豬傳染性胃腸炎滅活病毒口服免疫對(duì)仔豬消化道局部和全身免疫水平的影響
發(fā)布時(shí)間：2018-08-23 18:57
【摘要】：豬傳染性胃腸炎是由豬傳染性胃腸炎病毒引起的一種高度接觸性傳染病,主要通過消化道進(jìn)行傳播,能引起2周齡以下仔豬嚴(yán)重腹瀉、嘔吐、脫水和高死亡率。通過消化道口服免疫可直接切斷豬傳染性胃腸炎病毒的感染和傳播途徑,有效控制豬傳染性胃腸炎的發(fā)生。然而滅活全病毒單獨(dú)口服免疫不足以有效激發(fā)機(jī)體的免疫應(yīng)答,需選擇合適的黏膜佐劑來增強(qiáng)疫苗的免疫原性,進(jìn)而達(dá)到良好的免疫效果。近年來利用CpG作為黏膜免疫增強(qiáng)劑已成為疫苗領(lǐng)域的研究熱點(diǎn)。CpG作為有效的黏膜佐劑在小鼠上已有大量研究,但在豬上其相關(guān)報(bào)道較少。因此本試驗(yàn)通過體外和體內(nèi)試驗(yàn)研究探討CpG配合豬傳染性胃腸炎滅活病毒口服免疫對(duì)仔豬消化道局部和全身免疫水平的影響,為研究應(yīng)激性小、保護(hù)力高的豬傳染性胃腸炎口服疫苗奠定基礎(chǔ)。首先,通過體外試驗(yàn)檢測(cè)CpG對(duì)豬脾臟淋巴細(xì)胞的免疫刺激作用。其次,在仔豬腸結(jié)扎試驗(yàn)中,探討CpG配合豬傳染性胃腸炎滅活病毒對(duì)腸黏膜組織中細(xì)胞因子IL-6、IL-12和IFN-γmRNA水平的影響;在仔豬免疫試驗(yàn)中,選取20頭6周齡三元雜交仔豬,隨機(jī)分為5組進(jìn)行首次免疫,一周后進(jìn)行二次免疫,正常飼養(yǎng),首免后42天宰殺,應(yīng)用組織學(xué)切片技術(shù)檢測(cè)回腸CD3+T淋巴細(xì)胞、上皮內(nèi)淋巴細(xì)胞(IEL)的數(shù)目,以此評(píng)估CpG配合豬傳染性胃腸炎滅活病毒口服免疫對(duì)消化道局部細(xì)胞免疫水平的影響。另外,通過免疫組化技術(shù)檢測(cè)回腸組織中IgA分泌細(xì)胞的陽(yáng)性面積,間接ELISA法檢測(cè)糞便和小腸組織中豬傳染性胃腸炎病毒特異性SIgA抗體水平,探討豬傳染性胃腸炎滅活病毒口服免疫對(duì)消化道局部體液免疫水平的影響;通過檢測(cè)血清中豬傳染性胃腸炎病毒特異性IgG抗體水平來探討豬傳染性胃腸炎滅活病毒口服免疫對(duì)全身體液免疫水平的影響。本研究?jī)?nèi)容分為以下四個(gè)部分:1 CpG對(duì)豬脾臟淋巴細(xì)胞的增殖刺激作用用細(xì)菌CpG-DNA、人工合成的CpG-ODN、豬傳染性胃腸炎滅活病毒、CpG-DNA配合豬傳染性胃腸炎滅活病毒刺激豬脾臟淋巴細(xì)胞72 h,檢測(cè)幾種處理對(duì)豬脾臟淋巴細(xì)胞的增殖作用。結(jié)果發(fā)現(xiàn):CpG-DNA和CpG-ODN均能在體外72 h內(nèi)誘導(dǎo)豬脾臟淋巴細(xì)胞顯著增殖,且二者的刺激指數(shù)之間無顯著差異;CpG配合TGEV能刺激豬的脾臟淋巴細(xì)胞顯著增殖。本試驗(yàn)結(jié)果表明CpG對(duì)豬脾臟淋巴細(xì)胞具有良好的免疫刺激作用,為后續(xù)的動(dòng)物試驗(yàn)提供了重要參考。2豬傳染性胃腸炎滅活病毒口服免疫對(duì)豬消化道局部細(xì)胞免疫水平的影響仔豬回腸結(jié)扎后注射豬傳染性胃腸炎滅活病毒和CpG,6 h后宰殺采集回腸組織,應(yīng)用實(shí)時(shí)熒光定量PCR的方法檢測(cè)IL-6、IL-12和IFN-γ mRNA的表達(dá)量。結(jié)果表明:應(yīng)用CpG配合豬傳染性胃腸炎滅活病毒,小腸中IL-6、IL-12和IFN-γ mRNA水平顯著升高;單獨(dú)應(yīng)用CpG也能增加IL-6、IL-12和IFN-γmRNA的表達(dá)水平;單獨(dú)應(yīng)用豬傳染性胃腸炎滅活病毒不能使IL-6和IFN-γ mRNA水平增加,但能增加IL-12的mRNA表達(dá)量。仔豬口服免疫后42天宰殺,采集回腸組織進(jìn)行固定,石蠟包埋并制作組織學(xué)切片,應(yīng)用免疫組化法顯示回腸CD3+T淋巴細(xì)胞分布和數(shù)量變化,結(jié)果表明:應(yīng)用CpG配合豬傳染性胃腸炎滅活病毒口服免疫能夠顯著增加回腸絨毛處CD3+ T淋巴細(xì)胞的數(shù)量。應(yīng)用HE染色法顯示豬回腸黏膜上皮內(nèi)淋巴細(xì)胞分布和數(shù)量變化。結(jié)果表明:應(yīng)用CpG配合豬傳染性胃腸炎滅活病毒口服免疫后,回腸黏膜上皮內(nèi)淋巴細(xì)胞數(shù)目顯著增加。以上三個(gè)試驗(yàn)結(jié)果提示,應(yīng)用CpG配合豬傳染性胃腸炎滅活病毒口服免疫可有效誘導(dǎo)仔豬消化道局部細(xì)胞免疫應(yīng)答。3豬傳染性胃腸炎滅活病毒口服免疫對(duì)豬消化道局部體液免疫水平的影響應(yīng)用免疫組化法顯示仔豬回腸IgA分泌細(xì)胞。結(jié)果表明:應(yīng)用CpG配合豬傳染性胃腸炎滅活病毒口服免疫能夠顯著增加仔豬回腸IgA分泌細(xì)胞的面積。應(yīng)用間接ELISA法每周監(jiān)測(cè)糞便中豬傳染性胃腸炎病毒特異性SIgA抗體水平,結(jié)果表明:首免后前三周,應(yīng)用CpG配合豬傳染性胃腸炎滅活病毒口服免疫能夠顯著提高鼻糞便中特異性SIgA抗體水平;單獨(dú)應(yīng)用豬傳染性胃腸炎滅活病毒也能顯著提高糞便中特異性SIgA抗體水平,但效果不如CpG配合組。首免第四周時(shí),口服免疫組中豬傳染性胃腸炎病毒特異性SIgA抗體水平逐漸下降,各組之間無顯著差異。應(yīng)用間接ELISA法檢測(cè)回腸組織中特異性IgA抗體水平,結(jié)果表明:單獨(dú)應(yīng)用豬傳染性胃腸炎滅活病毒或與CpG配合口服免疫45天后,回腸組織中特異性IgA抗體的水平仍顯著高于對(duì)照組;皮下注射豬傳染性胃腸炎滅活病毒組腸組織中特異性IgA抗體水平無顯著變化。以上三個(gè)試驗(yàn)結(jié)果提示,應(yīng)用CpG配合豬傳染性胃腸炎病毒口服免疫可有效誘導(dǎo)仔豬消化道局部體液免疫應(yīng)答。4豬傳染性胃腸炎滅活病毒口服免疫對(duì)豬全身免疫水平的影響應(yīng)用間接ELISA法每周監(jiān)測(cè)血清中豬傳染性胃腸炎病毒特異性IgG抗體水平,結(jié)果表明:皮下注射TGEV滅活病毒組血清中IgG水平最高,且一直較穩(wěn)定;在免疫早期,CpG配合豬傳染性胃腸炎滅活病毒口服免疫能夠顯著提高血清中特異性IgG抗體的水平,效果接近皮下注射組;單獨(dú)應(yīng)用豬傳染性胃腸炎滅活病毒口服免疫效果不如CpG配合組;隨著仔豬日齡增長(zhǎng),各組血清中特異性IgG抗體水平持續(xù)下降,但在免疫后35天仍顯著高于對(duì)照組。結(jié)果提示,應(yīng)用CpG配合豬傳染性胃腸炎滅活病毒口服免疫能夠有效誘導(dǎo)全身免疫應(yīng)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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28
本文編號(hào)：2199612
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28
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