ANNUAL REPORT, 1934 77 



time they are removed from the incubator. An attempt has been made to do this 

 experimentally, and it has been found that when the regular field vaccine was 

 used 80 percent of the chickens died of acute infection of the cloaca and Bursa 

 of Fabricius. For the successful vaccination of baby chicks, an attenuated virus 

 of uniform virulence must be used; and since no practical way of producing 

 such a standard virus has been discovered, it is impossible to successfully vaccinate 

 baby chicks on a large scale against infectious laryngotracheitis. 



Experiments with chickens at other ages indicated that they were not able 

 to withstand the field vaccine until they were two months of age, and the best 

 results were secured at three and four months of age. 



Serological Experiments with Infectious Laryngotracheitis Virus. (C. S. 

 Gibbs.) The elementary bodies occurring in the epithelial cells of the larynx 

 and trachea of birds sick or dying of infectious laryngotracheitis may be liberated 

 to a limited extent by grinding the exudate in 10 percent NaOH solution in an 

 unglazed mortar with an unglazed pestle for half an hour or longer. After allow- 

 ing the cellular debris to settle, the supernatant fluid is decanted into a centrifuge 

 tube and centrifugalized at 3200 revolutions per minute until a clear, supernatant 

 fluid is obtained. The supernatant fluid is carefully pipetted off, the tube refilled 

 with carbolyzed saline, and shaken. The washing is repeated three or four times, 

 after which the tube is centrifugalized at a moderate speed of 1200 or 1500 rev- 

 olutions per minute to throw down as much of the cellular material as possible 

 without interfering with the free elementary bodies. Unless an opalescent fluid 

 is obtained the grinding, washing, or both have not been properly done. By 

 adding an appropriate amount of immune serum to a properly prepared suspen- 

 sion of elementary bodies a flocculation test may be obtained which is specific 

 for infectious laryngotracheitis, and not for normal serum, or the serum ot birds 

 suffering from pullorum disease, fowl typhoid, chicken pox, avian paralysis, or 

 leukosis. Sometimes a slight flocculation occurs in the first supernatant fluid 

 after the first washing, but it has never been found in the fluids of the other 

 washings. 



The elementary body suspension will bind complement, when it is properly- 

 prepared and balanced in a hemolytic system consisting of sheep's corpuscles, 

 rabbit serum amboceptor, and guinea pig complement. Because of technical 

 difficulties in preparing the antigen, these serological tests have not found any 

 practical use in the control of infectious laryngotracheitis, but they are of scientific 

 interest, because they support the view of Seifried and Burnet that these bodies 

 are the virus of this disease. 



Infectious Laryngotracheitis Virus in Eggs. (C. S. Gibbs.) Infectious laryn- 

 gotracheitis virus has been passed through a series of graded acetic cellodion 

 filters, and the filtrate inoculated into eggs. It has been found that the chorio- 

 allantoic membrane of the egg may be affected by the virus and the embryo 

 killed in three to five days. In no instance has the virus been carried through 

 the incubation period of the egg. Virus introduced into eggs at the beginning 

 of the incubation period, before the chorio-allantoic membrane is formed, does 

 not live longer than 24 hours in the incubator, but may live longer than 24 hours 

 if the egg is kept in cold storage. Small quantities of the virus-filtrate introduced 

 into the air sac during the last week of incubation do not infect the embryo. 

 Large quantities of the liquid kill the embryo, but it appears to die of causes 

 other than infectious laryngotracheitis. It is believed that these studies have 

 been advanced far enough to indicate that the danger of transmitting infectious 

 laryngotracheitis through the egg is remote. 



