Evolution, Medicine, and Public Health

Filoviruses, including the likely bat-borne Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fevers in humans. The 2013-2016 EBOV outbreak caused >11,000 human fatalities in Guinea, Liberia, and Sierra Leone. Nearby countries, including Ghana, have been under sampled for filoviruses relative to West African countries where large outbreaks have occurred. While there have been no reported EBOV disease cases in Ghana, there were two fatal MARV disease cases in 2022, suggesting that at least one filovirus is circulating in the country. In this study, we investigated filovirus circulation in fruit bats and humans in Ghana. We leveraged an extensive serological dataset collected from multiple fruit bat species (n = 6,874) and humans (n = 1,300) across a decade in Ghana (2010-2020), including both rural and urban regions. We observed evidence of MARV circulation in Rousettus aegyptiacus bats, a presumed reservoir, as well as occasional seropositivity in sympatric bat species, suggesting that these other bats are incidental, dead-end hosts. Multivariate analyses suggested that multiple, partially cross-reactive filoviruses are circulating among fruit bats in Ghana. Finally, people who reported spending time in caves and hunting bats had higher serological reactivity against EBOV and MARV relative to individuals with no direct bat exposure, indicating possible undetected filovirus spillover at the bat-human interface in Ghana.

Dormancy is a widespread life-history strategy that enables organisms to persist through periods of adverse environmental conditions. Despite its prevalence, the evolutionary forces shaping dormancy and the timing of reactivation remain poorly understood, particularly in pathogens facing predictable environmental fluctuations. Here, we investigate how seasonal variation can drive the joint evolution of pathogen dormancy and reactivation, and whether these traits are favoured to evolve as fixed or plastic strategies. Using a theoretical model of vector-borne disease transmission, we show when seasonality can promote plasticity in dormancy and reactivation. The optimal timing of transitions between active and dormant states depends critically on the environmental cues available to pathogens and on their reliability for predicting future transmission opportunities. Although motivated by the biology of relapsing malaria parasites, our results provide a general framework for understanding the evolution of dormancy as an adaptive response to periodic environmental fluctuations across diverse pathogen systems.

Doxycycline post-exposure prophylaxis (doxy-PEP) represents major advancements in sexually transmitted infection prevention for men who have sex with men (MSM), yet the joint long-term population-level impact on Neisseria gonorrhoeae transmission dynamics and antimicrobial resistance (AMR) together with a moderately effective vaccine remains uncertain. Here, we developed a deterministic transmission model calibrated to empirical surveillance data from England to evaluate the 15-year epidemiological interactions of implementing doxy-PEP and vaccination at the population level. The model incorporates the tetracycline- and ceftriaxone-susceptible, tetracycline-resistant (Tet-R), ceftriaxone-resistant (Cef-R), and dual-resistant (Dual-R) strains. Our simulations suggest that the unmitigated deployment of doxy-PEP provides only modest reductions in overall gonorrhoea burden, yielding a net programmatic efficiency of 0.072 (95% CrI: 0.018 - 0.23) averted infections per enrolment over the 15-year horizon. Although doxy-PEP reduces tetracycline- and ceftriaxone-susceptible infections, it consistently selects for Tet-R lineages. Conversely, standalone vaccination yields substantially greater epidemiological benefit, averting 0.73 (95% CrI: 0.17 - 1.63) overall infections per enrolment. When deployed in tandem, the dual intervention strategy achieves the greatest overall effectiveness to 0.83 (95% CrI: 0.22 - 1.71) averted infections per enrolment while mitigating the Tet-R selection pressure observed under standalone doxy-PEP. Long-term strain dynamics were found to be governed by background frontline ceftriaxone treatment failure rates rather than intervention uptake. Under a scenario of compromised ceftriaxone efficacy (20% treatment failure), doxy-PEP is projected to favour expansion of Dual-R strain, yielding 510 (95% CrI: 38 -258600) excess Dual-R infections across the 15-year horizon (146.18% [95% CrI: 31.30 - 920.86%] cumulative increase) and triggering sustained transmission exceeding the no-intervention baseline. Conversely, vaccination standalone or combined strategies consistently suppress overall transmission. By year 15, annual Cef-R and Dual-R infections are restricted to less than or equal to 19 infections under both vaccine-inclusive approaches. These findings suggest that the public health utility of antibiotic prophylaxis is heavily contingent upon the preserved efficacy of frontline therapeutics. Our work demonstrates that single-agent prophylaxis risks localized containment of some pathogens at the cost of driving multidrug-resistant selection in others and underscores the necessity of integrating non-selective tools like vaccines to manage N. gonorrhoeae.

Viral populations can experience a dramatic reduction in population size and genetic diversity during transmission between donor and recipient hosts. Transmission bottlenecks can therefore decrease the evolutionary potential of viral populations, slowing adaptation by increasing the strength of genetic drift and decreasing the strength of selection. Recent barcoded influenza experiments in guinea pigs showed that recipient animals receive a diverse viral inoculum but lose most of that diversity within one to two days. The resulting bottleneck therefore arises not at physical transfer but during early viral growth in the recipient. We develop a branching-process framework to quantify how much of this loss follows from stochasticity in early growth alone. Each transmitted lineage is treated as an independent stochastic process governed by measurable viral parameters. We recover these parameters from viral growth rates estimated from observed viral load. A residual filter for each animal then captures any additional loss imposed by the recipient host. Applied to twenty-four recipient animals, the model reveals two distinct groups. For roughly half of the animals, the stochastic extinction during early growth already accounts for the observed loss. For the remaining animals, the additional host filter is severe. Only about one in a hundred free virions pass through. This decomposition offers a quantitative entry point for future work on immune contributions to transmission bottlenecks.

The coexistence of competing pathogen strains is shaped by cross-immunity, the cross-protection that infection with one strain confers against another. Although cross-immunity is often asymmetric between strains, this asymmetry is often neglected in the literature on multi-strain coexistence. The effect on coexistence-exclusion outcomes of waning immunity\textemdash which is particularly relevant for antigenically evolving pathogens\textemdash is also poorly understood. To understand how these factors affect strain coexistence, here we analyze a status-based two-strain SIRS model with asymmetric cross-immunity and strain-specific rates for transmission, recovery, and waning of immunity. We derive explicit invasion thresholds that also determine the feasibility and local stability of a unique coexistence equilibrium. Thus, these thresholds allow us to characterize the region of stable strain coexistence, as a function of the cross-immunities and rates of waning immunity. We also obtain closed-form expressions for the strain prevalences at the coexistence equilibrium, showing that the total prevalence may vary non-monotonically as the basic reproduction number of one strain increases. Finally, we show that a transient reduction in transmission can move a coexisting strain pair across an invasion boundary, driving the weaker strain extinct. Applying this result to influenza B, our analysis offers a parsimonious explanation for the disappearance of the Yamagata lineage during the COVID-19 pandemic.

In the face of an epidemic where a population behaviour both influences disease transmission and reacts to it, social processes can generate norms to support socially beneficial behaviour. Most mathematical models of coupled behaviour disease dynamics treat norms as pre-existing rather than explaining how they are maintained. Here, we investigate whether altruistic punishment can sustain a social distancing norm when individuals may defect, cooperate without punishing, or cooperate while paying a cost to punish defectors. We couple a transmission model to an imitation model for these three strategies. Disease prevalence affects behavioural payoffs, while the behavioural composition modifies transmission. We also compare this coupled system with a control where behavioural decisions respond to a fixed prevalence. We find a wide parameter regime corresponding to the establishment of an injunctive social norm in support of social distancing, where the punisher strategy is widespread. Persistence may occur through stable states where punishers or dominant. Disease behaviour feedback can also create oscillations (where the three strategies succeed one another in response to epidemic waves) or tipping points (sharp transitions between all-defector and cooperative states). These effects do not occur in the uncoupled model, although there are still broad parameter regimes where a social norm persists. Our findings show that costly peer punishment can support persistence of social norms that mitigate disease transmission. More broadly, endogenous epidemic feedback can qualitatively change the conditions under which cooperation and punishment are sustained, producing tipping points and long-term behavioural epidemiological cycles that fixed-payoff models cannot capture.

Malaria molecular surveillance (MMS) is becoming increasingly common in endemic settings and has been proposed as a tool for monitoring parasite transmission to inform programmatic decision-making. However, the conditions under which parasite genetic metrics provide interpretable signals for broader use cases, such as assessing intervention impacts and detecting importation, remain under-characterized. We present EMOD with Full Parasite Genetics (FPG), a simulation framework designed to explore how parasite genetic metrics arise from transmission, intervention, importation, and sampling processes at programmatically relevant timescales. Using seasonal scenarios across a range of transmission intensities, we demonstrate three principal findings. First, genetic metrics can detect insecticide-treated net intervention impacts at seasonal and yearly timescales, but the strength, timing, and form of the relationship between genetic and epidemiological measures vary by metric and sampling timing. Second, importation can break the expected relationship between parasite genetic diversity from local transmission intensity at very low incidence, allowing low-transmission settings with substantial importation to maintain elevated diversity metrics. Third, convenience sampling practices, including sample size, collection timing, and the clinical composition of sampled populations, introduce non-random biases in genetic metric estimation in a way that obscures the true transmission signal. Together, these findings show that parasite genetic metrics can support operational surveillance, but that their interpretation depends on transmission context, importation, metric choice, and sampling design. EMOD FPG provides a framework for evaluating these dependencies in future setting-specific analyses and for guiding the interpretation of parasite genetic data across sites and over time.

Segregated variation between populations is a fundamental evolutionary process leading to parasite specialization, yet the resulting impacts on infection heterogeneity within populations are theoretically and empirically understudied. We asked whether the distribution of host susceptibility to infection within populations carries the signatures of geographic structure from pathogen local adaptation, maladaptation, or generalism in a nuclear polyhedrosis virus that infects the Gulf Fritillary butterfly Dione vanillae. For this virus, there is genetic support for two geographically distinct groups within San Diego County, based on whole genome sequencing of 16 virus isolates. Reciprocal laboratory infections showed evidence of two contrasting viral life history strategies: a generalist phenotype that consistently infected variable hosts and a specialist that performed slightly better in its local host population. As predicted by our theoretical model, the more consistent infection displayed by the generalist across populations corresponded to lower heterogeneity in susceptibility within populations, modeled as gamma distribution. Furthermore, the generalist phenotype was collected over a wider geographic range despite having a tenfold-lower mean infection rate than the specialist, suggesting that a strategy of more consistent infection provides key fitness advantages across diverse host populations. Intriguingly, when there is variation in host susceptibility, interpretations of pathogen local adaptation are dose-dependent. Measuring infectivity across multiple doses enables estimation of the whole distribution of susceptibility, which provides more reliable identification of pathogen specialization to its local host. Our work demonstrates how trait distributions and not only their mean values can carry quantifiable signatures of eco-evolutionary processes in interspecific interactions.

Heartland virus (HRTV) and Bourbon virus (BRBV) are emerging tick-borne arboviruses transmitted by the lone star tick (Amblyomma americanum) that have caused dozens of cases of human disease in the United States, including multiple fatalities. Despite their significance, entomological, clinical, and molecular surveillance remains sparse, limiting our understanding of HRTV and BRBV distribution and risk. The Nebraska Department of Health and Human Services and the Nebraska Public Health Laboratory expanded tick-borne pathogen surveillance to include HRTV and BRBV in A. americanum ticks beginning in 2024. Here, we report the first detections of HRTV and BRBV in Nebraska and present a multi-segment phylogenetic analysis of complete virus genomes. Using a newly developed amplicon-based whole genome sequencing strategy, we generated complete HRTV genomes from three PCR-positive A. americanum pools collected in two counties in eastern Nebraska. Additionally, we generated a complete BRBV genome from a single PCR-positive A. americanum pool. A time-calibrated phylogenetic analysis of the L segment containing all publicly available HRTV sequences determined that the 3 genomes from Nebraska form a monophyletic cluster that initially diverged from viruses isolated from Missouri in the early 2000s, corresponding with the expansion of A. americanum into Nebraska. A phylogenetic analysis of BRBV segment 2 indicates that the genome from Nebraska sits on a long branch and likely diverged from other genomes sequenced in the early 2010s. Topological concordance across each segment suggests minimal occurrences of reassortment among the HRTV and BRBV genome sequences. These findings document the expansion of HRTV and BRBV to the western margin of the A. americanum range and demonstrate the utility of enhanced surveillance and whole genome sequencing for characterizing the spread of tick-borne arboviruses.

Cancer prevalence varies across species, with traits such as litter/clutch size, gestation duration, carnivory, and adult mass, partly explaining this variation. Yet no coherent explanation exists for why shorter gestation and carnivory both correlate with cancer prevalence or risk. Given that carnivores sleep more than herbivores, sleep reportedly being a compensation for brain immaturity after gestation, longer sleep appearing in species with shorter gestation, and shorter gestation in species with higher cancer prevalence, we hypothesize that sleep may mediate the gestation-cancer association. We obtained neoplasia prevalence, cancer prevalence, cancer mortality risk (n [≥] 20 individuals/species), gestation duration, and sleep duration data across vertebrates. We tested whether sleep duration mediates the known gestation-cancer prevalence association, whether sleep duration is directly correlated with neoplasia/cancer prevalence or risk, and tested the known gestation-sleep duration association using more vertebrate species and phylogenetic generalized least squares analyses. Gestation and sleep duration were not correlated with neoplasia prevalence, cancer prevalence, or cancer mortality risk. Gestation and sleep duration were negatively correlated. These results highlight the complexity of understanding how multiple physiology variables explain the variation in cancer prevalence or risk across vertebrates, and the need to verify previously known associations with more powerful and robust statistical tools.

Until 2020, two lineages of the influenza B virus had co-circulated globally. Measures to control the COVID-19 pandemic led to a near-absence of influenza infections. While B/Victoria reemerged in late 2021, there have been no reports of B/Yamagata since the pandemic. To investigate which epidemiological and immunological factors were primarily responsible for the extinction of B/Yamagata, we developed a global model for the two influenza B lineages. To mimic the transmission impacts of the pandemic, we implemented a transient reduction in contacts and identified parameter values that recapitulated viral coexistence dynamic before the pandemic and the qualitative post-pandemic outcomes of B/Victoria (reemergence in late 2021) and B/Yamagata (extinction). Our results suggest that, rather than immunological or evolutionary mechanisms, the extinction of B/Yamagata was mainly driven by its lower basic reproduction number making the virus particularly vulnerable during the early phase of the pandemic. Stochastic simulations of our best-fitting model suggest that B/Victoria was also close to extinction during this period. We investigate the model to assess the feasibility of B/Victoria eradication through vaccination and the potential for a sustained re-emergence of B/Yamagata in the 2026-27 flu season, thus highlighting important considerations for biosafety.

In temperate regions around the world, bats travel long distances every winter to gather at hibernation sites. A longstanding hypothesis is that each new generation of bats learns about the locations of these sites (called hibernacula) from older individuals, yet clear and compelling evidence demonstrating social transmission of this knowledge has been lacking. Here, we compiled 30,882 observations from 1985 to 2023 of 13,852 Greater mouse-eared bats (Myotis myotis) that were banded and observed at summer roosts, winter hibernacula, or both. Our analyses revealed four lines of evidence that Greater mouse-eared bats find suitable hibernacula using social information acquired at summer roosts. First, naive yearlings were more likely to be seen sharing their first hibernacula with adults from their summer birth colony relative to a null model where bats moved independently. Second, adult bats were also more likely to co-switch together into the same hibernacula across winters than expected from independent movements. Third, bats that roosted together in the summer were more likely to share a different site as a hibernaculum during the winter: being observed together during a summer changed the probability of a pair being observed together during a winter from 5% to 12%. Finally, high-resolution tracking revealed an instance of tandem flights to hibernacula sites during the summer, demonstrating that yearlings can learn from experienced adult bats months before hibernation. Together, our findings show that maternity colonies serve as "information centers" where females acquire knowledge of suitable hibernation sites throughout their long lives.

An ongoing outbreak of Bundibugyo virus disease (BVD) in the Democratic Republic of the Congo was deemed a public health emergency of international concern in May 2026. To prevent cross-border importation, many countries, including the United States, Canada, India, Thailand, and Kenya have already proposed containment strategies, and others are likely to follow suit. How well (or poorly) are screening and quarantine containment measures are likely to work? We leverage established epidemiological theory and develop a mathematical model of traveler screening and post-arrival quarantine for BVD to answer this question. We find that traveler screening via symptom screening or molecular testing will miss the majority of infected travelers, and should be complemented by post-arrival quarantine and monitoring of sufficient duration to detect those with long incubation periods. Our findings underscore the limitations of border screening and the importance of complementary measures like post-arrival quarantine to prevent local importation of BVD.

Classic socio-ecological theory predicts that males and females experience different sources and mechanisms of social competition. Whether these differences translate into sex-specific structural properties of dominance hierarchies remains unclear. Here, we compiled 156 dominance interaction matrices from 80 published studies and extracted three commonly used metrics - hierarchy steepness, linearity and the directional consistency index - to investigate the structural characteristics of male and female dominance hierarchies across primates. All three metrics were strongly affected by methodological and demographic variables. Steepness increased with the number of recorded interactions and group size, linearity decreased as matrices became sparser, and directional consistency declined with increasing numbers of interactions. Steepness covaried positively with both linearity and directional consistency, indicating that groups with steeper hierarchies also exhibited more linear and more directionally consistent relationships. We found no sex differences in steepness, linearity or directional consistency. These results suggest that current metrics primarily reflect variation in the sampling effort and the rate of interaction of the recorded behaviour and appear therefore not to capture potential sex differences in the forms of competition. Our findings highlight the need for alternative measures of power asymmetries that are less confounded by sampling effort and demographic variation to better understand how competition and conflict are structured across primate societies.

Viral population dynamics are shaped by production and loss. For viruses of microbes, high standing levels of viral abundances are interpreted as evidence of high rates of viral-induced cellular loss and viral production, followed by rapid extracellular viral decay. Here we reassess assumptions of rapid extracellular decay in 17 curated datasets, finding that biphasic decay either fits better or is statistically indistinguishable from exponential decay in approximately half the datasets. In addition to intrinsic heterogeneity in decay rates, biphasic decay at population scales can arise generically through aggregation mechanisms, where single virions decay and viral aggregates are protected. Integrating aggregation-induced biphasic decay into a virus-host model reveals that accounting for aggregation can recapitulate joint observations of high virion abundances and low infection prevalence, without assuming significant levels of uniformly inefficient infection. Together, our results suggest that durable extracellular virion persistence is environmentally relevant in shaping virus-microbe population dynamics.

Background. Increasing evidence indicates that Ebola virus disease (EVD) survivors can remain a source of infection long after clinical recovery. Confirmed survivor-associated transmission events and genomic evidence linking the 2021 Guinea outbreak to viral lineages from the 2013-2016 West African epidemic have demonstrated that persistent infection in survivors can contribute to post-epidemic re-emergence. However, the population-level conditions under which survivor reservoirs may sustain recrudescence remain poorly understood. Methods. We developed an age-structured Bayesian transmission model to quantify survivor-driven recrudescence risk using historical Ebola outbreak data (1976-2022) and empirical viral persistence data from male survivors. Age-specific viral clearance probabilities were estimated for three age groups (less or equal to 25, 26-35, and >35 years). The recrudescence reproduction number (Rc) was derived using the next-generation matrix approach. Sensitivity analyses examined alternative assumptions regarding viral clearance and the potential contribution of female survivors. Results. The posterior mean recrudescence reproduction number remained below the persistence threshold (Rc=1) across all viral-clearance scenarios under the assumption of no female survivor contribution. Only by assuming the slowest rate of viral clearance and maximal female survivor contribution did the posterior mean for Rc exceed one (1.052; 95% CrI: 0.428-2.229), suggesting that survivor-driven transmission alone is unlikely to sustain Ebola re-emergence given our current understanding of recrudescence dynamics. Simulations showed that survivor-driven outbreak pressure (rate of survivor-initiated outbreaks) was driven primarily by outbreak size and clustering. Outbreaks involving less or equal to 5,000 EVD cases generally produced outbreak pressure below the estimated natural spillover rate, whereas outbreaks comparable in size to the 2013-2016 West African epidemic generated transient survivor-driven outbreak rates up to 7.8-fold higher than the natural spillover rate before declining to comparable levels within 3-7 years. Moreover, across all viral-clearance scenarios, older (>35 years) male survivors consistently exhibited the longest effective persistence durations and made the largest contribution to the recrudescence reproduction number. Conclusions. The human survivor reservoir represents a plausible complementary pathway for Ebola re-emergence, particularly following large epidemics and should be considered alongside zoonotic spillover as an important source of future outbreaks. Age-dependent viral clearance strongly shapes recrudescence dynamics, with older survivors contributing disproportionately to transmission potential. These findings support age-stratified survivor monitoring, extended persistence surveillance, and improved characterization of viral persistence in both male and female survivors to strengthen post-epidemic preparedness.

In many animal species, individuals acquire knowledge from others that enhances their survival and reproduction. However, among the many available cultural exemplars, not all provide reliable information. Consequently, individuals tend to choose their exemplars selectively. One widespread pattern is a preference for older individuals, who may have accumulated valuable knowledge through life. Yet empirical studies also show that individuals frequently learn from age peers, suggesting that copying elders is not universally optimal. The ecological and social conditions that favor learning from elders rather than peers, therefore, remain unclear. Here, we investigate the evolutionary drivers of age-biased exemplar choice in age-structured populations where individuals accumulate knowledge over their lifespan. We develop a model that captures the coevolution of exemplar age choice and age-specific investments in social learning, individual learning, and the use of acquired knowledge for energy extraction. We show that selection promotes a progressive shift from social to individual learning and from learning to energy extraction with age. Exemplar age choice, in turn, evolves through a trade-off between targeting knowledgeable individuals and accessible ones. This trade-off leads young learners to learn preferentially from relatively young exemplars, who are common and still able to provide substantial amounts of novel knowledge, given learners limited knowledge at early ages. As individuals age, encountering exemplars with substantially novel knowledge becomes increasingly difficult. Consequently, as they age, individuals are expected to shift toward learning from older individuals, who possess more knowledge. Population, environment, and knowledge characteristics can shift this balance, generating a wide range of strategies from learning primarily from peers to consistently targeting the oldest individuals. In particular, learning from age peers is favored in populations with strong within-cohort interaction structure, high mortality, or high encounter rates, in unstable environments with rapid knowledge loss, and when knowledge is easily acquired or transmitted.

Cooperation is foundational to complex sociality, yet presents profound evolutionary dilemmas - costs and benefits are rarely distributed evenly and the decision to collaborate or defect can involve a complex contextual calculus. These challenges are compounded when cooperation scales from pairs to groups. Group-level cooperation is fundamental to many species success, but how is it sustained and regulated in nature? One promising route to addressing this question is to examine how individuals reorganise their affiliative interactions in anticipation of group-level cooperation. We examine such pre-cooperative reorganisation using long-term data (2013-2018) from three neighbouring groups of wild chimpanzees at the Tai National Park, Cote dIvoire, who routinely cooperate as a collective to defend their territory against other groups. We found that chimpanzees adjusted the distribution of their social contacts in anticipation of risky and proactive territorial defence by forming more broadly connected, yet more diffuse, affiliative networks. Specifically, adult chimpanzees groomed and played with more group members on days of proactive territorial defence, and this pattern was temporally-sensitive, with increased affiliation occurring before, rather than after, the cooperative act. Chimpanzees accessed a broader range of partners through increased interaction efficiency by switching between more partners with shorter interactions per partner. This pattern suggests a shared evolutionary basis of dynamic social readjustment in preparation for group-level social dilemmas in hominids, potentially providing the foundation for the formalized systems of affiliation found in human societies.

The 17th Ebola outbreak in the Democratic Republic of the Congo, announced on 15 May 2026, was attributed to Bundibugyo ebolavirus (BDBV). Although case isolation is the main control strategy, its effectiveness is compromised when patients escape isolation facilities before recovery. Between 14 May and 17 June 2026, 175 individuals reportedly left isolation facilities without formal discharge across Ituri Province. We assessed how this "isolation leakage" affects community transmission. We refined the SEIHFR framework to distinguish undetected community infections, detected but not-yet-isolated cases, isolated individuals, leakage, funeral-associated transmission, and removals. Using Bayesian inference, we fitted the model to daily Ituri surveillance data, escapee counts, and isolation census records. We estimated the leakage rate, reporting and detection probabilities, and the transmission rate, while fixing other parameters based on the BDBV literature. The model reproduced confirmed cases, deaths, discharges, and escapees. We estimated R_0=3.67 (95% HDI: 2.0-5.7), a leakage rate of {rho} {approx} 0.034 day^-1 (0.022-0.051), and high contact-tracing-driven detection (p_d {approx} 0.91-0.99). Leakage increased the detection-dependent reproduction number [R](p_d) from approximately 3.2 to above 5. Eliminating leakage reduced cumulative infections by about one-third, from 1,120 to 764, while the minimum detection level required for control increased from p_d [≥] 0.73 without leakage to p_d [≥] 0.87 at the fitted leakage rate. Shortening time to isolation prevented the most infections (73.4%; 59-84), followed by reducing leakage (29.7%; 14-52) and re-isolating escapees (12.6%; 6-24). Delaying leakage reduction until week 4 reduced its benefit from about 27% to below 2%. Isolation leakage represents a major transmission pathway that has until now gone largely unmeasured. While rapid initiation of isolation is highly beneficial, it cannot compensate for permeable isolation; therefore, early, community-driven efforts to control leakage, embedded within a multilayered response, are critical.

Long-term winter dormancy in ectotherms (brumation) defines the annual cycle of many temperate-zone reptiles, yet the transcriptional regulation that supports survival across months of cold and aphagy remains poorly understood. We generated time-resolved transcriptomic profiles of liver and testis from male red-sided garter snakes (Thamnophis sirtalis parietalis) at five timepoints spanning the eight-month brumation cycle: pre-brumation, early, mid-, and late brumation, and post-arousal under continued aphagy. Time-course negative-binomial regression (maSigPro) followed by gene-set enrichment analysis identified 3,715 transcripts in liver and 5,828 in testis with significant temporal expression structure organized into five overarching temporal patterns: sustained downregulation, downregulation with post-arousal recovery, sustained upregulation, brumation-specific upregulation and cyclic modulation. Liver showed coordinated upregulation of fatty acid mobilization enzymes (ATGL, FOXO1, PPAR, CPT1A) and gluconeogenic regulators (CREBBP, PCK1) coincident with sustained low temperatures. Additionally, low temperature transcriptional activity was suggestive of a shift toward hepatic lipid mobilization and alanine-supported gluconeogenesis. Testis showed sustained suppression of meiosis, reproduction, and DNA-metabolism gene sets that did not fully recover at arousal consistent with this species dissociated reproductive pattern. Both tissues showed coordinated upregulation of stress-response pathways involving heat-shock proteins, HIF1 and a glutathione-based antioxidant defense. Interestingly, three vitellogenin transcripts and 17{beta}-hydroxysteroid dehydrogenases associated with estradiol-favoring steroid metabolism were upregulated in male liver during late brumation, which is not expected during natural physiology in adult males. Together these data support a framework in which temperature- and starvation-associated transcriptional programs contribute to survival of one of the longest, coldest brumations documented in a squamate. Summary statementA time-resolved transcriptomic analysis of liver and testis spanning eight months of winter brumation in Thamnophis sirtalis parietalis reveals gene expression patterns consistent with a temperature-associated shift toward hepatic lipid mobilization, sustained reproductive suppression, and vitellogenin response in males.