京海黄鸡柔嫩艾美耳球虫感染后盲肠转录组分析及其杂交配套系亲本的抗性评价

发布时间：2018-09-03 10:39

【摘要】：鸡球虫病是由一类寄生于鸡肠道内的寄生原虫所引起的疾病,以雏鸡高死亡率和出血性肠炎为主要特征。特别是鸡柔嫩艾美耳球虫病(E. tenella)在雏禽3周龄至2月龄时最易感染,发病率高达50%-70%,对我国养禽业造成了巨大的危害。本研究一方面为了寻找与鸡E. tenella抗性有关的关键基因和关键通路,以京海黄鸡为试验动物,在30日龄时高剂量感染柔嫩艾美耳球虫,采用RNA-seq技术分析感染后第8天感染鸡和非感染对照鸡盲肠组织的基因组表达水平,对部分差异基因通过qRT-PCR进行了验证,并对差异基因进行了GO、KEGG富集分析。另一方面为了探明京海黄鸡配套系亲本的球虫抗性,研究分别对配套系亲本进行了高剂量E. tenella感染、球虫疫苗免疫和疫苗免疫后高剂量E. tenella感染3个试验,采用存活率、体增重、卵囊脱落数、盲肠记分、抗球虫指数(ACI)等评价指标及一氧化氮(NO)、过氧化氢(CAT)、γ干扰素(IFN-γ)等10项血浆指标对其杂交配套系亲本BB、FF、WW对E. tenella抗性及球虫疫苗免疫后的免疫效果进行了评价。研究结果如下：1.转录组测序结果表明,鸡E. tenella感染组与对照组共有5477个表达差异显著的基因(Padj0.05),其中上调基因有2942个,下调基因有2535个。选取10个差异基因进行qRT-PCR验证,结果表明差异基因的表达倍数与RNA-seq的结果基本一致,说明试验结果真实可靠。2.GO和KEGG通路的富集分析表明差异基因主要与脂类代谢、细胞交流、炎症应答、免疫／防御、血管生成、信号转导等有关。鸡在应答E. tenella感染的过程中,机体产生了复杂的免疫应答,涉及黏着斑、细胞因子-受体相互作用、过氧化物酶体、细胞外基质-受体相互作用、过氧化物酶体增殖物激活受体等信号通路。这些通路中的关键差异基因有FAK、TGFB2、TGFB3、IL-6、IL-15、CD36等,这些基因均在宿主抵抗球虫感染过程中发挥重要作用。3.京海黄鸡杂交配套系亲本BB、FF、WW鸡在30日龄时高剂量感染E. tenella,感染后8天检测各项评价指标。其中WW感染组抗球虫指数(ACI)值最高为143.1,BB感染组ACI值为142.6,两者均为抗球虫低效,FF感染组ACI值最低为119.3,为抗球虫无效；ACI值与过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、白介素17(IL-17)和干扰素-γ(IFN-γ)4个免疫指标相关显著(P0.05)；BB感染组NO、白介素2(IL-2)、IL-17、SOD含量显著高于FF感染组(P0.05)。综合分析表明：京海黄鸡杂交配套亲本BB鸡对E. tenella抗性显著高于FF鸡,略高于WW鸡。4.京海黄鸡杂交配套系亲本BB、FF鸡在9日龄时经正典球虫四价活疫苗免疫,免疫后8天检测各项评价指标。其中BB鸡免疫组CAT、SOD和IL-17的含量显著高于FF鸡免疫组(P0.05)；BB免疫组CAT和SOD含量显著高于非免疫对照组(P0.05)；FF免疫组CAT和谷胱甘肽过氧化物酶(GSH-Px)含量均显著高于对照组(P0.05)。综合分析结果表明BB鸡免疫效果优于FF鸡群体,BB鸡E. tenella抗性优于FF鸡群体。5.京海黄鸡杂交配套系亲本BB、FF鸡在9日龄用两种球虫疫苗免疫,免疫后24天进行高剂量感染E. tenella,感染后8天检测各项指标。综合临床表现、盲肠病变、相对增重率及ACI,发现BB鸡群体E. tenella抗性优于FF鸡群体,免疫1组(500个活卵囊)优于免疫2组(正典球虫疫苗)；CAT、SOD的含量免疫1组鸡均显著高于免疫2组鸡(P0.05)；BB鸡中,IFN-y含量免疫1组鸡显著高于免疫2组鸡(P0.05)；FF鸡中,p胡萝卜素(p-C)含量免疫1组鸡显著高于免疫2组鸡(P0.05)。综合分析表明,500个E. tenella活卵囊免疫效果优于正典球虫疫苗,BB鸡群体E. tenella抗性优于FF群体。
[Abstract]:Chicken coccidiosis is a disease caused by a class of parasitic protozoa parasitized in chicken intestine. It is characterized by high mortality and hemorrhagic enteritis in chickens. Especially, E. tenella is the most susceptible to infection in chickens from 3 weeks to 2 months of age. The incidence of E. tenella is as high as 50%-70%. It has caused great harm to poultry industry in China. On the one hand, in order to find the key genes and key pathways related to E. tenella resistance in chickens, Jinghai yellow chickens were used as experimental animals. Eimeria tenella was infected at 30 days of age at high doses. RNA-seq technique was used to analyze the genomic expression level in cecum tissues of infected chickens and non-infected control chickens on the 8th day after infection. On the other hand, in order to prove the coccidiosis resistance of the parents of Jinghai Yellow Chicken, three tests were carried out, including high dose E. tenella infection, coccidiosis vaccine immunization and high dose E. tenella infection after vaccine immunization. Oocyst abscission number, cecum score, anti-coccidiosis index (ACI) and 10 plasma indices, such as nitric oxide (NO), hydrogen peroxide (CAT), interferon-gamma (IFN-gamma), were used to evaluate the immune effect of their parents BB, FF, WW on E.tenella resistance and coccidiosis vaccine immunization. The results were as follows: 1. Transcription group sequencing results table There were 5 477 differentially expressed genes (Padj0.05) in E. tenella infection group and control group, including 2 942 up-regulated genes and 2 535 down-regulated genes. Enrichment analysis of the pathway showed that the differentially expressed genes were mainly related to lipid metabolism, cellular communication, inflammatory response, immune/defense, angiogenesis and signal transduction. The key differentially expressed genes in these pathways are FAK, TGFB2, TGFB3, IL-6, IL-15 and CD36. These genes play an important role in host resistance to coccidiosis. 3. The parents BB, FF and WW of Jinghai Yellow Chicken crossbred lines were infected with E.ten at 30 days of age at high doses. The highest ACI value was 143.1 in WW infection group and 142.6 in BB infection group. The lowest ACI value was 119.3 in FF infection group, which was ineffective against coccidiosis. ACI value was associated with catalase (CAT), superoxide dismutase (SOD), interleukin 17 (IL-17) and interferon-gamma (IFN-17). The content of NO, IL-2, IL-17 and SOD in the BB infected group were significantly higher than those in the FF infected group (P The contents of CAT, SOD and IL-17 in the BB immunized group were significantly higher than those in the FF immunized group (P 0.05); the contents of CAT and SOD in the BB immunized group were significantly higher than those in the non-immunized control group (P 0.05); the contents of CAT and GSH-Px in the FF immunized group were significantly higher than those in the control group (P 0.05). The results showed that the immune effect of BB chickens was better than that of FF chickens, and the resistance of BB chickens to E. tenella was better than that of FF chickens. The results showed that E. tenella resistance of BB chickens was superior to that of FF chickens, and the immunization group 1 (500 live oocysts) was superior to that of immunization group 2 (canonical coccidiosis vaccine); the content of CAT and SOD in group 1 was significantly higher than that in group 2 (P 0.05); the content of IFN-y in group 1 was significantly higher than that in group 2 (P 0.05); and the content of p-C in FF chickens was immunized. The immune effect of 500 E.tenella oocysts was better than that of the classical coccidiosis vaccine, and the resistance of E.tenella in BB chickens was better than that of FF chickens.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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