Trichinellosis survey in the wild boar from the Toledo mountains in south-western Spain (2007-2008): molecular characterization of Trichinella isolates by ISSR-PCR.

In Spain, trichinellosis represents a public health problem, with an average of five outbreaks per year, wild boar meat being the main source of infection. A trichinellosis survey (2007-2008 hunting campaign) was carried out on wild boars in the Toledo Mountains (south-western Spain, EU) in the context of a surveillance programme on wildlife diseases. A total of 2216 wild boars from different locations of the region were examined. The examination was carried out by veterinarians in the local abattoir (Matadero Municipal de Toledo). The positive samples were sent to the Department of Parasitology (Facultad de Farmacia, UCM) for experimental isolation and specific identification by inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR). Using this technique we identified 17 isolates as Trichinella spiralis with an electrophoretic profile indistinguishable from the T. spiralis reference strain (ISS48). We confirmed that ISSR-PCR is a robust technique for the molecular identification of Trichinella isolates. According to our results, the prevalence of T. spiralis in wild boars from the Toledo Mountains (>800 m above sea level) during the hunting season was approximately 0.77%. The prevalence of T. spiralis (100% of our observations) is a good example of the persistence of this species in sylvatic conditions (coming from the domestic cycle), if a good wild host is abundant. Our observations confirm the major prevalence of T. spiralis over T. britovi in this region, as well as the risk to human health represented by the consumption of uninspected wild boar meat.

The addition of a new immunomodulator with the adjuvant adaptation ADAD system using fatty acid binding proteins increases the protection against Fasciola hepatica.

Fatty acid binding proteins (FABP) have shown protective immune response against Fasciola hepatica infection. We evaluated the protection induced by the Fh12 FABP from F. hepatica (Fh12) combined with the new immunomodulator the lipidic aminoalcohol OA0012 in the ADAD system in mice and sheep. In this work we introduced a lipidic aminoalcohol OA0012 as immunomodulator alone or in combination with the hydroalcoholic extract of Phlebodium pseudoaureum; PAL. Mice vaccinated with ADAD containing OA0012+Fh12 or OA0012+Qs+Fh12 had survival rates of 40-50%. Sheep ADAD-vaccinated with OA0012+Qs+Fh12 showed lower fluke recovery, less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Sheep ADAD-vaccinated with OA0012 combined PAL and Qs+Fh12 showed lower fluke recovery (42%), lower adult worms count (57%) lower faecal egg count (38%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the addition of a new immunomodulator of synthesis to ADAD system with FABPs increased the protection against F. hepatica.

Molecular characterization of Trichinella genotypes by inter-simple sequence repeat polymerase chain reaction (ISSR-PCR).

A bulk analysis of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) provides a quick, reliable, and highly informative system for DNA banding patterns that permit species identification. The present study evaluates the applicability of this system to Trichinella species identification. After a single amplification carried out on a single larva with the primer 816([CA]nRY) under high stringency conditions, which provide high reproducibility, we were able to identify by consistent banding patterns 5 sibling species: Trichinella spiralis (ISS48), 2 Trichinella britovi isolates (ISS11 and ISS86), Trichinella murrelli (ISS35), Trichinella nativa (ISS71), Trichinella nelsoni (ISS29); 3 additional Trichinella genotypes: T8 (ISS149), T9 (ISS408 and ISS409), and T6 (ISS34); and the nonencapsulated species Trichinella pseudospiralis (ISS13). Moreover, 33 new Trichinella isolates from 2 zoogeographical regions were unequivocally identified. All Trichinella isolates have shown an identical pattern with those produced by the reference strain. According to these data, we have demonstrated that ISSR-PCR is a robust technique that emerges as a useful new application for the molecular identification of Trichinella isolates in epidemiological studies.

Effect of piroxicam, metamizol, and S-adenosylmethionine in a murine model of experimental trichomoniasis.

Biological effects of piroxicam, metamizol, and S-adenosylmethionine (S-AMET) have been tested in NMRI mice infected intraperitoneally with Trichomonas vaginalis. An intraperitoneal treatment during ten preinfection days with piroxicam (10 mg/Kg/day), or metamizol (275 mg/Kg/day), but not with S-AMET (117 mg/Kg/day) induced a significant decrease of abdominal lesions and mortality, assessed by means of a pathogenicity index. The trichomonicidal activity of piroxicam, metamizol, and S-AMET was tested in vitro at the concentration of 300 microM, but found ineffective. These assays have shown the usefulness of the experimental trichomoniasis model for the study of the immunomodulating activity of synthetic drugs.

Vaccination of mice and sheep with Fh12 FABP from Fasciola hepatica using the new adjuvant/immunomodulator system ADAD.

We evaluate the ability of a Fasciola hepatica FABP native antigen (Fh12) with a new vaccination system called ADAD to protect mice and sheep against an experimental F. hepatica infection. The vaccination protocol consists of a set of two injections. The first injection contains a micelle in which two components are included, saponin from Quillaja saponaria (Qs) and/or Anapsos (A) a Polypodium leucotomos hydroalcoholic extract, both emulsified in a non-mineral oil (Montanide) in a water/oil emulsion (30/70). This is subcutaneously injected to achieve the "adaptation" of the immune system to subsequent stimuli. The second injection contains in addition the Fh12 antigen. Two different experiments were carried out using two mouse strains (BALB/c and CD-1). Mice vaccinated with Qs+A+Fh12 presented a survival rate of 40%, when compared with control groups. Furthermore, we evaluated the efficiency of the vaccination in sheep against an experimental F. hepatica challenge. The vaccinated sheep presented lower fluke recovery (24.5%), number of eggs in bile fluid (58.1%) and faeces (40.3%) than control groups. The recovered flukes were shorter (32.7%), immature (34.0%) and with lower body mass (31.6%) than non-complete vaccinated sheep. Thus, the new ADAD system could be a good alternative for future vaccination experiments against fasciolosis.

Trichomonas vaginalis: determination of acid phosphatase activity as a pharmacological screening procedure.

A simple method to screen trichomonacides, based on the quantification of acid phosphatase (AP) activity, has been designed. Using p-nitrophenyl phosphate as chromogenic substrate, we first determined the optimal conditions for enzyme reaction. After seeding, a linear correlation between number of trichomonads and optical densities at 405 nm was obtained at 24 hr but not at 48 hr. Then, the inhibitory effect of metronidazole was assessed both by microscope counts and by AP determination. Similar values for 50% inhibitory concentrations (2.6 microM), with 95% confidence limits of 1.91-3.33 for microscopic and 2.21-3.05 for colorimetric method, were obtained. We concluded that the colorimetric method described in this investigation is suitable for pharmacological studies and for the screening of new, potential antitrichomonal agents.

Modulation by Polypodium leucotomos extract of cytokine patterns in experimental trichomoniasis model.

The immunomodulating effects of Anapsos, an aqueous hydrosoluble extract obtained from the rhizomes of the fern Polypodium leucotomos, on both pathogenicity and cytokine levels in serum (IFN-gamma/IL-4) were assayed in a Trichomonas vaginalis experimental model (BALB/c mice infected with 10(7) trichomonads and examined at day 15 after infection). Doses of 20 mg/kg/day administered for 10 days before the infection with the parasite induced a decrease of the experimental pathogenicity approximately 10-20% compared to controls. Gross histopathologic changes at abdominal organs and mortality rate, as a consequence of pathogenicity of the protozoa and the immune response of the host, were evaluated. IFN-gamma and IL-4 cytokines were determined on days -5, 0, 5, 10, and 15 postinfection by indirect ELISA. Treatment with PAL before infection modulates and downregulates the IFN-gamma concentration, while anticipates and upregulates the IL-4 level. The assays performed have showed the utility of the murine model of experimental trichomoniasis for the evaluation of immunomodulatory activity of synthetic or natural products.

Effect of Anapsos in a murine model of experimental trichomoniasis.

Immunomodulator effect of Anapsos (Polypodium leukotomas extract) in NMRI (US Naval Medical Research Institute) outbred mice infected by the intraperitoneal route with 10(7) Trichomonas vaginalis has been tested. Gross histopathologic changes in abdominal organs and mortality rate, as a consequence of the pathogenicity of the protozoa and the immune response of the host, were evaluated. Among the different treatment regimes assayed, Anapsos at doses of 20 mg/Kg/day administered for 10 days before infection decreases the parasite pathogenicity index (PI) in the treated animals when compared to those of the untreated control group. The immunosuppressor treatments with azathioprine (100 mg/Kg/day x 1), cyclophosphamide (100 mg/Kg/day x 1), and FK-506 (10 mg/Kg/day x 10) significantly decreased the PI, while an immunostimulant treatment with glycophosphopeptical (13 mg/Kg/day x 10) increased it. These assays have shown the usefulness of the murine model of experimental trichomoniasis for the study of immunomodulator activity of natural or synthetic drugs.

Evaluation of a murine model of experimental trichomoniasis.

By using a reference strain of Trichomonas vaginalis and the intraperitoneal route for infecting animals, the influence of the strain of mice, the time observation and the inoculation doses were followed in order to standardize the optimal conditions for the evolution of experimental trichomoniasis. Our results suggest that the inoculation of BALB/c mice with 10(7) trichomonads and the semiquantitative assessment at day 15 postinfection of the gross-pathologic changes in the abdominal cavity--peritoneum, spleen, pancreas, stomach and liver--as well as the presence of ascitic fluid and mortality, maybe a suitable laboratory model of trichomoniasis.

Morphological and molecular identification of Tetratrichomonas flagellates from the giant anteater (Myrmecophaga tridactyla).

A tetratrichomonad flagellate found in the diarrhoeic faeces of a 5 years-old male giant anteater (Myrmecophaga tridactyla) was characterised by morphological and genetic analysis. This protozoan presents four anterior flagella of unequal length and a recurrent flagellum attached to the undulating membrane without a free end portion, and a broad axostyle projection. Numerous vacuoles of different sizes containing bacteria and digestion products were found. The complete sequence of the DNA coding for the 16S rRNA-ITS1-5.8S rRNA-ITS2 region was also obtained in order to compare this isolate with other tetratrichomonad species. The sequence obtained was identical to others previously obtained by other researchers from bovines and turtles (Geochelone sp.). It is not easily explainable how the same organism could be found in such different hosts and locations; however these results indicate that some tetratrichomonad species could have a wide host range and could survive in a wide range of environmental conditions.

Identification of Spanish Trichinella isolates by ISSR-PCR: intra-specific variability of Trichinella britovi.

In the present work, we investigated genetic variability of the Spanish Trichinella isolates by ISSR-PCR (inter-simple sequence repeat polymerase chain reaction), a technique that is being successfully used to study diversity among related populations. We recovered a total of 43 isolates from different host and geographic localization and identified them by molecular techniques (RAPD and multiplex-PCR) and by Western blot with monoclonal antibodies US5 and US9. Nineteen (44.2%) out of 43 were identified as Trichinella spiralis and 24 (55.8%) as Trichinella britovi. When these samples were analysed by the ISSR technique, all the T. spiralis isolates presented a pattern similar to the T. spiralis ISS116. By contrast, the ISSR-PCR analysis of the isolates identified as T. britovi, showed two different banding profiles compatible with the European T. britovi isolate pattern (ISS2), and the autochthonous Spanish T. britovi isolate (ISS11). Three of these 43 isolates were involved in human outbreaks; the three were identified as T. britovi and showed a pattern similar to the European isolate ISS2. As conclusion, we highlight that an intra-species variability within the Spanish T. britovi isolates analysed was observed, with a predominant group similar to T. britovi ISS2, while T. spiralis group isolates were more homogeneous. No correlations were found between the different ISSR-PCR T. britovi types and the host/geographical origin of the isolates.

Prevalence of Trichinella spp. in North Spain wild fauna and new variety of Trichinella britovi identification.

Trichinella spiralis and Trichinella britovi live in apparent sympatry among wild fauna of the Iberian Peninsula. In the present study 105 Trichinella isolates from wild mammals were typed by inter-sequence simple repeat PCR (ISSR-PCR). All isolates identified as T. spiralis were indistinguishable from the ISS48 reference strain. Among those belonging to T. britovi, four variations were clearly distinguishable; two of them, ISS11 C-76 and ISS86 MON, had been previously detected while the ISS2 reference strain and Trichinella Rioja 3, (MVUL/SP/02/R3) had not been reported before. The newly distinguished genotype of T. britovi was analyzed by ISSR-PCR, multiplex-PCR, UARR sequencing, and single larva cross-breeding with the other T. britovi genotypes including Trichinella T8 (ISS49). Among all of them, the ISS11 and ISS2 isolates were found to be the most frequent. The uniformity found within T. spiralis isolates is consistent with its recent introduction in Iberian Peninsula, whereas the presence of four variations within T. britovi suggests that this species is an endemic species. Orographical diversity of the West-End of Eurasian Region could act to preserve population diversity observed within T. britovi.

The Schistosoma bovis Sb14-3-3zeta recombinant protein cross-protects against Schistosoma mansoni in BALB/c mice.

Current control programs against schistosomiasis could be reinforced through the use of an effective vaccine. Schistosome 14-3-3 proteins have been proposed as candidates for vaccine against the respective infections, and were seen to elicit high protection levels against Schistosoma bovis in a previous work done by our group. We have therefore investigated the protective capacity of the 14-3-3 protein from S. bovis - Sb14zeta - against Schistosoma mansoni in mice. In addition, we have addressed the influence of the co-administration of three different immunomodulators with the 14-3-3 polypeptide. Protection was high when the Sb14zeta protein was combined in two independent experiments with the AA2829 and PAL immunomodulatory molecules as regards both the reduction of worm numbers (mean: 64.8%) and egg loads in liver (mean: 73.9%) or intestine (mean: 71.5%). In contrast, the degree of protection achieved with the Sb14zeta-CpG vaccine was very low (14.9% reduction in worm numbers, and 46.6% and 32% reduction in liver and intestinal egg loads). The immune responses observed in the vaccinated animals showed that the production of IFNgamma and the absence of IL-4, accompanied by a strong humoral response, are insufficient to elicit protection against S. mansoni.

Setting of a colorimetric method to determine the viability of Trypanosoma cruzi epimastigotes.

The method most commonly used in screening of drugs for the treatment of Chagas' disease, microscopic counting of viable trypanosomes, is time-consuming, labor-intensive, and dependent on the observer. Although the tetrazolium dye [MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is comparatively quick and accurate, it requires careful attention in design as well as in interpretation of the results. Therefore, we examined under various conditions the sensitivity and specificity of the MTT assay versus microscopic counting for determination of the viability of Trypanosoma cruzi for drug-screening purposes. We tested different concentrations of MTT in phenazine methosulfate (PMS) against T. cruzi epimastigotes of the Y strain in different stages of logarithmic growth. In our model, in tests of benznidazole and nifurtimox the optimal concentration of MTT was 2.5 mg/ml of PMS and the optimal incubation period was 75 min. This method detected parasite concentrations of approx. 500,000 epimastigotes/ml (P<0.01), and the linear correlation between absorbance values and numbers of epimastigotes per milliliter was very strong (approx. R = 0.99). The present MTT assay results in faster determination of the activity of compounds, is more objective, and enables testing of several drugs simultaneously.

Biological characterization of Trypanosoma cruzi strains.

Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans.

Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains.

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.

Evaluation of drug activity against intracellular forms of Trypanosoma cruzi employing enzyme immunoassay.

OBJECTIVE: To describe application of a new method for the evaluation of anti-Trypanosoma cruzi activity against intracellular forms. METHOD: Vero fibroblasts in 96-well tissue culture plates were infected with trypomastigote forms of T. cruzi. Amastigotes growth was estimated after 24 and 96 h both by microscopic counts of Giemsa-stained monolayers and enzyme-linked immunoIsorbent assay (ELISA). ELISA was performed directly on the fixed cultures using a rabbit anti-T. cruzi immunoIglobulin as the first antibody and a peroxidase-labelled antirabbit immunoglobulin as the second antibody. Three chemical series of structural analogous of gentian violet, thiadiazines and derivatives of 5-nitrothiophene-2-carbaldehyde as well as three reference compounds (nifurtimox, benznidazole and gentian violet) were then assayed. The anti-T. cruzi activity of all of them had been determined previously by microscopic counting of Giemsa-stained infected cultures. RESULTS: None of the assayed compounds showed better activity than the reference ones, but the application of the enzyme immunoassay to quantify the inhibition of growth amastigotes is of great interest, as it yielded results comparable with microscopic counts. CONCLUSION: ELISA can be applied to pharmacological screening, with some advantages over the microscopic examination, including possible automation, rapidity and objectivity in assessment.