Lilia Robert

TAENIA SOLIUM: DESCRIPTION OF THE INTESTINAL IMPLANTATION SITES IN EXPERIMENTAL HAMSTER INFECTIONS

Experimental infections in golden hamsters with viable Taenia solium metacestodes were used to study by light and electron microscopy the implantation site of the adult tapeworm in the intestinal wall. Implantation sites from 3-, 4-, 10-, and 40-day infections were located in the upper third of the duodenum, excised and fixed in Zenkers or Karnovskys solution, embedded in Polybed resin, and sectioned longitudinally to observe the position of the worm on the intestinal wall. The scolex of the tapeworm was situated between host villi, with the rostellum penetrating the intestinal wall and the suckers entrapping adjacent villi. Serial sections through several whole implantation sites revealed that the worm was anchored to the host by all 4 suckers simultaneously, each of which was located at a different level and had entrapped intestinal villi in its cavity. Host tissue within the suckers was damaged, exhibiting various degrees of cell lysis and necrosis of epithelial and submucosal cells. The tegumentary surface and microtriches of the scolex were well preserved, with occasional coalescence of tegumentary microvesicles in 10- and 40-day-old infections; microtriches were in direct contact with the damaged host tissue. This study is the first morphological and ultrastructural description of the attachment of T. solium to the intestinal wall employing an experimental model, the results of which may contribute to a better understanding of the biology of human tapeworm infections.

Ultrastructure of spermiogenesis and the spermatozoon in Taenia crassiceps strobilae WFU strain (Cestoda, Cyclophyllidea, Taeniidae) from golden hamsters

Strobilae from Taenia crassiceps (WFU strain) were obtained from outbred hamsters (Mesocricetus auratus) by feeding them viable metacestodes maintained by intraperitoneal passage in female Balb/c mice. Mature and gravid proglottids from strobilae were recovered from hamster intestines and fixed for light and electron microscopy. By light microscopy, the expected structure of taeniid proglottids was observed. Ultrastructural analysis of ten proglottids showed that testicular follicles and vas deferens contained filiform spermatids, with a single axoneme, and an elongated helicoidal nucleus inserted between the axoneme and the spiraled cortical microtubules. At the apical cone, a single crest-like body was found and mature spermatids also exhibited transverse intracytoplasmic walls. The morphology and characters of the spermatids in T. crassiceps conform to type III spermiogenesis, which has been described in other taeniids.

Taenia solium: Germinal Cell Precursors in Tapeworms Grown in Hamster Intestine

BACKGROUND In a previous study, it was shown that growth of evaginated metacestodes occurs in the germinative tissue of the neck by duplication of somatic stem cells. In these specimens, it was not possible to find the mitotic figures required to demonstrate duplication of germ cell lines. METHODS Taenia solium strobilae were collected from the intestinal lumen of outbred hamsters infected orally with 10 metacestodes dissected from naturally infected pigs. Animals were anesthetized 1-10 days postinfection, the small intestine excised, submerged in PBS, and cut open longitudinally. Live Taenias were incubated for 6-8 h in medium containing colchicine or 3H-thymidine, washed, and embedded for electron microscopy. For light microscopy and autoradiography, longitudinal sections were cut from whole blocks and mounted on glass slides. A population of large cells without nuclear membranes and containing discrete aggregates of chromatin were observed apposed to myofibrils in the germinative tissue of the neck. These cells were confirmed by electron microscopy as metaphase mitotic figures, with chromosomes attached to a microtubular spindle, embedded in cytoplasm, without a nuclear membrane, and with characteristic centrioles. RESULTS Only tapeworms in which 3H-thymidine was injected directly into the worm tissue by microsyringe were positive by autoradiography, demonstrating that in contrast to evaginated metacestodes, intestinal worms do not transport thymidine across the tegument. CONCLUSIONS The results show that differentiating T. solium worms have a subset of stem cells that require passage through a mammalian host to go into mitosis, and that tapeworms grown in an experimental animal do not take up 3H-thymidine in vitro.

Evagination and Infectivity of Taenia crassiceps Cysticerci in Experimental Animals

Cysticerci of Taenia crassiceps reproduce asexually by exogenous budding in the rodent intermediate host, and can experimentally develop to the adult stage within the small intestine of golden hamsters. In the present study, we report the loss of cysticercus infectivity for hamsters after maintaining the strain for 4 yr by consecutive peritoneal passage in mice. Larval infectivity was restored after a cysticercus from the WFU strain developed into a gravid tapeworm after being passaged through a dog. The eggs of this tapeworm were infective for mice, which subsequently developed cysticerci with renewed capability for infecting experimental hamsters. An in vitro evagination assay was also conducted using eleventh-generation WFU strain cysticerci, as well as second- and fourth-generation dog-derived cysticerci. Significantly higher (P < 0.0001) evagination was observed for 5-mo-old dog-derived and WFU infrapopulations when compared with respective evagination values for 9- and 12-mo-old infrapopulations. The extent of evagination was linked to the capacity of cysticerci to infect hamsters, so that greater evagination resulted in a higher infectivity for hamsters and vice versa.

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.

Discovery of an endemic area of Gnathostoma turgidum infection among opossums, Didelphis virginiana, in Mexico.

Abstract Gnathostomosis, caused by Gnathostoma binucleatum, is a serious public health issue in Mexico. Although 2 other Gnathostoma spp., G. turgidum and G. lamothei, have been found in wild animals, their natural life cycle or their relation to human disease remains unclear. While we were conducting an epidemiological survey on Gnathostoma spp. in Sinaloa State, Mexico, we found an endemic area for G. turgidum in common opossums, Didelphis virginiana, located in Tecualilla, Sinaloa. The species identification was carried out by morphological and molecular biological methods. This is the first record of an endemic area for G. turgidum infection in opossums, D. virginiana, in the Americas.

Ultrastructure of a spermatid transport system in the mature proglottids of experimental Taenia crassiceps (WFU strain)

In classical textbooks of parasitology, the mature proglottids of taeniids are depicted as structures in which the individual testis are connected to the vas deferens through the vas efferens system, usually depicted as a network of channels. From our morphological analyses of proglottids in the cestode Taenia crassiceps, we have been unable to identify this channel network. It is unclear how the spermatids are transported from the testes to the vas deferens, as is unresolved the location of the cells responsible for the production of testosterone (Leydig cells) or the possible equivalent of Sertoli cells, necessary for the differentiation process of these cells. In this experimental work, we have examined the ultrastructure of tissues in the vicinity of the vas deferens in mature proglottids obtained from the intestines of hamsters infected with cysticerci from the peritoneum of infected mice. Worm tissues were fixed, processed, and sectioned for transmission electron microscopy. Significant areas of the testis epithelia emitted cytoplasmic projections surrounded by extracellular matrix, where they appear as septated pockets enclosing free axonemes and spermatids. Vas efferens walls are made up of nucleated cells with cytoplasm annealing to each other through cell membrane junctions. Lodged between the junctions are membrane-bound pouches with dense granules found as aggregates or aligned in a semicircular array. The efferens wall exhibits numerous spermatids emerging into the lumen, an observation that suggests the epithelial wall may have the maturing functions of Sertoli cells of vertebrates. Large cells adjacent to the vas efferens contained prominent rough endoplasmic reticulum and large mitochondria, characteristics described for Leydig cells of vertebrates. Our observations suggest that taeniid spermatids are either transported from the testes to the vas system by epithelial pockets or that the epithelial pockets may be cross-sections of a highly coiled vas efferens system.

Antimicrobial peptides (Temporin A and Iseganan IB-367): Effect on the cysticerci of Taenia crassiceps

Taenia solium infections continue being a health problem in undeveloped countries, and few effective control measures against this parasite are being applied. Antimicrobial peptides (AMPs) belong to the innate immune response and capable of destroying pathogens. We tested the ability of two AMPs, Temporin A (TA) and Iseganan IB-367 (IB-367) to damage T. crassiceps cysticerci in vitro. Doses of 200 and 400 microg/ml of TA and IB-367 caused cysticerci to shrink, lose motility, the formation of macrovesicles in the tegument, as well as decreased evagination properties. These changes were observed as early as 3-6h and became more pronounced over 24h, when the morphological changes of the bladders became evident by both light and electron microscopy. Electron micrographs of cysticerci exposed to peptides showed initial changes as collapsed microvesicles in the tegument, with formation of large vesicles and extrusion of tegumentary tissues into the surrounding media, which led to complete loss of the tegument as well as shrinkage and complete loss of structure of parenchymal tissue after 24h. Peptides administered to cysticercotic mice one month post-infection in a single intraperitoneal dose of 200 or 400 microg, reduced the parasite load by 25% for IB-367, and 50% for TA. The humoral response of infected mice does not appear capable of killing surviving cysticerci. Our studies show that in vitro, AMPs severely damage the tegument and the scolex, and open a new pathway for biological drug design or the development of transgenic animals that over express these peptides capable of killing the cysticerci in vivo.

Apoptosis patterns in experimental Taenia solium and Taenia crassiceps strobilae from golden hamsters

Apoptosis or programmed cell death (PCD) patterns of two taeniid species, Taenia solium and Taenia crassiceps, were explored in adult tapeworms grown in golden hamsters. Animals were fed either ten viable T. solium cysticerci from naturally infected pigs or from T. crassiceps WFU strain maintained in Balb/c mice. Adult strobilae were recovered from the intestine at different times after infection and either frozen at −70°C or fixed in paraformaldehyde–glutaraldehyde. Frozen sections were processed using the DNA fragmentation fluorescent TUNEL reagents and examined in an epifluorescent microscope. Fixed tissues were processed for light and electron microscopy. Typical apoptotic cells were found in the central core of scolex and strobilar tissues, mainly in the germinal tissue and subtegumentary areas. By the TUNEL technique, cells exhibited the characteristic fluorescent images of condensed nuclear chromatin. By light microscopy of thick sections stained with toluidine blue, we found a number of small rounded cells which had lost their cytoplasmic bridges and had shrunken nuclei with aggregated chromatin, cells which were found interspersed with normal syncytial cells. Similar cell morphology was confirmed by electron microscopy. Stunted viable worms, recovered with longer mature specimens, had very short strobilae and exhibited a large number of apoptotic cells in the germinal neck tissues. The results are consistent with the syncytial nature of these parasites, and strongly suggest that cell proliferation and PCD in these adult cestodes are continuous processes of the germinal tissue and tegumentary cytons.

Analysis of the expression of cytoskeletal proteins of Taenia crassiceps ORF strain cysticerci (Cestoda)

The Taenia crassiceps ORF strain is used to generate a murine model of cysticercosis, which is used for diagnosis, evaluation of drugs, and vaccination. This particular strain only exists as cysticerci, is easily maintained under in vivo and in vitro conditions, and offers an excellent model for studying the cytoskeletons of cestodes. In this study, several experimental approaches were used to determine the tissue expression of its cytoskeletal proteins. The techniques used were microscopy (video, confocal, and transmission electron), one-dimensional (1D) and two-dimensional (2D) electrophoresis, immunochemistry, and mass spectrometry. The tissue expression of actin, tubulin, and paramyosin was assessed using microscopy, and their protein isoforms were determined with 1D and 2D electrophoresis and immunochemistry. Nineteen spots were excised from a proteomic gel and identified by liquid chromatography–tandem mass spectrometry and immunochemistry. The proteins identified were classic cytoskeletal proteins, metabolic enzymes, and proteins with diverse biological functions, but mainly involved in detoxification activities. Research suggests that most noncytoskeletal proteins interact with actin or tubulin, and the results of the present study suggest that the proteins identified may be involved in supporting the dynamics and plasticity of the cytoskeleton of T. crassiceps cysticerci. These results contribute to our knowledge of the cellular biology and physiology of cestodes.