Visualization and 3D reconstruction of flame cells of Taenia solium (cestoda).

BACKGROUND: Flame cells are the terminal cells of protonephridial systems, which are part of the excretory systems of invertebrates. Although the knowledge of their biological role is incomplete, there is a consensus that these cells perform excretion/secretion activities. It has been suggested that the flame cells participate in the maintenance of the osmotic environment that the cestodes require to live inside their hosts. In live Platyhelminthes, by light microscopy, the cells appear beating their flames rapidly and, at the ultrastructural, the cells have a large body enclosing a tuft of cilia. Few studies have been performed to define the localization of the cytoskeletal proteins of these cells, and it is unclear how these proteins are involved in cell function. METHODOLOGY/PRINCIPAL FINDINGS: Parasites of two different developmental stages of T. solium were used: cysticerci recovered from naturally infected pigs and intestinal adults obtained from immunosuppressed and experimentally infected golden hamsters. Hamsters were fed viable cysticerci to recover adult parasites after one month of infection. In the present studies focusing on flame cells of cysticerci tissues was performed. Using several methods such as video, confocal and electron microscopy, in addition to computational analysis for reconstruction and modeling, we have provided a 3D visual rendition of the cytoskeletal architecture of Taenia solium flame cells. CONCLUSIONS/SIGNIFICANCE: We consider that visual representations of cells open a new way for understanding the role of these cells in the excretory systems of Platyhelminths. After reconstruction, the observation of high resolution 3D images allowed for virtual observation of the interior composition of cells. A combination of microscopic images, computational reconstructions and 3D modeling of cells appears to be useful for inferring the cellular dynamics of the flame cell cytoskeleton.

Observaciones al microscopio electrónico de barrido del interior de un proglótido de un parásito adulto de taenia solium / Scanning electron microscopy observations of the cross-section of a taenia solium adult tapeworm

No existen, hasta el momento, imágenes que muestren la disposición de la citoarquitectura de parásitos adultos de Taenia solium, parásitos los cuales se encuentran en el intestino de portadores humanos asintomáticos. Las causas de ello podrían tener como base el que cuando se recuperan los parásitos, ellos han sufrido alteraciones debidas a la respuesta inmune de sus hospederos o bien, por el efecto que han producido en los parásitos los fármacos antihelmínticos que hayan sido usados en el tratamiento de los pacientes. Una de las alternativas que se han encontrado para la obtención de parásitos adultos, es la obtención de tenias a partir del modelo de teniosis experimental en hámsteres dorados e inmunosuprimidos y que gracias a este modelo se han podido efectuar diferentes tipos de estudios de los parásitos de esta fase infectiva. El propósito de este reporte es presentar imágenes de ultraestructura, obtenidas mediante Microscopía Electrónica de Barrido, de un corte transversal obtenido de un proglótido de una tenia recuperada de una infección experimental. Las imágenes se obtuvieron a diferentes aumentos y muestran aspectos relacionados con la superficie tegumentaria, el tegumento sincicial continuo, la capa germinal que incluye el soma de algunas células subtegumentarias y los ductos del sistema protonefridial tanto vacíos como llenos con corpúsculos calcáreos. Las imágenes ultraestructurales obtenidas muestran una forma de observación de la anatomía microscopica de los parásitos en estudio y ello contribuye a ampliar el conocimiento de los mismos en relación a aspectos de su biología celular y su fisiología.

There are no clear morphological evidences of the cytoarchitecture of intestinal adult tapeworms of Taenia solium recovered from infected humans. Parasites could be altered because of the host´s immunological response or by the direct action of drugs used for antihelminthic treatment. Experimental taeniosis in immunosuppressed golden hamsters is a useful way for recovering and studying adult parasites. The purpose of this report is to show images, taken at the ultrastructural level by Scanning Electron Microscopy (SEM), of a cross-sectioned strobilar chain from an adult tapeworm. The parasite was recovered from an experimental infection. Images were taken at several magnifications; they show the brush border tegumental surface, the syncytial tegument, the germinal layer, some cell bodies and the protonephridial system ducts: empty or filled with calcareous corpuscles. Ultrastructural images taken using SEM of T. solium adult parasites, recovered from experimental infections, could be a new way for observing the microscopic anatomy of these parasites and for increasing the knowledge of aspects related to their cellular biology and physiology.

[Observation on the ultrastructure of Taenia solium oncosphere].

OBJECTIVE: To observe the ultrastructure of Taenia solium oncospheres. METHODS: Patients infected with Taenia solium were de-wormed by decoction arecae and pumpkin seeds to get the mature proglottids and collect eggs. The eggs were treated with sodium hypochlorite to break the eggshells. Oncospheres were collected in Percoll (isoosmotic solution), and activated with artificial intestinal juice. The specimens were prepared with hot agar centrifugation for ultra-thin sections and observed with transmission electron microscopy (TEM) . RESULTS: T. solium oncosphere was in oval shape with a size of (14-17) microm x (10-13) microm. There were some irregular ecphymata or plicae on its surface. The hooks were composed of outer pellet layer, the middle fibrous layer and the central core marrow. Myoblasts, hook-forming cells and cerebral cortex cells were observed in the mature oncospheres. CONCLUSION: The ultrastructure of Taenia solium oncosphere is similar to that of Hymenolepis diminuta, with difference in hooks. There are binucleate cells which play a role in forming epithelium in the development of oncospheres.

Immunolocalization of Taenia solium gap junction innexins.

In previous studies, ultrastructural observations revealed a large number of gap junctions (GJs) in the neck and immature proglottid tissues of Taenia solium tapeworms. In these helminths, cytoplasmic glycogen sacs are connected by numerous discrete GJs to other cells throughout the maturing strobilar tissue. Discontinuous sucrose gradients were used to purify membrane fractions containing GJs, which were identified by ultrastructural analysis. A trans-membrane peptide sequence from a highly conserved innexin region was used to construct a 20-amino acid synthetic peptide and used to raise polyclonal antibodies in rabbits that recognized both a 55 and a 67 kDa protein in a Western blot of the GJ-enriched pellet. Immunohistochemistry of larval and adult worm sections incubated with antiserum to the synthetic peptide and a secondary anti-rabbit IgG bound to fluorescein, revealed strong binding to the tegumentary surface of the worm, as well as patchy fluorescent areas in the parenchyma. The results indicate that both the tegument of cysticerci and adult T. solium contain innexin-rich membranes, which may function as a tegumentary transport system for small molecules.

What triggers seizures in neurocysticercosis? A MRI-based study in pig farming community from a district of North India.

Colloidal and calcified cysts are considered responsible for seizure in neurocysticercosis (NCC); however, calcified cysts have also been reported in asymptomatic individuals. We carried out a MRI-based study in a rural pig farming community of North India to detect the various stages, locations and numbers of the cyst in asymptomatic individuals and compared them with symptomatic NCC cases to see its association with occurrence of seizures. A total of 107 asymptomatic family members of 29 symptomatic NCC confirmed cases were evaluated clinically, immunologically and by neuroimaging for NCC. MRI-based staging of the parasite was done in both groups, and compared to look for the differences, if any. Thirty-one (29.0%) asymptomatic family members of symptomatic cases were diagnosed to have NCC. There was no difference in proportion of colloidal/degenerating and calcified stages of the parasite between symptomatic and asymptomatic groups; however, significantly higher proportion of the asymptomatic populations had vesicular stage of the parasite (P=0.029). Our findings show that a large number of individuals harboring different stages of cysticerci in their brain remain symptoms free and question the belief that the degenerating/calcified stages of the parasite are primarily responsible for seizure occurrence in NCC.

Metabolite pattern of Cysticercus cellulosae metacestode from different predilection sites of swine using proton magnetic resonance spectroscopy.

Cysticercosis due to Taenia solium is one of the most common public health problems in various regions of the world. We have performed prolon magnetic resonance spectroscopy (1H MRS) experiments of the fluid aspirated from cysticerci excised from skeletal muscle (n = 16) and brain (n = 9) of infected swine to compare the metabolite pattern of cysticerci in different predilection sites. Perchloric acid extract of cysticercus cysts excised from skeletal muscles (n = 16) was also prepared to ascertain water-soluble, low molecular weight metabolites using 1H MRS. Absolute quantification and statistical analysis of different metabolites was done to look for any significant differences in different locations of cysts. The metabolite pattern of cysticerci was found to be similar in the various predilection sites. Metabolites observed were leucine, valine, alanine, lysine, glycine, lipid contents, lactate, glutamate, acetate, succinate, creatine, choline, and glucose. Concentration of creatine in cysticercus fluid of cysts removed from the muscle was found to be significantly higher (p = 0.001) than the cysts located in the brain. We conclude that the metabolite pattern in the cysticerci is not influenced by the surrounding tissue location; however concentration of certain metabolites may depend upon the tissue location.

A cysteine protease from Taenia solium metacestodes induce apoptosis in human CD4+ T-cells.

Here we investigated whether the depletion of CD4+ lymphocytes, observed in mononuclear cells incubated with Taenia solium metacestode E/S products or with living cysts was due to apoptosis. Using the deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), electron microscopy and DNA gel electrophoresis, we found signs of apoptosis in these cells. Results showed that cysteine protease activity was responsible for this effect, since E-64 prevented cell death in all cases. Electron microscopy studies showed that lymphocytes exhibited features of apoptosis such as cellular membrane integrity, strangling and fragmentation of nuclei, chromatin condensation, apoptotic bodies and loss of microvilli. In contrast, lymphocytes co-cultured with living metacestodes plus E-64 exhibited integrity of their structures. DNA fragmentation was detected by TUNEL assays and DNA gel electrophoresis. The results suggested that cell death induced by the cysteine protease from the T. solium metacestode may be involved in down-regulation of cell-mediated responses in infected hosts.

Ultrastructure of spermatogonia and spermatocyte lobules in Taenia solium strobilae (Cestoda, Cyclophyllidea, Taeniidae) from golden hamsters.

Golden hamsters ( Mesocricetus auratus) were infected with Taenia solium metacestodes dissected from infected pig meat. Adult worms were collected from hamster intestines of animals killed 5-60 days post-infection (dpi), incubated in RPMI 1640 medium with or without colchicine, fixed and processed for transmission electron microscopy (TEM). Sections for light microscopy from 40 different blocks with scolex, immature and mature proglottids were photographed. Thin sections were cut from 25 selected blocks, examined and photographed with TEM. Metaphase mitosis figures were observed in the subtegument of the germinative tissue and interpreted as germ cell precursors. In immature proglottids (20 dpi), discrete cell clusters of three to four cells surrounded by a thin cytoplasmic envelope were identified along the inner border of the lateral excretory ducts. These were also observed in more mature proglottids (40-60 dpi) as clusters of eight cells enclosed in a cytoplasmic envelope, with nuclei of spermatogonia exhibiting the synaptolems of primary meiotic cells. In mature proglottids from 45 dpi, a large number of spermatocyte lobules were found, exhibiting different stages of spermatogenesis from primary spermatocytes to mature filiform spermatids with a single axoneme, annular nucleus and spiral cortical microtubules, similar to spermatozoa described for type III spermiogenesis of species of the family Taeniidae. All mature spermatocyte lobules were enclosed in a highly organized cellular envelope and surrounded by a basal lamina. The envelopes contained a number of distinct organelles, seen in cross-section as discrete lattices of microtubules located between two layers of plasma membrane, as well as thickened furled cytoplasm with numerous strands of rough endoplasmic reticulum and pockets of microtubules.

Ultrastructure of smooth muscle, gap junctions and glycogen distribution in Taenia solium tapeworms from experimentally infected hamsters.

Taenia solium adults were grown in hamsters infected by feeding them with cysticerci from pig carcasses. Viable strobilae were collected from the hamster duodenum 20-60 days post-infection, fixed and processed for transmission electron microscopy (TEM). Fourteen strobilae were cut into pieces and embedded in individual blocks. Sections, stained with toluidine blue, were then photographed by light microscopy. Over 1,200 TEM images were obtained from selected blocks. Maturing proglottids exhibited a dense myofilament lattice of connecting fibers, each contained in sarcoplamsic extensions of myocytons and emitting cytoplasmic processes loosely attached to other cells, structures characterized as myocyton-myofilament-pseudopod units, which are interpreted as structures involved in the transport of cells and membrane-bound-glycogen from the germinative tissues to mature proglottids. Densely packed membrane-bound glycogen particles were found between the tegumentary cytons of the neck tissue, and as single-stranded particles between the tegumentary cytons of mature proglottids. These were wrapped around cell bodies in the parenchyma of maturing proglottids and as thin cytoplasmic strands between the testicular lobules of mature proglottids. A large number of cell-to-cell adhesions were identified as gap junctions connected to glycogen strands. We suggest that these are involved in the transport of glucose to differentiating tissues.

Morphological changes to early stage Taenia solium cysticerci following oxfendazole treatment.

The progressive morphological changes to early stage Taenia solium cysticerci following the treatment of pigs with a single therapeutic dose of oxfendazole (30 mg/kg), are described. On Day 1 after treatment, no obvious changes occurred in the general appearance of the larvae but alternations were seen by electron microscope, with an apparent reduction in the number of microtriches, and a complete disappearance of the tegument. Numerous granules were seen to have accumulated in the tegument cells. As treatment progressed, damage to the cysticerci was more serious and, by five days, all cysticerci were seen to be in an advanced stage of degeneration. By 45 days post-treatment, all cysts were calcified. These results suggest that oxfendazole is a highly effective drug against T. solium cysticerci in the early stages of development.