| In this paper, the ability and mechanism of antibacterial and immune defense ofChinese shrimp was studied using cytological, biochemical, molecular biological andproteomic methods. A sensitive and stable antibacterial and lysozyme assay methodswere selected. The effect of Vibrio anguillarum and laminarin (a kind of beta-1,3-glucan) on the immune ability of haemolymph of Chinese shrimp was analyzed.The changes of total haemocyte counts, antibacterial ability, lysozyme activity andlysosome membrane stability were detected. The cDNA sequences of lysozyme LYZ and arginine kinase (AK) were cloned and their expression was studied. Theresults showed that: Total haemocyte counts (THC) showed significant change in shrimp afterimmune stimulation. The THC showed sharply decrease after injection with 1 10-7bacteria. The lowest THC value was decreased by 73% relative to control group,which appeared at 45min (p<0.05) after dead V. anguillarum injection, then increasedand recovered to the non-injection level at 48h after injection. The THC of theshrimps injected with laminarin was higher than those of control group. The highestvalue appeared at 3h (p<0.05) after injection, which increased by 100% relative tocontrol group at the corresponding time point, then decreased and recovered to that ofthe control level at 12h after injection. Mock injection also caused increase in THC,however, a rapid return to the pre-injection levelafter injection. The in vitro effect of different dose of laminarin on stability of lysosomedemonstrated the NRR time showed increase when laminarin concentration between 1 V 10-5 mg/ml to 0.1mg/ml, the longest NRR time was 205min and the correspondingconcentration of laminarin was 1 10 3mg/ml. However, the NRR time was shorterthan that of control group (NRR time was 81 min) when laminarin concentration was1mg/ml. Injection with 0.1mg laminarin caused a significant increase in lysosomalstability. The longest neutral red retention time was shown at 3h (p<0.05) afterinjection with laminarin and the retention time was 180min. Then the neutral redretention time decreased, but it was still longer than that of the control group up to24h. The NRR time of the non-injected group was about 80min. Injection with shrimpsaline caused no significant variation of NRR time. The haemocyte lysosomemembrane stability decreased significantly after mixed dead Vibrio in vivo orinjection with it in vitro. The results of selection of sensitive antibacterial method showed that the mostsensitive antibacterial method was microtiter broth dilution assay, while inhibitionzone was the most insensitive method. However radial diffusion assay was proved tobe more effective to the shrimp plasma. Using improved antibacterial method, theantibacterial activities of haemocytes and plasma of Chinese shrimp were testedbefore and after injection with dead V. anguillarum. The results showed that the totalhaemocyte counts of F. chinensis significantly decreased and the antibacterial activityof haemocytes increased significantly (p<0.05) after challenging with V. anguillarum(p<0.05). However, there was not obviously changing in plasma (p>0.05). The resultssuggested that the expression of antibacterial factors in haemocytes were greatlyincreased after immunostimulation. The lysozyme-like activity was detectable in haemocyte, but no activity wasdetected in plasma of control group. Increased haemocyte lysozyme-like activity wasobserved in Chinese shrimp as a response to dead Vibrio, laminarin. Increasedlysozyme-like activity was detected in plasma after dead Vibrio injection. But noactivity was detectable in plasma after laminarin injection. Mock injection also causedincrease in lysozyme activity, however, no lysozyme activity was detected in plasma.Using Rotofor? cell, two lysozyme-like activity fractions with different isoelectronicpoint, abou...
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