DIAGNOSIS OF INFECTION WITH B. PULLORUM. 11 



From some preliminary tests it was found that the Uving test fluids 

 gave little better results. For this reason it was decided to carry out our 

 work, using the living organisms in preparing the various test fluids. 



The Test Fluid. 



Before preparing any of our test fluids for these macroscopic agglutina- 

 tion reactions, all strains of Bacterium pullorum were thoroughly tested 

 out to establish their pathogenic powers. The Bacterium 'pullorum mate- 

 rial had been isolated from 7 different sources, and was designated Si 

 (Strain No. 1) S2, S3, S4, 85, Sg and S7, antt represented cultures of Bacterium 

 pullorum isolated by the author from chicks which had died of the disease 

 from an infected flock of hens in western Massachusetts; from another 

 chick, dead of the disease; from an infected flock of more than 400 hens 

 from eastern Massachusetts ; from a fresh egg laid by a hen in this infected 

 flock; from the ovarian tissue of a badly infected hen in the State of Mary- 

 land; from a chick which had died after experimental inoculation with a 

 pure culture isolated from ovarian tissue ; from a strain isolated from Con- 

 necticut epidemics and furnished to the author three years ago by Dr. 

 Rettger of Yale University. The last, or Strain No. 7, was recovered 

 from a local epidemic. These strains were all carefully examined for pur- 

 ity, and after due time were obtained in a very active state of growth. 

 Strain No. 4 was finally not used because it appeared to have lost so 

 much of its virulence. 



For testing the virulence of these 6 strains of Bacterium 'pullorum 154 

 day-old chicks, hatched July 10, 1913, were used. They were divided 

 into 7 lots, 22 in each lot. Six of these sets were inoculated with Bad. 

 pullorum and the seventh was used for control. The chicks were fed 

 sterilized food and water and were retained in wire animal cages and 

 brooded with stone jugs containing hot water. The litter used was fine 

 shavings which had been sterilized and spread in a layer over floor of 

 cages prior to putting the chicks in. Each chick in the lots to be infected 

 received \ c.c. of a physiological saline suspension of the various strains 

 of Bacterium pullorum subcutaneously. The control lot received J c.c. 

 sterile physiological salt solution administered in the same manner. 

 Chicks in pen No. 1 were inoculated with Sr, chicks in pen No. 2, with S2; 

 chicks in pen No. 3, with S3; chicks in pen No. 4, with So; chicks in pen 

 No. 5, with Se; chicks in pen No. 6, with S7; and the chicks in pen No. 7 

 were the controls. 



As soon as chicks died they were carefulty autopsied and the liver, 

 heart, unabsorbed yolk and calcar examined for presence of Bacterium 

 pullorum. In Table 6 are arranged the mortality records which fur- 

 nish the evidence of the pathogenicity of these various strains. From 

 each chick, dead of the disease, cultures were retained, and in Table 

 6 P. signifies that the cultures were recovered from the respective 

 organs in an absolutely pure state. Wherever there is a denotation N.P. 

 it signifies that culture recovered was not pure. However, in no case did 



