Polioencephalopathy in Eurasier dogs.

BACKGROUND: Polioencephalopathies secondary to inborn errors of metabolism have been described in dogs, but few genetically characterized. OBJECTIVES: Clinically and genetically characterize polioencephalopathy in a family of Eurasier dogs. ANIMALS: Three Eurasier dogs (littermates) presented with early onset movement disorders (9 weeks in 2, 4-6 months in 1). Progressive gait abnormalities were detected in 2 of the dogs, persistent divergent strabismus in 1, whereas consciousness and behavior remained intact in all dogs. One dog was euthanized at 25 months. METHODS: Video footage was assessed in all dogs, and Dogs 1 and 2 had examinations and investigations performed. Whole genome sequencing of Dog 1 and further genetic analyses in the family were performed. A cohort of 115 Eurasier controls was genotyped for specific variants. RESULTS: Episodes were characterized by generalized ataxia, as well as a hypermetric thoracic limb gait, dystonia, and irregular flexion and extension movements of the thoracic limbs. Magnetic resonance imaging of the brain in Dogs 1 and 2 identified symmetrical, bilateral T2 and fluid attenuated inversion recovery hyperintense, T1 hypo to isointense, nonenhancing lesions of the caudate nucleus, lateral and medial geniculate nuclei, thalamus, hippocampus, rostral colliculus and mild generalized brain atrophy. Genetic analyses identified a homozygous mitochondrial trans-2-enoyl-CoA reductase (MECR) missense variant in all 3 dogs, and a homozygous autophagy-related gene 4D (ATG4D) missense variant in Dogs 1 and 2. CONCLUSIONS AND CLINICAL IMPORTANCE: We describe a presumed hereditary and progressive polioencephalopathy in a family of Eurasier dogs. Further research is needed to establish the role of the MECR gene in dogs and the pathogenic effects of the detected variants.

Clinical presentation, treatment, and outcome of 24 dogs with bacterial meningitis or meningoencephalitis without empyema (2010-2020).

BACKGROUND: Bacterial meningitis (BM) and meningoencephalitis (BMEM) are associated with high case fatality rates and neurologic sequelae in people, but limited data exists on outcome in dogs. HYPOTHESIS/OBJECTIVES: To report the clinicopathologic features, treatment and outcome of BM/BMEM in dogs, with a focus on clinical presentation, relapse and long-term neurological deficits. ANIMALS: Twenty-four client-owned dogs diagnosed with BM/BMEM without empyema. METHODS: Retrospective case series of dogs diagnosed with BM/BMEM from 5 veterinary referral hospitals between January 2010 and August 2020. RESULTS: Twenty-four dogs were included. Median duration of clinical signs was 2 days (range ≤24 hours to 30 days) and signs recorded included pyrexia (3) and cervical hyperesthesia (10). Neurological deficits were present in 18 dogs including altered mentation (12), ataxia (8), nonambulatory status (8), head tilt (8), and cranial nerve deficits (13). Intracellular bacteria were visualized on cerebrospinal fluid (CSF) analysis in 15/24 dogs, with positive CSF bacteriological culture in 8/21. Otitis media/interna (OMI) was diagnosed in 15/24 dogs, of which 6/15 dogs underwent total ear canal ablation and lateral bulla osteotomy. Twenty dogs survived to hospital discharge. Median duration of antibiotic administrations was 8 weeks (range, 2-16 weeks). Glucocorticoids were administered to 15 dogs. Median follow-up time was 92 days (range, 10-2233 days). Residual neurological deficits were reported in 9 dogs, with a single case of suspected relapse. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical signs were variable in dogs with BM/BMEM, the nidus of bacterial infection was often OMI and the majority of dogs made a full recovery with treatment.

The skeletal muscle phenotype of the DE50-MD dog model of Duchenne muscular dystrophy.

Background: Animal models of Duchenne muscular dystrophy (DMD) are essential to study disease progression and assess efficacy of therapeutic intervention, however dystrophic mice fail to display a clinically relevant phenotype, limiting translational utility. Dystrophin-deficient dogs exhibit disease similar to humans, making them increasingly important for late-stage preclinical evaluation of candidate therapeutics. The DE50-MD canine model of DMD carries a mutation within a human 'hotspot' region of the dystrophin gene, amenable to exon-skipping and gene editing strategies. As part of a large natural history study of disease progression, we have characterised the DE50-MD skeletal muscle phenotype to identify parameters that could serve as efficacy biomarkers in future preclinical trials. Methods: Vastus lateralis muscles were biopsied from a large cohort of DE50-MD dogs and healthy male littermates at 3-monthly intervals (3-18 months) for longitudinal analysis, with multiple muscles collected post-mortem to evaluate body-wide changes. Pathology was characterised quantitatively using histology and measurement of gene expression to determine statistical power and sample sizes appropriate for future work. Results: DE50-MD skeletal muscle exhibits widespread degeneration/regeneration, fibrosis, atrophy and inflammation. Degenerative/inflammatory changes peak during the first year of life, while fibrotic remodelling appears more gradual. Pathology is similar in most skeletal muscles, but in the diaphragm, fibrosis is more prominent, associated with fibre splitting and pathological hypertrophy. Picrosirius red and acid phosphatase staining represent useful quantitative histological biomarkers for fibrosis and inflammation respectively, while qPCR can be used to measure regeneration ( MYH3, MYH8), fibrosis ( COL1A1), inflammation ( SPP1), and stability of DE50-MD dp427 transcripts. Conclusion: The DE50-MD dog is a valuable model of DMD, with pathological features similar to young, ambulant human patients. Sample size and power calculations show that our panel of muscle biomarkers are of strong pre-clinical value, able to detect therapeutic improvements of even 25%, using trials with only six animals per group.

Multiplex in situ hybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics.

Dystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection of individual transcript molecules at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We instead multiplex within a single transcript, using probes targeted to the 5' and 3' regions of muscle dystrophin mRNA. Our approach shows this method can reveal transcriptional dynamics in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs in quantitative fashion (with the latter absent in dystrophic muscle, yet restored following therapeutic intervention). We show that even in healthy muscle, immature dystrophin mRNA predominates (60-80% of total), with the surprising implication that the half-life of a mature transcript is markedly shorter than the time invested in transcription: at the transcript level, supply may exceed demand. Our findings provide unique spatiotemporal insight into the behaviour of this long transcript (with implications for therapeutic approaches), and further suggest this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.

Single-transcript multiplex in situ hybridisation reveals unique patterns of dystrophin isoform expression in the developing mammalian embryo.

Background: The dystrophin gene has multiple isoforms: full-length dystrophin (dp427) is principally known for its expression in skeletal and cardiac muscle, but is also expressed in the brain, and several internal promoters give rise to shorter, N-terminally truncated isoforms with wider tissue expression patterns (dp260 in the retina, dp140 in the brain and dp71 in many tissues). These isoforms are believed to play unique cellular roles both during embryogenesis and in adulthood, but their shared sequence identity at both mRNA and protein levels makes study of distinct isoforms challenging by conventional methods. Methods: RNAscope is a novel in-situ hybridisation technique that offers single-transcript resolution and the ability to multiplex, with different target sequences assigned to distinct fluorophores. Using probes designed to different regions of the dystrophin transcript (targeting 5', central and 3' sequences of the long dp427 mRNA), we can simultaneously detect and distinguish multiple dystrophin mRNA isoforms at sub-cellular histological levels. We have used these probes in healthy and dystrophic canine embryos to gain unique insights into isoform expression and distribution in the developing mammal. Results: Dp427 is found in developing muscle as expected, apparently enriched at nascent myotendinous junctions. Endothelial and epithelial surfaces express dp71 only. Within the brain and spinal cord, all three isoforms are expressed in spatially distinct regions: dp71 predominates within proliferating germinal layer cells, dp140 within maturing, migrating cells and dp427 appears within more established cell populations. Dystrophin is also found within developing bones and teeth, something previously unreported, and our data suggests orchestrated involvement of multiple isoforms in formation of these tissues. Conclusions: Overall, shorter isoforms appear associated with proliferation and migration, and longer isoforms with terminal lineage commitment: we discuss the distinct structural contributions and transcriptional demands suggested by these findings.