鸡IL-18堆型艾美耳球虫3-1E-α-tubulin增强型核酸疫苗免疫保护机理
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摘要
鸡球虫病是由几种艾美耳球虫引起的一种肠道性疾病,是影响家禽生产性能、降低经济效益的重要寄生虫性疾病。实践证明,应用刺激宿主机体产生广泛的免疫反应来预防和治疗鸡球虫病是更有效的方法之一。核酸疫苗的显著优势是刺激机体产生广泛的细胞免疫和体液免疫应答及持久的免疫记忆,尤其是结合一定数量的细胞因子制成协同的核酸疫苗,更具有诱人的前景。基于此我们选择两种球虫主要抗原基因进行连接构建成融合基因,插入到真核表达载体中并和特定的细胞因子组成增强型核酸疫苗,检测鸡抗球虫攻击的免疫保护作用。
本研究选择禽类艾美尔球虫各虫种间保守性较高的3-1E基因和α-tubulin基因,以及新近发现的可作为免疫佐剂,具有增强免疫和治疗作用的多功能细胞因子鸡白细胞介素18(ChlL-18)为研究对象,以PVAX1为载体构建并鉴定了PVAX-α-tubulin真核表达质粒和PVAX-3-1E-α-tubulin融合基因真核质粒,通过基因免疫途径研究了5种真核表达质粒对免疫鸡的免疫调节机理以及对感染堆型艾美尔球虫(E.acervulina)鸡的免疫保护作用,试验内容与结果如下：
1以E.acervulina上海株(SH株)6h孢子化卵囊的总RNA为模板,应用RT-PCR技术扩增出含有完整开放阅读框架(ORF)的α-tubulin基因,该基因片段长为1362bp,经测序鉴定得知E.acervulina SH株α-tubulin基因cDNA核苷酸序列(HQ259108)与GeneBank参考株相应序列(X88776.1)同源性为99.4%,推导氨基酸序列同源性为99.5%。
2通过设计引物将3-1E基因的终止密码子删除,同时去除α-tubulin基因的起始密码子。应用重叠扩增PCR(Gene splicing overlap extension PCR,SOE PCR)技术成功地将E acervulina3-1E基因和α-tubulin基因片段进行连接,并在两个基因之间引入了柔性连接肽(G4S)3,获得融合基因3-1E-linker-α-tubulin。以PVAX1为载体成功构建了融合基因的真核表达质粒PVAX-3-1E-α-tubulin,融合基因质粒的体外瞬时转染试验表明,融合基因能够在细胞内进行高效表达,抗3-1E多克隆抗体和抗α-tubulin多克隆抗体分别能与融合基因表达的蛋白发生免疫学反应,表明构建的融合基因表达蛋白很好的保留了融合前两段基因蛋白的生物学活性,为后续研究融合基因对球虫感染的免疫保护机理提供了理论支持。
3实验分组：实验中构建5种真核表达质粒研究核酸疫苗对机体免疫保护效果,实验分为8组,分别为不免疫不攻虫组(B)、不免疫攻虫组(H)、PVAX空质粒免疫组(PVAX)、 PVAX-IL-18真核表达质粒免疫组(IL-18)、PVAX-3-1E真核表达质粒免疫组(3-1E)、PVAX-α-tubulin真核表达质粒免疫组(WDB、PVAX-3-1E-α-tubulin融合基因真核表达质粒免疫组(3W)、PVAX-3-1E-α-tubulin融合基因真核表达质粒+PVAX-IL-18真核表达质粒联合免疫组(3W+)。实验过程中,利用T淋巴细胞转化试验(MTT法)、B淋巴细胞转化试验(MTT法)、流式细胞仪及酶联免疫吸附试验(ELISA)对21、28、35、42、49日龄雏鸡的各项免疫指标进行了检测,最后用球虫攻击感染保护试验进一步比较了不同真核表达质粒对球虫感染鸡保护效果的差别,探讨免疫保护机理。
4实验结果：雏鸡脾脏、胸腺和外周血T淋巴细胞增殖功能检测试验中发现,各组雏鸡T淋巴细胞增殖功能在28日龄后均出现幅度不等的上升趋势,各实验组均表现出明显增殖活性。其中在49日龄时,3W+组数值最高,其次是3W组,单独免疫组3-1E、α-tubulin和IL-18组数值极显著低于3W+和3W组数值,该五组数值和对照组数值相比差异极显著。血液和胸腺T淋巴细胞增殖实验结果,趋势与脾脏结果类似。雏鸡脾脏和外周血B淋巴细胞增殖功能检测试验结果趋势与T细胞检测结果类似。
通过流式细胞仪对雏鸡脾脏、胸腺和外周血中CD4+T淋巴细胞数量进行检测发现,各雏鸡CD4+T淋巴细胞数量在28日龄后均出现幅度不等的上升趋势,各实验组均表现出CD4+T淋巴细胞数量增加。在42日龄时各实验组CD4+T淋巴细胞数量达到最高值,其中3W+组数值最高,其次是3W组,单独免疫组3-1E、α-tubulin(?)]IL-18组数值极显著低于3W+和3W组数值,该五组数值和对照组数值相比差异极显著。血液和胸腺CD4+T淋巴细胞数量变化实验结果变化趋势与脾脏结果类似。由此得出雏鸡免疫后,对CD4+T淋巴细胞数量增加有着明显促进作用,添加佐剂的3W+和未添加佐剂组3W相比,能够显著增加CD4+T淋巴细胞数量,使CD4+T淋巴细胞更好发挥其免疫学功能。雏鸡脾脏、胸腺和外周血中CD8+T淋巴细胞数量检测结果与CD4+T淋巴细胞检测结果类似。
本试验采用间接ELISA方法对抗3-1E,α-tubulin特异性IgG抗体进行检测。使用3-1E,α-tubulin蛋白作为抗原包被。通过对不同组别不同日龄的抗体检测结果表明,49日龄整体抗体水平最高,其中以3W+组抗体含量最高,其次是3W组、再次为α-tubulin组、3-1E组和IL-18组。说明CHIL18与3-1E和α-tubulin融合基因联合免疫可以有效地促进机体产生抗3-1E和α-tubulin的特异性抗体。
攻虫后对各实验组雏鸡的平均增重和相对增重、粪便卵囊排出数量、十二指肠病变计分及不同免疫组之间的免疫保护效率进行了比较。结果显示攻虫时,各真核表达质粒免疫组鸡体重与B组和H组两对照组相比组间差异不显著,即平均增重1无明显差异,由此说明DNA疫苗免疫对增重无不良影响；各实验组的平均增重2显著高于H组和PVAX组,其中3W+组和3W组鸡的平均增重2极显著高于其他疫苗实验组,说明该两组DNA疫苗对鸡抗球虫感染有良好的免疫保护作用；对于粪便卵囊排出量3-1E组、WDB组、IL18组、3W组和3W+组等五种DNA疫苗免疫组都能减少鸡卵囊排出量,各免疫组卵囊排出减少率分别为36.72、35.97、40.36、55.13和71.19,这表明融合基因免疫能显著减少球虫卵囊的排出量,当有IL-18因子参加联合免疫时,能进一步减少球虫卵囊排出量。在抗球虫综合指数方面,各实验组也明显优于H组和PVAX组,所有免疫组的ACI都大于160,H组的ACI只有130.61。3W组和3W+组ACI都大于180,3W+组的ACI最高,达到了194.32,表明有细胞因子协同后融合基因的免疫保护效果更明显。
Avian coccidiosis is an enteritic disease, which caused by protozoan parasites of Eimeriidae, Eimeria, affects chicken growth and production, and brings about significant economic loss. It was reported that, the application of coccidian vaccine to stimulate host immune responses is the main approaches to control coccidiosis. Nowadays, nucleic acid vaccine (DNA vaccine) has been demonstrated had the ability to improve host cellular immune response and keep long time of immune memory. Furthermore, DNA vaccine, collaborated with cytokine injection, could exert more significant immune response and provide better immune protection. In this paper, two kinds of main immune antigen genes of E. acervulina (3-1E and α-tubulin genes) were selected to construct eukaryotic expression plasmids, which contains fused two immune antigen genes, and immunized chickens with interleukin18(IL-18) collaboratively, and its immune modulate mechanism to E. acervulina infection was researched. The results were as follows:
1. The total RNA was extracted from sporozoites of E. acervulina SH strain, the a-tubulin gene was amplified by RT-PCR. The results showed that α-tubulin cDNA nucleotide of SH Strain of E. acervulina contained an open reading frame (ORF) and was1362bp in length. Sequence analysis demonstrated that it had99.4%and99.5%homology with the reference genes of E. acervulina strain (GenBank Accession Number HQ259108) in nucleotide acid and amino acid level, respectively.
2. With gene splicing overlap extension technology (SOE PCR), the3-1E and a-tubulin genes was jointed together with a flexible peptide (G4S-G4S-G4S) and constructed successfully the fused gene eukaryotic expression plasmid, PVAX1-3-lE-α-tubulin. In vitro transfect experiment with indirect immunofuorenscence technique, anti-3-1E and a-tubulin polyclonal antibodis were used respectively and specific positive responses were observed. It showed that constructed eukaryotic expression plasmid with fused genes could express these two proteins and kept their biological activities. This provide solid basis for the followed experiment.
3. Two hundreds of2-week-old SPF chickens were randomly divided into the following8groups with different treatments:Group1, as the control group, was injected intramuscularly in thigh muscle with PBS without coccidia challenge (B Group); Group2was injected with PBS with coccidia challenge (H Group); Group3immunized with PVAX empty vector (PVAX Group); Group4injected with PVAX-IL-18eukaryotic expression plasmid (IL-18Group); Group5immunized with PVAX-3-1E plasmid (3-1E Group); Group6immunized with PVAX-a-tubulin plasmid (WDB Group); Group7immunized with PVAX-3-1E-α-tubulin plasmid (3W Group); and Group8immunized with PVAX-3-lE-a-tubulin plasmid and injected with PVAX-IL-18collaboratively at the same time (3W+Group). Immunization was boosted with the respective constitute after one week. Seven days after booster immunization, SPF chickens were challenged with5×104E. acervulina sporulated oocysts. The immune protection efficacy for Eimeria infected chickens were observed, and immune protection mechanism was discussed.
3. The proliferation response of T and B-lymphocytes of spleen, thymus and peripheral blood showed the similar trends during the experiment period. After second vaccination, T and B lymphocyte proliferative function of chickens in group of IL-18,3-1E and WDB was all very significantly higher (p<0.01) than those in group B, H and PVAX. Furthermore,3W group was remarkably higher than those in group IL-18,3-1E and WDB (p<0.01). And the T lymphocyte proliferative function in group3W+was significantly higher than other groups, including the fused genes and single gene immunization groups (p<0.01). The results of T and B lymphocyte proliferative function showed that IL-18could improve the DNA vaccine immunization and enhance the T and B lymphocyte immunofunction.
In the experiment, T lymphocyte subpopulation in blood and immune organs of chickens ware assayed by flow cytometric analysis. The results showed that there were significant increase in the percentage of CD4+T lymphocyte subpopulation in spleen, thymus and peripheral blood after primary vaccination, and then kept the slow increase trends afterwards. Specifically,3W group kept the highest value among all groups. In which, The percentages of CD4+T cell in group IL-18,3-1E and WDB were remarkably higher than those in group B, H, and PVAX (p<0.01或p<0.05) from28days to the end of the experiment. The group3W was significantly higher than those of group IL-18,3-1E and WDB (p<0.01), and the group3W+was remarkably higher than all other groups (p<0.01or p<0.05)
The percentage of CD8+T lymphocyte subpopulation in blood and immune organs of chickens demonstrated different changes as those of CD4+T lymphocyte subpopulation. Namely, the significant increase of percentage of CD8+T lymphocyte subpopulation was observed at35days (not28days of CD4+T lymphocyte), and had small modification (even decrease) at42days, and had a small scale of increase at49days. Statistic analysis showed that the percentage of CD8+T lymphocyte subpopulation in blood and immune organs of chickens in group IL-18,3-1E and WDB was significantly higher than that in group B, H and PVAX (p<0.01) post primary vaccination, group3W was remarkably higher than that in group IL-18,3-1E and WDB (p<0.01) and group3W+showed highest value compared with other groups (p<0.01). The results of CD4+and CD8+T lymphocyte subpopulation assay showed that IL-18could enhance the CD4+and CD8+T lymphocyte number and modulate the host immune function. The following assay of peripheral blood specific antibody against3-1E and WDB with ELISA support the above results.
The immune protection efficacy of different kinds of eukaryotic plasmids for Eimeria infected chickens was evaluated. The average body weight gain of chickens before challenge in all groups, there was not significantly difference (p>0.05). However, after challenge, The average body weight gain of chickens in group3-1E, WDB,3W and3W+was significantly higher than those in group H and PVAX (p<0.05). Ocysts per gram in feces of infected chickens in group3-1E, WDB,3W and3W+was significant or very significantly lower than that in group H (p<0.05or p<0.01). And the ACI of group3-1E, WDB, IL-18,3W and3W+was respectively166.51,162.97,165.59,180.77,194.32, and was all higher than ACI in group H. The ACI in group3W was more than180, which indicate it could provide significant protection efficacy.
本研究选择禽类艾美尔球虫各虫种间保守性较高的3-1E基因和α-tubulin基因,以及新近发现的可作为免疫佐剂,具有增强免疫和治疗作用的多功能细胞因子鸡白细胞介素18(ChlL-18)为研究对象,以PVAX1为载体构建并鉴定了PVAX-α-tubulin真核表达质粒和PVAX-3-1E-α-tubulin融合基因真核质粒,通过基因免疫途径研究了5种真核表达质粒对免疫鸡的免疫调节机理以及对感染堆型艾美尔球虫(E.acervulina)鸡的免疫保护作用,试验内容与结果如下：
1以E.acervulina上海株(SH株)6h孢子化卵囊的总RNA为模板,应用RT-PCR技术扩增出含有完整开放阅读框架(ORF)的α-tubulin基因,该基因片段长为1362bp,经测序鉴定得知E.acervulina SH株α-tubulin基因cDNA核苷酸序列(HQ259108)与GeneBank参考株相应序列(X88776.1)同源性为99.4%,推导氨基酸序列同源性为99.5%。
2通过设计引物将3-1E基因的终止密码子删除,同时去除α-tubulin基因的起始密码子。应用重叠扩增PCR(Gene splicing overlap extension PCR,SOE PCR)技术成功地将E acervulina3-1E基因和α-tubulin基因片段进行连接,并在两个基因之间引入了柔性连接肽(G4S)3,获得融合基因3-1E-linker-α-tubulin。以PVAX1为载体成功构建了融合基因的真核表达质粒PVAX-3-1E-α-tubulin,融合基因质粒的体外瞬时转染试验表明,融合基因能够在细胞内进行高效表达,抗3-1E多克隆抗体和抗α-tubulin多克隆抗体分别能与融合基因表达的蛋白发生免疫学反应,表明构建的融合基因表达蛋白很好的保留了融合前两段基因蛋白的生物学活性,为后续研究融合基因对球虫感染的免疫保护机理提供了理论支持。
3实验分组：实验中构建5种真核表达质粒研究核酸疫苗对机体免疫保护效果,实验分为8组,分别为不免疫不攻虫组(B)、不免疫攻虫组(H)、PVAX空质粒免疫组(PVAX)、 PVAX-IL-18真核表达质粒免疫组(IL-18)、PVAX-3-1E真核表达质粒免疫组(3-1E)、PVAX-α-tubulin真核表达质粒免疫组(WDB、PVAX-3-1E-α-tubulin融合基因真核表达质粒免疫组(3W)、PVAX-3-1E-α-tubulin融合基因真核表达质粒+PVAX-IL-18真核表达质粒联合免疫组(3W+)。实验过程中,利用T淋巴细胞转化试验(MTT法)、B淋巴细胞转化试验(MTT法)、流式细胞仪及酶联免疫吸附试验(ELISA)对21、28、35、42、49日龄雏鸡的各项免疫指标进行了检测,最后用球虫攻击感染保护试验进一步比较了不同真核表达质粒对球虫感染鸡保护效果的差别,探讨免疫保护机理。
4实验结果：雏鸡脾脏、胸腺和外周血T淋巴细胞增殖功能检测试验中发现,各组雏鸡T淋巴细胞增殖功能在28日龄后均出现幅度不等的上升趋势,各实验组均表现出明显增殖活性。其中在49日龄时,3W+组数值最高,其次是3W组,单独免疫组3-1E、α-tubulin和IL-18组数值极显著低于3W+和3W组数值,该五组数值和对照组数值相比差异极显著。血液和胸腺T淋巴细胞增殖实验结果,趋势与脾脏结果类似。雏鸡脾脏和外周血B淋巴细胞增殖功能检测试验结果趋势与T细胞检测结果类似。
通过流式细胞仪对雏鸡脾脏、胸腺和外周血中CD4+T淋巴细胞数量进行检测发现,各雏鸡CD4+T淋巴细胞数量在28日龄后均出现幅度不等的上升趋势,各实验组均表现出CD4+T淋巴细胞数量增加。在42日龄时各实验组CD4+T淋巴细胞数量达到最高值,其中3W+组数值最高,其次是3W组,单独免疫组3-1E、α-tubulin(?)]IL-18组数值极显著低于3W+和3W组数值,该五组数值和对照组数值相比差异极显著。血液和胸腺CD4+T淋巴细胞数量变化实验结果变化趋势与脾脏结果类似。由此得出雏鸡免疫后,对CD4+T淋巴细胞数量增加有着明显促进作用,添加佐剂的3W+和未添加佐剂组3W相比,能够显著增加CD4+T淋巴细胞数量,使CD4+T淋巴细胞更好发挥其免疫学功能。雏鸡脾脏、胸腺和外周血中CD8+T淋巴细胞数量检测结果与CD4+T淋巴细胞检测结果类似。
本试验采用间接ELISA方法对抗3-1E,α-tubulin特异性IgG抗体进行检测。使用3-1E,α-tubulin蛋白作为抗原包被。通过对不同组别不同日龄的抗体检测结果表明,49日龄整体抗体水平最高,其中以3W+组抗体含量最高,其次是3W组、再次为α-tubulin组、3-1E组和IL-18组。说明CHIL18与3-1E和α-tubulin融合基因联合免疫可以有效地促进机体产生抗3-1E和α-tubulin的特异性抗体。
攻虫后对各实验组雏鸡的平均增重和相对增重、粪便卵囊排出数量、十二指肠病变计分及不同免疫组之间的免疫保护效率进行了比较。结果显示攻虫时,各真核表达质粒免疫组鸡体重与B组和H组两对照组相比组间差异不显著,即平均增重1无明显差异,由此说明DNA疫苗免疫对增重无不良影响；各实验组的平均增重2显著高于H组和PVAX组,其中3W+组和3W组鸡的平均增重2极显著高于其他疫苗实验组,说明该两组DNA疫苗对鸡抗球虫感染有良好的免疫保护作用；对于粪便卵囊排出量3-1E组、WDB组、IL18组、3W组和3W+组等五种DNA疫苗免疫组都能减少鸡卵囊排出量,各免疫组卵囊排出减少率分别为36.72、35.97、40.36、55.13和71.19,这表明融合基因免疫能显著减少球虫卵囊的排出量,当有IL-18因子参加联合免疫时,能进一步减少球虫卵囊排出量。在抗球虫综合指数方面,各实验组也明显优于H组和PVAX组,所有免疫组的ACI都大于160,H组的ACI只有130.61。3W组和3W+组ACI都大于180,3W+组的ACI最高,达到了194.32,表明有细胞因子协同后融合基因的免疫保护效果更明显。
Avian coccidiosis is an enteritic disease, which caused by protozoan parasites of Eimeriidae, Eimeria, affects chicken growth and production, and brings about significant economic loss. It was reported that, the application of coccidian vaccine to stimulate host immune responses is the main approaches to control coccidiosis. Nowadays, nucleic acid vaccine (DNA vaccine) has been demonstrated had the ability to improve host cellular immune response and keep long time of immune memory. Furthermore, DNA vaccine, collaborated with cytokine injection, could exert more significant immune response and provide better immune protection. In this paper, two kinds of main immune antigen genes of E. acervulina (3-1E and α-tubulin genes) were selected to construct eukaryotic expression plasmids, which contains fused two immune antigen genes, and immunized chickens with interleukin18(IL-18) collaboratively, and its immune modulate mechanism to E. acervulina infection was researched. The results were as follows:
1. The total RNA was extracted from sporozoites of E. acervulina SH strain, the a-tubulin gene was amplified by RT-PCR. The results showed that α-tubulin cDNA nucleotide of SH Strain of E. acervulina contained an open reading frame (ORF) and was1362bp in length. Sequence analysis demonstrated that it had99.4%and99.5%homology with the reference genes of E. acervulina strain (GenBank Accession Number HQ259108) in nucleotide acid and amino acid level, respectively.
2. With gene splicing overlap extension technology (SOE PCR), the3-1E and a-tubulin genes was jointed together with a flexible peptide (G4S-G4S-G4S) and constructed successfully the fused gene eukaryotic expression plasmid, PVAX1-3-lE-α-tubulin. In vitro transfect experiment with indirect immunofuorenscence technique, anti-3-1E and a-tubulin polyclonal antibodis were used respectively and specific positive responses were observed. It showed that constructed eukaryotic expression plasmid with fused genes could express these two proteins and kept their biological activities. This provide solid basis for the followed experiment.
3. Two hundreds of2-week-old SPF chickens were randomly divided into the following8groups with different treatments:Group1, as the control group, was injected intramuscularly in thigh muscle with PBS without coccidia challenge (B Group); Group2was injected with PBS with coccidia challenge (H Group); Group3immunized with PVAX empty vector (PVAX Group); Group4injected with PVAX-IL-18eukaryotic expression plasmid (IL-18Group); Group5immunized with PVAX-3-1E plasmid (3-1E Group); Group6immunized with PVAX-a-tubulin plasmid (WDB Group); Group7immunized with PVAX-3-1E-α-tubulin plasmid (3W Group); and Group8immunized with PVAX-3-lE-a-tubulin plasmid and injected with PVAX-IL-18collaboratively at the same time (3W+Group). Immunization was boosted with the respective constitute after one week. Seven days after booster immunization, SPF chickens were challenged with5×104E. acervulina sporulated oocysts. The immune protection efficacy for Eimeria infected chickens were observed, and immune protection mechanism was discussed.
3. The proliferation response of T and B-lymphocytes of spleen, thymus and peripheral blood showed the similar trends during the experiment period. After second vaccination, T and B lymphocyte proliferative function of chickens in group of IL-18,3-1E and WDB was all very significantly higher (p<0.01) than those in group B, H and PVAX. Furthermore,3W group was remarkably higher than those in group IL-18,3-1E and WDB (p<0.01). And the T lymphocyte proliferative function in group3W+was significantly higher than other groups, including the fused genes and single gene immunization groups (p<0.01). The results of T and B lymphocyte proliferative function showed that IL-18could improve the DNA vaccine immunization and enhance the T and B lymphocyte immunofunction.
In the experiment, T lymphocyte subpopulation in blood and immune organs of chickens ware assayed by flow cytometric analysis. The results showed that there were significant increase in the percentage of CD4+T lymphocyte subpopulation in spleen, thymus and peripheral blood after primary vaccination, and then kept the slow increase trends afterwards. Specifically,3W group kept the highest value among all groups. In which, The percentages of CD4+T cell in group IL-18,3-1E and WDB were remarkably higher than those in group B, H, and PVAX (p<0.01或p<0.05) from28days to the end of the experiment. The group3W was significantly higher than those of group IL-18,3-1E and WDB (p<0.01), and the group3W+was remarkably higher than all other groups (p<0.01or p<0.05)
The percentage of CD8+T lymphocyte subpopulation in blood and immune organs of chickens demonstrated different changes as those of CD4+T lymphocyte subpopulation. Namely, the significant increase of percentage of CD8+T lymphocyte subpopulation was observed at35days (not28days of CD4+T lymphocyte), and had small modification (even decrease) at42days, and had a small scale of increase at49days. Statistic analysis showed that the percentage of CD8+T lymphocyte subpopulation in blood and immune organs of chickens in group IL-18,3-1E and WDB was significantly higher than that in group B, H and PVAX (p<0.01) post primary vaccination, group3W was remarkably higher than that in group IL-18,3-1E and WDB (p<0.01) and group3W+showed highest value compared with other groups (p<0.01). The results of CD4+and CD8+T lymphocyte subpopulation assay showed that IL-18could enhance the CD4+and CD8+T lymphocyte number and modulate the host immune function. The following assay of peripheral blood specific antibody against3-1E and WDB with ELISA support the above results.
The immune protection efficacy of different kinds of eukaryotic plasmids for Eimeria infected chickens was evaluated. The average body weight gain of chickens before challenge in all groups, there was not significantly difference (p>0.05). However, after challenge, The average body weight gain of chickens in group3-1E, WDB,3W and3W+was significantly higher than those in group H and PVAX (p<0.05). Ocysts per gram in feces of infected chickens in group3-1E, WDB,3W and3W+was significant or very significantly lower than that in group H (p<0.05or p<0.01). And the ACI of group3-1E, WDB, IL-18,3W and3W+was respectively166.51,162.97,165.59,180.77,194.32, and was all higher than ACI in group H. The ACI in group3W was more than180, which indicate it could provide significant protection efficacy.
引文
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