Protective immunity against Taenia crassiceps murine cysticercosis induced by DNA vaccination with a Taenia saginata tegument antigen

This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.     

Inhibitory role of antibodies in the development of Taenia solium and Taenia crassiceps toward reproductive and pathogenic stages.

Untreated Taenia solium cysticerci obtained from different naturally infected pigs vary notably in their capacity to develop into intestinal tapeworms in prednisolone-treated hamsters, whereas cells derived from Taenia crassiceps cysticerci after 2 mo of infection almost always develop to cysticerci in the peritoneal cavity of susceptible BALB/cAnN mice. Preincubation of whole cysticerci or parasite cells with mice immunoglobulins raised against an 18-mer peptide epitope (GK-1) common to both parasites significantly interferes with both transformations. These crippling effects of antiparasite antibodies suggest new forms of immunological interference with parasite biology other than simple killing. Antibodies that cripple biological functions of the parasite, e.g., their development to reproductive or pathogenic stages, make them important protagonists in taeniasis/cysticercosis disease as classic parasitocidal antibodies. Different serum levels of crippling antibodies in the infected pigs could be responsible for the varied ability of cysticerci to convert to tapeworms. Antigens capable of inducing crippling antibodies, e.g., GK-1, could be useful as a therapeutic vaccine for pigs in order to reduce parasite transmission.     

Identification of loci controlling restriction of parasite growth in experimental Taenia crassiceps cysticercosis

Human neurocysticercosis (NC) caused by Taenia solium is a parasitic disease of the central nervous system that is endemic in many developing countries. In this study, a genetic approach using the murine intraperitoneal cysticercosis caused by the related cestode Taenia crassiceps was employed to identify host factors that regulate the establishment and proliferation of the parasite. A/J mice are permissive to T. crassiceps infection while C57BL/6J mice (B6) are comparatively restrictive, with a 10-fold difference in numbers of peritoneal cysticerci recovered 30 days after infection. The genetic basis of this inter-strain difference was explored using 34 AcB/BcA recombinant congenic strains derived from A/J and B6 progenitors, that were phenotyped for T. crassiceps replication. In agreement with their genetic background, most AcB strains (A/J-derived) were found to be permissive to infection while most BcA strains (B6-derived) were restrictive with the exception of a few discordant strains, together suggesting a possible simple genetic control. Initial haplotype association mapping using >1200 informative SNPs pointed to linkages on chromosomes 2 (proximal) and 6 as controlling parasite replication in the AcB/BcA panel. Additional linkage analysis by genome scan in informative [AcB55xDBA/2]F1 and F2 mice (derived from the discordant AcB55 strain), confirmed the effect of chromosome 2 on parasite replication, and further delineated a major locus (LOD = 4.76, p<0.01; peak marker D2Mit295, 29.7 Mb) that we designate Tccr1 (T. crassiceps cysticercosis restrictive locus 1). Resistance alleles at Tccr1 are derived from AcB55 and are inherited in a dominant fashion. Scrutiny of the minimal genetic interval reveals overlap of Tccr1 with other host resistance loci mapped to this region, most notably the defective Hc/C5 allele which segregates both in the AcB/BcA set and in the AcB55xDBA/2 cross. These results strongly suggest that the complement component 5 (C5) plays a critical role in early protective inflammatory response to infection with T. crassiceps.     

Population genetic structure of Taenia solium from Madagascar and Mexico: implications for clinical profile diversity and immunological technology

Taenia solium is a cestode parasitic of humans and pigs that strongly impacts on public health in developing countries. Its larvae (cysticercus) lodge in the brain, causing neurocysticercosis, and in other tissues, like skeletal muscle and subcutaneous space, causing extraneuronal cysticercosis. Prevalences of these two clinical manifestations vary greatly among continents. Also, neurocysticercosis may be clinically heterogeneous, ranging from asymptomatic forms to severely incapacitating and even fatal presentation. Further, vaccine design and diagnosis technology have met with difficulties in sensitivity, specificity and reproducibility. Parasite diversity underlying clinical heterogeneity and technological difficulties is little explored. Here, T. solium genetic population structure and diversity was studied by way of random amplified polymorphic DNA in individual cysticerci collected from pigs in Madagascar and two regions in Mexico. The amplification profiles of T. solium were also compared with those of the murine cysticercus Taenia crassiceps (ORF strain). We show significant genetic differentiation between Madagascar and Mexico and between regions in Mexico, but less so between cysticerci from different localities in Mexico and none between cysticerci from different tissues from the same pig. We also found restricted genetic variability within populations and gene flow was estimated to be low between populations. Thus, genetic differentiation of T. solium suggests that different evolutionary paths have been taken and provides support for its involvement in the differential tissue distribution of cysticerci and varying degrees of severity of the disease. It may also explain difficulties in the development of vaccines and tools for immunodiagnosis.     

Analysis of antigenic variation in cysticerci of Taenia solium

By studying the reactions and cross-reactions of antigen extracts from different collections of Taenia solium with their respective hyperimmune antisera, and by using numerical taxonomy to analyze the results, a measure of antigenic diversification within T. solium was established. Results varied somewhat depending on the immunological method employed. Double immunodiffusion and immunoelectrophoresis agreed in that all cysticerci were not identical, but shared approximately one third of their antigens. Double immunodiffusion recognized two groups of identical cysticerci: one included 50% of the cysticerci and the other, 21%. Immunoelectrophoresis was more discriminatory in that it recognized few extracts as identical. Electrophoretic data revealed that the most frequently shared antigen among cysticerci corresponded to that most frequently detected by immunologically responsive humans with cerebral cysticercosis. Antigen diversification of T. solium may provide a possible explanation of the pleomorphic immune response of man to this parasite.U     

Sex steroids and parasitism: Taenia crassiceps cisticercus metabolizes exogenous androstenedione to testosterone in vitro

Sex hormones are known to modulate immune responses and may be implicated in sex associated susceptibilities to infections. Taenia crassiceps cysticerci grow to larger numbers in female mice than in males. Gonadectomy alters the course of this infection and hormone replacement with 17beta-estradiol increases the parasite numbers. However, in chronic Taenia crassiceps cysticercosis the sex-hormone profile of males becomes more like that of the females' and progressively loose their sexual behavior. To have further insight in these outstanding endocrinological effects induced by the parasite upon the host, we investigated the parasite's capacity to produce sex steroids. In vitro experiments showed that Taenia crassiceps cysticerci transform 3H-Androstenedione to 3(H)-Testosterone, but not 3H-Pregnenolone. The production of 3H-Testosterone increased when the parasite numbers doubled. A recrystallisation procedure demonstrated that the metabolite identified by TLC was in fact testosterone. Thus, the cysticercus has the ability to use 3H-Androstenedione to make Testosterone possibly by a 17beta-Hydroxysteroid deshidrogenase-like activity in the parasite. In vivo, the parasite could use steroid precursors from the host to produce sex hormones, either accidentally or as needed for its own development, and thus alters the host's normal environment with sexual and immunological repercussions.     

Host-cell apoptosis in Taenia solium-induced brain granulomas in naturally infected pigs

To assess whether apoptosis occurs in pig brain granulomas due to Taenia solium cysticerci, brain tissues from 30 pigs naturally infected with T. solium cysticercosis were evaluated by terminal deoxynucleotidyl transferase-end labelling (TUNEL) staining. In addition, tissues were stained with CD3 marker to identify T lymphocytes. Examination of TUNEL-stained tissues showed apoptotic cells in early lesions that contained viable cysticerci. Apoptotic cells were primarily found interspersed with normal cell types, and were mostly located in the inflammatory infiltrate. Late or advanced granulomas with disintegrated scolices did not show TUNEL-positive cells. CD3+ cells were found in both early and advanced lesions and apoptosis mainly co-localized with CD3+ T lymphocytes. This suggests that these cells are constantly undergoing apoptosis and thus die as soon as they arrive at the site of infection. Apoptosis indeed may be one way by which T. solium cysticerci down-regulate the host's cellular immune response in early cysticercosis. Therefore, further research is needed to establish if other cells besides T-lymphocytes are also a target for destruction by cysticerci in early cysticercosis as well as studies to assess if cysteine protease is expressed by viable cysticerci in situ.     

Molecular mechanisms involved in the differential effects of sex steroids on the reproduction and infectivity of Taenia crassiceps

The in vitro exposure of Taenia crassiceps cysticerci to 17-beta estradiol (E2) and progesterone (P4) stimulated their reproduction and infectivity. Testosterone (T4) and dihydrotestosterone (DHT) inhibited their reproduction and reduced their motility and infectivity. E2 and P4 increased, whereas T4 and DHT reduced, the expression of parasite c-fos and c-jun and DNA synthesis. In vitro exposure of cysticerci to sex steroids before their inoculation into recipient noninfected mice resulted in large parasite loads when pretreated with E2 and P4 and in smaller loads when pretreated with T4 and DHT To determine the possible molecular mechanisms by which sex steroids affect T. crassiceps, sex steroid receptors were amplified. Taenia crassiceps expressed estrogen receptors (both alpha and beta isoforms) and androgen receptors but no P4 receptors. These results demonstrate that sex steroids act directly on parasite reproduction by binding to a classic and specific sex steroid receptor on the parasite. The differential response of cysticerci to sex steroids may also be involved in their ability to grow faster in the murine female or feminized male host. This is the first report of direct sex steroid effects on the parasite possibly through sex steroid receptors in the cysticerci.     

Taenia crassiceps cysticercosis in mice does not increase the carcinogenic effect of methyl-nitrosourea

An association between brain cysticercosis and malignant neoplasms in humans has recently been reported. To explore the possibility of a potentiating effect of cysticercosis on carcinogenesis mice infected with Taenia crassiceps cysticerci were exposed to the carcinogenic substance methyl-nitrosourea; 35% of them developed lymphoma, in contrast with 50% of control non-infected animals exposed to MNU. In this experimental model of cysticercosis we did not find a potentiating effect of peritoneal cysticercosis on the carcinogenicity of MNU.     

Taenia crassiceps cysticercosis: protective effect and immune response elicited by DNA immunization

The nucleotide sequence of a protective recombinant antigen of Taenia crassiceps cysticerci present in all stages of Taenia solium (KETc7), cloned into pcDNA3 plasmid with the signal peptide sequence of the beta-glycan receptor (pTc-sp7), has been shown to be effective in protecting mice against experimental infection of T. crassiceps. To explore further the possibilities of this form of immunization and the immune response induced, mice were injected intramuscularly (i.m.) or intradermally (i.d.) with 3 doses of pTc-sp7. Similar levels of resistance were found using either i.m. or i.d. immunization. Spleen cells from i.d. and i.m. DNA immunized mice induced a specific T-cell response to T. crassiceps antigens and to a synthetic peptide from the immunogen itself (GK-1). Proliferated cells were especially enriched in CD8+ CD4- T-lymphocytes. A clear increase in the percentage of CD3+ cells that produce gamma-interferon and interleukin-2 was detected when measuring the intracellular cytokine production, an indication of the pTc-sp7 capacity to induce an effective cellular response. These results provide encouraging information on the use of KETc7 in the prevention of cysticercosis as well as a first insight into the characterization of the immune response induced by pTc-sp7 that hints to the relevance of cellular immunity in protection.     

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.     

Molecular cloning and characterization of a 2-Cys peroxiredoxin from Taenia solium

A Taenia solium 2-Cys peroxiredoxin (Ts2-CysPrx) clone was isolated from a T. solium adult cDNA library. The clone encodes a polypeptide comprising 197 amino acids with a predictive Mr = 21,836. It has the 2 classical cysteine domains from the typical 2-Cys peroxiredoxins, and its primary amino acid sequence shows higher identity with 2 Echinococcus 2-Cys peroxiredoxins. Northern and Southern blot hybridizations exhibit an mRNA with a size of -1.0 kb, encoded by 1 gene. Ts2-CysPrx was expressed in Escherichia coli and purified by anion-exchange chromatography. Biochemical analysis showed Ts2-CysPrx is a dimer composed by monomers of -22 kDa that presented activity with hydrogen peroxide (H2O2) and cumene hydroperoxide. It presented the catalytic mechanism for a typical 2-CysPrx because the homodimeric oxidized form is reduced to a monomeric form by thioredoxin (Trx) and by dithiothreitol (DTT) and was converted to a homodimeric oxidized form by H2O2. Western blot studies using antibodies against Ts2-CysPrx revealed that the protein is expressed during the entire T. solium life cycle, as in other Taenia species. Immunohistochemical studies indicated that Ts2-CysPrx is localized on the tegument and in tegumentary and muscle cells of cysticerci. We also show that T. crassiceps cysticerci can tolerate H2O2 levels of 2.5 mM for 2.5 hr.     

The Asian Taenia and the possibility of cysticercosis

In certain Asian countries, a third form of human Taenia, also known as the Asian Taenia, has been discovered. This Asian Taenia seems to be an intermediate between Taenia solium and T. saginata since in morphological terms it is similar to T. saginata, yet biologically, as it uses the same intermediate host (pigs), it is more akin to T. solium. Taenia solium causes human cysticercosis, while T. saginata does not. It is not known whether the Asian taeniid is able to develop to the larval stage in humans or not. The arguments proposed by those authors who consider it unlikely that the Asian Taenia causes human cysticercosis are: (a) its molecular similarities with T. saginata; (b) the absence of cases of human cysticercosis in populations where the Asian adult is highly prevalent; and (c) the unsupporting results derived from an experimental infestation study. These three arguments are debated, although bearing in mind that at present there is still no clear scientific data to support that human cysticercosis can be caused by the Asian Taenia.     

Preferential growth of Taenia crassiceps cysticerci in female mice holds across several laboratory mice strains and parasite lines

A retrospective study of our 14-yr records on experimental Taenia crassiceps (ORF(fast) line) cysticercosis (n = 1,198) shows that in 16 of 17 different mice strains, female mice are more frequently infected and carry larger individual parasite loads than males. However, sexual differences in parasite loads significantly varies between strains in relation to their different genetic backgrounds (BALB > C57Bl = OTHERS > C3H). The coefficient of variation in all female mice is significantly smaller than that of all males, an indication of males' more potent, but erratically effective, restraint of cysticercus growth. Similar positive growth bias for female mice is shown by other lines of cysticerci, i.e., HYG(slow) and WFU(slow). These results contravene the usual expectation of female hosts being more resistant than males to parasite infections, and they point to the multiple factors that combined determine sex related differences of mice to experimental cysticercosis infection.     

Comparison of biochemical and immunochemical properties of myosin II in taeniid parasites

Type II myosins are highly conserved proteins, though differences have been observed among organisms, mainly in the filamentous region. Myosin isoforms have been identified in Taenia solium, a helminth parasite of public health importance in many developing countries. These isoforms are probably associated with the physiological requirements of each developmental stage of the parasite. In this paper we extend the characterization of myosin to several other Taenia species. Type II myosins were purified from the larvae (cysticerci) of Taenia solium, T. taeniaeformis and T. crassiceps and the adult stages of T. solium, T. taeniaeformis and T. saginata. Rabbit polyclonal antibodies against some of these myosins were specific at high dilutions but cross-reacted at low dilutions. ATPase activity was evaluated and kinetic values were calculated for each myosin. Homologous actin-myosin interactions increased both the affinity of myosin for ATP and the hydrolysis rate. The results indicate immunological and biochemical differences among taeniid myosins. This variability suggests that different isoforms are found not only in different taeniid species but also at different developmental stages. Further characterization of myosin isoforms should include determination of their amino acid composition.     

Taenia crassiceps: host treatment alters glycolisis and tricarboxilic acid cycle in cysticerci

Human cysticercosis by Taenia crassiceps is rare although it is considered of zoonotic risk, especially to immunocompromised individuals. Albendazole and praziquantel are widely used and effective in its treatment. Their active forms inhibit the glucose uptake by the parasite and induce muscle contractions that alter its glycogen levels interfering in the energetic metabolism of the parasite and leading to its death. The aim of this study was to evaluate alterations in glycolysis, the tricarboxylic acid cycle and glucose concentrations caused by low dosage treatments of the hosts with albendazole and praziquantel. Therefore, T. crassiceps intraperitoneally infected mice were treated by gavage feeding with 5.75 or 11.5 mg/kg of albendazole and 3.83 or 7.67 mg/kg of praziquantel. The treated mice were euthanized after 24 h and the cysticerci collected were morphologically classified into initial, larval or final phases. Concentrations of the organic acid produced and glucose were evaluated to detect alterations into the glycolysis and the tricarboxylic acid cycle pathways through chromatography and spectrophotometry. The low dosage treatment caused a partial blockage of the glucose uptake by the cysticerci in spite of the non significant difference between its concentrations. An activation of the tricarboxylic acid cycle was noted in the cysticerci that received the treatment due to an increase in the production of citrate, malate and α-ketoglutarate and the consumption of oxaloacetate, succinate and fumarate. The detection of α-ketoglutarate indicates that the cysticerci which were exposed to the drugs after host treatment present different metabolic pathways than the ones previously described after in vitro treatment.     

The 10 kDa protein of Taenia solium metacestodes shows genus specific antigenicity

Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.     

Fatal cysticercosis by Taenia crassiceps (Cyclophyllidea: Taeniidae) in a presumed immunocompromised canine host

Cysticercosis in a canine host (Canis familiaris) attributable to the taeniid cestode Taenia crassiceps is reported for the first time in North America. Numerous parent and daughter cysticerci occurred in a massive intrapleural and intraperitoneal infection in an apparently immunocompromised host. The largest cysticerci were ovoid to elongate, 5-9 mm in maximum length, and armed with 32-34 rostellar hooks in 2 rows; small hooks measured 114-143 microm long (x = 124+/-8.2 microm), and large hooks were 156-180 microm (x = 163+/-7.4 microm). Taenia crassiceps is widespread in boreal North America and, like a number of other taeniids, constitutes a potential risk as a zoonotic parasite. The immunological status of the host may be important in determining the outcome of infections for this and other taeniids in atypical hosts.     

Dogs as alternative intermediate hosts of Taenia solium in Papua (Irian Jaya), Indonesia confirmed by highly specific ELISA and immunoblot using native and recombinant antigens and mitochondrial DNA analysis

Serology (ELISA and immunoblot) using native glycoproteins, affinity purified glycoproteins, and a recombinant antigen is known to be highly specific to Taenia solium cysticercosis in humans and pigs. These techniques were applied for dogs in the highly endemic area of cysticercosis in Papua (Irian Jaya), Indonesia. Analysis of dog sera by both ELISA and immunoblot revealed 7 of 64 dogs were highly positive. Examination of two sero-positive dogs revealed cysticerci of T. solium in the brain and heart of these dogs. Mitochondrial DNA analysis confirmed that they were the same as T. solium previously confirmed from pigs and biopsies from local people from Irian Jaya. It is suggested that the life cycle of T. solium may be completed not only between humans and pigs but also between humans and dogs.