Detection and molecular characterization of astro and bocaviruses in dogs in Minnesota.

Canine astrovirus (CAstV) and canine bocavirus (CBoV) are involved in cases of mild, and sometimes severe, gastroenteritis in dogs. Fecal samples from two dead dogs with gastroenteritis were received at the University of Minnesota Veterinary Diagnostic Laboratory to determine the cause of death. Small round viruses of 20-35 nm diameter were observed by negative contrast electron microscopy. The samples were subjected to Illumina MiSeq sequencing. Both samples were strongly positive for CAstV; all viral reads were related to CAstV. In addition, sample number 1 had a few reads of CBoV. Two complete sequences of CAstV were identified (6625 and 6627 nt in length) with 95% nt identity. RT-PCR and PCR were used to confirm CAstV and CBoV infections in successive samples of canine gastroenteritis. Sanger sequencing was done on nucleic acids from positive samples. Of a total of ten samples, CAstV and CBoV infections were confirmed in six and eight animals, respectively. Four animals had mixed infection with both viruses. All sequences of ORF1b gene of CAstVs showed closest clusters in phylogenetic tree with 96-100% nucleotide and amino acids identity. On the other hand, identity between VP2 gene of different CBoV strains in this study ranged from 93%- 100%. All strains were located close to each other except the divergent MT078234 strain, which was arranged in a separate branch and was closer to reference strain JN648103/USA/2010. This study highlights the importance of electron microscopy and next generation sequencing for early detection and characterization of viruses associated with dog gastroenteritis.

Spongy Myelinopathy in Newborn Beef Calves Associated with Consumption of Corn Infected with Stenocarpella maydis.

Stillbirth and perinatal mortality with neurological signs and lesions were diagnosed in two calves following ingestion by their dams of corn infected with Stenocarpella maydis during the third trimester of gestation. Grossly, the brain and spinal cord were unremarkable. Microscopically, diffuse severe status spongiosis of the white matter was detected in the cerebral hemispheres, brainstem, spinal cord and cerebellum. To the best of our knowledge this is the first pathological description of congenital disease in calves associated with the consumption of S. maydis-infected corn; the findings resemble those reported for the naturally occurring and experimentally induced disease in lambs.

Systemic Amyloidosis in an African Tiger Snake (Telescopus semiannulatus).

An adult male African tiger snake (Telescopus semiannulatus) was diagnosed with disseminated mycobacteriosis and a hepatic biliary cystadenocarcinoma. Histologically, the spleen was largely replaced by extracellular deposits of eosinophilic, fibrillar to hyaline material. Similar material was also present in the testicular interstitium and occasional blood vessel walls. This material was congophilic with strong green birefringence under polarized light and emitted fluorescence when bound to the luminescent-conjugated oligothiophene, h-FTAA, an amyloid binding probe. Ultrastructurally, deposits were composed of aggregates of haphazardly arranged, non-branching fibrils up to 8 nm in diameter and of indeterminate length. These findings all supported a diagnosis of amyloidosis, most likely amyloid A (AA) type based on concurrent inflammatory disease in this snake. However, immunohistochemistry for serum amyloid A was negative. There are only rare previous reports of amyloidosis in reptiles and many have been incompletely characterized. This case presents a thorough investigation into an occurrence of systemic amyloidosis in a snake, including a novel use of luminescent-conjugated oligothiophene binding in a reptile to confirm the diagnosis.

Ocular Lesions in Red-Tailed Hawks ( Buteo jamaicensis) With Naturally Acquired West Nile Disease.

Ocular lesions are common in red-tailed hawks with West Nile (WN) disease. These lesions consist of pectenitis, choroidal or retinal inflammation, or retinal necrosis, but detailed investigation of the ocular lesions is lacking. Postmortem examination of the eyes of 16 red-tailed hawks with naturally acquired WN disease and 3 red-tailed hawks without WN disease was performed using histopathology, immunohistochemistry for West Nile virus (WNV) antigen, glial fibrillary acid protein, cleaved caspase-3, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. Retinal lesions were classified as type I or type II lesions. Type I lesions were characterized by lymphoplasmacytic infiltrates in the subjacent choroid with degeneration limited to the outer retina (type Ia lesion) or with degeneration and necrosis of the outer retina or outer and inner retina (type Ib lesion) while retinal collapse, atrophy, and scarring were hallmarks of type II lesions. Type II retinal lesions were associated with a more pronounced choroiditis. Although not statistically significant, WNV antigen tended to be present in larger quantity in type Ib lesions. Type I lesions are considered acute while type II lesions are chronic. The development of retinal lesions was associated with the presence of an inflammatory infiltrate in the choroid. A breakdown of the blood-retina barrier is suspected to be the main route of infection of the retina. Within the retina, virus appeared to spread via both neuronal and Müller cell processes.

Intracerebral Malignant Plasmacytoma in a Mule Deer (Odocoileus hemionus).

A wild, mature, gravid female mule deer (Odocoileus hemionus) was presented with marked neurological signs, including abnormal behaviour, circling and incoordination. The animal was humanely destroyed and submitted for diagnostic investigation. Grossly, a well-demarcated, 3 × 3 × 3 cm intracranial mass replaced the left olfactory bulb and frontal lobe. Histologically, there was a highly cellular, infiltrative and unencapsulated neoplastic mass of round cells with eccentrically located nuclei. Neoplastic cells were immunohistochemically labelled for lambda immunoglobulin light chain. Clusters of CD20- and CD79a-positive cells were scattered throughout the tumour and CD3- and Iba1-positive cells diffusely infiltrated the neoplasm. Ultrastructurally, the cytoplasm of the neoplastic cells had prominent rough endoplasmic reticulum with a variable degree of dilation. Histopathological, immunohistochemical and electron microscopical results were diagnostic for a solitary extramedullary plasmacytoma. Intracranial neoplasms are rarely diagnosed in wildlife species, but they should be included in the differential diagnosis for potential causes of central nervous system disease.

Evidence of the Primary Afferent Tracts Undergoing Neurodegeneration in Horses With Equine Degenerative Myeloencephalopathy Based on Calretinin Immunohistochemical Localization.

Equine degenerative myeloencephalopathy (EDM) is characterized by a symmetric general proprioceptive ataxia in young horses, and is likely underdiagnosed for 2 reasons: first, clinical signs overlap those of cervical vertebral compressive myelopathy; second, histologic lesions--including axonal spheroids in specific tracts of the somatosensory and motor systems--may be subtle. The purpose of this study was (1) to utilize immunohistochemical (IHC) markers to trace axons in the spinocuneocerebellar, dorsal column-medial lemniscal, and dorsospinocerebellar tracts in healthy horses and (2) to determine the IHC staining characteristics of the neurons and degenerated axons along the somatosensory tracts in EDM-affected horses. Examination of brain, spinal cord, and nerves was performed on 2 age-matched control horses, 3 EDM-affected horses, and 2 age-matched disease-control horses via IHC for calbindin, vesicular glutamate transporter 2, parvalbumin, calretinin, glutamic acid decarboxylase, and glial fibrillary acidic protein. Primary afferent axons of the spinocuneocerebellar, dorsal column-medial lemniscal, and dorsospinocerebellar tracts were successfully traced with calretinin. Calretinin-positive cell bodies were identified in a subset of neurons in the dorsal root ganglia, suggesting that calretinin IHC could be used to trace axonal projections from these cell bodies. Calretinin-immunoreactive spheroids were present in EDM-affected horses within the nuclei cuneatus medialis, cuneatus lateralis, and thoracicus. Neurons within those nuclei were calretinin negative. Cell bodies of degenerated axons in EDM-affected horses are likely located in the dorsal root ganglia. These findings support the role of sensory axonal degeneration in the pathogenesis of EDM and provide a method to highlight tracts with axonal spheroids to aid in the diagnosis of this neurodegenerative disease.

The Equine Movement Disorder "Shivers" Is Associated With Selective Cerebellar Purkinje Cell Axonal Degeneration.

"Shivers" is a progressive equine movement disorder of unknown etiology. Clinically, horses with shivers show difficulty walking backward, assume hyperflexed limb postures, and have hind limb tremors during backward movement that resembles shivering. At least initially, forward movements are normal. Given that neither the neurophysiologic nor the pathologic mechanisms of the disease is known, nor has a neuroanatomic locus been identified, we undertook a detailed neuroanatomic and neuropathologic analysis of the complete sensorimotor system in horses with shivers and clinically normal control horses. No abnormalities were identified in the examined hind limb and forelimb skeletal muscles nor the associated peripheral nerves. Eosinophilic segmented axonal spheroids were a common lesion. Calretinin-positive axonal spheroids were present in many regions of the central nervous system, particularly the nucleus cuneatus lateralis; however, their numbers did not differ significantly from those of control horses. When compared to controls, calretinin-negative, calbindin-positive, and glutamic acid decarboxylase-positive spheroids were increased 80-fold in Purkinje cell axons within the deep cerebellar nuclei of horses with shivers. Unusual lamellar or membranous structures resembling marked myelin decompaction were present between myelin sheaths of presumed Purkinje cell axons in the deep cerebellar nuclei of shivers but not control horses. The immunohistochemical and ultrastructural characteristics of the lesions combined with their functional neuroanatomic distribution indicate, for the first time, that shivers is characterized by end-terminal neuroaxonal degeneration in the deep cerebellar nuclei, which results in context-specific hypermetria and myoclonus.

Characterization of neuronal ceroid-lipofuscinosis in 3 cats.

Three young domestic shorthair cats were presented for necropsy with similar histories of slowly progressive visual dysfunction and neurologic deficits. Macroscopic examination of each cat revealed cerebral and cerebellar atrophy, dilated lateral ventricles, and slight brown discoloration of the gray matter. Histologically, there was bilateral loss of neurons within the limbic, motor, somatosensory, visual, and, to a lesser extent, vestibular systems with extensive astrogliosis in the affected regions of all 3 cases. Many remaining neurons and glial cells throughout the entire central nervous system were distended by pale yellow to eosinophilic, autofluorescent cytoplasmic inclusions with ultrastructural appearances typical of neuronal ceroid-lipofuscinoses (NCLs). Differences in clinical presentation and neurological lesions suggest that the 3 cats may have had different variants of NCL. Molecular genetic characterization in the 1 cat from which DNA was available did not reveal any plausible disease-causing mutations of the CLN1 (PPT1), CLN3, CLN5, CLN8, and CLN10 (CTSD) genes. Further investigations will be required to identify the mutations responsible for NCLs in cats.

Macroscopic, histologic, and ultrastructural lesions associated with avian keratin disorder in black-capped chickadees (Poecile atricapillus).

An epizootic of beak abnormalities (avian keratin disorder) was recently detected among wild birds in Alaska. Here we describe the gross, histologic, and ultrastructural features of the disease in 30 affected adult black-capped chickadees (Poecile atricapillus). Grossly, there was elongation of the rhamphotheca, with varying degrees of lateral deviation, crossing, and gapping between the upper and lower beak. Not uncommonly, the claws were overgrown, and there was alopecia, scaling, and crusting of the skin. The most prominent histopathologic features in the beak included epidermal hyperplasia, hyperkeratosis, and core-like intrusions of necrotic debris. In affected birds, particularly those with moderate to severe beak overgrowth, there was remodeling of premaxillary and mandibular bones and various dermal lesions. Lesions analogous to those found in beaks were present in affected claws, indicating that this disorder may target both of these similar tissues. Mild to moderate hyperkeratosis occurred in other keratinized tissues, including skin, feather follicles, and, occasionally, sinus epithelium, but typically only in the presence of microbes. We did not find consistent evidence of a bacterial, fungal, or viral etiology for the beak lesions. The changes observed in affected birds did not correspond with any known avian diseases, suggesting a potentially novel hyperkeratotic disorder in wild birds.

Spinal meningeal oligodendrogliomatosis in two boxer dogs.

Two Boxer dogs developed progressive ataxia in association with a neoplastic infiltration of the spinal leptomeninges. In the first dog, the leptomeningeal neoplasm encompassed the entire cord and the ventral aspect of the brainstem and extended bilaterally into the piriform lobes. In the second, the neoplasm surrounded the C1-C3 segments of the spinal cord and the brainstem without involvement of the brain or spinal cord parenchyma. In both dogs, the neoplastic cells had variably distinct cell borders, clear to eosinophilic cytoplasm, and a round to ovoid hyperchromatic nucleus. Neoplastic cells were immunopositive for Olig2 and doublecortin in both dogs and for vimentin in one dog but were immunonegative for glial fibrillary acidic protein, S-100, CD34, E-cadherin, cytokeratin, CD3, and CD20. The morphological and immunohistochemical features of the neoplastic cells were consistent with an oligodendrocyte lineage. This hitherto poorly recognized neoplasm in dogs is analogous to human leptomeningeal oligodendrogliomatosis.

Otitis interna induced by Cryptococcus neoformans var. grubii in a cat.

Cryptococcus neoformans var. grubii was identified at necropsy in a case of bilateral otitis interna in a 7-year-old, female, domestic shorthair cat with a 9-day history of acute onset of vestibular disease. Gross examination, including that of the middle and inner ears, was unremarkable. Histologically, the auricular vestibuli, cochleae, and semicircular canals were bilaterally affected by granulomatous inflammation with extracellular and intrahistiocytic yeasts. The yeasts and associated inflammation obstructed and disrupted perilymphatic and endolymphatic spaces of the inner ears. Disruption of the saccular and utricular maculae, cristae ampularis, and organ of Corti, as well as changes in the endolymphatic and perilymphatic fluids, probably impaired the vestibular and auditory functions of this cat. The route of infection was most likely hematogenous.

Purkinje cell heterotopy with cerebellar hypoplasia in two free-living American kestrels (Falco sparverius).

Two wild fledgling kestrels exhibited lack of motor coordination, postural reaction deficits, and abnormal propioception. At necropsy, the cerebellum and brainstem were markedly underdeveloped. Microscopically, there was Purkinje cells heterotopy, abnormal circuitry, and hypoplasia with defective foliation. Heterotopic neurons were identified as immature Purkinje cells by their size, location, immunoreactivity for calbindin D-28 K, and ultrastructural features. The authors suggest that this cerebellar abnormality was likely due to a disruption of molecular mechanisms that dictate Purkinje cell migration, placement, and maturation in early embryonic development. The etiology of this condition remains undetermined. Congenital central nervous system disorders have rarely been reported in birds.

Sarcocystis calchasi-associated neurologic disease in a domestic pigeon in North America.

Tissue cysts of a protozoan parasite were present in the skeletal muscle of a domestic pigeon (Columba livia f. domestica) with neurologic disease in Minnesota, USA. The animal had a severe granulomatous meningoencephalitis. The cysts were slender, up to 1 mm long and up to 0.03 mm in diameter. The cysts had a smooth wall without projections. Size and wall morphology were compatible with Sarcocystis calchasi. Polymerase chain reaction using S. calchasi-specific primers resulted in a specific amplicon from the skeletal muscle but not from the brain. Sequencing of the highly variable genomic regions ITS1 and D2 revealed 100% nucleic acid identity with the German strain of S. calchasi. Sarcocystis calchasi is the cause of an emerging lethal disease in pigeons in Germany. This is the first description of the parasite outside of Germany.

Comparative skeletal muscle histopathologic and ultrastructural features in two forms of polysaccharide storage myopathy in horses.

Polysaccharide storage myopathy (PSSM) has been found in more than 35 different horse breeds through identification of abnormal storage of polysaccharide in muscle biopsies. A dominant mutation in the glycogen synthase 1 gene (GYS1) accounts for a substantial proportion of PSSM cases in at least 17 breeds, including Quarter Horses, but some horses diagnosed with PSSM by muscle histopathologic analysis are negative for the mutation. We hypothesized that a second distinct form of glycogen storage disease exists in GYS1-negative horses with PSSM. The objectives of this study were to compare the histopathologic features, ultrastructure of polysaccharide, signalment, history, and presenting complaints of GYS1-negative Quarter Horses and related breeds with PSSM to those of GYS1-positive horses with PSSM. The total histopathologic score in frozen sections of skeletal muscle stained with hematoxylin and eosin, periodic acid Schiff (PAS) and amylase-PAS stains from 53 GYS1-negative horses did not differ from that of 52 GYS1-positive horses. Abnormal polysaccharide was fine granular or homogenous in appearance (49/53; 92%), often amylase-sensitive (28/53; 53%), more commonly located under the sarcolemma, and consisting of beta glycogen particles in GYS1-negative horses. However, in GYS1-positive horses, abnormal polysaccharide was usually coarse granular (50/52; 96%), amylase-resistant (51/52; 98%), more commonly cytoplasmic, and consisting of beta glycogen particles or, in some myofibers, filamentous material surrounded by beta glycogen particles. Retrospective analysis found that GYS1-negative horses (n = 43) were younger at presentation (4.9 +/- 0.6 years vs. 6.7 +/- 0.3 years for GYS1-positive horses) and were more likely to be intact males than GYS1-positive horses (n = 160). We concluded that 2 forms of PSSM exist and often have distinctive abnormal polysaccharide. However, because evaluation of the histologic appearance of polysaccharide can be subjective and affected by age, the gold standard for diagnosis of PSSM at present would appear to be testing for the GYS1 mutation followed by evaluating muscle biopsy for characteristic abnormal polysaccharide in those horses that are negative for the mutation.

Spontaneous and experimental glycoprotein storage disease of goats induced by Ipomoea carnea subsp fistulosa (Convolvulaceae).

Spontaneous and experimental poisoning with the swainsonine-containing and calystegine-containing plant Ipomoea carnea subsp fistulosa is described. Three of 8 goats presenting with emaciation, weakness, symmetrical ataxia, posterior paresis, proprioceptive deficits, abnormal posture, abnormal postural reaction, and muscle hypertonia were necropsied. I fistulosa was suspected to be the cause of the neurologic disease in all cases. An experiment was conducted to confirm the diagnosis using 12 goats and diets containing 3 different concentrations of the plant. All goats fed I fistulosa developed neurological signs that were similar to those observed in the spontaneous intoxication. Muscle atrophy and pallor were the only macroscopic changes observed in spontaneous and in experimental intoxication. Histological lesions of spontaneous and experimental animals were similar. The most prominent lesion was cytoplasmic vacuolation in neurons of the central and the autonomous nervous system, pancreatic acinar cells, hepatocytes, Kupffer cells, follicular epithelial cells of the thyroid gland, and macrophages of the lymphatic tissues. Neuronal necrosis, axonal spheroids formation, and astrogliosis were additionally observed in the brain. Ultrastructurally, the cytoplasmic vacuoles consisted of distended lysosomes surrounded by a single-layered membrane. Nonreduced end-rests or sequence of alpha-Man, alpha-Glc, beta(1-4)-GlcNAc, and NeuNAc on lysosomal membrane were revealed by lectin histochemistry. Samples of plants used in the experimental trial contained swainsonine and calystegine and their intermediary derivate. We conclude that I fistulosa induces a glycoprotein storage disease primarily based on the inhibition of the lysosomal alpha-mannosidase by the alkaloid swainsonine.

Induction of apoptosis by canine natural killer cells.

Besides a secretory pathway of canine natural killer (NK) cells, which results in necrosis of the target cell, a second pathway was demonstrated, which results in apoptosis of the target cell. Comparing the Chromium Release Assay (CRA) and the Rose Bengal Assay (RBA) for quantification of in vitro canine NK cell activity, a constant 10% higher NK cell activity was found in the RBA compared with the CRA. To find out the mechanism responsible for the different results of both tests, morphological studies of in vitro canine NK cell activity against epithelial and mesenchymal allogenic target cell lines were performed. Most target cells were undergoing necrosis as a result of NK cell killing, which was evidenced by transmission electron microscopy. However, besides necrotic target cells, shrunken target cells with dense cytoplasm, fragmented nuclei and disruption into membrane-bound bodies were detected, which are known as signs of apoptosis. Additionally, using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method, 13-23% of target cells presented a positive staining, indicative of apoptosis. These findings give evidence for the ability of canine NK cells to kill their target cells via two different pathways, which results either in apoptosis or necrosis.

Bilateral focal cerebral angiomatosis associated with nervous signs in a cat.

A case of cerebral angiomatosis in a cat was associated with neurologic signs characterized by clusters of severe generalized seizures. Bilaterally in the gray matter, most prominent in the cingulate gyrus, there was focal accumulation of garlandlike arrangements of blood vessels. Vessels exhibited activated, hypertrophic endothelial cells and thickening and progressive dystrophic mineralization of the basement membrane, with complete luminal obstruction of some affected vessels. Thickening of the basement membrane was due to accumulation of endothelium-derived proteins such as laminin and von Willebrand factor. Furthermore, moderate diffuse astrogliosis was observed. Findings indicate an idiopathic angiomatosis, with clinical signs possibly due to ischemia resulting from narrowing or complete obliteration of vessel lumina. Changes represent a unique endothelial cell-derived lesion within the brain not previously described in humans or domestic animals.