哈维氏弧菌引起的条纹海马表皮溃疡综合征的研究

Research on epidermis ulcer syndrome caused by Vibrio harveyi in Hippocampus erectus

摘要:

近年来，条纹海马的养殖在我国发展迅速，但由于多种养殖疾病的频繁暴发，严重制约了条纹海马养殖业的健康发展。自2017年以来，我国北方地区在条纹海马的养殖过程中，多次发生表皮溃疡综合征，患病海马游泳能力减弱，皮肤大面积溃疡，同时伴随烂尾症状，解剖后发现其肠道发白，肝脏及鳃颜色暗淡，死亡率较高，给海马养殖业造成巨大的经济损失。本研究从患病条纹海马的溃烂肌肉及肠道中分离出13株优势菌株，经回归感染实验证实菌株HDM-2可引起条纹海马出现表皮溃疡综合征的类似症状，且半数致死量（LD₅₀）约为2.89×10⁸ CFU/mL。为确定菌株HDM-2的分类地位，采用生理生化及基于16S rDNA的分子生物学方法对其进行鉴定，结果显示，HDM-2为哈维氏弧菌；为了解菌株HDM-2的菌体特征，采用透射电镜（TEM）对菌株HDM-2进行观察，结果显示，HDM-2为周生鞭毛，有荚膜，大小约2.20 μm×4.75 μm；为获知HDM-2的抗生素敏感性，采用纸片扩散法进行药敏试验，结果显示，菌株HDM-2对氟苯尼考和头孢他啶等较敏感；患病条纹海马的组织病理学研究表明，菌株HDM-2对条纹海马的皮肤、肝脏以及鳃组织损伤较严重。本文可为条纹海马表皮溃疡综合征的防治及深入研究提供基础资料。

Abstract:

Hippocampus spp. is unique marine fish with special body morphology and life history traits. Compared with other marine fishes, the most special part of Hippocampus spp. is male breeding and strict monogamy. In addition, its unique body morphology, including curvaceous torso and horse-shaped head, has given them higher ornamental value. The trade volume of Hippocampus spp. is huge in the world, and the majority of the trade in Hippocampus spp. is for traditional Chinese medicine. In recent years, the destruction of wild habitats and overfishing lead to a sharp decline of the wild populations. Aquaculture of Hippocampus spp. has been considered as an effective way to solve the problem of decline of the wild populations and the unsustainable trade for traditional Chinese medicine. Therefore, the development of the Hippocampus spp. aquaculture is essential. However, the threat of various diseases has seriously affected the healthy development of the aquaculture industry. Since 2017, in the breeding process of the H. erectus in northern China, epidermis ulcer syndrome has frequently occurred, the morbidity is rapid and the mortalily is high. Epidermis ulcer syndrome has become a high incidence disease in the aquaculture process. Diseased H. erectus showed obvious symptoms, such as large rot area in body surface, darker and lackluster of the gills and liver, diseased H. erectus would die within 3 days. If the diseased H. erectus were not removed in time, a large number of seahorses would be infected. Although most fish diseases can be treated with antibiotics, the overuse of antibiotics may produce many potential threats, which are not conducive to the sustainable and healthy development of the aquaculture industry. Therefore, it is essential to further study the pathogens of various fish diseases and then we can develop biological control on this basis. Accordingly, the priority of research should be disease treatment and health management, especially disease treatment. Specific vaccines can be developed from isolated pathogenic bacteria, which provide effective way for disease treatment. In this study, dominant strains were isolated from the lesion tissue and intestine of H. erectus with epidermis ulcer syndrome. In order to determine the pathogenic bacteria, we carried out artificial infection, the infection results suggested that strain HDM-2 was the pathogen causing epidermis ulcer syndrome, and the LD₅₀ of strain HDM-2 was about 2.89×10⁸ CFU/mL. We identified strain HDM-2 with physiological and biochemical identification and molecular biological identification methods, and we observed strain HDM-2 under transmission electron microscope. 16S rDNA gene sequence analysis by BLAST in GenBank suggested that the similarity between strain HDM-2 and V. harveyi reached 100%. The observation results showed that HDM-2 was about 2.2 μm×4.75 μm, with peritrichous flagella and capsule. In order to study antibiotic susceptibility of strain HDM-2, we implemented the antibiotic sensitivity test by using Kirby-Bauerdiffusion method, and results showed that strain HDM-2 was highly sensitive to florfenicol. In order to study the pathogenicity of the pathogen to the host, we carried out histopathological observation of the diseased H. erectus. Observation results suggested that skin, liver and gills were severely damaged by HDM-2. This study would lay a foundation for the prevention and further study of the epidermis ulcer syndrome of H. erectus.