E3 ubiquitin ligase CBLB regulates innate immune responses and bacterial dissemination during nontuberculous mycobacteria infection.

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens causing pulmonary infection to fatal disseminated disease. NTM infections are steadily increasing in children and adults, and immune-compromised individuals are at a greater risk of fatal infections. The NTM disease's adverse pathology and resistance to antibiotics have further worsened the therapeutic measures. Innate immune regulators are potential targets for therapeutics to NTM, especially in a T cell-suppressed population, and many ubiquitin ligases modulate pathogenesis and innate immunity during infections, including mycobacterial infections. Here, we investigated the role of an E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (CBLB), in immunocompromised mouse models of NTM infection. We found that CBLB is essential to prevent bacterial growth and dissemination. Cblb deficiency debilitated natural killer cells, inflammatory monocytes, and macrophages in vivo. However, Cblb deficiency in macrophages did not wane its ability to inhibit bacterial growth or production of reactive oxygen species or interferon γ production by natural killer cells in vitro. CBLB restricted NTM growth and dissemination by promoting early granuloma formation in vivo. Our study shows that CBLB bolsters innate immune responses and helps prevent the dissemination of NTM during compromised T cell immunity.

Canine thyroid carcinomas: A review with emphasis on comparing the compact subtype of follicular thyroid carcinomas and medullary thyroid carcinomas.

Canine thyroid carcinomas are relatively common malignant endocrine neoplasms in dogs derived from either thyroid follicular cells (forming follicular thyroid carcinomas) or medullary cells (parafollicular, C-cells; forming medullary thyroid carcinomas). Older and recent clinical studies often fail to discriminate between compact cellular (solid) follicular thyroid carcinomas and medullary thyroid carcinomas, which may skew conclusions. The compact subtype of follicular thyroid carcinomas appears to be the least differentiated subtype of follicular thyroid carcinomas and needs to be differentiated from medullary thyroid carcinomas. This review includes information on the signalment, presentation, etiopathogenesis, classification, histologic and immunohistochemical diagnosis, clinical management, and biochemical and genetic derangements of canine follicular and medullary carcinomas, and their correlates with human medicine.

Novel genetic variant associated with globoid cell leukodystrophy in a family of mixed breed dogs.

BACKGROUND: Globoid cell leukodystrophy (GCL) is a fatal autosomal recessive disease caused by variants in the galactosylceramidase (GALC) gene. Two dog breed-specific variants are reported. OBJECTIVES: Characterize the putatively causative GALC variant for GCL in a family of dogs and determine population allele frequency. ANIMALS: Four related mixed-breed puppies with signs of neurologic disease were evaluated. Subsequently, 33 related dogs were tested for genetic markers for parentage and the identified GALC variant. Additional GALC genotyping was performed on 278 banked samples from various breeds. METHODS: The 4 affected puppies had neurological exams and necropsies. DNA was isolated from blood samples. Variants in GALC were identified via Sanger sequencing. Parentage testing was performed using short tandem repeat markers. Prevalence of the GALC variant of interest was investigated in other breeds. RESULTS: GCL was confirmed histopathologically. A novel missense variant in GALC (NC_006590.4:g.58893972G>A) was homozygous in all affected animals (n = 4). A recessive mode of inheritance was confirmed by parentage testing as was variant linkage with the phenotype (LOD = 3.36). Among the related dogs (n = 33), 3 dogs were homozygous and 7 heterozygous. The variant allele was not detected in screening 278 dogs from 5 breeds. The novel variant is either unique to this family or has an extremely low allele frequency in the general population. CONCLUSIONS AND CLINICAL IMPORTANCE: A novel GALC variant was identified that likely explains GCL in this cohort. The identification of multiple causal variants for GCL in dogs is consistent with findings in humans.

Yellow Mealworm (Tenebrio molitor) and Lesser Mealworm (Alphitobius diaperinus) Proteins Slowed Weight Gain and Improved Metabolism of Diet-Induced Obesity Mice.

BACKGROUND: High-protein diets not only meet amino acid needs but also modulate satiety and energy metabolism. Insect-based proteins are sustainable, high-quality proteins. Mealworms have been studied, but limited information is known about their ability to impact metabolism and obesity. OBJECTIVE: We determined the effects of defatted yellow mealworm (Tenebrio molitor)- and whole lesser mealworm (Alphitobius diaperinus)-based proteins on the body weight (BW), serum metabolites, and liver and adipose tissue (AT) histology and gene expression of diet-induced obesity mice. METHODS: Male C57BL/6J mice were fed a high-fat diet (HFD; 46% kcal) to induce obesity and metabolic syndrome. Obese mice were then assigned to treatments (n = 10/group) and fed for 8 wk: HFD: HFD with casein protein; B50: HFD with 50% protein from whole lesser mealworm; B100: HFD with 100% protein from whole lesser mealworm; Y50: HFD with 50% protein from defatted yellow mealworm; Y100: HFD with 100% protein from defatted yellow mealworm. Lean mice (n = 10) fed a low-fat-diet (LFD; 10% kcal) were included. Longitudinal food intake, BW, body composition, and glucose response were measured. At time of killing, serum metabolites, tissue histopathology and gene expression, and hepatic triglycerides were analyzed. RESULTS: After 8 wk, HFD, B50, and B100 had greater (P < 0.05) weight gain than LFD, whereas Y50 and Y100 did not. Y50, B100, and Y100 had a lower (P < 0.05) BW change rate than HFD. Mealworm-based diets led to increased (P < 0.05) serum high-density lipoprotein (HDL) and reduced (P < 0.05) serum low-density lipoprotein (LDL) concentrations and reduced (P<0.05) LDL/HDL ratio. Mealworm-based diets led to increased (P < 0.05) hepatic expression of genes related to energy balance, immune response, and antioxidants and reduced (P < 0.05) AT expression of genes associated with inflammation and apoptosis. Mealworm-based diets altered (P < 0.05) hepatic and AT expression of glucose and lipid metabolism genes. CONCLUSIONS: In addition to serving as an alternative protein source, mealworms may confer health benefits to obese patients.

GM-CSF+ Tc17 cells are required to bolster vaccine immunity against lethal fungal pneumonia without causing overt pathology.

GM-CSF co-expressing T17 cells instigate pathologic inflammation during autoimmune disorders, but their function in immunity to infections is unclear. Here, we demonstrate the role of GM-CSF+Tc17 cells for vaccine immunity against lethal fungal pneumonia and the cytokine requirements for their induction and memory homeostasis. Vaccine-induced GM-CSF+ Tc17 cells are necessary to bolster pulmonary fungal immunity without inflating pathology. Although GM-CSF expressing Tc17 cells preferentially elevate during the memory phase, their phenotypic attributes strongly suggest they are more like Tc17 cells than IFNγ-producing Tc1 cells. IL-1 and IL-23, but not GM-CSF, are necessary to elicit GM-CSF+ Tc17 cells following vaccination. IL-23 is dispensable for memory Tc17 and GM-CSF+ Tc17 cell maintenance, but recall responses of effector or memory Tc17 cells in the lung require it. Our study reveals the beneficial, nonpathological role of GM-CSF+ Tc17 cells during fungal vaccine immunity.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Cytotoxicity of Cultured Canine Primary Hepatocytes Exposed to Itraconazole Is Decreased by Pre-treatment With Glutathione.

Objective: To identify the effect of glutathione (GSH) on cell survival in a novel in vitro model of itraconazole (ITZ)-associated hepatotoxicity using canine primary hepatocytes. Sample: Commercially sourced, cryopreserved male dog (Beagle) primary hepatocytes from a single donor. Procedures: Using a sandwich culture technique, canine primary hepatocytes were exposed to serial dilutions of ITZ. Calcein AM, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and neutral red were investigated as potential cell viability assays. Hepatocytes were then pre-incubated with GSH, exposed to serial dilutions of ITZ, and cell viability determined at 4 and 24 h post-ITZ exposure. Each condition was performed in technical triplicate and the effect of time, GSH concentration, and ITZ concentration on % cytotoxicity assessed using a multivariate linear regression model. Tukey's post-hoc test was used to detect individual differences. Results: The neutral red cell cytotoxicity assay was chosen based on its superior ability to detect dose-dependent changes in viability. Hepatocyte cytotoxicity significantly increased with ITZ concentration (P < 0.001) and time (P = 0.004) and significantly decreased with GSH treatment (P < 0.001). Conclusions and Clinical Relevance: This in vitro model demonstrates dose- and time-dependent ITZ-induced cytotoxicity, which is similar to clinical changes observed in canine patients and in in vivo rodent studies. Pre-treating with GSH is protective against in vitro cell death. These results suggest that GSH precursors may have a role in the management or prevention of ITZ-associated hepatotoxicity in dogs. Clinical trials are needed to evaluate their utility for this adverse drug reaction.

Development of a novel model of cholecystectomy in subsequently ovariectomized mice and characterization of metabolic and gastrointestinal phenotypes: a pilot study.

BACKGROUND: Cholecystectomy (XGB) is the most common abdominal surgery performed in the United States and is associated with an increased post-surgery incidence of metabolic and gastrointestinal (GI) diseases. Two main risk factors for XGB are sex (female) and age (40-50 yr), corresponding with onset of menopause. Post-menopausal estrogen loss alone facilitates metabolic dysfunction, but the effects of XGB on metabolic and GI health have yet to be investigated in this population. Study objectives were to (1) identify possible short-term effects of XGB and (2) develop a novel murine model of XGB in human menopause via subsequent ovariectomy (OVX) and assess longitudinal effects of OVX on metabolism, GI physiology, and GI microbiota in XGB mice. METHODS: Female C57BL/6 mice were utilized in two parallel studies (S1&S2). In S1, XGB mice were compared to a non-XGB baseline group after six wk. In S2, mice were XGB at wk0, either sham (SHM) or OVX at wk6, and sacrificed at wk12, wk18, and wk24. Body composition assessment and fresh fecal collections were conducted periodically. Serum and tissues were collected at sacrifice for metabolic and GI health endpoints. RESULTS: Compared to baseline, XGB increased hepatic CYP7A1 and decreased HMGCR relative expression, but did not influence BW, fat mass, or hepatic triglycerides after six wk. In S2, XGB/OVX mice had greater BW and fat mass than XGB/SHM. Cecal microbiota alpha diversity metrics were lower in XGB/OVX mice at wk24 compared the XGB/SHM. No consistent longitudinal patterns in fasting serum lipids, fecal microbial diversity, and GI gene expression were observed between S2 groups. CONCLUSIONS: In addition to developing a novel, clinically-representative model of XGB and subsequent OVX, our results suggest that OVX resulted in the expected phenotype to some extent, but that XGB may modify or mask some responses and requires further investigation.

Feasibility of hepatic fine needle aspiration as a minimally invasive sampling method for gene expression quantification of pharmacogenetic targets in dogs.

BACKGROUND: Quantifying hepatic gene expression is important for many pharmacogenetic studies. However, this usually requires biopsy (BX), which is invasive. OBJECTIVES: The objectives of this study were to determine the feasibility of using minimally invasive fine needle aspirate (FNA) to quantify hepatic gene expression and to assess expression variability between different sampling sites. METHODS: Biopsy and FNA samples were acquired from central and peripheral locations of the right and left lateral liver lobes of a dog. Relative expression of ABCB1, GSTT1 and CYP3A12 were measured via reverse transcriptase, quantitative PCR. The effect of sampling method, lobe and location within the lobe on gene expression was assessed using a three-way ANOVA. RESULTS: Relative expression of ABCB1 and GSTT1 were not statistically different between sampling methods but CYP3A12 expression was higher in samples collected by BX (p = .013). Lobe sampled affected ABCB1 expression (p = .001) and site within lobe affected ABCB1 (p = .018) and GSTT1 (p = .025) expression. CONCLUSIONS: FNA appears to be a feasible technique for minimally invasive evaluation of hepatic gene expression but results should not be directly compared to biopsy samples. Sampling location impacts expression of some targets; combination of FNAs from multiple sites may reduce variation.

Saccharomyces cerevisiae Fermentation Product Did Not Attenuate Clinical Signs, but Psyllium Husk Has Protective Effects in a Murine Dextran Sulfate Sodium-Induced Colitis Model.

BACKGROUND: Yeast products and psyllium husk may provide relief from clinical signs of colitis due to their ability to promote gut integrity, modulate gut microbiota, or positively affect immune responses, which have been demonstrated in several species. OBJECTIVE: The objective of this study was to investigate the effects of a Saccharomyces cerevisiae fermentation product (SCFP) and psyllium husk (PH) on cecal and fecal microbiota, colonic gene expression and histopathology, and mesenteric lymph node (MLN) immune cells in a dextran sulfate sodium (DSS)-induced colitis model. METHODS: Male C57BL/6J mice (n = 54) were assigned to a control, 5% SCFP, or 5% PH diet. After 2 wk of diet adaptation, mice were provided distilled water or 3% (wt:vol) DSS for 5 d ad libitum. Body weight, food and water intakes, and disease activity index (DAI) were recorded daily during the treatment period. Fresh fecal samples were collected before and during treatment for microbial analyses. After treatment, mice were killed, followed by tissue collection. Tissues were stored in proper solutions until further analyses. Data were analyzed using the Mixed Models procedure of SAS 9.4 (SAS Institute). RESULTS: Consumption of SCFP increased (P < 0.05) species richness of the gut microbiota and relative abundance of Butyricicoccus in fecal and cecal samples compared with control or PH mice. PH mice had greater (P < 0.05) gene expression of claudin (Cldn) 2, Cldn3, Cldn8, and occludin(Ocln) compared with control mice. DAI, MLN immune cell populations, colonic histopathology, and colonic gene expression were not affected (P > 0.05) by SCFP in DSS mice. DSS mice consuming PH had lower (P < 0.05) DAI compared with control or SCFP mice. CONCLUSIONS: Results suggest that, despite the modest changes it had on cecal and fecal microbiota, SCFP did not attenuate clinical signs associated with DSS-induced colitis in mice, while PH showed protective effects.

Ex vivo comparison of leakage pressures and leakage location with a novel technique for creation of functional side-to-side canine small intestinal anastomoses.

OBJECTIVE: To determine the ability of functional side-to-side small intestinal anastomoses (FSS-SIA) created with an electrothermal bipolar vessel sealing (EBVS) device to resist leakage. STUDY DESIGN: Experimental, ex vivo. SAMPLE POPULATION: Jejunal segments (n = 130) from 10 healthy canine cadavers. METHODS: Four types of anastomoses were created (two segments/construct and 15 constructs/group): EBVS (group A), EBVS + transverse stapling (group B), stapled (group C), and EBVS + suture augmentation (group D). Initial leakage pressure (ILP), initial leakage location (ILL), and maximal intraluminal pressure were compared between groups, and five group A constructs were analyzed histologically. RESULTS: Initial leakage pressure was greater in group D than in groups A, B, and C (P < .011). There was a difference in ILL among groups (P = .003). Leakage occurred at the side-to-side intestinal anastomosis fusion line in 13 of 15 (87%) constructs for groups A and B and in nine of 15 (60%) constructs for group D. Maximal intraluminal pressure was greater in group C than in groups A, B, and D (P < .004). Histological examination was consistent with collagenous fusion without cavitation defects. CONCLUSION: Functional side-to-side small intestinal anastomosis was consistently achieved with an EBVS device. Augmentation of EBVS anastomoses with simple interrupted sutures along the anastomotic fusion line increased ILP compared with stapled anastomoses. CLINICAL SIGNIFICANCE: Despite the success and feasibility of creating an FSS-SIA with an EBVS device, additional in vivo studies are required to determine the effectiveness of intestinal fusion prior to clinical implementation.

Pharmacokinetics of platinum and safety evaluation of carboplatin-impregnated calcium sulfate hemihydrate beads after implantation in healthy cats.

OBJECTIVE: To evaluate the pharmacokinetics (PK) of platinum (Pt) and safety of carboplatin-impregnated calcium sulfate hemihydrate (C-I CSH) beads after implantation in healthy cats. STUDY DESIGN: In vivo experimental study. ANIMALS: Six healthy adult cats. METHODS: Three C-I CSH beads were implanted in muscle pockets over the right and left hemithoraces of each cat (~3.9 mg/kg of Pt; 60.4 mg/m2 of calculated carboplatin). Hematology and blood chemistry were tested at baseline and 3, 7, 14, and 21 days postimplantation. Serum was analyzed for Pt at specific times from 1 hour to 21 days. Tissue was obtained for histopathology and analysis of Pt at 3, 7, 14, and 21 days at standardized distances from implantation sites. RESULTS: Platinum was detected in tissues at all times and distances (range, 0.1-4.19 µg/g). Serum Pt increased up to 2.6 hours (3.25 µg/mL) then decreased sharply. Samples containing muscle had higher Pt compared with samples without muscle (P = .004). Mild hypercalcemia was noted in four cats, and mild inflammatory reaction was noted on histopathology of all samples. CONCLUSION: Platinum was released from C-I CSH beads differentially into surrounding tissues over 21 days. Systemic absorption of Pt was minimal, but mild hypercalcemia occurred. CLINICAL SIGNIFICANCE: Implantation was well tolerated by healthy adult cats. Securing beads within muscle may limit Pt diffusion to targeted tissue. Although Pt concentrations did not achieve levels reported to be cytotoxic for feline sarcoma cells in culture, results provide evidence to support evaluation of efficacy in the tumor microenvironment of cats with locally invasive cancers.

MORTALITY INVESTIGATION OF MONITORED EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) IN CENTRAL ILLINOIS, USA, FROM 2016-18.

Mortality events in eastern box turtles (Terrapene carolina carolina) threaten conservation efforts across the species range. These events are often under-diagnosed and, when observed, predictive health factors are unavailable prior to death. At Kickapoo State Park in central Illinois, USA, ranaviruses caused observed mortality events in amphibians and chelonians in 2014 and 2015. Following these outbreaks, eastern box turtles (n=36) were affixed with radio transmitters and temperature data loggers to obtain repeated location and temperature data from spring 2016-spring 2018. Bimonthly, samples of blood and oral and cloacal swabs were collected to investigate health parameters (hematology and cytokine transcription) and presence of multiple pathogens. Deaths of instrumented turtles occurred in 2016 (n=5), 2017 (n=15), and 2018 (n=2). The largest single die-off occurred in February 2017 (n=7). Seventeen turtles were necropsied and multiple pathologic processes were identified, most frequently decreased adipose stores (n=6). Two turtles had pathologic findings consistent with multisystemic inflammation. In addition, infectious pathogens were identified in turtles prior to death, but no single agent was associated with each mortality event. Ranavirus was not detected in any turtle. Hot spot analysis revealed spatial clustering at the center and edges of the study area for body temperature as well as for relative cytokine transcription of interleukin-1 beta, tumor necrosis factor alpha, and interleukin-10 associated with turtle death. Though no single causal factor could be identified, the information from this mortality event can direct future chelonian mortality investigations by providing baseline longitudinal data prior to death and in surviving turtles.

Medulloblastoma in a 6 Year Old Mixed Breed Dog: Surgical Debulking and Chemotherapy.

A medulloblastoma was surgically debulked from a 6 year old American Staffordshire Terrier, who then received a modified lomustine (CCNU), vincristine, procarbazine, and prednisolone (LOPP) protocol. The dog improved significantly and continued to do well until deterioration and euthanasia 5 months following surgery. This is the first known published case report of surgical cytoreductive surgery of a medulloblastoma in a dog with documented response to surgery and chemotherapy. Medulloblastoma is a primitive neuroectodermal tumor that is the most common malignant central nervous system (CNS) tumor in children, though it is less common in adults. This case illustrates the value of considering human literature when creating treatment plans for uncommon brain tumors in veterinary patients. Medulloblastoma should be a differential for cerebellar tumors in young to middle aged dogs, and surgery and chemotherapy should be considered.

Basal cell enumeration does not predict malignancy in canine perianal gland tumor cytology.

BACKGROUND: Perianal (hepatoid) gland tumors are common in dogs, and the distinction between the benign and malignant forms is clinically important. Cytology of these tumors typically contains many large hepatoid cells and fewer small basal cells. OBJECTIVES: The objective of this study was to determine whether the proportion of the smaller basaloid reserve cells in cytologic samples from perianal tumors correlates with malignancy. METHODS: Eighty-three cases of cytologically diagnosed perianal gland tumors with corresponding histopathologic sections were identified from two separate institutions and included six (7.2%) malignant tumors and 77 (92.8%) benign tumors. The proportion of basal cells from each sample was evaluated. RESULTS: No difference between these groups was found, although the study was sufficiently powered to detect an approximately 1.5-fold change in basal cell proportion. CONCLUSIONS: This report found no evidence that the proportion of basal cells in canine perianal tumor cytology is an indication of the potential for malignancy. We, therefore, do not recommend citing this feature in cytologic reports or when communicating with clinicians.

Gut Microbiota in Human Systemic Lupus Erythematosus and a Mouse Model of Lupus.

Gut microbiota dysbiosis has been observed in a number of autoimmune diseases. However, the role of the gut microbiota in systemic lupus erythematosus (SLE), a prototypical autoimmune disease characterized by persistent inflammation in multiple organs of the body, remains elusive. Here we report the dynamics of the gut microbiota in a murine lupus model, NZB/W F1, as well as intestinal dysbiosis in a small group of SLE patients with active disease. The composition of the gut microbiota changed markedly before and after the onset of lupus disease in NZB/W F1 mice, with greater diversity and increased representation of several bacterial species as lupus progressed from the predisease stage to the diseased stage. However, we did not control for age and the cage effect. Using dexamethasone as an intervention to treat SLE-like signs, we also found that a greater abundance of a group of lactobacilli (for which a species assignment could not be made) in the gut microbiota might be correlated with more severe disease in NZB/W F1 mice. Results of the human study suggest that, compared to control subjects without immune-mediated diseases, SLE patients with active lupus disease possessed an altered gut microbiota that differed in several particular bacterial species (within the genera Odoribacter and Blautia and an unnamed genus in the family Rikenellaceae) and was less diverse, with increased representation of Gram-negative bacteria. The Firmicutes/Bacteroidetes ratios did not differ between the SLE microbiota and the non-SLE microbiota in our human cohort.IMPORTANCE SLE is a complex autoimmune disease with no known cure. Dysbiosis of the gut microbiota has been reported for both mice and humans with SLE. In this emerging field, however, more studies are required to delineate the roles of the gut microbiota in different lupus-prone mouse models and people with diverse manifestations of SLE. Here, we report changes in the gut microbiota in NZB/W F1 lupus-prone mice and a group of SLE patients with active disease.

Control of lupus nephritis by changes of gut microbiota.

BACKGROUND: Systemic lupus erythematosus, characterized by persistent inflammation, is a complex autoimmune disorder with no known cure. Immunosuppressants used in treatment put patients at a higher risk of infections. New knowledge of disease modulators, such as symbiotic bacteria, can enable fine-tuning of parts of the immune system, rather than suppressing it altogether. RESULTS: Dysbiosis of gut microbiota promotes autoimmune disorders that damage extraintestinal organs. Here we report a role of gut microbiota in the pathogenesis of renal dysfunction in lupus. Using a classical model of lupus nephritis, MRL/lpr, we found a marked depletion of Lactobacillales in the gut microbiota. Increasing Lactobacillales in the gut improved renal function of these mice and prolonged their survival. We used a mixture of 5 Lactobacillus strains (Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus gasseri), but L. reuteri and an uncultured Lactobacillus sp. accounted for most of the observed effects. Further studies revealed that MRL/lpr mice possessed a "leaky" gut, which was reversed by increased Lactobacillus colonization. Lactobacillus treatment contributed to an anti-inflammatory environment by decreasing IL-6 and increasing IL-10 production in the gut. In the circulation, Lactobacillus treatment increased IL-10 and decreased IgG2a that is considered to be a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype. These beneficial effects were present in female and castrated male mice, but not in intact males, suggesting that the gut microbiota controls lupus nephritis in a sex hormone-dependent manner. CONCLUSIONS: This work demonstrates essential mechanisms on how changes of the gut microbiota regulate lupus-associated immune responses in mice. Future studies are warranted to determine if these results can be replicated in human subjects.

Wischnewsky-like spots in fatal cases of canine hypothermia.

Two dogs with a history of being found dead within environments exposed to extremely cold weather were examined postmortem. In both cases, brown to red to black, round to irregularly shaped foci ranging from 0.5-15 mm in diameter were identified within the mucosa of the stomach. These foci are grossly and microscopically consistent with Wischnewsky spots that are reported in humans. These spots in the mucosa of the stomach are considered important findings in cases of hypothermia-related deaths in humans and are for the first time reported in the dog.