Crassiceps Metacestodes Research Articles

Chronic administration of cryptolepine nanoparticle formulation alleviates seizures in a neurocysticercosis model

Worldwide, neurocysticercosis remains an important cause of acquired epilepsy. We therefore seek to investigate the effectiveness of the nanoparticle formulation of cryptolepine in alleviating seizures in a neurocysticercosis model.A solid-lipid nanoparticle formulation of extracted cryptolepine was prepared. The parasites were maintained in T. crassiceps metacestode (ORF strain) - infected female BALB/c mice. Cryp (5 ​mg/kg), SLN-CRYP (5 ​mg/kg), ABZ (50 ​mg/kg) DXM (0.5 ​mg/kg), and PHE (30 ​mg/kg).were assessed for in vitro cysticidal, in vivo cysticidal and/or antiseizure activity in 70 mice that had developed seizures from infection with T. crassiceps. General pathologic processes were studied in the host tissue and inflammatory mediators were quantified from isolated mice brains.All treatments (CRYP, SLN-CRYP and ABZ) caused significantly reduced viability of T. crassiceps cysts. Treatment with SLN-CRYP significantly shrunk cysticerci and resolved ventricular expansion and deviation similar to albendazole on examination of encephala. SLN-CRYP inhibited hyperemia but was more effective against microgliosis, calcification, edema and meningitis. Mean seizure score was significantly reduced in models administered with SLN-CRYP (p ​< ​0.0001); as were frequency (p ​< ​0.0001) and duration (p ​< ​0.0001) of seizures. SLN-CRYP significantly reduced brain homogenate levels of IL-10 (p ​= ​0.0016) and IFN-γ (p ​< ​0.0001).Our study shows that the chronic administration of the nanoparticle formulation of cryptolepine is effective in alleviating seizures associated with neurocysticercosis in a mouse model.

Hippocampal sclerosis induced in mice by a Taenia crassiceps metacestode factor.

An experimental Taenia crassiceps mouse model was used to assess the role of Taenia solium metacestode factor (Fac) in human neurocysticercosis. Intraperitoneal infection with T. crassiceps metacestodes or subcutaneous inoculation with a T. crassiceps metacestode factor (Fac) produced significant impairment of performance (learning) in the Barnes maze and induced bilateral hippocampal sclerosis in mice. Several staining techniques revealed important cell dispersion, extensive apoptosis and cell loss in the dentate gyrus, hilus and CA1-CA3 regions of both hippocampi, as well as intense deterioration of the adjacent cortex. An outstanding disruption of its histoarchitecture in the surrounding tissue of all these regions and apoptosis of the endothelial cells were also observed.

Antigenic fractions from Taenia crassiceps metacestodes obtained by hydrophobicity for the immunodiagnosis of active and inactive forms of neurocysticercosis in human cerebrospinal fluid samples

This study aimed to evaluate the total extract of Taenia crassiceps metacestodes (TC) and its antigenic fractions obtained by Triton X-114 fractionation techniques, such as detergent (DC) and aqueous (AC), in the immunodiagnosis of human neurocysticercosis (NCC). Cerebrospinal fluid samples were divided into two groups: Group 1 (n=40), which was further divided into active (n=20) and inactive (n=20) NCC, and Group 2 (control group), which comprised 39 CSF samples from patients who had another neurological disorder, were suffering from other infectious diseases of the brain or had other parasitic infections. The total extracts and antigenic fractions were tested by enzyme-linked immunosorbent assay (ELISA) to detect human IgG anti-Taenia solium. T. crassiceps fractions (DC and AC) showed the same value of sensitivity (Se), 100%, for active and inactive NCC and a specificity (Sp) of 97.4%. The DS fraction obtained from T. solium showed 100% Se for active NCC, 95% Se for inactive NCC and a 92.3% Sp. The AS fraction obtained from T. solium showed 100% Se for both active and inactive NCC and a 94.9% Sp. There was a positive correlation between the total saline extract of T. crassiceps (TC) and T. solium (TS) and their fractions (DC, AC, DS and AS). Positive predictive value, negative predictive value, diagnostic efficiency and Youden index were calculated. In conclusion, these results demonstrated that detergent and aqueous fractions obtained from T. crassiceps metacestodes are important sources of specific antigens and are efficient for immunodiagnosis of active and inactive NCC.

Apoptosis of mouse hippocampal cells induced by Taenia crassiceps metacestode factor.

Seizures, headache, depression and neurological deficits are the signs and symptoms most frequently reported in human neurocysticercosis. However, the cause of the associated learning and memory deficits is unknown. Here, we used Taenia crassiceps infection in mice as a model of human cysticercosis. The effects of T. crassiceps metacestode infection or T. crassiceps metacestode factor (MF) treatment on mouse hippocampal cells were studied; control mice were included. At 45 days after infection or treatment of the mice with MF, all mice were anaesthetized and perfused transcardially with saline followed by phosphate-buffered 10% formalin. Then the brains were carefully removed. Coronal sections stained using several techniques were analysed. Extensive and significant apoptosis was found in the experimental animals, mainly in the dentate gyrus, CA1, CA2, CA3 and neighbouring regions, in comparison with the apparently intact cells from control mice (P < 0.01). These results suggest that neurological deficits, especially the learning and memory deficits, may be generated by extensive apoptosis of hippocampal cells.

Abnormalities in the WFU strain of Taenia crassiceps (Cyclophyllidea: Taeniidae) following years of propagation in mice.

Asexually proliferating Taenia crassiceps (Zeder, 1800) metacestodes isolated within past decades have been successfully sub-cultured under experimental conditions using Mus musculus Linnaeus, 1758 mice. However, during their development, morphological irregularities of scolex structures have been reported in two of the three strains of this cestode species maintained in mice - ORF and KBS. The main goal of this work is to describe the abnormalities observed in a sample of 118 cysticerci of the third T. crassiceps strain used at present - WFU. Morphological abnormalities were detected in 39.8% of the evaginated scoleces; they consisted of supernumerary suckers (n=2), duplicated (n=2) or absent rostellum (n=1), as well as absent or aberrant (n=29) hooks, which were significantly shorter when compared to the large and short hook lengths referred to in the literature.

ATaenia crassicepsmetacestode factor enhances ovarian follicle atresia and oocyte degeneration in female mice

The histopathological effects of Taenia crassiceps infection or T. crassiceps metacestode factor inoculation on the mouse ovary were determined using six female mice in three groups: infected mice, mice inoculated with the metacestode factor and control mice. The control group was subcutaneously inoculated with healthy peritoneal fluid. The infected group was intraperitoneally inoculated with 40 T. crassiceps metacestodes, and the metacestode factor group was subcutaneously inoculated with T. crassiceps metacestode factor (MF). Light and electron microscopy and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling) assays revealed a significant increase in ovarian follicular atresia (predominantly in antral/preovulatory stages of development), oocyte degeneration (P<0.05), and a decrease in the amount of corpus luteum in follicles of mice infected and inoculated with MF compared with the control group. Significant abnormalities of the granulosa cells and oocytes of the primordial, primary and secondary ovarian follicles occurred in both treated mouse groups (P<0.05) compared with no degeneration in the control group. These pathological changes in female mice either infected with T. crassiceps metacestodes or inoculated with T. crassiceps MF may have consequences for ovulation and fertility.

The Macrophage galactose-specific lectin (MGL) recognizes Taenia crassiceps antigens and plays a role in resistance to the infection (55.3)

Abstract C-type lectins are multifunctional sugar-binders on dendritic cells (DCs) and macrophages that internalize antigens for processing and presentation. MGL recognizes galactose and N-acetylgalactosamine and it is expressed on immature DCs and alternatively activated macrophages (AAM). Helminth parasites contain large number of glycosilated components which appears to play a role in the immuneregulation induced by such infections. To address whether MGL may influence the immune response and resistance to T. crassiceps we infected i.p. MGL deficient (MGL-/-, C57BL/6 background) and wildtype mice (MGL+/+) with 15 non-budding T. crassiceps metacestodes. Parasite burden, cellular and humoral responses were measured during 8 wks of infection. mMGL-/- DCs displayed lower ability to bind T. crassiceps antigens. Compared with wild-type mice, mMGL-/- mice failed to produce significant levels of inflammatory cytokines during the first two weeks of Taenia infection. MGL-/- mice developed a Th2-dominant whereas wild-type mice developed a Th1-dominant immune response after Taenia infection. Associated with the insufficient production of early inflammatory cytokines, mMGL-/- mice harbored significantly higher numbers of parasites whereas WT mice were resistant. Finally, FACS analysis showed overexpression of IL-4Rα, PDL1 and PDL2 on macrophage surface in MGL-/- group unrevealing AAM phenotypes. Thus, MGL may be involved in T. crassiceps recognition and subsequent innate immune activation.

Induction of immunoglobulin G1, interleukin-6 and interleukin-10 by Taenia crassiceps metacestode carbohydrates.

T helper type 2 (Th2) -polarized immune responses are characteristically dominant in helminth infections. Two murine models that show a Th1 to Th2 polarization with infection progression are those of Schistosoma mansoni and Taenia crassiceps. In both, an early Th1 response is replaced by a late Th2 response. We report that the nucleic acid-, protein- and lipid-free carbohydrate fraction of T. crassiceps metacestodes (denoted T-CHO) possesses Th2-like immunomodulatory activity. Immunization of two strains of rats (Dark Agouti and Albino Oxford) and BALB/c mice with chicken albumin in the presence of T-CHO resulted in selective enhancement of immunoglobulin G1 (IgG1) antibodies, considered to be associated with Th2 responses in both rats and mice. Interleukin-6 (IL-6) followed by IL-10 were the dominant cytokines detected in in vitro cultures of mouse spleen cells stimulated with T-CHO. IL-4 and IL-5 were not detected in these culture supernates. Furthermore, Taenia carbohydrates were mitogenic to spleen cells, activated serine phosphorylation of proteins and up-regulated the expression of the anti-apoptotic protein, Bcl-2. When mouse spleen cells were cultured in the presence of Taenia carbohydrates, a concentration-dependent down-regulation of IL-2 and an overlapping up-regulation of IL-6 secretion were seen.

Detection of IgG in cerebrospinal fluid for diagnosis of neurocysticercosis: evaluation of saline and SDS extracts from Taenia solium and Taenia crassiceps metacestodes by ELISA and immunoblot assay.

We compared saline (S) and sodium dodecyl sulphate (SDS) extracts from Taenia solium (homologous species - HO) and Taenia crassiceps (heterologous species - HE) metacestodes in order to detect IgG by ELISA and immunoblot assay (IBA) in cerebrospinal fluid (CSF) for the diagnosis of human neurocysticercosis (NC). CSF samples were obtained from 93 patients. Of these, 40 had NC, five had a diagnosis of probable NC, nine had central nervous system schistosomiasis or strongyloidiasis and 39 had other neurological alterations. Samples were analysed by ELISA and the results were compared with IBA in all samples with confirmed and probable NC diagnosis, in all samples with other central nervous system parasitic infection, and in 10 of those with another neurological alterations. ELISA sensitivity was 100%, 85%, 95% and 87.5% for the S-HO, S-HE, SDS-HO and SDS-HE extracts, respectively, and ELISA specificity was 100% for S-HO, S-HE, SDS-HO extracts and 97.9% for SDS-HE antigen. Immunodominant peptides detected by IBA were, by decreasing percentage of recognition: 64-68 and 45 kDa for S-HO; 108-114, 92-95, 64-68, 83 and 88 kDa for S-HE; 64-68, 108-114, 77 and 86 kDa for SDS-HO; and 108-114, 88 and 92-95 kDa for SDS-HE. Overall the homologous antigenic extracts showed higher sensitivity than the heterologous extracts in the diagnosis of NC in CSF samples. The heterologous extracts contained most of the immunodominant peptides presented in the homologous extracts, which are recognized by IgG antibodies in CSF samples.

Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96.4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose.

Apoptosis in human Jurkat T cells after culture with live Taenia crassiceps cysticerci in vitro.

This study examines the effects of Taenia crassiceps cysticerci on the viability of a human T lymphocyte cell line (Jurkat). Both budding and non-budding T. crassiceps metacestodes were cultured over 24 and 48 h in the presence of Jurkat cells. Cell viability decreased with increasing numbers of cysticerci, particularly budding cysticerci. Single cell gel electrophoresis (comet) analysis, which grades DNA damage, showed a significant increase in apoptosis at 24 and 48 h. The morphology of treated cells was determined using acridine orange with the classical morphology of apoptotic bodies seen to increase with increasing cysticerci numbers over time. These results indicate that parasite-induced apoptosis occurs during murine cysticercosis. Such a mechanism may be important in survival of other metacestode infections of medical or veterinary importance.

Immunodiagnosis of swine cysticercosis by indirect ELISA employing a heterologous antigen from Taenia crassiceps metacestode

C. Larralde et al. (1990, Arch. Pathol. Lab. Med., 114:926–928) demonstrated that heterologous antigen from the laboratory-adapted murine Taenia crassiceps metacestode may substitute those from Taenia solium in the immunodiagnosis of human cysticercosis by the indirect enzyme-linked immunosorbent assay (IE). This antigen is easily obtained at a laboratory level and solves the problem of T. solium cysticerci collection from naturally or experimentally infected swine. In this study an IE employing a heterologous antigen from the T. crassiceps metacestode was evaluated for the immunodiagnosis of swine cysticercosis. Sera from 300 swine free of T. solium cysticerci by post-mortem examination were employed to determine two IE cut- off values: 1) Mean ELISA values + 2 standard deviations (2σ cut-off) and 2) - Mean ELISA values + 3 standard deviations (3σ cut-off). The specificity of IE was 97% with the 2σ cut-off and 100% with the 3σ cut-off. When applied to ten sera from swine infected by cysticerci of T. solium by post-mortem examination, the sensitivity of IE was 100% independent of the cut off.

Bovine cysticercosis: demonstration in experimentally infected calves of serum IgG antibodies reactive with neutral glycolipids of Taenia saginata and T. crassiceps metacestodes.

The immunoreactivity of Taenia saginata and T. crassiceps metacestode neutral glyco(sphingo)lipids towards IgG antibodies derived from the sera of calves with experimental cysticercosis has been established. The glyco(sphingo)lipids are separable by normal-phase HPTLC (high-performance thin-layer chromatography) into groups of increasing sugar-chain length (lipid/ceramide mono-, di-, tri-, tetra- and > tetrasaccharides), with those corresponding to three and four hexoses being the main immunoreactive components (HPTLC immunostaining). In ELISA (enzyme-linked immunosorbent assay), reverse-phase HPTLC-isolated T. crassiceps metacestode glyco(sphingo)lipids equivalent to tri- and tetrahexoside allowed a discrimination between non-infected and infected calves (at least 80 metacestodes recovered). The formation of IgG antibodies was correlated with the infection, not with other non-specific inducing factors, as seen by the differential humoral response detected in experimentally infected (T. saginata) calves before and after Praziquantel treatment (HPTLC immunostaining and ELISA).

Immunization against Taenia crassiceps Cysticercosis: Identification of the Most Promising Antigens in the Induction of Protective Immunity

Cross immunity between Taenia solium and Taenia crassiceps parasites points to T. crassiceps cysticercosis as a convenient model to test promising antigens aimed at the development of a vaccine against T. solium cysticercosis. Since total antigens from T. crassiceps metacestodes induce significant levels of protection in pigs against T. solium cysticercosis, we initiated this work to identify the most interesting antigens involved in protection. Twelve different antigen fractions isolated from T. crassiceps cysticerci were evaluated with respect to their capacity to induce resistance against a challenge with 10 T. crassiceps cysticerci in male BALB/cAnN mice. Mice were intraperitoneally immunized with 2 doses of each antigen, 5 or 15 micrograms per mouse. The 12 antigen fractions were classified as protecting (200, 123, 74, 66, 56, 40-50, 27 and 8-14 kDa), facilitating (220-205 kDa), or irrelevant (150-160, 93, 108 kDa), according to their effect on the parasite load. The 3 most promising antigen fractions were reevaluated via subcutaneous immunization with Freund's complete adjuvant. A high level of protection was obtained when antigen fractions of 56, 66, and 74 kDa were used together. Interestingly, antigens with similar molecular weights were also detected in early steps of differentiation in T. solium cysticercosis. These observations may be helpful in the development of a synthetic or a recombinant vaccine against cysticercosis.

Taenia crassicepssurface immunoglobulins: parasite- or host-derived?

In common with other taeniid cestodes, host or host-like proteins, especially immunoglobulins, occur on the surface and in the cyst fluid of Taenia crassiceps metacestodes. Here, several approaches have been used to determine the origin of the immunoglobulins present on the tegument. Indirect IFAT showed that IgG was almost totally lost from the surface of bladders after 6 days culture in vitro. There was a rapid reacquisition of immunoglobulins following incubation of the cultured metacestodes with either normal mouse serum or mouse anti-T. crassiceps antiserum. Immunoprecipitation of in vitro translation products and biosynthetically labelled T. crassiceps proteins with a panel of anti-IgG antisera failed to positively identify any molecule with homology to mammalian immunoglobulins. These results suggest strongly that the immunoglobulins located on the surface of T. crassiceps are of host rather than parasite origin. The occurrence of a relatively low abundance receptor in the surface of the bladders, which binds non-specific host immunoglobulin, together with surface-bound specific anti-T. crassiceps antibodies can account for the presence of these host proteins. Freshly obtained bladders and metacestodes cultured in vitro for 6 days were transplanted into naive mice and the survival and development of the resulting parasites compared. In some individual mice there was a decrease in the number and volume of metacestodes and an increase in encapsulated parasites arising from cultured bladders. This was probably not related to the loss of host immunoglobulins from the parasite surface during culture as the reacquisition of these proteins after transplantation is likely to be far more rapid than any immune response could evoke in a naive host.

Taenia crassiceps Metacestode Vesicular Fluid Antigens Shared with the Taenia solium Larval Stage and Reactive with Serum Antibodies from Patients with Neurocysticercosis

After removal of host (mouse) serum proteins (albumin, transferrin, IgG and another five unidentified proteins) by immunoaffinity chromatography, the vesicular fluid of T. crassiceps metacestodes (TcVF) was immunoelectrophoretically examined for antigens recognized by rabbit antiserum to aqueous crude extract of T. solium cysticerci. Based on a precipitate pattern developed in electroimmunodiffusion, nine cross-reactive antigens could be demonstrated. In the ELISA, TcVF was shown to be a potent antigen for the demonstration of IgG antibodies in the sera of Mexican patients (n = 14) with confirmed neurocysticercosis (mean E 490 values ± SD: 0.39 ± 0.38) although it was less sensitive when compared to T. solium VF (0.95 ± 0.54). Sensitivity was much higher using cross-reactive TcVF antigens selected by immunoaffinity chromatography with rabbit IgG antibodies to larval T. solium crude extract (0.87 ± 0.57). SDS-PAGE fluorograph of cross-reactive, radioiodinated TcVF protein antigens and selected by antibodies of individual neurocysticercosis sera (n = 13), exhibited six to nine bands depending on the serum tested. Altogether ten 125I-labelled proteins (M r range from about 20000 to 200000) were recognized by neurocysticercosis antibodies. Four proteins (M r about 22000, 25000, 32000 and 45000) were detected by all sera.

Inhibition of in vitro and in vivo Mast Cell Degranulation by Taenia crassiceps Metacestodes in vitro Incubation Products

In vitro released products of T. crassiceps metacestodes (TcIP) harvested from the peritoneal cavity of NMRI mice were tested for inhibitory effects on the in vitro degranulation of peritoneal mast cells (MCs) of normal mice (NMRI) and rats (Wistar) and on the in vivo degranulation of rat (Wistar) skin MCs (PCA-assay). In vitro degranulation was elicited chemically (compound 48/80, polymyxin B or the bee venom peptide, mellitin). In vivo degranulation was triggered immunologically (anaphylactic systems ovalbumin/anti-ovalbumin or Fasciola hepatica crude fluke extract antigen/serum of fluke-infected rats (Wistar)). In vitro degranulation of murine peritoneal MCs or the in vitro histamine release of rat peritoneal MCs normally induced chemically was significantly inhibited when the MCs were preincubated with the TcIP or with serum of T. crassiceps-infected NMRI mice from day 35 post infection and thereafter. In vitro degranulation of peritonal MCs of infected mice was strongly inhibited beginning on day 10 after infection. Also in vivo degranulation of the IgE-sensitized rat skin MCs was significantly reduced by intradermal injection of the TcIP before (6, 3 and 1 h) antigen challenge and by preinjection (1 h) of serum from infected mice (day 80 p.i.) - The inhibitory effect was also demonstrated after immunoadsorption of mouse serum proteins naturally contaminating the TcIP. Heating (100°C/15 min), even in the presence of 0.25 M HCl, did not suppress the inhibitory activity.

Antibody levels in cattle naturally infected with Taenia saginata metacestodes in Britain

Excretory/secretory products of Taenia saginata metacestodes and an extract of whole T crassiceps metacestodes were found to be more specific antigens than a saline extract of T saginata proglottides for use with the enzyme linked immunosorbent assay in the detection of antibodies in serum samples from cattle in herds with a history of T saginata metacestode infection. It was possible to detect serum antibodies to Tsaginata in sera from three out of four herds of cattle with a history of T saginata metacestode infection on the farm.

Serodiagnosis of Taenia saginata cysticercosis in experimentally and naturally infected cattle by enzyme linked immunosorbent assay

The sensitivity and specificity of enzyme linked immunosorbent assay (ELISA) for the diagnosis of Taenia saginata cysticercosis was studied among laboratory reared experimentally infected calves and conventionally raised naturally infected cattle by using a T crassiceps metacestode antigen. The intensity of the metacestode burden in experimentally infected animals had a direct effect on the first appreciable rise in the spectrophotometric extinction values and also on the progressive elevation of these values during the later stages of infection. The animals harbouring less than 100 metacestodes were the most difficult to detect by ELISA. Serum from sheep infected with Cysticercus tenuicollis showed cross-reaction. Among the naturally infected cattle the sensitivity of ELISA was poor; only 37.5 per cent of the 24 proven cases could be diagnosed. One of the nine cattle harbouring natural infection of Fasciola hepatica showed cross-reaction. Of the 69 animals 4.3 per cent showed false positive reactions.

Taenia crassiceps: Immunity to metacestodes in BALB/c and BDF1 mice

The kinetics of primary and secondary infections with Taenia crassiceps larvae and the effects of immune serum on T. crassiceps larvae were studied in BALB/c and BDF1 mice. In both strains of mice a substantial degree of resistance to reinfection comparable to that previously reported in C3H mice can be induced by subcutaneous injection of three larvae 3 weeks prior to intraperitoneal challenge infection. Both early immune damage in the absence of adherent host cells and encapsulation by host cells are involved in rejection of larvae by BALB/c and BDF1 mice, but in both of these strains early immune damage is less pronounced and the cellular encapsulation response considerably more prominent than in the C3H mice studied previously. This difference is also reflected in the effect of immune serum on T. crassiceps metacestodes in vitro: immune serum from BALB/c and BDF1 mice is less effective than immune serum taken from C3H mice at comparable times after challenge infection in mediating damage to T. crassiceps larvae in vitro in the absence of host cells. These results suggest that genetically determined differences in immune capability can alter the state of equilibrium existing among different immune effector mechanisms without producing measurable effects upon overall host resistance to reinfection.