黄艾美球虫侵犯宿主组织的超微研究

利用透射电镜和扫描电镜相结合的技术对黄艾美球虫（E.flavescens）人工感染后的子孢子移动过程及被虫体寄生的宿主细胞形态学变化和粘膜形态学变化进行了观察。子孢子在进入小肠腺上皮过程中需白细胞介导，在白细胞内可发育成球形的滋养体，观察到有两个子孢子侵入同一白细胞的现象，但分别存在于各自的带虫空泡中，黄艾美球虫引起的肠粘膜损伤主要发生于感染177小时之后，其损伤主要表现在绒毛大片脱落，结缔组织裸露，表面可见到大量破损的孔洞及大量肠杆菌，被虫体寄生的腺上皮细胞基部膨胀，微绒毛脱落，细胞核膨大并部分包被虫体，有些细胞核裂解为二。     

鸡巨型艾美耳球虫卵囊呼吸作用的研究

采用纯种巨型艾美耳球虫(Eimeria maxima)卵囊,人工感染3周龄的石歧杂雏鸡,及时收集卵囊,以0.1N硫酸溶液悬浮,用微量呼吸检压仪检测卵囊呼吸作用。试验表明:孢子化过程中最初十小时内该球虫卵囊的耗氧量为3.59μl/小时/10~6个卵囊。耗氧量的变化与其细胞分裂过程相对应,耗氧量在三次核分裂前夕降低,但在分裂期升高。最高耗氧量出现于子孢子形成期,达18.91μl/小时/10~6卵囊。孢子化后卵囊的呼吸作用较缓慢,耗氧量为0.85μl/小时/10~6个卵囊。完成孢子化所需时间为26小时。     

鸡柔嫩艾美耳球虫不同抗药性虫株的种内多态性研究

应用RAPD技术进行柔嫩艾美耳球虫4个单一抗药性虫株与1个敏感株的基因组DNA多态性分析，发现4个抗药性虫株及敏感株之间的相似值均大于99%；OPAO3引物对抗盐霉素株扩增出一条约500bp的特异条带，OPH02引物对抗克球粉株和抗盐霉素株均扩增出了约1kb的第三条主带，这些特异条带的出现很可能与球虫抗药性基因有关，有可能用于抗药性虫株的诊断与鉴定。     

Sequence and Phylogenetic Analysis of rDNA ITS of Three Eimeria Species in Rabbit

In order to study whether the internal transcribed spacers (ITS) sequence could be used as a molecular marker for the species identification of rabbit coccidian, the rDNA ITS of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were amplified by polymerase chain reaction (PCR), and were cloned into pGEM-T Easy vector subsequently. The positive recombinant plasmids were identified by PCR and then sequenced. By sequence comparison and comparative analysis with the relative sequences of rabbit Eimeria spp. available in GenBank, the results showed that the lengths of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were 1065, 1009 and 1047 bp, respectively, and the sequence homologies with the same species sequences were 99.2%, 99.0% and 94.5%, respectively, while were 55.3% to 82.1% compared with corresponding sequences of other different species sequences. The phylogenetic analysis using software Mega 5.0 showed that all rabbit coccidia clustered together in a clade, which was divided into two sister lineages, corresponding to the presence or absence of oocyst residuum. The result demonstrated ITS could be used as a molecular marker for the species identification of rabbit coccidia.     

巨型艾美耳球虫偏菱形样蛋白EmRP免疫原性分析

在前期研究中,从巨型艾美耳球虫(Eimeria maxima)cDNA文库中筛选到了免疫保护性抗原偏菱形样蛋白EmRP,本研究为揭示该抗原的免疫保护机理,进一步检测了其免疫原性。以E.maxima卵囊cDNA为模板,用RT-PCR技术扩增EmRP,并构建真核表达质粒pVAX1-EmRP,将其经腿部肌肉注射2周龄的雏鸡,3周龄加强免疫。分别于首次免疫后和加强免疫后1周,采集血清,用ELISA方法检测血清特异性IgG水平,用荧光定量PCR方法(qPCR)检测细胞因子水平,用流式细胞术检测CD4^+T淋巴细胞和CD8^+T淋巴细胞的比例,分析EmRP诱导的免疫反应。序列分析表明,EmRP含有偏菱形样蛋白超家族成员保守区域,为非跨膜蛋白,分子量约为28.4 kD。真核表达质粒pVAX1-EmRP免疫鸡后,与对照组相比,鸡体IFN-γ、IL-2、IL-10和IL-17等细胞因子转录水平显著提升;CD8^+和CD4^+ T细胞比例显著提高,而血清特异性IgG水平与对照组差异不显著。结果表明,EmRP诱导的免疫保护作用主要是由其诱导的细胞免疫反应实现的,可以作为研制E.maxima新型疫苗的候选抗原。     

秦皇岛地区鸡球虫抗药性研究

应用艾维茵肉仔鸡，以最适抗球虫活性百分率（POAA）、病变记分减少率（RLS）和相对卵囊产量（ROP）为判定指标，检测了采自河北省秦皇岛地区的6种艾美耳球虫对8种常用抗球虫药物的敏感性。结果表明：6种混合艾美耳球虫对马杜霉素和盐霉素具有轻度抗药性；对拉沙洛菌素和氨丙淋具有中度抗药性；对球痢灵、克球多和常山酮具有重度抗药性；对地克珠利无抗药性。     

柔嫩艾美耳球虫感染雏鸡LPO含量和抗自由基酶活性的动力学

通过测定柔嫩艾美耳球虫 (Eimeria tenella)感染鸡的盲肠、肝脏、脾脏和法氏囊组织中脂质过氧化物 (L PO)含量和超氧化物歧化酶 (SOD)、谷胱甘肽过氧化物酶 (GXH- Px)及过氧化氢酶 (CAT)活性 ,研究雏鸡感染球虫后机体内自由基生成和抗自由基反应。结果表明 ,雏鸡感染 E.tenella后 ,盲肠、肝脏和脾脏组织中的 L PO含量在感染后显著 (P<0 .0 5 )或极显著 (P<0 .0 1)升高 ,T- SOD、Mn- SOD、Cu、Zn- SOD、GSH- Px和 CAT活性水平不同程度下降。试验结果说明在 E.tenella感染后 ,鸡体内的氧自由基生成反应加剧 ,氧自由基参与了球虫的致病过程。     

Comparative analysis of evaluation parameters in E. acervulina,E. maxima and E. tenella-infected broilers

Three parameters, body weight gain (BWG), intestinal lesion score (LS) and fecal oocyst shedding, were compared in broilers infected with major parasitic species; Eimeria acervulina, E. maxima, and E. tenella. First, two- and three-week-old chickens with Eimeria infection showed LS of approximately 3, but two-week-old chickens were more correlated with BWG. Second, significant differences in BWG were observed between male and female broilers challenged with Eimeria. Finally, E. maxima-infected broilers among three Eimeria species showed a higher relationship between BWG and LS, suggesting three considerations such as genders, age and Eimeria species for Eimeria experiments.     

常山提取物对人工感染鸡柔嫩艾美耳球虫病疗效的观察

为了探讨常山提取物对鸡球虫病的治疗效果,通过人工感染鸡柔嫩艾美耳球虫后在饲料中添加一定比例的药物,试验设常山提取物组(分别为200、100、50 mg/kg饲料)、鸡球虫散组(300 mg/kg饲料)、地克珠利组(1 mg/kg饲料)、感染对照组和健康对照组.结果显示,与感染对照组比较,所有给药组试验鸡盲肠和十二指肠肿胀明显减轻,血液性内容物明显减少,抗球虫指数分别为171.98、169.18、169.01、163.84和166.74,均达到中等抗球虫水平.结果表明,常山提取物具有良好的抗球虫效果,且作为一类中药组分,具有绿色环保、低毒低残留的特点,有望成为新型抗球虫药物.