trough inlets, flies were obtained from flytraps 

 placed about the feed rooms, and water insects 

 were collected in the river above the Leaven- 

 worth hatchery water inlet by routine collection 

 methods. The small mammals and portions of 

 all other collections were stored frozen; plank- 

 ton and insect samples were tested immediately 

 upon collection . TTie samples were ground with 

 levigated alumina, centrifuged, and the super- 

 natant solution diluted to approximately 1:100 

 dilution of the original sample. Quantities of 

 .05 ml. of this solution were then inoculated in- 

 traperitoneally into 10 healthy fingerlings reared 

 on the control diet. 



In September a small group of recovered 

 and a small group of healthy fish were taken 

 from the Leavenworth to the Winthrop station. 

 Here these fish and healthy and recovered finger- 

 lings reared at the Winthrop station were 

 inoculated with a bacteria -free virus filtrate 

 which varied in dilution from 1:10 to 1:10 

 and was obtained from moribund Winthrop fish . 

 Other experiments were carried out to study the 

 transmission of the disease in an attempt to es- 

 tablish the natural pattern of the virus infection. 

 Fingerlings which had survived the disease were 

 tested as possible carriers of the disease by 

 placing a group of healthy fish just below recov- 

 ered fish in the same trough. 



In the fall of the previous year the livers 

 of all adult fish spawned at the Leavenworth sta- 

 tion were tested for the virus by homogenizing 

 them with an equal quantity of nutrient broth and 

 inoculating 0.1 ml. of the homogenate into heal- 

 thy fingerlings . Thus, transmission of the virus 

 via the fertilized egg could be detected if it 

 occurred. Serial transfers, as previously de- 

 scribed, were made of those fish which became 

 moribund after inoculation with the adult Uver 

 extracts . These serial transfers were continued 

 until the first mortalities were occurring in four 

 days indicating the maximum degree of infectivity. 

 At this time the inocula, and further serial 

 transfer inocula, were filtered throu^ Mandler 

 filters prior to injection to demonstrate the 

 presence of an infectious, filterable agent. 



Concurrent with the laboratory studies 

 described, an extensive hematological study was 

 undertaken in an attempt to find a method of 

 early diagnosis of the virus infection. The re- 



sults of these studies have been reported else- 

 where (Watson et al 1956). 



RESULTS 



Leavenworth hatchery 



At Leavenworth the virus disease devel- 

 oped in only two troughs . The first outbreak 

 was recognized on May 12, the second in an 

 adjacent trou^ on June 18. The cumulative mor- 

 tality L-5/12 and L-6/18 respectively, is shown 

 in figure 1 . The fish had a length of one inch or 

 less and were four and one-half to five and one- 

 half months old. These troughs, each containing 

 about 14,000 fish, were nart of the group of 23 

 troughs which was receiving a diet containing 

 British Columbia sockeye salmon viscera. 



Winthrop hatchery 



At Winthrop the virus infection developed 

 among the fingerlings in three of the four ponds 

 retained on the viscera diet: the virus infection 

 did not develop among the fingerlings in four ponds 

 fed the control diet . Each pond was stocked with 

 about 50,000 fish. Fish in the first pond where 

 disease appeared were spawned at Lake Wenatchee 

 (Leavenworth) and the first increase in mortality 

 came on June 15; the second, stocked with Win- 

 throp-spawned fish, showed a mortality increase 

 on July 16; and the third on July 20 in a pond of 

 fingerlings spawned at Lake Wenatchee . The 

 cumulative mortality of these three ponds is shown 

 as W-6/15. W-7/16, and W-7/20 in figure 1. All 

 three ponds were receiving the diet containing 

 50 percent mixed salmon viscera . None of the 

 infected ponds was adjacent . It is improbable, 

 therefore, that the agent had been spread from 

 the first pond which became infected. 



At the time of the first outbreak the fish 

 were about five and one -half months old and 

 slightly over two inches long. By mid-July the 

 fish were seven and one -half months old and about 

 three inches in length . Confirmation of the virus 

 disease as the cause of the high mortalities in the 

 two troughs and three ponds was obtained by the 

 laboratory procedures previously described. 



