猪带绦虫六钩蚴TSOL18重组蛋白快速诊断试纸条研制

目的以猪带绦虫六钩蚴重组蛋白(TSOL18)为抗原,建立一种简便快速的猪囊虫抗体检测方法,评价其血清学诊断的价值。方法用胶体金颗粒标记纯化的TSOL18重组蛋白,将兔抗TSOL18-GST-IgG和TSOL18重组抗原分别喷涂于硝酸纤维素膜的质控线和检测线上,与PVC垫板等部件按顺序装配成猪囊虫抗体快速检测试纸条;用该试纸条检测猪血清样品测定敏感性和特异性。结果该试纸条与全囊虫抗原ELISA的相对敏感性和特异性分别为75.0%(12/16)和95.2%(40/42),与剖检计数法的相对敏感性达80.0%(12/15)。试纸条在4℃存放12个月,检测结果稳定,重复性好。结论基于TSOL18建立的胶体金免疫层析试纸条操作简单、快速敏感、稳定性好,可用于猪囊虫病感染的诊断和筛查。     

猪带绦虫TSOL18基因的表达、纯化和兔抗血清的制备

目的 构建猪带绦虫重组质粒pGEX-TSOL18,表达纯化TSOL18重组蛋白,制备兔抗重组蛋白血清。方法 全基因合成猪带绦虫TSOL18抗原编码基因,定向克隆到pGEX-1λT表达载体,构建重组质粒pGEX-TSOL18;转化大肠埃希菌ArcticExpress(DE3), IPTG诱导表达,SDS-PAGE电泳分析表达情况;亲和层析纯化获得重组蛋白,免疫兔制备抗血清;ELISA测定抗血清的效价,Western blot鉴定抗血清的特异性。结果 全基因扩增出393 bp的TSOL18抗原编码基因,酶切和测序鉴定证实TSOL18基因成功插入pGEX-1λT中;SDS-PAGE分析显示表达重组蛋白相对分子质量(Mr)约为41 kDa,经亲和层析后可获得纯度为75%的重组蛋白;ELISA测定抗血清的效价为1∶256 000,Western blot证实该抗血清能与重组蛋白发生特异性反应。结论 猪带绦虫TSOL18基因能在大肠埃希菌中高效表达,成功制备了高纯度的TSOL18重组蛋白和高滴度的兔抗重组蛋白血清,为猪带绦虫的疫苗研制奠定了基础。     

Antibody responses to the host‐protective Taenia solium oncosphere protein TSOL18 in pigs are directed against conformational epitopes

TSOL18 is a recombinant protein that has been shown in repeated experimental trials to be capable of protecting pigs against challenge infection with the cestode parasite Taenia solium. Antibodies raised by the vaccine are capable of killing the parasite in an in vitro culture and it is believed that antibody and complement‐mediated killing of invading parasites is the major protective immune mechanism induced by vaccination with TSOL18. Investigations were undertaken to characterize whether the principal antibody specificities raised by TSOL18 in pigs were against linear or conformational determinants. TSOL18 was expressed in two truncated forms representing either the amino terminal portion or the carboxy terminal portion, with the two truncations overlapping in sequence by 25 amino acids. The original protein (designated TSOL18N^?) and the two truncations (TSOL18N^?‐1 and TSOL18N^?‐2) were used in inhibition ELISA. TSOL18N^? was shown to be capable of completely inhibiting the binding of pig anti‐TSOL18N^? antibodies to TSOL18N^? in ELISA. However, neither TSOL18N^?‐1 nor TSOL18N^?‐2, either alone or when combined together, was capable of inhibiting any detectable amount of reactivity of pig anti‐TSOL18N^? antibodies with TSOL18N^?. It is concluded that the dominant antibody specificities, and probably the host‐protective specificities, of TSOL18 are conformational epitopes.     

Vaccines for prevention of cysticercosis

Neurocysticercosis due to Taenia solium infection is an important cause of human morbidity and mortality. Despite the availability of effective anthelmintics, the disease remains prevalent in many parts of the world and there is a need for new and improved measures for control of the infection. An effective vaccine to prevent infection in pigs, the parasite's natural intermediate host, would be a valuable new option to assist with T. solium control. Several approaches are being used currently towards the development of a T. solium vaccine and these approaches are reviewed briefly, with emphasis on the use of recombinant oncosphere antigens. Highly effective vaccines have been developed against cysticercosis in sheep and cattle caused by Taenia ovis and Taenia saginata, respectively. This success has encouraged the adoption of a similar strategy for T. solium. The recent finding that one oncosphere antigen, TSOL18, can induce complete protection against T. solium infection in pigs, highlights the potential for development of a practical vaccine. A vision is proposed for the development of a safe, effective, inexpensive vaccine for pigs, which can be administered in an edible form. Through an international collaborative effort, research is progressing towards the realisation of such a vaccine and its use to reduce the global burden of neurocysticercosis.     

猪带绦虫六钩蚴TSO45W基因工程疫苗研究进展

囊虫病是一种危害严重的人兽共患寄生虫病。研制有保护性作用的疫苗将是控制和消灭该病的有效方法,猪带绦虫保护性抗原的研究是猪囊虫病免疫的基础。45W蛋白是猪带绦虫六钩蚴时期的重要抗原,但天然45W抗原来源有限,限制了其应用,而基因工程重组抗原的应用可解决质量控制和抗原来源的问题。本文就近年来国内外对猪带绦虫六钩蚴TSO45W基因工程疫苗的研究进展作一综述。     

Vaccination of pigs to control human neurocysticercosis

Taenia solium taeniasis/cysticercosis is a zoonotic disease complex in which the pig is an obligate intermediate host. The infection is widespread, particularly in the developing world, and neurocysticercosis is a major cause of human neurologic disease where the parasite is endemic. Despite easy availability, effective anti-parasitic drugs have not been deployed effectively to control disease transmission. We have investigated a vaccine strategy to prevent parasite infection of the pig intermediate host. Such a strategy would interrupt the parasite's life cycle and eliminate the source of infection for humans. Two recombinant antigens selected from the parasite oncosphere life cycle stage were tested in vaccination trials in pigs that were challenged orally with Taenia solium eggs. Both antigens were highly effective in protecting the pigs against infection with the parasite (98.6% and 99.9% protection, respectively). No viable cysts were found in eight pigs vaccinated with one of the antigens. A recombinant subunit vaccine based on oncosphere antigens has the potential to improve the available control measures for T. solium and thereby reduce or eliminate neurocysticercosis.     

猪带绦虫TSOL18基因重组乳球菌疫苗的稳定性分析

目的 研究猪带绦虫TSOL18基因重组乳球菌(Lactococcus lactis,L.lactis)疫苗的人工传代稳定性。方法 将重组质粒pMG36e-TSOL18、pMG36e-SP-TSOL18电转入L.lactis,经PCR鉴定阳性菌株pMG36e-TSOL18/L.lactis、pMG36e-SP-TSOL18/L.lactis,在无红霉素抗性和含红霉素抗性条件下,连续人工传代20次,通过测定质粒遗传稳定率和双酶切鉴定,分析质粒pMG36e-TSOL18、pMG36e-SP-TSOL18在L.lactis中的稳定性情况。结果 质粒pMG36e-TSOL18、pMG36e-SP-TSOL18在L.lactis中连续传代20代后,在无红霉素抗性条件下,质粒稳定率均为100%,在含红霉素抗性条件下,质粒稳定率均为99%。将各代次的质粒采用限制性内切酶SacI/HindIII进行双酶切鉴定,琼脂糖凝胶电泳显示大小正确,连续传至20代时,与原代质粒相符。结论 提示质粒pMG36e-TSOL18、pMG36e-SP-TSOL18在L.lactis中连续传代20代时,均有较好的遗传稳定性,为猪带绦虫TSOL18基因重组乳球菌疫苗的进一步研究奠定了基础。     

Fact or hypothesis: Taenia crassiceps as a model for Taenia solium, and the S3Pvac vaccine

Research undertaken over the past 40 years has established many of the general principals concerning immunity to taeniid cestodes. Although much is well understood about the host-protective mechanisms against taeniids and this knowledge has been exploited in studies on vaccine development, many aspects require further investigation or confirmation. Some phenomena have come to be regarded as being well established, while careful analysis of the published data would suggest that they may be better regarded as hypotheses rather than established facts. This review considers one selected issue pertaining to immunity to cestode infections and examines carefully the nature of the evidence that is available to support conclusions that have been made in this area. The issue examined is the use of Taenia crassiceps as a model for cysticercosis in pigs caused by Taenia solium, together with the S3Pvac vaccine, which has been developed based on this model. Strong evidence is found to support the conclusion that defined T. crassiceps antigens can limit intraperitoneal proliferation of the ORF strain of T. crassiceps in mice; however, the potential for these antigens to affect T. solium infection in pigs requires further confirmation.     

猪带绦虫重组BCG-TSOL18疫苗构建及其表达

目的 构建猪带绦虫重组BCG-TSOL18疫苗,研究TSOL18基因在BCG中的表达情况。方法 通过酶切的方法 从重组质粒pGEX-TSOL18获取猪带绦虫TSOL18基因,将其定向克隆到大肠杆菌-分枝杆菌穿梭表达质粒pMV261中,构建猪带绦虫重组质粒pMV261-TSOL18,进行酶切、PCR和测序鉴定;再将其电穿孔转化入BCG,构建猪带绦虫重组BCG-TSOL18疫苗,进行PCR鉴定;SDS-PAGE和Western blot分析TSOL18基因在BCG中的表达情况。结果 通过酶切成功获得了393 bp的TSOL18基因片段;酶切、PCR和测序鉴定证明成功构建了猪带绦虫重组质粒pMV261-TSOL18;PCR鉴定证实猪带绦虫重组质粒pMV261-TSOL18成功转入BCG中,提示猪带绦虫重组BCG-TSOL18疫苗构建成功;SDS-PAGE分析发现在相对分子质量(Mr)约为14.7 kD处有明显的TSOL18目的蛋白条带,Western blot证实表达的TSOL18目的蛋白能被兔抗血清和囊虫病猪血清所识别。结论 成功构建了猪带绦虫重组BCG-TSOL18疫苗,TSOL18基因能够在BCG中成功表达,表达的TSOL18重组蛋白具有特异的抗原性,为该疫苗的进一步研究奠定了基础。     

Human Taeniasis and cysticercosis in Asia

CONTEXT: Human Taeniasis caused by the pork, Taenia solium, or beef, T saginata, tapeworm arises after eating pork or beef contaminated with metacestodes, the larval stage of these parasites. Taeniasis with T solium can lead to neurocysticercosis and threaten others by accidental ingestion of eggs released from asymptomatic Taeniasis patients. The 2003 World Health Assembly declared that T solium is of worldwide public-health importance, and that it is an eradicable parasitic disease worldwide. Adult taeniid tapeworms expelled from people in almost all Asian countries appeared to be T saginata (the so-called Asian Taenia), even though they ate pork. The organism is now named T asiatica, and has been found in Taiwan, Korea, China, Vietnam, and Indonesia. But it has been difficult to differentiate T saginata from beef and Asian Taenia from pork. STARTING POINT: Marshall Lightowlers and colleagues (Int J Parasitol 2003; 33: 1207-17) recently demonstrated that recombinant oncosphere vaccines against several taeniid cestodes, including T ovis, T saginata, T solium, and Echinococcus granulosus, are highly effective. Protection was almost 100%, in the laboratory and in the field. These researchers found several common features, including a predicted secretory signal sequence, and one or two copies of a fibronectin type III domain, each encoded by separate exons within the associated gene. WHERE NEXT? Molecular and immunological techniques, including vaccine research and development of animal models for differentiation of taeniid species in humans, have greatly advanced over the past decade. The clinical importance of infections by these taeniids, including T asiatica, in humans, and the potential for cysticercosis attributable to T asiatica in humans, needs further study.     

猪带绦虫六钩蚴表膜结合蛋白45WB2基因在毕赤酵母中的表达

目的 从猪带绦虫六钩蚴总RNA中扩增45WB2基因,并在甲醇酵母分泌表达载体pPIC9K中获得高效表达。 方法 采用逆转录-聚合酶链反应(RT-PCR), 从孵化激活的六钩蚴克隆获得459 bp的基因, 亚克隆至酵母分泌表达载体pPIC9K中, 经测序验证读码框正确后, 重组质粒电转化毕赤酵母SMD1168菌株。用PCR方法检测结果表明, 目的基因整合进毕赤酵母染色体DNA中。 重组菌株用1%甲醇诱导, 分别取诱导 72和96 h的上清和沉淀进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析。 结果 重组菌株在毕赤酵母中的表达蛋白分子量为Mr 16 000, 表达产物占上清总蛋白量的32.8%, 且能被猪囊尾蚴病患者血清识别。 结论 基因在毕赤酵母中成功表达, 表达产物有望作为免疫原用于猪囊尾蚴病的免疫预防。     

Genetic variability of the 45W gene family between Chinese and Mexican Taenia solium

Taenia solium 45W proteins are good candidates for development of anti-cysticercosis vaccines. However, the genetic characteristics of the 45 gene family are still unclear between different isolates. We investigated the polymorphism of the 45 gene family between Chinese and Mexican T. solium. Alignment showed that TSO45-4B and TSO45-1C antigens were conserved absolutely, whereas other TSO45 proteins varied between these two isolates. It is informative to guide using of recombinant 45W vaccines to control porcine cysticercosis caused by Asiatic or African/Latin American T. solium.     

Intestinal cestodes

PURPOSE OF REVIEW: This review summarizes the biology, clinical aspects, diagnosis, treatment and epidemiology for the common and rarer (zoonotic) intestinal cestodes of humans. RECENT FINDINGS: Mass drug application to eliminate Taenia solium carriers may have only temporary effects on cysticercosis transmission. At least two major world genotypes of T. solium have been identified and greater genetic heterogeneity may occur at the regional level. A new human taeniid T. asiatica has been confirmed which occurs sympatrically with T. saginata and T. solium in Southeast Asia. Coproantigen and PCR tests for Taenia spp. have greatly improved diagnostic efficacy and epidemiological studies. There appears to be an increase in human diphyllobothriasis in Europe, Japan and the Americas. SUMMARY: Human intestinal cestode infections are globally primarily caused by species in three genera: Taenia, Hymenolepis or Diphyllobothrium. Sporadic zoonotic infections caused by nontaeniids are usually food-borne or due to accidental ingestion of invertebrate hosts. Intestinal cestode infections generally result in only mild symptoms characterized chiefly by abdominal discomfort and diarrhoea. Most human intestinal cestode infections can be treated with a single oral dose of praziquantel or niclosamide.     

以HBc颗粒为呈现载体的猪囊虫疫苗的构建及其免疫学研究

目的构建以HBc为载体的带有三个猪囊虫抗原表位(n1,n2,n3)的重组融合表达质粒pET-Δc-3n,表达出融合蛋白并纯化,通过免疫小鼠研究其体液免疫效果。方法用PCR法将三个猪囊虫表位分别插入HBc序列的第78-79位之间以及149位之后,再将该序列克隆入pET28a载体中,构建重组表达质粒,用大肠杆菌BL21作宿主菌表达出融合蛋白,并命名为PCCE,纯化后免疫小鼠,检测小鼠的体液免疫应答。用绦虫卵攻击免疫小鼠,并观察疫苗的保护作用。结果测序结果表明重组质粒构建成功,SDSPAGE显示融合蛋白表达正确,并有多聚体形成现象,ELISA检测到高滴度抗体。小鼠体内绦虫卵攻击试验表明疫苗PCCE的相对保护率为89%。结论以HBc为呈现载体的猪囊虫疫苗被成功表达和纯化,该疫苗能诱导较强的体液免疫反应,免疫小鼠对绦虫卵攻击具有较好的免疫保护作用。提示该疫苗PCCE可能具有预防囊虫病的潜在价值。     

猪带绦虫重组双歧杆菌疫苗不同保存条件下的稳定性研究

目的 研究猪带绦虫TSO45W-4B基因、TSOL18基因和TSO45W-4B-TSOL18融合基因的重组双歧杆菌(Bb)疫苗在不同保存条件下的稳定性。方法 分别将猪带绦虫重组质粒pGEX-TSO45W-4B、pGEX-TSOL18和pGEX-TSO45W-4B-TSOL18电转入长双歧杆菌(B.longum),获得阳性菌株pGEX-TSO45W-4B/B.longum、pGEX-TSOL18/B.longum和pGEX-TSO45W-4B-TSOL18/B.longum,分别置于不同条件下保存,取出菌液,抽提质粒,电泳检测,进行稳定性分析。结果 阳性菌株pGEX-TSO45W-4B/B.longum、pGEX-TSOL18/B.longum和pGEX-TSO45W-4B-TSOL18/B.longum分别在常温下、4 ℃、-20 ℃、-80 ℃、-196 ℃(液氮中)分别保存48 h、1周、1月、2月,抽提质粒,电泳发现阳性菌株pGEX-TSO45W-4B/B.longum、pGEX-TSOL18/B.longum和pGEX-TSO45W-4B-TSOL18/B.longum在液氮中、-80 ℃、-20 ℃中比较稳定,而在普通的室温环境下,很快发生质粒丢失现象。结论 猪带绦虫TSO45W-4B基因、TSOL18基因和TSO45W-4B-TSOL18融合基因的重组Bb疫苗低温保存最稳定,常温最差,这为猪带绦虫重组Bb疫苗的进一步研究奠定了基础。     

Epidemiological study of Taenia solium taeniasis/cysticercosis in a rural village in Yucatan state, Mexico.

A survey to detect human taeniasis and cysticercosis was conducted in a community in Yucatan state, Mexico, an area endemic for Taenia solium. Information on the environmental, demographic and risk factors associated with transmission of T. solium within the community was recorded on questionnaires. Although no Taenia eggs or proglottides were found in the initial faecal samples collected from each of the 475 subjects, the results of a capture-ELISA for T. solium coproantigen were positive for 10 of the subjects (of both genders and various ages). After treatment with niclosamide, proglottides were detected in purge samples from seven of these 10 subjects. The prevalence of parasitologically confirmed taeniasis was therefore 1.5% (seven in 475). The other three ELISA-positive cases delayed supplying faecal material post-treatment, and it is nuclear whether they had expelled proglottides before providing the samples. All 10 ELISA-positive subjects became ELISA-negative after treatment. Seroprevalence of human cysticercosis, based on the detection in immunoblots of antibodies to antigens of 8- and 26-kDa from a crude saline extract of T. solium metacestodes, was 3.7% (i.e. five positives out of 134 subjects). None of the seropositive cases demonstrated clinical symptoms of infection. Again, the positive cases were of both genders and various ages. Although tongue palpation indicated that 17 (23%) of 75 pigs kept within the community had T. solium cysticercosis, the results of immunoblotting demonstrated antibodies to the 8- and/or 26-kDa antigens of T. solium in 26 (35%). The pigs allowed to roam throughout the community were far more likely to have cysticercosis than those kept in pens (odds ratio = 42, with a 95% confidence interval of 5.05-920.2; P < 0.00001). Not surprisingly, the risk factors associated with human taeniasis and cysticercosis included the eating of infected pork and close proximity to a carrier of T. solium. The main risk factor identified for porcine cysticercosis was free-range husbandry, permitting access to human faeces. This is the first comprehensive report of taeniasis and cysticercosis in a rural population from the Yucatan peninsula of Mexico.     

Taeniasis vs cysticercosis infection routes

Although cysticercosis caused by Taenia solium(T. solium) is considered a neglected disease, its life cycle has been well known for more than two centuries. T. solium not only causes cysticercosis but also taeniasis in humans. These two diseases have totally different infection routes. To acquire taeniasis(the presence of the adult stage of T. solium in the intestine), humans have to ingest the larval stage(cysticercus) that infects a variety of organs and viscera in pigs, its intermediate hosts. Therefore, taeniasis is acquired when eating raw or undercooked infected pork. The adult stage in the human intestine release eggs that contain a hexacanth embryo, the oncosphere. If humans accidentally ingest the eggs of T. solium, the released oncospheres penetrate the intestine and become cysticerci. Therefore, cysticercosis is acquired by the ingestion of eggs that contaminate water, vegetables, hands etc. These facts should not be forgotten to avoid misinformation in scientific publications.     

Cysticercosis/taeniasis in Asia and the Pacific

Three taeniid tapeworms infect humans in Asia and the Pacific: Taenia solim, Taenia saginata, and Taenia asiatica. Although there is continuing debate about the definition of a new species, phylogenetic analyses of these parasites have provided multiple lines of evidence that T. asiatica is an independent species and the sister species of T. saginata. Here we review briefly the morphology, pathology, molecular biology, distribution and control options of taeniasis/cysticercosis in Asia and the Pacific and comment on the potential role which dogs may play in the transmission of T. solium. Special attention is focused on Indonesia: taeniasis caused by T. asiatica in North Sumatra, taeniasis/cysticercosis of T. solium and taeniasis of T. saginata in Bali, and taeniasis/cysticercosis of T. solium in Papua (formerly Irian Jaya). Issues relating to the spread of taeniasis/cysticercosis caused by T. solium in Papua New Guinea are highlighted, since serological evidence suggests that cysticercosis occurs among the local residents. The use of modern techniques for detection of taeniasis in humans and cysticercosis in humans, pigs and dogs, with the possible adoption of new control measures will provide a better understanding of the epidemiology of taeniasis/cysticercosis in Asia and the Pacific and lead to improved control of zoonotic and simultaneously meat-borne disease transmission.     

抗猪带绦虫六钩蚴独特型抗体疫苗对猪体免疫保护作用的研究

目的　本研究在证实猪带绦虫六钩蚴抗原具有很高的免疫保护性的基础上 ,探讨六钩蚴抗独特型抗体作为六钩蚴抗原替代物对猪体的保护作用 ,为研制猪体囊虫疫苗提供理论基础。　方法　用六钩蚴粗制抗原免疫家兔 ,制备兔抗六钩蚴抗体 (Ab1) ,再用Ab1免疫昆明小白鼠 ,制备抗独特型抗体 (Ab2 ) ,免疫猪体 ,以 3节猪带绦虫孕卵节片攻击感染 ,90d后剖杀 ,观察感染情况。　结果　 5头试验猪只有 1头在膈肌发现 1个囊虫 ,而 2头阳性对照猪均被感染 ,各检查部位囊虫数平均为 4.5个 /10 0g和 3 .2个 /10 0 g。　 结论　抗猪带绦虫六钩蚴独特性抗体Ab2对猪体有较强的免疫保护作用。     

Identification of CD4+ T cell epitopes of Taenia solium paramyosin

T cell mediated response is involved in a protective immune response against experimental cysticercosis conferred by immunization with Taenia solium paramyosin (TPmy) to BALB/c mice. In this study, we analysed the TPmy amino acid sequence for predicted CD4+ T cells epitopes. Five different regions of this protein showed that the residues anchor to bind the I-Ad molecule, synthetic peptides containing these epitopes were evaluated for their ability to induce lymphoproliferative responses of spleen cells from TPmy immunized mice. Among them, Tp176 (amino acids 176-192 sequence DDLQRQMADANSAKSRL) was the immunodominant T cell epitope of TPmy. Delineation of this epitope should facilitate analysis of the role of CD4+ T cell response in experimental cysticercosis.