ABSTRACTS. 205

The period of absolute immunity is now reckoned to be about seven months, and not a year as Lomer thought. It has long been known that when two strains vary in virulence the immunity causea by the weaker may be broken down by the stronger. (" Berl. Tieriirzt. Woch.," Vol. XL., 1924, P·37.)

IMMUNISATION AGAINST TUBERCULOUS INFECTION. l

By CALMETTE, GUERIN, and WEILL-HALLE.

IN a series of researches pursued without interruption for more than twenty years the authors, after having studied the method of infection and the rOle of re-infections in the evolution of experimental tuberculosis, have demon· strated that it is possible by using as a virus-vaccine the living culture of a bacillus of bovine origin, artificially attenuated and deprived of any tuberculi­genic property, to confer on young non-infected animals a genuine immunity against either natural or artificial infection. The attenuation of the tubercle bacillus has been obtained by a method not previously employed for any other pathogenic organism. It consists in cultivating the bacilli in uninterrupted series in the presence of ox bile, the object being to modify hereditarily its physico-chemical constitution by causing it to develop in a medium that is extremely alkaline and particularly rich in lipoids (colesterin, lecithins, and neutral soaps).

After 230 successive cultures in thirteen years, on potato cooked in ox bile to which glycerine in the proportion of 5 per cent. has been added, the culture became inoffensive even in large doses for all species of animals, including the anthropoid apes. It no longer provoked the formation of tubercles by intravenous, intraperitoneal, or subcutaneous injection, or by ingestion.

In order to fix its qualities the attenuated bacilli were then cultivated on the usual media without the addition of bile. On these media it remains avirulent, but the bacilli which compose it are still toxic, secreting tuberculin like virulent bacilli, and determining in the system of animals into which it is injected the formation of anti-bodies which are detectable by the fixation of complement method of Bordet-Gengou. To the bacillus thus attenuated the authors apply the term BeG. They have never been able, by massive re-infections of animals already inoculated, to restore the power to determine the formation of tubercles, which appears to have been. permanently lost; it is perfectly tolerated by all tuberculous animals and by man, even with intravenous injections.

When it is injected under the skin or into the veins, or when it is administered by ingestion, even in massive doses, it proves perfectly harmless, so that even should it be eliminated or dispersed in the environ­ment it is incapable of injuring either man or any of the lower animals.

That is not the case with the bovine, equine, or avain bacilli which various authors have proposed to use as a vaccine for cattle or for the human subject. These bacilli, although more or less attenuated with regard to virulence, are all able to cause the formation of tubercles, and capable of spreading the tuberculous infection among other species of animals, especially the human

1 Communicated to the Acad. de Med., Paris.

206 ABSTRACTS.

(and above all in children), and also in the pig, the small rodents, or birds, which frequently act as vehicles of infection in byres or poultry houses.

In a great number of experiments on young calves, rabbits, guinea-pigs, and monkeys, the authors have observed that when the BeG bacillus is introduced in proper doses into the system of animals not previously infected it confers on these animals a manifest resistance to virulent artificial infections which are certainly fatal for control subjects.

In the case of bovine animals it has been shown that the injection of a single dose of 50 to 100 mg. of these living bacilli into the connective tissue of the dewlap produces in young subjects an immunity so strong that they will resist after three, six, twelve, or even eighteen months, the intravenous injection of 5 mg. of a virulent bovine bacillus. While a similar intravenous inoculation kills control subjects with an acute tuberculosis in six to eight weeks, in the vaccinated animals it causes no disturbance of health nor any tuberculous lesion.

Similar effects, but more fleeting, can be obtained with rabbits and guinea­pigs by causing them to absorb a sufficient dose of the BeG bacillus, either by subcutaneous or intravenous inoculation or by ingestion.

It has been established that the resistance thus acquired against virulent infections or re-infections is co-related with the symbiotic life of the bacilli with certain cellular elements. The result of this symbiotic life is the production of a sort of anatomous complex-the bacillised cell-comparable in some respects to a lichen, which is the product of the symbiosis of an alga and a fungus. The giant cell which is thus formed does not necessarily give rise to any tuberculous lesion. When this complex has been formed, and as long as its exists, the system of the animal reacts in a characteristic way to new doses of tuberculin or bacilli; it no longer tolerates the latter, even when they have been killed by heat, but tends to expel them outwardly. That is what has been called" Koch's phenomenon," because the observa­tion of this intolerance to re-infections led Koch to the discovery of tuberculin, although he had not at the time recognised the nature or the range of the phenomenon.

In cattle inoculated with this BeG bacillus the immunity is lost soon after the vaccinating symbiosis has itself disappeared, either because the bacilli of the vaccine have been destroyed by the normal process of cellular digestion, or because they have been eliminated by natural channels (bile, intestine, mammary glands). That happens in bovine animals towards the end of the second year after vaccination, and about the sixth month in the rabbit and guinea-pig. If any of the virulent bacilli of re-infection still remain intact, or if some are again introduced into the animal after the immunity has thus been extinguished, they re-acquire or retain all the patho­genic properties which they manifest with regard to a previously non-infected animal. .

The question is whether this is a case of genuine immunity, that is to say, a condition refractory to the tuberculous disease. In the opinion of the authors this question should have an affirmative answer, for the immunity is strictly analogous, with regard to its duration, to that which is set up by the living virus-vaccines, such as that of Jenner, the anti-anthrax vaccine, or the vaccines which are used against swine erysipelas or rabies. It is known that the immunity which follows vaccination against anthrax or against swine erysipelas hardly persists for more than a year, and that the immunity following vaccination with cow pox or the anti-rabic vaccination gradually disappears within about seven years. Why should it be otherwise after

ABSTRACTS. 207

artificial immunisation against tuberculosis? There is in this case a difficulty which is not encountered in the others, for it appears that immunity can only be produced in subjects that are perfectly free from infection. In animals already infected the injection of vaccine or of tubercle bacilli of any kind, whether attenuated or virulent, or even dead, determines an increased susceptibility to tuberculin and re·infection, as is evidenced by the occur­rence of" Koch's phenomenon."

It results from this that in France and in all the old civilised countries, where 98 per cent. of adult human beings and over 40 per cent. of cattle of five years old or more give a positive reaction to tuberculin, the anti­tuberculous vaccination which the authors have in view can only be applied to very young subjects, and in the case of calves to those that are new-born or only a few days old. Since the calves live in an environment that is more or less infected, at the end of a fortnight they have almost certainly had the opportunity to absorb some tubercle bacilli, which can be found arrested in one or more of their lymphatic glands or are fixed in some tuberculous lesion, with the result that the introduction of a large dose of bacillary vaccine into their system may set up the manifestations of intolerance.

The authors go on to say that, their laboratory experiments in connection with the new method of vaccination having furnished favourable results, they believed they were justified in applying it to young cattle in certain herds which had generously been offered for the purpose, and afterwards to monkeys and finally to infants.

Experiments on Anz·mals.-In the case of calves the vaccination has been carried out within fifteen days of birth, that is to say, at a time when they have not yet been extensively infected. After the operation they were left exposed to whatever risks of natural infection there might be, and no change was made in the manner in which they were kept.

The intention is to carry out this vaccination each year, and by gradual elimination of the non-vaccinated adults it is hoped to build up a non­infected herd within five years. In May 1924, 127 calves were thus vaccinated without any disturbance of health whatever.

The following examples are given of the effect of the vaccination of monkeys: On the 24th December 1923 three monkeys were put together in the same cage: one was vaccinated by causing it to ingest on five occasions (with intervals of forty-eight hours) 5 cgm. of BeG, the first dose being given on the 24th December 1923 and the last on the 1st January 1924. Another was given by the mouth virulent human tubercle bacilli on the 9th and 11th January 1924. The third animal was intended to serve as a control.

On the 17th April following, the control animal died, and the post-mortem examination showed gross lesions of abdominal tuberculosis, involving the lymphatic glands and the organs.

On the 2nd May the monkey infected with human tubercle bacilli died with almost identical lesions.

The monkey vaccinated with BCG vaccines remains in apparent good health.

Other experiments have shown that chimpanzees may be given by ingestion 25 cgm. of BCG, or a single subcutaneous injection of 4 cgm., without exhibiting any reaction except a micro-polyadenitis which appears about the eighth day and completely disappears after about a month. When

208 ABSTRACTS.

smaller doses, such as 1 cgm., are given by ingestion or inoculated under­the skin, not the slightest swelling of the lymphatic glands is observed.

Attempts to Immunise Young Children.-The authors desired to try the vaccination exclusively among the new-born children of tuberculous mothers, but that proved impossible. Some vaccinations were carried out in 1922, but the great majority have been practised during the current year.

Within the first few days of life (either the third, fifth, and seventh; or the fifth, seventh, and ninth days) the infants are given by the mouth three doses of 2 mgm. each of BeG bacilli, that is a total of 6 mgm. Two hundred and seventeen children have been thus treated, but owing to movements of the families the history of thirty-nine of these has been lost.

Of the remaining 178 chidren, nine died in the course of the first eighteen months. In two cases the cause of death was broncho-pneumonia and in a third case gastro-intestinal trouble. The cause of death in the other cases is not known.

The development of the remaining 169 children that have survived has been normal. The ingestion of the vaccine did not cause any disturbance whatever.

The cuti-reaction test was applied to fifty-three of the children, in most cases about three months after the ingestion of the third dose. It was negative in 88'7 per cent. of the cases and positive in the remainder. Five of the children in which the result of this test was negative had among their­immediate ancestors one tuberculous subject and were exposed to contagion. Out of the six which gave a positive result three were in contact with persons carrying bacilli. (Recueil de Med. Vet., Tome c., 1924, p. 385).

THE CULTIVATION OF THE VIRUS OF FOOT­AND-MOUTH DISEASE.

By H. DAHMEN.

THE author states that, at the time of writing, the work which led to the discovery of a method of cultivating the virus of foot-and-mouth disease had been in progress for about a year.

At the outset the author refers to previous claims made by Titze, Pfeiler, and others that they had actually succeeded in cultivating the virus outside the body, and gives reasons for rejecting the claims.

The author in his attempts to cultivate the virus in vitro was guided by his previous experience in connection with the cultivation of the organism of contagious bovine pleuro-pneumonia. In the first place, in order to free the fluid from vesicles from accidental bacteria it was passed through a Berkefeld filter, after which the virulence of the filtrate was tested. Small quantities of this filtrate were added to a fluid medium which the author­had used in his work with the pleuro-pneumonia virus, but the result of the experiment was negative. The fluid was modified by the addition of various substances, such as sugars of different kinds, salts, serum, blood, and plasma. Variations in the proportion of the different constituents and the effect of different degrees of alkalinity were also tried. The attempts at culture were made both under rerobic and anrerobic conditions. In some of the media employed a turbidity made its appearance, but inoculation