Russo Riccardo, Shoemaker Craig A, Panangala Victor S, Klesius Phillip H
Aquatic Animal Health Research Unit, United States Department of Agriculture, Agricultural Research Service, 990 Wire Road, Auburn, AL 36832, USA.
Fish Shellfish Immunol. 2009 Mar;26(3):543-52. doi: 10.1016/j.fsi.2009.02.011. Epub 2009 Feb 21.
Macrophages from catfish vaccinated with an Edwardsiella ictaluri vaccine and macrophages from non-vaccinated catfish were used in in vitro and in vivo studies with red-fluorescent E. ictaluri to assess phagocytic ability, reactive oxygen and nitric oxide production and bactericidal activity. In the in vitro experiment, macrophages were harvested from vaccinated and non-vaccinated fish and then exposed to red-fluorescent E. ictaluri. Results of this study showed that E. ictaluri can survive and replicate in macrophages from non-vaccinated catfish (relative percent killing, RPK, from 0.011 to 0.620 and from -0.904 to 0.042 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) even in the presence of reactive oxygen and nitrogen products. Macrophages from vaccinated fish were significantly (p < 0.05) more efficient in killing E. ictaluri (RPK from 0.656 to 0.978 and from 0.011 to 0.620 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) and produced significantly (p < 0.05) higher amounts of ROS (10-fold increase) and nitrogen oxide (about 10-fold increase) than macrophages from non-vaccinated fish. In the in vivo experiment, vaccinated and non-vaccinated catfish were injected with red-fluorescent E. ictaluri to allow the interaction between macrophages and other components of the immune system. After 6h, macrophages were harvested from the fish and seeded in glass chamber slides and bactericidal activity was measured in vitro. Results showed in vivo interaction of other components of the immune system enhanced bactericidal activity of macrophages from vaccinated fish. In another set of experiments, catfish were intraperitoneally injected with fluorescent bacteria opsonized with immune serum or non-opsonized and necropsied in the first 48 h after bacterial challenge to observe localization of E. ictaluri between vaccinated and non-vaccinated catfish. Vaccinated fish were able to control the dispersion of E. ictaluri in the body and red-fluorescent bacteria were observed only in the spleen, anterior and trunk kidney. In non-vaccinated fish E. ictaluri was able to replicate and invade all organs with the exception of the brain. We further determined that macrophages seeded with E. ictaluri could cause infection in non-vaccinated fish upon reinoculation with in vitro infected-macrophages. Overall, the results indicated that macrophages from vaccinated fish are activated and responsible for rapid clearance of infection upon re-exposure to virulent E. ictaluri.
用迟钝爱德华氏菌疫苗接种的鲶鱼巨噬细胞和未接种疫苗的鲶鱼巨噬细胞，被用于针对红色荧光迟钝爱德华氏菌的体外和体内研究，以评估吞噬能力、活性氧和一氧化氮的产生以及杀菌活性。在体外实验中，从接种疫苗和未接种疫苗的鱼体中采集巨噬细胞，然后使其接触红色荧光迟钝爱德华氏菌。本研究结果表明，迟钝爱德华氏菌能够在未接种疫苗的鲶鱼巨噬细胞中存活并繁殖（巨噬细胞与细菌比例分别为1:20和1:100时，相对杀菌率从0.011至0.620以及从-0.904至0.042），即便存在活性氧和氮产物。接种疫苗的鱼的巨噬细胞在杀灭迟钝爱德华氏菌方面显著更高效（巨噬细胞与细菌比例分别为1:20和1:100时，相对杀菌率从0.656至0.978以及从0.011至0.620），并且与未接种疫苗的鱼的巨噬细胞相比，产生的活性氧显著更高（增加了10倍），一氧化氮也显著更高（增加了约10倍）（p<0.05）。在体内实验中，给接种疫苗和未接种疫苗的鲶鱼注射红色荧光迟钝爱德华氏菌，以使巨噬细胞与免疫系统的其他成分相互作用。6小时后，从鱼体中采集巨噬细胞并接种到载玻片的玻璃小室中，然后在体外测量杀菌活性。结果显示，免疫系统其他成分的体内相互作用增强了接种疫苗的鱼的巨噬细胞的杀菌活性。在另一组实验中，鲶鱼腹腔注射经免疫血清调理或未经调理的荧光细菌，并在细菌攻击后的前48小时进行剖检，以观察接种疫苗和未接种疫苗的鲶鱼中迟钝爱德华氏菌的定位。接种疫苗的鱼能够控制迟钝爱德华氏菌在体内的扩散，仅在脾脏、前肾和躯干肾中观察到红色荧光细菌。在未接种疫苗的鱼中，迟钝爱德华氏菌能够繁殖并侵入除大脑以外的所有器官。我们进一步确定，接种迟钝爱德华氏菌的巨噬细胞在再次接种体外感染的巨噬细胞后可导致未接种疫苗的鱼感染。总体而言，结果表明接种疫苗的鱼的巨噬细胞被激活，并负责在再次接触强毒迟钝爱德华氏菌时迅速清除感染。
