Trichinella: differential expression of angiogenic factors in macrophages stimulated with antigens from encapsulated and non-encapsulated species.

The newborn larval stage of Trichinella spiralis enters the host striated skeletal muscle cell resulting in the formation of the nurse cell. Vascular Endothelial Growth Factor (VEGF) was detected in cells in the area immediately surrounding the nurse cells. However, no data are available on the antigens involved, the role of other angiogenic factors or the relationship of angiogenesis with Nitric Oxide (NO) production. Using macrophage cell culture we study the effect of different Trichinella L1 antigens from one encapsulated (T. spiralis) and one non-encapsulated (Trichinellapseudospiralis) on the expression of VEGF and basic Fibroblast Growth Factor (FGF2). Also, we investigate the relationship between the production of NO and angiogenic mediators. The results show that encapsulated and non-encapsulated Trichinella species are different in their capacity to stimulate the expression of VEGF and FGF2 from host macrophages. Finally, there is no relationship between angiogenic factors and NO production by T. spiralis antigen.

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.

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.

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.

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.

Pharmacological immunomodulation of surgical trauma.

Surgery and accidental trauma induce changes in the immune response, showing a predominant pattern of activation through the Th2 cell pathway to the detriment of Th1 cell pathway activation. Anapsos is a hydrosoluble extract obtained from Polypodium leucotomos. Anapsos has shown immunomodulating effects in vitro. On a rat experimental model (tibia and fibula fracture), cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, and IL-12) (enzyme-linked immunosorbent assay, ELISA) and cell percentages of CD4, CD8 CD25, CD122, and CD132 (monoclonal antibodies, MoAb) were determined in peripheral blood 7 days before surgery (PRE), 1 day after surgery (1PO), and 7 days after surgery (7PO). On postoperative day 1, rats undergoing fracture show an increase of CD8 percent expression and IL-6 and IL-10 levels, in contrast to rats undergoing fracture plus anapsos treatment. On postoperative day 7, rats undergoing fracture show an increase of IL-6 levels, whereas rats undergoing fracture plus anapsos do not. The IL-12 level decreases on postoperative day 7 in the group with fracture but not in the fracture plus anapsos group. Thus, we conclude that anapsos is able to modulate the immune response after trauma, inhibiting Th2 pathway activation with no effect on Th1 pathway activation. In trauma, Anapsos could prevent the shifting Th1/Th2 balance.

Genetic and immunological characterization of the 14-3-3xi molecule from Schistosoma bovis.

Currently available candidate vaccines against schistosomiasis elicit only partial protection. In addition, the type of immune response that could lead to the highest level of protection against schistosomes has not yet been described. Thus, efforts should be made in both the identification of novel proteins essential for the parasite cycle and in the modulation of immune responses against these novel candidates through the combined use of immunomodulatory molecules. Several parasites have 14-3-3 proteins, and these proteins are known to play a key role in parasite biology. In the present work, we report the isolation and characterization of a new 14-3-3 gene from Schistosoma bovis and offer new information regarding the genetic structure of the gene. In addition, we have produced the corresponding recombinant protein. Finally, we describe the immune responses elicited by this protein when combined with 4 different immunomodulators in immunized mice.

Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD.

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41-48 days post-infection. The mice vaccinated with ADAD only with PAL+Fh15 present a survival rate of 40-50% and those vaccinated with ADAD containing PAL+Qs+Fh15 had a survival rate of 50-62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL+Qs+Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines 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.

A new pharmacological screening assay with Trypanosoma cruzi epimastigotes expressing beta-galactosidase.

We have developed a new pharmacological screening assay for epimastigotes of Trypanosoma cruzi (clone CL-B5) that express the Escherichia coli LacZ gene. The assay is based on determining the activity of the cytoplasmic beta-galactosidase released into the culture on membrane lysis in the presence of the substrate chlorophenol red beta-D-galactopyranoside (CPRG). The experimental conditions were adjusted to find those in which the relationship between epimastigote number and CPRG absorbance was linear over the widest possible range. Absorbance was significantly correlated with the number epimastigote from 5x10(3) to 1.2x10(6) parasites/ml (r=0.98, P<0.01). The optimal final concentration of CPRG was 200 microM and the optimal incubation period was 6 h when parasites were incubated for 3 days. Once the assay was standardized, the trypanocidal activities of nifurtimox and benznidazole were determined both by CPRG assay and microscopic counting, demonstrating the methods utility for drug-screening. The efficacy obtained was comparable to that obtained with the manual method.

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.

Nematocidal activity of nitazoxanide in laboratory models.

The nematocidal activity of a broad-spectrum antiparasitic agent, nitazoxanide [( N-(5-nitrothiazol-2-gammal)salicylamide; NTZ], was evaluated in both in vitro and in vivo models using Caenorhabditis elegans, Heligmosomoides polygyrus and Trichinella spiralis. In vitro, NTZ (100 muM) exhibited a low activity against C. elegans and had no effect on embryonation and hatching of H. polygyrus eggs. At concentrations of 100 and 50 muM, the inhibition of excretion/secretion of acetylcholinesterase and acid phosphatase of adult H. polygyrus by NTZ was variable. The in vitro effects of mebendazole (5 muM), albendazole (1 muM) and levamisole (10 muM) were superior to those of NTZ. In mice, NTZ at 1 g/kg proved to be inactive against preadults of T. spiralis whereas mebendazole at 10 mg/kg reduced the worm burden by up to 83%. NTZ at 1 g/kg per day for 3 consecutive days showed a low activity against adults of H. polygyrus (21% reduction). Levamisole, at a single dose of 10 mg/kg, reduced the worm burden by up to 89.9%. The results of this study suggest that NTZ would not have met criteria of a candidate compound.

Heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53.

This study investigates the heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53. Western blotting analysis of several Trichinella isolates with the gp53-specific monoclonal antibodies (mAbs) US5 and US9, produced in Btkxid mice, revealed that gp53 from the species T. britovi, T. murrelli and genotype T8 had higher MW (60 kDa) than gp53 from T. spiralis, T. nelsoni and genotype T6 (53 kDa) and from T. nativa (55 kDa). mAb US5 reacted only with gp53 from T. spiralis. Experiments including immunoassays of gp53 binding by sera from T. spiralis-infected mice, in the presence of different potential inhibitors (recombinant gp53, US5, T. britovi-crude larval extract (CLE), and CLE N- and O-glycans), indicate (i) that gp53 from T. spiralis bears specific epitopes that induce antibody formation during infection; (ii) that the protein epitopes of gp53 are much more important (76 or 68% of total antibody reactivity in BALB/c and Swiss CD-1 mice, respectively) than the corresponding glycan epitopes including tyvelose (11 or 32% of total reactivity) for the induction of anti-gp53 circulating antibodies; and (iii) that the species-specific epitopes present on gp53 are differentially recognized in different mouse strains. Whereas in BALB/c mice US5- and non-US5-recognized species-specific epitopes on gp53 bind about 84% of circulating antibodies on day 80 post-infection, this percentage was only 38% in Swiss CD-1 mice. These data on the antigenicity of gp53 contrast with data for Trichinella CLE antigens, in that most circulating antibodies reactive with CLE antigens recognized tyvelose-containing epitopes (57% and 58% of circulating antibodies in BALB/c and Swiss CD-1 mice, respectively). Together these results demonstrate that gp53 is recognized during infection but is antigenically different from other Trichinella TSL-1 antigens.

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.

A comparison of antigenic peptides in muscle larvae of several Trichinella species by two-dimensional western-blot analysis with monoclonal antibodies.

The antigens recognised by mAb US5 specific to 53 kDa glycoprotein (gp 53) in T. spiralis L-1 muscle larvae (TSL1) antigens, mAb US9 specific to gp 53 in TSL1 from all encapsulated species and mAb US4 specific to a tyvelose containing tetrasaccharide present in TSL1, were investigated in crude extracts from muscle larvae of T. spiralis, T. nativa and T. britovi by 2D-electrophoresis and western-blot. At least four proteins of different p1 were recognised by mAb US5 on T. spiralis antigens. Recognition profile of mAb US9 on T. spiralis antigens exhibited some variation with regard to that of the US5. Polymorphism was apparent in gp 53. High reactivity was shown by the mAb US4 with the three species.

Possible presence of common tyvelose-containing glycans in Trichinella L1 larvae and embryonated eggs of several nematodes.

A monoclonal antibody (mAb US4) recognising an epitope containing tyvelose within the T. spiralis L-1 muscle larvae (TSL-1) antigens was tested in western-blot against various antigenic preparations from different stages of the following nematodes: T. spiralis (L1, adult), T. muris (egg, L1, L3, adult), Ascaris suum (egg, adult), Toxocara canis (egg, adult), Anisakis simplex (L3) and Haemochus contortus (egg). Positive reaction was present in antigen preparations from L1 larvae of T. spiralis and T. muris and from embryonated eggs of T. muris, A. seum, T. canis and H. contortus.

Comparison of IgG3 responses to carbohydrates following mouse infection or immunization with six species of Trichinella.

The IgG3 antibody responses to carbohydrate epitopes were compared in BALB/c mice infected or immunized with six species of Trichinella: T. spiralis (T1), T. nativa (T2), T. britovi (T3), T6, T. nelsoni (T7), and T8. The dynamics of IgG3 responses and antigen recognition following infection or immunization were measured by ELISA and Western blot respectively, using glycosylated and deglycosylated larval crude extracts (LCE) prepared from homologous isolates. A high degree of protein glycosylation was found in all species and with similar profiles. Deglycosylation was completely achieved only in LCE from T1 and T6 isolates. The dynamics of IgG3 responses following infection or immunization significantly differed whereas the antigen recognition profiles appeared similar. Variations in the levels and antigen recognition patterns of IgG3 among the different species were apparent. The highest IgG3 levels were recorded in infections by the T8 isolate and the lowest in infections by the T6 isolate, whereas for immunization the highest IgG3 response was induced by T7 and the lowest by T8. Following antigen deglycosylation, the IgG3 responses were significantly reduced or abrogated and the recognition patterns markedly modified or suppressed in the different species of Trichinella.

In vitro screening of american plant extracts on Trypanosoma cruzi and trichomonas vaginalis.

From the beginning of this decade and with the revival of the phytotherapy, biological research about immunomodulatory, anti-inflammatory and antiprotozoal effects of Central and South American plants have been in progress. Our objective was to determine the antiprotozoal activity of 79 extracts from different plant families, including Asteraceae, Araceae, Moraceae, Solanaceae, Rhamnaceae, Zingiberaceae, Leguminosae and Sapotaceae. Once matching with herbarium specimens authenticated the plants, selected parts were separated, dried carefully and reduced to powder. Most of the screened extracts were aqueous. Two protozoa with different metabolic pathways, Trypanosoma cruzi and Trichomonas vaginalis were used as experimental models. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose (LIT). Anti-Trichomonas activity was determined over cultures of the parasite in Diamond medium. In both cases, microscopic counting of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the cytocidal and cytostatic activities respect to control cultures. Of the nine extracts that showed antiprotozoal activity, those from Mikania cordifolia and Philodendron bipinnatifidum were then fractionated, and again, were assayed the organic and aqueous phases obtained.