感染溶藻弧菌对日本蟳肝胰腺免疫功能的影响

摘 要：为探讨日本蟳感染溶藻弧菌的致病机理，采用溶藻弧菌人工感染健康日本蟳，分析了感染前后日本蟳肝胰腺 PO、SOD和LSZ活性变化及注射免疫多糖后的免疫保护率，并研究了细胞超微结构的病理组织学。结果表明：日本蟳感染溶藻弧菌后，肝胰腺中PO、SOD和 LSZ的活性均受到不同程度的影响，感染6~12 h，3种酶的活性均有一个上升的过程，但之后随时间的延长呈现下降趋势；免疫感染组因感染弧菌前注射了免疫多糖，日本蟳肝胰腺中PO、SOD和LSZ酶活性均显著高于感染组的酶活性，感染7 d后的免疫保护率高达72.73％，表明免疫多糖可提高酶的活性，使机体的抗病菌能力增强。超微结构显示：对照组日本蟳肝胰腺细胞结构形态正常，上皮细胞表面的微绒毛排列整齐，各细胞器结构完整；免疫组内质网、糖原颗粒和线粒体丰富，微绒毛排列致密；感染组肝胰腺组织受到明显的损伤，细胞和部分微绒毛脱落、受损；线粒体和粗面内质网变形、水肿或仅剩一空泡；核高度异染色质化，核膜破裂。与感染组相比，免疫后感染组具有形态结构较正常的细胞核、整齐致密的微绒毛和丰富的溶酶体，但依然可见水肿的内质网和狭长畸变的线粒体。

Effect of Vibrio alginolyticus infection on immune function of Charybdis japonica hepatopancreas

Charybdis japonica is widely distributed along the coasts of China,Japan,Korea and Southeast Asia.Desired for its good taste as well as being rich in protein and low in cholesterol,this crab is currently one of the most important commercially farmed crabs in the world due to its rapid growth and easy culture methods.During the last two decades with the constantly expanding scale of aquaculture and the rapid development of intensive culture techniques in China,the environmental and health problems in C.japonica cultures have resulted in outbreak of infectious diseases.The worldwide shrimp/crab culture industry has suffered significant economic losses due to diseases mainly caused by vibrios,particularly Vibrio alginolyticus,one of the most detrimental pathogens widely disseminated in crabs.In recent years,the treatment of bacterial diseases of aquatic animals has relied on application of large quantities of antibiotics,which not only causes development of drug resistant pathogens but also presents a health risk to humans.Improving immunity and disease resistance in crustaceans is one of the fundamental measures for preventing and controlling disease.In this study,the pathogenic mechanism of a Gram-negative bacterium Vibrio alginolyticus in the stone crab Charybdis japonica was explored by investigating activities of enzymes related to innate immune function during infection,i.e.phenoloxidase(PO),superoxide dismutase(SOD) and lysozyme(LSZ).Four groups of crabs were tested by intramuscular injection of C.japonica:control group,normal saline; immunity group,0.5% polysaccharide; infected group,normal saline plus V.alginolyticus challenge 24 h later; immunized infected group,0.5% polysaccharide plus V.alginolyticus challenge 24 h later.After 6-12 h of exposure to V.alginolyticus,the activities of PO,SOD and LSZ increased in C.japonica hepatopancreas,but then decreased over longer infection time.Interestingly,enzyme activities in crabs treated with polysaccharides before V.alginolyticus challenge were higher than those in infected group,showing an immunoprotective rate of 72.73% at 7 days after exposure.This phenomenon suggested that the polysaccharides could enhance the organism's antiviral defenses by improving activities of immuno-enzymes.Morphological examination by transmission electron microscopy revealed significant damage in the hepatopancreas of V.alginolyticus- infected crabs,showing parts of the hepatopancreas epithelium and microvilli were reduced in number and size or damaged.Meanwhile,morphology of the mitochondria and rough endoplasmic reticulum(RER) changed.Moreover,heterochromatinized nuclei could be seen in hemocytes with ruptured nuclear membranes.The structure of hepatopancreas nuclei in immunized infected group was normal compared with that in infected group,and the microvilli arranged densely in neat rows.The lysosomes in hepatopancreas cell of immunized infected group increased; however,dropsical endoplasmic reticulum,thin cytoplasm and distorted mitochondria could still be seen.