Protocol for laboratory rearing and infection tracking of Rhodnius prolixus using 3D-printable designs
Human infections by Trypanosoma cruzi propagate via its blood-feeding triatomine vector. Investigating parasite-vector interactions depends upon robust techniques to rear insects and analyze infections. Here, we present a protocol for laboratory rearing and infection tracking of Rhodnius prolixus. We describe steps for housing, feeding, and sorting strategies using 3D-printable designs. We also detail procedures for gut dissection, fecal collection, and parasite re-isolation. This protocol describes techniques that support efforts to understand and mitigate vector-mediated Chagas disease transmission.
Ruby E Harrison, Ronald Drew Etheridge. STAR Protoc. 2025 Jun 13;6(3):103894. doi: 10.1016/j.xpro.2025.103894.
The importance of persistence and dormancy in Trypanosoma cruzi infection and Chagas disease
Trypanosoma cruzi typically establishes a life-long infection in its mammalian hosts, causing the destruction of muscle tissues and ultimately resulting in potentially fatal Chagas disease. In this review, we consider the array of avoidance mechanisms that allow for T. cruzi persistence, many of which are unconventional among protozoan pathogens but which collectively are highly effective in the face of otherwise potent host immune responses. We also reflect on the phenomenon of dormancy in T. cruzi amastigotes, which is likely not involved in the long-term persistence of infection. Lastly, we consider how these phenomena of persistence and dormancy complicate the effectiveness of potential therapeutic interventions to prevent Chagas disease.
Molly E Bunkofske, Fernando J Sanchez-Valdez, Rick L Tarleton. Curr Opin Microbiol. 2025 Jun 5:86:102615. doi: 10.1016/j.mib.2025.102615.
In the News: Rick Tarleton
Researchers secure funding to advance Chagas disease research (News-Medical.net)
Investigators are studying Chagas disease with a One Health approach (DVM360)
UGA and Texas A&M Researchers tackle Chagas disease in dogs and humans (WUGA)
Countable Labs Launches Single-Molecule DNA Counting System, PCR Application (GenomeWeb)
Scientists use ‘One Health’ model to fight Chagas disease
Supported by almost $4 million in new funding, researchers at the University of Georgia and Texas A&M are using improved detection and treatment methods to understand Chagas disease, a serious, often overlooked illness affecting both dogs and humans.
A team of researchers at the University of Georgia and Texas A&M University has received more than $4 million from federal and nongovernmental organizations to support research on Chagas disease.
The research will consist of multiple projects focused on the disease’s prevalence, diagnostics to detect the parasite that causes the disease, and treatment protocols to prevent infection and disease in dogs. The ultimate goal is to use the findings to help people as well.
Funded by a $3 million grant from the National Institutes of Health, UGA’s Rick Tarleton will co-lead a project focused on strategies to detect, treat and monitor treatment outcomes in dogs in Texas. The goal is to establish the best practices that prevent the development of cardiac disease, one severe potential side effect of Chagas disease, and to establish resistance to possible future infection.
The researchers will work with dogs that were naturally infected with Chagas disease. Because the disease presents similarly in dogs as in humans, dogs are a good model for examining the effectiveness of the treatment.
“There are a number of important questions related to treatment efficacy and the protection that cured subjects have from future infection that cannot be easily addressed in humans but can be in these dog populations that are under intense transmission pressure in Texas,” said Tarleton, Regents Professor in UGA’s Franklin College of Arts and Sciences.
A growing threat to dog, human health
Tens of millions of people across the Americas have Chagas disease.
Chagas disease is a largely neglected disease. The parasite that causes it, Trypanosoma cruzi, is spread by blood-sucking insects known as kissing bugs, which can be found throughout North, Central and South America.
The disease, which commonly develops in humans and dogs, as well as many other mammals, often goes unnoticed in early stages. But a chronic infection can lead to serious heart and digestive system problems, making early diagnosis and prompt treatment important.
Although most human cases of Chagas disease are reported from South and Central America and Mexico, the parasite and its insect vector are found in abundance in the southern United States. Outdoor pets — particularly working dogs — face especially high risks of infection.
“These areas we are working in have 20% to 30% rates of new infections per year,” Tarleton said. “Those tend to be severe infections where the dogs either die or develop a disease that makes them unable to work.”
Texas has become a hotspot of kissing bugs.
“Unfortunately, Texas has emerged as a hotspot of infected kissing bugs, infected wildlife and infected dogs across the landscape,” said Dr. Sarah Hamer, a professor in the Texas A&M College of Veterinary Medicine and a primary investigator on the projects.
“These projects will advance Chagas disease research to understand the process of natural infections, disease and effect of treatments,” Hamer said. “These projects combine many aspects of biomedical research. We’re conducting field and laboratory research, treating dogs, measuring clinical outcomes and studying ecological factors. It’s truly a ‘One Health’ approach.”
A One Health approach to Chagas disease research
Diagnosing Chagas is complicated — in both people and canines. False negatives aren’t unheard of, leading people to not know they or their pets are infected. And that delays treatment.
Even when the disease is diagnosed promptly, treating the condition can be challenging.
The go-to medications used to treat Chagas, as currently applied, are not reliably effective. But they’re currently the only treatment option. Tarleton’s previous work in mice and other species show that their effectiveness can be improved by altering the dosing regimen.
To address these issues, the researchers will track infected canines using a combination approach with sensitive tests to detect both the parasite DNA and the body’s response to infection. The team will simultaneously test a revised dosing strategy for the current antiparasitic treatment, providing fewer but high-level doses and extending the administration period to improve effectiveness.
Recording health information from such a large population of dogs will hopefully help us understand why the disease develops in different ways.” —Dr. Sarah Hamer, Texas A&M
“The drug we’re using is an existing treatment for Chagas disease in humans,” said Dr. Ashley Saunders, a professor in the Texas A&M College of Veterinary Medicine and a primary investigator on the projects. “But Dr. Tarleton has shown that the parasites aren’t susceptible to this drug when they’re dormant. By changing the drug delivery protocol to dosing over a longer period of time, when the dormant parasites become active again, they are killed by the drug.”
In a related study funded by the United States Department of Homeland Security, the researchers will also monitor DHS-owned working dogs that are often trained in areas where Chagas disease is prevalent. The goal is to understand how the dogs are exposed to the disease as well as the impacts it can have on the canines’ heart health, as well as to develop monitoring and treatment strategies for these working dogs.
“One of the reasons that monitoring dogs is so helpful is because Chagas disease can produce so many different subsets of health problems,” Saunders said. “Some dogs end up with a heart abnormality, but a large number continue living and working happily for many years. Others will die quite suddenly, before anyone knew they had the disease.”
“Recording health information from such a large population of dogs will hopefully help us understand why the disease develops in different ways,” Hamer said.
Advancing canine Chagas disease management
With continued support from the American Kennel Club Canine Health Foundation, the team will treat and monitor individual pet dogs brought to Texas A&M’s Small Animal Teaching Hospital while developing a staging system for Chagas disease in dogs.
“The staging system we develop will help us to categorize the severity of disease, making it easier to determine which dogs will benefit most from drug treatment,” Saunders said. “This scoring system will work hand-in-hand with our improved diagnostic and treatment plan.”
This story was originally published at UGA Today: https://news.uga.edu/scientists-use-one-health-model-to-fight-chagas-disease/
Lysosome and plasma membrane Piezo channels of Trypanosoma cruzi are essential for proliferation, differentiation and infectivity
Trypanosoma cruzi, the causative agent of Chagas disease, is a parasitic protist that affects millions of people worldwide. Currently there are no fully effective drugs or vaccines available. Contact of T. cruzi infective forms with their host cells or with the extracellular matrix increases their intracellular Ca2+ concentration suggesting a mechano-transduction process. We report here that T. cruzi possesses two distinct mechanosensitive Piezo channels, named TcPiezo1 and TcPiezo2, with different subcellular localizations but similarly essential for normal proliferation, differentiation, and infectivity. While TcPiezo1 localizes to the plasma membrane, TcPiezo2 localizes to the lysosomes. Downregulation of TcPiezo1 expression by a novel ligand-regulated hammerhead ribozyme (HHR) significantly inhibited Ca2+ entry in cells expressing a genetically encoded Ca2+ indicator while downregulation of TcPiezo2 expression inhibited Ca2+ release from lysosomes, which are now identified as novel acidic Ca2+ stores in trypanosomes. The channels are activated by contact with extracellular matrix and by hypoosmotic stress. The results establish the essentiality of Piezo channels for the life cycle and Ca2+ homeostasis of T. cruzi and a novel lysosomal localization for a Piezo channel in eukaryotes.
Guozhong Huang, Mayara S Bertolini, Justin Wiedeman, Ronald D Etheridge, Teresa Cruz-Bustos, Roberto Docampo. PLoS Pathog. 2025 Apr 23;21(4):e1013105. doi: 10.1371/journal.ppat.1013105.
Serial ‘deep-sampling’ PCR of fragmented DNA reveals the wide range of Trypanosoma cruzi burden among chronically infected human, macaque, and canine hosts, and allows accurate monitoring of parasite load following treatment
Infection with the protozoan parasite Trypanosoma cruzi is generally well-controlled by host immune responses, but appears to be rarely eliminated. The resulting persistent, low-level infection results in cumulative tissue damage with the greatest impact generally in the heart in the form of chagasic cardiomyopathy. The relative success in immune control of T. cruzi infection usually averts acute phase death but has the negative consequence that the low-level presence of T. cruzi in hosts is challenging to detect unequivocally. Thus, it is difficult to identify those who are actively infected and, as well, problematic to gauge the impact of treatment, particularly in the evaluation of the relative efficacy of new drugs. In this study, we employ DNA fragmentation and high numbers of replicate PCR reaction (‘deep-sampling’) and to extend the quantitative range of detecting T. cruzi in blood by at least three orders of magnitude relative to current protocols. When combined with sampling blood at multiple time points, deep sampling of fragmented DNA allowed for detection of T. cruzi in all infected hosts in multiple host species, including humans, macaques, and dogs. In addition, we provide evidence for a number of characteristics not previously rigorously quantified in the population of hosts with naturally acquired T. cruzi infection, including, a >6 log variation between chronically infected individuals in the stable parasite levels, a continuing decline in parasite load during the second and third years of infection in some hosts, and the potential for parasite load to change dramatically when health conditions change. Although requiring strict adherence to contamination-prevention protocols and significant resources, deep-sampling PCR provides an important new tool for assessing therapies and for addressing long-standing questions in T. cruzi infection and Chagas disease.
Brooke E White, Carolyn L Hodo, Sarah Hamer, Ashley B Saunders, Susana A Laucella, Daniel B Hall, Rick L Tarleton. Elife. 2025 Apr 15:14:RP104547. doi: 10.7554/eLife.104547.
Stable colonization of the model kissing bug Rhodnius prolixus by Trypanosoma cruzi Y strain
Trypanosoma cruzi is a single-celled eukaryotic parasite responsible for Chagas disease, a major cause of morbidity and mortality in Central and South America. While the host-pathogen interactions of T. cruzi have been extensively studied in vertebrate models, investigations into its interactions within its insect host remain limited. To address this gap and establish a genetically tractable system for studying parasite-vector dynamics, we conducted quantitative kinetic infection studies using the Y strain of T. cruzi and the model vector Rhodnius prolixus. We began by comparing parasite infection kinetics from two genetically diverse strains of T. cruzi, Brazil and Y, and demonstrated that ingested parasites from both strains transiently expand in the anterior regions of the insect digestive tract with stable colonization occurring in the hindgut over the long term. Notably, we demonstrated that the clonal Y strain, contrary to previous reports, can effectively infect and persist across multiple developmental stages of R. prolixus. Additionally, comparison of movement of parasites versus inert fluorescent microspheres introduced into artificial blood meals suggests that T. cruzi colonization of the R. prolixus gut occurs passively through peristaltic movement during digestion, rather than through active parasite-mediated chemotaxis. These findings highlight the T. cruzi Y strain – R. prolixus model system as a promising tool for the in-depth molecular characterization of parasite-vector interactions, potentially offering new insights into the biology of this neglected and deadly human pathogen.
Ruby E Harrison, Kevin J Vogel, Ronald Drew Etheridge. PLoS Negl Trop Dis. 2025 Mar 12;19(3):e0012906. doi: 10.1371/journal.pntd.0012906.
A limitation lifted: A conditional knockdown system reveals essential roles for Polo-like kinase and Aurora kinase 1 in Trypanosoma cruzi cell division
While advances in genome editing technologies have simplified gene disruption in many organisms, the study of essential genes requires development of conditional disruption or knockdown systems that are not available in most organisms. Such is the case for Trypanosoma cruzi, a parasite that causes Chagas disease, a severely neglected tropical disease endemic to Latin America that is often fatal. Our knowledge of the identity of essential genes and their functions in T. cruzi has been severely constrained by historical challenges in very basic genetic manipulation and the absence of RNA interference machinery. Here, we describe the development and use of self-cleaving RNA sequences to conditionally regulate essential gene expression in T. cruzi. Using these tools, we identified essential roles for Polo-like and Aurora kinases in T. cruzi cell division, mirroring their functions in Trypanosoma brucei. Importantly, we demonstrate conditional knockdown of essential genes in intracellular amastigotes, the disease-causing stage of the parasite in its human host. This conditional knockdown system enables the efficient and scalable functional characterization of essential genes in T. cruzi and provides a framework for the development of conditional gene knockdown systems for other nonmodel organisms.
J. Wiedeman, R. Harrison, & R.D. Etheridge, Proc. Natl. Acad. Sci. U.S.A. 122 (8) e2416009122, https://doi.org/10.1073/pnas.2416009122 (2025).
Inorganic Polyphosphate Is in the Surface of Trypanosoma cruzi but Is Not Significantly Secreted
Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that can lead to the development of cardiac fibrosis, which is characterized by the deposition of extracellular matrix (ECM) components in the interstitial region of the myocardium. The parasite itself can induce myofibroblast differentiation of cardiac fibroblast in vitro, leading to increased expression of ECM. Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that can also induce myofibroblast differentiation and deposition of ECM components and is highly abundant in T. cruzi. PolyP can modify proteins post-translationally by non-enzymatic polyphosphorylation of lysine residues of poly-acidic, serine-(S) and lysine (K)-rich (PASK) motifs. In this work, we used a bioinformatics screen and identified the presence of PASK domains in several surface proteins of T. cruzi. We also detected polyP in the external surface of its different life cycle stages and confirmed the stimulation of host cell fibrosis by trypomastigote infection. However, we were not able to detect significant secretion of the polymer or activation of transforming growth factor beta (TGF-β), an important factor for the generation of fibrosis by inorganic polyP- or trypomastigote-conditioned medium.
Logan P Crowe, Anna Gioseffi, Mayara S Bertolini, Roberto Docampo. Pathogens. 2024 Sep 9;13(9):776. doi: 10.3390/pathogens13090776.