634 STATE BOARD OF AGRICULTURE. 



the observation of Fabyan (1) that a large percentage of pigs wall develop 

 various comphcations in less than ten days after too heav}'- an inoculation, 

 which usually terminates in death before macroscopic changes appear in the 

 spleen and liver. 



rolloT\"ing the inoculations, sufficient time was permitted to elapse for the 

 development of characteristic lesions in the spleen and liver of the pigs. An 

 autopsy was then made, all anatomical changes were noted, all spleens and 

 livers were cultured for the presence of Bad. abortus and a blood sample was 

 taken for determining the presence of complement-fixing and agglutinating 

 antibodies for Bad. abortus. 



Inoculation. A loopful of each culture was planted into tubes of liver 

 infusion broth (pH. 6.6) and incubated at 37° for forty-eight hours. One- 

 half cubic centimeter of the broth culture was inoculated intra-abdominally 

 into each of two guinea pigs ranging from 300 to 600 grams in weight. The 

 pigs were placed in cages after inoculation and observed for a period of ap- 

 proximately twelve weeks. 



Autopsy. At the end of the foregoing period the pigs were chloroformed 

 and the abdominal and thoracic cavaties exposed, and all anatomical changes 

 occurring in the organs noted. 



Bacteriological Examination, Small bits of tissue from suspicious 

 lesions, and from the spleen and liver of each pig were streaked over the 

 surface of a liver agar (pH. 6.6) plate. The plates were placed in jars in 

 which 10 per cent of the air was replaced by CO2 gas, sealed and then incu- 

 bated at 37° C for three daj^s. The presence or absence of colonies of Bad. 

 abortus was noted in each case. 



Complement-Fixation Test. The antigen for the test was prepared by 

 growing several strains of Bad. abortus on liver agar for forty-eight hours. 

 The gro"«i:rh was then removed with sterile physiological salt solution to which 

 had been added 0.5 per cent phenol, and shaken mechanically for four hours. 

 The turbidit}' of the suspension was standardized to tube three of the Mc- 

 Farland (2) nephelometer and titrated for its anticomplementary dose. The 

 unit used in the test was usually one-third of the anticomplementary unit. 



All sera were inactivated in the water-bath at 56° C for one-half hour and 

 used in amounts of 0.1, 0.04, 0.02, and 0.005 c. c, and a fifth control tube of 

 0.1 c. c. 



The tests were conducted with the " sheep-rabbit hemolji^ic system using 

 two units of hemolj^sin and 0.5 c. c. of a 2 per cent suspension of sheep cells 

 in all titrations. 



The first and second incubations were conducted in the water-bath at 

 37° C for one-half hour. At the end of the second incubation the results were 

 recorded as positive in tubes showing no hemolysis, partial in tubes showing 

 any degree of hemolysis and negative in tubes showing complete hemolysis. 



Agglutination Test. The antigen for the agglutination test was pre- 

 pared b}' gro^^ing several strains of Bad. abortus on liver agar for fortj'-eight 

 hours, removing the growth \\dth sterile physiological salt solution to which 

 was added 0.5 per cent phenol, mechanically shaking for four hours and then 

 standardizing to a turbidity of three of the McFarland (2) nephelometer. 

 It might be mentioned here that the turbidity of the antigen is a very im- 

 portant factor in the agglutination test. An antigen of a turbidity less than 

 three is very unsatisfactor3' owing to the occurrence of "pro-agglutination". 



