Mr Berrys Mount Street L 0 N DON

Fig. 4. Label for one of many recommended rabies "cures," circa 1800. (Wellcome Library, London.)

been, but Fracastoro was able to develop them convincingly, and his chapters on specific diseases stand out for clarity of thought and the felicity of his writing.

Fracastoro's chapter on rabies certainly can be seen as a significant step on the way from the classics to the final conclusive nineteenth century developments in true understanding of the nature of the disease. He left his readers in no doubt that it could not be transmitted to humans by simple "contact, or by fomes, or at a distance, but only when the skin is so torn by the bite of a dog that blood is drawn, as though contagion takes place in the blood itself through contact with the teeth and foam from the mouth of the rabid animal" (Fig. 5). Writing more than a century before Leeuwenhoek (1632-1723) even began grinding his lenses, when Fracastoro mentioned "germs" or "seeds of disease," with the power "to propagate and engender what is similar to themselves," he certainly did not see them as living organisms, let alone animalculae, although he did add that animals dead of the disease no longer "preserved the contagion" because "germs of the contagion have perished together with the innate heat." [If read by pathologists about to perform post mortems, this might just be advice better ignored (Fracastoro, translated by W. C. Wright, 1930).]

For all the clarity of his thought, Fracastoro's ideas were not immediately of particular influence at a time when science relied on philosophy rather than experiment to prove its points, or as Nutton has memorably put it, "the hypothesis of causative seeds was a philosophical luxury for the intellectual practitioner" (Nutton, 1983). The nature of contagion, let alone of virus diseases in general and of rabies in particular, would continue to defeat the best efforts of medical practitioners and scientists for more than another three centuries. In the case of rabies.

Fig. 5. Man attempting to fend off a rabid dog. Details of French broadsheet, Paris, nineteenth century. (Wellcome Library, London.)

immoi'iioiiiK or mmsriti: in-. 1'iiniN knuack.

Fig. 5. Man attempting to fend off a rabid dog. Details of French broadsheet, Paris, nineteenth century. (Wellcome Library, London.)

development of Pasteur's postexposure prophylaxis predated any real understanding of its etiology by decades. In an exemplary translation, Wilmer Cave Wright (1930) allows Fracastoro to discuss the possibility of "immunization" against pestilence; this may be an overenthusiastic use of translator's poetic licence, since neither the context nor the Latin verbs used seem to suggest anything more than achieving a certain tolerance for the "germs" in question as in the case of some poisons such as arsenic.

Over subsequent centuries there was little progress in knowledge of either the clinical disease in humans or possible therapies, nor of how to protect human populations against roaming rabid dogs or wolves. However, there was no letup in the flow of literature on the subject; in addition to largely repetitive reports on remedies against the disease, there was a growing volume of papers detailing outbreaks of canine rabies throughout Europe and the Americas with numbers of recorded cases in epizootics and resulting human fatalities, as listed by George Fleming in 1872 and, more recently, by James H. Steele (1975 and 1991). A few descriptions of outbreaks through the centuries in greater detail can be found in Théodoridès's account in his definitive Histoire de la Rage (1986). There must, of course, remain doubts on the accuracy of early "statistics." The relatively small numbers of human victims dying after mad dog bites on the whole may be accepted; the considerable numbers of animal deaths recorded in individual outbreaks, whether of dogs, foxes, wolves, cattle, etc., could have been overestimated or, perhaps more likely, underestimated. Not surprisingly, few attempts were made to estimate numbers of animals affected in epizootics before 1700, even if they were reported. This has led some authors to assume that epizootics were rare until the Middle Ages; in any case, reports usually were restricted to single-animal attacks resulting in human fatalities that were duly counted and published: Around 900 a.d., a rabid bear bit 20 persons who attempted to kill it, of whom 6 unfortunates developed the disease and consequently were smothered to death over a 27-day period (Fleming, 1872; Steele, 1975, 1991) — a "treatment" still in use by the early eighteenth century when it was deplored and criticized in print by the father of Honoré de Balzac (discussed below).

In 1271, flocks of rabid wolves attacked herds of domestic animals in what was then Franconia; 30 human fatalities caused by bites were recorded. Epizootics of rabies were registered elsewhere in Europe in the sixteenth century; by 1604, there was a major outbreak of canine rabies in Paris, and from then on, European records show rabies to be present in frequent outbreaks among dogs, foxes, and wolves. It was known in England at least from the time of Edward, Second Duke of York (13737-1415), who wrote an account of the disease.

In Rabbis and Rabies, G. M. Baer et al. (1996) have claimed — without documentation — that Edward, Second Duke of York, "clearly described rabies in hounds and the manner of transmission" in 1420. Since this Second Duke is known to have died on the battlefield of Agincourt in 1415, this seemed to stretch credulity and warrant further investigation. Scrutiny of library catalogues has revealed that this colorful duke did indeed write the oldest English book on hunting, most of which was based on a translation of the Livre de la Chasse by Gaston III Phoebes, Comte de Foix, with an added five chapters by the duke himself, all written between 1406 and 1413. Chapter 13, written by the Duke, is entitled "Of the Sicknesses of Hounds and of [Their] Corrupcions [,sîc|." Here he described both "furious madness," in which the hounds howl and cry, escaping in their fury to "go everywhere, biting both men and women and all that they find before them.. .if they draw blood it shall go mad whatever thing it be " He also describes dumb madness, which in the language of his time he calls ragemuet, in which "...they neither bite nor run... and so they die..." The duke also mentions "remedies for men or for beast, including going to the sea and making nine waves, widening the wounds inflicted and letting a cock's vent suck all the venom of the biting," as well as the usual range of herbal remedies and decoctions. Long copied and circulated in manuscript form, the duke's book was printed in 1904, with a foreword by Theodore Roosevelt, who also took a particular interest in hunting.

By the mid-eighteenth century, reports of the presence of rabies appeared not only from the old world but also from locations throughout the new world; the practice of shooting all suspect dogs on sight and the introduction of quarantine arrangements began to spread from the 1752 outbreak in St. James, London, to rapidly growing colonial outposts (Fleming, 1872). According to Fleming, rabies was introduced into Argentina by the "sporting dogs" brought by English officers; it has remained enzootic there since. By then, canine rabies had been well established in colonial North America for more than 20 years, with serious outbreaks between 1785 and 1789, when a man having skinned a cow that died of rabies subsequently himself died of hydrophobia (Steele, 1975, 1991). From then on, and well into the following century, there were widespread outbreaks throughout Europe and other parts of the world, including the Americas and China and for the first time in Hong Kong in 1857, with both animal and human cases of rabies and hydrophobia.

One case abroad was reported by Fleming and later Steele and others: the death of Charles Lennox, Fourth Duke of Richmond (1764-1819), at the time governor-general of Canada, of hydrophobia, having been bitten 2 months earlier by a tame fox worried by dogs. J. D. Blaisdell (1992) questioned the diagnosis of hydrophobia, suggesting instead "hysterical rabies" brought on by an initial "mild cerebro-vascular accident," but his comments are not accepted by others with expertise in neurologic disorders, including rabies, as well as an interest in its history. Jackson, who in addition to his own expert knowledge of the disease has had access to contemporary case reports and papers held in the Public Archives of Canada, came to a different conclusion (Jackson, 1994). The records support and extend the nineteenth-century opinions: the 2-month incubation period following the fox bite and the details of the duke's clinical illness as recorded in the journals of the military officers who accompanied him. Jackson is convinced that it was a case of true rabies rather than the combination of an atypical presentation of two separate diagnoses (stroke and rabies hysteria) suggested by Blaisdell (1992) (A. C. Jackson, personal communication). After nearly two centuries, the diagnosis is confirmed: The duke died of hydrophobia and had not suffered from "hysterical rabies."

Occasional descriptions of episodes of hysterical rabies serve to underline the warnings of Bernard-François Balzac (1746-1826), father of the novelist, who was a hospital administrator at Tours. In a volume on the history and prevention of rabies, he — a stern critic of dogs, described as enemies of the public and of an "immoralité incurable" — recommended introduction of a tax on dogs, the amounts to depend on whether working dogs or mere pets (the tax was implemented 45 years later, long after his death). His main concern, however, was with those unfortunates who might be suffering only from imagined hydrophobia.

Balzac was anxious to protect such patients from the long-practiced form of euthanasia intended to end the distressing sufferings of victims of true rabies/ hydrophobia: strangling or suffocation, using the patient's own sheets and pillows, respectively (Balzac, 1810). Good intentions aside, Balzac feared that it might, in the wrong hands, prove "too tempting to the heirs or enemies of the patient."

While such episodes in Europe and North America show increasing concern with prevention and avoidance of unnecessary suffering, in Russia, serious outbreaks of rabies with inevitable hydrophobia casualties in poor and remote village communities in that vast country were recorded by Michele Marochetti, one of many European professionals working there during the nineteenth century. Personal physician to one Count Moszezensky, Marochetti spent years in the Ukraine and witnessed the consequences of "large hydrophobic dogs" terrorizing villagers. He wrote later: "On several occasions I was devastated when victims perished under my very eyes." His only suggestions for therapy were repetitions of the decoctions recommended through previous centuries (Marochetti, 1849).

In fact, both the case of the Duke of Richmond and Marochetti's notes serve to demonstrate how little progress had been made since the seventeenth century, despite the growing medical and scientific awareness of the century of enlightenment. For two centuries, research on rabies had been limited to attempts to reexamine recommended therapies of old, which in the case of rabies had by then held sway for so many centuries without being challenged. Already reports in the very first volume (1665-1666) of the Philosophical Transactions of the Royal Society, back in London after the disastrous year of the great plague, had touched briefly on age-old seawater treatment recommended for rabies (see above). The society had asked Robert Boyle (1627-1691) for answers to questions on a number of problems connected with seawater, among them, "What are the Medical Virtues of the sea, especially against Hydrophobia?" Despite his own later extensive writings on aspects of seawater, its saltiness at different depths, temperatures, and locations, even attempts to "sweeten" it — he never tried to answer this particular question.

However, elsewhere in his writings, where he considered "true and medical virtues" of other recommended therapies, Boyle was reluctant entirely to dismiss possible medicinal benefits of other time-honoured remedies. Some, valued for both prophylactic and possible curative properties since antiquity, were a variety of gemstones — "bezoars" or "madstones" — reputedly helpful against both plague and hydrophobia (Boyle, 1672). Boyle's contemporary, Nathaniel Hodges (1629-1688), a physician who remained in London bravely caring for his patients during the great plague of 1665 while others fled, was doubtful, considering the "Oriental Bezoar" overpriced and overrated (Holland, 2000). Sadly, after an impressive career and wide recognition, Hodges died in a debtors' prison. As far as the Royal Society was concerned, the belief in beneficial effects of "Bezoar-Stones" was laid to rest after Boyle's death by Frederick Slare (16477-1727) in his Experiments and Observations upon Oriental and Other BEZOAR-Stones, with the telling subtitle, "which prove them to be of no Use in Physic" (against hydrophobia or anything else) (Slare, 1715). Elsewhere, notably in the United States, belief in curative powers of "madstones" continued among Native Americans and others until modern times (Baer et al., 1996).

In the later seventeenth century there were signs of a growing realization that the animalculae observed by Antony van Leeuwenhoek (1632-1723) in his improved microscopes were indeed, as he claimed, "living creatures" and possibly could cause disease. Such ideas were reflected in the work of Francesco Redi (1626-1697), who in the late 1680s was the first to refute experimentally the long-held belief in spontaneous generation (Redi, 1688). His associates, Bonomo (d.1696) and Cestoni (1637-1718), then were able to implicate the mite acarus as the agent of scabies in humans. On the other hand, at this stage, the works of Leeuwenhoek and Redi had little impact on the literature on rabies. Terrifying as its manifestations were, rabies affected so few humans, or even their animals, when compared with the ravages of major scourges of the seventeenth and eighteenth centuries such as smallpox and bubonic plague in humans and cattle plague in their animals, that it could not be counted among the major problems of the age.

Curiously, one isolated example of an influence of the new ideas appeared in London in 1735 in the London Magazine, not a scientific publication. The anonymous author agreed with the general opinion that the rabid dog's saliva must be the seat of the disease but believed that the infection was caused by "minute particles or animalcules, mixt with saliva" that affected the brains of victims by incorporation into their "nervous juices." It was a bold statement for its time, which may perhaps account for its anonymity (Mullett, 1945).

Further advances in knowledge and understanding of the pathogenesis of rabies in humans and in animals progressed slowly, accelerating only in the nineteenth century in the wake of animal experiments and growing acceptance of the "germ theory." During the last decade of the nineteenth century, interest in research activities was increasing in medical and scientific societies everywhere. In England, the Literary and Philosophical Society of Manchester and the Society for the Improvement of Medical and Chirurgical Knowledge were founded in London in 1783 by John Hunter (1728-1793), surgeon and anatomist par excellence, and George Fordyce (1736-1802). In 1793, by happy coincidence, Samuel Argent Bardsley (1764-1851) in Manchester and Hunter's younger namesake and protégé, John Hunter, M.D. (1754-1809), each published papers that were to be seminal for work in the early nineteenth century. Bardsley had been appointed physician to the Manchester Infirmary in 1790; he remained there, as "the very model of a hospital physician" (Dictionary of National Biography, hence forth DNB, 1885), until he retired in 1823, continuing to contribute papers to the Memoirs of the Literary and Philosophical Society there. His 1793 observations on rabies and hydrophobia anticipated what was to follow in the next century by their clarity and confidence in disposing of age-old superstitions and replacing them with reason, facts, and common sense. He was convinced that neither rabies in dogs ("canine madness") nor hydrophobia in humans could occur spontaneously and ascribed apparent cases of the latter unconnected with animal bites to other diseases and "sometimes hysteria" (Bardsley, 1793).

John Hunter's paper had been published in 1793 in the Transactions of the London Society (Hunter, 1793). John Hunter, M.D., appeared as sole author, although his entry in the DNB, written by Charles Creighton (1847-1927), notes that it was "drawn up at the Society's request" — presumably other members of the society's total of 12 had been consulted. On the other hand, it was based largely on the younger John Hunter's own experiences in Jamaica, where he had been superintendent of military hospitals from 1781 to 1783, before settling down to practice in London. It also reflects Hunter's reputation, from the days of his Edinburgh thesis, for "correct reasoning" (DNB, 1819). His experiences in Jamaica had fired his interest in epidemiology and prevention of contagious diseases; he had written papers on outbreaks of typhus fever among poor London families before publishing, in 1788, Observations on the Diseases of the Army in Jamaica (Wilkinson, 1982). The latter contained important notes on remittent fever in the army but missed, as so many other observers did at the time, the role of mosquitoes in transmission, despite critical mention of the nuisance of their presence on the island.

Although in the paper on canine madness Hunter admitted having no direct proof that the disease could never occur spontaneously, his records from Jamaica at least provided circumstantial evidence: On that island full of dogs, in a hot climate, up to 40 years had elapsed with no occurrence of rabies. Before that, any outbreaks could be traced to dogs introduced from North America. On this basis, Hunter formed his conclusions, which contain suggestions pointing the way to the kind of animal experiments soon to become the mainstay of infectious disease research in the following century: experiments to be made "upon the poison," in this case of rabies, to determine its nature and path of transmission, including inoculating dogs and other species of animals with saliva from dogs suffering from canine rabies or even from "an hydrophobic patient." The inoculated animals were then to be observed, recording progress of the disease. Other suggestions included attempts to establish possible effects of "counter poisons" and of the length of time beyond which excision of the tissue around the inoculation site could not longer prevent development of the disease.

Nobody within the society, or elsewhere in the country, attempted to test the recommendations. Perhaps no suitable cases were available, or again, the death of their mentor, the John Hunter, in the same year may have removed their inspiration. The challenge was taken up 10 years later in Germany by Georg Gottfried Zinke (d. 1813), who carried out all the experiments suggested with one exception, the one involving saliva from a human patient. Zinke recorded his results in a volume published at Jena in 1804, the first description of experiments specifically intended to follow the path of transmission of the unknown agent of rabies. Zinke successfully produced rabies in healthy dogs, cats, rabbits, and fowl using a small brush to transfer saliva into incisions (Hunter had suggested the point of a lancet). With Hunter's suggestions and Zinke's experiments began the era of animal experiments and of the rapid development of comparative medicine and pathology (at the time referred to as "pathological physiology" or "pathologie expérimentale") in France and Germany in the nineteenth century (Wilkinson, 1992).

The missing experiment with saliva from a human case was carried out barely 10 years later in the year of Zinke's death in France by François Magendie (1783-1855), who published his results in 1821 in a paper that with gallic abandon and almost relish described a series of dramatic and dramatically presented experiments in a Parisian establishment for fighting dogs carried out between 1810 and 1820 with the assistance of medical students chosen for their "courage and sang-froid": The experiments involved transmission of rabies to mastiffs via saliva from a human case (Magendie, 1821). Others warned of the moral implications of such experiments; in London, Benjamin Mosely had written as early as 1808: "I have no doubt but deadly inoculation [with rabid saliva] might be performed in a way, which I do not think prudence would justify the mentioning — there is mischief enough already in the world" (Moseley, 1808). By the 1820s, however, rabies was well established as the first zoonosis to become a focus for research in comparative medicine.

In Berlin in the later 1820s, K. H. Hertwig (1798-1881) attempted, but failed, to induce rabies in healthy dogs by implantation of nervous tissue from rabid dogs (Hertwig, 1828), although like others before him he had some success with rabid saliva. It is the background and career of Hertwig, as of Magendie earlier, that point the way to further developments during the nineteenth century. Magendie was a pioneer neurophysiologist, architect with Sir Charles Bell (1774-1842), neuroanatomist, of the Bell-Magendie law (ventral spinal roots carry motor fibers and dorsal roots carry sensory fibers), and his involvement in rabies research at this stage reflects a new awareness of the neurotropic character of the disease. Hertwig, on the other hand, having qualified in medicine at Breslau in 1819, had gone on to supplement his medical degree with veterinary studies in Vienna, Munich, and Berlin, preparing himself for the opportunities now opening up for studies in comparative medicine and pathology. For the rest of his working life, Hertwig combined medical practice and research with teaching at the Berlin veterinary school. In this capacity he came to be an outstanding representative of the kind of cooperation between medical and veterinary science that was to carry comparative medicine into the future. It was made possible by the fact that education in medical schools could now be supplemented by courses at veterinary schools and colleges opening throughout Europe in rapid succession to the original French ones at Lyons in 1761-1762 and at Alfort in 1766 (Wilkinson, 1992).

As outbreaks of rabies became more common in Britain and the rest of Europe during the nineteenth century, the voice of the new veterinary profession also began to be heard. In London, William Youatt (1776-1847) took a particular interest in the disease and gave a course of lectures on "canine madness" in the 1830s. Toward the end of his life he wrote a manual on dogs, published posthumously, in which he included a chapter on rabies. Here he quotes Bardsley, agreeing absolutely that rabies is transmissible and never occurs spontaneously. Consequently, he also followed Bardsley's views on prevention. He wrote, "...if a species of quarantine could be established, and every dog confined separately for eight months, the disease would be annihilated in our country or could only reappear in consequence of the importation of some infected animal" (Youatt, 1851). Perhaps curiously for a veterinarian but showing a healthy respect for reality, he even shared the views of Balzac père in his critical attitude to prevailing numbers of "useless and dangerous dogs" and in particular to the practice of keeping "fighting-dogs" for "most brutal purposes." He referred to the "rabid virus" but only to admit that little was known of it and that at present it would be a "difficult process to analyse it." He was, however, the first to make a suggestion for further experiments that would prove to be of definitive importance for rabies research and the development of vaccines in France later in the century. Youatt's final words on the subject were, "I very much regret that I never instituted a course of experiments on the production and treatment of rabies in [the rabbit]. It would have been attended with little expense or danger, and some important discoveries might have been made."

Three decades later, the rabbit began its rapid rise to a position as the animal of choice in experiments on rabies in France. As Youatt knew, the rabbit develops the predominantly paralytic form of rabies and so could serve as a less dangerous and more convenient object for experiments than the dog with the furious form of the disease. Thirty years after the death of Youatt, it fell to Pierre-Victor Galtier (1846-1908), professor at the Lyons Veterinary School, to reintroduce Youatt's rabbit to the general world of science and veterinary medicine in Paris in 1879 (Galtier, 1879). Galtier had been educated at the Lyons school and subsequently spent all his working life there. Carrying out experiments on rabies, he emphasized the advantage of using rabbits a whole year before Louis Pasteur (1822-1895) began work on the subject. By then he was aware of the work of Galtier and had visited Lyons, where Chauveau had introduced him to the full volume of Galtier's publications. In December 1880 he was ready to begin his own experiments (Guiart, 1922).

Whatever the final verdict on Galtier's influence on Pasteur may be, there can be no doubt about the seminal influence of his original experiments with rabbits on all the work that followed after 1880 (Fig. 6). Théodoridès has given an extensive and definitive analysis of Galtier's contributions in 1986, unfortunately too late for Steele in 1975 (and repeated in 1991) to correct his misapprehensions

Fig. 6. Louis Pasteur holding two white laboratory rabbits. From Vanity Fair, 1887. (Wellcome Library, London.)

about the experiments of Galtier, whom he dismissed in a few lines, citing only his two pages of summary report delivered at the Académie des Sciences in 1879. On this basis, Steele remarked briefly that Galtier reported transmission of rabies to rabbits and from rabbit to rabbit "unfortunately without much needed detail." The details could have been found in the two original papers, published in separate veterinary journals, on which the report was based. There, in a total of 22 pages, Galtier provided all the information needed to evaluate his results, including his method of inoculating canine rabid saliva on the point of a lancet or directly by bite, introducing the ear of the live rabbit into the jaws of the rabid dog (Galtier, 1879).

Earlier in his chapter Steele had introduced "Bouchardat" (Apollinaire Bouchardat, 1806-1886) as being "among the first to think about inoculations against rabies and had an early influence on Pasteur. He attempted many experiments at the Lyon veterinary faculty." Since he had never worked at Lyons but was a pharmaceutical chemist attached to the Paris medical faculty and Hotel-Dieu, this is unlikely. In fact, the two papers by Bouchardat cited by Steele were written in response to a request for information during a serious outbreak of the disease — one of many in France in the nineteenth century — in the early 1850s. They contain evaluations of all the countless remedies against hydrophobia, mostly spurious, recommended over the centuries; Bouchardat made it clear that he had no reservations about the uselessness of any of them. Steele may have confused the texts and personalities of Galtier and Bouchardat (Bouchardat, 1852-1853, 1854-1855; Julien, 1977). The confusion is carried over, unchanged, into the updated chapter by Steele and Fernandez in the 1991 second edition of The Natural History of Rabies.

By the time Pasteur became aware of Galtier's contributions, he himself had already established the value and possibilities of prophylactic inoculation with attenuated material. It was a milestone not just in the history of rabies but in that of virus research in general and vaccine development in particular. Yet it was based on a chance observation, a case of serendipity to match Fleming's discovery of penicillin half a century later. But where Fleming did not quite have the courage of his convictions to take his discovery further at the time, Pasteur recognized the potential of his and immediately applied himself to its realization. Pasteur's fortuitous observation concerned a forgotten sealed flask of chicken cholera culture by mistake left undisturbed for a few weeks instead of the routine 24 hours. The observed attenuation of the agent responsible led, through the work of Toussaint (1847-1890) on defibrinated anthrax blood to the production of the first anthrax vaccine. The following year Pasteur addressed the International Medical Congress in London on vaccination, where he explained his use of the terms vaccination and vaccine, acknowledging Jenner's contribution (Pasteur, 1881).

Pasteur's development of the first rabies vaccine is, of course, the stuff of legend, all the more remarkable because the agent of the disease — the "virus" — was unknown at the time, unlike the bacillus of anthrax. However, this did not shake his determination to develop a vaccine, nor that of his colleagues, Emile Roux (1853-1933), who as the only man with a medical degree in the group was essential to Pasteur the chemist, Charles Chamberland (1851-1908), physiological chemist, Edmond Nocard (1850-1903), veterinarian, director of the Veterinary School at Alfort from 1889, and Louis Thuillier (1856-1883), who sadly died before he was 30, in pursuit of his duties, of cholera in the epidemic in Egypt that he had gone to investigate with Emile Roux and other members of the group. Pasteur pursued the work on rabies and decided that a vaccine against the disease should be the next major achievement in his laboratories.

In the case of rabies, Pasteur was faced with a disease agent about which, unlike the recently described anthrax bacillus, little, if anything, was known. Neither he nor anyone else had been able to see it in the microscope, let alone grow it in culture on artificial medium. Pasteur refused to give up; with characteristic ingenuity and determination, he bypassed established methods of culture and attenuation and turned to what he knew to be the natural habitat of the elusive "invisible virus": the spinal cords of his laboratory rabbits. Even then, he had first to confirm two necessary preconceptions: the neurotropic character of the virus, suspected certainly since the time of Magendie, and equally important and a concept totally his own, production of a ''virus fixe!' Working with Chamberland and Roux, he delivered the necessary proof of the neurotropic character of the virus; they also could show that the agent was present not only in the saliva of the rabid animal but also throughout its nervous system. The second important point was the production of a standardized form of the virus with a fixed incubation period. Initially, street virus was inoculated directly under the dura mater of dogs and then serially passed through rabbits until the length of the incubation period was shortened to a final limit of 6 to 7 days. Pasteur had achieved his goal: a fixed virus on which to base a vaccine (Fig. 7) (Pasteur et al., 1884; Pasteur, 1885).

After much additional laborious work, the vaccine in its final form was tried out on dogs and rabbits with encouraging results announced at the Académie des Sciences in 1885. Even then, Pasteur himself dreaded the first trial on a human patient, as he wrote to the Emperor of Brazil: ".. .however much I multiply my cases of protection in dogs I think my hand will tremble when I go on to mankind" (Vallery-Radot, 1900). This pained remark may to some extent also reflect Pasteur's experiences in May-June 1885, never published and only known now, more than 100 years later, when G. L. Geison was able to examine Pasteur's private laboratory notebooks and his correspondence with Dujardin-Beaumetz. The episode concerned two patients admitted to hospital as suspected rabies cases. The hospitals appealed to Pasteur as a last resort. One M. Girard was treated in May 1885 with "attenuated rabies virus." It was a curious case of on-and-off apparent recovery followed by repeated relapses, but after 3 weeks Girard was discharged, supposedly cured, from the Necker Hospital. His later fate is not known.

Within a month, 11-year-old Julie-Antoinette Poughon was admitted to the St. Denis Hospital. Bitten on the upper lip by her puppy sometime in May and admitted on June 22,1885, after 2 days of "severe headache," a diagnosis of rabies was confirmed. The girl was given one injection of an attenuated preparation immediately and a second at midnight, sadly to no avail. She died at 10:30 the following morning (Geison, 1995). A month later, Pasteur's hand was finally forced in the glare of publicity. A 9-year-old boy, Joesph Meister, was brought from Alsace with severe wounds from the bites of a mad dog, almost certainly fatal. Pasteur agonized, discussed ethical aspects of the case with trusted medical advisers, and finally decided that the only hope of saving the boy was a course of

Fig. 7. Statue in Paris celebrating the case of the shepherd boy J.-B. Jupille, the second survivor of rabies bites following treatment with Pasteur's vaccine in 1885. (Wellcome Library, London.)

postexposure prophylaxis. It was successful; the boy, despite his injuries, failed to develop rabies, and the vaccine was established. There were to be setbacks as well as many lives saved, but the principle of postexposure prophylaxis in rabies had been established (see Fig. 7), ready for the improvements and developments of the twentieth century (Paget, 1914).

The question of the nature of rabies virus was still, during the lifetime of Pasteur, left wide open. At the turn of the century, work on tobacco mosaic virus and foot-and-mouth disease opened the floodgates to a new era where "invisible," "filterable" viruses, including the virus of rabies, eventually would come into their own and play their role in molecular biology (Waterson and Wilkinson, 1978). Before that, the importance of Pasteur's work already was beginning to be reflected in developments at home and abroad. The Pasteur Institute (Fig. 8), funded by public subscriptions in Paris between 1886 and 1888, primarily to develop rabies vaccine, soon widened its horizons and was followed by a number of sister institutes with similar aims of vaccine production and research abroad [at Saigon (Cochin China) 1890, Tunis 1894, Indo-China 1895, and Algiers 1910] and at home (Lille 1895 and Nancy 1898) (Dedonder, 1985). In London, the British Institute of Preventive Medicine was incorporated in 1891, modeled on the Pasteur Institute with its commitments to both research and vaccine production.

Fig. 8. Chromolithograph celebrating Pasteur's work and discoveries. Paris, circa 1890. (Wellcome Library, London.)

Initially, rabies was not mentioned specifically among its aims, although Victor Horsley (1857-1916), among the experts consulted and an adherent of Pasteur's methods, must have been in favor of rabies vaccine production at home to avoid unnecessary and possibly fatal delays when patients were sent to Paris. He did, in fact, lose a laboratory assistant, bitten by a rabid cat, who died in spite of prophylactic treatment by Pasteur, having arrived in Paris too late (Bristowe and Horsley, 1889). In the event, rabies vaccine production at the institute in London did not begin until after the disease had been eradicated for the first time in 1902 (Wilkinson, 1992).

The early years of the twentieth century, before the outbreak of the Great War in 1914, were dominated by attempts in many laboratories to clarify the concepts of invisibility and filterability in the new type of infectious "microbes." In 1903, Paul Remlinger (1871-1964) was able to demonstrate, not without difficulty, the filterability of rabies virus, but like most of his colleagues in and out of the Pasteur Institutes at home and abroad, he was to remain reluctant to accept M. W. Beijerinck's theory of the contagium viviumfluidum (Remlinger, 1903). In the same year, Negri in Pavia contributed to the general confusion in early virus research when he described the eosinophilic inclusion bodies and identified them as protozoa causing the disease; he eventually named the putative organism Neurocytes hydrophobiae. On the other hand, over the years, Negri bodies, like Koch's tuberculin, have come into their own as an important diagnostic tool (Negri, 1903, 1909; Kristensson etal., 1996). Real progress in factual knowledge of morphology of the filterable viruses began with the introduction of the ultracentrifuge by The Svedberg and R. Fáhraeus in 1926, of electron microscopes in the 1930s, and with W. J. Elford's work on ultrafiltration beginning in 1928 (Waterson and Wilkinson, 1978). Helped by such techniques, perfected over the years, virologists had, by the 1960s and 1970s, for the first time a clear picture of the morphology, chemical composition, antigenic properties, and possibilities for cultivation. The virus of rabies finally could be defined as an RNA core contained in an envelope necessary for its infectivity with average overall dimensions of 180 x 75 mm and typically bullet-shaped (Topley and Wilson, 1984). Here the historian must come to a halt and leave the present and future to the authors of the following chapters, who are at the cutting edges of today's research. Except perhaps for a few concluding reflections on the continuing saga of outbreaks of rabies still occurring not only in endemic and underreported areas in Asia, the Middle East, Africa, and South America but also in countries in the Western world with access to the latest vaccines, such as the bioengineered V-RG (vaccinia-recombinant glycoprotein vaccine), developed by teams at the Wistar Institute in Philadelphia and the National Institutes of Health, cooperating with a French biotechnology firm. In 1984 this glycoprotein was found to be effective as an oral vaccine after outbreaks in racoons in the "Wistar's backyard" that began in 1982 (Findley, 1998).

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