PASTEUR TACKLES ANTHRAX—AND KOCH
From the late 1870s Pasteur applied all that he had discovered in his study of microbiology to the battle against infectious diseases. Yet another phase of his research had begun. Anthrax, a disease of sheep and cattle, was destroying the livelihood of French farmers who were powerless to save their dying animals. The debate over Germ Theory was still raging when Pasteur entered the anthrax arena. The cause of anthrax, a bacillus (a type of rod-shaped spore-producing bacteria), had first been identified in 1849. Robert Koch, the German physician, had succeeded in growing the bacillus in pure culture in 1877.
Definitive proof was still lacking, however, that the cultured bacillus, and not something else that may have entered Koch’s culture medium, was responsible for giving animals anthrax.[23] Pasteur provided the proof experimentally. He placed one drop of blood from a sheep dying of anthrax into 50 millilitres of sterile culture, grew up the bacterium, and then repeated this process 100 times. After the dilutions the last culture was as active as the first in producing anthrax. With this, Pasteur firmly established his Germ Theory of Disease.
Pasteur then applied what he had learnt from silkworm disease to help solve the mystery of how anthrax spread; why animals in one field remained healthy while animals in another succumbed. The answer: earthworms feeding on the carcasses of buried diseased sheep carried anthrax spores to the surface, contaminating the soil where healthy sheep graze and thus passing on the disease. Keeping animals away from contaminated fields would help control the spread of anthrax but was not the answer to preventing it.
‘Chance favours the prepared mind’ was an adage that Pasteur was fond of. A stroke of luck and Pasteur’s prepared mind combined to bring about a major breakthrough—the discovery of the method for the attenuation of virulent micro-organisms. Attenuation is the basis of vaccination. Pasteur was researching chicken cholera at the same time as anthrax. Another serious problem for farmers, this disease could wipe out an entire flock in as little as three days. The experiments Pasteur carried out on chicken cholera led him to the development of specific vaccines against chicken cholera, anthrax and swine erysipelas. Once he had mastered the method of attenuation Pasteur would apply the concept to rabies.
In experiments in his laboratory at Arbois, Pasteur was growing chicken cholera bacillus in pure culture. Chickens injected with the culture invariably died within 48 hours. On one occasion, Pasteur took a chance and injected two chickens with a culture that was several weeks old. The chickens became ill and then recovered. During the summer Pasteur returned to Paris leaving the cholera cultures stored in the laboratory. When the staff returned, they conducted some experiments using these cholera cultures on two chickens and were surprised when the chickens did not become infected. New cultures of the bacillus were made and tested on new birds plus the two healthy birds that had survived. The results were astonishing. The previously injected birds were unaffected by the bacillus, while the new birds all died.[24]
When Pasteur saw these results he immediately realised that in a sense he was repeating the studies Edward Jenner had conducted 80 years earlier when he gave humans immunity to smallpox by vaccinating individuals with the milder disease, cowpox. Pasteur hypothesised that pathogens can be attenuated, or weakened, by exposure to environmental conditions such as high temperature, oxygen and chemicals. He then grew the cholera bacillus at 42–43°C, at which temperature the bacillus is non-infectious, and these attenuated bacterial cultures were used to successfully vaccinate chickens against cholera.
Pasteur had the key to preventing anthrax. By using various techniques involving oxidation and ageing, Pasteur developed attenuated anthrax bacillus and in laboratory trials anthrax vaccines successfully protected sheep. When Pasteur reported his findings it was déjà vu: both the scientific community and the general populous were divided. A well-known veterinarian challenged Pasteur to conduct a controlled public trial of his anthrax vaccine. Pasteur did what he always did, met the challenge head on. Risking his reputation, Pasteur organised a public demonstration on a farm at Melun, south of Paris.
Twenty-five sheep were controls and another 25 were vaccinated by Pasteur. All 50 were injected with a lethal dose of anthrax. Pasteur affirmed that only 100 per cent success would prove his theory, which meant that all of the control sheep must die and all the vaccinated sheep must live. When Pasteur’s colleagues expressed their concern because the vaccines were still in the developmental stage, with bravado Pasteur declared that, ‘What succeeded with fourteen sheep in our laboratory will succeed with 50 at Melun,’[25] but he knew he was taking an incredible risk. He had not had a 100 per cent success rate in the laboratory. Pasteur chided himself for acting impetuously but the trial went ahead.
This experiment attracted international attention. People came from everywhere. Farmers and scientists had a vested interest, some of the latter hoping to see Pasteur humiliated while the general public came to an event that seemed curiously like a circus. Once the experiment had begun newspapers in France and England published daily bulletins. Two days after the final vaccination on 5 May 1882, all 25 of the control sheep were dead while the 25 vaccinated sheep remained alive and healthy. This was Pasteur’s greatest experiment. He was a scientific superstar. Foes became fans and Pasteur’s celebrity skyrocketed. At last the scientific elite and the hoi polloi believed in the existence of Pasteur’s microbes.
Within ten years of the trial at Melun, throughout France 3.5 million sheep and half a million cattle had been vaccinated. The mortality rate dropped to less than 1 per cent, an enormous saving for the French economy. Pasteur’s method of identifying the infectious agent, weakening it, and then using it to vaccinate a host was soon being applied to debilitating human diseases, by Pasteur and by others, thus ensuring the future salvation of millions. But if Louis Pasteur thought that finally all the sceptics and nay-sayers would be silent he expected too much. Pasteur was besieged by anti-vaccinators, who expressed similar views and objections to those who had opposed Jenner and his smallpox vaccinations.
Never able to avoid conflict for too long, Pasteur was soon embroiled again, this time with Robert Koch, who was working on contagious diseases. His publications in 1878 and 1879 had helped to confirm that bacteria were the cause and not the consequence of infection. An acrimonious dispute arose between Pasteur and Koch and his colleagues, who questioned Pasteur’s scientific methodology and harshly criticised his work on the attenuation of viruses and his conclusions on anthrax vaccination.
In an article published in 1983, H.H. Molleret describes the relationship between Koch and Pasteur as ‘hateful’.[26] The two great scientists first met in London in August 1881, during the International Congress of Medicine at which Pasteur presented his results on attenuation. The renowned Pasteur was 59, with a lifetime of work and discoveries behind him. Koch, who was only 38, already had impressive scientific credentials which Pasteur had previously acknowledged in April 1877 at the Science Academy in Paris when he referred to Koch’s discovery of the anthrax spore as a remarkable achievement. In England both men attended demonstrations in Joseph Lister’s laboratory at Cambridge University. When Koch presented the staining procedures that he had developed, a successful way of isolating micro-organisms, Pasteur again complimented Koch on a great scientific advance.
Molleret concludes that nothing in that first meeting justified the vicious attack that Koch launched in print a few months later. Koch discredited Pasteur’s theory on the role of the earthworm in anthrax, calling it naïve and untenable, and claimed that scientific knowledge of anthrax had not been enlarged in any way by Louis Pasteur. Koch’s colleague Friedrich Loeffler attacked Pasteur’s attenuated vaccines, claiming that the cultures of the chicken cholera bacillus prepared by Pasteur were not pure because they were not done on gelatin, a method recently introduced by Koch. He also claimed that Pasteur’s work on anthrax was based on poor science, and his experimental results were dependent on luck. Was there
anything left to denounce?
Pasteur was deeply wounded by what he referred to as the ‘strange ferociousness’ of these attacks. Instead of responding in print as he usually did Pasteur hoped to debate Koch in person in Geneva at the International Congress of Hygiene in September 1882. At the congress Koch was enjoying the notoriety of having recently discovered the cause of tuberculosis, a momentous medical breakthrough. Pasteur, determined to make his point, gave a lengthy presentation on vaccination against anthrax. He pointed out that ‘as brilliant as is demonstrated truth’ he had met with ‘contradictors’ in France and in foreign countries and publicly named Robert Koch.
The audience was astonished when Koch interrupted Pasteur’s speech. Koch dashed Pasteur’s hopes for a public discussion and instead made a declaration that he had come to the Geneva conference hoping to learn some new facts but that once again he had learnt nothing new from Pasteur. Discussion would prove fruitless Koch said, because he did not speak French and Pasteur did not know enough German and therefore he would reply to Pasteur only in medical journals. Koch concluded with a stinging barb, saying that when ‘Pasteur was celebrated as the second Jenner’ for his questionable work on anthrax, the praise had been premature. ‘Obviously in the desire to be enthusiastic it was forgotten that Jenner’s beneficial discovery was not in sheep but in humans.’[27]
In December 1882 Pasteur wrote his reply, ‘The Anthrax vaccination: Response to Dr Koch’s Memoir’. Written with a tone as cutting as Koch’s, Pasteur told the German doctor that he was indebted to French science and accused Koch of misrepresenting him in previous papers. It had become bitter claim and counterclaim. In 1884, after Koch had made another breakthrough, the discovery of the cholera germ, he visited hospitals and laboratories in Paris and Toulon but deliberately avoided Pasteur’s laboratory. Pasteur may have felt slighted but this did not prevent him some years later sending a telegram to Koch congratulating him on his discovery of the ‘remedy against tuberculosis’, which Koch announced at the Congress of Medicine in Berlin in August 1890.
Various suggestions as to the cause of the antagonism between Robert Koch and Louis Pasteur go beyond professional jealousy, brinkmanship and a personality clash. According to Molleret there was a climate of chauvinism and patriotism prevalent in the scientific community at the time. The rivalry over anthrax was an extension of an earlier patriotic quarrel that began well before Koch and Pasteur, with a debate over the discovery of the anthrax bacteria. The French attributed it to Rayer and Davaine in 1850 and the Germans credited Pollender who identified the bacteria in 1849 but did not publish his observations until 1855.
Another view is that the dispute between the two men and the scientific rivalry between the two countries was exacerbated by national loyalty and enmity between France and Germany after the Franco–Prussian War of 1870–71 which Germany won. Koch, who was anti-French, had volunteered for the Prussian Army in 1870 and had served in a military hospital near Orléans in France. Pasteur too was a passionate patriot who had wanted to join the National Guard. Pasteur’s colleague, the Russian scientist Elie Metchnikov, wrote about how the war saddened and troubled Pasteur. Pasteur’s own vitriolic words, written during the siege of Paris, reveal the depth of his animosity for the Germans, calling them ‘vandals’ who should ‘perish from cold, misery and sickness’ and vowing ‘Hatred of Prussia, Vengeance, Vengeance!’. In January 1871 Pasteur returned the honorary Doctor of Medicine from the University of Bonn that he had been awarded in 1868 and a bitter exchange of letters with the Dean of the Faculty of Medicine followed. Pasteur was undoubtedly a man of deep passions.
In Geneva in 1882 Robert Koch had alluded to the language barrier between himself and Pasteur. Misconceptions could have arisen because neither scientist could read the work of the other in its original language. Documents from the time, held in the museum at the Pasteur Institute, suggest that the relationship between these two scientific giants was in fact affected by a lack of fluency in each other’s language and that the altercation between them at the Congress of Geneva in 1882 may have resulted from a misinterpretation of one French word, recueil, which Koch interpreted as ‘pride’ rather than ‘collection’. The word for pride is orgueil.[28] Koch may have felt insulted and it was at that moment that he had interrupted the meeting.
But nothing could keep Pasteur from his single-minded purpose and the forward momentum of his research. In the face of all opposition Pasteur was determined to apply the science that he had spent a lifetime mastering to curing human disease. Buoyed by his successes with anthrax and fowl cholera, over the next few years Pasteur employed the fundamentals of microbiology in a new battle. In 1882, aged 60 and partially crippled by the stroke that had weakened his left leg, Pasteur began to work on what Elie Metchnikov referred to as his ‘swan-song’ and what others have called his ultimate triumph.
MAD DOG DISEASE
Rabies, like smallpox, is a disease that has been known and feared since ancient times. A disease of the nervous system, rabies causes acute encephalitis (inflammation of the brain) in both animals and humans. The word rabies comes from the Latin rabiere meaning ‘rage’, which in turn is derived from the Sanskrit word rabhas, ‘to be violent’. The Greeks call rabies lyssa, which means ‘madness’.[29] All these words reflect the horrendous visible effects that rabies has on its victims. People suffering from rabies were often depicted as raving mad and frothing at the mouth.
Although rabies did not have the apocalyptic connotation of smallpox or bubonic plague, to people of Pasteur’s time the horrifying disease evoked visions of victims raging, bound and howling, or being asphyxiated between two mattresses to subdue them, or having their bite wounds cauterised with a red-hot poker. Such treatments were as grotesque as the feared symptoms. A vaccine that cured the disease and eliminated such horrors would be considered nothing short of miraculous.
Rabies in humans is usually contracted after a bite from an infected animal because the virus can be present in the animal’s saliva. By causing the infected animal to be exceptionally aggressive, the virus ensures its transmission to the next host. The disease is often associated with the stereotype of rabid, aggressive dogs. Other animals can carry rabies, including cats, ferrets, skunks, foxes, bears and bats. With bats, rabies can be transmitted via airborne liquid particles from their mucous. Following a bite by a rabid animal, if the virus is not inactivated by an immune response it enters the peripheral nervous system and can avoid recognition by the immune system by travelling gradually from nerve cell to nerve cell. When it reaches the central nervous system the virus is hard to detect and at this point is invulnerable to an immune response induced by vaccination. Viruses have developed cunning survival mechanisms and this one seems particularly cunning. Once the rabies virus reaches the brain, it rapidly causes encephalitis; symptoms appear—cerebral dysfunction, anxiety, insomnia, confusion, agitation and hallucinations—and death is certain.
As a boy living in Jura, Pasteur had witnessed the ghastly effects of the disease on several townspeople who had contracted rabies from a dog. The later stages of the disease are the most horrendous. People produce large quantities of saliva and tears, are unable to speak or swallow and develop hydrophobia, the fear of water, another name by which rabies is known. The final phase is delirium. The period between infection and the onset of symptoms is normally three to twelve weeks but it can be as long as two years. Despite the long incubation period, however, death usually occurs two to ten days after the first symptoms appear. Until Louis Pasteur developed his vaccine in 1885, the handful of people who are known to have survived the disease, except for one recorded case, were all left with severe brain damage.
Pasteur was aware that defeating rabies would be a defining scientific achievement and could lead to the conquest of other human diseases. Knowing that there were inherent difficulties in taking up this challenge Pasteur set to work with his colleague Emile Roux. The first hurdle was the lack of evidence that rabies was caused b
y a micro-organism, due to the fact that the virus was too small to be seen by microscope. This threatened Pasteur’s Germ Theory, and was a point Koch would later use to attack Pasteur. However, convinced that an unseen microbe caused rabies, Pasteur followed his established procedures to find a way to weaken it.[30]
The work was slow and arduous. Pasteur and Roux initially attempted to transfer infection by injecting healthy dogs with saliva from rabid animals. The results were variable and unpredictable. Later, recognising that the active agent, even though it could not be seen, was in the spinal cord and brain, Pasteur and Roux applied extracts of rabid spinal cord directly to the brains of dogs. With this technique they could produce rabies in the test animals in a few days.
The aim, then, was to develop a vaccine that would provide protection before the virus moved from the bite site to the spinal cord and the brain. To do this Pasteur and Roux began by injecting dogs with spinal cord material taken from rabid rabbits. To attenuate the material it was air-dried over a twelve-day period. A strip of spinal cord was suspended from a hanger in the centre of what is now known as a Roux bottle. It had a hole at the top of the bottle and one on the lower side. Air entered from the bottom opening, passed over a drying agent and exited from the top.[31] The longer the cord was dried, the less potent was the tissue in producing rabies. Pasteur and Roux then injected the least potent preparation of minced spinal cord under the skin of their laboratory dogs. For the next twelve days the dogs were injected each day with an increasingly stronger extract, after which the animals were completely resistant to bites from rabid dogs.
Smallpox, Syphilis and Salvation Page 7