In late 2025, Germany faced a major outbreak of highly pathogenic influenza virus subtype H5N1 clade 2.3.4.4b, causing high mortality in migratory and resident wild birds and poultry. We examined 16 deceased birds, including Eurasian cranes, geese, a teal, and raptors found in western Germany between October and December 2025. Here, we provide a comparative analysis of light microscopic findings using histology and viral antigen distribution determined by immunohistochemistry, alongside the underlying viral phylogeny. Lesions and viral antigen distribution varied by species, with cranes showing infection in a broad organ spectrum, including the central nervous system (CNS), pancreas, spleen, liver, and kidneys, whereas other species exhibited a more targeted organ involvement. Phylogenetic analysis identified the EA-2024-DI.2.1 sublineage, with cranes forming a monophyletic cluster, which was consistent with the shared roosting/migratory behavior of cranes, although epidemiologic links were not formally demonstrated. In summary, our findings indicate species-specific lesion patterns and viral tissue tropism, which may influence transmission dynamics and environmental shedding.

Tumoral stroma as a key element of the tumor microenvironment has gained increased relevance in recent oncology studies. The tumor-stroma ratio (TSR) has been validated as a prognostic factor in human breast cancer. In feline mammary carcinomas (FMCs), few studies have delved into tumor stromal components, such as collagen and fibroblasts, and none have evaluated the TSR. Herein, multiple approaches for assessing tumor stroma and collagen in FMCs of 129 queens were used, including semi-quantitative evaluations in hematoxylin and eosin and in Picrosirius stainined sections and digital quantification by image analysis using ImageJ software. The desmoplasia-to-collagen ratio (DCR) was also computed using the image analysis results. A weak agreement was obtained between semi-quantitative and quantitative values of TSR and collagen amount; however, the majority of samples were scored below 50%, irrespective of the method, with a DCR above 50% in most of the carcinomas. Overall, clinical stage III and a more pronounced inflammatory infiltrate were consistently overrepresented in the increased stroma, total collagen, and desmoplasia groups. In the univariate survival analysis, a high amount of stroma, desmoplasia, and increased DCR were associated with shorter survival. Moreover, quantitative variables of TSR, desmoplasia, and DCR emerged as independent prognostic factors in multivariate models. The TSR and, specifically, the amount of collagen within the tumor stroma appear to have a role in the biological behavior of FMCs. Ancillary techniques for quantifying TSR, desmoplasia, and DCR provide additional prognostic information, and efforts should be made to include them in the routine post-surgical evaluations of FMC cases.

Fatty liver hemorrhagic syndrome (FLHS) is a common metabolic disorder in laying hens that severely impacts the poultry industry by reducing egg production and causing sudden mortality. To investigate the genetic factors underlying fatty liver development in chickens, we generated transgenic chimeric chickens by adenoviral injection into embryos and induced angiopoietin-like 3 (ANGPTL3) overexpression using a Tet-On system. The role of ANGPTL3 in the pathogenesis of FLHS was subsequently examined. Our results demonstrated that ANGPTL3 overexpression markedly increased liver weight and lipid deposition, aggravated oxidative stress and inflammatory responses, and altered the expression of lipid metabolism-related genes. These findings highlight a pivotal role of ANGPTL3 in the development of fatty liver in chickens and provide new insights into the molecular mechanisms underlying FLHS, offering potential targets for prevention and therapeutic intervention.

The bone marrow is a complex microenvironment essential for hematopoietic and mesenchymal cell production, with a central role in physiological homeostasis and disease. Reliable preservation is therefore critical for accurate morphological and molecular analyses in both clinical and preclinical settings. Conventional decalcification methods maintain tissue structure but can compromise protein and nucleic acid integrity. Here, we present an intact marrow extraction (IME) method for recovering bone marrow cores from murine femora, enabling processing as soft tissue without decalcification while preserving native architecture. Morphology, protein localization, and ribonucleic acid (RNA) quality were compared between ethylenediaminetetraacetic acid (EDTA)-decalcified samples (n = 9) and nondecalcified IME samples (n = 9). Histological assessment showed comparable preservation of cellular morphology across methods. However, EDTA decalcification reduced the detection of adipogenic (LEPR) and osteogenic (SP7) markers by immunohistochemistry or immunofluorescence and was associated with diminished RNA signal by in situ hybridization, particularly for Ubc. While the RNA integrity (DV200) was similar, RNA yields were substantially higher in IME samples. Overall, IME preserves morphology and antigenicity while improving RNA yield and reducing processing time, providing a robust alternative to decalcification for integrated histological and molecular analyses of bone marrow and other calcified tissues.

Image analysis in histologic assessments has increasingly gained traction in comparative pathology and biomedical research. However, application has been limited by a knowledge gap, particularly for veterinary pathologists without a strong background in coding-intensive image analysis. This study describes a quantitative image analysis pipeline for liver pathology in a special diet-induced metabolic dysfunction-associated steatohepatitis (MASH) mouse model, using the existing open-source, graphic user interface-based program QuPath. A machine learning-based pixel classifier was trained on annotations made in 7 digitized hematoxylin and eosin (H&E)-stained slides to detect normal hepatocytes, macrosteatosis, microsteatosis, vessels, and inflammation. Macrosteatosis annotations were then segmented via pixel thresholding to quantify lipid droplets. Post-processing pixel thresholding was applied to quantify fibrosis in picrosirius red-stained slides and lipid droplets in H&E-stained slides. Results were benchmarked against traditional manual grading by the nonalcoholic steatohepatitis activity score (NAS). Our results revealed a significantly larger macrosteatosis area percentage in MASH diet mice than in regular diet controls. The percentage areas for steatosis, inflammation, and fibrosis strongly correlated with increasing histopathologic scores. Unlike traditional ordinal severity scoring like NAS, this approach generates continuous, quantitative data suitable for parametric statistical analysis. This method is scalable and accessible, requires no coding expertise or commercial software, and offers a blueprint for generating truly quantitative histopathology data to veterinary pathologists and others engaged in MASH research.

The beagle dog is a commonly used nonrodent species in juvenile toxicity studies, supporting pediatric drug development, and is a relevant model in ophthalmic research. However, reference data on postnatal ocular development are limited, complicating the interpretation of histologic findings in immature tissues, especially when age-matched controls are unavailable. The present study provides a comprehensive histologic and immunohistochemical characterization of postnatal ocular development in 68 beagle dogs from birth to 6 months of age, at closely spaced timepoints. At birth, eyelids were fused, and all ocular structures were immature. Major developmental events occurred primarily within the first 2 months and included corneal epithelial stratification around eyelid opening (~postnatal day 14), regression of hyaloid and pupillary membrane vascular remnants, high proliferative indices across multiple structures, and retinogenesis. Notably, the retina still contained the fetal outer neuroblastic layer at birth. Histology and immunohistochemistry (rhodopsin, synaptophysin, glial fibrillary acidic protein, Ki-67, calbindin, cleaved caspase-3, and von Willebrand factor) showed that critical stages of retinogenesis occurred predominantly during the first 3 postnatal weeks, initiating within the visual streak and progressing in a central-to-peripheral gradient-paralleling human fetal retinal development, which begins in the foveal/parafoveal macula. Most ocular structures reached histomorphologic maturity between 2 and 6 months. These data provide essential references to distinguish normal developmental changes from test article-related effects in juvenile toxicity studies and enhance the translational relevance of the beagle dog for pediatric and ophthalmic research.

In a large-scale aging study of 31 inbred strains of mice, a variety of age-related lesions arose in and around the spine (vertebrae, spinal cord, and adjacent connective and muscle tissues), including lesions of intervertebral disks, vertebrae, muscles, soft tissues, nerves, ganglia, and spinal cord that were either sporadic or strain-specific. In most cases, the mice did not present with obvious clinical signs, as many lesions were mild. Lesions included herniated or displaced intervertebral disk fragments, which appeared to be sporadic in most strains but were common in others. Fibro-osseous lesions of bones were a systemic disease in female mice with no effect on disk disease. Rhabdomyosarcomas were common in A/J mice surrounding the vertebrae but also invaded into the spinal canal, sometimes almost transecting the cord. Lymphomas affected all AKR/J mice, sometimes infiltrating into the spinal canal. Muscular dystrophy of the musculature surrounding the spine, due to mutations in the dysferlin (Dysf) gene, was common in A/J and SJL/J mice. Coccygeal hemivertebra was a novel strain-specific lesion only in PL/J mice. Sporadic lesions in and around the spine included hemangiosarcomas, lymphomas (both enlarged lymph nodes and invading in and around the vertebrae), schwannomas, histiocytic sarcomas, poorly differentiated sarcomas, and epidermoid/dermoid cysts. Since these strains have all been sequenced and annotated, polymorphisms in genes known to affect the pathogenesis of some of these lesions will be reported in other publications.

The diagnosis of T-cell/natural killer (T/NK)-cell lymphoma in dogs may be challenging when neoplastic cells lack CD3 but express multiple myeloma oncogene-1 (MUM1), raising concerns for its misdiagnosis as a plasma cell tumor. We performed immunohistochemical and clonality analyses of 8 cases of canine digital CD30[+]MUM1[+] nonepitheliotropic lymphoma. Histologically, the diffuse or angiocentric infiltration of pleomorphic, large neoplastic lymphocytes was observed in the skin, often accompanied by granulation tissue and/or eosinophilic infiltration. Immunohistochemically, neoplastic lymphocytes expressed CD3 in 3 cases, but not in 5; however, all cases expressed at least 1 T/NK-cell marker, including CD8 or cytotoxic granules (granzyme B, perforin, or T-cell intracellular antigen-1). MUM1 was expressed in all cases, whereas immunoglobulin markers were negative. T-cell receptor gamma (TRG) clonality was detected in 5 cases and immunoglobulin heavy chain (IGH) clonality in 4 cases, limiting their diagnostic utility. These results demonstrate that canine digital, CD30[+]MUM1[+] nonepitheliotropic lymphomas retain T/NK-cell immunophenotypes and underscore the importance of comprehensive immunophenotyping for an accurate diagnosis.

Histological classification of mammary proliferative lesions in dogs and cats is essential for prognostic and predictive studies, as well as for comparative oncology; however, standardization of diagnoses is highly affected by substantial interobserver variability. In this study, a structured diagnostic algorithm based on the Davis-Thompson Foundation classification was assessed for the ability to improve interobserver agreement in the morphological diagnoses of mammary gland lesions in dogs and cats. Consensus diagnoses of 114 selected mammary lesions (91 canine, 23 feline) made by 16 veterinary pathologists with mammary tumor expertise were used as the gold standard, and 79 veterinary pathologists from 21 countries evaluated digitized whole slides in 2 rounds: a first reading without the algorithm and a second reading with the algorithm. Interobserver concordance was measured using unweighted kappa statistics for lesion category (hyperplastic/dysplastic, benign, malignant) and histological subtype. Across all lesions, agreement was higher for lesion category than for histological subtype. A slight improvement in concordance was observed when applying the algorithm, both for category (k = 0.72, before, and k = 0.73, after) and histotype classifications (k = 0.40, before to k = 0.42, after). Also, by using the algorithm, 73/79 raters (92%) modified at least 1 diagnosis and, among these, approximately two-thirds reached agreement with the gold standard in at least 1 case. Considering histotypes, malignant tumors with mixed epithelial and myoepithelial components showed persistently low agreement. Overall, the algorithm provided a reproducible diagnostic framework that may support training and improve standardization of canine and feline mammary tumor classification.

Systemic amyloidosis in felids is predominantly associated with amyloid A amyloidosis, whereas other forms remain poorly characterized. In this study, we describe a previously unrecognized form of amyloidosis in 6 genetically unrelated tigers (Panthera tigris) with involvement of the heart, synovium, kidneys, and spleen. Liquid chromatography-tandem mass spectrometry and immunohistochemistry identified odorant-binding protein (OBP), a lipocalin family protein, as the major amyloid precursor protein. Proteomic mapping of OBP-derived peptides together with in silico structural prediction suggested that low-complexity domains within the N-terminal region of tiger OBP may contribute to amyloid formation. Sequence analysis of OBP revealed no mutations shared among affected tigers, and no familial relationships were identified, suggesting that this disease may be associated with advanced age. These findings identify OBP amyloidosis as a novel amyloidosis with features suggestive of systemic distribution in tigers and provide insight into the potential role of N-terminal low-complexity domains in amyloid formation.

Quality assurance (QA) principles are well established in veterinary laboratory medicine. However, cytopathology reporting has not received equivalent systematic scrutiny. Reports vary in structure, terminology, and expressions of diagnostic confidence. These inconsistencies unintentionally lead to miscommunication among pathologists and between pathologists and clinicians, affecting report comprehension and subsequent case management. This commentary explores how the application of established QA principles to veterinary cytopathology reporting can strengthen its clinical utility. A review of existing veterinary and human cytopathology QA literature, combined with insights from published surveys of veterinary clinicians and clinical pathologists, highlight areas where core QA principles may enhance report quality and reproducibility, as well as advance clinical research. Key opportunities include optimizing report organization to improve readability, adopting more consistent diagnostic terminology, and incorporating statements about sample adequacy with suggestions for improvement when appropriate. Explicitly stating and explaining the level of interpretive confidence, while limiting and clearly defining confidence-modifying terms, can help reduce ambiguity and thus support clinical decision-making. Because cytopathology reports directly inform diagnosis as well as testing and therapeutic decisions, improving their clarity and consistency is both a clinical and an ethical priority. Applying QA principles to cytopathology reporting would bring this discipline into closer alignment with established practices in human medicine and other veterinary laboratory subdisciplines. Development of consensus recommendations will require multidisciplinary collaboration among pathologists and practitioners. An international task force is forming to develop standardized, broadly applicable recommendations that enhance the communication efficacy, transparency, and ultimate value of veterinary cytopathology reporting.

Papillomaviruses (PVs) are associated with a variety of skin lesions in cats. Herein, is reported 4 cats with unusual lesions that were associated with PV infection. For each case, the lesions were examined histologically, and p16[CDKN2A] protein immunolabeling was performed. Total DNA was extracted from the lesions, and both consensus and specific PCR primers were used to amplify PV DNA. In addition, in situ hybridization (ISH) was used to try to detect PV DNA within 2 of the lesions. Two cats had benign proliferations of sebaceous glands that contained histological changes suggestive of PV infection. Lesions from both cats were found to contain FcaPV5 DNA, although ISH was unable to localize PV DNA within either lesion. Another cat had multiple ulcerated lesions that were confined to the digits. Histology of one of these lesions was consistent with a viral plaque. The plaque contained PV-induced cell changes and FcaPV2 sequences. The fourth cat had a viral plaque that contained FcaPV8 DNA sequences. The epithelial cells within this plaque contained large spherical eosinophilic cytoplasmic bodies that have not been previously described and could therefore be unique to this FcaPV type. The findings from these 4 cats expand the range of lesions associated with FcaPVs in cats and suggest that careful evaluation of epithelial skin lesions for the possibility of an FcaPV cause is warranted. It is important to determine whether lesions are caused by PV infection due to the increased likelihood that multiple lesions will develop in affected cats.

Lymphoma is the most common nasal cavity tumor in cats. While most recent research efforts have characterized neoplastic cell markers in feline alimentary lymphoma, only few reports are dedicated to immunohistological features of nasal lymphoid tumors beyond T- or B-cell phenotyping. In comparison, feline lymphoplasmacytic chronic rhinitis represents chronic, progressive lymphoplasmacytic inflammation, causing cyclical tissue damage and repair. Sixty-five cases of lymphoplasmacytic rhinitis and 15 cases of feline diffuse, large cell, immunoblastic nasal lymphoma were selected and submitted for immunohistochemistry to detect expression of immune markers of cell origin (CD3, CD20/PAX5, and FOXP3), proliferation (PCNA), signal transducer and activator of transcription 5 (STAT5), DNA repair (MLH1, MSH2, and MSH6), and apoptosis (BCL2). Our results indicate that neoplastic lymphocytes were predominantly CD20+/PAX5+, suggesting a B-cell phenotype. Neoplastic lymphocytes were moderately to strongly STAT5+, MLH1+, MSH6+, MSH2+, PCNA+, and FOXP3+, as well as weakly or moderately BCL2+, in most cases (n = 14/15). In both lymphoma and rhinitis cases, inflammatory lymphocytes were predominantly CD3+, while expression of DNA repair markers MLH1, MSH2, and MSH6 was occasional, varying between weak and moderate. Our results suggest that MLH1, MSH2, MSH6, PCNA, and FOXP3 show potential as diagnostic markers in differentiating lymphoma from lymphoplasmacytic rhinitis, although a diagnostic panel with MLH1, MSH6, and PCNA may suffice for diagnosing most cases where lymphoma is suspected. These findings aid in distinguishing lymphoma from inflammation and identifying potential therapeutic targets. They also suggest a regulatory B-cell phenotype in nasal lymphoma, which may aid in immune evasion.