8" MASS. EXPERIMENT STATION BULLETIN 295 



were shaken vigorously for fifteen or twenty minutes until the fragments were 

 broken up as much as possible. The contents of the bottles were filtered through 

 several layers of sterile cheesecloth into sterile flasks. All of the coarse particles 

 of fibrous tissue, serous and mucous membranes, and trabeculae were thus re- 

 moved. 



Each suspension of tissue was divided into four parts. Part 1 was reserved 

 for control. To Part 2 was added 40 per cent formalin until a final concentration 

 of 0.1 per cent formaldehyde was attained, and the mixture shaken and placed in 

 the ice box for twenty hours before use. A solution of carbolic acid was added to 

 Part 3 until 0.5 per cent phenol was obtained. Glycerol was shaken into Part 4 

 until a homogeneous mixture was secured, containing about 25 per cent of the 

 fluid. Two chickens six weeks old were inoculated intratracheally with each 

 preparation until the tissues from the twelve original birds on the experiment had 

 been tested and the results noted. 



The virus was found in the blood and tissues of four of the chickens soon 

 after external symptoms were noticed, lingered for about 48 hours, and disappeared 

 so that it could not be determined by intratracheal inoculation outside of the 

 respiratory tract, l Measured by the comparative mortality of the chickens 

 in the various groups, the virus in the tracheal exudate was more virulent than 

 that in the tissues. Formalin and phenol apparently destroyed the virus in every 

 preparation in which they were used. The gylcerolized tracheal exudates main- 

 tained their virulence the best of all, but this preparation has already been stutlied, 

 and it is unnecessary to discuss it further. No evidence of the virus was found 

 either in the tissues or in the blood just before death as so often occurs in bacterial 

 diseases. The chickens on this experiment apparently died of asphyxiation, due 

 to plugging of either the larynx or syrinx by pseudomembrane. 



The results of this experiment indicate that it is impossible to prepare satis- 

 factory tissue vaccines for infectious laryngotracheitis by the methods used in 

 this investigation. 



HYPERIMMUNE SERUM 

 Intravenous Inoculations 



Hyperimmune serum was prepared by inoculating pullets intravenously with 

 tracheal exudates, which had been diluted 1-10 in saline solution and filtered 

 through Seitz E. K. Schichten filters. Two cc. were inoculated into the wing 

 veins at five-day intervals until six injections had been made. Eight days after 

 the last intravenous inoculation the birds were killed, the blood drawn into sterile 

 cylinders, and the clot allowed to form at room temperature. Later the clot 

 was lifted from the sides of the cylinders by means of sterile rods, and sterile 

 weights were placed on the surfaces of the clotted blood to press the serum out. 

 After standing in the ice box for forty-eight hours the clear serum was pipetted 

 from the cylinders into a sterile bottle, 0.5 per cent phenol added, and the bottle 

 shaken until a uniform mixture was obtained. The serum was stored in the ice box 

 until the chickens were ready for the experiment. 



For these experiments 42 pullets and cockerels three months old, which had 

 never been exposed to infectious laryngotracheitis, were secured. They were 

 divided into three groups of 14 birds each, and inoculated intratracheally with 

 infectious laryngotracheitis virus. Then 10 of the chickens in each group were 

 inoculated intravenously with hyperimmune serum, while four were left as un- 

 treated controls. The birds in Group 1 received 1 cc. of hyperimmune serum on 



' In these experiments the virus appeared to be associated with the erythrocytes, and its anti- 

 body with the leucocytes and serum. Investigation is under way to determine this point more fully. 



