ERADICATION OF PULLORUM DISEASE 1931-32 31 



inoculated intraperitoneally with 1 cc. of the suspension; Group II (3 birds, 

 37, 38, and 40) was fed 5 cc. of the suspension by introdu('ing the pipette well 

 into the esophagus; Group III (4 birds, 30, 32, 34, and 36) was exposed by in- 

 stilling two drops, 0.03 cc, into one eye; Group IV consisted of 1 bird. No. 33, 

 which was regarded as a control. The first exposure was on March 24, and 15 

 daUy consecutive doses were given. Agglutination tests, in dilutions of 1:10 and 

 higher, were made at frequent intervals. Dilutions of 1:10 and higher were 

 employed to determine the titer. Agglutinins were detected on the fourth day 

 after the first exposure, and on the eleventh day all birds posse-ssed agglutinins. 

 The titers in some birds of Group II and III showed a rapid diminution after 

 the third week. It appeared that the organism used as the infective agent had 

 lost some of its virulence since it had been transferred daily for a period of time. 

 On May 26, 4 birds (30, 34, 37, and 40) were given a second series of exposures. 

 Each bird received 10 consecutive daily inoculations by the intraperitoneal 

 method. A strain which was isolated from guinea fowl 949 and which was not 

 subjected to frequent transfers was employed. The suspension was prepared in 

 the same manner as reported earlier. All birds exposed for the second time 

 showed a marked response in agglutinin production. No clinical manifestations 

 as a result of inoculation were observed during the experiment. However, on 

 May 31, bird 39 displayed mild but typical symptoms of laryngotracheitis. 

 The diagnosis was confirmed by inoculation of susceptible chicks which showed 

 a mild form of the disease, and shortly after recovery Dr. C. S. Gibbs of this 

 Station found these chicks to be refractory to large doses of the pathogenic virus. 

 Table 9 shows that a rise in titer of bird 39 occurred following the attack of 

 laryngotracheitis. The control bird. No. 33, revealed slight agglutination in the 

 lower dilutions on four different tests, but this was regarded as non-specific 

 agglutination. Jellied samples also caused trouble at times, but they were almost 

 entirely eliminated towards the end by use of sodium citrate solution. 



Since the birds were maintained under such close confinement and unnatural 

 conditions one was led to suspect that they would not lay eggs. However, on 

 April 18, as Table 10 shows, the first egg was laid and on May 21, all but three 

 birds had attained production. The total number of eggs recorded was 171, and 

 of this number 148 were examined bacteriologicaUy. Some eggs were broken 

 and were unfit for examination. The technique in culturing the eggs was as 

 follows: The fresh eggs were placed at 37° C. for 7 days. Then the eggs were 

 bathed in a beaker containing 5 per cent phenol for approximately 5 minutes. 

 In removing the eggs from the container, the excess fluid was shaken off and care 

 was exercised not to touch the small end of the egg. This end was heated in the 

 flame and opened with a sterile forceps. The egg was then inverted on the mouth 

 of the bottle containing approximately 50 cc. of sterile broth. The broth and 

 egg contents were mixed thoroughly and incubated at 37 ° C. for 6 days. Trans- 

 fers were made to tubes of broth on the second, fourth, and sixth days. Only 

 growth that resembled S. pullorum was tested for its biochemical, tinctorial, and 

 agglutinable characteristics. Four eggs from two birds were found infected. 

 Bird 30 did not lay infective eggs until after it had received a second exposure. 

 The last egg from which the organism- was isolated was laid on July 10, five weeks 

 after the last ex-posure. It appears that the infection in the egg was the result 

 of established systemic infection rather than an elimination of the inoculated 

 suspension from the peritoneal cavity by way of the oviduct. The infective 

 egg accounted to bird No. 29 was laid on June 4. The percentage (2.7) of infec- 

 tive eggs detected is very small as compared to percentages commonly found 

 among eggs from reacting chickens. However, even though careful technique 

 was employed in the culture work, it may be possible that infection in some 

 eggs escaped our attention. 



