溶血性嗜水气单胞菌XF-6的分离鉴定及其对鲥鱼机体免疫功能的影响

Study on Separation of Hemolytic Aeromonas hydrophila Strain XF-6 from Tenualosa reevesii and Its Effect on Iron Metabolism

【目的】本文研究了湖北省武汉市一些养殖场鲥鱼发病原因以及致病菌对其免疫因子的影响。【方法】采用划线培养方法对染病鲥鱼进行了病原菌分离鉴定,从其肝脏中分离得到病原菌XF-6,经VITEK-32全自动细菌分析仪鉴定,为嗜水气单胞菌。用多重PCR方法确认嗜水气单胞菌XF-6属于溶血性嗜水气单胞菌,含有气溶素基因(aer A)和溶血素基因(ahh1)。同时研究了嗜水气单胞菌XF-6侵染对鲥鱼机体免疫功能的影响,通过腹腔注射嗜水气单胞菌XF-6,在侵染后不同时间点采集鲥鱼的血液和肝脏,分别采用分光光度计法和ICP-AES法检测血清和肝脏铁浓度,运用实时荧光定量PCR法对铁调素基因(hepc)、白细胞介素基因(il-6)、JAK3/STAT3信号通路的酪氨酸激酶3基因(jak3)、信号转导和转录激活因子3基因(stat3)的表达量进行检测。【结果】侵染嗜水气单胞菌XF-6后,鲥鱼血清中铁浓度明显降低,在12、24、48 h时显著低于对照组;血清中总铁结合力有所上升,但没有达到显著水平;肝脏中铁浓度相对于对照组没有达到显著性差异水平,但仍有升高。肝脏中hepc基因的表达量在6、12、24 h时显著上升;肝脏中il-6基因的表达量在12、24、48 h时显著上升、肝脏中jak3基因的表达量在12、24 h时显著上升,肝脏中stat3基因的表达量在6、12、24 h时显著上升。这4种基因表达量随后有所下降,但各时间点的表达量均高于对照。【结论】鲥鱼机体通过调节铁调素基因(hepc)、白细胞介素基因(il-6)、JAK3/STAT3信号通路的酪氨酸激酶3基因(jak3)、信号转导和转录激活因子3基因(stat3)等铁相关基因的表达,进而降低自身游离铁浓度和增加储存铁浓度来应对病原菌的侵染。

【Objective】The present paper aimed to study the causes of pathogenic bacteria and its effect on the immune factor.【Method】Using the streak plate culture,the pathogen named XF-6 was isolated and purified in the liver of Tenualosa reevesii through VITEK-32 automatic analyzer with detection of bacteria belonging to Aeromonas hydrophila.A multiple PCR method was used to confirm that Aeromonas hydrophila XF-6 contained the gas soluble gene（aer A） and the hemolysin gene（ahh1）,belonging to the hemolytic Aeromonas hydrophila.The effects on the immune function of Tenualosa reevesii with the infection of Aeromonas hydrophila XF-6 were confirmed by the injection of4.0 × 105 cfu/m L of Aeromonas hydrophila XF-6.The blood and liver of Tenualosa reevesii was collected after 6-96 hours infection,using spectrophotometric detection method to calculate the concentration of iron,using ICP-AES to calculate the iron concentration in liver,using the QR-PCR method to calculated the change of iron regulated genes,including hepcidin gene（hepc）,interleukin（il-6） gene and protein tyrosine kinase（Jak3） gene,transcription factor gene（stat3）.【Result】By the infection of Aeromonas hydrophila XF-6,the iron concentration in Tenualosa reevesii blood decreased in 12,24,48 hours; The concentration of blood iron protein increased but did not reach significant difference; The iron concentration in liver increased but did not reach significant difference.The expression of liver hepcidin gene reached significant difference in 6,12,24 hours; Liver expression of interleukin gene reached significantly in 12,24,48 hours;Liver tyrosine kinase gene expression reached significant differences in 12,24 hours; The expression of liver activator gene reached significant differences in 6,12,24 hours.【Conclusion】Therefore,after the infection of Aeromonas hydrophila XF-6 in Tenualosa reevesii,hepcidin gene（hepc）,interleukin（il-6）,protein tyrosine kinase（jak3） gene and transcription factor gene（stat3） expression were increased,thereby reducing the blood iron concentration,increasing blood ferritin,increasing hepatic iron concentration and improving the immune capacity.