Immunodiagnosis of human cysticercosis (Taenia solium) with antigens purified by monoclonal antibodies.

Monoclonal antibodies were generated from mice immunized with scolex protein antigen of Cysticercus cellulosae. Three monoclonal antibodies specific for cysticercal antigens, which did not show any cross-reactivity with Taenia solium or Taenia saginata antigens, were selected. Each monoclonal antibody coupled to Sepharose could purify one antigen, which appeared as a single band on polyacrylamide gel electrophoresis. When antigens purified by monoclonal antibodies were used to detect antibody in serum samples taken from patients with cysticercosis, taeniasis, and other parasitic infections in an enzyme-linked immunosorbent assay, cross-reactivity was observed until a serum dilution of 1:128 was reached. Since serum samples from unexposed subjects showed positive reactions until a dilution of 1:64 was reached, we chose a discriminative dilution (1:128) above which no cross-reaction was observed. The percent positive serum samples from cysticercosis patients was 100% by the enzyme-linked immunosorbent assay with any of the antigens purified by monoclonal antibodies.     

Comparative serological reactivity of Taenia crassiceps, Taenia solium and Taenia saginata metacestode neutral glycolipids to infection serum from Taenia crassiceps-infected mice.

A comparative survey was undertaken of the neutral fraction glycolipids from the metacestodes of 3 taeniid species, Taenia crassiceps, Taenia solium and Taenia saginata, to determine their chemical and serological staining patterns on separation by thin-layer chromatography. The orcinol-positive patterns of T. solium and T. saginata metacestodes exhibited a closer superficial resemblance to each other than to T. crassiceps or T. saginata adults. A comparison of component migration properties against standards of known structure indicated the main oligosaccharide chains to be mono-, di-, tri- and tetrasaccharides; however, in T. solium this was extended to at least a heptasaccharide. The multiple banding characteristic of each component is a consequence of lipid moiety heterogeneity. Serologically, the patterns of the 3 taeniid species neutral fraction glycolipids showed virtually the same immunological reactivity towards mouse normal serum, infection serum and a monospecific, polyclonal antibody directed against the trisaccharide component of T. crassiceps. The latter antibody was isolated from mouse infection serum by affinity chromatography on a column of glycolipid-bound octyl-Sepharose CL-4B. Immunochemically, the major common epitope expressed by the neutral fraction glycolipids of the 3 taeniid species is the same or very similar to the glycosphingolipid, neogalatriaosyl ceramide derived from the marine mollusc Turbo cornutus (Gal(beta 1-6) Gal(beta 1-6) Gal(beta 1-1)Cer). Host tissue neutral fraction glycolipids, porcine muscle and bovine muscle, as well as human spleen, were not immunoreactive.     

Taenia solium metacestode preparation in rural areas of sub-Saharan Africa: a source for diagnosis and research on cysticercosis

BackgroundTaenia solium metacestodes/cysts obtained from pig carcasses constitute a primary source for diagnostic tools used for the detection of human cysticercosis. Data on T. solium cyst preparation in Africa is still scarce but required to establish independent reference laboratories.ObjectivesThe aim of the present study is a) to present the likely yield of T. solium cyst material by the use of two different preparation methods in the field and b) to investigate its suitability for immunodiagnosis of human cysticercosis.MethodsIn Zambia, Uganda and Tanzania 670 pigs were screened for T. solium infection. Cysts were prepared by ‘shaking method’ and ‘washing method’. Generated crude antigens were applied in a standard western blot assay.Results46 out of 670 pigs (6.9%) were found positive for T. solium (Zambia: 12/367, 3.3%; Uganda: 11/217, 5.1%; Tanzania 23/86, 26.7%). Mean values of 77.7 ml whole cysts, 61.8 ml scolices/membranes and 10.9 ml cyst fluid were obtained per pig. Suitability of collected material for the use as crude antigen and molecular diagnostic techniques was demonstrated.ConclusionThis study clearly shows that T. solium cyst preparation in African settings by simple field methods constitutes an effective way to obtain high quality material as source for diagnostic tools and research purposes.     

Serological diagnosis of human cysticercosis by use of recombinant antigens from Taenia solium cysticerci.

A Taenia solium metacestode cDNA expression library in the lambda ZAPII vector was screened with pooled sera from patients with neurocysticercosis. Sixty primary clones were identified and shown to belong to two classes. The clones NC-3 and NC-9 did not reveal any significant homologies to sequences deposited in the databases and were further characterized. Both recombinant antigens were expressed as glutathione S-transferase fusion proteins and applied for serological diagnosis of human cysticercosis. An enzyme-linked immunosorbent assay was established and evaluated with 27 serum samples of La Réunion and Madagascar patients with cysticercosis. Diagnosis in these patients was established with radiological and serological procedures. For antigen NC-3 a sensitivity of 96.3% and a specificity of 91.5% for the serodiagnosis were achieved. In contrast, the sensitivity of antigen NC-9 was only 33.3%.     

Characterization and cloning of T24, a Taenia solium antigen diagnostic for cysticercosis

The third and final diagnostic antigen of the lentil lectin purified glycoproteins (LLGP) extracted from the larval stage of Taenia solium has been characterized, cloned, and expressed. T24 is an integral membrane protein that belongs to the tetraspanin superfamily. It migrates at a position corresponding to 24-kDa and as a homodimer at 42-kDa. Antibodies from cysticercosis patients recognize secondary structure epitopes that are dependent upon correctly formed disulfide bonds. A portion of T24, the large, extracellular loop domain, was expressed in an immunologically reactive form in insect cells. When tested in a Western blot assay with a large battery of serum samples, this protein, T24H, has a sensitivity of 94% (101/107), for detecting cases of cysticercosis with two or more viable cysts, and a specificity of 98% (284/290). The identification and expression of T24H sets the stage for the development of an ELISA suitable for testing single samples and for large-scale serosurveys that is not dependent upon the isolation and purification of antigens from parasite materials.     

Two epitopes shared by Taenia crassiceps and Taenia solium confer protection against murine T. crassiceps cysticercosis along with a prominent T1 response

Taenia crassiceps recombinant antigens KETc1 and KETc12 have been shown to induce high level of protection against experimental murine T. crassiceps cysticercosis, an experimental model successfully used to test candidate antigens for use in vaccination against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence, KETc1 and KETc12 were chemically synthesized in linear form. Immunization with KETc1 induced 66.7 to 100% protection against murine cysticercosis, and immunization with KETc12 induced 52.7 to 88.1% protection. The elicited immune response indicated that both peptides contain at least one B-cell epitope (as demonstrated by their ability to induce specific antibodies) and one T-cell epitope that strongly stimulated the proliferation of T cells primed with either the free peptide or total cysticercal T. crassiceps antigens. The high percentage of spleen cells expressing inflammatory cytokines points to the likelihood of a T1 response being involved in protection. The protective capacity of the peptides and their presence in all developmental stages of T. solium point to these two epitopes as strong candidates for inclusion in a polyepitopic synthetic vaccine against T. solium pig cysticercosis.     

The role of N-linked carbohydrates in the antigenicity of Taenia solium metacestode glycoproteins of 12, 16 and 18 kD

The glycoproteins of 12-28 kD from Taenia solium metacestodes provide a high specificity and sensitivity for the serological diagnosis of the central nervous system infection, neurocysticercosis. Their widespread use as antigens for routine serological assays will require their production in large and reproducible amounts. Prior to determining the ideal strategy to produce these antigens at a large scale, it is important to determine the contribution of the carbohydrates to the antigenicity of these molecules, given the uncertainty of reproducing saccharidic epitopes in recombinant expression systems. In this study we examined this issue. The chemical oxidation of the carbohydrates of the 12-28 kD glycoproteins with sodium metaperiodate, reduced the antigenicity of the molecules to variable extents, with the more notable changes being detected for the 18 and 28 kD antigens. This approach was complemented by purification of the 12, 16 and 18 kD antigens, followed by the enzymatic deglycosylation of their abundant N-linked oligosaccharides. Silver-stained SDS-PAGE analysis indicated that the three deglycosylated antigens now migrated as 7 kD products, suggesting a protein backbone with a similar size, but different extents of glycosylation. By Western blot, the antigenicity of these antigens was diminished. This was more notable for the 18 kD antigen, which is more heavily glycosylated than the 12 or 16 kD glycoproteins. These data suggest that the antigenicity of the glycoproteins of T. solium is due to a combination of carbohydrate and protein epitopes.     

Immunodiagnosis of human cysticercosis in cerebrospinal fluid. Antigens from murine Taenia crassiceps cysticerci effectively substitute those from porcine Taenia solium

Tapeworm antigens from Taenia crassiceps performed as well as those antigens from Taenia solium in an enzyme-linked immunosorbent assay for the detection of Cysticercus antibodies in 96 cerebrospinal fluid samples from patients with neurocysticercosis and in 96 CSF samples from patients with other varied neurological ailments. Thus, this manageable murine model of experimental cysticercosis solved the problem of antigen supply for clinical and epidemiological applications, and it provided an immediate means of abundant production of antigens for the wide distribution and standardization of immunodiagnostic tests for cysticercosis.     

Characterization and cloning of GP50, a Taenia solium antigen diagnostic for cysticercosis

GP50, a Taenia solium protein diagnostic for cysticercosis has been cloned, sequenced, and characterized. GP50 is one diagnostic component of the lentil lectin purified glycoprotein (LLGP) antigens that have been used for antibody-based diagnosis of cysticercosis in a Western blot assay for nearly 15 years. GP50 is a glycosylated and GPI-anchored membrane protein. The native protein migrates at 50kDa, but the predicted molecular weight of the mature protein is 28.9. Antigenically active recombinant GP50 has been expressed in a baculovirus expression system. The antigenic activity of both the native and recombinant proteins is dependent upon the correct formation of disulfide bonds. GP50, purified from cysticerci, has two homologs expressed in the adult worm, TSES33 and TSES38. Both are diagnostic for taeniasis. In spite of the amino acid similarities between GP50 and the TSES proteins, each appears to be a stage-specific antigen. A preliminary evaluation of recombinant GP50 in a Western blot assay showed 100% specificity for cysticercosis and 90% sensitivity for cysticercosis positive serum samples reactive with the GP50 component of LLGP.     

Characterization of the 8-kilodalton antigens of Taenia solium metacestodes and evaluation of their use in an enzyme-linked immunosorbent assay for serodiagnosis

The Western blot for cysticercosis, which uses lentil lectin purified glycoprotein (LLGP) antigens extracted from the metacestode of Taenia solium, has been the "gold standard" serodiagnostic assay since it was first described in 1989. We report that the diagnostic antigens at 14, 18, and 21 kDa, as well as some larger disulfide-bonded antigens, are actually all members of a very closely related family of proteins, the 8-kDa antigens. The genes for 18 unique, mature proteins have been identified. Nine of these were chemically synthesized and tested in an enzyme-linked immunosorbent assay with a battery of defined serum samples, including 32 cysticercosis-positive serum samples reactive with the 8-kDa antigens of LLGP on Western blotting, 34 serum samples from patients with other parasitic infections, and 15 normal human serum samples. One of the 8-kDa antigens, TsRS1, is 100% sensitive and 100% specific. TsRS1 will be one component of a cocktail of three to four synthetic or recombinant antigens, based on the diagnostic bands of the Western blot, which will be used for the serodiagnosis of cysticercosis.     

A review of the control of clonorchiasis sinensis and Taenia solium taeniasis/cysticercosis in China

Clonorchiasis sinensis and Taenia solium taeniasis/cysticercosis are major foodborne parasitoses. Clonorchiasis sinensis is actively transmitted in some areas of China, Korea, Russia, Vietnam, etc. Currently, it is estimated that more than 200 million people are at risk of infection, 15–20 million people are infected, and 1.5–2 million show symptoms or complications. In China, it is relatively heavily transmitted in Zhujiang River Delta, including Hong Kong and Macao, and Northeast China, where many Korean people live. The transmission is related to the unhealthy habits of residents who like to have raw fish or half-raw fish. The infection of Clonorchis sinensis could result in serious liver and biliary system damages, and chronic cases may induce liver and bile duct cancers. T. solium taeniasis/cysticercosis is distributed around the world except the areas where the residents have a taboo against pork for religious reasons. Recent years, the urban inhabitants infected with T. solium/Cysticercus are increasing in China. T. solium results in intestinal diseases, and cysticercosis is a very serious disease, especially nervous system cysticercosis. Its symptoms include headache, epilepsy, sudden death, etc. Health education and health promotion, environmental reconstruction, and chemotherapy are the main control measures for these diseases. Through several decades of efforts in China, the achievements of control of clonorchiasis and T. solium taeniasis/cysticercosis are great. For example, in one of the main clonorchiasis-endemic provinces, Shandong Province, clonorchiasis has been controlled. In 31?T. solium taeniasis/cysticercosis-endemic counties of Henan Province, through a 6-year control program, the decline rates of T. solium taeniasis and cysticercosis were 90.8 and 96.8?%, respectively. This paper reviews the researches on the control of clonorchiasis and T. solium taeniasis/cysticercosis in China past decades so as to provide references for other countries where these diseases are endemic to improve the control or elimination of clonorchiasis and T. solium taeniasis/cysticercosis.     

Immunosuppression and inhibition of inflammation in mice induced by a small Taenia solium RNA-peptide to implanted T. solium metacestodes

 Subcutaneous implantation of Taenia solium metacestodes in mice induces an inflammatory reaction made up mainly of neutrophils and eosinophils after 12 days. Administration of a small RNA-peptide (metacestode factor, MF) purified from T. solium metacestodes significantly reduces the inflammatory site in both size and composition, yielding a very low number of eosinophils. The metacestodes implanted in control mice were completely destroyed and their remnants were surrounded by an intense inflammation predominantly made up of neutrophils and eosinophils. In contrast, metacestodes implanted in mice treated with MF showed apparently intact suckers, rostellum, hooks, and tegument. Inhibition of inflammation around the parasites was also observed in mice immunized with T. solium metacestode antigens and inoculated simultaneously with MF. Mice immunized only with T. solium metacestode antigens produced a granulomatous process around metacestodes that destroyed most of the large metacestode structures: suckers, rostellum, hooks, and tegument-wall tissues. Furthermore, treatment of mice with MF or implanted metacestodes decreased the antibody (P<0.05) and cellular responses (P<0.05) to metacestode antigens. The antibody response was even lower when both of these treatments were given simultaneously. These findings support the idea that MF plays a key role in the down-regulation of the host immune response, contributing to the parasite’s survival. Received: 31 January 1996 / Accepted: 15 April 1996     

Comparative analysis of bovine extracts by immunoblotting and ELISA inhibition

Bovine epithelial and urine antigen extracts were compared using enzyme-linked immunosorbent (ELISA) inhibition assay and sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblotting. According to ELISA inhibition results, the two extracts share about 2% of their antigenic components. Urine-derived antigens seem to be antigenically closer to serum proteins than epithelial antigens, as determined by rabbit immune sera. The IgG responses of allergic farmers against epithelial antigens were directed mainly against a protein of 20 kD, while non-allergic farmers had only very weak reactions. Generally, the IgG response against urinary antigens seemed to be more heterogeneous than against epithelial antigens. Almost all cow-allergic farmers reacted with a band at 20 kD in IgE immunoblotting against urinary and epithelial antigen while all non-allergic farmers showed negative results.     

Characterization and protective potential of the immune response to Taenia solium paramyosin in a murine model of cysticercosis

Paramyosin has been proposed as a vaccine candidate in schistosomiasis and filariasis. However, limited information is available about its protective potential against cysticercosis and the immune response it induces. Immunization of mice with recombinant full-length paramyosin of Taenia solium (TPmy) results in about a 52% reduction in parasite burden after a subsequent challenge by intraperitoneal inoculation of Taenia crassiceps cysticerci. Immunization assays using recombinant fragments of TPmy, corresponding approximately to thirds on the amino, central, or carboxyl regions, suggest that protective epitopes are located mostly in the amino-end third. Proliferation assays using T cells obtained from mice immunized with the full-length recombinant TPmy also showed a preferential response to the amino-terminal fragment. In contrast, antibodies in the sera from these mice predominantly recognize epitopes located in the carboxyl-terminal fragment, being the immunoglobulin G1 subclass, the predominant antibody isotype. Characterization of the cellular immune response induced against the protective amino-terminal fragment reveals production of gamma interferon and interleukin-2, but not interleukin-4, suggesting a Th1-like profile.     

Accuracy of enzyme-linked immunosorbent assay using crude and purified antigens for serodiagnosis of melioidosis

Five enzyme-linked immunosorbent assays developed to detect antibodies to different Burkholderia pseudomallei antigen preparations were evaluated as diagnostic tests for melioidosis in northeast Thailand. The highest diagnostic indices were observed for an affinity-purified antigen (sensitivity, 82%; specificity, 72%) and crude B. pseudomallei antigen (sensitivity, 81%; specificity, 70%), an improvement over the indirect hemagglutination assay (sensitivity, 73%; specificity, 64%).     

Immunological evaluation of a 26-kDa antigen from Taenia solium larvae for specific immunodiagnosis of human neurocysticercosis

Human neurocysticercosis, due to infection of the central nervous system by cysticerci of Taenia solium, is a severe form of neurologic disease occurring in Central and South America. Specific proteins from scolex antigen from cysticerci were purified by polyacrylamide gel electrophoresis and electroelution and recognized in Western blots by antibodies present in sera from patients with neurocysticercosis. The proteins appeared as 13-, 17-, and 26-kDa bands on Coomassie blue-stained gels and proved to be specific to cysticerci of T. solium. No cross-reactivity with sera from patients with taeniasis or hydatidosis was observed. Enzyme-linked immunosorbent assay using the purified proteins of 13, 17, and 26 kDa demonstrated rates of 53%, 88%, and 100% specificity, respectively, at the cutoff serum dilution of 1:32 for the specific immunodiagnosis of␣human neurocysticercosis. Received: 10 May 1998 / Accepted: 21 July 1998     

Impairment of the inflammatory reaction on implanted Taenia solium metacestodes in mice by a T. solium RNA-peptide: a scanning electron microscopy study

Inhibition of inflammation by a Taenia solium RNA-peptide (metacestode factor, MF) was studied by scanning electron microscopy (SEM). Viable (96%) T. solium metacestodes obtained from a naturally infected pig were dissected and implanted in treated and control mice, removed at 6 and 12 days postimplantation (p.i.), and studied by SEM. At day 6, metacestodes in control mice showed vigorous inflammation, whereas in mice treated with MF they were apparently intact with exiguous inflammation. Mice immunized with T. solium metacestode antigens showed a moderate inflammation; those treated with both MF and T. solium antigens presented scanty inflammation. At day 12, metacestodes presented copious inflammation and severe damage to the sucker tissues in mice immunized with T. solium; in mice treated with either MF or MF and T. solium antigens there was only discrete inflammation. These observations illustrate the central role of MF in the inhibition of the early events leading to the parasite's destruction by means of an inflammatory response. Received: 17 February 1997 / Accepted: 19 August 1997     

Serodiagnosis of human cysticercosis by using antigens from vesicular fluid of Taenia crassiceps cysticerci.

Neurocysticercosis (NC), caused by the presence of Taenia solium metacestodes in tissues, is a severe parasitic infection of the central nervous system with universal distribution. To determine the efficiency of enzyme-linked immunosorbent assay (ELISA) and immunoblot with antigens of T. crassiceps vesicular fluid (Tcra) compared to standard techniques (indirect immunofluorescence test [IFT] and complement fixation test [CFT]) using T. solium cysticerci (Tso) for the serodiagnosis of NC, we studied serum samples from 24 patients with NC, 30 supposedly healthy individuals, 76 blood bank donors, 45 individuals with other non-NC parasitoses, and 97 samples from individuals screened for cysticercosis serology (SC). The sensitivity observed was 100% for ELISA-Tso and ELISA-Tcra, 91.7% for the IFT, and 87.5% for the CFT. The specificity was 90% for ELISA-Tso, 96.7% for ELISA-Tcra, 50% for IFT, and 63.3% for CFT. The efficiency was highest for ELISA-Tcra, followed by ELISA-Tso, IFT, and CFT. Of the 23 samples from SC group, which were reactive to ELISA-Tso and/or ELISA-Tcra, only 3 were positive to immunblot-Tcra (specific peptides of 14- and 18-kDa) and to glycoprotein peptides purified from Tcra antigen (gp-Tcra), showing the low predictive value of ELISA for screening. None of the samples from the remaining groups showed specific reactivity in immunoblot-Tcra. These results demonstrate that ELISA-Tcra can be used as a screening method for the serodiagnosis of NC and support the need for specific tests for confirmation of the results. The immunoblot can be used as a confirmatory test both with Tcra and gp-Tcra, with the latter having an advantage in terms of visualization of the results.