Actin expression in Taenia solium cysticerci (cestoda): tisular distribution and detection of isoforms

Identification, localization and partial biochemical characterization of actins expressed in the larval stage of the cestode parasite Taenia solium has been carried out. Frozen tissue sections of cysticerci, the larval stage of this parasite, were reacted with rhodamine-phalloidin, parasite actin was purified by polymerization in the presence of K(+), Mg(++) and ATP actin was analyzed by SDS-PAGE and two-dimensional gel electrophoresis, and immunoblotting of actin was performed in PVDF membranes and with commercial anti-actin monoclonal antibodies. Parasitic tissues showed different fibrous actin fluorescence patterns, which correlated with the expression of isoactins. Purified globular actin had a similar molecular mass to rabbit commercial actin (approximately 44 kDa). Actin was resolved into seven isoforms, indicating a family of actin genes.     

Muscular myosin isoforms of Taenia solium (Cestoda)

Type II myosin, the primary component of the thick filament of muscle fibers, is organized as a dimeric high molecular weight protein, and is composed of a pair of heavy chains (MHC) and two pairs of light chains. Myosin II transforms ATP energy into mechanical force. All type II myosins are conserved proteins but they have two variable regions that are located in different places of the molecule. Myosin molecules are encoded by a multigene family and many isoforms are generated. The expression of myosins depends on the developmental stage and on the type and degree of contractile activity and tissue, therefore several myosin isoforms are found in the same organism. Here we describe the use of different techniques that allowed demonstrating the presence of isoforms of the heavy chain type II myosin of Taenia solium cysticerci (larvae) and tapeworms (adults), a cestode parasite of importance in public health in many developing countries. Myosin was purified and used in comparative proteolytic fragmentation, ATPase activity, detection of antigenic differences and electrophoretic separation. The results obtained showed biochemical and immunochemical differences among cysticerci and tapeworms, and demonstrate the presence of myosin isoforms in T. solium that are probably associated to physiological requirements of each developmental stage.     

Taenia solium and Taenia saginata: identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies.     

Metabolism of steroid hormones by Taenia solium and Taenia crassiceps cysticerci

Previous in vitro experiments showed that both, Taenia crassiceps and Taenia solium cysticerci have the ability to metabolize exogenous androstenedione to testosterone. Here we evaluate on the capacity of both cysticerci to synthesize several sex steroid hormones, using different hormonal precursors. Experiments using thin layer chromatography (TLC) showed that both cysticerci were able to produce ³H-hydroxyprogesterone, ³H-androstenedione and ³H-testosterone when ³H-progesterone was used as the precursor. They also synthesized ³H-androstenediol and ³H-testosterone when ³H-dehydroepiandrosterone was the precursor. In addition, both cysticerci interconverted ³H-estradiol and ³H-estrone. These results, strongly suggest the presence and activity of the Δ4 and Δ5 steroid pathway enzymes, 3β-hydroxysteroid dehydrogenase/Δ^5-4 isomerase-like enzyme (3β-HSD), that converts androstenediol into testosterone; and the 17β-hydroxysteroid dehydrogenase that interconverts estradiol and estrone, in both types of cysticerci.     

Morphological types of Taenia solium cysticerci

In human brain cysticercosis, there are three morphological types of cysticercus. Teresa Robielo, Angelica Rivas and Ana Flisser describe the appearance and possible taxonomic position of these forms, and discuss their relation to the various pathologies of neurocysticercosis.     

Taenia solium taeniasis/cysticercosis in the Menoua division (West Cameroon)

The present study was carried out between August 1999 and April 2000 with the objective of determining the prevalence of Taenia solium taeniasis in two village communities of Bafou and Bamendou in the Menoua division (West Cameroon). Four (0.13%) out of 3,109 faecal samples were positive for Taenia spp. eggs using the flotation technique. Three of the four worms expelled were T. solium whereas the other one was T. saginata. Two cases of cysticercosis were diagnosed in one of the families with a T. solium carrier. Furthermore, coprological and serological investigations for T. solium taeniasis and cysticercosis were carried out among butchers and/or tongue inspectors (n = 137) of the city of Dschang. The results were compared with those of a control group (n = 198). Taenia spp. eggs were not detected by microscopic examination. The prevalence of cysticercosis in the two groups was relatively similar (3.6 and 4.5% respectively).     

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.     

Detection of genetic variation in Taenia solium

Genetic variability among Taenia solium isolates was studied in 160 cysticerci from 6 pigs, 4 from Mexico, 1 from Honduras, and 1 from Tanzania. Random amplified polymorphic DNA (RAPD) analysis performed with 4 commercial primers showed 88% polymorphic loci and an average heterozygosity of 0.077; however, several alleles were fixed within each isolate. Linkage disequilibrium analysis indicated that 3 of the 6 isolates had a random association of alleles, whereas the other 3 had a clonal structure. These results suggest the existence of local lineages in T. solium, with events of genetic recombination within them.     

State of the Art of Taenia solium as Compared to Taenia asiatica

Three species of tapeworms infect humans in their adult stage (Taenia solium, Taenia saginata and Taenia asiatica). The 3 are flat, opaque white or yellowish, and exceptional long segmented parasites, measuring 1 to 12 m in their adult stage. In this review, the development of the knowledge regarding the first species, mainly focused on understanding how the larval stage or cysticercus is transmitted to humans, is described. The second species is a cosmopolitan parasite that only causes taeniosis and not cysticercosis; therefore, it will not be included. Information on the third species, which is presently being produced, since this species was recognized as such only at the end of the 20th century, will be discussed at the end of this review.     

Characterization of glutathione S-transferase of Taenia solium.

A Taenia solium glutathione-S-transferase fraction (SGSTF) was isolated from a metacestode crude extract by affinity chromatography on reduced glutathione (GSH)-sepharose. The purified fraction displayed a specific glutathione S-transferase (GST) activity of 2.8 micromol/min/mg and glutathione peroxidase selenium-independent activity of 0.22 micromol/min/mg. Enzymatic characterization of the fraction suggested that the activity was closer to the mammalian mu-class GSTs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and enzyme activity analysis showed that the fraction was composed of a major band of Mr = 26 kd and that the active enzyme was dimeric. Immunohistochemical studies using specific antibodies against the major 26-kd band of the SGSTF indicated that GST protein was present in the tegument, parenchyma, protonephridial, and tegumentary cytons of the T. solium metacestode. Antibodies generated against the SGSTF tested in western blot showed cross-reactivity against GSTs purified from Taenia saginata, T. taeniaeformis, and T. crassiceps, but did not react with GSTs from Schistosoma mansoni, or mice, rabbit, and pig liver tissue. Furthermore, immunization of mice with SGSTF reduced the metacestode burden up to 74.2%. Our findings argue in favor of GST having an important role in the survival of T. solium in its hosts.     

Porphyrin content of the cysticercus of Taenia solium

The strong red fluorescence of the cysticercus of Taenia solium depends on the presence of several porphyrins in the vesicular fluid of the parasite: probably protoporphyrin IX, coproporphyin I or III, and 2 decarboxylated porphyrins intermediate between uroporphyrin and coproporphyrin. Cyst porphyrins associated to form conglomerates of high molecular weight that dissociated in acid solutions and were not antigenic themselves nor associated with antigenic molecules. An appreciable fraction of the porphyrins was capable of undergoing oxidation and reduction, indicating that some of the porphyrins were complexed with metal ions. The metabolic basis for the accumulation of porphyrins is unknown. Preliminary results suggest that conditions deleterious to the cysticercus cause release of porphyrins so that the appearance of porphyrins in the cerebrospinal fluid of neurocysticercotic patients may prove useful in monitoring therapeutic attacks on the parasite.     

Proteomic analysis of Taenia solium metacestode excretion-secretion proteins

The metacestode larval stage of Taenia solium is the causal agent of a zoonotic disease called cysticercosis. The disease has an important impact on pork trade (due to porcine cysticercosis) and public health (due to human neurocysticercosis). In order to improve the current diagnostic tools and to get a better understanding of the interaction between T. solium metacestodes and their host, there is a need for more information about the proteins that are released by the parasite. In this study, we used protein sequences from different helminths, 1DE, reversed-phase LC, and MS/MS to analyze the excretion-secretion proteins produced by T. solium metacestodes from infected pigs. This is the first report of the T. solium metacestode excretion-secretion proteome. We report 76 proteins including 27 already described T. solium proteins, 17 host proteins and 32 proteins likely to be of T. solium origin, but identified using sequences from other helminths.     

Morphologic and genetic identification of Taenia tapeworms in Tanzania and DNA genotyping of Taenia solium

Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined microscopically for helminth eggs and proglottids. Of these, 4 Taenia egg positive cases were identified, and the eggs were subjected to DNA analysis. Several proglottids of Taenia solium were recovered from 1 of the 4 cases. This established that the species were T. solium (n = 1) and T. saginata (n = 3). One further T. solium specimen was found among 128 fecal samples collected from Mbulu district in Arusha, and this had an intact strobila with the scolex. Phylegenetic analysis of the mtDNA cox1 gene sequences of these 5 isolates showed that T. saginata was basal to the T. solium clade. The mitochondrial cox1 gene sequences of 3 of these Tanzanian isolates showed 99% similarity to T. saginata, and the other 2 isolates showed 100% similarity to T. solium. The present study has shown that Taenia tapeworms are endemic in Kongwa district of Tanzania, as well as in a previously identified Mbulu district. Both T. solium isolates were found to have an "African/Latin American" genotype (cox1).     

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.     

Steroid hormone production by parasites: the case of Taenia crassiceps and Taenia solium cysticerci

Many examples of reciprocal endocrine interactions between parasites and hosts have been found in insects, arthropods and mammals. Cysticercosis produced by Taenia solium metacestodes is a widely distributed parasite infection that affects the human and the pig. Taenia crassiceps experimental murine cysticercosis has been used to explore the role of biological factors involved in host–parasite interactions. We had shown that T. crassiceps cysticercosis affects the serum concentration of steroid hormones and the reproduction behavior of the male mice host. In an effort to understand the biology of the parasite, we had investigated the parasite capacity to produce sex steroids. For this purpose, T. crassiceps cysticerci were incubated in the presence of different steroid precursors. TLC and recrystallization procedures showed that testosterone is produced from ³H-androstenedione in cysticerci. The conversion of ³H-testosterone to androstenedione, although present is much less significant. In addition, we had studied the production of testosterone by T. solium cysticerci. For this purpose, cysticerci were dissected from pork meat and incubated as above described. The results showed that T. solium cysticerci also produce testosterone. We have speculated about the importance of androgens in the growth of T. crassiceps cysticerci and found that the addition of the antiandrogen flutamide to the culture media of the parasites significantly decreased ³H-thymidine incorporation. We therefore hypothesized, that the ability of cysticerci to produce testosterone from steroid precursors might be important for the parasite growth and development.     

Isozyme analysis of Taenia solium isolates from Mexico and Colombia

Mexican and Colombian Taenia solium cysticerci and some species of Taenia adults were assayed using cellulose acetate electrophoresis to distinguish between isolates. Isozyme patterns for ARK, GOT, G3PD, GPI, and MPI were identical in all cysticerci suggesting homozygotic profiles. G6PD and MDH showed different patterns between Mexican and Colombian cysticerci, suggesting regional differences. ME activity was mainly detected in the adult stage suggesting that this enzyme is active in anaerobic environment, while MDH, detected in cysticerci, could be related to an environment that contains oxygen. Finally, the species of taeniid adults analyzed showed different patterns among them.