线纹海马体表溃烂病细菌的分离鉴定及致病性分析

Isolation, identification, and pathogenicity analysis of bacteria associated with skin ulceration in Hippocampus erectus

摘要:

线纹海马(Hippocampus erectus)是我国具有重要经济价值的主要养殖海马种类。在密集养殖环境下细菌性疾病频发，其中体表溃烂病是危害线纹海马养殖的主要疾病之一。溃烂病主要由弧菌属细菌引起，但其病原组成的复杂性和多样性尚未完全明确。目的 鉴定福建漳州地区养殖线纹海马溃烂病的分离菌，解析其致病性、耐药谱及毒力特征，为疾病防控提供科学依据。方法 从患体表溃烂病的海马病灶和内脏组织中分离病原菌，通过形态观察、生理生化检测、16S rRNA基因系统发育分析、回归感染以确定致病菌的种属；采用培养法检测分离菌株的溶血性、酪蛋白酶活性及盐度耐受性；通过PCR扩增确定10种毒力基因；采用纸片扩散法测试其对30种抗菌药物的耐药性；以斑马鱼为模型进行人工感染，确定半数致死量(LD₅₀)。结果 从患病海马各部位共分离出15株优势菌，其中3株(HCE003、HCE070、HCE098)均呈β溶血，总耐药率达到50.0%-56.7%。HCE003携带vvh、pPHDD1和hlyAch毒力基因，HCE070和HCE098携带hlyA、trh、hlyAch和vhh毒力基因，初步判定为强致病株，分别鉴定为弗氏柠檬酸杆菌(Citrobacter freundii)、海藻希瓦氏菌(Shewanella algae)和轮虫弧菌(Vibrio rotiferianus)。这3株菌在盐度15‰下可正常增殖，回归感染可引发线纹海马体表溃烂；人工腹腔感染斑马鱼的半致死浓度LD₅₀分别为1.71×10⁵、3.68×10⁵、2.51×10⁶ CFU/mL。结论 本研究在罹患溃烂病线纹海马中分离到具有多重耐药性及高毒力的弗氏柠檬酸杆菌和海藻希瓦氏菌，表明线纹海马溃烂病的致病菌除了已报道的弧菌，非弧菌属病原菌也可共同诱发海马体表溃烂，为海马养殖疾病防控及开发对应药物提供科学理论依据。

Abstract:

The lined seahorse (Hippocampus erectus) is a major cultured seahorse species with significant economic value in China. Bacterial diseases frequently occur in intensive aquaculture environments, among which skin ulceration is one of the most detrimental diseases affecting H. erectus farming. Skin ulceration is mainly caused by Vibrio spp., while the pathogen complexity and diversity remain unclear.Objective This study identified dominant bacterial strains from ulcerative lesions of H. erectus in Zhangzhou, Fujian and characterized their pathogenicity, antibiotic resistance profiles, and virulence traits, aiming to provide a scientific basis for disease prevention and control.Methods Bacteria were isolated from ulcerated and internal tissue samples of diseased seahorses. Species identification was performed via morphological observation, physiological-biochemical tests, 16S rRNA gene phylogenetic analysis, and reinfection of seahorses. The isolates were cultured for the measurement of hemolytic activity, caseinase production, and salinity tolerance. Ten virulence genes were detected by PCR. The susceptibility of the isolates to 30 antibiotics was tested via the disk diffusion method. Artificial infection was performed with zebrafish as a model to determine the median lethal dose (LD₅₀).Results Fifteen dominant strains were isolated from various tissue samples of diseased seahorses. Among them, three strains (HCE003, HCE070, and HCE098) exhibited β-hemolysis and high overall antibiotic resistance rates (50.0%-56.7%). HCE003 carried vvh, pPHDD1, and hlyAch, while both HCE070 and HCE098 carried hlyA, trh, hlyAch, and vhh. HCE003, HCE070, and HCE098 were preliminarily identified as highly pathogenic strains and were further characterized as Citrobacter freundii, Shewanella algae, and Vibrio rotiferianus, respectively. The three strains were capable of growing normally at the salinity of 15‰. Artificial challenge tests demonstrated that they could induce skin ulceration in H. erectus upon reinfection. The median lethal doses of HCE003, HCE070, and HCE098 in zebrafish were 1.71×10⁵ CFU/mL, 3.68×10⁵ CFU/mL, and 2.51×10⁶ CFU/mL, respectively.Conclusion This study is the first to report the isolation of multidrug-resistant and highly virulent C. freundii and S. algae from H. erectus with skin ulceration, indicating that non-Vibrio pathogens can also contribute to skin ulceration in seahorses. These findings provide scientific support for the development of targeted disease management strategies and therapeutic agents in seahorse aquaculture.