204 TECHNICAL BULLETIN 14 



peared on the comb and eyelids. Those birds, in particular, which de\eloped 

 local and generalized reactions after the vaccine injection, demonstrated a 

 complete immunity after check inoculation on the comb with the virus. 



This vaccine was labeled the triple streiigtli vaccine because it contained 

 three times as much virus per dose as that recommended by Beach (1). tfn- 

 attenuated vaccines of 10, 15, 20, and 25 milligrams of virus per dose were 

 also used. They failed to produce the same degree of immunity as did tho 

 triple strength vaccine. 



-Daily rectal temperatures and weiglit readings of the birds in the triple 

 strength vaccine experiment were taken. It was found that a prevaccinating 

 average normal temperature of 106.6° F. to 107° F. was increased, beginning 

 late on tiie day of vaccination and reaching a crisis of 108.6° F. to 109.5° F. 

 three days later, then decreasing until it iiad regained normal temperature 

 on the ninth day after the injection. A noticeable effect on the weiglit of the 

 birds occurred. The cockerels were from five to six months old and gaining 

 in weight on the average of from 10 to 20 grams per day prior to the vaccina- 

 tion. After the vaccination this gain in weight was either retarded for eleven 

 to twelve days or decreased from an average of 1480 grams to 1420 grams 

 during the same period. Thereafter the birds regained the average of 1480 

 grams and steadily increased in weiglit 



These reactions on the part of the l)od\ to tlie vaccine injection indicated 

 that the body was developing its protective forces against tlie disease and 

 from all probabilities these forces were specific antibodies. 



Complement-fixation Test. 



Anti(ien. 



Sweet (3) concluded, in his work with tlie complement-fixation test in con- 

 tagious epithelioma, that the antigen which he was using lacked marked anti- 

 genic properties. He did not attempt to impro\e his antigen in this respect. 



In this investigation a polyvalent, active virus was always used. An antigen 

 composed of 1 gram of the virus, thoroughly triturated in 100 cc. of pliysio- 

 logical saline solution and passed first through infusorial earth and next 

 through filter paper, was but slightly antigenic. Alcoholic extracts were pre- 

 pared from pulverized pox scabs and diphtheritic membranes, from pulverized 

 liver from pox infected birds, and from pulverized normal chicken hearts. 

 They were found to be without value as antigens. 



The antigen finally selected as most suitable and perhaps the only possible 

 reagent for this purpose, was 1.5 grams of jjowdered pox virus, thorougiily 

 triturated in 100 cc. of 0.5 per cent phenolized physiological saline solution 

 and passed through ordinary filter paper. Its hj^drogen ion concentration 

 was varied in an attempt to improve the antigenic properties. The original 

 concentration of approximately 5.8 to 6.0 was the most satisfactory. Prior to 

 its titration for antigenic and anticomplementary properties, it was heated 

 at 60° C. for forty-five minutes. 



A known four-plus positive serum was, of course, not available for use in 

 titrating the antigen for its antigenic properties. Varying doses of serum 

 and dilutions of antigen, and finally undiluted antigen, were used and a 

 degree of fixation obtained. It was necessary to use a two-plus serum in 

 the titration as a substitute for a four-plus serum. The reason for this will 

 be apparent when the results of the complement-fixation test are discussed. 

 In the antigenic titration of the antigen, 0.1 cc. of the undiluted antigen 

 was found to be the titre or unit dose. Four imits of the imdiluted antigen 

 were used as a working dose in the test. 



