Hemi-Nested PCR and RFLP Methodologies for Identifying Blood Meals of the Chagas Disease Vector, <i>Triatoma infestans</i>

<div><p><i>Trypanosoma cruzi</i>, the etiologic agent of Chagas disease, is transmitted by hematophagous reduviid bugs within the subfamily Triatominae. These vectors take blood meals from a wide range of hosts, and their feeding behaviors have been used to investigate the ecology and epidemiology of <i>T. cruzi</i>. In this study we describe two PCR-based methodologies that amplify a fragment of the 16S mitochondrial rDNA, aimed to improve the identification of blood meal sources for <i>Triatoma infestans</i>: a.- Sequence analyses of two heminested PCRs that allow the identification of mammalian and avian species, and b.- restriction fragment length polymorphism (RFLP) analysis from the mammalian PCR to identify and differentiate multi-host blood meals. Findings from both methodologies indicate that host DNA could be detected and the host species identified in samples from laboratory reared and field collected triatomines. The implications of this study are two-fold. First, these methods can be used in areas where the fauna diversity and feeding behavior of the triatomines are unknown. Secondly, the RFLP method led to the identification of multi-host DNA from <i>T. infestans</i> gut contents, enhancing the information provided by this assay. These tools are important contributions for ecological and epidemiological studies of vector-borne diseases.</p></div

Seroprevalencia de la infección por Trypanosoma cruzi y factores asociados en población adulta en una zona de alta endemicidad de Arequipa, Perú

Objective: determine the seroprevalence of infection by T. cruzi in the population above age 15 in the valley of Vítor, describing general characteristics and the presence of associated factors. Materials and Methods: 499 people above age 15 were randomly selected; epidemiological information was obtained through a survey. The diagnosis of the disease was made by ELISA and IIF. Results: the seroprevalence for Chagas reactivity was 10,2%, 10,35% in women and 10,0% in men. The median of age of the "typical" Chagas disease patient was 42±17,9, his/her time of residence in Vítor was 34±15,9 years; 51,0% had been born in the valley of Vítor. Agricultural workers made up 41,2%, of the general population, and 51,0% have a high school education. Rustic housing materials were associated with the infection. Conclusions: the valley of Vítor is an endemic area for Chagas disease. The infection equally affects men and women. The "typical" infected patient is native to the valley of Vítor, works in agriculture and has secondary level education. Rustic building materials is associated to the infection by T.cruzi because it favors the presence of the vector.Objetivo: determinar la seroprevalencia de infección por T. cruzi en pobladores mayores de 15 años del valle de Vítor, describiendo características generales y presencia de factores asociados a la infección. Material y Métodos: se seleccionaron aleatoriamente a 499 personas mayores de 15 años de edad, mediante encuesta se obtuvo la respectiva información epidemiológica. El diagnóstico serológico de la enfermedad se realizó mediante ELISA e IFI. Resultados: la seroprevalencia fue de 10,22%. En mujeres 10,35% y varones 10,04%. La mediana de edad del paciente chagásico fue de 42±17,86 años el tiempo de residencia fue de 34±15,95 años. El 50,98% es natural del valle de Vítor. Según la ocupación de la población general, agricultor 41,18%, ama de casa 31,37%. El 50,98% tiene instrucción secundaria. El único factor asociado a la infección fue el material rústico de las vivienda. Conclusion: el valle de Vítor es zona chagásica endémica. La infección afecta tanto a hombres como a mujeres. El poblador infectado es natural del valle de Vítor, se dedica a la agricultura y tiene secundaria como grado de instrucción. El material rústico de las viviendas se asocia a la infección por T.cruzi al favorecer la presencia del vector

Dispersal patterns of Trypanosoma cruzi in Arequipa, Peru.

Anthropogenic environmental alterations such as urbanization can threaten native populations as well as create novel environments that allow human pests and pathogens to thrive. As the number and size of urban environments increase globally, it is more important than ever to understand the dispersal dynamics of hosts, vectors and pathogens of zoonotic disease systems. For example, a protozoan parasite and the causative agent of Chagas disease in humans, Trypanosoma cruzi, recently colonized and spread through the city of Arequipa, Peru. We used population genomic and phylogenomic tools to analyze whole genomes of 123 T. cruzi isolates derived from vectors and non-human mammals throughout Arequipa to determine patterns of T. cruzi dispersal. The data show significant population genetic structure within city blocks-parasites in the same block tend to be very closely related-but no population structure among blocks within districts-parasites in neighboring blocks are no more closely related to one another than to parasites in distant districts. These data suggest that T. cruzi dispersal within a block occurs regularly and that occasional long-range dispersal events allow the establishment of new T. cruzi populations in distant blocks. Movement of domestic animals may be the primary mechanism of inter-block and inter-district T. cruzi dispersal

Immigration and establishment of Trypanosoma cruzi in Arequipa, Peru.

Changing environmental conditions, including those caused by human activities, reshape biological communities through both loss of native species and establishment of non-native species in the altered habitats. Dynamic interactions with the abiotic environment impact both immigration and initial establishment of non-native species into these altered habitats. The repeated emergence of disease systems in urban areas worldwide highlights the importance of understanding how dynamic migratory processes affect the current and future distribution and abundance of pathogens in urban environments. In this study, we examine the pattern of invasion of Trypanosoma cruzi-the causative agent of human Chagas disease-in the city of Arequipa, Peru. Phylogenetic analyses of 136 T. cruzi isolates from Arequipa and other South American locations suggest that only one T. cruzi lineage established a population in Arequipa as all T. cruzi isolated from vectors in Arequipa form a recent monophyletic group within the broader South American phylogeny. We discuss several hypotheses that may explain the limited number of established T. cruzi lineages despite multiple introductions of the parasite

Blood meal identification of field-collected <i>Triatoma infestans</i> by hemi-nested PCR reactions and restriction fragment length polymorphism (RFLP) analysis.

<p>+  =  positive for <i>T. cruzi</i> by microscopy.</p><p>−  =  negative for <i>T. cruzi</i> by microscopy.</p><p>n/a  =  not applicable, samples were negative for animal DNA by PCR.</p

Sexual reproduction in a natural Trypanosoma cruzi population.

BackgroundSexual reproduction provides an evolutionary advantageous mechanism that combines favorable mutations that have arisen in separate lineages into the same individual. This advantage is especially pronounced in microparasites as allelic reassortment among individuals caused by sexual reproduction promotes allelic diversity at immune evasion genes within individuals which is often essential to evade host immune systems. Despite these advantages, many eukaryotic microparasites exhibit highly-clonal population structures suggesting that genetic exchange through sexual reproduction is rare. Evidence supporting clonality is particularly convincing in the causative agent of Chagas disease, Trypanosoma cruzi, despite equally convincing evidence of the capacity to engage in sexual reproduction.Methodology/ principle findingsIn the present study, we investigated two hypotheses that can reconcile the apparent contradiction between the observed clonal population structure and the capacity to engage in sexual reproduction by analyzing the genome sequences of 123 T. cruzi isolates from a natural population in Arequipa, Peru. The distribution of polymorphic markers within and among isolates provides clear evidence of the occurrence of sexual reproduction. Large genetic segments are rearranged among chromosomes due to crossing over during meiosis leading to a decay in the genetic linkage among polymorphic markers compared to the expectations from a purely asexually-reproducing population. Nevertheless, the population structure appears clonal due to a high level of inbreeding during sexual reproduction which increases homozygosity, and thus reduces diversity, within each inbreeding lineage.Conclusions/ significanceThese results effectively reconcile the apparent contradiction by demonstrating that the clonal population structure is derived not from infrequent sex in natural populations but from high levels of inbreeding. We discuss epidemiological consequences of this reproductive strategy on genome evolution, population structure, and phenotypic diversity of this medically important parasite

Restriction fragment length polymorphism (RFLP) analysis of 16S rDNA from common Chagas mammalian and avian hosts (laboratory controls), 1.5% agarose gel electrophoresis.

<p>(a) Digestion with <i>Hae</i> III (b) Digestion with <i>Alu</i> I. (c) Simultaneous double digestion with <i>Hae</i> III and <i>Alu</i> I. (d) Key for fragment sizes following restriction enzyme digestion with <i>Hae</i> III, <i>Alu</i> I, and simultaneous digestion with both enzymes.</p

Distance Matrix and Pairwise Distances Between Different Mammalian Species, 16s mitochondrial rDNA locus.

<p>Species by scientific common and scientific names [GenBank accession numbers].</p

Restriction fragment length polymorphism (RFLP) analysis of 16S rDNA from field-collected <i>T. infestans</i> for blood meal analysis.

<p>(a) Single digestion with <i>Hae III</i> to show sample CH12 with defined pattern for <i>Mus musculus</i>. (b) Double digestion with <i>Hae III</i> and <i>Alu I</i>. Samples CH11 and CH16: rat; CH 13: guinea pig; samples CH15, CH17 and CH18: human. CH12 had DNA from mouse and human (arrows).</p