应用PPA—ELISA检测鸡毒支原体血清抗体

应用ＰＰＡ－ＥＬＩＳＡ检测鸡毒支原体血清抗体，其结果与血凝抑制试验和ＥＬＩＳＡ符合率为１００％，是一种特异，敏感，简单，快速的检测方法。     

用双抗体夹心ELISA法检测鸡病毒性关节炎病毒的研究

应用鸡病毒性关节炎病毒Ｕ．ＣｏｎｎＳ１１３３株，建立了检测鸡病毒性关节炎病毒抗原的双抗体夹心ＥＬＩＳＡ法。用该法检测三组接毒鸡的关节液，接毒后３天即可检出阳性，阳性率为５８．３％；发病严重的４～１１天阳性检出率达９５７％，１４天阳性率为５０％；１７天阳性率为３１．３％；２０天以后则全部为阴性。用该法对大肠杆菌（Ｅ．ＣｏＬｉ）、葡萄球菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ）、败血支原体（ＭＧ）、滑膜支原体（ＭＳ）、新城疫病毒（ＮＤＶ）、传染性支气管炎病毒（ＩＢＶ）、传染性脑脊髓炎病毒（ＡＥＶ）进行检测，结果均为阴性。试验表明，我们建立的双抗体夹心ＥＬＩＳＡ法，能早期、特异、敏感地检出鸡病毒性关节炎病毒抗原，从而解决了对该病早期诊断及类症鉴别带来的困难。     

抗鸡传染性法氏囊病病毒酶标抗体的研制与应用

该试验采用过碘酸钠氧化法研制成功了抗鸡传染性法氏囊病病毒酶标抗体。通过进行双抗体夹心法ＥＬＩＳＡ试验，对不同送检病料中的传染性法氏囊病病毒进行了检测。结果表明：用过碘酸钠氧化法制备酶标抗体方法简便、省时；用双抗体夹心法ＥＬＩＳＡ试验检测传染性法氏囊病病毒，具有灵敏度高、特异性强等优点。     

泰万菌素对白羽鸡鸡毒支原体阳性率控制试验

本试验采用血清学平板凝集法检测白羽肉种鸡鸡毒支原体抗体水平,通过鸡毒支原体抗体阳性率的变化,分析泰万菌素对白羽肉种鸡鸡毒支原体病的防控效果。试验设置两栋规模和日龄相同的白羽肉种鸡舍作为试验组和对照组,试验组在泰万菌素给药前1d及停药后1d随机抽取50只鸡,对照组同批次于另一鸡舍随机抽取50只鸡,由翅中静脉采全血后分离血清;采用血清平板凝集法检测样品中血清鸡毒支原体抗体水平,计算鸡毒支原体抗体阳性率。试验组给药前鸡毒支原体血清抗体阳性率为54%,给药后为34%;对照组给药前鸡毒支原体血清抗体阳性率为40%,给药后为38%。白羽肉种鸡给药泰万菌素后,能明显降低血清中鸡毒支原体抗体的阳性率,反映出泰万菌素对肉种鸡鸡毒支原体有很好的疗效。     

用双抗体夹心ELISA法检测鸡病毒性关节炎病毒的研究

应用鸡病毒性关节炎病毒Ｕ．Ｃｏｍ Ｓｕｎ株，建立了检测鸡病毒性关节炎病毒抗原的双抗体夹心ＥＬＩＳＡ法。用该法检测三组接毒鸡的关节液，接毒后３天即可检出阳性，阳性率为５８．３％；发病严重的４￣１１天阳性检出率达９５．７％；１４天阳性率为５０％；１７天阳性率为３１．３％；２０天以后则全部为阴性。用该法对大肠杆菌、葡萄球菌、败血支原体、滑膜支原体、新城疫病毒、传染性支气管炎病毒、传染性脑脊髓炎病毒进行检     

禽苗中外源性鸡白血病病毒检测方法的研究Ⅰ、免疫酶细胞片检测法的建立

已建立用直接或间接酶标组化法检测鸡蛋或鸡胚中污染的白血病／肉瘤病毒。该方法是将白血病／肉瘤病毒的代表毒株接种到已培养好的鸡胚单层成纤维细胞片上。继续培养一定时间，随后将固定好的细胞与特异性酶标抗体反应，经底物显色后，镜检。接种肉瘤病毒的细胞片在接毒６小时后就能检出少量的阳性细胞，接毒４８小时的细胞片中的阳性细胞则更为清楚，典型。将病毒液稀释至１０~（－７）时接种，其接毒细胞片在９６小时也能检测到典型的阳性细胞；同时，该方法也能检测Ａ、Ｂ两个亚群的另外四个毒株（ＲＡＶ－１、ＲＡＶ－２、ＲＳＶ－１、ＲＳＶ－２），而对禽的其它病原病毒则呈阴性反应。     

检测ＮＤＶ特异性ＩgG、IgM、ＩgA的间接ＥＬＩＳＡ方法的建立

本试验以新城疫病毒（ＮＤＶ）感染的鸡胚尿囊液经差速离心，庶糖密度梯度离心提纯的ＮＤＶ为包被抗原，以纯化的鸡ＩgG、IgM及ＩgA免疫，制备辣根过氧化物酶标记的兔抗鸡ＩgG、IgM及ＩgA，分别建立了检测ＤＮＶ特异性ＩgG、IgM、ＩgA的间接ＥＬＩＳＡ方法，抗原最适包被量为１．３４μg/孔。酶标兔抗鸡ＩgG、IgM、ＩgA的最佳稀释度分别为１：４００，１：４００，１：１００，血清样品检测ＩgG时，最适工作浓度为１：４００，检测IgM时为１：５０。该方法具有特异性强、灵敏度快，快速简便等优点，适合于对各类新城疫特异性抗体的动态监测和大批检测，同时也为城疫免疫要理研究及流行病学调查提供了可靠手段。     

应用Dot-ELISA检测鸡毒支原体血清抗体

应用Ｄｏｔ－ＥＬＩＳＡ检测鸡毒支原体血清抗体＠郭建华＠陈明勇＠陈德威￥中国农业大学实验动物研究所应用Ｄｏｔ┐ＥＬＩＳＡ检测鸡毒支原体血清抗体郭建华＊陈明勇陈德威（中国农业大学实验动物研究所,北京海淀１０００９４）鸡毒支原体（ＭＧ）为慢性呼吸道病（ＣＲＤ）的...     

鸡毒支原体平板凝集诊断抗原的研制

鸡毒支原体(mycoplasma gaujseptium，MG)主要引起鸡的慢性呼吸道疾病，是养禽业相当棘手的疾病问题之一。目前常用的抗体水平监测方法有：利用鸡毒支原体能凝集鸡或火鸡红细胞的特性，发展了血凝(HA)和血凝抑制(HI)试验，但此方法不够灵敏。酶联免疫吸附法(ELISA)灵敏度     

禽苗中外源性鸡白血病病毒检测方法的研究：Ⅰ.免疫酶细胞片检测法…

已建立用直接或间接酶标组化法检测鸡蛋或鸡胚中污染的白血病／肉瘤病毒，该方法是鸡胚是将白血病／肉瘤病毒的代表毒株接种到已培养好的鸡胚单层成纤维细胞片上，继续培养一定时间，随后将固定好的细胞与特异性酶标抗体反应，经底物显色，镜检，接种肉瘤病毒的细胞片在接毒６小时后就能检出少量的阳性细胞，接毒４８小时的细胞片中的阳性细胞则更为清楚，典型。将病毒液稀释至１０＾－７时接种，其接毒细胞片在９６小时时也能检测到     

间接血凝抑制试验检测血清和卵黄中鸡毒支原体抗体的研究

将13只正处于产蛋期的试验鸡在接种疫苗前后分别采血、分离血清；收集鸡蛋，分离蛋黄，并分别用16％NaCl、8％柠檬酸纳和氯仿处理后，检测鸡毒支原体(MG)间接血凝抑制(HI)抗体。结果表明：接种疫苗前的血清和16％NaCl、8％柠檬酸钠和氯仿处理卵黄抗体平均效价分别为4.7，4．4，4．1和4.2 log2；第一次接种疫苗后的血清和16％NaCl、8％柠檬酸纳和氯仿处理卵黄抗体平均效价分别为7．4、6．7、7．1、6．9log2；第二次接种疫苗后第1次采集的血清和16％NaCl、8％柠檬酸钠、氯仿处理卵黄抗体平均效价分别为10．4、9．6、10．1、9．710g2，第2次采集的血清和16％NaCl、8％柠檬酸钠、氯仿处理卵黄抗体平均效价分别为7．9、7.1、7．6和7．4log2。8％柠檬酸钠处理后的卵黄抗体效价最高，与血清抗体效价最接近。血清HI抗体效价与卵黄HI抗体效价具有良好的一致性和相关性，用卵黄可以代替血清进行HI试验。     

Investigation of survival time of some poultry mycoplasmas

Glucose-fermenting poultry mycoplasmas (Mycoplasma [M.] gallisepticum, M. pullorum, M. gallinaceum, M. gallopavonis) were tested in 2 experiments for their survival time at 20 degrees C and 37 degrees C on 18 different materials used on farms and in hatcheries. All mycoplasmas survived up to 16 days in egg yolk at both temperatures. On other materials, like egg shell, egg white, paper trails, feather, and others mycoplasmas generally survived 2 to 16 days at 20 degrees C. M. gallinaceum and M. gallopavonis proved more resistant to the environment than M. gallisepticum and M. pullorum.     

Comparison of culturing Mycoplasma gallisepticum from fresh eggs and 18-day-old embryos

Twenty-four 70-week-old and sixteen 27-week-old white leghorn hens were challenged with R strain Mycoplasma gallisepticum (MG) by injection into the caudal thoracic air sac and infraorbital sinus. Eggs were collected daily and cultured within 7 days or incubated for 18 days. Vitelline membranes of eggs were cultured directly; in 18-day-old embryos, cultures were taken from the yolk sac, air sacs, and oral cavity. Culture of vitelline membrane of eggs within 2 days was compared with culture of eggs stored 10 days post oviposition. The first MG-positive egg was laid 2 days postinfection (PI). Hens continued to lay positive eggs to the end of the experiments. There was no significant difference in MG recovery between eggs cultured within 2 days and those cultured 10 days post oviposition. MG was isolated at a significantly higher rate from eggs than from 18-day-old embryos. MG was isolated at a higher rate from the yolk sac of 18-day-old embryos than from the air sacs or oral cavity of the same embryos.     

不同品种、不同周龄鸡蛋营养成分比较研究

本试验选用北京油鸡、白来航、海兰褐3个品种,每个品种随机挑选17或19周龄(北京油鸡)、42周龄、70周龄母鸡收集鸡蛋进行蛋黄营养成分的测定。结果表明:不同品种3个产蛋时期的蛋黄比例、蛋白比例、蛋黄维生素E含量都有极显著差异(P<0.01)。其中白来航、海兰褐在不同产蛋时期的蛋黄、蛋白比例呈现相同的变化趋势,即蛋黄比例为70周龄蛋最高,蛋白比例为初产蛋最高。3个品种不同产蛋时期维生素E含量变化亦势趋相同,初产蛋最高。3个品种42周龄蛋中的必需氨基酸比例显著高于其他2个时间点(P<0.01)。商业品种与中国地方鸡种所产蛋在蛋黄、蛋白比例、维生素B2变化趋势迥异,无统一规律可循。     

Comparison of egg yolk and serum for the detection of Mycoplasma gallisepticum and M. synoviae antibodies by enzyme-linked immunosorbent assay

Egg yolk was evaluated in the enzyme-linked immunosorbent assay (ELISA) as an alternative source of antibodies for detection of Mycoplasma gallisepticum (MG) and M. synoviae (MS) infections in chickens. There was no statistically significant difference (P greater than 0.05) between the ELISA geometric mean titers (GMTs) of saline-diluted egg yolk and chloroform-extracted egg yolk, and both preparations had a high correlation coefficient (0.87 for MG; 0.97 for MS). The saline-diluted and chloroform-extracted yolk had a relative sensitivity of 90% and specificity of 98% in the MG ELISA; in MS ELISA they were 100% and 96%, respectively. Hemagglutination-inhibition (HI) results with chloroform-extracted samples were satisfactory, but those with saline-diluted samples were not. Neither preparation was satisfactory for use in the rapid plate agglutination (RPA) test. A 1-ml sample of yolk was compared with the whole-yolk method. The chloroform-extracted whole yolk yielded a significantly higher (P less than 0.05) GMT in the MG ELISA; however, there was no statistically significant difference (P greater than 0.05) between GMTs yielded by the two procedures in the MS ELISA. The correlation coefficients for the two sampling methods were 0.73 for MG ELISA and 0.63 for MS ELISA. ELISA detected no statistically significant difference (P greater than 0.05) between GMTs of serum and chloroform-extracted yolk from individual birds. Results with the HI test were comparable to those with ELISA on the same samples. The RPA test yielded comparable results on the serum samples. No statistically significant differences (P greater than 0.05) were observed in HI or ELISA antibody levels between egg-yolk samples and sera on random samples collected from nine flocks that were MG- and MS-free or were infected with MG, MS, or both; however, egg-yolk samples tended to have slightly higher titers than sera in both tests. The optimum screening dilution of chloroform-extracted yolk for detecting MG and MS antibodies by ELISA was 1:800.     

Effects of Egg Yolk Source on the Cryopreservation of Stallion Spermatozoa

Three experiments were conducted to determine whether replacement of chicken egg yolk, as a component of freezing extenders, with egg yolk from other avian species would improve the post-thaw motility and percentage of intact acrosomes of stallion spermatozoa. In the first experiment, substitution of chicken egg yolk with chukar egg yolk, as a component of the lactose-ethylenediaminetetraacetic acid extender, improved (P ≤ .05) the post-thaw motility of stallion spermatozoa. These results were not replicated in (IMV Technologies, Maple Grove, MN, USA) a more expansive study comparing 2%, 4%, 6%, or 8% egg yolk combined with INRA 96 when a “slow freeze” method was used, or the same substitution at levels ranging from 13% to 22% when egg yolk was combined with lactose-ethylenediaminetetraacetic acid for diluents used for a “fast freeze” method of cryopreservation. In the third study, egg yolks from regular and high omega-3 chicken eggs as well as from turkey, chukar, and mallard duck eggs were analyzed for lipid content and fatty acid profile. The yolk from the turkey eggs was higher (1,300 mg/100 g) and that from mallard ducks was lower (560 mg/100 g) in cholesterol as compared with the two types of chicken eggs and chukar egg yolk (range, 1,046-1,094 mg/100 g). In addition, the high omega-3 eggs did test higher for fatty acids (4.51 g/100 g) than other types of eggs (range, 0.28-0.73 g/100 g). Substitution of chicken egg yolk with turkey, but not duck, egg yolk resulted in higher post-thaw total motility (P ≤ .05) for spermatozoa obtained from two of the three stallions used in the third experiment.     

Detection of antibodies to Mycoplasma gallisepticum vaccine ts-11 by an autologous pMGA enzyme-linked immunosorbent assay

Mycoplasma gallisepticum is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for control of M. gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. With the use of specific monoclonal antibodies against M. gallisepticum strain S6 pMGA in immunoaffinity purification, the major membrane antigen of ts-11 was purified. An indirect enzyme-linked immunosorbent assay (ELISA) was developed with the purified antigen, and its potential for detection of antibodies induced after ts-11 vaccination was compared with an indirect ELISA with M. gallisepticum strain S6 pMGA. In the presence of high levels of ts-11-induced antibodies, both antigens detected similar numbers of positive sera. However, when lower levels of antibodies were present, ts-11 pMGA showed a higher sensitivity than S6 pMGA. Further examination of ts-11 pMGA with Mycoplasma synoviae-infected chicken sera revealed that ts-11 pMGA is specific for M. gallisepticum antibodies. With a panel of sera from ts-11-vaccinated or non-ts-11-vaccinated field chickens, the ts-11 pMGA ELISA was found to be more sensitive than the commercial rapid serum agglutination test in detecting antibodies to ts-11 vaccine. The results from this study suggest that the major membrane antigen of M. gallisepticum may have slightly different antigenic profiles in different strains, thereby necessitating the use of autologous antigens in serodiagnostic assays to increase sensitivity of the tests for mycoplasma antibodies. Thus, the low level of antibody response after ts-11 vaccination is, at least partially, due to the low ability of the current diagnostic antigens to bind ts-11 antibodies.     

犬瘟热病毒免疫蛋鸡血清抗体与卵黄抗体消长规律的研究

试验旨在了解产蛋鸡对犬瘟热病毒(canine distemper virus,CDV)抗原的免疫反应及其血清抗体和卵黄抗体的消长规律,为制备卵黄抗体提供依据。将CDV接种Vero细胞和DF1细胞进行传代并测定其病毒滴度,选取细胞病变出现早、病毒滴度高的Vero细胞毒作为接种病毒抗原免疫蛋鸡。每10 d免疫蛋鸡1次,第4次免疫后每30 d加强免疫1次。免疫前及免疫后每10 d采血分离血清,每5 d收集鸡蛋提取卵黄抗体,用琼脂扩散法和间接ELISA法测定其抗体水平。结果显示,血清抗体比卵黄抗体的滴度高,两者呈正相关性,卵黄抗体出现较血清抗体迟3~5 d,卵黄抗体在第3次免疫后3~5 d就已达到较高水平,此时即可开始收集鸡蛋制备卵黄抗体。     

不同储藏方式对鸡蛋品质的影响

 为探讨夏季常温保存和低温冷藏两种储藏方式对鸡蛋品质的影响,选择540个鸡蛋随机分为9组,1组当天测定蛋品质,3组置于28.31 ℃、湿度为68.13%的室温下保存,5组置于4~8 ℃的冷藏室保存,分别于第7、14、21、42和56 d测定蛋品质。结果表明,常温储藏的鸡蛋随着存放时间延长,鸡蛋品质不断下降,储藏21 d后,蛋重降低5.30%（P<0.05）,蛋壳厚度变薄（P<0.05）;哈氏单位随着储藏时间的延长迅速下降,第0 d比第21 d的哈氏单位值高59.18%（P<0.05）;蛋黄比率随储藏时间的延长有不断变大的趋势,第0 d的蛋黄比率比第21 d低15.58%（P<0.05）。低温冷藏的鸡蛋储藏到21 d时,仅哈氏单位比第0 d低6.78%（P<0.05）;冷藏到56 d时,蛋重和哈氏单位比第0 d分别降低5.63%和15.07%（P<0.05）,蛋黄比率提高了5.21%（P<0.05）。试验表明,鸡蛋在室温下的新鲜度适宜保存期不应超过21 d;低温冷藏时可以保存到56 d。     

鸡蛋暗斑对蛋品质及抗氧化能力的影响

[目的]研究鸡蛋暗斑对蛋品质及其抗氧化能力的影响。[方法]从饲喂同种日粮、饲养环境相同、同日龄褐壳蛋鸡同一批次所产的褐壳蛋中,用灯光照射法挑选出24枚典型正常蛋、24枚典型暗斑蛋进行试验,并分别对正常蛋和暗斑蛋进行蛋品质、蛋黄抗氧化指标的测定及比较。[结果]蛋品质比较:暗斑蛋与正常蛋相比,其蛋形指数、蛋壳强度、蛋重、哈氏单位、蛋白高度、鸡蛋等级、蛋黄颜色、蛋黄重、蛋壳重、蛋清重、蛋壳厚度、蛋黄比重、蛋壳比重、蛋清比重均差异不显著(P>0.05);暗斑蛋蛋壳颜色极显著深于正常蛋(P<0.01)。抗氧化指标比较:暗斑蛋蛋黄的总抗氧化能力(T-AOC)显著低于正常蛋蛋黄的总抗氧化能力(P<0.05),暗斑蛋蛋黄的丙二醛(MDA)含量与正常蛋蛋黄的丙二醛含量相比差异不显著(P>0.05)。[结论]与正常蛋相比,暗斑蛋的蛋壳颜色较深,总抗氧化能力较差。