Genetic diversity and DNA barcoding of the black fly (Diptera: Simuliidae) vectors of parasites causing human onchocerciasis in Guatemala.

Genetic variation based on mitochondrial cytochrome c oxidase I (COI) and II (COII) sequences was investigated for three black fly nominal species, Simulium metallicum Bellardi complex, S. callidum Dyar and Shannon, and S. ochraceum Walker complex, which are vectors of human onchocerciasis from Guatemala. High levels of genetic diversity were found in S. metallicum complex and S. ochraceum complex with maximum intraspecific genetic divergences of 11.39% and 4.25%, respectively. Levels of genetic diversity of these nominal species are consistent with species status for both of them as they are cytologically complexes of species. Phylogenetic analyses revealed that the S. metallicum complex from Guatemala divided into three distinct clades, two with members of this species from several Central and South American countries and another exclusively from Mexico. The Simulium ochraceum complex from Guatemala formed a clade with members of this species from Mexico and Costa Rica while those from Ecuador and Colombia formed another distinct clade. Very low diversity in S. callidum was found for both genes with maximum intraspecific genetic divergence of 0.68% for COI and 0.88% for COII. Low genetic diversity in S. callidum might be a consequence of the result being informative of only recent population history of the species.

Genetic diversity and DNA barcoding of the black fly (Diptera: Simuliidae) vectors of parasites causing human onchocerciasis in Guatemala

@#Genetic variation based on mitochondrial cytochrome c oxidase I (COI) and II (COII) sequences was investigated for three black fly nominal species, Simulium metallicum Bellardi complex, S. callidum Dyar & Shannon, and S. ochraceum Walker complex, which are vectors of human onchocerciasis from Guatemala. High levels of genetic diversity were found in S. metallicum complex and S. ochraceum complex with maximum intraspecific genetic divergences of 11.39% and 4.25%, respectively. Levels of genetic diversity of these nominal species are consistent with species status for both of them as they are cytologically complexes of species. Phylogenetic analyses revealed that the S. metallicum complex from Guatemala divided into three distinct clades, two with members of this species from several Central and South American countries and another exclusively from Mexico. The Simulium ochraceum complex from Guatemala formed a clade with members of this species from Mexico and Costa Rica while those from Ecuador and Colombia formed another distinct clade. Very low diversity in S. callidum was found for both genes with maximum intraspecific genetic divergence of 0.68% for COI and 0.88% for COII. Low genetic diversity in S. callidum might be a consequence of the result being informative of only recent population history of the species.

Strongyloides ratti: implication of mast cell-mediated expulsion through FcepsilonRI-independent mechanisms.

In order to examine whether FcepsilonRI-dependent degranulation of intestinal mast cells is required for expulsion of intestinal nematode Strongyloides ratti, CD45 exon6-deficient (CD45-/-) mice were inoculated with S. ratti. In CD45-/- mice, egg excretion in feces persisted for more than 30 days following S. ratti larvae inoculation, whereas in wild-type (CD45+/+) mice, the eggs completely disappeared by day 20 post-infection. The number of intestinal mucosal mast cells, which are known effector cells for the expulsion of S. ratti, was 75% lower in CD45-/- mice compared with that in CD45+/+ mice. Adoptive transfer of wild-type T cells from CD45+/+ mice into CD45-/- mice reduced the duration of S. ratti infection to comparable levels observed in CD45+/+ mice, with concomitant increases in intestinal mucosal mast cells. These results showed that CD45 is not involved in the effector function of intestinal mucosal mast cells against S. ratti infection. Since FcepsilonRI-dependent degranulation of mast cells is completely impaired in these CD45 knockout mice, we conclude that FcepsilonRI-dependent degranulation is not required in the protective function of intestinal mucosal mast cells against primary infection of S. ratti.

Molecular phylogeny of Trypanosoma cruzi from Central America (Guatemala) and a comparison with South American strains.

Molecular phylogenetic analysis was carried out for 21 strains of Trypanosoma cruzi, nine of which were obtained from Guatemala and 12 from South America. Phylogenetic trees were constructed using the nucleotide sequences of two nuclear gene regions, dihydrofolate reductase-thymidylate synthase (DHFR-TS) and trypanothione reductase (TR), and contiguous portions of two mitochondrial genes, cytochrome oxidase subunit II (COII) and reduced nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1). Possible genetic exchange between the rather divergent lineages of T. cruzi II from South America was suggested in the trees of the two nuclear genes. T. cruzi I strains obtained from Guatemala and Colombia were identical in all the genes examined, but other T. cruzi I isolates from South America were rather polymorphic in the DHFR-TS and mitochondrial genes. No genetic exchange was identified between T. cruzi I populations from Central and South America in the present study.

Strongyloides ratti: the role of interleukin-5 in protection against tissue migrating larvae and intestinal adult worms.

To determine the role of interleukin-5 (IL-5) and eosinophils in protection against Strongyloides ratti, mice treated with anti-IL-5 monoclonal antibody (mAb) were infected with S. ratti larvae. Strongyloides ratti egg numbers in faeces (EPG) in mAb treated mice were higher than those in control mice on days 6 and 7 after inoculation. The numbers of migrating worms in mAb treated mice 36 h after inoculation were higher than those observed in control mice. Intestinal worm numbers in mAb treated mice 5 days after inoculation were higher than those in control mice. These results show that eosinophils effectively protected the host against S. ratti infection by mainly the larval stage in primary infections. The involvement of eosinophils in protection against secondary infection was also examined. Before secondary infection, mice were treated with anti-IL-5 mAb and infected with S. ratti. Patent infections were not observed in either mAb treated or control Ab treated mice. The numbers of migrating worms in the head and lungs of mAb treated mice increased to 60% of that in primary infected mice. Intestinal worms were not found in mAb treated mice or in control mice after oral implantation of adult worms. Eosinophils were therefore mainly involved in protection against tissue migrating worms in secondary infections.

Nematocidal activities of thiabendazole and ivermectin against the larvae of Strongyloides ratti and S. venezuelensis.

With the aim of developing therapeutic agents for strongyloidosis, the disease caused by infection with Strongyloides stercoralis, we established a novel assay technique using S. ratti and S. venezuelensis as models for S. stercoralis. The newly developed assay technique was found to more accurately represent treatment-induced larval paralysis than existing assays. Our method uses paper disks impregnated with the test solution, which even allows materials that are sparingly soluble in water to be tested. An inverted microscope was used to observe the larval states, and these states were recorded using a digital camera. We observed the activities of ivermectin and thiabendazole against larvae and calculated larval motility and velocity. These two factors were then combined to determine the overall viability of larvae at selected concentrations. The activities of the anthelmintics were compared by calculating the concentrations at which 50% viability was demonstrated, or in other words, the concentration at which paralysis was caused in 50% of the individuals (50% paralysis concentration; PC(50)). Evaluations after 24h of exposure yielded the following reproducible PC(50) values for ivermectin and thiabendazole, respectively: S. ratti, 2.4 and 140 microM; and S. venezuelensis, 2.3 and 190 microM. After treatment with ivermectin, there was a tendency for larval motility to be greater than that of the controls at low concentrations, a result that might be associated with its mechanism of action.

Detection of kinetoplast DNA of Trypanosoma cruzi from dried feces of triatomine bugs by PCR.

It is important to clarify the distribution of infected triatomine bugs in the endemic area of Chagas' disease for proper control. In the present study, we tried to detect T. cruzi kinetoplast DNA by PCR from dried triatomine feces collected from the house wall of an endemic area to assess the distribution of infected bugs more easily. The primers (P35/P36) were chosen to amplify the conserved region within the minirepeats of T. cruzi kinetoplast minicircle DNA. The kinetoplast DNA of T. cruzi could be actually detected in the dried feces collected from the wall of a brick-built house in Santa Cruz, Bolivia. Next, we examined the stability of T. cruzi kinetoplast DNA in the feces exposed to artificial environments. T. cruzi DNA was also detected by PCR in the feces left for 26 weeks at 25 degrees C and in those left for 4 weeks at 40 degrees C. The present study indicates that examination of dried feces on the wall can be an effective tool for surveillance of the natural infection of triatomine bugs that live in houses.

IL-4 reduces resistance of mice to Trypanosoma cruzi infection.

The role of IL-4 has often been studied, especially in the Leishmania major infection model, but not in Trypanosoma cruzi infection. In the present study, the role of IL-4 in host defense against infection with the Tulahuen strain of T. cruzi was examined by depleting IL-4 with an anti-IL-4 monoclonal antibody in vivo. In both IL-4 depleted and control C57BL/6 mice, the parasitemia showed peaks on the 21st day of infection. Both parasitemia and mortality were decreased in IL-4 depleted mice compared with control mice when IFN-gamma and nitric oxide productions were increased in IL-4 depleted mice compared with control mice. The mice treated with N-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase, showed increased susceptibility to T. cruzi infection to the same level in both IL-4 depleted and control mice. Thus, it is suggested that endogenous IL-4 induces susceptibility to T. cruzi mainly by suppressing the production of IFN-gamma and nitric oxide, which has trypanocidal activity.

The effect of interleukin-4 on the induction of intestinal mast cells and chronological cytokine profiles during intestinal nematode Strongyloides ratti infection.

The effect of interleukin-4 (IL-4) on the induction of intestinal mast cells and cytokine profiles during Strongyloides ratti infection was studied using IL-4 knockout (IL-4 KO) mice. The antigen-specific proliferative response of mesenteric lymph node cells was not impaired in IL-4 KO mice. The number of intestinal mast cells induced in IL-4 KO mice during S. ratti infection was 2- to 3-fold lower than that observed in WT mice. Intestinal mastocytosis had disappeared in IL-4 KO mice by day 21 postinfection, when significant mastocytosis continued to be observed in WT mice. In mesenteric lymphnode of IL-4 KO, IL-3 production decreased and mice IFN-gamma production significantly increased as compared with those of WT mice. The numbers of eggs excreted per gram of feces (EPG) by IL-4 KO mice were greater than those excreted by WT mice on day 6 postinfection, but no difference was observed in the subsequent period. In conclusion, intestinal mast cells are induced during S. ratti infection in the absence of IL-4, and IL-4 is not essential for protection against intestinal adult worms of S. ratti.

Genetic structure of Trypanosoma cruzi in american continents: special emphasis on sexual reproduction in Central America.

Isozyme analysis (12 enzymes: 14 loci) was conducted on 99 isolates of Trypanosoma cruzi: 77 from Guatemala, 5 from Mexico and 17 from South American countries. Analyses of 4 population-genetic indices were undertaken to assess the possibility of genetic exchange occurring among Guatemalan isolates. The results provide evidence for a degree of genetic exchange occurring among isolates from this relatively small geographical area. Previous studies of population genetics on T. cruzi might have failed to detect this phenomenon because they tended to use isolates originating far from one another, rendering gene exchange unlikely for geographical reasons. Phylogenetic data, presented here, show considerable differences in genetic structure between Central and South American isolates, suggesting that different biological and clinical properties might be expected. For example, there are differences in clinical syndromes between Central and South America, a situation discussed further here.