The following sections are an account of the experimental work 

 on this disease. 



Methods and Materials 



Inoculum 



Source„=>-The original source of the infectious agent was from dead 

 or moribund fish from naturally infected hatchery populations. In the 

 majority of the experiments, the original source of the virus was from 

 the 19^2 epizootic at Cook, the 1953 epizootics at Issaquah and Leaven- 

 worth. The inoculum was prepared from moribund fish and those that died 

 during an epizootic and from artificially infected fish inoculated with 

 serial transfers of the infectious material. Samples of the livers and 

 some of the eggs of 2,000 apparently healthy adults which returned to spawn 

 in the Little Wenatchee and White Rivers in the fall of 1953 were tested- 



Preparation.— In general, the inoculum from dead or moribund fish was 

 prepared as follows % whole fish were blended in a Waring blender or an 

 Osterizer for 30 to 60 seconds; the blended suspension was centrifuged for 

 three to five minutes at about 2,000 r.p.m.j the supernatant was diluted 

 usually 1 to 100 with sterile tap water and passed through a bacterial 

 filter. The filtered suspension was tested for the presence of bacteria 

 by streaking on a nutrient agar plate. 



The bacterial filter used routinely in experiments was a 7-pound 

 Mandler filter. Other filters used less frequently included vacuum 

 Seitz, ultrafine sinter glass, No. I4. Pasteur -Chamberlin, and Millipore. 



Frequently, healthy fish were inoculated with various dilutions of 

 the infectious agent to determine the greatest dilution that would kill 

 healthy fish. This procedure is referred to as titration. Hundredfold 

 dilutions from 10"-'- to 10 were made when infectious material was 

 titrated,, Sterile tap water was used as the diluent fluid. 



To determine the organ specificity of the infectious agent, various 

 organs were removed from infected fish, homogenized in a 50-ml. tissue 

 blender, filtered through a Millipore filter, diluted from 10"^ to lO" 1 ^, 

 and each dilution inoculated into 25 fish. 



An attempt was made to determine whether adult sockeye salmon which 

 returned to spawn in the Little Wenatchee and White Rivers could be act- 

 ing as carriers of the agent responsible for the epizootics at the sockeye 

 hatcheries. Approximately 2,000 adults which returned to spawn in these 

 two rivers were tested for this possibility by making 76 pools of their 

 livers, blending and diluting each pool to 10"^-, and inoculating the un- 

 filtered suspension into 25 healthy fingerlings. Fish which died after 

 inoculation in ten groups of fingerlings were tested to determine if the 



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