New insights into developmental biology of Eimeria tenella revealed by comparative analysis of mRNA N6-methyladenosine modification between unsporulated oocysts and sporulated oocysts

Evidence showed that N6-methyladenosine(m^(6)A)modification plays a pivotal role in influencing RNA fate and is strongly associated with cell growth and developmental processes in many species.However,no information regarding m^(6)A modification in Eimeria tenella is currently available.In the present study,we surveyed the transcriptome-wide prevalence of m^(6)A in sporulated oocysts and unsporulated oocysts of E.tenella.Methylated RNA immunoprecipitation sequencing(MeRIP-seq)analysis showed that m^(6)A modification was most abundant in the coding sequences,followed by stop codon.There were 3,903 hypermethylated and 3,178 hypomethylated mRNAs in sporulated oocysts compared with unsporulated oocysts.Further joint analysis suggested that m^(6)A modification of the majority of genes was positively correlated with mRNA expression.The mRNA relative expression and m^(6)A level of the selected genes were confirmed by quantitative reverse transcription PCR(RT-qPCR)and MeRIP-qPCR.GO and KEGG analysis indicated that differentially m^(6)A methylated genes(DMMGs)with significant differences in mRNA expression were closely related to processes such as regulation of gene expression,epigenetic,microtubule,autophagy-other and TOR signaling.Moreover,a total of 96 DMMGs without significant differences in mRNA expression showed significant differences at protein level.GO and pathway enrichment analysis of the 96 genes showed that RNA methylation may be involved in cell biosynthesis and metabolism of E.tenella.We firstly present a map of RNA m^(6)A modification in E.tenella,which provides significant insights into developmental biology of E.tenella.

柔嫩艾美尔球虫厦门株(Eimeria tenella)生物学研究

采用单卵囊分离方法从厦门地区饲养的鸡盲肠中获得艾美尔球虫．经显微观察、测量和感染实验．从寄生部位、卵囊形态、孢子化时间和潜伏期等指标测定．实验结果：采用琼脂板方法分离和挑选单卵囊方法进行感染效果理想。单卵囊感染小鸡成功率为60％．该球虫寄生在鸡盲肠部位．其余部位未发现．卵囊形态长卵圆形．长25．80(23．5～28．5)．宽20．86(18．5～23．5)．长／宽：1．23(1．21～1．27)．27℃条件下孢子化时间为20h．．潜伏期为146h．与国内一些株进行比较存在差别．确定该艾美尔球虫为柔嫩艾美尔球虫(Eimeria tenella)，并定名为柔嫩艾美尔球虫厦门株．

不同表达条件对Eimeria tenella丙二酰单酰辅酶A:ACP转移酶活性的影响

为探讨不同原核表达载体、不同表达温度对重组表达Eimeria tenella丙二酸单酰辅酶A:ACP转移酶活性的影响,以生物信息学技术预测的E.tenellaⅡ型脂肪酸合成代谢途径中的丙二酰单酰辅酶A:ACP转移酶(Et-MCAT)编码序列为基础,分别设计引物扩增完整ORF,采取截除信号肽而保留转导肽以及切除信号肽和转导肽的不同策略,选择pMAL-c2x、pET-32a(+)和pProExHTa3种不同表达载体分别构建重组表达质粒,转化大肠杆菌Rosseta(DE3)后,在不同条件下诱导表达,利用酮戊二酸脱氢酶(KDH)偶联系统测定表达产物的酶活性,以评价不同表达策略对表达效果和表达产物活性的影响。表达水平和酶活性分析结果显示,惟切除信号肽和转导肽后的功能蛋白编码区能表达,Rosseta(DE3)/pMAL-c2x-mcat体系的可溶性表达量高于Rosseta(DE3)/pET-32a(+)-mcat,但二者表达产物的酶活性相似,且均随着诱导温度的降低其可溶性蛋白表达量增加。但Rosseta(DE3)/pProExHTa-mcat体系仅能表达不溶性蛋白,且复性后无酶活性,改变诱导温度对表达效果无明显影响。结果表明不同表达载体和诱导温度对大肠杆菌所表达EtMCAT的活性及其表达形式有显著影响,其中融合标签和表达温度可能是主要的影响因素,包涵体表达的重组酶即使复性处理仍难达到酶活性分析的要求。

柔嫩艾美球虫(Eimeria tenella)体外细胞培养

试验比较了玻璃珠层析法、ＤＥＡＥ纤维素层析法、Ｇ３漏斗过滤法３种提纯柔嫩艾美球虫（Ｅｉｍｅｒｉａｔｅｎｅｌｌａ）子孢子的方法。柱高８ｃｍ、直径２００μｍ的玻璃珠柱用ｐＨ８．０的Ｒｉｎｇｅｒ′ｓ缓冲液作洗脱液提纯子孢子，回收率为６４．１％，子孢子洗脱高峰期集中，且易于无菌操作；柱高５ｃｍ的ＤＥＡＥ纤维素柱用ｐＨ８．０的甘氨酸缓冲液作洗脱液，提纯子孢子回收率为６３．２％，但严格无菌操作困难；Ｇ３漏斗法易无菌操作，方法简便，但提纯效果不理想，回收率为２５％，有少量杂质。结果表明，玻璃珠柱层析法可作为细胞培养用提纯子孢子的首选方法。提纯的子孢子接种于体外培养的鸡原代肾细胞，在ＭＥＭ细胞形成培养液（含１０％的犊牛血清）和ＭＥＭ细胞维持培养液（含５％的犊牛血清和５％的水解乳蛋白）体外细胞培养系统中，能够发育到卵囊阶段；Ｅ．ｔｅｎｅｌａ在体外细胞培养中的发育与鸡体内发育的过程和时间基本相似，都经过子孢子→第一代裂殖体→第二代裂殖体→配子体→卵囊等阶段。试验也表明，水解乳蛋白作为营养补充物是Ｅ．ｔｅｎｅｌａ子孢子发育到卵囊的重要因素。

地克珠利、氨丙啉与左旋咪唑联合使用对Eimeria tenella的效力试验

目的研究地克珠利与氨丙啉、左旋咪唑的联合抗球虫效果。方法应用均匀设计法对地克珠利、氨丙啉和左旋咪唑的各6个不同剂量进行优化配伍。试验设立6个药物试验组，并设药物对照组、感染不给药组和不感染不给药组．除不感染不给药组外，其余全部试验鸡每只接种E．tenella孢子化卵囊1×10^5个，以抗球虫指数（ACI）判定药物的疗效。结果地克珠利5mg／kg＋氨丙啉240mg／kg＋左旋咪唑1．5mg／kg组的雏鸡存活率为100％，相对增重率97．5％，病变值15．5，其ACI 181．97；而地克珠利2mg／kg组的ACI仅为79．35：其余各组ACI均在150-170之间。结论本试验中以地克珠利5mg／kg＋氨丙啉240mg／kg＋左旋咪唑1．5mg/kg为最佳抗球虫药物组．

柔嫩艾美耳球虫(Eimeria tenella)BJ株SO7重组抗原的免疫试验

将柔嫩艾美耳球虫 ( Eimeria tenella) BJ株 SO7基因插入 p Thio His B载体中 ,构建了 1个表达载体 ,命名为 p Thio His SO7。将此表达载体转入大肠杆菌 DH5α,经 IPTG诱导表达后 ,用 SDS-PAGE检测表达产物。结果表明 ,含 p Thio His SO7质粒的工程菌具有明显的表达产物 ,表达效率为 1 7.1 %。用超声波粉碎仪裂解含重组抗原的工程菌 ,加入钾明矾作佐剂 ,用 2个剂量 ( 1 0、1 0 0μg)分别于 4日龄、1 1日龄、1 8日龄 3次免疫雏鸡 ,同时设置 SO7+弱毒株组、SO7+强毒株组、弱毒株组、强毒株组、未免疫未攻毒组和未免疫攻毒组作对照 ,于 2 5日龄用 3× 1 0 4个 E.tenella强毒卵囊进行攻毒。结果表明 ,用大剂量 SO7重组抗原免疫 ( 3次 ) ,鸡可获得部分保护力 ,其病变计分比未免疫攻毒组下降 3 1 %。

柔嫩艾美耳球虫(Eimeria tenella)感染鸡IgG生成细胞及血清IgG变化

利用免疫组化技术和 Dot-ELISA,分别检测了雏鸡初次感染柔嫩艾美耳球虫 ( E.tenella)后 ,盲肠局部和免疫器官中 Ig G生成细胞数的动态变化、循环血液中特异性 Ig G水平的动态变化、雏鸡母源抗体的动态变化以及不同抗体水平雏鸡的抗球虫能力。结果表明 ,( 1 )感染后 2～ 3d,盲肠粘膜、脾脏、法氏囊、盲肠扁桃体中的 Ig G生成细胞即开始增殖 ,9～ 1 2 d达峰值 ,随后开始下降 ,盲肠扁桃体中 Ig G生成细胞数在 2 2 d时仍高于对照组 ;( 2 )感染后 6 d即可在循环血液中检测到特异性 Ig G,1 8d达峰值 ,30 d降至感染后 7d时的水平 ;( 3)特异性母源抗体 Ig G水平高的雏鸡 ,抗球虫能力高 。

马杜霉素对柔嫩艾美耳球虫(Eimeria tenella)超微结构的影响

将马杜霉素按 0 .0 0 0 5 %的比例混入饲料 ,作用于鸡体内柔嫩艾美耳球虫 (Eimeria tenella) ,透射电镜观察 ,发现裂殖体变小 ,内含裂殖子的数目减少 ,裂殖子膨胀变为椭圆 ,裂殖子间 (带虫空泡内 )微管结构模糊、膨胀 ,裂殖体中有髓鞘样结构出现 ;细胞膜外突和细胞质分离形成空隙 ,细胞膜有破损、结构模糊 ;细胞质出现空泡化 ,内部出现附加体和髓鞘样结构 ;少数细胞核的外膜外突和内膜分离 ,有的部位的核膜模糊、破损。裂殖子顶体结构发生异常变化 ,棒状体消失 ,微线数目减少或者消失。线粒体表现为形态改变 ,肿大变圆 ,或者变为马蹄形 ,内膜和嵴脱落 ,内部形成空泡、膜状结构、螺旋状或者髓样结构 ,线粒体的嵴与长轴平行排列 ,且贯穿线粒体。作者推测 ,抗生素类抗球虫药的作用机理可能是 ,通过使虫体结构的完整性受损 ,抑制虫体的物质代谢及能量代谢等生理机能 ,达到杀死虫体或者抑制虫体生长、发育和繁殖的目的。

鸡Eimeria tenella致弱虫苗的实验研究及田间试验

对双重致弱球虫疫苗（DLV虫苗）进行了实验室的安全试验。通过对380羽3日龄小鸡的3批毒力测定，强毒虫株2.5×10^4接种小鸡，死亡率均超过50%；DLV虫苗2.5×10^4剂量接种3批小鸡，未见明显临床表现，在鸡体连续5次传代，也未出现明显返强现象，通过对260羽3日龄小鸡的3批免疫试验证明，DLV虫苗对10×10^5强毒株的攻击有100%的保护率，又用ELISA法测定抗体效价消长发现，免疫后7周左右抗体效价达高峰，至14周抗体效价仍可保持在一定高度，足以对再感染具有保护力。该虫苗易于保存，一般在4℃可保存6个月以上，仍有良好的免疫效果。通过在上海、浙江、江苏、安徽等地区的男单试验，发现免疫鸡的平均发病率为2.8%，死亡率为0.48%。常规用药鸡平均发病率为11.8%，死亡率为2.9%，空白对照鸡（不用苗，不用药）平均发病率为80%，死亡率为28%（经及时抢救治疗减少了死亡率）。免疫鸡比常规用药鸡节省耗药费用0.20元/羽。在大批量扩大免疫试验中，对24，800羽鸡用苗后的平均保护率可达99.66%，死亡率为0.34%，效果十分显著，通过实验室和现场应用试验，证明DLV虫苗可靠、安全、稳定，且有高度免疫原性和足够的抗体水平及免疫期。

球安等药物抗Eimeria tenella的效力

将 1日龄罗曼公雏于无球虫条件下饲养至 1 3日龄时随机分组 ,按组分别投予球安 ( Avatec,含 2 5 % L asalocid) 1 2 5 mg/ kg。球佳 (含 2 % Diclazuril) 1 mg/ kg,加福( Cygro,含 1 % Maduramycin) 5 mg/ kg和球净 ( Necoxine,含 2 5 % Nicabazine和 1 .6 %Ethopabata) 1 2 5 mg/ kg,并设立不给药不感染、不给药感染对照组。给药后第 3d,除不给药不感染对照组的鸡只外 ,其余全部试验鸡每只分别接种 E.tenella孢子化卵囊 1× 1 0 5个 ,根据各组鸡的存活率、相对增重率、盲肠病变计分、血便记分和抗球虫指数对 4种药物进行药物疗效判定。试验结果表明 :球安、球佳为高效抗球虫药 ,其存活率都达 1 0 0 % ,相对增重率分别为 1 0 3.1 2 5和 92 .5 ,平均血便记分为 0 .0 2和 0 .0 1 ,病变值 8.75和 6 .75 ,抗球虫指数 1 89.375和 1 80 .75 ;而加福和球净虽对雏鸡保护率为 1 0 0 % ,但血便和肠组织病变严重 ,抗球虫指数仅为 99.2 5和1 0 5 .75 ,表明抗球虫无效。

Synthesis and biological evaluation of ethyl 6-alkoxy-7-phenyl-4-hydroxy-3-quinolinecarboxylates against Eimeria tenella

A series of ethyl 6-alkoxy-7-phenyl-4-hydroxy-3-quinolinecarboxylates were designed and synthesized.Their structures were confirmed by ~1H NMR,^(13)C NMR,IR and HRMS.The biological activities were primarily evaluated against Eimeria tenella according to Anticoccidial Index(ACI) method in vivo.The results showed that compounds 5e,5f and 5i exhibited anticoccidial activities against E.tenella at 27 mg kg^(-1).

Expression of Chicken Toll-Like Receptors and Signal Adaptors in Spleen and Cecum of Young Chickens Infected with Eimeria tenella

Toll-like receptors （TLRs） are a group of highly conserved molecules which initiate the innate immune response to pathogens by recognizing structural motifs of microbes. Understanding the changes in chicken Toll-like receptors （ChTLRs） and signal adaptors expression that occur with Eimeria tenella infection will help to elucidate the molecular basis of immune control of coccidiosis caused by Eimeria. The present study detected the dynamic changes in the expression of ChTLRs and associated signal adaptors in the spleen and cecum ofE. tenella-infected chickens during the early stage of infection. The results showed that the expression peak for ChTLRs, MyD88 and TRIF occurred at 12 h post-infection （hpi）, ChTLR3, ChTLRI 5 and MyD88 mRNA expression in the spleen ofE. tenella infected chickens were significantly higher （P〈0.05） than that of negative control chickens, and there were similar tendencies of these molecules expression in the cecum and spleen of E. tenella-infected chickens. The expression of MyD88 was upregnlated at four time points in the cecum of E. tenella-infected chickens. The results of this study indicate that ChTLR3, ChTLR15 and MyD88 play a role in young chickens infected with E. tenella.

鸡柔嫩艾美耳球虫(Eimeria tenella)免疫应答的形态学观察

用柔嫩艾美耳球虫卵囊活苗对京白雏鸡进行一次免疫、二次免疫及涓滴免疫试验,对免疫后、雏鸡腔上囊、脾脏、胸腺及盲肠扁桃体等组织器官的免疫应答作了较为详细的观察。免疫后上述组织器官淋巴细胞明显增多,形成大量淋巴滤泡;通过对免疫组和对照组雏鸡上述免疫组织器官的对照检查,表明三种免疫方法免疫后腔上囊、脾脏、胸腺及盲肠扁桃体均呈现不同程度的增生,其中以涓滴免疫和二次免疫比较明显。试验结果提示,一次免疫、二次免疫及涓滴免疫均可刺激机体免疫组织器官淋巴细胞大量增生,产生有效的免疫应答反应,对强毒攻击表现有效的抵抗力。

柔嫩艾美耳球虫(Eimeria tenella)小配子发育超微结构的观察

利用透射电镜对柔嫩艾美耳球虫小配子发育的超微结构进行了观察 小配子母细胞和大配子母细胞于相邻的宿主细胞内相伴产生 ,系由末代裂殖子侵入后 ,长大变圆而形成 小配子的形成为直接分化型 首先细胞核分裂为多个 ,随后细胞核向周边移动 ,随之近核处限制膜外突 ,临近处限制膜下陷 中心粒在核上方形成并发育为基粒、鞭毛中的微管和附着微管 ,早期形成的小配子仍与小配子母细胞的残体相连 成熟小配子与残体分离 ,外型呈香蕉状 ,外被单位膜 ,内有一电子结构十分致密的细胞核 ,核头端侧面有一个巨大线粒体 ,小配子有鞭毛 2根 ,每根鞭毛内有微管 ,为 9+2结构 ,附着微管至少 6根 。

Analysis for Pathogenicity and Development of Transgenic Eimeria tenella Expressing both Yellow Fluorescent Protein and TgDHFR-TS Genes

In order to determine the effect of foreign genes on a transgenic parasite,the pathogenicity and development in chickens of transgenic E.tenella strain TE1,which expresses yellow fluorescent protein (YFP) and dihydrofolate reductase thymidylate synthase derived from Toxoplasma gondii(TgDHFR-TS), were compared with that of the parental strain BJ.Results indicated that the fecundity of the transgenic parasite(TE1) was reduced at least 4 times relative to that of the BJ strain.Low dosage of the TE1 strain induced less pathogenesis in chickens than did the BJ strain,but chickens inoculated with a higher dosage of TE1 oocysts displayed severe pathogenicity and mortality as did the BJ strain.In addition,trophozoites, the first generation and the second generation meronts and merozoites,microgamonts and macrogamonts of the transgenic parasite TE1 were seen by fluorescence microscopy.More interestingly,not all four sporonts in the sporulating transgenic oocysts express YFP.These findings suggest that the expression of YFP and TgDHFR-TS genes to some extent reduced pathogenicity and reproductive potential of transgenic E.tenella.Recombination between homologous or non-homologous chromosomes occurred during zygotic meiosis in E.tenella strain TE1.

The Effects of Exogenous Nitric Oxide on Eimeria tenella Oocysts

The effects of exogenous NO on Eimeria tenella oocysts were studied. The E. tenella oocysts freshly isolated from feces of experimental chickens infected with E. tenella and sporulated E. tenella oocysts were treated in vitro by the well-known NO-donors, SNP, SPER/NO and GSNO. The results showed that among the 3 NO donors, only GSNO had significant effects on the percentage of sporulated oocysts, the inhibiting rate of purified oocysts was from 93 to 97%, and reached 100% on the oocysts in the feces of chickens with E. tenella infection. GSNO also significantly decreased the virulence of E. tenella oocysts, and the oocysts treated by 2 - 8 mmol L-1 GSNO almost lost their pathogenicity to chickens.

Inhibitory Effects of S-Nitroso-Glutathione on Sporulation Process of Eimeria tenella Oocysts

To study the inhibiting effects of S-Nitroso-Glutathione （GSNO） on sporulation process of Eimeria tenella （E. tenella） oocysts by detecting the sporulation rate, GSNO as nitric oxide donor was used to treat E. tenella oocysts sporulated for different times; Several kinds of antioxidants and oxidants were used to investigate the inhibitory effects of GSNO on sporogony process of E. tenella oocysts. The results showed that GSNO still inhibited the oocysts sporulated 10 h, but had few inhibiting effect on the oocysts sporulated for 14 h; the antioxidants, such as Vitamin C （VC）, mannitol and sodium salicylate, could not eliminate the inhibitory effects of GSNO on oocysts; neither did the ferrous sulfate （FeSO4） nor Dithiothreitol （DTT）. However, the oxidants, potassium dichromate （K2Cr2OT） and potassium permanganate （KMnO4） could obviously repress the effects of GSNO on oocysts. At present, the inhibitory mechanism of GSNO on unsporulated oocysts was still unclear.

15株柔嫩艾美耳球虫Eimeria tenella对五种抗球虫药的抗药性调查研究

应用 AA 肉鸡,检测了采自我国12个省区的15株 Eimeria tenella 对5种抗球虫药的敏感性。根据最适抗球虫活性百分率、相对卵囊产量和病变记分减少率3项指标综合判定,对莫能霉素、氨丙啉、克球粉、氯苯胍和杀球灵具有不同程度抗药性的虫株分别占80%,66.6%,53.3%,53.3%和0%。不同地区虫株的抗药谱和抗药程度存在差异。多数虫株对2种或2种以上的抗球虫药具有复合抗药性。此外,研究制订了比较适用的检测球虫抗药性的判定方法和判定标准。

抗球虫剂对Eimeriatenella裂殖子的效力试验

利用鸡胚模型研究了三种聚醚类抗生素及七种化学抗虫剂对柔嫩艾美球虫第二代裂殖子的效果。纯化的裂殖子用抗球虫药作用３０分钟，用Ｈａｎｋ＇ｓ平衡盐溶液洗去药物后接种１０～１１天龄鸡胚，２．５天后收集鸡胚中的卵囊，以卵囊形成数判断药物的效果。结果证明，除Ａｍｐｒｏｌｉｕｍ（氨丙啉）外，大部分抗球虫剂对裂殖子是有效的。各种药物的最低有效浓度为：Ｌａｓａｌｏｃｉｄ（拉萨菌素）、Ｓａｌｉｎｏｍｙｃｉｎ（盐霉素）、Ｍｏｎｅｎｓｉｎ（莫能菌素）、Ｄｉｃｌａｚｕｒｉｌ（杀球灵）、Ｈａ１ｏｆｇｉｎｏｎｅ（常山酮）为１０μｇ／ｍｌ；Ｒｏｂｅｎｉｄｉｎｅ（氯苯胍）、Ｄｅｃｏｑｕｉｎａｔｅ（乙羟喹酯）为１０μｇ／ｍｌ；Ｃｌｏｐｉｄｏｌ（氯此酚）为５００μｇ／ｍｌ；Ｎｉｃａｒｂａｚｉｎ（尼卡巴嗪）为１０００μｇ／ｍｌ。药物剂量逐渐提高，裂殖子逐渐丧失其活性。当Ｌａｓａｌｏｃｉｄ、Ｓａｔｉｎ－ｏｍｙｃｉｎ、Ｍｏｎｅｎｓｉｎ、Ｄｉｃｌａｚｕｒｉｌ、Ｈａｌｏｆｕｇｉｎｏｎｅ、Ｒｏｂｅｎｉｄｉｎｅ分别达到３５０、１５０、１２０、２００、５００和７００μｇ／ｍｌ时，所有的裂殖子完全丧失活性。本文还比较了不同投药方法的优缺点，并讨论了药物的活性期。?

三株柔嫩艾美耳球虫(Eimeria tenella)的繁殖力和免疫原性的比较研究

分别用柔嫩艾美耳球虫早熟弱毒株、鸡胚适应株和强毒株孢子化卵囊１０００个／只，给１０日龄无球虫雏鸡免疫接种，逐日检查粪便并进行克粪便卵囊（Ｏ．Ｐ．Ｇ．）计数，结果发现早熟弱毒株和鸡胚适应株的潜隐期均有不同程度缩短，繁殖力亦有不同程度的下降。同时进行了柔嫩艾美耳球虫不同株球虫的免疫原性比较：分别用三株卵囊免疫雏鸡，免疫剂量为１０００个／只，免疫后１４ｄ各用１０００个／只强毒株卵囊攻虫，逐日进行Ｏ．Ｐ．Ｇ．计数，发现各组免疫鸡攻虫后的卵囊产量均大大下降，其中以强毒株免疫攻虫组的排出卵囊量最低，仅为未免疫组的１．２３％，早熟株和鸡胚株免疫鸡攻虫的排出卵囊量相近，分别为未免疫组的７．３５％和７．２３％。由此可见，三株柔嫩艾美尔球虫均表现出良好的免疫原性。