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.

Caroli-like disease is a rare congenital ductal plate malformation characterized by intrahepatic bile duct dilatation and has only rarely been reported in veterinary medicine. A 2-year-old castrated male small-breed dog (5.4 kg) was presented with jaundice, weight loss, and lethargy. Laboratory tests revealed elevated liver enzyme activities, hyperbilirubinemia, and coagulopathy. Radiography showed two mineralized opacities superimposed on the hepatic silhouette. Ultrasonography revealed cholecystolithiasis, gallbladder wall thickening, and biliary dilatation with choledocholithiasis, suggesting extrahepatic biliary obstruction. Computed tomography confirmed choledocholithiasis, diffuse biliary dilatation, and a 2- to 3-cm intrahepatic cystic structure containing calcified material and communicating with the intrahepatic bile ducts. Histopathological analysis revealed cholangiohepatitis with ductal proliferation, and immunohistochemical analysis confirmed the biliary epithelial origin of the lesion and supported a congenital ductal plate malformation. Despite surgical management, the patient died postoperatively. This case represents the first veterinary description of Caroli-like disease complicated by extrahepatic biliary obstruction in a young dog and highlights the fact that intrahepatic saccular dilatation may resemble gallbladder anomalies on ultrasonography, while computed tomography provides crucial diagnostic clarity. Recognition of such rare conditions may improve diagnostic accuracy and clinical decision-making in veterinary hepatobiliary disorders.

Severe combined immunodeficiency (SCID) pigs have become a promising large animal model for biomedical research, offering significant advantages over traditional mouse models due to their anatomical, physiological, and genetic similarities to humans. Humanized SCID pig models can potentially improve preclinical research in areas such as cancer immunotherapies, stem cell therapies, and transplantation methods, yet often lack significant lymphocyte development, including evidence of B cell and myeloid cell development. This work aims to increase the extent of humanization of the SCID pig. CRISPR guide RNAs were successfully developed for the RAG2 and IL2RG genes, and a double-knockout cell line (RAG2-/-IL2RG-/Y, RG) was established. Somatic cell nuclear transfer (SCNT) was then used to create cloned SCID fetuses, which were injected intraperitoneally with in vitro expanded human CD34+ umbilical cord cells at day 41-42 of gestation. Human leukocytes, including T, B, NK, and myeloid cell types, were detected in peripheral blood, spleen, bone marrow and within the thymus of neonatal animals using flow cytometry. Six of the twelve pigs injected had >5% human cells within the CD45+ cell thymic population. Histology of thymus tissues from multiple pigs showed substantial development of the cortex and medulla, which is absent in non-injected RG neonates. This work demonstrates an improvement in the spectrum of xenogenic immune cell lineages developed using an RG line injected with highly expanded CD34+ cells, yet functional analysis of these cell types is needed for further establishment of an in utero humanized SCID pig model.

Advanced in vivo imaging modalities, such as magnetic resonance imaging (MRI), are essential research tools to effectively detect tumors in preclinical mouse models. MRI provides detailed anatomical information on tumor location and size. However, this imaging modality is expensive and time-consuming. To overcome this access barrier without compromising imaging quality, a multianimal MRI protocol was implemented. This protocol uses a four-chamber bed insert to obtain high-resolution multianimal anatomical MRI scans within a single acquisition session. Simultaneously imagining multiple mice reduces the time and cost of this procedure. This protocol describes a multianimal MRI workflow designed to increase the efficiency of tumor detection and longitudinal tumor monitoring in a genetically engineered Kras-driven, p53-deleted (KPC) mouse model of pancreatic ductal adenocarcinoma. This well-established clinically relevant KPC model has provided invaluable insight into the molecular mechanisms underlying pancreatic carcinogenesis, tumor progression, and treatment resistance. As proof-of-concept, this protocol is applied to validate the therapeutic benefit of the standard-of-care chemotherapeutic agent gemcitabine in the KPC model. Future applications of this MRI-guided preclinical study design are briefly discussed to evaluate the therapeutic efficacy of combination therapies.

Breast cancer is the second leading cancer-related cause of death in women. While there are few proactive interventions for average-risk women, prophylactic mastectomy is the most effective, risk-reducing intervention for high-risk women. However, prophylactic mastectomy is an invasive procedure that removes all mammary epithelial cells along with the surrounding stroma, fat tissue, and/or musculature. Our overall research goal is to develop a non-invasive intraductal (ID) delivery procedure that locally kills the mammary epithelial cells by filling the entire ductal tree with an ablative solution. We previously demonstrated that ID delivery of ethanol as an ablative solution is effective in rodent models (mice and rats). This protocol presents an ID delivery of 10-70% ethanol solution containing iohexol (90-300 mg/mL) as an X-ray contrast agent into the multi-ductal tree system of the rabbit mammary gland. The mammary gland of a rabbit (Oryctolagus cuniculus) with a multi-duct system is more alike to the human breast than those of other large animals (e.g., cows, sheep). This rabbit protocol addresses technical challenges of scalability, real-time imaging, and ID delivery into a multi-ductal tree system in a large-animal intermediate model. This protocol establishes a fluoroscopy-guided multi-duct ID delivery with instruments, materials, and reagents that could be directly applied in the clinic. Tissue analysis enables optimizing the concentration of ethanol for maximal epithelial ablation and minimal collateral tissue damage as a starting point for future first-in-human evaluation of this ablative procedure for primary prevention of breast cancer.

Artificial intelligence (AI)-based decision support systems hold promise for enhancing diagnostic accuracy and efficiency in computational pathology. However, human-AI collaboration can introduce and amplify cognitive biases, like confirmation bias caused by false confirmation when erroneous human opinions are reinforced by inaccurate AI output. This bias may increase under time pressure, a ubiquitous factor in routine pathology, as it strains practitioners’ cognitive resources. We quantified confirmation bias triggered by AI-induced false confirmation and examined the role of time constraints in a web-based experiment, where trained pathology experts (n=28) estimated tumor cell percentages. Our results suggest that AI integration fuels confirmation bias, evidenced by a statistically significant positive linear-mixed-effects model coefficient linking AI recommendations mirroring flawed human judgment and alignment with system advice. Conversely, time pressure appeared to weaken this relationship. These findings highlight potential risks of AI in healthcare and aim to support the safe integration of clinical decision support systems.

In histological preparations of tumors, an important criterion for evaluation of malignancy is the variation of nuclear size (anisokaryosis), which is traditionally estimated by pathologists. To improve reproducibility, nuclear size measurements (morphometry) can be performed. In this study [1] we developed a segmentation-based nuclear morphometry algorithm and compared its prognostic value with pathologist’s estimates (routine method) and manual morphometry (gold standard method).

Gastric plasmacytoma is rare in dogs, with only four previous reports providing limited descriptions of abdominal ultrasound (AUS) and computed tomography (CT) findings. This study presents the AUS and CT imaging features of two histopathologically suspected gastric plasmacytomas. Combining the four previous and two new cases, these tumors predominantly occurred in the greater curvature and pyloric antrum as solitary round masses connected to the submucosal or transmural layers. Pre- and post-contrast tissue attenuation (Hounsfield unit; HU) differences exceeded 40, with variable AUS echogenicity and echotexture. Imaging features may resemble those of spindle cell tumors, requiring histological and immunohistochemical examinations for definitive diagnosis.

Modulating gene expression in macrophages can be used to improve tissue regeneration and redirect tumor microenvironments (TMEs) toward positive therapeutic outcomes. We have developed Bacillus subtilis as an engineered endosymbiont (EES) capable of residing inside the eukaryotic host cell cytoplasm and controlling the fate of macrophages. Secretion of mammalian transcription factors (TFs) from B. subtilis that expresses listeriolysin O (LLO; allowing the EES to escape destruction by the macrophage) modulated expression of surface markers, cytokines, and chemokines, indicating functional changes in a macrophage/monocyte cell line. The engineered B. subtilis LLO TF strains were evaluated in murine bone marrow-derived macrophages (BMDMs) by flow cytometry, chemokine/cytokine profiling, metabolic assays, and RNA-Seq delivery of TFs by the EES shifted BMDM gene expression, production of cytokine and chemokines, and metabolic patterns, indicating that the TF strains could guide primary macrophage function. Thereafter, the ability of the TF strains to alter the TME was characterized in vivo in an orthotopic murine model of triple-negative breast cancer to assess therapeutic effects. The TF strains altered the TME by shifting immune cell composition and attenuating tumor growth. Additionally, multiple doses of the TF strains were well-tolerated by the mice. The use of B. subtilis LLO TF strains as EES showed promise as a unique cancer immunotherapy by directing the immune function intracellularly. The uses of EES could be expanded to modulate other mammalian cells over a range of biomedical applications.

A captive 10-year-old male North American river otter (Lontra canadensis) was evaluated for a 2-week history of weight loss and intermittent vomiting and regurgitation. Exploratory surgery identified a raised nodule on the quadrate lobe of the liver and an irregular nodule in the left limb of the pancreas. Partial lobectomy of the quadrate and right medial hepatic lobes was performed to excise the masses, and the pancreatic nodule was removed. Histological and immunohistochemical results were consistent with a glucagonoma that had metastasized to the liver. The otter received palliative care until 42 days postoperatively when it was humanely euthanized due to recurrence of clinical signs. Glucagonoma is a rare neoplasm in all species, with only a few cases reported in dogs, cats and horses. To the authors' knowledge there are no other reports of glucagonoma in mustelid species.

Canine cutaneous mast cell tumors (ccMCTs) are frequent neoplasms with variable biological behaviors. Internal tandem duplication mutations in c-KIT exon 11 (c-KIT-11-ITD) are associated with poor prognosis but predict therapeutic response to tyrosine kinase inhibitors. In a previous work, deep learning algorithms managed to predict the presence of c-KIT-11-ITD on digitalized hematoxylin and eosin-stained histological slides (whole-slide images, WSIs) in up to 87% of cases, suggesting the existence of morphological features characterizing ccMCTs carrying c-KIT-11-ITD. This 3-stage blinded study aimed to identify morphological features indicative of c-KIT-11-ITD and to evaluate the ability of human observers to learn this task. 17 untrained pathologists first classified 8 WSIs and 200 image patches (highly relevant for algorithmic classification) of ccMCTs as either positive or negative for c-KIT-11-ITD. Second, they self-trained to recognize c-KIT-11-ITD by looking at the same WSIs and patches correctly sorted. Third, pathologists classified 15 new WSIs and 200 new patches according to c-KIT-11-ITD status. In addition, participants reported microscopic features they considered relevant for their decision. Without training, participants correctly classified the c-KIT-11-ITD status of 63%-88% of WSIs and 43%-55% of patches. With self-training, 25%-38% of WSIs and 55%-56% of patches were correctly classified. High cellular pleomorphism, anisokaryosis, and sparse cytoplasmic granulation were commonly suggested as features associated with c-KIT-11-ITD-positive ccMCTs, none of which showed reliable predictivity in a follow-up study. The results indicate that transfer of algorithmic skills to the human observer is difficult. A c-KIT-11-ITD-specific morphological feature remains to be extracted from the artificial intelligence model.

Chordomas are common in ferrets, but rare in other species. Only a handful of reports have been published about this neoplasm in the veterinary literature. During a routine physical examination on a breeder female sugar glider, a firm mass was palpated on the tail. Histopathological differential diagnosis of chondroma or chordoma was determined by cytology, and a preemptive caudectomy was performed. At the time of amputation, another firm mass was palpated just proximal to the original mass. Two chordomas separated by 2 mm were diagnosed based on an abundance of physaliferous cells and positive staining for vimentin and S‐100 protein with immunohistochemistry. Although metastasis of chordomas is uncommon in ferrets and surgical excision is usually curative; in most other species, chordomas are malignant. It is uncertain how chordomas behave in sugar gliders. In this case, euthanasia was elected due to post‐operative complications from self‐trauma, and thus, the prognosis remains unknown.

Background: Seminal vesiculitis is a disease characterized by inflammation of the seminal vesicles. It has been reported in stallions, bulls, rams, boars, rabbits, mice, and rats but has not been reported in golden hamsters. Methods: Golden hamsters (Mesocricetus auratus) with seminal vesiculitis diagnosed by histopathologic examination in a private animal hospital between February and December 2022 were eligible for inclusion. Results: Two golden hamsters were included in the study. Both patients presented with lethargy and hematuria. Ultrasonographic examination of the seminal vesicles showed less distinct lobulation, wall thickening, and hyperechoic fluid in 1 case. One patient was treated with trimethoprim-sulfamethoxazole. The hamster died 11 days after presentation. The other patient was treated with a seminal vesiculectomy and was euthanized 66 days after surgery due to congestive heart failure and a left atrial mass. Histopathologic examination of seminal vesicles from both patients showed dilation, transmural necrosis, and inflammation. Conclusions and clinical relevance: Seminal vesiculitis should be considered as a differential in male golden hamsters with hematuria. In the cases presented here, ultrasonography was useful to formulate a diagnostic suspicion. Treatment options require further investigation.

Lymphoglandular complexes are components of the gut-associated lymphoid tissue that are characterized by submucosal lymphoid aggregates invested by projections of mucosal epithelium. Reports of pathology involving these structures are rare in both human and veterinary literature. Here, the authors report 2 cases of rectal masses excised from dogs following a period of tenesmus and hematochezia. In both animals, the masses were composed of lymphoid tissue closely encompassing tubuloacinar structures. Immunohistochemistry and polymerase chain reaction antigen receptor rearrangement testing demonstrated that the lymphoid population was polyclonal, comprising T and B cells arranged in loosely follicular aggregates centered on the epithelial foci. In light of these findings, a diagnosis of lymphoglandular complex nodular hyperplasia was reported. To the authors’ knowledge, this is the first report of this condition in dogs.

Rodents are typically viewed as asymptomatic reservoirs for leptospirosis infection, as clinical disease in rodents is rarely described. This report includes three separate cases of leptospirosis in Patagonian maras (Dolichotis patagonum) over a 3-yr period in multiple locations within a single zoo. All three cases presented with varying clinical signs including lethargy, conjunctival hyperemia, hyperbilirubinemia, and presumed renal azotemia. Infection with Leptospira spp. was diagnosed antemortem by PCR on whole blood (n = 1, Case 1) or urine (n = 2, Cases 2 and 3). Leptospira antibody titers measured by serum microagglutination testing (n = 3) were elevated or increased in all three animals over a 1-3-wk period for Leptospira serovars Bratislava and Hardjo (Case 1) and Grippotyphosa (Case 2 and 3). Two of the three animals responded to treatment with penicillin and doxycycline and supportive care, whereas one animal did not respond to treatment. Postmortem findings in this individual included conjunctivitis, chemosis, dehydration, icterus, tricavitary serosanguinous effusions, necrotizing hepatitis, diffuse pulmonary congestion, and edema. Immunohistochemical examination identified scattered Leptospira organisms within hepatocytes and renal tubular epithelial cells. A wild raccoon (Procyon lotor) at the institution tested positive by PCR on kidney tissue for the same Leptospira spp. serovar and was the suspected source of infection. This case series highlights the clinical importance of leptospirosis as a differential for Patagonian maras presenting with lethargy, ocular signs, acute hepatic disease, and azotemia.

Lymphoma is one of the most common malignant neoplasms in cats. Historically, most feline lymphomas were associated with feline leukaemia virus infection, but due to routine vaccination against the virus, especially in developed countries, feline leukaemia virus-associated cases are dramatically reduced in numbers. More recently, the most common lymphoma in cats is intestinal lymphoma, with most common type being the enteropathy-associated T-cell lymphomas. Follicular lymphoma is recognised in cats, but is very uncommon. Primary colorectal follicular lymphoma is rare in humans and has been reported in three dogs. In this case report, we describe a primary colorectal lymphoma with histological and immunohistochemical features of a follicular lymphoma in a Savannah cat. To the authors' knowledge, this is the first case report of a primary colorectal follicular lymphoma in cats, and differential diagnosis of tumours in the colorectal area in cats should include follicular lymphoma.