Graduate Thesis Or Dissertation
 

Characteristics of two salmonid fish cell lines : glycolytic rates, CO₂ requirements and susceptibility to Reovirus and Reovirus-like agents

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  • The research presented in this thesis is concerned with the characterization of two salmonid cell lines, CSE 119 derived from embryos of coho salmon (Oncorhynchus kisutch) and STE 137 derived from embryos of steelhead trout (Salmo gairdneri). The rates of glucose utilization and lactic acid production during the most active growth phase of each cell line were determined and the results were compared to data from parallel experiments using a human embryonic cell line. The salmonid cells were grown at 23° C and the human cells at 35° C. All cells were grown in Eagle's minimal essential medium supplemented with 20% dialyzed newborn agamma calf The Q[superscript]O₂[subscript]CO₂ serum. values (equivalent microliters CO₂ produced per mg dry weight of cells per hour) were calculated for each cell type, and values of 7.97 and 4.50 were obtained for CSE 119 and STE 137, respectively. These values were somewhat lower than the value of 13.19 calculated for the human cells. In all three cell lines studied, the rates of glucose utilized and lactic acid produced per cell decreased over the growth periods observed. The decreases in the salmonid cells were most likely related to a concomitant increase in population density. In addition, it was shown that the very gradual fall in pH observed during the growth of the salmonid cells is apparently due in part, at least, to the smaller amount of lactic acid produced by these cells. During the course of the studies with the salmonid cell lines, an investigation of the CO₂ requirements of these cells was undertaken. For these experiments, Tris buffer was used to replace the bicarbonate in the medium and 20% dialyzed serum was again used. Results from the salmonid cells grown at 18° C were compared to parallel experiments using He La cells grown at 35° C. It was shown that growth under 2% (salmonid) and 3% (He La) atmospheric CO₂ was comparable to growth in stoppered cultures for CSE 119, STE 137 and the He La cells. The salmonid cells showed good growth in cul- tures that were open to the air (0.03% CO₂), but little or no growth occurred in the He La cells under the same conditions. To determine if CO₂ was actually required for growth of the salmonid cells, CO₂ free cultures were prepared using Conway microdiffusion dishes in which the cells were grown in the center well and a 10% solution of KOH was added to the outer well. The cultures were sealed with high vacuum grease. Under these conditions, both salmonid cell lines demonstrated a growth requirement for CO₂ comparable to that shown by the He La cells. Attempts were made to use oxalacetate to substitute for CO₂ in these cultures. Oxalacetate partially substituted for CO₂ in the He La cell cultures, but little or no growth occurred in the salmonid cell cultures under the same conditions. As part of a continuing effort to determine the viral susceptibility of salmonid cell lines, the coho cells (CSE 119) and the steelhead cells (STE 137) were tested for their susceptibility to Reovirus types 1 and 3, Infectious Pancreatic Necrosis (IPN) virus and Wound Tumor virus (WTV). Neither salmonid cell line nor He La cells were shown to produce infectious virus when inoculated with either reovirus type at 26° C. The salmonid cells would not tolerate 30° C for even short periods of time. Good replication of both reovirus types occurred in He La cells at 35° C. IPN virus replicated well in both salmonid cell lines at 18° C. Phase contrast studies of IPN infected salmonid cells showed an early webbing of the cytoplasm, followed by a rounding up of the cells, shrinkage of the nuclei and a heavy margination of nuclear chromatin. Because of its similarity to the reoviruses and its ability to replicate in an insect cell line, attempts were made to infect the salmonid cells with WTV at 23° C. No indications of WTV inclusion bodies or of a cytopathic effect were found in either salmonid cell line.
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