Inoculation against hydrophobia was rendered possible by the discovery of the fact that the rabies or hydrophobia virus is found in a pure condition, free from other microbes, in the nervous centers of animals. The material for inoculation is prepared from such nervous matter, the virulence of which is rendered fixe, as will be mentioned below.
The cholera microbe, which was subsequently named comma bacillus, was discovered by Koch in 1883, in the intestinal contents of cholera patients. Two years later cholera broke out in Spain, and Dr. James Ferran, a Spanish physician, began inoculating men with living cultures of comma bacillus taken from patients attacked with the disease. The procedure in its essential features corresponded to the pre-Jenner method of inoculation. The failure to fix the strength of the virus used for treatment rendered the method subject to the same uncertainty as that which was connected with inoculation with smallpox virus taken direct from patients. It was impossible to predict the effect of the injections. Comma bacilli taken from cholera patients may, under cultivation, show themselves extremely virulent, or, on the contrary, extremely mild. There are specimens which, when injected into a Guinea pig, even in an insignificant dose, will prove fatal to it, and there are others which will appear harmless when given in a dose seventy times greater. The immediate effect, and the protection caused by the inoculation, must, of course, vary accordingly. The attempt made by Ferran caused great interest, and a number of scientific commissions were sent to Spain from different countries of Europe to study the results of his work. They could, however, come to no conclusion, and the treatment speedily lost its position. Only some seven years later a method was found of fixing the strength of the cholera virus. I was connected with this stage of the work, and it may perhaps present some interest to the reader to relate the way in which the problem was solved, and to show how gradual is the development of ideas by which results in laboratory investigation are arrived at.
It has been mentioned already that the virulence of microbes changes under the influence of different agents in Nature—heat, light, chemicals. When a virus is first obtained from a patient or outside a patient its preceding history, its antecedents, the conditions under which it lived before, are extremely variable. Jenner’s method of cultivating the smallpox virus by transferring it from calf to calf secured for that virus uniform conditions of life, and its strength could thus be maintained unchanged for an indefinite length of time. Pasteur, in the preparation of hydrophobia vaccine, followed the same plan, and found in the successive inoculation from rabbit to rabbit a method of propagating the hydrophobia virus in a uniform condition. But attempts made to cultivate in a similar way the comma bacillus by transferring it from animal to animal failed.
The most susceptible animals for the cholera microbe are Guinea pigs. There are two principal methods of ingrafting upon them the virus: Koch’s method of administering it through the mouth and leaving it to develop in the intestines of the animal, and Pfeiffer’s of injecting it, not into the intestines, but into the abdominal or peritoneal cavity, where the intestines are lodged, by introducing there the virus with a hypodermic needle not allowed to penetrate into the intestines themselves. But by neither of these methods could the microbe be cultivated in an unbroken series of animals, as it became gradually weakened and soon lost its power of affecting such animals. For the purpose in question, cultivation in the peritoneal cavity had the advantage that in a healthy individual the peritoneum is free from other microbes, whereas in the intestines there are always present a large number of micro-organisms which interfere in variable ways with the growth of the particular microbes.
But when one inoculates the peritoneal cavity of a Guinea pig with a dose of cholera microbes sufficient to cause a fatal disease, it is found, when the animal dies, that the microbes have died also. Thus, the attempt to ingraft the virus from a first animal to a subsequent one is checked at the very beginning. This initial difficulty was overcome by merely giving to the first animal a dose larger than was necessary to cause a fatal effect. The animal then succumbs more rapidly, and the microbes have no time to disappear. At the post-mortem examination there is found, in the peritoneal cavity, a small amount of exudate liquid which contains large numbers of those microbes alive. When, however, that exudate is injected into the peritoneal cavity of a second animal that animal does not succumb to the infection, or even if it succumbs one finds that the microbes have again disappeared in this second animal. By starting with, a still larger initial dose one may have three, perhaps four, successive animals affected by the virus, but it invariably ends by being weakened, and finally dies out.
In trying to obviate this result I found, perhaps contrary to expectations, that the exudation liquid should be exposed to the air for a few hours before it is injected into a subsequent animal. This result was contradictory to the effect which Pasteur had found to be exercised by atmospheric oxygen on the virulence of microbes, and it requires at least some provisional explanation. The microbes of cholera differ from a certain number of other microbes in that they stand in need of a free and abundant access of air for growing and multiplying quite satisfactorily. They are deprived of this condition in the peritoneal cavity of an animal. It is possible, therefore, that a certain opposition between the maintenance or development of virulence on the one hand, and a lowering in vitality on the other, takes place while they are cultivated there, and a respite must be given them between each successive ‘passage’ through the Guinea pig by leaving them for a time in the free atmosphere. Be that explanation true or not, the result is that under such conditions the successive animals inoculated with the virus do succumb, and even in a shorter and shorter time, after the inoculation, the microbe apparently undergoing under such a treatment a progressive increase in virulence. A similar development up to a certain stage was observed by Pasteur when transferring the rabies virus from rabbit to rabbit. The last difficulty that presented itself was the following: The exudation liquid which is found in the peritoneal cavity post mortem varies in quantity; sometimes it is inconveniently large and diluted; sometimes, on the contrary, so scanty that it becomes difficult to collect and transfer it to another animal. I found that this variation stands in connection with the size of the animal, so that a diluted exudation fluid can be concentrated by injecting it into a small animal, while a too much concentrated exudate is rendered more dilute by transferring it to an animal of a larger size.
Thus, by the initial use of more than a fatal dose, by alternating cultivation in an animal with exposure to air, and by attention to the size of the animal employed, a material was obtained which, as mentioned, increased in intensity from the first and proved fatal to animals in a shorter and shorter time after inoculation. Later the virus reached a stage when it killed a Guinea pig of three hundred and fifty grammes weight in eight hours. After that, in each further inoculation the time of eight hours remained stationary, showing that the virus has reached the condition of a ‘virus fixe.’ These experiments were conducted by me in the Pasteur Institute, in 1889 to 1893, simultaneously on the cholera microbe and on the bacillus of typhoid. The two exhibited a number of common features in their nature, and the results as above detailed for the cholera microbe were found valid for the typhoid bacillus also.
Starting from the ‘virus fixe’ obtained as above, a method of double inoculation was worked out, one with an attenuated virus prepared from the ‘virus fixe,’ and another with the latter itself. The two ‘vaccines,’ when inoculated successively into Guinea pigs, protected them against all possible forms of cholera infection. The vaccines were cultivated on a solid medium, and when the crop of microbes was ready at the end of some twenty-four hours, they were washed off the surface of the medium and used as a kind of medicinal plant. It was found that the substances contained in the microbes preserved to a great extent their immunizing properties even when the microbes were killed by some delicate processes not affecting considerably their chemical constitution. The washings could, therefore, be prepared in dilute solutions of carbolic acid, and employed in the form of preserved vaccines. In 1892 and in the beginning of 1893 I made a series of experiments in Paris, in Netley, in London, in Cambridge, and in Calcutta, with these carbolized cholera vaccines, which had been preserved in sealed tubes for a period of six to seven months, and it was possible to show the protective effect of the method on animals as conclusively as Pasteur had done in the demonstration at Pouilly-le-Fort with anthrax. For the inoculation in man, however, I decided to use at first only unaltered living vaccines, as much more promising than the dead ones, especially from the point of view of the durability of the effect.
[To be concluded.]