加州鲈源鰤鱼诺卡氏菌的分离鉴定及致病性

Isolation, identification and pathogenicity of Nocardia seriolae from Micropterus salmoides

摘要:

[背景] 鰤鱼诺卡氏菌（Nocardia seriolae）是一种严重危害水产养殖业的病原菌，可引起以体表溃疡、出血及组织器官形成结节为特征的鱼类慢性肉芽肿疾病，目前尚无有效的防治方法。[目的] 明确引起安徽省临泉县某养殖场加州鲈（Micropterus salmonoides）结节病的病原菌，探讨其致病性，为该病的有效防治提供科学依据。[方法] 取肝脏结节病灶接种于TSB培养基分离优势细菌，利用表型检查结合分子生物学方法鉴定分离菌株。进一步通过检测分离菌株的毒力基因、测定其对加州鲈的半数致死量（LD₅₀）以及所感染加州鲈的组织病理学变化与组织载菌量，分析其致病性。[结果] 从病鱼体内分离到一株优势菌株NI，综合NI分离株的表型特性、16S rRNA基因序列与鰤鱼诺卡氏菌参考株相应序列的一致性以及特异性PCR扩增结果，确定其为鰤鱼诺卡氏菌。鰤鱼诺卡氏菌NI分离株携带毒力基因gapA、ibeA和mip，人工回归感染后加州鲈出现与自然病例相似的症状，其对加州鲈的LD₅₀为2.58×10⁶ CFU/尾。组织病理学观察到头肾、心脏、肝脏、胃和脾脏均出现慢性肉芽肿病变，肠管肌层疏松、肠绒毛脱落，肌肉组织中肌纤维疏松、间隙增宽。qPCR检测结果显示，组织中鰤鱼诺卡氏菌载量由高到低依次为头肾、心、肝、胃、脾、肠和肌肉。[结论] 鰤鱼诺卡氏菌是引起此次加州鲈结节病的病原菌，对该菌致病性的研究为加州鲈诺卡氏菌病的防控提供了理论依据。

Abstract:

[Background] Nocardia seriolae is the causative agent of chronic granulomatous disease in cultured fish characterized by skin ulcers, bleeding and nodular formation in tissues, which seriously harms aquaculture industry. So far, there is no effective prevention and treatment method for the disease. [Objective] To clarify the pathogen causing sarcoidosis of Micropterus salmonoides in an aquaculture farm in Linquan county, Anhui province and to explore its pathogenicity, so as to provide a scientific basis for the effective prevention and treatment of the disease. [Methods] The liver nodules were inoculated with TSB medium to isolate dominant bacterial strain, which was then identified by phenotypic examination combined with test of molecular biology. The pathogenicity of the isolated strain was analyzed through virulence gene identification, LD₅₀ to Micropterus salmonoides as well as the histopathological changes and tissue bacterial load. [Results] A dominant strain NI was isolated from diseased fish and identified as N. seriolae based on its phenotypic characteristics, the 16S rRNA gene sequence identity between the strain NI and N. seriolae reference strains, and the result of specific PCR amplification. Strain NI carried virulence genes gapA, ibeA and mip, and its LD₅₀ to Micropterus salmonoides was 2.58×10⁶ CFU/fish. The clinical signs of Micropterus salmonoides after artificial infection with the strain NI were in accordance with those of natural cases. Histopathologically, chronic granulomatous lesions in head kidney, heart, liver, stomach and spleen of diseased fish, loose intestinal muscle layer and shortened intestinal villi, loose and widened muscle fibers in the muscle tissue were observed. qPCR analysis found the descending order of N. seriolae load in the following infected tissues: head kidney, heart, liver, stomach, spleen, intestine and muscle. [Conclusion] Nocardia seriolae is the pathogen causing the outbreak of sarcoidosis in Micropterus salmonoides in the local aquaculture farm. Results of the study on pathogenicity of the isolated strain provided a theoretical basis for the prevention and control of nocardiosis in Micropterus salmonoides.