Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Characterization of Classical Sheep Scrapie in White-tailed Deer after Experimental Oronasal Exposure.

BACKGROUND: Classic scrapie is a prion disease of sheep and goats that is associated with accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the prion disease of cervids. This study was conducted to determine the susceptibility of white-tailed deer (WTD) to the classic scrapie agent. METHODS: We inoculated WTD (n = 5) by means of a concurrent oral/intranasal exposure with the classic scrapie agent from sheep or oronasally with the classic scrapie agent from goats (n = 6). RESULTS: All deer exposed to the agent of classic scrapie from sheep accumulated PrPSc. PrPSc was detected in lymphoid tissues at preclinical time points, and necropsies in deer 28 months after inoculation showed clinical signs, spongiform lesions, and widespread PrPSc in neural and lymphoid tissues. Western blots on samples from the brainstem, cerebellum, and lymph nodes of scrapie-infected WTD have a molecular profile similar to CWD and distinct from samples from the cerebral cortex, retina, or the original classic scrapie inoculum. There was no evidence of PrPSc in any of the WTD inoculated with classic scrapie prions from goats. CONCLUSIONS: WTD are susceptible to the agent of classic scrapie from sheep, and differentiation from CWD may be difficult.

Combinatorial treatment of brain samples from sheep with scrapie using sodium percarbonate, sodium dodecyl sulfate, and proteinase K increases survival time in inoculated susceptible sheep.

The agent of scrapie is resistant to most chemical and physical methods of inactivation. Prions bind to soils, metals, and various materials and persist in the environment confounding the control of prion diseases. Most methods of prion inactivation require severe conditions such as prolong exposure to sodium hypochlorite or autoclaving, which may not be suitable for field conditions. We evaluated the efficacy of a combinatorial approach to inactivation of US scrapie strain x124 under the mild conditions of treating scrapie-affected brain homogenate with sodium percarbonate (SPC), sodium dodecyl sulfate (SDS), or in combination followed by proteinase K (PK) digestion at room temperature. Western blot analysis of treated brain homogenate demonstrates partial reduction in PrPSc immunoreactivity. Genetically susceptible VRQ/ARQ Suffolk sheep were oronasally inoculated with 1 g of SPC (n = 1), SDS (n = 2), SDS + PK (n = 2), and SPC + SDS + PK (n = 4) treated brain homogenate. Sheep were assessed daily for clinical signs, euthanized at the development of clinical disease, and tissues were assessed for accumulation of PrPSc. Scrapie status in all sheep was determined by western blot, enzyme immunoassay, and immunohistochemistry. Mean incubation periods (IPs) for SPC (11.9 months, 0% survival), SDS (12.6 months, 0% survival), SDS + PK (14.0 months, 0% survival), and SPC + SDS + PK (12.5 months, 25% survival) were increased compared to positive control sheep (n = 2, 10.7 months, 0% survival) by 1.2, 1.9, 3.3, and 1.8 months, respectively. Treatment did not influence PrPSc accumulation and distribution at the clinical stage of disease. Differences in mean IPs and survival indicates partial but not complete reduction in scrapie infectivity.

Tralesinidase Alfa Enzyme Replacement Therapy Prevents Disease Manifestations in a Canine Model of Mucopolysaccharidosis Type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B; OMIM #252920) is a lethal, pediatric, neuropathic, autosomal recessive, and lysosomal storage disease with no approved therapy. Patients are deficient in the activity of N-acetyl-alpha-glucosaminidase (NAGLU; EC 3.2.150), necessary for normal lysosomal degradation of the glycosaminoglycan heparan sulfate (HS). Tralesinidase alfa (TA), a fusion protein comprised of recombinant human NAGLU and a modified human insulin-like growth factor 2, is in development as an enzyme replacement therapy that is administered via intracerebroventricular (ICV) infusion, thus circumventing the blood brain barrier. Previous studies have confirmed ICV infusion results in widespread distribution of TA throughout the brains of mice and nonhuman primates. We assessed the long-term tolerability, pharmacology, and clinical efficacy of TA in a canine model of MPS IIIB over a 20-month study. Long-term administration of TA was well tolerated as compared with administration of vehicle. TA was widely distributed across brain regions, which was confirmed in a follow-up 8-week pharmacokinetic/pharmacodynamic study. MPS IIIB dogs treated for up to 20 months had near-normal levels of HS and nonreducing ends of HS in cerebrospinal fluid and central nervous system (CNS) tissues. TA-treated MPS IIIB dogs performed better on cognitive tests and had improved CNS pathology and decreased cerebellar volume loss relative to vehicle-treated MPS IIIB dogs. These findings demonstrate the ability of TA to prevent or limit the biochemical, pathologic, and cognitive manifestations of canine MPS IIIB disease, thus providing support of its potential long-term tolerability and efficacy in MPS IIIB subjects. SIGNIFICANCE STATEMENT: This work illustrates the efficacy and tolerability of tralesinidase alfa as a potential therapeutic for patients with mucopolysaccharidosis type IIIB (MPS IIIB) by documenting that administration to the central nervous system of MPS IIIB dogs prevents the accumulation of disease-associated glycosaminoglycans in lysosomes, hepatomegaly, cerebellar atrophy, and cognitive decline.

Mitoapocynin Attenuates Organic Dust Exposure-Induced Neuroinflammation and Sensory-Motor Deficits in a Mouse Model.

Increased incidences of neuro-inflammatory diseases in the mid-western United States of America (USA) have been linked to exposure to agriculture contaminants. Organic dust (OD) is a major contaminant in the animal production industry and is central to the respiratory symptoms in the exposed individuals. However, the exposure effects on the brain remain largely unknown. OD exposure is known to induce a pro-inflammatory phenotype in microglial cells. Further, blocking cytoplasmic NOX-2 using mitoapocynin (MA) partially curtail the OD exposure effects. Therefore, using a mouse model, we tested a hypothesis that inhaled OD induces neuroinflammation and sensory-motor deficits. Mice were administered with either saline, fluorescent lipopolysaccharides (LPSs), or OD extract intranasally daily for 5 days a week for 5 weeks. The saline or OD extract-exposed mice received either a vehicle or MA (3 mg/kg) orally for 3 days/week for 5 weeks. We quantified inflammatory changes in the upper respiratory tract and brain, assessed sensory-motor changes using rotarod, open-field, and olfactory test, and quantified neurochemicals in the brain. Inhaled fluorescent LPS (FL-LPS) was detected in the nasal turbinates and olfactory bulbs. OD extract exposure induced atrophy of the olfactory epithelium with reduction in the number of nerve bundles in the nasopharyngeal meatus, loss of cilia in the upper respiratory epithelium with an increase in the number of goblet cells, and increase in the thickness of the nasal epithelium. Interestingly, OD exposure increased the expression of HMGB1, 3- nitrotyrosine (NT), IBA1, glial fibrillary acidic protein (GFAP), hyperphosphorylated Tau (p-Tau), and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-positive cells in the brain. Further, OD exposure decreased time to fall (rotarod), total distance traveled (open-field test), and olfactory ability (novel scent test). Oral MA partially rescued olfactory epithelial changes and gross congestion of the brain tissue. MA treatment also decreased the expression of HMGB1, 3-NT, IBA1, GFAP, and p-Tau, and significantly reversed exposure induced sensory-motor deficits. Neurochemical analysis provided an early indication of depressive behavior. Collectively, our results demonstrate that inhalation exposure to OD can cause sustained neuroinflammation and behavior deficits through lung-brain axis and that MA treatment can dampen the OD-induced inflammatory response at the level of lung and brain.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Temporospatial Development of Neuropathologic Findings in a Canine Model of Mucopolysaccharidosis IIIB.

Mucopolysaccharidosis (MPS) IIIB is a neuropathic lysosomal storage disease characterized by the deficient activity of a lysosomal enzyme obligate for the degradation of the glycosaminoglycan (GAG) heparan sulfate (HS). The pathogenesis of neurodegeneration in MPS IIIB is incompletely understood. Large animal models are attractive for pathogenesis and therapeutic studies due to their larger size, outbred genetics, longer lifespan, and naturally occurring MPS IIIB disease. However, the temporospatial development of neuropathologic changes has not been reported for canine MPS IIIB. Here we describe lesions in 8 brain regions, cervical spinal cord, and dorsal root ganglion (DRG) in a canine model of MPS IIIB that includes dogs aged from 2 to 26 months of age. Pathological changes in the brain included early microscopic vacuolation of glial cells initially observed at 2 months, and vacuolation of neurons initially observed at 10 months. Inclusions within affected cells variably stained positively with PAS and LFB stains. Quantitative immunohistochemistry demonstrated increased glial expression of GFAP and Iba1 in dogs with MPS IIIB compared to age-matched controls at all time points, suggesting neuroinflammation occurs early in disease. Loss of Purkinje cells was initially observed at 10 months and was pronounced in 18- and 26-month-old dogs with MPS IIIB. Our results support the dog as a replicative model of MPS IIIB neurologic lesions and detail the pathologic and neuroinflammatory changes in the spinal cord and DRG of MPS IIIB-affected dogs.

Diagnosis and clinical management of auricular chondritis in a dog presenting for evaluation of severe pain.

BACKGROUND: The aetiology and appropriate treatment for auricular chondritis in the dog are currently unclear. This report describes a unique presentation and successful treatment of a dog with auricular chondritis. CLINICAL SUMMARY: A 12-year-old, female spayed, Labrador retriever dog was presented for severe pain thought to be neurological in origin. The pain was located to the right pinna and two punch biopsies were acquired and evaluated, revealing lymphoplasmacytic to pyogranulomatous inflammation involving the auricular cartilage with no infectious agents. Treatment with systemic oral prednisone resulted in resolution of clinical signs within four weeks of initiation of treatment. The dog remained free of clinical signs for six months following discontinuation of treatment before being euthanized for an unrelated reason. CONCLUSIONS: Further evaluation of canine auricular chondritis is needed, yet pain may be a prominent finding; monotherapy with systemic prednisone may provide quick and complete resolution of clinical sysmptoms.

Histologic effects of MicroPulse™ transscleral cyclophotocoagulation in normal equine eyes.

OBJECTIVE: Determine the immediate post-operative effects of MicroPulse™ transscleral cyclophotocoagulation (MP-TSCPC) in healthy equine eyes. ANIMALS STUDIED: Ten adult horses. METHODS: MP-TSCPC was performed on sedated horses in 12 eyes (4 groups) using the following parameters (power, duration, duty cycle): (1) 2000 mW, 180 seconds, 31.3%; (2) 3000 mW, 180 seconds, 31.3%; (3) 3000 mW, 270 seconds, 31.3%; and (4) 3000 mW, 270 seconds, 50%. Three additional eyes were left untreated (control). Eyes were monitored clinically until euthanasia (mean = 3 hours post-procedure). Histologic sections were assessed with light microscopy and transmission electron microscopy (TEM). RESULTS: MP-TSCPC was well tolerated by sedated horses. Adverse effects were only noted in Group 4: ocular hypertension (n = 3/3), conjunctival burns (3/3), aqueous flare (2/3), and a corneal erosion (1/3). Histologic scoring of Group 4 was statistically greater than other treated groups (1-3) and control eyes (P ≤ .021). TEM showed subtle changes to the mitochondria and plasma membrane infoldings of the basilar surface of the nonpigmented epithelium in all treated eyes. CONCLUSIONS: MP-TSCPC does not cause immediate post-procedure adverse clinical effects or pronounced morphological changes to the ciliary body, except with the highest laser settings evaluated (power 3000 mW, duration 270 seconds, duty cycle 50%).

Long-term management of congestive heart failure secondary to mitral stenosis in a ring-tailed lemur (Lemur catta).

CASE DESCRIPTION: A 15-year-old sexually intact female ring-tailed lemur (Lemur catta) was evaluated for a heart murmur and progressive radiographic cardiomegaly. CLINICAL FINDINGS: The lemur was clinically normal at the time of initial evaluation. Results of transthoracic echocardiography performed when the animal was anesthetized indicated mitral valve stenosis and severe left atrial dilation. Three months later, signs of left-sided congestive heart failure (CHF; coughing, exercise intolerance, and tachypnea) were observed and confirmed by the presence of radiographic pulmonary edema. TREATMENT AND OUTCOME: Medical treatment that consisted of aspirin, benazepril, furosemide, pimobendan, spironolactone, and ultimately torsemide in lieu of furosemide successfully controlled the lemur's clinical signs for 33 months after the development of CHF. Euthanasia was then elected on the basis of perceived poor quality of life because tachypnea became refractory to progressively higher dosages of diuretic. Necropsy confirmed mitral stenosis with severe left atrial dilation and chronic pulmonary congestion. CLINICAL RELEVANCE: The present report described the long-term medical management of CHF secondary to mitral stenosis in a lemur. Mitral stenosis was suspected to be a congenital defect, similar to the cause of mitral stenosis reported for dogs and cats, rather than to be an acquired change in association with rheumatic heart disease as commonly occurs for people. The lemur's CHF was well managed for 33 months with treatment, including pimobendan, which was well tolerated.

Cautery Disbudding Iron Application Time and Brain Injury in Goat Kids: A Pilot Study.

Cautery disbudding is a painful procedure performed on goat kids to prevent horn growth that may result in brain injury. Thermal damage to the cerebral cortex of the brain and subsequent neurologic disease is a primary concern. Cautery iron application time may affect transmission of heat to the brain; however, research in this area is scarce. Therefore, the objective of this pilot study was to evaluate the effect of iron application time on brain injury of goat kids. A total of six buck and doe kids <9 days of age were obtained from a commercial dairy and transported to an Iowa State University research facility. Kids received a different randomly assigned application time (5, 10, 15, or 20s) on each horn bud. Kids were disbudded using an electric cautery iron (under isoflurane general anesthesia). After a 5-day observation period, the kids were euthanized, and magnetic resonance (MR) images were acquired to evaluate brain injury. Additionally, four of the six kids were presented for gross examination and two kids were selected for histopathologic examination. From the MR images, white matter edema was observed subjacent to four treated areas, representing application times of 5 s (one horn bud), 15 s (one horn bud), and 20 s (two horn buds). With the exception of the horn bud that received 5 s, which had white matter edema restricted to a single gyrus, the remaining three groups had a branching region of edema. No bone abnormalities were identified on any kids. Gross evidence of discoloration and hemorrhage on the cerebral hemispheres was observed on two horn buds that received 20 s, two horn buds that received 15 s, and one horn bud that received 10 s. Microscopic lesions consisting of leptomeningeal and cerebrocortical necrosis were observed in sections of brain from all groups. Lesions were most severe with 20 s. In conclusion, all application times used in this study resulted in some level of brain injury; however, using 15 s or more resulted in more severe and consistent brain injury. These results indicate that extended iron application time may increase the risk of brain injury in cautery disbudded kids.

ß-amyloid and tau pathology in the aging feline brain.

Domestic cats (Felis catus) are known to develop cognitive impairment, and several small series have demonstrated both ß-amyloid and tau aggregation, including neurofibrillary tangles, with age, making them a promising physiologic model of Alzheimer disease (AD). We therefore report the largest feline autopsy cohort to date of 32 cats ranging from 1.5 to 22.1 years of age, with systematic neuropathologic assessment according to NIA-Alzheimer's Association Criteria. Formalin-fixed paraffin-embedded tissue sections of brain were obtained retrospectively from cats autopsied at the Iowa State College of Veterinary Medicine. We found ß-amyloid staining, predominantly in Cortical Layers IV and VI in 27 of the 32 cats used in the study, with four of these animals showing tau-positive tangles and neuropil threads. In 75% of these cases (3/4), tau deposition was limited to entorhinal cortex, while one case showed diffuse positive staining throughout the hippocampal formation and neocortex. This last case showed positive staining for all phospho-tau-specific antibodies tested, similar to the pattern seen in human AD. Interestingly, we saw a higher ratio of pretangles to tangles than that in human AD, and none of the cases showed neuritic plaques on any of the stains used. Our findings indicate that aging domestic cats spontaneously develop both ß-amyloid and tau pathology similar, but not identical to that seen in human AD. This suggests that the domestic cat may serve as a potential model for mechanistic and therapeutic AD studies, but that additional research is needed to identify differences between the neuropathology of aging in humans and felines.

In Situ Temporospatial Characterization of the Glial Response to Prion Infection.

Mammalian transmissible spongiform encephalopathies (TSEs) display marked activation of astrocytes and microglia that precedes neuronal loss. Investigation of clinical parallels between TSEs and other neurodegenerative protein misfolding diseases, such as Alzheimer's disease, has revealed similar patterns of neuroinflammatory responses to the accumulation of self-propagating amyloids. The contribution of glial activation to the progression of protein misfolding diseases is incompletely understood, with evidence for mediation of both protective and deleterious effects. Glial populations are heterogeneously distributed throughout the brain and capable of dynamic transitions along a spectrum of functional activation states between pro- and antiinflammatory polarization extremes. Using a murine model of Rocky Mountain Laboratory scrapie, the neuroinflammatory response to prion infection was characterized by evaluating glial activation across 15 brain regions over time and correlating it to traditional markers of prion neuropathology, including vacuolation and PrPSc deposition. Quantitative immunohistochemistry was used to evaluate glial expression of iNOS and Arg1, markers of classical and alternative glial activation, respectively. The results indicate progressive upregulation of iNOS in microglia and a mixed astrocytic profile featuring iNOS expression in white matter tracts and detection of Arg1-positive populations throughout the brain. These data establish a temporospatial lesion profile for this prion infection model and demonstrate evidence of multiple glial activation states.

Sheep Are Susceptible to the Bovine Adapted Transmissible Mink Encephalopathy Agent by Intracranial Inoculation and Have Evidence of Infectivity in Lymphoid Tissues.

Transmissible mink encephalopathy (TME) is a food borne prion disease. Epidemiological and experimental evidence suggests similarities between the agents of TME and L-BSE. This experiment demonstrates the susceptibility of four different genotypes of sheep to the bovine adapted TME agent by intracranial inoculation. The four genotypes of sheep used in this experiment had polymorphisms corresponding to codons 136, 154, and 171 of the prion gene: V136R154Q171/VRQ, VRQ/ARQ, ARQ/ARQ, and ARQ/ARR. All intracranially inoculated sheep without comorbidities (15/15) developed clinical signs and had detectable PrPSc by immunohistochemistry, western blot, and enzyme immunoassay (EIA). The mean incubation periods in sheep with bovine adapted TME correlated with their relative genotypic susceptibility. There was peripheral distribution of PrPSc in the trigeminal ganglion and neuromuscular spindles; however, unlike classical scrapie and C-BSE in sheep, sheep inoculated with the bovine TME agent did not have immunohistochemically detectable PrPSc in the lymphoid tissue. To rule out the presence of infectivity, the lymph nodes of two sheep genotypes, VRQ/VRQ, and ARQ/ARQ, were bioassayed in transgenic mice expressing ovine prion protein. Mice intracranially inoculated with retropharyngeal lymph node from a VRQ/VRQ sheep were EIA positive (3/17) indicating that sheep inoculated with the bovine TME agent harbor infectivity in their lymph nodes despite a lack of detection with conventional immunoassays. Western blot analysis demonstrated similarities in the migration patterns between bovine TME in sheep, the bovine adapted TME inoculum, and L-BSE. Overall, these results demonstrate that sheep are susceptible to the bovine adapted TME agent, and the tissue distribution of PrPSc in sheep with bovine TME is distinct from classical scrapie.

Raccoons accumulate PrPSc after intracranial inoculation of the agents of chronic wasting disease or transmissible mink encephalopathy but not atypical scrapie.

Prion diseases are neurodegenerative diseases characterized by the accumulation of misfolded prion protein (PrPSc) in the brain and other tissues. Animal prion diseases include scrapie in sheep, chronic wasting disease (CWD) in cervids, and transmissible mink encephalopathy (TME) in ranch-raised mink. We investigated the susceptibility of raccoons to various prion disease agents and compared the clinicopathologic features of the resulting disease. Raccoon kits were inoculated intracranially with the agents of raccoon-passaged TME (TMERac), bovine-passaged TME (TMEBov), hamster-adapted drowsy (TMEDY) or hyper TME (TMEHY), CWD from white-tailed deer (CWDWtd) or elk (CWDElk), or atypical (Nor98) scrapie. Raccoons were euthanized when they developed clinical signs of prion disease or at study endpoint (<82 mo post-inoculation). Brain was examined for the presence of spongiform change, and disease-associated PrPSc was detected using an enzyme immunoassay, western blot, and immunohistochemistry. All raccoons inoculated with the agents of TMERac and TMEBov developed clinical disease at ~6.6 mo post-inoculation, with widespread PrPSc accumulation in central nervous system tissues. PrPSc was detected in the brain of 1 of 4 raccoons in each of the CWDWtd-, CWDElk-, and TMEHY-inoculated groups. None of the raccoons inoculated with TMEDY or atypical scrapie agents developed clinical disease or detectable PrPSc accumulation. Our results indicate that raccoons are highly susceptible to infection with raccoon- and bovine-passaged TME agents, whereas CWD isolates from white-tailed deer or elk and hamster-adapted TMEHY transmit poorly. Raccoons appear to be resistant to infection with hamster-adapted TMEDY and atypical scrapie agents.

Sheep With the Homozygous Lysine-171 Prion Protein Genotype Are Resistant to Classical Scrapie After Experimental Oronasal Inoculation.

Scrapie is a fatal neurodegenerative disease of sheep resulting from the accumulation of a misfolded form of the prion protein (PrPSc). Polymorphisms in the host prion protein gene ( PRNP) can affect susceptibility to the scrapie agent. Lysine (K) at codon 171 of PRNP is an inadequately characterized, naturally occurring polymorphism in sheep. We inoculated Barbado sheep with PRNP genotypes QQ171, QK171, or KK171 by either the intracranial (IC, n = 2-7 per genotype) or oronasal (ON, n = 5 per genotype) routes with a scrapie isolate to investigate the effect of lysine at codon 171 on susceptibility. When neurologic signs were observed or at the end of the experiment (70 months postinoculation [MPI]), sheep were necropsied and tissue collected for histopathologic, immunohistochemical, enzyme immunoassay and Western blot examination for PrPSc. All genotypes of sheep developed scrapie after IC inoculation. After ON inoculation, sheep with the QK171 genotype had prolonged incubation periods compared to the QQ genotype. During the experiment, 2 of 5 of the ON-inoculated QK genotype sheep developed neurologic signs and had PrPSc in the brain. The other 3 of 5 sheep were asymptomatic at 70 MPI but had detectable PrPSc in peripheral tissues. None of the ON-inoculated sheep of the KK171 genotype developed signs or had detectable PrPSc. Our experiments demonstrate that sheep with the KK171 genotype are resistant to scrapie via oronasal exposure and that sheep with the QK171 genotype have prolonged incubation relative to QQ171 sheep. The K171 prion protein allele may be useful to enhance scrapie resistance in certain breeds of sheep.

Acute and 28-day repeated dose toxicity evaluations of 2-hydroxybenzylamine acetate in mice and rats.

2-hydroxybenzylamine (2-HOBA), a compound naturally found in buckwheat, has been shown to protect cells and tissues from the damaging effects of oxidative stress. The purpose of this report was to evaluate 2-HOBA in preclinical oral rodent toxicity studies. This report includes the results from three oral toxicity studies in rodents: a preliminary 28-day feeding study in mice, a 14-day acute oral toxicity study in rats, and a 28-day repeated dose oral toxicity study in rats. The preliminary mouse feeding study showed no adverse effects of 2-HOBA at concentrations up to 0.456% by weight in feed, but decreased food intake and weight loss were observed at 1.56% 2-HOBA in the diet, likely due to poor palatability. In the acute dosing study, 2000 mg/kg BW 2-HOBA resulted in mortality in one of the six tested female rats, indicating a median lethal dose of 2500 mg/kg BW. In the 28-day repeated oral dose study, small differences were observed between 2-HOBA treated and control group rats, but none of these differences were determined to be of toxicological significance. Together, these studies support the lack of toxicity of oral administration of 2-HOBA acetate at doses up to 1000 mg/kg BW d-1 in rodents.

Concurrent renal amyloidosis and thymoma resulting in a fatal ventricular thrombus in a dog.

Thymoma-associated nephropathies have been reported in people but not in dogs. In this report, we describe a dog with thymoma and concurrent renal amyloidosis. A 7-year-old castrated male Weimaraner was presented for progressive anorexia, lethargy, and tachypnea. The dog was diagnosed with azotemia, marked proteinuria, and a thymoma that was surgically removed. Postoperatively, the dog developed a large left ventricular thrombus and was euthanized. Necropsy confirmed the presence of a left ventricular thrombus and histopathology revealed renal amyloidosis. We speculate that the renal amyloidosis occurred secondary to the thymoma, with amyloidosis in turn leading to nephrotic syndrome, hypercoagulability, and ventricular thrombosis. This case illustrates the potential for thymoma-associated nephropathies to occur in dogs and that dogs suspected to have thymoma should have a urinalysis and urine protein creatinine ratio performed as part of the pre-surgical database.