Bide Your Time With Bites: A Case of Rattlesnake Envenomation in Pennsylvania.

This case report represents a 26-year-old male with no significant past medical history who presented to the emergency department in western Pennsylvania following a western diamondback rattlesnake (Crotalus Atrox) bite to his hand. His initial swelling was mild, and his coagulation studies were unremarkable, with minimal changes on repeat studies, and poison control recommended against antivenom administration. He was discharged home with oral antibiotics and analgesics due to his stable clinical course. However, he returned to the emergency department about 12 hours later with worsening pain and swelling that extended to his elbow. He was then given antivenom and transferred to a larger center for higher-level care, ultimately having symptom resolution after further antivenom administration. This report serves to underline the importance of clinician education regarding envenomation management throughout the United States, including areas without indigenous venomous snakes.

Rattlesnake Crotalphine Analgesic Active on Tetrodotoxin-Sensitive Na+ Current in Mouse Dorsal Root Ganglion Neurons.

Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss.

The relevance of vascular adjustments to hemodynamic control in the face of temperature change in Crotalus durissus.

The presence of cardiac shunts in ectothermic tetrapods is thought to be consistent with active vascular modulations for proper hemodynamic support. Local control of blood flow modulates tissue perfusion and thus systemic conductance (Gsys) is assumed to increase with body temperature (Tb) to accommodate higher aerobic demand. However, the general increase of Gsys presses for a higher right-to-left (R-L) shunt, which reduces arterial oxygen concentration. In contrast, Tb reduction leads to a Gsys decrease and a left-to-right shunt, which purportedly increases pulmonary perfusion and plasma filtration in the respiratory area. This investigation addressed the role of compensatory vascular adjustments in the face of the metabolic alterations caused by Tb change in the South American rattlesnake (Crotalus durissus). Cardiovascular recordings were performed in decerebrated rattlesnake preparations at 10, 20 and 30°C. The rise in Tb increased metabolic demand, and correlated with an augmentation in heart rate. Although cardiac output increased, systemic stroke volume reduced while pulmonary stroke volume remained stable. Although that resulted in a proportionally higher increase in pulmonary blood flow, the R-L shunt was maintained. While the systemic compliance of large arteries was the most relevant factor in regulating arterial systemic blood pressure, peripheral conductance of pulmonary circulation was the major factor influencing the final cardiac shunt. Such dynamic adjustment of systemic compliance and pulmonary resistance for shunt modulation has not been demonstrated before and contrasts with previous knowledge on shunt control.

In-vitro exposure of feline red blood cells to rattlesnake venom causes echinocytosis.

The objective of this study was to investigate in-vitro exposure of healthy feline red blood cells to venom of four species of rattlesnakes and to evaluate the presence and type of echinocytes that form. Blood from 10 healthy felines was collected and within 30 min exposed to four species of rattlesnake venom. Cytologic evaluation for echinocytes was performed at 0-, 15-, and 30-min post-exposure to each of the species' venom. There was significant formation of echinocytes in a time dependent manner with in-vitro exposure of red blood cells to rattlesnake venom. Cytologic evaluation of blood smears at 15 and 30 min showed a progressive increase in echinocyte numbers over time, with a significantly higher number of echinocytes formed at both timepoints compared to the 0-min timepoint. Furthermore, the morphology of the echinocytes differed based on the length of time exposed to the venom, with type I echinocytes being significantly present early on post-exposure (0 and 15 min) and type III echinocytes being most prevalent later post-exposure (15 and 30 min). There was not a significant difference in the formation of echinocytes between the rattlesnake species. This study demonstrates that there is a time dependent formation of echinocytes in feline blood post-exposure to four species of rattlesnake venom in the in-vitro setting. Cytologic evaluation of blood smears in cats with suspected rattlesnake envenomation to evaluate for echinocyte populations may use to provide support for the diagnosis and to direct the timely administration of appropriate treatments.

Infections following rattlesnake envenomation and use of antibiotics.

BACKGROUND: There are 7000-8000 venomous snake bites annually in the USA. Antibiotics are commonly administered to bite victims because infection is difficult to differentiate from local tissue injury following envenomation. METHODS: The Arizona Poison and Drug Information Center (APDIC) in Tucson oversees antivenom administration for 14 Arizona counties. Records (1999-2021) were searched for antibiotic use and confirmed infections after a rattlesnake bite. RESULTS: There were 4160 calls to APDIC regarding rattlesnakes. After excluding bites to animals, 'dry bites', prisoners and records with missing data, 2059 records were evaluated. Systemic antibiotics were administered to 206 patients (10% of bite victims). Twenty patients (0.97%) had confirmed infections, including cellulitis (n=10), fasciitis (n=4), abscess (n=3) and osteomyelitis (n=3). Five of the victims had positive blood cultures. The presence of tissue necrosis, leukocytosis, fever and elevated fibrinogen levels did not discriminate between toxic effects of venom and infection. CONCLUSIONS: Confirmed infections following a rattlesnake bite are uncommon (0.97% of bites). Physicians should refrain from prescribing antibiotics, as they are not justified for most rattlesnake bite victims and the variety of pathogens encountered precludes use of any single effective antibiotic.

Exploring the protein profile and biological activity of Crotalus molossus venom against E. coli, P. aeruginosa and S. aureus bacteria and T47D breast carcinoma cells.

Mexico has the highest diversity of snake species in the world, following Australia when considering just venomous snakes. Specifically, in Sonora, the second largest state in the country, more than 15 highly venomous species occur, including the northern black-tailed rattlesnake (Crotalus molossus). This specie's venom has not been as thoroughly researched in contrast with other Mexican vipers, nevertheless some studies report its biological activity and even pharmacological potential with antibacterial and cytotoxic activity. In this study we identified the main protein components from a pool of C. molossus venom through a gel-free proteomics approach, reporting ∼140 proteins belonging to the SVMP (38.76%), PLA2 (28.75%), CTL (11.93%), SVSP (6.03%) and LAAO (5.67%) toxin families. To study its biological activities, we evaluated its hemolytic, antibacterial, and cytotoxic activity in red blood cells, Gram positive and negative bacteria and a luminal A breast carcinoma cell line (T47D), respectively, in vitro. We report that concentrations <100 µg/mL are potentially not hemolytic and reduced the bacteria viability of E. coli and S. aureus with an IC50 of 10.27 and 11.51 µg/mL, respectively. Finally, we determined the C. molossus venom as cytotoxic against the T47D breast carcinoma cell line, with an IC50 of 1.55 µg/mL. We suggest that the evaluated cytotoxicity was due to a high abundance of SVMPs and PLA2s, since it's been reported that they affect the extracellular matrix and membrane permeation. This may provide a useful tool for pharmaceutical screening in the future.

Twenty-five years of suspected rattlesnake encounters in Arizona.

INTRODUCTION: Rattlesnake (Crotalus spp., Sistrurus spp.) bites in the southwestern United States are associated with significant morbidity. This study aims to describe 25 years of rattlesnake encounters reported to the Arizona Poison and Drug Information Center to identify vulnerable populations and circumstances where encounters occur to create public education to reduce future bites. METHODS: Cases of suspected rattlesnake encounters in Arizona reported to the Arizona Poison and Drug Information Center between 1999 and 2023 were analyzed to identify populations and circumstances associated with encounters. RESULTS: A total of 3,808 cases were analyzed overall and by age subgroups. Most encounters occurred in men (69.9%), during the evening (16:00-21:59; 49.2%), in summer (41.9%), and close to home (38.2%). Most bites occurred to the lower extremity (51%). Children 0 to 12-years-old have more encounters than those 13-years-old and older in rural zip codes (27.7% versus 14.8%; P = 0.005), during spring (31.8% versus 22.3%; P = 0.0005), and during the evening (64.4% versus 48.1%; P < 0.001). DISCUSSION: Rattlesnakes are encountered when rattlesnake and human behavior patterns overlap. Many people spend time outside during evening hours in the summer, and valuable resources like food, water, and shelter can be found near houses where humans spend much of their time. Most age groups have similar encounter circumstances but encounters among children 0 to 12-years-old differ in time of day, season, and urbanization level than encounters of those 13-years-old and older. Limitations of this study include underreporting of encounters, incomplete case details, potential reporting bias, potential snake misidentification, and geographic coverage of the poison center. CONCLUSION: Prevention of rattlesnake bites by reducing encounters is the most effective way to reduce suffering and healthcare costs. Future steps include creating and disseminating targeted public health education using the data collected.

The tale of the rattle: Using rattle size to understand growth and sexual dimorphism in an insular population of rattlesnakes (Crotalus oreganus caliginis).

Islands have played a key role in our understanding of rapid evolution. A large body of literature has examined morphological changes in response to insularity and isolation, which has yielded useful generalizations about how animals can adapt to live in very small geographic areas. However, understanding the evolution of morphological variation in insular populations often requires detailed data sets on longitudinal patterns of growth and development, and such studies typically necessitate long-term mark-recapture on a large sample of individuals. Rattlesnakes provide a unique opportunity to address some of these difficulties because the addition of rattle segments to the rattle string occurs with regular periodicity and their size directly correlates with the body size of the snake at the time of the ecdysis cycle generating the segment. Here, we used a large database of rattle segment sizes recorded from island (Isla Coronado Sur, Baja California, Mexico) and mainland (Camp Pendleton, California, United States) populations of Western Rattlesnakes (Crotalus oreganus and C. o. caliginis) that separated approximately 10,000 years ago to compare body sizes at different ecdysis cycles, which allowed us to assess differences in growth rates and patterns of sexual size dimorphism. Our results show that rattlesnakes on Isla Coronado Sur appear to be born smaller and grow more slowly than their mainland counterparts, resulting in a "dwarfed" island population. However, despite significant differences in body size, both populations exhibited the same degree of sexual dimorphism. Our study demonstrates the potential to use rattle characteristics to recover detailed estimates of fundamental demographic parameters.

Clinical Management of North American Snake and Marine Envenomations.

Envenomations are the 23rd most common reason for calls to US poison control centers, with over 35,000 incidents reported annually. Snake bites account for over 20% of those calls, while marine envenomations are likely underreported at 3% to 4%.1 While these types of envenomations may not be encountered on a daily basis for many physicians, the different types of envenomations warrant unique management strategies based on the offending creature and symptom presentation. This text serves as a review of the epidemiology, clinical presentations, and management of endemic North American species of snakes and marine vertebrate and invertebrate envenomations.

Image Fusion Method Based on Snake Visual Imaging Mechanism and PCNN.

The process of image fusion is the process of enriching an image and improving the image's quality, so as to facilitate the subsequent image processing and analysis. With the increasing importance of image fusion technology, the fusion of infrared and visible images has received extensive attention. In today's deep learning environment, deep learning is widely used in the field of image fusion. However, in some applications, it is not possible to obtain a large amount of training data. Because some special organs of snakes can receive and process infrared information and visible information, the fusion method of infrared and visible light to simulate the visual mechanism of snakes came into being. Therefore, this paper takes into account the perspective of visual bionics to achieve image fusion; such methods do not need to obtain a significant amount of training data. However, most of the fusion methods for simulating snakes face the problem of unclear details, so this paper combines this method with a pulse coupled neural network (PCNN). By studying two receptive field models of retinal nerve cells, six dual-mode cell imaging mechanisms of rattlesnakes and their mathematical models and the PCNN model, an improved fusion method of infrared and visible images was proposed. For the proposed fusion method, eleven groups of source images were used, and three non-reference image quality evaluation indexes were compared with seven other fusion methods. The experimental results show that the improved algorithm proposed in this paper is better overall than the comparison method for the three evaluation indexes.

Snake eyes: Characterization of topical ocular exposures from rattlesnakes in Arizona.

Patients occasionally present with reports of ocular exposure to fluids from rattlesnakes, claiming or suspecting the substance to be venom. This study set out to evaluate and characterize reported cases of suspected venom-induced ophthalmia in humans. A retrospective review of rattlesnake exposures reported to the Arizona Poison and Drug Information Center over a 24-year period was conducted for ocular exposures. Recorded information included patient demographics, clinical course, laboratory results, and treatments. Documentation regarding interactions between patients and snakes was reviewed by Arizona Poison and Drug Information Center herpetologists to evaluate what substance was expelled from the snake resulting in ocular exposure. Our review of rattlesnake encounters found a total of 26 ocular exposure cases. Patient demographics were largely intentional interactions and involved the male sex. Symptoms ranged from asymptomatic to minor effects with 46.2% managed from home and treated with fluid irrigation. A review of cases by herpetologists concluded the exposure patients commonly experienced was to snake musk. Kinematics of venom expulsion by rattlesnakes conclude the venom gland must be compressed, fangs erected to ≥60o, and fang sheath compressed against the roof of the mouth for venom expulsion. Evidence suggests the chance of venom "spitting" by rattlesnakes is close to zero. Rattlesnakes are documented to forcefully expel airborne malodorous "musk" defensively. An important distinction to remember is musk has a foul odor and is usually colorless, while venom is comparatively odorless and yellow. Rattlesnake venom-induced ophthalmia is a rare event as venom expulsion requires the kinematics of feeding or defensive bites. If the rattlesnake is not in the process of biting or otherwise contacting some other object with its mouth, it is more biologically plausible patients are being exposed to snake musk as a deterrent. Whether it's venom or musk, topical exposure to the eyes should prompt immediate irrigation.

Fasciotomy following North American pit viper envenomation in Texas 2004-2021.

INTRODUCTION: North American pit viper envenomation occurs over 4,000 times annually in the United States, with polyvalent Fab antivenom being the primary treatment. Fasciotomy is occasionally performed due to concerns about compartment syndrome. We utilized our direct access to Texas Poison Center Network data to create a new snakebite abstraction form and database on relevant available information between 2004 and 2021 and to identify, describe, and estimate the incidence of fasciotomy following pit viper envenomation in Texas. METHODS: We searched the Texas Poison Center Network database for cases during 2004-2021 using keywords such as fasciotomy, surgery, compartment pressure, and compartment syndrome. Descriptive statistics summarized the data. RESULTS: Of 16,911 reported envenomations, 0.69 percent involved fasciotomies (n = 117). Most common bite sites were digits/hands and lower extremities. Patients who underwent fasciotomy were typically male, aged 20-59, and 10 years younger than the total snakebite population. Only 6 percent of reported compartment syndrome cases had a compartment pressure measurement. Antivenom was administered in 101 (86.3 percent) cases, 92 (91.1 percent) of which received only Fab antivenom product. Patients with bites from rattlesnakes (47.9 percent) were associated with most fasciotomies. DISCUSSION: Our findings suggest a potential increase in snakebite exposures, accompanied by a decrease in fasciotomies. Overall, copperheads constituted the majority of snakebites, but most fasciotomies were from rattlesnake envenomations (47.9 percent). In this cohort, compartment syndrome diagnosis and decisions regarding fasciotomy were primarily based on clinical evaluation/surgeon expertise without compartment pressure measurements. Despite the efficacy of antivenom, only 86.3 percent of patients in our study received antivenom. CONCLUSIONS: Fasciotomy after North American pit viper envenomation in Texas is uncommon (0.69 percent) and has decreased over time, possibly due to increased antivenom use or surgeon comfort with nonsurgical management.

The past, present, and future of predator-prey interactions in a warming world: Using species distribution modeling to forecast ectotherm-endotherm niche overlap.

Climate change has the potential to disrupt species interactions across global ecosystems. Ectotherm-endotherm interactions may be especially prone to this risk due to the possible mismatch between the species in physiological response and performance. However, few studies have examined how changing temperatures might differentially impact species' niches or available suitable habitat when they have very different modes of thermoregulation. An ideal system for studying this interaction is the predator-prey system. In this study, we used ecological niche modeling to characterize the niche overlap and examine biogeography in past and future climate conditions of prairie rattlesnakes (Crotalus viridis) and Ord's kangaroo rats (Dipodomys ordii), an endotherm-ectotherm pair typifying a predator-prey species interaction. Our models show a high niche overlap between these two species (D = 0.863 and I = 0.979) and further affirm similar paleoecological distributions during the last glacial maximum (LGM) and mid-Holocene (MH). Under future climate change scenarios, we found that prairie rattlesnakes may experience a reduction in overall suitable habitat (RCP 2.6 = -1.82%, 4.5 = -4.62%, 8.5 = -7.34%), whereas Ord's kangaroo rats may experience an increase (RCP 2.6 = 9.8%, 4.5 = 11.71%, 8.5 = 8.37%). We found a shared trend of stable suitable habitat at northern latitudes but reduced suitability in southern portions of the range, and we propose future monitoring and conservation be focused on those areas. Overall, we demonstrate a biogeographic example of how interacting ectotherm-endotherm species may have mismatched responses under climate change scenarios and the models presented here can serve as a starting point for further investigation into the biogeography of these systems.

Snake venom cysteine-rich secretory protein from Mojave rattlesnake venom (Css-CRiSP) induces acute inflammatory responses on different experimental models.

Snake venoms contain various molecules known for activating innate immunity and causing local effects associated with increased vascular permeability, such as vascular leakage and edema, common symptoms seen in snakebite envenomings. We have demonstrated that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability. This study aimed to explore the functional role of CRiSP isolated from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom (Css-CRiSP) on the activation of inflammatory responses in different models. We measured the release of inflammatory mediators in cultured human dermal blood endothelial cells (HDBEC), lymphatic endothelial cells (HDLEC) and monocyte-derived macrophages (MDM) at 0.5, 1, 3, 6, and 24 h after treatment with Css-CRiSP (1 µM). We also determined the acute inflammatory response in BALB/c mice 30 min after intraperitoneal injection of the toxin (2 µg/mouse). Css-CRiSP induced the production of IL-8 and IL-6, but not TNF-α, in HDBEC and HDLEC in a time-dependent manner. In addition, Css-CRiSP significantly enhanced the production of IL-6, TNF-α, IL-8, and IL-1ß in MDM. Moreover, it caused a remarkable increase of chemotactic mediators in the exudates of experimental mice. Our results reveal that Css-CRiSPs can promote a sustained release of inflammatory mediators on cell lines and an acute activation of innate immunity in a murine model. These findings contribute to the growing body of evidence supporting the involvement of svCRiSPs in the augmentation of envenomation effects, specifically, the role of svCRiSPs in inducing vascular dysfunction, initiating early inflammatory responses, and facilitating the activation of leukocytes and releasing mediators. These findings will lead to a better understanding of the pathophysiology of envenoming by Mojave rattlesnakes, allowing the development of more efficient therapeutic strategies.

Crotoxin Modulates Macrophage Phenotypic Reprogramming.

Macrophage plasticity is a fundamental feature of the immune response since it favors the rapid and adequate change of the functional phenotype in response to the pathogen or the microenvironment. Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has a long-lasting antitumor effect both in experimental models and in clinical trials. In this study, we show the CTX effect on the phenotypic reprogramming of macrophages in the mesenchymal tumor microenvironment or those obtained from the peritoneal cavity of healthy animals. CTX (0.9 or 5 µg/animal subcutaneously) administered concomitantly with intraperitoneal inoculation of tumor cells (1 × 107/0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT) modulated the macrophages phenotype (M1), accompanied by increased NO• production by cells from ascites, and was evaluated after 13 days. On the other hand, in healthy animals, the phenotypic profile of macrophages was modulated in a dose-dependent way at 0.9 µg/animal: M1 and at 5.0 µg/animal: M2; this was accompanied by increased NO• production by peritoneal macrophages only for the dose of 0.9 µg/animal of CTX. This study shows that a single administration of CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of cells from ascites, influencing events involved with mesenchymal tumor progression. These findings may favor the selection of new therapeutic targets to correct compromised immunity in different systems.

Fission-fusion dynamics in the social networks of a North American pitviper.

Many animal species exist in fission-fusion societies, where the size and composition of conspecific groups change spatially and temporally. To help investigate such phenomena, social network analysis (SNA) has emerged as a powerful conceptual and analytical framework for assessing patterns of interconnectedness and quantifying group-level interactions. We leveraged behavioral observations via radiotelemetry and genotypic data from a long-term (>10 years) study on the pitviper Crotalus atrox (western diamondback rattlesnake) and used SNA to quantify the first robust demonstration of social network structures for any free-living snake. Group-level interactions among adults in this population resulted in structurally modular networks (i.e., distinct clusters of interacting individuals) for fidelis use of communal winter dens (denning network), mating behaviors (pairing network), and offspring production (parentage network). Although the structure of each network was similar, the size and composition of groups varied among them. Specifically, adults associated with moderately sized social groups at winter dens but often engaged in reproductive behaviors-both at and away from dens-with different and fewer partners. Additionally, modules formed by individuals in the pairing network were frequently different from those in the parentage network, likely due to multiple mating, long-term sperm storage by females, and resultant multiple paternity. Further evidence for fission-fusion dynamics exhibited by this population-interactions were rare when snakes were dispersing to and traversing their spring-summer home ranges (to which individuals show high fidelity), despite ample opportunities to associate with numerous conspecifics that had highly overlapping ranges. Taken together, we show that long-term datasets incorporating SNA with spatial and genetic information provide robust and unique insights to understanding the social structure of cryptic taxa that are understudied.

Compartment Syndrome after Crotalid Envenomation in the United States: A Review of the North American Snakebite Registry from 2013 to 2021 on Behalf of the ToxIC Snakebite Study Group.

INTRODUCTION: Compartment syndrome (CS) is a rare but serious complication after crotalid envenomation in the United States. Few data are available regarding the epidemiology and management of these cases. Significant controversy and misunderstanding over best practices, including measurement of compartment pressures and use of fasciotomy, exist for this syndrome. This study aims to describe presentation and management of suspected CS cases after native snakebite reported to the North American Snakebite Registry (NASBR). METHODS: This is an analysis of snakebite cases reported to the Toxicology Investigators Consortium NASBR between January 1, 2013 and December 31, 2021. Cases of native snakebite with documented concern for CS were included. RESULTS: Over an 8-y period, 22 cases of suspected CS were identified, representing 1% of all cases reported to the NASBR. Fasciotomies were performed in 41% (n=9) of these cases, most commonly to the upper extremity (67%, n=6). In cases of suspected CS, intracompartmental pressures (ICPs) were rarely measured (23%, n=5) and fasciotomies were performed without measurement of ICPs frequently (56%, n=5). In 1 case, ICPs were measured and found to be low (8 mm Hg) and fasciotomy was avoided. CONCLUSIONS: Measurement of compartment pressures in cases of suspected CS was uncommon in cases reported to the NASBR. Fasciotomy was commonly performed without measurement of compartment pressures.

Refractory compartment syndrome after antivenom administration for an Eastern Diamondback Rattlesnake bite requiring fasciotomy for limb salvage: A case report.

Case: 56-year-old male who developed post-snakebite compartment syndrome (PSCS) of the upper extremity which was refractory to antivenom administration. He had elevated compartment pressure measurements in his upper extremity. He underwent open fasciotomy for the compartment syndrome, followed by delayed primary closure and skin grafting. He now has two years of follow-up with a functional upper extremity. Conclusion: This is a case of a patient who suffered post-snakebite compartment syndrome from his pet Eastern Diamondback Rattlesnake. This case highlights the importance of correctly diagnosing compartment syndrome and validates fasciotomy as a treatment measure for confirmed post-snakebite compartment syndrome (PSCS).

Diet of a threatened rattlesnake (eastern massasauga) revealed by DNA metabarcoding.

Characterizing the diet of imperiled species using minimally invasive methods is crucial to understanding their ecology and conservation requirements. Here, we apply a DNA metabarcoding approach to study the diet of the eastern massasauga rattlesnake (Sistrurus catenatus), a Federally Threatened snake found throughout the Great Lakes region. Eighty-three fecal samples collected across 10 different massasauga populations located in Michigan, USA, were sequenced, with 70 samples containing prey DNA. We used universal metazoan primers and developed a host-specific oligonucleotide blocker to characterize their diet. We identified at least 12 different prey species, with eastern massasaugas exhibiting opportunistic feeding and a strong preference towards small mammals. Meadow voles (Microtus pennsylvanicus) were the most common prey item (70% of diet) followed by the northern short-tailed shrew (Blarina brevicauda) and masked shrew (Sorex cinereus; 15.7% of diet each), along with occasional bird and snake prey. Adult individuals exhibited a more generalized diet, consuming a larger number of prey taxa on average. Younger snakes consumed a smaller variety of prey items and tended to consume smaller-sized mammals such as masked shrews (Sorex cinereus) and northern short-tailed shrews (Blarina brevicauda). We conclude that small mammals are a crucial part of eastern massasauga rattlesnake diet and recommend this be taken into consideration when conservation strategies are developed. The methods developed in this study can be applied to other reptile species, providing an accurate, minimally invasive, and thorough diet assessment for at-risk reptile species.