26 MASS. EXPERIMENT STATION BULLETIN 370 



were also the other reactions of fowl paralysis and neoplasia in the experimental 

 chickens which, as has been previously discussed, these workers believed due to a 

 common etiologic agent. 



Verne, Oberling, and Guerin (207) made tissue cultures of the bone marrow of 

 birds in which leukosis had been induced. One culture of the marrow of a bird 

 infected with the leukosis strain of Engelbreth-Holm was virulent on the 15th 

 day of incubation. Another culture was virulent on the eighth day only, although 

 a mild form of the disease was produced by this culture on the 15th day in one of 

 six chickens which received it. A culture of the marrow of a bird which had been 

 inoculated with the Oberling and Guerin strain of leukosis produced the disease 

 in one of two chickens inoculated on the eighth day. Material of the same culture 

 taken on the 15th day of incubation produced only a mild reaction of the blood 

 in three of six birds inoculated with it. Leukotic blood or marrow kept in sealed 

 tubes at the same temperature as the tissue cultures served as controls and none 

 of the birds receiving this material developed the disease. 



Furth and Stubbs (86) reported that tissue cultures of a tumor induced with 

 their Strain 13 agent were capable of producing sarcoma or a combination of 

 sarcoma and erythroblastic leukosis on the 12th, 23rd, 35th, and 67th days of 

 incubation at 39° C. More recently Furth and Breedis (84) have found that the 

 agent of this Strain 13 was active after 158 days in a culture of the sarcoma, 

 whereas in a culture of normal fibroblastic cells the agent died within two weeks. 

 A culture of the tumor produced by Strain 13 (osteochondrosarcoma) produced 

 fibroblast-like cells. When this culture was introduced into chickens after 91 

 days of cultivation it caused both the tumor and the reaction usually obtained 

 with the Strain 2 agent from which Strain 13 was derived. The cells in liquid 

 cultures of the bufTy coat of leukotic blood (the leukosis being produced by the 

 Strain 1 agent) were still able to cause erythroblastic leukosis after 32 days of 

 incubation. Cultures of the marrow of birds affected with Strain 1 yielded only 

 fibroblastic cells which were without ability to produce disease. The agent of 

 Strain 1 likewise perished in cultures of sarcoma cells (Strain 11). 



RuffiUi (174 a, 174 b) has reported that the myocardium from a chicken affected 

 with erythroblastic leukosis yielded a culture of fibroblasts which retained the 

 ability to produce leukosis in chickens for 44 passages over a period of 122 days. 

 Ruffilli (174 g) also made serial cultures, in normal plasma with 15 percent em- 

 bryonic extract, of marrow obtained from a chicken inoculated 38 hours pre- 

 viously with the leukosis agent of Oberling and Guerin. The marrow itself was 

 infective for chickens and on the tenth day of life in vitro (third culture passage) 

 the culture produced leukosis in one of two chickens into which it was implanted. 

 Ruffilli (174 h) also reported that a culture of fibroblasts seemed to carry the 

 leukosis agent which had been associated with it during cultivation. His experi- 

 ment was carried out by using normal plasma and 20 percent embryonal extract 

 as a nutritive fluid. Fragments of heart muscle from a newly hatched chick were 

 planted in the culture media and beside each portion of muscle was placed a bit 

 of the buffy coat of leukotic blood. Fibroblasts only were present in the cultures 

 after the tenth day in vitro (fourth transplant passage). On the twenty-second 

 day in vitro (ninth transplant passage) three cultures were injected into each of 

 two chickens. Both died with leukosis after 18 and 35 days. The use of cell free 

 leukosis agent instead of buffy coat in the above experiment would have ruled 

 out the possibility of the leukotic cells themselves becoming transformed into 

 the infective fibroblasts. 



The reaction of tissue cultures nourished with plasma from chickens with 

 leukosis and from chickens bearing Rous sarcoma was studied by Ruffilli (174 b, 



