Column chromatographic characterization of cytoplasmic proteins in Eimeria maxima oocysts from chickens

Cytoplasmic proteins from unsporulated and sporulated Eimeria maxima oocysts were analyzed by gel-filtration column chromatography. Unsporulated oocysts were characterized as having 3 major cytoplasmic proteins and sporulated oocysts as having 5 major cytoplasmic proteins. Molecular weights ranged from 5 x 10(3) to 1.4 x 10(6). Larger molecular weight proteins were detected in sporulated and unsporulated oocysts, but were associated more with sporocysts of sporulated oocysts.     

柔嫩艾美耳球虫Etron2基因在不同发育阶段转录水平差异的分析

为研究柔嫩艾美耳球虫不同发育时期Etron2基因的转录水平差异,选取E.tenella生活史中的未孢子化卵囊、孢子化卵囊、子孢子、第二代裂殖子,分别提取总RNA,反转录为cDNA;选择Etactin作为参照基因,Etron2为目的基因,设计实时荧光定量PCR的引物,分析在E.tenella生活史中不同发育阶段Etron2转录水平的差异。结果显示Etron2在未孢子化卵囊中转录水平最高,然后分别是裂殖子、孢子化卵囊和子孢子。在柔嫩艾美耳球虫发育过程中,Etron2基因可能在未孢子化卵囊阶段被转录储备,随着宿主细胞的刺激,得以大量表达,在入侵过程中发挥作用。     

球虫消毒剂的筛选及氨水对艾美耳球虫卵囊抑杀条件的优化

为了筛选有效的球虫消毒剂,本研究将柔嫩艾美耳球虫卵囊分别与16种常用消毒剂和1种高效抗球虫药物溶液（250 mg/L妥曲珠利溶液）混合作用2 h然后在2.5%重铬酸钾溶液中孢子化培养,或混合作用4 h后接种14日龄雏鸡,根据卵囊孢子化率、孢子化抑制率、每克粪便卵囊数量（oocysts per gram of feces,OPG）、病变计分和血便情况评价消毒剂和抗球虫药物抑杀球虫卵囊效果。结果显示,8%氨水对未孢子化卵囊和孢子化卵囊均表现为100%的抑杀效果;250 mg/L妥曲珠利、3%甲醛、4%苯酚、0.5%过氧乙酸、5% NaOH+10% NaCl和5% NaOH对球虫卵囊活性具有一定的抑制作用,孢子化抑制率超过10%,但是对孢子化卵囊抑杀作用不明显;而剩余10种常用消毒剂对球虫卵囊没有明显的抑杀作用。氨水抑杀球虫卵囊条件优化的试验结果显示,1%~8%氨水与未孢子化卵囊作用1 h以上,均可100%抑杀球虫卵囊活性。粪便干扰试验结果显示,粪便不影响2%和4%氨水对球虫卵囊的抑杀效果。本研究结果说明常用消毒剂和抗球虫药物不能有效杀灭球虫卵囊的活性,而氨水是一种高效球虫消毒剂。本研究结果可为开发新型高效球虫消毒剂和球虫病控制提供参考。     

柔嫩艾美耳球虫表面抗原（EtSAG）基因的分离及鉴定

利用本实验室前期获得的柔嫩艾美耳球虫（Eimeria tenella）孢子化卵囊和未孢子化卵囊差异表达ESTs序列,选取编号为BW4-C03的孢子化卵囊,采用RACE技术,获得该基因全长序列。经BLAST分析,该序列与柔嫩艾美耳球虫表面抗原有72%以上的同源性,命名为EtSAG。利用荧光定量PCR（Real-time PCR）检测发现该基因在孢子化卵囊的转录拷贝数最高,且随着孢子化时间的延长,转录拷贝数逐渐增加。采用原核表达载体pET-28C表达该基因,得到的融合蛋白大小约为36 kDa,符合预期大小。经Western blot分析,该重组蛋白可被兔抗柔嫩艾美耳球虫的多克隆抗血清识别,表明该蛋白具有较好的反应原性。本研究结果为进一步研究该基因的生物学功能奠定了基础。     

Differential Proteomic Analysis between Sporulated Oocyst and Unsporulated Oocyst of Cystoisospora suis

The differentially expressed proteins between sporulated oocyst and unsporulated oocyst of Cystoisospora suis strains were screened and compared. In this study, the C. suis infection model of piglets were established and a quantitative proteomic analysis between sporulated oocyst and unsporulated oocyst of C. suis were conducted by iTRAQ labeling and LC-MS/MS. Part of the iTRAQ results were validated by qRT-PCR. Total 174 proteins were identified, among which 40 proteins were differentially expressed, 38 proteins were upregulated and 2 proteins were downregulated. The differentially expressed proteins were mainly involved in metabolism, antibiotics biosynthesis, secondary metabolites biosynthesis and glycolysis/gluconeogenesis pathways. The results of this study provide reference for elucidating the developmental mechanism of C. suis sporulated oocyst and facilitated discovering a new biomarker for C. suis.     

猪囊等孢球虫孢子化与未孢子化卵囊的差异表达蛋白质组学分析

通过比较猪囊等孢球虫孢子化卵囊与未孢子化卵囊的差异表达蛋白质,以期从蛋白质组学角度研究其发育机制,并为此类球虫的发育机制及该病的免疫预防或化学防治找到新的“关键”分子奠定基础。首先建立哺乳仔猪的猪囊等孢球虫感染模型,然后运用iTRAQ技术,结合LC-MS/MS分析差异表达蛋白质,并采用荧光定量PCR验证iTRAQ数据。结果共鉴定出174个蛋白,差异表达蛋白40个,其中38个蛋白上调表达,2个蛋白下调表达,差异蛋白主要涉及代谢、抗生素的生物合成、次生代谢产物的生物合成和糖酵解/糖异生过程等通路。研究结果为阐明猪囊等孢球虫孢子化发育机制以及发现新的生物标志物提供参考。     

鸡柔嫩艾美耳球虫SO7基因的原核表达及动物免疫试验

为了研究鸡柔嫩艾美耳球虫SO7基因原核表达重组蛋白的免疫效力,分析其在鸡抗柔嫩艾美耳球虫感染中对鸡平均增重、抗球虫指数(ACI)、减少盲肠病变和卵囊数量中的作用,从柔嫩艾美耳球虫孢子化卵囊中提取总RNA作为模板,用RT-PCR扩增出SO7基因片段,再应用DNA重组技术将SO7基因克隆到原核表达载体pET32a(+)中并用IPTG诱导表达。SDS-PAGE结果显示,pET32a(+)-SO7表达的目的蛋白约为40ku,主要以包涵体形式存在。用纯化的重组蛋白进行动物免疫试验显示,SO7重组蛋白在鸡抗柔嫩艾美耳球虫感染中有较好的免疫保护作用,能够显著提高鸡柔嫩艾美耳球虫感染鸡的平均增重和抗球虫指数(ACI),对减少盲肠病变和卵囊数量也有部分作用。     

The prepatent and patent periods of Eimeria alabamensis and further description of the exogenous stages

Eimeria alabamensis infections were established in calves 5 to 6 weeks of age by adminestering 10 million, 80 million, or 100 million sporulated oocysts. The prepatent period was 6 to 8 days (mean 6.6). Oocyst discharges usually lasted for 2 to 3 days although a few calves passed oocysts throughout the rest of the 3-week observation period. Calves with oocyst discharge exceeding 1 million oocysts per g of feces had a moderate diarrhea at the time of peak oocyst discharge. No other clinical signs were observed in any of the infected calves. Reinoculations with 100 million sporulated oocysts given 3 weeks after the initial inoculations of 10 million or 80 million oocysts resulted in infections characterized by greatly reduced oocyst discharges. Sporulated oocysts of E. alabamensis were 16 to 24 μ by 12 to 16 μ and were usually ovoid. The oocyst wals consisted of two layers. Sporocysts were elongate-ellipsoid, had a distinct Stieda body, and were 10 to 12 μ by 4 to 6 μ. Completely sporulated oocysts were first observed after 5 days at 25 °C, and most were sporulated after 8 days. Oocysts did not sporulate at 4 °C, 33 °C, and 37 °C.     

Interaction of low-pathogenicity reoviruses and low levels of infection with several coccidial species

Various combinations of reoviruses and coccidia were studied to see if interactions would occur. Two reoviruses were used: virus 2035, a moderate to low pathogen, and virus 2177, a nonpathogen. Coccidia used were Eimeria acervulina, E. mitis, and E. maxima at dosages of 10(3) or 10(4) sporulated oocysts/chick and E. brunetti at 10(4) sporulated oocysts/chick. In Hubbard-Hubbard cockerels, a combination of virus 2035 and E. acervulina (10(4) oocysts/chick) or E. maxima (10(3) oocysts/chick) significantly (P less than or equal to 0.05) increased the frequency of stunting (% of chicks with body weight less than 80% of controls) and further depressed weight gain over that seen with either virus or coccidia alone. Conversely, virus 2177 ameliorated the same effects in Shaver-Arbor Acre cockerels given 10(4) oocysts/chick of E. mitis or E. maxima. The interaction could not be attributed to changes in the degree of coccidial infection based on oocyst production. Reovirus did not generally change the effect of coccidia on levels of plasma pigment and plasma protein. In Hubbard-Hubbard cockerels, coccidia-induced effects were not ameliorated by virus 2177, suggesting that breed difference in interaction can be expected.     

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。     

巨型艾美球虫gam56基因的截短原核表达及其产物的免疫保护效果

为评价gain56基因重组表达产物作为亚单位疫苗预防鸡巨型艾美球虫(E.maxima),感染的效果,以E.maxima NT株配子体总RNA为模板,RT.PCR扩增和克隆gain56基因,选择该基因两段丰富抗原表位的编码区片段.利用原核表达载体pGEX-6P-1在大肠杆菌中进行截短表达.以可溶性的GST-gain56-2融合蛋白为免疫原,设立高、中、低(1.0 mg、0.5 mg、0.25 mg)3个剂量,单独或使用弗氏完全佐剂,于5 d、12 d和19 d对雏鸡进行3次免疫,26 d用5 ×10~4个E.maxima孢子化卵囊进行攻虫,8 d后迫杀,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.结果显示:就相对增重率而言各免疫组比未免疫攻毒组的高27.6%以上,而各免疫组之间差异不显著(p>0.05);就卵囊减少率指标而言,各免疫组较未免疫攻毒组均有不同程度减少,但是均小于75%;就ACI指标而言,各免疫组较未免疫攻毒组均有不同程度增加,以中剂量蛋白佐剂组为最高.GST-gam56-2融合蛋白对于E.maxima感染具有一定的免疫保护效力.     

Inflammation-induced changes in serum modulate chicken macrophage function

Inflammation-induced changes in serum protein profiles and the effects of such serum on a chicken macrophage cell line HD11 were studied to find whether the changes in serum affect cellular immunity. Four-week-old male broiler chickens were injected subcutaneously with either olive oil or 50% croton oil mixed in olive oil to induce inflammation. The birds were bled at 48 h after injection, and serum protein profiles were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric evaluation. At 48 h post-injection the serum from croton oil-injected birds showed distinct changes in protein profiles characterized by a selective increase or decrease in levels of several serum proteins. The protein bands which showed increases had relative molecular weights (Mr) corresponding to 65 kilo Daltons (kD), 42 kD, and two or more proteins with Mr≥200 kD. The levels of serum albumin (49 kD), and a 56 kD protein were reduced in croton oil-injected birds. The modulating effects of such serum on HD11 cells were studied using bacterial lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) induced functional activation of these cells. The LPS-induced interleukin-6 (IL-6) production by HD11 cells was not affected by the presence of either olive oil-treated control or croton oil-treated inflammatory serum but nitrite production was enhanced by the inflammatory serum. Similarly, inflammatory serum also enhanced PMA-induced respiratory burst measured using dichlorofluorescein diacetate (DCF-DA) oxidation mediated by reactive oxygen intermediates. These results suggest that inflammatory serum can modulate macrophage function by influencing the production of reactive oxygen and nitrogen species which could affect their phagocytic and bactericidal activities.     

Demonstration of antibodies to Eimeria species in lambs by an enzyme-linked immunosorbent assay

Soluble extracts were prepared from sporulated oocysts, unsporulated oocysts and merozoites of Eimeria crandallis, E faurei and E ovinoidalis. They were assessed for antigenicity and specificity by ELISA using rabbit antisera to sporulated oocysts or merozoites. Antibody levels were examined in sera from colostrum-deprived coccidia-free lambs, conventionally reared lambs and lambs which had received experimental infections. Maternal antibody was demonstrated in colostrum and in serum taken at 24 hours from all conventionally reared animals but not colostrum-deprived animals. Antibody levels in conventional animals dropped over the first five weeks of life and rose again during the next five weeks. Antibody was not detected in coccidia-free animals. Monospecific infections of E faurei or E ovinoidalis demonstrated antibody responses to primary and secondary infections. Some specificity of response was suggested with E faurei infection. The antigen preparations showed considerable cross-reactions between species. These serum antibody responses, although appearing too late for individual diagnosis, may assist diagnosis on a flock basis.     

Anticoccidial effect of green tea-based diets against Eimeria maxima

Anticoccidial effects of green tea (GT)-based diets were evaluated in chickens following oral infection with Eimeria maxima an ubiquitous intestinal parasite of poultry that impairs the growth and feed efficiency of infected birds. Five-week-old chickens were assigned to four groups (GT 0.5%, GT 2.0%, untreated/infected and non-infected control) and each group consisted of 15 chickens. Chickens were fed a standard diet supplemented with ground green tea for 2 weeks prior to infection with E. maxima (10,000 sporulated oocysts per bird). The effects of green tea on E. maxima infection were assessed by two parameters, fecal oocyst shedding and body weight gain. The green tea-fed chickens produced significantly reduced fecal oocysts (P<0.05) when compared to the E. maxima-infected group fed standard diet. The green tea-based diet, however, did not improve body weight loss caused by E. maxima infection. This study is the first to demonstrate anticoccidial effect of green tea on Eimeria parasites.     

Experimental infections with coccidiosis and serum antibody quantitation in two breeds of goats

Two goat breeds, Anglonubian and Saanen were obtained at an early age and reared coccidia-free until they were 1 month old. They were then infected with 200,000 sporulated oocysts comprising mainly Eimeria christenseni (49%), E. apsheronica (29%) and lesser proportions of E. arloingi, E. hirci, E. ninakohlyakimovae and E. alijevi. Following patency, the number of faecal oocysts per gram were determined for each animal and body weights taken weekly. An ELISA was developed to assay antibody titres in pre- and post-infection sera of each individual kid. Cleaned unsporulated oocysts were sonified and the resulting soluble antigen used. Anglonubians shed significantly more oocysts daily (P less than 0.01) and also suffered greater retardation in growth. Initially both breeds had very similar growth rates, however post-infection weekly body weight gain among the Anglonubians was on average 1100 g compared to 1300 g for the Saanens. Antibody titres post-infection increased significantly among the Saanens (P less than 0.01) and also all but one among the Anglonubians recorded an increase in antibody titres. Unsuckled kid serum was devoid of any antibody activity.     

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.     

The sporulation time of Isospora suis oocysts from different sources

Feces containing Isospora suis oocysts were collected from naturally- and experimentally-infected pigs from four different areas of the United States. The unsporulated oocysts were cleaned, concentrated, mixed with 2.5% aqueous potassium dichromate solution, poured into petri dishes to a depth of 5 mm, and incubated at 25 degrees C. The oocysts were examined with a microscope at 12 h intervals and the stages of sporulation present were counted. Although a few oocysts were completely sporulated after 12 h of incubation, in most fecal samples the majority of the oocysts were not completely sporulated until 24 or 36 h. In the present study, the sporulation time of I. suis oocysts was considered to be less than or equal to 48 h. There were no major differences in the sporulation times of I. suis oocysts from the different sources.     

家蚕微孢子虫(Nosema bombycis)交叉感染的孢子形态变化及表面蛋白差异

试验探讨了微孢子虫的交叉感染性与孢子表面蛋白的变化。结果表明,家蚕微孢子虫(Nosema bombycis)转主感染桑尺蠖(Hemerophila atrilineata)后孢子形态变为细长,分离纯化感染前和感染后的孢子表面蛋白,通过SDS-PAGE电泳发现形态变异株出现了34 kD和67 kD的差异带,在相同条带17、30、47、77和114 kD表达量存在明显的差异,但33 kD和43 kD条带表达量没有差异。孢子表面蛋白的差异性表达可能与微孢子虫感染适应性有关,而33 kD和43 kD可能是微孢子虫共有的保守蛋白。     

ELISA检测DLV虫苗免疫鸡抗体消长规律的研究

将柔嫩、毒害、巨型艾美耳球虫的孢子化卵囊制成可溶性抗原，用酶联免疫吸附试验检测鸡球虫双重致弱多凤苗虫苗、为柔嫩、毒害、巨型艾美耳球虫三价苗免疫鸡血清抗球虫抗体的消长规律。     

硝唑尼特抗免疫抑制小鼠隐孢子虫活性试验

研究硝唑尼特抗隐孢子虫的活性,采用地塞米松抑制昆明鼠免疫功能,经口接种感染微小隐孢子虫孢子化卵囊,成功制备出微小隐孢子虫感染的动物模型。将感染微小隐孢子虫的小鼠随机分为感染对照组以及高、中、低剂量3个药物剂量组,连续经口灌服硝唑尼特治疗1周,观察每只小鼠每天排出微小隐孢子虫卵囊的数量,以感染抑制率来评价硝唑尼特抗微小隐孢子虫的活性。结果表明,200 mg/(kg.d)剂量组能显著减少小鼠排出隐孢子虫卵囊的数量,对治疗微小隐孢子虫感染具有较好的疗效。