Retrospective immunohistochemical investigation on dolphin morbillivirus infection by comparing the performance of heterologous monoclonal and polyclonal antibodies - Short communication.

Dolphin morbillivirus (DMV) is a pathogen of great concern in free-ranging cetaceans. Confirmation and staging of morbillivirus infections rely on histology and immunohistochemistry (IHC), following molecular detection. As at the present time no specific antibodies (Abs) against DMV are available, two heterologous Abs have been used worldwide for the examinations of morbillivirus infections of cetaceans. One is a monoclonal Ab (MoAb) prepared against the N protein of canine distemper virus (CDV), whereas the other is a polyclonal Ab raised in rabbits against rinderpest virus (RPV). Both Abs are known to show cross-reactivity with DMV. In this study we compared the labelling quality and the neuroanatomical distribution of staining with these two Abs by means of IHC analysis. To this end, serial sections of the target organs from ten free-ranging stranded cetaceans, previously diagnosed as being infected with DMV by PCR and/or serology, were subjected to IHC. The brain, lungs and lymph nodes of one animal were found to be positive with both Abs. From two other animals, the brain and the spleen, respectively, tested positive only with the polyclonal Ab. In the positive brain tissues, multifocal immunostaining was observed, with similar staining location and extent, with the two antibodies tested. Our results suggest that the polyclonal anti-RPV Ab might have a stronger binding activity to DMV than the anti-CDV MoAb. Nevertheless, the elaboration and use of specific anti-DMV Abs might be essential to guarantee conclusive results in diagnostic and pathogenetic investigations.

Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS.

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices.

Novel dolphin morbillivirus (DMV) outbreak among Mediterranean striped dolphins Stenella coeruleoalba in Italian waters.

An unusual mortality event (UME) of striped dolphins Stenella coeruleoalba occurred in the period July to December 2016 along the Italian Ionian coastline. We conducted a complete postmortem examination on 28 specimens and detected dolphin morbillivirus (DMV), by means of biomolecular analyses, in the target tissues of 17 animals. Unlike previous outbreaks occurring in the Mediterranean Sea in 2011 and 2013, we observed typical pathological changes suggestive of morbilliviral infection in an acute/subacute phase and immunohistochemical reactivity. The same findings were observed in 13 other specimens beached along the Italian coastline during 2016 with no temporal and geographical relationship with the ongoing epidemic outbreak. Molecular characterization and phylogenetic analysis showed that DMV sequences detected in Italy in 2016 clustered with those identified in Portugal and Galicia (Spain), representing a novel DMV strain of Atlantic origin which entered the Mediterranean Sea and affected a naïve striped dolphin population. DMV sequences detected in the previous Mediterranean outbreaks exhibited a marked genetic relatedness and diverged from those detected in cetaceans stranded along the Galician and Portuguese coasts since 2007.

Occlusive mycotic tracheobronchitis and systemic Alphaherpesvirus coinfection in a free-living striped dolphin Stenella coeruleoalba in Italy.

A juvenile female striped dolphin Stenella coeruleoalba live stranded on 4 March 2016 at Alassio, western Ligurian Sea coast, Italy. The dolphin died shortly after stranding, and a complete postmortem examination was performed. Necropsy revealed severe tracheal occlusion and unilateral bronchial stenosis with luminal accumulation of abundant green-yellow mucous-gelatinous material. Histological features suggestive of tracheobronchial aspergillosis were observed. Cultures of lung tissue and tracheo-bronchial exudate isolated Aspergillus fumigatus, identified by a Microseq D2 LSUrDNA fungal sequencing kit. A pan-Herpesvirus nested-PCR assay on frozen samples obtained from multiple organs was positive. Phylogenetic analysis on the partial DNA polymerase gene revealed that the striped dolphin isolate was closely related to known cetacean Alphaherpesvirus sequences from the same host species. Attempted virus isolation was unsuccessful. The tissue levels of different persistent organic pollutants and the toxicological stress, evaluated using a theoretical model, showed a severely impaired immune response. This study reports the first case of occlusive mycotic tracheobronchitis in a free-living cetacean and the first molecular identification of an Alphaherpesvirus in a free-ranging striped dolphin stranded on the coast of Italy.

Meningoencephalitis and Listeria monocytogenes, Toxoplasma gondii and Brucella spp. coinfection in a dolphin in Italy.

Listeria monocytogenes, Toxoplasma gondii and Brucella spp. can infect a wide range of species, including humans. In cetaceans, meningoencephalitis has been associated with T. gondii and Brucella spp. infection, whereas to our knowledge, L. monocytogenes infection has not previously been reported. Meningoencephalitis and L. monocytogenes, T. gondii and Brucella spp. were identified by means of both direct and indirect laboratory techniques in an adult female striped dolphin Stenella coeruleoalba found stranded in January 2015 on the Ligurian Sea coast, northwestern Italy. The animal was emaciated, and histopathology disclosed severe meningoencephalitis. The nature of the inflammatory response and intra-lesional protozoa were consistent with a mixed infection by L. monocytogenes, T. gondii and Brucella spp. We believe this is an unprecedented case of infection by 3 zoonotic pathogens and also the first bacteriologically confirmed case report of neurolisteriosis in cetaceans. Cerebral toxoplasmosis and neurobrucellosis may have led to the animal's disorientation and stranding, with L. monocytogenes having likely exacerbated the coinfection leading to the demise of this dolphin.

Ruminant rhombencephalitis-associated Listeria monocytogenes strains constitute a genetically homogeneous group related to human outbreak strains.

Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cow's milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.

Feasibility of 16S rRNA sequencing for cerebrospinal fluid microbiome analysis in cattle with neurological disorders: a pilot study.

Bacterial infection of the central nervous system (CNS) in cattle requires prompt and adequate antimicrobial treatment. The current gold standard for antemortem etiological diagnosis is cerebrospinal fluid (CSF) culture, which often yields false negative results. CSF has long been considered a sterile district in healthy patients, but this notion has been recently challenged. For this pilot study, we used 16S rRNA gene sequencing to investigate the microbial composition of CSF of cattle presenting with CNS disorders and to compare it between subjects with CNS infections and with CNS disorders of other nature. The study sample was 10 animals: 4 presenting with CNS infectious-inflammatory diseases and 6 with other CNS disorders, based on definitive diagnosis. Since the initial round of a standard 16S rRNA PCR did not yield sufficient genetic material for sequencing in any of the samples, the protocol was modified to increase its sensitivity. Bacterial genetic material was identified in 6 animals and 2 groups were formed: an infectious inflammatory (n = 3) and a noninfectious inflammatory group (n = 3). The most frequently expressed bacterial families were Pseudomonadaceae (44.61%), Moraxellaceae (19.54%), Mycobacteriaceae (11.80%); the genera were Pseudomonas (45.42%), Acinetobacter (19.91%), Mycobacterium (12.01%). There were no detectable differences in the CSF microbial composition of the samples from the two groups. Sequencing of bacterial DNA present in the CSF was possible only after increasing PCR sensitivity. The results of 16S rRNA sequencing showed the presence of a microbial community in the CSF in cattle with neurological disorders. Further studies, in which CSF samples from healthy animals and samples from the environment are included as controls, are needed.

A novel machine learning-based web application for field identification of infectious and inflammatory disorders of the central nervous system in cattle.

BACKGROUND: Central nervous system (CNS) infections in cattle are a major cause of economic loss and mortality. Machine learning (ML) techniques are gaining widespread application in solving predictive tasks in both human and veterinary medicine. OBJECTIVES: Our primary aim was to develop and compare ML models that could predict the likelihood of a CNS disorder of infectious or inflammatory origin in neurologically-impaired cattle. Our secondary aim was to create a user-friendly web application based on the ML model for the diagnosis of infection and inflammation of the CNS. ANIMALS: Ninety-eight cattle with CNS infection and 86 with CNS disorders of other origin. METHODS: Retrospective observational study. Six different ML methods (logistic regression [LR]; support vector machine [SVM]; random forest [RF]; multilayer perceptron [MLP]; K-nearest neighbors [KNN]; gradient boosting [GB]) were compared for their ability to predict whether an infectious or inflammatory disease was present based on demographics, neurological examination findings, and cerebrospinal fluid (CSF) analysis. RESULTS: All 6 methods had high prediction accuracy (≥80%). The accuracy of the LR model was significantly higher (0.843 ± 0.005; receiver operating characteristic [ROC] curve 0.907 ± 0.005 ) than the other models and was selected for implementation in a web application. CONCLUSION AND CLINICAL IMPORTANCE: Our findings support the use of ML algorithms as promising tools for veterinarians to improve diagnosis. The open-access web application may aid clinicians in achieving correct diagnosis of infectious and inflammatory neurological disorders in livestock, with the added benefit of promoting appropriate use of antimicrobials.

First Swedish case of fatal equine parasitic encephalitis by Halicephalobus gingivalis.

BACKGROUND: Halicephalobus gingivalis is a nematode with zoonotic potential which can cause fatal opportunistic infections in various mammals. The parasite has never been diagnosed in Sweden, in any species, prior to the presented case. CASE PRESENTATION: An imported 21-year-old Icelandic mare developed severe neurological signs. The horse was eventually euthanized and submitted for post-mortem examination where severe lesions in the kidneys were noted. Histopathology revealed the presence of H. gingivalis in both kidneys and the brain. Phylogenetic analysis of the parasite determined it to belong to Lineage 1. CONCLUSIONS: With the occurrence of H. gingivalis in Sweden, the disease should be added to the list of differential diagnoses in cases with acute onset of neurological disease in both horses and other mammals including humans.

Brucella ceti Infection in Striped Dolphins from Italian Seas: Associated Lesions and Epidemiological Data.

Brucella ceti infections have been increasingly reported in cetaceans. In this study, we analyzed all cases of B. ceti infection detected in striped dolphins stranded along the Italian coastline between 2012 and 2021 (N = 24). We focused on the pathogenic role of B. ceti through detailed pathological studies, and ad hoc microbiological, biomolecular, and serological investigations, coupled with a comparative genomic analysis of the strains. Neurobrucellosis was observed in 20 animals. The primary histopathologic features included non-suppurative meningoencephalitis (N = 9), meningitis (N = 6), and meningoencephalomyelitis (N = 5), which was also associated with typical lesions in other tissues (N = 8). Co-infections were detected in more than half of the cases, mostly involving Cetacean Morbillivirus (CeMV). The 24 B. ceti isolates were assigned primarily to sequence type 26 (ST26) (N = 21) and, in a few cases, ST49 (N = 3). The multilocus sequence typing (cgMLST) based on whole genome sequencing (WGS) data showed that strains from Italy clustered into four genetically distinct clades. Plotting these clades onto a geographic map suggests a link between their phylogeny and the topographical distribution. These results support the role of B. ceti as a primary neurotropic pathogen for striped dolphins and highlight the utility of WGS data in understanding the evolution of this emerging pathogen.

Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene.

Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP). Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type) and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002). Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.

Chronic Wasting Disease Monitoring in Italy 2017-2019: Neuropathological Findings in Cervids.

Chronic wasting disease (CWD) is a prion disease that affects cervids; it is classified under transmissible spongiform encephalopathies (TSEs). CWD is particularly contagious, making its eradication in endemic areas very difficult and creating serious problems for cervid conservation and breeding. It has recently become an emerging public health risk to be managed by health authorities. Starting in 2017, active CWD surveillance in Italy has intensified with the monitoring of wild and farmed cervids. The present study summarizes findings from a histopathological survey of the brains from wild ruminants collected via CWD monitoring between 2017 and 2019. A total of 113 brains from 62 red deer (Cervus elaphus) and 51 roe deer (Capreolus capreolus) were submitted for analysis at the National Reference Center for Animal Encephalopathies (CEA) to determine major patterns of neuropathological lesions and correlated pathogens. Brain lesions were detected in 20 animals, 10 brain samples were unsuitable for examination, and 83 presented no lesions. Neuropathological examination revealed non-suppurative encephalitis or meningoencephalitis in most cases (15/20). This brain study revealed evidence for the absence of CWD in Italy and provided a reference spectrum of neuropathological lesions for differential diagnosis in cervids.

Neuropathological Characterization of Dolphin Morbillivirus Infection in Cetaceans Stranded in Italy.

Cetacean morbillivirus (CeMV) is responsible for epidemic and endemic fatalities in free-ranging cetaceans. Neuro-inflammation sustained by CeMV is a leading cause of death in stranded cetaceans. A novel dolphin morbillivirus (DMV) strain of Atlantic origin circulating in Italian waters since early 2016 has caused acute/subacute lesions associated with positive immunolabelling of the virus. To date, myelin damage has not been fully documented and investigated in cetaceans. This study describes neuropathological findings in the brain tissue of 31 cetaceans found stranded along the Italian coastline and positive for DMV infection on molecular testing. Cell changes in the areas of myelinopathy were revealed by double indirect immunofluorescence. The most frequent DMV-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Myelin reduction and areas of demyelination were revealed by means of a specific myelin biomarker. Morbilliviral antigen immunolabelling was mainly observed in neurons and microglial cells, in association with a marked activation of microglia and astrocytes. These findings extend our knowledge of DMV-associated brain lesions and shed light on their pathogenesis.

Ticks infesting humans and associated pathogens: a cross-sectional study in a 3-year period (2017-2019) in northwest Italy.

BACKGROUND: Tick-borne diseases are common throughout Europe. Ticks transmit pathogens to the host while feeding and together with mosquitoes, they are major vectors of infectious agents worldwide. In recent years, there has been a marked increase in the incidence of tick-bite events and tick-borne disease in northwest Italy, but information on the prevalence of tick-borne pathogens in ticks removed from humans remains scarce. To fill this gap, we report here the prevalence of tick bites and tick-borne pathogens documented for humans in Piedmont, northwest Italy, in the 3-year period 2017-2019. METHODS: Ticks attached to humans during 2017-2019 were collected from residents of urban and rural area by physicians and veterinarians working with local veterinary agencies. All ticks (n = 1290) were morphologically identified to the species level. A subset of ticks removed from children (age 0-18 years) and the elderly (> 70 years), both age groups considered to be at-risk populations, was screened by biomolecular analysis to detect pathogens (e.g. Rickettsia spp., Borrelia spp., Anaplasma spp.). Pathogen identity was confirmed by Sanger sequencing. RESULTS: Ticks were taxonomically assigned to ten species of six genera (Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus). Most belonged to the genus Ixodes: 1009 ticks (78.22%) were classified as Ixodes ricinus. A subset of 500 ticks collected from the two at-risk populations were subjected to PCR assay to determine the presence of Rickettsia spp., Borrelia spp., and Anaplasma spp. The overall prevalence of infection was 22.8% (n = 114; 95% confidence interval [CI]: 19.19-26.73%), meaning that at least one pathogen was detected: Rickettsia spp. (prevalence 15%, n = 76; 95% CI 12.17-18.65%); Borrelia spp. (prevalence 6.4%, n = 32; 95% CI 4.42-8.92%); and Anaplasma spp. (prevalence 1.2%, n = 6; 95% CI 0.44-2.6%). CONCLUSIONS: Our data underline the importance of surveillance in the epidemiology of tick-borne diseases and the implementation of strategies to control tick infestation and associated pathogens.

Evidence for Unknown Sarcocystis-Like Infection in Stranded Striped Dolphins (Stenella coeruleoalba) from the Ligurian Sea, Italy.

Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of Toxoplasma gondii, additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large Sarcocystis-like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude cross-reaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols precluded a neural infection by Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi, and Neospora caninum. T. gondii coinfection was confirmed only in dolphin SD2. Sarcocystis sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of Sarcocystis-like tissue cysts in the brain of stranded cetaceans along with the first description of Sarcocystis sp. infection in muscle tissue of dolphins from the Mediterranean basin.

Variability in disease phenotypes within a single PRNP genotype suggests the existence of multiple natural sheep scrapie strains within Europe.

Variability of pathological phenotypes within classical sheep scrapie cases has been reported for some time, but in many instances it has been attributed to differences in the PRNP genotype of the host. To address this issue we have examined by immunohistochemistry (IHC) and Western blotting (WB) for the disease-associated form of the prion protein (PrP(d)), the brains of 23 sheep from five European countries, all of which were of the same ARQ/ARQ genotype. As a result of IHC examinations, sheep were distributed into five groups with different phenotypes and the groups were the same regardless of the scoring method used, 'long' or 'short' PrP(d) profiling. The groups made did not respond to the geographical origin of the cases and did not correlate with the vacuolar lesion profiles, which showed a high individual variability. Discriminatory IHC and WB methods coincided to detect a 'CH1641-like' case but otherwise correlated poorly in the classification of disease phenotypes. No other polymorphisms of the PRNP gene were found that could account for the pathological differences, except perhaps for a sheep from Spain with a mutation at codon 103 and a unique pathological phenotype. Preliminary evidence indicates that those different IHC phenotypes correlate with distinct biological properties on bioassay, suggesting that they are indicative of strain diversity. We therefore conclude that natural scrapie strains exist and that they can be revealed by detailed pathological examinations, which can be harmonized between laboratories to produce comparable results.

Olfactory system involvement in natural scrapie disease.

The olfactory system (OS) is involved in many infectious and neurodegenerative diseases, both human and animal, and it has recently been investigated in regard to transmissible spongiform encephalopathies. Previous assessments of nasal mucosa infection by prions following intracerebral challenge suggested a potential centrifugal spread along the olfactory nerve fibers of the pathological prion protein (PrP(Sc)). Whether the nasal cavity may be a route for centripetal prion infection to the brain has also been experimentally studied. With the present study, we wanted to determine whether prion deposition in the OS occurs also under field conditions and what type of anatomical localization PrP(Sc) might display there. We report here on detection by different techniques of PrP(Sc) in the nasal mucosa and in the OS-related brain areas of sheep affected by natural scrapie. PrP(Sc) was detected in the perineurium of the olfactory nerve bundles in the medial nasal concha and in nasal-associated lymphoid tissue. Olfactory receptor neurons did not show PrP(Sc) immunostaining. PrP(Sc) deposition was found in the brain areas of olfactory fiber projection, chiefly in the olfactory bulb and the olfactory cortex. The prevalent PrP(Sc) deposition patterns were subependymal, perivascular, and submeningeal. This finding, together with the discovery of an intense PrP(Sc) immunostaining in the meningeal layer of the olfactory nerve perineurium, at the border with the subdural space extension surrounding the nerve rootlets, strongly suggests a probable role of cerebrospinal fluid in conveying prion infectivity to the nasal submucosa.

Intraspecies transmission of BASE induces clinical dullness and amyotrophic changes.

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrP(TSE)), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrP(TSE) type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.

Intracranial squamous cell carcinoma in an Ovis aries.

Squamous cell carcinoma (SCC) is a malignant mucoepithelial tumor that affects pets and farm animals. Common sites are dorsal areas and/or areas of poor skin pigmentation exposed to mutagenic ultraviolet (UV) radiation. Novel ovine papillomavirus (OaPV3) was recently described in SCC lesions in Sardinia breed ovines. In 2017, a 7­year­old half­breed aries was presented with symptoms compatible with a vestibular syndrome. The animal was euthanized 1 month after the onset of clinical signs due to a lack of response to treatment and poor prognosis. A complete postmortem examination was performed. Necropsy revealed only a loss of incisors, associated with alveolar necrotic osteomyelitis, and left unilateral purulent nasal discharge. No other thoracic or abdominal lesions were observed. Opening of the skull revealed a cauliflower­like space­occupying mass. Histological examination showed trabecules and islands of squamous, neoplastic epithelial cells with the formation of concentric keratin layers. This raised the suspicion of SCC, which was confirmed with cytokeratin­positive immunostaining. Simplex PCR on the frozen tissue mass was negative for OaPV1, OaPV2, and OaPV3. This case report suggests that SCC, although rare, should be included in the differential diagnosis of cases of vestibular disorder.

Chondroitin Sulphate Proteoglycan 4 (NG2/CSPG4) Localization in Low- and High-Grade Gliomas.

BACKGROUND: Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines. METHODS: NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques. RESULTS: NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors. CONCLUSION: The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.