Phenotypic and genotypic characterization of a myelopathy in pugs : A journey from ’wobbly pugs’ to pug dog myelopathy

Spinal cord disease, myelopathy, in dogs have several aetiologies, all characterised by their clinical presentation. Pugs with a thoracolumbar myelopathy (PDM) differ from most dogs by developing a form of myelopathy that shows little clinical variability. Most pugs with PDM show slowly progressive paraparesis and ataxia, absence of obvious spinal pain and a high prevalence of incontinence.  The overall aims of the thesis were to investigate the prevalence of PDM, to clinically and pathologically characterize this unique disorder in pugs and to explore underlying aetiologies. The importance of congenital vertebral malformations (CVMs) was examined using advanced diagnostic imaging. A potential contribution of an autoimmune aetiology was studied by analysing biomarkers in blood and cerebrospinal fluid. The necrotizing meningoencephalitis (NME) susceptibility dog leucocyte (DLA) class II risk was assessed and a genetic contribution to PDM was investigated by molecular genetics.  In summary, gait abnormalities were common in Swedish pugs and the character of the abnormal gait suggesting an underlying spinal cord disorder. The lack of association between presence, or type, of CVMs in the thoracolumbar vertebral column and neurological deficits, paraparesis and ataxia, suggested that other or additional factors were important for the development of PDM. Neuropathological examination showed that the clinical disease, PDM, was caused by the formation of profound meningeal fibrosis that interfered with the vascular supply to the spinal cord as well as caused obstruction of cerebrospinal fluid (CSF) flow with subsequent parenchymal spinal cord destruction. In addition a considerable number of PDM pugs presented with inflammation in the central nervous system. A contribution of the immunological system on the development of PDM was supported by the finding of anti-GFAP autoantibodies in the CSF of pugs with PDM. Our results also indicated that the DLA class II risk haplotype for necrotizing meningoencephalitis (NME), even in a heterozygous state, may cause a more severe PDM phenotype. The potential relevance of multiple candidate genes to the pathogenesis of PDM, including those implicated with bone homeostasis, the differentiation of cartilage, inflammation and fibrotic scar tissue formation, should be confirmed in future studies

Vertebral and spinal malformations in small brachycephalic dog breeds: Current knowledge and remaining questions

Small brachycephalic dog breeds, such as the French bulldog, English bulldog and pug have become increasingly popular. These breeds are predisposed to a variety of vertebral and spinal malformations, including hemivertebra, caudal articular process dysplasia, transitional vertebra, cranial thoracic vertebral canal stenosis, spinal arachnoid diverticulum and meningeal fibrosis. Recent studies have provided new insights into the prevalence, anatomical characteristics, pathophysiology and treatment of these conditions. Thoracic hemivertebra, caudal articular process dysplasia, transitional vertebra, and cranial thoracic vertebral canal stenosis occur commonly in neurologically normal dogs. Although the clinical relevance of these vertebral anomalies has therefore been questioned, severe kyphosis and hemivertebra in pugs have been associated with an increased likelihood of neurological signs. Meningeal fibrosis is characterised by the formation of dense intradural fibrotic adhesions, constricting the spinal cord. This condition has been heavily associated with the pug breed. It is in pugs further common to observe multiple concurrent spinal disorder in association with chronic progressive pelvic limb gait abnormalities. This clinical presentation has been referred to as 'pug dog thoracolumbar myelopathy' and potential genetic risk factors have recently been identified. Despite our increased knowledge, many questions remain currently unanswered. This review discusses our current understanding and controversies surrounding vertebral and spinal malformations in small brachycephalic dog breeds

Prevalence of radiographic detectable intervertebral disc calcifications in Dachshunds surgically treated for disc extrusion

<p>Abstract</p> <p>Background</p> <p>An association between the occurrence of calcified discs, visible on radiographic examination (CDVR), and disc extrusions has been suggested in published literature over the past 10-20 years, mainly from Nordic countries. It has also been postulated that dogs without CDVR would not develop disc extrusions. Furthermore, inheritance of CDVR has been calculated and it has been postulated that, by selecting dogs for breeding with few, or no CDVR, the prevalence of disc extrusions in the Dachshund population may be reduced.</p> <p>Methods</p> <p>The prevalence of radiographic detectable intervertebral disc calcifications was calculated from one hundred surgeries for disc extrusion, performed in 95 Dachshunds, in order to determine if the disc causing clinically significant IVDD, had radiographic signs of calcification at the time of confirmed disc extrusion. Inclusion criteria, for each dog, included a complete physical, orthopedic and neurologic examination, radiographs of the entire vertebral column, a myelogram or magnetic resonance imaging examination indicating extradural spinal cord compression, and finally a surgical procedure confirming the diagnosis of a disc extrusion. In addition to descriptive statistics, age correlation with number of calcifications visible at radiographic examination and with CDVR at the surgery site was examined.</p> <p>Results</p> <p>We found that disc extrusions occur as frequently in discs that are found to have radiographic evidence of calcification as those discs that do not have signs of radiographic calcification, and that IVDD (intervertebral disc disease) requiring surgery does occur in the absence of any calcified discs on radiographic examination. We found that calcified discs were more frequent in our Dachshund population compared to previous studies suggesting that disc calcification might be a serious risk factor for developing disc extrusion. Further studies are needed to show, conclusively, if selection of breeding dogs based on CDVR in the Dachshund will reduce the incidence of IVDD. The presence of the calcifications of intervertebral disc should be evaluated with caution, as only part of the calcifications will be detected and the real extent of the disc degeneration may be underestimated.</p

Assessment of glial fibrillary acidic protein and anti-glial fibrillary acidic protein autoantibody concentrations and necrotising meningoencephalitis risk genotype in dogs with pug dog myelopathy

BackgroundPugs commonly present with thoracolumbar myelopathy, also known as pug dog myelopathy (PDM), which is clinically characterised by progressive signs involving the pelvic limbs, no apparent signs of pain and, often, incontinence. In addition to meningeal fibrosis and focal spinal cord destruction, histopathology has confirmed lymphohistiocytic infiltrates in the central nervous system (CNS) in a considerable number of pugs with PDM. Lymphohistiocytic CNS inflammation also characterises necrotising meningoencephalitis (NME) in pugs. This study aimed to investigate the potential contribution of an immunological aetiology to the development of PDM.MethodsThe concentrations of glial fibrillary acidic protein (GFAP) in serum and CSF and of anti-GFAP autoantibodies in CSF were measured with an ELISA. In addition, a commercial test was used for genetic characterisation of the dog leukocyte antigen class II haplotype, which is associated with NME susceptibility.ResultsThis study included 87 dogs: 52 PDM pugs, 14 control pugs, four NME pugs and 17 dogs of breeds other than pugs that were investigated for neurological disease (neuro controls). Anti-GFAP autoantibodies were present in 15 of 19 (79%) of the PDM pugs tested versus six of 16 (38%) of the neuro controls tested (p = 0.018). All 18 PDM pugs evaluated had detectable CSF GFAP. Serum GFAP was detected in two of three (67%) of the NME pugs and in two of 11 (18%) of the control pugs but not in any of the 40 tested PDM pugs. Male pugs heterozygous for the NME risk haplotype had an earlier onset of clinical signs (70 months) compared to male pugs without the risk haplotype (78 months) (p = 0.036).LimitationsThe study was limited by the lack of healthy dogs of breeds other than pugs and the small numbers of control pugs and pugs with NME.ConclusionsThe high proportion of PDM pugs with anti-GFAP autoantibodies and high CSF GFAP concentrations provide support for a potential immunological contribution to the development of PDM

Evaluation of antibodies in cerebrospinal fluid for the diagnosis of tick-borne encephalitis in dogs

Tick-borne encephalitis (TBE) is caused by the neurotropic tick-borne encephalitis virus (TBEV). In dogs, this virus may affect the central nervous system (CNS), causing meningoencephalitis, meningomyelitis, radiculitis or any combination of these. Diagnosis of TBE relies on a combination of clinical signs of CNS disease and laboratory findings, including CSF pleocytosis and serum TBEV antibody titers. Exposure to TBEV does not necessarily cause clinical disease, and seroprevalence has been reported as high as 40% in endemic areas. This causes concerns of over-diagnosing TBE in dogs with CNS disease. By examining TBEV antibodies in dogs with and without neurological disease in a TBEV endemic area, this study aimed to evaluate the diagnostic value of TBEV antibodies in the cerebrospinal fluid (CSF) in dogs. Eighty-nine dogs were included in the study, 56 with neurological disease and 33 neurologically normal control dogs. A positive TBEV CSF and serum IgG antibody titer (> 126 U/mL) was found in 3/89 dogs (3.4%). A positive serum TBEV antibody titer was found in 11 of the 89 dogs (12.4%). None of the control dogs showed a positive CSF antibody titer, whilst two showed positive serum concentrations. A positive CSF IgG antibody titer supports a clinical diagnosis of TBE in patients with acute onset of CNS disease and may help reduce the risk of over-diagnosis

Multiple Genetic Loci Associated with Pug Dog Thoracolumbar Myelopathy

Pug dogs with thoracolumbar myelopathy (PDM) present with a specific clinical phenotype that includes progressive pelvic limb ataxia and paresis, commonly accompanied by incontinence. Vertebral column malformations and lesions, excessive scar tissue of the meninges, and central nervous system inflammation have been described. PDM has a late onset and affects more male than female dogs. The breed-specific presentation of the disorder suggests that genetic risk factors are involved in the disease development. To perform a genome-wide search for PDM-associated loci, we applied a Bayesian model adapted for mapping complex traits (BayesR) and a cross-population extended haplotype homozygosity test (XP-EHH) in 51 affected and 38 control pugs. Nineteen associated loci (harboring 67 genes in total, including 34 potential candidate genes) and three candidate regions under selection (with four genes within or next to the signal) were identified. The multiple candidate genes identified have implicated functions in bone homeostasis, fibrotic scar tissue, inflammatory responses, or the formation, regulation, and differentiation of cartilage, suggesting the potential relevance of these processes to the pathogenesis of PDM

Mutations in the CYP27B1 gene cause vitamin D dependent rickets in pugs

Rickets is a disorder of bone development and can be the result of either dietary or genetic causes. Here, related pugs from 2 litters were included. Three pugs had clinical signs including, lameness, bone deformities, and dyspnea. One other pug was found dead. Radiographs of 2 affected pugs, 5 and 6 months old, showed generalized widening, and irregular margination of the physes of both the appendicular and the axial skeleton with generalized decrease in bone opacity and bulbous swelling of the costochondral junctions. Two pugs had low serum calcium and 1,25 (OH)(2)D-3 concentrations. Test results further indicated secondary hyperparathyroidism with adequate concentrations of 25-hydroxyvitamin D. Necropsy revealed tongue-like projections of cartilage extending into the metaphysis consistent with rickets, loss of metaphyseal mineralization and lung pathology. Vitamin D-dependent rickets was diagnosed. A truncating mutation in the 1 alpha-hydroxylase gene (CYP27B1) was identified by genome sequence analysis of the pugs with VDDR type 1A. Vitamin D-dependent rickets type 1A can occur in young pugs, and if left untreated is a life-threatening condition. Early medical intervention can reverse clinical signs and should be instituted as soon as possible

Basal Autophagy is altered in Lagotto Romagnolo Dogs with an ATG4D mutation

A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.Peer reviewe

Impaired NDRG1 functions in Schwann cells cause demyelinating neuropathy in a dog model of Charcot-Marie-Tooth type 4D

Mutations in the N-myc downstream-regulated gene 1 (NDRG1) cause degenerative polyneuropathy in ways that are poorly understood. We have investigated Alaskan Malamute dogs with neuropathy caused by a missense mutation in NDRG1. In affected animals, nerve levels of NDRG1 protein were reduced by more than 70% (p < 0.03). Nerve fibers were thinly myelinated, loss of large myelinated fibers was pronounced and teased fiber preparations showed both demyelination and remyelination. Inclusions of filamentous material containing actin were present in adaxonal Schwann cell cytoplasm and Schmidt-Lanterman clefts. This condition strongly resembles the human Charcot-MarieTooth type 4D. However, the focally folded myelin with adaxonal infoldings segregating the axon found in this study are ultrastructural changes not described in the human disease. Furthermore, lipidomic analysis revealed a profound loss of peripheral nerve lipids. Our data suggest that the low levels of mutant NDRG1 is insufficient to support Schwann cells in maintaining myelin homeostasis. (C) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license

Mystery of fatal 'staggering disease' unravelled: novel rustrela virus causes severe meningoencephalomyelitis in domestic cats

‘Staggering disease’ is a neurological disease entity considered a threat to European domestic cats (Felis catus) for almost five decades. However, its aetiology has remained obscure. Rustrela virus (RusV), a relative of rubella virus, has recently been shown to be associated with encephalitis in a broad range of mammalian hosts. Here, we report the detection of RusV RNA and antigen by metagenomic sequencing, RT-qPCR, in-situ hybridization and immunohistochemistry in brain tissues of 27 out of 29 cats with non-suppurative meningoencephalomyelitis and clinical signs compatible with’staggering disease’ from Sweden, Austria, and Germany, but not in non-affected control cats. Screening of possible reservoir hosts in Sweden revealed RusV infection in wood mice (Apodemus sylvaticus). Our work indicates that RusV is the long-sought cause of feline ‘staggering disease’. Given its reported broad host spectrum and considerable geographic range, RusV may be the aetiological agent of neuropathologies in further mammals, possibly even including humans