Spatial transcriptomic atlas of murine neurotoxocariasis reveals region-specific host responses and dysfunction in the brain

Spatial transcriptomic atlas of murine neurotoxocariasis reveals region-specific host responses and dysfunction in the brain | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Spatial transcriptomic atlas of murine neurotoxocariasis reveals region-specific host responses and dysfunction in the brain Guangxu Ma, Minyao Zou, Sishi Liu, Yi Chen, Zhiwei Xiong, Haiyan Wu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6743037/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Toxocara canis is a globally distributed parasite that infects dogs and other canids, shedding eggs into the environment. Humans become accidental hosts by ingesting these eggs, leading to syndromes such as visceral, ocular and neurotoxocariasis – the latter being poorly understood. Neurotoxocariasis has been associated with epilepsy, cognitive impairment and schizophrenia, but its pathomechanism remains unclear. Here, we combine spatial transcriptomics, single-nucleus RNA sequencing and behavioural assays in a mouse model to characterise the host–parasite interface in the brain. T. canis larvae caused region-specific brain damage, vascular remodelling and confined immune responses. Parasite gene expression was dominated by immunomodulatory excretory/secretory proteins. Infected mice exhibited impairments in motor learning and memory, coinciding with transcriptional changes in neurons and glia. These findings provide mechanistic insight into neurotoxocariasis and demonstrate the utility of the mouse model as a platform to investigate helminth-induced neurological disorders. Biological sciences/Microbiology/Parasitology/Parasite host response Biological sciences/Microbiology/Parasitology/Parasite immune evasion Health sciences/Pathogenesis/Infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Human toxocariasis is a globally distributed but under-recognised zoonotic parasitic disease caused by infection with larvae of Toxocara canis (dog roundworm) or T. cati (cat roundworm) 1 . People, particularly children, become infected following the accidental ingestion of embryonated eggs or infective larvae, typically from contaminated soil, food or water. Following ingestion, larvae hatch in the intestine, penetrate the intestinal wall, enter the bloodstream and disseminate to various organs, including the liver, lungs, central nervous system (CNS) and eyes. These events lead to syndromes broadly classified as visceral larva migrans (VLM), ocular larva migrans (OLM) and neurotoxocariasis 1, 2 . The first recognised case of human neurotoxocariasis was reported in 1951, when Toxocara larvae were identified in the brain of a child who had died from poliomyelitis 3 . Although CNS involvement is often subclinical, a wide spectrum of neurological manifestations – including eosinophilic meningitis, encephalitis, myelitis and cerebral vasculitis – has been documented 4,5 . Recent seroepidemiological studies suggest potential associations between Toxocara infection and neuropsychiatric and neurodegenerative disorders, including epilepsy, schizophrenia, cognitive decline, Parkinson's disease and Alzheimer's disease 6–8 . Although systemic immune responses to T. canis have been characterised in dogs, humans and mice, and are typically marked by Th2-skewed immunity and eosinophilia, the local immune response within the CNS remains poorly defined 9 . Migrating larvae excrete and secrete a complex array of immunomodulatory molecules – including mucins, lectins, protease inhibitors and other glycoproteins – that modulate host immunity and promote larval persistence during tissue migration 10–12 . Notably, pathological examination of murine brains harbouring larvae often reveals limited leukocytic infiltration, but marked transcriptomic and lipidomic alterations 13–17 , suggesting that the brain suppresses inflammatory responses to prevent bystander tissue damage. Despite these indications, the pathogenesis of neurotoxocariasis and the role of Toxocara in neurodegenerative disease remain elusive 7,18 . This work employed high-resolution spatial transcriptomics to define an in situ cellular and immune atlas of the murine brain during T. canis neuroinvasion, in order to characterise regional transcriptional landscapes and immune microenvironments. By integrating spatial transcriptomic data with behavioural assays of learning and memory in mice, we provide experimental evidence that T. canis neuroinvasion is associated with region-specific molecular perturbations and functional cognitive impairment. This study links spatially resolved host–parasite interactions with neurophysiological outcomes in a well-defined model of neurotoxocariasis. Results Spatial and temporal neuroinvasion by T. canis in mice To investigate the kinetics and spatial pattern of neuroinvasion, we orally infected specific pathogen-free (SPF) BALB/c mice with 1000 embryonated T. canis eggs each, and examined brain tissue at multiple time points after infection. Brains from six infected mice were assessed at 7, 14, 21, 28 and 35 days post-infection (dpi) using the tissue digestion method. On average, ~10% of the infective dose was recovered from the brains. Larval recovery peaked at 21 dpi and gradually declined thereafter, indicating a dynamic process of CNS invasion, persistence or clearance. At no stage during experimentation were larvae detected in the brains of uninfected control mice. At 21 dpi, the time point with the highest larval burden, macroscopic examination revealed focal parenchymal damage and haemorrhagic lesions, prompting detailed microscopic analysis of coronal brain slices. We examined the olfactory bulb, cerebrum and cerebellum. In the olfactory bulb, although parenchymal damage was evident in most slices, larvae were not consistently observed. In contrast, both haemorrhagic lesions and larvae were frequently detected in the cerebrum and cerebellum (Supplementary Fig. 1). These observations confirmed neuroinvasion by T. canis and revealed region-specific pathological changes in the murine brain, with the cerebrum and cerebellum being key target sites for larval migration and associated tissue injury 19 . Spatially resolved cell atlases for T. canis -invaded brain regions in mice To investigate the host–parasite interface in neurotoxocariasis, we applied Stereo-seq spatial transcriptomics to coronal sections of the olfactory bulb (slice S1), cerebrum (slices S2–S5) and cerebellum (slice S6) invaded by T. canis larvae (Fig. 1a; Supplementary Fig. 1). This enabled high-resolution spatial mapping of transcription in both host and parasite (Fig. 1b). Analysis of the data revealed widespread transcription across all brain regions, with 35,913 to 40,476 murine genes transcribed in slices S1 to S6, respectively (Table 1). Parasite-derived transcripts were also detected, corresponding to 27, 50, 54, 113, 56 and 60 T. canis genes, with molecular identity (MID) counts of 37, 80, 128, 7,717, 1,032 and 1,205 per 100 × 100-pixel spatial bin (Bin100) in slices S1, S2, S3, S4, S5, and S6, respectively (Table 1). Notably, the highest abundance of parasite transcripts was observed in the cerebrum (S4, S5) and cerebellum (S6), compared with the olfactory bulb (S1), where transcript counts were markedly lower. Using the gene transcription matrix generated from this analysis (Fig. 1c), we performed unsupervised clustering and manual cell type annotation (Fig. 1d). Spatially resolved transcription of canonical markers allowed the identification of astrocytes, basket cells, endothelial cells, ependymal cells, microglia, mural cells, neurons and oligodendrocytes. These atlases define the cellular architecture of brain regions invaded by T. canis , offering new insights into the spatial organisation of host responses to neuroinvasion (Fig. 1e). Localised immune recognition with limited leukocytic infiltration in T. canis -infected brains Spatially resolved atlases revealed focal accumulation of endothelial and mural cells at sites of parenchymal damage and haemorrhage in brain slices S1, S2 and S3 (Fig. 1e). Microglia and oligodendrocytes were localised primarily to the meninges and cortex, suggesting roles in damage response and vascular repair. In contrast, no such cell aggregation was observed in slices S4, S5 or S6, despite high levels of parasite-derived transcripts (Fig. 1b,e), indicating supressed host responses in tissue microenvironments associated with larvae or damage induced by migrating larvae. Notably, neutrophilic and eosinophilic granulocytes – known to mediate larval trapping and granuloma formation in peripheral tissues 20–22 – were absent from all regions examined, even in areas with visible larval migration or tissue disruption. These findings support previous evidence of limited leukocytic infiltration in T. canis -invaded rodent brains and raise the possibility of local immune suppression or evasion within the CNS 23 . To investigate further, we performed deeper cell type annotation using public reference data, identifying a broader repertoire of host cell types (Supplementary Figs. 2 and 3) – including adipocytes, astrocytes, Bergmann glia, endothelial and epithelial cells, fibroblasts, mesenchymal cells, microglia, mural cells, neurons, stromal cells, and particularly erythrocytes, dendritic cells, B cells, and T cells. Notably, we also identified macrophages and granulocytes not previously seen in healthy mouse brains 24–26 . The presence of antigen-presenting cells (microglia, macrophages, dendritic cells) and immune effectors (B cells, T cells, granulocytes) indicates an impairment of the blood-brain barrier; while immune recognition of T. canis occurs but is confined to discrete niches surrounding the larvae. To further explore this, we conducted snRNA-seq on sections adjacent to slices S4, S5 and S6 containing migrating T. canis larvae (Fig. 1b; Table 1; Supplementary Fig. 1). Microglia predominated in the dataset from the S4-adjacent sample (MID = 7,717), but not in S5- or S6-adjacent sample (MID = 1,032 and 1,205, respectively) (Supplementary Fig. 4). These findings indicate that key antigen-presenting cells are in close vicinity to migrating T. canis larvae. Taken together, the Stereo-seq and snRNA-seq data indicate that T. canis is recognised by the CNS immune system, but that immune responses remain highly localised, potentially to avoid broader neuroinflammation. Focal immune activation in T. canis -infected brains Due to variability in lesion location between animals, pairwise anatomical comparisons with uninfected controls were not feasible. Therefore, we focused our spatial transcriptomic analyses on larval microenvironments (~1,000 cells per region) in brain slices S4, S5 and S6, where T. canis transcripts were abundant (Fig. 1b). Within these regions, we observed focal infiltration of microglia, most prominently in S4, relative to the contralateral, parasite-free hemisphere (Fig. 2a). A localised accumulation of endothelial cells was also evident around migrating larvae, suggestive of angiogenesis or vascular injury. These patterns were not discernible on a tissue-wide level (cf. Fig. 1d,e), indicating that the T. canis larvae themselves induce discrete, spatially confined inflammatory and vascular responses. To investigate the associated molecular landscape, we compared transcriptional profiles between areas with ( n = 2) and without ( n = 2) visible larvae. A total of 970 genes were differentially transcribed (fold change > 2, p < 0.05) (Fig. 2b; Supplementary Table 1). These genes were significantly enriched ( p < 0.01) for pathways involved in metabolism (e.g., sphingolipid metabolism, non-canonical glycan degradation), environmental information processing (e.g., mitogen-activated protein kinase [MAPK], Rap1, Ras signalling), genetic information processing (e.g., mismatch repair, DNA replication), cellular processes (e.g., apoptosis), immune function (e.g., antigen presentation, C-type lectin receptor signalling, leukocyte trans-endothelial migration, platelet activation, circadian rhythm) and pathogenesis (e.g., glioma, nicotine addiction) (Fig. 2c). Notably, concurrent activation of MAPK signalling (pro-inflammatory) and glycan degradation pathways (immune evasion) indicates that T. canis neuroinvasion provokes a dual response: focal host immune activation, counterbalanced by parasite-driven modulation 22 . These findings define a spatially restricted but molecularly complex interface between host immunity and parasite persistence within the brain. Systemic host responses and parasite adaptation during T. canis infection To investigate systemic responses to T. canis infection beyond the CNS, we profiled lipid metabolites in the sera of infected and uninfected mice at 21 dpi. A total of 735 differential (fold change > 1.5, p < 0.05) lipid species were detected in the sera of infected mice compared with uninfected ones (Supplementary Fig. 5A). These metabolites were significantly enriched ( p < 0.01) for pathways involved in immune and inflammatory signalling (e.g., B and T cell receptor signalling, Th1, Th2 and Th17 differentiation, nuclear factor kappa-beta [NF-κB], MAPK and ErbB signalling), hormonal regulation (e.g., gonadotropin-releasing hormone [GnRH], oestrogen, insulin secretion, peroxisome proliferator-activated receptor PPAR signalling), metabolic function (e.g., bile secretion, lipid and cholesterol metabolism) and cellular homeostasis (e.g., neurotrophin and relaxin signalling, vitamin absorption, autophagy) (Fig. 2d; Supplementary Table 2). These lipidomic results indicate a broad systemic response to infection, complementing local immune responses in the brain 16,27 . To assess whether circulating host signals influence the parasite, we incubated third-stage T. canis larvae in serum from infected mice for 24 h. Transcriptomic analysis identified more than 350 T. canis genes as differentially transcribed (fold change > 1.5, p 350) were enriched ( p < 0.05) for pathways linked to signal transduction (e.g., neurotransmitter transport, G protein-coupled receptor [GPCR] signalling), immune evasion (e.g., O-glycan processing, collagen and cuticle remodelling), stress response (including dauer entry regulation) and motility (e.g., positive regulation of movement) (Fig. 2e). Key pathways included mucin-type O-glycan biosynthesis, PPAR signalling, neuroactive ligand–receptor interaction, fatty acid and cholesterol metabolism and MAPK signalling (Supplementary Table 3). These findings are consistent with molecular alterations in the T. canis -infected brain (Fig. 2e) and illustrate a coordinated parasite response to host immune pressure. Although infection elicits inflammatory and immunological responses in the CNS, the parasite counters with adaptive mechanisms, including glycan masking, cuticle remodelling, stress mitigation and neuromuscular modulation 10,28 . This reciprocal interaction highlights a dynamic host–parasite interface extending from the CNS microenvironment to systemic circulation. Stress diapause and endocrine regulation of T. canis larvae in the brain Analysis of brain-resident T. canis larvae revealed gene transcription profiles consistent with a survival strategy in response to the inflamed host environment (Supplementary Table 4). Transcripts included those encoding structural cuticle components (e.g., collagens, cuticulins), membrane transporters (e.g., transthyretin-like proteins, multidrug resistance protein 3 [MRP3], Niemann–Pick disease type C-associated cholesterol receptor [NPC1/NCR1]), GPCRs (e.g., FMRFamide receptor, RhoA, SRAB14), enzymes (e.g., β-galactosidase, acetyl-CoA synthetase, acyl-CoA thioesterase, peroxidase) and ion channels (e.g., the nicotinic acetylcholine receptor ACR-16, bestrophin, innexin). These transcriptional patterns reflect physiological adaptations to maintain cuticle integrity, nutrient acquisition, oxidative stress resistance and metabolic suppression – hallmarks of diapause or hypobiosis 29 . These signatures were corroborated in vitro : larvae exposed to serum from infected mice showed an enrichment of transcripts linked to dauer entry – a conserved stress-induced quiescent state in nematodes (Fig. 2e). In C. elegans , dauer entry is regulated by the dafachronic acid (DA) hormonal module, which includes DAF-9 (cytochrome P450), DA (a bile acid–like ligand), and DAF-12 (a nuclear hormone receptor) 30 . To investigate whether this module is conserved in T. canis and explore whether it plays a regulatory role in the immune evasion by T. canis in the CNS, we quantified key proteins in larvae exposed to serum from infected mice. Compared with controls, exposed larvae showed a reduced DAF-9 protein expression ( p < 0.01), decreased abundance of 25S-Δ7-DA ( p < 0.001) and diminished DAF-12 protein levels ( p < 0.05) (Fig. 3a–c), indicating dauer entry. The developmental arrest of larvae following migration in the CNS is likely a response to immune pressure, and indicates the involvement of hormone–immune interactions in neurotoxocariasis 31 . Notably, suppression of the DAF-9–DA–DAF-12 pathway was reversed when larvae were exposed to serum from perinatally infected mice, suggesting that a hormonal mechanism underpins exit from dauer or a reactivation of development (Fig. 3c–e; Supplementary Fig. 5c,d) 1 6 , 32 . This finding was supported by increased transcription of neuroactive ligand–receptor genes, including Tcan_08258 (encoding a GnRH receptor homologue) and Tcan_10138 (a predicted GPCR of unknown function) (Fig. 3f). Altered GnRH signalling in infected mice implicates Tcan_08258 in stress diapause and reactivation. Although the function of Tcan_10138 remains unclear, lipid profiling of negative, positive and perinatal-positive sera identified candidate ligands with reciprocal abundance. Stigmasta-4,22-dien-3-one, CE 22:6, cholic acid and DGDG O-19:2_24:6 were predicted to bind Tcan_10138 with high affinity (–8.8 to –7.5 kcal/mol) (Fig. 3g,h). These host-derived lipid molecules – particularly stigmasta-4,22-dien-3-one and CE 22:6 – may act analogously to ascarosides – small molecule regulators of dauer entry in free-living nematodes – which have also been shown to suppress type 2 inflammation in mammals 33 – 34 . Together, these findings indicate that T. canis larvae enter a transcriptionally regulated, hormonally modulated diapause-like state in the murine brain. Moreover, endocrine cues from the host appear capable of orchestrating both the initiation and termination of this stress-adaptive program. Immune evasion and modulation by T. canis in the brain Spatial transcriptomics revealed that more than 90% of T. canis transcripts detected in infected brain slices encode excretory/secretory (ES) proteins (Fig. 4a), including mucins (120 kDa), C-type lectins (32 kDa), phosphatidylethanolamine-binding proteins (PEBPs; 26 kDa), venom allergen-like proteins and chondroitin proteoglycans (Supplementary Table 4). These ES proteins are central to parasite immune evasion, with mucin transcripts being the most abundant. These mucins contribute to a “fuzzy surface coat” that shields larvae from host immune attack 28 . Despite structural homology with host proteins, T. canis ES proteins contain functional domains that are absent from mammalian orthologues. For instance, T. canis PEBP1 ( Tc -PEBP-1) includes a signal peptide and two ShKT toxin domains – features absent from murine PEBP1 ( Mm PEBP1) (Supplementary Fig. 6a,b) – suggesting disruption of immune cell signalling, particularly in T cells and macrophages. We focused on Tc- MUC-1, a mucin that interacts with cytoskeletal and signalling proteins in murine macrophages via ShKT domains 35 . Although direct interaction between Tc- MUC-1 and Mm PEBP1 was not confirmed in RAW264.7 cells, structural modelling predicted strong binding between Mm FABP5 and both Mm PEBP1 and Tc- PEBP-1, with binding free energies of –18.48 and –35.04 kcal/mol, respectively (Fig. 4b). These findings support the existence of Tc- MUC-1– Mm FABP5– Tc- PEBP-1 or mixed Tc – Mm protein complexes. As FABP5 regulates PPAR signalling 36 , Tc PEBP1 may influence macrophage polarisation and immune signalling. To assess the immunomodulatory role of Tc- PEBP-1, RAW264.7 macrophages were stimulated with recombinant Tc- PEBP-1 (r Tc- PEBP-1) (Supplementary Fig. 6c). At 5 μg/mL, r Tc- PEBP-1 induced a rounded morphology consistent with M1 polarisation, whereas 15 μg/mL induced elongation characteristic of M2 activation (Supplementary Fig. 4d). M1 markers ( iNOS , Ccl3 , Tnfa , Il1b ) were downregulated in a dose-dependent manner (Fig. 4c,d), whereas M2 markers ( Pparg , Arg1 ) were significantly upregulated at 15 μg/mL (Fig. 4e,f). r Tc- PEBP-1 also increased expression of NOD2, RIP2 and phosphorylated NF-κB p65 (Fig. 4g–i; Supplementary Fig. 6e–h), implicating the NOD2–RIP2–NF-κB pathway in downstream signalling (Fig. 4j). Together, these results indicate that T. canis ES proteins, including Tc PEBP1, function as immunomodulators: low levels of Tc- PEBP-1 promote proinflammatory activation, whereas higher levels of Tc- PEBP-1 facilitate an anti-inflammatory phenotype in host macrophages. Neurocognitive impact of T. canis neuroinvasion To assess the effects of T. canis neuroinvasion, we characterised region-specific cellular and transcriptional changes in the brains of infected mice. Using spatially resolved cell clusters and marker gene signatures, we identified distinct host responses in the olfactory bulb, cerebrum and cerebellum (Fig. 5a). In the olfactory bulb (slices S1), parenchymal damage was associated with the accumulation of adipocytes, astrocytes, fibroblasts and a distinct adipocyte cluster. Marker genes in these cells were enriched for biological processes such as gliogenesis, protein serine/threonine kinase activity and amoeboid-type migration, with associated pathways including PI3K–Akt signalling, aminoacyl-tRNA biosynthesis and focal adhesion. In the cerebrum (slices S2–S5), infiltrating adipocytes, endothelial cells, microglia, dendritic cells, B cells, T cells and fibroblasts were present at sites of tissue damage and haemorrhage. The transcriptional profiles of these cells were enriched for functions linked to axonogenesis, synapse organisation, cognition, dendritic spine formation and epithelial cell proliferation, and were associated with calcium signalling, cholinergic synapse activity, neuroactive ligand–receptor interaction, oxytocin signalling, long-term memory and neurodegeneration (Supplementary Fig. 7). In the cerebellum (slice S6), endothelial cells and B cells were detected within lesion sites, with gene enrichment in haemoglobin binding, peroxidase activity and amoeboid-type migration as well as in inflammatory regulation (e.g., interferon-γ and prostaglandin signalling) and immune evasion (e.g., insulin-like growth factor 2 and vitronectin signalling) characteristic of pathogens associated with sleeping sickness (African trypanosomiasis), cerebral malaria or the long-term potentiation in Parkinson’s disease 37 , 38 . These region-specific transcriptional responses suggest that T. canis induces localised immune activation, structural disruption and dysregulation of molecular pathways critical for sensory processing, learning, memory and motor coordination – processes likely underlying the cognitive and neuropsychiatric disturbances in the murine model and the progression of some neurodegenerative disorders in humans 6,7,14,18,27 . To evaluate behavioural outcomes, mice infected with 1000 embryonated T. canis eggs were subjected to rotarod, predator cue exposure (modified light/dark box) and Morris Water Maze (MWM) testing (Fig. 5b). Infected mice ( n = 8) exhibited significantly impaired motor skill learning in the rotarod assay compared with uninfected controls ( n = 6) ( p < 0.05; Fig. 5c). In the predator cue test at 35 dpi, infected mice showed a non-significant reduction in avoidance behaviour, spending slightly less time in the dark compartment containing rat scent or dog urine (Fig. 5d). In MWM, infected mice demonstrated progressively increased escape latency and swim path length between 42 and 48 dpi (Fig. 5e,f), followed by a significant reduction in time spent in the target quadrant at 49 dpi ( p < 0.01), consistent with spatial memory impairment (Fig. 5g). Taken together, these findings show that T. canis neuroinvasion results in region-specific inflammation and transcriptional disruption, leading to measurable deficits in motor coordination, learning and memory. Discussion This study provides the first spatially resolved, transcriptome-wide characterisation of T. canis neuroinvasion in a murine model, highlighting the mechanisms underlying its persistence and host modulation in the brain. Using integrated spatial transcriptomics, single-nucleus RNA sequencing and behavioural assays, we show that T. canis establishes a regionally restricted presence in the brain, modulates local immune responses and alters host gene transcription and networks relevant to neurological function, overcoming some technical limitations of dual (parasite/host) RNA-seq 39 . These findings improve our understanding of neurotoxocariasis beyond structural pathology to encompass molecular and functional consequences of infection. T. canis , in its larval stage, can infect a broad range of paratenic hosts, including humans, where it is responsible for a spectrum of disease syndromes 1,2 . Although interspecific differences in susceptibility, pathogenesis and immune responses are likely, the mouse provides a genetically tractable and experimentally reproducible model to interrogate mechanistic aspects of infection and disease 22,23,40 . Our findings using this system offer insights that might be broadly relevant across distinct host species and provide a foundation for comparative studies. Our results support the concept of immune privilege in the CNS, where, despite evidence of larval localisation and tissue disruption, leukocytic infiltration remains minimal, unless migrating larvae leave the tissue site. Instead, immune and inflammatory responses are localised to specific microenvironments, with focal involvement of resident immune and vascular cell types. This finding indicates a form of “compartmentalised immunity” that serves to limit collateral tissue damage while permitting chronic parasite persistence. The absence of robust granulocytic responses also suggests effective immune evasion and/or suppression strategies by the parasite. Transcriptomic analyses revealed that T. canis larvae in the brain predominantly transcribe genes encoding ES proteins, many of which are known to modulate host immune responses. These include mucins, lectins, and proteins with ShKT domains, such as Tc- PEBP-1, which we identified as a key immunomodulator 35 . Tc- PEBP-1 influenced macrophage polarisation in a concentration-dependent manner, shifting from pro-inflammatory to regulatory phenotypes, and engaged intracellular signalling via the NOD2–RIP2–NF-κB pathway. This dual functionality might allow the parasite to calibrate host responses for survival without inducing overt pathological changes. Our lipidomic data further support a systemic component to host–parasite communication. Serum from infected mice induced transcriptional responses in infective T. canis larvae in vitro , including an upregulation of genes involved in immune evasion, neurosensory processing and stress diapause. This dynamic host–parasite interplay was exemplified by the induction of dauer-like transcriptional signatures in brain-resident larvae, including the suppression of the DAF-9–DA–DAF-12 axis 30 . Hormonal signals from perinatally infected hosts reversed this state, pointing to a host-driven endocrine mechanism regulating a stress adaptation (hypobiosis) or reactivation in the parasite. The effects of T. canis neuroinvasion extended to host cellular architecture and behaviour. We observed transcriptional changes in host brain cells associated with synaptic organisation, neurodevelopment and degeneration, accompanied by impaired motor learning and spatial memory in mice. These data offer some experimental validation for reported associations between toxocariasis and neuropsychiatric or cognitive disorders in humans 41,42 . Our findings indicate that neurological disorders can be associated with neuroinflammation, down-regulation of cell migration and neuron projection (e.g., axonogenesis) as well as wound healing (linked to gliogenesis) in the brain, supporting the hypothesis that neurodegeneration in murine neurotoxocariasis has an immunopathogenic basis 23,43 . We also showed that lipid/cholesterol metabolism is tightly linked to host-parasite interactions (e.g., bile acid secretion pathway) and possibly immunopathological alterations (e.g., cellular response to alkaloids and high-density lipoprotein particles in oligodendrocytes) in murine neurotoxocariasis 17,27,44-46 . While here we explored neurotoxocariasis in mice over a short (3–7 week) period, future investigations are needed to study, in more detail, the complex interplay between infiltrated immune cells (e.g., microglia, dendritic cells, B cell and T cells), neuron cells and impaired motor learning and spatial memory in mice with chronic T. canis infection 46 – 50 . In conclusion, our findings reveal how T. canis exploits spatial and immunological features of the CNS to persist, modulate host responses and impair neural function. This work sets the scene for future studies aimed at resolving the molecular interface between neuroinvasive helminths and host brain circuits. A deeper understanding of host signalling processes and pathways that regulate developmental states in helminths may offer new insights into chronic parasitic infections and their long-term neurological impacts. Methods Ethics statement All experiments involving mice and rats followed the regulations of the Guide for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Welfare Ethics Committee at Zhejiang University, Hangzhou, China (permit no. ZJU20250029). Parasite collection and egg preparation Adult T. canis worms were collected from the faeces of naturally infected dogs at the Animal Hospital affiliated with the College of Veterinary Medicine, Southwest University. Embryonated eggs were harvested from the uteri of molecularly identified gravid females, incubated at 25 °C for eight weeks and hatched using glass beads. Infective larvae were purified via the Baermann technique 51 , cultured or snap-frozen in liquid nitrogen and stored at –80 °C until use. Murine infection model and brain sampling Six-week-old, helminth-free BALB/c mice were orally administered 1000 infective T. canis eggs each and maintained under pathogen-free conditions 52 . Infection was confirmed by IgG ELISA (Novatic Diagnostics, Germany). For larval recovery, at 7, 14, 21, 28 and 35 dpi, mice ( n = 6) were euthanised by cervical dislocation, and brains were collected and digested for larval identification and counting. For brain slices preparation, at 21 dpi, brains of infected mice were collected, frozen, and embedded in OCT (SAKURA, USA). Serial coronal slices (10 μm) were prepared and slices adjacent to larval migration sites or lesions – confirmed by haematoxylin and eosin (H&E) staining – were selected for spatial and transcriptomic profiling. Spatial transcriptomics Stereo-seq was performed on brain slices from the olfactory bulb (S1), cerebrum (S2–S5) and cerebellum (S6), which were mounted onto 1 × 1 cm Stereo-seq chips (BGI Research, Shenzhen) and processed following published protocols 53 . Transcriptomes were mapped to Mus musculus (GRCm38) and T. canis (WormBase PRJNA248777.WBPS17) reference genomes using SAW v1.0. Data were analysed using PCA, UMAP and Seurat v4.0.0 to cluster cells and identify marker genes. Pseudotime analysis was conducted with Monocle3 v1.0.0 54 , and annotation was performed using SingleR v1.6.1 55 . Differentially transcribed genes (fold change > 1, p < 0.05) were analysed for GO and KEGG pathway enrichment using ClusterProfiler v4.0.0. Single-nucleus RNA sequencing Single-nucleus RNA sequencing (snRNA-seq) was performed on slices adjacent to S4, S5, and S6 using the DNBelab C Series Single-Cell Library Prep Set (BGI Research, Qingdao). After droplet generation and cDNA library preparation, sequencing was performed on a DIPSEQ T1 platform (CNGB). Reads were filtered with scRNA_parse v1.0.1 and aligned to the Mus musculus genome using STAR v2.7.2b 56 . Transcript counts were computed with PISA v0.7. Clustering and annotation were conducted using Seurat v4.0.0, SciBet and SingleR v1.8.0 57 , 58 . Larval culturing and stimulation Third-stage T. canis larvae (n = 10,000) were cultured in RPMI-1640 medium (Vivacell, China) containing 1% antibiotics and 10% serum from uninfected, infected or pregnant/infected mice. After 24 h at 37 °C with 10% CO₂, larvae were washed thrice in saline, centrifuged and snap frozen for RNA or protein analysis. RNA-seq Total RNA was isolated using TRIzol reagent (Thermo Fisher), assessed via NanoDrop and Bioanalyzer 2100 (Agilent), and polyA-selected for cDNA library preparation. Sequencing was performed on an Illumina NovaSeq 6000 (LC-Bio, Hangzhou, China). Cutadapt v1.9 and FastQC v0.11.9 were used for data filtering. Clean reads were aligned against the draft genome of T. canis (PRJNA248777.WBPS17) with HISAT2 v2.2.1 and analysed using DESeq2 (fold change > 1.5, p < 0.05), GSEA v4.1.0 and MSigDB. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from stimulated larvae (n = 10,000) using TRIzol (Thermo Fisher), reverse-transcribed with the Primer-Script RT reagent kit (Takara), and analysed with TB Green qPCR Master Mix (Takara) on a CFX96 system (Bio-Rad). Relative transcript levels of the genes Tc-daf-9 and Tc-daf-12 were calculated using the 2 ⁻ΔΔCt method, normalised to T. canis gapdh transcription. Primer sequences are listed in Supplementary Table 1. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) Lyophilised larvae ( n = 50,000) were extracted in methanol, sonicated, centrifuged, then subjected to LC-MS/MS analysis using an AB Sciex 4500 MD system, using Δ4-DA and Δ7-DA standards (GlpBio, USA) for calibration. Each condition was assessed in triplicate. Lipidomic profiling To explore factors influencing the transition of migrating larvae of T. canis into diapause, serum (100 μL) from each uninfected, infected or pregnant/infected mouse ( n = 6) was extracted in methanol, vacuum-dried, resuspended and analysed by ultra-performance liquid chromatography (UPLC) (Thermo Fisher) coupled with TripleTOF 6600 (SCIEX). XCMS, CAMERA and metaX in R were used for data processing. Metabolites with fold change > 1.5, p < 0.05 were identified and annotated using KEGG and HMDB v5.0 59 , 60 . Structural modelling and protein–protein docking Signal peptides and domains of Tc -PEBP-1 were predicted using SignalP v6.0, SMART and InterProScan 61 – 63 . The tertiary structures of proteins (e.g., ShKT domains of Tc -MUC-1, Tc -PEBP-1, Mm PEBP1, and Mm FABP5) were predicted using AlphaFold2 v2.1.0, and docking simulations were performed with ClusPro v2.0 and HawkDock v2 64 - 66 . Protein complexes were visualised using ChimeraX v1.0. 67 Recombinant protein production and polyclonal antibodies To investigate the effect of host immune factors on T. canis larvae, and of T. canis ES proteins on host immune responses, RNA from 3,000 larvae was reverse-transcribed, and coding sequences of Tc-daf-9 , Tc-daf-12 and Tc-pebp-1 were cloned into pMD19-T, pET-28a or pCOLD-TF vectors, transformed into DH5α (TransGen Biotech, China), and expressed in BL21 (DE3) cells (TransGen Biotech, China). Protein induction was performed at 15 °C with 0.6 mM IPTG. r Tc- DAF-9, r Tc- DAF-12, and r Tc- PEBP-1 were purified via Ni-NTA columns (Sangon, China), endotoxin removed and quantified using the bicinchoninic acid (BCA) assay. 68 Western blot Protein extracts from stimulated T. canis larvae (n = 10,000) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes and probed with primary mouse antibodies against T. canis DAF-9 and DAF-12 (custom-generated), then the HRP-labelled goat anti-mouse IgG antibody (Beyotime, China). T. canis beta-actin was used as a loading control. Bands were quantified using ImageJ v2.0. Macrophage stimulation and cytokine assays To study the immune recognition of T. canis ES proteins by host immune cells, RAW264.7 cells (10⁶) were cultured in DMEM (Meilunbio, China) supplemented with 10% foetal bovine serum (Thermo Fisher) and Penicillin–Streptomycin (Thermo Fisher) at 37 °C, 5% CO₂. Cells were stimulated with r Tc- PEBP-1 (5–40 μg/mL), 1 μg/mL LPS (positive control; Solarbio, China), 50 nM small interfering RNA (siRNA) targeting Nod1 , Nod2 , or Rip2 mixed with Lipofectamine 3000 (Thermo Fisher), or vehicle (negative/irrelative control) for 24 h. Cell viability was assessed by trypan blue staining, relative mRNA levels of Nod1 , Nod2 , Rip2 , Arg1 , Ccl3 , Il1b , Il10, iNOS , Pparg , Tgfb , and Tnfa were measured using qRT-PCR with the 2 ⁻ΔΔCt method (normalised to gapdh transcription, and cytokine secretion (TNF-α, IL-1β, IL-10, TGF-β) was measured in supernatants using ELISA kits (Beyotime, China). For immunoblotting, protein extracts from RAW264.7 cells (10⁶) (Beyotime, China) were separated by SDS-PAGE, transferred to PVDF membranes and probed with primary antibodies against murine NOD1/2, RIP2, NF-κB p65 and phospho-p65, then secondary antibodies (Cell Signaling Technology, USA). Murine GAPDH was used as a loading control. Bands were quantified using ImageJ v2.0. Behavioural assays Behavioural (e.g., motor, learning and memory skills) assessments of infected (n = 8) and uninfected (n = 6) mice were conducted, by rotarod testing (motor coordination at 35 dpi), light–dark box (predator aversion at 35 dpi) and Morris Water Maze (MWM; learning and memory from 43 to 49 dpi) experiments 69 – 70 . Spatial memory performance was evaluated by escape latency, swim path length and time in the target quadrant. Statistical analysis and reproducibility All in vitro experiments included three technical replicates; at least six biological replicates were used for in vivo studies. Data were analysed using unpaired Student’s t-test or one-way ANOVA in GraphPad Prism 9. p < 0.05 was considered statistically significant. Declarations Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability All data supporting the findings of this study are available in the manuscript or Supplementary Information (Supplementary Figs. 1–7; Supplementary Tables 1–4). Sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1257458. The nucleotide sequences of T. canis genes daf-9 , daf-12 and pebp1 have been deposited in the NCBI GenBank under accession nos. PV590097, PV590098 and PV590067, respectively. Acknowledgments We are grateful to Professor Aifang Du from the College of Animal Sciences, Zhejiang University for constructive comments during the drafting of this manuscript. We thank the staff of The Shared Management Platform for Large Instrument and The Experimental Teaching Centre, College of Animal Sciences, Zhejiang University. This work is supported by the National Natural Science Foundation of China (nos. 32473050 and 32002304), the National Basic Research Program of China (2023YFD1801700), and the Key Research and Development Program of Zhejiang Province (no. 2023C02036). RBG’s research program is presently funded by the Australian Research Council and Oz Omics Pty Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contribution s Conceptualization: G.M., and R.B.G. Methodology: G.M., H.W., and R.Z. Investigation: M.Z., S.L., Y.C., Z.X., H.W. and F.L. Visualization: M.Z., S.L., Z.X., Y.Y., R.Z., and G.M. Funding acquisition: G.M., and R.B.G. Project administration: Y.Y., R.Z., and G.M. Supervision: G.M., and R.B.G. Writing—original draft: Y.C., and G.M. 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Table 1 Table 1 | Stereo-seq data summary of Toxocara canis -invaded mouse brain slices (S1 to S6). Descriptions S1 S2 S3 S4 S5 S6 Clean reads 1,894,168,033 2,588,324,830 2,114,853,521 2,582,671,676 2,232,624,519 2,915,787,774 Clean reads Q20 96.11% 96.69% 96.16% 96.78% 96.47% 97.11% Clean reads Q30 87.25% 88.70% 87.66% 89.10% 88.52% 90.02% Unique mapping reads 1,546,477,368 2,051,786,083 1,735,618,844 2,119,775,668 1,823,187,537 2,345,573,814 Unique mapping reads ratio 81.64% 79.27% 82.07% 82.08% 81.66% 80.44% Number of MID under tissue 88,484,150 151,430,550 281,694,844 322,204,437 302,226,886 226,650,184 Mouse genes under tissue 35,913 35,861 38,071 39,949 39,538 40,476 Number of MID of T. canis 37 80 128 7,717 1,032 1,205 Genes of T. canis 27 50 54 113 56 60 Q20: an error rate of 1 in 100; Q30: an error rate of 1 in 1000; MID: molecular identity Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTablelegends.docx TableS1.xlsx Supplementary Table 1 TableS2.xlsx Supplementary Table 2 TableS3.xlsx Supplementary Table 3 TableS4.xlsx Supplementary Table 4 TableS5.xlsx Supplementary Table 5 SupplementaryFigurelegends.docx SupplementaryFig.1.tif Supplementary Fig. 1 SupplementaryFig.2.tif Supplementary Fig. 2 SupplementaryFig.3.tif Supplementary Fig. 3 SupplementaryFig.4.tif Supplementary Fig. 4 SupplementaryFig.5.tif Supplementary Fig. 5 SupplementaryFig.6.tif Supplementary Fig. 6 SupplementaryFig.7.tif Supplementary Fig. 7 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6743037","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":467460760,"identity":"679d11b9-8af1-4194-ab59-8a1356de3071","order_by":0,"name":"Guangxu Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYLCChAoGZhAtQbyWB2dI1cL4sA3CIE6LfER24ofEeXXsBgeYD97mYbDLI6jF8MzZzRKJ29iYDQ6wJVvzMCQXE9bS3rsBqIUHqIXHTJqH4UBiA0EtzbybfyTOkQBq4f9GnBZ59t5tEokNBiBb2IjTYsBzdptFwrEEZsnDbMaWcwySibBlRu7mmz9q6pL5jjc/vPGmwo4IWw5A6GRIZBoQUg+yBWqoHRFqR8EoGAWjYKQCALxjOAu20uuVAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5929-7887","institution":"Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Guangxu","middleName":"","lastName":"Ma","suffix":""},{"id":467460761,"identity":"8c506e28-0a80-475d-b78a-36838b2a27bb","order_by":1,"name":"Minyao Zou","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Minyao","middleName":"","lastName":"Zou","suffix":""},{"id":467460762,"identity":"b20e6531-81cf-4d37-bdb5-30af149744e1","order_by":2,"name":"Sishi Liu","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Sishi","middleName":"","lastName":"Liu","suffix":""},{"id":467460763,"identity":"87bfdca5-309a-41f4-9428-fd99adb51ff0","order_by":3,"name":"Yi Chen","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Chen","suffix":""},{"id":467460764,"identity":"45adf3e3-0648-4609-8ceb-8d7bb076a842","order_by":4,"name":"Zhiwei Xiong","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Zhiwei","middleName":"","lastName":"Xiong","suffix":""},{"id":467460765,"identity":"12a90af6-2b6b-4ab2-86a9-bf7be3c50105","order_by":5,"name":"Haiyan Wu","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Wu","suffix":""},{"id":467460766,"identity":"c7ec049a-a1c3-4d52-bb78-b0bfe0f71c9e","order_by":6,"name":"Fang Li","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Li","suffix":""},{"id":467460767,"identity":"53bb0bfa-8ae3-4392-b4d1-f2fd4dd1904b","order_by":7,"name":"Yi Yang","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Yang","suffix":""},{"id":467460768,"identity":"8f7c5053-3c80-48e0-a32c-e776a3a8ab8a","order_by":8,"name":"Rongqiong Zhou","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Rongqiong","middleName":"","lastName":"Zhou","suffix":""},{"id":467460769,"identity":"5981f4ef-57f3-44c1-bc8e-b00a2acf464c","order_by":9,"name":"Robin B. Gasser","email":"","orcid":"https://orcid.org/0000-0002-4423-1690","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Robin","middleName":"B.","lastName":"Gasser","suffix":""}],"badges":[],"createdAt":"2025-05-25 10:00:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6743037/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6743037/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84209012,"identity":"ea725144-6860-4330-a5d7-ee4f55151dbf","added_by":"auto","created_at":"2025-06-09 09:43:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":14341741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial cell atlas of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eToxocara canis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-invaded murine brain slices. a\u003c/strong\u003e Haematoxylin and eosin-stained slices showing parenchymal damage and haemorrhagic lesions in \u003cem\u003eT. canis\u003c/em\u003e-invaded murine brains. \u003cstrong\u003eb\u003c/strong\u003e Fluorescence imaging of olfactory bulb (S1), cerebrum (S2–S5) and cerebellum (S6) slices; white pixels represent murine nucleic acids, red pixels (arrows) indicate \u003cem\u003eT. canis\u003c/em\u003e nucleic acids. \u003cstrong\u003ec\u003c/strong\u003eStereo-seq-derived transcript abundance matrix and spatial distribution (Bin100) across S1–S6, with colour indicating total transcript count (light green: 20,000; dark red: 60,000). \u003cstrong\u003ed\u003c/strong\u003e Cell clustering and manual annotation of astrocytes, endothelial cells, epithelial cells, microglia, neurons and oligodendrocytes, based on dimensionality reduction of transcript data using UMAP. \u003cstrong\u003ee\u003c/strong\u003e Spatial mapping of annotated cell types across brain slices S1–S6.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/4d7ba07853c8e074e332f6eb.png"},{"id":84209878,"identity":"1700007c-4895-4778-96f0-e3fefca586b6","added_by":"auto","created_at":"2025-06-09 09:51:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10736637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune microenvironment of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eToxocara canis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-invaded murine brains. a\u003c/strong\u003e Spatial localisation of two \u003cem\u003eT. canis\u003c/em\u003e larvae and their contralateral control regions across the sagittal plane of cerebrum slice S4. Locations of larvae, microglia, endothelial cells, neurons and oligodendrocytes are indicated. Inset shows haematoxylin and eosin-stained image of a larval niche with associated haemorrhagic lesion. \u003cstrong\u003eb\u003c/strong\u003eDifferential transcriptomic analysis between larval niches and matched control regions. Significantly upregulated (\u003cem\u003en\u003c/em\u003e = 475) and downregulated (\u003cem\u003en\u003c/em\u003e= 497) genes (fold change \u0026gt; 2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) are shown in a volcano plot (red and blue, respectively). \u003cstrong\u003ec\u003c/strong\u003e Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of differentially transcribed genes in larval niches. Enriched categories include metabolism (e.g., glycan degradation, sphingolipid metabolism), genetic information processing (e.g., mismatch repair), environmental signalling (e.g., MAPK, Ras, Rap1), cellular processes (e.g., apoptosis), immune pathways (e.g., antigen presentation, C-type lectin signalling, leukocyte trans-endothelial migration, circadian rhythm), and disease pathways (e.g., glioma, nicotine addiction). \u003cstrong\u003ed\u003c/strong\u003e KEGG pathways enriched among significantly altered lipid metabolites in sera of \u003cem\u003eT. canis\u003c/em\u003e-infected mice versus controls. Affected pathways include immune and inflammatory signalling, bile acid and lipid metabolism, endocrine regulation, and cellular defence. \u003cstrong\u003ee\u003c/strong\u003e KEGG pathway enrichment of lipid metabolites in migrating larvae stimulated \u003cem\u003ein vitro\u003c/em\u003e with host-positive serum. Enriched pathways reflect survival strategies, including cuticle remodelling, glycan masking, stress diapause (dauer entry), environmental signal transduction, fatty acid metabolism and locomotion.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/f2df4f0d8ffce7e8b3406514.png"},{"id":84209877,"identity":"2fb91b7b-9fd7-4868-a862-c663d57190df","added_by":"auto","created_at":"2025-06-09 09:51:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9674005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStress diapause of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eToxocara canis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e larvae in response to host immune signals. a\u003c/strong\u003e Relative transcript levels of \u003cem\u003edaf-9\u003c/em\u003e and \u003cem\u003edaf-12\u003c/em\u003ein larvae treated with positive versus negative murine serum. \u003cstrong\u003eb\u003c/strong\u003e Western blot analysis of DAF-9 and DAF-12 protein in larvae treated with or without positive serum. Quantified changes relative to negative serum-treated controls are shown. \u003cstrong\u003ec\u003c/strong\u003e Relative transcript levels of \u003cem\u003edaf-9\u003c/em\u003e and \u003cem\u003edaf-12\u003c/em\u003ein larvae treated with positive/prenatal versus positive serum. \u003cstrong\u003ed\u003c/strong\u003eWestern blot analysis of DAF-9 and DAF-12 protein in larvae treated with positive/prenatal versus positive serum, with quantification shown. \u003cstrong\u003ee\u003c/strong\u003eQuantification of Δ4- and Δ7-dafachronic acid (DA) in larvae treated with negative, positive or positive/prenatal serum. Data are shown as mean ± standard deviation. Student’s t-test used for comparisons; *, **, *** and ns denote \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 and not significant, respectively. \u003cstrong\u003ef\u003c/strong\u003e Gene set enrichment analysis identifying 77 and 63 downregulated genes in positive serum-treated larvae compared with negative and positive/prenatal serum-treated larvae, respectively. Consistently downregulated genes \u003cem\u003eTcan_08258\u003c/em\u003e and \u003cem\u003eTcan_10138\u003c/em\u003e encode G protein-coupled receptors (GPCRs) involved in neuroactive ligand–receptor interactions. \u003cstrong\u003eg\u003c/strong\u003e Heatmap of the top 50 variable lipid metabolites across negative, positive and positive/prenatal serum samples. \u003cstrong\u003eh\u003c/strong\u003e Molecular docking of GPCR encoded by \u003cem\u003eTcan_10138\u003c/em\u003e with stigmasta-4,22-dien-3-one, cholic acid and DGDG O-19:2_24:6, with predicted binding energies of –8.8, –8.0 and –7.9 kcal/mol, respectively.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/a0d5623e9a4a8e68ae931e8c.png"},{"id":84210647,"identity":"4e099d8a-fb79-4e5c-bf21-553afd4721c4","added_by":"auto","created_at":"2025-06-09 09:59:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5681116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune evasion and modulation by migrating \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eToxocara canis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e larvae. a\u003c/strong\u003e Proportion of protein families encoded by \u003cem\u003eT. canis\u003c/em\u003e genes in murine brain slices based on Stereo-seq data. Excretory/secretory (ES) protein-coding genes constitute 91.53% of total detected genes. Expression levels of mucins (TES-120), C-type lectins (TES-32), phosphatidylethanolamine-binding proteins (TES-26), venom allergen-like proteins and chondroitin proteoglycans are shown in a heatmap based on molecular identity (MID) counts. \u003cstrong\u003eb\u003c/strong\u003e Structural comparison of protein complexes comprising \u003cem\u003eT. canis\u003c/em\u003e MUC1 (\u003cem\u003eTc-\u003c/em\u003eMUC-1) ShKT domains, murine FABP5 (\u003cem\u003eMm\u003c/em\u003eFABP5) and PEBP1 (\u003cem\u003eMm\u003c/em\u003ePEBP1), versus \u003cem\u003eTc-\u003c/em\u003eMUC-1 ShKT domains, \u003cem\u003eT. canis\u003c/em\u003e PEBP1 (\u003cem\u003eTc\u003c/em\u003e-PEBP-1) and \u003cem\u003eMm\u003c/em\u003eFABP5. \u003cem\u003eTc\u003c/em\u003e-PEBP-1 shows stronger predicted binding to \u003cem\u003eMm\u003c/em\u003eFABP5 (-35.04 kcal/mol) than its murine orthologue (-18.48 kcal/mol). \u003cstrong\u003ec\u003c/strong\u003e Relative transcript levels of M1 marker genes (iNOS, \u003cem\u003eCcl3\u003c/em\u003e, \u003cem\u003eTnfa\u003c/em\u003e,\u003cem\u003e Il1b\u003c/em\u003e) in RAW264.7 cells stimulated with 0, 5, 10 and 15 µg/mL recombinant \u003cem\u003eT. canis\u003c/em\u003e PEBP1 (r\u003cem\u003eTc-\u003c/em\u003ePEBP-1), compared to LPS-stimulated controls. \u003cstrong\u003ed\u003c/strong\u003e Levels of secreted TNF-α and IL-1β in the same stimulated RAW264.7 cells. \u003cstrong\u003ee\u003c/strong\u003e Relative transcript levels of M2 marker genes (\u003cem\u003eArg1\u003c/em\u003e, \u003cem\u003ePparg\u003c/em\u003e, \u003cem\u003eTgfb\u003c/em\u003e, \u003cem\u003eIl10\u003c/em\u003e) in RAW264.7 cells stimulated with r\u003cem\u003eTc-\u003c/em\u003ePEBP-1. \u003cstrong\u003ef\u003c/strong\u003e Levels of secreted TGF-β and IL-10 in the same cells. \u003cstrong\u003eg–i\u003c/strong\u003e Western blot analysis and band intensity quantification of NOD2, RIP2, phosphorylated NF-κB p65 and GAPDH in RAW264.7 cells stimulated with r\u003cem\u003eTc-\u003c/em\u003ePEBP-1 for 6, 12, 24, 36 and 48 hours. Data are presented as mean ± standard deviation; statistical significance was assessed by one-way ANOVA, with different letters indicating \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. \u003cstrong\u003ej\u003c/strong\u003e Schematic model proposing that PEBP1 and other ES proteins released by \u003cem\u003eT. canis\u003c/em\u003e larvae promote polarisation of M0 macrophages into M2a macrophages via the NOD1/2–RIP2–NF-κB axis, enhancing Arg-1, TGF-β and IL-10 production, upregulating PPARγ signalling and phagocytic capacity.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/bf99314f822e775449be952c.png"},{"id":84209881,"identity":"4fed15ae-d0c5-444a-ad46-8c54a87207f2","added_by":"auto","created_at":"2025-06-09 09:51:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":14238988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathological consequences of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eToxocara canis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eneuroinvasion in mice. a\u003c/strong\u003e Annotation and spatial mapping of cell clusters in the olfactory bulb (S1), cerebrum (S2 and S3) and cerebellum (S6) slices. Unsymmetrical distributions of neuronal and immune cell populations are highlighted in black boxes. Associated Gene Ontology biological processes (e.g., ameboid-type migration, peroxidase activity, gliogenesis, cognition) and pathways (e.g., PI3K–Akt signalling, neurodegeneration, long-term memory) are summarised. \u003cstrong\u003eb\u003c/strong\u003e Schematic illustrating \u003cem\u003eT. canis\u003c/em\u003eneuroinvasion in the murine CNS and behavioural assays (rotarod, light/dark box, Morris Water Maze) used to assess motor learning, anxiety-like behaviour, and memory. \u003cstrong\u003ec\u003c/strong\u003e Motor coordination of \u003cem\u003eT. canis\u003c/em\u003e-infected and uninfected BALB/c mice assessed by rotarod test at 35 days post infection (dpi). \u003cstrong\u003ed\u003c/strong\u003e Time spent by infected and uninfected mice in the predator-scented area (anesthetised rat and dog urine) measured using a light/dark box test at 35 dpi. \u003cstrong\u003ee\u003c/strong\u003e Learning and memory performance in infected and uninfected mice assessed by Morris Water Maze at 43 dpi, evaluating time to reach the platform and swimming path length. \u003cstrong\u003ef\u003c/strong\u003eContinued Morris Water Maze assessment on days 45–48 dpi confirming learning and memory impairments. \u003cstrong\u003eg\u003c/strong\u003e Time spent in the target quadrant of the Morris Water Maze by infected and uninfected mice. Data are presented as mean ± standard error of the mean; statistical significance was determined by Student’s t-test. *, ** and ns denote \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and not significant, respectively.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/a7d7a80d3db4a5c49fcd7d7d.png"},{"id":84211170,"identity":"2063d5bc-0fb1-48fb-9225-e81f87997182","added_by":"auto","created_at":"2025-06-09 10:07:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":52548217,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/883ed4f2-6ea1-44ad-a3ea-d3f9f937a256.pdf"},{"id":84209003,"identity":"67c9d6e3-e8a9-48a2-a1af-c4d19d405dc5","added_by":"auto","created_at":"2025-06-09 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09:43:06","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":959124,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/85c3372c31f7fecda14e8092.xlsx"},{"id":84209016,"identity":"532d83cc-1c47-4141-8544-4e023c1bd753","added_by":"auto","created_at":"2025-06-09 09:43:07","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5158290,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3\u003c/p\u003e","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/0f9dce316e9c69d13d34f19c.xlsx"},{"id":84209009,"identity":"f2e71459-8ff7-4f04-8752-8a971c362a8e","added_by":"auto","created_at":"2025-06-09 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09:43:07","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":42387614,"visible":true,"origin":"","legend":"Supplementary Fig. 4","description":"","filename":"SupplementaryFig.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/d442915c73d0ffc4be1d287e.tif"},{"id":84209022,"identity":"4fbee70f-19e5-4974-9abe-9ae2e2579b3c","added_by":"auto","created_at":"2025-06-09 09:43:07","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":61470298,"visible":true,"origin":"","legend":"Supplementary Fig. 5","description":"","filename":"SupplementaryFig.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/86af37c6aedc50040a943ac8.tif"},{"id":84209023,"identity":"9779cf0f-9b6a-4725-8d3b-56eee4aeb572","added_by":"auto","created_at":"2025-06-09 09:43:07","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":43538318,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Fig. 6\u003c/p\u003e","description":"","filename":"SupplementaryFig.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/e1895f9dd6f28118ca2404bd.tif"},{"id":84209018,"identity":"3e82d1c8-abbb-4e01-be75-d638782c29ce","added_by":"auto","created_at":"2025-06-09 09:43:07","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":24531574,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Fig. 7\u003c/p\u003e","description":"","filename":"SupplementaryFig.7.tif","url":"https://assets-eu.researchsquare.com/files/rs-6743037/v1/032206aa3d07887f90b82dd0.tif"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Spatial transcriptomic atlas of murine neurotoxocariasis reveals region-specific host responses and dysfunction in the brain","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman toxocariasis is a globally distributed but under-recognised zoonotic parasitic disease caused by infection with larvae of \u003cem\u003eToxocara canis\u003c/em\u003e (dog roundworm) or \u003cem\u003eT. cati\u003c/em\u003e (cat roundworm)\u003csup\u003e1\u003c/sup\u003e. People, particularly children, become infected following the accidental ingestion of embryonated eggs or infective larvae, typically from contaminated soil, food or water. Following ingestion, larvae hatch in the intestine, penetrate the intestinal wall, enter the bloodstream and disseminate to various organs, including the liver, lungs, central nervous system (CNS) and eyes. These events lead to syndromes broadly classified as visceral larva migrans (VLM), ocular larva migrans (OLM) and neurotoxocariasis\u003csup\u003e1,\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe first recognised case of human neurotoxocariasis was reported in 1951, when \u003cem\u003eToxocara\u003c/em\u003e larvae were identified in the brain of a child who had died from poliomyelitis\u003csup\u003e3\u003c/sup\u003e. Although CNS involvement is often subclinical, a wide spectrum of neurological manifestations \u0026ndash; including eosinophilic meningitis, encephalitis, myelitis and cerebral vasculitis \u0026ndash; has been documented\u003csup\u003e4,5\u003c/sup\u003e. Recent seroepidemiological studies suggest potential associations between \u003cem\u003eToxocara\u003c/em\u003e infection and neuropsychiatric and neurodegenerative disorders, including epilepsy, schizophrenia, cognitive decline, Parkinson\u0026apos;s disease and Alzheimer\u0026apos;s disease\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e. Although systemic immune responses to \u003cem\u003eT. canis\u003c/em\u003e have been characterised in dogs, humans and mice, and are typically marked by Th2-skewed immunity and eosinophilia, the local immune response within the CNS remains poorly defined\u003csup\u003e9\u003c/sup\u003e. Migrating larvae excrete and secrete a complex array of immunomodulatory molecules \u0026ndash; including mucins, lectins, protease inhibitors and other glycoproteins \u0026ndash; that modulate host immunity and promote larval persistence during tissue migration\u003csup\u003e10\u0026ndash;12\u003c/sup\u003e. Notably, pathological examination of murine brains harbouring larvae often reveals limited leukocytic infiltration, but marked transcriptomic and lipidomic alterations\u003csup\u003e13\u0026ndash;17\u003c/sup\u003e, suggesting that the brain suppresses inflammatory responses to prevent bystander tissue damage. Despite these indications, the pathogenesis of neurotoxocariasis and the role of \u003cem\u003eToxocara\u003c/em\u003e in neurodegenerative disease remain elusive\u003csup\u003e7,18\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis work employed high-resolution spatial transcriptomics to define an \u003cem\u003ein situ\u003c/em\u003e cellular and immune atlas of the murine brain during \u003cem\u003eT. canis\u003c/em\u003e neuroinvasion, in order to characterise regional transcriptional landscapes and immune microenvironments. By integrating spatial transcriptomic data with behavioural assays of learning and memory in mice, we provide experimental evidence that \u003cem\u003eT. canis\u003c/em\u003e neuroinvasion is associated with region-specific molecular perturbations and functional cognitive impairment. This study links spatially resolved host\u0026ndash;parasite interactions with neurophysiological outcomes in a well-defined model of neurotoxocariasis.\u003c/p\u003e\n"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSpatial and temporal neuroinvasion by \u003cem\u003eT. canis\u003c/em\u003e in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the kinetics and spatial pattern of neuroinvasion, we orally infected specific pathogen-free (SPF) BALB/c mice with 1000 embryonated \u003cem\u003eT. canis\u003c/em\u003eeggs each, and examined brain tissue at multiple time points after infection. Brains from six infected mice were assessed at 7, 14, 21, 28 and 35 days post-infection (dpi) using the tissue digestion method. On average, ~10% of the infective dose was recovered from the brains. Larval recovery peaked at 21 dpi and gradually declined thereafter, indicating a dynamic process of CNS invasion, persistence or clearance. At no stage during experimentation were larvae detected in the brains of uninfected control mice.\u003c/p\u003e\n\u003cp\u003eAt 21 dpi, the time point with the highest larval burden, macroscopic examination revealed focal parenchymal damage and haemorrhagic lesions, prompting detailed microscopic analysis of coronal brain slices. We examined the olfactory bulb, cerebrum and cerebellum. In the olfactory bulb, although parenchymal damage was evident in most slices, larvae were not consistently observed. In contrast, both haemorrhagic lesions and larvae were frequently detected in the cerebrum and cerebellum (Supplementary Fig. 1). These observations confirmed neuroinvasion by \u003cem\u003eT. canis\u003c/em\u003e and revealed region-specific pathological changes in the murine brain, with the cerebrum and cerebellum being key target sites for larval migration and associated tissue injury\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpatially resolved cell atlases for \u003cem\u003eT. canis\u003c/em\u003e-invaded brain regions in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the host\u0026ndash;parasite interface in neurotoxocariasis, we applied Stereo-seq spatial transcriptomics to coronal sections of the olfactory bulb (slice S1), cerebrum (slices S2\u0026ndash;S5) and cerebellum (slice S6) invaded by \u003cem\u003eT. canis\u0026nbsp;\u003c/em\u003elarvae (Fig. 1a; Supplementary Fig. 1). This enabled high-resolution spatial mapping of transcription in both host and parasite (Fig. 1b).\u003c/p\u003e\n\u003cp\u003eAnalysis of the data revealed widespread transcription across all brain regions, with 35,913 to 40,476 murine genes transcribed in slices S1 to S6, respectively (Table 1). Parasite-derived transcripts were also detected, corresponding to 27, 50, 54, 113, 56 and 60 \u003cem\u003eT. canis\u003c/em\u003e genes, with molecular identity (MID) counts of 37, 80, 128, 7,717, 1,032 and 1,205 per 100 \u0026times; 100-pixel spatial bin (Bin100) in slices S1, S2, S3, S4, S5, and S6, respectively (Table 1). Notably, the highest abundance of parasite transcripts was observed in the cerebrum (S4, S5) and cerebellum (S6), compared with the olfactory bulb (S1), where transcript counts were markedly lower.\u003c/p\u003e\n\u003cp\u003eUsing the gene transcription matrix generated from this analysis (Fig. 1c), we performed unsupervised clustering and manual cell type annotation (Fig. 1d). Spatially resolved transcription of canonical markers allowed the identification of astrocytes, basket cells, endothelial cells, ependymal cells, microglia, mural cells, neurons and oligodendrocytes. These atlases define the cellular architecture of brain regions invaded by \u003cem\u003eT. canis\u003c/em\u003e, offering new insights into the spatial organisation of host responses to neuroinvasion (Fig. 1e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLocalised immune recognition with limited leukocytic infiltration in \u003cem\u003eT. canis\u003c/em\u003e-infected brains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpatially resolved atlases revealed focal accumulation of endothelial and mural cells at sites of parenchymal damage and haemorrhage in brain slices S1, S2 and S3 (Fig. 1e). Microglia and oligodendrocytes were localised primarily to the meninges and cortex, suggesting roles in damage response and vascular repair. In contrast, no such cell aggregation was observed in slices S4, S5 or S6, despite high levels of parasite-derived transcripts (Fig. 1b,e), indicating supressed host responses in tissue microenvironments associated with larvae or damage induced by migrating larvae. Notably, neutrophilic and eosinophilic granulocytes \u0026ndash; known to mediate larval trapping and granuloma formation in peripheral tissues \u003csup\u003e20\u0026ndash;22\u003c/sup\u003e \u0026ndash; were absent from all regions examined, even in areas with visible larval migration or tissue disruption. These findings support previous evidence of limited leukocytic infiltration in \u003cem\u003eT. canis\u003c/em\u003e-invaded rodent brains and raise the possibility of local immune suppression or evasion within the CNS\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo investigate further, we performed deeper cell type annotation using public reference data, identifying a broader repertoire of host cell types (Supplementary Figs. 2 and 3) \u0026ndash; including adipocytes, astrocytes, Bergmann glia, endothelial and epithelial cells, fibroblasts, mesenchymal cells, microglia, mural cells, neurons, stromal cells, and particularly erythrocytes, dendritic cells, B cells, and T cells. Notably, we also identified macrophages and granulocytes not previously seen in healthy mouse brains\u003csup\u003e24\u0026ndash;26\u003c/sup\u003e. The presence of antigen-presenting cells (microglia, macrophages, dendritic cells) and immune effectors (B cells, T cells, granulocytes) indicates an impairment of the blood-brain barrier; while immune recognition of \u003cem\u003eT. canis\u003c/em\u003e occurs but is confined to discrete niches surrounding the larvae.\u003c/p\u003e\n\u003cp\u003eTo further explore this, we conducted snRNA-seq on sections adjacent to slices S4, S5 and S6 containing migrating \u003cem\u003eT. canis\u003c/em\u003e larvae (Fig. 1b; Table 1; Supplementary Fig. 1). Microglia predominated in the dataset from the S4-adjacent sample (MID = 7,717), but not in S5- or S6-adjacent sample (MID = 1,032 and 1,205, respectively) (Supplementary Fig. 4). These findings indicate that key antigen-presenting cells are in close vicinity to migrating \u003cem\u003eT. canis\u003c/em\u003e larvae. Taken together, the Stereo-seq and snRNA-seq data indicate that \u003cem\u003eT. canis\u003c/em\u003e is recognised by the CNS immune system, but that immune responses remain highly localised, potentially to avoid broader neuroinflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFocal immune activation in \u003cem\u003eT. canis\u003c/em\u003e-infected brains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to variability in lesion location between animals, pairwise anatomical comparisons with uninfected controls were not feasible. Therefore, we focused our spatial transcriptomic analyses on larval microenvironments (~1,000 cells per region) in brain slices S4, S5 and S6, where \u003cem\u003eT. canis\u003c/em\u003e transcripts were abundant (Fig. 1b). Within these regions, we observed focal infiltration of microglia, most prominently in S4, relative to the contralateral, parasite-free hemisphere (Fig. 2a). A localised accumulation of endothelial cells was also evident around migrating larvae, suggestive of angiogenesis or vascular injury. These patterns were not discernible on a tissue-wide level (cf. Fig. 1d,e), indicating that the \u003cem\u003eT. canis\u003c/em\u003e larvae themselves induce discrete, spatially confined inflammatory and vascular responses.\u003c/p\u003e\n\u003cp\u003eTo investigate the associated molecular landscape, we compared transcriptional profiles between areas with (\u003cem\u003en\u003c/em\u003e = 2) and without (\u003cem\u003en\u003c/em\u003e = 2) visible larvae. A total of 970 genes were differentially transcribed (fold change \u0026gt; 2, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Fig. 2b; Supplementary Table 1). These genes were significantly enriched (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) for pathways involved in metabolism (e.g., sphingolipid metabolism, non-canonical glycan degradation), environmental information processing (e.g., mitogen-activated protein kinase [MAPK], Rap1, Ras signalling), genetic information processing (e.g., mismatch repair, DNA replication), cellular processes (e.g., apoptosis), immune function (e.g., antigen presentation, C-type lectin receptor signalling, leukocyte trans-endothelial migration, platelet activation, circadian rhythm) and pathogenesis (e.g., glioma, nicotine addiction) (Fig. 2c).\u003c/p\u003e\n\u003cp\u003eNotably, concurrent activation of MAPK signalling (pro-inflammatory) and glycan degradation pathways (immune evasion) indicates that \u003cem\u003eT. canis\u003c/em\u003e neuroinvasion provokes a dual response: focal host immune activation, counterbalanced by parasite-driven modulation\u003csup\u003e22\u003c/sup\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThese findings define a spatially restricted but molecularly complex interface between host immunity and parasite persistence within the brain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSystemic host responses and parasite adaptation during \u003cem\u003eT. canis\u003c/em\u003e infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate systemic responses to \u003cem\u003eT. canis\u003c/em\u003e infection beyond the CNS, we profiled lipid metabolites in the sera of infected and uninfected mice at 21 dpi. A total of 735 differential (fold change \u0026gt; 1.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) lipid species were detected in the sera of infected mice compared with uninfected ones (Supplementary Fig. 5A). These metabolites were significantly enriched (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) for pathways involved in immune and inflammatory signalling (e.g., B and T cell receptor signalling, Th1, Th2 and Th17 differentiation, nuclear factor kappa-beta [NF-\u0026kappa;B], MAPK and ErbB signalling), hormonal regulation (e.g., gonadotropin-releasing hormone [GnRH], oestrogen, insulin secretion, peroxisome proliferator-activated receptor PPAR signalling), metabolic function (e.g., bile secretion, lipid and cholesterol metabolism) and cellular homeostasis (e.g., neurotrophin and relaxin signalling, vitamin absorption, autophagy) (Fig. 2d; Supplementary Table 2). These lipidomic results indicate a broad systemic response to infection, complementing local immune responses in the brain\u003csup\u003e16,27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo assess whether circulating host signals influence the parasite, we incubated third-stage \u003cem\u003eT. canis\u003c/em\u003e larvae in serum from infected mice for 24 h. Transcriptomic analysis identified more than 350 \u003cem\u003eT. canis\u003c/em\u003e genes as differentially transcribed (fold change \u0026gt; 1.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) compared with larvae incubated in control serum from uninfected mice (Supplementary Fig. 5b). These genes (n \u0026gt; 350) were enriched (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) for pathways linked to signal transduction (e.g., neurotransmitter transport, G protein-coupled receptor [GPCR] signalling), immune evasion (e.g., O-glycan processing, collagen and cuticle remodelling), stress response (including dauer entry regulation) and motility (e.g., positive regulation of movement) (Fig. 2e). Key pathways included mucin-type O-glycan biosynthesis, PPAR signalling, neuroactive ligand\u0026ndash;receptor interaction, fatty acid and cholesterol metabolism and MAPK signalling (Supplementary Table 3).\u003c/p\u003e\n\u003cp\u003eThese findings are consistent with molecular alterations in the \u003cem\u003eT. canis\u003c/em\u003e-infected brain (Fig. 2e) and illustrate a coordinated parasite response to host immune pressure. Although infection elicits inflammatory and immunological responses in the CNS, the parasite counters with adaptive mechanisms, including glycan masking, cuticle remodelling, stress mitigation and neuromuscular modulation\u003csup\u003e10,28\u003c/sup\u003e. This reciprocal interaction highlights a dynamic host\u0026ndash;parasite interface extending from the CNS microenvironment to systemic circulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStress diapause and endocrine regulation of \u003cem\u003eT. canis\u003c/em\u003e larvae in the brain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of brain-resident \u003cem\u003eT. canis\u003c/em\u003e larvae revealed gene transcription profiles consistent with a survival strategy in response to the inflamed host environment (Supplementary Table 4). Transcripts included those encoding structural cuticle components (e.g., collagens, cuticulins), membrane transporters (e.g., transthyretin-like proteins, multidrug resistance protein 3 [MRP3], Niemann\u0026ndash;Pick disease type C-associated cholesterol receptor [NPC1/NCR1]), GPCRs (e.g., FMRFamide receptor, RhoA, SRAB14), enzymes (e.g., \u0026beta;-galactosidase, acetyl-CoA synthetase, acyl-CoA thioesterase, peroxidase) and ion channels (e.g., the nicotinic acetylcholine receptor ACR-16, bestrophin, innexin). These transcriptional patterns reflect physiological adaptations to maintain cuticle integrity, nutrient acquisition, oxidative stress resistance and metabolic suppression \u0026ndash; hallmarks of diapause or hypobiosis\u003csup\u003e29\u003c/sup\u003e. These signatures were corroborated \u003cem\u003ein vitro\u003c/em\u003e: larvae exposed to serum from infected mice showed an enrichment of transcripts linked to dauer entry \u0026ndash; a conserved stress-induced quiescent state in nematodes (Fig. 2e). In \u003cem\u003eC. elegans\u003c/em\u003e, dauer entry is regulated by the dafachronic acid (DA) hormonal module, which includes DAF-9 (cytochrome P450), DA (a bile acid\u0026ndash;like ligand), and DAF-12 (a nuclear hormone receptor)\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo investigate whether this module is conserved in \u003cem\u003eT. canis\u003c/em\u003e and explore whether it plays a regulatory role in the immune evasion by \u003cem\u003eT. canis\u003c/em\u003e in the CNS, we quantified key proteins in larvae exposed to serum from infected mice. Compared with controls, exposed larvae showed a reduced DAF-9 protein expression (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), decreased abundance of 25S-\u0026Delta;7-DA (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and diminished DAF-12 protein levels (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Fig. 3a\u0026ndash;c), indicating dauer entry. The developmental arrest of larvae following migration in the CNS is likely a response to immune pressure, and indicates the involvement of hormone\u0026ndash;immune interactions in neurotoxocariasis\u003csup\u003e31\u003c/sup\u003e. Notably, suppression of the DAF-9\u0026ndash;DA\u0026ndash;DAF-12 pathway was reversed when larvae were exposed to serum from perinatally infected mice, suggesting that a hormonal mechanism underpins exit from dauer or a reactivation of development (Fig. 3c\u0026ndash;e; Supplementary Fig. 5c,d)\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e32\u003c/sup\u003e. This finding was supported by increased transcription of neuroactive ligand\u0026ndash;receptor genes, including \u003cem\u003eTcan_08258\u003c/em\u003e (encoding a GnRH receptor homologue) and \u003cem\u003eTcan_10138\u003c/em\u003e (a predicted GPCR of unknown function) (Fig. 3f). Altered GnRH signalling in infected mice implicates \u003cem\u003eTcan_08258\u003c/em\u003e in stress diapause and reactivation. Although the function of \u003cem\u003eTcan_10138\u003c/em\u003e remains unclear, lipid profiling of negative, positive and perinatal-positive sera identified candidate ligands with reciprocal abundance. Stigmasta-4,22-dien-3-one, CE 22:6, cholic acid and DGDG O-19:2_24:6 were predicted to bind \u003cem\u003eTcan_10138\u003c/em\u003e with high affinity (\u0026ndash;8.8 to \u0026ndash;7.5 kcal/mol) (Fig. 3g,h).\u003c/p\u003e\n\u003cp\u003eThese host-derived lipid molecules \u0026ndash; particularly stigmasta-4,22-dien-3-one and CE 22:6 \u0026ndash; may act analogously to ascarosides \u0026ndash; small molecule regulators of dauer entry in free-living nematodes \u0026ndash; which have also been shown to suppress type 2 inflammation in mammals\u003csup\u003e33\u003c/sup\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e\u003csup\u003e34\u003c/sup\u003e. Together, these findings indicate that \u003cem\u003eT. canis\u003c/em\u003e larvae enter a transcriptionally regulated, hormonally modulated diapause-like state in the murine brain. Moreover, endocrine cues from the host appear capable of orchestrating both the initiation and termination of this stress-adaptive program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmune evasion and modulation by \u003cem\u003eT. canis\u003c/em\u003e in the brain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpatial transcriptomics revealed that more than 90% of \u003cem\u003eT. canis\u003c/em\u003e transcripts detected in infected brain slices encode excretory/secretory (ES) proteins (Fig. 4a), including mucins (120 kDa), C-type lectins (32 kDa), phosphatidylethanolamine-binding proteins (PEBPs; 26 kDa), venom allergen-like proteins and chondroitin proteoglycans (Supplementary Table 4). These ES proteins are central to parasite immune evasion, with mucin transcripts being the most abundant. These mucins contribute to a \u0026ldquo;fuzzy surface coat\u0026rdquo; that shields larvae from host immune attack\u003csup\u003e28\u003c/sup\u003e. Despite structural homology with host proteins, \u003cem\u003eT. canis\u003c/em\u003e ES proteins contain functional domains that are absent from mammalian orthologues. For instance, \u003cem\u003eT. canis\u003c/em\u003e PEBP1 (\u003cem\u003eTc\u003c/em\u003e-PEBP-1) includes a signal peptide and two ShKT toxin domains \u0026ndash; features absent from murine PEBP1 (\u003cem\u003eMm\u003c/em\u003ePEBP1) (Supplementary Fig. 6a,b) \u0026ndash; suggesting disruption of immune cell signalling, particularly in T cells and macrophages.\u003c/p\u003e\n\u003cp\u003eWe focused on \u003cem\u003eTc-\u003c/em\u003eMUC-1, a mucin that interacts with cytoskeletal and signalling proteins in murine macrophages via ShKT domains\u003csup\u003e35\u003c/sup\u003e. Although direct interaction between \u003cem\u003eTc-\u003c/em\u003eMUC-1 and \u003cem\u003eMm\u003c/em\u003ePEBP1 was not confirmed in RAW264.7 cells, structural modelling predicted strong binding between \u003cem\u003eMm\u003c/em\u003eFABP5 and both \u003cem\u003eMm\u003c/em\u003ePEBP1 and \u003cem\u003eTc-\u003c/em\u003ePEBP-1, with binding free energies of \u0026ndash;18.48 and \u0026ndash;35.04 kcal/mol, respectively (Fig. 4b). These findings support the existence of \u003cem\u003eTc-\u003c/em\u003eMUC-1\u0026ndash;\u003cem\u003eMm\u003c/em\u003eFABP5\u0026ndash;\u003cem\u003eTc-\u003c/em\u003ePEBP-1 or mixed \u003cem\u003eTc\u003c/em\u003e\u0026ndash;\u003cem\u003eMm\u003c/em\u003e protein complexes. As FABP5 regulates PPAR signalling\u003csup\u003e36\u003c/sup\u003e, \u003cem\u003eTc\u003c/em\u003ePEBP1 may influence macrophage polarisation and immune signalling.\u003c/p\u003e\n\u003cp\u003eTo assess the immunomodulatory role of \u003cem\u003eTc-\u003c/em\u003ePEBP-1, RAW264.7 macrophages were stimulated with recombinant \u003cem\u003eTc-\u003c/em\u003ePEBP-1 (r\u003cem\u003eTc-\u003c/em\u003ePEBP-1) (Supplementary Fig. 6c). At 5 \u0026mu;g/mL, r\u003cem\u003eTc-\u003c/em\u003ePEBP-1 induced a rounded morphology consistent with M1 polarisation, whereas 15 \u0026mu;g/mL induced elongation characteristic of M2 activation (Supplementary Fig. 4d). M1 markers (\u003cem\u003eiNOS\u003c/em\u003e, \u003cem\u003eCcl3\u003c/em\u003e, \u003cem\u003eTnfa\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e) were downregulated in a dose-dependent manner (Fig. 4c,d), whereas M2 markers (\u003cem\u003ePparg\u003c/em\u003e, \u003cem\u003eArg1\u003c/em\u003e) were significantly upregulated at 15 \u0026mu;g/mL (Fig. 4e,f). r\u003cem\u003eTc-\u003c/em\u003ePEBP-1 also increased expression of NOD2, RIP2 and phosphorylated NF-\u0026kappa;B p65 (Fig. 4g\u0026ndash;i; Supplementary Fig. 6e\u0026ndash;h), implicating the NOD2\u0026ndash;RIP2\u0026ndash;NF-\u0026kappa;B pathway in downstream signalling (Fig. 4j). Together, these results indicate that \u003cem\u003eT. canis\u003c/em\u003e ES proteins, including \u003cem\u003eTc\u003c/em\u003ePEBP1, function as immunomodulators: low levels of \u003cem\u003eTc-\u003c/em\u003ePEBP-1 promote proinflammatory activation, whereas higher levels of \u003cem\u003eTc-\u003c/em\u003ePEBP-1 facilitate an anti-inflammatory phenotype in host macrophages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurocognitive impact of \u003cem\u003eT. canis\u003c/em\u003e neuroinvasion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the effects of \u003cem\u003eT. canis\u003c/em\u003e neuroinvasion, we characterised region-specific cellular and transcriptional changes in the brains of infected mice. Using spatially resolved cell clusters and marker gene signatures, we identified distinct host responses in the olfactory bulb, cerebrum and cerebellum (Fig. 5a). In the olfactory bulb (slices S1), parenchymal damage was associated with the accumulation of adipocytes, astrocytes, fibroblasts and a distinct adipocyte cluster. Marker genes in these cells were enriched for biological processes such as gliogenesis, protein serine/threonine kinase activity and amoeboid-type migration, with associated pathways including PI3K\u0026ndash;Akt signalling, aminoacyl-tRNA biosynthesis and focal adhesion. In the cerebrum (slices S2\u0026ndash;S5), infiltrating adipocytes, endothelial cells, microglia, dendritic cells, B cells, T cells and fibroblasts were present at sites of tissue damage and haemorrhage. The transcriptional profiles of these cells were enriched for functions linked to axonogenesis, synapse organisation, cognition, dendritic spine formation and epithelial cell proliferation, and were associated with calcium signalling, cholinergic synapse activity, neuroactive ligand\u0026ndash;receptor interaction, oxytocin signalling, long-term memory and neurodegeneration (Supplementary Fig. 7). In the cerebellum (slice S6), endothelial cells and B cells were detected within lesion sites, with gene enrichment in haemoglobin binding, peroxidase activity and amoeboid-type migration as well as in inflammatory regulation (e.g., interferon-\u0026gamma; and prostaglandin signalling) and immune evasion (e.g., insulin-like growth factor 2 and vitronectin signalling) characteristic of pathogens associated with sleeping sickness (African trypanosomiasis), cerebral malaria or the long-term potentiation in Parkinson\u0026rsquo;s disease\u003csup\u003e37\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e38\u003c/sup\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThese region-specific transcriptional responses suggest that \u003cem\u003eT. canis\u003c/em\u003e induces localised immune activation, structural disruption and dysregulation of molecular pathways critical for sensory processing, learning, memory and motor coordination \u0026ndash; processes likely underlying the cognitive and neuropsychiatric disturbances in the murine model and the progression of some neurodegenerative disorders in humans\u003csup\u003e6,7,14,18,27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo evaluate behavioural outcomes, mice infected with 1000 embryonated \u003cem\u003eT. canis\u003c/em\u003e eggs were subjected to rotarod, predator cue exposure (modified light/dark box) and Morris Water Maze (MWM) testing (Fig. 5b). Infected mice (\u003cem\u003en\u003c/em\u003e = 8) exhibited significantly impaired motor skill learning in the rotarod assay compared with uninfected controls (\u003cem\u003en\u003c/em\u003e = 6) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; Fig. 5c). In the predator cue test at 35 dpi, infected mice showed a non-significant reduction in avoidance behaviour, spending slightly less time in the dark compartment containing rat scent or dog urine (Fig. 5d). In MWM, infected mice demonstrated progressively increased escape latency and swim path length between 42 and 48 dpi (Fig. 5e,f), followed by a significant reduction in time spent in the target quadrant at 49 dpi (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), consistent with spatial memory impairment (Fig. 5g). Taken together, these findings show that \u003cem\u003eT. canis\u003c/em\u003e neuroinvasion results in region-specific inflammation and transcriptional disruption, leading to measurable deficits in motor coordination, learning and memory.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides the first spatially resolved, transcriptome-wide characterisation of \u003cem\u003eT. canis\u003c/em\u003e neuroinvasion in a murine model, highlighting the mechanisms underlying its persistence and host modulation in the brain. Using integrated spatial transcriptomics, single-nucleus RNA sequencing and behavioural assays, we show that \u003cem\u003eT. canis\u003c/em\u003e establishes a regionally restricted presence in the brain, modulates local immune responses and alters host gene transcription and networks relevant to neurological function, overcoming some technical limitations of dual (parasite/host) RNA-seq\u003csup\u003e39\u003c/sup\u003e. These findings improve our understanding of neurotoxocariasis beyond structural pathology to encompass molecular and functional consequences of infection.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eT. canis\u003c/em\u003e, in its larval stage, can infect a broad range of paratenic hosts, including humans, where it is responsible for a spectrum of disease syndromes\u003csup\u003e1,2\u003c/sup\u003e. Although interspecific differences in susceptibility, pathogenesis and immune responses are likely, the mouse provides a genetically tractable and experimentally reproducible model to interrogate mechanistic aspects of infection and disease\u003csup\u003e22,23,40\u003c/sup\u003e. Our findings using this system offer insights that might be broadly relevant across distinct host species and provide a foundation for comparative studies.\u003c/p\u003e\n\u003cp\u003eOur results support the concept of immune privilege in the CNS, where, despite evidence of larval localisation and tissue disruption, leukocytic infiltration remains minimal, unless migrating larvae leave the tissue site. Instead, immune and inflammatory responses are localised to specific microenvironments, with focal involvement of resident immune and vascular cell types. This finding indicates a form of \u0026ldquo;compartmentalised immunity\u0026rdquo; that serves to limit collateral tissue damage while permitting chronic parasite persistence. The absence of robust granulocytic responses also suggests effective immune evasion and/or suppression strategies by the parasite.\u003c/p\u003e\n\u003cp\u003eTranscriptomic analyses revealed that \u003cem\u003eT. canis\u003c/em\u003e larvae in the brain predominantly transcribe genes encoding ES proteins, many of which are known to modulate host immune responses. These include mucins, lectins, and proteins with ShKT domains, such as \u003cem\u003eTc-\u003c/em\u003ePEBP-1, which we identified as a key immunomodulator\u003csup\u003e35\u003c/sup\u003e. \u003cem\u003eTc-\u003c/em\u003ePEBP-1 influenced macrophage polarisation in a concentration-dependent manner, shifting from pro-inflammatory to regulatory phenotypes, and engaged intracellular signalling via the NOD2\u0026ndash;RIP2\u0026ndash;NF-\u0026kappa;B pathway. This dual functionality might allow the parasite to calibrate host responses for survival without inducing overt pathological changes.\u003c/p\u003e\n\u003cp\u003eOur lipidomic data further support a systemic component to host\u0026ndash;parasite communication. Serum from infected mice induced transcriptional responses in infective \u003cem\u003eT. canis\u003c/em\u003e larvae \u003cem\u003ein vitro\u003c/em\u003e, including an upregulation of genes involved in immune evasion, neurosensory processing and stress diapause. This dynamic host\u0026ndash;parasite interplay was exemplified by the induction of dauer-like transcriptional signatures in brain-resident larvae, including the suppression of the DAF-9\u0026ndash;DA\u0026ndash;DAF-12 axis\u003csup\u003e30\u003c/sup\u003e. Hormonal signals from perinatally infected hosts reversed this state, pointing to a host-driven endocrine mechanism regulating a stress adaptation (hypobiosis) or reactivation in the parasite.\u003c/p\u003e\n\u003cp\u003eThe effects of \u003cem\u003eT. canis\u003c/em\u003e neuroinvasion extended to host cellular architecture and behaviour. We observed transcriptional changes in host brain cells associated with synaptic organisation, neurodevelopment and degeneration, accompanied by impaired motor learning and spatial memory in mice. These data offer some experimental validation for reported associations between toxocariasis and neuropsychiatric or cognitive disorders in humans\u003csup\u003e41,42\u003c/sup\u003e. Our findings indicate that neurological disorders can be associated with neuroinflammation, down-regulation of cell migration and neuron projection (e.g., axonogenesis) as well as wound healing (linked to gliogenesis) in the brain, supporting the hypothesis that neurodegeneration in murine neurotoxocariasis has an immunopathogenic basis\u003csup\u003e23,43\u003c/sup\u003e. We also showed that lipid/cholesterol metabolism is tightly linked to host-parasite interactions (e.g., bile acid secretion pathway) and possibly immunopathological alterations (e.g., cellular response to alkaloids and high-density lipoprotein particles in oligodendrocytes) in murine neurotoxocariasis\u003csup\u003e17,27,44-46\u003c/sup\u003e. While here we explored neurotoxocariasis in mice over a short (3\u0026ndash;7 week) period, future investigations are needed to study, in more detail, the complex interplay between infiltrated immune cells (e.g., microglia, dendritic cells, B cell and T cells), neuron cells and impaired motor learning and spatial memory in mice with chronic \u003cem\u003eT. canis\u0026nbsp;\u003c/em\u003einfection\u003csup\u003e46\u003c/sup\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e\u003csup\u003e50\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, our findings reveal how \u003cem\u003eT. canis\u003c/em\u003e exploits spatial and immunological features of the CNS to persist, modulate host responses and impair neural function. This work sets the scene for future studies aimed at resolving the molecular interface between neuroinvasive helminths and host brain circuits. A deeper understanding of host signalling processes and pathways that regulate developmental states in helminths may offer new insights into chronic parasitic infections and their long-term neurological impacts.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments involving mice and rats followed the regulations of the Guide for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Welfare Ethics Committee at Zhejiang University, Hangzhou, China (permit no. ZJU20250029).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParasite collection and egg preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult \u003cem\u003eT. canis\u003c/em\u003e worms were collected from the faeces of naturally infected dogs at the Animal Hospital affiliated with the College of Veterinary Medicine, Southwest University. Embryonated eggs were harvested from the uteri of molecularly identified gravid females, incubated at 25 °C for eight weeks and hatched using glass beads. Infective larvae were purified via the Baermann technique\u003csup\u003e51\u003c/sup\u003e, cultured or snap-frozen in liquid nitrogen and stored at –80 °C until use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMurine infection model and brain sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-week-old, helminth-free BALB/c mice were orally administered 1000 infective \u003cem\u003eT. canis\u003c/em\u003e eggs each and maintained under pathogen-free conditions\u003csup\u003e52\u003c/sup\u003e. Infection was confirmed by IgG ELISA (Novatic Diagnostics, Germany). For larval recovery, at 7, 14, 21, 28 and 35 dpi, mice (\u003cem\u003en\u003c/em\u003e = 6) were euthanised by cervical dislocation, and brains were collected and digested for larval identification and counting. For brain slices preparation, at 21 dpi, brains of infected mice were collected, frozen, and embedded in OCT (SAKURA, USA). Serial coronal slices (10 μm) were prepared and slices adjacent to larval migration sites or lesions – confirmed by haematoxylin and eosin (H\u0026amp;E) staining – were selected for spatial and transcriptomic profiling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpatial transcriptomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStereo-seq was performed on brain slices from the olfactory bulb (S1), cerebrum (S2–S5) and cerebellum (S6), which were mounted onto 1 × 1 cm Stereo-seq chips (BGI Research, Shenzhen) and processed following published protocols\u003csup\u003e53\u003c/sup\u003e. Transcriptomes were mapped to \u003cem\u003eMus musculus\u003c/em\u003e (GRCm38) and \u003cem\u003eT. canis\u003c/em\u003e (WormBase PRJNA248777.WBPS17) reference genomes using SAW v1.0. Data were analysed using PCA, UMAP and Seurat v4.0.0 to cluster cells and identify marker genes. Pseudotime analysis was conducted with Monocle3 v1.0.0\u003csup\u003e54\u003c/sup\u003e, and annotation was performed using SingleR v1.6.1\u003csup\u003e55\u003c/sup\u003e. Differentially transcribed genes (fold change \u0026gt; 1, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) were analysed for GO and KEGG pathway enrichment using ClusterProfiler v4.0.0.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-nucleus RNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-nucleus RNA sequencing (snRNA-seq) was performed on slices adjacent to S4, S5, and S6 using the DNBelab C Series Single-Cell Library Prep Set (BGI Research, Qingdao). After droplet generation and cDNA library preparation, sequencing was performed on a DIPSEQ T1 platform (CNGB). Reads were filtered with scRNA_parse v1.0.1 and aligned to the \u003cem\u003eMus musculus\u003c/em\u003e genome using STAR v2.7.2b\u003csup\u003e56\u003c/sup\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eTranscript counts were computed with PISA v0.7. Clustering and annotation were conducted using Seurat v4.0.0, SciBet and SingleR v1.8.0\u003csup\u003e57\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLarval culturing and stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThird-stage \u003cem\u003eT. canis\u003c/em\u003e larvae (n = 10,000) were cultured in RPMI-1640 medium (Vivacell, China) containing 1% antibiotics and 10% serum from uninfected, infected or pregnant/infected mice. After 24 h at 37 °C with 10% CO₂, larvae were washed thrice in saline, centrifuged and snap frozen for RNA or protein analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using TRIzol reagent (Thermo Fisher), assessed via NanoDrop and Bioanalyzer 2100 (Agilent), and polyA-selected for cDNA library preparation. Sequencing was performed on an Illumina NovaSeq 6000 (LC-Bio, Hangzhou, China). Cutadapt v1.9 and FastQC v0.11.9 were used for data filtering. Clean reads were aligned against the draft genome of \u003cem\u003eT. canis\u003c/em\u003e (PRJNA248777.WBPS17) with HISAT2 v2.2.1 and analysed using DESeq2 (fold change \u0026gt; 1.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), GSEA v4.1.0 and MSigDB.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from stimulated larvae (n = 10,000) using TRIzol (Thermo Fisher), reverse-transcribed with the Primer-Script RT reagent kit (Takara), and analysed with TB Green qPCR Master Mix (Takara) on a CFX96 system (Bio-Rad). Relative transcript levels of the genes \u003cem\u003eTc-daf-9\u0026nbsp;\u003c/em\u003eand \u003cem\u003eTc-daf-12\u003c/em\u003e were calculated using the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method, normalised to \u003cem\u003eT. canis\u003c/em\u003e \u003cem\u003egapdh\u003c/em\u003e transcription. Primer sequences are listed in Supplementary Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiquid chromatography with tandem mass spectrometry (LC-MS/MS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLyophilised larvae (\u003cem\u003en\u003c/em\u003e = 50,000) were extracted in methanol, sonicated, centrifuged, then subjected to LC-MS/MS analysis using an AB Sciex 4500 MD system, using Δ4-DA and Δ7-DA standards (GlpBio, USA) for calibration. Each condition was assessed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipidomic profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore factors influencing the transition of migrating larvae of \u003cem\u003eT. canis\u003c/em\u003e into diapause, serum (100 μL) from each uninfected, infected or pregnant/infected mouse (\u003cem\u003en\u003c/em\u003e = 6) was extracted in methanol, vacuum-dried, resuspended and analysed by ultra-performance liquid chromatography (UPLC) (Thermo Fisher) coupled with TripleTOF 6600 (SCIEX). XCMS, CAMERA and metaX in R were used for data processing. Metabolites with fold change \u0026gt; 1.5, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 were identified and annotated using KEGG and HMDB v5.0\u003csup\u003e59\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e60\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural modelling and protein–protein docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignal peptides and domains of \u003cem\u003eTc\u003c/em\u003e-PEBP-1 were predicted using SignalP v6.0, SMART and InterProScan\u003csup\u003e61\u003c/sup\u003e\u003csup\u003e–\u003c/sup\u003e\u003csup\u003e63\u003c/sup\u003e. The tertiary structures of proteins (e.g., ShKT domains of \u003cem\u003eTc\u003c/em\u003e-MUC-1, \u003cem\u003eTc\u003c/em\u003e-PEBP-1, \u003cem\u003eMm\u003c/em\u003ePEBP1, and \u003cem\u003eMm\u003c/em\u003eFABP5) were predicted using AlphaFold2 v2.1.0, and docking simulations were performed with ClusPro v2.0 and HawkDock v2\u003csup\u003e64\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e66\u003c/sup\u003e. Protein complexes were visualised using ChimeraX v1.0.\u003csup\u003e67\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecombinant protein production and polyclonal antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of host immune factors on \u003cem\u003eT. canis\u003c/em\u003e larvae, and of \u003cem\u003eT. canis\u003c/em\u003e ES proteins on host immune responses, RNA from 3,000 larvae was reverse-transcribed, and coding sequences of \u003cem\u003eTc-daf-9\u003c/em\u003e, \u003cem\u003eTc-daf-12\u003c/em\u003e and \u003cem\u003eTc-pebp-1\u003c/em\u003e were cloned into pMD19-T, pET-28a or pCOLD-TF vectors, transformed into DH5α (TransGen Biotech, China), and expressed in BL21 (DE3) cells (TransGen Biotech, China). Protein induction was performed at 15 °C with 0.6 mM IPTG. r\u003cem\u003eTc-\u003c/em\u003eDAF-9, r\u003cem\u003eTc-\u003c/em\u003eDAF-12, and r\u003cem\u003eTc-\u003c/em\u003ePEBP-1 were purified via Ni-NTA columns (Sangon, China), endotoxin removed and quantified using\u0026nbsp;the bicinchoninic acid\u0026nbsp;(BCA)\u0026nbsp;assay.\u003csup\u003e68\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein extracts from stimulated \u003cem\u003eT. canis\u003c/em\u003e larvae (n = 10,000) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes and probed with primary mouse antibodies against \u003cem\u003eT. canis\u003c/em\u003e DAF-9 and DAF-12 (custom-generated), then the HRP-labelled goat anti-mouse IgG antibody (Beyotime, China). \u003cem\u003eT. canis\u003c/em\u003e beta-actin was used as a loading control. Bands were quantified using ImageJ v2.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMacrophage stimulation and cytokine assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the immune recognition of \u003cem\u003eT. canis\u003c/em\u003e ES proteins by host immune cells, RAW264.7 cells (10⁶) were cultured in DMEM (Meilunbio, China) supplemented with 10% foetal bovine serum (Thermo Fisher) and Penicillin–Streptomycin (Thermo Fisher) at 37 °C, 5% CO₂. Cells were stimulated with r\u003cem\u003eTc-\u003c/em\u003ePEBP-1 (5–40 μg/mL), 1 μg/mL LPS (positive control; Solarbio, China), 50 nM small interfering RNA (siRNA) targeting \u003cem\u003eNod1\u003c/em\u003e, \u003cem\u003eNod2\u003c/em\u003e, or \u003cem\u003eRip2\u003c/em\u003e mixed with Lipofectamine 3000 (Thermo Fisher), or vehicle (negative/irrelative control) for 24 h. Cell viability was assessed by trypan blue staining, relative mRNA levels of \u003cem\u003eNod1\u003c/em\u003e, \u003cem\u003eNod2\u003c/em\u003e, \u003cem\u003eRip2\u003c/em\u003e, \u003cem\u003eArg1\u003c/em\u003e, \u003cem\u003eCcl3\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eIl10, iNOS\u003c/em\u003e, \u003cem\u003ePparg\u003c/em\u003e, \u003cem\u003eTgfb\u003c/em\u003e, and \u003cem\u003eTnfa\u003c/em\u003e were measured using qRT-PCR with the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method (normalised to \u003cem\u003egapdh\u003c/em\u003e transcription, and cytokine secretion (TNF-α, IL-1β, IL-10, TGF-β) was measured in supernatants using ELISA kits (Beyotime, China). For immunoblotting, protein extracts from RAW264.7 cells (10⁶) (Beyotime, China) were separated by SDS-PAGE, transferred to PVDF membranes and probed with primary antibodies against murine NOD1/2, RIP2, NF-κB p65 and phospho-p65, then secondary antibodies (Cell Signaling Technology, USA). Murine GAPDH was used as a loading control. Bands were quantified using ImageJ v2.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioural assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBehavioural (e.g., motor, learning and memory skills) assessments of infected (n = 8) and uninfected (n = 6) mice were conducted, by rotarod testing (motor coordination at 35 dpi), light–dark box (predator aversion at 35 dpi) and Morris Water Maze (MWM; learning and memory from 43 to 49 dpi) experiments\u003csup\u003e69\u003c/sup\u003e\u003csup\u003e–\u003c/sup\u003e\u003csup\u003e70\u003c/sup\u003e. Spatial memory performance was evaluated by escape latency, swim path length and time in the target quadrant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis and reproducibility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll \u003cem\u003ein vitro\u003c/em\u003e experiments included three technical replicates; at least six biological replicates were used for \u003cem\u003ein vivo\u003c/em\u003e studies. Data were analysed using unpaired Student’s t-test or one-way ANOVA in GraphPad Prism 9. \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eReporting summary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available in the manuscript or Supplementary Information (Supplementary Figs. 1\u0026ndash;7; Supplementary Tables 1\u0026ndash;4). Sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1257458. The nucleotide sequences of \u003cem\u003eT. canis\u003c/em\u003e genes \u003cem\u003edaf-9\u003c/em\u003e, \u003cem\u003edaf-12\u003c/em\u003e and\u003cem\u003e\u0026nbsp;pebp1\u003c/em\u003e have been deposited in the NCBI GenBank under accession nos. PV590097, PV590098 and PV590067, respectively.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Professor Aifang Du from the College of Animal Sciences, Zhejiang University for constructive comments during the drafting of this manuscript. We thank the staff of The Shared Management Platform for Large Instrument and The Experimental Teaching Centre, College of Animal Sciences, Zhejiang University.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis work is supported by the National Natural Science Foundation of China (nos. 32473050 and 32002304), the National Basic Research Program of China (2023YFD1801700), and the Key Research and Development Program of Zhejiang Province (no. 2023C02036). RBG\u0026rsquo;s research program is presently funded by the Australian Research Council and Oz Omics Pty Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: G.M., and R.B.G. Methodology: G.M., H.W., and R.Z. Investigation: M.Z., S.L., Y.C., Z.X., H.W. and F.L. Visualization: M.Z., S.L., Z.X., Y.Y., R.Z., and G.M. Funding acquisition: G.M., and R.B.G. Project administration: Y.Y., R.Z., and G.M. Supervision: G.M., and R.B.G. Writing\u0026mdash;original draft: Y.C., and G.M. Writing\u0026mdash;review \u0026amp; editing: G.M., and R.B.G.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u0026nbsp;\u003c/strong\u003eThe online version contains supplementary material available at XXXXX.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to Guangxu Ma.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMa, G., et al. 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Short and long-term motor skill learning in an accelerated rotarod training paradigm. \u003cem\u003eNeurobiol Learn Mem.\u003c/em\u003e \u003cstrong\u003e81,\u003c/strong\u003e 211\u0026ndash;216 (2004).\u003c/li\u003e\n\u003cli\u003eBromley-Brits, K., Deng, Y., \u0026amp; Song, W. Morris water maze test for learning and memory deficits in Alzheimer\u0026rsquo;s disease model mice. \u003cem\u003eJ. Vis. Exp.\u003c/em\u003e\u003cstrong\u003e53,\u003c/strong\u003e 2920 (2011).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e|\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eStereo-seq data summary of\u003cem\u003e\u0026nbsp;Toxocara canis\u003c/em\u003e-invaded mouse brain slices (S1 to S6).\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"548\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescriptions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eS6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eClean reads\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1,894,168,033\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2,588,324,830\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2,114,853,521\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2,582,671,676\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2,232,624,519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2,915,787,774\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eClean reads Q20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e96.11%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e96.69%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e96.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e96.78%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e96.47%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e97.11%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 113px;\"\u003e\n \u003cp\u003eClean reads Q30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e87.25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e88.70%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e87.66%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e89.10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e88.52%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e90.02%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eUnique mapping reads\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1,546,477,368\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e2,051,786,083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e1,735,618,844\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2,119,775,668\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1,823,187,537\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e2,345,573,814\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eUnique mapping reads ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e81.64%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e79.27%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e82.07%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e82.08%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e81.66%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e80.44%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eNumber of MID under tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e88,484,150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e151,430,550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e281,694,844\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e322,204,437\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e302,226,886\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e226,650,184\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eMouse genes under tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e35,913\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e35,861\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e38,071\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e39,949\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e39,538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e40,476\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eNumber of MID of \u003cem\u003eT. canis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e7,717\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1,032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e1,205\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eGenes of \u003cem\u003eT. canis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eQ20: an error rate of 1 in 100; Q30: an error rate of 1 in 1000; MID: molecular identity\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6743037/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6743037/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eToxocara canis\u003c/em\u003e is a globally distributed parasite that infects dogs and other canids, shedding eggs into the environment. Humans become accidental hosts by ingesting these eggs, leading to syndromes such as visceral, ocular and neurotoxocariasis – the latter being poorly understood. Neurotoxocariasis has been associated with epilepsy, cognitive impairment and schizophrenia, but its pathomechanism remains unclear. Here, we combine spatial transcriptomics, single-nucleus RNA sequencing and behavioural assays in a mouse model to characterise the host–parasite interface in the brain. \u003cem\u003eT. canis\u003c/em\u003e larvae caused region-specific brain damage, vascular remodelling and confined immune responses. Parasite gene expression was dominated by immunomodulatory excretory/secretory proteins. Infected mice exhibited impairments in motor learning and memory, coinciding with transcriptional changes in neurons and glia. These findings provide mechanistic insight into neurotoxocariasis and demonstrate the utility of the mouse model as a platform to investigate helminth-induced neurological disorders.\u003c/p\u003e","manuscriptTitle":"Spatial transcriptomic atlas of murine neurotoxocariasis reveals region-specific host responses and dysfunction in the brain","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 09:43:02","doi":"10.21203/rs.3.rs-6743037/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"15e43a62-a0be-40b8-baa4-61fda51b88e1","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49632307,"name":"Biological sciences/Microbiology/Parasitology/Parasite host response"},{"id":49632308,"name":"Biological sciences/Microbiology/Parasitology/Parasite immune evasion"},{"id":49632309,"name":"Health sciences/Pathogenesis/Infection"}],"tags":[],"updatedAt":"2026-04-08T06:46:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-09 09:43:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6743037","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6743037","identity":"rs-6743037","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}