428 THE PARATYPHOID A. BACILLUS 



agglutinates the paratyphoid A bacillus only in low dilution (1-20 to 1-40). 

 Co-agglutinins for related organisms and especially for the typhoid bacillus are 

 as a rule also present in the serum : the amount of the co-agglutinin is very 

 variable often exceeding the titre of the specific agglutinin, and moreover may 

 be present to the exclusion of the latter. Absorption tests will alloiv of a correct 



7. Complement fixation. 



The serum of animals immunized with the paratyphoid A bacillus contains 

 an immune body which is fixed by both the paratyphoid A and typhoid bacilli, 

 but the converse does not hold good ; the immune body in the serum of animals 

 immunized with the typhoid bacillus is not fixed by the paratyphoid A 

 bacillus (Rieux and Sacquepee). 



In healthy men inoculated against enteric fever, specific amboceptors for 

 the paratyphoid A bacillus were present in amounts apparently equal to the 

 specific amboceptors for the typhoid bacillus, though in no case had the 

 individuals tested suffered from paratyphoid A fever (Grattan and Wood). 



If the dosage of antigen and antibody be carefully determined, the para- 

 typhoid A bacillus can be clearly and absolutely differentiated from the 

 typhoid and paratyphoid B bacilli by the complement fixation method 

 (H. R. Dean). 



Dean's method. Preparation of extract. Agar cultures were sown in Roux bottles, 

 incubated at 37 C. for 48 hours and then emulsified in 20 c.c. of distilled water. 

 The emulsion was tubed in quantities of 5 c.c., and in some cases heated to 60 C. 

 (this heating had no effect on the properties of the extract) ; then after being 

 thoroughly shaken it was frozen hard, and subsequently thawed slowly at room 

 temperature. After freezing and thawing twice the emulsion was shaken all night 

 in a shaking machine, again frozen and thawed twice, shaken again all night and 

 then centrifuged until the supernatant liquid was clear or only slightly opalescent. 

 The extract was then stored in the cold room. 



Antiserum. Rabbits were inoculated intra-venously four or five times at intervals 

 of 4 or 5 days with saline emulsions of 24-hour agar cultures. The animals were 

 tested on the eighth day after the last inoculation and if the serum was satisfactory 

 were bled on the ninth or tenth day. .The serum was heated at 56 C. for half an 

 hour and stored in quantities of 2 c.c. 



Complement. The guinea-pig serum was prepared on the day of use. 



Hsemolytic system. Sheep red cells and rabbit-sheep serum. 



Experimental data. The bulk of fluid in each tube was 2 '5 c.c. 0'5 c.c. of diluted 

 bacterial extract (antigen), 0'5 c.c. of diluted serum (antibody) and 0'5 c.c. of a 1 in 

 10 dilution of fresh guinea-pig serum (complement). After incubation there were 

 added 0'5 c.c. of the dilution of haemolytic serum (determined on the day of the 

 experiment) and 0'5 c.c. of a 1 in 20 suspension of washed sheep cells. 



The dilution of antiserum necessary for a differentiation experiment can be 

 ascertained by the titration of the antiserum with the homologous extract. As a 

 rule satisfactory differentiation can be obtained with the greatest dilution of anti- 

 serum which is found to give a thoroughly satisfactory reaction with the homologous 

 extract. 



With low dilutions of antiserum a marked group reaction is obtained and dif- 

 ferentiation is impossible. The group antibodies can however be removed by 

 absorption (vide supra). 



SECTION IV. THE DIAGNOSIS OF PARATYPHOID A. INFECTIONS. 



The isolation and identification of the bacillus. 



The diagnosis of a paratyphoid A infection should be based upon the 

 demonstration of the specific organism in the tissues or excreta. 



