Molecular population genetics and phylogeography of the Chagas disease vector Triatoma infestans in South America.

Knowledge of the genetic variability, population structure, and evolutionary history of Triatoma infestans may be useful for developing rational vector control strategies. A 661-bp fragment of the mitochondrial gene cytochrome oxidase I (COI) was sequenced and analyzed in bugs from Argentina, Uruguay, Peru, and Bolivia, including peridomestic, domestic, Andean, and Chaco sylvatic bugs. A total of 48 polymorphic sites among 37 haplotypes were described. Nucleotide variation fluctuated among samples, with the highest nucleotide diversity observed in seven Argentinean provinces. Within this group, some populations showed patterns of variability compatible with population expansions and/or fine-scale population structure, whereas others suggested population bottlenecks and/or population admixture processes. A maximum parsimony analysis of the haplotypes showed the presence of a Bolivian/Peruvian and an Argentinean/Uruguayan clade. Bolivian sequences were further divided in Chaco sylvatic and Andean domestic and sylvatic. Two different nested clades were found within the Argentinean/Uruguayan cluster. Analysis of molecular variance (AMOVA) and K(ST)* analysis supported a strong population structure in Argentina, where genetic differentiation was correlated with geographic distance. Departures from neutrality expectations and a nested cladistic analysis suggest a recent population expansion of T. infestans in Argentina, followed by restricted gene flow and patterns of isolation by distance. This expansion could have taken place as a two-wave process, as was shown by the phylogenetic analysis and signatures of population admixture in the southern most Argentinean populations.

Genetic structure of Triatoma infestans populations in rural communities of Santiago del Estero, northern Argentina.

To gain an understanding of the genetic structure and dispersal dynamics of Triatoma infestans populations, we analyzed the multilocus genotype of 10 microsatellite loci for 352 T. infestans collected in 21 houses of 11 rural communities in October 2002. Genetic structure was analyzed at the community and house compound levels. Analysis revealed that vector control actions affected the genetic structure of T. infestans populations. Bug populations from communities under sustained vector control (core area) were highly structured and genetic differentiation between neighboring house compounds was significant. In contrast, bug populations from communities with sporadic vector control actions were more homogeneous and lacked defined genetic clusters. Genetic differentiation between population pairs did not fit a model of isolation by distance at the microgeographical level. Evidence consistent with flight or walking bug dispersal was detected within and among communities, dispersal was more female-biased in the core area and results suggested that houses received immigrants from more than one source. Putative sources and mechanisms of re-infestation are described. These data may be use to design improved vector control strategies.

Impact of community-based vector control on house infestation and Trypanosoma cruzi infection in Triatoma infestans, dogs and cats in the Argentine Chaco.

The relative impact of two community-based vector control strategies on house infestation by Triatoma infestans and Trypanosoma cruzi infection in bugs, domestic dogs and cats was assessed in two neighboring rural areas comprising 40 small villages and 323 houses in one of the regions most endemic for Chagas disease in northern Argentina. The prevalence and abundance of domestic infestation were 1.5- and 6.5-fold higher, respectively, in the area under pulsed, non-supervised control actions operating under the guidelines of the National Vector Control Program (NCVP) than in the area under sustained, supervised surveillance carried out jointly by the UBA research team and NCVP. The prevalence of infestation and infection varied widely among village groups within each area. In the pulsed control area, the prevalence of infection in bugs, dogs and cats was two- to three-fold higher than in the area under sustained surveillance, most of the infected animals qualified as autochthonous cases, and evidence of recent transmission was observed. Infection was highly aggregated at the household level and fell close to the 80/20 rule. Using multiple logistic regression analysis clustered by household, infection in dogs was associated positively and significantly with variables reflecting local exposure to infected T. infestans, thus demonstrating weak performance of the vector surveillance system. For high-risk areas in the Gran Chaco region, interruption of vector-mediated domestic transmission of T. cruzi requires residual insecticide spraying that is more intense, of a higher quality and sustained in time, combined with community participation and environmental management measures.

Identification and characterization of microsatellite markers in the Chagas disease vector Triatoma infestans (Heteroptera: Reduviidae).

Triatoma infestans, the main vector of Chagas disease in the southern cone countries, is the principal target of a regional elimination program. A better understanding of its dispersal, sources of reinfestation, and insecticide resistance is key to an effective control program. To address such problems, we identified and characterized 13 microsatellite loci of T. infestans. For each locus, primer sequences and PCR conditions are presented. Allele variability and frequency were analyzed in 59 T. infestans specimens from different rural communities in northwestern Argentina; nine loci were considered suitable for population genetic studies. Departure from Hardy-Weinberg equilibrium was detected in 10/13 loci with F(IS) values ranging from 0.04 to 0.91, indicating heterozygote deficit and a possible grade of sub-structure in the sample analyzed. Presence of null alleles in some loci cannot be discarded. The present work provides a promising tool to develop a population genetic study of natural populations of T. infestans in tandem with field studies and analyses of bug dispersal and the reinfestation process.

Differential detection of Blastocrithidia triatomae and Trypanosoma cruzi by amplification of 24salpha ribosomal RNA genes in faeces of sylvatic triatomine species from rural northwestern Argentina.

Flagellates indistinguishable from Trypanosoma cruzi were detected by microscopy in faecal samples of 2/110 Triatoma guasayana and 2/283 Triatoma garciabesi captured in a rural area of northwestern Argentina. Inoculation of faecal homogenates to mice followed by xenodiagnosis, haemoculture, histopathology and culture from cardiac homogenates, and PCR based on T. cruzi minicircle and nuclear sequences failed to detect T. cruzi infection, pointing to another trypanosomatidean. A PCR strategy targeted to the D7 domain of 24salpha ribosomal DNA genes amplified a 250 bp sequence from one T. guasayana and one T. garciabesi faecal lysate. Sequence analysis revealed 100% identity with 24salpha rDNA amplicons from Blastocrithidia triatomae obtained from faeces of reared Triatoma infestans bugs. Phylogenetic analysis clustered this sequence with C. fasciculata and L. major, separated from the Trypanosoma branch (bootstrap: 968/1000), in concordance with a Neighbour-joining dendrogram based on 18s rDNA sequences. This PCR procedure provides a rapid sensitive tool for differential diagnosis of morphologically similar trypanosomatids in field surveys of Chagas disease vectors and laboratory-reared triatomines used for xenodiagnosis.

Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 225 free-range chickens (Gallus domesticus) from Portugal was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 61 chickens with titers of 1:5 in 8, 1:10 in 6, 1:20 in 3, 1:40 in 23, 1:80 in 5, 1:160 in 4, 1:320 in 8, and 1:640 or higher in 4. Hearts, leg muscles, and brains of 15 seropositive (MAT 1:10 or higher) chickens were bioassayed individually in mice. Tissue from 38 chickens with titers of 1:5 or less were pooled and fed to a T. gondii-free cat. Feces of the cat were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 16 of 19 chickens with MAT titers of 1:10 or higher. Genotyping of 12 of these 16 isolates with polymorphisms at the SAG2 locus indicated that 4 were type III, and 8 were type II. None of the isolates was lethal for mice. Phenotypically, T. gondii isolates from chickens from Portugal were different from those of T. gondii isolates from chickens from Brazil.

Feeding rates, nutritional status and flight dispersal potential of peridomestic populations of Triatomainfestans in rural northwestern Argentina.

Triatoma infestans, the main vector of Chagas disease in Southern Cone countries, frequently infests peridomestic structures housing domestic animals. A total of 814 T. infestans collected from 35 different peridomestic sites in rural northwestern Argentina over 1-4 consecutive seasons was examined for recent blood meals and nutritional status. Bugs from goat or pig corrals had lower qualitative nutritional status and mean weight to length ratios (W/L) than those captured in chicken coops. Males systematically had lower qualitative nutritional status and W/L than females. Using logistic multiple regression, the daily feeding rates of T. infestans were significantly associated with season and stage but not ecotope, whereas the proportion of well-fed bugs varied significantly with all three factors. The seasonal trends in feeding rates and nutritional status were consistent with the local availability and breeding timing of domestic animals. The observed data fed into an empirical model predicted that the probability of flight initiation would peak in summer from pig or goat corrals, not chicken coops, and be insignificant in all ecotopes in spring and fall. Male T. infestans outnumbered females as potential fliers. This is the first study conducted in well-defined habitat units that shows significant heterogeneities in the feeding rates and nutritional status of triatomine populations linked to host demographics and management, and how these affect flight dispersal potential over seasons. Peridomestic bug populations are of great relevance as a source of domestic reinfestation and for the elimination of T. infestans.

Genetic and biologic characteristics of Toxoplasma gondii infections in free-range chickens from Austria.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in free-range chickens (Gallus domesticus) from 11 Bio-farms in Austria was determined. Antibodies to T. gondii assayed by the modified agglutination test (MAT) were found in 302 of 830 (36.3%) chickens with titers of 1:10 in 50, 1:20 in 69, 1:40 in 53, 1:80 in 40, 1:160 or higher in 90. Hearts of 218 chickens with MAT titers of 10 or higher were bioassayed individually in mice. Tissues from 1183 chickens were pooled and fed to 15, T. gondii-free cats. Feces of the cats were examined for oocysts; 11 cats shed T. gondii oocysts. T. gondii was isolated from 56 chickens by bioassay in mice. Thus, there were 67 isolates of T. gondii from these chickens. Genotyping of these 67 isolates using the SAG2 locus indicated that all 33 were Type II. Phenotypically and genetically these isolates were different from T. gondii isolates from Brazil. None of the isolates was virulent for mice. This is the first report of isolation of T. gondii from chickens from Austria.

Genetic and biologic characteristics of Toxoplasma gondii isolates in free-range chickens from Colombia, South America.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 77 free-range chickens (Gallus domesticus) from Colombia, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 32 (44.4%) of 72 chickens with titers of 1:5 in 4, 1:10 in 3, 1:20 in 1, 1:40 in 1, 1:80 in 8, 1:160 in 8, 1:320 in 3, and 1:640 or higher in 4. Hearts and brains of 31 seropositive chickens were pooled and bioassayed in mice. Tissues from 32 (16+16) seronegative chickens were pooled and fed to two, T. gondii-free cats, and tissues from nine chickens without matching sera were fed to one T. gondii-free cat. Feces of cats were examined for oocysts. T. gondii oocysts were excreted by a cat that was fed tissues of 16 seronegative chickens. T. gondii was isolated by bioassay in mice from 23 chickens with MAT titers of 1:20 or higher. All infected mice from 16 of the 23 isolates died of toxoplasmosis. Overall, 82 (81.1%) of 101 mice that became infected after inoculation with chicken tissues died of toxoplasmosis. Genotyping of these 24 isolates using polymorphisms at the SAG2 locus indicated that seven T. gondii isolates were Type I, 17 were Type III, and none was Type II. Phenotypically, T. gondii isolates from chickens from Colombia were similar to isolates from Brazil but different from the isolates from North America; most isolates from chickens from Brazil and Colombia were lethal for mice whereas isolates from North America did not kill inoculated mice. Genetically, none of the T. gondii isolates from Colombia and Brazil was SAG2 Type II, whereas most isolates from chickens from North America were Type II. This is the first report of genetic characterization of T. gondii isolates from Colombia, South America.

Toxoplasma gondii infections in chickens from Venezuela: isolation, tissue distribution, and molecular characterization.

The prevalence of Toxoplasma gondii, in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 46 free-range chickens (Gallus domesticus) from Venezuela was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 16 (32%) chickens with titers of 1:5 in 1, 1:10 in 2, 1:40 in 2, 1:80 in 2, 1:160 in 2, 1:320 in 3, 1: 640 in 2, and 1:1,280 or higher in 2. Hearts, pectoral muscles, and brains of 13 chickens with MAT titers of 1:40 or more were bioassayed individually in mice. Tissues of each of 3 chickens with titers of 1:5 or 1:10 were pooled and bioassayed in mice. Tissues from the remaining 30 seronegative chickens were pooled and fed to 1 T. gondii-free cat. Feces of the cat were examined for oocysts; it did not shed oocysts. Toxoplasma gondii was isolated from 12 of 13 chickens with MAT titers of 1:40 or more. Toxoplasma gondii was isolated from pooled tissues of 1 of 2 chickens with titers of 1:10. Eight of these 13 isolates were virulent for mice. Genotyping of 13 of these isolates using the SAG2 locus indicated that 10 were type III, and 3 were type II. Phenotypically and genetically these isolates were different from T. gondii isolates from North America and Brazil. This is the first report of isolation of T. gondii from chickens from Venezuela.

Prevalence of viable Toxoplasma gondii in beef, chicken, and pork from retail meat stores in the United States: risk assessment to consumers.

The prevalence of viable Toxoplasma gondii was determined in 6,282 samples (2,094 each of beef, chicken, and pork) obtained from 698 retail meat stores from 28 major geographic areas of the United States. Each sample consisted of a minimum of 1 kg of meat purchased from the retail meat case. To detect viable T. gondii, meat samples were fed to T. gondii-free cats and feces of cats were examined for oocyst shedding. Initially, 100 g of meat from 6 individual samples of a given species were pooled (total, 600 g), fed to a cat over a period of 3 days, and feces were examined for oocysts for 14 days; the remaining meat samples were stored at 4 C for 14 days (until results of the initial cat fecal examination were known). When a cat fed pooled samples had shed oocysts, 6 individual meat samples from each pool were bioassayed for T. gondii in cats and mice. Toxoplasma gondii isolates were then genetically characterized using the SAG2 locus and 5 hypervariable microsatellite loci. In all, 7 cats fed pooled pork samples shed oocysts. Toxoplasma gondii oocysts were detected microscopically in the feces of 2 of the cats; 1 isolate was Type II and the second was Type III. Analyzed individually, T. gondii was detected by bioassay in 3 of the 12 associated samples with genetic data indicating T. gondii isolates present in 2. The remaining 5 pooled pork samples had so few oocysts that they were not initially detected by microscopic examination, but rather by mouse bioassay of cat feces. Two were Type I, 1 was Type II, and 2 were Type III. None of the cats fed chicken or beef samples shed oocysts. Overall, the prevalence of viable T. gondii in retail meat was very low. Nevertheless, consumers, especially pregnant women, should be aware that they can acquire T. gondii infection from ingestion of undercooked meat, and in particular, pork. Cooking meat to an internal temperature of 66 C kills T. gondii.

Zn adsorption by different fractions of Galician soils.

To evaluate the contribution of organic matter, oxides, and clay fraction to Zn adsorption in six soils from Galicia (Spain), after soil characterization, adsorption isotherms were obtained by adding nine solutions containing between 20 and 500 mg L(-1) concentrations of Zn(NO(3))(2). Distribution coefficients were obtained from the data of adsorption isotherms. Zn adsorption isotherms corresponding to untreated soil and to the organic matter removed samples and organic matter and oxides removed samples were compared with curves pattern and adjusted to Langmuir and Freundlich empirical models. Untreated soils described L-curves whereas when soils were deprived of any component, the curves described were S-type. Distribution coefficients allowed knowing the Zn adsorption capacity of the untreated soil, and of the organic matter, oxides, and clay fraction. Soil organic matter is the main component that affects Zn adsorption as long as soil pH is near neutrality. At acid pH, the oxides are the main component that affects Zn adsorption, although to a much smaller extent than the organic matter near neutral conditions. So soil pH is the main soil factor that determines Zn adsorption, before any other soil property.

Effect of the Prestige oil spill on salt marsh soils on the coast of Galicia (northwestern Spain).

At four estuarine sites on the coast of Galicia (northwestern Spain), all of which were affected by the Prestige oil spill, soil samples were taken from polluted and unpolluted areas and their petroleum hydrocarbon contents, heavy metal contents, and other chemical and physical characteristics were measured. Oil pollution altered both chemical and physical soil properties, aggregating soil particles in plaques, lowering porosity, and increasing resistance to penetration and hydrophobicity. The chromium, nickel, copper, iron, lead, and vanadium contents of polluted soils were between 2 and 2500 times higher than those of their unpolluted counterparts and the background concentrations in Galician coastal sediments. In the cases of Cr, Cu, Ni, Pb, and V, their origin in the polluting oil was corroborated by the high correlation (r >/= 0.74) between the concentrations of these metals and the total petroleum hydrocarbon (TPH) content of the polluted soils. Soil redox potentials ranged from -19 to -114 mV in polluted soils and 112 to 164 mV in unpolluted soils, and were negatively correlated with TPH content (p < 0.01). The low values in the polluted soils explain why the soluble fractions of their total heavy metal contents were very small (generally less than 3%, and in many cases undetectable).

Genetic variability, phylogenetic relationships and gene flow in Triatoma infestans dark morphs from the Argentinean Chaco.

The recent discovery of sylvatic populations of Triatoma infestans outside the Andean Valleys of Bolivia prompted an evolutionary question about the putative ancestral area of origin and dispersal of the species, and an epidemiological question regarding the possible role of these sylvatic populations in the recolonization process of insecticide-treated houses. The finding of a population of sylvatic melanic T. infestans (dark morphs) in the Argentinean dry Chaco at 7 km from a peridomestic bug population of typical coloration gave us the opportunity to test both questions simultaneously by employing phylogenetic and population genetic approaches. For this purpose we analyzed sylvatic and peridomestic bugs using sequence-based mitochondrial and nuclear markers (mtCOI and ITS-1) and microsatellites. Sylvatic bugs were confirmed to be T. infestans and not hybrids, and showed high levels of genetic variability and departures from neutral expectations for mtCOI variation. New ITS-1 and mtCOI haplotypes were recorded, as well as haplotypes shared with peridomestic and/or domestic bugs from previous records. The peridomestic population was invariant for ITS-1 and mtCOI, but showed variability for microsatellites and signatures of a population bottleneck, probably due to a limited number of founders. Phylogenetic analyses were consistent with the presence of ancestral haplotypes in sylvatic bugs. According to F-statistics and assignment methods there was a significant differentiation between sylvatic and peridomestic bugs and gene flow was low and asymmetric, with more bugs moving from the peridomicile to the sylvatic environment. These results support the hypothesis of the Chaco region as the area of origin of T. infestans, and a limited role of sylvatic melanic T. infestans in peridomestic infestation in the Argentinean Chaco.

Seasonal variations in active dispersal of natural populations of Triatoma infestans in rural north-western Argentina.

The flight dispersal of Triatoma infestans Klug (Hemiptera: Reduviidae) is one of the main mechanisms determining community re-infestation after control interventions. An empirical model of flight initiation coupled with data from a longitudinal study predicted that the flight dispersal of T. infestans would peak in summer. To test this prediction, longitudinal light trap collections were conducted during 3-8 nights in March (late summer), July (winter) and November (spring) 2003, and in March 2004 in a rural community in north-west Argentina. Following each light-trapping collection date, all peridomestic sites around light traps were inspected to assess the relative abundance and nutritional status of T. infestans at each site. A total of 21 adult and five nymph T. infestans, six Triatoma guasayana Wygodzinsky & Abalos, and nine Triatoma garciabesi Carcavallo et al. were collected in 96 light-trapping nights, whereas 696 T. infestans were collected from the peridomestic sites that surrounded the light traps. The arrival of T. infestans in the light traps occurred in 64% of catch stations and peaked in the summer surveys (10-14 bugs) compared with spring and winter surveys. When winds were < 5 km/h, the arrival of adult T. infestans at the light traps was significantly associated with maximum temperature and relative humidity. This is the first field report of seasonal variations in the flight dispersal activity of T. infestans.

PCR-based screening and lineage identification of Trypanosoma cruzi directly from faecal samples of triatomine bugs from northwestern Argentina.

This study applied improved DNA extraction and polymerase chain reaction strategies for screening and identification of Trypanosoma cruzi lineages directly from faeces of triatomines collected in a well-defined rural area in northwestern Argentina. Amplification of the variable regions of the kinetoplastid minicircle genome (kDNA-PCR) was performed in faecal lysates from 33 microscope (MO)-positive and 93 MO-negative Triatoma infestans, 2 MO-positive and 38 MO-negative Triatoma guasayana and 2 MO-positive and 73 MO-negative Triatoma garciabesi. kDNA-PCR detected T. cruzi in 91% MO-positive and 7.5% MO-negative T. infestans, which were confirmed by amplification of the minicircle conserved region. In contrast, kDNA-PCR was negative in all faecal samples from the other triatomine species. A panel of PCR-based genomic markers (intergenic region of spliced-leader DNA, 24Salpha and 18S rRNA genes and A10 sequence) was implemented to identify the parasite lineages directly in DNA lysates from faeces and culture isolates from 28 infected specimens. Two were found to be infected with TCI, 24 with TCIIe, 1 with TCIId and 1 revealed a mixed TCI+TCII infection in the faecal sample whose corresponding culture only showed TCII, providing evidence of the advantages of direct typing of biological samples. This study provides an upgrade in the current diagnosis and lineage identification of T. cruzi in field-collected triatomines and shows T. cruziII strains as predominant in the region.

Study of the refirming effect of a plant complex.

Loss of skin elasticity is one of the main problems of ageing. This is a mechanical property influenced by elastin, a protein in the dermis which, together with collagen and glycosaminoglycans, makes up the connective tissue. This tissue is affected by a large number of events (such as cutaneous ageing, pregnancy, slimming processes and cellulitis) which eventually cause it to change. At the same time, the metabolism of the proteins of the connective tissue decreases and there is an ever greater presence of enzymes, principally elastases and collagenases, which are responsible for breaking down the elastin and the collagen. One way to prevent such a loss of elasticity is to use active ingredients that are able to inhibit elastase enzymes. A plant complex was prepared using the following plants: lady's thistle (Silybum marianum GAERTN), alchemilla or yarrow (Alchemilla vulgaris L.), horsetail (Equisetum arvense L.) as well as germinated seeds (Glycine soja Siebold and Zucc., Triticum vulgare Vilars, Medicago sativa L., Raphanus sativus L.). The complex was standardized to give the corresponding active principles, silybin, tannins, silicon and peptides, respectively, and in vitro enzymatic tests were carried out to establish its ability to inhibit elastase. The study of enzymatic inhibition was carried out using two enzymes: (1) porcine pancreatic elastase (PPE), and (2) human leukocyte elastase (HLE). The results showed that the plant complex presents non-competitive inhibition in the order of 41.0% against PPE and 50.0% against HLE. An in vivo test was made alongside the in vitro test using an SEM 474 Cutometer (Courage & Khazaka) to study the elasticity of the skin, and positive effects were obtained when applying a cosmetic formulation containing 5% of the plant complex. Image analysis of duplicates of the cutaneous surface, before and after treatment began with a product containing 5% of plant complex and showed that wrinkles were decreased by 36.7%.