Habitat Management to Reduce Human Exposure to Trypanosoma cruzi and Western Conenose Bugs (Triatoma protracta).

Chagas disease, which manifests as cardiomyopathy and severe gastrointestinal dysfunction, is caused by Trypanosoma cruzi, a vector-borne parasite. In California, the vector Triatoma protracta frequently colonizes woodrat (Neotoma spp.) lodges, but may also invade nearby residences, feeding upon humans and creating the dual risk of bite-induced anaphylaxis and T. cruzi transmission. Our research aimed to assess T. cruzi presence in woodrats in a previously unstudied northern California area, statistically evaluate woodrat microhabitat use with respect to vegetation parameters, and provide guidance for habitat modifications to mitigate public health risks associated with Tr. protracta exposure. Blood samples from big-eared woodrats (N. macrotis) trapped on rural private properties yielded a T. cruzi prevalence of 14.3%. Microhabitat analyses suggest that modifying vegetation to reduce understory density within a 40 meter radius of human residences might minimize woodrat lodge construction within this buffer area, potentially decreasing human exposure to Tr. protracta

Molecular Diversity of Trypanosoma cruzi Detected in the Vector Triatoma protracta from California, USA.

BACKGROUND: Trypanosoma cruzi, causative agent of Chagas disease in humans and dogs, is a vector-borne zoonotic protozoan parasite that can cause fatal cardiac disease. While recognized as the most economically important parasitic infection in Latin America, the incidence of Chagas disease in the United States of America (US) may be underreported and even increasing. The extensive genetic diversity of T. cruzi in Latin America is well-documented and likely influences disease progression, severity and treatment efficacy; however, little is known regarding T. cruzi strains endemic to the US. It is therefore important to expand our knowledge on US T. cruzi strains, to improve upon the recognition of and response to locally acquired infections. METHODOLOGY/PRINCIPLE FINDINGS: We conducted a study of T. cruzi molecular diversity in California, augmenting sparse genetic data from southern California and for the first time investigating genetic sequences from northern California. The vector Triatoma protracta was collected from southern (Escondido and Los Angeles) and northern (Vallecito) California regions. Samples were initially screened via sensitive nuclear repetitive DNA and kinetoplast minicircle DNA PCR assays, yielding an overall prevalence of approximately 28% and 55% for southern and northern California regions, respectively. Positive samples were further processed to identify discrete typing units (DTUs), revealing both TcI and TcIV lineages in southern California, but only TcI in northern California. Phylogenetic analyses (targeting COII-ND1, TR and RB19 genes) were performed on a subset of positive samples to compare Californian T. cruzi samples to strains from other US regions and Latin America. Results indicated that within the TcI DTU, California sequences were similar to those from the southeastern US, as well as to several isolates from Latin America responsible for causing Chagas disease in humans. CONCLUSIONS/SIGNIFICANCE: Triatoma protracta populations in California are frequently infected with T. cruzi. Our data extend the northern limits of the range of TcI and identify a novel genetic exchange event between TcI and TcIV. High similarity between sequences from California and specific Latin American strains indicates US strains may be equally capable of causing human disease. Additional genetic characterization of Californian and other US T. cruzi strains is recommended

Native rodent species are unlikely sources of infection for Leishmania (Viannia) braziliensis along the Transoceanic Highway in Madre de Dios, Peru.

An estimated 2.3 million disability-adjusted life years are lost globally from leishmaniasis. In Peru's Amazon region, the department of Madre de Dios (MDD) rises above the rest of the country in terms of the annual incidence rates of human leishmaniasis. Leishmania (Viannia) braziliensis is the species most frequently responsible for the form of disease that results in tissue destruction of the nose and mouth. However, essentially nothing is known regarding the reservoirs of this vector-borne, zoonotic parasite in MDD. Wild rodents have been suspected, or proven, to be reservoirs of several Leishmania spp. in various ecosystems and countries. Additionally, people who live or work in forested terrain, especially those who are not regionally local and whose immune systems are thus naïve to the parasite, are at most risk for contracting L. (V.) braziliensis. Hence, the objective of this study was to collect tissues from wild rodents captured at several study sites along the Amazonian segment of the newly constructed Transoceanic Highway and to use molecular laboratory techniques to analyze samples for the presence of Leishmania parasites. Liver tissues were tested via polymerase chain reaction from a total of 217 rodents; bone marrow and skin biopsies (ear and tail) were also tested from a subset of these same animals. The most numerous rodent species captured and tested were Oligoryzomys microtis (40.7%), Hylaeamys perenensis (15.7%), and Proechimys spp. (12%). All samples were negative for Leishmania, implying that although incidental infections may occur, these abundant rodent species are unlikely to serve as primary reservoirs of L. (V.) braziliensis along the Transoceanic Highway in MDD. Therefore, although these rodent species may persist and even thrive in moderately altered landscapes, we did not find any evidence to suggest they pose a risk for L. (V.) braziliensis transmission to human inhabitants in this highly prevalent region

Molecular Diversity of Trypanosoma cruzi Detected in the Vector Triatoma protracta from California, USA.

BackgroundTrypanosoma cruzi, causative agent of Chagas disease in humans and dogs, is a vector-borne zoonotic protozoan parasite that can cause fatal cardiac disease. While recognized as the most economically important parasitic infection in Latin America, the incidence of Chagas disease in the United States of America (US) may be underreported and even increasing. The extensive genetic diversity of T. cruzi in Latin America is well-documented and likely influences disease progression, severity and treatment efficacy; however, little is known regarding T. cruzi strains endemic to the US. It is therefore important to expand our knowledge on US T. cruzi strains, to improve upon the recognition of and response to locally acquired infections.Methodology/principle findingsWe conducted a study of T. cruzi molecular diversity in California, augmenting sparse genetic data from southern California and for the first time investigating genetic sequences from northern California. The vector Triatoma protracta was collected from southern (Escondido and Los Angeles) and northern (Vallecito) California regions. Samples were initially screened via sensitive nuclear repetitive DNA and kinetoplast minicircle DNA PCR assays, yielding an overall prevalence of approximately 28% and 55% for southern and northern California regions, respectively. Positive samples were further processed to identify discrete typing units (DTUs), revealing both TcI and TcIV lineages in southern California, but only TcI in northern California. Phylogenetic analyses (targeting COII-ND1, TR and RB19 genes) were performed on a subset of positive samples to compare Californian T. cruzi samples to strains from other US regions and Latin America. Results indicated that within the TcI DTU, California sequences were similar to those from the southeastern US, as well as to several isolates from Latin America responsible for causing Chagas disease in humans.Conclusions/significanceTriatoma protracta populations in California are frequently infected with T. cruzi. Our data extend the northern limits of the range of TcI and identify a novel genetic exchange event between TcI and TcIV. High similarity between sequences from California and specific Latin American strains indicates US strains may be equally capable of causing human disease. Additional genetic characterization of Californian and other US T. cruzi strains is recommended

PCR assay flowsheet¹ to identify <i>Trypanosoma cruzi</i> discrete typing units (DTUs).

<p>Directional arrows indicate assay order and stop signs denote when sufficient data was gathered to theoretically identify the DTU. The final assay (<i>GPI</i>) is included as a confirmatory step, but is not required for DTU identification. ¹Modified from Lewis et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.ref037" target="_blank">37</a>] ²The large subunit rDNA assay is also referred to as the 24sα rRNA gene assay. ³An additional band of approximately 125bp may or may not be visible in combination with the 110bp band. ⁴Heat Shock Protein-60 (HSP60) results in an amplicon of 432-462bp, which upon RFLP with <i>Eco</i>V restriction enzyme yields the following patterns: 1 band (432–462), 2 bands (118–148 + 314), or 3 bands (118–148 + 314 + 432–462). ⁵This PCR used a pool of three primers to amplify a portion of the non-transcribed intergenic region of the tandemly repeated mini-exon gene. ⁶Glucose Phosphate Isomerase (GPI) results in an amplicon of approximately 1264bp, which upon RFLP with <i>Hha</i>I restriction enzyme yields the following patterns: 2 bands (447 + 817), 3 bands (253 + 447 + 490), or 4 bands (253 + 447 + 490 + 817). TcIV will display 2 or 3 bands for North American and South American strains, respectively.</p

Phylogeny for 62 <i>Trypanosoma cruzi</i> concatenated 786 bp cytochrome oxidase II-NADH 1 (<i>COII-ND1</i>) sequences.

<p>The TcI clade is condensed in this figure and contains the majority of the sequences obtained in this study (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.g005" target="_blank">Fig 5</a> for expanded version). Depicted is the Neighbor-Joining (NJ) tree constructed in MEGA6 with evolutionary distances computed via the Maximum Composite Likelihood method and the scale bar indicating the number of nucleotide substitutions per site. The numbers above or below the nodes represent the bootstrap confidence levels for 2,000 NJ replicates (1^st value) and 500 Maximum Likelihood replicates run under the Tamura 3-parameter model (due to slightly incongruent topology, ML bootstrap values are only shown at three nodes) for those values <b>≥</b> 50%. Only one sequence obtained in this study (Esc19 = Escondido 19) was grouped as TcIV based on mitochondrial gene sequences. All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country of origin indicated in parentheses. Tree is outgroup rooted with <i>T</i>. <i>cruzi marinkellei</i> (GenBank #AF359054).</p

Phylogeny for 26 <i>Trypanosoma cruzi</i> 350 bp RNA-Binding Protein-19 (<i>RB19</i>) sequences.

<p>Neighbor-Joining tree constructed in MEGA6 with evolutionary distances computed via Maximum Composite Likelihood. The numbers above the nodes represent bootstrap confidence levels for 2,000 replicates. Only values ≥ 50% are shown. The only TcI isolate not obtained in this study is represented by a square. Sequences obtained in this study are indicated by a triangle (Esc = Escondido, SoCal = Southern California, Vall = Vallecito). All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country origin indicated in parentheses. The scale bar indicates the number of nucleotide substitutions per site. Tree is outgroup rooted with <i>T</i>. <i>cruzi marinkellei</i> (TcMark CONTIG 1404).</p