A Study of Cryptosporidium parvum Genotypes and Population Structure

Genetic evidence for the occurrence of two Cryptosporidium parvum subgroups is presented. This evidence is based on restriction fragment length polymorphism analysis of several independent loci. Sequence analysis of the b -tubulin intron revealed additional polymorphism. The stability of the genetic profiles following passage of C. parvum isolates between different hosts was investigated.

Genetic structure of Triatoma venosa (Hemiptera: Reduviidae): molecular and morphometric evidence

Triatoma venosa presents a restricted geographical distribution in America and is considered as a secondary vector of Chagas disease in Colombia and Ecuador. A total of 120 adult insects were collected in domestic and peridomestic habitats in an endemic area of the department of Boyacá, Colombia, in order to determine their genetic structure through morphometric and molecular techniques. The head and wings of each specimen were used for the analyses of size, shape, and sexual dimorphism. A significant sexual dimorphism was found, although no differences in size among the studied groups were detected. Differences were found in the analyzed structures except for male heads. DNA was extracted from the legs in order to carry out the internal transcriber space-2 (ITS-2) amplification and the randon amplified polymorphic DNA (RAPD) analyses. Length polymorphisms were not detected in the ITS-2. Fst and Nm values were estimated (0.047 and 3.4, respectively). The high genetic flow found among the insects captured in the domicile and peridomiciliary environment does not permit a genetic differentiation, thus establishing the peridomicile as an important place for epidemiological surveillance.

Genetic variability of Aedes aegypti in the Americas using a mitochondrial gene: evidence of multiple introductions

To analyze the genetic relatedness and phylogeographic structure of Aedes aegypti, we collected samples from 36 localities throughout the Americas (Brazil, Peru, Venezuela, Guatemala, US), three from Africa (Guinea, Senegal, Uganda), and three from Asia (Singapore, Cambodia, Tahiti). Amplification and sequencing of a fragment of the mitochondrial NADH dehydrogenase subunit 4 gene identified 20 distinct haplotypes, of which 14 are exclusive to the Americas, four to African/Asian countries, one is common to the Americas and Africa, and one to the Americas and Asia. Nested clade analysis (NCA), pairwise distribution, statistical parsimony, and maximum parsimony analyses were used to infer evolutionary and historic processes, and to estimate phylogenetic relationships among haplotypes. Two clusters were found in all the analyses. Haplotypes clustered in the two clades were separated by eight mutational steps. Phylogeographic structure detected by the NCA was consistent with distant colonization within one clade and fragmentation followed by range expansion via long distance dispersal in the other. Three percent of nucleotide divergence between these two clades is suggestive of a gene pool division that may support the hypothesis of occurrence of two subspecies of Ae. aegypti in the Americas.

No evidence of association of MUC-1 genetic polymorphism with embryo implantation failure

Pregnancy loss can be caused by several factors involved in human reproduction. Although up to 50% of cases remain unexplained, it has been postulated that the major cause of failed pregnancy is an error of embryo implantation. Transmembrane mucin-1 (MUC-1) is a glycoprotein expressed on the endometrial cell surface which acts as a barrier to implantation. The gene that codes for this molecule is composed of a polymorphic tandem repeat of 60 nucleotides. Our objective was to determine if MUC-1 genetic polymorphism is associated with implantation failure in patients with a history of recurrent abortion. The study was conducted on 10 women aged 25 to 35 years with no history of successful pregnancy and with a diagnosis of infertility. The control group consisted of 32 patients aged 25 to 35 years who had delivered at least two full-term live children and who had no history of abortions or fetal losses. MUC-1 amplicons were obtained by PCR and observed on agarose and polyacrylamide gel after electrophoresis. Statistical analysis showed no significant difference in the number of MUC-1 variable number of tandem repeats between these groups (P > 0.05). Our results suggest that there is no effect of the polymorphic MUC-1 sequence on the implantation failure. However, the data do not exclude MUC-1 relevance during embryo implantation. The process is related to several associated factors such as the mechanisms of gene expression in the uterus, specific MUC-1 post-translational modifications and appropriate interactions with other molecules during embryo implantation.

Molecular evidence of mother-to-child transmission of HTLV-IIc in the Kararao Village (Kayapo) in the Amazon Region of Brazil

Blood samples from native Indians in the Kararao village (Kayapo), were analysed using serological and molecular methods to characterize infection and analyse transmission of HTLV-II. Specific reactivity was observed in 3/26 individuals, of which two samples were from a mother and child. RFLP analysis of the pX and env regions confirmed HTLV-II infection. Nucleotide sequence of the 5' LTR segment and phylogenetic analysis showed a high similarity (98%) between the three samples and prototype HTLV-IIa (Mot), and confirmed the occurrence of the HTLV-IIc subtype. There was a high genetic similarity (99.9%) between the mother and child samples and the only difference was a deletion of two nucleotides (TC) in the mother sequence. Previous epidemiological studies among native Indians from Brazil have provided evidence of intrafamilial and vertical transmission of HTLV-IIc. The present study now provides molecular evidence of mother-to-child transmission of HTLV-IIc, a mechanism that is in large part responsible for the endemicity of HTLV in these relatively closed populations. Although the actual route of transmission is unknown, breast feeding would appear to be most likely.

Geographic pattern of genetic diversity in natural populations of Rosewood (Aniba rosaeodora), in the Central Amazonia

Rosewood (Aniba rosaeodora Ducke, Lauraceae) is an Amazonian evergreen tree and a source of the purest linalool, the main component of its essential oil, which is very valuable in the international perfumery market. After decades of over-exploitation it is currently considered as threatened. We evaluated the genetic diversity and its distribution in four populations in Central Amazonia. Thirty-five reliable RAPD markers were generated, of which 32 were polymorphic (91.4%). Variation was higher within the populations (76.5%; p < 0.0001) and geographic distribution contributed to population differentiation (23.4%; p < 0.0001). The Amazon River had a small influence on gene flow (3.3%; p < 0.0001), but we identified evidence of gene flow across the river. There were significant differences in marker frequencies (p < 0.05), in agreement with the low gene flow (Nm = 2.02). The correlation between genetic distance and gene flow was - 0.95 (p = 0.06) and between geographic distance and gene flow was -0.78 (p = 0.12). There was a geographic cline of variability across an East-West axis, influenced as well by the Amazon River, suggesting the river could be a barrier to gene flow. Although threatened, these Rosewood populations retain high diversity, with the highest levels in the Manaus population, which has been protected for over 42 years in a Reserve.

Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group 1/2. These results suggest different origins for these strains.

Genetic variability analysis among clinical Candida spp. isolates using random amplified polymorphic DNA

The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV). These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.

Biological and genetic aspects of experimental hybrids from species of the Phyllosoma complex (Hemiptera: Reduviidae: Triatominae)

The present work is a thorough investigation of the degree of reproductive isolation between Meccus mazzottii and Meccus longipennis, Meccus picturatus, Meccus pallidipennis and Meccus bassolsae, as well as between M. longipennis and M. picturatus. We examined fertility and segregation of morphological characteristics in two generations of hybrids derived from crosses between these species. The percentage of pairs with (fertile) offspring was highest in the set of crosses between M. longipennis and M. picturatus, and lowest between M. mazzottii and M. picturatus. Most first-generation (F1) individuals from crosses involving M. mazzottii were morphologically similar to this species, while only F1 x F1 progeny of parental crosses between M. mazzottii and M. longipennis had offspring second generation that looked like M. mazzottii. The results indicate that different degrees of reproductive isolation apparently exist among the species of the Phyllosoma complex examined in this study. The biological evidence obtained in this study does not support the proposal that M. longipennis and M. picturatus are full species. It could indicate on the contrary, that both could be considered as subspecies of a single polytypic species. On the other hand, biological evidence supports the proposal that M. mazzottii is a full species.

Candidate gene analysis of ocular toxoplasmosis in Brazil: evidence for a role for toll-like receptor 9 (TLR9)

Toxoplasma gondii infection is an important mediator of ocular disease in Brazil more frequently than reported from elsewhere. Infection and pathology are characterized by a strong proinflammatory response which in mice is triggered by interaction of the parasite with the toll-like receptor (TLR)/MyD88 pathway. A powerful way to identify the role of TLRs in humans is to determine whether polymorphisms at these loci influence susceptibility to T. gondii-mediated pathologies. Here we report on a small family-based study (60 families; 68 affected offspring) undertaken in Brazil which was powered for large effect sizes using single nucleotide polymorphisms with minor alleles frequencies > 0.3. Of markers in TLR2, TLR5 and TLR9 that met these criteria, we found an association Family Based Association Tests [(FBAT) Z score = 4.232; p = 1.5 x 10-5; p corrected = 1.2 x 10-4] between the C allele (frequency = 0.424; odds ratio = 7; 95% confidence interval 1.6-30.8) of rs352140 at TLR9 and toxoplasmic retinochoroiditis in Brazil. This supports the hypothesis that direct interaction between T. gondii and TLR9 may trigger proinflammatory responses that lead to severe pathologies such as the ocular disease that is associated with this infection in Brazil.

Genetic and biological characterization of a densovirus isolate that affects dengue virus infection

Brevidensoviruses have an encapsidated, single-stranded DNA genome that predominantly has a negative polarity. In recent years, they have received particular attention due to their potential role in the biological control of pathogenic arboviruses and to their unnoticed presence in cell cultures as contaminants. In addition, brevidensoviruses may also be useful as viral vectors. This study describes the first genetic and biological characterization of a mosquito densovirus that was isolated in Brazil; moreover, we examined the phylogenetic relationship between this isolate and the other brevidensoviruses. We further demonstrate that this densovirus has the potential to be used to biologically control dengue virus (DENV) infection with in vitro co-infection experiments. The present study provides evidence that this densovirus isolate is a fast-spreading virus that affects cell growth and DENV infection.

Biological and genetic aspects of crosses between species of the genus Meccus (Hemiptera: Reduviidae Triatominae)

The degree of reproductive isolation between Meccus phyllosomus and the remaining five species of the genus Meccus, as well as between Meccus bassolsae and Meccus pallidipennis, Meccus longipennis and Meccus picturatus, was examined. Fertility and the segregation of morphological characteristics were examined in two generations of hybrids from crosses between these species. The percentage of couples with offspring (fertile) was high in the vast majority of sets of crosses, with the exception of that between ♀M. phyllosomus and ♂Meccus mazzottii. In sets of crosses involving M. bassolsae specimens, no first-generation (F1) individuals were morphologically similar to M. bassolsae, but instead shared the morphology of the other parental species. A similar phenomenon was observed in most sets of crosses involving M. phyllosomus. These results indicated that different degrees of reproductive isolation exist among the species of Meccus involved in this study. The biological evidence obtained in this study does not support the proposal that M. bassolsae is a full species. It could indicate that, on the contrary, it should be considered a subspecies of a single polytypic species. The biological evidence does support the proposal that M. phyllosomus is a full species.

Genetic polymorphisms of NAT2, CYP2E1 and GST enzymes and the occurrence of antituberculosis drug-induced hepatitis in Brazilian TB patients

Isoniazid (INH), one of the most important drugs used in antituberculosis (anti-TB) treatment, is also the major drug involved in hepatotoxicity. Differences in INH-induced toxicity have been attributed to genetic variability at several loci, such as NAT2, CYP2E1, GSTM1 and GSTT1, that code for drug-metabolising enzymes. Our goal was to examine the polymorphisms in these enzymes as susceptibility factors to anti-TB drug-induced hepatitis in Brazilian individuals. In a case-control design, 167 unrelated active tuberculosis patients from the University Hospital of the Federal University of Rio de Janeiro, Brazil, were enrolled in this study. Patients with a history of anti-TB drug-induced acute hepatitis (cases with an increase to 3 times the upper limit of normal serum transaminases and symptoms of hepatitis) and patients with no evidence of anti-TB hepatic side effects (controls) were genotyped for NAT2, CYP2E1, GSTM1 and GSTT1 polymorphisms. Slow acetylators had a higher incidence of hepatitis than intermediate/rapid acetylators [22% (18/82) vs. 9.8% (6/61), odds ratio (OR), 2.86, 95% confidence interval (CI), 1.06-7.68, p = 0.04). Logistic regression showed that slow acetylation status was the only independent risk factor (OR 3.59, 95% CI, 2.53-4.64, p = 0.02) for the occurrence of anti-TB drug-induced hepatitis during anti-TB treatment with INH-containing schemes in Brazilian individuals.

Molecular evidence for a single taxon, Anopheles nuneztovari s.l., from two endemic malaria regions in Colombia

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F ST) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study.

The relationship between genetic variability and the susceptibility of Biomphalaria alexandrina snails to Schistosoma mansoni infection

In the present study, Biomphalaria snails collected from five Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta), as well as reference control Biomphalaria alexandrina snails from the Schistosome Biological Supply Center (SBSC) (Theodor Bilharz Research Institute, Egypt), were subjected to species-specific polymerase chain reaction (PCR) assays to identify the collected species. All of the collected snails were found to be B. alexandrina and there was no evidence of the presence of Biomphalaria glabrata. Randomly amplified polymorphic DNA (RAPD)-PCR assays showed different fingerprints with varying numbers of bands for the first generation (F1) of B. alexandrina snail populations (SBSC, Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta). The primer OPA-1 produced the highest level of polymorphism and amplified the greatest number of specific bands. The estimated similarity coefficients among the B. alexandrina populations based on the RAPD-PCR profiles ranged from 0.56 (between SBSC and Ismailia snails) to 0.72 (between Ismailia and Kafr El-Sheikh snails). Experimental infection of the F1 of progeny from the collected snails with Schistosoma mansoni (SBSC strain) showed variable susceptibility rates ranging from 15% in the Fayoum snail group to 50.3% in SBSC snails. A negative correlation was observed between the infection rates in the different snail groups and the distances separating their corresponding governorates from the parasite source. The infection rates of the snail groups and their similarity coefficients with SBSC B. alexandrina snails were positively correlated. The variations in the rates of infection of different B. alexandrina groups with S. mansoni, as well as the differences in the similarity coefficients among these snails, are dependent not only on the geographical distribution of the snails and the parasite, but also on the genetic variability of the snails. Introduction of this variability into endemic areas may reduce the ability of the parasite to infect local hosts and consequently reduce schistosomiasis epidemiology.