不同佐剂和免疫途径对柔嫩艾美耳球虫SO7抗原免疫保护效果的影响

利用PGEX-6P-1融合表达系统将SO7基因在大肠杆菌中进行融合表达,对表达产物进行初步纯化和复性,制备免疫原。分别使用不同剂量的人参总皂甙和重组γ-干扰素以及弗氏完全佐剂作为免疫佐剂,于5日龄、12日龄和19日龄对雏鸡进行3次免疫,同时设立蛋白口服免疫组、蛋白皮下注射免疫组、卵囊口服免疫组、未免疫未攻毒组和未免疫攻毒组作对照,于26日龄用105个柔嫩艾美耳球虫卵囊进行攻毒,8d后扑杀,对各组的存活率、相对增重率、病变减少率、相对卵囊产量和抗球虫指数ACI等指标进行统计分析,比较免疫保护效果。与非免疫攻毒组以及不加佐剂的免疫攻毒组相比,佐剂使用后各组的增重、病变记分、相对卵囊产量、ACI等指标均有一定的改善,对柔嫩艾美耳球虫的人工感染可以提供部分保护。3种佐剂中,γ-干扰素和人参总皂甙能增强重组蛋白的免疫保护效果,且佐剂的剂量对免疫保护的效果有一定的影响,以5000Uγ-干扰素效果最佳,效果与E.tenella活卵囊免疫相当,弗氏完全佐剂的免疫增强效果不明显。单独使用重组蛋白皮下注射或口服免疫,虽有一定的保护作用,但不明显。一定剂量的γ-干扰素对SO7抗原的免疫保护效果起明显的增强作用,是一个具有很好临床应用前景的新型佐剂。     

鸡巨型艾美耳球虫早熟株免疫效果试验

为确定使雏鸡获得良好免疫效果的最小巨型艾美耳球虫(E.maxima)早熟株接种量,分别于4日龄对免疫攻毒组以不同剂量进行首免,11日龄以首免2倍剂量对二次免疫攻毒组鸡进行二免。一次或二次免疫后7 d或10 d用同源亲本株进行攻毒,检测免疫攻毒鸡相对增重率、卵囊减少率、病变记分减少率(RLS)和抗球虫指数(ACI)。结果显示,一次免疫剂量≥400个孢子化卵囊/只或两次免疫的一免剂量和二免剂量≥100个孢子化卵囊/只和200个孢子化卵囊/只时,各免疫攻毒鸡的相对增重率≥85%,RLS≥70%,卵囊减少率≥75%,相对保护率≥90%,ACI≥170,均达到良好免疫效果。故将E.maxima早熟株的一次免疫最小免疫剂量定为400个孢子化卵囊/只,两次免疫最小免疫剂量定为一免100个孢子化卵囊/只和二免200个孢子化卵囊/只。     

柔嫩艾美耳球虫Serpin基因的原核表达及其免疫保护效果研究

为评价Serpin基因重组表达产物对预防鸡柔嫩艾美耳球虫(Eimeria tenella,E.tenella)感染的效果,克隆和表达E.tenella Serpin基因,对表达产物进行初步纯化和复性,制备免疫原.设计了重组蛋白肌肉注射组、重组蛋白口服组、重组蛋白滴鼻组3个试验组以及红、白对照组.分别于7、14和28日龄对雏鸡进行3次免疫,35日龄时用3×104个E.tenella孢子化卵囊攻虫,第7天末宰杀,对各组的存活率、相对增重率、卵囊减少率、ACI等指标进行统计分析.结果表明,各免疫组的平均增重与红对照组相比均有显著增加(P＜0.05),其中以重组蛋白肌肉注射组增重最多,相对增重率为66.73％,优于其他免疫组;各免疫组卵囊产量较红对照组均有显著的下降,其中重组蛋白肌肉注射组卵囊减少率最高,为30.01％;各免疫组ACI均明显高于红对照组,其中以重组蛋白口服组最高,但仍低于160,暗示Serpin蛋白并不是一个理想的球虫疫苗保护性抗原.     

巨型艾美球虫重组蛋白Gam82-Y和重组质粒pcDNA-gam82的免疫保护效果研究

为研究巨型艾美球虫NT株重组蛋白Gam82-Y和重组质粒pcDNA-gam82对鸡的免疫保护效果,本研究以平均增重、相对增重率及卵囊减少率等作为评价指标,检测结果显示:中剂量重组蛋白佐剂组(0.5 mg-FCA)和中剂量重组蛋白组(0.5 mg)的免疫保护力均低于卵囊免疫组(p＜0.05),但比其他各免疫组高(p＜0.05);重组质粒(pcDNA-gam82)免疫组的平均增重、相对增重率、卵囊减少率等方面与重组蛋白免疫组相同,两者均优于未免疫攻虫组(p＜0.05),但仍未能够达到卵囊免疫组的免疫保护水平(p＜0.05).     

鸡球虫重组蛋白rEnApiAP2对鸡免疫保护效果的观察

将毒害艾美耳球虫重组蛋白rEnApiAP2单独免疫(单免)或分别与柔嫩艾美耳球虫重组蛋白rEtGAM56或rEtGAM59联合免疫(联免)雏鸡,同时设rEtGAM56单免、rEtGAM59单免、未免疫攻虫和空白组为对照,分别攻毒害艾美耳球虫或柔嫩艾美耳球虫,以成活率、排出血便数、平均增重、相对增重率、病变记分、卵囊减少率、抗球虫指数(ACI)和血清抗体水平为指标,评价rEnApiAP2单免及与rEtGAM联免的免疫保护效果。结果显示：各试验组的成活率均为100%;与未免疫攻虫组比较,各免疫组鸡排血便堆数减少、平均增重增加。在rEnApiAP2单免组间,低剂量组的相对增重率(82.36%)、卵囊减少率(73.88%)和抗球虫指数(155.26)均为最高,平均病变记分最低(1.71)。攻毒害艾美耳球虫后,联免组的ACI值均大于相应的单免组,其中rEnApiAP2与rEtGAM56联免组的ACI值(169.83)最高。攻柔嫩艾美耳球虫后,rEnApiAP2单免组的ACI值(128.37)低于rEtGAM56、rEtGAM59单免组(130.12、151.88),rEnApiAP2与rEtGA...     

EtMIC-2表达产物对鸡三种艾美耳球虫的保护试验

鸡艾美耳球虫病由隶属顶复门7种艾美耳球虫（E.tenella、Enecatrix、E.acervulina、E.naxima、E.brunetti、E.praecox、E.mitis）引起，常混合感染，有很高的发病率和死亡率，轻者则影响鸡的增重和饲料转化率。微线是顶器的结构之一，位于子孢子和裂殖子前端，EtMIC-2是其50kDa左右的酸性蛋白，在虫体入侵时表达，该蛋白可能在虫体入侵时起重要作用。用Pet载体经原核表达该蛋白，7、14日龄经口服、肌注不同剂量工程活菌和菌体裂解蛋白二次免疫鸡，免疫后1周用3种艾美耳球虫（E.tenella、E.acervulina、E.maxima）攻虫，发现相对增重率随菌体蛋白含量增加呈现增加趋势。E.tenella攻虫表现最有效的是口服PBS200ug活菌组为96%，E.ac-ervulina、E.maxima分别为96%、87%。从攻虫后7～14d卵囊排出曲线看，每天排卵囊数和相对卵囊产量均低于红对照；相对卵囊产量,E.tenella攻虫后，口服PBS 200ug活菌组最低，为17%，E.acervulina、E.maxima攻虫后，口服PBS 200ug裂解菌组最高，分别为90%、93%。从增重与卵囊产量来看，EtMIC-2对E.tenella、E.ac-ervulina和E.maxima这3种球虫有一定的免疫保护作用，尤其口服200ugPBS活菌时，对E.tenella保护作用最明显。说明来源于E.tenella子孢子的Etmic-2基因的表达产物对E.tenella、E.acervulina和E.maxima3种球虫有一定的免疫保护作用。     

表达柔嫩艾美耳球虫HMGB1基因质粒DNA的免疫效果分析

旨在评价表达柔嫩艾美耳球虫(E.tenella)HMGB1基因质粒DNA免疫对同源株攻虫的免疫保护效果。将EtHMGB1基因插入pcDNA3.1(-)/myc-His A真核表达载体中,并在Hela细胞中进行体外瞬时表达。设计了pcDNA3.1(-)/myc-EtHMGB1免疫组、pcDNA3.1(-)/myc-His A免疫组、重组蛋白质+FCA佐剂免疫组、pcDNA3.1(-)/myc-EtHMGB1+重组蛋白质联合免疫组、FCA佐剂免疫组、未免疫攻虫组和未免疫未攻虫组7个试验组。分别于14和21日龄对鸡进行两次免疫,28日龄时除未免疫未攻虫组外各组用1×104个E.tenella孢子化卵囊攻虫,以平均增重、卵囊产量和病变记分作为免疫保护效果的评价指标。结果显示,成功构建了pcDNA3.1(-)/myc-EtHMGB1重组质粒,转染后该质粒可在Hela细胞中进行瞬时表达。该重组质粒单独免疫、重组蛋白质单独免疫或重组质粒与重组蛋白质联合免疫均对鸡肠道病变改善效果不明显,但可显著提高增重,降低卵囊产量,尤以重组质粒与重组蛋白质联合免疫效果最佳,显示Prime-boost免疫策略可提高该重组质粒的免疫保护效果。上述结果显示HMGB1可作为未来球虫疫苗研制的良好候选抗原之一。     

巨型艾美耳球虫不同地理株交叉免疫保护性研究

筛选出与巨型艾美耳球虫(E.maxima)山东分离株(Msd)免疫原性差异大的早熟弱毒株,为研制具有良好免疫原性的高效多价鸡球虫疫苗提供理论依据。4日龄雏鸡免疫E.maxima山东分离早熟株(PMsd),免疫后7 d分别攻击E.maxima Msd、上海分离株(Msh)、北京分离株(Mbj)、山西分离株(Msx);14日龄雏鸡分别免疫E.maxima Mbj和Msx,免疫后7 d,Mbj免疫组分别攻击Msh和Msx,Msx免疫组分别攻击Msh和Mbj。免疫PMsd攻击Msd组鸡只达到良好的免疫保护,而免疫PMsd攻击Msx组鸡只除病变记分减少率(28.6%)高于Mbj组(23.8%)外,相对增重率(74.3%)、卵囊减少率(9.7%)、抗球虫指数(159.3)均低于其他各组;免疫Msx攻击Mbj和Msh组鸡只卵囊减少率、抗球虫指数、相对增重率均大于免疫Mbj攻击Msx和Msh组。4种不同地理株的巨型艾美耳球虫中,Msx与Msd的免疫原性差异最大,且较Mbj更为广谱。     

3株鸡巨型艾美耳球虫抗原性分析

本研究对3株鸡巨型艾美耳球虫(Eimeria maxima)福建株、上海株和广东株进行免疫原性分析,其中广东株属于早熟株。将7日龄雏鸡分别免疫3株E.maxima孢子化卵囊,12 d后分别攻击相同剂量的同源及异源虫株孢子化卵囊,设不免疫攻虫对照组和不免疫不攻虫对照组,17d开始收集粪便直至无卵囊排出为止,每日计数卵囊,计算卵囊相对减少率。结果:雏鸡免疫后以同源虫株攻击,保护率均在98%以上,而雏鸡免疫后以异源虫株攻击,其交叉保护率在40.78%⁷2.37%之间。提示:E.maxima对同源虫株的保护率明显高于对异源虫株的保护率,说明各分离株间抗原性存在差异。     

柔嫩艾美耳球虫杨凌株3-1E基因表达产物的初步免疫效果研究

用柔嫩艾美耳球虫(Eimeria tenella子孢子表面抗原基因原核细胞表达产物免疫雏鸡,观察其对球虫攻击的免疫保护作用.将可溶性重组蛋白和包涵体重组蛋白经肌肉注射分别于7日龄、14日龄两次免疫罗曼小公雏,同时设攻虫对照组和空白对照组,于第2次免疫后1周(28日龄)用1 ×104个E.tenella卵囊进行攻毒,观察其诱导产生的保护力.结果表明,3-1E可溶性蛋白组免疫无论从卵囊计数、盲肠病变计分和相对增重均优于包涵体免疫组.说明3-1E基因在大肠杆菌中的表达产物对鸡柔嫩艾美耳球虫的攻击有一定的免疫保护作用.     

Comparison of disease susceptibility and subclass-specific antibody response in SC and FP chickens experimentally inoculated with Eimeria tenella, E. acervulina, or E. maxima

Susceptibility to disease and the subclass-specific antibody response to Eimeria tenella, E. acervulina, and E. maxima were compared in two inbred strains of chickens, FP (B15B21) and SC (B2B2). FP strain was more susceptible to coccidiosis than SC chickens based on oocyst production, lesion score, and clinical signs. FP chickens infected with E. tenella had more severe cecal lesions and a significantly lower hematocrit level than SC chickens. FP chickens infected with E. acervulina excreted five times as many oocysts at 6 days postinfection as SC and showed a 71% reduction in plasma carotenoid level compared with controls (56% reduction in SC chickens). Body-weight change did not correlate with other signs of disease. Both SC and FP chickens produced high levels of serum IgM and IgG and biliary IgA. Although SC chickens had a slightly higher antibody response than FP chickens at 7 days postinoculation, both strains maintained high levels of IgM, IgG, and IgA for a prolonged period post primary inoculation. Although SC and FP chickens show different disease susceptibility to coccidiosis, they demonstrate similar antibody response.     

Coccidiosis immunization: effects of mushroom and herb polysaccharides on immune responses of chickens infected with Eimeria tenella

An experiment was conducted to investigate the effects of polysaccharide extracts (E) of two mushrooms, Lentinus edodes (LenE) and Tremella fuciformis (TreE), and an herb, Astragalus membranaceus (AstE), on the immune responses of chickens infected with Eimeria tenella. A total of 180 broiler chickens were assigned to nine groups: three groups were fed with each of the extracts (LenE, TreE, and AstE), three groups were fed with the extracts and immunized with live oocyst vaccine (LenE+V, TreE+V, and AstE+V), a group was immunized with the vaccine only, and there were two controls (E. tenella-infected and noninfected groups). The oocyst vaccine was given at 4 days of age, and the extracts (1 g/kg of the diet) were supplemented from 8 to 14 days of age. At 18 days of age, all birds except those in the noninfected group were infected with 9 x 10(4) sporulated oocysts. The results showed that at 7 days postinfection (p.i.), birds fed the extracts without vaccination had lower body weight (BW) gain than those given the vaccine only. However, the extracts in conjunction with the vaccine significantly enhanced BW gain of the infected chickens compared with the vaccine group. Of the three extracts, LenE and TreE showed a better growth-promoting effect. The extracts largely increased oocyst excretion of droppings during the primary response postvaccination. The cecal peak oocyst output and lesion scores measured at 7 days p.i. were higher in the groups fed the extracts than in the group immunized with the vaccine only, whereas those of the groups fed with the extracts and immunized with the vaccine were not significantly different from the vaccine group. Of the three extracts, both LenE- and AstE-fed groups showed lower cecal oocyst output. Thus, as compared with the extracts, the live, attenuated vaccine showed better results with significantly increased immune response in coccidial infected birds. The polysaccharide extracts may prove useful against avian coccidiosis, and, particularly when they are used in conjunction with vaccine, they have shown preliminary promise against the experimental coccidial infection.     

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.     

Resistance to intestinal coccidiosis following DNA immunization with the cloned 3-1E Eimeria gene plus IL-2, IL-15, and IFN-gamma

A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-gamma. Vaccination efficacy was assessed by increased serum anti-3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina. When used alone, anti-3-1E antibody titers were transiently, but reproducibly, increased at 2 wk and 3 wk posthatching in a dose-dependent manner. Similarly, significantly reduced oocyst shedding and increased weight gain were observed at relatively high-dose 3-1E vaccinations (> or =25 microg/egg). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-gamma genes induced higher serum antibody responses compared with immunization with 3-1E alone. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-gamma significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis.     

Eimeria praecox infection ameliorates effects of Eimeria maxima infection in chickens

The effect of Eimeria praecox on concurrent Eimeria maxima infection was studied in susceptible chickens. Clinical signs of coccidiosis were assessed in single E. praecox or E. maxima infections and compared to dual infection with both Eimeria species. Groups infected solely with 10(4)E. maxima oocysts displayed weight gains that were 48% of weight gain in uninfected controls. Weight gain in chickens infected only with 10(4)E. praecox oocysts was 90% of uninfected controls. Average weight gain in chickens infected with both E. maxima and E. praecox was 79% of controls, and showed no significant difference (P>0.05) from weight gain in E. praecox-infected chickens. Feed utilization (feed conversion ratio, FCR) in chickens infected with both species showed no significant difference (P>0.05) from FCR in non-infected controls or chickens infected with E. praecox alone; all showing a significant difference (P<0.05) from FCR in chickens infected solely with E. maxima. Although E. praecox did not appear to have a negative effect on weight gain and FCR, it did cause a significant decrease in serum carotenoids. Analysis of oocysts excreted by chickens during dual infection showed little effect of E. praecox on E. maxima oocyst production.     

Immunization of young chickens by trickle infection with Eimeria tenella.

Immunization of chickens was attempted with low levels of Eimeria tenella oocysts (50 oocysts per day) over the first 1 or 2 weeks of life--the "trickle infection" (TI) method. When chickens were immunized by TI at 0-13 days of age, no cecal lesions and a reduced number of oocysts in ceca were observed after challenge at 17 days of age. TI at 0-6 days of age conferred better protection against challenge with E. tenella than did TI at 7-13 days. However, cecal lesions were observed in almost all of these chickens. These findings indicated that TI for 2 weeks (0-13 days of age) provided better immunity than TI for 1 week (0-6 or 7-13 days of age).     

Effect of ibuprofen on coccidiosis in broiler chickens

Ibuprofen (IBU)-a nonsteroidal anti-inflammatory drug-inhibits the biosynthesis of prostaglandins with pro-inflammatory and immunosuppressive properties and is therefore proposed as a candidate molecule for the treatment of coccidiosis in broiler chickens. In all experiments, IBU was administered via drinking water. In a first experiment, chickens were infected at 10 or 21 days of age with oocysts of Eimeria acervulina (5 X 10(4)), Eimeria maxima (3 X 10(4)), and Eimeria tenella (7.5 X 10(3)) and medicated with IBU at a dose of 15 mg/kg body weight (BW). In a second experiment, chickens were infected at 6 days of age with 10(4) oocysts of E. acervulina and medicated with IBU at a dose of 100 mg/kg BW. In the third experiment, an inoculum consisting of 5 x 10(4) or 10(5) E. acervulina oocysts was administered at 6 days of age to chickens medicated with IBU at a dose of 100 mg/kg BW. In a fourth experiment, the effect of IBU on sporulation and infectivity of E. acervulina oocysts was studied. Coccidial lesion scores (CLSs), oocyst shedding, and weight gain were used as evaluation parameters in all experiments except the fourth, where weight gain was not taken into account. In addition, the sporulation percentage was determined in the last experiment. No influence of IBU on the indicated parameters was observed after providing the drug at a dose of 15 mg/kg BW, whereas CLSs and oocyst shedding were reduced when IBU was provided at a dose of 100 mg/kg BW. However, IBU did not significantly show any effect on the degree of sporulation and infectivity of E. acervulina oocysts at a dose of 100 mg/kg BW.     

11株巨型艾美耳球虫繁殖力的比较研究

用巨型艾美耳球虫扬州株、南通株、连云港株、苏州株、凤阳株、青岛株、福州株、龙岩株、广州株、上海株和美国株孢子化卵囊100个/只或10 000 个/只,经嗉囔接种7 日龄或21 日龄无球虫黄羽肉公雏,以麦克马斯特法对感染后第6 天至第14 天间每天24 h排出的卵囊计数并进行统计分析。结果表明,各虫株的排卵囊量有差异,100个/只卵囊感染7日龄雏鸡时,广州株和凤阳株排卵囊量最多,上海株和苏州株排卵囊量最少;100个/只卵囊感染21 日龄雏鸡时,扬州株排卵囊量最多,上海株排卵囊量最少;10 000 个/只卵囊感染21 日龄雏鸡时,苏州株排卵囊量最多,连云港株和扬州株的排卵囊量最少;排卵囊量随着感染剂量的增加或鸡日龄的增大而增多;在同一感染剂量时,福州株和连云港株增加最多,上海株和苏州株增加最少;在同一感染鸡日龄时,苏州株和上海株增加最多,南通株和扬州株增加最少;各虫株的排卵囊规律极相似,在感染后第7 天排卵囊量达峰值,第8 天、第9 天排出的卵囊量显著减少,第10天排出的卵囊量仅占总量的2%,第14天接近于0%;第6天至第8天排出的卵囊量占总量的87%~99%。