| Vibrio has been widely recognized as a primary pathogen of many commercially cultured invertebrate species the world over. Shrimp culture problems associated with Vibrio has a direct effect on production, feed efficiency, and growth performance, which may limit shrimp culture development. Litopenaeus vannamei is very important in shrimp culture. With the rapid development of shrimp culture, aquaculture environment has been deteriorating, and vibriosis has become the main factors to limit the development of shrimp-culture industry.This study was divided into two parts,The first part based on the Cox proportional hazard model evaluated the effects of bacterial dosage, infection route and Vibrio strain in experimental challenges of Litopenaeus vannamei. While considerable research has revealed many important factors affecting outbreaks of vibriosis, many additional aspects of the dynamics of associated mechanisms need to be explored. A greater understanding of the interaction between Vibrio and shrimp mortality has been achieved, which should prove to be helpful when attempting to control the risk factors associated with the experimental challenge. In this work, Cox model was first used to analyze Vibrio hazards in aquaculture and it was the first model to capture the dynamics shrimp survivals under Vibrio experimental challenges. For this purpose a new numerical method was developed, and to study the quantitative contribution of seemingly-obvious risk factors that lead to shrimp vibriosis. A prognostic index (PI), constructed from the Cox model, would be of great assistance in evaluating and choosing experimental treatments. Results indicate that:1.Based on Kaplan-Meier estimates of Vibrio dosage, infection route, and Vibrio strain, we were able to construct survival curves that display the survival distribution of shrimps in challenge tests. Survival curves were compared by means of applying the log-rank test. The infection event resulted in continuous and extensive mortality of shrimps in high dose group (108cfu/mL) than low dose group (107cfu/mL, P=0.038); infection rates following injection were significantly different from those following immersion (P< 0.05); Vh had a significantly higher virulence in this test than the case for Vp (P=0.042).2.Injection was found to be the more efficient method for infecting shrimp (HR=40.4;P=0.000), which means that the mortality risk is 40.4 times greater than that obtained following immersion. Relative to the low dose, of 107 cfu/shrimp, the high dose group, of 108 cfu/shrimp, was regarded as having a higher hazard risk in terms of causing shrimp mortality (HR=5.9, P< 0.000). The virulence of Vh was found to be 4.8 times that of Vp(P< 0.000).3. In the experimental challenge for estimating shrimp survival, the infection route had the higher effect on mortality (P=3.698, P< 0.05), followed by dose (β=1.778, P< 0.05) and Vibrio strain (0=1.561, P< 0.05).The second part studied on the pathological changes and transcriptional response to the immersion infection by different doses Vibrio harveyi in shrimp Litopenaeus vannamei mid-gut. The dynamic changes in intestinal morphology were explored. Changes in expression of anti-lipopolysaccharide factor gene (ALF), penaeidin gene (Pen-4c), crustin gene (Cru), lysozyme gene (LZM) and fatty acid binding protein gene (Fabp) were quantified by qPT-PCR, while the number of Vibrio in intestine and water was monitored. The results list following,1.Immersion is an efficient method in terms of Vibrio colonization and intestinal infections. The intestinal infections could be divided into two levels:mild infection and intense infection. The mild infection occurred in middle/terminal phase of low dose group and middle dose group. It also occurred in early phase of high dose group. In mild infection, the intestinal epithelial cells appeared degradation and the mucosal folds occurred shrinkage and separation. The intense infection occurred in terminal phase of low dose group and middle dose group. It was also common in middle phase of high dose group. And then, the intestinal epithelial cells appeared complete collapsing and escaping, and the muscular layer was also damaged. As the dose of Vibrio increased and infection time added, the intestinal tissue damaged severely. Within 40h post infection, shrimp gut morphology deteriorated continuously, and the tissue was not repaired.2.At 8-16h after infection by V. harveyi, the expression levels of ALF, Pen-4c, Cru and LZM up-regulated immediately and were back to the initial level at the end of 32h. Cru has the biggest increase, while the expression level of Fabp did not significantly increase (P<0.05).3.The number of vibrio in water of three treatments was decreasing post infection, and no multiplication occurred. At the end of 32h, the population of vibrio in water was stable. In three treatments, the cumulative bacteria uptake was increasing in intestine via immersion at 0-8h and balanced at 8-24h. |
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