Purification of larval Taenia solium antigens by gel filtration.

Crude larval Taenia solium extracts were fractionated by Sephacryl S-200 gel filtration into four fractions (W1-W4). The sensitivities of the fractions to rabbit and pig antiserum against Taenia solium were tested by double immunodiffusion, immunoelectrophoresis, and ELISA. Fraction W2 which was highly sensitive to antisera was shown by immunoblotting to contain antigen B (95 and 105 kDa). The four fractions were shown to contain antigenic determinants common with pig serum proteins and crude extracts of other Platyhelminthes (especially Taenia hydatigena). Fraction W2 has the potential to be used as a serodiagnostic antigen.     

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。     

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.     

Countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats

The procedure of countercurrent immunoelectrophoresis in the diagnosis of Taenia hydatigena cysticercosis in goats was carried out for antemortem diagnosis of T. hydatigena cysticercosis in experimentally and naturally infected goats. The antigens of cyst fluid, scolex and membrane of T. hydatigena metacestodes were purified and compared. The sensitivity of the test in experimentally and naturally infected goats was 57.1 and 52.5%, respectively, whereas its specificity using antisera raised against T. solium cysticercosis, hydatid cyst and Fasciola gigantica was 66.7 and 83.4% with partially purified and fractionated antigens, respectively. Of all three antigens, the cyst fluid antigen was found to be most reactive. The test could be employed for antemortem diagnosis of T. hydatigena cysticercosis using purified antigen.     

Comparative immunoelectrophoretic analysis of Echinococcus granulosus, Taenia hydatigena and Taenia pisiformis cyst fluid antigens by hyperimmune rabbit sera.

Cyst fluid antigens of Echinococcus granulosus, Taenia hydatigena and T pisiformis were examined by electrophoresis using homologous and heterologous hyperimmune rabbit sera to these antigens. While arc 5 forming antibodies were identified in sera from rabbits immunised with E granulosus and T hydatigena cyst fluids, antibodies responsible for forming precipitating antigen B band were detected in rabbit antisera to E granulosus, T hydatigena and T pisiformis antigens. T hydatigena cyst fluid appears to contain antigen similar to E granulosus antigen 5 and probably antigen B while T pisiformis cyst fluid has mainly an antigen close to hydatid antigen B.     

Comparison of enzyme-linked immunosorbent assay with dot immunobinding assay for detection of antibodies against Pasteurella multocida in turkeys

A dot immunobinding assay (DIA) was developed to detect antibodies against Pasteurella multocida in turkey serum. Five coating antigens, namely, whole-cell (WC) antigen, sonicated cell lysate (SCL), crude capsular extract (CE), formalin extract (FE), and heat-stable antigen (HSA), were compared by enzyme-linked immunosorbent assay (ELISA) and DIA using reference antisera against P. multocida organisms. WC and SCL antigens showed higher sensitivity, whereas FE and HSA antigens were more specific coating antigens in both assays. The specificity of DIA was greater than ELISA by comparing the P/N ratios of HSA against serum prepared from heterologous serotype of P. multocida. The DIA had also several distinct advantages over the ELISA, which included reduction of the manipulation time and more uniform binding of coating antigens onto the nitrocellulose membranes compared with binding of coating antigens to microtiter plates for ELISA.     

Taenia solium oncosphere antigens induce immunity in pigs against experimental cysticercosis

Immunity to Taenia solium infection was investigated using an experimental intramuscular oncosphere infection assay (IMOA) model in pigs. Three naturally infected pigs with cysticercosis were treated with oxfendazole (OFZ), a drug demonstrated to kill cysts in porcine muscle. These animals were then challenged with oncospheres but did not develop any cysts while three uninfected pigs that were similarly challenged, did develop intramuscular cysts. In another study, two groups of three pigs each were immunized with crude T. solium oncosphere and metacestode antigens, respectively, and tested with the IMOA. Immunization with crude oncosphere antigens (OAs) induced 100% protection, while metacestode antigens provided only partial protection. Immunoblots showed that pigs with complete immune protection to oncosphere intramuscular challenge had antibodies to two OAs at 31.3 and 22.5 kDa, respectively. Antibody to these two antigens was absent in pigs immunized with metacestodes or in uninfected control pigs. This study demonstrated the presence of two antigens that are unique to the oncosphere. Although, antibody to these two antigens is consistently present in pigs that are protected from an oncosphere intramuscular challenge their role in preventing infection by T. solium larval cysts is still hypothetical.     

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.     

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.     

Characterization of Mycobacterium paratuberculosis antigenic proteins

The characterization of a purified antigen from Mycobacterium paratuberculosis, recently made commercially available for use in serodiagnosis by enzyme-linked immunosorbent assay (ELISA), of paratuberculosis in cattle was described. This assay had 89% specificity and 83% sensitivity for M paratuberculosis infection. The protein/polypeptide composition of the purified antigen was compared with that of a crude protoplasmic extract of strain 18 M paratuberculosis used in the agar-gel immunodiffusion test and ELISA and with that of sonicated strain 19698 M paratuberculosis organisms grown on Dorset-Henley synthetic liquid medium. The sonicated M paratuberculosis contained 27 major proteins/polypeptides; the crude protoplasmic extract, 18; and the purified antigen contained 14 proteins/polypeptides, using sodium dodecyl-sulfate polyacrylamide-gel electrophoresis analysis. The serologic reactivity of these proteins/polypeptides were defined, using the enzyme-linked immuno-electrotransfer blot technique. The sonicated M paratuberculosis contained 20 serologically reactive proteins/polypeptides (34,000 to 84,000 daltons); the crude protoplasmic extract contained 3 (37,000 to 45,000 daltons); and the purified extract contained a diffuse polypeptide band (34,000 to 38,000 daltons). Identification by enzyme-linked immuno-electrotransfer blot technique of M paratuberculosis antigens reactive in the ELISA will allow us to further study these antigens in the ELISA to improve sensitivity and specificity of the diagnostic test.     

Evaluation of pseudorabies viral antigens in the agar gel immunodiffusion test.

Procedures designed to extract pseudorabies viral (PRV) antigens from PRV-infected tissue cultures were investigated to determine whether differences in extraction method had an effect upon the final concentrated antigenic product. All four of the preparations made from PRV-infected tissue culture cells (trypsin extract and disrupted cells) or entire PRV-infected cultures (polysorbate 80 extract and (NH4)2SO4 precipitate) contained relatively large amounts of the same antigen, whereas cell-free PRV-infected tissue culture fluids did not contain significant amounts of this antigen. Specific antibody directed against this antigen was present in all PRV antisera tested. Two other antigens were observed in some of the preparations, but PRV antisera varied in their ability to precipitate with these antigens. Therefore, the number of precipitation lines observed in agar gel immunodiffusion between PRV preparations and PRV-positive antisera depended both upon the extraction method used to obtain the antigen and upon the specificity of the selected antiserum.     

Haematological and serological data from dogs raised worm-free and monospecifically infected with helminths

Pups free from helminth infection were obtained by treating the bitch prior to parturition, and the bitch and her pups after whelping, with oxfendazole and praziquantel. They were then housed in an area free from contamination with helminth eggs. Freedom from infection was confirmed by necropsy examinations and by serological tests using the Enzyme Linked Immunosorbent Assay (ELISA). Groups of pups were infected with Toxocara canis, Echinococcus granulosus, Taenia ovis, T. pisiformis and T. hydatigena and bled at 5-day intervals. Haematological examinations and measurement of serum proteins were performed on blood samples obtained throughout 30 days of infection. Levels of serum proteins were not significantly altered in any infection. Pups infected with T. canis showed a marked increase in numbers of circulating eosinophils during the phase of larval migration through the liver and lungs; they also showed marked antibody responses to T. canis larval ES antigen. Haematological values were not significantly altered in any other pups.     

Evaluation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis purified proteins of Mycoplasma gallisepticum and M. synoviae as antigens in a dot-enzyme-linked immunosorbent assay

Selected immunogenic proteins of Mycoplasma gallisepticum (MG) strain R and M. synoviae (MS) isolate F10-2AS were purified from sodium dodecyl sulfate-polyacrylamide gels. Purified MG proteins of 65 to 63 (p64) kilodaltons (kDa), and 26 and 24 (p26/24) kDa, and purified MS proteins of 53 (p53) kDa, 41 (p41) kDa, and 22 (p22) kDa were evaluated as potential antigens for an enzyme-linked immunosorbent assay (ELISA). Chicken antisera to MG, MS, or oil-emulsion vaccines were used to evaluate these purified proteins as antigens in a dot-ELISA. MG antigen p64 detected antibodies 3 days after the serum plate agglutination (SPA) test and 7 days before the hemagglutination-inhibition (HI) test. Antigen p64 detected antibodies to 12 MG isolates, and in sera from field outbreaks of MG. No cross-reactions with MS-positive antisera were seen with antigen p64. MG antigen p26/24 did not perform as well as p64. MS antigen p41 detected antibodies 5 days after the SPA test and at least 11 days before the HI test, and in sera from field outbreaks of MS. However, some MG-positive antisera reacted with p41. MS antigens p53 and p22 did not perform well.     

Pasteurella haemolytica: purification of saline-extractable proteins by isoelectrofocusing

A procedure was developed for separating antigens associated with a saline extract of Pasteurella haemolytica serotype 1. Seven antigens were identified by immunoelectrophoresis to be associated with the extract. The extract was subjected to preparative isoelectrofocusing in a pH range of 3-10. The majority of extracted proteins were found to have pI's of 4-6, whereas the carbohydrate antigen(s) were distributed over a pI range of 3.0-8.0. The fractions that were of interest were pooled and refocused in a narrower pH range to improve resolution of the protein antigens. Specific antigens from defined pH ranges were pooled to form 6 antigen groups. These antigen groups were examined further by immunoelectrophoresis, analytical isoelectrofocusing, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molecular weights of the proteins found in the capsular extracts ranged from 33 k to greater than 80 k. Injection of mice with capsular extract or antigen Groups 1-6 in Freund's incomplete adjuvant resulted in a serum antibody response to the various antigens as detected by an enzyme-linked immunosorbent assay. Significant protection (P less than 0.05) against challenge with virulent P. haemolytica was seen in mice injected with antigen Groups 2 and 4. Six calves were immunized with saline extract. These calves had greater resistance to experimental pneumonic pasteurellosis than did 6 non-vaccinated calves. A serum antibody response to the crude extract and to each antigen group was detected in vaccinated calves by an enzyme-linked immunosorbant assay.     

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.     

Detection of Taenia hydatigena copro-antigens by ELISA in dogs

A sandwich-ELISA was developed for the detection of soluble Taenia hydatigena antigens in fecal samples of dogs. Affinity-purified polyclonal catching antibodies and alkaline phosphatase-conjugated detecting antibodies were employed, which had been obtained from rabbits hyperimmunized with excretory/secretory antigens derived from in vitro maintained adult Taenia hydatigena. The assay allowed the detection of 800 ng T. hydatigena antigen g-1 of feces as a lower limit. Six helminth-free dogs were each infected with 10 T. hydatigena cysticerci isolated from Swiss sheep. After prepatent periods ranging from 57 to 71 days, the dogs started to excrete Taenia eggs and/or proglottids. The ELISA detected Taenia antigens in all six dogs during the prepatent period starting individually between Day 18 and 45 post-infection (p.i.). Anthelmintic treatment of three dogs at Day 95 p.i. resulted in elimination of the cestodes and within the 5 following days in the disappearance of Taenia antigens from feces. The specificity of the assay was evaluated by testing crude antigens derived from helminths or bacteria. Four Taenia species showed cross-reactivity at concentrations of 5 micrograms protein ml-1. Conversely, no cross-reactions occurred with various antigen batches derived from Echinococcus granulosus, E. multilocularis, Dipylidium caninum, Mesocestoides corti, Diphyllobothrium sp., Toxocara canis and bacterial antigens (Salmonella and Escherichia). Moreover, fecal samples from dogs naturally infected with T. canis (n: 13), hookworms (n: 2), Trichuris vulpis (n: 13) and of 10 dogs with mixed infections with these three nematode groups were tested, and results confirmed the high degree of specificity. The Taenia antigens detectable by this ELISA remained immunologically stable in native feces stored at +25 degrees, +4 degrees or at -20 degrees C for at least 5 days.     

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.     

Purification of a 28 kDa Babesia (Theileria) equi antigen and a 29 kDa spurious erythrocyte antigen from in vitro culture through ion exchange chromatography.

An extract of in vitro cultivated Babesia equi was fractionated using a MonoQ anion exchange column. Separation of a 28 kDa B. equi antigen from a 29 kDa spurious erythrocyte antigen, both of which were intensely immunoreactive, was achieved by chromatography of the infected erythrocyte proteins. Using tricine-SDS-PAGE, the 28 kDa antigen of B. equi showed multiple band resolution, while the 29 kDa antigen was consistently resolved as a single band. The 29 kDa antigen was identified in both infected and non-infected erythrocyte extracts. Moreover, B. equi antiserum recognised this antigen in the non-infected erythrocyte extract, and conversely serum from horses not infected with babesia detected the antigen in infected erythrocyte extract. This 29 kDa antigen could represent a horse erythrocyte isoantigen. The purified 28 kDa antigen is specifically recognised by B. equi antisera and therefore could be useful for the production of the recombinant replica and to employ these in further test systems.     

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.     

Antigenic analyses of tissues and excretory and secretory products from Strongylus vulgaris.

Rabbit antisera were prepared against veronal buffered saline extracts of L4 and L5 Strongylus vulgaris, adult S. vulgaris and adult Strongylus equinus retrieved from naturally infected horses. In agar gel diffusion with these antisera, adult S vulgaris and S. equinus each appeared to have at least one unique antigen; larval S. vulgaris appeared to have two species-specific and two stage-specific antigens. There were several common antigens. Excretory and secretory products were collected also from L4 and L5 an maintained over several days in tissue culture fluid. In agar gel diffusion against the above rabbit antisera, a stage-specific antigen was found also in excretory and secretory products. In addition, excretory and secretory products had three antigens in common with adult and larval S. vulgaris, but only one of these was common to adult S. equinus. The excretory and secretory products appear, therefore, to have two species-specific and one stage-specific antigens.