Whole genome sequencing reveals a 7 base-pair deletion in DMD exon 42 in a dog with muscular dystrophy.

Dystrophin is a key cytoskeletal protein coded by the Duchenne muscular dystrophy (DMD) gene located on the X-chromosome. Truncating mutations in the DMD gene cause loss of dystrophin and the classical DMD clinical syndrome. Spontaneous DMD gene mutations and associated phenotypes occur in several other species. The mdx mouse model and the golden retriever muscular dystrophy (GRMD) canine model have been used extensively to study DMD disease pathogenesis and show efficacy and side effects of putative treatments. Certain DMD gene mutations in high-risk, the so-called hot spot areas can be particularly helpful in modeling molecular therapies. Identification of specific mutations has been greatly enhanced by new genomic methods. Whole genome, next generation sequencing (WGS) has been recently used to define DMD patient mutations, but has not been used in dystrophic dogs. A dystrophin-deficient Cavalier King Charles Spaniel (CKCS) dog was evaluated at the functional, histopathological, biochemical, and molecular level. The affected dog's phenotype was compared to the previously reported canine dystrophinopathies. WGS was then used to detect a 7 base pair deletion in DMD exon 42 (c.6051-6057delTCTCAAT mRNA), predicting a frameshift in gene transcription and truncation of dystrophin protein translation. The deletion was confirmed with conventional PCR and Sanger sequencing. This mutation is in a secondary DMD gene hotspot area distinct from the one identified earlier at the 5' donor splice site of intron 50 in the CKCS breed.

Osteopontin is linked with AKT, FoxO1, and myostatin in skeletal muscle cells.

INTRODUCTION: Osteopontin (OPN) polymorphisms are associated with muscle size and modify disease progression in Duchenne muscular dystrophy (DMD). We hypothesized that OPN may share a molecular network with myostatin (MSTN). METHODS: Studies were conducted in the golden retriever (GRMD) and mdx mouse models of DMD. Follow-up in-vitro studies were employed in myogenic cells and the mdx mouse treated with recombinant mouse (rm) or human (Hu) OPN protein. RESULTS: OPN was increased and MSTN was decreased and levels correlated inversely in GRMD hypertrophied muscle. RM-OPN treatment led to induced AKT1 and FoxO1 phosphorylation, microRNA-486 modulation, and decreased MSTN. An AKT1 inhibitor blocked these effects, whereas an RGD-mutant OPN protein and an RGDS blocking peptide showed similar effects to the AKT inhibitor. RMOPN induced myotube hypertrophy and minimal Feret diameter in mdx muscle. DISCUSSION: OPN may interact with AKT1/MSTN/FoxO1 to modify normal and dystrophic muscle. Muscle Nerve 56: 1119-1127, 2017.

Sparing of the dystrophin-deficient cranial sartorius muscle is associated with classical and novel hypertrophy pathways in GRMD dogs.

Both Duchenne and golden retriever muscular dystrophy (GRMD) are caused by dystrophin deficiency. The Duchenne muscular dystrophy sartorius muscle and orthologous GRMD cranial sartorius (CS) are relatively spared/hypertrophied. We completed hierarchical clustering studies to define molecular mechanisms contributing to this differential involvement and their role in the GRMD phenotype. GRMD dogs with larger CS muscles had more severe deficits, suggesting that selective hypertrophy could be detrimental. Serial biopsies from the hypertrophied CS and other atrophied muscles were studied in a subset of these dogs. Myostatin showed an age-dependent decrease and an inverse correlation with the degree of GRMD CS hypertrophy. Regulators of myostatin at the protein (AKT1) and miRNA (miR-539 and miR-208b targeting myostatin mRNA) levels were altered in GRMD CS, consistent with down-regulation of myostatin signaling, CS hypertrophy, and functional rescue of this muscle. mRNA and proteomic profiling was used to identify additional candidate genes associated with CS hypertrophy. The top-ranked network included α-dystroglycan and like-acetylglucosaminyltransferase. Proteomics demonstrated increases in myotrophin and spectrin that could promote hypertrophy and cytoskeletal stability, respectively. Our results suggest that multiple pathways, including decreased myostatin and up-regulated miRNAs, α-dystroglycan/like-acetylglucosaminyltransferase, spectrin, and myotrophin, contribute to hypertrophy and functional sparing of the CS. These data also underscore the muscle-specific responses to dystrophin deficiency and the potential deleterious effects of differential muscle involvement.

MIP-3ß/CCL19 is associated with the intrathecal invasion of mononuclear cells in neuroinflammatory and non-neuroinflammatory CNS diseases in dogs.

BACKGROUND: Chemokines such as MIP-3ß/CCL19 are important factors in the mechanism of cell migration and pathogenesis of central nervous system (CNS) inflammatory reactions. The hypothesis of this study is that CCL19, also known as MIP-3ß, is involved in the pathogenesis of inflammatory and non-inflammatory CNS diseases of dogs. Experiments were performed on cerebrospinal fluid (CSF) and serum samples of dogs affected with steroid responsive meningitis-arteritis (SRMA) during the acute phase as well as during treatment. Dogs with SRMA were compared to dogs with presumed meningoencephalomyelitis of unknown origin (MUO), and both groups sub-categorized into dogs receiving no therapy and with patients receiving prednisolone therapy. Idiopathic epilepsy (IE), a group with normal CSF cell count, was used as a control. Additionally, dogs with intervertebral disc disease (IVDD) of varying severity were analyzed. Chemokine concentrations were determined by enzyme linked immunosorbent assay. Migration assays were performed on seven selected CSF samples using a disposable 96-well chemotaxis chamber. RESULTS: CCL19 was detectable in CSF samples of all dogs. Dogs with untreated SRMA and MUO displayed pronounced CCL19 elevations compared to the control group and patients receiving glucocorticosteroid treatment. CSF cell counts of untreated SRMA and MUO patients were significantly positively correlated with the CCL19 CSF concentration. IVDD patients also had elevated CCL19 concentration compared to controls, but values were considerably lower than in inflammatory CNS diseases. Selected CSF samples displayed chemotactic activity for mononuclear cells in the migration assay. CONCLUSIONS: CCL19 CSF concentrations were markedly elevated in patients affected with the neuroinflammatory diseases SRMA and MUO and showed a strong correlation with the CSF cell count. This chemokine may play an important role in the pathogenesis of SRMA and MUO. The elevation of CSF CCL19 in IVDD suggests that it may also be involved in the secondary wave of spinal cord injuries.

Evaluation of standard magnetic resonance characteristics used to differentiate neoplastic, inflammatory, and vascular brain lesions in dogs.

Magnetic resonance (MR) imaging characteristics are commonly used to help predict intracranial disease categories in dogs, however, few large studies have objectively evaluated these characteristics. The purpose of this retrospective study was to evaluate MR characteristics that have been used to differentiate neoplastic, inflammatory, and vascular intracranial diseases in a large, multi-institutional population of dogs. Medical records from three veterinary teaching hospitals were searched over a 6-year period for dogs that had diagnostic quality brain MR scans and histologically confirmed intracranial disease. Three examiners who were unaware of histologic diagnosis independently evaluated 19 MR lesion characteristics totaling 57 possible responses. A total of 75 dogs with histologically confirmed intracranial disease were included in analyses: 51 with neoplasia, 18 with inflammatory disease, and six with cerebrovascular disease. Only strong contrast enhancement was more common in neoplasia than other disease categories. A multivariable statistical model suggested that extra-axial origin, T2-FLAIR mixed intensity, and defined lesion margins were also predictive of neoplasia. Meningeal enhancement, irregular lesion shape, and multifocal location distinguished inflammatory diseases from the other disease categories. No MR characteristics distinguished vascular lesions and these appeared most similar to neoplasia. These results differed from a previous report describing seven MR characteristics that were predictive of neoplasia in dogs and cats. Findings from the current study indicated that the high performance of MR for diagnosing canine intracranial diseases might be due to evaluator recognition of combinations of MR characteristics vs. relying on any one MR characteristic alone.

Canine models of Duchenne muscular dystrophy and their use in therapeutic strategies.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder in which the loss of dystrophin causes progressive degeneration of skeletal and cardiac muscle. Potential therapies that carry substantial risk, such as gene- and cell-based approaches, must first be tested in animal models, notably the mdx mouse and several dystrophin-deficient breeds of dogs, including golden retriever muscular dystrophy (GRMD). Affected dogs have a more severe phenotype, in keeping with that of DMD, so may better predict disease pathogenesis and treatment efficacy. Various phenotypic tests have been developed to characterize disease progression in the GRMD model. These biomarkers range from measures of strength and joint contractures to magnetic resonance imaging. Some of these tests are routinely used in clinical veterinary practice, while others require specialized equipment and expertise. By comparing serial measurements from treated and untreated groups, one can document improvement or delayed progression of disease. Potential treatments for DMD may be broadly categorized as molecular, cellular, or pharmacologic. The GRMD model has increasingly been used to assess efficacy of a range of these therapies. A number of these studies have provided largely general proof-of-concept for the treatment under study. Others have demonstrated efficacy using the biomarkers discussed. Importantly, just as symptoms in DMD vary among patients, GRMD dogs display remarkable phenotypic variation. Though confounding statistical analysis in preclinical trials, this variation offers insight regarding the role that modifier genes play in disease pathogenesis. By correlating functional and mRNA profiling results, gene targets for therapy development can be identified.

Screening for Viral Nucleic Acids in the Cerebrospinal Fluid of Dogs With Central Nervous System Inflammation.

Central nervous system (CNS) inflammation is a common cause of neurological dysfunction in dogs. Most dogs with CNS inflammation are diagnosed with presumptive autoimmune disease. A smaller number are diagnosed with an infectious etiology. Additionally, at necropsy, a subset of dogs with CNS inflammation do not fit previously described patterns of autoimmune disease and an infectious cause is not readily identifiable. Because viral infection is a common cause of meningoencephalitis in people, we hypothesize that a subset of dogs presented with CNS inflammation have an occult viral infection either as a direct cause of CNS inflammation or a trigger for autoimmunity. The goal of this research was to screen cerebrospinal fluid from a large number dogs with CNS inflammation for occult viral infection. One hundred seventy-two dogs with neurological dysfunction and cerebrospinal fluid (CSF) pleocytosis were identified. Of these, 42 had meningoencephalitis of unknown origin, six had steroid-responsive meningitis-arteritis, one had eosinophilic meningoencephalitis, five had documented infection, 21 had and undetermined diagnosis, and 97 had a diagnosis not consistent with primary inflammatory disease of the CNS (e.g., neoplasia). CSF samples were subsequently screened with broadly reactive PCR for eight viral groups: adenovirus, bunyavirus, coronavirus, enterovirus, flavivirus, herpesvirus, paramyxovirus, and parechovirus. No viral nucleic acids were detected from 168 cases screened for eight viral groups, which does not support occult viral infection as a cause of CNS inflammation in dogs. La Crosse virus (LACV) nucleic acids were detected from four cases in Georgia. Subclinical infection was supported in two of these cases but LACV could not be ruled-out as a cause of infection in the other two cases, suggesting further research is warranted to determine if LACV is an occult cause of CNS inflammation in dogs.

Identification of risk loci for necrotizing meningoencephalitis in Pug dogs.

Due to their unique population structure, purebred dogs have emerged as a key model for the study of complex genetic disorders. To evaluate the utility of a newly available high-density canine whole-genome array with >170,000 single nucleotide polymorphisms (SNPs), genome-wide association was performed on a small number of case and control dogs to determine disease susceptibility loci in canine necrotizing meningoencephalitis (NME), a disorder with known non-Mendelian inheritance that shares clinical similarities with atypical variants of multiple sclerosis in humans. Genotyping of 30 NME-affected Pug dogs and 68 healthy control Pugs identified 2 loci associated with NME, including a region within dog leukocyte antigen class II on chromosome 12 that remained significant after Bonferroni correction. Our results support the utility of this high-density SNP array, confirm that dogs are a powerful model for mapping complex genetic disorders and provide important preliminary data to support in depth genetic analysis of NME in numerous affected breeds.

Clinical progression of X-linked muscular dystrophy in two German Shorthaired Pointers.

CASE DESCRIPTION: 2 full-sibling male German Shorthaired Pointer (GSHP) puppies (dogs 1 and 2) with X-linked muscular dystrophy and deletion of the dystrophin gene (gene symbol, DMD) each had poor growth, skeletal muscle atrophy, pelvic limb weakness, episodic collapse, and episodes of coughing. CLINICAL FINDINGS: Initial examination revealed stunted growth, brachygnathism, trismus, and diffuse neuromuscular signs in each puppy; clinical signs were more severe in dog 2 than in dog 1. Immunohistochemical analysis revealed a lack of dystrophin protein in both dogs. During the next 3 years, each dog developed hyperinflation of the lungs, hypertrophy of the cervical musculature, and hypertrophy of the lateral head of the triceps brachii muscle. TREATMENT AND OUTCOME: Monitoring and supportive care were provided at follow-up visits during an approximately 7-year period. No other specific treatment was provided. Neuromuscular signs in both dogs remained stable after 3 years of age, with dog 2 consistently more severely affected than dog 1. The dogs had multiple episodes of aspiration pneumonia; dogs 1 and 2 were euthanatized at 84 and 93 months of age, respectively. CLINICAL RELEVANCE: The clinical course of disease in these dogs was monitored for a longer period than has been monitored in previous reports of dystrophin-deficient dogs. The clinical progression of muscular dystrophy in the 2 GSHPs was compared with that for other breeds and species with dystrophin-deficient conditions, and the potential basis for the phenotypic variation observed between these littermates, along with potential therapeutic ramifications for dogs and humans, was evaluated.

Intraaxial spinal cord hemorrhage secondary to atlantoaxial subluxation in a dog.

A 1-year-old, 3.5-kg, spayed female, toy poodle was presented for acute-onset tetraplegia and neck pain. Neuroanatomical diagnosis was consistent with a first through fifth cervical (C(1) through C(5)) spinal cord lesion. Radiographs of the cervical vertebral column revealed atlantoaxial (AA) subluxation. Magnetic resonance imaging revealed abnormalities consistent with intraaxial spinal cord hemorrhage at the level of the AA articulation. The dog was treated with external coaptation. After 8 days, the dog regained voluntary motor function in all four limbs. Surgical stabilization was pursued. Postoperatively, the dog regained the ability to ambulate. This report details the imaging findings and management of a dog with intraaxial spinal cord hemorrhage secondary to AA subluxation.

Broadly reactive pan-paramyxovirus reverse transcription polymerase chain reaction and sequence analysis for the detection of Canine distemper virus in a case of canine meningoencephalitis of unknown etiology.

Despite the immunologic protection associated with routine vaccination protocols, Canine distemper virus (CDV) remains an important pathogen of dogs. Antemortem diagnosis of systemic CDV infection may be made by reverse transcription polymerase chain reaction (RT-PCR) and/or immunohistochemical testing for CDV antigen; central nervous system infection often requires postmortem confirmation via histopathology and immunohistochemistry. An 8-month-old intact male French Bulldog previously vaccinated for CDV presented with multifocal neurologic signs. Based on clinical and postmortem findings, the dog's disease was categorized as a meningoencephalitis of unknown etiology. Broadly reactive, pan-paramyxovirus RT-PCR using consensus-degenerate hybrid oligonucleotide primers, combined with sequence analysis, identified CDV amplicons in the dog's brain. Immunohistochemistry confirmed the presence of CDV antigens, and a specific CDV RT-PCR based on the phosphoprotein gene identified a wild-type versus vaccinal virus strain. This case illustrates the utility of broadly reactive PCR and sequence analysis for the identification of pathogens in diseases with unknown etiology.

Heritability and transmission analysis of necrotizing meningoencephalitis in the Pug.

Necrotizing meningoencephalitis (NME) in the Pug is an invariably fatal disease with an early age of onset whose cause remains unknown. Breed predilection strongly suggests genetic component(s), and viral etiology proves negative in studied cases. The current study was undertaken as the first analysis of the heritable component(s) involved in NME in the Pug. Complete medical records, individual characteristics, and pedigree information were collected for 58 affected dogs with data pertaining to 4698 dogs analyzed. A high inbreeding coefficient with differences across gender and significant differences across coat color classes and variable expression was evident. Median onset age was 19months and median survival time 23days. Screening for herpes-, adeno-, and parvoviruses was negative. The data demonstrate a strong familial inheritance of NME in the Pug. This investigation provides parameters of disease from the largest Pug NME cohort analyzed to date and offers evidence of previously unrecognized familial inheritance.

Clinicopathologic and magnetic resonance imaging characteristics associated with polioencephalomyelopathy in a Shih Tzu.

CASE DESCRIPTION: A 17-month-old 7-kg (15.4-lb) Shih Tzu was evaluated because of progressive thoracic limb weakness of 3 months' duration. CLINICAL FINDINGS: Neuroanatomic diagnosis was consistent with a lesion affecting the cervicothoracic (C6 through T2) spinal cord segments. Electrophysiologic testing revealed abnormal spontaneous activity in the thoracic limbs. Via magnetic resonance (MR) imaging, a lesion in the spinal cord that extended from the C5 through C7 vertebrae was detected, as were symmetric lesions in the cranial portion of the cervical spinal cord, caudal colliculi, and vestibular and cerebellar nuclei. Tests to detect metabolites indicative of inborn errors in metabolism revealed no abnormalities. TREATMENT AND OUTCOME: Prior to undergoing MR imaging, the dog received clindamycin (14 mg/kg [6.4 mg/lb], PO, q 12 h), trimethoprim-sulfadiazine (17 mg/kg [7.7 mg/lb], PO, q 12 h), and prednisone (1 mg/kg [0.45 mg/lb], PO, q 24 h). Because of its deteriorating condition, the dog was euthanized. During necropsy, gross lesions were identified in the cervical spinal cord, caudal colliculi, and vestibular and cerebellar nuclei (corresponding to lesions detected via MR imaging). Microscopic evaluation of the brain and spinal cord revealed polioencephalomyelopathy; there was severe spongiosis of the neuropil with reactive astrocytes (many with high numbers of swollen mitochondria) and preservation of large neurons. CLINICAL RELEVANCE: The form of polioencephalomyelopathy in the Shih Tzu of this report was similar to that described for Australian Cattle dogs; the similarity of findings in dogs with those in humans with Leigh disease is suggestive of a mitochondrial defect.

Adverse effects and outcome associated with dexamethasone administration in dogs with acute thoracolumbar intervertebral disk herniation: 161 cases (2000-2006).

OBJECTIVE: To determine complications and neurologic outcomes associated with dexamethasone administration to dogs with surgically treated thoracolumbar intervertebral disk herniation, compared with dogs not receiving dexamethasone. DESIGN: Retrospective case series. ANIMALS: 161 dogs with surgically confirmed thoracolumbar disk herniation. PROCEDURES: Medical records from 2 hospitals were used to identify dogs that had received dexamethasone < 48 hours prior to admission (dexamethasone group dogs), dogs that received glucocorticoids other than dexamethasone < 48 hours prior to admission (other-glucocorticoid group dogs), and dogs that received no glucocorticoids (nontreatment group dogs). Signalment, neurologic injury grade, laboratory data, and complications were extracted from medical records. RESULTS: Dexamethasone group dogs were 3.4 times as likely to have a complication, compared with other-glucocorticoid or nontreatment group dogs. Dexamethasone group dogs were 11.4 times as likely to have a urinary tract infection and 3.5 times as likely to have diarrhea, compared with other-glucocorticoid or nontreatment group dogs. No differences in neurologic function at discharge or recheck evaluation were detected among groups. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that treatment with dexamethasone before surgery is associated with more adverse effects, compared with treatment with glucocorticoids other than dexamethasone or no treatment with glucocorticoids, in dogs with thoracolumbar intervertebral disk herniation. In this study population, no difference in outcome was found among groups. These findings suggest that the value of dexamethasone administration before surgery in dogs with thoracolumbar disk herniation should be reconsidered.

Corneal ulceration associated with naturally occurring canine herpesvirus-1 infection in two adult dogs.

CASE DESCRIPTION: An 8-year-old Labrador Retriever with diabetes mellitus in which bilateral phacoemulsification had been performed 3 weeks earlier was evaluated for acute onset of blepharospasm, and a 7-year-old Miniature Schnauzer with chronic immune-mediated thrombocytopenia was reevaluated for keratoconjunctivitis sicca that had been diagnosed 4 weeks earlier. CLINICAL FINDINGS: Dendritic corneal ulcerations were detected in both dogs. Canine herpesvirus-1 (CHV-1) was isolated from corneal swab specimens obtained during the initial evaluation of each dog and during recheck examinations performed until the ulcerations were healed. Canine herpesvirus-1 serum neutralization titers were detected in both dogs. Results of virus isolation from oropharyngeal and genital swab specimens were negative for both dogs. The isolated viruses were identified as CHV-1 via immunofluorescence, transmission electron microscopy, PCR assay, and gene sequencing. Negative controls for PCR assay and virus isolation included conjunctival swab specimens from 50 dogs without extraocular disease and corneal swab specimens from 50 dogs with corneal ulcers, respectively. TREATMENT AND OUTCOME: Lesions resolved in both dogs after topical administration of idoxuridine or trifluridine and discontinuation of topically administered immunosuppressive medications. CLINICAL RELEVANCE: To the authors' knowledge, this is the first report of corneal ulcerations associated with naturally occurring CHV-1 infection and may represent local ocular recrudescence of latent CHV-1 infection. The viruses isolated were identified as CHV-1, and the morphology, antigenicity, and genotype were similar to those for CHV-1 isolates obtained from a puppy that died from systemic CHV-1 infection.

Evaluation of cytospin precision in low cellularity canine cerebrospinal fluid.

The cell count and differential of cerebrospinal fluid (CSF) cytologic examination classify CSF as inflammatory or not. The cytospin cell yield is related to cell count, but to our knowledge a relationship has not been characterized and cytospin precision is undocumented in any species. The objective of our study was to calculate intra-assay precision of cellular yield and differential on cytocentrifuged canine CSF, determine the factors that may affect precision, and predict the number of cytospins necessary to confirm mild neutrophilic pleocytosis. Ten concurrent replicate cytospins were created from nonhemorrhagic CSF, obtained from 60 dogs in other terminal studies, with either a manual or calibrated pipetting technique. Up to 500 cells per cytospin were counted and classified on each slide. Coefficient of variation (CV), multiple regression, and probabilities were calculated for relationships between cell yield and independent factors including technique, total nucleated cell count, cell differential, and total protein. Manual and calibrated pipetting had similar CVs (average 31%) for total cell yield, but the calibrated technique had fewer foamy macrophages. CV for neutrophil percentage among low cellularity samples with any neutrophils was 146%. Probability based on linear regression showed that 1 cytospin is sufficient to identify samples with >3% neutrophils. Occasional neutrophils, eosinophils, mitotic figures, phagocytic cells, and ependymal cells were seen in many low cellularity canine CSF samples. Canine CSF cytospin cell yield and differential evaluations are imprecise. Calibrated rather than manual pipetting is recommended.

Polymerase chain reaction screening for DNA viruses in paraffin-embedded brains from dogs with necrotizing meningoencephalitis, necrotizing leukoencephalitis, and granulomatous meningoencephalitis.

The objective of this investigation was to determine whether or not herpesvirus (herpes-), adenovirus (adeno-), or canine parvovirus DNA is present in the brains of dogs with necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE), and granulomatous meningoencephalitis (GME). Paraffin-embedded brain specimens from 12 histopathologically confirmed dogs with NME, 3 with NLE, and 7 with GME were screened for viral DNA with degenerate herpes- and adenovirus polymerase chain reaction (PCR) and a canine parvovirus-specific PCR. Positive-control specimens included genomic viral DNA and paraffin-embedded tissues from dogs with confirmed herpes-, adeno-, or canine parvovirus infections. Herpes-, adeno-, or canine parvovirus DNA was amplified by PCR from the corresponding positive-control specimens. Negative controls included 7 dogs with various brain disorders and produced no viral amplicons. The 22 dogs with NME, NLE, and GME were negative for viral DNA. Additional studies testing for other viruses or inherited genetic mutations are warranted to gain insight into the etiologies of NME, NLE, and GME. We discuss potential etiologies and provide a clinical and histopathologic overview of these common canine encephalitides.

Meningioangiomatosis in young dogs: a case series and literature review.

Meningioangiomatosis (MA) is a proliferative disorder of the central nervous system (CNS) that has been reported rarely in humans and sporadically in dogs. Meningioangiomatosis may occur in the brainstem or cervical spinal cord of young dogs and can be identified tentatively by magnetic resonance imaging. The histopathologic hallmark of MA is a leptomeningeal plaque that extends along the CNS microvasculature and invades the adjacent neural parenchyma. This case series describes the neurologic signs, clinical progression, diagnostic imaging, and neuropathology of 4 dogs with MA. The 4 dogs with MA are compared and contrasted with 4 previously reported cases in dogs as well as with their human counterpart.

Polymerase chain reaction (PCR) amplification of parvoviral DNA from the brains of dogs and cats with cerebellar hypoplasia.

Cerebellar hypoplasia in cats is caused most commonly by an in utero or perinatal infection with feline panleukopenia virus (parvovirus). Cerebellar hypoplasia has been reported infrequently in dogs, but no viral etiology has been identified to date. DNA was extracted from archival, paraffin-embedded, cerebellar tissue from 8 cats and from 2 canine littermates with cerebellar hypoplasia, 2 canine littermates with cerebellar cortical abiotrophy, 6 dogs with congenital cerebellar vermal defects, 1 dog with congenital hydranencephaly, and 15 dogs and cats with various encephalitdes. The DNA extracted from each cerebellum was subject to polymerase chain reaction (PCR) amplification by 3 primer pairs specific for parvovirus DNA. Sequence analysis of PCR products from each of the 8 cats and 2 dogs with cerebellar hypoplasia confirmed their identity with parvoviral DNA. The 6 dogs with cerebellar vermal defects, 2 dogs with cortical abiotrophy, 1 dog with congenital hydranencephaly, and all control samples were PCR negative for parvovirus. Parvoviral structural proteins were not identified by immunohistochemistry in either dog with cerebellar hypoplasia. This study shows that parvoviral DNA can be amplified from feline and canine archival brain tissue and that cerebellar hypoplasia in dogs might be associated with in utero parvovirus infection.

Use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs.

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the detection of Toxoplasma gondii and Neospora caninum DNA in canine and feline biological samples. SAMPLE POPULATION; Biological samples from 7 cats with systemic (n = 4) or CNS (3) toxoplasmosis, 6 dogs with neospora- or toxoplasma-associated encephalitis, and 11 animals with nonprotozoal disease. PROCEDURE: Primers for T gondii, N caninum, and the canine ferritin gene (dogs) or feline histone 3.3 gene (cats) were combined in a single PCR assay. The DNA was extracted from paraffin-embedded brain tissue, CSF, or skeletal muscle. The PCR products with positive results were cloned, and sequence identity was confirmed. RESULTS: Of 7 cats and 4 dogs with immunohistochemical or serologic evidence of toxoplasmosis, PCR results were positive for all cats and 3 dogs for T gondii, and positive for T gondii and N caninum for 1 dog. Another dog had negative PCR results for both parasites. Of 2 dogs with immunohistochemical or serologic evidence of neosporosis, PCR results were positive for 1 for N caninum and positive for the other for T gondii. All negative-control samples yielded negative results for T gondii and N caninum on the PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: Standard tests for toxoplasmosis or neosporosis associated with the CNS rely on serologic, histologic, or immunohistochemical analysis and can be difficult to interpret. The multiplex PCR assay with built-in control reactions could be a complementary clinical tool for the antemortem diagnosis of toxoplasmosis or neosporosis associated with the CNS.