Some Achievements in the Prevention of Disease
IN introducing the lecturer the Society's Chairman. Professor Henry Barcroft, said: "You will all notice the absence, this evening, of our President, Lord Halsbury, who is indisposed and has sent his apologies for the fact that he could not come today.
Now it is my very great pleasure to be introducing Sir John Boyd who, of course, needs no introduction at all, but I would just like to say how very much we owe him in one special respect and that is the way he has actually worked for the cause of Research Defence. He has taken an enormous amount of trouble, first of all in writing the article in connection with the Littlewood Report, which many of you will have seen in Conquest and secondly, having discharged his duties for so many years, he has spent, I am sure, a very great deal of time this year in preparing his lecture for us this evening, and we owe him a special debt of gratitude for this. I will now ask Sir John if he will give his address:
Some Achievements in the Prevention of Disease
By SIR JOHN BOYD, O.B.E., M.D., F.R.C.P.. F.R.S
The Stephen Paget Memorial Lecture, which 1 have the honour of delivering this evening, was instituted to perpetuate the memory of that remarkable man Stephen Paget, to whom all who in the course of their work make use of animal experiment, owe a very real debt. He it was who founded the Research Defence Society in 1908. Throughout his life he championed the cause of research workers. He actively resisted the attacks of antivivisectionists by writing letters to the press, by taking part in debates and by holding forth in the "People's Forum" at Marble Arch, notably at a time during World War I when his opponents were endeavouring to persuade members of the Forces to refuse antityphoid inoculation. His enthusiasm and singleness of purpose is something never to be forgotten.
One of the functions of this Society which he founded is to keep the general public well informed concerning these advances in the prevention and treatment of disease which depend in large measure on animal experiments. Some of these ideas and methods have become so well established that they are taken for granted, and the ups and downs encountered in the earlier stages of their evolution are forgotten. Because of this 1 have thought it would be of interest to dip back into history and to recall the story of the emergence of some of these concepts and of their pursuit in the face of formidable difficulties and frequent disappointment. In particular I have in mind the subject of active immunisation.
The empirical conception of active immunisation is an old one, best exemplified in the case of smallpox. It is a long recognised fact that one attack of smallpox confers life-long immunity. To avert severe attacks, attempts were made to induce mild attacks by artificial infection. Lymph from a pustule on what appeared to be a mild case was scratched into the skin of the hitherto uninfected person in the hope that this would produce an equally mild attack. It did not always work out this way, and the ensuing illness could be severe. This procedure, which was a common practice inTurkey, was introduced intoBritainin the early part of the 18th century by Lady Montagu, wife of the British Ambassador inConstantinople. It never became popular, and later in the century was replaced by vaccination with cowpox lymph, introduced by Edward Jenner. This story is so well known that I will not repeat it.
No serious attempt was made to investigate or develop the underlying principles of these procedures until the time of Louis Pasteur, in the latter half of the 19th century. While carrying out some investigations into the organism which causes chicken cholera, Pasteur found that one of his cultures had become attenuated and non-virulent. Whether by accident or design, some chicks which had been inoculated with this non-virulent strain were given an injection of a virulent strain, and, much to Pasteur's interest and surprise, survived. This observation was the key to his further research.
Around 1880 Pasteur turned his attention to anthrax, a disease then common among farmyard animals, and the cause of many deaths. The responsible organism, the anthrax bacillus, had been isolated and cultured by Robert Koch a few years previously. Inspired by his findings in chicken cholera, Pasteur attempted to produce an avirulent culture of the anthrax bacillus, and finally succeeded, his method being to add certain antiseptics to the culture and to incubate the treated cultures in shallow layers at a temperature of 42° to 43°C. In eight days the bacilli became innocuous. He then found that by injecting animals—both laboratory and farmyard animals—with a dose of attenuated organisms, followed 12 days later by a dose of more virulent organisms, he could produce immunity to fully virulent bacteria. This observation was clinched by a classical experiment. Needless to say, Pasteur's novel and, as they then appeared, far-fetched theories were ridiculed by the lay public and challenged by his colleagues, particularly members of the veterinary profession, who regarded him as an interfering chemist. Among his critics was a M. Rossignol, a leading veterinary practitioner in the district, who despite his criticism was Pasteur's good friend. Rossignol and Pasteur between them conceived the idea of carrying out a large controlled trial, and Rossignol, being a man of some influence, raised the necessary funds and placed the resources of his large farm at Pouilly le Fort at Pasteur's disposal. Pasteur was supplied with 24 sheep, one goat and six cows, which he inoculated with vaccines of graded virulence, first on 5th May, 1881, and again on 17th May. On 31st May these animals were transported to Mr. Rossignols farm at Pouilly le Fort, where a control batch of 24 sheep, one goat, and four cows were assembled, together with a considerable gathering of critical spectators from all walks of life. In front of this audience Pasteur injected into all the animals, both immunised and controls, a portion of a culture of virulent anthrax bacilli. On 2nd June the critics reassembled to see the outcome of the experiment. All the immunised animals were alive and well. The control goat and 21 of the control sheep were dead, and two more sheep died during the day. The control cows did not die, but were febrile and suffered from extensive local lesions. Next day—3rd June—one of the immunised animals died, but autopsy revealed the cause of death to be a dead foetus in utero, and not anthrax. Pasteur's claims were thus irrefutably established. Inoculation against anthrax is now widely practised, but with a more sophisticated vaccine, in areas where the disease is rife, and has produced a notable fall in the incidence and mortality rate.
With this convincing demonstration of the possibilities of active immunisation, Pasteur's active brain turned to its application in other infective conditions, and in particular, to rabies. Of all the diseases which can affect mankind, none is more horrible, more fearsome in every way, or more distressing to the patient and to all who have to come in contact with him. Infection is conveyed by the bite of a rabid animal—dog, jackal, wolf, mongoose, cat, vampire-bat and many others, including man, for the virus is present in the saliva of victims of this disease. In Britain, mainly as the result of strict quarantine measures, rabies is now extinct, but the possibility of its re-introduction is very real, for a pet-loving people is hard to convince of the necessity for the rigid application of these regulations. The danger lies in the long incubation period, as weeks or months may elapse between the time of the infective bite and the appearance of symptoms. Over most of the world rabies is endemic, and in some rural districts, especially parts of the American continent, the menace is very real.
Convinced that the disease was caused by some living agent, Pasteur made many unsuccessful attempts, to culture the organism, whatever it might be, on artificial media. These failures forced him to the conclusion that the agent would grow only in living tissue, and that he must attempt to culture it in the brain of the dog. Here I must quote a translation of an account given by Roux, a colleague with whom Pasteur worked in close co-operation, of the first time this experiment was carried out
Roux on first transmission of rabies to the dog by the intracerebral route: “Ordinarily an experiment once conceived and talked over was carried out without delay. This one, on which we counted so much, was not begun immediately, for Pasteur felt a veritable repugnance towards vivisection. He was present without too much squeamishness at simple operations, such as a subcutaneous inoculation, and yet, if the animal cried a little, he immediately felt pity and lavished on the victim consolation and encouragement which would have been comical had it not been touching. The thought that the skull of the dog was to be perforated was disagreeable to him: he desired intensely that the experiment should be made, but he dreaded to see it undertaken. I performed it one day in his absence; the next day, when I told him that the intracranial inoculation presented no difficulty, he was moved with pity for the dog. 'Poor beast. Its brain is certainly badly wounded, it must be paralysed'. Without replying, I went below to look for the animal, and had him brought into the laboratory. Pasteur did not love dogs, but when he saw this one full of life, curiously ferreting about everywhere, he showed the greatest satisfaction, and straightway lavished on him the kindest words. He felt an infinite liking for the dog which had endured trephining without complaint and had thus relieved him of scruples concerning the operation".
However, the experiment was successful. The dog developed rabies in 14 days.
From the infected brain of this dog it proved possible to pass on the disease to the rabbit, again by intracerebral injection, which was of course much simpler in this smaller animal. At first the incubation period in the rabbit was variable but after a number of passages it settled down, and symptoms appeared regularly on the sixth day. This so-called "fixed" virus became an invaluable standard for further experiment.
Following the principle embodied in his anthrax experiments, Pasteur tried, and for a time in vain, to lower the virulence of the infective agent. Then in Roux's laboratory Pasteur one day observed an experiment in which Roux was attempting to kill the infective agent of rabies by desiccation. Pasteur modified Roux's technique by suspending the spinal cord of a rabbit which had died of rabies in a jar, at the bottom of which he placed caustic potash. In 14 days the cord dried up and became non-infective, and further experiments showed that the progress of attenuation from full virulence to non-infectivity was a gradual one. Now Pasteur had his immunising material. He injected experimental animals daily with doses of cord suspension of increasing virulence—first 14 days dried, then 13 days and so on throughout the series. At the end of the course the animals were challenged by intracerebral injection of virulent material, and remained unaffected. They had been successfully immunised.
Could this process be applied to the prevention of rabies in human beings who had been bitten by rabid dogs? Because of the length of the incubation period it seemed possible that it might be successful, but as might be expected the idea of inoculating human beings with this potentially infective material was violently opposed, among others even by Roux. However, in time a willing volunteer came forward, a boy called Joseph Meister fromAlsace, who had been bitten by a rabid dog on the hands, legs, and thigh 60 hours before treatment was started. He was given 14 graded daily injections, the last being of untreated virulent cord. This last injection did not produce any symptoms, so clearly the boy had been immunised by the earlier treatment. But of course whether he had been infected by the bites from the dog is a question which can never be answered, for it is a well established fact that the bite of a rabid animal is not invariably infective.
The further history of Joseph Meister is of some interest. In adult life he became doorkeeper of the Pasteur Institute inParis, and is said to have committed suicide in 1940 to avoid being forced by the invading Germans to open the crypt in which Pasteur is buried.
A second case, a shepherd boy aged 15. Jean Baptiste Jupelle, severely bitten by a dog which was killed and proved to be rabid, subsequently came forward. He was treated and survived. Thereafter, though slowly, the treatment became generally and widely adopted, and appeared to achieve some success, though, for the reason already mentioned, this could not be unequivocally confirmed.
In the years which have passed since Pasteur's discovery, vast improvements have been made in the preparation of anti-rabies vaccine. One of the first was a changeover from dried rabbits cord to carbolised sheep's brain vaccine. This made possible the preparation of large quantities of stable vaccine that could be sent by post to wherever it was needed, so obviating the necessity of treating all patients at one centre. But both of these vaccines, because of the nervous tissue they contained, had the grave disadvantage of producing occasional cases of degenerative changes in the nervous system and subsequent paralysis. To avoid this complication, virus for vaccine production has in recent years been successfully cultured in fertile hen's eggs and with better results, in fertile duck's eggs. Later still, vaccines made from virus grown in tissue culture have been prepared and are being tested.
What has been achieved by anti-rabies vaccine? There is no doubt that it produces immunity to subsequent infection, but when administered after the potentially infective bite can it overtake and destroy the virus before it establishes itself? This is difficult to prove in a way that will satisfy the statisticians, but those who have extensive experience believe that it can.
In recent years a high-litre antiserum has been produced, and there is good evidence that when this antiserum is administered soon after the infective bite it provides a passive immunity which affords protection during the build up of the active immunity induced by the antirabic vaccine.
However, this is only the human side of the case. In those countries where rabies is endemic, it is of common occurrence in wild animals and, stemming from them, in domestic animals, particularly dogs, cattle, sheep, etc. Indeed, it is much more important as a veterinary than as a human disease. And while under normal conditions one would not think of pre-immunising a community of human beings, such preventive immunisation is commonly practised in domestic animals, particularly in dogs (including pet dogs) and farmyard animals. The protection conferred is excellent and has gone far to reduce the incidence of rabies among these animals and, as a corollary, among human beings. Thus inU.S.A.from 1946 onwards there has been an active campaign along these lines combined with greater control of stray dogs. In 1946, some 8,384 cases of rabies in dogs and 10,883 cases in cattle, sheep, pigs, goats and horses were reported. In 1965 the corresponding figures were 412 and 3.457. In terms of human infections, the incidence fell from 33 in 1946 to 1 in 1963, 1 in 1964 and 2 in 1965, so that in this indirect way the risk of human infection has been almost eliminated. Unfortunately, as long as, in an endemic area, susceptible animals exist in their wild state, rabies can never be eradicated.
Those of us whose memories go back 40 years or more will remember that in those days distemper, a disease with a considerable mortality rate, was rife among the canine population, and was a particularly serious hazard in kennels, among packs of hounds, and at shows such as Crafts, where one infected animal might start off a widespread epidemic.
Following the discovery that communicable diseases could be caused by infection with microorganisms, repeated attempts—all unsuccessful —were made to identify a bacterium as the cause of canine distemper. An organism known as Bacillus bronchisepticus came under grave suspicion but the case against it was never proved. In the early years of this century Carre inFrance produced evidence which indicated that the causative agent was a filter-passing virus, but for one reason or another his work was largely disregarded. In general, research on distemper was desultory and inconclusive. In 1922 the veterinary authorities in this country succeeded in exciting the interest and enlisting the support of the Editor of the sporting journal, The Field. With powerful assistance from this source a fund was raised sufficient to finance research on a substantial scale. The task was undertaken by P. P. Laidlaw and G. W. Dunkin, research workers on the staff of the National Institute of Medical Research, and was carried out in a brilliantly logical and methodical fashion which will long be a model for investigations of this kind.
Their early work made use of the long-standing observation, based on epidemiological and "clinical" evidence, that the ferret, like the dog, suffers from and is highly susceptible to distemper. As will be seen, this observation was fully confirmed by experimental evidence produced at a subsequent stage in this investigation. The ferret is, of course, a particularly useful experimental animal, being easy to maintain and handle. Because of the wide prevalence and high infectivity of distemper, great care had to be taken to exclude from the experiments any animal which by previous exposure to the disease had acquired some degree of immunity, and also to protect susceptible animals destined for experimental use from accidental infection. To achieve this, only animals locally bred from "clean" stock were used in the investigation, and all animals were specially housed. To avoid airborne infections, small huts were built throughout the grounds, separated from each other and from the research laboratories by not less than 50 yards. Entrance to the huts containing "clean" animals was restricted, and the attendants were never allowed to come in contact with sick ferrets or dogs, or to keep dogs of their own. The animals were fed on raw horsemeat and milk, which likewise was never handled by attendants who came into contact with sick ferrets or dogs. In the specially designed laboratory stringent precautions were taken to prevent the spread of infection from the "dirty" to the "clean" side. Entrance to every section was through a pool of lysol, the attendants wearing rubber boots and gloves and long rubber coats which could be swilled down with disinfectant.
These precautions were successful. On only one occasion did cross-infection occur, when a gale blew virus from an infected hut to a clean hut down wind.
Work began with an exhaustive investigation into distemper in ferrets. It was found that infection, which under natural conditions is airborne or the result of direct contact, could be produced at will by the injection of small quantities of blood or diseased tissue taken from an animal suffering from distemper. The course of the illness, the symptoms, and the histopathology were studied in detail. The mortality rate was high—somewhere around 90°0. Two points of fundamental importance emerged. First, although cultures of heart's blood were invariably bacteriologicaily sterile, the blood was nevertheless infective, and therefore contained the infective agent, presumably an ultramicroscopic virus. Second, injections of infected ferret's blood produced typical distemper in puppies. Thus it was firmly established that ferret distemper and dog distemper were identical diseases produced by the same agent, so that the ferret was available as a reference animal in experiments on dog distemper.
The research then passed on to the study of distemper in dogs. As in the case of the ferret investigations, strict precautions had to be taken to use only susceptible animals. The original carefully selected stock was removed from the breeding hut as soon as "clean" puppies became available, and all subsequent breeding was from this clean stock. Experimental animals were kept in small runs scattered throughout the grounds, and were attended with the same precautions as were used in caring for the ferrets.
The symptoms of uncomplicated dog distemper, unlike those of the disease in the ferret, were found to be rather indefinite. The animals developed a fairly high temperature, and at the onset there was usually running from the nose. Frequently the temperature was diphasic, and there was a gastro-intestinal disturbance of greater or lesser severity. Symptoms of variable severity resulting from secondary infection developed in the respiratory system. Nothing characteristic was to be found at autopsy.
Laboratory investigations along the usual lines proved that the infecting agent was a filter-passing virus and not a bacterium, and that it was present in high concentration in the spleen.
The next step taken was to immunise ferrets against dog distemper virus, and this was successfully accomplished by injecting clean ferret stock with a formalised suspension of spleen taken from an infected dog—which contained a high concentration of dead virus. To be effective a large dose was required, and further experiment showed that it was advantageous to consolidate the immunity thus established with a subsequent dose of living virus, following which immunity was found to be solid and probably lifelong. Thus the possibility of conferring active immunity by means of a virus vaccine—first dead virus, then live virus—was established. Finally the problem of immunising dogs was tackled, applying the experience gained in the ferret experiments, and it was found that the same methods were successful. A first injection of formalised dog distemper virus, followed by a second injection of living virus, produced solid immunity.
As might be expected, these results were received with some scepticism by the veterinary profession, but experience, and particularly the experience of veterinary practitioners responsible for the health of packs of foxhounds, showed that this method of immunisation passed the crucial test—it worked.
For some years all went well, and this method of immunisation was accepted and widely practised, but, as it turned out, this was by no means the end of the story. In the years following the last war, things began to go wrong. Dogs which had been immunised by the standard method became infected with a distemper-like disease in which nervous symptoms were predominant. As such nervous symptoms had been a rare occurrence in distemper as it was known in the past, it was concluded that the causative agent was not true distemper virus, though it might be a related organism. Later it was observed that dogs suffering from this distemper-like illness had a peculiar hardening of the pads of the feet, and the disease came to be known as "hard-pad" disease. Unlike the distemper virus, the hard-pad virus, injected into the ferret, produced an illness with a relatively low mortality rate. On the balance of evidence, it was concluded that hard-pad virus was related to distempervirus, and was almost certainly a variant of some kind. Many theories have been elaborated to explain the appearance inBritainof hard-pad disease, which caused severe and widespread epidemics similar to those caused in the past by the true distemper virus. The most favoured is that the hard-pad virus is a continental variant of the distemper virus which reachedBritainin dogs smuggled in by troops returning from the war, just as rabies was introduced after the First World War. Once established it flourished in a canine population which had no immunity to this particular strain.
Meantime, however, progress had been made in the method of vaccine production. Foxes, like dogs, suffer severely from distemper, and in fox farms distemper is a serious problem. Researches carried out on fox distemper virus showed that, after repeated passages through ferrets, this virus gradually lost its virulence for foxes. Somewhat similar results were obtained when dog distemper virus was repeatedly passed through ferrets, but this modified virus still produced symptoms of sorts, particularly in dogs which were not kept under optimum conditions. Because of this it was not considered suitable for making a vaccine for unrestricted use.
Following the lead given by studies of the yellow fever virus which I shall discuss later, dog distemper virus was successfully cultured in fertilised hen's eggs, and after a number of passages in this unnatural host became attenuated and lost its virulence, though retaining its antigenic properties, that is, its ability to immunise. This was a notable advance. The attenuated virus, grown in fertilised eggs, was prepared as a vaccine, and stored in freeze-dried form. Administered in a single dose, it conferred solid immunity. This single-dose technique was, of course, much more convenient than the original two-dose method. And there was an additional and unexpected bonus. It also conferred immunity against hard-pad virus.
I cannot quote any massive statistical figures to demonstrate the benefits that have accrued from the use of distemper vaccine, but. slightly altering the wording of a famous inscription, I might say: "* If you would see what has been accomplished, look around". Distemper is no longer a menace in kennels; dogs can safely be sent for exhibition at Crufts and elsewhere; and. most important of all, the innumerable owners of dogs throughout the country are relieved of all anxiety on the score of this once dreaded disease.
The next subject I have selected is yellow fever, and here, in terms of life-saving, we have a spectacular achievement. To most of the present generation yellow fever is little more than a name, perhaps associated in the minds of those who have to travel through certain regions in the tropics with another of those tiresome compulsory inoculations. Until the early years of this century, however, yellow fever was one of the important killing diseases in the world, occurring in vast explosive epidemics which in the course of a few weeks could decimate the cities in which it appeared. The early symptoms are those of an acute febrile illness, with rigors, headaches, loin pains and perhaps some nausea
*"If you would see his monument, look around". To Sir Christopher Wren. Inscription over the interior of the North Door inSt. Paul's Cathedral,London.
and vomiting. After three or four days there is a remission, and in many cases convalescence sets in at this stage, with rapid recovery. In other cases there is an early recurrence of symptoms with developing jaundice, multiple haemorrhages, giving in most cases the characteristic black vomit, kidney and cardiac involvement, and death. The mortality rate varies. It may be as low as 5%, or may reach 30% to 40%. Based in the CaribbeanIslandsand adjoining parts of Central and South America, yellow fever was carried far and wide in ships sailing from this endemic area. Spainand Portugalpaid a high tribute for the trading monopoly they once enjoyed with the South American continent: Cadiz, Malaga, Seville, Lisbon, Cartagena, Barcelona—to mention but a few—were among the cities which suffered. As an example. 6,000 people died in Lisborrin 1857 during a short but explosive outbreak. Nor were the ports of the North American continent in the Gulf of Mexicoand along the Atlantic seaboard any less vulnerable. In New Orleansin 1853 just under 8,000 died in an epidemic in a population of about 30,000, while in Memphisin 1879 over 5,000 deaths occurred in some 17,000 reported cases. These are but a few isolated instances— many similar disasters could be quoted. Even Britainwas briefly visited, when in 1865, carried by the sailing ship Hecla fromCuba, yellow fever was introduced intoSwansea and caused some 13 deaths and an undetermined number of nonfatal cases among the dock-workers, the size of the outbreak being limited by the relatively unsuitable environmental conditions.
The conquest of the disease, for conquest it is, falls into two distinct phases: the first, the incrimination of the mosquito as the vector of the disease, the second, the isolation of the virus and the preparation of a safe and effective vaccine.
The first of these phases is packed with drama. During the latter part of the 19th century there was in Havanaa very remarkable medical practitioner called Carlos Juan Finlay, who became intensely interested in the epidemiology of yellow fever. As a result of his studies, his ideas, by 1881, were sufficiently crystallised to enable him to read before a scientific society a paper entitled "The mosquito hypothetically considered as the agent in the transmission of Yellow Fever". He even suggested that the mosquito in question was *Stegomyia fasciata. The conception that a mosquito could transmit disease was revolutionary. It anticipated by a decade Manson's dis-
*Now known as Aedes aegypli.
covery that another species of mosquito is the vector of the worm which causes elephantiasis and, of course, Ross's discovery, in 1897, that anopheline mosquitoes transmit malaria. Needless to say it was ridiculed, particularly as various research workers from high places were intent on discovering a bacterium as the etiological agent. Nothing daunted. Finlay pursued his investigations, and even went so far as to test his theory by endeavouring to transmit the infection to volunteers (Spanish soldiers and Jesuit priests) by the bites of mosquitoes which had been fed on patients suffering from yellow fever. For reasons which will be made clear later, his results were equivocal. In 1894, before a Convention of Hygiene and Demography held in Budapest, he read another paper in which he said that, to prevent yellow fever, mosquitoes should be kept from biting patients suffering from the disease, that "contaminated" mosquitoes should be destroyed, as mosquitoes fed on yellow fever patients may remain alive up to 40 days, and that preventive measures should be maintained until that period is passed, as a contaminated mosquito remains infective during its lifetime. These recommendations are in complete accord with our present-day knowledge of the transmission of yellow fever. But he remained unheeded.
Meanwhile various commissions and individual research workers were applying themselves to solving the problem of the etiology of the disease, but without success. An Italian worker, Sanarelli, isolated a bacillus from the black vomit so characteristic of yellow fever, and this organism for a time was highly suspect. In 1900 the U.S. Army Medical Department sent to Cuba a team destined to make history, the so-called Walter Reed Commission, consisting of Major Walter Reed (in charge) and three civilian research workers. Agremont. Carroll and Lazear. As a first task they examined the possibility of transmission by direct contagion and investigated the claims of Sanarelli's bacillus. They soon were able to show conclusively that this organism was not the cause of the disease. At this point Walter Reed had to return toU.S.A.for a time. Carroll and Agremonte were left to pursue general lines of investigation, but Lazear, who had established a close liaison with Finlay. was given permission to explore Finlay's theory. With the latter's co-operation he hatched out a batch of mosquitoes and caused them to feed, first on a yellow fever patient, and later on healthy volunteers. Nothing happened. In no way discouraged, he repeated the experiment, but this time there were only two volunteers, his colleague Carroll, and an American soldier who was working with them, and who was one of those who jeered at Finlay's theory. Both developed yellow fever. The American soldier died, and Carroll narrowly escaped a similar fate. A few days later Lazear himself fell ill with yellow fever and died: whether through accidental infection or as the result of an unrecorded experiment is something which was never discovered. These dramatic events brought Walter Reed hurrying back, to attempt to solve a very difficult problem. Why had one experiment failed, and the other been so strikingly successful? Fortunately, Lazear had kept the most meticulous notes of all his experiments, and a careful study of these provided a possible explanation. In the unsuccessful experiment the mosquitoes had been fed on a patient then in the later stages of his illness, and had been allowed to bite the volunteers very shortly afterwards. In the successful experiment the mosquitoes were fed on a patient who had been ill for a short time, and were not made to bite the volunteers for about 14 days. Subsequent experiments confirmed that patients suffering from yellow fever are infective to mosquitoes only during the first few days of their illness, and that the agent has to undergo development in the mosquitoes for at least 12 days before they became capable of transmitting the disease. These facts, and also the fact that the agent was a filter-passing virus, were elucidated at dire risk to the lives of further human volunteers.
The confirmation of Finlay's hypothesis produced immediate results. Stegomyia is a "domestic" mosquito which breeds in casual collections of water around human dwellings, and is comparatively easy to eradicate. In a very short timeHavana was "cleaned up", and yellow fever disappeared. Good results were achieved in many other large cities, particular attention being paid to seaports in the endemic zone, so that major shipborne epidemics no longer occurred. However, because of the enormous extent of the country in which the disease was endemic, in particular the countries in the northern part of South America, it was long before satisfactory control was established everywhere, and even a quarter of a century later outbreaks, some of which were of considerable magnitude, were still occasionally occurring in some of the larger cities.
As time went on, too, it became obvious that the whole story of yellow fever epidemiology had not been revealed. Outbreaks, some small, some quite extensive, were notified from locations where there were no Stegomyia fasciata. By 1938 there was considerable evidence to incriminate a mosquito of the genus Haemagogus as the vector, but much difficulty was encountered in breeding this mosquito in the laboratory, and no convincing proof was forthcoming. In 1940 a fortunate incident occurred. Some members of a team investigating the yellow fever problem in the jungle stopped to watch woodmen felling a tall tree. As it crashed down large numbers of mosquitoes, which proved to be Haemogogus were shaken from the top branches and promptly attacked the workmen and the members of the yellow fever team. Clearly this mosquito had its normal habitat in the forest canopy. With this information, methods of breeding Haemogogm in captivity were devised, and in due course it was shown to be a potential vector of the yellow fever virus. The problem was finally solved when virus was isolated from marmosets which, like Haemagogus, lived in the forest canopy. Here was the source of outbreaks of silvan yellow fever. In the vast and sparsely inhabited forests ofSouth America eradication of this source of yellow fever is impracticable, but by adopting measures to detect cases of the disease at the earliest moment and to contain small local outbreaks as they occur, it has been kept more or less under control.
I must now return to phase 2, the virus and the vaccine. Such information as was available about the virus in those early stages had been obtained at the cost of some human lives and grave risk to others who volunteered as subjects for experiment. Clearly some susceptible laboratory animal must be found as a first step in its isolation. However, for years all attempts to find such an animal were unsuccessful, and the research hung fire. In 1925 the International Health Division of the Rockefeller Foundation sent a strong team, known as the West African Yellow Fever Commission, to West Africato study yellow fever as it occurs in that country, and to make further attempts to isolate the virus. They soon succeeded in locating some cases of yellow fever, but were no more successful than others had been in the past in transmitting the infection to any laboratory animal or any of the local monkeys. A suggestion was then made that, as yellow fever does not occur in India, rhesus monkeys from that country might have no natural immunity and be susceptible. Rhesus monkeys were accordingly imported from India, and success was immediate. One of these monkeys, inoculated with blood from a youth called Asibi, who was suffering from a mild attack of yellow fever, developed typical symptoms, and the infection was readily passed on to other monkeys of this species. But again at a cost to those handling this dangerous material, Adrian Stokes, a brilliant microbiologist seconded to the commission from Guy's Hospital because of his special knowledge of leptospirosis, was accidentally infected and died. Noguchi, an American bacteriologist, met a similar fate (the reason for the presence of Stokes and Noguchi in this team is a most intriguing story which I have no time to relate). Finally Young, who performed an autopsy on Noguchi and presumably infected himself in the process, also sickened and died. Three deaths. However, the necessary "breakthrough" had been accomplished, and from this point onwards progress was uninterrupted if slow. The research was transferred to a laboratory in the Rockefeller Institute in New York. It was found that mice could be infected by the intracerebral injection of virus (obtained in the first instance from the monkey) and this enabled a test to be devised by which the past incidence of yellow fever in a community could be determined. Next, from infected mouse brain, in vitro cultures were obtained in a medium of minced mouse embryo, and, a further step ahead, in medium of minced chick embryo. In such cultures the neurotropic properties of the virus were maintained, and a successful attempt to rid the virus of these properties was made by growing it in a medium of minced chick embryos from which the brain and spinal cord had been removed. Several lines of virus were maintained by regular subculture in the particular medium used for each line. In the line grown on minced chick embryo minus brain and cord, somewhere between the 89th and 114th subculture, mutation occurred—a gift from the gods. When routinely tested at the 89th subculture the virus was in possession of its original virulence: at the 114th subculture, it was practically non-virulent, though still in possession of its antigenic, immunity producing properties. And so it has remained, this so-called 17D strain, to this day. Attempts to repeat this process of mutation have all been unsuccessful. From 17D have come the millions of doses of attenuated virus vaccine which have been used, with complete success, in the intervening years. The immunity conferred by the 17D vaccine is solid. At first it was thought to last four years, then six years, now the figure is placed at 10 years, but there is little reasonable doubt that, like the immunity conferred by an attack of yellow fever, it is lifelong. What has the use of this vaccine achieved? Ft has of course afforded complete protection to those who have to live in or to travel through endemic areas. More important, it has eliminated the risk of yellow fever being imported into uninfected territories. Certain mysteries in the epidemiology of yellow fever remain unsolved. Though endemic inWest Africa, it has never made its way to the East coast. Thus in the days when the only method of getting from Africa to tropical Asia, and particularly India, was by sea, there was never any danger of yellow fever finding its way to these territories, where conditions for its spread appear to be ideal. This has been completely altered by the advent of air travel, which makes it theoretically possible for a person to be infected in West Africa and landed inIndia before symptoms develop. Before an effective vaccine was available this possibility caused much concern. Nowadays all aerodromes in the yellow fever zone are maintained in a mosquito-free state. Every aeroplane touching down in one of these aerodromes is sprayed with insecticide, and all passengers are required to produce a valid certificate of vaccination against yellow fever. So far these precautions have been successful; and what could have been a major catastrophe has been averted.
Two instances may be quoted which emphasize the protective value of the vaccine. In the late 1940's, an epidemic of yellow fever broke out among howler and spider monkeys in the tropical forests ofCentral America. It spread slowly northwards through the Isthmus, and finally about 1956 burnt itself out in the forests ofBrazil. The monkeys of these areas were all but exterminated, but because of the inoculation of the human population along this route only a few isolated human cases occurred.
By way of contrast, inEthiopiawhere there was no inoculation, and where the disease was not at first identified, an outbreak of yellow fever occurred between 1960 and 1962 in which there were an estimated 200,000 cases with 30,000 deaths.
Of the diseases I have discussed, none is in any sense man-made, by which I mean the result of overcrowding, or insanitary working conditions, or inadequate diet or other human errors. They were, and still are, part of the unending and ruthless challenge of untamed nature, and remain a menace to the well-being of men and domestic animals. It is no exaggeration to say that striking success has been achieved in meeting and overcoming this challenge. In reaching this goal, innumerable experiments have been carried out on various animals—dogs, rabbits, ferrets, monkeys, mice, and others, including in some cases and with occasional fatal results, man. In terms of contemporary knowledge could this goal have been achieved without recourse to animal experiment? The answer is a firm negative. Whether or not, if the necessary techniques had been known, viruses could have been isolated in (live) in vitro preparations of cells or tissues or organs direct from the naturally infected animal, the nature of any virus obtained in this way could have been confirmed with certainty only by reversing the procedure and testing the capacity of the cultured virus to reproduce the disease in a susceptible animal (human or otherwise). Further, the safety and potency of a virus vaccine must needs be tested by animal inoculation. In the case of an attentuated virus vaccine, elaborate tests at regular intervals must be carried out on the seed virus from which each batch of vaccine is prepared, and samples from every completed batch must be similarly tested to ensure that the virus has not undergone a back-mutation to virulence. This is particularly necessary in the case of yellow fever vaccine where any reversion to virulence could have disastrous consequences.
In parenthesis, it is interesting to note that an off-shoot of the antivivisection group is clamouring for animal experiment to be replaced by techniques based on cell-culture, tissue-culture and organ culture, and by tests carried out on primitive creatures such as protozoa. In doing so they are giving the impression that these methods have been devised by their colleagues and are being neglected by those whom they call '"vivisectors". In fact, everything worthwhile that is known of these techniques stems from the observation and work of those self-same vivisectors, who for many reasons are just as anxious to by-pass animal experiment as are their detractors, and who can be relied on to pursue investigations along these lines in an intelligent and critical fashion.
The path which the research worker must follow is clear. Pasteur's reaction to animal experiment, as described by Roux, has been shared by many others, and in particular by animal-lovers who have followed a research career. However, as Pasteur discovered, reality can, and usually does, fall far short of the picture painted by an active but uninformed imagination. This absolves no one from adopting every possible means of reducing to a minimum any discomfort or pain caused by an experimental procedure, and one can say that in this country the exercise of such care is the rule. But if progress in the prevention and cure of disease is to be maintained, there is, within our present knowledge, no alternative to animal experiment, however distasteful this may be. The benefits which have accrued to man, and, let it not be forgotten, to his domestic animals and pets, must, in this vexed question, be paramount.
The Chairman called upon Professor A. D. Macdonald to pass a vote of thanks to the lecturer:
"Mr. Chairman, Ladies and Gentlemen, My chairman and I have regularly invited an elder statesman of R.D.S. to propose a vote of thanks to our Paget Memorial Lecturer. But I felt 1 would like to try myself on this occasion, since during the greater part of his Chairmanship of Council and my Secretaryship we have worked together.
Sir John Boyd has been utterly unsparing of himself in his service to our Society and particularly during the sessions of the Littlewood Committee and after the publication of its report he devoted much of his time and energy to our cause. And since then he has produced this unique justification of the work of the experimentalist which constituted the bulk of the 1969 issue of Conquest 'Experiments on Animals and the Littlewood Report’. My personal sense of debt is. I am sure, shared by all who have studied this pamphlet.
And now we have this admirable lecture for which to return thanks. When I conveyed Council's invitation to deliver it to Sir John it was with some anxiety if not apprehension for he had just suffered a great personal tragedy. Characteristically, he did not allow that or the fact of his recent retirement with the chance to devote himself to his golf and to his garden to come between his leisure and what he accepted as his duty. I hope the garden has flourished in this fine late summer. I hope all his drives have finished in the middle of the fairways and all his putts in the middle of the holes. Last time I played—with a son whose No. 3 irons are longer than my best woods f started to deplore my lost length but was told ‘Remember your age’ and that 'when you can afford to lose golf balls you can't hit them that far'. May none of these sadnesses come to our lecturer! He has provided us almost with a new charter which will live on and inspire us long after its publication. In our gratitude, our respect, our admiration and affection for our past chairman we are a very united Society. Our continued best wishes. Sir John, for your future".