In Vitro Study of Taenia solium Postoncospheral Form

Abstract

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.

Fig 1. Light micrographs of development of Taenia solium postoncospheral form in HCT-8 cell monolayer.

(a) Left: T. solium activated oncosphere at 1 day of culture viewed at 10x objective; Right: oncosphere with typical hooks. (b) Left: T. solium postoncospheral forms at 15 day of culture viewed at 10x objective; Right: nucleus and cells were present in small postoncospheral forms. (c) Left: T. solium postoncospheral forms at 30 days of culture viewed at 10x objective; Right: protuberance was observed in one end of the body. (d) T. solium postoncospheral forms at 60 days of culture viewed at 10x objective; structure like a belly button observed in the middle of the body.

Fig 2. Fluorescence microscopy of T. solium postoncospheral forms stained with DAPI stain.

Number of nuclei in T. solium activated oncosphere (a) compared with the number of nuclei in T. solium postoncospheral forms at 15 (b), 30 (c) and 60 (d) days of culture.

Fig 3. Scanning electron micrographs of T. solium postoncospheral forms.

(a) T. solium activated oncosphere showing elongated microvilli. (b) 15 day postoncospheral forms showing the protuberance at one end of the body. (c) Close-up of the tegument of 30 day postoncospheral form showing microtriches. (d) 30 day postoncospheral form showing vesicles.

Fig 4. Scanning electron micrographs of the protuberance forming in T. solium postoncospheral forms at three growth points.

(a) Microtriches and (b) vesicles (arrows) observed in the surface of the protuberance at one end of the body at 15 (I), 30 (II) and 60 (III) days PO forms.

Fig 5. Morphology of T. solium 30 day postoncospheral form.

(a) Haematoxylin stain of 30 day postoncospheral form showing a large central cavity and the tegument. (b) Transmission electron microscopy of the tegument of 30 day postoncospheral form showing microtriches and cells.

Fig 6. Rat brain infected with T. solium postoncospheral forms.

(a) Rat brain 4 months post infection showing two extraparenchymal cysticerci (arrows). (b) Coronal sections of the brain containing an intraparenchymal cysticercus stained with Masson’s Trichrome staining showing tegument and scolex.

Fig 7. Western blot of T. solium antigens at different forms.

(a) Using rabbit anti-oncosphere sera. (b) Using rabbit anti-cysticerci sera.