Hydrophobic fraction of Taenia saginata metacestodes, rather than hydrophilic fraction, contains immunodominant markers for diagnosing human neurocysticercosis.

INTRODUCTION: Considering that alternative antigens for diagnosing neurocysticercosis continue to be a challenge because of the increasing difficulty in obtaining parasites from naturally infected pigs for preparation of Taenia solium homologous antigen, the aim of the present study was to evaluate the detergent (D) and aqueous (A) fractions from saline extract of Taenia saginata metacestodes for diagnosing neurocysticercosis. METHODS: Taenia saginata was obtained from naturally infected bovines in the Triângulo Mineiro region, State of Minas Gerais, Brazil. The carcasses came from cold storage units and had been slaughtered in accordance with the inspection technique recommended by the Federal Inspection Service. The D and A fractions were obtained by using Triton X-114 (TX-114). Serum samples were obtained from 40 patients with a diagnosis of neurocysticercosis, 45 with other parasitic diseases and 30 from apparently normal individuals. IgG antibody levels were evaluated using the ELISA and immunoblotting assays. RESULTS: The ELISA sensitivity and specificity were 95% and 73.3%, when using saline extract; 95% and 82.6% for the D fraction; and 65% and 61.3% for the A fraction, respectively. The immunoblotting assay confirmed the ELISA results, such that the D fraction was more efficient than the other extracts, and the 70-68 kDa component was immunodominant among neurocysticercosis patients. CONCLUSIONS: These results demonstrated that the D fraction from Taenia saginata metacestodes obtained using TX-114 can be used as a heterologous antigenic fraction in the immunoblotting assay for serologically diagnosing human neurocysticercosis, given its ability to select immunodominant antigens.

Hydrophobic fraction of Taenia saginata metacestodes, rather than hydrophilic fraction, contains immunodominant markers for diagnosing human neurocysticercosis / Fração hidrofóbica de metacestódeos de Taenia saginata, ao contrário da fração hidrofílica, contém marcadores imunodominantes para o diagnóstico de neurocisticercose humana

INTRODUCTION: Considering that alternative antigens for diagnosing neurocysticercosis continue to be a challenge because of the increasing difficulty in obtaining parasites from naturally infected pigs for preparation of Taenia solium homologous antigen, the aim of the present study was to evaluate the detergent (D) and aqueous (A) fractions from saline extract of Taenia saginata metacestodes for diagnosing neurocysticercosis. METHODS: Taenia saginata was obtained from naturally infected bovines in the Triângulo Mineiro region, State of Minas Gerais, Brazil. The carcasses came from cold storage units and had been slaughtered in accordance with the inspection technique recommended by the Federal Inspection Service. The D and A fractions were obtained by using Triton X-114 (TX-114). Serum samples were obtained from 40 patients with a diagnosis of neurocysticercosis, 45 with other parasitic diseases and 30 from apparently normal individuals. IgG antibody levels were evaluated using the ELISA and immunoblotting assays. RESULTS: The ELISA sensitivity and specificity were 95 percent and 73.3 percent, when using saline extract; 95 percent and 82.6 percent for the D fraction; and 65 percent and 61.3 percent for the A fraction, respectively. The immunoblotting assay confirmed the ELISA results, such that the D fraction was more efficient than the other extracts, and the 70-68kDa component was immunodominant among neurocysticercosis patients. CONCLUSIONS: These results demonstrated that the D fraction from Taenia saginata metacestodes obtained using TX-114 can be used as a heterologous antigenic fraction in the immunoblotting assay for serologically diagnosing human neurocysticercosis, given its ability to select immunodominant antigens.

INTRODUÇÃO: Considerando que antígenos alternativos para o diagnóstico da neurocisticercose (NC) continua sendo um desafio devido ao aumento da dificuldade em se obter parasitas de suínos naturalmente infectados, para a preparação do antígeno homólogo de Taenia solium, o objetivo do presente estudo foi avaliar frações detergente (D) e aquosa (A), do extrato salino de metacestódeo de Taenia saginata para diagnóstico da NC. MÉTODOS: Bovinos, naturalmente infectados com Taenia saginata, procedentes da região do Triângulo Mineiro, Estado de Minas Gerais, Brasil, foram obtidos de frigoríficos e abatidos de acordo com a técnica de inspeção recomendada pelo Serviço de Inspeção Federal. As frações D e A foram obtidas utilizando Triton X-114 (TX-114). Amostras de soro foram obtidas de 40 pacientes com diagnóstico de NC, 45 com diagnóstico de outras doenças parasitárias e 30 de indivíduos aparentemente normais. Níveis de IgG foram avaliados pelos testes ELISA e Imunoblotting. RESULTADOS: A sensibilidade e especificidade do teste ELISA foram 95 por cento e 73,3 por cento, quando utilizado o extrato salino, 95 por cento e 82,6 por cento para fração D, e 65 por cento e 61,3 por cento para a fração A, respectivamente. O ensaio Imunoblotting confirmou os resultados do teste ELISA, sendo a fração D mais eficiente que os outros extratos, observando-se que o componente 70-68kDa se comportou como imunodominante para os pacientes com NC. CONCLUSÕES: Estes resultados demonstraram que a fração D de metacestódeo de Taenia saginata obtida com TX-114 pode ser utilizada como fração antigênica heteróloga pelo Imunoblotting para o diagnóstico sorológico da NC humana, considerando sua habilidade para selecionar antígenos imunodominantes.

IgA detection in human neurocysticercosis using different preparations of heterologous antigen.

Neurocysticercosis (NC) is the most important neurological disease of parasitic origin in humans. IgA and IgG detection in serum from neurocysticercosis patients was tested using some antigenic preparations of total saline extract from Taenia saginata: detergent (D) and aqueous (A) phases extracted with Triton X-114 and the jacalin bound (JBF) and unbound fractions (JUF) obtained by affinity chromatography using jacalin column. Samples were obtained from 45 patients with definitive NC, who were subdivided into active-NC group and inactive-NC group; 35 patients with other parasitoses; and 30 apparently healthy individuals. Sensitivity and specificity were calculated. Specificity to detect IgA and IgG for D phase, respectively, were 89.8% and 86.9% and for IgG detection 91.3% and 76.8% when using D phase and JUF, respectively. D phase and JBF proved to be specific and efficient and could be efficiently utilized as an alternative antigen for IgA detection in NC, with comparable results with IgG.

Taenia saginata metacestode antigenic fractions without affinity to concanavalin A are an important source of specific antigens for the diagnosis of human neurocysticercosis.

Taenia saginata metacestode antigens have been constituted a useful alternative antigen for neurocysticercosis (NC) serodiagnosis, particularly due to an increasing difficulty to obtain Taenia solium homologous antigen. Cross-reactivity with Echinococcus granulosus infection occurs in homologous and heterologous antigens and could be avoided by using different purified methods. The present study evaluated antigen fractions obtained from saline extracts of T. saginata metacestodes purified by affinity chromatography with jacalin or concanavalin A (ConA) lectins to detect IgG antibodies by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis to diagnose human NC. Serum samples were collected from 142 individuals: 40 of them were diagnosed with NC, 62 presented Taenia sp. and other parasites, and 40 were apparently healthy individuals. The jacalin- and ConA-unbound fractions demonstrated sensitivity and specificity higher than those of bound fractions. Among unbound fractions, ConA demonstrated statistically higher sensitivity and specificity by ELISA (90% and 93.1%, respectively). By immunoblot assay, the 64- to 68-kDa component from the ConA-unbound fraction showed 100% sensitivity and specificity, making this component suitable for use as a specific antigen for diagnosis of NC. To our knowledge, this is the first report showing the relevance of using the unbound ConA fraction of T. saginata metacestodes to diagnose NC. In conclusion, the results obtained herein clearly demonstrate that antigenic fractions without affinity to ConA, obtained from T. saginata metacestodes, are an important source of specific peptides and are efficient in the diagnosis of NC when tested by immunoblot assay.

Saline extract of Taenia saginata metacestodes as an alternative antigen for the immunodiagnosis of neurocysticercosis in human cerebrospinal fluid.

The aim of the present research was to test the application of Taenia saginata metacestodes as an alternative antigen for use in enzyme-linked immunosorbent assay (ELISA) and Western Blotting (WB) tests compared with the use of metacestodes antigen of Taenia solium in cerebrospinal fluid (CSF) samples. The samples were obtained from 35 patients with definitive neurocysticercosis (NCC)-group 1-and 44 patients with other neurological disorders (control)-group 2. The sensitivity and specificity of ELISA, using antigen obtained from T. solium, applied to the patients of group 1 yielded results of 100%. When the tests were conducted using T. saginata metacestodes, results were 100% and 93.2%, respectively. The 47-52-, 64-68-, and 70-kDa antigens showed high frequencies in CSF samples from patients with NCC when WB was conducted with both antigens. The results indicate that T. saginata metacestodes can be used as an alternative antigen for the diagnosis of human NCC in CSF samples.

The Taenia saginata homologue of the major surface antigen of Echinococcus spp. is immunogenic and 97% identical to its Taenia solium homologue.

The TEG-Tsag gene of Taenia saginata is homologous to the genes expressing the two major surface antigens of Echinococcus spp. (EM10 and EG10). Surface antigens of parasites are logical candidates for vaccines, and in this paper we demonstrate that cattle vaccinated with the recombinant TEG-Tsag protein, either used singly or in conjunction with the recombinant HP6-Tsag protein, the major 18 kDa surface/secreted antigen of T. saginata oncospheres, produce excellent antibody responses to both these recombinant proteins. Thus TEG-Tsag may have utility as a vaccine and also as a diagnostic tool for bovine cysticercosis. In addition, as we now demonstrate a 97% homology between TEG-Tsag and its Taenia solium homologue, TEG-Tsol, this latter molecule may have similar potential in the control of human and porcine cysticercosis. The TEG molecule is characterized by an N-terminal FERM domain and a C-terminal ERM domain which are found in a number of cytoskeletal-associated proteins located at the interface between the plasma membrane and the cytoskeleton and in proteins that interact with lipid membranes. The FERM domain is also postulated to bind to adhesion proteins, in a PIP2-regulated fashion, providing a link between cytoskeletal signals and membrane dynamics. Thus TEG protein may play a role in tegument function and interaction with the host.

Wrapping the alpha-crystallin domain fold in a chaperone assembly.

Small heat shock proteins (sHsps) are oligomers that perform a protective function by binding denatured proteins. Although ubiquitous, they are of variable sequence except for a C-terminal approximately 90-residue "alpha-crystallin domain". Unlike larger stress response chaperones, sHsps are ATP-independent and generally form polydisperse assemblies. One proposed mechanism of action involves these assemblies breaking into smaller subunits in response to stress, before binding unfolding substrate and reforming into larger complexes. Two previously solved non-metazoan sHsp multimers are built from dimers formed by domain swapping between the alpha-crystallin domains, adding to evidence that the smaller subunits are dimers. Here, the 2.5A resolution structure of an sHsp from the parasitic flatworm Taenia saginata Tsp36, the first metazoan crystal structure, shows a new mode of dimerization involving N-terminal regions, which differs from that seen for non-metazoan sHsps. Sequence differences in the alpha-crystallin domains between metazoans and non-metazoans are critical to the different mechanism of dimerization, suggesting that some structural features seen for Tsp36 may be generalized to other metazoan sHsps. The structure also indicates scope for flexible assembly of subunits, supporting the proposed process of oligomer breakdown, substrate binding and reassembly as the chaperone mechanism. It further shows how sHsps can bind coil and secondary structural elements by wrapping them around the alpha-crystallin domain. The structure also illustrates possible roles for conserved residues associated with disease, and suggests a mechanism for the sHsp-related pathogenicity of some flatworm infections. Tsp36, like other flatworm sHsps, possesses two divergent sHsp repeats per monomer. Together with the two previously solved structures, a total of four alpha-crystallin domain structures are now available, giving a better definition of domain boundaries for sHsps.

Tsp36, a tapeworm small heat-shock protein with a duplicated alpha-crystallin domain, forms dimers and tetramers with good chaperone-like activity.

Small heat shock proteins (sHSPs), which range in monomer size between 12 and 42 kDa, are characterized by a conserved C-terminal alpha-crystallin domain of 80-100 residues. They generally form large homo- or heteromeric complexes, and typically have in vitro chaperone-like activity, keeping unfolding proteins in solution. A special type of sHSP, with a duplicated alpha-crystallin domain, is present in parasitic flatworms (Platyhelminthes). Considering that an alpha-crystallin domain is essential for the oligomerization and chaperone-like properties of sHSPs, we characterized Tsp36 from the tapeworm Taenia saginata. Both wild-type Tsp36 and a mutant (Tsp36C-->R) in which the single cysteine has been replaced by arginine were expressed and purified. Far-UV CD measurements of Tsp36 were in agreement with secondary structure predictions, which indicated alpha-helical structure in the N-terminal region and the expected beta-sandwich structure for the two alpha-crystallin domains. Gel permeation chromatography and nano-ESI-MS showed that wild type Tsp36 forms dimers in a reducing environment, and tetramers in a non-reducing environment. The tetramers are stabilized by disulfide bridges involving a large proportion of the Tsp36 monomers. Tsp36C-->R exclusively occurs as dimers according to gel permeation chromatography, while the nondisulfide bonded fraction of wild type Tsp36 dissociates from tetramers into dimers under nonreducing conditions at increased temperature (43 degrees C). The tetrameric form of Tsp36 has a greater chaperone-like activity than the dimeric form.