In Vitro Study of Taenia solium Postoncospheral Form.

BACKGROUND: The transitional period between the oncosphere and the cysticercus of Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages. METHODOLOGY/PRINCIPAL FINDINGS: T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development--days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15-30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages. CONCLUSIONS/SIGNIFICANCE: This is the first report of an in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite's immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations.

Standardization of a fluorescent-based quantitative adhesion assay to study attachment of Taenia solium oncosphere to epithelial cells in vitro.

To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment.

Utility of a protein fraction with cathepsin L-Like activity purified from cysticercus fluid of Taenia solium in the diagnosis of human cysticercosis.

Neurocysticercosis, an endemic parasitic disease in most developing countries, is caused by Taenia solium and compromises the human central nervous system. Cathepsin L-like proteases are secreted by several parasites including T. solium and constitute important antigens for immunodiagnostics. A protein fraction with cathepsin L-like activity was purified from the cysticercus fluid by size exclusion and ion exchange chromatography. Cathepsin L-like activity was measured fluorometrically by detecting the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC. The purified protein fraction included antigens of 53 and 25 kD that were tested in a Western immunoblot and in an enzyme-linked immunosorbent assay (ELISA) for detection of human cysticercosis. The sensitivity of the Western immunoblot was 96% for patients infected with multiple cysts and 78% for patients with a single cyst. Specificity was 98%. The sensitivity of the ELISA was 98% in patients with multiple cysts and 84% in patients with a single cyst. Specificity was 92.7%.

Taenia solium oncosphere adhesion to intestinal epithelial and Chinese hamster ovary cells in vitro.

The specific mechanisms underlying Taenia solium oncosphere adherence and penetration in the host have not been studied previously. We developed an in vitro adhesion model assay to evaluate the mechanisms of T. solium oncosphere adherence to the host cells. The following substrates were used: porcine intestinal mucosal scrapings (PIMS), porcine small intestinal mucosal explants (PSIME), Chinese hamster ovary cells (CHO cells), epithelial cells from ileocecal colorectal adenocarcinoma (HCT-8 cells), and epithelial cells from colorectal adenocarcinoma (Caco-2 cells). CHO cells were used to compare oncosphere adherence to fixed and viable cells, to determine the optimum time of oncosphere incubation, to determine the role of sera and monolayer cell maturation, and to determine the effect of temperature on oncosphere adherence. Light microscopy, scanning microscopy, and transmission microscopy were used to observe morphological characteristics of adhered oncospheres. This study showed in vitro adherence of activated T. solium oncospheres to PIMS, PSIME, monolayer CHO cells, Caco-2 cells, and HCT-8 cells. The reproducibility of T. solium oncosphere adherence was most easily measured with CHO cells. Adherence was enhanced by serum-binding medium with >5% fetal bovine serum, which resulted in a significantly greater number of oncospheres adhering than the number adhering when serum at a concentration less than 2.5% was used (P < 0.05). Oncosphere adherence decreased with incubation of cells at 4 degrees C compared with the adherence at 37 degrees C. Our studies also demonstrated that T. solium oncospheres attach to cells with elongated microvillus processes and that the oncospheres expel external secretory vesicles that have the same oncosphere processes.

Prevalence of antibodies to unique Taenia solium oncosphere antigens in taeniasis and human and porcine cysticercosis.

The presence of two oncosphere antigens (OAs) of 22.5 and 31.3 kD in whole and excretory/secretory (ES) OA preparations of both Taenia solium and T. saginata or in antigen preparations from T. solium metacestodes or immature tapeworms was assessed. This included an evaluation of whether antibodies to other cestodes cross-reacted to these OAs. The OAs were present in whole oncosphere extract and E/S antigens of T. solium, but were not present in other stages (immature tapeworm or metacestode) or in OAs of T. saginata. The majority (95%) of T. solium tapeworm carriers had antibodies to these OAs, while only 20% of active neurocysticercosis cases were positive. No antibodies to the OAs were found in healthy controls, subjects infected with Hymenolepis nana, patients with hydatid disease, T. saginata tapeworm carriers, hamsters infected with immature T. solium tapeworms, or dogs infected with Echinococcus granulosus. The OAs are stage and species specific to T. solium and antibodies to OAs are usually present in tapeworm carriers.

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.