基于溶藻弧菌感染的虾苗细菌性玻化症(BVS)及其防控中药筛选和药效学评价

Chinese herbal compound screening and its pharmacodynamic efficacy evaluation against shrimp postlarva bacterial vitrified syndrome (BVS) induced by Vibrio alginolyticus

中文关键词: 凡纳对虾  细菌性玻化症(BVS)  病理模型  中药复方  药效学

英文关键词: Penaeus vannamei  Bacterial vitrified syndrome (BVS)  Pathological model  Chinese herbal compound  Pharmacodynamics

中文摘要:

      为了筛选可用于防治虾苗细菌性玻化症(bacterial vitrified syndrome, BVS)的中药复方，本研究首先通过牛津杯法和二倍稀释法进行体外抑菌实验，从50种中药中筛选出对BVS致病菌溶藻弧菌(Vibrio alginolyticus)和副溶血弧菌(V. parahaemolyticus)具有良好抑菌效果的中药。然后，以不同浓度的溶藻弧菌浸浴感染虾苗，建立BVS病理模型；再通过体内药效学实验从死亡率、组织病理学和超微组织病理学角度比较不同中药复方对于患BVS虾苗的防治效果。体外抑菌实验结果显示，五味子(Schisandrae chinensis fructus)、马鞭草(Verbenae herba)、乌梅(Mume fructus)等中药对溶藻弧菌的抑菌圈直径可达12 mm以上；诃子(Chebulae fructus)、五味子、牡丹皮(Moutan cortex)等中药对副溶血弧菌的抑菌圈直径可达13 mm以上。五倍子(Galla chinensis)、诃子、五味子、丁香(Caryophylli flos)对溶藻弧菌和副溶血弧菌的最小抑菌浓度(MIC)均≤12.5 mg/mL，最小杀菌浓度(MBC)均≤50 mg/mL。结合香附(Cyperi rhizoma)、栀子(Gardeniae fructus) 2味中药组成3种中药复方，处方1、处方2和处方3；以5×104 CFU/mL的溶藻弧菌感染虾苗成功建立BVS病理模型，在此基础上采用药物治疗患病虾苗7 d，各组死亡率由低至高依次为空白对照组、处方1组、20%氟苯尼考粉对照组、处方3组、处方2组和阳性对照组。处方1组虾苗死亡率显著低于阳性对照组(P<0.05)；处方2组、处方3组和20%氟苯尼考对照组虾苗死亡率低于阳性对照组，但差异不显著(P>0.05)。由此可见，处方1可显著降低患病虾苗死亡率。组织病理学和超微组织病理学研究显示，相比于其他感染组，处方1组虾苗肝胰腺病变程度明显较轻，肠道结构较为完整；虾苗肝小管上皮细胞的细胞膜、细胞核正常，线粒体、内质网丰富且基本正常。表明口服处方1可对患病虾苗的肝胰腺和肠道组织起到良好的保护作用，并优于处方2、处方3和20%氟苯尼考粉。综上所述，处方1 (诃子30 g、五味子20 g、香附20 g)对溶藻弧菌感染的虾苗保护和治疗效果良好。本研究结果可为研制防控虾苗BVS专用中药提供依据，助力对虾养殖绿色高质量发展。

英文摘要:

      Penaeus vannamei is an important breeding shrimp species in China. Moreover, the quality of the larvae is the cornerstone for the sustainable development of the shrimp industry. In recent years, shrimp postlarva bacterial vitrified syndrome (BVS) has caused huge economic losses in the shrimp postlarva breeding industry, with epidemiological characteristics of rapid onset and high mortality, occurring at 2–3 days post-hatch. The diseased postlarva symptoms include emaciation, decreased activity, and an empty intestinal tract and stomach. The hepatopancreas shows atrophy, blurring of contour, paleness, and even vitrified syndrome. The causative agents of the disease are Vibrio alginolyticus and Vibrio parahaemolyticus. Furthermore, antibiotics are often used to treat BVS; however, frequent and inappropriate use of antibiotics can lead to bacterial resistance and drug residues. With the advantages of antibacterial activity, immunity regulation, low toxicity, few side effects, and lack of drug resistance and drug residues, Chinese herbal medicines play an important role in aquaculture. There are currently no effective drugs available to prevent and treat BVS. It is urgent and important that effective Chinese herbal compounds are developed for BVS prevention and treatment. In this study, first, an antibacterial test was carried out in vitro by the Oxford cup and double dilution methods. Overall, 50 types of Chinese herbal medicines were screened, and those with a good bacteriostasis effect on V. alginolyticus and V. parahaemolyticus were identified. Then, a pathological model of BVS was constructed with different concentrations of V. alginolyticus. The efficacies of the Chinese herb compounds were evaluated based on the mortality, histopathology and ultrastructural pathology characteristics of the postlarvae. The bacterial inhibition test showed that the inhibition zone of V. alginolyticus could be > 12 mm with the application of one of six types of Chinese herbs, including Schisandrae chinensis fructus, Verbenae herba, and Granati pericarpium. The inhibition zone of V. parahaemolyticus could be > 13 mm with the application of one of five types of Chinese herbs, including Chebulae fructus, S. chinensis fructus, Moutan cortex and Sanguisorbae radix. Then, 11 types of antibacterial and two types of immunological drugs were selected for further analysis. Moreover, the MIC and MBC of G. chinensis, C. fructus, Caryophylli flos, and S. chinensis fructus against V. alginolyticus and V. parahaemolyticus were ≤ 12.5 mg/mL and ≤ 50 mg/mL, respectively. Then, three types of compounds were used for in vivo pharmacodynamics tests by combining Chinese antibacterial medicine with Chinese immunological medicine (Cyperi rhizoma and Gardeniae fructus), which were named prescription 1 (C. fructus 30 g, S. chinensis fructus 20 g, C. rhizoma 20 g), prescription 2 (C. fructus 40 g, G. pericarpium 30 g, C. flos 30 g), and prescription 3 (C. fructus 40 g, G. chinensis 30 g, G. fructus 30 g), respectively. Florfenicol was used as the positive drug control, and V. alginolyticus infection without treatment was used as the positive control. The BVS model indicated that V. alginolyticus caused the same vitrification symptoms in shrimp postlarvae as a natural infection. The LD50 of shrimp postlarvae was 2.82 × 105 CFU/mL after 72 h of infection with V. alginolyticus. The pathological model of BVS was constructed by immersing shrimp postlarvae with 5 × 104 CFU/mL V. alginolyticus. Based on this, the infected shrimp postlarvae were treated with drugs for 7 days. After this period, the mortality rates of each group from low to high were: blank control < prescription 1 < florfenicol < prescription 3 < prescription 2 < positive control. The mortality rate of the shrimp postlarvae in the prescription 1 group was significantly lower than that in the positive control group (P < 0.05). The mortality rate of the shrimp postlarvae in the prescription 2 and 3 groups and the florfenicol group was lower than that in the positive control group, but the difference was not significant (P > 0.05). Thus, prescription 1 can significantly reduce the mortality of BVS-positive shrimp postlarvae. Histopathology observation showed that after 7 days of administration, the hepatopancreas and intestinal epithelium of larvae in the prescription 1 group were more intact, with more hepatic tubules, less exfoliation and necrosis of the epithelial cells, and more abundant and well-arranged intestinal epithelial cells. Ultrastructural pathology observation showed that after 7 days of administration, the cell membrane and nucleus of the liver tubule epithelial cells of the diseased shrimp postlarvae in the prescription 1 group were normal, and the mitochondria and endoplasmic reticulum were abundant. Histopathology observation showed that the hepatopancreas and intestinal tract lesions of the shrimp postlarvae in the prescription 1 treatment were significantly less than those in the other groups. In summary, Chinese herbal compounds (C. fructus 30 g, S. chinensis fructus 20 g, C. rhizoma 20 g) had the best effect for preventing and treating shrimp postlarva BVS caused by V. alginolyticus. The results of this study lay a scientific foundation for the development of specialized herbal compounds for the prevention and treatment of bacterial infections and contribute to the green and high-quality development of the shrimp industry.