接种抗原后欧洲鳗鲡免疫组织形态学变化及抗体产生细胞分析
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摘要
欧洲鳗鲡是我国最主要的鳗鲡养殖品种。自引进中国后,养殖规模和密度不断扩大,随之,日渐多发的病害严重影响了养殖效益。为了探知欧洲鳗鲡的免疫生物学特点,本研究结合组织形态学观察和ELISPOT方法,以嗜水气单胞菌-MOMP ISCOMs疫苗作为抗原接种欧洲鳗鲡,分析免疫器官组织形态学变化和抗体产生细胞(Antibody Secreting Cell,ASC)增殖情况,同时了解血清中非特异性免疫物质的变化规律。研究结果如下:
1.嗜水气单胞菌-MOMP的提取及ISCOMs的制备:根据实验室现有的嗜水气单胞菌-MOMP提取方法进行优化,采用SDS-PAGE对所提取的产物进行电泳分析,同时利用透射电镜对制备的ISCOMs进行观察,确定该方法可以成功制备嗜水气单胞菌-MOMP ISCOMs抗原。
2.接种抗原后欧洲鳗鲡免疫器官组织形态学变化观察:①接种后脾脏仅出现白髓增殖现象,未见其他明显病理症状。②肠道结构良好,绒毛整齐。③抗原经过口服和腹腔注射接种后欧洲鳗鲡肠上皮内淋巴细胞数量变化规律不同。口服接种组表现为:前、中肠上皮内淋巴细胞数量均先下降后上升,第21d达到实验期峰值(P<0.01);后肠接种第7d小幅上升后持续下降。注射接种组表现为:前、中、后肠上皮内淋巴细胞数量在第一次接种后略微上升,第7d下降至低于对照组;二次接种后淋巴细胞数量再次增加,第14d达到峰值后下降(P<0.01),而后肠上皮内淋巴细胞数量在第21d维持较高水平(P<0.01)。④口服和腹腔注射接种ISCOMs抗原后欧洲鳗鲡肠上皮内杯状细胞数量总体上增加。口服接种组肠道上皮内杯状细胞均表现为先下降后达到峰值,前肠上皮内杯状细胞在第14d达到峰值(P<0.01),中肠在第21d达到峰值(P<0.01),后肠则在第7d达到峰值(P<0.01)。注射组表现为接种后第1d前、中肠上皮内杯状细胞极显著高于对照组(P<0.01),后肠显著高于对照组(P<0.05);经过二次注射接种,第14d前、中、后肠上皮内杯状细胞均和对照组差异均极显著(P<0.01)。
3.接种抗原后欧洲鳗鲡ASC的增殖规律:①口服和注射接种后欧洲鳗鲡肾脏中分离的细胞数量均有增加。口服组肾脏中ASC和特异性ASC第14d都极显著高于对照组(P<0.01),第28d都显著高于对照组(P<0.05);注射组第14d肾脏中ASC维持正常水平(P>0.05),特异性ASC显著高于对照组(P<0.05),第28d肾脏ASC和特异性ASC都极显著高于对照水平(P<0.01)。②口服接种后皮肤中分离的细胞数量持续增长;而注射组先增长,第28d降至正常。口服接种至第14、28d皮肤中ASC都极显著高于对照组(P<0.01);皮肤中特异性ASC在第14d显著高于对照组(P<0.05),第28d恢复正常(P>0.05)。注射组皮肤中ASC和特异性ASC在第14d极显著高于对照组(P<0.01),第28d显著高于对照组水平(P<0.05)。
4.接种抗原后欧洲鳗鲡血清中非特异性免疫指标消长情况:口服和注射接种后欧洲鳗鲡血清中溶菌酶、转铁蛋白(Tf)和碱性磷酸酶(ALP)的含量均得到提高,且注射接种后血清中干扰素(IFN)含量也上升;同时检测到血清中产生特异性抗体。
综上所述,嗜水气单胞菌-MOMP ISCOMs疫苗副作用较小,使用安全。口服和注射接种均能不同程度影响欧洲鳗鲡肠道上皮内淋巴细胞和杯状细胞数量,诱导局部免疫应答反应;提高皮肤和肾脏中的淋巴细胞数量、ASC和特异性ASC数量,刺激机体产生免疫保护,同时鱼体非特异性免疫能力得到增强。研究结果进一步阐明了嗜水气单胞菌致病机理和鳗鲡免疫应答体系,为预防鳗鲡病害,合理使用疫苗提供可靠的科学依据。
Recently, Anguilla Anguilla is one of the most popular breed of eel cultivated in China. After being imported, the scale and density of farming operations have been expanded rapidly. At same time, health securities of the eel are sometimes threatened by diseases, which dramatically decreases the benefits of farmers. Aeromonas hydrophilia is a common pathogen prevailed in eel farms. In order to explain the pathogenesis of Aeromonas Hydrophila, determine the immune reactions and provide scientific basis for future vaccination in practice of eels, the immune tissues morphology was observed, antibody secreting cells (ASC) were determined with ELISPOT, and nonspecific immune factors were measured after Anguilla anguilla vaccinated orally or injection with Aeromonas Hydrophila-ISCOMs. The results are listed below:
1. Isolation of MOMP of Aeromonas Hydrophila and ISCOMs preparation: MOMP of Aeromonas Hydrophila was first isolated through optimized extraction conditions and ISCOMs was prepared with the method currently used in our laboratory. The MOMP and ISCOMs were tested by using SDS-PAGE and an electron microscope, persisting in their feasibilities and validating for making Aeromonas hydrophila-MOMP ISCOMs.
2. Observation of the immune tissue morphological changes of Anguilla Anguilla after vaccination with ISCOMs, compared with control group:①The white pulp in the spleen is increased remarkably, but no other obvious pathological changes could be observed.②Villi are normal and the guts are remained structu -rally sound.③The number of lymphocytes in intestinal epithelium are different between oral vaccination and injection vaccination. In oral vaccinated group, the number of lymphocytes in the intestinal epithelium of the foregut and mid-gut are declined initially, and ascended to peak at the 21th day after vaccination (P<0.01). The number of lymphocytes in the hindgut are ascended slightly at the 7th day after vaccination, and then declined steadily. In injection vaccinated group, the number of lymphocytes in the intestinal epithelium of the foregut, mid-gut and hindgut are slight increased initially, and declined at the 7th day after the first vaccination, and then they are increased again to peak at 14th day, and then they are declined (P<0.01), except the number of lymphocytes of the hindgut are still maintained at high level to the 21th day (P<0.01), after the second vaccination.④The number of goblet cells in the intestinal epithelium of the foregut, mid-gut, and hindgut are all increased significan -tly in both oral vaccination and injection vaccination.
3. Observations of the ASC of Anguilla Anguilla after vaccinations:①the number of ASC and specific ASC are higher in groups of both oral vaccination and injection vaccination than in control at the 14th day, and the 28th day. In kidney, the differences of the number of ASC and specific ASC are significant greatly at the14th day (P<0.01) and at 28th day (P﹤0.05) respectively after oral vaccination. In the group of injection vaccination, compared to control group, the number of ASC in kidney are kept in the normal level (P>0.05), and the specific ASC are increased significantly (P﹤0.05) at the 14th day. And then the number of ASC and specific ASC in kidney are increased significantly (P﹤0.01) at 28th day.②Compared with control group, the number of the cells in skin are increased continuously after oral vaccination, and the number of the cells in skin are increased in early stage, then declined to normal level at the 28th day after injection vaccination. The number of ASC in skin are increased significantly at the 14th day and the 28th day (P<0.01) after oral vaccination. The number of specific ASC in skin are increasted significantly at 14th day (P﹤0.05), and declined to normal level at the 28 day (P>0.05). The number of ASC and specific ASC in skin are increased significantly at the14th day (P<0.01)and at the 28th day(P﹤0.05) after injection vaccination.
4. Non-specific immune factors in blood of Anguilla Anguilla after vaccination: the content of lysozyme, transferrin (Tf), and alkaline phosphatase (ALP) in the serum are higher in groups of oral and injected vaccination than the control. The interferon (IFN) titer in serum was higher in the injected group than in the control. Specific antibody secretion can be induced by either oral vaccination or injected vaccination. In conclusion, Aeromonas hydrophila-ISCOMs shows less irritation and no side effects are observed. The number of lymphocytes and goblet cells in the intestinal epithelium are both increased in different degree by oral vaccination or injected vaccination, indicating that local immune responses are induced by vaccination of aeromonas hydrophila-ISCOMs. The number of ASC and specific ASC of kidney and skin in vaccinated groups are obviously higher than that in the control, and activities of non-specific immune factors (lysozyme, transferring, alkaline phospha -tase, interferon) in blood of vaccinated groups are evidently enhanced, compared with control group, showing that immunoreactions are improved after vaccination with Aeromonas Hydrophila -ISCOMs.
1.嗜水气单胞菌-MOMP的提取及ISCOMs的制备:根据实验室现有的嗜水气单胞菌-MOMP提取方法进行优化,采用SDS-PAGE对所提取的产物进行电泳分析,同时利用透射电镜对制备的ISCOMs进行观察,确定该方法可以成功制备嗜水气单胞菌-MOMP ISCOMs抗原。
2.接种抗原后欧洲鳗鲡免疫器官组织形态学变化观察:①接种后脾脏仅出现白髓增殖现象,未见其他明显病理症状。②肠道结构良好,绒毛整齐。③抗原经过口服和腹腔注射接种后欧洲鳗鲡肠上皮内淋巴细胞数量变化规律不同。口服接种组表现为:前、中肠上皮内淋巴细胞数量均先下降后上升,第21d达到实验期峰值(P<0.01);后肠接种第7d小幅上升后持续下降。注射接种组表现为:前、中、后肠上皮内淋巴细胞数量在第一次接种后略微上升,第7d下降至低于对照组;二次接种后淋巴细胞数量再次增加,第14d达到峰值后下降(P<0.01),而后肠上皮内淋巴细胞数量在第21d维持较高水平(P<0.01)。④口服和腹腔注射接种ISCOMs抗原后欧洲鳗鲡肠上皮内杯状细胞数量总体上增加。口服接种组肠道上皮内杯状细胞均表现为先下降后达到峰值,前肠上皮内杯状细胞在第14d达到峰值(P<0.01),中肠在第21d达到峰值(P<0.01),后肠则在第7d达到峰值(P<0.01)。注射组表现为接种后第1d前、中肠上皮内杯状细胞极显著高于对照组(P<0.01),后肠显著高于对照组(P<0.05);经过二次注射接种,第14d前、中、后肠上皮内杯状细胞均和对照组差异均极显著(P<0.01)。
3.接种抗原后欧洲鳗鲡ASC的增殖规律:①口服和注射接种后欧洲鳗鲡肾脏中分离的细胞数量均有增加。口服组肾脏中ASC和特异性ASC第14d都极显著高于对照组(P<0.01),第28d都显著高于对照组(P<0.05);注射组第14d肾脏中ASC维持正常水平(P>0.05),特异性ASC显著高于对照组(P<0.05),第28d肾脏ASC和特异性ASC都极显著高于对照水平(P<0.01)。②口服接种后皮肤中分离的细胞数量持续增长;而注射组先增长,第28d降至正常。口服接种至第14、28d皮肤中ASC都极显著高于对照组(P<0.01);皮肤中特异性ASC在第14d显著高于对照组(P<0.05),第28d恢复正常(P>0.05)。注射组皮肤中ASC和特异性ASC在第14d极显著高于对照组(P<0.01),第28d显著高于对照组水平(P<0.05)。
4.接种抗原后欧洲鳗鲡血清中非特异性免疫指标消长情况:口服和注射接种后欧洲鳗鲡血清中溶菌酶、转铁蛋白(Tf)和碱性磷酸酶(ALP)的含量均得到提高,且注射接种后血清中干扰素(IFN)含量也上升;同时检测到血清中产生特异性抗体。
综上所述,嗜水气单胞菌-MOMP ISCOMs疫苗副作用较小,使用安全。口服和注射接种均能不同程度影响欧洲鳗鲡肠道上皮内淋巴细胞和杯状细胞数量,诱导局部免疫应答反应;提高皮肤和肾脏中的淋巴细胞数量、ASC和特异性ASC数量,刺激机体产生免疫保护,同时鱼体非特异性免疫能力得到增强。研究结果进一步阐明了嗜水气单胞菌致病机理和鳗鲡免疫应答体系,为预防鳗鲡病害,合理使用疫苗提供可靠的科学依据。
Recently, Anguilla Anguilla is one of the most popular breed of eel cultivated in China. After being imported, the scale and density of farming operations have been expanded rapidly. At same time, health securities of the eel are sometimes threatened by diseases, which dramatically decreases the benefits of farmers. Aeromonas hydrophilia is a common pathogen prevailed in eel farms. In order to explain the pathogenesis of Aeromonas Hydrophila, determine the immune reactions and provide scientific basis for future vaccination in practice of eels, the immune tissues morphology was observed, antibody secreting cells (ASC) were determined with ELISPOT, and nonspecific immune factors were measured after Anguilla anguilla vaccinated orally or injection with Aeromonas Hydrophila-ISCOMs. The results are listed below:
1. Isolation of MOMP of Aeromonas Hydrophila and ISCOMs preparation: MOMP of Aeromonas Hydrophila was first isolated through optimized extraction conditions and ISCOMs was prepared with the method currently used in our laboratory. The MOMP and ISCOMs were tested by using SDS-PAGE and an electron microscope, persisting in their feasibilities and validating for making Aeromonas hydrophila-MOMP ISCOMs.
2. Observation of the immune tissue morphological changes of Anguilla Anguilla after vaccination with ISCOMs, compared with control group:①The white pulp in the spleen is increased remarkably, but no other obvious pathological changes could be observed.②Villi are normal and the guts are remained structu -rally sound.③The number of lymphocytes in intestinal epithelium are different between oral vaccination and injection vaccination. In oral vaccinated group, the number of lymphocytes in the intestinal epithelium of the foregut and mid-gut are declined initially, and ascended to peak at the 21th day after vaccination (P<0.01). The number of lymphocytes in the hindgut are ascended slightly at the 7th day after vaccination, and then declined steadily. In injection vaccinated group, the number of lymphocytes in the intestinal epithelium of the foregut, mid-gut and hindgut are slight increased initially, and declined at the 7th day after the first vaccination, and then they are increased again to peak at 14th day, and then they are declined (P<0.01), except the number of lymphocytes of the hindgut are still maintained at high level to the 21th day (P<0.01), after the second vaccination.④The number of goblet cells in the intestinal epithelium of the foregut, mid-gut, and hindgut are all increased significan -tly in both oral vaccination and injection vaccination.
3. Observations of the ASC of Anguilla Anguilla after vaccinations:①the number of ASC and specific ASC are higher in groups of both oral vaccination and injection vaccination than in control at the 14th day, and the 28th day. In kidney, the differences of the number of ASC and specific ASC are significant greatly at the14th day (P<0.01) and at 28th day (P﹤0.05) respectively after oral vaccination. In the group of injection vaccination, compared to control group, the number of ASC in kidney are kept in the normal level (P>0.05), and the specific ASC are increased significantly (P﹤0.05) at the 14th day. And then the number of ASC and specific ASC in kidney are increased significantly (P﹤0.01) at 28th day.②Compared with control group, the number of the cells in skin are increased continuously after oral vaccination, and the number of the cells in skin are increased in early stage, then declined to normal level at the 28th day after injection vaccination. The number of ASC in skin are increased significantly at the 14th day and the 28th day (P<0.01) after oral vaccination. The number of specific ASC in skin are increasted significantly at 14th day (P﹤0.05), and declined to normal level at the 28 day (P>0.05). The number of ASC and specific ASC in skin are increased significantly at the14th day (P<0.01)and at the 28th day(P﹤0.05) after injection vaccination.
4. Non-specific immune factors in blood of Anguilla Anguilla after vaccination: the content of lysozyme, transferrin (Tf), and alkaline phosphatase (ALP) in the serum are higher in groups of oral and injected vaccination than the control. The interferon (IFN) titer in serum was higher in the injected group than in the control. Specific antibody secretion can be induced by either oral vaccination or injected vaccination. In conclusion, Aeromonas hydrophila-ISCOMs shows less irritation and no side effects are observed. The number of lymphocytes and goblet cells in the intestinal epithelium are both increased in different degree by oral vaccination or injected vaccination, indicating that local immune responses are induced by vaccination of aeromonas hydrophila-ISCOMs. The number of ASC and specific ASC of kidney and skin in vaccinated groups are obviously higher than that in the control, and activities of non-specific immune factors (lysozyme, transferring, alkaline phospha -tase, interferon) in blood of vaccinated groups are evidently enhanced, compared with control group, showing that immunoreactions are improved after vaccination with Aeromonas Hydrophila -ISCOMs.
引文
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