Enveloped and non-enveloped viral-like particles in Trypanosoma cruzi epimastigotes.

Electron microscopy is routinely used to identify viral infections in protozoan parasites. These viruses have been described as non-enveloped and icosahedral structures with a diameter of 30-60 nm. Most of them are classified within the non-segmented dsRNA Totiviridae family. We observed virus-like particles (VLPs) through transmission electron microscopy in the cytoplasm of Trypanosoma cruzi epimastigotes grown in cultures. Clusters of electrodense enveloped VLPs having a diameter of 48 nm were also observed. These clusters appear to have been released from distended Golgi cisternae. Furthermore, a paracrystalline array of electrodense, non-enveloped VLPs (with a diameter of 32 nm) were found in distended Golgi cisternae or as smaller clusters at a distance from the RE or Golgi. We cannot rule out that the 48 nm enveloped VLPs belong to the ssRNA Flaviviridae family because they are within its size range. The localization of enveloped VLPs is consistent with the replication strategy of these viruses that transit through the Golgi to be released at the cell surface. Due to the size and shape of the 32 nm non-enveloped VLPs, we propose that they belong to the dsRNA Totiviridae family. This is the first description of cytoplasmic enveloped and non-enveloped VLPs in T. cruzi epimastigotes.

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.

Etiopathogenesis of Burkitt's lymphoma: a lesson from a BL-like in CD1 mouse immune to Plasmodium yoelii yoelii.

INTRODUCTION: There is a jaw cancer that develops in children five to eight years old in holoendemic malaria regions of Africa, associated to malaria and Epstein Barr virus infections (EBV). This malignancy is known as endemic Burkitt's lymphoma, and histopatologically is characterized by a starry sky appearance. To date, no histopathologic expression of Burkitt's lymphoma has been reported in non-genetically manipulated experimental animals. The purpose of the study is to describe the case of a mouse immune to Plasmodium yoelii yoelii (Pyy) that developed a Burkitt's lymphoma-like neoplasm after repeated malaria infections. RESULTS: Immune mouse 10 (IM-10) developed neoplasms at eight months of age, after receiving three Pyy inoculations. At autopsy eight subcutaneous tumors were found of which the right iliac fosse tumor perforated the abdominal wall and invaded the colon. The histopathologic study showed that all neoplasms were malignant lymphomas of large non-cleaved cells also compatible with variants or previous states of development of a Burkitt's lymphoma-like. The thymus, however, showed a typical starry sky Burkitt's lymphoma-like neoplasm. CONCLUSIONS: Neoplasm development in CD1 mouse is associated to both, immunity against malaria and continuous antigenic stimulation with living parasites.It is the first observation of a histopathologically expressed Human Burkitt's lymphoma-like neoplasm in a non-genetically manipulated mouse.Chronic immune response associated to neoplasms development could probably be not an exclusive expression of malaria-host interaction but, it could be a pattern that can bee applied also to other agent-host interactions such as host-bacteria, fungus, virus and other parasites.

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.

Intrahepatic growth and maturation of Gnathostoma turgidum in the natural definitive opossum host, Didelphis virginiana.

Gnathostoma turgidum is a gastric nematode parasite of opossums found in the Americas. We recently found that G. turgidum juveniles appear in the liver of the opossums where they become mature adults and almost synchronously move to the stomach during certain months of the year, suggesting the importance of the liver for the growth and maturation of this species in the final hosts. In this study we attempted to detect G. turgidum larvae in the liver of opossums, Didelphis virginiana that are the natural final hosts. The results show that tiny (<3mm in length) third stage larvae (L3) appeared in the liver of opossums around November and December. Also in the liver, we found large L3 of up to about 10mm in length together with juveniles and mature adults from February to March. In spite of their length, large L3 have 4 rows of hooklets, and their gonads remained undeveloped. Morphological features of the small and large L3 of G. turgidum are described including scanning electron microscope images. The seasonal switching of the several growth stages of G. turgidum from small L3 to adult worms in the liver and eventual migration to the stomach in opossums suggests the unique feature of G. turgidum utilizing the liver as the maturation site.

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.

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

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.

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.

Characterization of Taenia solium cysticerci microsomal glutathione S-transferase activity.

Glutathione S-transferase activity has been shown to be associated with the microsomal fraction of Taenia solium. Electron microscopy and subcellular enzyme markers indicate the purity of the microsomal fraction that contains the glutathione S-transferase activity. T. solium microsomes were solubilized under conditions used to solubilize integral microsomal proteins. This procedure proved necessary to obtain enzymatic activity. To characterize this parasite enzyme activity, several substrates and inhibitors were used. The optimum activity for microsomal glutathione S-transferase was found to be pH 6.6, with a specific enzyme activity of 0.9, 0.1, 0.067, 0.03, and 0.05 micromol min(-1) mg(-1) with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 2,4-hexadienal, and trans-2-nonenal, respectively. No activity of glutathione peroxidase was observed. T. solium microsomes had an appKm (GSH)=0.161 microM, appKm (CDNB)=14.5 microM, and appVmax of 0.15 and 27.9 micromol min(-1) mg(-1) for GSH and CDNB, respectively. T. solium microsomes were inhibited by several glutathione S-transferase enzyme inhibitors, and it was possible to establish a simple inhibition system as well as corresponding Ki's for each inhibitor. These results indicate that the T. solium microsomal glutathione S-transferase is different from the parasite cytoplasmic enzymes that catalyze similar reactions.

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.

Taeniid tapeworm responses to in vitro glucose.

Experimental taeniid strobilae from Taenia solium and T. crassiceps (WFU strain) were incubated for 0-72 h in 0, 5 or 20 mM glucose solutions and further exposed for 15 min to the gap junction fluorochrome Lucifer Yellow. Frozen sections were obtained from each worm and observed under an epifluorescent microscope. Worm sections from strobilae incubated with glucose, revealed intense fluorescence in the base of the tegumentary surface, suggesting that this tissue behaves as a gap junction complex. Fluorescence intensity differences between control worms not exposed to glucose and worms incubated with glucose, were highly significant. The results demonstrate that under in vitro conditions, glucose is taken up along the whole strobilar tegument in both taeniid species, suggesting, that although taeniids attached to the duodenum probably take up most of their nutrients directly from the mucosal wall, the capacity for absorbing glucose along the tegumentary surface is always active and may increase the survival capacity of these intestinal worms by promoting glucose absorption at other points in the intestinal lumen.

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 degrees 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.

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.

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.

Las helmintiasis intestinales en la República Mexicana / Intestinal helminthiases in the Mexican Republic

En la República Mexicana se han realizado escasos trabajos dirigidos a determinar la frecuencia de las helmintiasis intestinales en humanos. Sin embargo, con los pocos trabajos confiables de que se dispone, realizados de 1981 a 1992, es posible señalar que la ascariasis, tricocefalosis, uncinariasis e himenolepiasis, se encuentran presentes en forma significativa con porcentajes de infección del 11,2 por ciento, 1,7 por ciento, 0,15 por ciento y 1,8 por ciento, respectivamente. Con la información vertida en este artículo y obtenida de las encuestas realizadas en el período mencionado anteriormente, se puede señalar que las infecciones intestinales por helmintos están presentes en forma significativa en la República Mexicana. Sin lugar a dudas, esto es debido a que aún persisten los factores que contribuyen a la diseminación de las helmintiasis, tales como fecalismo, pobreza, malos hábitos higiénicos, manejo inadecuado de alimentos y bebidas, y en general deficiencias en las condiciones de saneamiento ambiental

Frecuencia de las protozoosis intestinales en la República Mexicana / Frequence of intestinal protozooses at Mexican Republic

Es de hacer notar que las frecuencias con que se reportan las distintas protozoosis intestinales en las diversas localidades de la República Mexicana en los últimos 10 años, son incompletas y poco fidedignas en general, a lo qe hay que agregar que han sido escasas las encuestas epidemiológicas realizadas en el país. Sin embargo, por los pocos estudios confiables efectuados en el país vemos que la amibiasis (30,6 por ciento), giardiasis (22,3 por ciento), criptosporidiosis (39,1 por ciento) se presentan con porcentajes de infección muy significativos, ya que afectan a núcleos de población numerosos. Los estudios que aquí se muestran conducen a afirmar que las infecciones por protozoos intestinales del hombre en México, siguen persistiendo considerablemente, de acuerdo a la población total del país, y que esto se debe entre otras causas, a que continuan presentándose, en las distintas zonas factores que favorecen la presencia y persistencia de parásitos intestinales, que teniendo como un marco general serias deficiencias en la cultura higiénica de numerosas comunidades, tales como el fecalismo al aire libre, inadecuados hábitos higiénicos y alimenticios, así como condiciones ambientales propicias para la sobrevivencia de estos protozoos

Estado actual de los conocimientos sobre la enfermedad de Chagas en la República Mexicana / Current status of knowledges on Chagas disease in Mexican Republic

Se presenta una minuciosa revisión de la literatura médica nacional e internacional acerca de la enfermedad de Chagas en la República Mexicana, desde 1939, cuando el Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, fue reportado por primera vez, la cual ha hecho posible presentar el panorama general sobre esta parasitosis en el país, desde 1939 hasta 1991. Mediante una serie de tablas y figuras se presentan las localidades del país en donde se han encontrado casos humanos, reservorios y vectores de T. cruzi. Se hacen comentarios en relación a los resultados reportados y se señala la importancia de incrementar los estudios sobre la enfermedad de Chagas en México