特异性高免卵黄抗体与鸡柔嫩艾美尔球虫致病关系的研究

为了探讨体液免疫在鸡球虫病中的作用,通过制备柔嫩艾美尔球虫(E.tenella)特异性高免卵黄抗体,检测卵黄抗体在鸡盲肠中的代谢规律,对E.tenella进行体内外抑制和杀灭实验,观察特异性高免卵黄抗体对E.tenella的作用。结果表明:(1)口服的卵黄抗体在鸡消化道较稳定;(2)口服卵黄液可使感染E.tenella鸡的死亡率降低33.34%,增重率和抗球虫指数分别提高39.19%和73.50;(3)特异性高免卵黄抗体对E.tenella的子孢子具有明显的溶解和致弱作用,但对卵囊的孢子化速率和孢子化球虫卵囊的致病性没有明显影响。     

E.tenella感染鸡CD4+、CD8+T细胞的动态变化研究

用免疫组化ABC法检测了柔嫩艾美耳球虫(E.tenella)感染雏鸡后各免疫器官和盲肠局部的T淋巴细胞亚群CD4 淋巴细胞和CD8 T淋巴细胞数的动态变化。结果表明:(1)雏鸡初次感染E.tenella后,免疫器官盲肠扁桃体、脾脏、胸腺和盲肠黏膜中的CD4 T淋巴细胞均于第2天开始增殖,第6天~8天达到峰值;二次感染后第2天有短暂的下降,第5天开始缓慢回升,第8天二次达到峰值,但第二个峰值比第一个峰值低,说明CD4 T淋巴细胞积极参与启动免疫应答和抵抗初次感染。(2)雏鸡初次感染E.tenella后,免疫器官法氏囊、盲肠扁桃体、脾脏、胸腺和盲肠黏膜中的CD8 T淋巴细胞都于第2天开始增殖,第8天达到峰值;二次感染后立即回升,第5天达到峰值,然后缓慢下降,且第二个峰值比第一个峰值高,表明CD8 T淋巴细胞是抵抗再感染的主力。     

金黄色葡萄球菌特异性卵黄抗体的制备及其影响因素

目的:考察金黄色葡萄球菌特异性卵黄抗体制备过程中的影响因素.方法:以灭活的金黄色葡萄球菌为抗原,采用不同的浓度(108cfu/mL,109cfu/mL,1010cfu/mL)、在不同免疫佐剂(商品弗氏佐剂和自制弗氏佐剂)作用下,对不同日龄(120天和300天)的蛋鸡进行免疫.免疫后收集鸡蛋,蛋黄用6倍体积水稀释,采用ELISA法测定抗体效价.结果:抗原浓度为109cfu/mL所得抗体效价最高;免疫120日龄蛋鸡获得抗体效价高于免疫300日龄蛋鸡所得抗体效价;在抗原浓度和蛋鸡日龄相同的情况下,商品弗氏佐剂比自制具有更好的免疫增强作用.结论:特异性卵黄抗体制备受多种因素的影响,抗原浓度、蛋鸡日龄、佐剂质量均对卵黄中特异性抗体水平有影响.     

柔嫩艾美耳球虫安徽肥西(AHFX)株的分离与鉴定

目的 分离并鉴定安徽肥西病鸡盲肠内的柔嫩艾美耳球虫.方法 通过雏鸡进行单卵囊接种与增殖试验,对所获卵囊用形态学方法进行初步鉴定;运用PCR方法扩增该分离株的ITS-1基因序列,并与相关序列进行比对、构建系统进化树.结果 该球虫分离株孢子化卵囊的平均大小为21.1 μm ×18.0 μm,卵形指数为1.17,潜隐期为140 h;其ITS-1基因序列与柔嫩艾美耳球虫(Eimeria tenella)GQ856310相似性达98.2％,二者的亲缘关系非常接近.结论 该球虫分离株为柔嫩艾美耳球虫(E.tenella),暂命名为柔嫩艾美耳球虫安徽肥西(AHFX)株.     

鸡卵黄抗眼镜王蛇毒抗体IgY的理化特性

目的 研制抗眼镜王蛇毒鸡卵黄抗体并研究该抗体的理化特性。方法 拟用减毒后的眼镜王蛇毒免疫母鸡后,从卵黄中制备抗眼镜王蛇毒抗体IgY。采用间接ELISA法对此抗体的理化特性进行研究。结果 ELISA显示免疫后12天开始出现特异性抗体,且抗体滴度逐渐升高,约在免疫后60天左右,最高可达1：100000,此最高滴度可维持30天,以后滴度逐渐降低,至免疫后100～110天,滴度仍可维持在最高滴度的一半(1：50000)。此抗体在10～65℃温度范围内活性保持稳定;在pH为4～12范围内,抗体活性稳定;此抗体经胰蛋白酶处理1h内,活性下降不明显,但1h以后活性迅速下降。结论从卵黄中制备抗眼镜王蛇毒抗体IgY,是可行的,稳定的。     

柔嫩艾美耳球虫(Eimeria tenella)早熟株与亲本株致病性和繁殖力的研究

通过对经15代选育的柔嫩艾美耳球虫(E. tenella)山西株的早熟株与其亲本株的繁殖力和致病性进行比较研究,证实早熟株的潜隐期比亲本株缩短21 h,繁殖力下降40%左右;对致病性的研究显示,早熟株感染后对鸡只增重、AC I的影响较小,对11日龄雏鸡的半数感染量和半数致死量较亲本株增大,肠道病变记分较亲本株下降。由此认为,该早熟株符合球虫早熟株的特性,可用于鸡球虫病早熟苗的制作。     

鸡Leptin基因正确性的免疫验证

①取20只240日龄的粤黄鸡种母鸡平分为处理组和对照组,分别在第3、31、63和84d对之接种鸡Leptin重组融合蛋白(处理组)或BSA(对照组)1mL/羽。每天记录产蛋率,每10天测定采食量,每两周称重,在试验的第1、31、53、78和91d采血。结果表明Leptin处理组的血浆抗Leptin抗体水平和Leptin水平在免疫后显著高于对照组(P<0.01);第3和4次免疫后处理组产蛋率比对照组显著低约50%(P<0.05);在第98d试验结束时,处理组腹脂量为(115.60±15.07)g,显著高于对照组[(77.98±5.70)g,P<0.05];试验期间处理组的采食量略低于对照组,增重则略高于对照组,但两者差异均不显著(P>0.05);处理组血浆总T3和总T4水平与对照组差异不显著(P>0.05)。②取12只50日龄的粤黄鸡后备小母鸡,平分成处理组和对照组,分别肌注含抗Leptin抗体的卵黄提取液和生理盐水6mL/只,处理组在2、4、6h的累积采食量显著高于对照组(P<0.05),但在8h的累积采食量与对照组差异不显著(P>0.05)。两个试验通过免疫重组鸡Lep tin融合蛋白,使鸡表现出Leptin功能的丧失,证明目前所克隆的鸡Leptin基因在家鸡中的确存在和其正确性。     

鸡血清与卵黄中抗中华眼镜蛇毒IgY动态变化研究

目的探索特异性IgY的产生和变化规律。方法用眼镜蛇毒原毒免疫产蛋母鸡,ELISA定期检测卵黄中的抗体效价变化,小鼠体外中和实验检测其生物活性。第1次免疫40周后,眼镜蛇毒攻击已免疫母鸡,检测攻击前后鸡血清中抗体效价变化情况,未经眼镜蛇毒免疫的母鸡作阴性对照。结果经免疫后第7天蛋黄中即可检测到抗体,经多次加强免疫,40周时蛋黄中还能保持高效价的抗体,通过分离纯化,此抗体可保护实验小鼠免受4 LD50眼镜蛇毒的攻击;同时,鸡血清中也保留着较高效价的抗体,可中和4 LD50以上的眼镜蛇毒。结论用眼镜蛇毒免疫鸡,经多次加强免疫,卵黄和鸡血清中可持久保持高效价的特异性抗体,初步检测此抗体可中和4 LD50的蛇毒。     

柔嫩艾美耳球虫早熟系的选育及其生物学特性研究

选育制备弱毒疫苗所需的柔嫩艾美耳球虫早熟系并对其生物学特性进行研究。运用Jeffers建立的早熟系选育方法对柔嫩艾美耳球虫山西株（E．tenella SX010323）进行早熟选育,并对其内生发育、致病性、繁殖力、免疫原性、稳定性进行观察和检测。结果表明：经过海兰白雏鸡15次传代之后,E．tenella SX010323潜隐期由141h缩短至120h,卵囊明显缩小。选育得到的柔嫩艾美耳球虫山西株早熟系（E．tenella SX010323P15）其内生发育表现第二代裂殖生殖不完全;亲本株与早熟系对11日龄雏鸡的半数致死量分别为7．52&#215;10^4个／只、27．64&#215;10^4个／只,早熟系在致病性与繁殖力上均较母株大幅度降低,但保留了母株的免疫原性,经早熟系免疫的雏鸡可抵抗亲本株半数致死量的攻击。对选育的早熟系进行放松选择传代10次,其潜隐期、OPG、致病性均保持了早熟系的特征。提示通过早熟选育得到了遗传相对稳定的柔嫩艾美耳球虫早熟系。     

天蚕卵黄原蛋白的合成、运转与沉积

系统测定了天蚕Antheraea yamamai吐丝结茧至成虫期脂肪体、血淋巴和卵巢中卵黄蛋白和可溶性蛋白总含量的动态变化。结果表明,脂肪体是卵黄原蛋白(Vg)合成场所,Vg合成始于吐丝结茧后第4天;脂肪体、血淋巴中Vg滴度在吐丝结茧后第4天开始上升,化蛹后第6天或第8天达高峰,成虫羽化第1天则明显下降。卵巢对Vg摄取始于化蛹第1天,此后随蛹日龄逐渐上升,并渐趋平稳。同一卵巢管中卵黄蛋白(Vt)含量自顶端至基端随卵室增大而逐渐升高,不同日龄蛹中相应序号卵室的Vt含量以日龄大者为高;卵室中Vt含量与卵室体积大小呈正线性关系。电镜观察表明,Vg被卵母细胞摄入后以卵黄体形式存在,不同发育阶段卵巢中卵母细胞内卵黄体大小不同,以早期者为小;同一卵巢管中不同卵母细胞内卵黄体以顶端为小,基端明显增大,且卵黄体呈网状。     

Field Application of a Subunit Vaccine against an Enteric Protozoan Disease

Background

Coccidiosis is a major global veterinary health problem in intensively reared chickens. It is caused by apicomplexan parasites of the genus Eimeria.

Principal Findings

A subunit vaccine composed of purified antigens from the gametocytes of Eimeria maxima was used to stimulate the production and transfer of maternal antibodies between breeding hens and their hatchlings. The vaccine was injected into hens twice before they began laying eggs. Immunization had no adverse affects on egg laying or health of the hens and resulted in high antibody levels throughout the life of the hens. Progeny of immunized hens excreted significantly less oocysts of various species of Eimeria in their faeces than chicks from unvaccinated hens. Furthermore, the offspring of vaccinated hens developed stronger natural immunity to Eimeria, so that they were resistant to challenge infection even at 8 weeks of age, well after all maternal antibodies had left their circulation. Field trials were conducted in South Africa, Brazil and Thailand, involving at least 1 million progeny of vaccinated hens and at least 1 million positive control birds (raised on feed containing anticoccidial drugs or immunized with a live vaccine) in each country. Additionally, trials were carried out in Israel involving 60 million progeny of vaccinated hens and 112 million positive control birds. There were no significant differences in growth rate, feed conversion ratios or mortality in the offspring of vaccinated hens compared with the positive control chickens in any of these countries regardless of different management practices, different breeds of chickens or climate.

Conclusions

These results demonstrate that a vaccine composed of antigens purified from the gametocytes of Eimeria can be used safely and effectively to prevent the deleterious effects of coccidiosis. It is the first subunit vaccine against any protozoan parasite to be successfully applied on a commercial scale.     

Mouse monoclonal antibodies to chicken VH idiotypic determinants reactivity with B and T cells

We have prepared mouse monoclonal antibodies against idiotypic (Id) determinants on chicken antibodies to N-acetylglucosamine (NAGA) and p-aminobenzoic acid (PABA) made by inbred line EL 6(3) birds. The monoclonal anti-NAGA Id antibody, termed CId-1, reacted with affinity purified antibodies to NAGA, but not with antibodies specific for PABA, arsanilic acid (Ars), phosphorylcholine (PC), or with normal chicken IgG and IgM. The monoclonal anti-PABA ID antibody, termed CId-2, reacted with anti-PABA antibodies and to a lesser extent with anti-Ars antibodies, but not with anti-NAGA, anti-PC, and normal IgG and IgM. The Id determinants were found among antibodies to NAGA and PABA made by outbred and inbred lines of White Leghorn chickens. The binding of the CId-1 and CId-2 antibodies to intact homologous anti-NAGA and anti-PABA antibodies, respectively, was not hapten-inhibitable in either case. Both anti-Id antibodies reacted specifically with isolated homologous heavy chains, suggesting VH Id specificities. The monoclonal CId-1 and CId-2 antibodies were reactive by immunofluorescence with approximately 0.9 and 0.2%, respectively, of the circulating lymphocytes and with approximately 0.4 and 0.15 of plasma cells. CId-1+ and CId-2+ bursal cells were first detected on the 16th and 14th days of incubation, respectively; both reached maximal frequencies by the 17th day of incubation. The CId-2 antibody reacted exclusively with immunoglobulin-positive cells. The CId-1 antibody also reacted with a subpopulation (0.4%) of immunoglobulin-negative lymphocytes from normal and agammaglobulinemic chickens, and thus would appear to recognize an idiotypic determinant expressed by certain clones of B and T cells.     

富硒板党对老龄大鼠免疫功能的影响及其机制

目的：研究富硒板党（BCPA）注射液对老龄大鼠免疫功能的影响及其机制。方法：2月龄、12月龄和22月龄大鼠（雌雄各半）60只,分别作为年轻组、中年组、老龄组。每个年龄段大鼠随机分为对照组和BCPA实验组（n=10）。BCPA实验组每天腹腔按7．2g/kg注射BCPA,对照组注射等量生理盐水,常规饲养连续45d。BCPA给药后第15天,进行免疫注射。第22天,迟发型免疫应答（DTH）诱导。第15天、30天及45天尾静脉采集各年龄组大鼠血液,抗原特异性IgG、IgGl及IgG2α检测。第45天,取脾细胞分析主要T细胞亚群,并增殖脾细胞。结果：年轻、中年组BCPA实验组与对照组比较DTH反应均无显著差异,老龄BCPA实验组DTH反应则明显增强。特异性IgG、IgGl及IgG2α抗体年轻BCPA实验组与对照组比较无显著性差异,老龄BCPA实验组则明显增高（P〈0．05）。CD3＋T淋巴细胞、CD4＋辅助T淋巴细胞在年轻组中比较无显著差异,中年、老龄组中有显著提升（P〈0．05）。年轻BCPA实验组脾细胞有很强的增殖反应（P〈0．05）。结论：BCPA能增强老龄大鼠DTH反应,增强抗KLM特异性IgG、IgGl和IgG2α抗体的产生,可提高对抗原的反应性,提高CD4＋T细胞数量,促进免疫应答,在对抗免疫衰老,提高抗氧化能力方面有重要的作用。     

Prevalence, antigenic specificity, and bactericidal activity of poultry anti-Campylobacter maternal antibodies

Poultry are considered the major reservoir for Campylobacter jejuni, a leading bacterial cause of human food-borne diarrhea. To understand the ecology of C. jejuni and develop strategies to control C. jejuni infection in the animal reservoir, we initiated studies to examine the potential role of anti-Campylobacter maternal antibodies in protecting young broiler chickens from infection by C. jejuni. Using an enzyme-linked immunosorbent assay (ELISA), the prevalence of anti-C. jejuni antibodies in breeder chickens, egg yolks, and broilers from multiple flocks of different farms were examined. High levels of antibodies to the organism were detected in serum samples of breeder chickens and in egg yolk contents. To determine the dynamics of anti-Campylobacter maternal antibody transferred from yolks to hatchlings, serum samples collected from five broiler flocks at weekly intervals from 1 to 28 or 42 days of age were also examined by ELISA. Sera from the 1-day and 7-day-old chicks showed high titers of antibodies to C. jejuni. Thereafter, antibody titers decreased substantially and were not detected during the third and fourth weeks of age. The disappearance of anti-Campylobacter maternal antibodies during 3 to 4 weeks of age coincides with the appearance of C. jejuni infections observed in many broiler chicken flocks. As shown by immunoblotting, the maternally derived antibodies recognized multiple membrane proteins of C. jejuni ranging from 19 to 107 kDa. Moreover, in vitro serum bactericidal assays showed that anti-Campylobacter maternal antibodies were active in antibody-dependent complement-mediated killing of C. jejuni. Together, these results highlight the widespread presence of functional anti-Campylobacter antibodies in the poultry production system and provide a strong rationale for further investigation of the potential role of anti-C. jejuni maternal antibodies in protecting young chickens from infection by C. jejuni.     

Comparison of immunospecific antibody response in young and old chickens immunized with human IgG

Three groups of 18-month-old chickens and three groups of 5-month-old chickens were immunized with human immunoglobulin G (IgG) using one of three adjuvants in the first injection (Freund's Complete Adjuvant (FCA), Freunds Incomplete Adjuvant (FIA) and Hunter's TiterMax (HTM)) following the same immunization scheme. The specific antibody response in serum was measured by ELISA. In both older and younger chickens the serum antibody response in the FCA group reached a significantly higher level (P < 0.01) than in the FIA group and in the HTM group on week 5. The FCA group also had a significantly higher (P < 0.01) response on week 10 compared to the HTM group. Other than that, there was no significant difference between the three adjuvant groups in specific serum antibody response in older chickens. In the younger chickens the specific serum antibody response in the FCA group was significantly higher (P < 0.05) than the response in the HTM group. There was no significant difference in the chicken serum antibody response between the FCA and the FIA groups, nor was there a significant difference between the FIA and the HTM groups. Comparing the younger chickens and the older chickens immunized using the same adjuvant, the older chickens had consistently higher titres than the younger chickens, although the difference was not always significant.     

Clinical and Serological Response after Experimental Inoculation with Babesia Divergens of Newborn Calves with and without Maternal Antibodies

The influence of maternal antibodies on clinical and serological response after experimental inoculation with Babesia divergens of newborn calves was studied. Five calves, born to dams seropositive for B.divergens, (Group 1) had specific maternal antibodies when tested 12 h after their first feeding of colostrum. At that point they were inoculated i.v. with B.divergens infected erythrocytes. Five other calves, born to dams seronegative for B.divergens, (Group 2) had no Babesia specific maternal antibodies when inoculated at the same age. Babesia divergens organisms were demonstrated in blood smears from calves in both groups at some point 5 to 10 days p.i. All calves in both groups had B.divergens specific IgM antibodies at 7 to 17 days p.i. as shown by a modified IF-test. Specific IgG antibodies, transferred by colostrum, were found in all calves of Group 1 before inoculation of B.divergens. The IgG titre of these animals increased by a doubling dilution step at 11–25 days p.i. Among calves of Group 2 specific IgG antibodies were found at first between day 9 and 15 p.i. Both IgM and IgG antibody titres had to be investigated since demonstrated IgG antibodies can originate both from maternally transferred antibodies and from actively produced antibodies after an infection. There was no difference in clinical parameters; parasitaemia, PCV, Hb, and rectal temperature between the groups. This experiment gives evidence that there can be a resistance to bovine babesiosis in newborn calves independent of maternal antibodies.     

Development of an effective polyvalent vaccine against both Marek's and Newcastle diseases based on recombinant Marek's disease virus type 1 in commercial chickens with maternal antibodies

An earlier report (M. Sakaguchi et al., Vaccine 16:472-479, 1998) showed that recombinant Marek's disease virus type 1 (rMDV1) expressing the fusion (F) protein of Newcastle disease virus (NDV-F) under the control of the simian virus 40 late promoter [rMDV1-US10L(F)] protected specific pathogen-free chickens from NDV challenge, but not commercial chickens with maternal antibodies against NDV and MDV1. In the present study, we constructed an improved polyvalent vaccine based on MDV1 against MDV and NDV in commercial chickens with maternal antibodies. The study can be summarized as follows. (i) We constructed rMDV1 expressing NDV-F under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10P(F)]. (ii) Much less NDV-F protein was expressed in cells infected with rMDV1-US10P(F) than in those infected with rMDV1-US10L(F). (iii) The antibody response against NDV-F and MDV1 antigens of commercial chickens vaccinated with rMDV1-US10P(F) was much stronger and faster than with rMDV1-US10L(F), and a high level of antibody against NDV-F persisted for over 80 weeks postvaccination. (iv) rMDV1-US10P(F) was readily reisolated from the vaccinated chickens, and the recovered viruses were found to express NDV-F. (v) Vaccination of commercial chickens having maternal antibodies to rMDV1-US10P(F) completely protected them from NDV challenge. (vi) rMDV1-US10P(F) offered the same degree of protection against very virulent MDV1 as the parental MDV1 and commercial vaccines. These results indicate that rMDV1-US10P(F) is an effective and stable polyvalent vaccine against both Marek's and Newcastle diseases even in the presence of maternal antibodies.     

Maternal antibodies against tetanus toxoid do not inhibit potency of antibody responses to autologous antigen in newborn rhesus monkeys

Background

Our previous study suggested newborns have competent immune systems with the potential to respond to foreign antigens and vaccines. In this study, we examined infant immune responses to tetanus toxoid (TT) vaccination in the presence of maternal antibody to TT.

Methods

We examined changes in plasma levels of tetanus toxoid‐specific IgG1 (anti‐TT IgG1) in a total of eight infant rhesus macaques from birth through 6 months of age using a commercial Monkey Anti‐TT IgG1 ELISA kit.

Results

A significant correlation between anti‐TT IgG1 levels in vaccinated dams and their paired newborn infants was detected in control (non‐vaccinated) infants as previously reported. Maternal anti‐TT IgG1 levels declined rapidly within 1 month of birth in non‐vaccinated infants (n=4). In four infants vaccinated with TT at birth, we found two had rapid and robust antibody responses to vaccination. Interestingly, the other two first showed declining TT antibody levels for 2 weeks followed by increasing levels without additional vaccine boosts, indicating all four had good antibody responses to primary TT vaccination at birth, despite the presence of high levels of maternal antibodies to TT in all four infants.

Conclusions

Our data indicate that newborn macaques have competent immune systems that are capable of generating their own primary antibody responses to vaccination, at least to tetanus antigens. Maternal antibodies thus do not significantly impair antibody response to the vaccination, even when received on the day of birth in infant rhesus macaques.     

携带新城疫病毒DNA疫苗的鼠伤寒沙门氏菌及其安全性与免疫效力

根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计一对引物,通过RTPCR扩增出鹅源新城疫病毒分离株JS5F基因,测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1F。将pVAX1F转化减毒鼠伤寒沙门氏菌SL7207,构建成携带DNA疫苗的重组沙门氏菌SL7207(pVAX1F)。重组菌以不同剂量口服免疫1日龄雏鸡,结果表明,细菌对雏鸡具有良好的安全性,且不影响鸡的增重。将SL7207(pVAX1F)分别以108CFU和109CFU的剂量3次口服免疫1日龄商品代伊莎褐蛋鸡,抗体检测结果显示,在三免后1周,SL7207(pVAX1F)109CFU剂量组的血清抗体效价与空载体组之间存在显著性差异(p<0.05)。重组菌两个剂量组在首免后2周开始出现粘膜抗体,并于二免后2周和3周达到较高水平。免疫保护试验结果显示,SL7207(pVAX1F)109CFU剂量组的保护率为77.27%,与空载体组之间存在显著性差异(p<0.05)。     

白术多糖对小鼠免疫功能调节的研究

目的将白术多糖作为抗原刺激小鼠免疫系统,探讨其对免疫功能调节作用,为多糖在免疫调节功能方面的应用开发及在药物鉴定方面的应用提供科学依据。方法分别给予小鼠自术多糖、可溶性淀粉多糖、伤寒多糖抗原刺激,检测小鼠血清中的相应抗体及交叉抗体,探讨补益类水溶性多糖对小鼠免疫功能的调节作用。结果多糖抗原均能刺激机体产生特异性IgG类抗体（P〈0.05）;也能在一定程度上激发非特异性IgG类抗体即交叉抗体的产生;但不激发病理性抗体产生（RF阴性）。结论补益类水溶性多糖能激发免疫反应与其他多糖可能有共同的途径和机制。