Rabid

by Monica Murphy

  Pasteur did not commit himself. He cultured a figure-eight-shaped microbe from the blood of the dead rabbits in veal broth and tested its virulence in more rabbits and also in dogs. Again, it swiftly dispatched its recipients. With further investigation, Pasteur and his assistants found that they could isolate and culture this organism from patients who were hospitalized with illnesses completely different from rabies—even from healthy adults. Pasteur named the microbe pneumococcus and declared that he was “absolutely ignorant of any connection that there may be between this new disease and hydrophobia.”

  Critics seized on this as evidence of the slipperiness of germ theory. Pasteur claims to be working on one disease, they sneered, but instead is working on another. To this notion Pasteur responded indignantly, “This is indeed a new disease produced by a new microbe; neither the microbe nor the disease has been described before. This tenacity in research, Monsieur, is the honor of our work, and it was because we, my collaborators and myself, pursued these experimental combinations that we were able to demonstrate that the new disease existed in the buccal mucus of children who had died of the same disease as well as in the saliva of perfectly healthy persons. It was then, and only then, that I had the right to assert that the new disease had no relation with rabies.”

  If rabies was not pneumococcus, then what was it, exactly? Despite a thorough investigation using all the tools of the Pasteur laboratory, no combination of methods and media available to Pasteur and his assistants would yield a microbial cause for rabies. Even as Pasteur’s team discovered that the infectious principle for rabies resided in the central nervous system as well as in the salivary glands, they failed to culture a pathogen from either location. Thanks largely to the work of Pasteur himself, it was by this time a basic tenet of medical science that infectious diseases are caused by specific demonstrable microorganisms. Robert Koch’s famous “postulates,” first articulated in 1880, had made clear the relationship between microorganisms and disease, defining a disease-causing microbe as one that appears exclusively in diseased individuals; that can be isolated and cultured from a diseased host; that will cause disease when next introduced into a susceptible host; and that can be subsequently recovered from the experimental host and shown to be identical in culture to the microbe originally isolated. For rabies, not a single one of these conditions had been met. Koch’s precepts have often been summed up with the phrase “one disease, one microbe,” and Pasteur concurred with this view, but his vision saw a third term in this equation: one vaccine. He believed that every disease-causing microbe, once isolated, could be attenuated so as to safely confer immunity on a potential host. But it was hard to see how this equation could hold true unless a pathogen could be isolated, identified, trapped under glass, and then tamed.

  Pasteur referred to the unseen—and apparently unseeable—agent of rabies as a virus. As his biographer Patrice Debré observed a century later, the word “virus” had until that point been associated with a darkly mysterious etiology: with miasmas, with poisons, with plagues. Rabies behaved as though it were a microbic contagion, and so Pasteur maintained absolute faith that it was one, even though he could neither culture it in broth nor observe it under the light microscope. The word “virus” conveyed his uncertainty of rabies’ specific form and characteristics. It was not until 1898 that a “virus” was scientifically defined as a microbe that is invisible under the light microscope and can pass through a filter designed to trap bacteria; it was not until 1903 that it was experimentally demonstrated that the agent of rabies fit squarely within this category.

  Despite the confounding invisibility of rabies, despite the fact that it seemed to violate the scientific principles of the day to do so (principles that Pasteur himself had played no small part in establishing), Pasteur persevered in his work on a vaccine. His intellectual flexibility in the face of unexpected results allowed him to conclude early on that trying to cultivate the agent of rabies using existing laboratory methods would be fruitless. Instead, he nimbly refocused his attention and that of his assistants on inducing immunity, in animals and eventually humans, to what would remain an obscure, intangible foe.

  Bourrel’s two rabid dogs were part of a surge of rabies cases in Paris during 1880, and so the Pasteur laboratory would have no trouble obtaining infectious material. They got it from kennels of the national veterinary school at Maisons-Alfort and from private veterinary offices around the city. Because rabies could not be cultured on a plate or in a vial, it had to be maintained in living tissue. In the 1880s, this meant within the corporeal cells of a living afflicted animal. The maintenance of rabid animals within the modest rooms and basement of the Pasteur laboratory was discomforting to the personnel. There was the ever-present risk of contracting rabies—either directly from the jaws of the animal, or at the bench top, where infected tissues and sharp instruments could combine to do harm. Meanwhile, the researchers were forced to weather the public fury of the antivivisectionists, who denounced their work as senseless torture of innocent creatures.

  In order to create and test a vaccine against rabies, the Pasteur team first had to develop a strain of rabies that behaved more reliably than the natural infection. Early studies relied on the crude method of one animal biting another, followed by an anxious wait over weeks or longer to see whether infection had been transmitted. The Pasteurians developed a preference for inoculating their subjects, rather than exposing them to the competing risks that accompanied the bite from a rabid dog: trauma, sepsis, fright. However, this technique involved the dangerous collection of rabid saliva from a raging animal. Pasteur’s son-in-law, René Vallery-Radot, recalled one such dramatic scene:

  “We absolutely have to inoculate the rabbits with this slaver,” said M. Pasteur. Two helpers took a cord with a slip knot and threw it at the dog as one throws a lasso. The dog was caught and pulled to the edge of the cage. They seized it and tied its jaws together. The dog, choking with rage, its eyes bloodshot, and its body racked by furious spasms, was stretched out on a table while M. Pasteur, bending a finger’s length away over this foaming head, aspirated a few drops of slaver through a thin tube. It was…at the sight of this awesome tête-à-tête that I saw M. Pasteur at his greatest.

  Such exertions would soon prove unnecessary. Careful experiments showed that rabies could be as readily communicated with material from the affected animal’s brain stem as with its saliva. “The seat of the rabic virus,” wrote Pasteur, “is therefore not in the saliva only: the brain contains it to a degree of virulence at least equal to that of the saliva of rabid animals.” Whether the Pasteurians were inoculating nervous tissue or slaver, uncertainty during a prolonged incubation period remained a problem, as not all animals would manifest signs of rabies following inoculation and the interval before onset of signs was still quite variable. “It is torture for the experimenter to be condemned to wait for months on end for the result of an experiment,” lamented Pasteur.

  The Pasteur team soon found it was able to improve the infection rate and shorten the incubation period by administering chloroform anesthesia to the recipient animal, trepanning a hole in its skull, and then inoculating the rabid nervous tissue directly onto the dura mater, the connective tissue that covers the brain. Pasteur, disturbed by the invasive nature of this procedure, initially resisted the widespread implementation. But he was soon reassured by the vigorous, happy appearance of his laboratory’s first subject dog, only one day post-trepanation. The method was perhaps more stressful for the experimenters themselves, as remembered by Emile Roux’s niece:

  [Roux], [Charles] Chamberland, and [Louis] Thuillier bent down around a table. A large dog was tied down on it, its muscles contracted and its fangs bared…. If the animal, despite all the precautions, had caused them to make a false move, if one of them had cut himself with his scalpel, and if a small piece of the rabid spinal cord had penetrated into the cut, there would have been weeks and weeks filled with the anguished question: will he or will he not co
me down with rabies?…They were no longer just “researchers” absorbed in the meticulous work of their laboratory; they were pioneers, adventurers of science.

  Using the trepanation technique, Pasteur’s assistants successfully transmitted rabies to the healthy animal in every case attempted. Signs of disease were apparent in the inoculated animal in less than two weeks—a significantly shorter time than with natural infection—and death concluded within a month. As canine rabies was thereby passed to a rabbit, and from one rabbit to another rabbit, and from that rabbit to still another rabbit, and so on in successive passages, the incubation period became reliably shorter. Once twenty-one passages had been made, brain to brain, one rabbit to another, the incubation period had decreased to eight days. Here it became fixed and constant, so that subsequent passages in rabbits produced no further change.

  Shortened incubation period is associated with increased virulence. The enhanced virulence of rabies following intracranial serial passage in the rabbit was plainly observable when the virus was inoculated back into a canine host: dogs infected with the rabbit virus were even more catastrophically affected than those afflicted with natural strains. Through persistent repetition, Pasteur could now induce a consistently deadly version of the volatile virus at will. Even if he could not culture it in a tube like a bacterium, or coax it to shine in the eyepiece of his light microscope like a spore, the rabies virus was now finally under his control.

  The next step would be attenuation: the deliberate weakening of the virus in order to induce immunity without causing disease. From the beginning, Pasteur had sought a strain with a sure and fast-acting immunity that could be applied after exposure to rabies had already occurred. Much more challenging to achieve than the already-established method of prophylaxis, the vaccine strain would race to establish immunity against a natural rabies infection as it made its murderous way from bite wound to brain. If the infection inhabited the brain before protective immunity had taken hold, the patient’s death from rabies would be as certain as ever. But Pasteur hoped that an extremely robust yet attenuated rabies vaccine would provoke a sufficiently quick, vigorous immune response to interrupt the progress of the infection and spare the brain—indeed would drive the virus from the body altogether. The postexposure application of a paradoxically “weak strong” rabies vaccine strain would require innovation beyond Jennerian-Pasteurian vaccine principles. In fact, it would necessitate the creation of an entirely new branch of science: immunology.

  Pasteur would create his highly immunogenic but determinately safe rabies vaccine strain through a two-stage process: a first stage that would carefully hone the virulence of the virus, and a second that would deliberately blunt it. The second stage, like the first stage, relied on the ingenious manipulation of postmortem nervous tissue from rabid animals. It also, like the first stage, was directly carried out by Pasteur’s most trusted assistants: Chamberland, Thuillier, Adrien Loir, and, especially, Roux. Roux, in fact, probably invented the Pasteurian method of attenuating the most dangerous strains of rabies by aging the dissected spinal cords of rabbits that had succumbed to the virus in specialized flasks for desiccation (although Pasteur himself would take much of the credit for this). As they had done with so many other methodologies devised in the Pasteur laboratory, the research team perfected and verified this protocol through numerous repetitions. Soon they had gone on to demonstrate the powerful effectiveness of their attenuated-virulent strain as a vaccine—both as a prophylaxis against future exposure to rabies in dogs and, ultimately, as a postexposure immunization therapy that would prevent rabies in dogs already exposed to the deadly virus.

  In September 1884, Pasteur received a letter from the emperor of Brazil, inquiring when a vaccine would become available for human victims of a rabid bite. He replied:

  Until now I have not dared to attempt anything on men, in spite of my own confidence in the result and the numerous opportunities afforded to me since my last reading at the Academy of Sciences. I fear too much that a failure might compromise the future, and I want first to accumulate successful cases in animals. Things in that direction are going very well indeed; I have already several examples of dogs made refractory after a rabietic bite. I take two dogs, cause them both to be bitten by a mad dog; I vaccinate the one and leave the other without any treatment: the latter dies and the first remains perfectly well.

  But even when I shall have multiplied examples of the prophylaxis of rabies in dogs, I think my hand will tremble when I go on to Mankind.

  It was only six months later, in March 1885, that Pasteur wrote in a letter to his friend Jules Vercel, “I have not yet dared to treat human beings after bites from rabid dogs; but the time is not far off, and I am much inclined to begin with myself—inoculating myself with rabies, and then arresting the consequences; for I am beginning to feel very sure of my results.”

  Perhaps he continued to weigh the possibility, but Pasteur never did submit himself to this terrifying trial of his own vaccine. It was never necessary, as there were always unfortunate dog-bite victims whose physicians and families would offer them up for experimentation. Pasteur’s notes indicate that he allowed himself to be persuaded more than once to administer a vaccine to humans already in the throes of hydrophobia. These patients received no benefit from vaccination, as the natural virus had already infected their brains at the time of inoculation. Pasteur drew the necessary scientific conclusions and then made sure that these false starts were never publicized. He continued to believe that under the right circumstances the rabies vaccine would succeed, and he was determined that the vaccine’s public debut in humans would be nothing less than triumphant—providing the world with a broadly persuasive argument for the lifesaving potential of vaccines.

  It was the destiny of Joseph Meister, a boy of nine, to provide Pasteur with a sufficiently compelling experimental case to deploy his fledgling vaccine. While walking alone to school on the outskirts of his small Alsatian village, Meister was viciously attacked by a grocer’s dog. The animal knocked him to the ground and tore at his flesh while he cowered, holding his hands over his face in vain. By the time a nearby bricklayer reached the scene and fended off the dog with two iron bars, Meister had suffered fourteen penetrating wounds to his thighs, legs, and hand. Later that day, after cauterizing the bite wounds with carbolic acid, Meister’s local physician sent the boy to distant Paris for consultation with the famous Louis Pasteur.

  Pasteur proceeded cautiously. He was touched by his initial meeting with the stricken boy and his mother but did not agree to treat Meister until he had conferred with Alfred Vulpian, one of France’s most respected physicians and a member of the government’s official Commission on Rabies, and Jacques-Joseph Grancher, the head of the pediatric clinic at the Paris Children’s Hospital. The two esteemed medical men agreed that experimental treatment with Pasteur’s vaccine would offer Meister his best hope for survival given the extremely grave nature and severity of his wounds. Vulpian and Grancher provided not only an ethical sounding board for Pasteur but also very necessary practical assistance as he proceeded with his trial. Louis Pasteur had never been trained as a doctor, did not have a medical license, and so was prohibited from holding the syringe as it administered the first modern laboratory vaccine for humans, even though he himself had overseen every aspect of its creation.

  Meister received his first injection immediately. “On 6 July, at eight o’clock in the evening, sixty hours after the bites of 4 July, and in the presence of Drs. Vulpian and Grancher, we inoculated into a fold of skin over young Meister’s right hypochondrium half a Pravaz syringe of the spinal cord from a rabbit dead of rabies on 21 June; the cord had since then—that is, for fifteen days—been kept in a flask of dry air,” recorded Pasteur in his laboratory notebook. The full, ten-day treatment would consist of thirteen inoculations, all delivering postmortem spinal tissue from a rabid rabbit. Each successive injection would contain a section of cord that had been exposed to air for a shorter tim
e than the one before it, so that as the series proceeded, the vaccine would become less attenuated.

  Throughout treatment, Meister and his mother were housed adjacent to Pasteur’s lab at Collège Rollin. While Meister made himself happily at home among the laboratory chickens, rabbits, guinea pigs, and mice, Pasteur’s dauntless confidence in the rabies vaccine wavered as the inoculations he dispensed became more and more virulent. “My dear children,” began a letter from Mme Pasteur, “your father has had another bad night; he is dreading the last inoculations on the child. And yet there can be no drawing back now! The boy continues in perfect health.”

  On July 16, at eleven o’clock in the morning, Meister received his final inoculation. This preparation contained the most virulent tissue of all: rabid spinal cord from a dog that had been infected with a strain of rabies virus maximally strengthened by serial passage in the rabbit and harvested only one day prior to injection. Such a dangerous inoculation would provide a convincing test of Meister’s immunity: a naive recipient of this shot would be expected to show signs of rabies within several days. Pasteur’s son-in-law describes the fateful occasion as tense:

  Cured from his wounds, delighted with all he saw, gaily running about as if he had been in his own Alsatian farm, little Meister, whose blue eyes now showed neither fear nor shyness, merrily received the last inoculation; in the evening, after claiming a kiss from “dear Monsieur Pasteur,” as he called him, he went to bed and slept peacefully. Pasteur spent a terrible night of insomnia; in those slow dark hours of night when all vision is distorted, Pasteur, losing sight of the accumulation of experiments which guaranteed his success, imagined that the little boy would die.