Nov. 10, 1913 
Diagnosis of Dourine 
103 
In place of preparing suspensions of the trypanosomes, however, an 
antigen was made of the blood and macerated spleens of rats killed at the 
height of surra infection. This material was placed in a bottle containing 
glass beads and shaken for six hours, filtered through gauze, and car- 
bolized. The results from this antigen proved satisfactory, and it was 
used repeatedly on the blood of the horses affected with dourine that 
were left of the Iowa shipment. 
The smallest quantity of the serum which gave a positive reaction with 
the antigen was 0.05 c. c.; however, the various comparative tests indi¬ 
cated that fixation in tubes containing 0.2 c. c. of serum is sufficient for 
diagnostic purposes. Sera from normal animals, also those affected with 
various other diseases, failed to give a reaction. This antigen proved 
active on 10 consecutive days, but failed to produce fixation of complement 
on subsequent tests. Later attempts by the same procedure also resulted 
less satisfactorily, and it was therefore deemed advisable to try other 
methods in order to procure an antigen of more uniform action. 
The following procedure was next employed: 
After successive examinations of the blood of a dog infected with 
surra, about 200 c. c. of blood were drawn from the jugular vein when 
the microscopic examination revealed an extreme infestation with the 
parasite. The blood was drawn into a 1 per cent potassium-citrate 
solution in large centrifuge tubes of 100 c. c. capacity. A quantity of 
potassium-citrate solution was used equal to the amount of blood drawn 
into each tube, and 0.5 gram of saponin was added to each tube in order 
to dissolve the red blood corpuscles. After a thorough shaking and after 
complete hemolysis had taken place, the tube was centrifuged for 30 
minutes at 2,500 revolutions, and the supernatant fluid was siphoned off. 
The residue, which was of an opaque color and consisted principally of 
trypanosomes, was then thoroughly mixed and shaken up with salt solu¬ 
tion, when it was again placed in the centrifuge; this washing was 
repeated three times. After the last washing the thrown-down opaque 
mass was emulsified with 50 c. c. of salt solution and titered as to its 
merits as an antigen for dourine tests. The results were highly satisfac¬ 
tory, and the titer was established at 0.5 c. c. of this emulsion per tube. 
However, the disadvantages of this method—namely, the difficulty in the 
preparation of this antigen and also the small quantity which was obtain¬ 
able from a single bleeding of a dog—were soon apparent. 
In July, 1912, the outbreak of dourine in Montana was discovered, as 
already mentioned. Several samples of blood sera from clinical cases 
were forwarded by the State authorities to the Bureau of Animal Indus¬ 
try for verification. Positive reactions were obtained in numerous 
instances with antigens thus prepared, establishing conclusively the 
presence of the disease in that State, as well as suggesting the possibilities 
of the test as a means of its eradication. It was not long before dis- 
