Metacestode Cyst Research Articles

In Vitro and in Vivo Effect of Mapk Signal Transduction Pathway Inhibitors on Echinococcus multilocularis

To evaluate the effect of mitogen-activated protein kinase (MAPK) signal transduction pathway inhibitors against alveolar echinococcosis in vitro and in vivo, Echinococcus multilocularis metacestode cysts and protoscolices were obtained from infected mice. Protein chip technology was utilized to screen for key highly expressed target proteins in the MAPK pathway in this parasite and their corresponding inhibitors. Four-week-old Balb/c female mice used for the in vivo experiment underwent inoculation of E. multilocularis by intraperitoneal injection, as well as intragastric administration of MAPK inhibitors for 6 wk. We included 6 groups of mice: a phosphate-buffered saline (PBS) group (negative control); an albendazole-treated group (positive group); and 4 experimental groups treated with TRx0237 mesylate, GDC-0994, pifithrin-β hydrobromide, or Selonsertib. Echinococcus multilocularis protoscolices were collected and cultured in 1066 medium with penicillin/streptomycin and 10% fetal bovine serum. The in vitro experiment included a PBS group (negative control), a dimethyl sulfoxide-treated group (solvent group), and 4 inhibitor-treated groups as in the in vivo experiment (experimental groups). Each inhibitor group received 4 drug concentrations (5, 30, 55, and 80 μM), and the experiment was performed in triplicate per sample. Fluorescence microscopy was used to evaluate the survival rate of the protoscolices every 48 hr beginning from the first 24 hr. The same grouping was used to evaluate cytotoxicity on E. multilocularis germinal cells and L02 cells. The average weights of E. multilocularis metacestode cyst tissue from each group of the in vivo experiment were 873 mg (PBS), 335 mg (albendazole), 323 mg (TRx0237 mesylate), 420 mg (GDC-0994), 340 mg (pifithrin-β hydrobromide), and 642 mg (Selonsertib). Results showed albendazole, TRx0237 mesylate, and pifithrin-β hydrobromide had significant inhibitory effects on inhibition of E. multilocularis. We found a positive correlation between drug concentrations and the inhibitory effects seen in the in vitro experiment, with the differences in contrast with the control group becoming statistically significant after 72 hr of treatment ( P < 0.05). The inhibition rates of TRx0237 mesylate to germinal cells by drug concentration were 23.73, 46.59, 74.71, and 77.44%. Other drugs had no effect on germinal cells. All the inhibitors had low toxicity on L02 cells. Inhibitors of the MAPK signal transduction pathway showed significant inhibitory effects on E. multilocularis, suggesting these may be potential candidates for the treatment of alveolar echinococcosis.

Identification of Six Introns in a Partial Sequence of Echinococcus granulosus Paramyosin Gene

Paramyosin is a major protein produced by the metacestode cyst of Echinococcus granulosus, the causative agent of cystic hydatid disease. This protein has been shown to play an important role in modulating host immune responses. In this study, we attempted to characterize the noncoding sequence of the paramyosin gene. Genomic DNA was isolated from G1 Iranian hydatid cysts. A DNA fragment of 3200 bp in length was amplified from the paramyosin gene. The polymerase chain reaction (PCR) product was cloned to the pTZ57T vector and sequenced by M13 primers and then compared with unique cDNA coding sequences of E. granulosus (Z21787) and Taenia solium (AY034087). Six introns I (107 bp), II (75 bp), III (47 bp), IV (921 bp), V (19 bp), and VI (456 bp) were identified in the partial sequence of the paramyosin gene. Some nucleotide changes were observed in three exons I, IV, and VI. This data could help scientists in better understanding the possible alternative splicing and designing a real-time PCR technique for the evaluation of the transcription levels of paramyosin in the stages of the Echinococcus sp. life cycle.

Anatomy and development of the larval nervous system in Echinococcus multilocularis

BackgroundThe metacestode larva of Echinococcus multilocularis (Cestoda: Taeniidae) develops in the liver of intermediate hosts (typically rodents, or accidentally in humans) as a labyrinth of interconnected cysts that infiltrate the host tissue, causing the disease alveolar echinococcosis. Within the cysts, protoscoleces (the infective stage for the definitive canid host) arise by asexual multiplication. These consist of a scolex similar to that of the adult, invaginated within a small posterior body. Despite the importance of alveolar echinococcosis for human health, relatively little is known about the basic biology, anatomy and development of E. multilocularis larvae, particularly with regard to their nervous system.ResultsWe describe the existence of a subtegumental nerve net in the metacestode cysts, which is immunoreactive for acetylated tubulin-α and contains small populations of nerve cells that are labeled by antibodies raised against several invertebrate neuropeptides. However, no evidence was found for the existence of cholinergic or serotoninergic elements in the cyst wall. Muscle fibers occur without any specific arrangement in the subtegumental layer, and accumulate during the invaginations of the cyst wall that form brood capsules, where protoscoleces develop. The nervous system of the protoscolex develops independently of that of the metacestode cyst, with an antero-posterior developmental gradient. The combination of antibodies against several nervous system markers resulted in a detailed description of the protoscolex nervous system, which is remarkably complex and already similar to that of the adult worm.ConclusionsWe provide evidence for the first time of the existence of a nervous system in the metacestode cyst wall, which is remarkable given the lack of motility of this larval stage, and the lack of serotoninergic and cholinergic elements. We propose that it could function as a neuroendocrine system, derived from the nervous system present in the bladder tissue of other taeniids. The detailed description of the development and anatomy of the protoscolex neuromuscular system is a necessary first step toward the understanding of the developmental mechanisms operating in these peculiar larval stages.

Detection of polymorphism in AgB1 gene from sheep, cattle and human isolates of echinococcus granulosus by SSCP

Antigen B (AgB) is a major protein produced by the metacestode cyst of Echinococcus granulosus, the causative agent of cystic hydatid disease. E. granulosus AgB is a gene family of at least five gene loci (B1–B5), each one consisting of several minor variants. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and polymorphism of AgB1 in 99 isolates which the 43 were from cattle, 25 of sheep and 31 of human. All samples were analyzed with 12S rRNA-PCR for the strain detection and all of were identified as G1–G3 cluster (E. granulosus sensu stricto). The 16 human, 35 cattle and 25 sheep isolates were yielded the 102bp band by PCR and these samples were tested by SSCP. As results of the SSCP, different band profiles were detected one each of cattle and human isolates while the other 74 isolates showed same band patterns. The DNA sequence analysis was performed for these two isolates and the other selected 4 isolates and polymorphism was confirmed.

The Echinococcus granulosus Antigen B Gene Family Comprises at Least 10 Unique Genes in Five Subclasses Which Are Differentially Expressed

BackgroundAntigen B (EgAgB) is a major protein produced by the metacestode cyst of Echinococcus granulosus, the causative agent of cystic hydatid disease. This protein has been shown to play an important role in modulating host immune responses, although its precise biological function still remains unknown. It is generally accepted that EgAgB is comprised of a gene family of five subfamilies which are highly polymorphic, but the actual number of genes present is unknown.Methodology/Principal FindingsBased on published sequences for the family, we designed specific primers for each subfamily and used PCR to amplify them from genomic DNA isolated from individual mature adult worms (MAW) taken from an experimentally infected dog in China and individual larval protoscoleces (PSC) excised from a single hydatid cyst taken from an Australian kangaroo. We then used real-time PCR to measure expression of each of the genes comprising the five EgAgB subfamilies in all life-cycle stages including the oncosphere (ONC).Conclusions/SignificanceBased on sequence alignment analysis, we found that the EgAgB gene family comprises at least ten unique genes. Each of the genes was identical in both larval and adult E. granulosus isolates collected from two geographical areas (different continents). DNA alignment comparisons with EgAgB sequences deposited in GenBank databases showed that each gene in the gene family is highly conserved within E. granulosus, which contradicts previous studies claiming significant variation and polymorphism in EgAgB. Quantitative PCR analysis revealed that the genes were differentially expressed in different life-cycle stages of E. granulosus with EgAgB3 expressed predominantly in all stages. These findings are fundamental for determining the expression and the biological function of antigen B.

A recombinant chimeric antigen toward a standardized serodiagnosis of Taenia solium neurocysticercosis

Neurocysticercosis (NC) invokes formidable neurological problems worldwide. Previous proteomic analyses revealed most of the low-molecular-weight proteins might derive from two macromolecules of 120 kDa (consisting of 14-38 kDa subunits) and 150 kDa (7-15 kDa subunits) of Taenia solium metacestode (TsM) cyst fluid (CF). We characterized serological properties of these two proteins and established an immunopotent chimera. The 120 and 150 kDa proteins harbored 54-81 and 94-98% of the antibody-binding activity of the crude CF with minimal antigenic cross-reactivity to each other. The expression and immune recognition of the 150 kDa subunits were relatively constant, regardless of the different geographical origins of the CF collected, while those of the 120 kDa subunits varied by their origins (Asia vs. America). We cloned four representative proteins (one from the 120 kDa and three from the 150 kDa) that showed different epitope specificities, generated a chimera, and demonstrated that this regimen may bolster serodiagnostic reliability. Overall sensitivity and specificity, against sera from active-/mixed-stage NC and those from other infections, and healthy-controls, were determined to be 97.5% (156/160 samples) and 97.8% (265/271 cases). Patient sera from adult taeniases, sparganosis, and fascioliasis showed weak cross-reactions. Micro-ELISA showed similar results. This chimera may prove useful in the construction of standardized platform for NC serodiagnosis.

Isolation of a 14 kDa antigen from Taenia solium cyst fluid by HPLC and its evaluation in enzyme linked immunosorbent assay for diagnosis of porcine cysticercosis

A fraction with a major band of 14 kDa was obtained from crude cyst fluid of Taenia solium cysticerci by 2-step chromatography. A first fraction isolated by gel filtration (Sephacryl S-300 high resolution) was purified using an anion exchange column (Mono Q HR 5/5) on high performance liquid chromatography. Evaluation of the analytic sensitivity of this fraction (F3) was carried out in an antibody enzyme linked immunosorbent assay (Ab-ELISA-F3) using serum samples from pigs experimentally infected with different doses of T. solium eggs. The cross-reactivity of F3 was evaluated with serum samples from pigs that were naturally or experimentally infected with Taenia hydatigena, Taenia saginata asiatica, Fasciola hepatica, Trichinella spiralis, Metastrongylus apri, Trypanosoma congolense and Sarcoptes scabiei, and with serum samples of rabbits hyper-immunised with metacestode cyst fluid of T. hydatigena and T. solium. Antibody titres of lightly or heavily infected pigs differed in their kinetics. However, the increase in F3-specific antibodies could not be related to the infection level. Analysis of the specificity of the F3 showed that serum samples of pigs infected with other parasites did not recognise this antigen. Cross-reaction with T. hydatigena occurred in ELISA using cyst fluid as antigen, but the F3 antigen fraction was not recognized by rabbit hyper-immune serum samples to T. hydatigena. Evaluation of the diagnostic sensitivity and specificity of the Ab-ELISA-F3 was done by a non-parametric receiver operating characteristic (ROC) analysis using 66 serum samples from Zambian village pigs. The total number of cysticerci of these pigs was determined by dissection (28 pigs harboured T. solium cysticerci and 38 were negative at dissection). In addition, 58 serum samples from Cameroonian pigs (28 pigs from cysticercosis-free farms and 30 pigs with cysticerci at tongue inspection) were used in a separate ROC analysis. The results from the ROC analysis yielded a low diagnostic value (area under ROC curve = 0.48) with the sera from the Zambian pigs while a relatively high diagnostic value was obtained with the sera from Cameroonian pigs (area under ROC curve = 0.78). The main factor contributing to a low diagnostic value based on the Zambian serum samples seemed to be the false-positive reactions that were likely caused by the occurrence of transient antibodies in the non-infected animals.

Occurrence and biochemical characteristics of cestode lymphocyte mitogens.

The cestodes Echinococcus granulosus, Taenia multiceps, T. pisiformis, T. hydatigena, Hymenolepis diminuta, Moniezia expansa and Anoplocephala perfoliata all produced substances that stimulated thymidine incorporation by whole blood lymphocyte cultures. This mitogenic activity was demonstrated in metacestode cyst fluids, live protoscoleces and scoleces, parasite culture supernatants, and extracts of adult parasites. T. multiceps metacestode cyst fluid mitogen adhered to, but would not pass through, cellulose dialysis tubing. This adherence was reduced or prevented by D-glucose and by proteins. The mitogen was weakly anionic. With Sephadex G75 gel filtration, its elution volume was greatly decreased when the elution buffer contained 0.5 M D-glucose. Mitogenic activity was protease resistant and could be separated by gel filtration from all the cyst fluid proteins.

Investigation of effects of some immunoadjuvants and routes of antigen administration in raising antibodies in rabbits against metacestode cyst antigens of taenia multiceps

The relative efficiency of depot (Freund's complete adjuvant (FCA) and saponin), and non-depot ( Corynebacterium parvum strain P. acnes I, C. parvum strain IMI585, and levamisole) immunoadjuvants were compared in raising agglutinating and precipitating antibodies against the metacestode fluid of Taenia multiceps of sheep origin (TMCF). Haemagglutinating but not precipitating antibodies were detectable during the period of primary immunisation. Precipitating antibodies of higher titre were produced for longer when TMCF was injected with immunoadjuvant than when the same volume of TMCF was injected alone. Significantly ( P < 0.05) higher indirect haemagglutination (IHA) titres were produced during the primary response following C. parvum treatment than after FCA treatment. FCA treated rabbits showed significantly ( P < 0.05) higher IHA titres than those receiving C. parvum treatment on day 42 only, during the anamnestic response. Ulcers formed at the sites of injection of depot immunoadjuvants. Significantly ( P < 0.05) higher IHA titres were produced when FCA with TMCF was injected intradermally into 10–12 sites than when the same volume was injected into 4 sites. C. parvum strain IM1585 was a better immunoadjuvant in this system than the strain P. acnes I. The intravenous route of administration yielded better titres than the intraperitoneal route when C. parvum strain P. acnes I was used.