Red Blood Cell Analysis Identifies Trypanosoma cruzi in Wild Pennsylvania Bats

Rationale: We have previously identified nucleic acid sensors on bat red blood cells (RBCs) and demonstrated that human RBCs can bind and harbor microbial genetic material, providing diagnostic insights into pathogen presence. Based on these findings, we hypothesized that bat RBCs could serve as a reservoir for pathogen nucleic acids. Chagas disease, caused by Trypanosoma cruzi ( T. cruzi ), is a leading cause of heart failure worldwide. While primarily endemic to Central and South America, its incidence in the United States is increasing due to both immigration from endemic regions and local transmission. Here, we report the presence of T. cruzi in wild big brown bats of Pennsylvania and demonstrate the feasibility of using bat RBCs for pathogen RNA detection. Methods: All studies were approved by the Institutional Animal Care and Use Committee. Terminal blood samples were obtained from wild-caught big brown bats ( Eptesicus fuscus ) admitted to Pennsylvania wildlife rehabilitators and deemed non-releasable. RBCs were isolated by centrifugation, washed in PBS, and preserved in RNA/DNA shield (Zymo Research). Total nucleic acid was extracted using the Quick-DNA/RNA Pathogen Magbead kit (Zymo Research), followed by RNA extraction of a 20uL aliquot using DNase according to the manufacturer's protocol. The NEBNext Ultra II Library Prep kit (New England Biolabs) was used to reverse transcribe RNA into cDNA which was used to construct and barcode metagenomic libraries for paired-end sequencing on a NextSeq2000 (Illumina). To confirm the presence of T. cruzi, DNA was extracted from distinct RBC aliquots from four bats with and without sequencing-confirmed T. cruzi (n=2/group). Quantitative PCR (qPCR) using T. cruzi -specific primers and probes was conducted, followed by gel electrophoresis to isolate amplicons followed by Sanger sequencing. Histological analysis of cardiac tissue was performed to assess the presence of T. cruzi parasites. Results: Metagenomic sequencing identified partial T. cruzi genomes in 5 of 10 bat RBC samples. DNA extraction and qPCR from packed RBCs showed amplification with T. cruzi -specific primers and probes in bats previously identified as T. cruzi-positive by RNA sequencing. Sanger sequencing of the amplicons showed the highest alignment with the T. cruzi CL strain, the causative agent of Chagas disease. Histological evaluation of cardiac tissue from one study bat confirmed the presence of a T. cruzi amastigote nest. Conclusion: The identification and confirmation of T. cruzi in wild Pennsylvania bats suggest a shift in the perceived geographic distribution of this pathogen. However, whether bats contribute to human transmission remains unclear. These findings underscore the need for increased Chagas disease screening in regions as far north as Pennsylvania. Additionally, this study reinforces the concept that RBCs serve as a reservoir for pathogen detection, suggesting that DNA extraction from small RBC volumes could be a valuable surveillance tool for Chagas and other diseases. ITMAT Pilot PiCAB grant (JCE, NSM)- NIH/NCATS UL1TR001878 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.