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BACKGROUND: The blink reflex excitability, assessed through paired electrical stimuli responses, has been modulated using traditional non-invasive neurostimulation techniques. Recently, transcranial Alternating Current Stimulation (tACS) emerged as a tool to modulate brain oscillations implicated in various motor, perceptual, and cognitive functions. This study aims to investigate the influence of 20-Hz and 10-Hz tACS sessions on the primary motor cortex and their impact on blink reflex excitability. MATERIALS AND METHODS: Fifteen healthy volunteers underwent 10-min tACS sessions (intensity 1 mA) with active/reference electrodes placed over C4/Pz, delivering 20-Hz, 10-Hz, and sham stimulation. The blink reflex recovery cycle (BRrc) was assessed using the R2 amplitude ratio at various interstimulus intervals (ISIs) before (T0), immediately after (T1), and 30 min post-tACS (T2). RESULTS: Both 10-Hz and 20-Hz tACS sessions significantly increased R2 ratio at T1 (10-Hz: p = 0.02; 20-Hz: p < 0.001) and T2 (10-Hz: p = 0.01; 20-Hz: p < 0.001) compared to baseline (T0). Notably, 20-Hz tACS induced a significantly greater increase in blink reflex excitability compared to sham at both T1 (p = 0.04) and T2 (p < 0.001). CONCLUSION: This study demonstrates the modulatory effect of tACS on trigemino-facial reflex circuits, with a lasting impact on BRrc. Beta-band frequency tACS exhibited a more pronounced effect than alpha-band frequency, highlighting the influential role of beta-band oscillations in the motor cortex on blink reflex excitability modulation.
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BACKGROUND: A wealth of evidence underscores the bioactive properties of nutraceuticals and functional foods in addressing oxyinflammatory-based diseases with implications at both peripheral and central levels. Opuntia ficus-indica (OFI) is well-documented for its health-promoting attributes, though its fruit (OFIF) remains relatively understudied. Not only poses Metabolic Syndrome (MetS) cardiometabolic risks but also contributes significantly to cognitive impairment, especially in crucial brain areas such as hippocampus and hypothalamus. METHODS: Following 8 weeks of HFD to induce MetS, rats received OFIF oral supplementation for 4 weeks to evaluate cognitive and affective modifications using behavioural paradigms, i.e. open field, burrowing, white-dark box, novelty-suppressed feeding, and object recognition tests. Our investigation extended to biochemical evaluations of lipid homeostasis, central and peripheral oxidative stress and neurotrophic pathways, correlating these measures together with circulating leptin levels. RESULTS: Our data revealed that OFIF modulation of leptin positively correlates with systemic and brain oxidative stress, with markers of increased anxiety-like behaviour and impaired lipid homeostasis. On the other hand, leptin levels reduced by OFIF are associated with improved antioxidant barriers, declarative memory and neurotrophic signalling. DISCUSSION: This study underscores OFIF neuroactive potential in the context of MetS-associated cognitive impairment, offering insights into its mechanisms and implications for future therapeutic strategies.
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Cognição , Dieta Hiperlipídica , Modelos Animais de Doenças , Frutas , Metabolismo dos Lipídeos , Síndrome Metabólica , Opuntia , Estresse Oxidativo , Animais , Estresse Oxidativo/efeitos dos fármacos , Opuntia/química , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/metabolismo , Masculino , Cognição/efeitos dos fármacos , Ratos , Dieta Hiperlipídica/efeitos adversos , Metabolismo dos Lipídeos/efeitos dos fármacos , Ratos Wistar , Leptina/sangue , Leptina/metabolismo , Extratos Vegetais/farmacologia , Antioxidantes/farmacologia , Comportamento Animal/efeitos dos fármacos , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/tratamento farmacológicoRESUMO
Background: Meningioma is the most common tumor of the central nervous system of dogs. For this tumor, surgery remains the treatment of choice, either alone or in combination with radiotherapy. Unfortunately, chemotherapeutic strategies are practically absent in dogs and palliative therapies are the only option to surgery. Somatostatin receptor subtype 2 (SSTR2) is expressed in canine meningioma. Since the potent cell-proliferation inhibiting effect of somatostatin (SST), the aim of this study was to investigate in vitro the effects of octreotide, as SST analog, in the viability of canine meningioma. Methods: Four surgical canine meningiomas were used in this study to establish cell cultures. Expression of SSTR2 was verified with immunolabelling in FFPE samples and cell cultures. The effects of octreotide on cell viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). After 24 hours they were exposed to different concentrations of octreotide (0.1 nM, 1 nM, 10 nM, 100 nM) for 24 and 48 hours. Results: All meningiomas consisted of grade I tumors. The cultured neoplastic cells expressed SSTR2 from 80% to 100%. Octreotide significantly increased cell death after 48 hours of continuous exposure, with 10 and 100 nM octreotide doses. The percentage of cell viability was 80.92â ±â 4.9 and 80.49â ±â 3.61, compared to the control, respectively, consistent with decreased cell viability of about 20% for both doses. Conclusions: Octreotide reduced the alive neoplastic cultured cells of low-grade canine meningioma in a dose-dependent pattern with continuous exposition for 48 hours. These results support an alternative systemic treatment of meningioma with octreotide in the dog.
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BACKGROUND: Simulating the post-traumatic continuity defect of small human peripheral nerves, we compared the effectiveness of fibrin glue with neurorrhaphy for nerve gap restoration. METHODS: In twenty-four male Wistar rats, a fifteen mm defect in one sciatic nerve only was made and immediately repaired with an inverted polarity autograft. According to the used technique, rats were divided into Group A (Control), using traditional neurorrhaphy, and Group B (Study), using fibrine glue sealing; in total, 50% of rats were sacrificed at 16 weeks and 50% at 21 weeks. Before sacrifice, an assessment of motor function was done through Walking Track Analysis and an electroneurophysiological evaluation. After sacrifice, selected muscle mass indexes and the histology of the regenerated nerves were assessed. All data were evaluated by Student's t test for unpaired data. RESULTS: No significant differences were found between the two groups, with only the exception of a relative improvement in the tibialis anterior muscle's number of motor units in the study group. CONCLUSION: Despite the fact that the use of fibrin glue as a nerve sealant is not superior in terms of functional recovery, its effectiveness is comparable to that of microsurgical repair. Hence, the faster and technically easier glueing technique could deserve broader clinical application.
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In felines, ocular and nonocular melanomas are uncommon tumors that represent a diagnostic challenge for pathologists, especially when amelanotic. To date, the immunohistochemical diagnostic panel in cats is based on specific melanocytic markers (Melan-A and PNL2) and a nonspecific but sensitive marker (S100). In human medicine, SOX-10 is reported to be a sensitive antibody for the detection of melanoma micrometastasis in the lymph node. TRP-1, an enzyme involved in melanogenesis, has recently been used in humans and dogs as a specific melanocyte marker. The aim of this study was to evaluate the cross-reactivity and the expression of SOX-10 and TRP-1 antibodies in feline normal tissue and melanocytic tumors. Thirty-one cases of ocular, cutaneous, and oral melanomas were retrospectively evaluated and confirmed by histopathological examination and by immunolabeling with Melan-A and/or PNL2. SOX-10 nuclear expression in normal tissues was localized in epidermal, subepidermal, hair bulb, and iridal stromal melanocytes and dermal nerves. In melanomas, nuclear expression of SOX-10 was detected in ocular (11/12; 92%), oral (6/7; 86%), and cutaneous sites (12/12; 100%). TRP-1 cytoplasmic immunolabeling in normal tissue was observed in epidermal and bulbar melanocytes and in the lining pigmented epithelium of the iris and in its stroma. Its expression was positively correlated to the degree of pigmentation in the tumor and was observed in 75% of ocular (9/12), 43% of oral (3/7), and 33% of cutaneous melanomas (4/12). This study demonstrated the cross-reactivity of SOX-10 and TRP-1 antibodies in feline non-neoplastic melanocytes and their expression in ocular and nonocular melanomas.
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BACKGROUND: Vascularized nerve grafts (VNGs) have been proposed as a superior alternative to free nerve grafts (FNGs) for complex nerve defects. A greater regenerative potential has been suggested by clinical and experimental studies, but conclusive evidence is still lacking. METHODS: In this experimental study, 10 adult male Wistar rats received a non-vascularized orthotopic sciatic nerve graft on their right side, and a vascularized orthotopic sciatic nerve graft nerve on their left side. Functional outcome following nerve regeneration was evaluated through electrodiagnostic studies, target muscles weight and histomorphology, and data of VNGs and FNGs were compared. RESULTS: The results of this study showed a significant difference in the motor unit number of Gastrocnemius Medialis (GM) estimated by MUNE in the VNG side compared to the FNG side. No other significant differences in axonal regeneration and muscle reinnervation were evident at either electrodiagnostic, histomorphology studies or muscle weight. CONCLUSIONS: This experimental model showed slight differences in nerve regeneration between VNGs and FNGs, but cannot support a high clinical advantage for VNGs. The results of this study show that VNGs are not strongly superior to FNGs in the rat model, even in avascular beds. Clinical advantages of VNGs are likely to be limited to extensive and thick nerve defects and can only be assessed on experimental model with bigger animals. Also, we showed that the MUNE technique provided a reliable and reproducible evaluation of functional outcomes in the rat sciatic nerve and defined a reproducible protocol for functional evaluation of muscle reinnervation.
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Introduction: Nonalcoholic fatty liver disease (NAFLD), characterized by lipid accumulation within hepatocytes exceeding 5% of liver weight, is strongly related to metabolic disorders, obesity, and diabetes and represents a health emergency worldwide. There is no standard therapy available for NAFLD. Lifestyle intervention, including phytonutrient intake, is key in preventing NAFLD development and progression. Methods: We used a rat model of NAFLD to evaluate the effect of dietary supplementation with red tomato (RT) and golden tomato (GT)-a patented mix of fruit with varying degrees of ripeness and particularly rich in naringenin and chlorogenic acid-after steatosis development. We assessed the effects on body weight, metabolic profile, and hepatic steatosis. Results and discussion: We found a correlation between the amelioration of all the parameters and the liver gene expression. Our results showed that, together with the reversion of steatosis, the consumption of RT and GT can cause a significant reduction in triglycerides, low-density lipoprotein-cholesterol, fasting glucose, and homeostasis model assessment index. Meanwhile, we observed an increase in high-density lipoprotein-cholesterol according to the amelioration of the general lipidic profile. Regarding hepatic gene expression, we found the upregulation of Gk and Hnf4α involved in metabolic homeostasis, Lepr involved in adipokine signaling, and Il6 and Tnf involved in inflammatory response. Taken together, our results suggest that dietary intake of red and golden tomatoes, as a nutraceutical approach, has potential in preventing and therapeutics of NAFLD.
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Mosquito-borne flaviviruses are emerging pathogens with zoonotic potential. Due to the recent climate and environmental changes, they are spreading across Europe, becoming a major threat for public and veterinary health. West Nile virus (WNV) and Usutu virus (USUV) are arboviruses that are responsible for multiple disease outbreaks in different species of birds, reptiles, and mammals, including humans. This review reports and compares the clinical signs as well as the gross and microscopic pathological features during natural infection with WNV and USUV in wild and domestic animals, as well as in humans. The main objective of this comparative review is to delineate the common features and the specific differences that characterize WNV- and USUV-induced diseases in each group of species and to highlight the main gaps in knowledge that could provide insight for further investigation on the pathogenesis and neurovirulence of these viruses.
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Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus causing mortality in Eurasian blackbirds (Turdus merula) in Europe. In dead blackbirds, avian malaria co-infection due to mosquito-borne hemosporidians (e.g., Plasmodium spp.) has been reported. In humans, a similar co-infection of a flavivirus, Dengue virus, and Plasmodium spp. is causing increased severity of clinical disease. Currently, the effects of co-infection of arboviruses and hemosporidians in blackbirds remain unclear. This study investigates the rate of USUV and Plasmodium spp. co-infection in found-dead blackbirds (n = 203) from 2016 to 2020 in the Netherlands. Presence of Plasmodium spp. was evaluated by cytology (43/203; 21,2%), histopathology (94/186; 50,5%) and qPCR (179/203; 88,1%). The severity of histological lesions in USUV and Plasmodium spp. co-infected dead blackbirds (121/203; 59,6%) were compared with those in Plasmodium spp. single-infected cases. Additionally, since no knowledge is present on the infection rate on live birds and mosquitoes in the Netherlands, a small group of live blackbirds (n = 12) and selected in the field-collected mosquito pools (n = 96) in 2020 were tested for the presence of Plasmodium spp. The latter was detected in the tested live blackbirds by qPCR (8/10; 80%), and cytology (3/11; 27,3%) and in the mosquito pools by qPCR (18/96; 18,7%). For this study, co-infection between USUV and Plasmodium spp. was observed only in the dead blackbirds. The high Plasmodium spp. presence, associated with lower lesions score, in single infected found dead birds suggest a predominantly smaller pathogenic role as single agent. On the other hand, the higher histological lesion scores observed in USUV and Plasmodium spp. co-infected birds suggests a major pathogenic role for the virus or an increased severity of the lesions due to a possible interplay of the two agents.
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Pseudoneglect phenomenon refers to a condition in which healthy subjects tend to perceive the left side of exactly bisected lines as being slightly longer than the right one. However, behavioural data showed that athletes practising an open-skill sport display less pseudoneglect than the general population. Given the fact that so-called exergames (also known as active video games) are platforms designed to fully mimic sport activity, this work intends to investigate whether and how a one-week training period of exergame open-skill sport can determine a similar decrease in pseudoneglect. Fifteen healthy participants (non-athletes) responded to a visuospatial attention task and a control memory task in basal conditions (t0: Pre-game) and after a short period (one week, one hour/day) of tennis exergaming (t1: Post-game). In the Post-game condition, subjects from this experimental group (ExerGame group: EG) reduced leftward space overestimation and made significantly fewer leftward errors compared to the Pre-game condition. Additionally, two other experimental groups were employed: one evaluated within the same conditions of the main experiment but using a non-exergame (Non-Exergame groups: NEG) and the other one without any video game stimulus (Sedentary group: SE). Our findings suggest that daily training of a tennis exergame seems to be able to improve visuospatial attention isotropy by reducing leftward space overestimation, whereas outcomes from non-exergaming and sedentary activity do not modify subjects' performance.
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Tomato fruits defined as "golden" refer to a food product harvested at an incomplete ripening stage with respect to red tomatoes at full maturation. The aim of this study is to explore the putative influence of "golden tomato" (GT) on Metabolic Syndrome (MetS), especially focusing on the effects on redox homeostasis. Firstly, the differential chemical properties of the GT food matrix were characterized in terms of phytonutrient composition and antioxidant capacities with respect to red tomato (RT). Later, we assessed the biochemical, nutraceutical and eventually disease-modifying potential of GT in vivo in the high-fat-diet rat model of MetS. Our data revealed that GT oral supplementation is able to counterbalance MetS-induced biometric and metabolic modifications. Noteworthy is that this nutritional supplementation proved to reduce plasma oxidant status and improve the endogenous antioxidant barriers, assessed by strong systemic biomarkers. Furthermore, consistently with the reduction of hepatic reactive oxygen and nitrogen species (RONS) levels, treatment with GT markedly reduced the HFD-induced increase in hepatic lipid peroxidation and hepatic steatosis. This research elucidates the importance of food supplementation with GT in the prevention and management of MetS.
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Glioblastoma multiforme (GBM) is a primary brain tumor that is very aggressive, resistant to treatment, and characterized by a high degree of anaplasia and proliferation. Routine treatment includes ablative surgery, chemotherapy, and radiotherapy. However, GMB rapidly relapses and develops radioresistance. Here, we briefly review the mechanisms underpinning radioresistance and discuss research to stop it and install anti-tumor defenses. Factors that participate in radioresistance are varied and include stem cells, tumor heterogeneity, tumor microenvironment, hypoxia, metabolic reprogramming, the chaperone system, non-coding RNAs, DNA repair, and extracellular vesicles (EVs). We direct our attention toward EVs because they are emerging as promising candidates as diagnostic and prognostication tools and as the basis for developing nanodevices for delivering anti-cancer agents directly into the tumor mass. EVs are relatively easy to obtain and manipulate to endow them with the desired anti-cancer properties and to administer them using minimally invasive procedures. Thus, isolating EVs from a GBM patient, supplying them with the necessary anti-cancer agent and the capability of recognizing a specified tissue-cell target, and reinjecting them into the original donor appears, at this time, as a reachable objective of personalized medicine.
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Neoplasias Encefálicas , Vesículas Extracelulares , Glioblastoma , Humanos , Glioblastoma/metabolismo , Linhagem Celular Tumoral , Neoplasias Encefálicas/metabolismo , Recidiva Local de Neoplasia/metabolismo , Vesículas Extracelulares/metabolismo , Microambiente TumoralRESUMO
Toxoplasma gondii and Neospora caninum are apicomplexan protozoa of major concern in livestock and T. gondii is also considered one of the major threats and a public health concern. These protozoa have a wide range of intermediate hosts, including birds. The present work aimed to assess the prevalence of these cyst-forming parasites in migratory and sedentary birds of prey. The skeletal muscle and myocardium of 159 birds of prey from Central Italy, belonging to 19 species and recovered across 6 Wildlife Recovery Centers/Care structures along the Italian migratory route, were collected specifically for molecular (PCR) and for histopathological analysis to detect T. gondii and N. caninum. For the molecular analysis, genomic DNA was extracted. The DNA was tested by sequence typing, targeting GRA6, 529 bp repeated element, B1, PK1, BTUB, SAG2, alt.SAG2, and APICO genes for T. gondii and to end-point PCR targeting NC5 gene for N. caninum. Thirty-seven out of the one hundred and fifty-nine analyzed samples tested positive for T. gondii with a prevalence of 23.27% and nine for N. caninum, with a prevalence of 5.66%. Thirty-two sequences were obtained from the thirty-seven isolates of T. gondii. Among these, 26 presented alleles compatible with type I strain in 1 or more loci, 4 with type II strain and 2 consisted of atypical strains. Toxoplasma gondii genetic variability in birds of prey confirms previous findings of wildlife as reservoirs of atypical strains. Results from the histology showed few protozoal tissue cysts in skeletal muscle (n. 4) and hearts (n. 2).
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A captive-kept adult male tiger presented with a large cutaneous and subcutaneous mass on the thigh with a fistula. During sedation, multiple nodules were detected and samples for a histopathological exam were collected. Histologically, granulomatous panniculitis and dermatitis were seen around dense aggregates of pigmented fungal hyphae, and a diagnosis of phaeohyphomycosis was made; considering the clinical features, it was classified as a eumycotic mycetoma. This is a rarely reported subcutaneous fungal infection in humans and animals, caused by dematiaceous fungi. Clinically, it is characterized by tumefaction, fistulous sinus tracts, and the formation of macroscopically visible grains. In the literature, only a few infections in wild felids have been reported. In this case, Fontana-Masson staining better showed pigmentation and panfungal PCR and sequencing identified Madurella pseudomyectomatis (OP623507) as the causative agent. Systemic therapy with oral administration of itraconazole was planned, but the patient died during the first period of treatment. The animal was not submitted for post-mortem examination. Visceral dissemination of the agent cannot be excluded. To the authors' knowledge, this is the first report of eumycotic mycetoma by Madurella pseudomycetomatis in a captive tiger.
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Indicaxanthin (IX) from Opuntia Ficus Indica (OFI) has been shown to exert numerous biological effects both in vitro and in vivo, such as antioxidant, anti-inflammatory, neuro-modulatory activity in rodent models. Our goal was to investigate the eventual neuro-active role of orally assumed fruits containing high levels of IX at nutritionally-relevant amounts in healthy subjects, exploring cortical excitability and plasticity in the human motor cortex (M1). To this purpose, we applied paired-pulse transcranial magnetic stimulation and anodal transcranial direct current stimulation (a-tDCS) in basal conditions and followed the consumption of yellow cactus pear fruits containing IX or white cactus pear fruits devoid of IX (placebo). Furthermore, resting state-functional MRI (rs-fMRI) preliminary acquisitions were performed before and after consumption of the same number of yellow fruits. Our data revealed that the consumption of IX-containing fruits could specifically activate intracortical excitatory circuits, differently from the placebo-controlled group. Furthermore, we found that following the ingestion of IX-containing fruits, elevated network activity of glutamatergic intracortical circuits can homeostatically be restored to baseline levels following a-tDCS stimulation. No significant differences were observed through rs-fMRI acquisitions. These outcomes suggest that IX from OFI increases intracortical excitability of M1 and leads to homeostatic cortical plasticity responses.
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Excitabilidade Cortical , Córtex Motor , Opuntia , Estimulação Transcraniana por Corrente Contínua , Humanos , Potencial Evocado Motor/fisiologia , Frutas , Córtex Motor/fisiologia , Plasticidade Neuronal/fisiologiaRESUMO
In 2017, respiratory disease and low mortality were reported in domestic flying pigeons (Columba livia var. domestica) trained as hunting live bait in a breeding farm in Umbria, Italy. Clinically, open beak breathing, dropped wings, and pharyngeal and laryngeal hyperaemia were observed. Three birds were submitted for necropsy. Gross pathological evaluation revealed in all cases diffuses hyperaemia of the tracheal mucosa in association with mild emaciation and multiorgan congestion. Microscopically, diffuse epithelial hyperplasia of the trachea (n = 3) and diffuse lymphocytic infiltration of the lamina propria (n = 3) were observed. No lesions were reported in other organs. Based on reported clinical signs and lesions, Mycoplasma spp. were suspected, and molecular detection was performed on tracheal specimens leading to the identification of Mycoplasma columbinasale. Immunohistochemistry was subsequently performed to localize the microorganism within tissue lesions. Immunohistochemistry confirmed the presence of Mycoplasma species on the tracheal epithelial cells of all birds. Following tylosin administration, complete resolution of the clinical condition and lack of recurrence of clinical signs were reported in the breeding farm. These findings suggest that M. columbinasale could potentially have a role in the respiratory disease and low mortality in domestic pigeons.
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This study aimed to describe the specific localization and anatomical pattern of 24 feline lymphomas of the nervous system for which the immunophenotype was identified by immunohistochemistry investigations to support the potential specific correlation between subtypes and anatomical patterns. In total 10 tumors affected the spinal cord, eight the brain, four the peripheral nerves, one involved both the brain and the spinal cord, and one simultaneously the brain and the optic nerves. Twenty two tumors were primary lymphomas. The affected animals were 8 years of mean age. Tumors developed as an extra-axial mass (11 cases), intra-axial mass (six cases), leptomeningeal lymphomatosis (three cases), and neurolymphomatosis (five cases). One of them expressed both leptomeningeal lymphomatosis and neurolymphomatosis patterns. Two intra-axial brain lymphomas showed an angiotropic pattern. The optic chiasm was the most involved site for neurolymphomatosis. Immunolabeling was performed using anti-CD3, CD20, CD79a, PAX5, MUM-1, CD56, and anti-CD44 antibodies. In total, 12 tumors consisted of B cell lymphomas, and six of T cell lymphomas, two cases were double-reactive lymphomas while two cases consisted of non-B non-T lymphomas. B cell lymphoma affected animals of 6.4 years of mean age, while the T cell lymphoma affected older animals (mean age of 11.1 years). Extra-axial tumors mainly consisted of B cell lymphomas (8/11). Neurolymphomatosis expressed different immunophenotypes, and the B cell phenotype was the most prevalent in the optic chiasm. Two leptomeningeal lymphomatoses expressed T cell immunophenotype. For the first time, plasmacytoid differentiation was found for angiotropic lymphoma and neurolymphomatosis. All the cases, except one, were CD56-negative. CD44-expression confirmed a common malignant potential for all the anatomical patterns of the nervous system lymphoma in cats. Immunophenotype of feline lymphoma of the nervous system and its potential association with specific anatomical patterns should be strongly required in the diagnostic workup and clinical approach to this tumor especially when its primary origin is confirmed.
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West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that have been associated with neurological diseases in humans and wild birds. Wild bird rescue centers are potential significant hot spots for avian infection surveillance, as recognized in the Italian Integrate National Surveillance Plan for Arboviruses. Here we report the results of a post-mortem active monitoring study conducted from November 2017 to October 2020 on animals hosted in five wild bird rescue centers of Central Italy. Five hundred seventy-six (n = 576) wild birds were tested by real-time polymerase chain reaction (RT-PCR) for the presence of WNV or USUV RNA fragments. No birds tested positive for USUV RNA (n = 0; 0.00%). Evidence of WNV RNA (Ct value = 34.36) was found in one bird (n = 1; 0.17%), an adult little grebe (Tachybaptus ruficollis subsp. ruficollis), that tested WNV positive in December 2019. This study highlights the strategic role of wildlife rescue centers in monitoring both the introduction and circulation of avian emerging zoonotic diseases. In addition, the presence of WNV during the cold season evidences the possible role of birds in overwintering mechanisms in the Italian territory and requires further investigations.