Host-associated microbiomes are compositionally stable across most of the life span, yet undergo consistent and marked deterioration during aging, a phenomenon linked to metabolic dysfunction and disease. What drives thi...Host-associated microbiomes are compositionally stable across most of the life span, yet undergo consistent and marked deterioration during aging, a phenomenon linked to metabolic dysfunction and disease. What drives this late-life collapse remains poorly understood, in part because the mechanisms by which hosts actively construct and maintain the microbial niche during adulthood remain incompletely characterized. This Unsolved Mystery integrates evidence from immunology and ecosystem ecology to investigate the role of immunosenescence in age-associated dysbiosis, raising the possibility of interventions that restore immune surveillance capacity alongside ecologically informed microbiome management, rather than targeting community composition in isolation.
Pseudomonas aeruginosa and Klebsiella pneumoniae are gram-negative opportunistic pathogens that frequently colonize the human body and are major causes of infection. These bacteria are often co-isolated in polymicrobial...Pseudomonas aeruginosa and Klebsiella pneumoniae are gram-negative opportunistic pathogens that frequently colonize the human body and are major causes of infection. These bacteria are often co-isolated in polymicrobial urinary tract and lung infections, the latter of which is associated with increased disease severity and worse clinical outcomes. Despite their overlapping niches and clinical relevance, little is known about how these two pathogens interact and how those interactions influence human health. Given the growing recognition that microbial interactions are key drivers of disease, we investigated how P. aeruginosa and K. pneumoniae influence one another. We discovered an antagonistic interaction in which P. aeruginosa restricts the growth of K. pneumoniae. This inhibition is driven by phenazine production in P. aeruginosa, specifically the secondary metabolites pyocyanin and pyorubin, which are both necessary and sufficient to suppress K. pneumoniae growth. Using a diverse set of clinical isolates, we found that this antagonism is strain-dependent. Both the susceptibility of K. pneumoniae to phenazines and the ability of P. aeruginosa to restrict K. pneumoniae growth varies between strains. Moreover, the necessity of phenazine production is specific to the site of infection. Together, these findings demonstrate that strain background and environmental context are critical determinants of pathogen interactions. These findings reveal that both strain background and environmental redox conditions govern the ecological rules of pathogen interaction, providing a framework for predicting outcomes.
Several SARS-CoV-2 proteins have been shown to counteract the host innate immune response, mostly using in vitro protein expression, which may not fully reflect their role in the context of viral infection. In addition,...Several SARS-CoV-2 proteins have been shown to counteract the host innate immune response, mostly using in vitro protein expression, which may not fully reflect their role in the context of viral infection. In addition, while each viral protein was characterized in a different experimental system, its relative contribution to immunosuppression remains unclear. Here we used a SARS-CoV-2 bacterial artificial chromosome with en passant mutagenesis to recover a panel of 12 infectious recombinant SARS-CoV-2 viruses, each with mutations in either NSP1, NSP2, NSP3, NSP6, NSP12, NSP13, NSP14, NSP15, NSP16, ORF3a, ORF6, or ORF8. We used the interferon-stimulated response element (ISRE)-driven luciferase assay in 293T-ACE2/TMPRSS2 cells to test the panel, demonstrating that mutations in many proteins, especially in NSP1 and NSP15, increased the type I interferon response relative to the parental wild-type virus. RNA-seq analysis of mutant-virus infected Calu-3 cells showed that the mutations in NSP1 or NSP15 lead to higher expression of multiple genes involved in innate immune response, cytokine-mediated signaling, and regulation of lymphocyte proliferation. Furthermore, mutations in either NSP1 or NSP15 resulted in a greater maturation of human monocyte-derived dendritic cells in vitro. Infection of K18 hACE2 transgenic mice with either NSP1 or NSP15 mutated viruses demonstrated attenuated respiratory tract replication. Analysis of lung immune cells from infected mice by single-cell RNA-seq identified 15 populations of major myeloid and lymphoid cells with changes in the pattern of their activation associated with viral infection. The effects of mutations in NSP1 or NSP15 on these responses are consistent with differences in the immunosuppressive mechanisms utilized by the two proteins. Overall, these data demonstrate different and redundant mechanisms of innate immune antagonism by SARS-CoV-2 and suppression of activation of antigen-presenting cells and T and B lymphocytes mediated by multiple viral proteins.
Pseudomonas aeruginosa employs host translation inhibition as a virulence-enhancing strategy. We previously showed that the bacterium induces cleavage of Caenorhabditis elegans large ribosomal RNA at helix 69 (H69), part...Pseudomonas aeruginosa employs host translation inhibition as a virulence-enhancing strategy. We previously showed that the bacterium induces cleavage of Caenorhabditis elegans large ribosomal RNA at helix 69 (H69), part of a central intersubunit bridge and the ribosomal decoding center. In this study, we demonstrate that a previously uncharacterized ribonuclease, Ribocin, is necessary and sufficient for H69 cleavage. Recombinant Ribocin cuts H69 in worm and mammalian ribosomes, indicating that H69 cleavage by P. aeruginosa is phylogenetically conserved. In worms, mammalian cells, and rabbit reticulocyte lysates, H69 cleavage results in translation inhibition. Furthermore, Ribocin contributes to bacterial virulence toward C. elegans, triggers a major host response to translation inhibition, and operates in parallel with Exotoxin A-mediated translation inhibition. These findings unveil the first known nuclease that cleaves eukaryotic ribosomes at H69 and expand the understanding of host translation-inhibition by establishing targeted rRNA cleavage as a mechanism of host attack.
Crosstalk between hepatocellular carcinoma (HCC) and the tumor microenvironment (TME) is pivotal for the initiation and management of HCC. The infiltration and function of natural killer (NK) cells in the TME are frequen...Crosstalk between hepatocellular carcinoma (HCC) and the tumor microenvironment (TME) is pivotal for the initiation and management of HCC. The infiltration and function of natural killer (NK) cells in the TME are frequently hindered. However, it is unclear whether a crucial regulatory factor originating from HCC cells directly modulates NK cell activity to evade immune surveillance. In this study, we found that mannose-binding lectin 2 (MBL2) expression was markedly decreased in HCC and positively correlated with HCC prognosis. MBL2 inhibited the proliferation and migration of HCC cells intracellularly. Human and murine co-culture systems of HCC and NK cells were established to demonstrate that secreted MBL2 recruited and activated NK cells in the TME, particularly upregulating the infiltration of NKp46+ NK cells. Furthermore, secreted MBL2 promoted the production of IL-13 and IL-25 by NK cells, resulting in a decrease in exhausted cytotoxic T lymphocytes. Mechanistically, MBL2 interacts with the integrin β1 receptor, activating the FAK/AKT pathway and increasing PD-L1 expression on NK cells. Our discovery identifies MBL2 as an NK cell-activating cytokine, initiating the integrin β1/FAK/AKT pathway in NK cells and reshaping an immune-activated microenvironment of HCC. Strategies to up-regulate MBL2 may enhance the anti-PD-L1 immunotherapy efficacy and serve as a potential therapeutic approach for HCC.
Marine fish play a significant yet understudied role in the oceanic carbon cycle through the production of magnesium-rich calcium carbonate (CaCO3) precipitates known as ichthyocarbonates. These deposits form in the gut...Marine fish play a significant yet understudied role in the oceanic carbon cycle through the production of magnesium-rich calcium carbonate (CaCO3) precipitates known as ichthyocarbonates. These deposits form in the gut of marine teleost fish in response to salinity, serving as part of their osmoregulation strategy. Through this, marine fish may contribute as much as 9.04 Pg of CaCO3 per year in global new carbonate production, being equivalent to or potentially higher than the production by coccolithophores and pelagic foraminifera. Despite their ecological relevance, the biological mechanisms driving ichthyocarbonate precipitation remain to be fully resolved. Intriguingly, bacteria are consistently found in intimate association with ichthyocarbonate precipitates. Given the widespread capacity of prokaryotes to mediate CaCO₃ precipitation, this association points to a previously unexplored microbial contribution to the process. To investigate the potential role of bacteria in ichthyocarbonate production, we subjected Gulf toadfish (Opsanus beta) to salinity treatments common to their native range and known to elicit changes in CaCO3 precipitation. To assess the respective contributions of the host and its microbiota to ichthyocarbonate formation in the gut, we characterized the microbiome across the toadfish gut and performed meta-transcriptomic analysis. Across the toadfish gut, we identify a high abundance of vibrios associated with ichthyocarbonates with the metabolic potential for CaCO3 precipitation. Specifically, we observe the expression of the transcriptional activator of urease (ureR) by Photobacterium damselae subsp. damselae, which can induce the precipitation of CaCO3 via the production of bicarbonate. We demonstrate that CaCO₃ precipitation in marine fish may not solely be a host-driven process, but potentially the result of a functional symbiosis with gut-associated Vibrio bacteria. We hypothesize that just as photosymbionts enable corals to build reefs, fish hosts, along with their microbial partners, may synergistically contribute to oceanic carbonate production. This discovery, if confirmed, expands the role of symbiosis in marine biomineralization and underscores its broader influence on global biogeochemical cycles.
Sixty years after the concept of population coding in neuronal networks was introduced, we still lack a comprehensive understanding of its performance limits and the role of neuronal physiology. Here, we use dynamic gain...Sixty years after the concept of population coding in neuronal networks was introduced, we still lack a comprehensive understanding of its performance limits and the role of neuronal physiology. Here, we use dynamic gain analysis in a general model of population coding and demonstrate that disparate parameters of neurons and populations determine how accurately they can encode information. These are cell number, cell size, and the correlation time of the background noise. We experimentally test and confirm these predictions on neurons of excitatory populations in the mouse barrel cortex. Surprisingly, dendrite size and background correlations are precisely matched with the number of neurons in layer 4, such that even a single thalamocortical spike at the input is reliably reflected in the population output. However, this encoding performance can be modulated by the channels that mediate M-current, suggesting that coding in layer 4 may vary as a function of brain state.
The scavenger receptor CD163 detoxifies free hemoglobin released on erythrocyte lysis to prevent oxidative damage. The best understood route for hemoglobin detoxification involves the formation of haptoglobin-hemoglobin...The scavenger receptor CD163 detoxifies free hemoglobin released on erythrocyte lysis to prevent oxidative damage. The best understood route for hemoglobin detoxification involves the formation of haptoglobin-hemoglobin complexes that bind CD163 and are internalized into macrophages, resulting in hemoglobin degradation. However, during conditions such as sickle cell anemia or malaria, haptoglobin is depleted. CD163 can then act as a lower-affinity receptor for free hemoglobin. Previous studies revealed that CD163 forms a multimeric "base," which presents "arms" that form a binding site for haptoglobin-hemoglobin. In this study, we use cryogenic electron microscopy to reveal how human CD163 binds hemoglobin tetramers in a process that, unlike haptoglobin-hemoglobin uptake, requires a full trimeric CD163 assembly to achieve sufficient binding. We reveal how flexibility at the calcium-mediated base, combined with a hinge between receptor domains 2 and 3, allows the arms to wrap around diverse ligands. This brings together multiple small binding surfaces from different domains to form cradles for different ligands. These adaptations allow the scavenger receptor to be promiscuous, protecting us from oxidative damage caused by hemoglobin release in various pathological conditions.
A common misconception is that increasing longevity reflects slower aging. Instead, most longevity gain comes from medical advances that allow survival with disease, rather than changes to the biology of aging itself, ch...A common misconception is that increasing longevity reflects slower aging. Instead, most longevity gain comes from medical advances that allow survival with disease, rather than changes to the biology of aging itself, challenging how we study aging and health.
Decisions are driven by perception, but also by non-perceptual factors. It remains an open question, however, whether frontal brain regions involved in perceptual decision-making tend to uniquely reflect the perception o...Decisions are driven by perception, but also by non-perceptual factors. It remains an open question, however, whether frontal brain regions involved in perceptual decision-making tend to uniquely reflect the perception of an animal, or the final choice of action driven by perceptual and non-perceptual factors. Using functional ultrasound imaging (fUSI), we investigated how the premotor cortex (PMC) in ferrets represents stimuli in a Go/NoGo auditory categorization task, varying the difficulty in order to manipulate the rates of perceptual errors relative to non-perceptual errors. We found that on Easy error trials, where error in perception was less likely, PMC activity was similar to correct answers for the same stimulus category. In contrast, on Difficult error trials, PMC activity more closely reflected the choice the animal made, being similar to correct answers for the opposite category. These results together are consistent with PMC activity reflecting the reward-predictive perceptual category. Perceptual errors could be refined further by assessing licking patterns, but licking patterns alone did not explain the effect. This study advances our understanding of the functional role of the frontal cortex in decision-making, suggesting that the PMC integrates sensory inputs to guide behavior based on perceptual information, rather than motivational information.
Photothermal therapy (PTT) serves as a complementary strategy to conventional cancer treatments. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA)-approved photothermal agent. However, its clini...Photothermal therapy (PTT) serves as a complementary strategy to conventional cancer treatments. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA)-approved photothermal agent. However, its clinical application is hindered by poor stability, short blood half-life, and lack of tumor targeting. Herein, we developed biosynthetic gas vesicles (GVs) covalently conjugated with ICG (ICG-GVs) for remotely controlled, visibly acoustic delivery of ICG to tumors in a subcutaneous xenograft model of MB49 murine bladder cancer in C57BL/6 mice. The resulting ICG-GVs exhibit uniform morphology (~200 nm) with an ICG loading rate of 58%, good colloidal stability, and enable trimodal imaging (ultrasound, near-infrared fluorescence, and photoacoustic) for real-time visualization of delivery. Pharmacokinetic analysis revealed that ICG-GVs significantly prolong ICG circulation half-life and increase AUC. Ultrasound-triggered GV cavitation enhanced intratumoral ICG delivery, achieving tumor temperatures >60 °C upon laser irradiation, leading to complete tumor regression and prolonged survival without detectable toxicity. This study provides a clinically translatable strategy for precise and effective ICG-based PTT.
RIG-I and STING are critical for mediating the RIG-I and cGAS-STING signaling pathways that guard against viral infection. Here, we report that ubiquitin-specific peptidase 39 (USP39) positively regulates the RIG-I and c...RIG-I and STING are critical for mediating the RIG-I and cGAS-STING signaling pathways that guard against viral infection. Here, we report that ubiquitin-specific peptidase 39 (USP39) positively regulates the RIG-I and cGAS-STING pathways to induce antiviral innate immunity in vitro and in vivo. The USP39 deficiency impaired the antiviral immune response of macrophages, leading to low type I IFNs expression, and high RNA and (e.g., VSV, H1N1 PR8) DNA virus (e.g., HSV-1) replication. Moreover, USP39-deficient mice were more sensitive to VSV or HSV-1 infection than control mice. Conversely, USP39 overexpression promoted the antiviral immune response. Mechanistically, we found that USP39 regulates RIG-I protein expression by promoting pre-RIG-I mRNA splicing and maturation. In addition, we also revealed that USP39 interacts with and stabilizes STING protein by deubiquitinating K48-linked polyubiquitin of STING at K288. These data show that USP39 positively regulates RNA and DNA-virus-induced RIG-I and cGAS-STING signaling, respectively, by promoting post-transcriptional control of RIG-I and stabilization of STING. These data provide new insights and potential therapeutic targets to control viral infections.
Aging has effects on the immune system that are similar in men and women, but also reshapes their immune systems in unique, sex-specific ways. These sex-specific patterns of immune aging influence disease susceptibility,...Aging has effects on the immune system that are similar in men and women, but also reshapes their immune systems in unique, sex-specific ways. These sex-specific patterns of immune aging influence disease susceptibility, vaccine effectiveness, cancer survival, and responses to pharmacological therapies, and have direct implications for preventive medicine and clinical care. However, these differences in susceptibilities and responses are rarely considered in research, clinical trials, or treatment guidelines. By integrating knowledge of sex-specific immune aging with real-world outcomes from vaccines, cancer immunotherapy, and pharmacovigilance studies, this Essay argues that accounting for both sex and age is essential to advance personalized medicine.
Trimethylation of histone 3 lysine 4 (H3K4me3) is a mark of active transcription, and its regulatory role in RNA polymerase II-mediated transcription has been well-studied. However, if and how this mark regulates RNA pol...Trimethylation of histone 3 lysine 4 (H3K4me3) is a mark of active transcription, and its regulatory role in RNA polymerase II-mediated transcription has been well-studied. However, if and how this mark regulates RNA polymerase I (RNA Pol I) is not known. Here, we used customized genome assemblies for rDNA to demonstrate that KMT2A and KMT2F bind to entire rDNA loci. The binding of these enzymes was mirrored by the binding of H3K4me2 and H3K4me3 marks. Using biochemical assays, we demonstrate the interaction of KMT2-specific subunits with RNA Pol I transcriptional machinery. Our findings reveal KMT2F as the primary KMT depositing the H3K4me3 on rDNA. Loss of H3K4me3 adversely affects the epigenetic landscape and leads to repression of the rDNA locus. Mechanistically, using mammalian cells as a model system, we demonstrate that KMT2F promotes the formation of the pre-initiation complex by RNA Pol I. Our findings highlight the thus far undiscovered role of H3K4me3 in the transcriptional initiation of rDNA genes.
Humans show remarkable flexibility in adapting their behaviour to constantly changing environments. This flexibility relies on the ability to regulate motivation in response to changing motivational demands. Typically, t...Humans show remarkable flexibility in adapting their behaviour to constantly changing environments. This flexibility relies on the ability to regulate motivation in response to changing motivational demands. Typically, the amount of effort required to achieve a certain goal is not precisely signalled by the environment but needs to be learnt from experience. By contrast, prior work examining motivated choices has usually directly instructed effort requirements. It therefore remains unclear how healthy individuals estimate and flexibly regulate effort and how they might achieve this in dynamically changing environments. In the current study, we examine how effort learning is shaped by different types of environmental uncertainty when motivational requirements are not explicitly instructed. Analogous to tasks in the reward learning domain, we designed a novel effort learning task that systematically manipulated two key sources of uncertainty: volatility and noise. Participants were asked to exert effort by squeezing hand-held dynamometers. We characterised effort learning across different stages of the effort production process (e.g., initiation of effort production, effort expectation, error-driven adjustment), which allowed us to capture the dynamics underlying effort estimation and regulation over time. Our findings reveal that humans are able to learn effort requirements by integrating both effort priors and sensorimotor feedback. We further show that effort learning is modulated by environmental statistics, with slower force initiation, weaker priors, slower learning, and faster within-trial force adjustments in high noise environments, but slower learning and slower within-trial force adjustments in high volatility environments. In summary, when effort information is not instructed, different sources of uncertainty about an action's required effort are integrated into participants' internal priors to flexibly guide effort exertion. Our work may provide a useful framework for understanding motivational disorders where abnormal effort learning and estimation may underlie the reduced willingness to exert effort for reward.
Pangolins have become emblematic of the global wildlife trade crisis due to intense trafficking for consumption and traditional medicine. Coupled with habitat loss, the illicit trade in pangolins has severely threatened...Pangolins have become emblematic of the global wildlife trade crisis due to intense trafficking for consumption and traditional medicine. Coupled with habitat loss, the illicit trade in pangolins has severely threatened wild populations. Genetic identification of distinct pangolin populations is an imperative step toward guiding effective and informed conservation management. These populations can serve as a reference for assigning seized individuals to their geographic origins, and thus tracing trafficking networks. However, pangolin population genetics studies have been hindered by limited sampling of geo-referenced individuals, largely due to the species' elusive nature. To address this, we developed a tailored gene-capture approach targeting 671 loci totaling 627 kb with high evolutionary and adaptive value across all eight pangolin species. We optimized the approach for low-quality DNA, including samples from museum collections and wildlife trade, such as bushmeat and scale seizures. We reassessed range-wide population delineations for the three most traded species, the white-bellied (Phataginus tricuspis), Sunda (Manis javanica), and Chinese (M. pentadactyla) pangolins, highlighting the need for biogeographically consistent lineage nomenclature and spatially aware analyses to support coherent conservation planning. The unprecedented geo-referenced DNA database for the three species yielded snapshot insights into pangolin trafficking hotspots and trade dynamics of both domestic markets and global trade seizures, the former providing novel insights into bushmeat trade. Domestic trade reflects local and occasional cross-border sourcing, averaging 454 km across the three species, while international trafficking seizures in mostly scales point to broader, regional procurement. However, common sourcing regions between the two trade market types indicate their interconnectivity, suggesting that local trade may contribute to international trade supply. Our study identified significant international trade hotspots for the white-bellied, Sunda, and Chinese pangolins, centered around southwestern Cameroon, southwestern Borneo Island, and Myanmar, respectively. Addressing geo-referenced sampling gaps and increasing local-to-global seizure data over time may offer deeper spatiotemporal insights into pangolin trade dynamics. Our study design may serve as a replicable model for enabling authorities and practitioners to implement intelligence-driven, geographically targeted interventions, by identifying the key regions most implicated in pangolin trafficking.
G protein-coupled receptor 174 (GPR174), a key modulator of autoimmune responses, maintains immune homeostasis through distinct G protein signaling pathways, particularly Gs and Gi. Although the structural mechanism of l...G protein-coupled receptor 174 (GPR174), a key modulator of autoimmune responses, maintains immune homeostasis through distinct G protein signaling pathways, particularly Gs and Gi. Although the structural mechanism of lysophosphatidylserine (LysoPS)-activated GPR174 in the Gs pathway has been characterized, how hydration-mediated interactions influence GPR174 activation and signaling selectivity remains unclear. Here, we determined high-resolution cryo-electron microscopy (cryo-EM) structures of LysoPS-activated human GPR174 bound to Gs (2.0 Å) and Gi (3.4 Å), revealing a continuous hydration-mediated signal transduction network that bridges the sodium-binding pocket, the NPxxY and DRY motifs, and the G protein-binding interface. This network stabilizes the active-state conformation of GPR174 and dynamically reshapes the intracellular cavity, thereby enabling differential engagement of Gs and Gi. Molecular dynamics simulations and functional assays demonstrated that the hydration network is essential for receptor activation and selectively modulates G protein coupling. To evaluate its conservation, we performed sequence alignments and structural analyses across class A GPCRs, defining three hydration cavities: the conserved water cavity (CWC), the junctional water cavity (JWC), and the extended water cavity (EWC), whose hydration is determined by residue properties at position 5.58. Together, our study reveals a hydration-driven molecular mechanism that underlies the activation of GPR174 and its dual G protein selectivity. These findings advance the understanding of hydration-mediated signaling in GPR174 and provide a framework for investigating water-mediated regulation across class A GPCRs.
Cryptococcus neoformans is an environmental pathogen that remodels its cellular physiology to survive within mammals and, in susceptible hosts, cause life-threatening meningoencephalitis. Of the many distinctions between...Cryptococcus neoformans is an environmental pathogen that remodels its cellular physiology to survive within mammals and, in susceptible hosts, cause life-threatening meningoencephalitis. Of the many distinctions between the external environment and mammalian tissues, CO2 concentration in the host is two orders of magnitude higher than in the environment and represents a critical stress for C. neoformans. C. neoformans strains that do not replicate at host CO2 concentrations are less virulent in mouse models of infection, further supporting CO2 tolerance as a virulence trait. To further understand the genetic determinants of C. neoformans CO2 tolerance, we performed a near genome-wide screen for deletion mutants with altered CO2 fitness using a competitive growth assay. A total of 301 of 4,692 deletion mutants showed altered CO2 tolerance (245 reduced fitness; 56 increased fitness) demonstrating the global effect of host CO2 on C. neoformans physiology. Based on this data set as well as a metabolomic analysis of C. neoformans adaptation to host CO2, we show that remodeling of central carbon metabolism, oxidative stress buffering, and membrane homeostasis represent an integrated response to CO2 stress that is mediated in part by the TOR-Ypk1 signaling axis. We propose that CO2-induced capsule formation leads to reduced cellular glucose which, in turn, triggers remodeling of central carbon metabolism toward utilization of alternative carbon sources and increased mitochondrial respiration/reactive oxygen generation. Thus, these data provide a near genome-wide profile of the genetic determinants of C. neoformans CO2 tolerance as well as a model for how this important environmental human fungal pathogen alters its physiology to proliferate in the host.
Transmission routes of highly pathogenic H5N1 between cows or to humans remain unclear due to limited data from affected dairy farms. We performed air, farm wastewater, and milk sampling on 14 H5N1-positive dairy farms a...Transmission routes of highly pathogenic H5N1 between cows or to humans remain unclear due to limited data from affected dairy farms. We performed air, farm wastewater, and milk sampling on 14 H5N1-positive dairy farms across two different California regions. Infectious virus was detected in the air in milking parlors and in wastewater streams, while viral RNA was found in exhaled breath of cows. Sequence analysis of infectious H5N1 virus from air and wastewater samples on one farm revealed viral variants relevant for potential human susceptibility. Longitudinal analysis of milk from the individual quarters of cows revealed a high prevalence of subclinical H5N1-positive cows. Additionally, a heterogeneous distribution of infected quarters that maintained a consistent pattern over time was observed, inconsistent with shared milking equipment serving as the sole transmission mode. The presence of subclinically infected cows was further supported by detection of antibodies in the milk of animals that exhibited no clinical signs during the H5N1 outbreak on one farm. Our data highlight additional sources and potential modes of H5N1 transmission on dairy farms.