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。     

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     

Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory- secretory antigen

An excretory-secretory (ES) antigen was used in a serodiagnostic enzyme-linke immunosorbent assay (ELISA) for swine trichinosis. ELISA procedures included a double- antibody test, using either an anti-swine IgG or a protein A enzyme conjugate, and a triple-antibody test using a pig IgG heavy-chain specific second antibody with a conjugated third antibody. The ES antigen was effective in eliminating all false-positive reactivity in sera from farm-raised hogs. The triple-antibody procedure was more sensitive and demonstrated a greater efficiency in detecting positive animals and early seroconversions. Naturally-infected pigs with worm burdens as low as 0.01 larvae per gram (LPG) of diaphragm were seropositive using these procedures. Seroconversion in experimentally-infected animals receiving low doses of muscle larvae (500) occurred considerably later than in animals receiving high doses (10000). This might account for false-negative reactions in naturally-infected animals with very low (less than 0.1 LPG) worm burdens.     

Immunogenetic analysis of Trichinella spiralis infections in swine

The immune responses of outbred swine, inoculated with several different low doses of Trichinella spiralis muscle larvae (ML), was followed over 5-6 weeks of primary infection, in order to determine an inoculation dose which could be used to identify genetic controls on the response to this helminth parasite. Reproducible infections were established when swine were inoculated with 100-300 ML. Humoral antibody responses to different larval stages were evident at 4 weeks using enzyme-linked immunosorbent assay (ELISA) of antibody-binding to excretory-secretory (ES) products of ML, and flow cytometric (FCM) analysis of antibody-binding to newborn larvae. T-cell blastogenesis to T. spiralis ML antigens was predominantly in the CD4+, class II restricted, T-cell subset. Having established an appropriate inoculation dose, swine leukocyte antigen (SLA) inbred miniature swine were then inoculated with this low dose of T. spiralis ML, to determine whether major histocompatibility complex (MHC) genes regulate swine immune responses to T. spiralis, as has been found in rodent models. Preliminary evidence indicated that swine of the SLA c/c haplotype may exhibit a lower burden of T. spiralis larvae in the tongue and diaphragm. This lower muscle burden correlated with the earlier development of a humoral antibody response in these genetically-defined swine.     

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.

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.     

Field evaluation of the enzyme-linked immunosorbent assay for swine trichinosis: efficacy of the excretory-secretory antigen

A field evaluation of an enzyme-linked immunosorbent assay (ELISA) for swine trichinosis was done with sera obtained from 5 herds experiencing ongoing transmission of Trichinella spiralis. Epizootiologic studies conducted on these herds offered an opportunity to evaluate the accuracy of an ELISA, using larval T spiralis excretory-secretory antigens. Sera from 162 infected pigs and 143 serum samples from noninfected pigs originating from the same farms were tested. The infection status of the pigs was determined by digestion of diaphragm or tongue muscle samples. Two criteria were established to classify the ELISA optical density (OD) readings: Criterion I stated that an OD greater than or equal to 5 times the mean OD of several normal swine sera pools was positive; criterion II stated that a OD greater than or equal to 4 times the normal sera values was positive. The results obtained did not reveal obvious serologic variations among infected herds located in the 4 states involved. Overall, the test detected 93% (criterion I) and 96% (criterion II) of infected pigs. The majority of false-negative sera was from hogs that had less than 5 larvae/g of muscle; 1 hog had 73.8 larvae/g of diaphragm muscle. The false-positive rates were 8% for criterion I and 9% for criterion II. The actual rate for these false-positive samples may have been overestimated, because generally, only small tissue samples (0.4 to 10 g) were digested; larger sample sizes might have altered the results. The relevance of this qualification is that these pigs originated from herds with prevalence rates greater than 50%. Other factors that may account for occasional false-positive sera or false-negative sera in the swine trichinosis ELISA are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunity in swine inoculated with larvae or extracts of a pig isolate and a sylvatic isolate of Trichinella spiralis

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.     

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。     

Evaluation of ELISA and Western Blot Analysis using three antigens to detect anti-Trichinella IgG in horses

We assessed a serological method for detecting Trichinella infection in horses, specifically, an ELISA using three antigens to detect anti-Trichinella IgG (i.e. a synthetic tyvelose glycan-BSA (stg-BSA) antigen, an excretory/secretory (ES) antigen, and a crude worm extract (CWE) antigen). Serum samples were collected from 2502 horses (433 live horses from Romania and 2069 horses slaughtered in Italy and originating from Italy, Poland, Romania, and Serbia). Serum samples were also taken from horses experimentally infected with different doses of T. spiralis and T. murrelli larvae, as controls. The cut-off value of ELISA was determined on serum samples from 330 horses from Trichinella-free regions of Italy, which were also examined by artificial digestion of preferential-muscle samples. In the experimentally infected horses, the stg-BSA and ES antigens were less sensitive than the CWE antigen. Trichinella spiralis showed a higher immunogenicity than T. murrelli, and the IgG immunoresponse was dose-dependent. The kinetics of anti-Trichinella IgG were similar among all experimentally infected horses. No circulating antibodies were detected 4-5 months after experimental infection, although these horses still harbored infective larvae. Depending on the antigen used, for 4-7 of the 330 horses from Trichinella-free areas, the optical density (OD) of the serum sample was higher than the cut-off value, yet these samples were negative when subjected to Western Blot. Similar results were obtained for the 1739 horses slaughtered in Italy (originating from Italy, Poland, Romania, and Serbia) and the 433 live Romanian horses. Of the 4 horses with muscle larvae, only one was positive by ELISA and Western Blot. Because the anti-Trichinella IgG remain circulating for only a short period of time, whereas the larvae remain infective for longer periods, serology cannot be used for either diagnosing Trichinella infection in horses or estimating the prevalence of infection. Artificial digestion of at least 5 g of preferential-muscle tissue continues to be the method of choice at the slaughterhouse for preventing equine-borne trichinellosis in humans.     

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。     

Evaluation of different antigens in a seroepizootiological survey of trichinellosis by enzyme-linked immunosorbent assay

Antigen isolated from the large-particle fraction of the muscle Trichinella spiralis larvae (PAW), excretory/secretory (E/S) and crude worm extract (CWE) antigens were evaluated in a seroepizootiological survey of trichinellosis by the enzyme-linked immunosorbent assay (ELISA). The ELISA using PAW antigen yielded 16 positive animals (1.6%), E/S antigen revealed 21 (2.1%) positive, and the highest number of positive (23 or 2.3%) were obtained using CWE antigen. Parasitological post-mortem examination of all seropositive animals showed five and seven false-positive animals when E/S and CWE antigens were used, respectively.     

肌旋毛虫功能性抗原实验研究

本实验应用生化萃取技术和体外人工培养技术,制备了肌旋毛虫匀浆(HO)抗原和排泄—分泌物(ES)抗原,并对两种不同来源抗原的部分生化特性和免疫学特性进行了比较。HO抗原通过SephadexG—200又分为第一峰(FP)和第二峰(SP)。在5～20％的聚丙烯酰胺梯度凝胶电泳和薄层聚丙烯酰胺凝胶等电聚焦电泳上,ES和FP抗原出现许多条电泳区带。ES、FP和HO抗原对小鼠的保护力(肌幼虫减少率)分别为78％,76％和46％。     

旋毛虫肌幼虫ES Ag单克隆抗体的制备与特性鉴定

目的获得能够用于制备旋毛虫病快速检测试纸条的EsAg的单克隆抗体。方法应用旋毛虫肌幼虫排泄分泌抗原（ESAg）免疫诱导Balb／c小鼠,使小鼠产生较强的免疫应答,将免疫小鼠的脾细胞与NS0瘤细胞融合,利用Es45、ES49抗原通过间接ELISA对大量杂交瘤细胞培养上清的筛选,筛选出分泌高亲和力单克隆抗体的4株杂交瘤细胞。结果ES45和ES49（分子质量分别为45ku,49ku）分别被Ts-2D4、Ts-4C5和Ts-4H6、Ts-2G8单克隆抗体识别,与猪肺丝虫（Metastrongylus pudendotectus．MP）、囊虫（Cysticercus cellulosae,CC）、细颈囊尾蚴（Cysticercus tenuicollis,CT）、蛔虫（Ascaris suum,AS）、弓形虫（Toxoplasma gondii,TG）、住肉孢子虫（Sarcocystis miescheriana,SM）抗原反应测试表明,4株单抗与参试抗原均无交叉反应,所有单抗上清ELISA平均效价为1：1120,腹水ELISA平均效价为1．1×10^6,亲和力常数的平均值为6．12×10^9 L／mol。以金标免疫吸附试纸条原理为基础,利用制备的单抗成功研制了旋毛虫病快速检测试纸务,操作快速、无需设备及试剂,可以作为旋毛虫病的实时监测工具。结论ESAg单抗用于制备旋毛虫病快速诊断试纸条是理想的试剂。     

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.     

Evaluation of the ELISA for the serological diagnosis of trichinosis in Canadian swine

An ELISA using a Trichinella spiralis spiralis excretory-secretory antigen was evaluated as a procedure for the diagnosis of trichinosis in swine in Canada. Field and experimental trials were carried out using both indirect serological (ELISA) and direct parasitological (pepsin-digestion) methods concurrently on serum and musculature, respectively, from each animal. The ELISA is a sensitive and specific test for the detection of Trichinella antibodies in porcine sera when present. The development of Trichinella antibodies appears to be dependent on the magnitude of the infection established, age of the infection when the animal is tested and the immunocompetence or response to infection of individual animals. False negative reactions were recorded in both field and experimental trials. In the field study, five of the 1009 swine examined were parasitologically positive with light infections ranging from 0.01 to 0.046 larvae per gram (la/g) of musculature yet all were serologically negative. Experimentally it was shown that Trichinella antibodies develop slowly, at least two to three months postinfection, in pigs with very light infections. Even in pigs which developed infections of 33 to 55 la/g of musculature, seroconversion occurred greater than 23 and less than 30 days postinfection. The immunocompetence or response to infection of individual pigs was variable as illustrated by one pig inoculated with 3000 infective larvae which had consistently lower titers compared to others in the same group despite the establishment of a muscle infection of 8.5 la/g of musculature. One false positive reaction was recorded in the experimental trial in an animal which had received 100 larvae and seroconverted at about three months postinfection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seroepidemiology of trichinellosis in Florida swine

Surveys for swine trichinellosis, conducted in Florida using a commercial enzyme-linked immunosorbent assay (ELISA) kit with a modified protocol for screening and Western blot as a definitive test, revealed significantly different seroprevalences of 0.3% (four of 1294 samples) and 2.8% (five of 179 samples) in domestic breeding swine and feral swine, respectively.

Seropositive swine were identified in four of 114 (3.5%) domestic swine herds and in three of six feral swine regions. The location of two domestic herds with seropositive swine within approximately 2 miles of one another, and adjacent to a region where a seropositive feral hog was identified, suggests that a focus of trichinellosis exists in this area. The occurrence of singleton reactors in domestic herds may be explained by the absence of the common modes of transmission and suggests that swine were infected incidentally by feeding on the infected carcasses of small wild mammals or rodents. Although the presence of Trichinella spiralis spiralis isolates cannot be excluded, it is more likely that a sylvatic Trichinella isolate occurs in Florida swine. This conclusion is supported by the low seroprevalence in domestic swine, the failure to detect larvae in domestic or feral swine and the high prevalence in Florida panthers.     

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.     

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...     

抗旋毛虫单克隆抗体细胞林的建立及其特性鉴定

为筛选旋毛虫保护性抗原,建立了4株分泌抗旋毛虫McAb的细胞株。经连续培养30余代,仍稳定地分泌抗体。其中,2G3和2C10的靶抗原为旋毛虫幼虫表膜。免疫球蛋白 型及亚 鉴定表明,2G3和2C10为IgG_1,1D5为IgG_3,1C9为IgM。除2C10和IC9对伊氏锥虫可溶性抗原呈现阳性反应外,未见对猪圆线虫、猪囊尾蚴、伊氏锥虫表膜抗原和正常猪肉反应。经ELISA测定,2G3、2C10、1D5、1C9均可与施毛虫幼虫可溶性抗原作用,其腹水效价分别为1:50000、1:10000、1:1000、1:800。各McAb均与旋毛虫幼虫排泄分泌物抗原(ES抗原)作用,经ELISA测定表明,2G3腹水效价达1:320000。     

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.