鸭瘟病毒gC基因疫苗在雏鸭体内的抗原表达时相和分布规律
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摘要
本研究围绕鸭瘟病毒(DPV)的纯化、DPV gC抗原检测方法的建立和优化、DPVgC基因疫苗(pcDNA-DPV-gC)在雏鸭体内的抗原表达时相和分布规律及DPV弱毒疫苗在雏鸭体内的增殖和分布规律等4个方面开展以下研究：①DPV的纯化和超显微结构观察；②特异性检测DPV gC抗原的间接免疫组化和间接免疫荧光方法的建立和优化；③将pcDNA-DPV-gC分别以每只6μg、3μg、1μg基因枪轰击和200μg、100μg、50μg肌肉注射免疫20日龄的雏鸭；④将100μg脂质体/DNA基因疫苗和壳聚糖/DNA基因疫苗以肌注、口服和滴鼻等不同免疫途径免疫20日周龄的雏鸭；⑤在对照组中,将0.5mL DPV弱毒疫苗、100μg空白质粒和0.5mL生理盐水以皮下和肌注等不同免疫途径免疫20日龄的雏鸭,免疫后不同的时间点(4h、12h、1d、3d、5d、7d、2w、4w、6w和10 w),随机采集雏鸭的心、肝、脾、肺、肾、胰、脑、食道、胸腺、法氏囊、哈氏腺、十二指肠、盲肠、直肠和注射部位等组织,通过建立的间接免疫组化和间接免疫荧光方法检测pcDNA-DPV-gC在雏鸭体内的抗原表达时相和分布规律及DPV弱毒疫苗在雏鸭体内的增殖和分布规律。获得结果如下：
1.利用差速离心和蔗糖密度梯度离心法纯化DPV,在电镜下可观察到多量、纯净的病毒粒子,纯化DPV具有典型的疱疹病毒的形态结构；将纯化DPV抗原多次免疫家兔可获得效价为1：32的兔抗DPV高免血清,经饱和硫酸铵粗提和High-Q柱纯化可获得较纯的兔抗DPV IgG。
2.建立的间接免疫组化和间接免疫荧光方法具有主观,敏感性强,特异性高等优点,是进行DPV gC抗原检测和定位的可靠方法,pcDNA-DPV-gC免疫后1d即可在雏鸭体内检测到DPV gC蛋白且持续表达至10 w。
3.基因枪轰击和肌肉注射免疫pcDNA-DPV-gC在雏鸭体内的抗原表达时相和分布规律：各剂量免疫组第1d在肝、十二指肠、盲肠、直肠和注射部位检测到DPV gC蛋白,其中注射部位的阳性信号最强；第2w时各组织中的抗原表达量达到高峰,随着时间推移,阳性信号以不同速度逐渐衰减；第10w时各剂量免疫组仍在肝、法氏囊、脑、十二指肠、盲肠和直肠检测到持续表达DPV gC蛋白；而胰在所有时间点均未检出阳性信号；pcDNA-DPV-gC在不同组织的表达量也存在差异,肝、脾、法氏囊、脑、十二指肠、盲肠和直肠是DPV gC抗原主要的分布器官；阳性信号主要出现在肝脏的肝细胞、脾脏白髓或红髓淋巴细胞、法氏囊皮质或髓质淋巴细胞、脑皮质神经胶质细胞及肠黏膜上皮细胞和固有层细胞等部位。
4.脂质体/DNA基因疫苗和壳聚糖/DNA基因疫苗在雏鸭体内的抗原表达时相和分布规律：肌注组免疫后1d,肝脏、法氏囊、十二指肠、盲肠和直肠出现阳性信号；滴鼻组免疫12 h,肺脏出现阳性反应细胞,免疫后1 d,哈氏腺和法氏囊检测到DPV gC蛋白；口服组免疫雏鸭12 h,食道出现中等强度的阳性染色,免疫后1d,法氏囊、十二指肠、盲肠和直肠检测到DPV gC蛋白。其中肺脏、食道、哈氏腺、法氏囊、十二指肠、盲肠和直肠阳性信号较强,且持续时间长。阳性信号主要出现在肺脏和食道的上皮细胞、法氏囊和哈氏腺淋巴细胞、肠黏膜上皮细胞和固有层细胞等部位。
5.不同剂量pcDNA-DPV-gC在雏鸭体内的抗原表达时相和分布规律：不同剂量pcDNA-DPV-gC免疫雏鸭后,各组织的抗原表达量呈现的总体规律为6μg组>3μg组>1μg组,200μg组>100μg组>50μg组,说明基因疫苗pcDNA-DPV-gC的免疫剂量与DPV gC抗原的表达和分布呈现一定的正相关,但非等比例增长。
6.不同免疫途径免疫pcDNA-DPV-gC在雏鸭体内的抗原表达时相和分布规律：基因枪轰击和肌肉注射免疫pcDNA-DPV-gC后,DPV gC抗原可在雏鸭体内广泛表达和分布。免疫早期,基因枪组各组织的DPV gC蛋白表达量大于肌注组,尤其是皮肤的DPV gC蛋白表达量高于注射部位肌肉；免疫中期,基因枪组和肌注组的阳性信号都有强烈的增加,肌注组略高于基因枪组,但差别不明显；免疫后期,基因枪组和肌注组各组织的阳性信号不断减少,免疫后10 w大部分组织检测不到阳性信号。肌注、滴鼻和口服免疫脂质体/DNA基因疫苗和壳聚糖/DNA基因疫苗后,阳性信号在雏鸭不同组织的分布也产生一定的差异,如滴鼻组哈氏腺的阳性信号比肌注组和口服组高,口服组食道的阳性信号比肌注组和口服组高,DPV gC抗原在雏鸭各组织中的表达量和持续时间的总体规律为肌注组>滴鼻组>口服组。
7.不同免疫佐剂包裹的pcDNA-DPV-gC在雏鸭体内的抗原表达时相和分布规律：免疫佐剂脂质体和壳聚糖都能促进pcDNA-DPV-gC在雏鸭体内抗原的表达和分布。壳聚糖/DNA组在雏鸭各组织中的阳性信号强度比脂质体组更强,持续时间也比脂质体组长。因此壳聚糖可作为pcDNA-DPV-gC免疫佐剂的首选。
8.DPV弱毒疫苗在雏鸭体内增殖和分布规律：DPV弱毒疫苗免疫雏鸭后,第12 h肝脏和脾脏检测到DPV抗原,随着时间推移,阳性信号在1 d-7 d呈现上升趋势,2w-4 w达到最大值,但免疫后10 w仍可在雏鸭大部分组织检测到DPV抗原。鸭瘟疫苗病毒对雏鸭各组织表现出强烈的泛嗜性,肝脏,法氏囊,胸腺、十二指肠、盲肠和直肠是DPV主要的易感器官,而心脏、胰脏和食道的检测率低。
In this paper, the study concentrates on the purification of duck plague virus (DPV), the detecting methods of DPV gC antigens, antigenic expression phase and distribution regularity of DPV gC gene vaccine, and proliferation and distribution regularity of attenuated DPV vaccine.①purification and microstructure morphology observation of DPV;②development and application of an indirect immunohistochemical method (IHC) and an indirect immunofluorescence assay (IFA) for detection of DPV gC antigens in paraffine sections;③the 20-day-old ducklings were immunized with different doses of pcDNA-DPV-gC gene vaccine via gene gun bombardment (6μg,3μg and 1μg) and intramuscular injection (200μg,100μg and 50μg), respectively;④the 20-day-old duckings were immunized with 100μg liposome/DNA gene vaccine and chitosan/DNA gene vaccine via intramuscular injection, oral administration and nasal administration, respectively;⑤In the control groups, the 20-day-old ducklings were subcutaneouly immunized 0,5 mL attenuated DPV vaccine, and other groups were intramuscularly inoculated with 0.5 mL sterile physiologic saline and 100μg blank plasmid. At intervals of 4h,12h,1d,3d,5d,7d,2w,4w,6w and 10w post-vaccination (p.v.),two ducklings were randomly euthanatized from the immunization group.After euthanasia, heart, liver, spleen, lung, kidney, pancreas, brain, esophagus, thymus, bursa of Fabricus (BF), Harderian gland, duodenum, caecum, rectum and injection site were collected. The control groups were processed in the same way.Development and application of the IHC and IFA were detected antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine in the vaccinated ducklings, and the same methods were prepared to detect proliferation and distribution regularity of attenuated DPV vaccine. The results were showed as follow:
1.The purification of DPV could be operated by conventional differential centrifugation and discontinuous sucrose-density gradient centrifugation. Many purification virus were examined by negative staining microscopy under transmission electron microscope(TEM). Some definite structures of mature DPV particles could be clearly observed with typical morphous structure of Herpervirus. Rabbit anti-DPV polyclonal serum was obtained from the rabbits immunized with purified DPV antigens, and the titers of specific antibody were more than 1:32 in the final injection. The DPV IgG was extracted by saturated ammonium sulfate method and purified through High-Q chromatograghy.
2.Development and application of the IHC and IFA are subjective, sensitive and specificity, which is a reliable method for detecting dynamic distribution of DPV gC antigens, and the DPV gC protein could be distributed in the tissues of the vaccinated ducklings at 1 d p.v.,and exist in those at least 10 w p.v..
3.The antigenic expression phase and distribution regularity of pcDNA-DPV-gC with gene gun bombardment and intramuscular injection:The positive immunoreactivity in different dose groups was found in the liver, duodenum, caecum, rectum and injected spot at 1 d p.v., and the immunoreactivity of injected spot was stronger than other organs.In addition, a drastic reduction of DPV gC antigen levels were observed after the highest immunoreactivity at 2 w p.v.,but still had a detectable vaccine antigen level in the liver, brain, duodenum, caecum and rectum at 10 w p.v.. Interestingly, the pancreas was scarcely detected positive signals at different time points; The antigen levels have a conspicuous discrepancy among the different tissues.The positive immunogenicity was mainly found in the liver, spleen, bursa of Fabricus, brain, duodenum, caecum and rectum, which served as the principal sites for antigen localization. The target cells had a ubiquitous distribution, especially in the hepatocyte of liver, the lymphocyte of splenic white pulp and red pulp, the lymphocyte of cortical substance and punctate substance of bursa of Fabricius, the neuroglia cells of brain pallium, cellula epithelialis and lamina propria mucosae of intestinal tract.
4.The antigenic expression phase and distribution regularity of liposome/DNA gene vaccine and chitosan/DNA gene vaccine:The positive signals were observed in the liver, bursa of Fabricius (BF),duodenum, caecum and rectum in the intramuscular injection group at 1 d p.v.,and in the nasal administration group, the positive staining reaction were found in the lung at 12 h p.v.,and the DPV gC proteins were observed in the Harderian gland and BF at 1 d p.v. Moreover,the positive staining were firstly found in the esophagus in the oral administration group at 12 h p.v.,and the DPV gC proteins were observed in BF, duodenum, caecum and rectum at 1 d p.v. The positive immunogenicity was mainly found in the lung, esophagus, Harderian gland, BF, duodenum, caecum and rectum. The target cells had a ubiquitous distribution, especially in the cellula epithelialis of lung and esophagus, the lympholeukocyte of BF and Harderian gland, lamina propria and cellula epithelialis of intestinal tract.
5.The antigenic expression phase and distribution regularity in the different doses of pcDNA-DPV-gC gene vaccine:The signal intensity and duration time of different doses were in below order:6μg groups>3μg groups>1μg groups,200μg groups>100μg groups>50μg groups. The results explained that there were the positive dependablity between the immunization doses and the dynamic expression regularity of DPV gC antigens.
6.The antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine in the different immunization-routes:The DPV gC antigens could distribute in the tissues of ducklings via the different immunization routes(gene gun bombardment and intramuscular injection).The expression level of DPV gC antigens in the gene gun groups were higher than the intramuscular injection groups in the early immunization period, especially DPV gC proteins of the skin were more than the muscle of injection site. The distinction could not be obvious because the number of positive immunoreactivity in all tissues of the gene gun groups and intramuscular injection groups had dramatically increased in the intermediate immunization stage. However, a drastic reduction of DPV gC antigen levels were observed, and most of tissues could not found the positive signals in the gene gun groups and intramuscular injection groups at 10 w p.v.. The antigen levels have a conspicuous discrepancy among the different tissues when the ducklings were immunized liposome/DNA gene vaccine and chitosan/DNA gene vaccine via the different immunization routes (intramuscular injection, oral administration and nasal administration). For example, the positive immunoreactivity of Harderian gland in the nasal administration groups were higher than the intramuscular injection groups and oral administrations groups, and the antigen levels of esophagus in the oral administration groups were more than the intramuscular injection groups and nasal administration groups. The signal intensity and duration time of different doses were in below order:intramuscular injection groups> nasal administration groups>oral administration groups.
7. The antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine in the different immunological adjuvant:The different immunization adjuvant could promote antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine. The result showed that the persistence time and the positive signal intensity in the chitosan groups were more than the liposome groups. Chitosan was better immunological adjuvant as pcDNA-DPV-gC gene vaccine.
8.The proliferation and distribution regularity of attenuated DPV vaccine:The first signals of DPV-specific antigens were observed in the liver and spleen at 12 h p.v., but no positive staining cells could be observed in the controls birds.The number of positive immunoreactivity in all tissues had dramatically increased to reach peak levels between 1 d and 7 d p.v.. The highest levels of positive staining reaction were found between 2 w and 4 w p.v.. The attenuated DPV vaccine appeared intense pantropic to the tissues of the vaccinated ducklings. The liver, BF, thymus, duodenum, caecum, rectum were served as the principal sites for antigens localization, but the heart, pancreas and esophagus had lower detection ratio.
1.利用差速离心和蔗糖密度梯度离心法纯化DPV,在电镜下可观察到多量、纯净的病毒粒子,纯化DPV具有典型的疱疹病毒的形态结构；将纯化DPV抗原多次免疫家兔可获得效价为1：32的兔抗DPV高免血清,经饱和硫酸铵粗提和High-Q柱纯化可获得较纯的兔抗DPV IgG。
2.建立的间接免疫组化和间接免疫荧光方法具有主观,敏感性强,特异性高等优点,是进行DPV gC抗原检测和定位的可靠方法,pcDNA-DPV-gC免疫后1d即可在雏鸭体内检测到DPV gC蛋白且持续表达至10 w。
3.基因枪轰击和肌肉注射免疫pcDNA-DPV-gC在雏鸭体内的抗原表达时相和分布规律：各剂量免疫组第1d在肝、十二指肠、盲肠、直肠和注射部位检测到DPV gC蛋白,其中注射部位的阳性信号最强；第2w时各组织中的抗原表达量达到高峰,随着时间推移,阳性信号以不同速度逐渐衰减；第10w时各剂量免疫组仍在肝、法氏囊、脑、十二指肠、盲肠和直肠检测到持续表达DPV gC蛋白；而胰在所有时间点均未检出阳性信号；pcDNA-DPV-gC在不同组织的表达量也存在差异,肝、脾、法氏囊、脑、十二指肠、盲肠和直肠是DPV gC抗原主要的分布器官；阳性信号主要出现在肝脏的肝细胞、脾脏白髓或红髓淋巴细胞、法氏囊皮质或髓质淋巴细胞、脑皮质神经胶质细胞及肠黏膜上皮细胞和固有层细胞等部位。
4.脂质体/DNA基因疫苗和壳聚糖/DNA基因疫苗在雏鸭体内的抗原表达时相和分布规律：肌注组免疫后1d,肝脏、法氏囊、十二指肠、盲肠和直肠出现阳性信号；滴鼻组免疫12 h,肺脏出现阳性反应细胞,免疫后1 d,哈氏腺和法氏囊检测到DPV gC蛋白；口服组免疫雏鸭12 h,食道出现中等强度的阳性染色,免疫后1d,法氏囊、十二指肠、盲肠和直肠检测到DPV gC蛋白。其中肺脏、食道、哈氏腺、法氏囊、十二指肠、盲肠和直肠阳性信号较强,且持续时间长。阳性信号主要出现在肺脏和食道的上皮细胞、法氏囊和哈氏腺淋巴细胞、肠黏膜上皮细胞和固有层细胞等部位。
5.不同剂量pcDNA-DPV-gC在雏鸭体内的抗原表达时相和分布规律：不同剂量pcDNA-DPV-gC免疫雏鸭后,各组织的抗原表达量呈现的总体规律为6μg组>3μg组>1μg组,200μg组>100μg组>50μg组,说明基因疫苗pcDNA-DPV-gC的免疫剂量与DPV gC抗原的表达和分布呈现一定的正相关,但非等比例增长。
6.不同免疫途径免疫pcDNA-DPV-gC在雏鸭体内的抗原表达时相和分布规律：基因枪轰击和肌肉注射免疫pcDNA-DPV-gC后,DPV gC抗原可在雏鸭体内广泛表达和分布。免疫早期,基因枪组各组织的DPV gC蛋白表达量大于肌注组,尤其是皮肤的DPV gC蛋白表达量高于注射部位肌肉；免疫中期,基因枪组和肌注组的阳性信号都有强烈的增加,肌注组略高于基因枪组,但差别不明显；免疫后期,基因枪组和肌注组各组织的阳性信号不断减少,免疫后10 w大部分组织检测不到阳性信号。肌注、滴鼻和口服免疫脂质体/DNA基因疫苗和壳聚糖/DNA基因疫苗后,阳性信号在雏鸭不同组织的分布也产生一定的差异,如滴鼻组哈氏腺的阳性信号比肌注组和口服组高,口服组食道的阳性信号比肌注组和口服组高,DPV gC抗原在雏鸭各组织中的表达量和持续时间的总体规律为肌注组>滴鼻组>口服组。
7.不同免疫佐剂包裹的pcDNA-DPV-gC在雏鸭体内的抗原表达时相和分布规律：免疫佐剂脂质体和壳聚糖都能促进pcDNA-DPV-gC在雏鸭体内抗原的表达和分布。壳聚糖/DNA组在雏鸭各组织中的阳性信号强度比脂质体组更强,持续时间也比脂质体组长。因此壳聚糖可作为pcDNA-DPV-gC免疫佐剂的首选。
8.DPV弱毒疫苗在雏鸭体内增殖和分布规律：DPV弱毒疫苗免疫雏鸭后,第12 h肝脏和脾脏检测到DPV抗原,随着时间推移,阳性信号在1 d-7 d呈现上升趋势,2w-4 w达到最大值,但免疫后10 w仍可在雏鸭大部分组织检测到DPV抗原。鸭瘟疫苗病毒对雏鸭各组织表现出强烈的泛嗜性,肝脏,法氏囊,胸腺、十二指肠、盲肠和直肠是DPV主要的易感器官,而心脏、胰脏和食道的检测率低。
In this paper, the study concentrates on the purification of duck plague virus (DPV), the detecting methods of DPV gC antigens, antigenic expression phase and distribution regularity of DPV gC gene vaccine, and proliferation and distribution regularity of attenuated DPV vaccine.①purification and microstructure morphology observation of DPV;②development and application of an indirect immunohistochemical method (IHC) and an indirect immunofluorescence assay (IFA) for detection of DPV gC antigens in paraffine sections;③the 20-day-old ducklings were immunized with different doses of pcDNA-DPV-gC gene vaccine via gene gun bombardment (6μg,3μg and 1μg) and intramuscular injection (200μg,100μg and 50μg), respectively;④the 20-day-old duckings were immunized with 100μg liposome/DNA gene vaccine and chitosan/DNA gene vaccine via intramuscular injection, oral administration and nasal administration, respectively;⑤In the control groups, the 20-day-old ducklings were subcutaneouly immunized 0,5 mL attenuated DPV vaccine, and other groups were intramuscularly inoculated with 0.5 mL sterile physiologic saline and 100μg blank plasmid. At intervals of 4h,12h,1d,3d,5d,7d,2w,4w,6w and 10w post-vaccination (p.v.),two ducklings were randomly euthanatized from the immunization group.After euthanasia, heart, liver, spleen, lung, kidney, pancreas, brain, esophagus, thymus, bursa of Fabricus (BF), Harderian gland, duodenum, caecum, rectum and injection site were collected. The control groups were processed in the same way.Development and application of the IHC and IFA were detected antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine in the vaccinated ducklings, and the same methods were prepared to detect proliferation and distribution regularity of attenuated DPV vaccine. The results were showed as follow:
1.The purification of DPV could be operated by conventional differential centrifugation and discontinuous sucrose-density gradient centrifugation. Many purification virus were examined by negative staining microscopy under transmission electron microscope(TEM). Some definite structures of mature DPV particles could be clearly observed with typical morphous structure of Herpervirus. Rabbit anti-DPV polyclonal serum was obtained from the rabbits immunized with purified DPV antigens, and the titers of specific antibody were more than 1:32 in the final injection. The DPV IgG was extracted by saturated ammonium sulfate method and purified through High-Q chromatograghy.
2.Development and application of the IHC and IFA are subjective, sensitive and specificity, which is a reliable method for detecting dynamic distribution of DPV gC antigens, and the DPV gC protein could be distributed in the tissues of the vaccinated ducklings at 1 d p.v.,and exist in those at least 10 w p.v..
3.The antigenic expression phase and distribution regularity of pcDNA-DPV-gC with gene gun bombardment and intramuscular injection:The positive immunoreactivity in different dose groups was found in the liver, duodenum, caecum, rectum and injected spot at 1 d p.v., and the immunoreactivity of injected spot was stronger than other organs.In addition, a drastic reduction of DPV gC antigen levels were observed after the highest immunoreactivity at 2 w p.v.,but still had a detectable vaccine antigen level in the liver, brain, duodenum, caecum and rectum at 10 w p.v.. Interestingly, the pancreas was scarcely detected positive signals at different time points; The antigen levels have a conspicuous discrepancy among the different tissues.The positive immunogenicity was mainly found in the liver, spleen, bursa of Fabricus, brain, duodenum, caecum and rectum, which served as the principal sites for antigen localization. The target cells had a ubiquitous distribution, especially in the hepatocyte of liver, the lymphocyte of splenic white pulp and red pulp, the lymphocyte of cortical substance and punctate substance of bursa of Fabricius, the neuroglia cells of brain pallium, cellula epithelialis and lamina propria mucosae of intestinal tract.
4.The antigenic expression phase and distribution regularity of liposome/DNA gene vaccine and chitosan/DNA gene vaccine:The positive signals were observed in the liver, bursa of Fabricius (BF),duodenum, caecum and rectum in the intramuscular injection group at 1 d p.v.,and in the nasal administration group, the positive staining reaction were found in the lung at 12 h p.v.,and the DPV gC proteins were observed in the Harderian gland and BF at 1 d p.v. Moreover,the positive staining were firstly found in the esophagus in the oral administration group at 12 h p.v.,and the DPV gC proteins were observed in BF, duodenum, caecum and rectum at 1 d p.v. The positive immunogenicity was mainly found in the lung, esophagus, Harderian gland, BF, duodenum, caecum and rectum. The target cells had a ubiquitous distribution, especially in the cellula epithelialis of lung and esophagus, the lympholeukocyte of BF and Harderian gland, lamina propria and cellula epithelialis of intestinal tract.
5.The antigenic expression phase and distribution regularity in the different doses of pcDNA-DPV-gC gene vaccine:The signal intensity and duration time of different doses were in below order:6μg groups>3μg groups>1μg groups,200μg groups>100μg groups>50μg groups. The results explained that there were the positive dependablity between the immunization doses and the dynamic expression regularity of DPV gC antigens.
6.The antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine in the different immunization-routes:The DPV gC antigens could distribute in the tissues of ducklings via the different immunization routes(gene gun bombardment and intramuscular injection).The expression level of DPV gC antigens in the gene gun groups were higher than the intramuscular injection groups in the early immunization period, especially DPV gC proteins of the skin were more than the muscle of injection site. The distinction could not be obvious because the number of positive immunoreactivity in all tissues of the gene gun groups and intramuscular injection groups had dramatically increased in the intermediate immunization stage. However, a drastic reduction of DPV gC antigen levels were observed, and most of tissues could not found the positive signals in the gene gun groups and intramuscular injection groups at 10 w p.v.. The antigen levels have a conspicuous discrepancy among the different tissues when the ducklings were immunized liposome/DNA gene vaccine and chitosan/DNA gene vaccine via the different immunization routes (intramuscular injection, oral administration and nasal administration). For example, the positive immunoreactivity of Harderian gland in the nasal administration groups were higher than the intramuscular injection groups and oral administrations groups, and the antigen levels of esophagus in the oral administration groups were more than the intramuscular injection groups and nasal administration groups. The signal intensity and duration time of different doses were in below order:intramuscular injection groups> nasal administration groups>oral administration groups.
7. The antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine in the different immunological adjuvant:The different immunization adjuvant could promote antigenic expression phase and distribution regularity of pcDNA-DPV-gC gene vaccine. The result showed that the persistence time and the positive signal intensity in the chitosan groups were more than the liposome groups. Chitosan was better immunological adjuvant as pcDNA-DPV-gC gene vaccine.
8.The proliferation and distribution regularity of attenuated DPV vaccine:The first signals of DPV-specific antigens were observed in the liver and spleen at 12 h p.v., but no positive staining cells could be observed in the controls birds.The number of positive immunoreactivity in all tissues had dramatically increased to reach peak levels between 1 d and 7 d p.v.. The highest levels of positive staining reaction were found between 2 w and 4 w p.v.. The attenuated DPV vaccine appeared intense pantropic to the tissues of the vaccinated ducklings. The liver, BF, thymus, duodenum, caecum, rectum were served as the principal sites for antigens localization, but the heart, pancreas and esophagus had lower detection ratio.
引文
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