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1.
根据GenBank中旋毛虫53000抗原基因的序列设计引物，用PCR技术直接从旋毛虫肌幼虫cDNA文库中扩增靶基因p53cDNA，将其克隆到pMD-18T载体，转化至大肠埃希氏菌NovaBlue后测序分析，结果表明，克隆到1176bp的p53cDNA，共编码391个氨基酸。序列分析表明，p53cDNA序列与GenBank中的旋毛虫p53基因序列相比共有12个核苷酸发生改变，二者的同源性为98％，所编码蛋白质氨基酸序列的同源性为98％。将其克隆到原核表达载体pET28a并转化至表达菌BL21star（DE3），经IPTG诱导表达后获得约48000的重组蛋白。Western blotting检测证实，p53重组蛋白可以被旋毛虫感染猪血清识别，具有很好的抗原性。 相似文献
2.
Cloning and analysis of a novel cDNA from Trichinella spiralis encoding a protein with an FYVE zinc finger domain 总被引：2，自引：0，他引：2
Fu BQ Liu MY Kapel CM Meng XP Lu Q Wu XP Chen QJ Boireau P 《Veterinary parasitology》2005,132(1-2):27-30
A cDNA library from Trichinella spiralis adults 3 days post-infection was screened with a cDNA probe, designated T 54, derived from a newborn larvae subtracted cDNA library. Sequence analysis showed that the positive clone contained a cDNA insert of 1464 bp in length with a single open reading frame of 1290 bp, which encoded a protein of 429 amino acids with a putative molecular mass of 49.9 k Da. Database analysis predicted the deduced protein had a leucine zipper motif and an FYVE zinc finger domain. The recombinant fusion protein was expressed and rabbit anti-recombinant protein sera reacted with a single peptide migrating at approximately 55 k Da in crude worm extract from muscle larvae, adults and newborn larvae stages. 相似文献
3.
应用旋毛虫感染猪血清，对旋毛虫新生幼虫cDNA文库进行了免疫筛选。对阳性克隆pBK-cMV-WN10的序列分析结果表明。cDNA全长为1352bp。含有1个1218bp的完整的开放阅读框架(ORF)，编码的多肽由406个氨基酸残基组成，其相对分子质量理论推导值为45900，等电点为5．43，N末端的信号肽及糖基化位点(NCS)表明其可能为分泌性糖蛋白，氨基酸序列19～156与158～295为重复区域，相似性为74％．C末端有1个半胱氨酸蛋白酶抑制剂结构域，但旋毛虫p46000抗原与其他线虫的半胱氨酸蛋白酶抑制蛋白结构有很大差异，可能已经失去半胱氨酸蛋白酶抑制蛋白的功能。PCR结果显示。从旋毛虫新生幼虫、肌幼虫、3日龄成虫和5日龄成虫cDNA中均扩增出此基因，表明此基因在旋毛虫各个时期均有表达。 相似文献
4.
Comparative assessment of a double antibody enzyme immunoassay test kit and a triple antibody enzyme immunoassay for the diagnosis of Trichinella spiralis spiralis and Trichinella spiralis nativa infections in swine. 总被引：1，自引：1，他引：0 
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下载免费PDF全文 Enzyme immunoassays using the triple antibody enzyme linked immunosorbent assay (ELISA) with both Trichinella spiralis spiralis and T. spiralis nativa excretory-secretory (ES) antigens and a commercial Trichinella spiralis enzyme immunoassay test kit were carried out on sera from pigs that were infected with light, moderate and high doses of infective T. spiralis spiralis and T. spiralis nativa respectively. Seroconversion occurred in all pigs given infective Trichinella larvae although no trichinae were recovered from pigs given T. spiralis nativa larvae and examined between days 92 and 99 postinfection by pepsin digestion. Anti-Trichinella antibodies were detected in pigs infected with T. spiralis spiralis and T. spiralis nativa by ELISA using either the homologous or heterologous ES antigen. The commercial Trichinella spiralis enzyme immunoassay test kit also detected anti-Trichinella antibodies in both the T. spiralis spiralis and T. spiralis nativa infected pigs. The commercial test kit did not appear to be as sensitive as the triple antibody ELISA since it usually took two to three days longer for seroconversion to be detected by the former procedure. Finally seroconversion occurred more rapidly in swine infected with T. spiralis spiralis than with pigs receiving comparable doses of T. spiralis nativa. 相似文献
5.
旋毛虫新生幼虫WN1抗原基因的克隆及表达 总被引：1，自引：0，他引：1
应用旋毛虫人工感染猪血清对旋毛虫新生幼虫cDNA文库进行免疫筛选，对阳性克隆WN1序列进行生物信息学分析。结果表明，WN1 cDNA全长为474bp，含有1个354bp的完整的开放阅读框架(ORF)，编码的多肽由117个氨基酸残基组成，其相对分子质量理论推导值为13200，等电点为4．25，N-末端的信号肽表明其可能为分泌性蛋白。GenBank数据库检索结果显示，此序列为一个新基因的全长cDNA。经PCR扩增WN1基因，将其克隆到原核表达载体pET28a，重组质粒经鉴定后，转化大肠杆菌BL21 star(DE3)并诱导表达出相对分子质量为15000的重组蛋白，与理论设计完全相符。 相似文献
6.
7.
Immunity in swine inoculated with larvae or extracts of a pig isolate and a sylvatic isolate of Trichinella spiralis 总被引：2，自引：0，他引：2
A Marinculic H R Gamble J F Urban D Rapic T Zivicnjak H J Smith K D Murrell 《American journal of veterinary research》1991,52(5):754-758
Inoculation of swine with a sylvatic isolate of Trichinella spiralis, designated T s nativa, resulted in low numbers of muscle larvae, compared with muscle larvae accumulation in swine inoculated with a pig type of T s spiralis. Despite low infectivity of T s nativa for swine, primary inoculation resulted in high levels of immunity against challenge infection with T s spiralis. This immunity was expressed in accelerated expulsion of challenge adults from the intestine and reduced numbers of muscle larvae. Pigs inoculated with T s nativa developed cellular and humoral responses similar to those in pigs inoculated with T s spiralis. However, in immunoblots, sera from pigs inoculated with T s nativa recognized additional proteins in muscle larvae excretory-secretory (ES) products, compared with sera from pigs inoculated with T s spiralis. Active immunization of pigs with ES products from T s nativa resulted in numerically higher, but not significantly different levels of immunity, compared with pigs immunized with ES from T s spiralis. The highest levels of immunity were obtained in pigs immunized with a T s spiralis newborn larval extract. The combination of ES products and newborn larval extract did not result in additive levels of immunity. These results indicate that the major immune effector response to Trichinella sp in pigs is against the newborn larvae, regardless of the genetic type of Trichinella sp. 相似文献
8.
R D Hanbury P B Doby H O Miller K D Murrell 《Journal of the American Veterinary Medical Association》1986,188(10):1155-1159
In a herd of approximately 1,000 hogs, evaluation of muscle specimens collected at various intervals during a 12-year period (1973 to 1985) indicated continuous transmission of Trichinella spiralis. The farm's rat population and the incidence of trichinosis in the rats was high during 1974, but diminished markedly by 1978. In January 1984, a longitudinal investigation, using tracer pigs, was performed to determine whether rodents and/or other wild animals were involved in transmission of T spiralis on this farm. Tracer pigs exposed to rodents and wild animals did not become infected with T spiralis. The rodent population on the farm was small and none of the rodents trapped and examined were found to be infected. Hog cannibalism also was evaluated as a mode of T spiralis transmission. Results of the investigation indicated that hog cannibalism was the mode of transmission for trichinosis in the herd. 相似文献
9.
Bokken GC van Eerden E Opsteegh M Augustijn M Graat EA Franssen FF Görlich K Buschtöns S Tenter AM van der Giessen JW Bergwerff AA van Knapen F 《Veterinary parasitology》2012,184(2-4):126-132
The aim of this study was to examine the dynamics of parasite specific antibody development in Trichinella spiralis and Toxoplasma gondii co-infections in pigs and to compare these with antibody dynamics in T. spiralis and T. gondii single infections. In this experiment, fifty-four pigs were divided into five inoculated groups of ten animals, and one control group of four animals. Two groups were inoculated with a single dose of either T. gondii tissue cysts or T. spiralis muscle larvae, one group was inoculated simultaneously with both parasites and two groups were successively inoculated at an interval of four weeks. Specific IgG responses to the parasites were measured by ELISA. T. gondii burden was determined by MC-PCR carried out on heart muscle and T. spiralis burden by artificial digestion of diaphragm samples. Specific IgG responses to T. gondii and T. spiralis in single and simultaneously inoculated animals showed a respective T. gondii and T. spiralis inoculation effect but no significant interaction of these parasites to the development of specific antibodies with the serum dilutions used. Moreover, our data showed that the specific IgG response levels in groups of animals successively or simultaneously co-infected were independent of a respective previous or simultaneous infection with the other parasite. Additionally, no differences in parasite burden were found within groups inoculated with T. gondii and within groups inoculated with T. spiralis. Conclusively, for the infection doses tested in this experiment, the dynamics of specific antibody development does not differ between single and simultaneous or successive infection with T. gondii and T. spiralis. However, lower parasitic doses and other ratios of doses, like low-low, low-high and high-low of T. gondii and T. spiralis in co-infection, in combination with other time intervals between successive infections may have different outcomes and should therefore be studied in further detail. 相似文献
10.
旋毛虫新生幼虫期特异性T668全长cDNA的克隆及序列分析 总被引：6，自引：4，他引：2
利用旋毛虫新生幼虫期特异性 c DNA片段 T6 6 8- SS2作为核酸探针 ,在新生幼虫 c DNA文库中筛选出 10个阳性克隆。序列测定及分子生物学软件分析表明 ,克隆 G10 - 6的 c DNA片段全长 16 0 0 bp,含 12 90 bp的开放阅读框架 ,编码 1个由 4 30个氨基酸残基组成的多肽 ,其相对分子质量理论推导值为 4 6 84 0 ,等电点 (PI)为 9.6 5。主导氨基酸为Ser(11.39% )和 Thr(10 .93% )。开放阅读框架中具有丝氨酸蛋白酶保守功能区及酶活性位点结构 ,其 N末端的信号肽及 2个糖基化位点表明 ,其为分泌性糖蛋白 ,预示其可能在细胞外起着重要作用。DNA同源性分析表明 ,该 c DNA为一新的 c DNA分子。 相似文献
11.
Franssen FF Fonville M Takumi K Vallée I Grasset A Koedam MA Wester PW Boireau P van der Giessen JW 《Veterinary research》2011,42(1):113
ABSTRACT: Domestic pigs are the main representatives of the domestic cycle of Trichinella spiralis that play a role in transmission to humans. In Europe, backyard pigs of small household farms are the most important risks for humans to obtain trichinellosis. Rats might play a role in the transmission of Trichinella spiralis from domestic to sylvatic animals and vice versa. In order to be able to investigate the role of wild rats in the epidemiology of T. spiralis in The Netherlands, we studied the dynamics of antibody response after T. spiralis infections in experimental rats, using infection doses ranging from very low (10 muscle larvae, ML, per rat) to very high (16 000 ML per rat). To evaluate the feasibility of rats surviving high infection doses with T. spiralis, clinical and pathological parameters were quantified. Serological tools for detecting T. spiralis in rats were developed to quantitatively study the correlation between parasite load and immunological response. The results show that an infection dose-dependent antibody response was developed in rats after infection with as low as 10 ML up to a level of 10 000 ML. A positive correlation was found between the number of recovered ML and serum antibody levels, although specific measured antibody levels correspond to a wide range of LPG values. Serum antibodies of rats that were infected even with 10 or 25 ML could readily be detected by use of the T. spiralis western blot 2 weeks post infection. We conclude that based on these low infection doses, serologic tests are a useful tool to survey T. spiralis in wild rats. 相似文献
12.
为了研究PCR检测感染小鼠血液中旋毛虫DNA的敏感性,应用旋毛虫1.6 kb重复序列为扩增靶序列对旋毛虫(T1)、乡土旋毛虫(T2)、布氏旋毛虫(T3)、伪旋毛虫(T4)和南方旋毛虫(T7)肌幼虫DNA进行PCR扩增,并检测小鼠感染20、100、300条T1肌幼虫后不同时间的外周血.结果表明,T1、T4和T7肌幼虫可扩增出特异性目的条带(510 bp),而T2和T3无扩增产物;1、0.04和0.02条T1、T4和T7肌幼虫均能扩增到清晰的目的条带(510 bp).20条幼虫感染小鼠后5 d～6 d,PCR阳性率均为7.69%;100条幼虫感染小鼠后5 d～12 d可检出旋毛虫DNA,其中感染后5 d～7 d的阳性率分别为30.77%、38.46%及30.77%;300条幼虫感染小鼠后5 d～15 d可检出旋毛虫DNA,感染后7 d的阳性率为61.54%,感染后6 d与8 d～10 d的阳性率均为53.85%. 3组旋毛虫感染小鼠PCR阳性率间的差异有统计学意义(p<0.01),PCR阳性率随感染剂量的增加而升高(p<0.01),100条与300条感染小鼠感染后不同时间的PCR阳性率与检测时间有相关性(p<0.01).以上实验结果表明PCR检测感染小鼠血液中旋毛虫DNA的敏感性与感染程度和检测时间有关,对感染早期旋毛虫抗体阴性宿主有一定诊断价值. 相似文献
13.
Trichinella spiralis spiralis infections were established in sheep by administering infective larvae via gavage or feeding infected musculature. Trichinella spiralis nativa infective larvae had a low infectivity for sheep although light infections may be established in some animals with large infective doses. For the most part, sheep were averse to ingesting musculature mixed in a grain ration unless it was camouflaged with molasses. The heaviest infections usually occurred in the masseter muscle. The fact that sheep are averse to ingesting muscle tissue may reduce the likelihood of trichinosis. Anti-Trichinella antibodies to both T: spiralis spiralis and T. spiralis nativa were produced as demonstrated by the enzyme-linked immunosorbent assay. Seroconversion occurred in several sheep challenged with T. spiralis nativa even though larvae were not recovered from the musculature by pepsin-digestion. 相似文献
14.
旋毛虫起源、进化及其宿主的感染来源与途径 总被引：1，自引：1，他引：0
毛形属在距今27 500万年前从毛形科中正式分化出来,各旋毛虫种间的遗传差异形成于距今的1 500万~2 000万年间。对亚洲和欧洲的共110个Trichinella spiralis地理隔离株的线粒体基因分析结果显示,欧洲地理株间仅存1个碱基差异,具有高度的一致性,而亚洲地理株间则具有8个碱基差异,存在明显的种内差异。依据线粒体累积变异速率计算,T.spiralis由亚洲传播至欧洲的时期大约在60 00年~16 000年以前。不同种旋毛虫对动物的感染具有一定的异嗜性,而宿主感染旋毛虫的途径则主要来源于水平传播,即食用感染有虫体的肉类;机械性传播,即经粪便、土壤、废水、食腐性昆虫的机械性传播;垂直传播,目前在人类、豚鼠中有检出报道。 相似文献
15.
旋毛虫肌幼虫RNA的提取及目的基因的PCR扩增 总被引：9，自引：0，他引：9
用改进的ＡＧＰＣ法成功地提取出旋毛虫肌幼虫总ＲＮＡ。每０．１ｍｌ压积虫体可获得１００μｇ的ＲＮＡ，其Ａ２６０与Ａ２８０及Ａ２６０与Ａ２３０的比值均接近于２，变性琼脂糖凝胶电泳显示，旋毛虫肌幼虫总ＲＮＡ缺少大部分真核生物所具有的２８Ｓ ｒＲＮＡ．通过比较研究，筛选出理想的虫体处理方法。用聚合酶链式反应直接从总ＲＮＡ中进行目的基因的扩增，得到一分子量为０．７ｋｄ的ＤＮＡ。通过限制性内切酶对其消化鉴定， 相似文献
16.
A Jiménez-González C De Armas-Serra A Criado-Fornelio N Casado-Escribano F Rodríguez-Caabeiro J C Díez 《Veterinary parasitology》1991,39(1-2):89-99
Tubulin was estimated to account for 0.3% of the total soluble protein in Trichinella spiralis cytosolic fractions. Tubulin from T. spiralis was partially purified by precipitation with either taxol or vinblastine sulphate. Immunoblotting with alpha- and beta-tubulin monoclonal antibodies revealed the presence of tubulin in T. spiralis partially purified preparations. Electrophoretic mobility of T. spiralis tubulin in sodium dodecyl sulphate-polyacrylamide gels was very similar to that shown by pig brain tubulin. Further studies with colchicine binding assays indicated that T. spiralis tubulin has binding features similar to that of tubulin from other nematodes: colchicine association constant = 8.1 x 10(-4) M and competitive inhibition of colchicine binding by podophyllotoxin, with an inhibition constant of 1.3 x 10(-6) M. Finally, inhibition of colchicine binding by several benzimidazoles (mebendazole, fenbendazole, oxibendazole and albendazole) was investigated. All the benzimidazoles inhibited colchicine binding in a competitive manner, with inhibition constant values ranging from 1.4 x 10(-7) M (mebendazole) to 3.9 x 10(-6) M (fenbendazole). 相似文献
17.
B R Berends J F Smeets A H Harbers F van Knapen J M Snijders 《The Veterinary quarterly》1991,13(4):190-198
In the framework of the Dutch field trial 'Integrated Quality Control (IQC) for finishing pigs' ELISA (screening) techniques were used to detect animals seropositive for Toxoplasma gondii and/or Trichinella spiralis. The aim was to determine whether farms which consistently delivered seropositive pigs could be detected and monitored (defined as 'problem farms'). The investigation involved 120 farms and three slaughterhouses, and a total of 23,348 serum samples were examined. In addition, all pigs were also screened for the presence of Trichinella spiralis with the digestion method (pooled samples). The prevalence of seropositivity for Trichinella spiralis and Toxoplasma gondii was 0.3% and 2.1% respectively. Parasitological examinations concerning T. spiralis were negative. Considering the characteristics of the used methodology, the conclusion was drawn that there were no parasitological or serological indications for T. spiralis infections, and that with respect to T. gondii the infection rate seemed to be equally low for all farms involved. In addition, a longitudinal pilot study during a whole finishing period was undertaken at two finishing farms. Animals seropositive for Toxoplasma gondii were found from the earliest days of the finishing period. Housing and management may (still) play an important role in the prevention of contact with this parasite. 相似文献
18.
Characterisation of Trichinella isolates from Bulgaria by molecular typing and cross-breeding 总被引：1，自引：0，他引：1
Kurdova R Müller N Tsvetkova N Michov L Georgieva D Ivanova M Gottstein B 《Veterinary parasitology》2004,123(3-4):179-188
Trichinella spp. larvae were collected from domestic and wild-life animals in association with 15 human trichinellosis outbreaks registered between 1999-2002 in Bulgaria. Furthermore, Trichinella spp. isolates were obtained from 62 naturally infected wild animals and of a rat. All isolates were subjected to speciation by both multiplex PCR and cross-breeding experiments. Epidemiological and clinical data were collected and analysed using standard protocols for epidemiological surveillance and control of outbreaks. Only two species were identified-Trichinella britovi and Trichinella spiralis. Results obtained by molecular typing fully matched those of cross-breeding. More specifically, parasite isolates obtained upon 15 epidemic outbreaks revealed the predominance of T. britovi (n = 10) when compared to T. spiralis (n = 5). With regard to host origin, the predominant species detected among wild boar was T. britovi (n = 4), and T. spiralis was identified in one wild boar sample only. Among the isolates obtained from domestic pig products, T. britovi was found in five cases and T. spiralis in four cases, respectively. In the naturally infected wild animals not related to epidemics, only T. britovi was demonstrated. The present results provide a strong indication that both T. britovi and T. spiralis operate within domestic and sylvatic cycles in Bulgaria. Geographically, the distribution of T. britovi appears to include Central, Southern, Eastern and Western parts of the country, and wildlife animals from the Mid Balkan Mountains and Mid Sredna Gora Mountains, T. spiralis was found in Western and Southwestern Bulgaria, only. 相似文献
19.
用消化法所得的猪旋毛虫、犬旋毛虫、旋毛形线虫 (Trichinellaspiralis)和本地毛形线虫 (Trichinellanativa)分别感染健康猪。结果表明 :4个旋毛虫隔离种对猪的感染性存在着明显差异 ,猪旋毛虫和T .spiralis对猪易感 ,其繁殖力指数 (RCI)分别为 385 .6 8± 41.5 1和 30 0 .5 5± 12 .45 ;而犬旋毛虫和T .nativa对猪不易感 ,RCI分别是 0 .0 6 4± 0 .0 31和 0 .0 33± 0 .0 33。结果揭示黑龙江省猪旋毛虫相当于T .spiralis,犬旋毛虫相当于T .nativa ;犬旋毛虫和T .nativa很难通过猪的感染而对人体健康构成威胁。 相似文献
20.
旋毛虫感染小鼠对p46 000重组抗原的抗体应答 总被引：1，自引：0，他引：1
分别以旋毛虫肌幼虫ES抗原和p46000重组蛋白作为抗原，对小鼠人工感染旋毛虫后的抗体应答进行了ELISA检测。结果表明，以肌幼虫200条／只经口感染小鼠后，肌幼虫ES抗原在感染后9d可检出抗体，并于感染后35～42d达到最高水平；应用重组抗原检测时，感染后10d可检出抗体，抗体水平略低于用ES抗原，但是其消长规律基本一致．而且与阴性血清相比差异明显；抗体在117d后仍维持于较高水平。 相似文献