| Aquaculture industry for many years suffered from the harm of bacteria and viruses, and the aquaculture industry is caused great economic losses. The Litopenaeus vannamei is breed in large-scale around the word because of its high performance. But with the expansion of the scale of farming, many pathogenic frequently emerge.This research includes three parts. (Ⅰ) Analyze the culturable quantity and composition of bacteria which in artificial cultivative prawn larvae. (2) Impact of coinfection with Vibrio parahaemolyticus and white spot syndrome virus (WSSV) on the survival of penaeid shrimp Litopenaeus vannamei. (3) Impact of coinfection with different virulence of two Vibrio parahaemolyticus and white spot syndrome virus (WSSV) on the survival of penaeid shrimp Litopenaeus vannamei.1. Analyze the culturable quantity and composition of bacteria which in artificial cultivative prawn larvae:Prawn larvae is the foundation of prawn aquaculture, and the health prawn larvae is the precondition for breeding success. Both bacterial and viral disease influent the shrimp industry seriously, thus study the qualitative and quantitative of the bacteria in prawn and the virus diction has important implications. Bacterial flora in the prawn larvae, Litopenaeus vannamei and Fenneorpenaeus chinensis, cultured in part of breeding field of Shandong, Tianjin and Zhejiang, were investigated and the isolated strains were purified and identified by conventional method and also 16SrDNA sequence analysis method. We also detected the white spot syndrome virus (WSSV) and Infectious hypodermal & haematopoietic necrosis virus (IHHNV) by PCR method. The result showed that, the total number of cultured bacteria in the prawn larvae, L. vannamei and F. chinensis, was 105～107CFU/g. The isolated bacteria belonged to the genus of Vibrio, Photobacterium, Bacillus, Halomonas, Biaionia, Marinobacter, Polaribacter, Planococcus. Vibrio was the highest proportion among the dominant bacteria, which was from 40.00% to 90.23%. Uneven distribution of bacterial presented the feature that less bacteria species category and more individual species distribution. There were some samples of both L. vannamei and F. chinensis were WSSV & IHHNV positive. WSSV positive rate was 33.33%, IHHNV was 22.22%. detected the two virus at the same time was 11.11%. The total number of bacteria in the prawns larvae carried just WSSV or both of WSSV, IHHNV were from1.23×107 CFU/g to 4.14×107 CFU/g, and vibrios number was about 107 CFU/g, both which was significantly higher than other samples(P<0.05). The number of vibrios in the prawn larvae which carried IHHNV or did not carry virus were between 104 to 106 CFU/g. The research showed that vibrios widely exist in the prawns larvae, L. vannamei and F. chinensis. And the prawn larvae carry WSSV may cause the number of vibrios increasing. But it can’t be confirm whether this could increase the risk of shrimp culture. For the further investigation didn’t be carried out.2. Impact of coinfection with Vibrio parahaemolyticus and white spot syndrome virus (WSSV) on the survival of penaeid shrimp Litopenaeus vannamei:White spot syndrome virus (WSSV) is an important viral pathogen infecting farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus (V. parahaemolyticus) infection to shrimp farming has become increasingly severe too. Viral and bacterial cross or superimposed infection may induce more shrimp death. The present study either used feeding method to firstly infect the Litopenaeus vannamei (L. vannamei) with WSSV and then injected a low dose of V. parahaemolyticus (WSSV + Vp), or firstly infected the L. vannamei with low-dose injection of V. parahaemolyticus and then fed the shrimp with WSSV to achieve viral infection (Vp+WSSV). The impact of coinfection with V. parahaemolyticus and WSSV on the survival of L. vannamei was evaluated by comparing the cumulative mortality rates between experimental and control groups. We also spread the thiosulfate citrate bile salts sucrose agar (TCBS) plate to determine the number of Vibrio in the hemolymph of L. vannamei, and absolute quantitative PCR method was used to determine the copy number of WSSV in the gill of L. vannamei; the expression level of LvMyD88 and Lvakt genes in the gill of L. vannamei was detected by real-time PCR. From these we may analyze the cause of differential mortality rates. Our results showed that,(1) the accumulative mortality rates of L. vannamei in WSSV + Vp and group reached 100% on the 10th day after WSSV infection, while the accumulative mortality rates of L. vannamei in Vp + WSSV group and WSSV alone control approached 100% on the 11th and 13rd day of infection respectively. (2)The number of Vibrio in group infected with V. parahaemolyticus alone gradually declined, other groups showed significantly increased number of Vibrio (P<0.05). (3)The copy numbers of WSSV in the gill of WSSV + Vp. Vp + WSSV, and WSSV alone groups increased from 105/mg tissue to 107/mg tissue at 72,96, and 144 h, respectively. These results suggested that V. parahaemolyticus infection accelerated the proliferation of WSSV in L. vannamei, and vise versa; the combined accelerated proliferation of both V. parahaemolyticus and WSSV led to a massive death of L. vannamei.3. Impact of coinfection with different virulence of two Vibrio parahaemolyticus and white spot syndrome virus (WSSV) on the survival of penaeid shrimp Litopenaeus vannamei: The present study infected the Litopenaeus vannamei (L. vannamei) by injecting WSSV and V. parahaemolyticus. Either firstly infected the L. vannamei with V. parahaemolyticus and then injected WSSV, or firstly infected the L. vannamei with WSSV and then injected V. parahaemolyticus. The impact of coinfection with V. parahaemolyticus and WSSV on the survival of L. vannamei was evaluated by comparing the cumulative mortality rates between experimental and control groups. We also spread the thiosulfate citrate bile salts sucrose agar (TCBS) plate to determine the number of Vibrio in the hemolymph of L. vannamei, and absolute quantitative PCR method was used to determine the copy number of WSSV in the gill of L. vannamei. The result shows that:(1) WSSV firstly infect and then V. parahaemolyticus secondary infection can accelerate the death of L. vannamei. And the V. parahaemolyticus secondary infection in connection with the density and virulence of V. parahaemolyticus. The copy number of WSSV in the gill in connection with the virulence of V. parahaemolyticus, but the number of V. parahaemolyticus is not. (2) When V. parahaemolyticus firstly infect and then WSSV secondary infection, the death rate of L. vannamei and the copy number of WSSV in the gill have no relation to the virulence of V. parahaemolyticus. |
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