\u3cem\u3eTrypanosoma Cruzi\u3c/em\u3e in Wild Raccoons and Opossums from Kentucky

Only 6 autochthonous cases of human Chagas disease have been documented in the U.S.A., however, as many as 5% of immigrants from Latin America may be infected with the etiologic agent, Trypanosoma cruzi. The parasite has been isolated from a variety of wild mammals, particularly in the southeastern region of the U.S.A. The goal of our study was to determine if the sylvatic cycle of T. cruzi infection occurs in Kentucky, and, if present, to assess the prevalence of infection in Warren and Barren counties. Raccoons and opossums were live-trapped between June and December, 2007. Animals were anesthetized with isoflurane, and blood samples were collected using a vacutainer system. Sera were frozen at -80oC for subsequent analysis, and whole blood samples were inoculated, in duplicate, into liver infusion tryptose (LIT) medium and cultured at 27oC. Seventeen T. cruzi isolates from raccoons have been positively identified by hemoculture. A total of 25/44 (57%) raccoon samples were found to be positive by hemoculture or serological analysis. In Warren County 18/25 (72%) of raccoons tested positive for T. cruzi and 7/19 raccoons (37%) in Barren County were positive for the parasite. Eighteen of 43 (42%) of the sera collected from opossums in Warren County and 3 of 5 (60%) from Barren County were judged to be positive by either enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay tests (IFAT). There were no positive hemoculture results for the opossum samples. The infection rates found in the current study for raccoons were slightly higher than those reported in previous studies in the U.S.A. However, the overall prevalence of T. cruzi in opossums (determined by serological analysis) was consistent with previous studies performed in the southeast. To our knowledge, this is the first report of T. cruzi from the state of Kentucky

Trypanosoma cruzi strain TcIV infects raccoons from Illinois

BACKGROUND The northern limits of Trypanosoma cruzi across the territory of the United States remain unknown. The known vectors Triatoma sanguisuga and T. lecticularia find their northernmost limits in Illinois; yet, earlier screenings of those insects did not reveal the presence of the pathogen, which has not been reported in vectors or reservoir hosts in this state. OBJECTIVES Five species of medium-sized mammals were screened for the presence of T. cruzi. METHODS Genomic DNA was isolated from heart, spleen and skeletal muscle of bobcats (Lynx rufus, n = 60), raccoons (Procyon lotor, n = 37), nine-banded armadillos (Dasypus novemcinctus, n = 5), Virginia opossums (Didelphis virginiana, n = 3), and a red fox (Vulpes vulpes). Infections were detected targeting DNA from the kinetoplast DNA minicircle (kDNA) and satellite DNA (satDNA). The discrete typing unit (DTU) was determined by amplifying two gene regions: the Spliced Leader Intergenic Region (SL), via a multiplex polymerase chain reaction, and the 24Sα ribosomal DNA via a heminested reaction. Resulting sequences were used to calculate their genetic distance against reference DTUs. FINDINGS 18.9% of raccoons were positive for strain TcIV; the rest of mammals tested negative. MAIN CONCLUSIONS These results confirm for the first time the presence of T. cruzi in wildlife from Illinois, suggesting that a sylvatic life cycle is likely to occur in the region. The analyses of sequences of SL suggest that amplicons resulting from a commonly used multiplex reaction may yield non-homologous fragments

Genetic diversity of Trypanosoma cruzi infecting raccoons (Procyon lotor) in 2 metropolitan areas of southern Louisiana: implications for parasite transmission networks

Trypanosoma cruzi, the aetiological agent of Chagas disease, exists as an anthropozoonosis in Louisiana. Raccoons are an important reservoir, as they demonstrate high prevalence and maintain high parasitaemia longer than other mammals. Given the complex nature of parasite transmission networks and importance of raccoons as reservoirs that move between sylvatic and domestic environments, detailing the genetic diversity of T. cruzi in raccoons is crucial to assess risk to human health. Using a next-generation sequencing approach targeting the mini-exon, parasite diversity was assessed in 2 metropolitan areas of Louisiana. Sequences were analysed along with those previously identified in other mammals and vectors to determine if any association exists between ecoregion and parasite diversity. Parasites were identified from discrete typing units (DTUs) TcI, TcII, TcIV, TcV and TcVI. DTUs TcII, TcV and TcVI are previously unreported in raccoons in the United States (US). TcI was the most abundant DTU, comprising nearly 80% of all sequences. All but 1 raccoon harboured multiple haplotypes, some demonstrating mixed infections of different DTUs. Furthermore, there is significant association between DTU distribution and level III ecoregion in Louisiana. Finally, while certain sequences were distributed across multiple tissues, others appeared to have tissue-specific tropism. Taken together, these findings indicate that ongoing surveillance of T. cruzi in the US should be undertaken across ecoregions to fully assess risk to human health. Given potential connections between parasite diversity and clinical outcomes, deep sequencing technologies are crucial and interventions targeting raccoons may prove useful in mitigating human health risk

Disease surveillance and projected expansion in climatic suitability for Trypanosoma cruzi, the Etiological agent of Chagas disease, in Oklahoma.

The vector-borne hemoflagellate parasite Trypanosoma cruzi infects seven million people globally and causes chronic cardiomyopathy and gastrointestinal diseases. Historically, T. cruzi was endemic to Central and South America, but is now found throughout the southern United States and across 43 countries globally. There are three reports of T. cruzi in wild raccoons and dogs in Oklahoma, but its endemicity in the state is poorly studied. We suspect Mexican free-tailed bats (Tadarida brasiliensis) contribute to the endemicity of T. cruzi in Oklahoma by their annual migration from Central America to North American maternity roosts. During the summer of 2017, we sampled 361 Mexican free-tailed bats at three maternity roosts in Oklahoma for T. cruzi. We collected wing tissues and extracted DNA, amplified target T. cruzi DNA by PCR using the primers TCZ1/TCZ2, and observed amplification by gel electrophoresis. We detected T. cruzi DNA in one juvenile Mexican free-tailed bat resulting in a prevalence of 0.27% in the 361 sampled bats. The positive sample was sequenced at Eton Biosciences, confirmed as T. cruzi, and uploaded to GenBank (MG869732). This finding is the first reported detection of a wild bat naturally infected with T. cruzi in Oklahoma, suggests Mexican free-tailed bats can contribute to T. cruzi endemicity via migration between endemic foci, and provides insight on the endemicity of T. cruzi in underrepresented endemic areas. To better understand the potential impact of global climate change on the future epidemiology of T. cruzi in Oklahoma, we used the program MaxEnt to develop an ecological niche model for T. cruzi and five widespread Triatoma vectors based on 19 bioclimatic variables and 546 published localities within the United States. We modeled regions of current potential T. cruzi and Triatoma distribution, and regions projected to have suitable climatic conditions under a Representative Concentration Pathway (RCP 8.5) scenario by 2070. Regions with potential suitable climatic conditions for T. cruzi, T. indictiva, T. lecticularia, T. protracta, and T. sanguisuga are predicted to increase within the United States and Oklahoma by 2070. Regions with potential suitable climatic conditions for T. gerstaeckeri are predicted to increase within the United States but not in Oklahoma by 2070. Our findings agree with previous literature and confirm that climate change will influence the expansion of T. cruzi and important Triatoma vectors in Oklahoma and the United States

38th Annual WKU Student Research Conference

Western Kentucky University 38th Annual Student Research Conference program and student abstracts. Saturday, April 12, 2008, Carroll Knicely Conference Center, Bowling Green, Kentucky

Molecular Analysis of \u3ci\u3eTrypanosoma cruzi\u3c/i\u3e Isolates Obtained from Raccoons (\u3ci\u3eProcyon lotor\u3c/i\u3e) in Warren and Barren Counties of Kentucky

Trypanosoma cruzi, the etiologic agent of Chagas disease, infects a variety of wild mammals in the southern United States, but it has only recently been isolated from raccoons trapped in the state of Kentucky. The purpose of the present study was to use a molecular genotyping approach, followed by DNA sequencing to determine the genotypes (type I, or types IIa-IIe) of 15 of the Kentucky isolates. DNA samples were prepared from 15 T. cruzi- isolates using a Qiagen mini kit, and PCR amplification was performed using published primers for the 24S α rDNA sequence (D71 and D72), the non-transcribed spacer of the mini-exon genes (TC, TC1, and TC2), the 18S rDNA sequence (V1 and V2), and TCZ1 and TCZ2 primers that amplify a 188-base pair segment of the repetitive 195-bp nuclear DNA sequence of T. cruzi. DNA sequencing (ABI 3130 Genetic Analyzer) was performed on all amplification products obtained from the PCR analysis of the RW2 and RB12 isolates (randomly selected to represent both Warren and Barren counties of Kentucky; the number started with an “R” which stood for raccoon, a “W” for Warren County or a “B” for Barren County, followed by a number which represented the order in which animal was trapped). The resulting sequences were edited before analysis using the BLAST database of the National Center for Biotechnology Information (NCBI) Genbank. All 15 isolates were positively confirmed as T. cruzi based upon PCR amplification of a 195 bp repetitive genomic DNA sequence, and all 15 isolates showed identical PCR amplification results with all 4 sets of T. cruzi-specific primers. Two positive PCR samples were randomly selected for further DNA sequence analysis, and all samples were positively identified as the type IIa genotype of T. cruzi with max identities ranging from 94%-99%. The results of this study confirm that all hemoculture isolates obtained from raccoons trapped in Warren and Barren counties of Kentucky are T. cruzi. Furthermore, all BLAST comparisons of amplicon DNA sequences showed high sequence identity to type IIa strains of T. cruzi. The type IIa strain of T. cruzi is the most commonly reported genotype from raccoons trapped in the U.S.A