In this thesis, we examined the effects of the exposures to anthropogenic
pollutants on the fish, primarily juvenile chinook salmon, immune system using newly
and recently developed immune assays. In addition, we developed a new assay for
measuring immunocompetence of fish. In the first chapter, the Alamar Blue assay was
developed to quantify the proliferation of chinook salmon (Oncorhynchus tshawytscha)
leukocytes. Isolated splenic and pronephric leukocytes were stimulated with different
concentration of mitogens (LPS, PWM, and ConA) for various incubation times.
Optimum cell culture conditions (cell density, mitogen concentration, and incubation
time) for the Alamar Blue assay were evaluated by comparison with flow cytometric
analysis. The Alamar Blue dye was non-toxic for leukocytes, and the assay proved to be
able to quantify the mitogenic responses using LPS, but PWM and ConA.
In the second chapter, we determined the effects and mechanisms by which p,p'-
DDE exposure might affect the immune system of chinook salmon (Oncorhynchus
tshawytscha). Isolated salmon splenic and pronephric leucocytes were incubated with
different concentrations of p,p'-DDE, and cell viability, induction of apoptosis, and
mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p'-
DDE significantly reduced cell viability and proliferation and increased apoptosis. The
effect of p,p'-DDE on pronephric leukocytes was more severe than on splenic leukocytes,
likely because pronephric leucocytes had a higher proportion of granulocytes, cells that
appear more sensitive to p,p'-DDE. The effect of p,p'-DDE on leucocytes appeared to
vary between developmental stages or season. The mitogenic response of leukocytes of
chinook salmon exposed to p,p'-DDE in vivo exhibited a biphasic dose-response
relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p'-DDE had a
significantly lower percentage of Ig+ blasting cells than controls. Our results support the
theory that exposure to chemical contaminants could lead to an increase in disease
susceptibility and mortality of fish due to immune suppression.
In the third chapter, we evaluated the direct effects of in vitro exposures to
tributyltin (TBT), widely used biocide, on the cell mediated immune system of chinook
salmon (Oncorhynchus tshawytscha). Splenic and pronephric leukocytes isolated from
juvenile chinook salmon were exposed for 6, 24, or 96 hr to a concentration range of 0.03
0.1 mg TBT 1����� in cell cultures. Effects of TBT on cell viability, induction of apoptosis,
and mitogenic responses were measured by flow cytometry. Splenic and pronephric
leukocytes in the presence of TBT experienced a concentration-dependent decrease in the
viability in cell cultures following the induction of apoptosis. In addition, pronephric
lymphocytes exhibited a greater sensitivity to TBT exposure than pronephric
granulocytes. The functional ability of splenic B-cells to undergo blastogenesis upon
LPS stimulation was also significantly inhibited in the presence of 0.05, 0.07, or 0.10 mg
1����� of TBT in the cell cultures. Flow cytometric assay with the fluorescent conjugated
monoclonal antibody against salmon surface immunoglobulin was employed for the
conclusive identification of B-cell in the chinook salmon leukocytes. Our findings
suggest that adverse effects of TBT on the function or development of fish immune
systems could lead to an increase in disease susceptibility and its subsequent ecological
implications. / Graduation date: 2004
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/32271 |
| Date | 24 July 2003 |
| Creators | Misumi, Ichiro |
| Contributors | Schreck, Carl B. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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