Effects Of Stress On Non-Specific Immune Cells In Pelteobagrus Fulvidraco

This is the first to study effects of stress on non-specific immune cells in yellowcatfish (Pelteobagrus fulvidraco) systematically. The normal haematological andultrastructural features of the peripheral blood cells, the histological characteristics andthe ontogenetic development of the pronephros, as well as the ultrastructural features ofthe pronephos immune cells were investigagated by both light and transmission electronmicroscope (TEM). The effects of transport stress on the percentage and function of theperipheral leucocytes were monitored Using serum cortisol concentration as main stressindicator. The pronephros macrophages were separated by Percoll continuous densitygradient centrifugation, both structural and ultrastructural features and phagocytosis of thepronephros macrophages were observed, and the effects of exogenous cortisol,macrophage activating factors (MAF) and lipopolysaccharide (LPS) on the respiratoryburst of the pronephros macrophages were further studied. The peritonitis was elicited byperitoneal injection of liquid paraffin, the types of peritoneal exudate cells (PEC) andchanges in numbers and function of PEC during the process of peritonitis wereinvestigated, and the effects of exogenous cortisol on PEC were further studied. Theeffect of exogenous COltisol on the growth and peripheral blood cell numbers in fingerlingyellow catfish was also investigated. The main results and conclusions were summarizedas follows.1. Erythrocytes, thrombocytes, lymphocytes, neutrophils and monocytes were identifiedin blood smears and confirmed by TEM. Immature erythrocytes and occasionally dividingerythrocytes were also present in smears. The thrombocytes were the most abundant celltype after erythrocytes, representing 78% in differential leucocyte counts (DLC). Thelymphocytes constituted 17.5% in DLC and had the highest nuclear: cytoplasmic ratio of all the leucocytes observed. A unique lymphocyte that was morphologically similar to thechannel catfish nonspecific cytotoxic cells (NCCs) was observed. Neutrophils, the onlytype of granulocytes identified in yellow catfish, had the largest mean cell diameter of allleucocytes and constituted 3.7% in DLC. The only type of the specific granules in theneutrophils contained fibrillous material and were ovoid or rod-like in shape. Monocytesrepresented less than 1% in DLC.2. Cessation of feeding was apparent during the first 4d to 7d after transport. All fishresumed feeding by 8d and adapted to daily management by 15d. Blood cortisol andglucose concentrations were significantly high immediately after transport (0d) and at 1d,2d and 3d. Elevated blood cortisol and glucose significantly decreased by 7d andmaintained at low levels at 21d, 28d and 35d. No significant differences were found incirculating erythrocyte numbers. Compared to values of each parameter for peripheralblood leucocytes (PBL)at 21d, 28d and 35d, lymphocytopenia was found at 3d and 7d,neutrophilia was detected at 0d, ld, 2d and 3d. Significantly decreased phagocytosis andphagocytic index of PBL were found at 7d and 14d. The above results indicate (1) thattransport induced characteristic stress responses in the adult yellow catfish, (2) thattransport resulted in significant alterations in the percentage of peripheral bloodlymphocytes and neutrophils and suppression in the phagocytic function of PBL in theadult yellow catfish, (3) that a minimum of three weeks needed for the earth-pond-rearedadult yellow catfish to completely recover from transport stress in the present laboratorysystem.3. Only renal tubules were observed on 1d post hatching (dph), undifferentiated cells andlymphocytes were observed between renal tubules on 2dph and 7dph respectively,henceforth lymphocytes and haemopoietic cells increased gradually in number. Renaltubules atrophied gradually and disappeared at last from 26dph to 43dph, and thepronephros transformed from excretory organ to lymphoid organ. The pronephros wasdevoid of capsule and rich in veins, sinuses and all types of immune cells. The adrenaltissue cells were seen around the portal vein and small vein. The pronephros of young fish contained more eosinophilic granular cells (EGC) than adult fish, while the pronephros ofadult fish contained more melanomacrophages, which usually aggregated asmelanomacrophage center near veins. The major ultrastructural characteristics of immunecells in pronephros are as follows: The ratio of nuclear to plasma and the number ofmitochondrion varied greatly among lymphocytes. One type of granulocyte and its specialgranules were the same as peripheral neutrophils, while the other type and its specialgranules were the same as EGC or mast cells reported in other fish. Monocytes andmacrophages all contained numerous lysozymes and other orgalles such as mitochondria,rough endothelia reticulae, however the latter in different functional state contained largenumbers of phagosomes of different lizes. Melanomacrophages had irregular granules,which characterized with fingerprints.4. A large number of (1.2-1.6mL per fish) high purity pronephros macrophages at aconcentration of 1×10~7cells/mL can be easily obtained by Percoll continuous densitygradient centrifugation. About 95-98% cells adhered and spread out on cover slips after3h-18h in culture were macrophages. The obtained pronephros macrophages showed themorphological characteristics of macrophages and above 95% of them had the ability tophagocytose (F-SA). PMA (phorbol-12-myristate-13-acetate) induced respiratory burst inthe obtained pronephros macrophages, which was inhibited both in vitro and in vivo aftera relatively long exposure of the pronephros macrophages to a relatively high dose ofexogenous cortisol.5. MAF-containing supernatant was produced by stimulating pronephros leucocytes fromone individual yellow catfish with 10μg/mL Concanavalin-A (ConA) and 10ng/mL PMA.The respiratory burst activity of pronephros macrophages was significantly increasedafter 24h exposure to the MAF-containing supernatant diluted by 1:4. All threeconcentrations of 12.5, 25 and 50μg/mL LPS up-regulated the respiratory burst activity ofpronephros macrophages after 24h incubation, and the respiratory burst activity increasedwith the increase of LPS levels in the medium. 6. PEC elicited by peritoneal injection of liquid paraffin included neutrophils,macrophages and EGC. Neutrophils were composed of 61±1.1% of the total neutrophilsand macrophages at 2d after injection, while macrophages constituted 77.8±2.8% and82.3±1.1% of the total neutrophils, macrophages and EGC at 6d and 10d after injectionrespectively. EGC was not seen until 6d after liquid paraffin injection and increased innumbers thereafter. The percentage of phagocytozed F-SA and nitroblue tetrazolium(NBT) reaction positive phagocytes (including peritoneal exudate neutrophils andmacrophages) increased gradually during the time of elicited peritonitis. The oraladministration of exogenous cortisol (100mg/kg diets) significantly reduced thepercentage of peritoneal exudate macrophages and the percent phagocytosis of theperitoneal phagocytes, while increased the percentage of peritoneal exudate EGC at 6dafter liquid paraffin injection.7. The main effects of long-term oral administration of exogenous cortisol (100mg/kgdiets) on fingerling yellow catfish include (1) reduced growth by reduction of intestinalmucosal folds and enhancement of gluconeogenesis, (2) reduced the number of peripheralblood leucocytes and the lymphocyte percentage in DLC, but increased the percentage ofneutrophils and monocytes in DLC.