旋毛虫丝氨酸蛋白酶抑制剂定位及免疫保护效果评估

探讨旋毛虫丝氨酸蛋白酶抑制剂(Ts-serpin)的抗原性、定位及免疫保护性。将本实验室前期构建的旋毛虫丝氨酸蛋白酶抑制剂的原核表达载体进行大量表达纯化。利用不同感染时间的猪旋毛虫阳性血清,通过Western blot方法对纯化后的重组丝氨酸蛋白酶抑制剂(rTs-serpin)进行反应原性鉴定,并制备多克隆抗体,用间接免疫荧光法检测Ts-serpin在旋毛虫肌幼虫中的定位。随后将rTs-serpin免疫小鼠进行免疫保护效果评估。结果显示:rTs-serpin可被不同感染时间的猪旋毛虫阳性血清特异性识别,表明rTsserpin是高反应原性抗原;Western blot结果显示旋毛虫排泄分泌物和旋毛虫虫体粗提取物均存在Ts-serpin,间接免疫荧光显示Tsserpin定位在旋毛虫肌幼虫表皮中;免疫保护试验结果显示rTs-serpin免疫后的小鼠旋毛虫肌幼虫减虫率约为32.2%。综上所述,Tsserpin主要定位在旋毛虫肌幼虫表皮,rTs-serpin具有较强的抗原性,且对小鼠具有免疫保护作用。     

旋毛虫肌幼虫p53cDNA的克隆及鉴定

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。     

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。     

旋毛虫鸟氨酸脱羧酶对旋毛虫抗酸能力调控的研究

为探究旋毛虫体内是否存在鸟氨酸依赖性抗酸系统(AR5)及旋毛虫鸟氨酸脱羧酶(TsODC)对旋毛虫抗酸能力的调控作用，本实验分别采用p H1.5、p H2.5、p H4.5、p H6.6的培养液以及PBS (pH7.4)作为对照组分别培养0.5 h、1 h和2 h后，通过统计旋毛虫肌幼虫的存活率、采用荧光定量PCR (qPCR)和western blot，分别筛选旋毛虫肌幼虫体外最适酸处理条件。结果显示，在酸度为p H2.5培养2 h时，肌幼虫的存活率为51%(此时死亡数与存活数基本相等)及Ts ODC基因的转录和表达水平最高。因此，p H2.5培养2 h为旋毛虫肌幼虫体外最佳酸处理条件。于培养液中分别添加不同浓度的精氨酸、Ts ODC多抗血清、姜黄素和雷帕霉素，以PBS作为对照组分别培养12 h、24 h和48 h后，采用上述最佳酸处理条件处理后，通过计算肌幼虫存活率，采用q PCR检测Ts ODC基因的转录水平并初步筛选不同制剂的最佳培养浓度及时间，并在各最佳浓度和时间培养后，再经最佳酸处理条件处理，采用western blot检测Ts ODC蛋白的表达，分析各制剂对旋毛虫抗酸能力的...     

不同发育时期旋毛虫脂肪酸组成的研究

为探讨旋毛虫生长发育过程中脂肪酸组分的变化及外界环境对其影响,本研究采用贝尔曼氏装置分别收集旋毛虫肌幼虫和成虫,同时,将少量骨骼肌和肠黏膜进行酯化处理,通过气相色谱质谱联用技术进行分析。结果显示:肌幼虫和成虫的脂肪酸组分涵盖了C12～C22的22种,主要为16、18和20碳脂肪酸,但旋毛虫肌幼虫与成虫的各脂肪酸相对含量有明显差异(p0.05)。分别对旋毛虫肌幼虫和成虫及其各自寄生部位骨骼肌和肠黏膜中的脂肪酸成份进行比较分析,结果显示:各脂肪酸组成相似,但在相对含量上却有显著差异(p0.05)。表明不同发育时期旋毛虫的脂肪酸组成在种类和相对含量上均存在明显差异,这可能与其所处的不同宿主环境和不同发育时期虫体的特殊生理结构有关。     

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     

旋毛虫P53ES抗原基因的克隆及真核表达

应用RT-PCR方法扩增旋毛虫ES抗原P53基因,构建P53基因绿色荧光蛋白表达载体pEGFP-N1-P53,利用脂质体法将其转染哺乳动物细胞COS-7.于转染24 h后在荧光显微镜下观察到发绿色荧光的转染细胞;同时通过westen blot分析证实了P53基因表达产物的抗原性.结果表明,P53基因在COS-7细胞中表达获得了表达,而且其融合蛋白具有免疫原性.     

旋毛虫感染小鼠对p46 000重组抗原的抗体应答

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。     

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。     

旋毛虫P53ES重组蛋白对小鼠的免疫保护作用

为进一步探讨旋毛虫P53基因原核表达重组蛋白的免疫保护作用,本实验采用纯化的重组蛋白作为抗原免疫小鼠,免疫剂量为50μg/100μL,每隔10d免疫1次,共3次。末次免疫后10d,每只小鼠以200条旋毛虫肌幼虫攻虫。分别检查感染后7d小鼠肠道内成虫数量、体外培养雌虫所产新生蚴数量以及感染后35d肌肉荷虫量。结果显示:小鼠肠道内成虫、新生蚴和肌幼虫的减虫率分别为61.9%、53.45%和71.48%,表明旋毛虫表达P53基因的重组蛋白对小鼠产生了较好的免疫保护效果。     

Anti-Trichinella antibodies detected in chronically infected horses by IFA and Western blot, but not by ELISA

In the Balkan countries, where trichinellosis is a re-emerging zoonosis, it is of great importance to determine Trichinella infection prevalence among the major hosts, including horses. One method for monitoring prevalence is serological surveillance; however, the validity of serological methods in horses is not well understood. The dynamics of anti-Trichinella IgG production and circulating excretory/secretory (ES) antigens were investigated in three horses experimentally-infected with Trichinella spiralis. Horses were slaughtered at 32 week post infection (p.i.). Low worm burdens were found in all three animals. Anti-Trichinella IgG was detected up to 32 weeks p.i. by an indirect immunofluorescence assay (IFA) and by Western blot (Wb), but not by ELISA. The ELISA test detected antibodies for only a short period of time (up to 18 weeks p.i. using ES antigen or up to 20 weeks p.i. using tyvelose-BSA antigen). The presence of circulating muscle larvae ES antigen in sera of infected horses was observed by dot blot from the 4th week p.i. up to the 32nd week p.i.     

青海省部分地区商品猪旋毛虫感染的血清学调查

以旋毛虫肌幼虫排泄分泌)抗原作为检测抗原,采用间接酶联免疫吸附试验(ELISA)法,分别对采集自青海省西宁市张氏生猪屠宰场、互助县屠宰场、平安县屠宰场的商品猪血清进行旋毛虫抗体检测,共检查猪血清1 065份.结果阳性血清为19份,阳性率为1.78％.可见在青海省商品猪中旋毛虫具有一定的感染率,动物卫生监督以及肉品检疫部...     

Persistence of reactivity against the 45 k Da glycoprotein in late trichinellosis patients

Over the years, the opinions of clinicians on the existence of the so-called chronic trichinellosis or late sequelae of infection have differed. However, the persistence of a humoral immune response against Trichinella in these late-stage patients has been confirmed using specific tests such as the competitive inhibition assay (CIA). We evaluated sera from late-stage trichinellosis patients (2--8 years from acute infection), for their reactivity against Trichinella spiralis antigens. The following tests were carried out: (i) indirect immunofluorescence assay (IFA), performed on muscle sections from mice, 30 days following synchronous infection by intramuscular injection with T. spiralis newborn larvae (NBL); (ii) enzyme immunoassay, employing a synthetic beta-tyvelose antigen conjugated to bovine serum albumin (BSA-Ag); and (iii) western blot (WB) with both an "in house" kit and a commercial kit. The results of IFA obtained by confocal laser microscopy showed that sera reacted against both surface and internal structures of L(1) larvae but at varying levels. Employing the synthetic antigen, EIA showed that 50% of sera tested were positive for the presence of specific antibodies against beta-tyvelose. By WB, all sera were reactive with the 45 k Da glycoprotein (45 gp). These data suggest that reactivity against the beta-tyvelosylated 45 gp persists even in very late stages of human trichinellosis.     

五种旋毛虫抗原对猪的免疫保护作用研究

本实验研究了旋毛虫肌幼虫可溶性粗抗原、排泄分泌抗原（ＥＳ）、表面抗原（ＳＡ）及成虫ＥＳ、ＳＡ５种抗原对猪的免疫保护作用。结果５种抗原对猪均具有一定程度的免疫原性，可诱导猪体产生对攻击感染的抵抗力（减虫率），其中肌幼虫粗抗原为５５．２０％；肌幼虫ＥＳ为４２．５６％，肌幼虫ＳＡ为７２．２１％；成虫ＥＳ为３２．９２％；成虫ＳＡ为４２．１７％。免疫５种抗原后用肌幼虫“Ｂ”抗原、新生幼虫可溶性抗原及成虫可溶性抗原进行ＥＬＩＳＡ检测，均可测出血清抗体应答反应，其中以相应抗原测出的抗体应答较强烈。免疫５种抗原后猪外周血液中Ｂ淋巴细胞减少，Ｔｈ及Ｔｓ增加，Ｔｈ／Ｔｓ比值降低，呈暂时的细胞免疫抑制现象。     

Differential detection of Trichinella papuae, T. spiralis and T. pseudospiralis by real-time fluorescence resonance energy transfer PCR and melting curve analysis

Trichinellosis caused by nematodes of Trichinella spp. is a zoonotic foodborne disease. Three Trichinella species of the parasite including Trichinella spiralis, Trichinella papuae and Trichinella pseudospiralis, have been etiologic agents of human trichinellosis in Thailand. Definite diagnosis of this helminthiasis is based on a finding of the Trichinella larva (e) in a muscle biopsy. The parasite species or genotype can be determined using molecular methods, e.g., polymerase chain reaction (PCR). This study has utilized real-time fluorescence resonance energy transfer PCR (real-time FRET PCR) and a melting curve analysis for the differential diagnosis of trichinellosis. Three common Trichinella species in Thailand were studied using one set of primers and fluorophore-labeled hybridization probes specific for the small subunit of the mitochondrial ribosomal RNA gene. Using fewer than 35 cycles as the cut-off for positivity and using different melting temperatures (T(m)), this assay detected T. spiralis, T. papuae and T. pseudospiralis in muscle tissue and found the mean T(m) ± SD values to be 51.79 ± 0.06, 66.09 ± 0.46 and 51.46 ± 0.09, respectively. The analytical sensitivity of the technique enabled the detection of a single Trichinella larva of each species, and the detection limit for the target DNA sequence was 16 copies of positive control plasmid. A test of the technique's analytical specificity showed no fluorescence signal for a panel of 19 non-Trichinella parasites or for human and mouse genomic DNA. Due to the sensitivity and specificity of the detection of these Trichinella species, as well as the fast and high-throughput nature of these tools, this method has application potential in differentiating non-encapsulated larvae of T. papuae from T. spiralis and T. pseudospiralis in tissues of infected humans and animals.     

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.     

Re-evaluation of patients involved in a trichinellosis outbreak caused by Trichinella britovi 15 years after infection

This study re-evaluates 13 out of 48 subjects involved in a trichinellosis outbreak that occurred in Central Italy (Umbria Region) in 1988 resulting from the consumption of raw boar meat harboring Trichinella britovi. During the outbreak, 28 of 48 serologically positive subjects were asymptomatic, whereas 20 subjects presented one or more clinical signs including but not limited to fever, myalgia, periorbital oedema and conjunctivitis. Several patients were hospitalized with severe clinical signs requiring treatment with mebendazole and corticosteroids. Upon re-evaluation of 13 patients, none presented clinical signs; however, three still had increased CPK or LDH serum levels with some signs of electromyographic changes. In this study, enzyme immunoassays (EIA) were used to test the 13 positive sera for reactivity with T. britovi antigens using both excretory/secretory (E/S) antigens and a synthetic antigen composed of beta-tyvelose conjugated to bovine serum albumin. Western blots (WB) were also carried out using a commercial kit. Studies using EIA with E/S antigen identified five positive sera; however, using beta-tyvelose as antigen, only one positive sample was identified. Nearly all sera reacted positively with one or more Trichinella antigens when analyzed by WB, in particular to the 45 k Da beta-tyvelose containing glycoprotein. Results indicate that T. britovi, though less pathogenic than other Trichinella species, is clearly capable of inducing sustainable sequelae.     

旋毛虫排泄/分泌(ES)抗原致鼠胸腺淋巴细胞凋亡的研究

本试验首次以体外培养的小鼠胸腺淋巴细胞为实验对象,加入旋毛虫（Trichinella spiralis）肌幼虫ES抗原做刺激物,通过对鼠胸腺淋巴细胞DNA损伤、凋亡水平的检测,进而证明旋毛虫肌幼虫ES抗原能够诱导鼠胸腺淋巴细胞发生调亡。掌握这种免疫细胞凋亡（apoptosis）发生的过程,对分析免疫应答的特点和调控,以及探索旋毛虫病（Trichinosis）的发病机制和提供防治对策都具有重要意义。