Laboratory diagnosis of canine GM2-gangliosidosis using blood and cerebrospinal fluid.

In the present study, laboratory techniques were used to diagnose canine GM2-gangliosidosis using blood and cerebrospinal fluid (CSF) that can be collected noninvasively from living individuals. Lysosomal acid beta-hexosaminidase (Hex) was measured spectrofluorometrically using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl 7-(6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside) as substrates. Main isoenzymes A and B of Hex in leukocytes were also analyzed using cellulose acetate membrane electrophoresis. GM2-ganglioside in CSF was detected and determined quantitatively by using thin-layer chromatography/enzyme-immunostaining method with anti-GM2-ganglioside antibody. In normal dogs, Hex activities could be determined in leukocytes, serum, and CSF and the total activities were markedly reduced in all the enzyme sources in a dog with Sandhoff disease. Electrophoresis of a leukocyte lysate from a normal dog showed that the Hex A and Hex B were not separated distinctively with formation of a broad band, whereas there were no bands in electrophoresis of a lysate from a dog with Sandhoff disease, showing a deficiency in the total enzyme activity. GM2-ganglioside could be detected and determined quantitatively in as little as 100 microl of canine CSE GM2-ganglioside in CSF in a dog with Sandhoff disease increased to 46 times the normal level. In conclusion, the methods in the present study are useful for diagnosis of canine GM2-gangliosidosis. These techniques enable definitive and early diagnosis of canine GM2-gangliosidosis even if tissues and organs cannot be obtained.     

Detection of Nesopora caninum-Specific DNA from Cerebrospinal Fluid by Polymerase Chain Reaction in a Dog with Confirmed Neosporosis

A one-month male Greyhound dog presented with a swinging gait of the hindlimbs, and later developed muscular atrophy of the femoral region and hyperextension of hindlimbs. The dog had positive serum IFAT titers to Neospora caninum, but a negative titer in the cerebrospinal fluid (CSF). N. caninum-specific DNA was amplified from the CSF using a semi-nested polymerase chain reaction assay. Clusters of protozoa in biopsied muscle fibers were subsequently confirmed as N. caninum tachyzoites by immunohistochemical examination. Early recognition and treatment are necessary for effective recovery of clinical canine neosporosis, but antemortem diagnosis is difficult. We suggest that the detection of parasite deoxyribonucleic acid in the CSF is a useful antemortem diagnostic method in facilitating treatment of this disease.     

Cytologic detection of Toxoplasma gondii in the cerebrospinal fluid of a dog and in vitro isolation of a unique mouse-virulent recombinant strain

Parasites resembling Neospora caninum or Toxoplasma gondii were detected by cytologic examination of cerebrospinal fluid (CSF) from a dog with neurologic disease. The dog became severely ill and was euthanized. Canine tissue homogenates were used for direct parasite isolation in cell culture, bioassay in 2 mouse lineages, and PCR. T. gondii was isolated in monkey kidney cells, and species identity was confirmed by PCR. Inoculated parasites were highly virulent for mice, which developed clinical signs and were euthanized immediately. PCR-RFLP for T. gondii using the cultured isolate (TgDgBA22) was conducted with 12 genetic markers, and a unique recombinant strain was identified. Detection of T. gondii by CSF cytology, although described in humans, had not been reported previously in dogs, to our knowledge, and was crucial for the diagnosis of toxoplasmosis in the examined dog.     

Detection of an autoantibody from Pug dogs with necrotizing encephalitis (Pug dog encephalitis).

An autoantibody against canine brain tissue was detected in the cerebrospinal fluid (CSF) and serum of two Pug dogs (Nos. 1 and 2) by indirect immunofluorescence assay (IFA). Dog No. 1, a 2-year-old male, exhibited severe depression, ataxia, and generalized seizures and died 2 months after the onset of symptoms. Dog No. 2, a 9-month-old male, exhibited severe generalized seizures and died 17 months after the onset of symptoms. Histopathologic examination revealed a moderate to severe multifocal accumulation of lymphocytes, plasma cells, and a few neutrophils in both the gray and white matter of the cerebrum in dog No. 1. In dog No. 2, the cellular infiltrates were mild, but there was a severe, diffuse, and multifocal necrosis in the cerebral cortex with prominent astrocytosis. With the aid of IFA using fluorescein isothiocyanate-labeled antidog IgG goat serum and a confocal imaging system, specific reactions for glial cells were detected in the CSF of these Pug dogs but not in six canine control CSF samples. Double-labeling IFA using CSF from these Pug dogs and a rabbit antiserum against glial fibrillary acidic protein (GFAP) revealed that the autoantibody recognized GFAP-positive astrocytes and their cytoplasmic projections. By immunoblot analysis, the autoantibody from CSF of these Pug dogs recognized two common positive bands at 58 and 54 kd, which corresponded to the molecular mass of human GFAP. The role of this autoantibody for astrocytes is not yet clear. However, if the presence of the autoantibody is a specific feature of Pug dog encephalitis, it will be a useful clinical diagnostic marker and a key to the pathogenesis of this unique canine neurologic disease.     

Suppurative spinal meningomyelitis in a dog with intra-neutrophilic cerebrospinal fluid cells Ehrlichia canis morulae

A 2-year-old castrated male Creole Shepherd mixed dog was presented for non-ambulatory paraparesis of the pelvic limbs. The magnetic resonance imaging and cerebrospinal fluid (CSF) analysis were consistent with meningomyelitis. Positive serology for Ehrlichia canis/Ehrlichia ewingii suggested exposure to a pathogen; qPCR on the serum and the CSF confirmed active infection. Ehrlichial morulae were observed within CSF and peripheral blood polymorphonuclear neutrophils; a species-specific PCR confirmed E. canis infection. This is an interesting report of E. canis infection in a dog with morulae observed in neutrophils both in the peripheral blood and CSF.     

Disseminated protothecosis diagnosed by evaluation of CSF in a dog

A 5-year-old female spayed Shetland Sheepdog Mix dog was evaluated for a history of recent seizure activity, progressive hind limb ataxia, polyuria, and polydipsia and no history of gastrointestinal signs. Physical examination findings included conscious proprioceptive deficits, ataxia, and anterior uveitis along with a hypermature cataract in the right eye. Results of a CBC, serum biochemical profile, urinalysis, and computed tomography scan of the brain were unremarkable. Cerebrospinal fluid (CSF) analysis revealed marked eosinophilic pleocytosis and rare organisms consistent with Prototheca spp within neutrophils and macrophages. On postmortem histologic examination, mononuclear inflammation and numerous intralesional algal organisms, similar to those seen on the cytologic preparation of CSF, were found in the brain, eyes, kidneys, and heart. Abnormalities were not detected on gross and histologic examination of the gastrointestinal tract. Cultures of CSF and subdural/olfactory bulb, but not intestinal tract, yielded growth of Prototheca spp, and PCR analysis and DNA sequencing confirmed the organism as Prototheca zopfii genotype 2. We have reported a rare case of disseminated protothecosis that was diagnosed by evaluation of CSF in a dog presented with neurologic signs and no overt enteric disease. Protothecosis should be considered as a rare cause of seizures, even in the absence of obvious enteric signs, and should be included in the differential diagnosis of eosinophilic pleocytosis.     

Tick-borne encephalitis virus as a possible cause of optic neuritis in a dog

A 3-year-old spayed female Siberian Husky was presented due to acute vision loss. Examination revealed bilateral optic neuritis and lymphocytic meningoencephalitis. The serum (1:800) and cerebrospinal fluid (CSF; 1:200) immunoglobulin (Ig)G titers for tick-borne encephalitis virus (TBEV) were elevated as were the serum IgG titer for Anaplasma phagocytophilum (1:640) and serum IgM titer for Toxoplasma gondii (1:20). Intracytoplasmic inclusion bodies such as ehrlichial or anaplasmal morulae were not observed in the CSF or blood smear. The dog was treated with methylprednisone and doxycycline. The left eye regained vision; the right eye remained blind. Anti-inflammatory therapy was stopped on day 18 after diagnosis. Four days later the dog showed evidence of hyperesthesia in the cervical region. Analysis of CSF showed no abnormalities and CSF IgG titers for TBEV and A. phagocytophilum were negative. Funduscopic evidence of active papillitis was absent on day 22 in the left eye and on day 86 in the right eye. On day 243, the dog was presented again with lethargy, ataxia, disorientation and temporary head tilt. The IgG titer for TBEV was again elevated in the CSF (1:800) and in serum (1:400). After interpretation of all findings, we assume that meningoencephalitis and optic neuritis in this patient was caused by TBEV and associated immune-mediated inflammation. In endemic areas, TBEV should be considered as cause of optic neuritis in dogs.     

Progressive tetraparesis and laryngeal paralysis in a young Rottweiler with neuronal vacuolation and axonal degeneration: an Australian case

A 5-month-old female Rottweiler dog was diagnosed to have a neurodegenerative disease that has been recently report ed in Rottweilers from North America and Europe. The dog was presented with progressive signs of ataxia, tetraparesis and inspiratory stridor. The clinical investigation included analysis of CSF, radiography, myelography and electrophysiolog-ical testing. No evidence of vertebral malformation or inflammatory CNS disease was identified. Bilateral laryngeal paraly sis was identified in the lightly anaesthetised dog. Electromyography showed abnormal spontaneous activity from the intrin sic musculature of the larynx. At necropsy there were no gross abnormalities of the nervous system but there was atrophy of the dorsal cricoarytenoid muscles of the larynx. There were widespread histological abnormalities throughout the ner vous system including neuronal vacuolation, spongiform changes in the neuropil and axonal degeneration which was most prominent in the spinal cord. These clinical and pathological findings are consistent with the diagnosis of a new neurode-generative disease reported from North America and Europe. This diagnosis is of particular significance in Australia where transmissible spongiform encephalopathies have not been identified.     

Cerebrospinal fluid flow on time‐spatial labeling inversion pulse images before and after treatment of congenital hydrocephalus in a dog

A 3‐month‐old male cross‐breed dog presented with signs of progressive diffuse brain disease. Noncommunicating congenital hydrocephalus concurrent with cervical syringomyelia was diagnosed on magnetic resonance images. On time‐spatial labeling inversion pulse (Time‐SLIP) images CSF flow through the mesencephalic aqueduct was poorly defined and there was flow into the syrinx across the craniocervical junction. After percutaneous ventricular drainage and ventriculoperitoneal shunting, CSF flow through the aqueduct was clearly detected and flow into the syrinx disappeared. In addition, CSF flow in the subarachnoid space at the pons and ventral aspect of the cervical subarachnoid space was restored. Signs of neurological dysfunction improved after ventriculoperitoneal shunting and the cerebral parenchyma was increased in thickness on 2‐year follow‐up computed tomography images. Patterns of CSF flow on Time‐SLIP images before and after CSF drainage or ventriculoperitoneal shunting aid in clarifying disease pathogenesis and confirm effects of CSF drainage.     

Cerebrospinal fluid from a 6-year-old dog with severe neck pain

A 6-year-old, intact female, Labrador Retriever/Terrier cross was presented to the University Veterinary Hospital, University College Dublin with a 3-week history of therapy-resistant cervical pain and intermittent fever. Physical examination findings included marked cervical pain resulting in neck extension and vocalization. Examination of the CSF revealed mild pleocytosis (total nucleated cells = 0.009 x 10(9)/L, reference interval <0.005 x 10(9)/L). Cytocentrifuged preparations of the CSF were of low cellularity, containing predominantly macrophages and occasional small lymphocytes. Several small- to medium-sized fragments of a slightly granular, amorphous, eosinophilic substance were observed. The majority of mononuclear cells were located within this material, in small groups of 3-13 cells. The amorphous foamy material stained positive with Luxol fast blue, suggestive of myelin-like material. The dog was euthanized and postmortem examination revealed intervertebral disk protrusion between C2 and C3. Hematoxylin- and Luxol fast blue-stained histopathologic sections of brain and spinal cord revealed only mild hemorrhage. The extracellular material in the CSF of this dog may have been caused by myelin degeneration or leakage of phospholipids from damaged cells. Because no histologic evidence of demyelination was observed with the disk extrusion, the myelin-like material in this case was thought to be the product of phospholipid breakdown from damaged cellular membranes. Three cases of dogs with spinal cord disease and myelin-like material in the CSF have been reported previously. The clinical significance of this finding is still unknown.     

Syndrome of inappropriate antidiuretic hormone secretion concurrent with liver disease in a dog

A 5-year-old female Chihuahua was presented for acute collapse. Laboratory examinations showed markedly elevated levels of hepatobiliary enzymes. Empiric antibiotic therapy for bacterial infection of the liver was ineffective. The clinical signs worsened with the development of hyponatremia with hypoosmolality and elevated urine sodium levels. The dog was suspected of having acute cholangiohepatitis associated with an immune-mediated disease. Subsequently, it was diagnosed with syndrome of inappropriate antidiuretic hormone secretion (SIADH) on the basis of the specific disease criteria. Further tests showed normal function of the adrenal and thyroid glands, and MRI and cerebrospinal fluid (CSF) analysis did not show any intracranial diseases. Immunosuppressive therapy and water restriction resolved the clinical signs and improved the SIADH in this dog. This case indicates that SIADH can occur concurrently with suspected immune-mediated liver disease in dogs.     

Neuronal-Visceral GM1 Gangliosidosis in Portuguese Water Dogs

Three female siblings in a litter of seven Portuguese Water dogs (PWDs) showed clinical signs of ataxia and/or lameness at 5 months of age. Signs of cerebellar dysfunction (intention tremors, ataxia, widebased stance, dysmetria, and/or nystagmus) and mild limb weakness developed rapidly. Results of hemograms (three dogs), blood chemistry profiles (two dogs), urinalyses (two dogs), electroencephalograms (two dogs), and radiographs of the limbs or pelvis (three dogs), vertebrae (two dogs), and skull (one dog) were unremarkable except for an absolute lymphocytosis in one dog. Routine cerebrospinal fluid (CSF) analyses were normal in all three dogs. However, the CSF creatine kinase concentration was elevated in the one dog in which it was measured. Mucopolysacchariduria was present in all three dogs. Due to the rapid progression of clinical signs and a poor prognosis, all three dogs were euthanatized between 6 and 7 months of age. Histopathologic and electron microscopic studies showed neuronal cytoplasmic inclusions, vacuolated hepatocytes, and vacuolated renal tubular epithelial cells, compatible with the diagnosis of a storage disease. Beta-galactosidase activities in leukocytes, serum, and brain homogenates were reduced when compared with that in normal dogs and the stored product was identified as GM1 ganglioside, confirming GM1 gangliosidosis.     

Increased concentration of GM1-ganglioside in cerebrospinal fluid in dogs with GM1- and GM2-gangliosidoses and its clinical application for diagnosis.

GM1- and GM2-gangliosidoses are lethal lysosomal diseases that are caused by a defect of acid hydrolases, resulting in the intralysosomal accumulation of the specific physiological substrates, GM1- and GM2-gangliosides, respectively. In the present study a method for the diagnosis of canine GM1-gangliosidosis was established using canine cerebrospinal fluid (CSF). The concentration of GM1-ganglioside in CSF was determined by thin-layer chromatography-enzyme immunostaining using biotin-conjugated cholera toxin B, which specifically binds with GM1-ganglioside. The concentration of CSF GM1-ganglioside was increased in Shiba dogs with GM1-gangliosidosis, and the increased level was approximately proportional to the age of the dogs. The concentration was high in the affected dog even at 5 months of age, when Shiba dogs with GM1-gangliosidosis first manifest neurologic signs. In addition, the concentration of CSF GM1-ganglioside in a dog with the GM2-gangliosidosis 0 variant (Sandhoff disease) was also 7 times the normal level. From these results it was concluded that this laboratory technique enables a definitive and early diagnosis of canine GM1-gangliosidosis even if tissues and organs cannot be obtained. However, because GM1-ganglioside can also be elevated in cases of GM2-gangliosidosis, it is necessary to assay for specific enzyme deficiencies to definitively separate GM1- from GM2-gangliosidosis.     

Pyogranulomatous meningoencephalitis due to Actinomyces sp. in a dog

Actinomyces sp. are commensal, filamentous, gram-positive, acid-fast-negative bacteria that can cause pyogranulomatous inflammation in animals and humans. Central nervous system (CNS) disease is a rare presentation of actinomycosis and is usually due to extension from infected wounds or seeding from distant sites. A dog with progressive, poorly localized neurologic signs had primary CNS actinomycosis without history or evidence of previous trauma or other organ involvement. Histologically, there was a severe pyogranulomatous meningoencephalitis with intralesional filamentous bacteria that were also visible on cytology of the cerebral spinal fluid (CSF) postmortem. Actinomyces sp. was cultured postmortem from the CSF, confirming the diagnosis. This case demonstrates that Actinomyces sp. can be a causative agent of primary CNS disease in dogs.     

INTRACRANIAL INTRA-ARACHNOID CYST WITH INTRACYSTIC HEMORRHAGE IN TWO DOGS

Clinical signs, magnetic resonance imaging (MRI) features, treatment, and outcome of two adult dogs with neurologic dysfunction resulting from hemorrhage into a quadrigeminal intracranial intra-arachnoid cyst are described. In dog 1, the cyst was hyperintense to cerebrospinal fluid (CSF) on T1-weighted MRI and hypointense to CSF on T2-weighted images. In dog 2, the cyst was isointense to CSF on T1- and T2-weighted images. Both dogs were treated with craniotomy and cyst fenestration. A large blood clot was removed from the lumen of the cyst in each dog. Dog 1 is clinically normal 3.5 years post-surgery and has a persistent cyst. Dog 2 had a good initial response to therapy but was euthanized 2.5 years post-operatively due to generalized seizures. The late onset of clinical signs in these dogs most likely resulted from hemorrhage into the cyst. Surgical fenestration and hematoma removal appear to provide a satisfactory treatment for adult dogs with an intracranial intra-arachnoid cyst and intracystic hemorrhage. Persistence of the cyst may occur in some dogs.     

Magnetic resonance imaging in two dogs with central nervous system disease

Accurate localization of the lesions in two dogs with progressive neurological disease was demonstrated with magnetic resonance imaging (MRI). The first dog had unilateral cerebellar signs with associated paradoxical vestibular symptoms. The CSF tap and clinical localization suggested a right-sided cerebellar tumour and this was confirmed with MRI scanning. The second dog had predominantly asymmetrical fore-brain signs with circling, personality changes, seizures and contralateral proprioceptive deficits. CSF analysis suggested an inflammatory or neoplastic condition. MRI showed a diffuse oedematous lesion of the left cerebral hemisphere which corresponded exactly with the lesions seen at necropsy. The advantages of MRI over CT scans are discussed.     

Eosinophilic cerebrospinal fluid pleocytosis associated with neural Angiostrongylus vasorum infection in a dog

A 1‐year‐old, female intact Pug dog was presented to the Small Animal Teaching Hospital of the University of Liverpool with a 4‐week history of progressive multifocal intracranial signs. Magnetic resonance imaging (MRI) detected multiple hemorrhagic lesions in the brain. The Baermann and zinc sulfate flotation tests with centrifugation, performed on fecal samples, were positive for lungworm larvae and an antigenic test confirmed Angiostrongylus vasorum infection. Anthelmintic treatment was started with a consequent marked clinical improvement. Seventy days later, the dog was clinically normal, and no larvae were detected on the Baermann test. Repeat MRI of the brain revealed marked improvement of the hemorrhagic lesions. Cerebrospinal fluid analysis (CSF) showed marked eosinophilic pleocytosis, and anthelmintic treatment was restarted. A follow‐up CSF analysis 4 months after the first presentation revealed resolution of the eosinophilic pleocytosis. This is the first case report of marked eosinophilic pleocytosis associated with neural A vasorum infection in a dog. The CSF eosinophilic pleocytosis persisted for several weeks after treatment, even in the absence of concurrent clinical signs and with a negative A vasorum Baermann test.     

Ventricular pneumocephalus and septic meningoencephalitis secondary to dorsal rhinotomy and nasal polypectomy in a dog

CASE DESCRIPTION: A 4-year-old sexually intact female French Bulldog was evaluated because of lethargy, anorexia, and chronic rhinitis-sinusitis. The dog had nasal discharge of 18 months' duration; dorsal rhinotomies were performed 3 months and 2 weeks prior to referral. CLINICAL FINDINGS: On initial evaluation, intraventricular pneumocephalus and sinusitis were diagnosed; CSF analysis revealed high total protein concentration and mononuclear pleocytosis. The dog's condition improved with treatment. Two weeks after discharge, it was treated by a local veterinarian because of upper airway obstruction; 3 days later, the dog was referred because of seizures. Computed tomography revealed a large fluid-filled, left lateral ventricle and a soft tissue mass protruding through a cribriform plate defect. The mass was histologically consistent with brain tissue. Findings of clinicopathologic analyses were unremarkable. Results of cytologic examination of a CSF sample were indicative of septic, suppurative inflammation, and bacteriologic culture of CSF yielded Escherichia coli. TREATMENT AND OUTCOME: Amputation of the herniated olfactory bulb and antimicrobial treatment resolved the septic meningoencephalitis, but neurologic deficits recurred 6 weeks later. Definitive correction of the cribriform plate defect with bone and fascial grafts was attempted. Postoperative rotation of the bone graft resulted in cerebral laceration and hemorrhage, and the dog was euthanized. CLINICAL RELEVANCE: Findings suggest that following dorsal rhinotomy and nasal polypectomy surgery, the dog developed herniation of the left olfactory bulb, intra-ventricular pneumocephalus, and septic meningo-encephalitis because of a cribriform plate defect. Care must be taken to prevent rotation of bone grafts used in cribriform defect repair.     

IMAGING DIAGNOSIS—NECROTIZING MENINGOMYELITIS AND POLYARTHRITIS

A vaccinated 2-year-old female neutered Weimaraner had bilateral pelvic limb ataxia that progressed over 12 h. The dog became nonambulatory, with signs of pain on palpation of the lumbar spine. The dog also developed multiple joint effusions. On magnetic resonance (MR) imaging, there was a diffuse, asymmetric T2-hyperintensity in the thoracolumbar spinal cord which was characterized by contrast enhancement. Lumbar cerebrospinal fluid (CSF) analysis had an elevated white blood cell count and protein. On the basis of MR images and CSF analysis, a presumptive diagnosis of diffuse myelitis was made. The dog became paraplegic and was euthanized. Postmortem examination confirmed the presence of myelitis with vasculitis and nonerosive polyarthritis.     

Cerebrospinal fluid from a dog with hind limb ataxia

A 9-year-old spayed female German Shepherd dog with a history of orthopedic disease was presented to the North Carolina State University Veterinary Teaching Hospital for evaluation of recent, progressive, bilateral, hindlimb ataxia. Analysis of cisternal and lumbar cerebrospinal fluid (CSF) samples revealed normal total nucleated cell counts and a mild increase in protein concentration in the lumbar sample. In cytocentrifuged specimens of both CSF samples, aggregates of refractile, angular to irregular, pale blue to colorless, crystalline material were observed in the background. Some of the material appeared birefringent under polarized light. Differentials for the material included contrast agent, epidural anesthetics or other pharmacologic agents, or artifact introduced through sample processing, collection, or handling. Based on investigation of clinical and laboratory processes it was determined that tubes used to collect CSF in the hospital recently had been changed from additive-free glass tubes to silica-coated shatter-resistant plastic tubes (BD Vacutainer Plus serum tubes, silicone-coated, Becton Dickinson). A cytocentrifuged preparation of saline placed in a silica-coated tube contained crystalline material identical to that observed in the CSF samples; saline placed in an additive-free glass tube contained no material. In this case, we document the microscopic appearance of highly concentrated silica particles in cytocentrifuged preparations of CSF and underscore the importance of recognizing and identifying this artifact in cytologic preparations.