Neurodegeneration in a domestic rabbit with severe malnourishment and low hepatic copper.

Copper is a trace element that plays an essential role in neurodevelopment and neurologic function. Acquired copper deficiency has a range of neurologic manifestations, with myelopathy being the most common association. We describe here the clinical, radiologic, histopathologic, and toxicologic findings of a rabbit with malnutrition, neurodegeneration, and suspected copper deficiency. A stray, adult female dwarf rabbit cross (Oryctolagus cuniculus) in poor body condition developed ataxia and pelvic limb weakness progressing to lateral recumbency and urine retention. The clinical findings suggested multifocal brainstem disease with right-sided central vestibular involvement; however, microscopic examination identified thoracic and lumbosacral spinal cord myelopathy. Differentials for the spinal cord changes included neurodegenerative disease, nutritional deficiency, neurotoxin, trauma to the lumbosacral region, and ischemia. Hepatic copper levels were suboptimal at 18 ppm dry weight (RI: 24-150 ppm dry weight). While speculative, copper-deficiency myelopathy is a treatable cause of non-compressive myelopathy that may occur in this species.

A fresh look at the SarcoFluor antibody test for the detection of specific antibodies to Sarcocystis neurona for the diagnosis of equine protozoal myeloencephalitis.

Equine protozoal myeloencephalitis (EPM) is a challenging disease to diagnose in horses with neurological signs. To optimize contemporary diagnostic testing, including the use of serum:CSF antibody ratios, the SarcoFluor antibody test for Sarcocystis neurona requires revalidation. The SarcoFluor, a previously validated immunofluorescent antibody test (IFAT) for the detection of antibodies specific to S. neurona in serum and cerebrospinal fluid (CSF) of naturally infected horses was analyzed using recent data and considering a serum:CSF antibody ratio threshold. Utilization of serum and CSF phosphorylated neurofilament heavy protein (pNfH) concentrations in support of an EPM diagnosis was also evaluated. 172 horses were divided into three groups: EPM-positive horses (EPM+, n=42), neurological non-EPM horses (n=74) confirmed with non-EPM neurological diseases (cervical vertebral compressive myelopathy, equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy), and control horses (control, n=56) without neurological signs and neurological abnormalities on histology. Logistic regression was used to compare EPM diagnostic regimens. Specifically, EPM+ horses were compared with neurological non-EPM horses showing neurological signs. To consider diagnostic utility, post-test probabilities were calculated by titer. When differentiating between EPM and other neurological diseases, the combination of serum and CSF SarcoFluor testing added more information to the model accuracy than either test alone. Using serum and CSF for pNfH in support of an EPM diagnosis did not identify cutoffs with statistically significant odds ratios but increased the overall model accuracy when used with the IFAT. Utilization of IFAT titers against S. neurona in serum and CSF result in a high post-test probability of detecting EPM+ horses in a clinical setting.

Miliary osteoma cutis in a climbing mantella frog (Mantella laevigata).

A climbing mantella frog (Mantella laevigata) was presented with nodular thickened skin. Histological examination revealed dermal nodules composed of differentiated bone consistent with miliary osteoma cutis, a non-neoplastic condition where bone is abnormally deposited within the skin. This is the first report of idiopathic osteoma cutis in an amphibian.

Equine neuroaxonal dystrophy/degenerative myeloencephalopathy in Gypsy Vanner horses.

BACKGROUND: Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a neurodegenerative disease that primarily affects young, genetically predisposed horses that are deficient in vitamin E. Equine NAD/EDM has not previously been documented in Gypsy Vanner horses (GVs). OBJECTIVES: To evaluate: (1) the clinical phenotype, blood vitamin E concentrations before and after supplementation and pedigree in a cohort of GV horses with a high prevalence of neurologic disease suspicious for eNAD/EDM and (2) to confirm eNAD/EDM in GVs through postmortem evaluation. ANIMALS: Twenty-six GVs from 1 farm in California and 2 cases from the Midwestern U.S. METHODS: Prospective observational study on Californian horses; all 26 GVs underwent neurologic examination. Pre-supplementation blood vitamin E concentration was assessed in 17- GVs. Twenty-three were supplemented orally with 10 IU/kg of liquid RRR-alpha-tocopherol once daily for 28 days. Vitamin E concentration was measured in 23 GVs after supplementation, of which 15 (65%) had pre-supplementation measurements. Two clinically affected GVs from California and the 2 Midwestern cases had necropsy confirmation of eNAD/EDM. RESULTS: Pre-supplementation blood vitamin E concentration was ≤2.0 µg/mL in 16/17 (94%) of GVs from California. Post-supplementation concentration varied, with a median of 3.39 µg/mL (range, 1.23-13.87 µg/mL), but only 12/23 (52%) were normal (≥3.0 µg/mL). Normalization of vitamin E was significantly associated with increasing age (P = .02). Euthanized horses (n = 4) had eNAD/EDM confirmed at necropsy. CONCLUSIONS AND CLINICAL IMPORTANCE: GVs could have a genetic predisposition to eNAD/EDM. Vitamin E supplementation should be considered and monitored in young GVs.

Clinicopathological and pedigree investigation of a novel spinocerebellar neurological disease in juvenile Quarter Horses in North America.

BACKGROUND: In 2020, a novel neurologic disease was observed in juvenile Quarter Horses (QHs) in North America. It was unknown if this was an aberrant manifestation of another previously described neurological disorder in foals, such as equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM). HYPOTHESIS/OBJECTIVES: To describe the clinical findings, outcomes, and postmortem changes with Equine Juvenile Spinocerebellar Ataxia (EJSCA), differentiate the disease from other similar neurological disorders, and determine a mode of inheritance. ANIMALS: Twelve neurologically affected QH foals and the dams. METHODS: Genomic DNA was isolated and pedigrees were manually constructed. RESULTS: All foals (n = 12/12) had a history of acute onset of neurological deficits with no history of trauma. Neurological deficits were characterized by asymmetrical spinal ataxia, with pelvic limbs more severely affected than thoracic limbs. Clinicopathological abnormalities included high serum activity of gamma-glutamyl transferase and hyperglycemia. All foals became recumbent (median, 3 days: [0-18 days]), which necessitated humane euthanasia (n = 11/12, 92%; the remaining case was found dead). Histological evaluation at postmortem revealed dilated myelin sheaths and digestion chambers within the spinal cord, most prominently in the dorsal spinocerebellar tracts. Pedigree analysis revealed a likely autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL IMPORTANCE: EJSCA is a uniformly fatal, rapidly progressive, likely autosomal recessive neurological disease of QHs <1 month of age in North America that is etiologically distinct from other clinically similar neurological disorders. Once the causative variant for EJSCA is validated, carriers can be identified through genetic testing to inform breeding decisions.

Primary and secondary leptomeningeal gliomatosis in dogs.

Leptomeningeal gliomatosis (LG) is characterized by extensive dissemination of neoplastic glial cells in the subarachnoid space either without an intraparenchymal glioma (primary LG or PLG) or secondary to an intraparenchymal glioma (secondary LG or SLG). Given the low frequency of LG in human and veterinary medicine, specific diagnostic criteria are lacking. Here, we describe 14 cases of canine LG that were retrospectively identified from 6 academic institutions. The mean age of affected dogs was 7.3 years and over 90% of patients were brachycephalic. Clinical signs were variable and progressive. Relevant magnetic resonance image findings in 7/14 dogs included meningeal enhancement of affected areas and/or intraparenchymal masses. All affected dogs were euthanized because of the poor prognosis. Gross changes were reported in 12/14 cases and consisted mainly of gelatinous leptomeningeal thickening in the brain (6/12 cases) or spinal cord (2/12 cases) and 1 or multiple, gelatinous, gray to red intraparenchymal masses in the brain (6/12 cases). Histologically, all leptomeningeal neoplasms and intraparenchymal gliomas were morphologically consistent with oligodendrogliomas. Widespread nuclear immunolabeling for OLIG2 was observed in all neoplasms. The absence of an intraparenchymal glioma was consistent with PLG in 3 cases. The remaining 11 cases were diagnosed as SLG.

Genetic polymorphisms in vitamin E transport genes as determinants for risk of equine neuroaxonal dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with vitamin E deficiency. In humans, polymorphisms in genes involved in vitamin E uptake and distribution determines individual vitamin E requirements. HYPOTHESIS/OBJECTIVES: Genetic polymorphisms in genes involved in vitamin E metabolism would be associated with an increased risk of eNAD/EDM in Quarter Horses (QHs). ANIMALS: Whole-genome sequencing: eNAD/EDM affected (n = 9, postmortem [PM]-confirmed) and control (n = 32) QHs. VALIDATION: eNAD/EDM affected (n = 39, 23-PM confirmed) and control (n = 68, 7-PM confirmed) QHs. Allele frequency (AF): Publicly available data from 504 horses across 47 breeds. METHODS: Retrospective, case control study. Whole-genome sequencing was performed and genetic variants identified within 28 vitamin E candidate genes. These variants were subsequently genotyped in the validation cohort. RESULTS: Thirty-nine confirmed variants in 15 vitamin E candidate genes were significantly associated with eNAD/EDM (P < .01). In the validation cohort, 2 intronic CD36 variants (chr4:726485 and chr4:731082) were significantly associated with eNAD/EDM in clinical (P = 2.78 × 10-4 and P = 4 × 10-4 , respectively) and PM-confirmed cases (P = 6.32 × 10-6 and 1.04 × 10-5 , respectively). Despite the significant association, variant AFs were low in the postmortem-confirmed eNAD/EDM cases (0.22-0.26). In publicly available equine genomes, AFs ranged from 0.06 to 0.1. CONCLUSIONS AND CLINICAL IMPORTANCE: Many PM-confirmed cases of eNAD/EDM were wild-type for the 2 intronic CD36 SNPs, suggesting either a false positive association or genetic heterogeneity of eNAD/EDM within the QH breed.

High-Grade, Stage 2 Mast Cell Tumors: Outcome in Dogs With Local and Systemic Therapy.

Canine mast cell tumors (MCTs) have highly variable clinical behavior, and predicting outcomes in individual dogs remains challenging. Many studies combine dogs with varying tumor grades, clinical stage, or treatments, confounding those results. The purpose of this retrospective study was to determine outcome and prognostic factors in a specific subset of dogs with high-grade, stage 2, cutaneous MCTs treated with adequate local control via surgery with or without radiation therapy and adjuvant cytotoxic chemotherapy. Seventeen dogs met the inclusion criteria, and the median survival time was 259 days. Development of local recurrence, tumor location, and presence of ulceration were all associated with shorter survival times. Tumor size, mitotic count, chemotherapy protocol, lymph node classification, and radiation therapy were not significantly associated with outcome. In this study, a specific population of dogs characterized by high-grade MCTs with local lymph node metastasis who received aggressive local and systemic therapy had a median survival of about 8.5 mo. Dogs with ulcerated tumors, recurrent tumors, or tumors located on the head had a worse outcome despite aggressive therapy. These results may serve as a basis of comparison for future research exploring alternative treatment combinations in this specific population of dogs.

Intra- and Intertumoral Microglia/Macrophage Infiltration and Their Associated Molecular Signature Is Highly Variable in Canine Oligodendroglioma: A Preliminary Evaluation.

The goal of this study was to define the glioma-associated microglia/macrophage (GAM) response and associated molecular landscape in canine oligodendrogliomas. Here, we quantified the intratumoral GAM density of low- and high-grade oligodendrogliomas compared to that of a normal brain, as well as the intratumoral concentration of several known GAM-derived pro-tumorigenic molecules in high-grade oligodendrogliomas compared to that in a normal brain. Our analysis demonstrated marked intra- and intertumoral heterogeneity of GAM infiltration. Correspondingly, we observed significant variability in the intratumoral concentrations of several GAM-associated molecules, unlike what we previously observed in high-grade astrocytomas. However, high-grade oligodendroglioma tumor homogenates (n = 6) exhibited an increase in the pro-tumorigenic molecules hepatocyte growth factor receptor (HGFR) and vascular endothelial growth factor (VEGF), as we observed in high-grade astrocytomas. Moreover, neoplastic oligodendrocytes displayed robust expression of GAL-3, a chimeric galectin implicated in driving immunosuppression in human glioblastoma. While this work identifies shared putative therapeutic targets across canine glioma subtypes (HGFR, GAL-3), it highlights several key differences in the immune landscape. Therefore, a continued effort to develop a comprehensive understanding of the immune microenvironment within each subtype is necessary to inform therapeutic strategies going forward.

Oculosystemic pneumocystosis in 2 sibling Chihuahuas.

Sibling female and male Chihuahuas were evaluated for a 9-month history of tachypnea that failed to respond to fenbendazole, doxycycline, amoxicillin-clavulanate, and prednisone. Physical examination identified tachypnea, hyperpnea, and harsh bronchovesicular lung sounds. Fundic examination disclosed diffuse chorioretinitis, manifested as multifocal chorioretinal granulomas in the female dog and occasional chorioretinal scars in the male dog. Thoracic radiographs indicated moderate to severe interstitial to broncho-interstitial infiltrates in both dogs. Serum and urine antigen and antibody testing in the female dog failed to identify infectious agents, but cytologic assessment of hepatic lymph node, liver, and splenic aspirates identified Pneumocystis trophozoites. Infection was confirmed in both dogs by 28S rRNA PCR sequencing from multiple tissue samples. The female dog responded well to trimethoprim-sulfamethoxazole, but the male dog was euthanized because of liver failure, presumably related to antimicrobial treatment.

Fatal balamuthosis in a Siberian tiger and a literature review of detection options for free-living amoebic infections in animals.

Free-living amoebae are rare causes of morbidity and mortality in humans and animals around the globe. Because the route of exposure and clinical progression of disease caused by different species of amoebae may vary in people and animals, determining the species of amoeba present is important. We describe here a fatal infection by the free-living amoeba Balamuthia mandrillaris in a Siberian tiger (Panthera tigris altaica). The 17-y-old patient had a rapid clinical decline after a peracute onset of severe lethargy, dull mentation, and anorexia. Autopsy did not identify a cause of death. Histology revealed inflammation associated with amoebic trophozoites in the brain, lungs, and iris of one eye. These amoebae were confirmed to be B. mandrillaris based on a PCR assay and sequencing. Although there are subtle morphologic differences between cyst stages of Acanthamoeba spp., B. mandrillaris, and Naegleria fowleri when present and identified on routine staining, other modalities, including PCR, immunofluorescence, electron microscopy, and immunohistochemistry, are typically utilized to confirm the pathogen involved in these cases. We review the reports of balamuthosis in animals.

Lung and lymph node explants to study the interaction between host cells and canine distemper virus.

Canine distemper virus (CDV, family Paramyxoviridae) is a widely known fatal disease in unvaccinated dogs and wild carnivores. The virus enters via the respiratory tract and rapidly spreads to the lymphoid organs. To investigate viral entry into these tissues, a dog tissue explant model was developed for lung and lymph nodes. Canine lung explants were cultured with CDV for three days. During this time CDV antigens were visible on alveolar cells, which were CD163-positive and SLAM-positive (signaling lymphocytic activation molecule), demonstrating that they were macrophages. The lymph node explants were maintained for five days. During this time the viral replication increased progressively by each day post infection and syncytia were observed by day three, post exposure. The microscopic distribution of CDV-positive cells in the lymph nodes, including the syncytia, and co-expression of CD163 and SLAM, demonstrated that they were macrophages. These findings suggest that alveolar macrophages are the first cells in the lung to become infected during CDV infection, and lymph node explants showed similar replication rates and virus-cell interactions as seen in experimental live animals. This demonstrates the utility of canine respiratory and lymphoid explant model to evaluate cell entry and viral replication of CDV and other morbilliviruses in dogs or other susceptible carnivores.

Case series: Pleural effusion caused by urinary ultrafiltrate in two cats without evidence of urinary obstruction, trauma, or simultaneous perinephric pseudocysts.

Objectives: To describe the diagnostic techniques, surgical treatments, and outcomes of two cats with recurrent pleural transudate caused by urinary ultrafiltrate. Animals: Two cats without evidence of trauma, urinary tract obstruction, or concurrent perinephric pseudocysts that were evaluated and treated for recurrent pleural transudate caused by urinary ultrafiltrate. Study design: Short case series. Methods: Multiphase contrast CT scan revealed leakage of contrast media from the kidneys bilaterally into the retroperitoneal spaces in both cats. Renal scintigraphy performed in one cat revealed progressive accumulation of 99mTc diethylenetriamine penta-acetic acid (Tc-DTPA) in the pleural space. Exploratory laparotomy localized the leakage of fluid to renal capsular defects bilaterally in both cats. The retroperitoneum was incised bilaterally to promote fluid drainage into the peritoneum, and nephropexies were performed. Results: One cat had long-term survival with recurrent, though decreasing volumes of, pleural effusion. The second cat was euthanized 16 days postoperatively for progressive renal disease. Conclusion: The diagnosis of spontaneous urinary ultrafiltrate accumulation in the pleural space of cats without evidence of trauma, urinary tract obstruction, or concurrent perinephric pseudocysts has not previously been reported. The surgical correction described reduced but did not completely eliminate the rate of pleural effusion accumulation.

Inhaled recombinant human IL-15 in dogs with naturally occurring pulmonary metastases from osteosarcoma or melanoma: a phase 1 study of clinical activity and correlates of response.

PURPOSE: Although recombinant human interleukin-15 (rhIL-15) has generated much excitement as an immunotherapeutic agent for cancer, activity in human clinical trials has been modest to date, in part due to the risks of toxicity with significant dose escalation. Since pulmonary metastases are a major site of distant failure in human and dog cancers, we sought to investigate inhaled rhIL-15 in dogs with naturally occurring lung metastases from osteosarcoma (OSA) or melanoma. We hypothesized a favorable benefit/risk profile given the concentrated delivery to the lungs with decreased systemic exposure. EXPERIMENTAL DESIGN: We performed a phase I trial of inhaled rhIL-15 in dogs with gross pulmonary metastases using a traditional 3+3 cohort design. A starting dose of 10 µg twice daily × 14 days was used based on human, non-human primate, and murine studies. Safety, dose-limiting toxicities (DLT), and maximum tolerated dose (MTD) were the primary objectives, while response rates, progression-free and overall survival (OS), and pharmacokinetic and immune correlative analyses were secondary. RESULTS: From October 2018 to December 2020, we enrolled 21 dogs with 18 dogs reaching the 28-day response assessment to be evaluable. At dose level 5 (70 µg), we observed two DLTs, thereby establishing 50 µg twice daily × 14 days as the MTD and recommended phase 2 dose. Among 18 evaluable dogs, we observed one complete response >1 year, one partial response with resolution of multiple target lesions, and five stable disease for an overall clinical benefit rate of 39%. Plasma rhIL-15 quantitation revealed detectable and sustained rhIL-15 concentrations between 1-hour and 6 hour postnebulization. Decreased pretreatment lymphocyte counts were significantly associated with clinical benefit. Cytotoxicity assays of banked peripheral blood mononuclear cells revealed significant increases in peak cytotoxicity against canine melanoma and OSA targets that correlated with OS. CONCLUSIONS: In this first-in-dog clinical trial of inhaled rhIL-15 in dogs with advanced metastatic disease, we observed promising clinical activity when administered as a monotherapy for only 14 days. These data have significant clinical and biological implications for both dogs and humans with refractory lung metastases and support exploration of combinatorial therapies using inhaled rhIL-15.

Case Report: MRI, Clinical, and Pathological Correlates of Bromethalin Toxicosis in Three Dogs.

Bromethalin toxicosis is an increasingly common clinical presentation in dogs that may be fatal depending on the extent of intoxication. Antemortem diagnosis of bromethalin toxicosis was achieved in three dogs by demonstration of the active metabolite desmethylbromethalin in fat or serum. Magnetic resonance imaging (MRI) findings were consistent with a diffuse leukoencephalopathy with restricted diffusion and prominent involvement of the corticospinal motor tracts on T2-weighted and diffusion-weighted sequences. Imaging findings were confirmed in one non-surviving dog at necropsy. Resolution of MRI abnormalities was demonstrated in one surviving dog that was consistent with the associated resolution of clinical signs. Initial findings in these dogs support further investigation of specific MRI patterns in cases of leukoencephalopathy to aid differential diagnosis. While antemortem detection of bromethalin and its metabolites confirms exposure, quantitation may be informative as a prognostic biomarker.

R132H IDH1 sensitizes glioma to the antiproliferative and cytotoxic effects of BET inhibition.

INTRODUCTION: Mutations in isocitrate dehydrogenase 1/2 (IDHmut) identify a subset of gliomas that exhibit epigenetic dysregulation via aberrant DNA methylation. These tumors are ultimately fatal and lack effective therapeutic strategies. Considering the epigenetic dysregulation of IDHmut gliomas, we hypothesized that epigenetic-targeting drugs may yield therapeutic benefits in gliomas bearing IDHmut. One set of targets includes the bromodomain and extraterminal (BET) family of transcriptional coactivators. METHODS: We used TCGA data from glioma patients to determine whether BET proteins affect patient survival differently based on IDH status. Follow-up experiments using a set of IDH wildtype/mutant glioma cultures, as well as an IDH wildtype glioblastoma cell line expressing exogenous R132H IDH1, focused on cell health assays to investigate whether IDHmut was associated with increased sensitivity to the BET inhibitor JQ1. Immunoblots were used to evaluate the molecular response to JQ1 in these cultures. RESULTS: We identified that high BRD4 expression associated with decreased survival only in IDHmut glioma patients. Cell viability analysis showed that IDHmut sensitized glioma cells to delayed cytotoxicity (10 days) in response to JQ1. Early effects of JQ1 (3 days) were primarily antiproliferative, with IDHmut glioma exhibiting a modest increase in sensitivity. Finally, exogenous R132H IDH1 expression in a resistant IDH wildtype cell line recapitulated the JQ1-mediated delayed cytotoxicity seen in our endogenous IDHmut glioma cells. CONCLUSION: Overall, these data suggest that BRD4 enhances malignancy primarily in gliomas bearing IDHmut and is associated with greater sensitivity to BET inhibition. The finding that BET inhibition primarily exhibits delayed cytotoxicity may be overlooked in conventional short endpoint dose-response assays. Follow-up mechanistic and animal studies will help address the translational potential of these findings.

IDH1 mutant glioma is preferentially sensitive to the HDAC inhibitor panobinostat.

INTRODUCTION: A large subset of diffusely infiltrative gliomas contains a gain-of-function mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2mut) which produces 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent DNA demethylases, thereby inducing widespread DNA and histone methylation. Because histone deacetylase (HDAC) enzymes are localized to methylated chromatin via methyl-binding domain proteins, IDH1/2mut gliomas may be more dependent on HDAC activity, and therefore may be more sensitive to HDAC inhibitors. METHODS: Six cultured patient-derived glioma cell lines, IDH1wt (n = 3) and IDH1mut (n = 3), were treated with an FDA-approved HDAC inhibitor, panobinostat. Cellular cytotoxicity and proliferation assays were conducted by flow cytometry. Histone modifications and cell signaling pathways were assessed using immunoblot and/or ELISA. RESULTS: IDH1mut gliomas exhibited marked upregulation of genes associated with the HDAC activity. Glioma cell cultures bearing IDH1mut were significantly more sensitive to the cytotoxic and antiproliferative effects of panobinostat, compared to IDH1wt glioma cells. Panobinostat caused a greater increase in acetylation of the histone residues H3K14, H3K18, and H3K27 in IDH1mut glioma cells. Another HDAC inhibitor, valproic acid, was also more effective against IDH1mut glioma cells. CONCLUSION: These data suggest that IDH1mut gliomas may be preferentially sensitive to HDAC inhibitors. Further, IDH1mut glioma cultures showed enhanced accumulation of acetylated histone residues in response to panobinostat treatment, suggesting a direct epigenetic mechanism for this sensitivity. This provides a rationale for further exploration of HDAC inhibitors against IDH1mut gliomas.

Encephalopathy caused by Talisia esculenta intoxication in pregnant ewes and their newborn lambs.

An outbreak of acute encephalopathy occurred in pregnant ewes and their newborn lambs associated with consumption of Talisia esculenta fruits and bark. Clinical signs in 5 adult pregnant ewes included drooling, bloat, tachypnea, depression, ataxia, body shaking, difficulty in rising, and recumbency. Three neonatal lambs born to some of those ewes had similar clinical signs. No significant gross abnormalities were observed on autopsy. Histologically, neuronal necrosis, axonal and dendritic swelling, and loss of Purkinje neurons were observed in the cerebellum. The observation of similar neurologic clinical signs and lesions in pregnant ewes and their neonatal lambs suggests that the toxic principle of T. esculenta crosses the placenta and reaches the fetus.

Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM. HYPOTHESIS/OBJECTIVES: Based on the association of α-tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α-tocopherol, but not γ-tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM-affected horses. ANIMALS: Vitamin E metabolism: Proof of concept (POC) study; eNAD/EDM-affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM-affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM-affected (n = 12) and age- and sex-matched control (n = 11-12) horses. METHODS: The rates of α-tocopherol/tocotrienol and γ-tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative reverse-transcriptase PCR (qRT-PCR) and droplet digital (dd)-PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog. RESULTS: Metabolic rate of α-tocopherol was increased in eNAD/EDM horses (POC,P < .0001; validation, P = .03), with no difference in the metabolic rate of γ-tocopherol. Horses with eNAD/EDM had increased expression of the CYP4F2 equine orthologue (P = .02) but no differences in copy number. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased α-tocopherol metabolism in eNAD/EDM-affected QHs provides novel insight into alterations in vitamin E processing in eNAD/EDM and highlights the need for high-dose supplementation to prevent the clinical phenotype in genetically susceptible horses.