IMMUNOLOGY OF LARYNGOTRACHEITIS 15 



virus in the bursa of Fabricius, although the bursal fold was closed with adhesions 

 in 2 of the roosters so that they could not be tested for the carrier stage. One 

 pullet died before the experiment was closed. Necropsy revealed the bursal fold 

 constricted so that the opening from the oviduct into the cloaca was obstructed, 

 and the bird could not lay. Two completely formed eggs were found in the 

 oviduct. 



Bacteria in Tracheal Exudates Complicate Bursal Vaccinations 



In order to study the importance of bacteria in bursal vaccinations, 36 chickens 

 six weeks old were divided into three groups of 12 each. Group 1 was inoculated 

 with Seitz-filtered infectious laryngotracheitis virus. Group 2 with streptococci 

 isolated from the tracheas of chickens and fowls (Gibbs, 1933b), and Group 3 

 with staphylococci from the tracheas of normal fowls which showed pathogenic 

 properties when the mucous membrane was injured as in infectious laryngo- 

 tracheitis (Gibbs, 1930). The same degree of infection occurred in Groups 1 

 and 2, and in three of the chickens in Group 3, so that it was impossible to dis- 

 tinguish the bacterial from the virus inoculation or "take." It was concluded 

 from this experiment that bacteria in the tracheal exudate may complicate vac- 

 cinations in the bursa of Fabricius and interfere with the determination of success- 

 ful takes. For this reason it was felt that considerable care should be exercised 

 in the selection and cultivation of tracheal exudate for vaccine. 



In the routine preparation of infectious laryngotracheal exudate for vaccine, 

 the virus should be filtered in order to free it from bacteria. It should always be 

 prepared from chickens one month old, which have been hatched in incubators 

 and reared under especially sanitary conditions away from all other birds. 



The filtration of infectious laryngotracheitis virus presents some difficult prob- 

 lems. However, if a modification of Ward's (1928) method is used the virus 

 should pass Seitz and Berkefeld filters more readily than if the exudate is tritur- 

 ated in physiological saline solution or yeast water alone. In applying the modi- 

 fied Ward method to the filtration of infectious laryngotracheitis virus, the 

 exudates were ground up in mortars with fragments of Pyrex glass, and suspended 

 in hormone broth. Before pouring the mixture into the filters the surfaces of the 

 Seitz disks were covered with a layer of broken glass. Extra fragments of 

 glass were not added to the Berkefeld filters because they could not be held 

 in place before pouring in the exudate and starting the suction. The hormone 

 broth is a modification of Huntoon's method, and it was prepared as follows: — 



Four hundred grams of minced chicken heart free from fat, ten grams of pep- 

 tone, and five grams of salt were boiled for fifteen minutes in one liter of water. 

 The meat particles were removed by straining through cheesecloth, and the 

 broth autoclaved at 15 pounds extra pressure for half an hour. It was allowed to 

 stand over night in the ice box. The next morning the supernatant fluid was de- 

 canted off^, 25 cc. to a flask, and autoclaved for 15 minutes. 



Table 11 shows the results of this experiment with Seitz and Berkefeld V, N, and 

 W filters, and indicates that hormone broth plus Ward's method of filtration is more 

 efficacious than either physiological saline solution or yeast water alone. If this 

 method of filtration had been known when the experiments on the intravenous and 

 subcutaneous inoculation of infectious laryngotracheitis were made, the results 

 might have been more encouraging. Discrepancies of this sort are to be expected 

 in a project which has not passed out of the experimental stage. 



