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In a retrospective metatranscriptomics study, we identified tick-borne encephalitis virus (TBEV) to be the causative agent for a fatal non-suppurative meningoencephalitis in a three-week-old Dalmatian puppy in Switzerland. Further investigations showed that the two other littermates with similar signs and pathological lesions were also positive for TBEV. By using an unbiased approach of combining high-throughput sequencing (HTS) and bioinformatics we were able to solve the etiology and discover an unusual case of TBEV in three young puppies. Based on our findings, we suggest that a vector-independent transmission of TBEV occurred and that most likely an intrauterine infection led to the severe and fulminant disease of the entire litter. We were able to demonstrate the presence of TBEV RNA by in situ hybridization (ISH) in the brain of all three puppies. Furthermore, we were able to detect TBEV by RT-qPCR in total RNA extracted from formalin-fixed and paraffin embedded (FFPE) blocks containing multiple peripheral organs. Overall, our findings shed light on alternative vector-independent transmission routes of TBEV infections in dogs and encourage veterinary practitioners to consider TBEV as an important differential diagnosis in neurological cases in dogs.

ObjectiveTo report a digital workflow for use and long-term outcome of cranioplasty with a 3D-printed patient-specific Polyetheretherketone (PEEK) implant in a 12-y-old German Shepherd dog after surgical removal of an extensive occipital bone multilobular osteochondrosarcoma (MLO).Study designRetrospective case report.AnimalA 12-year-old neutered female German Shepherd dog was presented with facial deformity, blindness, tetraparesis, and ataxia. Magnetic resonance imaging (MRI) and computed tomography (CT) identified a large skull-based mass extending extra-and intracranially with severe compression of the cerebellum and occipital lobes of the cerebrum.MethodsOne-stage decompressive craniectomy using virtual surgical planned 3D-printed craniotomy cutting guides and the Misonix BoneScalpel® and reconstruction with a patient-specific 3D-printed PEEK cranial implant.Results3D-printed craniectomy cutting guides allowed an adequate fit of the cranial implant to the original skull. Misonix BoneScalpel® allowed performing a safe and extensive craniectomy. Postoperative CT (8 weeks after surgery) confirmed the PEEK cranial implant to be in place and without implant rejection. Clinically, the neurological examination identified only a right-hind limb delay in proprioception 8 weeks postoperatively, which remained unchanged at 18 months after surgery. Adjunctive treatment included metronomic chemotherapy. Eighteen months after surgery the dog passed away for reasons unrelated to the MLO, no implant-related complications were reported.Conclusion3D-printed craniectomy cutting guides, patient-specific PEEK cranial implant, and metronomic chemotherapy can lead to a successful long-term outcome in dogs with extensive skull MLO.Clinical significancePEEK is an alternative biomaterial that can be used successfully for skull reconstruction.

Abstract Objectives The standard treatment for canine and feline meningiomas includes radiotherapy, surgical excision or combined therapy. However, new therapeutic approaches are required due to the possible recurrence or progression of meningiomas despite initial therapy. Adjunctive therapy with synthetic long‐acting somatostatin (SST) analogues has been described in humans with SST‐expressing tumours. The expression of SST receptors (SSTRs) by feline meningiomas is currently unknown, and there are little data about canine meningiomas. We hypothesized that SSTR is expressed by canine and feline meningiomas (S1). Methods Seven canines and 11 felines with histologically confirmed meningiomas underwent STTR screening. RNA expressions of SSTR1, SSTR2, SSTR3 and SSTR5 (canine) and SSTR1–SSTR 5 (feline) in fresh frozen and formalin‐fixed and paraffin‐embedded (FFPE) samples were investigated using real‐time (RT)‐qPCR. The expression of SSTR1 and SSTR2 in FFPE samples was evaluated using immunohistochemistry (IHC). The specificity of applied antibodies for canine and feline species was confirmed by western blotting. Results In canine meningiomas (n = 7), RNA expression of SSTR1, SSTR2 and SSTR5 was detected in all samples; SSTR3 RNA expression was detected in only 33% of samples. In feline meningiomas (n = 12), RNA expression of SSTR1, SSTR4, SSTR5 and SSTR2 was detected in 91%, 46%, 46% and 36% of samples, respectively; SSTR3 was not expressed. Overall, the detection rate was lower in FFPE samples. IHC revealed the expression of SSTR1 and SSTR2 in all samples from both species. However, it is important to exercise caution when interpreting IHC results due to the presence of diffuse background staining. Conclusions SSTRs are widely expressed in canine and feline meningiomas, thereby encouraging further studies investigating SSTR expression to conduct trials about the effect of adjunctive therapy with long‐acting SST‐analogues.

The high recurrence rate of feline meningioma despite the generally benign histomorphology warrants additional markers of clinical aggressiveness. The Ki-67 index is commonly used as prognostic marker for meningioma recurrence in people. Osteopontin (OPN) is a protein involved in tumor progression and may be a potential malignancy marker. To date, osteopontin expression has not been investigated in feline meningioma. The aim of this study was to evaluate the extent of Ki-67 and osteopontin immunostaining of feline meningioma and to find possible associations with WHO (World Health Organization) grades and subtypes. Fifty-three feline meningioma samples were graded according to the human WHO classification and underwent immunohistochemical examination for Ki-67 and OPN. Fifty samples were classified as WHO grade I and three as WHO grade II. The mean Ki-67 ratio was 9.19 ± 9.47. Osteopontin expression was correspondingly high with a mean OPN IHC score of 150.17 (0–242.8), and a median Allred score of 7 (0–8). There was no significant correlation with Ki-67 index, osteopontin expression, WHO grades, or subtypes. The overall high expressions of osteopontin and Ki-67 may help explain the tendency for recurrence of feline meningioma. The human WHO grading system may not be sufficient to accurately estimate the clinical behavior of meningioma in this species.

Abstract Feline morbillivirus (FeMV) is a recently discovered morbillivirus of the family Paramyxoviridae, which include several highly contagious viruses with zoonotic potential. In this case report we describe the detection of FeMV in archived brain tissue of a 2‐month‐old Bengal cat with nonsuppurative encephalitis from the year 2011 in Switzerland by high‐throughput sequencing (HTS). Our metagenomics approach was able to obtain a full‐length sequence covering the entire FeMV genome. Phylogenetic analysis showed that our FeMV strain clustered within FeMV genotype 1. We were able to detect FeMV RNA by in situ hybridization (ISH) in brain sections with inflammatory lesions and demonstrated its potential neurotropism and association with encephalitis. Our results provide further insight into this recently discovered morbillivirus and encourage further investigations into the pathogenesis and epidemiology of associated diseases in cats and potentially other species.

Monitoring neoplasms in standardized registries facilitates epidemiologic studies of risk factors for tumor development and predisposition. In an observational study, we determined incidence rates (IR) and malignant tumor incidence rate ratios (IRR) by age, sex, and breed in Swiss dogs using demographic data from the official Swiss dog registration database Amicus. The dataset analyzed included 54'986 tumors diagnosed by histology and cytology in four Swiss veterinary pathology laboratories between 2008 and 2020. Diagnoses were coded according to the Vet-ICD-O-canine-1 system. Most tumors occurred in the skin (n = 19'045; 34.64%), soft tissues (n = 11'092; 20.17%), and mammary glands (n = 7'974; 14.50%). The IRs for all and for malignant tumors were 775/100'000 dog-years at risk (95%CI 764-777) and 338/100'000 dog-years at risk (95%CI 333-342), respectively. Females (850; 95%CI 834-853) had a higher overall tumor IR than males (679; 95%CI 666-684). The highest tumor IR was found at 11 years of age (1'857; 95%CI 1'780-1'867). Potential novel breed-specific predispositions were uncovered, with high IRs for several benign and malignant tumors in Polski Owczarek Nizinnys (overall IR: 3'303; 95%CI 2'502-3'864) and high IRs for malignant tumors in Russian Black Terriers (melanomas: 345; 95%CI 138-708), Field Spaniels (adenocarcinomas: 376; CI95% 138-817), Dogo Argentinos (mast cell tumors: 844; CI95% 591-1'169), King Charles Spaniels and Manchester Terriers (lymphomas: 319; CI95% 137-627 and 302; CI95% 98-704, respectively), Landseers (osteosarcomas: 74; CI95% 15-216), Bouvier des Flandres (hemangiosarcomas: 127; CI95% 26-371), and Bearded Collies and Cane Corso Italianos (gliomas: 91; CI95% 45-162 and 34; CI95% 7-99, respectively). Nordic hunting dogs had the highest (8.08; CI95% 3.55-16.7) and Chihuahueno the lowest cancer IRRs (0.42; 95%CI 0.31-0.57) compared to mixed breeds. In conclusion, the calculated IRs and IRRs revealed previously unknown predisposing factors, including novel breed-specific susceptibilities. The results may have implications for cancer screening, diagnostic work-up, breeding management and oncologic and translational research.

Abstract Background Listeria monocytogenes (Lm) is a bacterial pathogen of major concern for humans and ruminants due to its neuroinvasive potential and its ability to cause deadly encephalitis (neurolisteriosis). On one hand, polymorphonuclear neutrophils (PMN) are key players in the defense against Lm, but on the other hand intracerebral infiltration with PMN is associated with significant neural tissue damage. Lm-PMN interactions in neurolisteriosis are poorly investigated, and factors inducing PMN chemotaxis to infectious foci containing Lm in the central nervous system (CNS) remain unidentified. Methods In this study, we assessed bovine PMN chemotaxis towards Lm and supernatants of infected endogenous brain cell populations in ex vivo chemotaxis assays, to identify chemotactic stimuli for PMN chemotaxis towards Lm in the brain. In addition, microglial secretion of IL-8 was assessed both ex vivo and in situ. Results Our data show that neither Lm cell wall components nor intact bacteria elicit chemotaxis of bovine PMN ex vivo. Moreover, astrocytes and neural cells fail to induce bovine PMN chemotaxis upon infection. In contrast, supernatant from Lm infected microglia readily induced chemotaxis of bovine PMN. Microglial expression and secretion of IL-8 was identified during early Lm infection in vitro and in situ, although IL-8 blocking with a specific antibody could not abrogate PMN chemotaxis towards Lm infected microglial supernatant. Conclusions These data provide evidence that host-derived rather than bacterial factors trigger PMN chemotaxis to bacterial foci in the CNS, that microglia have a primary role as initiators of bovine PMN chemotaxis into the brain during neurolisteriosis and that blockade of these factors could be a therapeutic target to limit intrathecal PMN chemotaxis and PMN associated damage in neurolisteriosis.

Abstract Background Oligodendroglioma (OG) accounts for 22% of primary brain tumors in dogs. Oligodendroglioma in dogs is graded as low‐grade (II) or high‐grade (III), based on the presence of microvascular proliferation and necrosis. Objective To investigate if magnetic resonance imaging (MRI) features differ between OG II and III in dogs. Animals Thirty‐two dogs with histological diagnosis of intracranial OG and MRI. Methods Retrospective descriptive study. Histology was reviewed to grade OG according to the revised classification. Brain MRI results were reviewed following criteria including contrast enhancement (CE) pattern, presence of cystic structures, gradient‐recalled‐echo (GRE) signal voids, and necrosis based on signal intensity, as well as diffusion‐weighted imaging characteristics. The MRI features were compared between OG II and III using Fisher's exact tests and logistic regression models. Results Histology identified 8 dogs with OG II (25%) and 24 with OG III (75%). All OG III showed moderate‐to‐marked CE including 18/24 (75%) with a ring pattern. These features were not seen in OG II. Heterogeneity, cystic structures, GRE signal voids, and necrosis were associated with OG III. No difference in diffusion characteristics was detected between OG II and III. Conclusion and Clinical Importance Moderate‐to‐marked CE and ring pattern were present in dogs with OG III but not in OG II. The presence of cystic structures, GRE signal voids, and necrosis was strongly associated with OG III. Although the importance of brain tumor grading in dogs with regard to prognosis and treatment options remains unknown, the results indicate that MRI reflects the histological features used for grading OG in dogs.

Abstract Background Lymphoma is the most common spinal cord neoplasm and second most common intracranial tumor in cats, but description of specific magnetic resonance imaging (MRI) features is lacking. Objective Describe the clinical and MRI features of lymphoma affecting the central (CNS) or peripheral (PNS) nervous system or both in cats. Animals Thirty‐one cats with confirmed cytological or histopathological diagnosis or both of lymphoma involving the CNS or PNS or both, and MRI findings of the lesions. Methods Multicenter retrospective descriptive study. Signalment and medical information were recorded. Magnetic resonance imaging findings were reviewed by 3 observers following a list of predefined criteria and consensus was sought. Frequency distributions of the different categorical data were reported. Results Median duration of clinical signs at time of presentation was 14 days (range, 1‐90). Neurological examination was abnormal in 30/31 cats. On MRI, lesions affecting the CNS were diagnosed in 18/31 cats, lesions in both CNS and PNS in 12/31, and lesions in the PNS only in 1/31. Intracranial lesions were diagnosed in 22 cats (extra‐axial, 7/22; intra‐axial, 2/22; mixed, 13/22), and spinal lesions were diagnosed in 12 (6/12 involving the conus medullaris and lumbosacral plexuses). Infiltration of adjacent extra‐neural tissue was present in 11/31 cases. Contrast enhancement was seen in all lesions, being marked in 25/30. Meningeal enhancement was present in all but 2 cases. Several distinct MRI patterns were observed. Conclusions and Clinical Importance Nervous system lymphoma in cats has a wide range of MRI features, of which none is pathognomonic. However, together with clinical data and cerebrospinal fluid (CSF) analysis, MRI may provide a strong tentative antemortem diagnosis.

Abstract Background The majority of emerging infectious diseases in humans are of animal origin, and many of them are caused by neuropathogenic viruses. Many cases of neurological disease and encephalitis in livestock remain etiologically unresolved, posing a constant threat to animal and human health. Thus, continuous extension of our knowledge of the repertoire of viruses prone to infect the central nervous system (CNS) is vital for pathogen monitoring and the early detection of emerging viruses. Using high-throughput sequencing (HTS) and bioinformatics, we discovered a new retrovirus, bovine retrovirus CH15 (BoRV CH15), in the CNS of a cow with non-suppurative encephalitis. Phylogenetic analysis revealed the affiliation of BoRV CH15 to the genus Betaretrovirus. Results BoRV CH15 genomes were identified prospectively and retrospectively by PCR, RT-PCR, and HTS, with targeting of viral RNA and proviral DNA, in six additional diseased cows investigated over a period of > 20 years and of different geographical origins. The virus was not found in brain samples from healthy slaughtered control animals (n = 130). We determined the full-length proviral genomes from six of the seven investigated animals and, using in situ hybridization, identified viral RNA in the cytoplasm of cells morphologically compatible with neurons in diseased brains. Conclusions Further screening of brain samples, virus isolation, and infection studies are needed to estimate the significance of these findings and the causative association of BoRV CH15 with neurological disease and encephalitis in cattle. However, with the full-length proviral sequences of BoRV CH15 genomes, we provide the basis for a molecular clone and further in vitro investigation. Graphical Abstract

The term “meningoencephalitis of unknown origin” (MUO) describes a group of different encephalitides in dogs in which no infectious agent can be identified and a multifactorial etiology is suspected. Among others, genetic factors and unknown triggers seem to be involved. Included are necrotizing leukoencephalitis (NLE), necrotizing meningoencephalitis (NME), and granulomatous meningoencephalitis (GME). In this case series, we describe the histopathological findings of four toy breed dogs with focal or multifocal necrotizing encephalitis and mainly lymphocytic perivascular infiltrates on histopathological examination. At the same time, however, in all dogs, focal or multifocal high-grade angiocentric granulomatous inflammatory lesions were evident with focal histiocytic perivascular infiltrates in the brain. The former changes are typical for NLE and NME. In contrast, the latter changes are indicative of GME. This case series shows that the boundaries between the necrotizing and granulomatous variants of MUO might be smooth and suggests that NLE, NME, and GME are not as distinct as previously described. This finding could be a crucial piece of the puzzle in the study of the pathogenesis of MUO as individual susceptibility and specific triggers could be responsible for the manifestation of the different MUO subtypes.

Abstract Background Osteomyelitis is relatively frequent in young pigs and a few bacterial species have been postulated to be potential causative agents. Although Actinobacillus (A.) pleuropneumoniae has been sporadically described to cause osteomyelitis, typically, actinobacillosis is characterized by respiratory symptoms. Nevertheless, subclinical infections are a challenging problem in pig herds. To the authors’ knowledge, this is the first case description that reports clinical, diagnostic imaging, pathological and histopathological findings of vertebral osteomyelitis in a pig and first describes A. pleuropneumoniae as the causative agent identified by advanced molecular methods. Case presentation An eight-week-old female weaner was presented with a non-ambulatory tetraparesis. The neurological signs were consistent with a lesion in the C6-T2 spinal cord segments. Imaging studies revealed a collapse of the seventh cervical vertebral body (C7) with a well demarcated extradural space-occupying mass ventrally within the vertebral canal severely compressing the spinal cord. Post-mortem examination identified an abscess and osteomyelitis of C7 and associated meningitis and neuritis with subsequent pathological fracture of C7 and compression of the spinal cord. In the microbiological analysis, A. pleuropneumoniae was identified using PCR and DNA sequence analysis. Conclusions A. pleuropneumoniae can be responsible for chronic vertebral abscess formation with subsequent pathological fracture and spinal cord compression in pigs.

Abstract Background The ingestion of grapes or raisins has been reported to cause acute kidney injury (AKI) in dogs, with a clinical picture dominated by early gastrointestinal signs and rapidly developing uremia. Ataxia is mentioned in a few reports, but not further characterized. Objectives To evaluate the clinical, laboratory, and pathological features of dogs diagnosed with grape or raisin toxicosis (GRT) with emphasis on renal and neurological manifestations, compared to a control group of dogs with AKI from other causes. Animals Fifteen client‐owned dogs with GRT and 74 control dogs. Methods Retrospective study over 17 months. Results All dogs with GRT were presented with severe AKI (grade 4, n = 5; grade 5, n = 10). Eleven dogs (73%) had marked forebrain, cerebellar, or vestibular signs. These manifestations dominated the clinical picture in some dogs, but were not associated with the severity of azotemia or the presence of systemic hypertension. Eight dogs (53%) survived, and 5 dogs experienced a complete neurological recovery. Causes of death were unrelated to the neurological manifestations. Neuropathological examination of 4 dogs did not identify any structural central nervous system abnormality. Only 2 control dogs (3%) displayed neurological signs with seizures unrelated to the AKI; 42 control dogs (57%) survived. Conclusions and Clinical Importance Severe forebrain, cerebellar, or vestibular signs may be an important feature of GRT and dominate the early clinical picture. The described features suggest a reversible functional brain injury specific to GRT and unrelated to uremia.

The apicomplexan parasite Neospora (N.) caninum causes neosporosis in numerous host species. There is no marketed vaccine and no licensed drug for the prevention and/or treatment of neosporosis. Vaccine development against this parasite has encountered significant obstacles, probably due to pregnancy-induced immunomodulation hampering efficacy, which has stimulated the search for potential drug therapies that could be applied to limit the effects of neosporosis in dams as well as in offspring. We here investigated, in a pregnant neosporosis mouse model, the safety and efficacy of a combined vaccination-drug treatment approach. Mice were vaccinated intramuscularly with 1 × 107 CFU of our recently generated Listeria (L.) monocytogenes vaccine vector expressing the major N. caninum tachyzoite surface antigen NcSAG1 (Lm3Dx_SAG1). Following mating and experimental subcutaneous infection with 1 × 105N. caninum (NcSpain-7) tachyzoites on day 7 of pregnancy, drug treatments were initiated using the bumped kinase inhibitor BKI-1748 at 20 mg/kg/day for 5 days. In parallel, other experimental groups were either just vaccinated or only treated. Dams and offspring were followed-up until day 25 post-partum, after which all mice were euthanized. None of the treatments induced adverse effects and neither of the treatments affected fertility or litter sizes. Cerebral infection in dams as assessed by real-time PCR was significantly reduced in the vaccinated and BKI-1748 treated groups, but was not reduced significantly in the group receiving the combination. However, in non-pregnant mice, all three treatment groups exhibited significantly reduced parasite burdens. Both, vaccination as well BKI-1748 as single treatment increased pup survival to 44 and 48%, respectively, while the combination treatment led to survival of 86% of all pups. Vertical transmission in the combination group was 23% compared to 46 and 50% in the groups receiving only BKI-treatment or the vaccine, respectively. In the dams, IgG titers were significantly reduced in all treatment groups compared to the untreated control, while in non-pregnant mice, IgG titers were reduced only in the group receiving the vaccine. Overall, vaccine-linked chemotherapy was more efficacious than vaccination or drug treatment alone and should be considered for further evaluation in a more relevant experimental model.

The aim of the present study was to evaluate the use of a novel intraoperative cone-beam computed tomography (CBCT)-based automated registration system for frameless stereotactic brain biopsy in companion animals. An experimental cadaveric study evaluated thalamic and piriform lobe target site needle placement error in three dogs and three cats without a history of intracranial disease. Diagnostic accuracy and diagnostic yield were prospectively evaluated in twenty-four client-owned dogs and four cats with intracranial disease. Twenty-one procedures were performed post mortem (eighteen dogs and three cats), and seven biopsy procedures were performed in alive patients (six dogs and one cat). Procedural duration was evaluated in ten post mortem and four living patients. Outcome was evaluated in six dogs and one cat. In dogs, the calculated median needle placement error was 1.8 mm (range 0.71–2.84 mm) and 1.53 mm (range 1.45–1.99 mm) for piriform lobe and thalamus target sites, respectively. In cats, the calculated median needle placement error was 0.79 mm (range 0.6–1.91 mm) for the piriform lobe target site and 1.29 mm (range 0.47–2.69 mm) for the thalamic target site. The diagnostic yield was 96.4% (95% CI 0.81–0.99), the diagnostic accuracy was 94.4% (95% CI 0.72–0.99). Median total procedural duration for post mortem biopsies was 57.5 min (range 41–69 min). Median total procedural duration for intra vitam biopsies was 122.5 min (range 103–136 min). Three dogs were discharged 1 day after biopsy and one dog after 6 days. Two dogs and one cat were euthanized 24 and 48 h after biopsy. Intraoperative CBCT-based automated image registration for frameless stereotactic biopsies in companion animals is capable of providing diagnostic brain biopsy specimens independent of skull size and morphology with diagnostic yield and accuracy comparable to published values for diverse frameless and frame-based stereotaxy systems used in veterinary medicine. Duration of the procedure is not negatively affected and within the published range with other systems. Mobile intraoperative CBCT-based registration combined with neuronavigation delivers diagnostic brain biopsies in companion animals.

Neospora caninum is an apicomplexan parasite that causes abortion and stillbirth in cattle. We employed the pregnant neosporosis mouse model to investigate the efficacy of a modified version of the attenuated Listeria monocytogenes vaccine vector Lm3Dx_NcSAG1, which expresses the major N. caninum surface antigen SAG1. Multivalent vaccines were generated by the insertion of gra7 and/or rop2 genes into Lm3Dx_NcSAG1, resulting in the double mutants, Lm3Dx_NcSAG1_NcGRA7 and Lm3Dx_NcSAG1_NcROP2, and the triple mutant, Lm3Dx_NcSAG1_NcGRA7_NcROP2. Six experimental groups of female BALB/c mice were inoculated intramuscularly three times at two-week intervals with 1 × 107 CFU of the respective vaccine strains. Seven days post-mating, mice were challenged by the subcutaneous injection of 1 × 105N. caninum NcSpain-7 tachyzoites. Non-pregnant mice, dams and their offspring were observed daily until day 25 post-partum. Immunization with Lm3Dx_NcSAG1 and Lm3Dx_NcSAG1_NcGRA7_NcROP2 resulted in 70% postnatal pup survival, whereas only 50% and 58% of pups survived in the double mutant-vaccinated groups. Almost all pups had died at the end of the experiment in the infection control. The triple mutant was the most promising vaccine candidate, providing the highest rate of protection against vertical transmission (65%) and CNS infection. Overall, integrating multiple antigens into Lm3Dx_SAG1 resulted in lower vertical transmission and enhanced protection against cerebral infection in dams and in non-pregnant mice.

Listeria monocytogenes (LM) has been proposed as vaccine vector in various cancers and infectious diseases since LM induces a strong immune response. In this study, we developed a novel and safe LM-based vaccine vector platform, by engineering a triple attenuated mutant (Lm3Dx) (ΔactA, ΔinlA, ΔinlB) of the wild-type LM strain JF5203 (CC 1, phylogenetic lineage I). We demonstrated the strong attenuation of Lm3Dx while maintaining its capacity to selectively infect antigen-presenting cells (APCs) in vitro. Furthermore, as proof of concept, we introduced the immunodominant Neospora caninum (Nc) surface antigen NcSAG1 into Lm3Dx. The NcSAG1 protein was expressed by Lm3Dx_SAG1 during cellular infection. To demonstrate safety of Lm3Dx_SAG1 in vivo, we vaccinated BALB/C mice by intramuscular injection. Following vaccination, mice did not suffer any adverse effects and only sporadically shed bacteria at very low levels in the feces (

Case summary A 4-month-old cat was presented with acute paraplegia after the referring veterinarian performed a subcutaneous injection (cefovecin and dexamethasone) in the caudodorsal thoracic area, during which the cat suddenly became uncooperative. A complete neurological examination performed 1 day after the injection revealed paraplegia without deep pain perception and reduced segmental spinal reflexes in the pelvic limbs. Findings were consistent with either an L4–S3 myelopathy or a T3–L3 myelopathy with subsequent spinal shock. MRI showed swelling of the spinal cord from T1 to L1 with heterogeneous T2-weighted intramedullary hyperintensity and no contrast enhancement. A centrally located intraspinal signal void was visible in T2*-weighted images. These changes were compatible with a suspected traumatic intraspinal injection. Despite intensive supportive care over 4 days, neurological status did not improve and the cat was euthanased. Gross pathology findings revealed severe intramedullary haemorrhage and myelomalacia in the T10–L1 spinal cord segments. Histopathology of the spinal cord after haematoxylin and eosin staining revealed a severe intramedullary space-occupying haemorrhage with focal malacia. A trajectory-like, optically empty cavity containing some eosinophilic droplets at the edges was detected. Although no further evidence of trauma was noted in the surrounding structures, the spinal cord changes were compatible with a perforating trauma. Relevance and novel information To our knowledge, this is the first report of thoracic intraspinal injection causing myelomalacia defined by an ante-mortem MRI and confirmed post mortem by histopathology. The traumatic myelopathy appeared to be most compatible with an intraspinal injection causing vascular rupture.

Background: Listeria rhombencephalitis, infection of the brainstem with Listeria monocytogenes, occurs mainly in humans and farmed ruminants and is associated with high fatality rates. Small ruminants (goats and sheep) are a large animal model due to neuropathological similarities. The purpose of this study was to define magnetic resonance imaging (MRI) features of listeria rhombencephalitis in naturally infected small ruminants and correlate them with histopathology. Secondly, the purpose of this study was to compare the results with MRI findings reported in humans.Methods: Twenty small ruminants (13 sheep and 7 goats) with listeria rhombencephalitis were prospectively enrolled and underwent in vivo MRI of the brain, including T2-weighted, fluid attenuation inversion recovery, and T1-weighted sequences pre- and post-contrast administration and postmortem histopathology. In MRI, lesions were characterized by location, extent, border definition, signal intensity, and contrast enhancement. In histopathology, the location, cell type, severity, and chronicity of inflammatory infiltrates and signs of vascular damage were recorded. In addition, histopathologic slides were matched to MRIs, and histopathologic and MRI features were compared.Results: Asymmetric T2-hyperintense lesions in the brainstem were observed in all animals and corresponded to the location and pattern of inflammatory infiltrates in histopathology. Contrast enhancement in the brainstem was observed in 10 animals and was associated with vessel wall damage and perivascular fibrin accumulation in 8 of 10 animals. MRI underestimated the extension into rostral brain parts and the involvement of trigeminal ganglia and meninges.Conclusion: Asymmetric T2-hyperintense lesions in the brainstem with or without contrast enhancement can be established as criteria for the diagnosis of listeria rhombencephalitis in small ruminants. Brainstem lesions were similar to human listeria rhombencephalitis in terms of signal intensity and location. Different from humans, contrast enhancement was a rare finding, and abscessation was not observed.

The apicomplexan parasite Neospora caninum is the worldwide leading cause of abortion and stillbirth in cattle. An attenuated mutant Listeria monocytogenes strain (Lm3Dx) was engineered by deleting the virulence genes actA, inlA, and inlB in order to avoid systemic infection and to target the vector to antigen-presenting cells (APCs). Insertion of sag1, coding for the major surface protein NcSAG1 of N. caninum, yielded the vaccine strain Lm3Dx_NcSAG1. The efficacy of Lm3Dx_NcSAG1 was assessed by inoculating 1 × 105, 1 × 106, or 1 × 107 CFU of Lm3Dx_NcSAG1 into female BALB/c mice by intramuscular injection three times at two-week intervals, and subsequent challenge with 1 × 105N. caninum tachyzoites of the highly virulent NcSpain-7 strain on day 7 of pregnancy. Dose-dependent protective effects were seen, with a postnatal offspring survival rate of 67% in the group treated with 1 × 107 CFU of Lm3Dx_NcSAG1 compared to 5% survival in the non-vaccinated control group. At euthanasia (25 days post-partum), IgG antibody titers were significantly decreased in the groups receiving the two higher doses and cytokines recall responses in splenocyte culture supernatants (IFN-γ, IL-4, and IL-10) were increased in the vaccinated groups. Thus, Lm3Dx_NcSAG1 induces immune-protective effects associated with a balanced Th1/Th2 response in a pregnant neosporosis mouse model and should be further assessed in ruminant models.

Spongy degeneration with cerebellar ataxia (SDCA) is a genetically heterogeneous neurodegenerative disorder with autosomal recessive inheritance in Malinois dogs, one of the four varieties of the Belgian Shepherd breed. Using a combined linkage and homozygosity mapping approach we identified an ∼10.6 Mb critical interval on chromosome 5 in a Malinois family with four puppies affected by cerebellar dysfunction. Visual inspection of the 10.6 Mb interval in whole-genome sequencing data from one affected puppy revealed a 227 bp SINE insertion into the ATP1B2 gene encoding the β2 subunit of the Na+/K+-ATPase holoenzyme (ATP1B2:c.130_131insLT796559.1:g.50_276). The SINE insertion caused aberrant RNA splicing. Immunohistochemistry suggested a reduction of ATP1B2 protein expression in the central nervous system of affected puppies. Atp1b2 knockout mice had previously been reported to show clinical and neurohistopathological findings similar to the affected Malinois puppies. Therefore, we consider ATP1B2:c.130_131ins227 the most likely candidate causative variant for a second subtype of SDCA in Malinois dogs, which we propose to term spongy degeneration with cerebellar ataxia subtype 2 (SDCA2). Our study further elucidates the genetic and phenotypic complexity underlying cerebellar dysfunction in Malinois dogs and provides the basis for a genetic test to eradicate one specific neurodegenerative disease from the breeding population in Malinois and the other varieties of the Belgian Shepherd breed. ATP1B2 thus represents another candidate gene for human inherited cerebellar ataxias, and SDCA2-affected Malinois puppies may serve as a naturally occurring animal model for this disorder.

Abstract Listeriosis is a severe disease caused by the opportunistic bacterial pathogen Listeria monocytogenes (L. monocytogenes). Previous studies indicate that of the four phylogenetical lineages known, lineage I strains are significantly more prevalent in clinical infections than in the environment. Among lineage 1, sequence type (ST1) belongs to the most frequent genotypes in clinical infections and behaves hyperinvasive in experimental in vitro infections compared to lineage II strains suggesting that yet uncharacterized virulence genes contribute to high virulence of certain lineage I strains. This study investigated the effect of four specific lineage I genes encoding surface proteins with internalin‐like structures on cellular infection. CNS derived cell lines (fetal bovine brain cells, human microglia cells) and non‐CNS derived cell lines (bovine macrophage cells, human adenocarcinoma cells) that represent the various target cells of L. monocytogenes were infected with the parental ST1 strain and deletion mutants of the four genes. Despite their association with lineage I, deletion of the four genes investigated did not dampen the hyperinvasiveness of the ST1 strain. Similarly, these genes did not contribute to the intracellular survival and intercellular spread of L. monocytogenes ST1, indicating that these genes may have other functions, either during the infection process or outside the host.

Spongy degeneration with cerebellar ataxia (SDCA) is a severe neurodegenerative disease with monogenic autosomal recessive inheritance in Malinois dogs, one of the four varieties of the Belgian Shepherd breed. We performed a genetic investigation in six families and seven isolated cases of Malinois dogs with signs of cerebellar dysfunction. Linkage analysis revealed an unexpected genetic heterogeneity within the studied cases. The affected dogs from four families and one isolated case shared a ∼1.4 Mb common homozygous haplotype segment on chromosome 38. Whole genome sequence analysis of three affected and 140 control dogs revealed a missense variant in the KCNJ10 gene encoding a potassium channel (c.986T>C; p.Leu329Pro). Pathogenic variants in KCNJ10 were reported previously in humans, mice, and dogs with neurological phenotypes. Therefore, we consider KCNJ10:c.986T>C the most likely candidate causative variant for one subtype of SDCA in Malinois dogs, which we propose to term spongy degeneration with cerebellar ataxia 1 (SDCA1). However, our study also comprised samples from 12 Malinois dogs with cerebellar dysfunction which were not homozygous for this variant, suggesting a different genetic basis in these dogs. A retrospective detailed clinical and histopathological analysis revealed subtle neuropathological differences with respect to SDCA1-affected dogs. Thus, our study highlights the genetic and phenotypic complexity underlying cerebellar dysfunction in Malinois dogs and provides the basis for a genetic test to eradicate one specific neurodegenerative disease from the breeding population. These dogs represent an animal model for the human EAST syndrome.

The metabolic profiling of tissue biopsies using high-resolution–magic angle spinning (HR-MAS) 1H nuclear magnetic resonance (NMR) spectroscopy may be influenced by experimental factors such as the sampling method. Therefore, we compared the effects of two different sampling methods on the metabolome of brain tissue obtained from the brainstem and thalamus of healthy goats by 1H HR-MAS NMR spectroscopy—in vivo-harvested biopsy by a minimally invasive stereotactic approach compared with postmortem-harvested sample by dissection with a scalpel. Lactate and creatine were elevated, and choline-containing compounds were altered in the postmortem compared to the in vivo-harvested samples, demonstrating rapid changes most likely due to sample ischemia. In addition, in the brainstem samples acetate and inositols, and in the thalamus samples ƴ-aminobutyric acid, were relatively increased postmortem, demonstrating regional differences in tissue degradation. In conclusion, in vivo-harvested brain biopsies show different metabolic alterations compared to postmortem-harvested samples, reflecting less tissue degradation. Sampling method and brain region should be taken into account in the analysis of metabolic profiles. To be as close as possible to the actual situation in the living individual, it is desirable to use brain samples obtained by stereotactic biopsy whenever possible.

An 8-year-old alpaca was admitted to the emergency service of the Clinic for Ruminants in Bern due to a reduced general condition and progressive neurological signs. Despite supportive treatment, its condition deteriorated and the animal had to be euthanized. Histopathological analysis revealed a severe non-suppurative polioencephalomyelitis with neuronal necrosis, most likely of viral origin. We detected abundant neuronal labelling with antibodies directed against two different epitopes of Bovine Astrovirus CH13/NeuroS1 (BoAstV-CH13/NeuroS1), which is a common viral agent associated with non-suppurative encephalitis in Swiss cattle. These findings were further verified by detection of viral RNA by use of in-situ hybridization and real-time RT-PCR. Next generation sequencing revealed that the detected virus genome had a pairwise identity of 98.9% to the genome of BoAstV-CH13/NeuroS1. To our knowledge, this is the first report of an astrovirus-associated polioencephalomyelitis in an alpaca. These results point to the possibility of an interspecies transmission of BoAstV-CH13/NeuroS1.

Japanese encephalitis virus (JEV) is primarily transmitted between mosquitoes and birds but can also infect pigs. Here the authors demonstrate that JEV, which was thought to be spread exclusively by mosquitoes, can be transmitted between pigs through a direct contact.

We observed a hereditary phenotype in Alaskan Huskies that was characterized by polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV). The affected dogs developed a progressive severe ataxia, which led to euthanasia between 8 and 16 months of age. The pedigrees were consistent with a monogenic autosomal recessive inheritance. We localized the causative genetic defect to a 4 Mb interval on chromosome 19 by a combined linkage and homozygosity mapping approach. Whole genome sequencing of one affected dog, an obligate carrier, and an unrelated control revealed a 218-bp SINE insertion into exon 7 of the RAB3GAP1 gene. The SINE insertion was perfectly associated with the disease phenotype in a cohort of 43 Alaskan Huskies, and it was absent from 541 control dogs of diverse other breeds. The SINE insertion induced aberrant splicing and led to a transcript with a greatly altered exon 7. RAB3GAP1 loss-of-function variants in humans cause Warburg Micro Syndrome 1 (WARBM1), which is characterized by additional developmental defects compared to canine POANV, whereas Rab3gap1-deficient mice have a much milder phenotype than either humans or dogs. Thus, the RAB3GAP1 mutant Alaskan Huskies provide an interesting intermediate phenotype that may help to better understand the function of RAB3GAP1 in development. Furthermore, the identification of the presumed causative genetic variant will enable genetic testing to avoid the nonintentional breeding of affected dogs.

Listeria monocytogenes is a foodborne pathogen that causes abortion, septicemia, gastroenteritis and central nervous system (CNS) infections in ruminants and humans. L. monocytogenes strains mainly belong to two distinct phylogenetic groups, named lineages I and II. In general, clinical cases in humans and animals, in particular CNS infections, are caused by lineage I strains, while most of the environmental and food strains belong to lineage II. Little is known about why lineage I is more virulent than lineage II, even though various molecular factors and mechanisms associated with pathogenesis are known. In this study, we have used a variety of whole genome sequence analyses and comparative genomic tools in order to find characteristics that distinguish lineage I from lineage II strains and CNS infection strains from non-CNS strains. We analyzed 225 strains and identified single nucleotide variants between lineages I and II, as well as differences in the gene content. Using a novel approach based on Reads Per Kilobase per Million Mapped (RPKM), we identified 167 genes predominantly absent in lineage II but present in lineage I. These genes are mostly encoding for membrane-associated proteins. Additionally, we found 77 genes that are largely absent in the non-CNS associated strains, while 39 genes are especially lacking in our defined “non-clinical” group. Based on the RPKM analysis and the metadata linked to the L. monocytogenes strains, we identified 6 genes potentially associated with CNS cases, which include a transcriptional regulator, an ABC transporter and a non-coding RNA. Although there is not a clear separation between pathogenic and non-pathogenic strains based on phylogenetic lineages, the presence of the genes identified in our study reveals potential pathogenesis traits in ruminant L. monocytogenes strains. Ultimately, the differences that we have found in our study will help steer future studies in understanding the virulence mechanisms of the most pathogenic L. monocytogenes strains.

Endocannabinoids (ECs) are involved in immunomodulation, neuroprotection and control of inflammation in the central nervous system (CNS). Activation of cannabinoid type 2 receptors (CB2) is known to diminish the release of pro-inflammatory factors and enhance the secretion of anti-inflammatory cytokines. Furthermore, the endocannabinoid 2-arachidonoyl glycerol (2-AG) has been proved to induce the migration of eosinophils in a CB2 receptor-dependent manner in peripheral blood and activate neutrophils independent of CB activation in humans. The aim of the current study was to investigate the influence of the endocannabinoid system in two different CNS inflammatory diseases of the dog, i.e. Steroid-Responsive Meningitis-Arteritis (SRMA) and Intraspinal Spirocercosis (IS). The two main endocannabinoids, anandamide (AEA) and 2-AG, were quantified by mass spectrometry in CSF and serum samples of dogs affected with Steroid- Responsive Meningitis-Arteritis in the acute phase (SRMA A), SRMA under treatment with prednisolone (SRMA Tr), intraspinal Spirocercosis and healthy dogs. Moreover, expression of the CB2 receptor was evaluated in inflammatory lesions of SRMA and IS and compared to healthy controls using immunohistochemistry (IHC). Dogs with SRMA A showed significantly higher concentrations of total AG and AEA in serum in comparison to healthy controls and in CSF compared to SRMA Tr (p

Cold shock-domain family proteins (Csps) are highly conserved nucleic acid binding proteins regulating the expression of various genes including those involved in stress resistance and virulence in bacteria. We show here that Csps are involved in virulence, cell aggregation and flagella-based extracellular motility of Listeria monocytogenes. A L. monocytogenes mutant deleted in all three csp genes (ΔcspABD) is attenuated with respect to human macrophage infection as well as virulence in a zebrafish infection model. Moreover, this mutant is incapable of aggregation and fails to express surface flagella or exhibit swarming motility. An evaluation of double csp gene deletion mutant (ΔcspBD, ΔcspAD and ΔcspAB) strains that produce single csp genes showed that there is redundancy as well as functional differences among the three L. monocytogenes Csps in their contributions to virulence, cellular aggregation, flagella production, and swarming motility. Protein and mRNA expression analysis further showed impaired expression of key virulence and motility genes in the csp mutants. Our observations at protein and mRNA level suggest Csp-dependent expression regulation of these genes at transcriptional and post-transcriptional levels. In a mutant lacking all csp genes (ΔcspABD) as well as those possessing single csp genes (ΔcspBD, ΔcspAD, and ΔcspAB) we detected reduced levels of proteins or activity as well as transcripts from the prfA, hly, mpl, and plcA genes suggesting a Csp-dependent transcriptional regulation of these genes. These csp mutants also had reduced or completely lacked ActA proteins and cell surface flagella but contained elevated actA and flaA mRNA levels compared to the parental wild type strain suggesting Csp involvement in post-transcriptional regulation of these genes. Overall, our results suggest that Csps contribute to the expression regulation of virulence and flagella-associated genes thereby promoting host pathogenicity, cell aggregation and flagella-based motility processes in L. monocytogenes.

Toxin−antitoxin systems (TASs) are widely distributed in prokaryotes and encode pairs of genes involved in many bacterial biological processes and mechanisms, including pathogenesis. The TASs have not been extensively studied in Listeria monocytogenes (Lm), a pathogenic bacterium of the Firmicutes phylum causing infections in animals and humans. Using our recently published TASmania database, we focused on the known and new putative TASs in 352 Listeria monocytogenes genomes and identified the putative core gene TASs (cgTASs) with the Pasteur BIGSdb-Lm database and, by complementarity, the putative accessory gene TAS (acTASs). We combined the cgTASs with those of an additional 227 L. monocytogenes isolates from our previous studies containing metadata information. We discovered that the differences in 14 cgTAS alleles are sufficient to separate the four main lineages of Listeria monocytogenes. Analyzing these differences in more details, we uncovered potentially co-evolving residues in some pairs of proteins in cgTASs, probably essential for protein−protein interactions within the TAS complex.

European sporadic bovine encephalitis (ESBE) is a frequent diagnosis in neurologically diseased cattle, but its etiology remained unresolved. Using in-situ hybridization, we have detected a recently discovered neurotropic bovine astrovirus in historical tissues in a high proportion of brain samples of affected cattle. Our results suggest that astroviruses were already involved in the pathogenesis of the disease several decades ago, but have gone undetected.

[This corrects the article DOI: 10.1371/journal.pgen.1005427.].

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

The first case of spongiform encephalopathy in a zebu (Bos indicus) was identified in a zoo in Switzerland. Although histopathologic and immunohistochemical analyses of the central nervous system indicated a diagnosis of bovine spongiform encephalopathy (BSE), molecular typing showed some features different from those of BSE in cattle (B. taurus).

An inherited polyneuropathy (PN) observed in Leonberger dogs has clinical similarities to a genetically heterogeneous group of peripheral neuropathies termed Charcot-Marie-Tooth (CMT) disease in humans. The Leonberger disorder is a severe, juvenile-onset, chronic, progressive, and mixed PN, characterized by exercise intolerance, gait abnormalities and muscle atrophy of the pelvic limbs, as well as inspiratory stridor and dyspnea. We mapped a PN locus in Leonbergers to a 250 kb region on canine chromosome 16 (Praw = 1.16×10-10, Pgenome, corrected = 0.006) utilizing a high-density SNP array. Within this interval is the ARHGEF10 gene, a member of the rho family of GTPases known to be involved in neuronal growth and axonal migration, and implicated in human hypomyelination. ARHGEF10 sequencing identified a 10 bp deletion in affected dogs that removes four nucleotides from the 3'-end of exon 17 and six nucleotides from the 5'-end of intron 17 (c.1955_1958+6delCACGGTGAGC). This eliminates the 3'-splice junction of exon 17, creates an alternate splice site immediately downstream in which the processed mRNA contains a frame shift, and generates a premature stop codon predicted to truncate approximately 50% of the protein. Homozygosity for the deletion was highly associated with the severe juvenile-onset PN phenotype in both Leonberger and Saint Bernard dogs. The overall clinical picture of PN in these breeds, and the effects of sex and heterozygosity of the ARHGEF10 deletion, are less clear due to the likely presence of other forms of PN with variable ages of onset and severity of clinical signs. This is the first documented severe polyneuropathy associated with a mutation in ARHGEF10 in any species.

Next-generation sequencing (NGS) has opened up the possibility of detecting new viruses in unresolved diseases. Recently, astrovirus brain infections have been identified in neurologically diseased humans and animals by NGS, among them bovine astrovirus (BoAstV) CH13/NeuroS1, which has been found in brain tissues of cattle with non-suppurative encephalitis. Only a few studies are available on neurotropic astroviruses and a causal relationship between BoAstV CH13/NeuroS1 infections and neurological disease has been postulated, but remains unproven. Aiming at making a step forward towards assessing the causality, we collected brain samples of 97 cases of cattle diagnosed with unresolved non-suppurative encephalitis, and analyzed them by in situ hybridization and immunohistochemistry, to determine the frequency and neuropathological distribution of the BoAstV CH13/NeuroS1 and its topographical correlation to the pathology. We detected BoAstV CH13/NeuroS1 RNA or proteins in neurons throughout all parts of the central nervous system (CNS) in 34% of all cases, but none were detected in cattle of the control group. In general, brain lesions had a high correlation with the presence of the virus. These findings show that a substantial proportion of cattle with non-suppurative encephalitis are infected with BoAstV CH13/NeuroS1 and further substantiate the causal relationship between neurological disease and astrovirus infections.

Emerging Microbes & Infections (2017) 6, e71; doi:10.1038/emi.2017.56; published online 9 August 2017

Tyrolean Grey cattle represent a local breed with a population size of ∼5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.

The pathobiology of atypical scrapie, a prion disease affecting sheep and goats, is still poorly understood. In a previous study, we demonstrated that atypical scrapie affecting small ruminants in Switzerland differs in the neuroanatomical distribution of the pathological prion protein (PrP(d)). To investigate whether these differences depend on host-related vs. pathogen-related factors, we transmitted atypical scrapie to transgenic mice over-expressing the ovine prion protein (tg338). The clinical, neuropathological, and molecular phenotype of tg338 mice is similar between mice carrying the Swiss atypical scrapie isolates and the Nor98, an atypical scrapie isolate from Norway. Together with published data, our results suggest that atypical scrapie is caused by a uniform type of prion, and that the observed phenotypic differences in small ruminants are likely host-dependant. Strikingly, by using a refined SDS-PAGE technique, we established that the prominent proteinase K-resistant prion protein fragment in atypical scrapie consists of two separate, unglycosylated peptides with molecular masses of roughly 5 and 8 kDa. These findings show similarities to those for other prion diseases in animals and humans, and lay the groundwork for future comparative research.

We examined 66 cats with salinomycin intoxication. Salinomycin caused different LMN signs of varying degrees of severity in all cases. Changes in blood work were unspecific, with the most frequent being increased serum creatine kinase activity, leukocytosis, and increased liver enzymes. Pathological electrodiagnostic findings: fibrillation potentials and positive sharp waves were detected in 10 cases, motor nerve conductance velocity was mildly decreased in 8/12 cats, and sensory nerve conductance velocity and repetitive nerve stimulation were normal in all examined cases. In five cases the peripheral neuropathy was confirmed by pathohistology. Fluid therapy and supportive care were used as therapy and 52 cats recovered completely. The probability for complete remission was significantly different between mildly and severely affected cases. It seems that the severity of clinical signs and prognosis correlate well with the amount of toxin ingested. We conclude that early recognition and decontamination combined with supportive care results in complete recovery.

Scrapie is a transmissible spongiform encephalopathy (TSE) in sheep and goats. In recent years, atypical scrapie cases were identified that differed from classical scrapie in the molecular characteristics of the disease-associated pathological prion protein (PrP(sc)). In this study, we analyze the molecular and neuropathological phenotype of nine Swiss TSE cases in sheep and goats. One sheep was identified as classical scrapie, whereas six sheep, as well as two goats, were classified as atypical scrapie. The latter revealed a uniform electrophoretic mobility pattern of the proteinase K-resistant core fragment of PrP(sc) distinct from classical scrapie regardless of the genotype, the species, and the neuroanatomical structure. Remarkably different types of neuroanatomical PrP(sc) distribution were observed in atypical scrapie cases by both western immunoblotting and immunohistochemistry. Our findings indicate that the biodiversity in atypical scrapie is larger than expected and thus impacts on current sampling and testing strategies in small ruminant TSE surveillance.