Immunoelectrophoretic analysis of major component proteins in cystic fluid of Taenia solium metacestodes.

When cystic fluid of Taenia solium metacestodes (CF) was filtrated through Sephacryl S-300 Superfine, major proteins were in fractions III and IV. Major protein in fraction III was Band C protein of 150 kDa and that in fraction IV was Band N protein (Choi et al., 1990). When CF was electrophoresed in 0.9% agarose gel and reacted with anti-CF rabbit serum (RACF), two main bands, a long outer and a short inner band, were precipitated, together with 8 minor bands. RACF reacted with fraction III forming the long outer band whereas RACF formed the short inner band with fraction IV in immunoelectrophoresis (IEP). The long outer precipitin band of CF fraction III was similar to antigen B in hydatid fluid (HF) of Oriol et al. (1971), while the short inner band of CF fraction IV was similar to HF antigen 5 of Capron et al. (1967). When HF was reacted with RACF, the short inner band was immunoprecipitated without forming the long outer band. Common antigenicity between CF and HF seemed to exist in fraction IV rather than in fraction III of CF. Patient sera of neurocysticercosis reacted more frequently with fraction III than with fraction IV.     

Measurement of 150 kDa protein of Taenia solium metacestodes by antibody-sandwich ELISA in cerebrospinal fluid of neurocysticercosis patients.

An antigenic protein in cystic fluid of Taenia solium metacestodes (CF) of 150 kDa was measured by antibody-sandwich ELISA in serum and cerebrospinal fluid (CSF) of neurocysticercosis patients. Capture antibodies were rabbit antisera against CF (RACF) and a monoclonal antibody (MAb) against 150 kDa protein in CF. Lower limit of antibody-sandwich ELISA was 8 ng/ml of the protein. Except CF, no tested helminths extracts reacted. Levels of the protein in 351 sera from 255 patients (55 surgery confirmed and 202 antibody and CT/MRI confirmed) were below sensitivity of the assay. Of 276 CSF from 212 patients, 31 samples (11.2%) showed positive findings. This assay, therefore, was not sensitive enough to be a diagnostic. Instead, the 150 kDa protein appeared in CSF in such situations as in 2 days after praziquantel treatment, or as in a patient infected with a racemose cysticercus with degenerated cyst wall. Of cases whose follow-up CSF were assayed, 2 cases showed that the protein appeared intermittently. These results suggest strongly that appearance of free 150 kDa protein is associated with cyst wall rupture. In CSF which contained the 150 kDa protein over 61 ng/ml, the protein was recognized in SDS-PAGE before and after immunoprecipitation.     

Cross-reacting and specific antigenic components in cystic fluid from metacestodes of Echinococcus granulosus and Taenia solium

Sera from confirmed patients of 5 hydatidosis, 67 neurocysticercosis and 89 other parasitic diseases were tested for specific antibody (IgG) levels by ELISA to cystic fluid antigens from metacestodes of Echinococcus granulosus (HF) and Taenia solium (CF). All hydatidosis sera reacted positively to both HF and CF while neurocysticercosis sera did in 49.3% to HF and 85.1% to CF. The frequencies of cross-reactions were lower in other parasitic diseases to both antigens. By SDS-PAGE, protein bands of 64, 35, 22 and 7 kilodaltons (kDa) were found common in HF and CF. SDS-PAGE/immunoblot exhibited that hydatidosis sera reacted crossly to CF at 135, 110, 100, 86, 64, 45, 39, 35 and 24 kDa bands while neurocysticercosis sera did to HF at 135, 100, 86, 64, 52, 39, 35, 29 and 24 kDa bands. These results indicated that protein bands of 135, 100, 86, 64, 39, 35 and 24 kDa were major common components in HF and CF. Protein bands of 7 kDa in HF and 15, 10 and 7 kDa in CF did not react crossly and were specific components in respective antigens.     

Immunoblot findings of calcareous corpuscles binding proteins in cyst fluid of Taenia solium metacestodes

After collecting calcareous corpuscles from plerocercoid of Spirometra mansoni (sparganum), we evaluated the antigenic values of calcareous corpuscles binding proteins obtained from the cyst fluid of Taenia solium metacestodes. Immunoblot analysis revealed that cysticercosis patient sera strongly recognized 10 and 95 kDa calcareous corpuscles binding proteins. This result demonstrated that calcareous corpuscles are bound with major secretory antigenic proteins, which is possibly involved in the secretory pathways of the 10 and 95 kDa proteins presenting in the cyst fluid of T. solium metacestodes.     

Immunolocalization of the 150 kDa protein in cyst fluid of Taenia solium metacestodes

The 150 kDa protein of cyst fluid (CF) of Taenia solium metacestodes was purified by ammonium sulfate fractionation and Superose 6 HR gel filtration chromatography. The purified protein consisted of three subunits (15, 10 and 7 kDa proteins), which were analyzed with the use of a 7.5-15% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunofluorescence study was carried out by using immunize specific polyclonal antibody. Positive reactions were noticed at bladder walls, calcareous corpuscles, granules of cyst fluid and some host tissue surrounding the bladder wall of the metacestodes. These results suggest that the 150 kDa protein was secreted into host tissues, inducing immune responses in the host, and it may play important roles in the cellular physiology of the parasites.     

Immune response to different fractions of Taenia solium cyst fluid antigens in patients with neurocysticercosis

The immunopathogenesis of neurocysticercosis (NCC) largely remains unknown. We analyzed the immune response to different fractions of Taenia solium cyst fluid antigens in patients with NCC. Lymphocytes were separated from 48 patients with NCC-related active epilepsy and 30 healthy controls. T. solium (isolated from pig muscles) antigens (crude lysate, CL; cyst wall, CW and cyst fluid, CF) at 20 μg/well concentrations were used to stimulate the cells in a lymphocyte transformation test (LTT). Only CF antigen stimulated cell proliferation significantly greater than control (p < 0.001), hence cyst fluid antigens were further studied. The CF antigens were electro-blotted on nitrocellulose membrane (NC), cut at 0.5 cm distance and particulate antigens were prepared. A total of 12 fractions, designated F1 to F12 according to molecular weight were tested in-vitro for LTT. After 72 h of stimulation by the different fractions, Th1 (IL-1β, TNF-α, IL-2) and Th2 (IL-4, IL-10) cytokine responses were determined in culture supernatants by ELISA. Low molecular weight fractions F1 through F4 (Mol. wt. < 25 kDa) were found to be potent inducers of cytokines. Fractions F1, F3 and F4 induced the production of Th1 (IL-1β, TNF-α, IL-2), whereas F2 induced the production of Th2 (IL-4 and IL-10) cytokine. The study shows that the low molecular weight fractions of CF antigens are immuno-dominant. Most of these fractions (F1, F3, F4) induce strong Th1 immune response except F2 which induces Th2 response. Further studies are needed to identify the different antigens present in these fractions to determine the molecules responsible for the immune response.     

Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J_unbound) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ_unbound) and aqueous (AJ_unbound) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J_unbound, 92.5% and 93.5% for DJ_unboundand 82.5% and 82.6% for AJ_unbound. By immunoblot, the DJ_unboundfraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ_unboundfraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.     

Genetic polymorphism in Taenia solium metacestodes from different Brazilian geographic areas

The aim of the present study is to investigate genetic polymorphisms in Taenia solium metacestodes from different Brazilian geographical areas and to relate them to antibody recognition in serum samples of neurocysticercosis (NC) patients. Metacestodes were obtained from the Distrito Federal (DF), Bahia, Minas Gerais (MG) and S?o Paulo (SP) regions of Brazil. Samples of human sera from 49 individuals with NC, 68 individuals with other helminthiasis and 40 healthy volunteers were analysed (157 individuals in total). Antigens were prepared and used in enzyme-linked immunosorbent assay and western blotting assays to detect specific immunoglobulin G antibodies. Genetic distances between metacestode populations were analysed using random amplified polymorphic DNA (RAPD) analysis. Our results show that there was a higher frequency of reactivity in the DF region in the sera from NC patients (p < 0.05), while discrimination between active and inactive NC was seen only in extracts from the MG and SP regions (p < 0.05). Using RAPD, the sample from the DF region presented a greater increase compared to the other regions. A relationship between genetic polymorphisms among T. solium metacestodes from different areas in Brazil and the differences in antibody detection in patients with NC were established.     

In situ detection of antigenic glycoproteins in Taenia solium metacestodes

Taenia solium has a complex life cycle. Its cysticercus can lodge in the brain, causing neurocysticercosis (NCC), and the adult tapeworm's survival in the intestine results in taeniasis. In this study, the in situ detection of previously described glycoprotein antigens used for serological diagnosis of NCC and the detection of other glycoconjugates was explored in cysticerci and the surrounding porcine tissue to understand their potential role in pathogenesis. Immunohistochemistry with an antiserum specific for glycoprotein antigens rich in N-linked carbohydrates and in situ histochemistry with a battery of lectins that have affinity to a variety of glycoconjugates were performed. The glycoconjugates rich in N-linked carbohydrates were detected in the vesicular fluid and tegument of the vesicular membrane and scolex, where the parasite has direct contact with the host tissues during cysticercosis and taeniasis, respectively. Additionally, as the inflammatory response progressed, the parasite's antigenic glycoproteins were also detected in the cytoplasm of inflammatory cells in the surrounding granuloma. In contrast, the spiral canal tegument, which will be exposed to intestinal enzymes in taeniasis, had N-acetyl-galactosamine-rich mucins. Thus, the differential saccharidic composition in T. solium metacestode structures may be important for the survival of the parasite in different host sites.     

TH2 profile in asymptomatic Taenia solium human neurocysticercosis

Neurocysticercosis (NC), a parasitic disease caused by Taenia solium, may be either asymptomatic or have mild to severe symptoms due to several factors. In this study, the immunological factors that underlie NC pleomorphism were studied. Ten of the 132 inhabitants of a rural community in Mexico (Tepez) had a computerized tomography (CT) scan compatible with calcified NC, and all were asymptomatic. Their immunological profiles were compared with those of 122 CT scan negative (non-NC) subjects from the same village. NC was associated with a TH2 response (IgG4, IL-4, IL-5, IL-13). Subjects from Tepez had higher levels of specific antibodies (IgG1, IgG2, IgG4, IgE) and specific cell proliferation than subjects from an area with low exposure (Ensenada). This suggests that non-NC subjects from Tepez had been exposed to T. solium and resisted infection in the brain. Distinct immunological profiles in equally exposed individuals differing in outcome of infection support the hypothesis of host-related factors in resistance to and pathogenesis of NC. This is the first study reporting the immunological profile associated with the asymptomatic form of NC.     

Partial characterization of a 29 kDa cysteine protease purified from Taenia solium metacestodes

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.     

Crystals of virus-like particles in the metacestodes of Taenia solium and T. crassiceps

Crystals of virus-like particles (VLP) are described as occurring in the nuclei of damaged tegumentary cytons from carcasses of Taenia solium metacestodes that had been stripped of their teguments. The VLP are grouped as parallel lines of round particles in an hexagonal packaging of spheroids forming small or large crystals. The individual particles have an external diameter of 36-37 nm and a wall of 5-6 nm thick, which surround a cavity of lower electron density. As identical crystals were also observed in normal tissues of T. solium and of T. crassiceps, it is suggested that both species of cysticerci are normal carriers of a similar species of virus. The possible biological implications of this condition are discussed.     

Taenia solium metacestode immunodominant peptides recognized by IgG antibodies in cerebrospinal fluid and serum paired samples from patients with active and inactive neurocysticercosis

The aim of this study was to test if serological distinction between patients with active and inactive neurocysticercosis (NCC), could be accomplished by the recognition of immunodominant peptides in total saline antigenic extract of Taenia solium metacestodes by IgG antibody in cerebrospinal fluid (CSF) and serum paired samples. CSF and serum samples of 10 each, active NCC patients, inactive NCC, and individuals with other neurological disorders, were used to recognize the antigenic peptides by western blot (WB). In the active NCC the 28-32 and 39-42 kDa peptides were more frequently detected in CSF than in sera (p < 0.05). The 47-52, 64-68, and 70 kDa antigens showed high frequencies in both samples from patients with active NCC. All the CSF samples of inactive NCC and other neurological disorder (control) patients tested negative, while serum samples from these last two groups recognized mainly the 80, 86, 95, and 98 kDa bands. This finding eliminates the use of the high molecular weigh bands (>or= 80 kDa) for diagnosis of NCC. The final conclusions were that the difference between active and inactive NCC may be done with the detection of peptides only in the CSF samples and that the 47-52, 64-68, and 70 kDa bands may be included as specific markers for active NCC when detected in CSF samples by WB using total saline extract of T. solium metacestode.     

The 10 kDa protein of Taenia solium metacestodes shows genus specific antigenicity

Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.     

Bioactive molecules of Taenia solium metacestode,a causative agent of neurocysticercosis

Neurocysticercosis (NC), an infection of the CNS with Taenia solium metacestode, exemplifies formidable public health concerns associated with significant morbidity and mortality. The disease is a complex phenomenon involving molecular cell biological cross-talks between the parasite and human host. To effectively combat NC, specific diagnosis and proper management are prerequisites. Bioactive molecules implicated in host–parasite interactions and parasitic homeostasis should be elucidated. This article provides an overview of currently available serological biomarkers, especially those comprising low-molecular-weight proteins, and discusses available immunoproteomics for identification of such molecules. T. solium metacestode bioactive molecules, which might be critically implicated in the progression of NC disease, are summarized. Comprehensive understanding of the biochemical properties and biological functions of bioactive molecules may contribute to the development of novel intervention strategies against NC.     

Clinical signs for identification of neurocysticercosis in swine naturally infected with Taenia solium

Taenia solium infection is a zoonotic disease and swine is the natural intermediate host. Till date no literatures have described clinical signs in swine indicative of brain involvement by cysticerci. In the present study we describe such clinical signs of porcine neurocysticercosis (NCC). These signs were excessive salivation, excessive blinking and tearing, and subconjunctival nodule. A total of 30 swine (18 with 2 or all 3 clinical signs and 12 without any sign) underwent magnetic resonance imaging (MRI). All 18 swine with above signs had NCC on MRI along with variable involvement of other organs that were subsequently confirmed by ex vivo MRI, necropsy and histopathology, while none of the 12 animals without any sign had NCC. As development of a porcine NCC model has proved difficult, we propose that naturally infected swine can be identified on the basis of these clinical signs and thus used as a model for further research on NCC.     

Annexin B1 from Taenia solium metacestodes is a newly characterized member of the annexin family

We previously reported cloning of the Taenia solium annexin B1 gene from a metacestode cDNA expression library and demonstrated that it acts as a protective antigen for effective vaccine development against cysticercosis. In the present study we produced recombinant annexin B1 and antiserum against the protein to investigate its structural and functional properties. Western blotting of metacestode fractions indicated that T. solium annexin B1, similar to vertebrate annexins, associates with acid phospholipids in the presence of Ca(2+). This property was confirmed by the recognition of apoptotic cells by labeled annexin B1. CD spectroscopy results demonstrated that alpha-helices are the main secondary structures of the protein. Ca(2+) binding increases the alpha-helix content and causes significant thermal stabilization with a melting temperature increase of approximately 10 degrees C. Functional Ca(2+)-dependent phospholipid binding sites of annexin B1 were investigated using mutant proteins. By changing a conserved acidic amino acid residue that putatively combines Ca(2+) in each domain of annexin B1 singly or in combination, we found that Ca(2+) binding in the first domain is more important than that at the other Ca(2+) binding sites. Annexin B1 is a metacestode stage-specific antigen, with the protein being mainly localized in the teguments and surrounding cyst wall of T. solium metacestodes, suggesting a role in the parasite-host interaction.     

Bioactive molecules of Taenia solium metacestode, a causative agent of neurocysticercosis

Neurocysticercosis (NC), an infection of the CNS with Taenia solium metacestode, exemplifies formidable public health concerns associated with significant morbidity and mortality. The disease is a complex phenomenon involving molecular cell biological cross-talks between the parasite and human host. To effectively combat NC, specific diagnosis and proper management are prerequisites. Bioactive molecules implicated in host-parasite interactions and parasitic homeostasis should be elucidated. This article provides an overview of currently available serological biomarkers, especially those comprising low-molecular-weight proteins, and discusses available immunoproteomics for identification of such molecules. T. solium metacestode bioactive molecules, which might be critically implicated in the progression of NC disease, are summarized. Comprehensive understanding of the biochemical properties and biological functions of bioactive molecules may contribute to the development of novel intervention strategies against NC.     

Oncospheres of Taenia solium and T. saginata asiatica develop into metacestodes in normal and immunosuppressed mice.

Normal and immunosuppressed mice were infected with oncospheres of Taenia saginata asiatica and T. solium. Although normal ICR mice were not susceptible to these two parasites, cysticerci were recovered from the immunosuppressed ones following venous injection. For T. s. asiatica, immunosuppressed ICR mice had an infection rate of 12.5% and six cysticerci of this parasite were recovered from three males. After injection of T. solium oncospheres, a high infection rate of 57% was obtained and 23 cysticerci were collected from 13 male immunosuppressed ICR mice. The immunosuppressed C57 mice had the highest infection rate (100%) and cysticercus recovery rate (2.4%) for T. solium. The infection rate and cysticercus recovery rate in six normal C57 mice were 40% and 3% respectively. The immunosuppressed ICR, Balb/c and C3H mice were also susceptible to T. s. asiatica.     

Genetic variation in the Cytb gene of human cerebral Taenia solium cysticerci recovered from clinically and radiologically heterogeneous patients with neurocysticercosis

Neurocysticercosis (NC) is a clinically and radiologically heterogeneous parasitic disease caused by the establishmentof larval Taenia solium in the human central nervous system. Host and/or parasite variations may be related to this observed heterogeneity. Genetic differences between pig and human-derived T. solium cysticerci have been reported previously. In this study, 28 cysticerci were surgically removed from 12 human NC patients, the mitochondrial gene that encodes cytochrome b was amplified from the cysticerci and genetic variations that may be related to NC heterogeneity were characterised. Nine different haplotypes (Ht), which were clustered in four haplogroups (Hg), were identified. Hg 3 and 4 exhibited a tendency to associate with age and gender, respectively. However, no significant associations were found between NC heterogeneity and the different T. solium cysticerci Ht or Hg. Parasite variants obtained from patients with similar NC clinical or radiological features were genetically closer than those found in groups of patients with a different NC profile when using the Mantel test. Overall, this study establishes the presence of genetic differences in the Cytb gene of T. solium isolated from human cysticerci and suggests that parasite variation could contribute to NC heterogeneity.