Validating a minipig model of reversible cerebral demyelination using human diagnostic modalities and electron microscopy.

BACKGROUND: Inflammatory demyelinating diseases of the central nervous system, such as multiple sclerosis, are significant sources of morbidity in young adults despite therapeutic advances. Current murine models of remyelination have limited applicability due to the low white matter content of their brains, which restricts the spatial resolution of diagnostic imaging. Large animal models might be more suitable but pose significant technological, ethical and logistical challenges. METHODS: We induced targeted cerebral demyelinating lesions by serially repeated injections of lysophosphatidylcholine in the minipig brain. Lesions were amenable to follow-up using the same clinical imaging modalities (3T magnetic resonance imaging, 11C-PIB positron emission tomography) and standard histopathology protocols as for human diagnostics (myelin, glia and neuronal cell markers), as well as electron microscopy (EM), to compare against biopsy data from two patients. FINDINGS: We demonstrate controlled, clinically unapparent, reversible and multimodally trackable brain white matter demyelination in a large animal model. De-/remyelination dynamics were slower than reported for rodent models and paralleled by a degree of secondary axonal pathology. Regression modelling of ultrastructural parameters (g-ratio, axon thickness) predicted EM features of cerebral de- and remyelination in human data. INTERPRETATION: We validated our minipig model of demyelinating brain diseases by employing human diagnostic tools and comparing it with biopsy data from patients with cerebral demyelination. FUNDING: This work was supported by the DFG under Germany's Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy, ID 390857198) and TRR 274/1 2020, 408885537 (projects B03 and Z01).

Severe Neurologic Disease in a Horse Caused by Tick-Borne Encephalitis Virus, Austria, 2021.

As evidenced by sero-epidemiological studies, infections of horses with the tick-borne encephalitis virus (TBEV) occur frequently in TBEV-endemic areas. However, there are only very few reports of clinical cases. A possible underreporting may be due to a variety of diagnostic challenges. In this study, ELISA and neutralization tests were applied to serum samples. Brain tissue samples were investigated for the presence of nucleic acids of TBEV, Equid alphaherpesvirus 1, Borna disease virus 1, West Nile and Usutu viruses, rustrela virus, as well as Eastern, Western, and Venezuelan equine encephalitis viruses with RT-qPCR, RT-PCR, and qPCR, respectively. TBEV-specific amplification products were subjected to Sanger sequencing. In addition, a direct fluorescent antibody test for rabies was performed. Clinical and patho-histological findings are reported. Using specific RT-qPCR and RT-PCR assays, TBEV nucleic acids were demonstrated in brain tissue samples. Sequencing revealed the Western (formerly Central) European subtype of TBEV as the etiological agent. A high titer of TBEV-specific neutralizing antibodies was found in the serum. RNAscope in situ hybridization revealed TBEV RNA confined to neuronal cell bodies and processes. No other pathogens or nucleic acids thereof could be detected. Diagnostic procedures need to be carried out early after the onset of neurological signs to allow for a final etiological diagnosis of acute TBEV infections in horses.

Relationship between magnetic resonance imaging findings and histological grade in spinal peripheral nerve sheath tumors in dogs.

BACKGROUND: Peripheral nerve sheath tumors (PNSTs) are a group of neoplasms originating from Schwann cells or pluripotent cell of the neural crest. Therapeutic options and prognosis are influenced by their degree of malignancy and location. HYPOTHESIS/OBJECTIVES: Identify magnetic resonance imaging (MRI) features predictive of PNST histologic grade. ANIMALS: Forty-four dogs with histopathological diagnosis of spinal PNSTs and previous MRI investigation. METHODS: A multicenter retrospective study including cases with (a) histopathologic diagnosis of PNST and (b) MRI studies available for review. Histologic slides were reviewed and graded by a board-certified pathologist according to a modified French system (FNCLCC) for grading soft tissue sarcomas. The MRI studies were reviewed by 2 board-certified radiologists blinded to the grade of the tumor and the final decision on the imaging characteristics was reached by consensus. Relationships between tumor grade and histological and MRI findings were assessed using statistical analysis. RESULTS: Forty-four cases met inclusion criteria; 16 patients were PNSTs Grade 1 (low-grade), 19 were PNSTs Grade 2 (medium-grade), and 9 were PNSTs Grade 3 (high-grade). Large volume (P = .03) and severe peripheral contrast enhancement (P = .04) were significantly associated with high tumor grade. Degree of muscle atrophy, heterogeneous signal and tumor growth into the vertebral canal were not associated with grade. CONCLUSIONS AND CLINICAL IMPORTANCE: Grade of malignancy was difficult to identify based on diagnostic imaging alone. However, some MRI features were predictive of high-grade PNSTs including tumor size and peripheral contrast enhancement.

Long-term follow-up of cats in complete remission after treatment of feline infectious peritonitis with oral GS-441524.

OBJECTIVES: Feline infectious peritonitis (FIP), a common disease in cats caused by feline coronavirus (FCoV), is usually fatal once clinical signs appear. Successful treatment of FIP with oral GS-441524 for 84 days was demonstrated recently by this research group. The aim of this study was to evaluate the long-term outcome in these cats. METHODS: A total of 18 successfully treated cats were followed for up to 1 year after treatment initiation (9 months after completion of the antiviral treatment). Follow-up examinations were performed at 12-week intervals, including physical examination, haematology, serum biochemistry, abdominal and thoracic ultrasound, FCoV ribonucleic acid (RNA) loads in blood and faeces by reverse transciptase-quantitative PCR and anti-FCoV antibody titres by indirect immunofluorescence assay. RESULTS: Follow-up data were available from 18 cats in week 24, from 15 cats in week 36 and from 14 cats in week 48 (after the start of treatment), respectively. Laboratory parameters remained stable after the end of the treatment, with undetectable blood viral loads (in all but one cat on one occasion). Recurrence of faecal FCoV shedding was detected in five cats. In four cats, an intermediate short-term rise in anti-FCoV antibody titres was detected. In total, 12 cats showed abdominal lymphadenomegaly during the follow-up period; four of them continuously during the treatment and follow-up period. Two cats developed mild neurological signs, compatible with feline hyperaesthesia syndrome, in weeks 36 and 48, respectively; however, FCoV RNA remained undetectable in blood and faeces, and no increase in anti-FCoV antibody titres was observed in these two cats, and the signs resolved. CONCLUSIONS AND RELEVANCE: Treatment with GS-441524 proved to be effective against FIP in both the short term as well as the long term, with no confirmed relapse during the 1-year follow-up period. Whether delayed neurological signs could be a long-term adverse effect of the treatment or associated with a 'long FIP syndrome' needs to be further evaluated.

An Inflammatory Myopathy in the Dutch Kooiker Dog.

The Dutch Kooiker dog (het Nederlandse Kooikerhondje) is one of nine Dutch dog breeds. As of 1960, a number of heritable diseases have been noted in this breed. One is an inflammatory myopathy that emerged in 1972, with numbers of affected dogs gradually increasing during the last few decades. The objective of this paper is to describe clinical signs, laboratory results, electromyography and histopathology of the muscle biopsies of the affected dogs. Method: Both retrospectively as well as prospectively affected Kooiker dogs were identified and categorized using a Tiered level of Confidence. Results: In total, 160 Kooiker dogs-40 Tier I, 33 Tier II and 87 Tier III-were included. Clinical signs were (1) locomotory problems, such as inability to walk long distances, difficulty getting up, stiff gait, walking on eggshells; (2) dysphagia signs such as drooling, difficulty eating and/or drinking; or (3) combinations of locomotory and dysphagia signs. CK activities were elevated in all except for one dog. Histopathology revealed a predominant lymphohistiocytic myositis with a usually low and variable number of eosinophils, neutrophils and plasma cells. It is concluded that, within this breed, a most likely heritable inflammatory myopathy occurs. Further studies are needed to classify this inflammatory myopathy, discuss its treatment, and unravel the genetic cause of this disease to eradicate it from this population.

Investigation of the presence of specific neural antibodies in dogs with epilepsy or dyskinesia using murine and human assays.

BACKGROUND: Autoimmune mechanisms represent a novel category for causes of seizures and epilepsies in humans, and LGI1-antibody associated limbic encephalitis occurs in cats. HYPOTHESIS/OBJECTIVES: To investigate the presence of neural antibodies in dogs with epilepsy or dyskinesia of unknown cause using human and murine assays modified for use in dogs. ANIMALS: Fifty-eight dogs with epilepsy of unknown cause or suspected dyskinesia and 57 control dogs. METHODS: Serum and CSF samples were collected prospectively as part of the diagnostic work-up. Clinical data including onset and seizure/episode type were retrieved from the medical records. Screening for neural antibodies was done with cell-based assays transfected with human genes for typical autoimmune encephalitis antigens and tissue-based immunofluorescence assays on mouse hippocampus slices in serum and CSF samples from affected dogs and controls. The commercial human und murine assays were modified with canine-specific secondary antibody. Positive controls were from human samples. RESULTS: The commercial assays used in this study did not provide unequivocal evidence for presence of neural antibodies in dogs including one dog with histopathologically proven limbic encephalitis. Low titer IgLON5 antibodies were present in serum from one dog from the epilepsy/dyskinesia group and in one dog from the control group. CONCLUSION AND CLINICAL IMPORTANCE: Specific neural antibodies were not detected using mouse and human target antigens in dogs with epilepsy and dyskinesia of unknown origin. These findings emphasize the need for canine-specific assays and the importance of control groups.

A loss-of-function variant in canine GLRA1 associates with a neurological disorder resembling human hyperekplexia.

Hereditary hyperekplexia is a rare neuronal disorder characterized by an exaggerated startle response to sudden tactile or acoustic stimuli. In this study, we present a Miniature Australian Shepherd family showing clinical signs, which have genetic and phenotypic similarities with human hereditary hyperekplexia: episodes of muscle stiffness that could occasionally be triggered by acoustic stimuli. Whole genome sequence data analysis of two affected dogs revealed a 36-bp deletion spanning the exon-intron boundary in the glycine receptor alpha 1 (GLRA1) gene. Further validation in pedigree samples and an additional cohort of 127 Miniature Australian Shepherds, 45 Miniature American Shepherds and 74 Australian Shepherds demonstrated complete segregation of the variant with the disease, according to an autosomal recessive inheritance pattern. The protein encoded by GLRA1 is a subunit of the glycine receptor, which mediates postsynaptic inhibition in the brain stem and spinal cord. The canine GLRA1 deletion is located in the signal peptide and is predicted to cause exon skipping and subsequent premature stop codon resulting in a significant defect in glycine signaling. Variants in GLRA1 are known to cause hereditary hyperekplexia in humans; however, this is the first study to associate a variant in canine GLRA1 with the disorder, establishing a spontaneous large animal disease model for the human condition.

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.

Novel association of gluten sensitivity with immune-mediated neuromuscular syndrome in a dog.

Gluten-related disorders in humans comprise different entities, including coeliac disease. Patients typically have measurable titers of anti-gliadin IgG or IgA (AGAs) and anti-transglutaminase-2 IgA (TG2). In addition to intestinal symptoms, human patients often show various neurological complications. In dogs, the neurological manifestation is rarely reported. Here we describe the muscle and nerve biopsies of an 11-year-old, male Border Terrier presenting with lower motor neuron signs submitted for histological examination. Examination of the biopsies showed an oligofocal lymphohistiocytic and plasmocytic myositis and a diffuse neuropathy of mixed nodo-paranodal and demyelinating type. Suspecting a neuromuscular form of breed-related gluten hypersensitivity, measurements of AGAs and TG2 antibodies were performed. Both titers ranged above control values. Hence, a gluten-related neuromyopathy was diagnosed. A gluten-free diet was prescribed and a complete disappearance of clinical signs was observed. Gluten-related disorders should be considered as a differential diagnosis in dogs with intestinal and neuromuscular signs.

Case report: Atypical and chronic masticatory muscle myositis in a 5-month old Cavalier King Charles Spaniel. Clinical and diagnostic findings, treatment and successful outcome.

Masticatory muscle myositis (MMM) is the second most common inflammatory myopathy diagnosed in dogs, but it is rarely described in puppies. The disease is characterized by the production of autoantibodies against 2M myofibers contained in masticatory muscle, although the cause of this production is still unclear. The aim of the present case report was to describe the clinical presentation, diagnostic findings, treatment, and follow-up of an atypical case of chronic masticatory muscle myositis in a very young dog. A 5-month old Cavalier king Charles Spaniel (CKCS) was presented to the AniCura Istituto Veterinario Novara with a two weeks, progressive history of lethargy and difficulty in food prehension. Neurological examination revealed bilateral masticatory muscle atrophy, mandibular ptosis with the jaw kept open, inability to close the mouth without manual assistance, jaw pain, and bilateral reduction of palpebral reflex and menace reaction; vision was maintained. A myopathy was suspected. Computed tomography (CT), magnetic resonance imaging (MRI), enzyme-linked immunosorbent assay test for 2M antibodies, and histopathological examination of masticatory muscle biopsy confirmed the diagnosis of MMM. Glucocorticoids treatment was started and clinical signs promptly improved. To the authors' knowledge, this is the first case describing mandibular ptosis in a dog affected by chronic MMM, successfully managed with medical treatment and the first report describing the CT and MRI findings in a young CKCS affected by MMM.

Clinical Follow-Up and Postmortem Findings in a Cat That Was Cured of Feline Infectious Peritonitis with an Oral Antiviral Drug Containing GS-441524.

This is the first report on a clinical follow-up and postmortem examination of a cat that had been cured of feline infectious peritonitis (FIP) with ocular manifestation by successful treatment with an oral multicomponent drug containing GS-441524. The cat was 6 months old when clinical signs (recurrent fever, lethargy, lack of appetite, and fulminant anterior uveitis) appeared. FIP was diagnosed by ocular tissue immunohistochemistry after enucleation of the affected eye. The cat was a participant in a FIP treatment study, which was published recently. However, 240 days after leaving the clinic healthy, and 164 days after the end of the 84 days of treatment, the cured cat died in a road traffic accident. Upon full postmortem examination, including histopathology and immunohistochemistry, there were no residual FIP lesions observed apart from a generalized lymphadenopathy due to massive lymphoid hyperplasia. Neither feline coronavirus (FCoV) RNA nor FCoV antigen were identified by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively, in any tissues or body fluids, including feces. These results prove that oral treatment with GS-441524 leads to the cure of FIP-associated changes and the elimination of FCoV from all tissues.

Case Report: Clinical Use of a Patient-Individual Magnetic Resonance Imaging-Based Stereotactic Navigation Device for Brain Biopsies in Three Dogs.

Three-dimensional (3D) printing techniques for patient-individual medicine has found its way into veterinary neurosurgery. Because of the high accuracy of 3D printed specific neurosurgical navigation devices, it seems to be a safe and reliable option to use patient-individual constructions for sampling brain tissue. Due to the complexity and vulnerability of the brain a particularly precise and safe procedure is required. In a recent cadaver study a better accuracy for the 3D printed MRI-based patient individual stereotactic brain biopsy device for dogs is determined compared to the accuracies of other biopsy systems which are currently used in veterinary medicine. This case report describes the clinical use of this 3D printed MRI-based patient individual brain biopsy device for brain sampling in three dogs. The system was characterized by a simple handling. Furthermore, it was an effective and reliable tool to gain diagnostic brain biopsy samples in dogs with no significant side effects.

Detection of Feline Coronavirus Variants in Cats without Feline Infectious Peritonitis.

(1) Background: This study aimed to detect feline coronavirus (FCoV) and characterize spike (S) gene mutation profiles in cats suffering from diseases other than feline infectious peritonitis (FIP) using commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) and reevaluating results by sequencing. (2) Methods: In 87 cats in which FIP was excluded by histopathology and immunohistochemistry, FCoV 7b gene and S gene mutation RT-qPCR was performed prospectively on incisional biopsies and fine-needle aspirates of different organs, body fluids, and feces. Samples positive for S gene mutations or mixed FCoV underwent sequencing. (3) Results: In 21/87 cats, FCoV RNA was detectable. S gene mutations were detected by commercial RT-qPCR (and a diagnostic algorithm that was used at the time of sample submission) in at least one sample in 14/21 cats (66.7%), with only mutated FCoV in 2/21, only mixed in 1/21, and different results in 11/21 cats; in the remaining 7/21 cats, RNA load was too low to differentiate. However, sequencing of 8 tissue samples and 8 fecal samples of 9 cats did not confirm mutated FCoV in any of the FCoV RNA-positive cats without FIP. (4) Conclusions: Sequencing results did not confirm results of the commercial S gene mutation RT-qPCR.

SLC25A12 Missense Variant in Nova Scotia Duck Tolling Retrievers Affected by Cerebellar Degeneration-Myositis Complex (CDMC).

We investigated two litters of distantly related Nova Scotia Duck Tolling Retrievers (NSDTR), of which four puppies were affected by cerebellar signs with or without neuromuscular weakness. The phenotype was termed cerebellar degeneration­myositis complex (CDMC). We suspected a heritable condition and initiated a genetic analysis. The genome of one affected dog was sequenced and compared to 565 control genomes. This search yielded a private protein-changing SLC25A12 variant in the affected dog, XM_038584842.1:c.1337C>T, predicted to result in the amino acid change XP_038440770.1:(p.Pro446Leu). The genotypes at the variant co-segregated with the phenotype as expected for a monogenic autosomal recessive mode of inheritance in both litters. Genotyping of 533 additional NSDTR revealed variant allele frequencies of 3.6% and 1.3% in a European and a North American cohort, respectively. The available clinical and biochemical data, together with current knowledge about SLC25A12 variants and their functional impact in humans, mice, and dogs, suggest the p.Pro446Leu variant is a candidate causative defect for the observed phenotype in the affected dogs.

Biopsy Characteristics, Subtypes, and Prognostic Features in 107 Cases of Feline Presumed Immune-Mediated Polyneuropathy.

Inflammatory polyradiculoneuropathy (IMPN) is one of the causes of sudden onset of neuromuscular signs such as para-/tetraparesis in young cats. Even though most cases have a favorable outcome, persistent deficits, relapses, and progressive courses are occasionally seen. As clinical presentation does not always appear to predict outcome and risk of recurrence, this study was initiated to screen for prognostic biopsy findings in a large cohort of histologically confirmed IMPN cases with clinical follow-up. In total, nerve and muscle specimens of 107 cats with biopsy diagnosis of presumed autoreactive inflammatory polyneuropathy and 22 control cases were reviewed by two blinded raters for a set of 36 histological parameters. To identify patterns and subtypes of IMPN, hierarchical k-means clustering of 33 histologic variables was performed. Then, the impact of histological parameters on IMPN outcome was evaluated via an univariate analysis to identify variables for the final multivariate model. The data on immediate outcome and follow-up were collected from submitting neurologists using a purpose-designed questionnaire. Hierarchical k-means clustering sorted the tissues into 4 main categories: cluster 1 (44/129) represents a purely inflammatory IMPN picture, whereas cluster 2 (47/129) was accompanied by demyelinating features and cluster 3 (16/129) by Wallerian degeneration. Cluster 4 (22/129) reflects normal tissues from non-neuropathic control cats. Returned questionnaires provided detailed information on outcome in 63 animals. They were categorized into recovered and non-recovered. Thereby, fiber-invasive infiltrates by mononuclear cells and mild fiber loss in intramuscular nerve branches correlated with higher probabilities of recovery. Remyelination in semithin sections, on the other hand, is correlated with a less favorable outcome. Animals grouping in cluster 1 had a tendency to a higher probability of recovery compared to other clusters. In conclusion, diagnosis of feline IMPN from nerve and muscle biopsies allowed for the identification of histologic features that were positively or negatively correlated with outcome.

Clinical Course and Diagnostic Findings of Biopsy Controlled Presumed Immune-Mediated Polyneuropathy in 70 European Cats.

There is a paucity of information on the clinical course and outcome of young cats with polyneuropathy. The aim of the study was to describe the clinical features, diagnostic investigations, and outcome of a large cohort of cats with inflammatory polyneuropathy from several European countries. Seventy cats with inflammatory infiltrates in intramuscular nerves and/or peripheral nerve biopsies were retrospectively included. Information from medical records and follow up were acquired via questionnaires filled by veterinary neurologists who had submitted muscle and nerve biopsies (2011-2019). Median age at onset was 10 months (range: 4-120 months). The most common breed was British short hair (25.7%), followed by Domestic short hair (24.3%), Bengal cat (11.4%), Maine Coon (8.6%) and Persian cat (5.7%), and 14 other breeds. Male cats were predominantly affected (64.3%). Clinical signs were weakness (98.6%) and tetraparesis (75.7%) in association with decreased withdrawal reflexes (83.6%) and, less commonly, cranial nerve signs (17.1%), spinal pain/hyperesthesia (12.9%), and micturition/defecation problems (14.3%). Onset was sudden (30.1%) or insidious (69.1%), and an initial progressive phase was reported in 74.3%. Characteristic findings on electrodiagnostic examination were presence of generalized spontaneous electric muscle activity (89.6%), decreased motor nerve conduction velocity (52.3%), abnormal F-wave studies (72.4%), pattern of temporal dispersion (26.1%) and unremarkable sensory tests. The clinical course was mainly described as remittent (49.2%) or remittent-relapsing (34.9%), while stagnation, progressive course or waxing and waning were less frequently reported. Relapses were common and occurred in 35.7% of the cats' population. An overall favorable outcome was reported in 79.4% of patients. In conclusion, young age at the time of diagnosis and sudden onset of clinical signs were significantly associated with recovery (p < 0.05). Clinical and electrodiagnostic features and the remittent-relapsing clinical course resembles juvenile chronic inflammatory demyelinating polyneuropathy (CIDP), as seen in human (children/adolescents), in many aspects.

Pig models for Duchenne muscular dystrophy - from disease mechanisms to validation of new diagnostic and therapeutic concepts.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal muscles and heart. Animal models are essential for preclinical evaluation of novel diagnostic procedures and treatment strategies. Gene targeting/editing offers the possibility of developing tailored pig models for monogenic diseases. The first porcine DMD model was generated by deletion of DMD exon 52 (DMDΔ52) in cultured kidney cells, which were used for somatic cell nuclear transfer to produce DMDΔ52 offspring. The animals resembled clinical, biochemical, and pathological hallmarks of DMD, but died before sexual maturity, thus preventing their propagation by breeding. This limitation was overcome by the generation of female heterozygous DMDΔ52 carrier pigs, which allowed the establishment of a large breeding colony. In this overview, we summarize how porcine DMD models have been used for dissecting disease mechanisms, for validating multispectral optoacoustic tomography as an imaging modality for monitoring fibrosis, and for preclinical testing of a CRISPR/Cas9 based approach to restore an intact DMD reading frame. Particular advantages of porcine DMD models include their targeted design and the rapid disease progression with early cardiac involvement, facilitating translational studies in reasonable time frames.