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The study investigated the protective activity of red grape seeds (Vitis vinifera L, Burgund Mare variety) (BM) extracts in vivo on multiple doses of ultraviolet radiation (UV)-B-induced deleterious effects in SKH-1 mice skin. Eighty 8-weeks-old female SKH-1 mice were divided into 8 groups: control, vehicle, UV-B irradiated, vehicle+UV-B irradiated, BM 2.5mg polyphenols (PF)/cm(2)+UV-B irradiated, BM 4 mg PF/cm(2)+UV-B irradiated, UV-B+BM 2.5mg PF/cm(2), UV-B+BM 4 mg PF/cm(2). The extract was applied topically before or after each UV-B exposure (240 mJ/cm(2)), for 10 days consecutively. The antioxidant activity of BM extract is higher than gallic acid (k(BM)=0.017, k(gallic acid)=0.013). Multiple doses of UV-B generated the formation of cyclobutane pyrimidine dimers (CPDs) and sunburn cells, increased glutathione peroxidase (GPx) and catalase (CAT) activities respectively glutathione (GSH) and IL-1β levels in skin. In group treated with 2.5mg PF/cm(2) before UV-B irradiation BM extract inhibited UV-B-induced sunburn cells, restored the superoxide dismutase (MnSOD) activity, increased insignificantly CAT and GPx activities and reduced IL-1β level. The BM 4.0 mg PF/cm(2) treatment decreased GSH level and reduced the percentage of CPDs positive cells in skin. Both doses of BM extract administered after UV-B irradiation increased the MnSOD and GPx activities and reduced the formation of sunburn cells in skin. Our results suggest that BM extract might be a potential chemo-preventive candidate in reducing the oxidative stress and apoptosis induced by multiple doses of UV-B in skin.

Few studies were conducted to investigate the pathogenesis and to characterize the histopathological alterations in the intestinal mucosa of kids with cryptosporidiosis. In our study, 58 kids were presented for necropsy, and the faeces of another 83 goat kids were studied. Animals were aged between 1 day and 3 weeks. Infection was confirmed by oocyst identification in the faeces (Ziehl-Neelsen stain modified by Henriksen). Gross examination was followed by examination of the tissue using histopathological procedures (Hematoxylin-Eosin stain), scanning electron microscopy (SEM) and immunofluorescence. Pathological changes occurred mainly in the ileum, but also jejunum and caecum of the infected animals, at the apical surface of intestinal epithelial cells. Oocysts appeared as round or oval formations, with a diameter 4.5 m, sometimes covering the atrophic and denudated free border of intestinal villi. Most histological and morphological characteristics of infection with C. parvum are common on the whole to other Cryptosporidium spp. Massive dentification of cryptospodiosis in kids indicates the possible role of these animals as disease reservoirs for other animals and for humans. This is the first study to characterize the Cryptosporidium infection by confocal technique and SEM in Romania.

Chinchillas are primarily used as laboratory animals in auditory pathways research, it is necessary to acquire detailed neuroanatomical data on other encephalon segments, in order to assess whether this species might also contribute as olfactory pathways experimental models. Thus, our research focused on providing a gross and microscopic description of the chinchilla’s olfactory bulbs. Five chinchilla encephalon samples were harvested from commercially slaughtered young adults. The pairs of olfactory bulb were isolated by dissection, macroscopically examined while still attached, then detached, fixed in buffered formalin, serially cut and stained using a modified Luxol-Cresyl protocol. Gross and microscopic features were assessed and compared to literature descriptions of related species such as the rat and the rabbit. Chinchillas presented large olfactory bulbs with an average maximal diameter of 3 mm each, positioned in two corresponding deep grooves of the cribriform plate, orally to the anterior limit of the cerebral hemispheres. They decreased in girth ventro-aborally towards their base, which was located on the ventral side of the telencephalon. Histologically, they presented a laminar structure: an olfactory nerve layer, a glomerular layer, an external plexiform layer, a mitral cell layer, an internal plexiform layer and a granule cell layer. The proportionally well developed olfactory bulbs and the existence of the accessory olfactory bulbs demonstrates that C. lanigera is a macrosmatic species. The olfactory bulbs in the chinchilla present the gross anatomy and histological outline concurring with literature rodent descriptions, with certain particularities (accessory bulb topography, olfactory ventricle topography, glomerular size and shape).

Introduction. The cardiac ectopia is a congenital disorder, characterized by the location of the heart outside the thoracic cavity. This disorder results due to an error during the maturation of the medial line of the mesoderm and of the formation of the ventral wall of the body during the embryonic development. The etiology is not well known being considered the involving of the genetic and environmental factors. The most important genetic factors involved in the heart ecopia are considered family Hox genes and the proto-oncogenes which are generally involved in the induction and the differentiation of the embryonic structures and the family Pax genes which are involved in the growing and development of the organs, being considered as master genes, controlling the activity of other subordinate genes. The location of ecopia is very often ventral cervical, but there were also recorded cases of sternal (thoracical) and very rare the abdominal one. (Abi, 2008, Wyrost, 1981). Aims . The purpose of this study is to emphasize the clinical signs, diagnosis of the cervical heart ectopia and to establish the possible genetic prevention methods of this disorder. Materials and methods . The study was carried out on a two days old calf, which presented the clinical signs of heart ectopia. Results . At palpation, the clinical examination emphasized a modified area in volume at the cervical level and the auscultation revealed the heart beats. At necropsy patent ductum arteriosus was diagnosed , together with the stenosis of the aorta and bilateral heart hypertrophia. Conclusions. In order to avoid the congenital pathological modifications of this type it is very important the checking of the sperm quality, both in the case of artificial and natural inseminations. It is very important, especially in the case of beef breeds to avoid the breeding between half-sisters or brothers or between the parents and their offspring. In order to test the genitors for their potential of gene carriers the test-cross is recommended and the elimination of the carriers from the genitor group.

Graphene-oxide (GO) and its most encountered derivatives, thermally reduced graphene oxide (TRGO) and nitrogen-doped graphene (N-Gr), were synthesized and structurally characterized by spectroscopic techniques, like Raman and (13)C MAS solid state NMR. Several biological effects (cytotoxicity, oxidative stress induction, and cellular and mithocondrial membrane alterations) induced by such graphene-based materials on human dental follicle stem cells were investigated. Graphene oxide shows the lowest cytotoxic effect, followed by the nitrogen-doped graphene, while thermally reduced graphene oxide exhibits high cytotoxic effects. Graphene oxide induces oxidative stress without causing cell membrane damage. Nitrogen-doped graphene shows a slight antioxidant activity; however, at high doses (20 and 40 μg/ml) it causes membrane damage. Both graphene oxide and nitrogen-doped graphene seem to be valuable candidates for usage in dental nanocomposites.

Background Metallic phosphides are extremely toxic pesticides that are regulated in their usage. Information concerning the impact of metallic phosphides on human health is abundant. Data regarding the clinical pathology of phosphide poisoning in humans or domestic and wild animals is largely incomplete with only a few cases of metallic phosphide poisoning being reported every year, especially in humans. For the majority of cases reported in dogs the data are vague or incomplete. Here we report a complete and detailed description of pathological changes in a case of intentional metallic phosphide poisoning in a dog including an exhaustive examination of the brain. Case presentation A 1 year old, male, Belgian Shepherd crossbreed dog with a clean medical history and no observed clinical signs prior to death, was submitted for post mortem examination. The dog was found dead by the owner. Near the body a suspect mix of bread, fat and a blackish powder was found. The owner announced the authorities and submitted the animal and the possible bait for forensic examination. At necropsy, multisystemic necrotic and degenerative lesions were observed. Histological exam confirmed the presence of necrotic and degenerative lesions of variable severity in all of the examined organs. The toxicological forensic examination revealed the presence of the phosphine gas in the gastric content and the bait. Conclusion Metallic phosphide poisoning is a rarely reported entity, since the diagnosis of intentional poisoning with these compounds is a great challenge for forensic pathologists and toxicologists. To our knowledge, this is the first study describing the lesions completely in veterinary forensic toxicology. We assume that the toxic shows systemic endotheliotropism and damage of the endothelial cells responsible for the hemorrhagic lesions and for the secondary ischemic necrosis in various organs. This report will contribute to a better understanding of the pathogenesis in cases of acute metallic phosphide exposure in animals.

The prevalence of Toxoplasma gondii, an economically important zoonotic protozoan, was investigated in horses slaughtered for export and human consumption in the North of Romania. Pairs of samples, sera and heart tissues, were collected from 82 slaughtered horses. Examination of horse sera by ELISA at a dilution of 1:10, and by modified agglutination test (MAT) at a dilution of 1:6, revealed that 32 (39%) and 31(37.8%) horses, respectively, had antibodies against T. gondii. Using polymerase chain reaction (PCR) analysis, T. gondii DNA was not found in any heart sample collected from horses. By bioassay in mice, we obtained viable isolates of T. gondii from two of ten horses determined to be strongly positive by serological assay/ELISA. The prevalence estimated in horses highlighted the potential risk for human contamination by consumption of raw or undercooked meat.

Aims. The aim of this experimental study is to assess the bone healing phenomenon produced in the presence of several dental materials: a polycarboxylate cement, a glass-ionomer cement, a composite resin and MTA (mineral trioxide aggregate) based cement. Methods. The biocompatibility of four root-end fillings materials, used in periapical surgery was investigated after intra-osseous implantation of the materials in rats’ calvaria. Tissue reaction was studied at 2, 4, 6, 8, 10 and 12 weeks after implantation. We took into consideration the presence of inflammatory cells (polymorphonuclear leukocytes, macrophages, plasma cells, lymphocytes and giant cells) and classified the aspects of the histological samples according to the following scale: 0 - no inflammation, 1 – mild, isolated inflammation, 2 - moderate, localized inflammatory reaction, 3 - severe, diffuse and intense inflammatory reaction.Results. The inflammatory reaction was present at the six intervals for all the tested materials, but at 12 week interval, the reaction was minimal in all cases. Also, a dissolution reaction was observed for all the materials, less intense for glass-ionomer cement and polycarboxilate cement.Conclusions. At the end of the experimental period, glass-ionomer cement and polycarboxilate cement suffered a lesser dissolution reaction as compared to the second group of tested materials.

The study investigated the effects of the combined treatment Parecoxib (Pcox) and 5,10,15,20-tetra-sulphonato-phenyl-porphyrin(TSPP)-mediated photodynamic therapy on Walker 256 carcinosarcoma.Five groups of male Wistar rats were used: the control group, treated with TSPP, group 2, irradiated 24 h thereafter, group 3, treated with Pcox and irradiated 24 h thereafter, groups 4 and 5 treated with combined therapies, TSPP and Pcox before irradiation, and Pcox 24 h after TSPP and irradiation respectively. Tumour inflammation, growth and non-growth factors, apoptosis/necrosis rate and oxidative/nitrosative stress markers were investigated.Malondialdehyde levels and cyclooxygenase (COX)-2 expression increased significantly in the group treated with Pcox after TSPP-PDT when compared with TSPP + IR group (p0.05, p0.001 respectively), in correlation with a decrease in glutathione levels (p0.05). The quantification of apoptosis, based on the TUNEL-assay, and necrosis rate revealed an increase of apoptotic/necrotic index in the same group (p0.05). On the other hand, Pcox administered before irradiation showed a significant increase in both vascular endothelial growth factor (VEGF) and COX-2 levels (p0.05) and in nitric oxide production (p0.01), when compared with the control group.The administration of Pcox after TSPP-mediated PDT showed promising antitumoural effects, leading to an increase in oxidative and nitrosative stress as well as apoptosis/necrosis rate in tumour tissue. These results show that combined regimens that involve selective COX-2 inhibitors administration after irradiation may improve the therapeutic effectiveness of PDT.

Canine leishmaniasis is a vector-borne zoonotic disease caused by the protozoan parasite Leishmania infantum. In Romania between 1955 and 2013, no cases of human autochthonous visceral leishmaniasis were reported. Data regarding canine leishmaniasis is similarly scarce. Since the first report of clinical autochthonous canine leishmaniasis in 1935, there were only three sporadic reports of positive dogs all without any clinical signs. Our study reports the first clinical case of autochthonous canine leishmaniasis in the last 80 years, stressing the importance of a targeted surveillance of Leishmania infection, as infected dogs act as the primary reservoir for zoonotic visceral leishmaniasis.

Because polycystic ovarian syndrome (PCOS) is a risk factor for type 2 diabetes, the affected women can present frequently prediabetic states such as impaired fasting glycaemia and/or impaired glucose tolerance. The purpose of our study is to explore the effect of antiandrogenic spironolactone on glucose metabolism and oxidative stress (OS) parameters in oestradiol valerate (OV) induced PCOS rat model.72 female Wistar rats were distributed either to PCOS group (n=65, OV dissolved in sesame oil, 5 mg/0.4 ml), or to non-PCOS control group (n=7, sesame oil, 0.4 ml). After a month, ultrasound was performed to assess the ovarian morphology, and the results of an initial oral glucose tolerance test (OGTT) were used to identify the animals with altered glucose metabolism (AGM). Glucose transporter 4 (GLUT4) was evaluated from muscle biopsies, OS parameters were assessed from blood and muscle samples, and ovaries of 3 rats were removed for histopathological examination. Afterwards, the AGM group was divided in a treated PCOS group denoted as Sp+D (per os spironolactone dissolved in DMSO, 2 mg/0.2 ml), and a PCOS control treated with DMSO (0.2 ml). After one month of daily treatment, a final OGTT was performed. GLUT4 and OS parameters were again evaluated and ovaries were removed for histopathological examination.As compared to the values prior to the treatment, Sp+D reversed fasting hyperglycaemia (p

The study aims to investigate the mechanisms involved in the in vitro effect of UVB on endothelial vascular cells (HUVECs) pretreated with a photochemopreventive agent, the Calluna vulgaris (Cv) extract. Two concentrations of Cv, below the limit of cytotoxicity IC50 (2.5 and 7.5 μg GAE/ml) and two doses of UVB (50 and 100 mJ/cm(2)) were used. Oxidative stress parameters were quantified at 1 h and 24 h after irradiation and apoptosis, DNA damage and the induction/activation of NF-κB were evaluated at 24 h. UVB exposure led to the formation of lipid peroxides in a dose dependent manner (p0.001), induced apoptosis, increased the γ-H2AX levels and the activation of NF-κB. Pretreatment with 2.5 μg GAE/ml Cv improved the antioxidant defense, protected against DNA lesions and was able to decrease cellular death at low dose of irradiation. 7.5 μg GAE/ml Cv was prooxidant, favored the formation of DNA lesions, amplified the NF-κB activation UVB-induced (p0.01) and led to high levels of cellular death. Both doses of Cv inhibited caspase-3 activation. The modulatory effect of Cv extract on endothelial cells exposed to UVB depend on the concentration of Cv used. This study provides insides into the mechanisms triggered by UVB and antioxidants on skin endothelial cells.

To evaluate the influence of an extract of Genista tinctoria L. herba (GT) or methylparaben (MP) on histopathological changes and 2 biomarkers of oxidative stress in rats subchronicly exposed to bisphenol A (BPA).Adult female Wistar rats were orally exposed for 90 d to BPA (50 mg/kg), BPA+GT (35 mg isoflavones/kg) or BPA+MP (250 mg/kg). Plasma and tissue samples were taken from liver, kidney, thyroid, uterus, ovary, and mammary gland after 30, 60, and 90 d of exposure respectively. Lipid peroxidation and in vivo hydroxyl radical production were evaluated by histological analysis along with malondialdehyde and 2,3-dihydroxybenzoic acid detection.The severity of histopathological changes in liver and kidneys was lower after GT treatment than after BPA or BPA+MP treatment. A minimal thyroid receptor antagonist effect was only observed after BPA+MP treatment. The abnormal folliculogenesis increased in a time-dependent manner, and the number of corpus luteum decreased. No significant histological alterations were found in the uterus. The mammary gland displayed specific estrogen stimulation changes at all periods. Both MP and GT revealed antioxidant properties reducing lipid peroxidation and BPA-induced hydroxyl radical generation.GT L. extract ameliorates the toxic effects of BPA and is proved to have antioxidant potential and antitoxic effect. MP has antioxidant properties, but has either no effect or exacerbates the BPA-induced histopathological changes.

To date there is no defined pharmacologic treatment protocol available against cutaneous protothecosis, which is difficult to combat using conventional drugs.Our experiment aimed to comparatively investigate the effect of two essential oils (Mentha piperita and Saturenja hortensis) against cutaneous protothecosis experimentally induced by Prototheca zopfii in mice.Immunosuppressed BALB/c female mice, were divided into six experimental groups, infected with P. zopfii, and then treated for 21 days against the infection. The effectiveness of the different treatments was assessed clinically and histologically by quantifying the degree of inflammation (immunohistochemical quantification of macrophages, T lymphocytes and neutrophils) and fibrosis.Skin lesions in experimental protothecosis from non-treated mice were more severe as compared to the four groups of treated animals. Both M. piperita and S. hortensis have proved to be efficient in vivo in the treatment of cutaneous protothecosis by reducing the clinical signs and significantly reducing the degree of inflammation (P0.05 for the number of macrophages, T lymphocytes and neutrophils) and fibrosis as compared to untreated animals.Interestingly, our study shows that M. piperita and S. hortensis could represent a potential source of natural antimicrobial products in the treatment of cutaneous protothecosis.

UVB is a major cause of nonmelanoma skin cancer in humans. Photochemoprevention represents an important strategy in protecting the skin against the detrimental effects of ultraviolet B (UVB). We investigated the activity of Calluna vulgaris (Cv) delivered via a hydrogel on 3 main pathways (oxidative stress, inflammation, DNA damage) on skin exposed to multiple doses of UVB in SKH-1 mice. Fifty female mice were divided randomly into 5 groups: control, vehicle, UVB irradiated, Cv + UVB irradiated, and Cv + vehicle + UVB irradiated. The extract was applied topically on the skin in a dose of 4 mg polyphenols/cm2 30 minutes before each UVB (240 mJ/cm2) exposure over 10 consecutive days. Malondialdehyde, reduced glutathione, tumor necrosis factor-α, interleukin-6, cyclobutane pyrimidine dimer (CPD) levels, sunburn cell formation and epidermal thickness, and the number of epidermal cell layers in skin were evaluated 24 hours after the last treatment. UVB increased cytokine levels (P < 0.001), formation of CPDs (P < 0.001) and sunburn cells (P < 0.001), and the epidermal thickness and number of epidermal cell layers (P < 0.001) compared with the control group. The topical application of Cv protected the skin against inflammation and DNA damage, as shown by a decreased number of CPDs (P < 0.001) and sunburn cells (P < 0.001). The administration of Cv via hydrogel may be a viable method for chemoprevention..

This study assesses the impact of equine infectious anaemia virus (EIAV) infection on the oxidant/antioxidant equilibrium of horses. Blood samples from 96 Romanian horses aged 1-25 years, were divided into different groups according to their EIAV-infection status, age, and time post-seroconversion. The effect of infection on oxidative stress was estimated by measuring enzymatic antioxidants (superoxide dismutase [SOD], glutathione peroxidase [GPx] and catalase), non-enzymatic antioxidants (uric acid and carotenoids), and lipid peroxidation (malondialdehyde [MDA]). Infection modified the oxidant/antioxidant equilibrium in the horses, influencing GPx and uric acid levels (P0.05). Time post-seroconversion also contributed to oxidative stress imbalance, exhibiting a significant influence on both SOD and MDA concentrations in the blood (P0.05). Animal age did not have a significant influence on oxidative stress. Recently infected horses (1 year following seroconversion), and horses5 years old, represented the most vulnerable category in terms of oxidative stress, followed by recently infected animals5 years old. The results of this study are novel in implicating EIAV infection in the development of oxidative stress in horses.

To identify by ultrasonography (DermaScan C, Cortex Technology1) the reference points necessary to monitor the horizontal bone resorbtion and to assess the accuracy of the measurements by comparing with Cone Beam Computed Tomography images, having direct microscopic section measurements as a gold standard.We performed the examinations on the lingual side of the alveolar bone of 4 pig mandibles. We tried to identify the enamel-cementum junction, the root, the periodontal space at the appearance of the tooth from the alveolar bone, the edge of cortical bone at its coronary limit (in order to assess the horizontal bone resorption). We measured the distance between the enamel-cementum junction and the coronal edge of cortical bone.There was a strong positive correlation between the alveolar bone level measured with the 3 techniques. Correlation parametric R2 tests were statistically significant for DermaScan measurements compared to microscopy ones (p0.0001). Linear regression showed statistically significant correlations for alveolar bone level measurements made by CBCT compared to microscopy (p0.05).The cementum-enamel junction may be identified on ultrasonic images with high accuracy according to the tooth anatomical convexities, therefore this method can be used for monitoring the periodontium.

EQUINE INFECTIOUS ANAEMIA VIRUS-INDUCED PULMONARY INTERSTITIAL DAMAGE: WHAT ABOUT AEROSOL TRANSMISSION? P. Bolfa*, J.L. Cadore y, J.F. Mornex z, C. Catoi*, A. Gal*, M. Taulescu* and C. Lerouxz *Pathology Department, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania, yInternal Medicine Department, VetAgro Sup, Marcy-l’Etoile and zUMR754 INRA University Lyon 1, Retrovirus and Comparative Pathology, France Introduction: Equine infectious anaemia (EIA) is considered a blood-borne disease primarily transmitted iatrogenically or by haematophagous insects and the virus infects cells of the monocyte/ macrophage lineage. Alternative routes of transmission have not been explored, although there is evidence that EIA was spread via aerosolized particles during the 2006 EIA virus (EIAV) outbreak in Ireland. Materials and Methods: Haematoxylin and eosin-stained sections of lung from 77 EIAV seropositive Romanian horses were scored based on lymphocyte infiltration, (peri) bronchiolar inflammation and thickness of the alveolar septa. Immunolabelling for p26 EIAV capsid protein expression and smooth muscle actin was performed. Results: 52% of the EIAV-positive horses displayed a mild inflammation around the bronchi; 22%, moderate inflammation with inflammatory cells inside the wall and epithelial bronchiolar hyperplasia; and 6.5%, moderate to severe inflammation, with destruction of the bronchiolar epithelium and accumulation of smooth muscle cells within the pulmonary parenchyma. Interestingly, EIAV p26 was expressed in the cytoplasm of cells compatible by morphology and localization with alveolar and bronchiolar epithelial cells. Conclusions: The observed lesions were compatible with the interstitial diffuse pneumonia observed during other lentiviral infections. The presence of EIAV capsid in lung epithelial cells suggests that EIAV might be responsible for the bronchointerstitial damage observed. THE NOVEL HUMAN CORONAVIRUS EMC CAUSES DISEASE IN MACAQUES BUT NOT IN FERRETS J.M.A. van den Brand, L. Wiersma, S.L. Smits, L. Provacia, V.S. Raj, A.D.M.E. Osterhaus, T. Kuiken and B.L. Haagmans Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands Introduction: In 2012 a novel human coronavirus (HCoV-EMC) causing lower respiratory tract infection was discovered in human patients. The clinical course of the disease was similar to that seen in severe acute respiratory syndrome (SARS). However, the virulence and pathogenesis of the virus is not yet known and no animal models for human disease have been established. Materials and Methods: Cynomolgus macaques and ferrets were inoculated with HCoV-EMC. At 1 or 4 days after inoculation the animals were killed and pathological, immunohistochemical, in-situ hybridization, virological and serological analyses were performed on samples from those animals. To determine the presence of the receptor for the HCoV-EMC dipeptidyl peptidase 4 (DDP4) in different cell types of the respiratory tissue, immunohistochemical analysis was used. Results: HCoV-EMC caused mild respiratory disease in macaques, with non-ciliated bronchiolar epithelial cells as important target cells. Ferrets were not productively infected. Conclusions: Our results confirm HCoV-EMC as the cause of respiratory disease in a non-human primate model. This suggests that the cynomolgus macaque is a suitable experimental animal species to model this disease in man.

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A 2-year-old female llama ( Lama glama), from a private zoological park, with anorexia, ataxia, dyspnea, ascites, and emaciation, was necropsied. Gross inspection, and cytological, histological, and immunohistochemical analyses were performed. A firm, white, multinodular mass, 25 cm in diameter was found in the liver parenchyma. Similar nodules up to 3 cm were found in lymph nodes, lung, diaphragm, and peritoneum. Histologically, the affected organs were replaced by an infiltrative growth of undifferentiated neoplastic cells. Immunohistochemically, neoplastic cells were positive for pancytokeratin (panCK), CK20, and CK19, and negative for thyroid transcription factor 1, CK7, and carcinoembryonic antigen. A diagnosis of poorly differentiated metastatic cholangiocarcinoma was made.