| Recent years, shrimp culture suffered great damage from viral and bacterial diseases, so its sustainable development was serious constrainted. Recognizing the negative effect of antibiotic, emphasis was transfered to stimulate the immune system of shrimp. Immunological capability and health of shrimp is highly correlated to each other, fortunately, immunostimulants can enhance the immunocompetence of animal, which can contribute to health of animal, making them more immune to many kinds of diseases. So how to screen immunostimulants of shrimp became the focus of research.This study mainly included five contents. The intestinal flora of Penaeus chinensis in various growth stages and artificial infection test, and the probiotic bacteria isolated from intestinal of shrimps and its probiotics function. The purpose of these two parts was to establish the foundation of screening and combining of probiotic bacteria or bacteria which have immune activation effect. The variation rules of immune parameters of Litopenaeus vannamei challenged by white spot syndrome virus (WSSV), thus we can establish the sensitive indexes on evaluation of immunoreactive substances. At the same time, we discussed whether Procambarus clarkia can be regarded as a kind of pattern animals for screening immunostimulants of shrimp. In addition, we researched on the immunological protection of Vibrio anguillarum vaccine to Penaeus chinensis infected with Vibrio anguillarum.The intestinal flora of Penaeus chinensis in various growth stages. This experiment detected quantity and constitution of intestinal flora of Penaeus chinensis in various growth stages. The result suggested that the quantity of intestinal flora of Penaeus chinensis increased unceasingly from egg to brood shrimp in 3×107-4×108 cfu/g. The category of intestinal flora of Penaeus chinensis also increased unceasingly. The major bacteria in intestinal flora from egg to postlarvae were Bacillus. Only Bacillus contained from egg to nauplius. The bacteria category increased until zoea, Vibrio parahaemolyticus and Vibrio alginolyticus contained. Pseudoalteromonas and Flexibacter maritimus contained until mistress. Root nodule bacteria and Vibrio harveyi contained until postlarvae. The major bacteria in intestinal flora in adult shrimp and brood shrimp were Vibriol and Bacillus. Vibrio campbellii, Pseudoalteromonas, Photobacterium, Exiguobacterium Collins and Sphingomonas paucimobilis were found in these stages.Artificial infection test of the intestinal flora isolated from Penaeus chinensis. In the present study, we injected intestinal bacteria (bacterial strains PC070460, PC070461, PC070462, PC070463, PC070368, PC070370, PC070373, PC070374, PC070375 and PC070376) isolated from healthy Penaeus chinensis to Penaeus chinensis. The result suggested that the higher dose of bacterial strains PC070461 and PC070374 were pathogenic to Penaeus chinensis (P<0.05), however the lower dose reduced the cumulative mortality of Penaeus chinensis, and played a protective role to Penaeus chinensis. The bacterial strains PC070462 and PC070373 also played a protective role to Penaeus chinensi, and increased the survival rate of Penaeus chinensis obviously (P<0.05). The lower dose of bacterial strain PC070463 played a protective to Penaeus chinensis (P<0.05), while the higher dose has no protective effect to Penaeus chinensis. The bacterial strain PC070376 was a weak pathogenic bacterium, and it was pathogenic to Penaeus chinensis to a certain extent.Screening and identification of four probiotic bacteria isolated from intestinal of shrimps. We isolated a total of 96 bacterial strains from intestinal of shrimps of different growth stages, and then tested their inhibitory activities. Four stains were selected by their inhibitory activities to three pathogenic indicating vibrios, Vibrio anguillarum, Vibrio harveyi and Vibrio parahaemolyticus were tested. Confirm their inhibitory activities by solid diffusion assay and liquid blend assay. Artificial infection test showed that the four strains have no pathogenicity to shrimps. The traditional physiological and biochemical methods were applied in the bacterial classification. 16S rRNA sequence of strains were sequenced and compared with that of the correlative strains. Based on the results of molecular phylogenetic dendrogram, physiology and biochemistry characteristics, the bacterial strains LV060806, LV060808 were identified as Vibrio natriegens, the bacterial strains LV060810, LV060815 were identified as Vibrio alginolyticus.Analysis of immunological characteristics of Litopenaeus vannamei during WSSV infection. Took the Litopenaeus vannamei which challenged by white spot syndrome virus (WSSV) as materials, compared the phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (UL), antimicrobial peptides activity of the serum of shrimps in different time with chemical methods and Real-time quantitative PCR method respectively. The result suggested all the immune parameters have obviously stress reaction along with healthy degree of shrimp, and the change is remarkable. When challenged with the lower dose WSSV, PO, ACP, UL, AKP, antimicrobial peptides activity of each group were higher (P<0.05) than the control group in five days, and then, were lower than the control group or at the same level with the control group. When challenged with the higher dose WSSV, PO, UL activity of each group were higher (P<0.05) than the control group until the end of the experiment. ACP, AKP, antimicrobial peptides activity of each group were higher (P<0.05) than the control group in one day, and then, were lower than the control group. The results confirmed that the phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (UL), antimicrobial peptides activity of the serum of shrimps were all changed obviously after infected with WSSV, and the total change tendency was increased and then decreased. But the maintenance increased time varied along with different immune parameters and different infection dose. The results prompted that these immune parameters, especially gene expression of lysozyme and antimicrobial peptides, can be used as sensitive index of WSSV infection, research on the molecular mechanism of WSSV infection, and can be used as basis of screening immunoreactive substances of shrimps.Effects of WSSV on haemocyte's phagocytosis activity and hepatopancrea's phosphatase activity of Procambarus clarkia. This experiment detected the effect of White Spot Syndrome Virus (WSSV) on impacting phagocytosis activity of haemocyte, and acid phosphatase (ACP), alkaline phosphatase (AKP) activity of hepatopancreas of Procambarus clarkia. It provided the basis of changes of immune level after affected with pathogeny. The result indicated that these immune parameters of Procambarus clarkia could change along with the time of WSSV infected, and phagocytosis activity could reflect the health of shrimp indirectly. But the variational rules of ACP and AKP were not obvious, and its variational rules were various in different infect stage.The immunological protection of Vibrio anguillarum vaccine to Penaeus chinensis infected with Vibrio anguillarum. In the present study, we injected the Vibrio anguillarum vaccine killed by formalin and antibody derived from immuned Paralichthys olivaceus to Penaeus chinensis respectively, and then, injected Vibrio anguillarum to Penaeus chinensis. Calculated cumulative mortality, cumulative survival and relative percent survival(%) of Penaeus chinensis, and research the immunological protection of Vibrio anguillarum vaccine and antibody to Penaeus chinensis. The result suggested that the Vibrio anguillarum vaccine of formalin-killed and antibody both have the function of immunological protection to Penaeus chinensis, and the cumulative mortality was 25.82%, 9.09% respectively, each group were lower (P<0.05) than the control group. The immunological protection of antibody to Penaeus chinensis was obvious especially, and the relative percent survival of antibody was 80.45%.
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