During infection in epithelial cells, after invading the cytosol, multiplying, and spreading, Listeria monocytogenes (Lm) ceases to produce ActA and becomes trapped in Listeria-containing vacuoles (LisCVs). These persistence acidic vacuoles harbor bacterial subpopulations that resist to stress in a metabolically dormant state. Although LisCVs have been proposed as a hallmark of Lm persistence in epithelial cells, their prevalence across strains and the bacterial factors underlying their formation remain uncharacterized. Given the significant genetic diversity within the species, it is important to consider this variability when studying persistence phenotype. Therefore, we screened over one hundred Lm isolates spanning two major evolutionary lineages and belonging to 23 clonal complexes from diverse ecological origins. Strikingly, the vast majority of strains, including both clinical and environmental isolates, were capable of forming LisCVs, suggesting that vacuolar persistence is a widespread and conserved feature of Lm pathogenesis. Nevertheless, among the group of hypo-virulent strains mostly associated with food and carrying a truncated InlA, we identified four isolates with an altered persistence phenotype. Two of them showed defects in the early stages of infection and carried mutations in key virulence genes (hly and gshF). The other two, instead, were specifically affected in the persistence stage by showing a reduced ability to form LisCVs. Comparative genomic analysis revealed that a mutation in the folP gene, required for folate biosynthesis, was responsible for impaired persistence. Live-imaging and microscopy analysis highlighted a reduced bacterial motility and intercellular spreading of the folP mutant, although the level of ActA at the bacterial surface was increased. Together, our work identifies folate biosynthesis as a critical metabolic pathway governing Lm persistence by regulating ActA levels and activity. Downregulation of ActA at the bacterial surface is therefore a crucial event for the establishment of the intracellular persistent niche during long-term infection of epithelial cells.

Intracranial neoplasms are rare in cattle. Bovine ependymomas have been reported only sporadically, with limited morphologic descriptions and, to our knowledge, without immunohistochemical characterization. Here, we describe the gross, histologic, and immunophenotypic features of a papillary ependymoma in the brain of a 7.5-y-old cow that was a bovine spongiform encephalopathy suspect. Grossly, a large, intraparenchymal, well-demarcated mass occupied the left cerebral hemisphere, extending from the frontal to the occipital lobe and communicating with the left lateral ventricle. Histologically, the neoplasm was composed of elongated-to-columnar cells arranged predominantly in perivascular pseudorosettes with prominent papillary architecture. Immunohistochemically, >95% of neoplastic cells had strong cytoplasmic glial fibrillary acidic protein immunolabeling, which was accentuated in perivascular processes; ~20% of neoplastic cells were also immunolabeled for vimentin. Neoplastic cells lacked immunolabeling for pan-cytokeratin, S100, oligodendrocyte transcription factor 2, NeuN, synaptophysin, and E-cadherin. The combined morphologic and immunophenotypic findings supported a diagnosis of papillary ependymoma and excluded morphologic mimics.

A 2-year-old Boxer dog was examined for a 2-week history of progressive, painful right-lateralized paraparesis. Magnetic resonance imaging showed a focal ovoid mass with suspected intramedullary, or less likely, intradural location at the level of L3 vertebral body. Based on the imaging features, neoplasia, and specifically, a nephroblastoma, was suspected. Surgical excision found a difficult to excise mass with indistinct borders. The dog showed marked deterioration postoperatively and was euthanized 3 days later due to suspected myelomalacia. Histopathology and immunohistochemistry of the biopsy revealed that the spinal cord was replaced by a poorly organized proliferation of cells that consisted of both neuronal and glial components, embedded within an edematous neuropil. Immunohistochemistry of the lesion showed positivity for neuronal and astrocytic markers. These findings were consistent with a ganglioglioma, a rare neoplasm in dogs.

Gliomatosis cerebri (GC) represents an antemortem diagnostic challenge in the absence of histopathology. Proton magnetic resonance spectroscopy (1H-MRS) features of the disease in humans include elevated myo-inositol (mI)-to-creatine and decreased N-acetyl-aspartate (NAA)-to-creatine ratios. Brain 1H-MRS findings at 3 Tesla (3 T) field strength in dogs with GC have not yet been described. A 12-year-old West Highland White Terrier was presented with a progressive history of multifocal encephalopathy. A 3 T MRI revealed a diffuse, bilateral, ill-defined, intra-axial white matter lesion that was T2W and FLAIR hyperintense, T1W iso- to hypointense, showed no contrast enhancement, and was associated with moderate mass effect. 1H-MRS with voxel positioning at the left parietal area showed highly elevated mI and decreased NAA levels compared to healthy control dogs measured using the same protocol in the thalamus. GC was confirmed by stereotactic brain biopsy. Comparable 1H-MRS changes to those reported in humans were identified in a dog with GC.

Listeria monocytogenes is a human and veterinary pathogen, one of the most common agents of foodborne infections worldwide. It can cause severe complications such as meningitis or miscarriage. Anti-virulence therapies, which target virulence factors such as pore-forming toxins, offer an alternative approach to combating infections. In this study, cholesterol-containing liposomal nanotraps effectively neutralized L. monocytogenes exotoxins, particularly listeriolysin O (LLO), thereby protecting mammalian cells. Notably, toxin neutralization was observed under both neutral and acidic conditions, where LLO activity is optimized to facilitate bacterial escape from the phagosome. Liposomal nanotraps were phagocytosed by macrophages and colocalized with intracellular Listeria, increasing the clearance rate of intracellular bacteria. These findings expand the potential use of broad-spectrum liposomal nanotrap therapy, which could be employed alongside current standard of care treatments to assist the immune system in controlling virulent pathogens.

Gliomatosis cerebri (GC) represents an antemortem diagnostic challenge in the absence of histopathology. Proton magnetic resonance spectroscopy (1H-MRS) features of the disease in humans include elevated myo-inositol (mI)-to-creatine and decreased N- acetyl-aspartate (NAA)-to-creatine ratios. Brain 1H-MRS findings at 3 Tesla (3 T) field strength in dogs with GC have not yet been described. A 12-year-old West Highland White Terrier was presented with a progressive history of multifocal encephalopathy. A 3 T MRI revealed a diffuse, bilateral, ill-defined, intra-axial white matter lesion that was T2W and FLAIR hyperintense, T1W iso- to hypointense, showed no contrast enhancement, and was associated with moderate mass effect. 1H-MRS with voxel positioning at the left parietal area showed highly elevated mI and decreased NAA levels compared to healthy control dogs measured using the same protocol in the thalamus. GC was confirmed by stereotactic brain biopsy. Comparable 1H-MRS changes to those reported in humans were identified in a dog with GC.

BACKGROUND:Ascending-descending myelomalacia (ADMM) is a progressive softening of the spinal cord observed in dogs after spinal cord injury (SCI). On histopathology, areas of hemorrhagic necrotic material are found in the central canal and dorsal funiculi. HYPOTHESIS/OBJECTIVES:We investigated if hemorrhagic necrotic material dorsal to the central canal can be identified on magnetic resonance imaging (MRI). We hypothesized that signal changes are seen in dogs with ADMM, but not in those without ADMM. ANIMALS:Twenty-six dogs with pathologically confirmed ADMM, focal myelomalacia (FM) and 10 control dogs. METHODS:Retrospective case-control study comparing intramedullary signal dorsal to the central canal in dogs with ADMM, FM, and control dogs. RESULTS:A hypointense signal dorsal to the central canal on transverse T2-weighted fast spin echo and gradient echo images was observed. If present in both T2-d T2*-weighted sequences, it was significantly associated with ADMM (p = 0.004; specificity, 81%; sensitivity, 100%). If the T2-weighted hypointense focus was identified at a distance ≥ 3 vertebral bodies from the initial site of spinal cord injury, it was strongly associated with ADMM (p = 0.01) with a specificity of 100% and sensitivity of 78%. CONCLUSION AND CLINICAL IMPORTANCE:A dorsal intramedullary T2-weighted hypointense focus likely represents hemorrhagic necrotic material in the dorsal funiculi. If present at a distance of ≥ 3 vertebral bodies away from the initial site of SCI, it might aid in the diagnosis of ADMM in dogs by MRI.

Bacterial infections of the central nervous system (CNS) pose a significant threat to public health, especially with the growing challenge of antimicrobial resistance. Among these, Listeria monocytogenes (Lm) stands out as a key pathogen, responsible for often fatal neurolisteriosis in humans and cattle. Emerging evidence highlights the distinct roles played by microglia, the resident macrophages of the CNS, and infiltrating monocyte-derived macrophages (MDM) during neuroinflammation. Using bovine models, we investigated the interactions between these two macrophage populations and Lm during infection. Our results show that Lm thrives in the cytosol of microglia, driving productive infection and facilitating bacterial spread. In contrast, MDM effectively sequesters Lm within the phagolysosomal system, limiting its replication and inducing a viable but non-culturable (VBNC) state without completely eliminating the pathogen. Listeriolysin O contributes to the dichotomy of Lm fate, determining whether Lm escapes into the cytosol or transitions to the VBNC state. These findings underscore the complexity of Lm-host dynamics in neurolisteriosis, emphasizing the distinct yet complementary roles of microglia and MDM in shaping CNS infection. By elucidating these mechanisms, our study offers new perspectives on the neurolisteriosis pathogenesis and opens avenues for innovative therapeutic approaches to combat bacterial neuroinfections.

The bacterium Listeria monocytogenes (Lm) causes listeriosis in humans and ruminants. Acute lesions are predominantly infiltrated by polymorphonuclear neutrophils (PMNs), considered to be the efficient bactericidal arm of innate immunity. However, recent evidence suggests that PMNs cannot achieve antilisterial sterilizing immunity and that Lm may persist within PMNs. Despite this, interactions between PMNs and Lm remain poorly understood. In this study, we characterized the listericidal activity and interaction dynamics of bovine PMNs with Lm ex vivo. Phagocytosed Lm failed to escape into the PMN cytosol and was primarily targeted by phagolysosomal mechanisms. However, PMNs enabled prolonged intravacuolar survival of a resilient Lm subpopulation, largely as viable but non-culturable (VBNC) bacteria. This resilient Lm population could spread from PMNs to a cell line, resuscitate, and complete its canonical life cycle, thereby perpetuating the infection. Therefore, we identify PMNs as a mobile niche for Lm survival and provide evidence that PMNs harbor VBNC bacteria, potentially facilitating Lm dissemination within the host. IMPORTANCE:Listeria monocytogenes (Lm) is a significant foodborne pathogen responsible for high hospitalization rates in humans, especially vulnerable groups such as the elderly, pregnant women, and immunocompromised individuals. In animals like ruminants, Lm infection leads to severe disease manifestations, notably brainstem encephalitis. This study uncovers a novel mechanism by which bovine neutrophils (PMNs) harbor Lm in a viable but non-culturable (VBNC) state, enabling the bacteria to hide in the host. PMNs, traditionally viewed as bacteria killers, may serve as Trojan horses, allowing Lm to persist and spread within the host. This discovery has broad implications for understanding Lm's persistence, its role in recurrent infections, and the development of new therapeutic strategies targeting VBNC forms of Lm to improve treatment outcomes and disease control.

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.

We investigated a syndromic disease comprising blindness and neurodegeneration in 11 Saarlooswolfdogs. Clinical signs involved early adult onset retinal degeneration and adult-onset neurological deficits including gait abnormalities, hind limb weakness, tremors, ataxia, cognitive decline and behavioral changes such as aggression towards the owner. Histopathology in one affected dog demonstrated cataract, retinal degeneration, central and peripheral axonal degeneration, and severe astroglial hypertrophy and hyperplasia in the central nervous system. Pedigrees indicated autosomal recessive inheritance. We mapped the suspected genetic defect to a 15 Mb critical interval by combined linkage and autozygosity analysis. Whole genome sequencing revealed a private homozygous missense variant, PCYT2:c.4A>G, predicted to change the second amino acid of the encoded ethanolamine-phosphate cytidylyltransferase 2, XP_038402224.1:(p.Ile2Val). Genotyping of additional Saarlooswolfdogs confirmed the homozygous genotype in all eleven affected dogs and demonstrated an allele frequency of 9.9% in the population. This experiment also identified three additional homozygous mutant young dogs without overt clinical signs. Subsequent examination of one of these dogs revealed early-stage progressive retinal atrophy (PRA) and expansion of subarachnoid CSF spaces in MRI. Dogs homozygous for the pathogenic variant showed ether lipid accumulation, confirming a functional PCYT2 deficiency. The clinical and metabolic phenotype in affected dogs shows some parallels with human patients, in whom PCYT2 variants lead to a rare form of spastic paraplegia or axonal motor and sensory polyneuropathy. Our results demonstrate that PCYT2:c.4A>G in dogs cause PCYT2 deficiency. This canine model with histopathologically documented retinal, central, and peripheral neurodegeneration further deepens the knowledge of PCYT2 deficiency.

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.

Listeria monocytogenes is an ubiquitous environmental saprophytic bacterium causing listeriosis in domestic animals, humans, and occasionally wildlife. In animals, this foodborne zoonotic disease mainly occurs in ruminants and it is rare in carnivores. Seven red foxes (Vulpes vulpes) and one Eurasian lynx (Lynx lynx) were diagnosed with listeriosis between 2010 and 2021 at the Institute for Fish and Wildlife Health, Bern, Switzerland. Necropsy and histopathology revealed meningitis (six of seven red foxes), hepatitis (six of seven red foxes), pneumonia (five of seven red foxes), splenitis (two of seven red foxes) and splenomegaly (the Eurasian lynx, two of seven red foxes). Listeria monocytogenes was isolated from either lung, spleen, liver, or kidney of all animals. Serotyping detected L. monocytogenes serotype 1/2a in five red foxes and the Eurasian lynx and serotype 4b in two red foxes. Six red foxes were positive for canine distemper virus (CDV) by polymerase chain reaction, whereas the Eurasian lynx and one red fox were negative. One red fox that was positive for CDV and listeriosis was also diagnosed with salmonellosis. The identified L. monocytogenes serotypes are among the three most frequently isolated serotypes (1/2a, 1/2b, and 4b) from food or the food production environment and those that cause most listeriosis cases in humans and animals. Coinfection with CDV in six red foxes questions the role of CDV as potential predisposing factor for septicemic listeriosis. The detection of listeriosis in the regionally endangered Eurasian lynx and in carnivores highly abundant in urban settings, such as red foxes, reinforces the importance of wildlife health surveillance in a One Health context and adds the Eurasian lynx to the list of carnivores susceptible to the disease. Further investigations are required to assess the prevalence and epidemiology of L. monocytogenes in free-ranging carnivores and its interaction with CDV.

ObjectivesThe 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).MethodsSeven 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.ResultsIn 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.ConclusionsSSTRs 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. center dot Canine and feline meningiomas express somatostatin receptors (SSTRs).center dot RNA analysis showed SSTR1, SSTR2 and SSTR5 expressions in canine meningiomas.center dot Feline meningiomas exhibited SSTR1, SSTR4, SSTR5 and SSTR2 expressions.center dot Immunohistochemistry confirmed SSTR1 and SSTR2 expressions in both species.center dot There might be a potential for adjunctive therapy with long-acting somatostatin analogues, but follow-up studies with larger groups are required. Potentials of analogues are not evaluated in this study. image

A 9-month-old Cavalier King Charles Spaniel with 3-month history of paroxysmal episodes of cervical pain and scratching had a magnetic resonance imaging revealing a large intra-axial cystic lesion in the left frontal lobe, caudal transtentorial and foraminal brain herniation, and cervical syringomyelia. Despite suspected high-grade glioma, clinical signs improved under palliative treatment. Follow-up magnetic resonance imaging 2.5 years later showed a similar lesion size, with no radiological evidence of increased intracranial pressure. The differential diagnosis was reconsidered to possibly porencephaly, congenital cystic malformation or parasitic cyst. The dog remained clinically stable for 19 months before developing acute generalised, single, self-limiting seizures, deteriorating to cluster seizures, stupor, cardiovascular arrest and death. Postmortem magnetic resonance imaging revealed lesion progression with ventricle invasion. Neuropathology confirmed a high-grade oligodendroglioma with subarachnoid and intraventricular drop metastasis. This is the first report describing long-term clinical course and magnetic resonance imaging lesion progression under palliative treatment of a dog ultimately diagnosed with high-grade oligodendroglioma.

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.

Canine and human brain tumours exhibit similar incidence rates and prognoses. Recent studies have demonstrated that extracellular vesicles derived from human patients (PDEVs) can be loaded with contrast agents and exhibit tumour tropism in murine models. We showed in a previous study that gadolinium-labelled EVs derived from canine gliomas (cPDEVs) can selectively targets murine glioblastoma cells in animal models. As a further step, we investigated the potential heterologous and cross-species tumour tropism of cPDEVs with brain tumours. With the perspective of imminent clinical application as both markers and drug delivery tools, we have successfully established the isolation protocol for cPDEVs and confirmed the aseptic conditions of the procedure and therefore the sterility of the isolated EVs. To assess the functionality of cPDEVs as drug delivery tool, they were loaded with indocyanine green (ICG) and injected into murine models of cancer for in vivo fluorescence biodistribution studies. Biodistribution analysis in mice revealed that ICG-loaded cPDEVs injected into murine models of subcutaneous tumours accumulated exclusively in the neoplastic tissue, even when evaluated 24 h post-injection, thus showing the cross-species and heterologous selective tumour tropism of the nanoparticles. With these tests, we have established a safe protocol for isolating and loading autologous cPDEVs with various markers, thereby paving the way for the clinical testing phase. These significant findings suggest the potential use of cPDEVs as a theranostic tool in the management of canine brain tumours, with promising implications for translational medicine applications in the future.