C.—Bacteriolytic Action.
If we bring into contact with a 1 per cent. solution of Cobra-venom, rendered aseptic by filtration through porcelain, sensitive micro-organisms, such as the cholera vibrio, or the bacterium of anthrax in a very young non-sporulated culture, or in its non-spore-producing variety, we find that these microbes are dissolved by the solution of venom in varying periods of time.
On making a direct microscopical examination we see that Koch’s vibrios become immovable, then break up into granulations and disappear in the liquid. The bacteriolysis is even more distinct in the case of the bacterium. The enveloping membrane seems to dissolve, and the microbe appears as though composed of a series of granulations placed end to end, which finally disperse and disappear.
By my instructions this bacteriolytic property of venom with respect to different species of micro-organisms was studied by Noc. It was especially clearly seen with the non-spore-producing bacterium of anthrax, the cholera vibrio, Staphylococcus aureus, the bacillus of diphtheria, and B. subtilis in a young culture; it is less distinct with B. pestis, B. coli, and B. typhi, is almost nil with the pyocyanic bacillus and B. prodigiosus, and nil with B. tuberculosis.
Investigations have likewise been made by Noc, and subsequently by Goebel,[58] in order to determine whether cobra-venom dissolves Trypanosomes. These hæmatozoa are more resistant than bacteria, but they nevertheless end by being dissolved after twenty to thirty minutes’ contact in the 1 per cent. solution.
The bacteriolytic substance in venom is distinct from that which produces proteolysis, for the latter is destroyed at 80° C., while the former only disappears with a temperature of and beyond 85° C. maintained for half an hour. It is likewise distinct from the hæmolysin, for this resists temperatures considerably higher than 85° C. Moreover, venom which has dissolved microbes until the saturation point has been reached, is found to have preserved in its entirety its hæmolytic power upon the red corpuscles of the horse. Neither does it act upon the microbes owing to the presence of a cytase or alexin. The well-known characteristics of alexins are not met with here—destruction at 55° to 56° C., sensitivity to light, rapid alteration at ordinary temperatures, &c.
We cannot, again, compare the bacteriolytic action of venom to that of rat-serum, which dissolves B. anthracis by aid of a substance distinct from vibrionicide alexin. According to the researches of Malvoz and Y. Pirenne, the lysin of rat-serum appears to be a basic substance, the neutralisation of which destroys its activity. Now Cobra-venom in a very active solution is perfectly neutral to sensitive litmus papers, while these are turned blue by rat-serum. Moreover, venom acts not only upon microbes of the same kind, but also on very different species which are not affected by rat-serum, especially upon B. pestis, for which, on the contrary, this serum, when fresh, proves a favourable culture medium. The bacteriolytic power of Cobra-venom therefore constitutes a special property of venom.
“In their work on the cytolysins of venom, S. Flexner and Noguchi have shown that animal cells, when heated to 55° C. and rendered inactive, do not undergo complete dissolution under the influence of venoms which destroy the fresh cells. The authors in question infer the existence of cellular receptors (endo-complements, according to the theory of Ehrlich), which fix the amboceptors of venom. Pursuing the same order of ideas, I have observed that bacteria killed by heating for one hour at 60° C. do not undergo total disintegration as do living bacteria. But, while Flexner and Noguchi infer the plurality of the cytolysins in venom for different animal cells, I have not been able to prove the same thing with regard to the bacteriolysin; venom saturated with cholera vibrios to such an extent that vibrios added at repeated intervals are no longer dissolved, is incapable of dissolving another highly sensitive species of microbe, such as the asporogenous bacterium, and vice versâ. Besides, it would be difficult to understand the existence in venom of cytolysins specific for a whole series of species of micro-organisms” (Noc).[59]
Antivenomous serum, in a dose of 0·01 or 0·05 c.c., neutralizes the bacteriolytic action of 1 milligramme of Cobra-venom, while normal serum heated, even in larger doses, is without effect. The lysin and the antivenomous serum appear also to enter into stable combination; by heating to 80° C., after dilution of the mixture neutral antivenomous serum + venom, the property of dissolving is not restored to the latter.
Pursuing his researches upon the bacteriolytic actions, Noc has also shown that the fresh serums of the rabbit, horse, guinea-pig, rat, and man are capable of destroying them completely. We must conclude from this that venom has the property of fixing the alexin of these fresh serums, and in fact it is easy to show that this fixation takes place by experimenting with hæmolytic alexin, which is much more easy to study; it is sufficient to eliminate the intervention of the hæmolysin proper to Cobra-venom.
With this object, Noc employed horse-corpuscles (which are readily dissolved by fresh rat-serum), and neutralised the hæmolysin proper to the venom by antivenomous serum, which has no effect upon fresh horse-corpuscles and upon the alexin of rat-serum.
For experimental purposes six tubes are prepared with contents as follows:—
(1) 0·5 c.c. of fresh rat-serum.
(2) 0·5 c.c. of fresh rat-serum + 0·5 milligramme of Cobra-venom (0·5 c.c. of a solution of 1 in 1,000).
(3) 0·5 c.c. of fresh rat-serum + 1 milligramme of venom (after fifteen minutes’ contact of the venom with the alexin in tubes 2 and 3 the venom is neutralised by 1 c.c. of antivenomous serum in the case of tube 2, and by 2 c.c. in that of tube 3).
(4) 1 milligramme of venom.
(5) 1 c.c. of antivenomous serum.
(6) 0·5 c.c. of fresh rat-serum + 1 c.c. of antivenomous serum.
To each tube 2 drops of defibrinated horse-blood are added, and the tubes are placed in the stove at a temperature of 35° C.
In tubes 1 and 6, which contain fresh rat-serum alone, and fresh serum + antivenomous serum, hæmolysis appears in a few minutes. In tube 4, which received venom alone, hæmolysis is also produced in one hour. It is not produced at all in tubes 2 and 3, which received the neutral mixture of fresh serum and venom, proving that the hæmolytic alexin has been fixed by the venom. The latter, therefore, here plays the part of a true fixator or amboceptor.
Venom behaves, in short, after the manner of extracts of organs. The fixation of hæmolytic alexin by extracts of organs, the tissues, and animal cells (liver, spleen, spermatozoids, &c., &c.), has already been demonstrated by V. Dungern, P. Müller, Levaditi, and E. Hoke. The same fact is also observed with solutions of peptone. The fixation of alexin is therefore a general property of certain albuminoid molecules.
It was interesting to endeavour to reproduce, with Cobra-venom, J. Bordet’s experiments upon alexins and anti-alexins. It was to be hoped that we had in this substance an anti-alexic body capable of being preserved for an indefinite time and constant in its activity, which would enable us easily to measure the dose of alexin contained in a small quantity of a serum, or other liquid of leucocytic origin.
The experiment proved to Noc that, contrary to the ideas of Ehrlich and his pupils, and conformably to the results obtained by Bordet with serums and toxins, the neutralisation of venom takes place in a variable ratio.
If a dose A of fresh serum is capable of neutralising exactly 5 milligrammes of Cobra-venom with regard to a sensitive microbe, on employing a dose of the strength of 2 A we ought to find a bactericidal dose, 1 A, in the excess of serum, according to the theory of definite proportions. No such bactericidal action is seen, however; the serum, on the other hand, acts in the contrary direction by means of its nutritive substances, and in the mixture 2 A + venom we obtain a larger number of colonies of micro-organisms than in the mixture A + venom.
We see, then, that the property of cells of fixing in excess the active substance in serums, discovered by Bordet for the hæmolysins (staining phenomena), is met with again in the case of extracts of organs, at least with regard to the bacteriolytic substance of Cobra-venom.
It results, then, from the foregoing facts that Cobra-venom contains a cytolysin, which acts upon micro-organisms and is capable of fixing the alexin of normal serums.
The application of these data to the living animal is evidently full of difficulties, by reason of the complexity of the substances that come into play. Let us see, however, to what extent they are capable of serving to explain the phenomena that are produced as the result of poisoning.
It was observed by Kaufmann that the cadavers of animals which have died from snake-bite are very rapidly invaded by the bacteria of putrefaction. Welch and Ewing, referring to these phenomena of rapid putrefaction in cases of death from venom, explained them as being due to the loss of the bactericidal power of the serum. In hot countries, even when snake-bites are not fatal, they are frequently complicated by local suppuration or gangrene, occasioned by micro-organisms introduced at the time of the bite. The minute analysis of the phenomena of poisoning shows, in reality, that the organism undergoes different modifications according to the quantity of venom injected and its channel of penetration.
When the dose of venom is rapidly lethal, whether because it penetrates into the veins or because a larger amount of it is diffused beneath the skin, it occasions a transient hypoleucocytosis, which is, moreover, a reaction common to injections of venom, pro-peptone, extracts of organs, and microbic toxins (Delezenne, Nolf). It follows that blood collected a short time after the injection may be totally bereft of its bactericidal power, in consequence of the disappearance of the leucocytes, which have migrated into the organs.
Thus it was observed by S. Flexner and H. Noguchi that the serum of a rabbit, treated with 10 milligrammes of Cobra-venom, showed, fifty-seven minutes after the injection, a great loss of bactericidal properties. But it is impossible to conclude, from the diminution of bactericidal power in this experiment, that the alexin becomes fixed by the venom. Since the secretion of alexin is connected with the presence of leucocytes, the hypoleucocytosis due to the venom is sufficient to explain the loss of bactericidal power.
Nevertheless, the action of venom is not confined to these physiological phenomena; in diffusing itself through the organism it stays more especially in parts where the circulation has become slower, in the capillaries of the organs where the leucocytes that have disappeared from the general circulation are already to be found agglomerated and altered. Here the cytolysins of the venom, continuing their effects, are capable of neutralising the alexins set at liberty by the destruction of the leucocytes, and thus the rapid multiplication of the bacteria of putrefaction, which have come from the intestine or were carried in with the bite, is easily explained. In the same way, we can account for the suppuration that is met with as a complication of non-lethal bites, in spite of the hyperleucocytosis consequent upon the penetration of a weak dose of venom; immediate neutralisation of the alexin set at liberty at the level of the wound has sufficed to enable micro-organisms to multiply.