三株柔嫩艾美球虫野外分离株的致病性分析

用每鸡4×104和8×104个孢子化卵囊的剂量感染黄羽肉雏,以临床症状、肉眼病变、平均增重、病变记分和死亡率为判定指标,对柔嫩艾美球虫EtCZ-1、EtCZ-2、EtCZ-3株的致病性进行了比较分析。结果显示:各虫株引起的临床症状和肠道肉眼病变基本相似,致病性随着感染剂量的增加而增强。各感染组的增重与对照组相比均有显著差异(P<0.50),相对增重率分别下降了74.4%～83.2%、68.7%～72.2%和70.6%～74.4%;感染后鸡出现血便的时间较一致,即第108小时开始排血便,在120h～144h达高峰;病变值和卵囊值均随着感染剂量的增加而增大;除EtCZ-1株高剂量组的存活率是90%以外,其余各组均为100%。显示3株球虫的致病性相似。     

外源性一氧化氮对鸡柔嫩艾美尔球虫的作用

用酸化亚硝酸钠作为一氧化氮(NO)供体处理新收取的柔嫩艾美尔球虫未孢子化和已孢子化卵囊,来探讨外源性NO对球虫卵囊的抑杀作用。另外通过内服亚硝基谷胱甘肽(GSNO)试验来观察外源性NO对球虫病的防治作用。结果表明,酸化亚硝酸钠溶液(pH=1)对球虫卵囊的孢子生殖具有明显的抑制作用,20mmol/L酸化亚硝酸钠溶液对卵囊孢子生殖的抑制率可达100%;但不经酸化的亚硝酸钠溶液和相同pH的盐酸溶液对卵囊的孢子生殖没有明显影响。经20mmol/L以上浓度酸化亚硝酸钠处理的孢子化卵囊也失去了对雏鸡的致病力,但直接内服GSNO溶液则没有明显的抗球虫作用。这提示外源性NO对鸡柔嫩艾美尔球虫未孢子化和孢子化卵囊均有抑杀作用,但直接内服没有明显的抗球虫病作用。     

不同地理株柔嫩艾美耳球虫(E.tenela) 杂交虫株(F1)的培育

通过用单卵囊接种技术培育了山东株(S)和黑龙江株(H),然后将山东株(S)和黑龙江株(H)的柔嫩艾美耳球虫的孢子化卵囊同时喂给雏鸡,收获卵囊,提取基因组DNA,再利用RAPD技术对它们基因组DNA进行分析,分析了19株柔嫩艾美耳球虫卵囊的DNA分子,确定了1株为山东株和黑龙江株的杂交株(F1)。这一研究结果为深入研究球虫的免疫机制奠定了基础。     

表达黄色荧光蛋白和乙胺嘧啶抗性基因的转基因柔嫩艾美耳球虫的发育和致病性分析

为了研究外源基因表达对转基因柔嫩艾美耳球虫(Eimeria tenella)生物学特性的影响,对转基因球虫(TE1)和BJ株(野生强毒)的发育和致病性进行比较分析.结果显示,相对于BJ株,转基因球虫TE1株繁殖力下降约4倍,低剂量感染时致病性下降明显,但是高剂量感染时依然保持较强的致病性.另外,在荧光显微镜下发现,TE1有滋养体、第一代裂殖体/裂殖子、第二代裂殖体/裂殖子和大小配子体等内生发育虫体.孢子化卵囊内4个孢子囊并非全部表达黄色荧光蛋白.这些结果说明黄色荧光蛋白和乙胺嘧啶抗性基因的表达在一定程度上降低了转基因柔嫩艾美耳球虫致病性和繁殖力.在柔嫩艾美耳球虫合子减数分裂过程中,存在同源或异源染色体重组现象.     

Protective effects of sugar cane extracts (SCE) on Eimeria tenella infection in chickens

The effects of oral administration of sugar cane extracts (SCE) on Eimeria tenella oocysts infection in chickens were studied with 2 different experiments. In Experiment 1, 3-week-old inbred chickens (MHC; H.B15) were inoculated into the crop with SCE (500 mg/kg/day) for 1 day or 3 consecutive days, and then challenged with E. tenella sporulated oocysts (2 x 10(4) cells/chicken). In Experiment 2, 1-week-old chickens were orally administered SCE at the same dose for 3 consecutive days, and then initially infected with E. tenella sporulated oocysts (2 x 10(3) cells/chicken). At 2 and 3 weeks of age, these chickens were immunized intravenously with the mixed antigens of sheep red blood cells (SRBC) and Brucella abortus (BA). At 4 weeks of age, chickens were challenged with E. tenella sporulated oocysts (1 x 10(5)/chicken). Challenged chickens with E. tenella oocysts showed markedly decreased body weight gain/day, severe hemorrhage and great number of shedding oocysts in feces and high lesion scores. Oral administration of SCE and initial infection with oocysts (2 x 10 (3)/chicken) resulted in a remarkable improvement in body weight gain/day, hemorrhage, the number of shedding oocysts and lesion score, compare to other infected groups. In addition, SCE-inoculated chickens with the initial infection showed a significant increase in antibody responses against SRBC and BA and also improvement in decreased relative proportions of Bu-1a(+) and CD4( )cells in cecal tonsil lymphocytes of E. tenella-challenged chickens. Cecal tissues of chickens administered SCE and initially infected with E. tenella oocysts showed lower numbers of schizonts, gametocytes and oocysts than those of infected control chickens. These results suggest that SCE have immunostimulating and protective effects against E. tenella infection in chickens.     

球抗预防鸡柔嫩艾美耳球虫病效果的观察

采用接种孢子化柔嫩艾美耳球虫卵囊的方法,复制鸡球虫病,观察并计算球抗对柔嫩艾美耳球虫病鸡的存活率、相对增重率、血便记分、盲肠病变记分和卵囊值的影响,并求出球抗的抗球虫指数.结果发现球抗能提高球虫病鸡的存活率和相对增重率,减少血便记分和盲肠内容物卵囊数,减轻盲肠病变.表明球抗对鸡柔嫩艾美耳球虫病有一定的预防作用.     

Evaluation of interspecific hybrids of the chicken, guinea fowl, and Japanese quail for innate resistance to coccidia

Experimental chicken/guinea fowl hybrids, guinea fowl, and chickens were orally inoculated with Eimeria acervulina or E. tenella, which are specific for chickens, or with E. grenieri, which is specific for guinea fowl. No intact oocysts were found in feces within 24 hr of inoculation, suggesting that excystation occurred in the normal and abnormal hosts. No oocysts were found in the feces of hybrids during a 9-day postinoculation period. The guinea fowl passed oocysts of guinea fowl coccidia (E. grenieri) but not those of chicken coccidia, and the chickens passed oocysts of chicken coccidia (E. acervulina and E. tenella) but not those of guinea fowl coccidia. Some asexual development (schizogony) occurred in hybrids inoculated with E. tenella, but sexual development (gametogony) did not. In contrast, quail/chicken hybrids became infected with oocysts of chicken coccidia (E. acervulina, E. tenella, and E. maxima) and quail coccidia (E. bateri) and passed a few oocysts during the normal patent period; control chickens and quails became heavily infected with oocysts of chicken and quail coccidia, respectively.     

Isolation of five species ofeimeria from chickens in Bangladesh

Five species of Eimeria, namely E. acervulina, E. tenella, E. maxima, E. brunetti and E. necatrix were identified in chickens in Bangladesh on the basis of lesions seen at post-mortem examinations of naturally infected birds, and on the dimensions of oocysts and the lesions seen in chicks experimentally infected with single oocyst derived strains. The use of filter top, polycarbonate cages permitted the isolation of strains without sophisticated animal isolators and is appropriate for use in laboratories throughout the developing world. Responses to a questionnaire sent to all known intensive poultry farms suggested that coccidiosis is a major disease. For control, producers rely mostly on management procedures and the tactical use of sulphonamides; in-feed chemoprophylaxis is not widely used. These control measures are unsatisfactory and recent coccidiosis outbreaks were reported from seven of 16 farms. There was evidence of a seasonal incidence in clinical coccidiosis.     

Differential diagnosis of five avian Eimeria species by polymerase chain reaction using primers derived from the internal transcribed spacer 1 (ITS-1) sequence

Chicken coccidia are protozoan parasites of the genus Eimeria. They cause economical losses in the poultry industry globally. The various species can be distinguished on the basis of the morphology of the oocysts and parasitic site in intestine, but these criteria sometimes are unreliable. Therefore, a species-specific polymerase chain reaction (PCR) was developed. Based on variable sequence regions, specific primers were constructed for the differentiation of five Eimeria species (Eimeria acervulina, E. brunette, E. maxima, E. necatrix, and E. tenella). PCR products were amplified from coccidian vaccine (coccivac-D and coccivac-B) and E. tenella and were subsequently sequenced. Similarities of the five species sequences between the vaccines and Genbank were 94-100%. Analysis of the E. tenella internal transcribed spacer 1 (ITS-1) partial sequence from Taiwan and from Genbank indicated that the similarity was 99.6%. The PCR sensitivity test of E. tenella in Taiwan is 50 oocysts. The five sets of primers will not amplify any non-specific bands of the chicken genome or its intestinal contents. Therefore, the five sets of specifically designed primers are guaranteed to be useful for differential diagnosis of avian coccidiosis caused by Eimeria spp.     

Distribution of oocysts, sporocysts and sporozoites of Eimeria tenella and Eimeria maxima in the digestive tract of chicken.

The distribution of oocysts, sporocysts and sporozoites of Eimeria tenella and Eimeria maxima in the digestive tract of chicken and in excreta was investigated. At 1 h after the oral inoculation of E. tenella oocysts, the number of sporocysts in the cecum was 3.4 x 10(6) and decreased gradually thereafter, and the number of sporozoites in the cecum increased and remained at a high level until 12 h after the inoculation. Small numbers of sporocysts and sporozoites of E. tenella were found in other intestinal sites. A great number of E. maxima sporozoites was found, especially in the jejunum, 2 h after the inoculation. The findings that the largest populations of sporozoites of E. tenella and E. maxima were found in the cecum and the jejunum, respectively, indicate that the site specificity of sporozoite invasion for each species is determined before the invasion takes place.     

鸡胚接种柔嫩艾美耳球虫和堆形艾美耳球虫活卵囊的免疫保护试验

给18日龄鸡胚接种一定剂量的柔嫩艾美耳球虫(Eim eria tenella)和/或堆形艾美耳球虫(E.acervulina)孢子化卵囊,出雏后在无球虫环境中笼养,1~10日龄每天收集各组粪便样本,计数克粪便卵囊数(OPG),并于14日龄时以大剂量同源孢子化卵囊攻虫,以相对增重率(RWG)、饲料转化率(FCR)、相对卵囊产量(ROP)评价免疫保护效果。结果显示,以E.tenella或E.acervulina卵囊免疫18日龄鸡胚,其卵囊排出的潜隐期及达到峰值的时间与1日龄雏鸡接种组相一致,有相似的排卵囊曲线,提示其诱导免疫的建立是在出雏后开始建立的。攻虫后各免疫组的RWG由攻虫对照组的31.9%~51.7%提高到了76.5%~83.6%,RCR由攻虫对照组的4.11~4.89改善为2.72~2.96,ROP降至4.7%~23.5%。结果表明以一定剂量E.tenella和E.acervulina卵囊单独或混合经羊膜腔免疫18日龄鸡胚都可以建立起针对出雏后14日龄同源攻虫的良好免疫保护力。比较混合免疫E.tenella和E.acervulina卵囊组与单一接种E.tenella或E.acervulina卵囊组的免疫效果发现,混合免疫组的各项指标均稍优于后者。     

Development of protective immunity against Eimeria tenella and E. acervulina in White Leghorn chickens inoculated repeatedly with high doses of turkey coccidia

Repeated inoculation (immunization) of 2-week-old white leghorn chickens with 10(6) oocysts of the turkey coccidia Eimeria adenoeides or E. meleagrimitis partially protected chickens against moderate challenge with E. tenella or E. acervulina oocysts, but not with E. necatrix oocysts. After challenge, mean weight gains of the immunized chickens and the unchallenged controls did not differ significantly, but weight gains of unimmunized chickens were significantly lower. The mean feed-conversion ratio of the immunized challenged chickens was 3.14, as compared with 4.42 for unimmunized challenged control chickens. In general, immunization did not markedly reduce intestinal lesions. Repeated inoculation of chickens with the turkey coccidium E. gallopavonis failed to produce statistically significant protection against challenge with E. tenella, E. acervulina, or E. necatrix, as determined by weight gain, feed-conversion efficiency, and lesion scores. Antibody profiles of individual chickens did not correlate with protection.     

球虫抗药性分子生物学检测技术的建立

选择在敏感虫株中沉默、抗药虫株中表达的特异序列AGD5设计检测引物，采用RT—PCR方法。以总RNA反转录的cDNA为模板，RT—PCR方法能够鉴别马杜霉素抗药虫株和3株马杜霉素敏感虫株（2株对所有药物敏感的不同地理株，1株地克珠利抗药性虫株对马杜霉素敏感），此鉴定结果与鸡体试验的结果一致，说明敏感虫株和抗药虫株在该序列上的差异发生在转录水平。而以卵囊总DNA为模板的PCR方法不能鉴别敏感和马杜霉素抗药性虫株。     

GSNO对E．tenella内LDH、G-6-PD、ACO和SOD活性的影响

亚硝基谷胱甘肽（GSNO）对鸡柔嫩艾美耳球虫（E．tenella）卵囊的孢子生殖具有不可逆地抑制作用，但其作用机理尚不清楚。本研究采用聚丙烯酰胺凝胶电泳来分析GSNO对E．tenella卵囊内与糖代谢有关的乳酸脱氢酶（LDH）、葡萄糖．6．磷酸脱氢酶（G．6．PD）、乌头酸酶（ACO）以及GSNO对卵囊内与抗氧化有关的超氧化物歧化酶（SOD）活性的影响，电泳后经特异染色显示酶活性。结果表明：GSNO处理的未孢子化与孢子化卵囊中均有LDH、（3-6．PD、ACO及SOD活性，这提示外源性NO对球虫孢子生殖的抑制并没有灭活卵囊中LDH、G．6．PD、ACO和SOD的活性或NO对这些酶的作用是可逆的。     

柔嫩艾美耳球虫早熟株免疫剂量研究

为了确定柔嫩艾美耳球虫（Eimenatenella）早熟株合适的免疫剂量,本文设立7个早熟株免疫攻虫组、1个不免疫攻虫组和1个不免疫不攻虫组,免疫组的免疫剂量为孢子化卵囊100、200、400、600、800、1000和2000个／羽,经嗉囊感染,7日龄首次免疫,14日龄以同等剂量进行第二次免疫,21日龄以8×10^4个／羽的同源母株进行攻虫,28日龄结束试验,以存活率、增重、肠道病变记分、血便数量、卵囊减少率为观测指标。对免疫保护效果较好的3个免疫剂量进行重复试验,同时设置商品化球虫疫苗对照组,免疫方法、试验周期、试验指标同第一批试验。结果显示：攻虫后,不免疫攻虫组出现5％死亡,而各免疫组来出现死亡;各免疫组卵囊减少率在61．57％～69．52％;200～2000免疫组的增重与不免疫不攻虫组差异不具备显著统计学意义（P〉0．05）;600～2000免疫组的肠道病变记分和血便数量均明显少于不免疫攻虫组（P〈0．05）。用600、800和1000进行重复试验,三个免疫组攻虫期间均来出现死亡,而不免疫组和疫苗对照组均出现5％死亡;三个免疫组的增重均明显高于不免疫攻虫组和疫苗对照组（P〈0．05）;早熟株免疫组的肠道病变记分和血便数量明显低于不免疫攻虫组（P〈0．05）,而疫苗对照组与不免疫攻虫组的相当（P〉0．05）;卵囊减少率在66.30％-78．75％,高于疫苗对照组的51．82％。结果表明,该柔嫩艾美耳球虫早熟株保持了良好的免疫原性,不同免疫剂量均能诱发鸡产生免疫保护力,其中600、800和1000个／羽的免疫效果均优于疫苗对照组,可考虑以600个／羽作为该早熟株在疫苗制备中的推荐免疫剂量。     

柔嫩艾美耳球虫耐药株与鸡3种球虫的同工酶的研究

对不同离子载体抗生素具有抗药性的柔嫩艾美耳球虫和药物敏感的柔嫩区美耳球虫、布氏匀美耳球虫和堆型艾耳球虫的孢子化卵囊,采用聚安凝胶垂直板电泳,进行了乳酸脱氢酶（ＬＤＨ）、葡萄糖磷酸异构酶（ＧＰＩ）和葡萄糖－６－磷酸脱氢酶（Ｇ６ＰＤ）等同工酶华不同离子载体抗生素具有抗药性的柔嫩艾美耳球虫和药物同工酶酶谱可以反应出球虫种间差异;而柔嫩艾耳球虫抗性虫株的ＬＤＨ酶谱是２条带,敏感虫株是３条带,抗性株比敏感株     

Isolation and selection of ionophore-tolerant Eimeria precocious lines: E. tenella, E. maxima and E. acervulina

Eimeria parasites were isolated from Nanhai Guangdong province (southern China) and studied in chickens in wire cages to evaluate their drug resistance against commonly used ionophores: monensin (100 mg/kg of feed), lasolacid (90 mg/kg), salinomycin (60 mg/kg), maduramicin (5 mg/kg) and semduramicin (25 mg/kg). Chinese Yellow Broiler Chickens were infected with 40,000 crude sporulated Eimeria oocysts at 15 days of age and prophylactic medication commenced a day prior to infection. Drug resistance was assessed for each ionophore drug by calculating the anticoccidial index (ACI) and percentage optimum anticoccidial activity (POAA) based on relative weight gain, rate of oocyst production and lesion values. Results revealed that Nanhai Eimeria oocysts comprising of E. tenella, E. maxima and E. acervulina, were resistant to monensin, sensitive to both salinomycin and lasolacid and partially sensitive to maduramicin and semduramicin. By selection for early development of oocysts during passage through chickens, the prepatent time of E. tenella, E. maxima and E. acervulina were reduced by 49, 36 and 22 h, respectively. The precocious lines are less pathogenic than the parent strains from which they were selected and conferred a satisfactory protection for chickens against coccidiosis. These ionophore-tolerant precocious lines could have wider applications in the development of anticoccidial vaccines for sustainable control of coccidiosis.     

Effects of clopidol on sporulation and infectivity of Eimeria tenella oocysts

Feed additive anticoccidials currently used in Japan were examined for possible effects on oocyst sporulation of Eimeria tenella. Monensin, salinomycin, lasalocid, amprolium plus ethpabate, amporolium plus ethopabate plus sulfaquinoxaline, clopidol, or nicarbazin were given to chickens continuously via the feed at the recommended use level or one-half of that level. Oocysts discharged in feces 7-8 days post inoculation (PI) were collected and aerated for sporulation. Low sporulation rate was noted, when clopidol at 62.5 mg kg-1 was given from 4 to 7 days PI. These oocysts were as infective as oocysts from controls, based on weight gain, feed efficiency, gross lesion score of cecae, and oocyst count 7 days PI. The results of the study indicated that the second schizogony and gametogony are vulnerable to clopidol, as evidenced by oocyst sporulation, but infectivity of these sporulated oocysts was not affected.     

Influence of inoculation dose, inoculation schedule, chicken age, and host genetics on disease susceptibility and development of resistance to Eimeria tenella infection

Various parasite- and host-related factors influencing disease susceptibility and development of protective immunity against Eimeria tenella infection were investigated in two inbred strains of chickens. Chickens that received a primary inoculation of 10(3), 10(4), or 10(5) oocysts showed a significant reduction in packed cell volume and produced significantly more oocysts than chickens inoculated with fewer oocysts. Younger chickens were as susceptible as older chickens to identical parasite doses. However, upon a secondary inoculation 5 weeks following primary inoculation, FP chickens 1 to 21 days old at the time of primary inoculation developed resistance to reinfection, whereas SC chickens less than 3 weeks old at the time of primary inoculation were highly susceptible to secondary infection. Flow cytometric analysis of spleen lymphocytes showed a substantial reduction in T-cell number in 1-day-old SC but not FP chickens. Furthermore, 1-week-old SC chickens showed depressed mitogenic responses to concanavalin A compared with 1-week-old FP chickens. There was no significant difference between SC and FP chickens in speen B-cell number, regardless of age.     

13株柔嫩艾美耳球虫分离株对地克珠利及盐霉素耐药情况分析

为了解不同地区球虫对地克珠利(DIC)与盐霉素(SAL)的耐药情况,收集了来自江苏、山东、福建、安徽、河南地区共13个鸡场的球虫样本,对其进行了DIC与SAL的耐药性分析。试验时对每个虫株分别设药物防治组、感染不用药组和空白对照组,以抗球虫指数、病变记分减少率、相对卵囊产量、最适抗球虫活性百分率为指标进行综合评定。结果:来自江苏南通地区虫株中有85%对DIC表现出不同程度的耐药性,100%对SAL耐药;河南开封分离株对2种药物表现完全耐药;山东地区仅有1株对DIC轻度耐药,剩余虫株对2种药物均敏感。研究表明,目前南通与开封地区应避免使用DIC和SAL进行球虫病的防治,而山东、福建、安徽可放心使用这2种药物。