April i6, 1896] 



NA TURE 



571 



further important evidence has thus been obtained in favour of 

 the reality of the protection to which I have referred. 



Sewall, in 1886, undertook an investigation with the object of 

 determining if immunity against the fatal effects of rattlesnake 

 venom could be produced by the inoculation of repeated doses, 

 each too small to produce ill-effects. The experiments were 

 made on pigeons, and he succeeded in proving that immunity 

 could be secured to the extent, at least, of protection against 

 seven times the minimum-lethal dose. Kanthack made a similar 

 series of experiments in 1891, which allowed him to conclude 

 that rabbits may be accustomed to resist lethal doses of cobra 

 venom. Working with the venom of vipers, Kaufmann in 1891, 

 and Phisalix and Bertrand in 1893, obtained experimental evi- 

 dence of the possibility of producing a definite, though not high 

 degree of resistance against the toxic effects of this venom. In 

 the following year, Calmette, continuing some earlier obser- 

 vations which had led him to express the opinion that protection 

 against snake venom could not be produced, published evidence 

 confirming the results of previous investigators, but also showing 

 that a higher degree of protection could be secured than they 

 had obtained, for he succeeded in administering to each of 

 several rabbits, within a period of eight months, a total quantity 

 of from 30 to 35 milligrammes of venom. 



In 1894, also, both Phisalix and Bertrand and Calmette ob- 

 tained evidence of the power of the blood-serum of protected 

 animals to counteract the effects of venom. Calmette at the 

 same time claimed that hypochlorite and chloride of calcium 

 were antidotes of considerable value ; and in a later publication, 

 he showed that the blood-serum of animals immunised by the 

 administration of venom possesses a certain degree of antidotal 

 efficacy against the toxines of several diseases. 



In the case of many of the venoms which I have had the good 

 fortune to obtain, the quantity at my disposal was not sufficient 

 for experimental examination on the plan that seemed desirable, 

 and, besides, the examination of each of them would require 

 several months of work. The venoms that have as yet been 

 used are four in number, those, namely, of the cobra of India 

 (Naja triptidians), of the Crotalits horridtts of America, of a 

 large colubrine snake, probably a species of Diemenia from 

 Queensland, Australia, and of the Sepedon lueinachates <A .\frica. 

 They are, therefore, tho.se of the most deadly of the poisonous 

 serpents of Asia, America, Australia, and Africa, respectively ; 

 and, further, they are representative of the chief differences that 

 occur in the composition and action of venoms, for they are derived 

 from members af the two greac groups of the colubrine and 

 viperine serpents. My supply of cobra venom, however, being 

 much larger than that of any of the others, this venom was 

 chiefly used in the experiments. 



An essential preliminary to exact investigations with active 

 substances must always be the determination of the activity of 

 the substances. The only convenient method for doing this is 

 to define the smallest dose capable of producing death for any 

 given weight of animal— that is, the minimum-lethal dose. 

 The venoms in their natural liquid state are unstable, and 

 they are also inconstant in activity, mainly because of variations 

 in the quantity of the water which they contain. Dried venoms 

 have therefore been used in all the experiments. The cobra 

 venom has, however, nearly always been received in the form 

 of a dry solid ; but when this was not so, it has been dried in 

 vacuo over sulphuric acid. 



Experiments were made with it on several animals — as the 

 frog, guinea-pig, rabbit, white rat, cat, and the innocuous grass 

 snake of Italy (7'ropedoiiotus natrix). Very considerable 

 differences were found to occur in the minimum-lethal dose for 

 each of these animals. For the guinea-pig, the minimum-lethal 

 dose per kilogramme wa-. -00018 grm.; for the frog, 0002 grm. ; 

 for the rabbit, -000245 grm. ; for the white rat, 00025 g"""!- 5 

 for the cat, somewhat less than -005 grm. ; and for the grass 

 snake, the relatively large dose of -03 grm. ' Cobra venom thus 

 takes a position among the most active of knf)wn substances, 

 rivalling in its lethal power the most potent of the vegetable 

 active principles, such as aconitine, strophanthin or acokantherin. 



These facts having been ascertained, attempts were next made 

 to render animals proof against lethal doses, by administering to 

 them a succession of gradually increasing non-lethal doses. 

 These were, for the first few doses, in some of the experiments, 

 one-tenth of the minimum-lethal, in others one-fifth, in others 



G 



Frog, 

 Rabbit. 

 White rat, 



pig, nearly \ 

 nearly \ 



NO. I 38 I, 



millig. 



Kitten (6 weeks), a millig. 

 Cat, 5 .. . 



Grass snake, 3 centig. 



VOL. 53] 



one-half of the minimum-lethal, and in others almost as great as 

 the minimum-lethal. At varying intervals, the doses were 

 repeated, and by-and-by gradually increased, until the actual 

 minimum-lethal had been attained. The .subsequent doses, by 

 gradual increments, exceeded the minimum-lethal, and after five 

 or six times the minimum-lethal had been reached, it was found 

 that the increments could be increased so that each became 

 twice, four times, and latterly even five times the minimum- 

 lethal, and still the animal suffered little, and, in many cases, no 

 appreciable injury. 



This brief statement, however, does not represent the experi- 

 mental difficulties that were encountered. It describes the 

 course of events in the altogether successful experiments. Non- 

 success, however, was frequent, and many failures occurred 

 before experience indicated the precautions and conditions that 

 are necessary for success. 



Serpents' venom exerts what may broadly be described as a 

 duplex action. It produces functional disturbances una.sso- 

 ciated with visible structural changes, and it also produces 

 obvious structural changes. The latter are of a highly irritative 

 character, causing intense visceral congestions in the lungs, 

 kidneys, and other organs, and when the venom is given by 

 subcutaneous injection, on all the structures of the skin and 

 subjacent parts. There are apparently also some definite changes 

 produced in the blood, with regard to which several important 

 facts have been discovered by Dr. Martin, of the University of 

 Sydney, and by Surgeon-Colonel Cunningham, of Calcutta. 

 Irritative effects are obviously produced by cobra venom, 

 even in non-lethal doses, and with greatly increased viru- 

 lence by doses that exceed the minimum-lethal ; but, in 

 respect to this action, the other three venoms used are greatly 

 more active than the venom of the cobra. Evidence w-as 

 obtained to indicate that in the process of immunisation a 

 diminution occurs in the intensity of these local actions ; but 

 this diminution does not proceed so rapidly as that in the unseen 

 functional or other changes which are the more direct causes of 

 death ; and, further, the local irritative changes, after having 

 been produced, are slower to disappear than the unseen 

 functional disturbances. Until these facts had been appreciated, 

 and, indeed, even with the adoption of precautions suggested by 

 them, frequent failures occurred. The apparently contradictory 

 results, accordingly, were obtained of the production, by 

 gradually increasing doses, on the one hand, of a protection 

 against quantities much above the minimum-lethal, so perfect that 

 no apparent injury was caused ; and, on the other hand, when 

 the intervals of time separating successive doses had been too brief, 

 of an intolerance so decided that death was produced by the 

 last of a succession of gradually increasing doses, no one of 

 which was so great as the minimum-lethal. The latter unfor- 

 tunate event was frequently displayed in frogs and guinea-pigs, 

 and attempts to carry immunisation in them to a high point 

 usually resulted in failure. 



Notwithstanding these difficulties, however, such gralifymg 

 results have been obtained as that rabbits could at last receive, 

 by subcutaneous injection, so much as ten, twenty, thirty, and 

 even the remarkable quantity of fifty times the minimum-lethal 

 dose, without manifesting any obvious symptoms of poisoning. 



Almost the only observable phenomena were a rise in the 

 body temperature, which continued for a few hours after the 

 injection, and which contrasts with the fall that occurs after the 

 administration of even non-lethal doses, in non-protected 

 animals ; and a loss of appetite, which usually, though not in- 

 variably, occurred, and was probably the cause of a temporary 

 fall in weight during the day or two days succeeding each 

 injection. On the other hand, during the process of successful 

 immunisation, the animals increased in weight, fed well, and 

 appeared to acquire increased vigour and liveliness (Fig. l). 



It is marvellous to observe these evidences of the absence of 

 injurious effects, and even of the production of l)enefit in an 

 animal which, for instance, has received in one single dose a 

 quantity of venom sufficient to kill, in less than six hours, fifty 

 animals of the same weight, and in the course of five or six 

 months a total quantity of venom sufficient to destroy the lives 

 of 370 animals of the same species and weight. 



With the cobra venom, I have also immunised cats and while 

 rats, both by sul)Cutaneous and by stomach administration ; but 

 the significance of the latter method of administration will be 

 afterwards considered. A horse has also been immunised ; and 

 I have to express my obligations to Principal Williams and Prof. 

 W. Owen Williams for granting me the accommotlation of their 



