旋毛虫病免疫诊断抗原的筛选研究

本文报道了旋毛虫肌幼虫匀浆抗原(HO抗原),排泄-分泌物抗原(ES抗原),可溶性杆细胞颗粒相关抗原(S_3抗原),葡聚糖凝胶G200柱层第一峰(FP)和第二峰(SP)抗原,亲和层析提纯抗原(PAW)的制备及其用于ELISA和Dot-ELISA诊断旋毛虫病的价值。证实亲和层析堤纯抗原的特异性高、敏感性强,可用于旋毛虫病的免疫诊断和流行病学调查。     

旋毛虫肌幼虫排泄/分泌物抗原的研究

为了研究在特定条件下经体外培养的旋毛虫肌幼虫排泄/分泌物(ES)抗原的特性,通过体外培养将所收集到的ES抗原经聚丙烯酰胺凝胶电泳(SDS-PAGE)、琼脂扩散试验进行分析。结果经SDS-PAGE分析出现了比较明显的2条蛋白带,其分子量为45 ku、49 ku;经琼脂扩散试验ES抗原与犬旋毛虫阳性血清出现了明显清晰的沉淀线,而与犬蛔虫阳性血清、犬绦虫阳性血清之间未出现沉淀线,ES抗原具有较强的特异性。表明研究结果为开展ES抗原的分子生物学的研究、分子生物学基因工程苗的研制打下了良好的基础。     

旋毛虫肌幼虫排泄／分泌物抗原的研究

为了研究在特定条件下经体外培养的肌旋毛虫幼虫排泄／分泌物（ES）抗原的特性,通过体外培养将所收集到的ES抗原经聚丙烯酰胺凝胶电泳（SDS—PAGE）、琼脂扩散试验进行分析。结果经SDS—PAGE分析出现了比较明显的2条蛋白带,其分子量为45ku、49ku;经琼脂扩散试验ES抗原与犬旋毛虫阳性血清出现了明显清晰的沉淀线,而与犬蛔虫阳性血清、犬绦虫阳性血清之间未出现沉淀线,ES抗原具有较强的特异性。表明研究结果为开展ES抗原的分子生物学的研究、分子生物学基因工程苗的研制打下了良好的基础。     

旋毛虫S_3抗原的提取及其抗独特型抗体制备

通过匀浆、差速离心等方法提取旋毛虫肌幼虫S3抗原,并对其进行了琼脂双向双扩散、免疫电泳、聚丙烯酰胺梯度凝胶电泳、薄层等电聚焦电泳、肌幼虫孵化试验等免疫学和生化特性分析。以S3抗原免疫兔,再将其抗体免疫同种异体兔,经亲和层析共获得3株抗独特型抗体(R1、R2、R3)。固相放射免疫竞争抑制试验表明,随着抗独特型抗体浓度的升高,其对125I标记的S3抗原-兔Ab1系统及人Ab1系统抑制率均升高,其中R1株对以上两系统的最高抑制率分别达92%和68%。进而证实,3株抗独特型抗体可变区结构与旋毛虫肌幼虫S抗原的决定簇或其邻近结构是相似的,为种间交叉反应型抗独特型抗体。     

旋毛虫肌幼虫可溶性抗原,排汇分泌抗原的电泳分析

枉文报道了应用十二烷基硫酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ），聚丙烯酰胺凝胶等电聚焦（ＩＥＦ）电泳及二维电泳（ＩＥＦ／ＳＤＳ－ＰＡＧＥ）对旋毛虫肌幼虫排汇分泌（ＥＳ）抗原和肌幼虫可溶性抗原的分析结果。肌幼虫ＥＳ抗原经ＳＤＳ－ＰＡＧＥ后用考马斯亮蓝染色蛋白质，结果显示１６条蛋白带，分子量范围２１￣８０ＫＤ，其中主带９条。ＩＥＦ电泳后分别用ＰＡＳ染多糖、考马斯亮蓝Ｒ－２５０染蛋白质、Ｎｉｌｅ＇     

几种旋毛虫抗原PAPS诊断液的效果比较试验

将几种旋毛虫抗原以同一浓度（0.6mg/mL）与PAPS微球交联,制成诊断试剂,进行旋毛虫阳性血清检测效果比较试验。结果显示,成虫、成虫ES、新生幼虫、肌幼虫B峰4川抗原均出现非特异性反应,而肌幼虫、肌幼虫ES、肌幼虫A峰抗原具有很高的敏感性,三者效价均达1:640。将此3种抗原所制得的诊断试剂对几种寄生虫阳性血清进行交叉试验,显示肌幼虫ES抗原对血吸虫、锥虫、弓形虫、肺吸虫、牛肝片吸虫、羊肝片吸虫阳性血清均不出现交叉反应,而肌幼虫A峰抗原、肌幼虫抗原对牛、羊肝片吸虫均出现交叉反应,对其他血清则不出现。表明肌幼虫ES抗原效果最佳,特异性最好,有很高的应用价值。     

蓝马鸡和藏马鸡血清淀粉酶和酯酶同工酶酶谱

采用聚丙烯酰胺凝胶垂直平板电泳法对19只蓝马鸡和15只藏马鸡血清淀粉酶和血清酯酶同工酶酶谱进行了研究。结果发现:(1)蓝马鸡和藏马鸡血清淀粉酶有AMY1,AMY2,AMY3三种同工酶,其中AMY1和AMY2存在多态性;(2)两种马鸡的血清酯酶均存在ES1,ES2和ES3三种同工酶;蓝马鸡的ES1同工酶存在显现酶活性的ES1A(36.8%)和不显现酶活性的ES10(63.2%)2种表型,而藏马鸡全部为ES10型。     

蓝马鸡和藏马鸡的血清酯酶同工酶酶谱

采用聚丙烯酰胺凝胶垂直平板电泳法对 1 9只蓝马鸡和 1 5只藏马鸡的血清酯酶同工酶酶谱进行了研究。结果发现 :(1 ) 2种马鸡的血清酯酶存在ES1 ,ES2和ES3 3种同工酶 ;(2 )蓝马鸡的ES1同工酶存在显现酶活性的ES1A (36 8% )和不显现酶活性的ES1O (63 2 % ) 2种表型 ,而藏马鸡全部为ES1O型 ;(3)ES2同工酶只有一条浓染的酯酶区带 ;(4)ES3同工酶存在ES3ABC ,ES3AB和ES3BC 3种表型 ,蓝马鸡和藏马鸡均以ES3ABC为优势表型 (分别为 68 4%和 60 0 % )。     

旋毛虫不同发育时期排泄分泌物抗原在免疫学诊断中的应用研究

[目的]系统了解并比较旋毛虫不同发育时期排泄分泌物(ES)抗原的免疫学特性,探索可用于临床检测出栏猪旋毛虫感染的血清学诊断技术。[方法]分别以旋毛虫肠道期10 h肌幼虫(10 h ML)、肠道期30 h成虫(30 h Ad)、3 d成虫(Ad3)、6 d成虫与新生幼虫混合(Ad6+NBL)以及肌幼虫(ML)五个不同发育时期的ES作为包被抗原,应用ELISA方法,检测感染不同剂量、不同天数的猪血清中的抗旋毛虫抗体Ig M和Ig G水平,绘制抗体消长规律曲线并进行数据分析。[结果]10 h ML ES和ML ES作为包被抗原适合检测不同感染剂量、感染35 d之前的猪抗旋毛虫Ig M抗体,低剂量感染10 d左右可以检出,高剂量感染5 d也可以检出;Ad3 ES作为包被抗原对高剂量感染35 d之前的猪抗旋毛虫Ig M抗体检测敏感;Ad3和ML的ES作为包被抗原可检测不同剂量、感染35 d之后的猪抗旋毛虫Ig G抗体,其中Ad3 ES抗原检测低剂量感染的效果优于ML ES抗原。[结论]肠道期肌幼虫、成虫和肌幼虫的ES抗原可用于检测旋毛虫的早期感染,成虫和肌幼虫的ES抗原可用于检测出栏猪的旋毛虫感染。本研究为进一步合理有效利用旋毛虫不同发育时期的ES抗原,建立更有效的检测屠宰动物旋毛虫感染的方法提供了重要理论基础和参考。     

伊氏锥虫同工酶、蛋白质和抗原组分的比较研究

本文采用生化技术对八个中国伊氏锥虫株及一个布氏锥虫株的同工酶、蛋白质和抗原组分进行比较研究。根据同工酶电泳结果,可将伊氏锥虫和与其形态上不能区分的布氏锥虫区别开来,亦可将伊氏锥虫分为两个酶株群(Z1、Z2)。根据SDS-聚丙烯酰胺凝胶电泳、等电聚焦电泳和免疫印迹试验的结果,可将酶株群Z1分成五个不同多肽群(株)。本研究结果表明,中国伊氏锥虫遗传变异程度较低,是一个相对稳定的种群。     

PAPS免疫微球快速诊断猪旋毛虫病的研究

应用新型的聚醛化聚苯乙烯（ＰＡＰＳ）载体微球与最佳的旋毛虫抗原共价交联制备成特异性强、敏感性高、重复性好的快速诊断试剂,应用于猪旋毛虫病的生前诊断。我们对旋毛虫各个发育期的虫体和分泌排泄抗原进行了分析研究,并以同一蛋白质浓度（０．６ｍｇ／ｍＬ）的成虫,新生幼虫、肌幼虫、成虫ＥＳ、肌幼虫ＥＳ、肌幼虫Ａ峰、肌幼虫Ｂ峰等七种抗原分别与ＰＡＰＳ载体微球共价交联制成各种快诊试剂,然后进行效果比较试验,其结果     

多头绦虫抗原特性的研究──多头绦虫节片抗原及原头节ES抗原的SDS-PAGE分析

为了探明多头绦虫成虫节片抗原及多头蚴原头节排泄分泌（ES）抗原的多肽组分，以供今后在免疫诊断与预防脑多头蚴病的应用，本试验应用SDS-PAGE首次分析了多头绦虫成虫节片抗原及多头蚴原头节ES抗原的多肽组分。应用12．5％凝胶的连续系统，垂直板型电泳，考马斯亮兰R－250染色的结果表明，成虫节片抗原共有16条多肽带，其分子量范围29～154kD，其中主带4条，分别为73、88、128、134kD，原头节ES抗原共6条多肽带，分子量范围33～132kD，其中主带2条，分别为33和108kD。本试验初步阐明多头绦虫成虫节片抗原和原头节ES抗原的多肽组分，为进一步研究多头绦虫抗原的特性奠定了基础。     

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied.     

Evaluation of excretory-secretory antigens for the serodiagnosis of swine trichinellosis

Groups of hog sera from endemic and non-endemic areas for swine trichinellosis in Yugoslavia were tested by ELISA using excretory-secretory (ES) antigens collected from T. spiralis muscle larvae maintained in vitro for 24, 48 or 72 h. The 24-h ES had the highest level of specificity for T. spiralis infection. Antigen preparations recovered after 48 or 72 h yielded an increasing rate of false-positive reactions. Additional antigens occurred in the 48- and 72-h ES preparations as determined by gel electrophoresis and monoclonal antibody binding. The occurrence of false-negative reactions was directly correlated with T. spiralis worm burdens. Hogs with muscle larvae densities greater than 10 larvae per gram were all positive by ELISA. Among 17 hogs with less than 10 larvae per gram, only one hog was negative by ELISA with 24-h ES antigen; the false-negative rate was higher with 48- and 72-h ES. These results show that ES antigen produced during the first 24 h of in vitro cultivation is highly specific for the immunodiagnosis of swine trichinellosis.     

Immunodiagnosis of experimental Parelaphostrongylus tenuis infection in elk

Elk infected with the meningeal worm, Parelaphostrongylus tenuis (Protostrongylidae), do not consistently excrete larvae in feces, making the current method of diagnosing live animals using the Baermann fecal technique unreliable. Serological diagnosis could prove more useful in diagnosing field-infected animals but depends on the identification and availability of good quality antigen. To mimic field infections, 2 elk were inoculated with 6 infective L3 larvae of P. tenuis, and another 2 with 20 L3 larvae. Fecal samples were examined for nematode larvae using the Baermann technique and serum samples taken were tested for anti-P. tenuis antibody with ELISAs by using the excretory-secretory (ES) products of L3, and sonicated adult worms as antigens. One animal passed first-stage larvae in its feces 202 days postinoculation, but passed none thereafter. The remaining 3 inoculated animals did not pass larvae. In contrast to parasite detection, antibodies against larval ES products were detected in all animals starting from 14 to 28 days postinoculation and persisted until the termination of the experiment on day 243 in 2 animals that harbored adult worms. Antibodies against somatic antigens of the adult worm were not detected until day 56 but also persisted until the end of the experiment in the animals with adult worms. In 2 elk that had no adult worms at necropsy, anti-ES antibodies were detected transiently in both, while anti-adult worm antibodies were present transiently in one. These findings confirm the superiority of P. tenuis larval ES products over somatic adult worm antigens as serodiagnostic antigens, as previously observed in studies of infected white-tailed deer, and extend the application of the newly developed ELISA test in diagnosing and monitoring cervids experimentally infected with P. tenuis.     

Development of a mixed antigen agar gel enzyme assay (AGEA) for the detection of antibodies to poxvirus in chicken and turkey sera

A mixed-antigen agar gel enzyme assay (AGEA) was developed to detect antibodies to poxviruses in chicken and turkey sera. The assay combines the principles of immunodiffusion and enzyme assay. For the detection of antibodies to fowl poxvirus (FP), pigeon poxvirus (PP) and turkey poxvirus (TP) in turkey serum samples, the three antigens were combined to form a mixed-antigen assay. To screen for antibodies to FP and PP in chicken serum samples, the two antigens were combined. When FP and PP viruses were combined as antigens, the sensitivity for chicken sera was 64% but the sensitivity of the agar gel precipitation test (AGPT) was 34% (P<0.001). When antibodies were detected in turkey sera using the mixed antigens, the AGEA had a sensitivity of 66.4% while that of AGPT was 25% (P<0.001).     

Diagnostic tests for Trichinella spiralis infection in pigs. A comparative study of ELISA for specific antibody and histamine release from blood cells in experimental infections.

A study on the histamine release test (HR) for the demonstration of infections with Trichinella spiralis in pigs was carried out on 18 pigs, six infected with 200 larvae, six infected with 5000 larvae and six non-infected (control group). The results obtained by HR during a 7 week infection were compared with those of the enzyme-linked immunosorbent assay (ELISA). All inoculated pigs were found to be positive on Day 40 post-inoculation (p.i.) by necropsy examination of selected muscle groups, with mean recoveries of 7.9 and 225 larvae g-1 of tissue in the low- and high-dose group, respectively. At this time, all animals of the high-dose group and five out of six animals of the low-dose group were antibody positive in ELISA with any of three coating antigens employed (a crude muscle larva extract, an excretory/secretory (ES) antigen and a purified 45 kDa antigen). HR performed on whole blood was positive in four out of six pigs of the high-dose group and one out of six pigs of the low-dose group. The earliest ELISA seroconversions took place at Day 15 p.i. with crude and ES antigens. The earliest measurable reaction in HR performed on whole blood was found on Day 19 p.i. There was considerable individual variation regarding which test was the most sensitive for the early detection of infection. Washing of the blood cells prior to antigen provocation led to a markedly improved sensitivity of HR, all animals of the high-dose and three out of six animals of the low-dose group being positive by Day 40 p.i. The time course of the development of ELISA titres and HR reactivity indicated that this effect is due to the removal of blocking antibodies.     

Comparison of an indirect haemagglutination assay and an ELISA for diagnosing Fasciola hepatica in experimentally and naturally infected sheep.

An enzyme-linked immunosorbent assay (ELISA) with somatic (S) or excretory-secretory antigens (ES) was compared with an indirect haemagglutination assay (IHA) for ability to detect antibodies against Fasciola hepatica in sheep. The specificity of both assays was determined by testing sera collected from sheep experimentally or naturally mono-infected with Fasciola hepatica, Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Taenia ovis, Eimeria spp., Trichostrongylus vitrinus, Trichostrongylus colubriformis or Nematodirus battus respectively. With S or ES antigens the specificity of the ELISA was 98% or 95% respectively, whereas the specificity of the IHA was 86%. Antibodies directed against Fasciola hepatica were detected by the ELISA with S or ES antigens from 2 weeks after infection until the end of the experiment, whereas the IHA detected antibodies from week 3. We conclude that the ELISA with S antigens compares favourably with the IHA and can be used for the serodiagnosis of ovine fasciolosis in the Netherlands.     

Sensitivity and specificity of Mycoplasma gallisepticum agglutination antigens prepared from medium with artificial liposomes substituting for serum

Three batches of strain A5969 Mycoplasma gallisepticum (MG) serum-plate-agglutination (SPA) antigen grown in regular Frey's medium with 12% swine serum, three batches grown in Frey's medium containing artificial liposomes instead of serum, and one commercial SPA antigen were evaluated for sensitivity and specificity. Sensitivity was measured using chickens exposed to MG by intraocular and intranasal inoculation. Specificity was measured in uninoculated controls and in groups inoculated with the oil-emulsion vaccines Haemophilus paragallinarum, infectious bursal disease inactivated virus vaccine, or Staphylococcus aureus. Sera were tested 1 to 8 weeks postinoculation. All SPA antigens had a perfect sensitivity score, except one liposome-grown antigen batch (LC). The two other liposome-grown antigen batches (LA and LB) maintained significantly higher specificity by yielding significantly (P less than 0.01) fewer false positive (FP) agglutination reactions than did the other antigens. The three antigen batches produced in medium with serum had intermediate levels of FP agglutination reactions. When known MG-negative sera were tested, MG SPA antigens LC and commercial SPA antigen yielded significantly (P less than 0.01) higher numbers of FP agglutination reactions than the other SPA antigens.     

Partial protection of lambs against Haemonchus contortus by vaccination with a fractionated preparation of the parasite

Florida Native lambs, less than 6 months of age, were successfully vaccinated against Haemonchus contortus with a high mol. wt fraction (greater than 30,000 daltons) derived from a somatic extract of H. contortus larvae (SEL) and excretions and secretions (ES) of larvae isolated during in vitro development from the infective 3rd to 4th stage. A 59% reduction in adult worm numbers was obtained in vaccinates compared to naive lambs following challenge. The protection in vaccinated lambs was similar to that seen in lambs exposed to a primary infection of H. contortus larvae which had been cleared with anthelmintic prior to the challenge infection. The unfractionated SEL/ES preparation and a low mol. wt fraction gave no significant protection against challenge infection.