9株鱼源鰤诺卡氏菌生物学特征和致病性比较

Comparative on characteristics and pathogenicity of Nocardia seriolae isoalted from 9 fishes

摘要:

[背景] 鰤诺卡氏菌（Nocardia seriolae）是一种革兰氏阳性好氧菌，易引起鲈形目鱼类结节病发生，近几年对大口黑鲈、杂交鳢等名优鱼类的危害越来越大。[目的] 比较分析9株不同年份、不同地区患结节病的不同淡水鱼品种分离的鰤诺卡氏菌的种间同源关系、生长特性、致病性以及药物敏感性差异，为进一步研究制定相应的防控与治疗措施提供基础数据和科学依据。[方法] 在9株试验菌生理生化实验鉴定结果基础上，采用16S rRNA基因序列比对、限制性片段长度多样性比较菌株间的同源性；扩增看家基因secA1基因序列进行不同来源菌株种内相似度比对；通过体外培养比较不同菌株的生长情况；人工注射感染分析9株试验菌对大口黑鲈的致病性差异；采用微量肉汤二倍稀释法测试9株试验菌对13种抗菌药物的最小抑菌浓度；运用PCR技术检测菌株携带毒力基因和耐药基因的情况。[结果] 生理生化结果显示9株试验菌与鰤诺卡氏菌的基本理化特征相符；9株试验菌聚类分析聚为一类，种内相似度高，限制性片段长度多样性酶切图谱相同，生长特性无明显差异；9株试验菌对大口黑鲈致病性无显著差异，但毒力基因携带情况略有不同，其均携带mce1A、mig和pup基因，而dop和whiB3只在部分菌株检出；9株试验菌均对氟甲喹和氨苄西林耐药，除NS1外，都对磺胺间甲氧嘧啶耐药，对大部分抗菌药物敏感，主要携带bla_TEM和sul1耐药基因。[结论] 不同时间、不同地区及不同宿主分离的9株鰤诺卡氏菌在生物学特性、致病性和药物敏感性等方面无明显差异；同源性相近，提示华南地区感染淡水鱼的鰤诺卡氏菌可能为同一流行株型。研究结果为鰤诺卡氏菌的发病机制及防控技术的进一步研究奠定了基础。

Abstract:

[Background] Nocardia seriolae is a kind of Gram-positive aerobic bacterium, which may cause sarcoidosis in both freshwater and marine fish, among them, perciformes are the most susceptible to the disease. In recent years, serious losses caused by N. seriolae in freshwater fishes such as Micropterus salmoides and Channa argus have been increasing. [Objective] To compare and analyze the homology, growth characteristics, pathogenicity and antimicrobial susceptibilities, 9 N. seriolae strains isolated from different freshwater fish species suffering from sarcoidosis in different years and regions were determined in this study, and to further develop a reliable scientific measures to control and treat this disease. [Methods] 16S rRNA gene and the housekeeping gene secA1 were amplified and sequenced for identification and phylogenetic analysis of N. seriolae after biological characteristics and phylogenetic analysis. PCR-restriction fragment length polymorphism (PCR-RFLP) was developed to analyze the homoology of different origins of N. seriolae. Growth curve of 9 strains was determined by in vitro culture. Pathogenicity of 9 N. seriolae strains was characterized by artificial injection against largemouth bass. Antimicrobial susceptibility testing was carried out to determine the phenotypes of 13 antimiocribal agents by microbroth double dilution method. Virulence genes and drug resistance genes of 9 strains were also detected in this study. [Results] Based on the biological characteristics, most of the phenotypes in 9 strains were similar to Nocardia seriolae. Nine strains were identified to be N. seriolae and clustered into one category with high intraspecies similarity. PCR-RFLP digestion patterns were identical and the growth curvre of 9 strains showed no significant difference of growth characteristics. A variety of virulence genes were detcted in 9 strains. Three genes of mce1A, mig and pup were all detected in 9 strains, whereas dop and whiB3 genes were only carried in some of the strains. There was no significant difference in the pathogenicity of 9 N. seriolae to largemouth bass. All the strains were resistant to sulfamonomethoxine, flumequine and ampicillin, while most of the strains were susceptible to other antimicrobial agents tested. bla_TEM gene was detected in 9 N. seriolae strains, some of which also carried sul1 gene. [Conclusion] Nine strains of N. seriolae isolated from diferent origins showed similar growth characteristics, pathogenicity and antimicrobial susceptibilities. It implied that N. seriolae was the predominant pathogen of sarcoidosis prevailed in freshwater fish and with similar homology. The results of the study provide foundation of further research on the pathogenesis, prevention and control technology of N. seriolae.