Validation of the design of a high-sensitivity and high-resolution PET system for a preclinical PET/EPR hybrid scanner.

We report the development of a high-sensitivity and high-resolution PET subsystem for a next-generation preclinical PET/EPR hybrid scanner for investigating and improving hypoxia imaging with PET. The PET subsystem consists of 14 detector modules (DM) installed within a cylindrical supporting frame whose outer and inner diameters are 115mm and 60mm, respectively. Each DM contains eight detector units (DU) in a row and each DU is made of a 12×12 array of 1×1×10mm3 LYSO crystals (with a 1.05mm pitch) coupled to a 4×4 silicon photomultiplier (SiPM) array that has a 3.2mm pitch (Hamamatsu multi-pixel photon counter (MPPC) array 14161-3050HS-04). The PET subsystem has a 104mm axial field-of-view (AFOV) that is sufficient for full-body mouse imaging, therefore enabling temporal and spatial correlation studies of tumor hypoxia between PET and EPR. It employs 1mm-width crystals to support sub-millimeter image resolution that is desired for mouse imaging. Al-though a DM contains 1,152 LYSO crystals, by use of a newly devised signal readout method only six outputs are produced. Recently a partial prototype of this subsystem consisting of four DMs is built. In this paper, we present performance measurement results obtained for the developed DMs and initial imaging results obtained by the prototype. The developed DMs show uniformly superior performance in identifying the hit crystal and detector unit, in energy resolution, and in coincidence time resolution. The images obtained for a 22Na point source and a 18F-filled U-shaped tube source show an image resolution of about 1.1mm and 1.2mm FWHM in the transverse and axial directions respectively, and demonstrate successful imaging over the entire 104mm AFOV of the prototype. This estimated image resolution however includes the contribution by the source size.

DOI- and TOF-capable PET array detector using double-ended light readout and stripline-based row and column electronic readout.

We investigate a highly multiplexing readout for depth-of-interaction (DOI) and time-of-flight PET detector consisting of an N×N crystals whose light outputs at the front and back ends are detected by using silicon photomultipliers (SiPM). The front N×N SiPM array is read by using a stripline (SL) configured to support discrimination of the row position of the signal-producing crystal. The back N×N SiPM array is similarly read by an SL for column discrimination. Hence, the detector has only four outputs. We built 4×4 and 8×8 detector modules (DM) by using 3.0×3.0×20 mm3 lutetium-yttrium oxyorthosilicates. The outputs were sampled and processed offline. For both DMs, crystal discrimination was successful. For the 4×4 DM, we obtained an average energy resolution (ER) of 14.1%, an average DOI resolution of 2.5 mm, a non DOI-corrected coincidence resolving time (CRT), measured in coincidence with a single-pixel reference detector, of about 495 ps. For the 8×8 DM, the average ER, average DOI resolution and average CRT were 16.4%, 2.9 mm, and 641 ps, respectively. We identified the intercrystal scattering as a probable cause for the CRT deterioration when the DM was increased from 4×4 to 8×8.

A Preclinical Positron Emission Tomography (PET) and Electron-Paramagnetic-Resonance-Imaging (EPRI) Hybrid System: PET Detector Module.

We report the design and experimental validation of a compact positron emission tomography (PET) detector module (DM) intended for building a preclinical PET and electron-paramagnetic-resonance-imaging hybrid system that supports sub-millimeter image resolution and high-sensitivity, whole-body animal imaging. The DM is eight detector units (DU) in a row. Each DU contains 12×12 lutetium-yttrium oxyorthosilicate (LYSO) crystals having a 1.05 mm pitch read by 4×4 silicon photomultipliers (SiPM) having a 3.2 mm pitch. A small-footprint, highly-multiplexing readout employing only passive electronics is devised to produce six outputs for the DM, including two outputs derived from SiPM cathodes for determining event time and active DU and four outputs derived from SiPM anodes for determining energy and active crystal. Presently, we have developed two DMs that are 1.28×10.24 cm2 in extent and approximately 1.8 cm in thickness, with their outputs sampled at 0.7 GS/s and analyzed offline. For both DMs, our results show successfully discriminated DUs and crystals. With no correction for SiPM nonlinearity, the average energy resolution for crystals in a DU ranges from 14% to 16%. While not needed for preclinical imaging, the DM may support 300-400 ps time-of-flight resolution.

Application of 3D-printed prosthetic lower beak in an Oriental stork (Ciconia boyciana).

To our knowledge, ours is the first case of applying a 3D-printed prosthetic beak to an Oriental stork (Ciconia boyciana). A stork in captivity underwent several surgeries for beak fractures, but the lower-mandible fractures failed to be repaired. Therefore, we applied a patient-specific beak prosthesis of titanium alloy and nylon. Because the prosthetic beak could not be maintained due to mandible and soft-tissue inflammation, the stork was euthanized. Still, we confirmed typical behavior and feeding for ~3 months after surgery. This report highlights some of the challenges we encountered and identifies process improvements required for a more successful surgery.

The role of AI in prostate MRI quality and interpretation: Opportunities and challenges.

Prostate MRI plays an important role in imaging the prostate gland and surrounding tissues, particularly in the diagnosis and management of prostate cancer. With the widespread adoption of multiparametric magnetic resonance imaging in recent years, the concerns surrounding the variability of imaging quality have garnered increased attention. Several factors contribute to the inconsistency of image quality, such as acquisition parameters, scanner differences and interobserver variabilities. While efforts have been made to standardize image acquisition and interpretation via the development of systems, such as PI-RADS and PI-QUAL, the scoring systems still depend on the subjective experience and acumen of humans. Artificial intelligence (AI) has been increasingly used in many applications, including medical imaging, due to its ability to automate tasks and lower human error rates. These advantages have the potential to standardize the tasks of image interpretation and quality control of prostate MRI. Despite its potential, thorough validation is required before the implementation of AI in clinical practice. In this article, we explore the opportunities and challenges of AI, with a focus on the interpretation and quality of prostate MRI.

A case of rickets in an artificially raised white-tailed eagle (Haliaeetus albicilla) chick at a zoo.

A two-week-old white-tailed eagle presented with an inability to stand and flex its limbs. Despite hatching naturally and owing to lack of parental attention, the bird was raised indoors by zookeepers with no access to sunlight. Palpation and radiographic examination of the bilateral tibiotarsus and femur bone revealed pronounced deformation and curvature, and bilateral decreased bone densities, respectively. The reduced calcium concentration in the blood was treated with calcium gluconate injections and calcium-supplemented feeds. Chopped mouse tails were fed directly, and whole pink-skinned nude mice were fed weekly. The zookeeper also gently massaged the bird and dressed it with a bandage. Sunlight exposure was provided daily. Saliva containing chicken feed was obtained from the mother. The bird could stand properly after four weeks of treatment, and the blood calcium concentration was restored to normal levels.

Successful repair of coracoid and femur fractures in a Eurasian eagle owl (Bubo bubo) and its post-release survival.

To our knowledge, this is the first case of concurrent diaphyseal left coracoid and left femoral fractures in a Eurasian eagle owl and its post-release survival in Korea. The femur was surgically repaired using an external skeletal fixator-intramedullary (IM) pin tie-in method, and the coracoid was repaired solely with an IM pin on day 6 after femur surgery. The eagle owl underwent a gradual rehabilitation process. The bird was successfully rehabilitated and released 101 d after initial presentation. The bird was monitored using a wildlife tracking device and was confirmed to have survived for over 5 mon in the wild.

Pulse Sequence Dependence of a Simple and Interpretable Deep Learning Method for Detection of Clinically Significant Prostate Cancer Using Multiparametric MRI.

RATIONALE AND OBJECTIVES: Multiparametric magnetic resonance imaging (mpMRI) is increasingly used for risk stratification and localization of prostate cancer (PCa). Thanks to the great success of deep learning models in computer vision, the potential application for early detection of PCa using mpMRI is imminent. MATERIALS AND METHODS: Deep learning analysis of the PROSTATEx dataset. RESULTS: In this study, we show a simple convolutional neural network (CNN) with mpMRI can achieve high performance for detection of clinically significant PCa (csPCa), depending on the pulse sequences used. The mpMRI model with T2-ADC-DWI achieved 0.90 AUC score in the held-out test set, not significantly better than the model using Ktrans instead of DWI (AUC 0.89). Interestingly, the model incorporating T2-ADC- Ktrans better estimates grade. We also describe a saliency "heat" map. Our results show that csPCa detection models with mpMRI may be leveraged to guide clinical management strategies. CONCLUSION: Convolutional neural networks incorporating multiple pulse sequences show high performance for detection of clinically-significant prostate cancer, and the model including dynamic contrast-enhanced information correlates best with grade.

The optimal 18F-fluoromisonidazole PET threshold to define tumor hypoxia in preclinical squamous cell carcinomas using pO2 electron paramagnetic resonance imaging as reference truth.

PURPOSE: To identify the optimal threshold in 18F-fluoromisonidazole (FMISO) PET images to accurately locate tumor hypoxia by using electron paramagnetic resonance imaging (pO2 EPRI) as ground truth for hypoxia, defined by pO2 [Formula: see text] 10 mmHg. METHODS: Tumor hypoxia images in mouse models of SCCVII squamous cell carcinoma (n = 16) were acquired in a hybrid PET/EPRI imaging system 2 h post-injection of FMISO. T2-weighted MRI was used to delineate tumor and muscle tissue. Dynamic contrast enhanced (DCE) MRI parametric images of Ktrans and ve were generated to model tumor vascular properties. Images from PET/EPR/MRI were co-registered and resampled to isotropic 0.5 mm voxel resolution for analysis. PET images were converted to standardized uptake value (SUV) and tumor-to-muscle ratio (TMR) units. FMISO uptake thresholds were evaluated using receiver operating characteristic (ROC) curve analysis to find the optimal FMISO threshold and unit with maximum overall hypoxia similarity (OHS) with pO2 EPRI, where OHS = 1 shows perfect overlap and OHS = 0 shows no overlap. The means of dice similarity coefficient, normalized Hausdorff distance, and accuracy were used to define the OHS. Monotonic relationships between EPRI/PET/DCE-MRI were evaluated with the Spearman correlation coefficient ([Formula: see text]) to quantify association of vasculature on hypoxia imaged with both FMISO PET and pO2 EPRI. RESULTS: FMISO PET thresholds to define hypoxia with maximum OHS (both OHS = 0.728 [Formula: see text] 0.2) were SUV [Formula: see text] 1.4 [Formula: see text] SUVmean and SUV [Formula: see text] 0.6 [Formula: see text] SUVmax. Weak-to-moderate correlations (|[Formula: see text]|< 0.70) were observed between PET/EPRI hypoxia images with vascular permeability (Ktrans) or fractional extracellular-extravascular space (ve) from DCE-MRI. CONCLUSION: This is the first in vivo comparison of FMISO uptake with pO2 EPRI to identify the optimal FMISO threshold to define tumor hypoxia, which may successfully direct hypoxic tumor boosts in patients, thereby enhancing tumor control.

Two Cases of Mange Mite (Sarcoptes scabiei) Infestation in Long-Tailed Goral (Naemorhedus caudatus) in Republic of Korea.

The long-tailed goral, Naemorhedus caudatus (Mammalia: Bovidae), is one of the endangered animals in the Republic of Korea (Korea). Sarcoptic mange mites infested in diverse species of mammals, including humans, but no case has been reported in long-tailed gorals. We report 2 cases of mange mite, Sarcoptes scabiei, infestation in long-tailed gorals. Mange mites were sampled in the skin legions of the 2 long-tailed gorals, which were rescued in 2 different regions, Uljin-gun, Gyeongsangbuk-do and Cheorwon-gun, Gangwon-do, Korea. Our results showed that the ectoparasite was the itch mite that burrowed into skin and caused scabies on the morphological inspection and placed within the phylogenetic relations of the species. The present study confirmed for the first time in Korea that mange mites are pathogenic scabies of long-tailed goral. Closer surveillance of this pathogenic ectoparasite in zoonotic and infectious ecosystems is warranted.

Multiplexing Readout for Time-of-Flight (TOF) PET Detectors Using Striplines.

A recent trend in PET instrumentation is the use of silicon photomultipliers (SiPMs) for high-resolution and time-of-flight (TOF) detection. Due to its small size, a PET system can use a large number of SiPMs and hence effective and scalable multiplexing readout methods become important. Unfortunately, multiplexing readout generally degrades the fast timing properties necessary for TOF, especially at high channel reduction. Previously, we developed a stripline (SL) based readout method for PET that uses a time-based multiplexing mechanism. This method maintains fast timing by design and has been successfully used for TOF PET detectors. In this paper, we present a more systematic study in which we examine how two important design parameters of the readout - the number of inputs on an SL (n SL) and the pathlength between adjacent input positions (Δℓ) - affect its detection performance properties for PET. Our result shows that, up to n SL = 32 the readout can achieve accurate pixel discrimination and causes little degradation in the energy resolution. The TOF resolution is compromised mildly and a coincidence resolving time on the order of 300 ps FWHM can be achieved for LYSO- and SiPM-based detectors. We also discuss strategies in using the readout to further reduce the number of electronic channels that a PET system would otherwise need.

Microparticle-Based Biochemical Sensing Using Optical Coherence Tomography and Deep Learning.

Advancing continuous health monitoring beyond vital signs to biochemistry will revolutionize personalized medicine. Herein, we report a biosensing platform to achieve remote biochemical monitoring using microparticle-based biosensors and optical coherence tomography (OCT). Stimuli-responsive, polymeric microparticles were designed to serve as freely dispersible biorecognition units, wherein binding with a target biochemical induces volumetric changes of the microparticle. Analytical approaches to detect these submicron changes in 3D using OCT were devised by modeling the microparticle as an optical cavity, enabling estimations far below the resolution of the OCT system. As a proof of concept, we demonstrated the 3D spatiotemporal monitoring of glucose-responsive microparticles distributed throughout a tissue mimic in response to dynamically fluctuating levels of glucose. Deep learning was further implemented using 3D convolutional neural networks to automate the vast processing of the continuous stream of three-dimensional time series data, resulting in a robust end-to-end pipeline with immense potential for continuous in vivo biochemical monitoring.

Improving Tumor Hypoxia Location in 18F-Misonidazole PET with Dynamic Contrast-enhanced MRI Using Quantitative Electron Paramagnetic Resonance Partial Oxygen Pressure Images.

Purpose: To enhance the spatial accuracy of fluorine 18 (18F) misonidazole (MISO) PET imaging of hypoxia by using dynamic contrast-enhanced (DCE) MR images as a basis for modifying PET images and by using electron paramagnetic resonance (EPR) partial oxygen pressure (pO2) as the reference standard. Materials and Methods: Mice (n = 10) with leg-borne MCa4 mammary carcinomas underwent EPR imaging, T2-weighted and DCE MRI, and 18F-MISO PET/CT. Images were registered to the same space for analysis. The thresholds of hypoxia for PET and EPR images were tumor-to-muscle ratios greater than or equal to 2.2 mm Hg and less than or equal to 14 mm Hg, respectively. The Dice similarity coefficient (DSC) and Hausdorff distance (d H ) were used to quantify the three-dimensional overlap of hypoxia between pO2 EPR and 18F-MISO PET images. A training subset (n = 6) was used to calculate optimal DCE MRI weighting coefficients to relate EPR to the PET signal; the group average weights were then applied to all tumors (from six training mice and four test mice). The DSC and d H were calculated before and after DCE MRI-corrected PET images were obtained to quantify the improvement in overlap with EPR pO2 images for measuring tumor hypoxia. Results: The means and standard deviations of the DSC and d H between hypoxic regions in original PET and EPR images were 0.35 mm ± 0.23 and 5.70 mm ± 1.7, respectively, for images of all 10 mice. After implementing a preliminary DCE MRI correction to PET data, the DSC increased to 0.86 mm ± 0.18 and the d H decreased to 2.29 mm ± 0.70, showing significant improvement (P < .001) for images of all 10 mice. Specifically, for images of the four independent test mice, the DSC improved with correction from 0.19 ± 0.28 to 0.80 ± 0.29 (P = .02), and the d H improved from 6.40 mm ± 2.5 to 1.95 mm ± 0.63 (P = .01). Conclusion: Using EPR information as a reference standard, DCE MRI information can be used to correct 18F-MISO PET information to more accurately reflect areas of hypoxia.Keywords: Animal Studies, Molecular Imaging, Molecular Imaging-Cancer, PET/CT, MR-Dynamic Contrast Enhanced, MR-Imaging, PET/MR, Breast, Oncology, Tumor Mircoenvironment, Electron Paramagnetic ResonanceSupplemental material is available for this article.© RSNA, 2021.

Equivariant Spherical Deconvolution: Learning Sparse Orientation Distribution Functions from Spherical Data.

We present a rotation-equivariant self-supervised learning framework for the sparse deconvolution of non-negative scalar fields on the unit sphere. Spherical signals with multiple peaks naturally arise in Diffusion MRI (dMRI), where each voxel consists of one or more signal sources corresponding to anisotropic tissue structure such as white matter. Due to spatial and spectral partial voluming, clinically-feasible dMRI struggles to resolve crossing-fiber white matter configurations, leading to extensive development in spherical deconvolution methodology to recover underlying fiber directions. However, these methods are typically linear and struggle with small crossing-angles and partial volume fraction estimation. In this work, we improve on current methodologies by nonlinearly estimating fiber structures via self-supervised spherical convolutional networks with guaranteed equivariance to spherical rotation. We perform validation via extensive single and multi-shell synthetic benchmarks demonstrating competitive performance against common base-lines. We further show improved downstream performance on fiber tractography measures on the Tractometer benchmark dataset. Finally, we show downstream improvements in terms of tractography and partial volume estimation on a multi-shell dataset of human subjects.

Q-space Conditioned Translation Networks for Directional Synthesis of Diffusion Weighted Images from Multi-modal Structural MRI.

Current deep learning approaches for diffusion MRI modeling circumvent the need for densely-sampled diffusion-weighted images (DWIs) by directly predicting microstructural indices from sparsely-sampled DWIs. However, they implicitly make unrealistic assumptions of static q-space sampling during training and reconstruction. Further, such approaches can restrict downstream usage of variably sampled DWIs for usages including the estimation of microstructural indices or tractography. We propose a generative adversarial translation framework for high-quality DWI synthesis with arbitrary q-space sampling given commonly acquired structural images (e.g., B0, T1, T2). Our translation network linearly modulates its internal representations conditioned on continuous q-space information, thus removing the need for fixed sampling schemes. Moreover, this approach enables downstream estimation of high-quality microstructural maps from arbitrarily subsampled DWIs, which may be particularly important in cases with sparsely sampled DWIs. Across several recent methodologies, the proposed approach yields improved DWI synthesis accuracy and fidelity with enhanced downstream utility as quantified by the accuracy of scalar microstructure indices estimated from the synthesized images. Code is available at https://github.com/mengweiren/q-space-conditioned-dwi-synthesis.

Hierarchical geodesic modeling on the diffusion orientation distribution function for longitudinal DW-MRI analysis.

The analysis of anatomy that undergoes rapid changes, such as neuroimaging of the early developing brain, greatly benefits from spatio-temporal statistical analysis methods to represent population variations but also subject-wise characteristics over time. Methods for spatio-temporal modeling and for analysis of longitudinal shape and image data have been presented before, but, to our knowledge, not for diffusion weighted MR images (DW-MRI) fitted with higher-order diffusion models. To bridge the gap between rapidly evolving DW-MRI methods in longitudinal studies and the existing frameworks, which are often limited to the analysis of derived measures like fractional anisotropy (FA), we propose a new framework to estimate a population trajectory of longitudinal diffusion orientation distribution functions (dODFs) along with subject-specific changes by using hierarchical geodesic modeling. The dODF is an angular profile of the diffusion probability density function derived from high angular resolution diffusion imaging (HARDI) and we consider the dODF with the square-root representation to lie on the unit sphere in a Hilbert space, which is a well-known Riemannian manifold, to respect the nonlinear characteristics of dODFs. The proposed method is validated on synthetic longitudinal dODF data and tested on a longitudinal set of 60 HARDI images from 25 healthy infants to characterize dODF changes associated with early brain development.

A framework to construct a longitudinal DW-MRI infant atlas based on mixed effects modeling of dODF coefficients.

Building of atlases plays a crucial role in the analysis of brain images. In scenarios where early growth, aging or disease trajectories are of key importance, longitudinal atlases become necessary as references, most often created from cross-sectional data. New opportunities will be offered by creating longitudinal brain atlases from longitudinal subject-specific image data, where explicit modeling of subject's variability in slope and intercept leads to a more robust estimation of average trajectories but also to estimates of confidence bounds. This work focuses on a framework to build a continuous 4D atlas from longitudinal high angular resolution diffusion images (HARDI) where, unlike atlases of derived scalar diffusion indices such as FA, statistics on dODFs is preserved. Multi-scalar images obtained from DW images are used for geometric alignment, and linear mixed-effects modeling from longitudinal diffusion orientation distribution functions (dODF) leads to estimation of continuous dODF changes. The proposed method is applied to a longitudinal dataset of HARDI images from healthy developing infants in the age range of 3 to 36 months. Verification of mixed-effects modeling is obtained by voxel-wise goodness of fit calculations. To demonstrate the potential of our method, we display changes of longitudinal atlas using dODF and derived generalized fractional anisotropy (GFA) of dODF. We also investigate white matter maturation patterns in genu, body, and splenium of the corpus callosum. The framework can be used to build an average dODF atlas from HARDI data and to derive subject-specific and population-based longitudinal change trajectories.

Raptor genomes reveal evolutionary signatures of predatory and nocturnal lifestyles.

BACKGROUND: Birds of prey (raptors) are dominant apex predators in terrestrial communities, with hawks (Accipitriformes) and falcons (Falconiformes) hunting by day and owls (Strigiformes) hunting by night. RESULTS: Here, we report new genomes and transcriptomes for 20 species of birds, including 16 species of birds of prey, and high-quality reference genomes for the Eurasian eagle-owl (Bubo bubo), oriental scops owl (Otus sunia), eastern buzzard (Buteo japonicus), and common kestrel (Falco tinnunculus). Our extensive genomic analysis and comparisons with non-raptor genomes identify common molecular signatures that underpin anatomical structure and sensory, muscle, circulatory, and respiratory systems related to a predatory lifestyle. Compared with diurnal birds, owls exhibit striking adaptations to the nocturnal environment, including functional trade-offs in the sensory systems, such as loss of color vision genes and selection for enhancement of nocturnal vision and other sensory systems that are convergent with other nocturnal avian orders. Additionally, we find that a suite of genes associated with vision and circadian rhythm are differentially expressed in blood tissue between nocturnal and diurnal raptors, possibly indicating adaptive expression change during the transition to nocturnality. CONCLUSIONS: Overall, raptor genomes show genomic signatures associated with the origin and maintenance of several specialized physiological and morphological features essential to be apex predators.

Role of HIF-1α in response of tumors to a combination of hyperthermia and radiation in vivo.

PURPOSE: Mild temperature hyperthermia (MTH) increases blood flow and oxygenation in tumours. On the other hand, high-dose-per-fraction irradiation damages blood vessels, decreases blood flow and increases hypoxia in tumours. The radiation-induced hypoxia in tumours activates hypoxia-inducible factor-1α (HIF-1α) and its target genes, such as vascular endothelial growth factor (VEGF), promoting revascularization and recurrence. In the present study, we examined the hypothesis that MTH inhibits radiation-induced upregulation of HIF-1α and its target genes by increasing tumour oxygenation. MATERIALS AND METHODS: FSaII fibrosarcoma tumours grown subcutaneously in the legs of C3H mice were used. Tumours were irradiated with 15 Gy using a 60Co irradiator or heated at 41 °C for 30 min using an Oncothermia heating unit. Blood perfusion and hypoxia in tumours were assessed with Hoechst 33342 and pimonidazole staining, respectively. Expression levels of HIF-1α and VEGF were determined using immunohistochemical techniques. Apoptosis of tumour cells was quantitated via TUNEL staining and the effects of treatments on tumour growth rate were assessed by measuring tumour diameters. RESULTS: Irradiation of FSaII tumours with a single dose of 15 Gy led to significantly decreased blood perfusion, increased hypoxia and upregulation of HIF-1α and VEGF. On the other hand, MTH at 41 °C for 30 min increased blood perfusion and tumour oxygenation, thereby suppressing radiation-induced HIF-1α and VEGF in tumours, leading to enhanced apoptosis of tumour cells and tumour growth delay. CONCLUSION: MTH enhances the anti-tumour effect of high-dose irradiation, at least partly by inhibiting radiation-induced upregulation of HIF-1α.

Serological evidence of H5-subtype influenza A virus infection in indigenous avian and mammalian species in Korea.

In Korea, H5-subtype highly pathogenic avian influenza (HPAI) has caused huge economic losses in poultry farms through outbreaks of H5N1 since 2003, H5N8 since 2013 and H5N6 since 2016. Although it was reported that long-distance migratory birds may play a major role in the global spread of avian influenza viruses (AIVs), transmission from such birds to poultry has not been confirmed. Intermediate hosts in the wild also may be a potential factor in viral transmission. Therefore, a total of 367 serum samples from wild animals were collected near major migratory bird habitats from 2011 to 2016 and tested by AIV-specific blocking ELISA and hemagglutination inhibition (HI) test. Two mammalian and eight avian species were seropositive according to the ELISA test. Among these, two mammalian (Hydropotes inermis and Prionailurus bengalensis) and three avian (Aegypius monachus, Cygnus cygnus, and Bubo bubo) species showed high HI titres (> 1,280) against one or two H5-subtype AIVs. As H. inermis (water deer), P. bengalensis (leopard cat), and B. bubo (Eurasian eagle owl) are indigenous animals in Korea, evidence of H5-subtype AIV in these animals implies that continuous monitoring of indigenous animals should be followed to understand interspecies transmission ecology of H5-subtype influenza viruses.