猪传染性胃肠炎灭活病毒口服免疫对仔猪消化道局部和全身免疫水平的影响

发布时间：2018-08-23 18:57

【摘要】：猪传染性胃肠炎是由猪传染性胃肠炎病毒引起的一种高度接触性传染病,主要通过消化道进行传播,能引起2周龄以下仔猪严重腹泻、呕吐、脱水和高死亡率。通过消化道口服免疫可直接切断猪传染性胃肠炎病毒的感染和传播途径,有效控制猪传染性胃肠炎的发生。然而灭活全病毒单独口服免疫不足以有效激发机体的免疫应答,需选择合适的黏膜佐剂来增强疫苗的免疫原性,进而达到良好的免疫效果。近年来利用CpG作为黏膜免疫增强剂已成为疫苗领域的研究热点。CpG作为有效的黏膜佐剂在小鼠上已有大量研究,但在猪上其相关报道较少。因此本试验通过体外和体内试验研究探讨CpG配合猪传染性胃肠炎灭活病毒口服免疫对仔猪消化道局部和全身免疫水平的影响,为研究应激性小、保护力高的猪传染性胃肠炎口服疫苗奠定基础。首先,通过体外试验检测CpG对猪脾脏淋巴细胞的免疫刺激作用。其次,在仔猪肠结扎试验中,探讨CpG配合猪传染性胃肠炎灭活病毒对肠黏膜组织中细胞因子IL-6、IL-12和IFN-γmRNA水平的影响;在仔猪免疫试验中,选取20头6周龄三元杂交仔猪,随机分为5组进行首次免疫,一周后进行二次免疫,正常饲养,首免后42天宰杀,应用组织学切片技术检测回肠CD3+T淋巴细胞、上皮内淋巴细胞(IEL)的数目,以此评估CpG配合猪传染性胃肠炎灭活病毒口服免疫对消化道局部细胞免疫水平的影响。另外,通过免疫组化技术检测回肠组织中IgA分泌细胞的阳性面积,间接ELISA法检测粪便和小肠组织中猪传染性胃肠炎病毒特异性SIgA抗体水平,探讨猪传染性胃肠炎灭活病毒口服免疫对消化道局部体液免疫水平的影响;通过检测血清中猪传染性胃肠炎病毒特异性IgG抗体水平来探讨猪传染性胃肠炎灭活病毒口服免疫对全身体液免疫水平的影响。本研究内容分为以下四个部分:1 CpG对猪脾脏淋巴细胞的增殖刺激作用用细菌CpG-DNA、人工合成的CpG-ODN、猪传染性胃肠炎灭活病毒、CpG-DNA配合猪传染性胃肠炎灭活病毒刺激猪脾脏淋巴细胞72 h,检测几种处理对猪脾脏淋巴细胞的增殖作用。结果发现:CpG-DNA和CpG-ODN均能在体外72 h内诱导猪脾脏淋巴细胞显著增殖,且二者的刺激指数之间无显著差异;CpG配合TGEV能刺激猪的脾脏淋巴细胞显著增殖。本试验结果表明CpG对猪脾脏淋巴细胞具有良好的免疫刺激作用,为后续的动物试验提供了重要参考。2猪传染性胃肠炎灭活病毒口服免疫对猪消化道局部细胞免疫水平的影响仔猪回肠结扎后注射猪传染性胃肠炎灭活病毒和CpG,6 h后宰杀采集回肠组织,应用实时荧光定量PCR的方法检测IL-6、IL-12和IFN-γ mRNA的表达量。结果表明:应用CpG配合猪传染性胃肠炎灭活病毒,小肠中IL-6、IL-12和IFN-γ mRNA水平显著升高;单独应用CpG也能增加IL-6、IL-12和IFN-γmRNA的表达水平;单独应用猪传染性胃肠炎灭活病毒不能使IL-6和IFN-γ mRNA水平增加,但能增加IL-12的mRNA表达量。仔猪口服免疫后42天宰杀,采集回肠组织进行固定,石蜡包埋并制作组织学切片,应用免疫组化法显示回肠CD3+T淋巴细胞分布和数量变化,结果表明:应用CpG配合猪传染性胃肠炎灭活病毒口服免疫能够显著增加回肠绒毛处CD3+ T淋巴细胞的数量。应用HE染色法显示猪回肠黏膜上皮内淋巴细胞分布和数量变化。结果表明:应用CpG配合猪传染性胃肠炎灭活病毒口服免疫后,回肠黏膜上皮内淋巴细胞数目显著增加。以上三个试验结果提示,应用CpG配合猪传染性胃肠炎灭活病毒口服免疫可有效诱导仔猪消化道局部细胞免疫应答。3猪传染性胃肠炎灭活病毒口服免疫对猪消化道局部体液免疫水平的影响应用免疫组化法显示仔猪回肠IgA分泌细胞。结果表明:应用CpG配合猪传染性胃肠炎灭活病毒口服免疫能够显著增加仔猪回肠IgA分泌细胞的面积。应用间接ELISA法每周监测粪便中猪传染性胃肠炎病毒特异性SIgA抗体水平,结果表明:首免后前三周,应用CpG配合猪传染性胃肠炎灭活病毒口服免疫能够显著提高鼻粪便中特异性SIgA抗体水平;单独应用猪传染性胃肠炎灭活病毒也能显著提高粪便中特异性SIgA抗体水平,但效果不如CpG配合组。首免第四周时,口服免疫组中猪传染性胃肠炎病毒特异性SIgA抗体水平逐渐下降,各组之间无显著差异。应用间接ELISA法检测回肠组织中特异性IgA抗体水平,结果表明:单独应用猪传染性胃肠炎灭活病毒或与CpG配合口服免疫45天后,回肠组织中特异性IgA抗体的水平仍显著高于对照组;皮下注射猪传染性胃肠炎灭活病毒组肠组织中特异性IgA抗体水平无显著变化。以上三个试验结果提示,应用CpG配合猪传染性胃肠炎病毒口服免疫可有效诱导仔猪消化道局部体液免疫应答。4猪传染性胃肠炎灭活病毒口服免疫对猪全身免疫水平的影响应用间接ELISA法每周监测血清中猪传染性胃肠炎病毒特异性IgG抗体水平,结果表明:皮下注射TGEV灭活病毒组血清中IgG水平最高,且一直较稳定;在免疫早期,CpG配合猪传染性胃肠炎灭活病毒口服免疫能够显著提高血清中特异性IgG抗体的水平,效果接近皮下注射组;单独应用猪传染性胃肠炎灭活病毒口服免疫效果不如CpG配合组;随着仔猪日龄增长,各组血清中特异性IgG抗体水平持续下降,但在免疫后35天仍显著高于对照组。结果提示,应用CpG配合猪传染性胃肠炎灭活病毒口服免疫能够有效诱导全身免疫应答。
[Abstract]:* * transmissible gastroenteritis is a highly contagious disease caused by transmissible gastroenteritis virus. It is mainly transmitted through the alimentary canal. * it can cause severe diarrhea, vomiting, dehydration and high mortality in piglets less than 2 weeks of age. Through oral immunization of the digestive tract, the infection and transmission route of porcine transmission gastroenteritis virus can be directly cut off. * to control the occurrence of transmissible gastroenteritis in pigs. However, oral inactivation of whole virus alone is not enough to stimulate the immune response of the body. It is necessary to select suitable mucosal adjuvants to enhance the immunogenicity of the vaccine and achieve good immune effect. In recent years, using CpG as mucosal immune enhancer has become a research hotspot in the field of vaccines..CpG As an effective mucosal adjuvant, a lot of studies have been carried out in mice, but there are few reports on pigs. Therefore, this * * * in vitro and in vivo studies the effects of oral immunization with CpG on inactivation of the transmissible gastroenteritis virus on the local and systemic immune level of piglets. First, the immunostimulatory effect of CpG on pig spleen lymphocytes was detected by * in vitro test. Secondly, the effects of CpG combined with transmissible gastroenteritis inactivated virus on the levels of cytokines * IL-6, IL-12 and IFN- * mRNA in intestinal mucosa were investigated in piglet intestinal ligation test. 20 * 6 week old three yuan cross piglets were randomly divided into 5 groups. They were immunized for the first time. After two weeks of immunization, they were fed normally and 42 days after the first immunization. The number of ileum CD3+T lymphocytes and intraepithelial lymphocytes (IEL *) was detected by histological sections, and the CpG immunization against the inactivated virus of transmissible gastroenteritis was evaluated. In addition, the positive area of IgA secreting cells in ileum tissue was detected by immunohistochemistry. Indirect ELISA was used to detect the level of specific SIgA antibody in fecal and small intestine tissues. The * * of inactivated virus in pigs was detected by oral immunization against the local body fluid in the alimentary canal. The influence of immune level was investigated. The influence of * * transmissible gastroenteritis virus specific IgG antibody level in serum on the immune level of whole body fluid of swine infectious gastroenteritis inactivated virus was investigated. The study is divided into four parts: 1 * CpG, the proliferation and stimulation of splenic lymphoid cells in pigs, using bacteria CpG-DNA, artificial labor. The * * CpG-ODN, transmissible gastroenteritis inactivated virus, CpG-DNA and swine infectious gastroenteritis inactivated virus stimulated 72 * h of pig spleen lymphocytes. The proliferation of porcine spleen lymphocytes was detected by several treatments. It was found that both CpG-DNA and CpG-ODN could induce significant proliferation of porcine splenic lymphocytes in 72 h in vitro, and two stimuli. There was no significant difference between the index *; CpG and TGEV could stimulate the proliferation of splenic lymphocytes in pigs * the results showed that CpG had a good immune stimulation effect on porcine splenic lymphocytes, and provided an important reference for subsequent animal experiments..2 * transmissible gastroenteritis inactivated virus was used for oral immunization to local cellular immune water in pigs. The influence of flat infection on infective * * and CpG of transmissible gastroenteritis after piglet ileostomy was killed. After 6 h, the ileal tissues were slaughtered and the expression levels of IL-6, IL-12 and IFN- * mRNA were detected by real-time fluorescence quantitative PCR. The results showed that IL-6, IL-12 and IFN- gamma levels in the small intestine were significantly increased by using CpG combined with inactivated swine transmissible gastroenteritis virus. High level; the application of CpG alone can increase the expression level of IL-6, IL-12 and IFN- * mRNA; the inactivation of virus by using transmissible gastroenteritis alone can not increase IL-6 and IFN- * mRNA levels, but can increase the mRNA expression of IL-12. 42 days after oral immunization, the piglets were slaughtered, and the ileum tissues were fixed, paraffin embedded and made histological sections. The distribution and quantity of CD3+T lymphocytes in ileum were revealed by the histochemical method. The results showed that * oral immunization with CpG inactivated with transmissible gastroenteritis virus could significantly increase the number of CD3+ * T lymphocytes in ileum villi. The distribution and quantity of lymphoid cells in the ileum mucosa of pigs were shown by HE staining. After oral immunization with G * inactivated virus of transmissible gastroenteritis virus, the number of lymphocytes in the ileum mucosa increased significantly. The above three * * results suggest that oral immunization with CpG combined with inactivated virus of transmissible gastroenteritis can effectively induce local cellular immunity in the digestive tract of piglets, and the.3 transmissible gastroenteritis inactivated virus is free. The effect of pestilence on the local humoral immunity level in the digestive tract of pigs was investigated by immunohistochemical method. The results showed that oral immunization with CpG combined with inactivated virus of transmissible gastroenteritis could significantly increase the area of IgA secreting cells in the ileum of piglets. Indirect IgA was used to monitor transmissible gastroenteritis in feces weekly by indirect ELISA. The level of specific SIgA antibody showed that: the first three weeks after the first immunization, the application of CpG combined with inactivated virus of swine transmissible gastroenteritis virus could significantly increase the level of specific SIgA antibody in nasal excrement. The application of inactivated swine transmissible gastroenteritis virus could significantly increase the level of specific SIgA antibody in feces, but the effect was not as good as that of CpG. In the first week, the level of specific SIgA antibody of transmissible gastroenteritis virus in the oral immunization group decreased gradually. There was no significant difference between the groups. Indirect ELISA was used to detect the level of specific IgA antibody in the ileum tissues. The results showed that the virus was inactivated only by transmissible transmissible gastroenteritis virus or 45 days after oral immunization with CpG. The level of specific IgA antibody in intestinal tissue was still significantly higher than that in control group * there was no significant change in the level of specific IgA antibody in intestinal tissue of subcutaneous injection of transmissible gastroenteritis inactivated virus group. The above three * * results suggest that oral immunization with CpG and swine transmissible gastroenteritis virus can effectively induce local humoral immunity in the digestive tract of piglets. The effect of oral immunization against.4 * * transmissible gastroenteritis inactivated virus on the whole body immune level of pigs * the indirect ELISA method was used to monitor the level of specific IgG antibody of swine transmissible gastroenteritis virus in serum every week. The results showed that the level of IgG in the serum of the inactivated TGEV group was the highest and remained stable. Oral immunization with inactivated virus in patients with dyed gastroenteritis can significantly increase the level of specific IgG antibody in serum * the effect is similar to that in subcutaneous injection group; the effect of oral immunization with inactivated * transmissible gastroenteritis virus alone is not as good as that in CpG matching group; with the increase of piglet age, serum specific IgG antibody levels in each group continue to decline, but after immunization 35 The results indicate that oral immunization with CpG and inactivated virus * can effectively induce systemic immune response.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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