Chemical characterization and in vitro anthelmintic activity of Citrus bergamia Risso and Citrus X paradisii Macfad essential oil against Haemonchus contortus Kirby isolate.

Haemonchus contortus, a blood-sucking parasite of small ruminants, produces very important economic losses in the productive sector. This abomasum parasite has become resistant to most commercial drugs worldwide, and alternatives to fight this problem are urgently needed. Essential oils (EO) are a complex mixture of volatile secondary metabolites, composed mainly by terpenoids and phenolic compounds, from plants that have several pharmacological properties, including anthelmintic activity. Particularly, citrus peel is a source of cold-pressed EO, where limonene is its major component, and can be used as an additional food component for ruminants. The aim of the present work was to determine the in vitro anthelmintic activity of EO from Citrus bergamia (EOB), C. x paradisii (EOG) and limonene against the benzimidazole-susceptible Kirby isolate of H. contortus, using the egg hatch test (EHT) and the exsheathed third stage larval motility test (XLMT) using a WMicroTracker equipment. Albendazole (ABZ) and monepantel (MON) were used as positive controls. The 50% inhibitory concentrations (IC50) in XLMT were 8.77 and 13.88 µg/ml for EOB and EOG respectively, after an incubation of 72 h. An interesting observation on XLMT resulted when the positive controls were tested on the same plate, but in different well of the EOB. The volatile components of the EO significantly influenced (P < 0.05) the percentage of larval motility, reducing values from 66.9 to 19.6% for ABZ, and from 72.8 to 33.7% for MON, when comparing the activity of positive controls in a control plate without EO. The in vitro anthelmintic activity of EOB and EOG shows that they could be interesting candidates for nematode control. It is still necessary additional studies against the adult stage of H. contortus in efficacy trials in infected animals to validate their anthelmintic activity.

Purification of native M. vogae and H. contortus tubulin by TOG affinity chromatography.

Microtubules are non-covalent cylindrical polymers formed by alpha- and beta-tubulin heterodimer units, crucial for cell division, intracellular transport, motility and differentiation. This makes them very attractive pharmacological targets exploited to develop different drugs such as anthelmintics, antifungals, and antineoplastics. In this work, in order to establish an in vitro target-based screen to integrate to the search for new anthelmintics, we explored the extraction of native assembly-competent tubulin from two helminth parasites: Mesocestoides vogae tetrathyridia (syn. corti, Cestoda: Cyclophyllidea), a useful cestode biological model, and Haemonchus contortus, a sheep gastrointestinal nematode of interest in livestock production. For this purpose, a novel tubulin affinity chromatography procedure was employed, based on the binding capacity of TOG (Tumor Overexpressed Gene) domain from MAPs (microtubule-associated proteins). The TOG domain of the protein Stu2 from Saccharomyces cerevisiae fused to GST (glutathione S- transferase) were produced in E. coli, and the immobilized recombinant proteins allowed for native tubulin extraction from parasites. The binding capacity of TOG1 affinity column (3.6%) was estimated using commercial porcine brain tubulin. A total amount of up to 126 µg of M. vogae tubulin was purified, whereas H. contortus tubulin co-eluted with glutamate dehydrogenase enzyme. The identity of tubulins was confirmed by western blotting and mass spectrometry. The abundance of tubulin estimated in M. vogae was 10% soluble extract, which probably could explain differences observed between tubulin purification results of both helminth parasites.

Mucin-type O-glycosylation in Mesocestoides vogae (syn. corti).

Protein glycosylation is an important post-translational modification underlying host-parasite interactions, which may determine the outcome of infection. Although Mesocestoides vogae represents an important model for investigating the various aspects of cestode biology, virtually no information is available about the structure and synthesis of glycans in this parasite. In this work, focused on the initiation pathway of mucin-type O-glycosylation in M. vogae, we characterized O-glycoproteins bearing the simple mucin-type cancer-associated Tn and sialyl-Tn antigens, and the expression and activity of ppGalNAc-T, the key enzyme responsible for the first step of mucin-type O-glycosylation. Using immunohistochemistry, Tn and sialyl-Tn antigens were detected mainly in the tegument (microtriches) and in parenchymal cells. Tn expression was also observed in lateral nerve cords. Both Tn and sialyl-Tn antigens were detected in in vitro cultured parasites. Based on their electrophoretic mobility, Tn- and sialyl-Tn-bearing glycoproteins from M. vogae were separated into several components of 22 to 60 kDa. The observation that Tn and sialyl-Tn glycoproteins remained in the 0.6N perchloric acid-soluble fraction suggested that they could be good candidates for characterizing mucin-type glycosylation in this parasite. O-glycoproteins were purified and initially characterized using a proteomic approach. Immunohistochemical analysis of the tissue distribution of ppGalNAc-T revealed that this enzyme is expressed in the sub-tegumental region and in the parenchyma of the parasite. In M. vogae cultured in vitro, ppGalNAc-T was mainly detected in the suckers. Using a panel of 8 acceptor substrate synthetic peptides, we found that M. vogae ppGalNAc-T preferentially glycosylate threonine residues, the best substrates being peptides derived from human mucin MUC1 and from Trypanosoma cruzi mucin. These results suggest that M. vogae might represent a useful model to study O-glycosylation, and provide new research avenues for future studies on the glycopathobiology of helminth parasites.

Characterization of the carcinoma-associated Tk antigen in helminth parasites.

Expression of Tk antigen, a truncated carbohydrate antigen, was examined in helmith parasites. Using the monoclonal antibody LM389, this antigen was detected in extracts from Taenia hydatigena, Mesocestoides vogae (syn corti), and Taenia crassiceps. No reactivity was observed in Thysanosoma spp., Dipylidium caninum, Fasciola hepatica, and Nyppostrongylus brasiliensis. On the basis of their electrophoretic mobility, different patterns of Tk-bearing glycoproteins were observed among T. hydatigena, M. corti and T. crassiceps by immunoblotting, with certain components resolved as broad bands typical of mucin-like glycoproteins. Most Tk-reactive material remained in the 0.6 N perchloric acid-soluble fraction, confirming that Tk epitopes are carried by mucin-type glycoproteins. Immunohistochemical analysis revealed that in T. hydatigena, Tk antigen is mainly expressed in the tegument, whereas in M. corti the reactivity was principally observed in the subtegumental parenchyma. The presence of a novel tumor-associated carbohydrate antigen in invertebrates, contributes to strengthen the notion that truncated mucin-type O-glycosylation is a normal phenomenon in parasitic worms and may help identify new biological characteristics of helminth parasites.

Synthesis and biological evaluation of simplified mycothiazole analogues.

Thiazoline and oxazoline analogues of the natural product mycothiazole were synthesized from a common intermediate and evaluated in vitro against HCT-15 colon cancer cells and L(4) larvae of nematode Nippostrongylus brasiliensis. The nature of the heterocyclic moiety seems to modulate the cytotoxic or anthelmintic activity.

The toxicity of praziquantel against Mesocestoides vogae (syn. corti) tetrathyridia can be assessed using a novel in vitro system.

We recently standardised Mesocestoides vogae (syn. corti) tetrathyridia cultures in the presence of sodium taurocholate. Parasite clustering and segmentation were observed as taurocholate-dependent effects in biphasic and monophasic media, respectively, and both were inhibited by a specific minimum inhibitory concentration (m.i.c.) of the cestocidal drugs albendazol and praziquantel. In the present study, we analysed the relationship between clustering inhibition and drug toxicity using praziquantel and a mouse experimental infection. In an "in vitro-in vivo" trial, a significant (ANOVA, P<0.05) reduction was observed in the infectivity of tetrathyridia previously cultured with praziquantel m.i.c. (0.06 micro g/ml) for 10 days. In an "in vivo-in vitro" trial, the clustering of tetrathyridia recovered from mice treated with praziquantel was found to be markedly reduced: 22%, compared with 83% cluster-containing wells of parasites from control mice. These results show that the outcome of infection and the suppression of taurocholate-induced clustering provide consistent indications of praziquantel toxicity against M. vogae, an observation confirmed by histological studies. The easily recorded clustering inhibition of M. vogae tetrathyridia in biphasic medium is a potentially useful system for the assessment of drug toxicity against cestode larvae.