104 
Journal of Agricultural Research 
Vol. I, No. a 
covery was made that the disease was quite widely spread in Montana 
owing to the previous failure to recognize it. In an endeavor to comply 
with the request of the State authorities to make diagnoses in a large 
number of animals, it was soon apparent that a different method would 
necessarily have to be devised in order to make the desired progress. 
PREPARATION OF ANTIGEN 
Various organs from rats just dead from surra were tried out in both 
fresh and preserved states, and the results which were obtained from the 
fresh suspension of the macerated spleen of a rat just dead from surra 
were the most promising. In order to establish whether such an antigen 
would constantly, or at least in most instances, give the results desired, 
it was repeatedly tested on positive sera of horses affected with dourine, 
as well as on horse serum known to be free from immune bodies of 
dourine. After repeated tests on horses clinically affected with dourine 
had shown the antigen to be uniformly constant in its action, the pro¬ 
cedure of diagnosing dourine by this method was definitely adopted. 
It was at this time that our present method of preparing antigen was 
first employed, which is as follows: 
Gray or white rats are infected with surra by the injection of 0.2 c. c. 
of blood from a rabbit infected with that disease. Since tests have to be 
made every day to keep up with the large number of cases submitted 
and as the antigen proves effective only when prepared fresh, it was 
arranged that at least two rats should die daily with the disease. When 
the rats appeared to be at the point of death late in the afternoon it was 
found that placing such rats in the ice chest until they died furnished a 
better antigen than when they have died in the cage during the night 
and have to be used the following morning. 
The spleens of the rats are removed, placed in a mortar, and ground up 
with a small amount of salt solution to a pulpy mass. From time to 
time more of the salt solution is added, and the suspension thus obtained 
is filtered twice through a double layer of gauze into a test tube. The 
quantity of the suspension from each spleen is made up to 40 c. c. by 
dilution with salt solution. 
This suspension constitutes the antigen for the tests of the suspected 
dourine sera. Dr. Jacob Traum, who was temporarily assigned to this 
work, found that when the suspension was titered against sera in gradu¬ 
ated quantities from a known positive and a known negative case the 
best results were obtained, and this method has since been adopted. The 
quantity of antigen employed is double the amount necessary to pro¬ 
duce complete fixation with positive serum. The following table gives 
the method practiced in titrating the antigen: 
