Fig. 1. Peripheral total blood cell counts (A) from tilapia control (0.0 mg/kg), 10.0 and 50.0 mg/kg azathioprine dose groups. Percent lymphocytes (B) for each dose group from leukocyte differential counts performed from whole blood smears are reported. Numbers are expressed as mean ± SEM from a representative experiment with five fish per dose group (^*p < 0.05, Dunnett's t-test).

Fig. 2. Splenic/body weight ratios (A) from tilapia exposed to 10.0 or 50.0 mg/kg azathioprine. Total cell spleen counts (B) are reported for the 10.0 and 50.0 mg/kg azathioprine dose groups. Each bar reflects mean ± SEM for five fish per group. (^*p < 0.05, Dunnett's t-test).

Fig. 3. Pronephros total cell counts from tilapia exposed to 10.0 mg/kg and 50.0 mg/kg azathioprine dose groups. Each bar represents the mean ± SEM of five fish per group (^*p < 0.05, Dunnett's t-test).

Fig. 4. Light scatter analysis of the isolated pronephros leukocytes showed a decline in progenitor cells of azathioprine exposed fish. The above histograms are representative of the control (A) and 25.0 mg/kg dose azathioprine lymphoprep-separated pronephros leukocytes (B). The y-axis depicts cell number (counts) and the x-axis depicts cell size (μm). The cursors are gated on 6.14–10.55 μm size range, and values are reported as a percentage of the total cells counted.

Fig. 5. Cyto-centrifugation preps from pronephros isolated leukocytes. (A) The leukocyte population in the pronephros of the control and 10.0 mg/kg dose groups were not remarkably different. Numerous macrophages (M), lymphocytes (L), and progenitor cells (PC) were observed. Two of the apoptotic cells seen in controls (arrows) are shown. (B) The pronephric leukocytes from the 50 mg/kg dose group demonstrated significant morphological changes, including great variability in the appearance of the progenitor cells (PC), and vastly increased numbers of apoptotic cells (arrows). Modified Wright Stain; original magnification 200x.

Fig. 6. Determination of apoptosis with 7-AAD in 2 h cultured isolated pronephric and splenic leukocytes. Representative histograms (A) show peak fluorescence from forward scatter to side scatter histogram. The y-axis represents cell number and the x-axis represents fluorescent staining intensity. The percentage of viable (low intensity), early apoptosis (moderate intensity) and late apoptosis/necrosis (high intensity) cells correspond to gates B, C, and D, respectively.

Fig. 7. (A) Histopathology of a control spleen. Red pulp (RP) and while pulp (WP) areas are readily distinguishable. The RP is dominated by erythrocytes, and the WP by stromal cells. A melanomacrophage (MC) center is present, adjacent to a small artery (A) and vein (V). (B) Representative spleen from a 50 mg/kg – exposed fish. The WP is greatly expanded at the expense of the RP, reflected by an increase of stromal white pulp cells and a loss of erythrocytes. Vacuolization and cavitation of many of the stromal white pulp cells is evident, and pyknotic nuclei of several necrotic stromal cells (arrows) are also visible. Several isolated melanomacrophages (M) are also present, but no organized melanomacrophage center is present in this section. Masson's trichrome; original magnification 80x.

Fig. 8. (A) Histopathology of control pronephros. The tissue is densely cellular and even, with few areas of tissue fracture. Lymphoid elements (L) are numerous, with adjacent centers becoming confluent and forming lymphoid cords (C) within the pronephric stroma. Groups of erythrocytes (E) are visible among mingled stromal elements supporting cells of non-lymphoid lines. Isolated melanomacrophages (M) are also visible. (B) A representative pronephros from a 50 mg/kg-exposed fish, the cellular layer is fractured in several places. The general appearance of the tissue is darker than the control, due to the distribution of cells within the stromal areas that are smaller in size and denser-staining than in control (arrows). Lymphoid tissue is greatly reduced, and cords of lymphoid tissue are not seen. A large melanomacrophage center (MC) is present. Masson's trichrome; original magnification 80x.