Villamil L, Figueras A, Aranguren R, Novoa B
Instituto de Investigaciones Marinas, CSIC, Vigo, Spain.
J Fish Dis. 2003 Jun;26(6):321-9. doi: 10.1046/j.1365-2761.2003.00464.x.
The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo. The in vitro treatment of head kidney (HK) macrophages with viable V. pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response. In vivo, the intraperitoneal injection of V. pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response. The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V. pelagius (5 x 10(3) and 5 x 10(4) bacteria mL(-1)) and higher doses of the heat-killed bacteria (5 x 10(4)-5 x 10(6) bacteria mL(-1)). In both cases, the NO inhibitor N-omega -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments. In contrast, incubation with ECPs at higher doses caused a reduction in NO production. In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection. Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection. In addition, viable V. pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria. As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay. The results showed that growth of V. pelagius was significantly inhibited using SNAP at a high concentration (1 mM).
在大菱鲆幼鱼大规模死亡期间分离出的致病性浮游弧菌，对大菱鲆（Scophthalmus maximus (L.)）巨噬细胞非特异性免疫反应的影响进行了体内和体外研究。与未处理的巨噬细胞相比，用活的浮游弧菌体外处理头肾（HK）巨噬细胞会导致化学发光（CL）反应显著抑制，而与热灭活细菌孵育则不影响该反应。在体内，与对照鱼相比，腹腔注射浮游弧菌导致感染后第1天和第4天感染鱼的CL反应显著抑制。体外与中等剂量的活浮游弧菌（5×10³和5×10⁴个细菌/mL⁻¹）和较高剂量的热灭活细菌（5×10⁴ - 5×10⁶个细菌/mL⁻¹）孵育可增强HK巨噬细胞一氧化氮（NO）的产生。在这两种情况下，NO抑制剂N-ω-硝基-L-精氨酸能够下调与细菌处理孵育引起的特异性NO诱导。相反，用较高剂量的胞外产物（ECPs）孵育会导致NO产生减少。在体内，感染后第10天巨噬细胞上清液中也观察到NO产生显著增强。注射后第4天和第10天，实验感染鱼血清中的溶菌酶浓度也显著增加。此外，活的浮游弧菌及其ECPs在体外显著降低HK巨噬细胞活力，而与热灭活细菌孵育期间活力未观察到显著差异。由于实验感染鱼中NO产生增强，在无细胞试验中体外测试了NO供体S-亚硝基乙酰青霉胺（SNAP）的抑制作用。结果表明，高浓度（1 mM）的SNAP可显著抑制浮游弧菌的生长。
