images of one another. His vivid imagination leaped to the conclusion that the two types of crystals were composed of different forms of tartaric acid, the molecules of one form being mirror images of the other. Therefore the crystals too were mirror images, since, as Pasteur guessed, the shapes of the crystals resulted from the shapes of the molecules.

By painstakingly separating the tiny right-handed crystals from the left-handed ones, Pasteur obtained a pure solution of right-handed molecules, and this solution rotated polarized light. The left-handed crystals, when dissolved, produced the opposite rotation! Pasteur ran from the laboratory, embraced the first person that he met in the hall, and exclaimed: “I have just made a great discovery! I am so happy that I am shaking all over, and I am unable to set my eyes again to the polarimeter.”

Jean Baptiste Biot, the founder of the field of polarimetry, was sceptical when he heard of Pasteur’s results; and he asked the young man to repeat the experiments so that he could see the results with his own eyes. Under Biot’s careful supervision, Pasteur separated the two types of crystals of racemic acid, and put a solution of the left-handed crystals into the polarimeter.

“At the first sight of the color tints presented by the two halves of the field”, Pasteur wrote, “and without having to make a reading, Biot recognized that there was a strong rotation to the left. Then the illustrious old man, who was visibly moved, seized me by the hand and said: ‘My dear son, all my life I have loved science so deeply that this stirs my heart!’”

As he continued his work with right- and left-handed molecules, Pasteur felt that he was coming close to an understanding of the mysteries of life itself, since, as Biot had shown, the molecules which rotate polarized light are almost exclusively molecules produced by living organisms. He soon discovered that he could make an optically active solution of tartaric acid in another way: When he let the mould penicillium glaucum grow in a solution of racemic acid, the left-handed form disappeared, and only the right-handed form remained. In this way, Pasteur became interested in the metabolism of microscopic organisms. Pasteur’s work on crystallography and optical activity had made him famous among chemists, and he was appointed Professor of Chemistry at the University of Strasbourg. He soon fell in love with and married the daughter of the Rector of the university, Marie Laurent. This marriage was very fortunate for Pasteur. In the words of Pasteur’s assistant, Emil Roux, “Madame Pasteur loved her husband to the extent of understanding his studies... She was more than an incomparable companion for her husband: She was his best collaborator”. She helped him in every way that she could - protecting him from everyday worries, taking dictation, copying his scientific papers in her beautiful handwriting, discussing his experiments and asking intelligent questions which helped him to clarify his thoughts.

After a few years at Strasbourg, Pasteur was appointed Dean of the Faculty of Sciences at the University of Lille. In appointing him, the French government explained to Pasteur that they expected him to place the Faculty of Sciences of the university at the service of the industry and agriculture of the district.

Pasteur took this commission seriously, and he soon put his studies of microorganisms to good use in the service of a local industry which produced alcohol from beet juice. He was able to show that whenever the vats of juice contained bacteria, they spoiled; and he showed the local manufacturers how eliminate harmful bacteria from their vats. As a result of this work, the industry was saved.

His work on fermentation put Pasteur into conflict with the opinions of the most famous chemists of his time. He believed that it was the action of the living yeast cells which turned sugar into alcohol, since he had observed that the yeasts were alive and that the amount of alcohol produced was directly proportional to the number of yeasts present.

On the other hand, the Swedish chemist, Jøns Jakob Berzelius (1779- 1848), had considered fermentation to be an example of catalysis, while Justus von Liebig (1805-1875) thought that the yeasts were decaying during fermentation, and that the breakdown of the yeast cells somehow assisted the conversion of sugar to alcohol. (Both Pasteur and Berzelius were right! Although the fermentation observed by Pasteur was an example of the action of living yeasts, it is possible to extract an enzyme from the yeasts which can convert sugar to alcohol without the presence of living cells.)

Pasteur studied other fermentation processes, such as the conversion of sugar into lactic acid by the bacilli which are found in sour milk, and the fermentation which produces butyric acid in rancid butter. He discovered that each species of microorganism produces its own specific type of fermentation; and he learned to grow pure cultures of each species.

At the suggestion of Napoleon III, Pasteur turned his attention to the French wine industry, which was in serious difficulties. He began to look for ways to get rid of the harmful bacteria which were causing spoilage of the wine. After trying antiseptics, and finding them unsatisfactory, Pasteur finally found a method for killing the bacteria, without affecting the taste of the wine, by heating it for several minutes to a temperature between 50 and 60 degrees centigrade. This process (“Pasteurization”) came to be applied, not only to wine, but also to milk, cheese, butter, beer and many other kinds of food.

Pasteur developed special machines for heat-treating large volumes of liquids. He patented these, to keep anyone else from patenting them, but he made all his patents available to the general public, and refused to make any money from his invention of the Pasteurization process. He followed the same procedure in patenting an improved process for making vinegar, but refusing to accept money for it.

Pasteur was now famous, not only in the world of chemists and biologists, but also in the larger world. He was elected to membership by the French Academy of Sciences, and he was awarded a prize by the Academy for his research refuting the doctrine of spontaneous generation.

The germ theory of disease

In 1873, Louis Pasteur was elected to membership by the French Academy of Medicine. Many conservative physicians felt that he had no right to be there, since he was really a chemist, and had no medical “union card”. However, some of the younger doctors recognized Pasteur as the leader of the most important revolution in medical history; and a young physician, Emil Roux, became one of Pasteur’s devoted assistants. When he entered the Academy of Medicine, Pasteur found himself in the middle of a heated debate over the germ theory of disease. According to Pasteur, every contagious disease is caused by a specific type of microorganism. To each specific disease there corresponds a specific germ.

Pasteur was not alone in advocating the germ theory, nor was he the first person to propose it. For example, Varro (117 B.C. - 26 B.C.), believed that diseases are caused by tiny animals, too small to be seen, which are carried by the air, and which enter the body through the mouth and nose.

In 1840, Jacob Henle, a distinguished Bavarian anatomist, had pointed out in an especially clear way what one has to do in order to prove that a particular kind of germ causes a particular disease: The microorganism must be found consistently in the diseased tissue; it must be isolated from the tissue and cultured; and it must then be able to induce the disease consistently. Finally, the newly-diseased animal or human must yield microorganisms of the same type as those found originally.

Henle’s student, Robert Koch (1843-1910), brilliantly carried out his teacher’s suggestion. In 1872, Koch used Henle’s method to prove that anthrax is due to rodlike bacilli in the blood of the infected animal. Koch’s pioneering contributions to microbiology and medicine were almost as great as those of Pasteur. Besides being the first person to prove beyond doubt that a specific disease was caused by a specific microorganism, Koch introduced a number of brilliant technical improvements which paved the way for rapid progress in bacteriology and medicine.

Instead of using liquids as culture media,Koch and his assistant, Petri, pioneered the use of solid media. Koch developed a type of gel made from agar-agar (a substance derived from seaweed). On the surface of this gel, bacteria grew in tiny spots. Since the bacteria could not move about on the solid surface, each spot represented a pure colony of a single species, derived from a single parent. Koch also pioneered techniques for staining bacteria, and he introduced the use of photography in bacteriology. He was later to isolate the bacillus which causes tuberculosis, and also the germ which causes cholera.

When Koch’s work was attacked in the French Academy of Medicine, Pasteur rushed to his defense. In order to demonstrate that it was living bacilli in the blood of a sheep with anthrax which transmitted the disease, and not something else in the blood, Pasteur took a drop of infected blood and added it to a large flask full of culture medium. He let this stand until the bacteria had multiplied; and then he took a tiny drop from the flask and transferred it to a second flask of nutrient broth. He did this a hundred times, so that there was no possibility that anything whatever remained from the original drop of sheep’s blood. Nevertheless, a tiny amount of liquid from the hundredth flask was just as lethal as fresh blood drawn from a sheep with anthrax.

Vaccines

Pasteur read and reread the papers of Jenner on immunization against smallpox. He searched continually for something analogous to smallpox vaccination which could be applied to other diseases. Finally, the answer came by chance.

Pasteur and his assistants had been studying chicken cholera, an invariably fatal disease of chickens. Roux and Chamberland were carrying out a series of experiments where they made a fresh culture of chicken cholera bacteria every day. When they injected a bit of liquid from any of these cultures into a chicken, the chicken always died. It was summer, and the young men went off for two weeks of vacation.

When they came back, they took their two-week-old culture of chicken cholera out of the cupboard and injected it into a hen; but the hen didn’t die. They decided that while they had been on vacation, the culture must have lost its strength; and after some effort, they obtained a new specimen of active chicken cholera bacteria, which they injected into their hens. All the hens died except one. The hen which had previously been inoculated with two-week-old culture didn’t even get sick!

When Pasteur returned to his laboratory, the two young men hesitated to tell him about this strange result because they were afraid that he might be angry with them for going off on a holiday and breaking off the series of experiments. However, they finally confessed what had happened, and added the strange detail about the chicken which had not died. In the middle of their apologies, Pasteur raised his hand. “Please be quiet for a moment”, he said, “I want to think”. After a few moments of silence, Pasteur looked at Roux and Chamberland and said, “That’s it! The hen that didn’t die was vaccinated by the old culture!”

This was the big breakthrough - a turning point in medical history. Pasteur, Roux and Chamberland had discovered by chance a method of weakening a culture of bacteria so that it would not produce the fatal disease with which it was usually associated; but on the other hand, it was still able to alert the body’s defense mechanisms, so that the inoculated animal became immune. This great discovery was made by chance, but, as Pasteur was fond of saying, “In research, chance favors the prepared mind”.

Pasteur, Roux and Chamberland dropped everything else and began a series of experiments to find the best way of weakening their cultures of chicken cholera. They found that the critical factor was the proper amount of exposure to air. (Probably the culture contained a few mutant bacteria, able to grow well in air, but not able to produce chicken cholera; and during the exposure of a culture, these mutants multiplied rapidly, until the entire population was composed of mutants.)

Pasteur now began research on a vaccine against anthrax - a disease which was causing serious economic loss to farmers, and which could affect humans as well as animals. With anthrax, the problem was to keep the bacilli from forming spores. After much experimentation, the group found that if they held their anthrax cultures at a temperature between 42 C and 43 C, the bacilli would still grow, but they did not form spores.

Pasteur and his coworkers allowed their cultures to grow at 42 C in shallow dishes, where there was good contact with the air. They found that after two weeks, the cultures were weakened to the point where they would make a sheep sick, but not kill it. They developed a method for inoculating animals in two stages - first with a very much weakened culture, and later with a stronger one. After the second inoculation, the animals could stand an injection of even the most virulent anthrax bacilli without becoming ill.

When Pasteur published these results, there was much sarcasm among veterinarians. The editor of the Veterinary Press, a surgeon named Rossignol, wrote: “Monsieur Pasteur’s discovery, if it were genuine, should not be kept in the laboratory”. Rossignol proposed a public trial of the anthrax vaccine, and he started a campaign to collect money for the purchase of experimental animals.

Pasteur’s friends warned him against accepting the risk of a public trial at such an early stage. He had not tested his vaccine sufficiently, and a failure would make him the laughing stock of Europe. However, Pasteur saw the trial as a chance to focus public attention on microorganisms and vaccines. Like Galileo, Pasteur had a flair for dramatic gestures and public debate; and the impact of his career was greatly enhanced by his ability to attract widespread attention.

A farm near Melun called Pouilly le Fort was chosen as the site for the experiment; and sixty sheep, together with several cows, were put at Pasteur’s disposal. Thousands of people made the journey from Paris to Melun to watch the first injections, which were made on May 5, 1881. Twelve days later, the same sheep were inoculated with a stronger vaccine. Then, on May 31, the big test was made - both the vaccinated and unvaccinated animals were inoculated with a highly lethal culture of anthrax. Pasteur went back to Paris. There was nothing to do but wait.

The next afternoon, a telegram from Rossignol shattered Pasteur’s confidence: It said that one of the vaccinated sheep was dying. Pasteur spent a sleepless night. The following morning, however, at nine o’clock, another telegram arrived from Rossignol: All the vaccinated sheep were well, even the one which had seemed to be dying; and all the unvaccinated sheep were either dying or already dead! Rossignol, who had been Pasteur’s enemy, was completely converted; and his telegram ended with the words, “Stunning success!” When the aging

Pasteur limped onto the field at Pouilly le Fort that afternoon, a great cheer went up from the thousands of people present.

Rabies

The next disease which Pasteur attempted to conquer was rabies, the terrifying and invariably fatal disease which often follows the bite of a mad dog. The rabies virus travels slowly through the body from the wounds to the spinal cord, where, after one or two months, it attacks the nervous system. If a victim is offered water and attempts to swallow, his head jerks back in terrible spasms, which make rabies extremely frightening, both for the victim and for the onlooker. For this reason, the disease is sometimes called hydrophobia - fear of water.

Pasteur and his coworkers soon discovered that even with their best microscopes, they were unable to see the organism which causes rabies. In fact, the disease is caused by a virus, much too small to be seen with an optical microscope. Thus the aging Pasteur was confronted with an entirely new technical problem, never before encountered in microbiology.

He soon found that it was impossible to culture the rabies virus in a flask or dish, as he was in the habit of doing with bacteria. Absorbed in his research, he forgot his wedding anniversary. Marie Pasteur, however, remembered; and she wrote in a letter to her daughter:

“Your father is absorbed in his thoughts. He talks little, sleeps little, rises at dawn, and in a word, continues the life which which I began with him this day thirty-five years ago.”

Besides being technically difficult, the work on rabies was also dangerous. When Pasteur, Roux and Chamberland took samples of saliva from the foaming jaws of mad dogs, they risked being bitten by accident and condemned to an agonizing death from the convulsions of rabies. Since they could not culture the rabies virus in a dish or a flask of nutrient fluid, they were forced to grow it inside the nervous systems of experimental animals. After four years of difficult and hazardous work, they finally succeeded in developing a vaccine against rabies. In the method which finally proved successful, they took a section of spinal cord from a rabbit with rabies and exposed it to air inside a germproof bottle. If the section of spinal cord remained in the bottle for a long time, the culture was very much weakened or “attenuated”, while when it was exposed to air for a shorter time, it was less attenuated. As in the case of anthrax, Pasteur built up immunity by a series of injections, beginning with a very much attenuated culture, and progressing to more and more virulent cultures.

At last, Pasteur had a method which he believed could be used to save the lives of the victims of mad dogs and wolves; and he found himself faced with a moral dilemma: Everyone who developed rabies died of it; but not everyone who was bitten by a mad dog developed rabies. Therefore if Pasteur gave his vaccine to a human victim of a mad dog, he might harm someone who would have recovered without treatment.

He had published the results of his research, and he was inundated with requests for treatment, but still he hesitated. If he treated someone, and the person afterward died, he might be accused of murder; and all the work which he had done to build up public support for the new movement in medicine might be ruined.

Finally, on July 6, 1885, Pasteur’s indecision was ended by the sight of a man and woman who had come to him with their frightened nineyear- old son. The boy, whose name was Joseph Meitner, had been severely bitten by a mad dog. It was one thing to write letters refusing requests for treatment, and another thing to look at a doomed and frightened child and turn him away.

Pasteur felt that he had to help the boy. He consulted Alfred Vulpian, a specialist in rabies, and Vulpian assured him that Joseph Meitner had been bitten so severly that without treatment, he would certainly develop rabies and die. Pasteur also consulted Dr. Granchier, a young physician who had joined his staff, and together the three men agreed that there was no time to lose - they would have to begin inoculations immediately if they were to save the boy’s life. They decided to go ahead. To Pasteur’s great joy, Joseph Meitner remained completely well.

The second rabies victim to be treated by Pasteur was a fourteenyear- old shepard named Jupille. He had seen a mad dog about to attack a group of small children, and he had bravely fought with the maddened animal so that the children could escape. Finally he had managed to tie its jaws together, but his hands were so badly bitten that without treatment, he was certain to die. Like Joseph Meitner, Jupille was saved by the Pasteur treatment. A statue of Jupille in front of the Pasteur Institute commemorates his bravery.

Pasteur had now grown so old, and was so worn out by his labors that he could do no more. The task of winning a final victory over infectious diseases was not finished - it was barely begun; but at least the feet of researchers had been placed on the right road; and there were younger men and women enthusiastically taking up the task which Pasteur laid down.

On December 27, 1892, physicians and scientists from many countries assembled in Paris to celebrate Pasteur’s seventieth birthday. The old man was so weak that he was unable to reply in his own words to the address of Sir Joseph Lister and to the cheers of the crowd; but his words were read by his son. Pasteur spoke to the young men and women who would take his place in the fight against disease:

“Do not let yourselves be discouraged by the sadness of certain hours which pass over nations. Live in the serene peace of your laboratories and libraries. Say to yourselves first, ‘What have I done for my instruction?’, and as you gradually advance, ‘What have I done for my country?’, until the time comes when you may have the intense happiness of thinking that you have contributed in some way to the progress and good of humanity.”

Chapter 11: ATOMS IN CHEMISTRY.

Suggestions for further reading

1. Clifford Dobell (editor), Antony van Leeuwenhoek and his Little Animals, Dover, New York (1960).
2. Paul de Kruif, Microbe Hunters, Pocket Books Inc., New York (1959).
3. René Dubos, Pasteur and Modern Science, Heinemann, London (1960).
4. A.P.Waterson and Lise Wilkinson, An Introduction to the History of Virology, Cambridge University Press (1978).
5. P.E. Baldry, The Battle Against Bacteria, Cambridge University Press (1965).
6. L. Wilkinson, Animals and Disease; An Introduction to the History of Comparative Medicine, Cambridge University Press, (1992).
7. Arthur Rook (editor), The Origins and Growth of Biology, Penguin Books Ltd. (1964).

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