its Patholofji/ and Method* of Protective Inoculation. 299 



lation was repeated. The temperature began to be elevated on the 

 fourteenth day, and it died of horse-sickness seven days later. The 

 primary inoculation in this case, while being ineffectual to induce the 

 disease, had evidently lowered the susceptibility, so that a fresh 

 stimulus, by the same virus, was sufficient to overcome the resist- 

 ance entirely. 



Having recognised that the blood of animals which lived beyond 

 the ordinary period at which horses usually die from horse-sickness 

 was lowered in virulence, I determined to attempt to produce this 

 change in vitro. 



Having, 'therefore, prepared bottles containing citrate solution, and 

 having thoroughly sterilised them, selected animals were bled under 

 the most rigid aseptic management, and the blood received in the 

 bottles which, after being replugged, were incubated at a temperature 

 of 102 F. during ten days. In one such experiment, out of a total of 

 fourteen bottles, thirteen remained perfectly free from extraneous 

 organisms. Such blood after incubation was then preserved and 

 tested. 



I found, in this manner, that it was possible to produce an atten- 

 uated virus equally suitable for inoculation as that obtained from the 

 donkey or the ox. 



While, howeA^er, these experiments demonstrated that it was possible 

 to protect horses by repeated inoculations of an attenuated virus, they 

 equally demonstrated the irregularity of action, owing to the varying 

 susceptibility to the disease in its attenuated form which obtains 

 among horses. 



Several important facts, however, which were elucidated, are deserv- 

 ing of careful consideration, viz. : 



Death in cases of horse-sickness cannot directly be ascribed to 

 hyperpyrexia, inasmuch as several horses have recovered after having 

 experienced temperatures of over 107 F. ; while others, which have 

 died, and, in which characteristic lesions have been found, have not 

 had a temperature exceeding 105 F. 



Protection can be arrived at without the production of very great 

 reaction, provided that a number of inoculations are made into the 

 animal, and that these have been so arranged as to proceed very 

 gradually to the highest degree of virulence. 



3. It is exceedingly difficult to determine the exact degree of 

 attenuation in any particular sample of an attenuated virus. 

 I have usually attempted this by the inoculation of the virus 

 into one or, at most, two horses; but if the susceptibility of 

 such animals happens to be of a low grade, then the reaction 

 produced may not obtain in other horses for which it 

 may subsequently be used. In other words, to determine 



