Cross-reactions between crude antigens of larval Taenia solium (Cysticercus cellulosae) and other helminths of pigs

A crude antigen extract of larval Taenia solium was shown by immunodiffusion (ID) and immunoelectrophoresis (IEP) to cross-react with rabbit antisera against pig serum proteins and larval T. hydatigena, and by enzyme-linked immunosorbent assay (ELISA) with antisera against pig serum proteins, Fasciolopsis buski, larval T. hydatigena, hydatid cyst, Hymenolepis diminuta and Dipylidium caninum. Immunoblotting demonstrated that the crude antigens extract contained epitopes of pig serum proteins of 48 and 66 kDa. The crude extract also contained a subunit of antigen B (95 kDa) which was also found in T. hydatigena and H. diminuta. Immunoperoxidase and indirect immunofluorescence studies showed that cross-reacting antigens were distributed mainly on the tegument of T. solium.     

猪带绦虫六钩蚴45W-4B重组蛋白的免疫原性研究

目的观察猪带绦虫六钩蚴45W-4B抗原对仔猪的免疫保护作用。方法用IPTG诱导表达融合蛋白GST-45W-4B。用GST柱进行纯化，以Montanide ISA 206为佐剂制成疫苗免疫仔猪。间接ELISA检测仔猪血清抗45W-4B IgG抗体水平，MTT法进行外周血淋巴细胞殖试验。免疫1个月后全部实验组经口攻击感染25000彬头猪带绦虫虫卵，攻虫3个月后剖检，计算减虫率及保护率。结果免疫组注射疫苗的第2周起，抗体为阳性，45W-4B组持续升高至第8周，淋巴细胞增殖明显高于未免疫感染组。用该抗原制备的疫苗获得了95％的减虫率和33％的保护率。结论该抗原对猪囊尾蚴病具有很好的免疫保护作用。     

猪囊尾蚴重组抗原和基因工程疫苗的研究进展

猪囊尾蚴病（Cysticercosis）是由猪带绦虫的幼虫囊尾蚴寄生于人或猪等而引起的人畜共患寄生虫病,是公认的世界经济病之一。严重威胁着人体健康,并给畜牧业造成重大经济损失,猪囊尾蚴病的免疫防治势在必行。然而在猪囊尾蚴病疫苗研究中,疫苗抗原的选择和来源一直困扰着兽医工作者。该文就近年来猪囊尾蚴病诊断重组抗原和基因工程疫苗的分子生物学研究进展进行了综述。     

Isolation of a 14 kDa antigen from Taenia solium cyst fluid by HPLC and its evaluation in enzyme linked immunosorbent assay for diagnosis of porcine cysticercosis

A fraction with a major band of 14kDa was obtained from crude cyst fluid of Taenia solium cysticerci by 2-step chromatography. A first fraction isolated by gel filtration (Sephacryl S-300 high resolution) was purified using an anion exchange column (Mono Q HR 5/5) on high performance liquid chromatography. Evaluation of the analytic sensitivity of this fraction (F3) was carried out in an antibody enzyme linked immunosorbent assay (Ab-ELISA-F3) using serum samples from pigs experimentally infected with different doses of T. solium eggs. The cross-reactivity of F3 was evaluated with serum samples from pigs that were naturally or experimentally infected with Taenia hydatigena, Taenia saginata asiatica, Fasciola hepatica, Trichinella spiralis, Metastrongylus apri, Trypanosoma congolense and Sarcoptes scabiei, and with serum samples of rabbits hyper-immunised with metacestode cyst fluid of T. hydatigena and T. solium. Antibody titres of lightly or heavily infected pigs differed in their kinetics. However, the increase in F3-specific antibodies could not be related to the infection level. Analysis of the specificity of the F3 showed that serum samples of pigs infected with other parasites did not recognise this antigen. Cross-reaction with T. hydatigena occurred in ELISA using cyst fluid as antigen, but the F3 antigen fraction was not recognized by rabbit hyper-immune serum samples to T. hydatigena. Evaluation of the diagnostic sensitivity and specificity of the Ab-ELISA-F3 was done by a non-parametric receiver operating characteristic (ROC) analysis using 66 serum samples from Zambian village pigs. The total number of cysticerci of these pigs was determined by dissection (28 pigs harboured T. solium cysticerci and 38 were negative at dissection). In addition, 58 serum samples from Cameroonian pigs (28 pigs from cysticercosis-free farms and 30 pigs with cysticerci at tongue inspection) were used in a separate ROC analysis. The results from the ROC analysis yielded a low diagnostic value (area under ROC curve=0.48) with the sera from the Zambian pigs while a relatively high diagnostic value was obtained with the sera from Cameroonian pigs (area under ROC curve=0.78). The main factor contributing to a low diagnostic value based on the Zambian serum samples seemed to be the false-positive reactions that were likely caused by the occurrence of transient antibodies in the non-infected animals.     

Serodiagnosis of porcine cysticercosis by enzyme-linked immunosorbent assay (ELISA) using fractionated antigens

The sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Taenia solium cysticercosis was evaluated in experimentally and naturally infected pigs, using T. solium larval scoleces and its fractionated 1st and 2nd peaks on Sephadex G-200 as antigens. First peak antigen gave maximum sensitivity and highest antibody titres. The overall sensitivity of this test was found to be 91.5, 95.8 and 70.8% with scolex, 1st and 2nd peak antigens, respectively. False positive reactions occurred in 9.09% of uninfected pigs with scolex and 1st peak antigens and cross-reactions occurred in 25% of Taenia hydatigena-infected animals using scolex and 2nd peak antigens. No cross-reaction was observed using 1st peak antigen. The specificity of the test was 92.3, 96.2 and 92.3% with scolex, 1st and 2nd peak antigens, respectively.     

The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs

Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.     

Risk factors associated with porcine cysticercosis in selected districts of Eastern and Southern provinces of Zambia

To determine the risk factors associated with Taenia solium transmission in humans and pigs in the rural areas of Eastern and Southern provinces of Zambia, a questionnaire was administered in 788 households from 155 villages. Pigs were examined from 800 households. Tongue examination and enzyme-linked immunosorbent assay (Ag-ELISA) for the detection of circulating antigens of T. solium cysticerci were used to measure infection in pigs. A snowballing technique was utilised to select households with pigs. Prevalence of households with pigs infected with T. solium on tongue examination by district ranged from 12.7% to 32.1% with Ag-ELISA having a range of 30.0-51.7%. Of the total number of households visited, 18.8% and 37.6% had at least one pig positive for porcine cysticercosis on tongue examination and Ag-ELISA, respectively. Risk factors associated with T. solium infection were lack of pork inspection at slaughter (96.7%), consumption of pork with cysts (20.1%), selling of pork infected with T. solium cysticerci (18.3%), free-range husbandry system (83.2%) and absence of latrines (58.0). Free-range husbandry system (OR=1.68; 95% CI=1.36-2.07) was a significant risk factor for porcine cysticercosis in the surveyed areas. The result that pigs were mostly kept on free-range and semi-intensive husbandry systems may have permitted them to have access to eating human faeces that could be contaminated with tapeworm eggs. This study has shown that T. solium infection poses a high public health risk in the study areas and urban areas as well. We recommend that a human survey be conducted to verify the human exposure to taeniasis and/or cysticercosis in Zambia.     

Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96. 4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose.     

异源性抗原抗猪囊尾蚴感染的研究

本文报道了泡状带绦虫活化六钩蚴的超声裂解抗原可以诱导猪体产生抗猪带绦虫攻击感染的交叉保护作用。猪囊尾蚴匀浆抗原也使猪体产生了较强的抗猪带绦虫攻击感染的保护作用。泡状带绦虫六钩蚴超裂抗原免疫组与猪囊尾蚴匀浆抗原免疫组的保护情况是相似的,这表明异源免疫也可使猪体产生较好的抗猪囊尾蚴感染的免疫。由于制备异源性抗原的泡状带绦虫能够从狗的体内获得,因此在体外培养猪带绦虫未获成功之前可以解决从人体获取猪带绦虫的困难。     

Detection of Taenia solium Cysticercosis in Pigs by ELISA with an Excretory-Secretory Antigen

Serodiagnosis of Taenia solium cysticercosis in pigs was conducted by an enzyme-linked immunosorbent assay with an excretory-secretory antigen. The antigen was obtained by in vitro cultivation of the cysticerci in a synthetic medium RPMI 1640. The sensitivity and specificity of the ELISA in detecting infection in pigs reared on free range was 92% and 100%, respectively. In addition, 33.33% of pigs in which infection could not be detected at meat inspection were found positive by ELISA. However, none of the sera from a group of farm-reared pigs were positive. No cross reactions were observed in pigs that contained either the cysticerci of Taenia hydatigena or hydatid cysts.     

Prevalence of Taenia solium porcine cysticercosis in the Eastern, Southern and Western provinces of Zambia

Tongue examination and detection of circulating antigen (Ag-ELISA) were used to establish the prevalence of Taenia solium porcine cysticercosis in free-range pigs in selected districts of Eastern, Southern and Western provinces of Zambia, and to determine if prevalence of porcine cysticercosis was associated with age, breed and sex. Households with pigs were identified using the snowballing technique. A total of 1691 pigs were examined out of which 183 (10.8%) were positive on tongue examination. Ag-ELISA gave a sero-prevalence of 23.3%. When considering the factors in a logistic regression analysis, only breed type was significantly associated with porcine cysticercosis (OR=0.72; 95%CI=0.63-0.81). The crossbred pigs were 72% more likely to have had cysticercosis than the Nsenga (dwarf local) breed as determined by Ag-ELISA. The result that crossbred pigs had a higher prevalence of T. solium cysticercosis suggests that pig breeds may display different susceptibility to cysticercosis. The limited use of latrines in these areas implies that people use the nearby bush for defecation, resulting in pigs having access to human faeces. Therefore, investigation of taeniosis and cysticercosis in humans is warranted to better comprehend the local epidemiology and transmission risks. This should then be followed by extension programs to communities so that the control plans that could be instituted are more sustainable.     

Vaccination against Taenia solium cysticercosis in underfed rustic pigs of México: roles of age, genetic background and antibody response

Vaccination of pigs of mixed genetic make-up, raised as rustically as done in rural Mexico, resulted in effective protection to experimental challenge against Taenia solium cysticercosis. Maximum protection was achieved if pigs were immunized at 70 days of age. There was large variation of viable parasite load within vaccinated pigs and controls, which is suggestive of significant genetic factors influencing susceptibility, besides immunization. Our results strengthen the advisability of pig vaccination for control of T. solium cysticercosis, since it lowers the number of viable cysticerci capable of transforming into tapeworms.     

Humoral responses in rabbits immunized with two fractions of Haemonchus contortus: the antigen specificity of such antibodies.

Humoral responses were examined in rabbits immunized with either 28-40 kDa (Fraction 1) or a 19-24 kDa (Fraction 2) antigenic fraction from soluble antigens (Sol L3 Ag) from infective larvae (L3) of Haemonchus contortus. These fractions were eluted from electrophoretically separated Sol L3 Ag. Immunoblots revealed antibodies to Fraction 1 (fr. 1) or Fraction 2 (fr. 2) polypeptides as well as to several other molecular weight polypeptides of the Sol L3 Ag. The latter antibodies were shown by absorption studies not to be Sol L3 Ag cross-reactive anti-bacterial rabbit antibodies. When Sol L3 Ag was affinity-purified using monoclonal antibody to phosphorylcholine (PC) and the resulting fractions were further analysed by immunoblotting using rabbit anti fr. 1 or anti fr. 2 antiserum, the PC antigen was found to be shared between fr. 1 and other polypeptides of Sol L3 Ag. Using the rabbit antibody fractions eluted from nitrocellulose membranes containing fr. 1 or 2 polypeptides, it was found that these fractions contained antibody that bound mainly to fr. 1 and only to fr. 2 polypeptides of Sol L3 Ag. It is concluded that, from the present immune rabbit sera, antibodies specific for either fr. 1 or fr. 2 may be isolated and then used to purify small amounts of the corresponding antigens.     

Evaluation of tongue inspection and serology for diagnosis of Taenia solium cysticercosis in swine: usefulness of ELISA using purified glycoproteins and recombinant antigen

Evaluation of serology using glycoproteins (GPs) purified by preparative isoelectric focusing (pH 8.8) and recombinant chimeric antigen (RecTs) of Taenia solium was carried out using (1) blood samples on filter papers from pigs infected with different doses of eggs of T. solium in Mexico, (2) serum samples from pigs found infected naturally in Vietnam and Ecuador and (3) serum samples from pigs suspected to be infected with T. solium by tongue inspection in Tanzania. Antibody responses (IgG) were detectable in experimentally infected pigs confirmed harbouring 16 or more cysts at necropsy from 30 days after egg inoculation. One of three pigs naturally infected and harbouring 2.5 cysts/kg muscle and most of pigs harbouring=5.0 cysts/kg were also seropositive by ELISA. Although pigs may be infected with other taeniid species such as Taenia hydatigena, pigs harbouring this parasite were negative in ELISA. Approximately, 76 and 78% of sera from pigs having nodule(s) in the tongue (positive tongue inspection) were serologically positive by both ELISA and immunoblot, respectively. Furthermore, approximately 34 and 18% of sera from pigs having no nodules in the tongue (negative tongue inspection) were also seropositive by ELISA and immunoblot, respectively. ELISA using the two antigens was more sensitive than immunoblot and reliable for differentiation of pigs infected with cysticerci of T. solium from those either uninfected or infected with other taeniid species. Pigs without nodule by tongue inspection should be checked serologically in endemic areas.     

Epidemiologic observations on porcine cysticercosis in a rural community of Michoacan State, Mexico.

The prevalence of and risk factors for Taenia solium infection (cysticercosis) in pigs were studied in a rural community in Michoacan State, Mexico. Visual inspection of the tongues of 216 pigs revealed cysticerci in 14 (6.5%). The prevalence was slightly higher in male (10/105) than female pigs (4/110) (P = 0.06) and increased with age (P less than 0.05). The most important risk factors for infection in pigs were access to human feces at a public washing area (P = 0.004), the presence of an indoor latrine (P = 0.05) and indiscriminate disposal of human feces around the pig owner's household (P less than 0.1). Effective and long-lasting control of the transmission of T. solium from humans to pigs must include measures to deny pigs access to human feces, a change which is likely to be resisted because of the traditional and functional aspects of established pig-rearing practices.     

青海省猪囊尾蚴病血清学调查

猪囊尾蚴病又称猪囊虫病,是由人的有钩绦虫(猪带绦虫)的幼虫——猪囊尾蚴寄生在猪的肌肉中所引起一种人畜共患寄生虫病。为了摸清青海省猪囊尾蚴感染情况,对来自10个县市的788份猪血清样品采用ELISA方法进行了血清学检测,结果检出阳性样品17份,阳性率为2.16%。     

Performance of the ELISA test for swine cysticercosis using antigens of Taenia solium and Taenia crassiceps cysticerci

Studies were conducted to evaluate antigens of Taenia solium (Tso) and Taenia crassiceps (Tcra) cysticerci in the ELISA test for the diagnosis of swine cysticercosis. The samples analyzed were cysticercosis positive and negative control sera and heterologous sera. Four antigens were assayed: vesicular fluid (VF) and crude (T) Tcra and scolex (S) and crude (T) Tso. All antigens showed good performance, but VF-Tcra was the best followed by T-Tcra. Sensitivity rates of ELISA were respectively, in 2nd and 3rd standard deviation cut-offs, 96.0 and 80.0% for the VF antigen and specificity of 97.5 and 100.0%. Cross-reactivity was verified only for hidatidosis and ascaridiosis. Due to the high performance observed, the ELISA test using Tcra antigens should be recommended for the diagnosis of swine cysticercosis.     

A Slaughter Slab Survey for Extra-Intestinal Porcine Helminth Infections in Northern Tanzania

A survey on extra-intestinal porcine helminths was conducted at three slaughter slabs that receive pigs from Mbulu, a district endemic for porcine cysticercosis in northern Tanzania. Seventy carcases of pigs between 1 and 2 years old were examined between December 1997 and March 1998. The examination involved ante-mortem lingual examination for Taenia solium cysticercosis followed by post-mortem inspection. In addition, a laboratory procedure was performed to determine whether any of these domestic pigs were infected with Trichinella species. Parasites detected were Ascaris suum (44.3%), Echinococcus granulosus (4.3%) and Taenia hydatigena (1.4%). The lack of cases of porcine cysticercosis in this study compared to previous studies suggests that pig traders are conducting their own ante-mortem lingual examinations before purchasing pigs in the rural communities where the parasite is still highly prevalent. It is concluded that improved meat inspection could prove useful in reducing the local population's risk of infection with these parasites. The results of this study have revealed the parasites of agricultral and public health importance in the targeted communities. Further epidemiological investigations are required to better determine parasite prevalence and impact in order to formulate appropriate and cost-effective strategies for control.     

Impact of naturally acquired Taenia solium cysticercosis on the hormonal levels of free ranging boars

In chronically infected BALBc/AnN male mice, Taenia crassiceps cysticercosis induces changes in the host's sex steroids hormone that lead to their estrogenization and deandrogenization, with possible repercussions on their susceptibility to infections. Here reported are the serum steroid levels in free range cysticercotic male boars. Therefore, the possible effects of Taenia solium cysticerci over the pig steroid levels were evaluated. Herein are described the sex steroids and cortisol levels of non-cysticercotic (n=25) and cysticercotic (n=22) adult boars, as diagnosed by tongue inspection, all free-ranging in a typical village of an endemic rural area in Mexico. A significant reduction of testosterone (P=0.022) and a likely one of 17beta-estradiol (P=0.08) levels were found in the cysticercotic boars in comparison with those non-cysticercotic, whilst no significant differences in the cortisol and DHEA levels were detected. Serum levels of specific antibodies did not correlate with infection nor with the levels of any of the hormones measured. Results suggest that T. solium cysticercosis significantly affects the hormonal status of its porcine host independently of their antibody response.     

Vaccination against cestode parasites: anti-helminth vaccines that work and why

Highly effective recombinant vaccines have been developed against the helminth parasites Taenia ovis, Taenia saginata and Echinococcus granulosus. These vaccines indicate that it is possible to achieve a reliable, high level of protection against a complex metazoan parasite using defined recombinant antigens. However, the effectiveness of the vaccines against the taeniid cestodes stands in contrast to the more limited successes which characterise attempts to develop vaccines against other platyhelminth or nematode parasites. This review examines the features of the host-parasite relationships among the taeniid cestodes which have formed the basis for vaccine development. Particular consideration is given to the methodologies that have been used in making the cestode vaccines that might be of interest to researchers working on vaccination against other helminths. In developing the cestode vaccines, antigens from the parasites' infective larval stage contained within the egg (oncosphere) were identified as having the potential to induce high levels of protection in vaccinated hosts. A series of vaccination trials with antigen fractions, and associated immunological analyses, identified individual protective antigens or fractions. These were cloned from cDNA and the recombinant proteins expressed in Escherichia coli. This strategy was independently successful in developing vaccines against T. ovis and E. granulosus. Identification of protective antigens for these species enabled rapid identification, cloning and expression of their homologues in related species and thereby the development of effective vaccines against T. saginata, E. multilocularis and, more recently, T. solium. The T. saginata vaccine provides an excellent example of the use of two antigen components, each of which were not protective when used individually, but when combined they induce a reliable, high level of protection. One important contributing factor to the success of vaccine development for the taeniid cestodes was the concentration on studies seeking to identify native host-protective antigens, before the adoption of recombinant methodologies. The cestode vaccines are being developed towards practical (commercial) application. The high level of efficacy of the vaccines against T. solium cysticercosis and hydatid disease suggests that they would be effective also if used directly in humans.