84 EXPERIMENT STATION EECORD. 



In view of the necessity for a ready means of diagnosis, tlie application of 

 the complement fixation method has been attempted, using some dis- 

 eased horses sent to the experiment station at Bethesda, Md. The problem of 

 greatest difficulty has been the question of an appropriate antigen. " From time 

 to time, as these animals died, certain tissues were obtained which it was sus- 

 pected might furnish the desired results, but although shake extracts of the 

 spleens, livers, kidneys, and bone marrow, as well as alcoholic and acetone prep- 

 arations, were employed under various conditions, the results were rather dis- 

 couraging." 



From the literature it appears that the best results may be obtained from the 

 use of suspensions of pure trypanosomes, " In place of the specific trypanosome 

 of dourine being utilized, the writers selected the surra organism, as it had 

 been previously ascertained by several investigators that the reaction obtained 

 was not absolutely si^ecific for any one trypanosome infection but was rather 

 of a group nature." In a part of this work, instead of straight suspensions an 

 antigen was made of the blood and macerated spleens of rats killed at the height 

 of surra infection. The material was placed in a bottle containing glass beads, 

 then shaken for 6 hours, filtered through gauze, and carbolic acid added to the 

 filtrate. 



The smallest quantity of serum from horses which gave a positive reaction 

 was 0.05 cc, but the various comparative tests indicated that fixation in tubes 

 containing 0.2 cc. of serum was sufficient for diagnostic purposes. The sera 

 from normal animals, or those affected with diseases other than trypanoso- 

 miasis, did not react. As the method of preparing antigen described above did 

 not later always give satisfactory results, antigen was prepared by drawing the 

 blood of an infected dog into centrifuge tubes containing an equal amount of 

 1 per cent potassium citrate solution. The red blood corpuscles were cytolyzed 

 with saponin, the mixture centrifuged, and the supernatant fluid drawn off. The 

 opaque mass or residue after repeated washing with sodium chlorid solution 

 was emulsified and titrated. This antigen proved very satisfactory with the 

 blood from the horses in the Montana outbreak, but a more rapid method proved 

 essential. 



" 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. . . . After repeated tests on horses clinically affected with dourine 

 had shown the antigen to be uniformly constant in its action, the procedure of 

 diagnosing dourine by this method was definitely adopted. . . . 



" Gray or white rats are infected with surra by the Injection of 0.2 cc. 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 2 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 cc. by dilution with salt solution. 

 This suspension constitutes the jintigen for the tests of the suspected dourine 

 sera. . . . Occasionally the antigen does not prove satisfactory for the test 



