Chapter XV: Protective Vaccinations (2)

As the basis of all the new methods for vaccinating pigs against erysipelas is the preparation of serums capable of preventing the pathogenic effect of the bacilli, the question of the determination of the protective power of these serums comes to be one of considerable importance. At first one was satisfied with certain approximate estimations, but later the necessity was felt of having a more exact measurement. Leclainche is persuaded that of all the laboratory animals capable of being used for these experiments the pigeon is the only one that can usefully fulfil this rôle; very susceptible to the passage virus, it is killed by the bacillus after a regular incubation and invasion period, and the chronic form of the erysipelas, so troublesome in the rabbit and even in the pig, is met with in the pigeon in very exceptional cases only. Leclainche commenced his experiments by inoculating into the pectoral muscles of the pigeon mixtures of serum and virulent cultures. The pigeon received 1 c.c. of a culture of a passage virus mixed with variable quantities of serum. The serum is ready for use in the vaccination of pigs when the pigeons resist the injection of a mixture of ½ a c.c. of serum with 1 c.c. of a virus which kills the control pigeons in 60 to 72 hours.

At the Frankfort Institute of Experimental Therapeutics another method of testing devised by Marx[796] is used. In it injections, below the skin of a series of grey mice, are made of progressively increasing doses of the serum the strength of which it is desired to determine. Twenty-four hours later a virulent culture of the bacillus of swine erysipelas is introduced into the peritoneal cavity of the same mice. The virus is so chosen that the control mice die in about 72 hours. Marx finds that this method gives results which are much more constant and exact than any other; this opinion is confirmed at Höchst, the largest factory of serums in Germany.

VIII. _Vaccinations against bovine pleuropneumonia._ This infective disease is one of the most dreaded scourges of bovine animals. Very contagious, it has spread from central Europe not only into all the other countries of the European continent, but into Africa, America, and almost every quarter of the globe. The virus of this disease was discovered in the serous exudation of hepatised lungs long before the microbiological period of the Medical Sciences had begun.

[Sidenote: [500]]

Dr Willems of Harselt, who made an experimental investigation, remarkable for the time at which it was carried out (more than half a century ago), demonstrated at once the great virulence of the pulmonary serous fluid; he found also that the effects of the inoculation of the virus varied much according to the seat of inoculation. When made into the trunk, the neck, or the shoulders, the inoculations are usually fatal; at the periphery, the lower part of the limbs, at the extremity of the ears or of the tail, the inoculation ordinarily produces merely an inflammatory tumefaction of small extent, which is absorbed in a few weeks; after this the animal is refractory to the natural disease. Willems concluded from this that we may vaccinate against pleuropneumonia by inoculating the virulent serous fluid of the lung into the tail. Willems’ method of inoculation became a part of current practice 50 years ago.

For the carrying out of a large number of vaccinations it is necessary to have at one’s disposal an adequate quantity of virus; it was therefore to meet this requirement that researches were first carried out. The serous fluid was withdrawn from the hepatised lungs of animals that had succumbed to the disease and was inoculated into normal Bovidae as soon as possible, so as to avoid contamination of the fluid. In fact this pulmonary serous fluid often contains foreign germs capable of multiplying rapidly so that it putrefies very quickly. Pasteur showed that it was possible to remedy these drawbacks by a very simple method by which he could obtain a large quantity of rigorously pure virus. All that is necessary is to inoculate a little of the pleuropneumonic virus below the skin of a weaned calf, behind the shoulder. At the seat of inoculation there is an abundant exudation of virulent serous fluid into the cellular tissue, from which we are enabled to collect large quantities of pure virus.

In some countries, as in Germany and in Australia, institutions have been founded for the production by this method of the virulent serous fluid necessary for these inoculations.

The virus should be inoculated into the tip of the tail of animals that it is desired to immunise, because the temperature in this situation is relatively low and the connective tissue is dense and not very abundant. The inoculation is made with a lancet or a Pravaz syringe. The vaccination is generally borne well, in spite of the reaction phenomena which are manifested about two weeks after the introduction of the virus. At that time a febrile condition is set up and a swelling manifests itself at the point of inoculation, which, however, soon retrogresses and then disappears.

[Sidenote: [501]]

The immunity conferred by Willems’ method is substantial and lasting (for one or two years and even longer); this explains its great success in the hands of breeders and veterinarians. Accidents following its use are rare, and the mortality does not exceed 1 per cent.

In spite of all these advantages a new method was still desirable, a method which would allow of the preparation of large quantities of virus of a suitable and uniform activity under conditions of irreproachable purity. Thanks to the discovery of the micro-organism of pleuropneumonia which we owe to Nocard and Roux[797] this object has been achieved. With the collaboration of Borrel, Salimbeni, and Dujardin-Beaumetz, they succeeded in demonstrating and isolating this micro-organism, the smallest of all known living organisms. The first steps in these researches were very laborious, but later the organism of pleuropneumonia was cultivated on fluid and solid media: Martin’s broth (prepared with pigs’ stomachs) or agar with the addition of a certain quantity (about 5%) of fresh ox serum. The serum-broth, sown with pure pneumonic serous fluid, gives only a moderate growth, which becomes only slightly turbid and contains micro-organisms so small that it is impossible to distinguish them individually. They can be made out only when massed together in irregular clumps. The minuteness of this micro-organism is evidenced by the ease with which it passes through a Berkefeld filter, and even through certain Chamberland candles (F). This feature enables us to obtain the pure virus easily, a fact very important in connection with the isolation of the micro-organism.

[Sidenote: [502]]

Once in possession of pure cultures of the micro-organism of pleuropneumonia, Nocard and Roux attempted to make use of it in practical vaccination. They showed that the organism separated by them is capable of producing typical pleuropneumonia when it is inoculated into the appropriate regions of the body of bovine animals. But when inoculated subcutaneously or into the skin of the tail, it produces merely a mild and transient disease which confers an immunity quite as effectual as that set up by the inoculation of the virulent serous fluid. It may be readily understood that, under these conditions, pure cultures may be much more serviceably employed in the practice of vaccination than can Willems’ virus from the fact that it is easy to obtain large quantities of absolutely pure cultures. It is easy to predict that the new method will soon replace the old one, very great as are the services the latter has rendered to agriculture. Up to the present, vaccinations with pure cultures have been made in several districts in France with very favourable results. The Pasteur Institute and the Veterinary School at Alfort have already distributed to veterinary surgeons more than 5,000 vaccinal doses of culture; the protective action of these inoculations has been at least equal to that of the inoculations by Willems’ method and the resulting accidents have been reduced in the proportion of 20 to 1[798].

The serum of animals hyperimmunised against pleuropneumonia possesses a very distinct _protective_ action, but too little marked and of too short duration to be of any use in practice; it has also a _curative_ action arresting the invading march of a pleuropneumonic congestion; but here it is necessary to intervene early, before the appearance of fever, and to inject large quantities of serum.

The inoculation of a mixture of virus and serum produces no congestion; but it does not confer any immunity; the animal remains just as susceptible as the control to the inoculation of the pure virus.

[Sidenote: [503]]

IX. _Vaccinations against typhoid fever._ In the preceding sections I have treated more especially of the vaccination of domestic animals against several infective diseases. The information collected on this subject is marked by its great exactness, as it is easy to apply to animals the most rigorous experimental method. In the case of the human subject this is not such an easy matter. As it is impossible to submit him to experimental proof we are obliged to be satisfied with observation, controlled by statistical data. The experience of more than 100 years has, however, been sufficient to demonstrate the great utility of vaccinations against small-pox with the virus of cow-pox which is innocuous for the human subject. In the case of antirabic vaccinations we have to deal with injections into the human subject, first of weakened viruses and then of virulent viruses. Here, however, it is a question of the preservation of the already infected human organism, which, very often, only comes under treatment during the incubation stage of rabies. One can readily understand the hesitation to inoculate even weakened viruses into the human subject, especially when we are not dealing with altogether exceptional cases such as we have in the protection against rabies. We have, therefore, but few examples in which the methods of vaccination by micro-organisms have been applied to man. Such injections were first tried by Ferran[799] against Asiatic cholera. Having succeeded in vaccinating guinea-pigs against experimental cholera septicaemia, the Spanish investigator attempted to inoculate cholera vibrios into the subcutaneous tissue of man, hoping thus to vaccinate him against true cholera. In this way he was able to demonstrate that the subcutaneous injection of living vibrios never sets up symptoms of cholera. The injection is followed by a general reaction in the form of fever, pains in the back and inflammation at the point of inoculation, in a word, transient phenomena of little gravity. Encouraged by these initial results Ferran, profiting by the outbreak of cholera in the province of Valentia, injected into more than 20,000 persons living cultures of Koch’s vibrio. The results published by him did not, however, furnish any real proof of the possibility of conferring immunity against intestinal cholera by means of subcutaneous injections. Later Haffkine[800] modified Ferran’s primitive method somewhat, and instead of living vibrios he injected vibrionic cultures killed by heat or by antiseptics. During the cholera epidemic of 1892 and 1893 he tried the inoculation of these killed vibrios into man, with the object of vaccinating against Asiatic cholera. Later he went to Calcutta in order to try his method on a large scale. He was there enabled to inoculate a great number of persons, and the statistics which he collected appeared to him to be favourable.

[Sidenote: [504]]

But studies on the pathogenesis of Asiatic cholera shook the foundations of Ferran’s method. The injections of vibrios, living or killed, were found quite capable of vaccinating animals against vibrionic peritonitis and septicaemia, but they appear to exert no influence whatever against poisoning by the cholera toxin. When it had been learnt how to set up true intestinal cholera in young rabbits Ferran’s and other similar methods of vaccination were used in vain to prevent the incidence of this disease, which is very similar to Asiatic cholera of man. An experiment[801] made at the Pasteur Institute in Paris upon two persons vaccinated by Haffkine, showed that they were not protected against the choleriform diarrhoea set up by the ingestion of the cholera vibrios. A third person, who had never been “vaccinated” and who served as “control,” after the ingestion of the same cholera culture, behaved exactly as did the other two.

From all these data the conclusion was drawn that in order to prevent intestinal cholera it is necessary to use not cultures of vibrios, living or dead, but antitoxic serums. In fact, the majority of young rabbits vaccinated with these serums and afterwards submitted to infection by the cholera virus through the mouth were found to be vaccinated against intestinal cholera. It has not been possible, as yet, to apply this method to man, hence we are unable to give a decided opinion. Moreover, as the methods based on Ferran’s principle have now been abandoned I have not deemed it necessary to devote a special section to anticholera vaccinations. I could not, however, pass it by in silence, since the attempts to vaccinate man against cholera have led to the trial of a similar method against typhoid fever.

Pfeiffer and Kolle[802] were the first to inoculate man with typhoid coccobacilli sterilised by heat. They observed that these injections caused fever, pretty violent pains in the back accompanied by vertigo, shivering and pain at the point of inoculation, without, however, being in any way serious to health. At the same time they found that the blood serum of inoculated persons acquired a very marked protective power (for guinea-pigs injected into the peritoneal cavity with lethal doses of typhoid cultures) quite comparable to the properties discovered by them in the serum of persons who had recovered from typhoid fever. Pfeiffer and Kolle believed that they thus had a proof of the refractory condition of the individuals whom they had submitted to these injections.

[Sidenote: [505]]

These experiments were continued by Wright, Professor of Pathology at Netley, and it is owing to his unwearied efforts that science finds herself in possession of very important evidence on the subject of protective inoculations against typhoid fever in man. According to a verbal communication made to me by Wright, he has up to the present distributed more than 300,000 doses of his antityphoid vaccine. This vaccine he prepared in the following way[803]. The typhoid coccobacillus is sown in carefully neutralised broth containing 1% of peptone. The flasks of culture are kept in the incubator at about 37° C. for two or three weeks, after which their contents are transferred to large flasks in order to be submitted to a temperature of 60° C. This temperature is quite sufficient to kill all the coccobacilli, but for greater surety Wright added to his cultures one-tenth of their volume of a 5% solution of carbolic acid or of lysol. The vaccine, thus prepared, is examined as to its toxicity for the guinea-pig by means of subcutaneous injections. Wright injects into man a dose of vaccine which is sufficient to kill 100 grammes of guinea-pig (of the weight of 250 to 300 grammes). This dose often amounts to half a cubic centimetre, but it may have to be increased to 1 c.c. and even 1·5 c.c.

The inoculations are made below the skin of the flank or in the shoulder. They are followed by a rise of temperature which commences as early as two or three hours after the injection. This fever is accompanied by pains in the back, nausea, and want of appetite. There may even be collapse; this led Wright to keep his patient in bed for some time after the vaccinal injection. Besides this reaction, there occurs, at the seat of inoculation, a swelling and redness, accompanied by pain; as a rule all these symptoms have disappeared by the end of 48 hours.

[Sidenote: [506]]

Wright convinced himself that the blood serum of individuals treated by his vaccine, at the end of a certain time acquires the property of agglutinating typhoid coccobacilli in a variable, but usually very marked degree. He even thought that this property might up to a certain point serve as the measure of the immunity acquired against typhoid fever. His own researches, however, showed him that this supposition could not be maintained, and that the agglutinative power, varying greatly in strength, might sometimes be absent where the immunity could not be denied. On the other hand, he clearly showed, especially by the experiments with serum collected at the period which precedes the relapses, that the agglutinative property might be highly developed, in spite of the absence of immunity. Wright then set himself to study the bactericidal property of the serum of individuals who had been injected with his vaccine. He devised a very ingenious method of gaining with a minimum loss of time some idea of the fluctuations of this power of the body fluids to kill the typhoid coccobacillus. In the first place he demonstrated that the bactericidal property is not at all parallel to the agglutinative power, and this has further confirmed him in his opinion that there may be no direct relation between it and acquired immunity. He has found further that the power of the blood serum to destroy the typhoid coccobacillus is very variable in persons vaccinated by his method. After injections of large quantities of these killed bacilli this power may even be diminished for a very long period. On the other hand, medium or small doses of the vaccine first set up a negative stage, during which the bactericidal property is very feeble, and later they bring about an increase of this property, often very marked. Wright does not think that the bactericidal power can serve as the measure of the immunity acquired by the vaccinated individuals, but he hopes that some day a method may be found suitable for the examination of the blood which will give us information as to the degree of immunity conferred by the antityphoid vaccination. For the present the only basis upon which we can form any opinion on this subject is furnished by statistics. Now we know that it is often very difficult to collect data that are sufficiently exact. Hence during the war in South Africa, where one-fifth of the English troops, that is to say about 50,000 persons, were submitted to vaccinations by Wright’s method, it is only in certain cases that the statistical information can be utilised. Many of the patients attacked by slight fevers are omitted from the statistics, because from the absence of a precise diagnosis it is not known whether they should come under the category of typhoid patients or not. In other cases the secondary complications divert the attention of the doctors and prevent the registration of a proper diagnosis.

[Sidenote: [507]]

Of the data collected amongst the English troops in South Africa, Wright considers that those which were collected during the siege of Ladysmith were the most exact, on account of the facility with which it was possible to study and register all the cases of typhoid fever under these conditions of complete isolation. Now it has been recognised that, amongst the vaccinated soldiers and officers, there occurred scarcely one-eighth as many cases of typhoid fever as occurred amongst the unvaccinated (1,499 cases in 10,529 unvaccinated, and 35 cases in 1,705 vaccinated). The mortality amongst the vaccinated was also very much lower. The difference to the credit of the vaccinations should in reality be even greater, for amongst the unvaccinated are counted many persons who having already had an attack of typhoid fever were not submitted to vaccination.

The testimony of the majority of the medical men who followed the results of Wright’s method closely is also favourable to the vaccinations. Thus Henry Cayley[804] reports that the staff of a Scotch Hospital of the Red Cross, almost all of whom (57 persons out of 61) had received two vaccinal inoculations, escaped typhoid fever, in spite of the numerous opportunities afforded for the contraction of the disease. This very favourable example is also instructive in that it testifies to the value of two consecutive vaccinations. In many other cases where one has had to be satisfied with a single protective inoculation the results were less brilliant. According to Howard Tooth, who made his observations at Bloemfontein, the vaccinations according to Wright’s method must be regarded as very useful.

Outside South Africa this method has been employed on a fairly large number of persons in British India, in Egypt, and in Cyprus. According to the earlier statements from India the incidence amongst the vaccinated persons was one-third that of the unvaccinated. The most recent statistics[805] show still more favourable results. Thus at Meerut the incidence amongst vaccinated persons from Oct. 1899 to Oct. 1900 was one-eleventh that of the unvaccinated (2 cases of typhoid fever in 360 vaccinated, and 11 cases of the same disease in 179 unvaccinated): the mortality (one case amongst the former, six amongst the latter) was less than one-twelfth that of the unvaccinated.

In Egypt and in Cyprus according to the statistics communicated to Dr Wright[806] by Col. Fawcett these vaccinations have given even better results. In 2,669 unvaccinated persons there occurred 68 cases of typhoid fever with 10 deaths, whilst amongst the 720 vaccinated there was only a single case of this disease, this single case succumbing. Here, however, we have to do with a patient who must have received the vaccinal inoculation during the period of incubation, the disease breaking out soon after the vaccination. This would represent in all the cases a morbidity only one-seventeenth as intense amongst the vaccinated.

[Sidenote: [508]]

A few isolated voices only have not pronounced in favour of the antityphoid vaccinations and their opinion is formulated in a very undecided fashion. Amongst the most important of these adversaries, if indeed we may term them such, must be cited Washbourn[807], on account of his experience in microbiology. Attached as a doctor to the Yeomanry Hospital at Deelfontein in South Africa, he witnessed many cases of typhoid fever and was greatly struck by the death of two persons amongst the vaccinated patients. But he himself confesses that it is as yet premature to judge Wright’s method, and in support of his sceptical attitude does not offer any other satisfactory observation.

Outside the English colonies vaccinations against typhoid fever have been tried in Russia by Wyssokowitch[808]. He inoculated 235 soldiers of a regiment encamped at Kiew, amongst whom an epidemic of typhoid fever had broken out. The vaccinations were carried out by means of cultures killed with carbolic acid. We are unable to judge of the efficacy of the method because the number of persons vaccinated was too small and the epidemic too limited. It may be noted, however, that amongst these individuals not one took typhoid fever, whilst amongst the unvaccinated three cases of the disease were registered.

The antityphoid vaccinations have as yet only a very short history, and it is, perhaps, premature to express any decided opinion on the matter. We may, however, consider the results already obtained as offering encouragement to continue our experiments. Everything, indeed, tends to a recognition of the utility of vaccinations by means of killed typhoid cultures. The statistics are as a rule good; the danger from the protective inoculation is _nil_ or quite trifling. With the exception of the discomfort of which we have spoken and which is transitory, no untoward result has ever been observed.

[Sidenote: [509]]

To all this must be added the fact that from the point of view of the pathogenesis of typhoid fever, all the probabilities point in favour of the vaccinations. Whilst in Asiatic cholera we have to deal with an intoxication, from the alimentary canal, an intoxication set up by vibrionic products, against which the subcutaneous inoculation of micro-organisms can not be effective, in typhoid fever we have to do with a real infection. The micro-organism, although developed at first in the small intestine, becomes generalised throughout the system. Thanks to improved methods it can always, or almost always, be found in the blood of the patient, and its constant localisation in the spleen furnishes a real evidence of this. Under these conditions it is quite natural to suppose that everything which is able to prevent the penetration of the typhoid coccobacillus into the blood and the internal organs ought at the same time to contribute to the protection of the individual.

We are fully aware that science has not yet said its final word upon this question. We are coming more and more to the conclusion that it is necessary to make two injections instead of one. It is possible that we may have recourse to certain improvements of the method by combining with it the injections of antityphoid serums as a protective measure. The near future will doubtless bring us the solution of these very important questions.

X. _Vaccinations against human plague._ Plague, which for so long was looked upon as the greatest scourge of humanity, has until recently remained almost unknown from the scientific point of view. But from the moment that it became possible to apply to its study the immense advances realised by microbiology the thick veil which had hidden its nature fell at a single stroke and science found itself in possession of effective means of fighting against it. Amongst these means one of the most important is protective vaccination.

[Sidenote: [510]]

When the last pandemic of plague broke out in Bombay and in the East Indies in general, Haffkine was there engaged in applying his method of vaccination against Asiatic cholera of which we have spoken in the preceding section. Well acquainted with the results of the bacteriological researches made on bubonic plague by Kitasato, and especially by Yersin, he, in 1896, began to study this disease. After the discovery made by Yersin, Borrel, and Calmette[809], who showed that animals susceptible to human plague could be easily vaccinated against the micro-organism which gives rise to it, Haffkine[810] endeavoured to find a practical method for the vaccination of man. He set up a laboratory at Bombay and, after some preliminary experiments on rabbits, he commenced to inject human beings with pure cultures of the plague coccobacillus. From 1897 up to the present he was able to vaccinate a very large number of individuals, and the results obtained have encouraged him to continue the application of his method. The principle of this method is that which had guided him in the preparation of anticholera vaccines and which is used for the vaccines against typhoid fever. It consists in the employment of pure cultures of the specific organism killed by heat. The cultures are grown in large flasks containing peptonised broth and sown with a small quantity of the plague coccobacilli. A little sterile butter or cocoanut oil is poured on the surface of the fluid. Under these conditions the organism grows abundantly and produces growths which hang down into the fluid, reminding us of the stalactites in a grotto. This mode of development forms one of the most typical characters of the micro-organism of human plague. The culture flasks are kept at a temperature of about 30° C. for five to six weeks, at the end of which period a large number of the bodies of the micro-organisms have fallen to the bottom of the flask, allowing much of their toxic contents to escape. The fatty layer on the surface favours a surface development of the coccobacilli, the number of micro-organisms in a flask being thus greatly increased.

After growing for 35 to 42 days under these conditions the cultures are heated at 65°–70° C. for from one to three hours with the object of killing all the micro-organisms and so rendering their injection innocuous. To make sure of the effectiveness of this heating care is taken to remove a small portion of the fluid and to sow it in a suitable medium. Should this medium remain sterile the vaccine may be used. Into adult men it is injected in a dose of 3 c.c., whilst women, children, and adolescents receive 2–2·5 c.c., into the subcutaneous tissue.

[Sidenote: [511]]

Some hours after the injection of the vaccine the temperature rises above normal, reaching 38°·5 to 39° C., and sometimes even 40°–40°·5 C. This febrile condition lasts from 15 to 48 hours. It is soon accompanied by pain, redness, and swelling at the point of inoculation. These symptoms persist for from three to five days. The _malaise_ which follows the vaccinations is sometimes very uncomfortable or even painful, but never serious. Only in exceptional cases is the formation of abscesses observed, and this is due, undoubtedly, to contamination of the vaccines by foreign micro-organisms. The English Commission sent to India to study plague found other micro-organisms than the plague coccobacilli fairly frequently in the vaccine culture flasks, but, with very rare exceptions, these micro-organisms were found to be innocuous. By rigorously following the rules to be observed in making pure cultures it should not be difficult to avoid this complication.

Haffkine used every effort to induce his patients to be vaccinated a second time, being justly persuaded that two injections are capable of ensuring a more certain and more stable immunity than is a single injection.

[Sidenote: [512]]

From what moment immunity may be considered to be acquired has been a matter for great discussion. From very numerous experiments upon animals of various species, as well as many observations on man, it is now agreed that a period of several days (5–8) from the injection of the vaccine is required before immunity is manifested. It is for this reason that cases of plague which have broken out before this period has elapsed cannot be looked upon as contraindicating the efficacy of the method.

A large amount of evidence, coming from persons who have made their observations on the spot, is almost unanimous in endorsing the fact that Haffkine’s vaccination protects man against plague. It is often difficult to compile exact statistics in surroundings where so many factors contribute to deceive even the careful observer. In spite of this a certain amount of evidence has been collected which may be accepted as affording us fairly satisfactory information. One of the best groups of statistics was that collected at Damaun, a Portuguese possession in India, into which plague was imported from Bombay in 1897, and where a large number of vaccinations were carried out. From the report of Haffkine and Lyons[811], in a population of 8230 persons, rather more than one-fourth (2197) were vaccinated, the greater majority (6033) remaining uninoculated. Amongst the former only 36 died from plague, which corresponds to 1·6 per cent.; whilst amongst the unvaccinated persons the disease carried off 1482 persons or 24·6 per cent. Vaccination, therefore, according to these statistics, must have brought down the mortality to one-fifteenth. The German Commission[812], two members of which, Koch and Gaffky, went to Damaun to be present at the vaccinations and to observe their efficacy, pronounced in favour of Haffkine’s method. The English Commission[813] made reservations and criticised the statistics of Haffkine and Lyons (who amongst others attribute all the cases of deaths that occurred amongst the unvaccinated to plague), but in the end this Commission also recognised the utility of the vaccinations at Damaun.

The data collected with regard to the vaccinations at Undhera, Hubli, and several other places in British India confirm the results obtained at Damaun. The statistics collected at these localities are certainly open to criticism, but the result as a whole is none the less encouraging as regards this method of vaccination. According to the conclusions of the English Commission the “inoculations had a considerable effect in warding off plague attacks from the inoculated.... The protection afforded by inoculation seems, however, never to be absolute[814].” We do not, as yet, know the duration of the immunity produced by Haffkine’s vaccinations; it cannot be very long to judge from the experiments on animals, but it may last for several weeks, probably even for months.

The vaccinations by killed cultures may be especially useful when it is a question of limiting the extension of an epidemic that is already established. The ease with which the vaccine can be prepared renders it possible to obtain very large quantities of it in a short time, with which it is possible to immunise the entire population of towns or districts. But, as the immunity by this method requires several days for its development and as the injections of micro-organisms, even when killed, may be very injurious during the incubation period of plague or immediately before the infection, it is necessary to limit the vaccinations to persons who are not in intimate contact with the sick, or who are, from the beginning, exposed to infection[815].

[Sidenote: [513]]

Lustig and Galeotti[816] have described another method of preparing antiplague vaccine which can be utilised where it is of importance to obtain a large quantity of vaccine in a very short time. Instead of allowing the cultures to grow for five or six weeks as required by Haffkine’s method, the Italian observers make use of cultures on agar which have grown for two days only. The micro-organisms, removed from the surface of the agar, are treated with a weak solution of potash (0·75%–1%) which dissolves the bodies of the coccobacilli. This phenomenon has sometimes occurred by the end of twenty minutes, but it often requires an hour or more. The contact of the micro-organisms with the alkali must never exceed three hours. The viscous mass thus obtained is then treated with acetic acid, when a precipitate is thrown down. This precipitate, after being washed, is used for the vaccinations. When injected in large quantities into animals, Lustig and Galeotti’s product sets up necrosis, but a weak dose is well borne and confers immunity against plague. In man it is sufficient to inject two or three milligrammes of this substance diluted with water. The vaccinal nuclein of the Italian observers has been but little employed for the immunisation of man in India, but it is largely used in this country for the inoculation of horses from which to obtain an antiplague serum.

[Sidenote: [514]]

The serotherapeutics against human plague were inaugurated by the researches of Yersin, Borrel, and Calmette (_l.c._), who demonstrated that animals susceptible to the plague bacillus can be vaccinated and even cured of experimental plague. The preparation of antiplague serum has since been energetically pursued under Roux’s direction at the Pasteur Institute. After several trials, some of which were very encouraging, others, on the contrary, somewhat unfavourable, they succeeded in obtaining a serum which is capable of curing plague after it has broken out and has become grave. As in this treatise we intentionally leave aside everything connected with healing we shall speak only of the antiplague serum as a protective agent.

Whilst vaccinations by killed plague cultures have been practised principally in the East Indies, the immunisation with antiplague serum has been employed in Europe, especially at the time of the epidemics of Oporto in 1899 and of Glasgow in 1900. In all these cases use was made of the serum from the Pasteur Institute, up to the present the most active of all those prepared. It is a serum obtained from horses treated for a long period with cultures of the plague bacillus and with the toxin of the same organism. Treatment is begun by injecting plague coccobacilli killed by heat (70° C.). These injections are made into the veins, with the object of avoiding the local lesions which are observed after the subcutaneous introduction of micro-organisms. When the horses have been rendered refractory by this treatment with dead micro-organisms, the next step is to inject (also into the veins) small quantities of living cultures. The doses of these cultures are gradually increased, and end by conferring upon the animal a very strong immunity, which is strengthened by injections of products of cultures passed through a Chamberland filter.

Calmette and Salimbeni[817] injected prophylactically more than 600 persons menaced by plague at Oporto. These comprised the doctors and the staffs of the laboratories of hygiene and of the disinfection services, the firemen who removed the sick persons and the dead, the families of those who were attacked, the members of the French colony, etc. Into each person 5 c.c. of serum was injected below the skin of the abdomen. These vaccinations in some cases caused nettle-rash, eruptions similar to those so often observed after the injection of the other kinds of serums. Of the total number injected two persons contracted plague: the unfortunate Doctor Camera Pestana and his assistant. The former succumbed to the disease, but the second only contracted a very mild form of it. The study of these 600 cases, as well as of experiments on animals, demonstrated that the immunity conferred by the antiplague serum is set up immediately after its injection but is not of long duration. It is probable that it lasts for 8 or 10 days, or at furthest a fortnight only.

Similar results were obtained at Glasgow. Van Ermengem[818], who has published a report on the epidemic in this town, mentions that more than 70 persons in good health were inoculated with the serum; each one received 10 c.c. beneath the skin of the belly. Of these 70 persons one was attacked with a fairly mild plague 8 days after the vaccination, and another, a housekeeper, was attacked, 9 days after the injection, with a congestion of the cervical glands induced by the plague bacillus. Both cases recovered. All the other vaccinated persons, in spite of constant exposure to the plague infection, remained unaffected. Van Ermengem was of opinion that the two persons treated with the serum were already infected when they were vaccinated.

[Sidenote: [515]]

The Belgian observer points out, further, the frequency of secondary accidents which were produced in the persons vaccinated at Glasgow. Van Ermengem himself went through the ordeal after being injected with 10 c.c. of serum as a protective measure and this gave occasion to several critics to attack the Pasteur Institute. This is how Van Ermengem himself puts the matter. “The accidents after the immunising injections ... were very numerous, they were observed 33 times in 72 cases. Sometimes they were even fairly serious, to the point of causing great suffering to the patient and of disquieting those around them. We could describe them from thorough knowledge, since we experienced them, but they scarcely differ from those which are observed from time to time after the injection of antidiphtheria serum, and, like them, they disappear without leaving the least trace” (_l.c._ p. 18).

In spite of these accidents and the necessity of renewing frequently (every ten or fifteen days) the protective injections of serum, their use is quite advisable in certain circumstances. They may render great service on board infected vessels or in lazarettos (as in the case which occurred at Frioul after the arrival at Marseilles of Arab stokers suffering from plague), in docks, warehouses, and stores where contaminated merchandise is found. They should also be employed to vaccinate those coming into immediate contact with plague cases in hospitals and in private houses. In a word, vaccinations by serum, owing to their power of conferring a very rapid immunity, should be practised wherever there is more or less immediate and imminent danger. Under these conditions they are of very great service in localising the disease.

The methods of vaccination against plague that have been employed up to the present may undoubtedly be improved. Calmette and Salimbeni (_l.c._) have already published the results of experiments on animals undertaken with the object of studying the effect of a combined method of vaccination with antiplague serum and killed cultures of the plague bacillus. But even in their present form the methods used for protecting individuals against this disease deserve to be regarded as conferring great benefits on humanity.

[Sidenote: [516]]

XI. _Vaccinations against tetanus._ Tetanus unlike plague is not a contagious disease, nor is it capable of becoming epidemic. It constitutes, however, a very formidable disease against which all therapeutic methods have only a very limited effect. This is a further reason for drawing the whole attention of medical and veterinary men to the prevention of tetanus by vaccinal injections. Tetanus is a disease in which the intoxication plays an altogether dominant part. The tetanus bacilli do not develop, at the point where they are introduced into the body, unless favoured by auxiliary conditions, such as the multiplication of other micro-organisms. Even then the organism of tetanus reproduces itself with difficulty, and without becoming generalised throughout the body. The poison which it secretes is however sufficient to produce a very grave intoxication, ending most frequently in death. In certain countries tetanus, as a sequel to various wounds, is very frequently met with in man and in certain domestic animals, such as the horse, donkey, pig, etc.

It is only since the discovery by von Behring and Kitasato of an effective method of immunisation against tetanus that it has been possible to consider the practical application of antitetanus vaccinations. These observers demonstrated that the tetanus poison, when treated with trichloride of iodine, had its toxic action weakened and was transformed into an effective vaccine. Roux and Vaillard found that the addition of Lugol’s iodo-iodurated solution to the tetanus poison renders it capable of vaccinating all kinds of susceptible animals. It was shown later, that even with modified active tetanus toxin, we can still obtain good results when care is taken to inject the poison with great circumspection.

But it is not these vaccines obtained from tetanus cultures that have come to be used in practice. The best results are obtained by the use of antitetanus serums. After von Behring and Kitasato’s discovery of the power of the serum of animals immunised against tetanus to neutralise the action of the tetanus poison, very numerous experiments were made on the same subject. It has now become possible by treating horses with large quantities of tetanus toxin to obtain specific serums of extraordinary activity. Thus several serums are capable of preserving mice against a lethal dose of tetanus poison if we inject into them a quantity of serum equal to the one-thousand-millionth of their weight.

Serums of this strength protect domestic animals against tetanus. We know that many operations on horses, sheep, goats, pigs, and other mammals are very often followed by a tetanus which is usually fatal. Castration, amputation of the tail, the ablation of proud flesh or tumours, the operation for cryptorchitis or hernias, etc. are often complicated by tetanus. Moreover, tetanus may frequently appear in horses that have received wounds in the foot or in the lower parts of the limbs, “Clous de rue,” farrier’s punctures, wire-heels, blows, etc.

[Sidenote: [517]]

With the object of remedying this state of things Nocard[819] distributed to veterinarians about 70 litres of antitetanus serum to be employed for protective purposes. The majority of the animals treated (horses, donkeys, mules, bulls, rams, lambs, and pigs) received two injections of serum at an interval of 10–12 days, 20 c.c. for large animals and 6–10 c.c. for sheep and pigs. Of 3088 animals which received the first injection of serum immediately after the operation not a single one contracted tetanus. Of 400 animals which received the first injection at a later period, 1–4 days and more after the accidental wound of which they had been the victims, one horse only, treated five days after the accident (farrier’s puncture), was seized with mild tetanus, but it soon recovered. In the same localities where the results of the vaccination were so brilliant, 314 cases of grave and fatal tetanus occurred amongst animals operated upon or injured that were not submitted to the serum treatment.

It may be readily understood with these facts before us why the practice of protective vaccinations of animals against tetanus should have spread so rapidly amongst veterinarians. The demand for antitetanus serum from the Pasteur Institute of Paris for veterinary use increases every year at a great ratio. Thus in 1896 there were sent out only 1511 bottles of 10 c.c. each, in 1898 the number rose to 24,959 bottles, in 1900 it exceeded 43,000.

The efficacy of the antitetanus serum employed as a protective agent can no longer be questioned, but it must not be forgotten that its injection does not render the treatment of the wounds unnecessary. These wounds should receive a rigorous antiseptic cleansing. All foreign bodies should be carefully extracted; otherwise the prolonged presence of tetanus spores might set up a late tetanus after the disappearance of the transient immunity due to the serum.

[Sidenote: [518]]

The protective injections of antitetanus serum into men likely to contract tetanus are also beginning to spread. It often happens that bicyclists, in falling, receive injuries which are contaminated by horse-dung or other matters which may contain the spores of tetanus. In these cases, as in many other forms of injury, vaccination with antitetanus serum is indicated. Thus it happens from time to time at the Pasteur Institute that injured persons come and ask for a protective injection of serum. Several medical men and surgeons are now accustomed to vaccinate such of their patients as have had their wounds contaminated by earth or dung. All the cases of this treatment which have come to our knowledge have been followed by very good results.

XII. _Vaccinations against diphtheria._ Antidiphtheria vaccinations have been the subject of much discussion since the discovery of the antidiphtheria serum and its introduction into routine practice. A large number of works were published for and against the application of serum in protective treatment against diphtheria, especially in the early years of its use. Later the controversy has subsided somewhat, and at present very few writers are found who continue to decry antidiphtheria vaccinations.

The antidiphtheria serum was discovered in 1890 by von Behring working in collaboration with Kitasato; these observers demonstrated in laboratory animals its neutralising action upon the diphtheria toxin. A little later von Behring began to apply it in the treatment of diphtheria, but the early results were far from satisfactory, and von Behring soon recognised that it was necessary to obtain much more active serum. Along with Ehrlich of the Institute for Infective Diseases at Berlin he set to work to study this problem. In collaboration with several investigators, among whom I may cite Wernicke, Wassermann, and Kossel, he succeeded in obtaining very encouraging results as regards the antitoxic strength of the serums and their therapeutic action on children attacked by diphtheria.

At this time, also, Roux in Paris began, assisted by Martin and Chaillou, to study the same question. These observers prepared serums which for that period were very active and made a very effective application of them upon more than 300 diphtheria patients.

From the year 1894 the use of serum began to spread in all countries, and it was then that an attempt was made to apply it to the protection of children in good health, but who had been specially exposed to contagion.

[Sidenote: [519]]

It was necessary to have at command large supplies of antidiphtheria serum; this was prepared by injecting into horses repeated doses of the toxin manufactured by the diphtheria bacillus. The serums thus obtained were first tested as to their protective, antitoxic, and curative action on guinea-pigs, animals very susceptible to diphtheria. The necessity of finding some means of measuring the strength of the serum soon arose. Von Behring and Wernicke at first standardised it on the basis of the number of grammes of guinea-pig which could be protected by one gramme of serum. Later, von Behring[820] introduced the principle of the “normal serum,” that is to say, a serum of which 0·1 c.c., mixed with 10 lethal doses of diphtheria toxin, is capable of preventing every morbid symptom in a guinea-pig weighing 300 to 400 grammes.

Ehrlich[821] perfected this method in the following way: to tubes, each containing 10 lethal doses of a standard toxin, are added different amounts of serum. These mixtures are brought to the same volume of 4 c.c. by the addition of physiological saline solution, and each is immediately injected below the skin of a guinea-pig. If 0·1 c.c. of a serum completely neutralises the 10 lethal doses of toxin, the serum retains its name of normal serum; in the case where 0·05 c.c. is sufficient to bring about the same result the serum is designated double normal serum. When 0·001 c.c. gives the same results, a hundred times normal serum, and so on. A cubic centimetre of normal serum (that is to say a dose capable of neutralising 100 lethal doses of standard toxin) constitutes an “immunising unit” (Immunisirungseinheit (I.E.) of Ehrlich). As it was soon recognised that toxins, even when kept under the best conditions, lose more or less of their toxic power, Ehrlich had to modify his method of standardising serum. He now makes use of a standard antidiphtheria serum, kept in a dry condition, which is much more constant than are the toxins. Solutions of this standard serum are prepared and compared with the serum whose strength has to be determined. Ehrlich has given a detailed description of the method of procedure required to obtain exact results.

[Sidenote: [520]]

At the Pasteur Institute Ehrlich’s method has been adopted, supplemented however by another test for the estimation of the strength of antidiphtheria serums, a method allied to von Behring’s old method. Various doses of the serum to be examined are injected subcutaneously into guinea-pigs, and 24 hours later these guinea-pigs receive a quantity of a living culture of diphtheria bacilli which kills control animals in 30 hours. The protective power of the serum in relation to the weight of the animal is thus determined. For example, a serum which is said to be active at 1/100,000 has the power, in a quantity equal to 1/100,000th of the weight of the inoculated guinea-pig, of preventing a fatal result. It was thought, at first, that the protective power, measured in this way, would be proportional to the antitoxic property determined according to Ehrlich’s method. But as the results given by these two methods were often widely different, it was resolved at the Pasteur Institute to examine by both methods all the serums intended for use in practice. This led to the conclusion formulated by Roux[822], in his report communicated to the International Congress of Hygiene, held at Paris in 1900, that a serum possessing a very high protective power (against the living diphtheria bacillus) might be only feebly antitoxic, and _vice versâ_.

[Sidenote: [521]]

This result is explained by the fact that the antidiphtheria serums are very complex fluids, containing several superposed properties of very variable strength. Marx[823], of the Frankfort-on-Main Institute, tried to shake Roux’s conclusions, bringing forward his experiments made on guinea-pigs and rabbits injected with antidiphtheria serum into the peritoneal cavity and into the veins. He wished in this way to avoid the introduction of the serum into the subcutaneous tissues, whence the absorption of the antitoxin must take place in a very irregular fashion. In Marx’s experiments, thus carried out, the protective power of the serums was always found to run parallel with their antitoxic power, from which he concluded that Roux’s view was incorrect. It must not be forgotten, however, that this view was founded on experiments in which the antitoxin had been injected into the subcutaneous tissue before or simultaneously with the toxin or the diphtheria bacillus. Under these conditions the protective power is often found to be altogether disproportionate to the antitoxic power. This fact has been observed so carefully and with such exactness that it is impossible to deny it. Now it is undoubted that the conditions of the experiments upon which Roux relies correspond much more closely with those that are realised in vaccination of man against diphtheria than with the conditions met with in Marx’s experiments. In these vaccinations antidiphtheria serum is injected below the skin of persons whom it is wished to protect against the action of the diphtheria bacillus.

With the object of bringing about a unification of the methods of estimating serums used in different countries the International Congress of Hygiene, held at Madrid in 1898, appointed a special Commission to settle this problem. But when the Congress met again at Paris in 1900 this Commission had not completed the task allotted to it. The representatives of the various methods had exchanged ideas, but in applying the same method the results obtained in various places and by various observers presented differences too great to allow of any understanding being arrived at. It is evident that we have here a very complicated problem. The serums are tested on living animals in which of course nothing like the constancy of a chemical reaction can be obtained.

Possibly the methods of breeding and the races of the same animals in the different countries may be quite sufficient to explain the divergencies in the results obtained. Whatever may be the reason the unification of serum estimation has not yet been obtained, and it is difficult to anticipate that any better result is to be arrived at.

From all this we may draw the conclusion that the possibility of attaining a too rigorous precision in the standardisation of serum has been exaggerated. Our object must be to obtain results as favourable as possible in the application of the antidiphtheria serums, and for that purpose it is necessary to inject greater quantities than those which may be indicated by any method of estimation. This rule is applied as far as is possible at the Pasteur Institute.