488 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 



states, and may occur in such various and atypical forms as to render 

 clinical diagnosis difficult. 



Complement-fixation methods of diagnosis have been tried by Pav- 

 losvici, Winkler and Wyschelersky, Moller, Watson, Brown, and in a 

 large series of cases with good results by Moller, Eichhorn, and Buck. 1 

 These last-named investigators examined 8657 specimens of blood from 

 horses in Montana and North and South Dakota, and of these, 1076 

 yielded positive reactions. 



In most of these experiments the results were corroborated by clinical 

 and pathologic findings, and the investigators conclude that the com- 

 plement-fixation test is of great value, especially in countries where only 

 one of these protozoan diseases exists. 



Preparation and Standardization of Antigen. This is the most 

 difficult part of the technic, because the trypanosome is not readily 

 grown on artificial culture-media. Watery, alcoholic, and acetone 

 extracts of various organs of horses dead of the disease do not yield 

 satisfactory antigens. Since the reaction is a group reaction, and 

 dourine is the only trypanosome infection in this country, Moller, 

 Eichhorn- and Buck selected the surra organism for the preparation of 

 antigen. After infecting a dog and at the height of infection with- 

 drawing 200 c.c. of blood into potassium citrate and hemolyzing with 

 0.5 gram of saponin, the trypanosomes were secured after thorough 

 centrifugalization and washed three times. After the last washing the 

 trypanosomes were emulsified in 50 c.c. of salt solution and preserved 

 with phenol. This antigen yielded highly satisfactory results, but the 

 difficulty of preparing it, and the small quantity secured, made it 

 necessary that another method be used. 



An extract of the spleen of a rat just dead of surra was found to yield 

 a satisfactory antigen. The extract does not keep well, and must be 

 prepared freshly every few days and carefully standardized. Gray or 

 white rats are infected with surra by injecting 0.2 c.c. of blood from a 

 rabbit with this disease. If a large number of tests are to be made, the 

 rats should be so infected that one or two are available each day for the 

 preparation of the antigen. 



The spleen from a rat with a small amount of salt solution added is 

 ground in a mortar until a pulpy mass results. More of the salt solution 

 is added from time to time, and the suspension thus obtained is filtered 

 twice through a double layer of gauze and diluted with salt solution to 

 40 c.c. 



1 Amer. Jour. Veter. Med., 1913, viii, 581. 



