PROTOCOL FOR HUMAN EXPOSURE TO OPIOIDS AND CONCENTRATED MEDETOMIDINE USED IN FIELD APPLICATIONS.

Wildlife professionals routinely use potent sedatives and anesthetics when chemically immobilizing wildlife and zoo species in remote environments. Accidental exposure to these prescription veterinary drugs is rare but could be rapidly fatal. Commonly used agents include opioids and α2 adrenoreceptor agonists. These drugs can be reversed with specific antagonists; however, they are often not approved for human use. The protocol created here can be used by wildlife health professionals in a field setting with basic human emergency medical response training in coordination with local Emergency Medical Services (EMS). Key components include, building local relationships between EMS and wildlife professionals, focused EMS training, administering opioid and α2 adrenergic antagonists off label, and local evacuation procedures. This framework could allow wildlife management agencies or zoos to mitigate the risk of human exposures to these commonly used drugs, significantly improving occupational safety in an otherwise high-risk environment.

Predicting climate-change induced heat-related illness risk in Grand Canyon National Park visitors.

BACKGROUND: The climate crisis is the greatest public health threat of the 21st century. Excessive heat is responsible for more deaths than any other extreme weather event, and the frequency, intensity, and duration of extreme heat events are increasing globally due to climate change. Exposure to excessive heat can result in heat related illnesses (HRIs) and long-term poor health outcomes. Physical exertion, sudden exposure to excessive heat, and the lack of physical or behavioral adaptation resources are all associated with greater HRI risk, which is expected to increase for visitors to Grand Canyon National Park (GCNP) and other public lands as climate change worsens. OBJECTIVES: Our objectives were to understand 1) the relationship between weather and HRI in GCNP visitors, 2) how future HRI rates may change, and 3) how land management agencies can update risk mitigation strategies to match changing risk and better manage an increased HRI burden. METHODS: We utilized previously published data on HRI in GCNP visitors, and records of daily visitation, temperatures, and maximum and minimum daily humidity from the same study period to develop a model estimate for HRI risk. We then used future climate projections from the World Climate Research Programme's Coupled Model Intercomparison Project phase 5 multi-model dataset to model future HRI risk under different climate scenarios. RESULTS: The incidence of HRI was significantly associated with maximum daily temperature and minimum relative humidity, and was more common in the shoulder season months. We estimated that HRI will increase 29%-137% over 2004-2009 levels through 2100, assuming no change in visitation. DISCUSSION: Climate change will continue to increase HRI risk for GCNP visitors and poses risks to public land managers' mission to provide for safe recreation experiences for the benefit of this and future generations in places like GCNP. Excessive risk during the shoulder season months presents an opportunity to increase preventative search and rescue and education efforts to mitigate increased risk.

Patterns and Ecological Mechanisms of Tick-Borne Disease Exposure Risk in Acadia National Park, Mount Desert Island, Maine, United States.

National parks are unique and significant vector-borne pathogen transmission settings, engaging over 300 million people in outdoor recreation per year. In this study, we integrated vector surveys and ecological habitat feature data in spatial models to characterize tick-borne disease exposure risk in Acadia National Park (ANP), Maine. To determine the broad-scale patterns of blacklegged tick Ixodes scapularis Say (Acari: Ixodidae) densities in ANP, we conducted host-seeking tick collections at 114 sites across the park over two years. Using these tick survey data and geospatial landscape feature data (i.e., land cover, elevation, forest patch size, and aspect) we developed a random forest model of nymphal tick density. We found that host-seeking tick density varies significantly across the park and is particularly high in areas characterized by deciduous forest cover and relatively low elevation. To explore potential fine-scale ecological drivers of tick density spatial patterns, we quantified microclimate conditions, host activity, and vegetation characteristics at a subset of 19 sites. We identified significant differences in microclimate conditions but not host activity or vegetation metrics across broad-scale landscape feature classes. Mean temperature and mean humidity were correlated to nymphal densities and therefore may provide a mechanistic link between landscape features and blacklegged tick densities. Finally, we detected multiple tick-borne pathogens in both ticks and small mammals sampled in ANP, including Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum. Our findings demonstrate the value of using ecological metrics to estimate vector-borne disease exposure risk and provide insight into habitat characteristics that may drive tick-borne disease exposure risk.

Rabies as a Potential Cause of Mass Mortality Events in North American Bat Species.

Rabies-association bat mortality events are rarely reported, suggesting that rabies is not a significant cause of bat mass mortality. Three bat die-offs in National Park Service units were attributed to rabies, highlighting the value of including rabies, and rabies virus strain spillover events, as a differential in mass mortality events.

Lake Michigan insights from island studies: the roles of chipmunks and coyotes in maintaining Ixodes scapularis and Borrelia burgdorferi in the absence of white-tailed deer.

Deer management (e.g., reduction) has been proposed as a tool to reduce the acarological risk of Lyme disease. There have been few opportunities to investigate Ixodes scapularis (blacklegged tick) and Borrelia burgdorferi sensu stricto dynamics in the absence of white-tailed deer (Odocoileus virginianus) in midwestern North America. A pair of islands in Lake Michigan presented a unique opportunity to study the role of alternative hosts for the adult stage of the blacklegged tick for maintaining a tick population as a deer herd exists on North Manitou Island but not on South Manitou Island, where coyotes (Canis latrans) and hares (Lepus americanus) are the dominant medium mammals. Additionally, we were able to investigate the maintenance of I. scapularis and B. burgdorferi in small mammal communities on both islands, which were dominated by eastern chipmunks (Tamias striatus). From 2011 to 2015, we surveyed both islands for blacklegged ticks by drag cloth sampling, bird mist netting, and small and medium-sized mammal trapping. We assayed questing ticks, on-host ticks, and mammal biopsies for the Lyme disease pathogen, B. burgdorferi. We detected all three life stages of the blacklegged tick on both islands. Of the medium mammals sampled, no snowshoe hares (Lepus americanus, 0/23) were parasitized by adult blacklegged ticks, but 2/2 coyotes (Canis latrans) sampled on South Manitou Island in 2014 were parasitized by adult blacklegged ticks, suggesting that coyotes played a role in maintaining the tick population in the absence of deer. We also detected I. scapularis ticks on passerine birds from both islands, providing support that birds contribute to maintaining as well as introducing blacklegged ticks and B. burgdorferi to the islands. We observed higher questing adult and nymphal tick densities, and higher B. burgdorferi infection prevalence in small mammals and in adult ticks on the island with deer as compared to the deer-free island. On the islands, we also found that 25% more chipmunks were tick-infested than mice, fed more larvae and nymphs relative to their proportional abundance compared to mice, and thus may play a larger role compared to mice in the maintenance of B. burgdorferi. Our investigation demonstrated that alternative hosts could maintain a local population of blacklegged ticks and an enzootic cycle of the Lyme disease bacterium in the absence of white-tailed deer. Thus, alternative adult blacklegged tick hosts should be considered when investigating deer-targeted management tools for reducing tick-borne disease risk, especially when the alternative host community may be abundant and diverse.

Tularemia (Francisella tularensis) in a Black-Tailed Prairie Dog (Cynomys ludovicianus) Colony.

Tularemia is a bacterial zoonosis caused by Francisella tularensis. We conducted a serosurvey of black-tailed prairie dogs (Cynomys ludovicianus) in Devils Tower National Monument, Wyoming, US, following an epizootic in voles ( Microtus spp.) due to F. tularensis. Only 1 of 44 (2%) sampled prairie dogs was seropositive for F. tularensis, providing evidence of survival and potentially limited spread among free-ranging prairie dogs.

GREEN TREE FROG ( HYLA CINEREA) AND GROUND SQUIRREL ( XEROSPERMOPHILUS SPILOSOMA) MORTALITY ATTRIBUTED TO INLAND BREVETOXIN TRANSPORT AT PADRE ISLAND NATIONAL SEASHORE, TEXAS, USA, 2015.

: On 16 September 2015, a red tide ( Karenia brevis) bloom impacted coastal areas of Padre Island National Seashore Park, Texas, US. Two days later and about 0.9 km inland, 30-40 adult green tree frogs ( Hyla cinerea) were found dead after displaying tremors, weakness, labored breathing, and other signs of neurologic impairment. A rainstorm accompanied by high winds, rough surf, and high tides, which could have aerosolized brevetoxin, occurred on the morning of the mortality event. Frog carcasses were in good body condition but contained significant brevetoxin in tissues. Tissue brevetoxin was also found in two dead or dying spotted ground squirrels ( Xerospermophilus spilosoma) and a coyote ( Canis latrans) found in the area. Rainwater collected from the location of the mortality event contained brevetoxin. Green tree frog and ground squirrel mortality has not been previously attributed to brevetoxin exposure and such mortality suggested that inland toxin transport, possibly through aerosols, rainfall, or insects, may have important implications for coastal species.

Prevalence and Diversity of Tick-Borne Pathogens in Nymphal Ixodes scapularis (Acari: Ixodidae) in Eastern National Parks.

Tick-borne pathogens transmitted by Ixodes scapularis Say (Acari: Ixodidae), also known as the deer tick or blacklegged tick, are increasing in incidence and geographic distribution in the United States. We examined the risk of tick-borne disease exposure in 9 national parks across six Northeastern and Mid-Atlantic States and the District of Columbia in 2014 and 2015. To assess the recreational risk to park visitors, we sampled for ticks along frequently used trails and calculated the density of I. scapularis nymphs (DON) and the density of infected nymphs (DIN). We determined the nymphal infection prevalence of I. scapularis with a suite of tick-borne pathogens including Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum, and Babesia microti. Ixodes scapularis nymphs were found in all national park units; DON ranged from 0.40 to 13.73 nymphs per 100 m2. Borrelia burgdorferi, the causative agent of Lyme disease, was found at all sites where I. scapularis was documented; DIN with B. burgdorferi ranged from 0.06 to 5.71 nymphs per 100 m2. Borrelia miyamotoi and A. phagocytophilum were documented at 60% and 70% of the parks, respectively, while Ba. microti occurred at just 20% of the parks. Ixodes scapularis is well established across much of the Northeastern and Mid-Atlantic States, and our results are generally consistent with previous studies conducted near the areas we sampled. Newly established I. scapularis populations were documented in two locations: Washington, D.C. (Rock Creek Park) and Greene County, Virginia (Shenandoah National Park). This research demonstrates the potential risk of tick-borne pathogen exposure in national parks and can be used to educate park visitors about the importance of preventative actions to minimize tick exposure.

A One Health Message about Bats Increases Intentions to Follow Public Health Guidance on Bat Rabies.

Since 1960, bat rabies variants have become the greatest source of human rabies deaths in the United States. Improving rabies awareness and preventing human exposure to rabid bats remains a national public health priority today. Concurrently, conservation of bats and the ecosystem benefits they provide is of increasing importance due to declining populations of many bat species. This study used a visitor-intercept experiment (N = 521) in two U.S. national parks where human and bat interactions occur on an occasional basis to examine the relative persuasiveness of four messages differing in the provision of benefit and uncertainty information on intentions to adopt a rabies exposure prevention behavior. We found that acknowledging benefits of bats in a risk message led to greater intentions to adopt the recommended rabies exposure prevention behavior without unnecessarily stigmatizing bats. These results signify the importance of communicating benefits of bats in bat rabies prevention messages to benefit both human and wildlife health.

The role of one health in wildlife conservation: a challenge and opportunity.

Numerous emerging infectious diseases (EIDs) have arisen from or been identified in wildlife, with health implications for both humans and wildlife. In the practice of wildlife conservation, to date most attention has focused on the threat EIDs pose to biodiversity and wildlife population viability. In the popular media and public eye, however, wildlife is often only portrayed as the cause of EIDs and resultant human health impacts. There is little coverage on the roles of human-induced habitat destruction or wildlife population stress in EID spread, nor the negative impacts of disease on wildlife. Here, we focus on a little-studied and seldom discussed concern: how real and perceived risks of wildlife-associated diseases for human and companion animal health might erode public support for wildlife conservation. We believe that wildlife-associated EIDs and public perceptions of these risks are among the most important threats to wildlife conservation. In light of this concern, we explore the challenges and opportunities for addressing this situation in a One Health context that emphasizes the interdisciplinary collaboration and the inextricable nature of human and animal health and disease.

Lipid modulation of calcium flux through CaV2.3 regulates acrosome exocytosis and fertilization.

Membrane lipid regulation of cell function is poorly understood. In early development, sterol efflux and the ganglioside GM1 regulate sperm acrosome exocytosis (AE) and fertilization competence through unknown mechanisms. Here, we show that sterol efflux and focal enrichment of GM1 trigger Ca(2+) influx necessary for AE through CaV2.3, whose activity has been highly controversial in sperm. Sperm lacking CaV2.3's pore-forming α1E subunit showed altered Ca(2+) responses, reduced AE, and a strong subfertility phenotype. Surprisingly, AE depended on spatiotemporal information encoded by flux through CaV2.3, not merely the presence/amplitude of Ca(2+) waves. Using studies in both sperm and voltage clamp of Xenopus oocytes, we define a molecular mechanism for GM1/CaV2.3 regulatory interaction, requiring GM1's lipid and sugar components and CaV2.3's α1E and α2δ subunits. Our results provide a mechanistic understanding of membrane lipid regulation of Ca(2+) flux and therefore Ca(2+)-dependent cellular and developmental processes such as exocytosis and fertilization.

Associations between serum levels of polybrominated diphenyl ether (PBDE) flame retardants and environmental and behavioral factors in pregnant women.

Polybrominated diphenyl ethers (PBDE) are flame retardants that were previously used in upholstery, fabrics, and household appliances. PBDEs have been linked to adverse health outcomes, including neurotoxicity, thyroid hormone dysregulation, endocrine disruption, and poor semen quality. Because PBDEs pass into placental circulation, maternal exposures can approximate fetal exposures. Our objectives were to determine whether diet and specific human behaviors were significantly associated with PBDE exposures in a cohort of pregnant women. Women between the 34th and 38th week of pregnancy were given a questionnaire about behavioral, environmental, and dietary factors and asked to provide blood samples. Serum PBDE levels were measured using GS-MS and lipid adjusted. An adjusted ordinary least squares regression model was run to identify potential associations between behaviors and serum PBDE levels. Serum concentrations of BDEs 47, 99, 100, and 153 were found above the limit of detection in at least 50% of study participants and used in our models. Associations with serum PBDEs were observed with self-reported hand-to-mouth behaviors, including biting nails and licking fingers. Serum BDE levels of 47, 99, 153, and total PBDEs were also significantly higher in those individuals owning a large-screen TV compared with those who did not. Serum PBDE levels were comparable to levels reported in the general population. Hand-to-mouth behaviors may influence serum PBDE concentrations in adults. Household electronics such as large-screen TVs appear to serve as a significant source of PBDEs in pregnant women. Together, hand-to-mouth behaviors and TV ownership may serve as a route of exposure to PBDEs in adults.

Exposures to endocrine-disrupting chemicals and age of menarche in adolescent girls in NHANES (2003-2008).

BACKGROUND: The observed age of menarche has fallen, which may have important adverse social and health consequences. Increased exposure to endocrine-disrupting compounds (EDCs) has been associated with adverse reproductive outcomes. OBJECTIVE: Our objective was to assess the relationship between EDC exposure and the age of menarche in adolescent girls. METHODS: We used data from female participants 12-16 years of age who had completed the reproductive health questionnaire and laboratory examination for the Centers for Disease Control and Prevention's National Health and Nutrition Examination Survey (NHANES) for years 2003-2008 (2005-2008 for analyses of phthalates and parabens). Exposures were assessed based on creatinine-corrected natural log urine concentrations of selected environmental chemicals and metabolites found in at least 75% of samples in our study sample. We used Cox proportional hazards analysis in SAS 9.2 survey procedures to estimate associations after accounting for censored data among participants who had not reached menarche. We evaluated body mass index (BMI; kilograms per meter squared), family income-to-poverty ratio, race/ethnicity, mother's smoking status during pregnancy, and birth weight as potential confounders. RESULTS: The weighted mean age of menarche was 12.0 years of age. Among 440 girls with both reproductive health and laboratory data, after accounting for BMI and race/ethnicity, we found that 2,5-dichlorophenol (2,5-DCP) and summed environmental phenols (2,5-DCP and 2,4-DCP) were inversely associated with age of menarche [hazard ratios of 1.10; 95% confidence interval (CI): 1.01, 1.19 and 1.09; 95% CI: 1.01, 1.19, respectively]. Other exposures (total parabens, bisphenol A, triclosan, benzophenone-3, total phthalates, and 2,4-DCP) were not significantly associated with age of menarche. CONCLUSIONS: Our findings suggest an association between 2,5-DCP, a potential EDC, and earlier age of menarche in the general U.S. population.

Characterization of Animal Exposure Calls Captured by the National Poison Data System, 2000-2010.

OBJECTIVE: Our objective was to characterize the data captured in all animal exposure calls reported to the National Poison Data System (NPDS), a national poison center reporting database, from 1 January 2000 through 31 December 2010 and identify Poison Center usage and needs in animal exposure calls. DESIGN: We calculated descriptive statistics characterizing animal type, exposure substance, medical outcome, year and month of call, caller location, and specific state for all animal exposure call data in NPDS from 1 January 2000 to 31 December 2010. SAS version 9.2 was used for the analysis. RESULTS: There were 1,371,095 animal exposure calls out of 28,925,496 (4.7%) total human and animal exposure calls in NPDS during the study period. The majority involved companion animal exposures with 88.0% canine exposures and 10.4% feline exposures. Pesticides were the most common exposure substance (n=360,375; 26.3%), followed by prescription drugs (n=261,543; 18.6%). The most common outcome reported was 'Not followed, judged as nontoxic exposure or minimal clinical effects possible' (n=803,491; 58.6%), followed by 'Not followed, judged potentially toxic exposure' (n=263,153; 19.2%). There were 5,388 deaths reported. Pesticide exposures were responsible for the greatest number of deaths (n=1,643; 30.4%). CONCLUSIONS AND CLINICAL RELEVANCE: Approximately 1 in 20 calls to PCs are regarding potentially toxic exposures to animals, suggesting a need for veterinary expertise and resources at PCs. Pesticides are one of the greatest toxic exposure threats to animals, both in numbers of exposures and severity of clinical outcomes, and is an important area for education, prevention, and treatment.

Toxicology, environmental health, and the "One Health" concept.

The One Health concept promotes collaboration among veterinarians, physicians, scientists, and other professions to promote human, animal, and ecosystem health. One Health illustrates the interconnectedness and interdependence of human, animal, and ecosystem health. This concept has traditionally focused on zoonoses that are infectious diseases, not on chemical- or poison-related illnesses in animals and their relationship to the detection and prevention of human illness. The purpose of this article is to describe key experiences of scientists in the Health Studies Branch within the National Center for Environmental of Health of the Centers for Disease Control and Prevention in which the study of animal illness facilitated a public health investigation into an outbreak of chemical-associated human disease. The experiences highlight how utilizing the One Health approach may improve chemical-associated outbreak investigations and facilitate appropriate intervention strategies. An appropriate One Health approach in toxicology and environmental health in outbreak settings should include consideration of the common environments and food sources shared by humans and animals and consideration of the potential for contaminated animal products as food sources in human exposures.

Mechanisms underlying the micron-scale segregation of sterols and GM1 in live mammalian sperm.

We demonstrate for the first time that a stable, micron-scale segregation of focal enrichments of sterols exists at physiological temperature in the plasma membrane of live murine and human sperm. These enrichments of sterols represent microheterogeneities within this membrane domain overlying the acrosome. Previously, we showed that cholera toxin subunit B (CTB), which binds the glycosphingolipid, G(M1), localizes to this same domain in live sperm. Interestingly, the G(M1) undergoes an unexplained redistribution upon cell death. We now demonstrate that G(M1) is also enriched in the acrosome, an exocytotic vesicle. Transfer of lipids between this and the plasma membrane occurs at cell death, increasing G(M1) in the plasma membrane without apparent release of acrosomal contents. This finding provides corroborative support for an emerging model of regulated exocytosis in which membrane communications might occur without triggering the "acrosome reaction." Comparison of the dynamics of CTB-bound endogenous G(M1) and exogenous BODIPY-G(M1) in live murine sperm demonstrate that the sub-acrosomal ring (SAR) functions as a specialized diffusion barrier segregating specific lipids within the sperm head plasma membrane. Our data show significant differences between endogenous lipids and exogenous lipid probes in terms of lateral diffusion. Based on these studies, we propose a hierarchical model to explain the segregation of this sterol- and G(M1)-enriched domain in live sperm, which is positioned to regulate sperm fertilization competence and mediate interactions with the oocyte. Moreover, our data suggest potential origins of subtypes of membrane raft microdomains enriched in sterols and/or G(M1) that can be separated biochemically.

Biochemical characterization of membrane fractions in murine sperm: identification of three distinct sub-types of membrane rafts.

Despite enormous interest in membrane raft micro-domains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components--sterols, phospholipids, and proteins--or additional raft-associating lipids such as the ganglioside, G(M1). Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in G(M1) and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly used detergent-based approaches, we utilized a non-detergent-based method, separating membrane fractions that were reproducibly distinct based on sterol, G(M1), phospholipid, and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible sub-types of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub-types, but that multiple membrane micro-domain sub-types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either "raft" or "non-raft" oversimplifies the actual biochemical complexity of cellular membranes.

GM1 dynamics as a marker for membrane changes associated with the process of capacitation in murine and bovine spermatozoa.

We previously showed that in live murine and bovine sperm heads, the ganglioside G(M1) localizes to the sterol-rich plasma membrane overlying the acrosome (APM). Labeling G(M1) using the pentameric cholera toxin subunit B (CTB) induced a dramatic redistribution of signal from the APM to the sterol-poor postacrosomal plasma membrane (PAPM) upon sperm death. We now show a similar phenomenon in the flagellum where CTB induces G(M1) redistribution to sterol-poor membrane subdomains of the annulus and flagellar zipper. Because sterol efflux from the plasma membrane is required for capacitation, we examined whether G(M1) localization might be useful to detect membrane changes associated with capacitation and/or acrosomal exocytosis. First, incubation of murine and bovine sperm with their respective stimuli for capacitation did not change G(M1) distribution in live cells. However, incubation of sperm of both species with specific stimuli for capacitation, followed by the use of specific fixation conditions, induced reproducible, stimulus-specific patterns of G(M1) distribution. By assessing changes in G(M1) distribution in response to progesterone-induced AE, we show that these patterns reflect the response of murine sperm populations to capacitating stimuli. These data suggest that G(M1) localization can be used as a diagnostic tool for evaluating sperm response to stimuli for capacitation and/or AE. Such information could be useful when deciding between technologies of assisted reproduction or when screening for male fertility. Furthermore, stimulus-specific changes in G(M1) distribution showed that sperm could respond to NaHCO(3) or mediators of sterol efflux independently, thereby refining existing models of capacitation.

Visualization of GM1 with cholera toxin B in live epididymal versus ejaculated bull, mouse, and human spermatozoa.

The organization of membrane subdomains in mammalian sperm has recently generated controversy, with several reports describing widely differing localization patterns for the ganglioside GM1. Using the pentameric B subunit of cholera toxin (CTB), we found GM1 to be restricted to the plasma membrane overlying the acrosome in the heads of live murine sperm. Interestingly, CTB had minimal binding to live bovine and human sperm. To investigate whether this difference in GM1 localization was because of species differences or differences between collection from the epididymis (mouse) or an ejaculate (bull, human), we examined epididymal bovine and human sperm. We found that GM1 localized to the plasma membrane overlying the acrosome in sperm from these species. To determine whether some component of seminal plasma was interfering with the ability of CTB to access GM1, we incubated epididymal mouse sperm with fluid from murine seminal vesicles and epididymal bull sperm with bovine seminal plasma. This treatment largely abolished the ability of the CTB to bind to GM1, producing a fluorescence pattern similar to that reported for the human. The most abundant seminal plasma protein, PDC-109, was not responsible for this loss. As demonstration that the seminal plasma was not removing GM1, sperm exposed to seminal plasma were fixed before CTB addition, and again displayed fluorescence over the acrosome. These observations reconcile inconsistencies reported for the localization of GM1 in sperm of different species, and provide evidence for the segregation of GM1 to a stable subdomain in the plasma membrane overlying the acrosome.