Veterinary Pathology

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an increasingly serious global health issue. The establishment of accurate animal models is crucial for elucidating its pathogenesis and developing effective therapeutic strategies. Although mouse models are widely used in MASLD research, they exhibit significant differences from humans in metabolic traits and disease progression, limiting their ability to fully recapitulate key pathological features of MASLD. In this study, we established an MASLD model in Apodemus peninsulae using a high-fat diet (HFD) and compared it with the commonly used C57BL/6J mouse model. The results showed that A. peninsulae developed marked lipid metabolism disorders and liver function impairment as early as week 4 with an HFD intervention. Histological analysis revealed progressive steatosis, inflammatory infiltration, and early fibrosis from weeks 8 to 16, which was confirmed by oil red O, Masson’s trichrome, and Sirius red staining. In contrast, pathological progression in C57Bl/6J mice was slower, with milder fibrosis. Immunolabeling and inflammatory cytokine expression further indicated a more intense inflammatory response in A. peninsulae. Overall, A. peninsulae offers advantages, such as rapid disease induction and stable phenotypes, making it a promising animal model for studying early-stage MASLD. Its shorter modeling period and more pronounced steatosis and liver injury suggest it more closely mimics the early pathological changes seen in human MASLD.

Canine intestinal T-cell lymphoma (ITCL) may arise from intraepithelial lymphocyte (IEL) subsets expanded in chronic enteropathy (CE). To investigate this potential cell of origin, we performed flow cytometry, immunohistochemistry, and RNA in situ hybridization on IELs using biopsy samples from 62 dogs, including 6 large-cell lymphomas (LCLs), 9 small-cell lymphomas (SCLs), 31 CEs with increased IELs (IEL⁺CE), and 16 CEs without increased IELs (IEL^–CE). IELs in ITCLs were predominantly CD4⁻CD8α⁻ (LCL, 5/6; SCL, 4/9), and 4/31 IEL⁺CEs had a higher proportion of CD4⁻CD8α⁻ IELs than the other CE cases. Forward scatter values of CD4⁻CD8α⁻ IELs were higher in IEL⁺CEs compared with IEL^–CEs, correlating positively with the Ki-67 index, indicating proliferation of this population. An increase in IELs lacking T-cell receptor expression was observed in 1/4 LCL, 2/5 SCL, and 1/13 IEL⁺CE cases. Lack of surface CD3 was observed in 2/9 SCL cases. One of 5 LCL cases partially expressed NKp46, whereas the proportion of NKp46⁺ IELs was low (< 4%) in 32/33 CE cases examined. In addition, 3/6 LCL cases expressed NKp46 mRNA without detectable protein expression. Clonality analysis of isolated IELs yielded at least 1 clonal peak in 1/2 LCLs, 4/7 SCLs, 12/23 IEL⁺CEs, and 4/13 IEL^–CEs, underscoring the limited diagnostic utility on distinguishing ITCL, particularly SCL, from IEL⁺CE. These findings suggest that most canine ITCLs have a CD4⁻CD8α⁻ immunophenotype and that unconventional IELs in IEL⁺CE may be the cells of origin. Moreover, the distinction between ITCL and CE remains challenging, emphasizing the importance of integrated diagnostic approaches.

Neuroaxonal dystrophy is a group of genetic neurodegenerative diseases that are histopathologically characterized by swollen axons (spheroids). We previously established a novel rat model of neuroaxonal dystrophy harboring a V95E missense mutation in the heat shock protein family A (HSP70) member 8 (Hspa8) gene that originally emerged by ENU-induced random mutagenesis. Hspa8 ^V95E mutant rats show marked spheroid formation in the central nervous system and develop progressive hind limb ataxia. However, the detailed pathology of Hspa8 ^V95E mutant rats remains to be elucidated. In the current study, we examined the characteristics of gait abnormalities and the detailed distribution of spheroids in the central nervous system of Hspa8 ^V95E knock-in (KI) rats. Using footprint tests, we demonstrated the increased ratio of step width to step length (outward step) of the hind limbs in Hspa8 ^V95E KI rats from 3 weeks of age. In Hspa8 ^V95E KI rats aged 15 weeks, spheroids were predominantly distributed in the proprioceptive sensory pathway from the lower body, including the gracile fasciculus and the nucleus. The number of spheroids increased with age and was accompanied by glial reactions in the dorsal funiculus. Moreover, we observed the accumulation of kinesin light chain 1; kinesin heavy chain isoforms 5A, -5B, and -5C; and microtubule-associated protein light chain 3 B in the spheroids and degenerated axons of Hspa8 ^V95E KI rats at 13 weeks of age. Collectively, our data suggest the involvement of impaired axonal transport and autophagy in spheroid formation in Hspa8 ^V95E mutant rats.

The clinical relevance of pancreatic histologic changes in cats is unclear. Determination of clinical relevance is complicated by the focal to multifocal nature of these lesions, variable clinical presentation of affected cats, and common comorbidities. Examining the histologic changes on serial pancreatic sections from a cohort of young adult shelter cats without externally observable clinical disease could inform what should be considered normal in cats. Pancreata were obtained from 48 cats euthanized for population control. While hypoalbuminemia was common, liver enzyme activity and gastrointestinal panel results were largely unremarkable. The pancreas was sectioned every 1 cm, routinely processed, and evaluated by a board-certified veterinary pathologist. Pancreatic histologic changes were uncommon in this cohort of cats. Hematoxylin and eosin-stained sections that showed lymphocytic infiltrates were recut for CD3 and CD20 immunohistochemistry. The distribution of CD3 and CD20 positive cells and their percentages were recorded. Of 48 cats, 13 cats (27%) had localized lymphocytic infiltrates with a median percentage of affected sections of 14% (range: 7%–92%). There were no differences in observed percentages or distribution of T- or B-cells. Scattered CD3+ T-cells were incidentally identified in intralobular parenchyma. As mild lymphocytic infiltration was uncommon in this group of cats, it may not indicate a physiological response, as the anticipated response to antigenic stimulation would be expected to occur more diffusely and more commonly. Thus, the role of these infiltrates may reflect bystander inflammation or a primary pathological process (pancreatitis) and likely varies depending on the specific disease context.

A 12-year retrospective search identified 106 sheep and goats diagnosed with Clostridium perfringens type D enterotoxemia, and in which the heart was histologically examined. Twenty cases (20/106, 19%), including 10 sheep and 10 goats, had cardiac lesions that were presumed to be associated with enterotoxemia. The lesions included myocardial degeneration and/or necrosis (n = 16, 80%), hemorrhage (n = 17, 85%), and proteinaceous interstitial edema (n = 6, 30%). Myocardial degeneration and/or necrosis was more frequent in goats (10/10, 100%) compared with sheep (6/10, 60%). Hemorrhage was more frequent in sheep (10/10, 100%) compared with goats (7/10, 70%). Myocardial proteinaceous interstitial edema was exclusive to sheep. Cardiac lesions occur in spontaneous cases of C. perfringens type D enterotoxemia in small ruminants and may play a role in the clinical signs and/or the demise of the animal.

Negri bodies (NBs) are cytoplasmic inclusions associated with rabies virus infection and are a characteristic histopathologic feature of rabies. Although their morphology has been described in humans and experimental animal models, detailed characterization in naturally infected dogs remains limited. The objective of this study was to characterize the histopathologic, immunophenotypic, and ultrastructural features of NBs in naturally infected dogs from the Philippines. Brain tissues from 24 dogs were examined by routine histology, special stains, immunohistochemistry, and indirect immunofluorescence. Transmission electron microscopy was performed in 6 selected cases. In the cerebrum, hippocampus, and cerebellum, NBs were predominantly composed of abundant matrix material and occasionally contained inner bodies (classic NBs), with variable association with bullet-shaped virions and ribosomes. These inclusions were immunoreactive for rabies virus nucleoprotein and phosphoprotein and stained positively with luxol fast blue. Inner bodies were immunoreactive for glycoprotein and matrix protein and were positive with silver impregnation, Nissl, and Bodian stains. In contrast, small inclusions (lyssa bodies) identified in the thalamus and brainstem consisted mainly of immature viral particles with minimal matrix material. Collectively, these findings indicate region-dependent ultrastructural variation in rabies virus-associated inclusions in naturally infected dogs, while supporting a shared viral origin among the different morphologic forms. This characterization further supports the diagnostic value of these inclusions in veterinary neuropathology.

Four dogs with neutrophilic cholangitis were identified with intracholangiocytic apicomplexan protozoa. Two cases were biopsies, and both cases had gallbladder involvement. The other two cases were unexpected findings on autopsy. Immunohistochemistry against Toxoplasma gondii on all cases labeled the zoites. Internal transcribed spacer-1 polymerase chain reaction (PCR) performed on formalin-fixed paraffin-embedded samples was successful in the 2 biopsy cases, and subsequent sequencing identified >99% sequence identity to Hammondia sp. Of the biopsied dogs, 1 dog recovered successfully after antiprotozoal treatment while the other dog was euthanized due to poor response. The 2 autopsy cases were given immunosuppressive medication. Hammondia are within the Sarcocystidae family of apicomplexan parasites and are closely related to Neospora caninum. This study and the current literature support that Hammondia, specifically Hammondia heydorni, an organism that was previously considered nonpathogenic or minimally pathogenic, can rarely exhibit tropism to the biliary system and cause neutrophilic cholangitis, cholangiohepatitis, and cholecystitis in dogs.

Lysosomal storage diseases (LSDs) are a diverse group of inherited disorders characterized by the accumulation of undegraded macromolecules within lysosomes, leading to cellular dysfunction and progressive organ damage. Neuronal ceroid lipofuscinosis (NCL) is a subset of LSDs defined by the accumulation of autofluorescent lipopigments in neurons and, in some cases, peripheral tissues. While naturally occurring LSDs have been documented in domestic and laboratory species, reports in nonhuman primates (NHPs) remain rare. This study describes an LSD in 5 related rhesus macaques (Macaca mulatta) at the California National Primate Research Center, with clinical and histopathologic features supportive of NCL. Affected animals exhibited progressive cognitive and motor deficits, along with an atypical musculoskeletal phenotype including coarse facial features. Histologic examination revealed neuronal cytoplasmic vacuolation, primarily in the cerebellum and brainstem, with storage material exhibiting autofluorescence, positive staining with Sudan black, and luxol fast blue, and a positive periodic acid-Schiff reaction. Transmission electron microscopy identified structures resembling curvilinear inclusions and granular osmiophilic deposits, supporting a NCL diagnosis. This report expands the spectrum of NCL presentations in NHPs, reinforcing their potential as an alternative model for LSD research. The identification of this naturally occurring NCL model in NHPs may enhance our understanding of translational models for therapeutic strategies.

Deep learning-based automated image analysis (DL-AIA) has been shown to outperform trained pathologists in tasks related to feature quantification. Related to these capabilities, the use of DL-AIA tools is currently extending from proof-of-principle studies to routine applications, including the evaluation of patient samples (diagnostic pathology), regulatory safety assessment (toxicologic pathology), and recurrent research tasks. To ensure that DL-AIA applications are safe and reliable, it is critical to conduct a thorough and objective generalization performance assessment to evaluate an algorithm’s ability to accurately predict patterns of interest and possibly evaluate model robustness (ie, the algorithm’s capacity to maintain predictive accuracy on images from different sources). In this article, we review the practices for performance assessment in veterinary pathology publications by which 2 main approaches were identified: (1) exclusive visual performance assessment (ie, eyeballing algorithmic predictions) plus validation of the model’s application utilizing secondary performance indices and (2) statistical performance assessment (alongside other methods), which requires creation of a test set with ground truth labels that is a hold-out from model development. This article compares the strengths and weaknesses of statistical and visual performance assessment methods. Furthermore, we discuss relevant considerations for rigorous statistical performance evaluation including metric selection, test data set image composition, ground truth label quality, resampling methods such as bootstrapping, statistical comparison of multiple models, and evaluation of model stability. It is our conclusion that visual and statistical evaluation have complementary strengths and a combined approach provides the greatest insight into the DL model’s performance and sources of error.

Automated measurements of anisokaryosis in canine cutaneous mast cell tumors (ccMCTs) have been shown to be predictive of survival, but questions remain regarding the intratumoral distribution of anisokaryosis. Whole-slide images of 96 ccMCTs were analyzed with a deep learning-based segmentation algorithm to quantify anisokaryosis using the standard deviation (SD) of the nuclear area. In 35/96 cases, >5% of the non-overlapping 256 × 256 µm² regions were hotspots (SD ≥11.5 µm²). Regions selected by 7 pathologists within these 35 cases matched hotspots in 32% of the instances. Outcome analysis (tumor-related death) based on single tumor regions yielded an area under the curve (AUC) of 0.901 for pathologist-selected hotspots, falling between random region selection (AUC: 0.862) and 90th-percentile targeted selection (AUC: 0.956). Whole-slide analysis of the hotspot proportion predicted survival with an AUC of 0.956, with 20% of hotspots as a prognostically meaningful threshold. Whereas pathologists-selected tumor regions are prognostically meaningful for nuclear morphometry, whole-slide analysis may provide additional prognostic information.

Brucellosis is an infectious disease that affects a wide range of animals, caused by bacteria belonging to 13 species and several biovars within the Brucella genus (B. melitensis, B. abortus, B. suis, B. ovis, B. neotomae, B. canis, B. ceti, B. pinnipedialis, B. microti, B. inopinata, B. papionis, B. vulpis, B. nosferati, and unclassified strains). Brucella spp. infects small ruminants, cattle, swine, dogs, wild mammals, reptiles, amphibians, and fish, and most Brucella spp. have zoonotic potential. Characterization of lesions in animal brucellosis remains paramount in the diagnosis in endemic regions and even for countries free of brucellosis, where passive surveillance plays an indispensable role in upholding and sustaining their status. Pathology also contributes to understanding the pathogenesis and associating emerging pathogenic Brucella spp. with disease in animals. Here, we review the pathology of brucellosis in animals with an emphasis on cross-species transmission of “classical” Brucella spp. to nonpreferential hosts and “atypical” Brucella spp. We conclude that Brucella is an expansive genus identified in numerous animals, but there are still significant gaps in the knowledge of the pathology of brucellosis in unusual hosts and emerging Brucella spp. This is a great opportunity for veterinary pathologists to spearhead advancements in the knowledge of the pathogenesis and diagnosis of Brucella spp. infections. The isolation of a Brucella spp. and the correlation with lesions should follow molecular diagnostics to define the genetic signature of the isolate, providing a better understanding of epidemiology and contributing to the control of brucellosis across animal and human populations.

Chronic wasting disease (CWD) and scrapie are transmissible spongiform encephalopathy diseases caused by prions, infectious forms of the prion protein. Currently, immunohistochemistry (IHC) is the sole approved diagnostic method for confirming these prion infections in formalin-fixed tissues. Evaluation of prion IHC requires specially trained veterinary pathologists to assess multiple quality control parameters as well as characteristic chromogen immunolabeling patterns. This manual slide review creates a significant bottleneck for laboratories needing to rapidly scale up surveillance during periods of increased testing demand. Given the repetitive and standardized nature of prion IHC slide review, this assay represents an ideal candidate for computer-assisted diagnostics. To address this challenge, we developed a deep learning-based image analysis approach tailored to review slides from large-scale veterinary prion disease surveillance. Our training dataset included 143 prion IHC whole-slide images containing a total of 3296 annotations. Annotated images were segmented into nonoverlapping tiles and used to fine-tune a pretrained convolutional neural network, enhancing the model’s ability to recognize prion-specific quality control parameters and labeling features. When tested on a separate, blinded testing dataset of 50 CWD IHC slides, the model achieved 100% concordance for chromogenic labeling when compared with evaluation by a trained veterinary pathologist. The overarching objective of this project is to automate the initial review of prion IHC slides using deep learning-based image analysis to substantially reduce the time needed for evaluation. Implementation of this technology should enhance diagnostic consistency, improve efficiency, and provide scalable capabilities essential for comprehensive prion surveillance throughout the veterinary diagnostic laboratory network.

While most cases of human muscle-invasive bladder cancer (MIBC) are diagnosed as conventional urothelial carcinoma (UC), there is growing recognition of histologic subtypes and divergent differentiation within conventional UC. In this study, 31 dogs with UC (33 slides) were evaluated for histologic subtypes and divergent differentiation, as specified by the recent World Health Organization update on the classification of urinary bladder cancers. Slides were reviewed by a medical uropathologist and 3 board-certified veterinary pathologists and assessed for expression of uroplakin III, vimentin, and E-cadherin. All tumors were classified as UC. Fifteen cases were identified as conventional UC. Among the remainder, 8 displayed glandular differentiation, 4 were classified as sarcomatoid UC, 2 showed squamous differentiation, 1 case was classified as a large nested subtype, and 1 case was classified as a tubular/microcystic subtype. This study found a higher frequency of certain histologic subtypes and divergent differentiation in canine UC, particularly sarcomatoid UC and UC with glandular differentiation, compared to previous reports of both canine UC and human MIBC. The relatively high prevalence of the sarcomatoid UC and UC with glandular differentiation in dogs observed in this study suggests that canine UC may serve as a valuable translational model for evaluating novel therapeutic agents, particularly for these rare and aggressive subtypes in humans.

Schistosomatidae is a family of trematodes that generally have an indirect life cycle, with snails as intermediate hosts in avian schistosomiasis and aquatic birds as definitive hosts. Although multiple studies report avian schistosomes, there are only a few reports describing gross and histological lesions of neuroschistosomiasis in aquatic birds. Herein, a retrospective study including 12 aquatic birds with neuroschistosomiasis was conducted to further characterize the clinical, gross, and histologic findings and to identify the trematodes. The median age of birds was 12 years, and schistosomes were observed in the brain, with or without granulomatous inflammation. Neuroschistosomiasis was likely related to death in 5 out of 12 birds. Twelve formalin-fixed paraffin-embedded (FFPE) samples containing brain, 1 FFPE sample containing intestine, and 1 fresh brain sample were used for polymerase chain reaction (PCR) and sequencing to identify the schistosomes. Dendritobilharzia pulverulenta was identified from 3 out of 13 FFPE samples and 1 fresh brain sample. Neuroschistosomiasis presumptively associated with Dendritobilharzia sp. infection has been reported in swans and geese; however, the identification was based on the clinical signs, lesions, location within the hosts, and the morphology of adult worms without molecular analysis. To the authors’ knowledge, the histologic description and molecular identification of schistosomes that cause neuroschistosomiasis in multiple aquatic bird species other than swans and geese have not been reported.

Fibromatous epulis of periodontal ligament origin (FEPLO), also known as peripheral odontogenic fibroma (POF), is a benign gingival lesion that occurs frequently in dogs. Common use of both names is cumbersome and confusing yet persists due to disagreement regarding pathogenesis. With the objective of clarifying the histogenesis and pathogenesis, 100 canine FEPLO/POF lesions that had been surgically excised en bloc were evaluated histologically to characterize the lesion’s relationship to surrounding anatomic structures and to document concurrent pathologies in those tissues. No lesions involved the periodontal ligament space, although 88/100 (88%) lesions attached to alveolar bone, similar to the normal anatomy of the gingival ligament. The lesions consist of fibrous tissue with osteogenic potential, and 54/100 (54%) lesions had bone or cemento-osseous matrix. Odontogenic epithelium was evident in 63/100 (63%) lesions, and the paradental location of the epithelium suggests that the origin of most odontogenic epithelium in FEPLO/POF lesions is a result of hyperplastic junctional epithelium extending into the lesion. Hypercellularity was interpreted as a feature of reactive lesions since 15/17 (88%) hypercellular lesions were ulcerated and 15/17 (88%) were inflamed. Frequent concurrent histologic findings included hypercementosis at the cementoenamel junction of the adjacent tooth (79/89, 89%), reactive alveolar bone (90/100, 90%), and inflammation of alveolar bone (67/100, 67%). While the pathogenesis cannot be determined with absolute certainty, available evidence supports a reactive, hyperplastic process rather than neoplastic. The authors believe that fibromatous hyperplasia of the gingival ligament is an anatomically correct name that accurately reflects the likely etiopathogenesis.

Whereas reproducibility of studies is a prerequisite for trustworthy deep learning (DL) in veterinary histopathology and microscopy, the actual degree of methodological transparency that exists in the literature remains uncertain. We performed a Preferred Reporting Items for Systematic Reviews and Meta-Analyses-guided systematic review to quantify the degree to which supervised DL and supervised machine learning studies report reproducibility-critical details. Using a veterinary-journal-restricted Boolean search executed in PubMed and Scopus, we screened 180 unique records and included 50 primary research articles for full-text analysis. Based on a recently published guideline for the development of DL models in veterinary pathology, we extracted information for each study across 5 dimensions: (1) study and task characterization, (2) data transparency, (3) experimental design and data-leakage control, (4) model and training details, and (5) performance evaluation and reporting. Among the included studies, private data sets predominated, with 90% of studies relying on private data. Sharing of code was uncommon (3%). Key training details such as augmentation and hyperparameters were often incompletely reported; augmentation was not reported in 56% of studies, and key hyperparameters were absent in 40% of studies. It was often not clear whether patient-level stratification (necessary to avoid data leakage) was performed. In summary, these results highlight major deficits in the reporting of details and experimental design necessary for reproducing DL results in veterinary histopathology. This review provides a practical baseline and reporting roadmap to support more transparent and reproducible research in veterinary computational pathology.