544 THE TETANUS BACILLUS 



amorphous, extremely virulent residue. 90 per cent, alcohol dissolves a 

 small quantity of this residue, and leaves after evaporation a whitish, non- 

 toxic substance having a waxy smell. The remainder of the residue not 

 dissolved by alcohol is readily soluble in water and produces typical symptoms 

 of tetanus in guinea-pigs : it can be precipitated from its aqueous solution 

 by alcohol. The active substance contained in the residue dialyzes slowly. 



Tetanolysin. Ehrlich and Madsen have demonstrated the presence of an 

 haemolysin in filtered cultures of the tetanus bacillus. Tetanolysin differs 

 from tetanus toxin : it is highly unstable and is destroyed if heated for 20 

 minutes at 50 C. or for some hours at 20 C. 



It dissolves the red cells of domestic animals and especially those of the 

 rabbit and horse. Animals immunized with filtered cultures rich in haemo- 

 lysin elaborate an antitetanolysin simultaneously with tetanus antitoxin. 



Detection of toxin in the living tissues. When, in a case of traumatic 

 tetanus, the bacillus cannot be found, an attempt may be made to demon- 

 strate the presence of toxin in the blood by inoculating a few cubic centi- 

 metres of the patient's serum into a mouse. The animal may die but the 

 method is not to be relied upon and should not in any case be adopted when 

 the patient has been treated with antitoxin. 



3. Vaccination. 



(i) Behring and Kitasato failed in their attempts to immunize animals 

 by repeated inoculations of small doses of tetanus toxin. They obtained 

 more satisfactory results, however, when they inoculated rabbits with a 

 mixture of toxin and iodine terchloride. 



Brieger, Wassermann and Kitasato by inoculating repeated and pro- 

 gressively increasing doses of cultures attenuated by growing on thymus- 

 broth only obtained unreliable results. Tizzoni and Cattani succeeded in 

 immunizing rabbits by inoculating them with attenuated cultures. 



Vaillard was able to immunize rabbits with toxin which had been partially 

 deprived of its toxic properties by heat. Rabbits were inoculated intra- 

 venously on several occasions with 10 c.c. of toxin heated for 1 hour first 

 at 60 C. then at 55 C. and finally at 50 C. To increase the degree of 

 immunization, progressively increasing doses of non-heated toxin were then 

 given viz. : first 5 c.c. then 10 c.c. followed by doses of 15 and 30 c.c. Guinea- 

 pigs were immunized in the same way. 



(ii) Roux and Vaillard prefer to use, for the purpose of vaccination, toxin 

 to which a solution of iodine has been added -(vide Diphtheria). Their toxin 

 is obtained as described above and it should kill mice in doses of 0*00025 c.c. 

 It is mixed with Gram's solution immediately before use. The immuniza- 

 tion of a rabbit and of a horse will be described in illustration of the method. 



Immunization of a rabbit. Inoculations given sub-cutaneously : 



1st day 3 c.c. of toxin mixed with 1 c.c. Gram's solution. 

 5th ; , 5 c.c. of toxin + 2 c.c. of Gram's solution. 

 9th 12 c.c. of toxin + 3 c.c. of Gram's solution. 



On the seventeenth day the immunization of the animal is complete. Its serum 

 is antitoxic. It can then be inoculated at intervals of a week with progressive 

 doses of 5, 10, 15, 20, 30 and 40 c.c. of pure toxin. Later, the inoculations may 

 be given intra-venously or intra-peritoneally, and finally, a dose of as much as 

 100 c.c. of toxin can be given at a single inoculation. 



Immunization of an horse. The treatment is begun with a dose of 1-5 c.c. of a 

 mixture of equal parts of toxin and Gram's solution sub-cutaneously. The inocula- 

 tions are repeated every 3 or 4 days. At the end of a fortnight 10 c.c. of a mixture 

 of 2 parts toxin and 1 part iodine solution are inoculated and the quantity is gradu- 

 ally increased until about the twenty-fifth day the injection of pure toxin is com- 



