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Made available in DSpace on 2022-05-01T15:46:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: The thymus is necessary for the differentiation of T cells, a process that is regulated by the type of antigens found in thymocytes, the environment of surrounding cells and the thymus architecture. There is evidence that infectious diseases may result in morphological changes in this organ, such as premature atrophy and decreased thymocyte proliferation, that can affect the immune response. Objectives: We characterised the morphology and tissue distribution of haematopoietic and stromal cells in the thymuses of dogs naturally infected with Leishmania infantum, with the aim to determine the changes that may contribute to the pathophysiology of the disease. Methods: Thymus samples were collected from 15 animals (aged 6 months to 5 years) ELISA-positive for leishmaniasis and from 10 dogs from non-endemic regions for leishmaniasis whose death was not related to infectious causes. The samples were submitted to histological processing and staining with Haematoxylin-Eosin to assess thymic morphometry and histopathological changes. Masson's trichrome staining was used to quantify the connective tissue present (collagen). The immunohistochemical method was used to determine the cellular constitution of the thymus, using antibodies that aimed at marking T lymphocytes (anti-CD3), B lymphocytes (anti-CD79a), macrophages (anti- MAC387), mesenchymal cells (anti-vimentin), epithelial cells (anti-cytokeratin), cells in mitosis (anti-Ki67) and cells in apoptosis (anti-caspase-3). Results: The histopathological evaluation of infected dogs showed more signs consistent with thymus atrophy, including decreased parenchyma, infiltration of adipose and connective tissue near the capsule and between the lobules, lymphoid rarefaction mainly in the cortical region and loss of the cortical-medullary demarcation. In addition, we observed a decrease in the amounts of CD3 + T lymphocytes, macrophages (MAC387) and Ki67-positive cells and an increase in the number of cells positive for cytokeratin and CD79a (B lymphocytes). Finally, the parasite was detected in 46% of infected thymuses and may contribute for the observed changes. Conclusions: Apparently, leishmaniasis, like other infectious diseases, causes atrophy of the thymus and depletion of thymocytes with a relative increase in thymus epithelial cells. These morphological changes in the normal organisation of the thymus by mechanisms not yet well known may result in the abnormal release of T cells, with consequent damage to the host's immune response. Universidade Estadual Paulista Júlio Mesquita Filho (UNESP) Faculdade de Medicina Veterinária de Araçatuba (FMVA), SP Universidade Estadual Paulista Júlio Mesquita Filho (UNESP) Faculdade de Ciências Agrárias e Veterinárias (FCAV), SP Universidade Estadual Paulista Júlio Mesquita Filho (UNESP) Faculdade de Medicina Veterinária de Araçatuba (FMVA), SP Universidade Estadual Paulista Júlio Mesquita Filho (UNESP) Faculdade de Ciências Agrárias e Veterinárias (FCAV), SP FAPESP: #2016/02384-9 FAPESP: #2017/26964-7

Made available in DSpace on 2022-04-29T08:32:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 The route used in the transplantation of mesenchymal stem cells (MSCs) can directly affect the treatment success. The transplantation of MSCs via the intrathecal (IT) route can be an important therapeutic strategy for neurological disorders. The objective of this study was to evaluate the safety and feasibility of the IT transplantation of autologous (Auto-MSCs) and allogeneic (Allo-MSCs) bone marrow mesenchymal stem cells (BM-MSCs) in healthy dogs. Based on neurodisability score, cerebrospinal fluid (CSF) and magnetic resonance imaging (MRI), no significant differences from the control group were observed on day 1 or day 5 after IT Auto- or Allo-MSCs transplantation (P > 0.05). In addition, analysis of matrix metalloproteinase (MMP)-2 and MMP-9 expression in the CSF revealed no significant differences (P > 0.05) at 5 days after IT transplantation in the Auto- or Allo-MSCs group when compared to the control. Intrathecal transplantation of BM-MSCs in dogs provides a safe, easy and minimally invasive route for the use of cell-based therapeutics in central nervous system diseases. Department of Veterinary Clinics School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Animal Reproduction and Veterinary Radiology São Paulo State University (UNESP) Department of Veterinary Surgery and Anesthesiology São Paulo State University (UNESP) Department of Clinics Surgery and Animal Reproduction São Paulo State University (UNESP) Department of Veterinary Clinics School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Department of Animal Reproduction and Veterinary Radiology São Paulo State University (UNESP) Department of Veterinary Surgery and Anesthesiology São Paulo State University (UNESP) Department of Clinics Surgery and Animal Reproduction São Paulo State University (UNESP)

A polioencefalomalácia (PEM) é uma doença neurológica caracterizada pelo diagnóstico morfológico de necrose liquefativa (malácia) da substância cinzenta do córtex cerebral. Trata se de uma condição multifatorial que acomete ruminantes promovendo alterações irreversíveis no metabolismo das células nervosas e consequentemente morte celular. As principais causas conhecidas são: deficiência de tiamina, meningoencefalite por herpesvírus bovino tipo 5 (BoHV-5), intoxicação por sal associada a privação de água e intoxicação por chumbo, enxofreou plantas que produzem tiaminases.

Made available in DSpace on 2018-12-11T16:51:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Visceral leishmaniosis (VL) is a multisystem disease that affects domestic dogs and can have several clinical manifestations, including some rare reports of neurological clinical signs, or it may remain asymptomatic, depending on the individual immune response against the Leishmania parasite. VL involves immune system sensors, such as the Toll-like receptors (TLRs), that are related to innate immunity and inflammation. Previously, we have reported the presence of brain inflammation in infected dogs. Here, we investigated the gene expression profile of TLRs 1–10 in the brain and the spleen of infected dogs, along with the production of proinflammatory cytokines (TNF-α, IFN-γ, IL-1β and IL-6) with the aim of explaining the origin of brain inflammation. The gene expression of TLRs has varied according to the tissue evaluated. In the brain, TLR-4 was only up-regulated in a small subpopulation of infected dogs, while in the spleen, we detected an increase in TLR-5 and TLR-9 transcripts, as well as a reduction in TLRs 2-4 and TLR-10. All cytokines except IL-6 were detected in infected dogs. Moreover, we detected Leishmania DNA in all infected dogs in both tissues evaluated. In the histopathological analysis, we observed a predominance of lymphoplasmacytic infiltrate, mainly in leptomeninges and choroid plexuses, ranging from mild to intense. This study provides the first insight into the TLRs profile in the brain and the spleen during canine VL and provides support to confirm the involvement of sensors of the innate immune system sensors against L. infantum parasites. Faculdade de Medicina Veterinária de Araçatuba UNESP – Univ. Estadual Paulista, Rua Clóvis Pestana, 793 Laboratoire des Processus Infectieux à Trypanosomatidés Département Infection et Epidemiologie Institut Pasteur, 25-28 Rue du Dr Roux Faculdade de Medicina Veterinária de Araçatuba UNESP – Univ. Estadual Paulista, Rua Clóvis Pestana, 793 FAPESP: 2013/25498-1 FAPESP: 2016/02384-9 CNPq: 420970/2016-9

Made available in DSpace on 2019-10-06T17:15:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01. Added 1 bitstream(s) on 2019-10-09T18:35:52Z : No. of bitstreams: 1 S0100-736X2019000600416.pdf: 2101882 bytes, checksum: b16fb22e7949f5359d411182dd845685 (MD5) Mesothelioma is considered a malignant neoplasm caused by the proliferation of mesothelial cells mostly from the pleura, peritoneum and pericardium. Here we described a case of fatal hemothorax caused by pleural mesothelioma in a lion by means of necropsy, histopathology and immunohistochemistry. Gross inspection of the thoracic cavity showed hemothorax with about 4 liters of blood. Microscopically, numerous, randomly distributed, soft, red-pink, irregular masses with up to 1cm in diameter were observed in both visceral and parietal pleurae. Microscopically, a papillary structure pattern was observed in the thoracic masses, composed mainly by one layer of cubic mesothelial cells, which presented eosinophilic cytoplasm, central nucleus and evident nucleolus, supported by a low cellular fibrovascularstroma. Neoplastic cells were positive for both cytokeratin and vimentin by immunohistochemistry. This seems to be the first report of fatal hemothorax caused by pleural mesothelioma in a lion. Centro de Pesquisas Universidade Paulista (Unip), Avenida José Maria Whitaker 290 Faculdade de Medicina Veterinária Universidade Estadual Paulista 'Julio de Mesquita Filho' (Unesp), Rua Clóvis Pestana 793 Zoológico Municipal de Araçatuba, Rua do Fico s/n Faculdade de Medicina Veterinária Universidade Estadual Paulista 'Julio de Mesquita Filho' (Unesp), Rua Clóvis Pestana 793

Made available in DSpace on 2022-05-01T12:09:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 The aim of this study was to evaluate the histopathological changes to the mammary gland that occur in female dogs with visceral leishmaniosis and to correlate the findings with the parasite load, inflammatory cell profile in mammary tissue and serum progesterone levels. For this, 20 adult female dogs that were naturally infected with Leishmania infantum, not spayed, not pregnant and free from mammary tumors were used. They were divided into two groups: G1 (n = 9) with high serum progesterone levels and G2 (n = 11) with low serum progesterone levels. The parasite load and the immunophenotype of leukocytes infiltrated into the mammary tissue (CD3, CD4, CD8 and MCA874) were evaluated using the immunohistochemical technique. In the mammary gland, chronic inflammatory infiltrate was mainly found in G1, sometimes associated with granulomatous inflammation, higher parasite load and higher density of cells immunolabeled for CD3, CD4, CD8 and MCA874. A significant positive correlation (p < 0.05) was observed between the parasite load and the immunolabeled leukocytes. The influence of the serum progesterone level in the mammary gland of infected female dogs can contribute to the maintenance of an immunosuppressive cell profile and favor the persistence of the parasite in this site. School of Agrarian and Veterinary Sciences São Paulo State University (UNESP), Jaboticabal Zoonosis Control Center University Center of Rio Preto (UNIRP), São José do Rio Preto School of Veterinary Medicine São Paulo State University (UNESP) School of Agrarian and Veterinary Sciences São Paulo State University (UNESP), Jaboticabal School of Veterinary Medicine São Paulo State University (UNESP)

International audience; ABSTRACT: Some studies have shown the role played by matrix metalloproteinases and their inhibitors in doxorubicin cardiotoxicity. In this study, we sought to investigate how plasma and myocardial MMP 2 and 9 perform in rabbits with doxorubicin-induced cardiomyopathy, searching for a correlation between the activity of these collagenases and cardiac remodeling. Cardiomyopathy was induced by doxorubicin given intravenously twice a week for six consecutive weeks. Plasma MMP activity and the echocardiogram were assessed at baseline, and at 15 and 45 days after first injection of doxorubicin. The myocardial activity of these enzymes was solely evaluated in nine rabbits at 45 days, and results were compared with nine healthy controls. We only identified the full-length forms of both MMP 2 and 9 throughout the study. The plasma pro-MMP 2 reduced along the deterioration of cardiac function, while the pro-MMP 9 increased significantly at T45 as compared to baseline and T15. A negative significant correlation was found to exist between the plasma activity of pro-MMP 2 and mitral E-to-mitral septal annular early diastolic velocity ratio, which is an estimate of mean left atrial pressure and congestion. Only pro-MMP 2 was found in myocardial samples, and mean activity of such enzyme was statistically lower than that recorded for healthy controls. Although no active form was documented for either collagenase, the duration of the treatment with doxorubicin played a role in the alteration of plasma pro-forms activity. However, these changes could not be associated with most echocardiographic parameters that are supportive of cardiac remodeling.; RESUMO: Alguns estudos já demonstraram o papel exercido pelas metaloproteinases de matriz e seus inibidores na cardiotoxicidade promovida pela doxorrubicina. Assim, este estudo teve como objetivo investigar o comportamento das MMPs 2 e 9 plasmáticas e miocárdicas em coelhos com cardiomiopatia induzida pela doxorrubicina, buscando determinar se há correlação entre a atividade dessas colagenases e o remodelamento cardíaco. A cardiomiopatia foi induzida pela doxorrubicina aplicada por via intravenosa duas vezes por semana ao longo de seis semanas consecutivas. A atividade plasmática das MMPs e o ecocardiograma foram avaliados no momento basal e aos 15 e 45 dias após a primeira aplicação da doxorrubicina. A atividade miocárdica dessas enzimas foi quantificada em apenas nove coelhos aos 45 dias e os resultados comparados com outros nove controles saudáveis. Foram identificadas apenas as formas inativas das MMPs 2 e 9 durante todo o estudo. A pro-MMP 2 plasmática reduziu à medida que a função cardiaca se deteriorou, enquanto a pro-MMP 9 aumentou significativamente em T45 quando comparada aos momentos basal e T15. Houve correlação negativa significativa entre a atividade plasmática da pro-MMP 2 e a relação entre E mitral e a velocidade anular mitral no início da diástole, um parâmetro que permite estimar a pressão atrial esquerda média e a congestão. Apenas a pro-MMP 2 foi documentada nas amostras miocárdicas dos coelhos com cardiomiopatia e atividade media dessa enzima foi estatisticamente menor que aquela observada nos controles saudáveis. Embora a forma ativa de ambas as colagenases não tenha sido identificada, o tempo de tratamento com doxorrubicina interferiu na atividade das formas inativas plasmáticas. Contudo, essas alterações não se associaram com a maioria dos parâmetros ecocardiográficos que indicam remodelamento cardíaco.

Osteosarcoma is the most common bone cancer in dogs. It has a high invasion capacity, commonly metastasize and it mainly arises in the medullary canal of long bones of the appendicular and axial skeleton, rarely affecting primarily extra-skeletal sites. Osteosarcoma of primary meningeal origin is extremely rare in human and veterinary medicine. A male, mixed breed dog was admitted at the Veterinary Hospital with a history of being overly excited, having decreased neurological reflexes and development of seizures. As the situation evolved, the animal was euthanized. The animal was referred to the Veterinary Pathology Service for necroscopic examination. The objective of this report was to describe a case of primary extra-skeletal osteosarcoma of the meninges in a dog, which presented clinical signs compatible with a disease originating in the central nervous system and which was diagnosed by histopathological and immunohistochemical analysis.

Made available in DSpace on 2018-12-11T17:08:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-12-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Visceral leishmaniasis (VL) is a disease causing several clinical manifestations in dogs, including neurological disorders. Nevertheless, there are few studies related to the evaluation of the brain alterations during VL. Evidences of the involvement of cerebral barriers in infected dogs was reported, including the presence of brain inflammatory infiltrate, with a predominance of CD3+ T cells. Therefore, the aim of this study was to determine the immunophenotypes of T lymphocytes in the cerebrospinal fluid (CSF), as well as in peripheral blood, and to correlate with brain alterations in dogs with VL. We detected elevated percentages of double negative (DN) and double positive (DP) T cells in the CSF, with a predominance of TCRαb. In the histopathological analysis, we observed a predominance of lymphoplasmacytic infiltrate, mainly in leptomeninges, ranging from mild to intense, and we observed a positive correlation between the intensity of inflammation in the subependymal area and the DN T cells of the CSF. Thus, the DN T cells seem be acting as villains of the immune system through pro-inflammatory mechanisms. Further, the proportion of the different population of CSF T cells did not differ from those observed in the blood, which provides us with more evidence of blood-CSF barrier breakdown. Together, the results provide more explanation to the inflammation observed in the brain of dogs with VL, which the DN T cells contribute to the origin and progression of the neurological disease. This study provides insight into the immunophenotypes of T lymphocytes in the CSF during canine visceral leishmaniasis. Laboratório de Patologia Aplicada (LApap) College of Veterinary Medicine UNESP—Univ Estadual Paulista, Rua Clóvis Pestana, 793 Araçatuba/SP Laboratoire des Processus Infectieux à Trypanosomatidés Département Infection et Epidemiologie Institut Pasteur, 25-28 Rue du Dr Roux College of Veterinary Medicine UNESP—Univ Estadual Paulista, Rua Clóvis Pestana, 793 Araçatuba/SP Laboratório de Patologia Aplicada (LApap) College of Veterinary Medicine UNESP—Univ Estadual Paulista, Rua Clóvis Pestana, 793 Araçatuba/SP College of Veterinary Medicine UNESP—Univ Estadual Paulista, Rua Clóvis Pestana, 793 Araçatuba/SP FAPESP: 2011/03504-4 FAPESP: 2012/01540-0

International audience; A female adult mixed-breed stray dog presented with hind limb paraparesis and clinical signs of visceral leishmaniasis. The cerebrospinal fluid presented signs of blood-brain barrier disruption. Both spleen and brain were positive for Leishmania spp. DNA. Besides inflammation, in situ hybridization and immunohistochemistry (IHC) revealed the presence of intracellular amastigotes in the choroid plexus (CP). Despite other studies that revealed parasite DNA, the current study describes the presence of Leishmania within the brain of a naturally infected dog, specifically in CP, with no previous reports in the Americas, and suggests the CP as a possible pathway to parasite entry into the brain.

Made available in DSpace on 2019-10-06T16:10:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-01 PD-1 is a negative costimulator of chronic infectious diseases In this study, we investigated the expression of PD-1 and its ligands in the spleen of dogs with visceral leishmaniasis and lymphoproliferative response to soluble antigen, in lymph node cells in the presence or absence of antibodies blocking PD-1 and its ligands. Our results showed expression of PD-1 and its ligands is higher after L. infantum infection and in the spleen of infected dogs, PD-1 blockage was able to restore the antigen-dependent lymphoproliferative response and regulated production of the cytokines IL-4 and IL-10 and NO production. We concluded that L. infantum infection modulates PD-1 and its ligands expression in canine VL and that blockage of PD-1 restores the immune response. Thus, blockage of PD-1 is a target for therapeutic drug development. Cellular Immunology Laboratory Department of Medicine Surgery and Animal Reproduction School of Veterinary Medicine of Araçatuba (FMVA) Universidade Estadual Paulista “Julio de Mesquita Filho” (UNESP) Cellular Immunology Laboratory Department of Medicine Surgery and Animal Reproduction School of Veterinary Medicine of Araçatuba (FMVA) Universidade Estadual Paulista “Julio de Mesquita Filho” (UNESP)

Visceral leishmaniosis (VL) remains a serious public health problem in Brazil. Dogs are the main hosts of the parasite, developing canine leishmaniosis (CanL), hence the importance of an accurate diagnosis of the animals. Recently, the application of qPCR method to non-invasive samples obtained from dogs with CanL has shown high sensitivity. Thus, we analyzed by qPCR blood, hair (from healthy zones and cutaneous lesions) and cerumen of 16 dogs with confirmed leishmaniosis from Aracatuba, a Brazilian endemic area. Cerumen-qPCR showed the highest sensitivity (87.5%), followed by hair (lesions: 78.57%, healthy skin: 62.5%), and blood (68.75%). We also analyzed blood, hair and cerumen of 5 healthy dogs from a non-endemic area, obtaining 100% of specificity in all samples. The use of cerumen and hair for qPCR analysis provides high reliability, taking into account the sensitivity and total specificity of the method. The non-invasive sampling procedure without the need of specific conditions of storage and transport support the usefulness of hair and cerumen for the diagnosis of CanL.

Activation of astrocytes or astrogliosis is a prominent component of the inflammatory response and an indicator of injury in the brain. These astrocytes produce a large array of inflammatory mediators, growth and neuroprotective factors. This study was an investigation from astrocyte (GFAP) response and TGF- ? 1 involvement during VSV acute encephalitis using immunohistochemistry to verify relation between astrocytes and TGF- ? 1. Animals developed symptoms around 6 th day after VSV inoculation. Viral proteins were mainly detected at olfactory bulb, ventricular cell layer and disseminated to hippocampus, mesencephalon and diecephalon areas and brain stem. Also at 6 th day post inoculation GFAP and TGF- ? 1 staining was observed in good association with virus-detected areas of brain. However, in mice with severe symptoms we observed reduction in the intensity of GFAP labeling at the same areas where TGF- ? 1 upregulation was observed. These areas show correlation with areas of necrosis and where are astrocytes with degenerative aspect. We observed TGF- ? 1 staining in damaged astrocytes, suggesting an effort of those cells in controlling inflammation in acute phase of VSV encephalitis.

Made available in DSpace on 2018-12-11T17:19:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The skin is the first organ to be infected by the parasite in canine visceral leishmaniasis. The enzyme matrix metalloproteinase (MMP) acts towards degradation of the extracellular matrix (ECM) and modulation of the inflammatory response against many kinds of injuries. The aims of this study were to evaluate the expression of MMP-2 and MMP-9 through immunohistochemistry and zymography on the skin (muzzle, ears, and abdomen) of dogs that were naturally infected by Leishmania spp. and to compare these results with immunodetection of the parasite and with alterations to the dermal ECM. Picrosirius red staining was used to differentiate collagen types I and III in three regions of the skin. The parasite load, intensity of inflammation, and production of MMP-2 (latent) and MMP-9 (active and latent) were higher in the ear and muzzle regions. MMP-9 (active) predominated in the infected group of dogs and its production was significantly different to that of the control group. Macrophages, lymphocytes, and plasma cells predominated in the dermal inflammation and formed granulomas in association with degradation of mature collagen (type I) and with discrete deposition of young collagen (type III). This dermal change was more pronounced in dogs with high parasite load in the skin. Therefore, it was concluded that the greater parasite load and intensity of inflammation in the skin led consequently to increased degradation of mature collagen, caused by increased production of MMPs, particularly active MMP-9, in dogs with visceral leishmaniasis. This host response profile possibly favors systemic dissemination of the parasite. Department of Veterinary Medicine Rio Preto University Center (UNIRP) Department of Infection and Epidemiology (IE) Pasteur Institute Department of Clinical Medicine Surgery and Animal Reproduction Araçatuba School of Veterinary Medicine (FMVA) São Paulo State University (UNESP) Department of Veterinary Pathology School of Veterinary and Agrarian Sciences (FCAV) São Paulo State University (UNESP) Departamento de Patologia Veterinária FCAV - Universidade Estadual Paulista (UNESP), Via de Acesso Prof. Paulo Donato Castellane, s/n Department of Pathology School of Veterinary Medicine and Animal Science (FMVZ) University of São Paulo (USP) Department of Clinical Medicine Surgery and Animal Reproduction Araçatuba School of Veterinary Medicine (FMVA) São Paulo State University (UNESP) Department of Veterinary Pathology School of Veterinary and Agrarian Sciences (FCAV) São Paulo State University (UNESP) Departamento de Patologia Veterinária FCAV - Universidade Estadual Paulista (UNESP), Via de Acesso Prof. Paulo Donato Castellane, s/n FAPESP: 2009/07815-4

International audience; ABSTRACT: The aim of this study was to investigate the occurrence of Tritrichomonas foetus in cats in the area surrounding the city of Araçatuba municipality, State of São Paulo, Brazil. Fecal samples from 129 cats were collected by rectal flush technique. It was compared two diagnosis methods, direct examination of feces and PCR. The presence of T. foetus DNA was verified using PCR by amplification of 347-bp fragment from the primers TFR3 and TFR4 and amplicons of positive cases were sequenced. Statistical analyses were performed investigating the associations between T. foetus infection with gender, age, breed, presence of diarrhea and/or history of diarrhea, previous treatment, lifestyle, origin, environment, and co-infection. T. foetus was observed in one sample (n=129) by direct microscopic examination of feces while PCR was positive in five samples (3.9%). Giardia species and Cryptosporidium species co-infection was also observed. Statistical analyses showed no significant associations between T. foetus infection and all listed factors, although most positive cats were asymptomatic and lived in multi-cat household. The isolates of T. foetus showed 100% identical sequences with other T. foetus isolates from cats around the world. So, the occurrence of T. foetus was confirmed in cats in Araçatuba city (Brazil). This parasite must be considered as a differential diagnosis in cats with diarrhea and also in asymptomatic carriers as source of infection in multi-cat environments.; RESUMO: O objetivo deste estudo foi investigar a ocorrência de Tritrichomonas foetus em gatos na região do município de Araçatuba, SP, Brasil. Foram coletadas amostras fecais de 129 gatos através da técnica de lavado retal. Dois métodos diagnósticos foram comparados, o exame direto das fezes e a PCR. A presença de DNA de T. foetus foi verificada por meio da PCR através da amplificação de 347 pares de bases a partir dos iniciadores específicos TFR3 e TFR4. Posteriormente, os resultados amplificados das amostras positivas foram sequenciadas. Também foi feita análise estatística a fim de investigar a correlação entre infecção por T. foetus e sexo, idade, raça, presença e/ou histórico de diarreia, tratamento prévio, coinfecção, estilo de vida, origem e tipo de ambiente. O protozoário pôde ser observado em uma amostra através do exame direto das fezes e à PCR foram detectadas cinco amostras positivas (3.9%). Foram detectadas coinfecções por Giardia spp. e Cryptosporidium spp. Não foram observadas correlações entre infecção por T. foetus e todos os fatores listados anteriormente, embora a maioria dos felinos positivos fossem assintomáticos e vivessem em ambientes multigatos. O resultado do sequenciamento genético dos isolados das amostras positivas mostrou 100% de similaridade com outros isolados de felinos no mundo. Assim, a ocorrência de T. foetus foi confirmada em gatos em Araçatuba, São Paulo, Brasil. Sendo assim, o parasito deve considerado como diagnóstico diferencial em gatos com diarreia assim como em portadores assintomáticos como fontes de infecção em ambientes multigatos.

Made available in DSpace on 2018-12-11T16:44:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-02 Animal experimentation is a controversial topic, especially among the general public and the scientific community. Thirty-eight undergraduate students attending the College of Veterinary Medicine–São Paulo State University in the municipality of Araçatuba, São Paulo, Brazil, were followed up between 2008 and 2011 and were asked to complete an annual questionnaire focused on different aspects of animal experimentation, including the animal species involved, the objectives of the research, ethics, animal welfare and euthanasia. Most students agreed that animal testing is not morally incorrect, and the dynamics of students’ attitudes were notable: undergraduates tended to change their opinion over time, with junior students opposing animal experimentation more than seniors do, indicating that the more scientific knowledge the students acquire, the more favourable to animal experimentation they become. Nevertheless, they agreed that research must consider the basic principles of animal welfare. Department of Animal Clinics Surgery and Reproduction College of Veterinary Medicine UNESP–Univ Estadual Paulista Department of Production and Animal Health College of Veterinary Medicine; UNESP–Univ Estadual Paulista Laboratoire des Processus Infectieux à Trypanosomatidés Département Infection et Epidemiologie Institut Pasteur Department of Animal Clinics Surgery and Reproduction College of Veterinary Medicine UNESP–Univ Estadual Paulista Department of Production and Animal Health College of Veterinary Medicine; UNESP–Univ Estadual Paulista

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:27Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:28:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-18 In canine visceral leishmaniasis (CVL), the abnormalities most commonly observed in clinical examination on the animals are lymphadenomegaly and skin lesions. Dogs are the main domestic reservoir for the protozoon Leishmania (L.) chagasi and the skin is the main site of contamination by the vector insect. Some protozoa use apoptosis as an immunological escape mechanism. The aim of this study was to correlate the presence of apoptosis with the parasite load and with the inflammatory response in the skin and lymph nodes of dogs naturally infected with Leishmania (L.) chagasi. Thirty-three dogs from the municipality of Araçatuba (São Paulo, Brazil) were used, an endemic area for CVL. Muzzle, ear and abdominal skin and the popliteal, subscapular, iliac and mesenteric lymph nodes of symptomatic (S), oligosymptomatic (O) and asymptomatic (A) dogs were analyzed histologically. The parasite load and percentage apoptosis were evaluated using an immunohistochemical technique. Microscopically, the lymph nodes presented chronic lymphadenitis and the skin presented plasmacytic infiltrate and granulomatous foci in the superficial dermis, especially in the ear and muzzle regions. The inflammation was most severe in group S. The parasite load and apoptotic cell density were also greatest in this group. The cause of the lymphoid atrophy in these dogs was correlated with T lymphocyte apoptosis, thus leaving the dogs more susceptible to CVL. The peripheral lymph nodes presented the greatest inflammatory response. Independent of the clinical picture, the predominant inflammatory response was granulomatous and plasmacytic, both in the skin and in the peripheral lymph nodes. The ear skin presented the greatest intensity of inflammation and parasite load, followed by the muzzle skin, in group S. The ear skin area presented a non-significant difference in cell profile, with predominance of macrophages, and a significant difference from group A to groups O and S. It was seen that in these areas, there were high densities of parasites and cells undergoing apoptosis, in group S. The association between apoptosis and parasite load was not significant in the lymph nodes, but in the muzzle regions and at the ear tips, a positive correlation was seen between the parasite load and the density of cells undergoing apoptosis. The dogs in group S had the highest parasite load and the greatest number of apoptotic cells, thus suggesting that the parasite had an immune evasion mechanism, which could be proven statistically in the skin. © 2012 Elsevier B.V. Departamento de Patologia Veterinária Faculdade de Ciências Agrárias e Veterinárias (FCAV) UNESP, Jaboticabal, São Paulo, 14884-900 Departamento de Ciências Exatas FCAV UNESP, Jaboticabal, São Paulo Departamento de Clínica Cirurgia e Reprodução Animal Faculdade de Medicina Veterinária de Araçatuba (FMVA) UNESP, Araçatuba, São Paulo Departamento de Patologia Veterinária Faculdade de Ciências Agrárias e Veterinárias (FCAV) UNESP, Jaboticabal, São Paulo, 14884-900 Departamento de Ciências Exatas FCAV UNESP, Jaboticabal, São Paulo Departamento de Clínica Cirurgia e Reprodução Animal Faculdade de Medicina Veterinária de Araçatuba (FMVA) UNESP, Araçatuba, São Paulo

Visceral leishmaniosis is a zoonotic disease that is transmitted by Lutzomyia longipalpis sandflies. Dogs are the main peri-urban reservoir of the disease, and progression of canine leishmaniosis is dependent on the type of immune response elaborated against the parasite. Type 1 immunity is characterized by effective cellular response, with production of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). In contrast, Type 2 immunity is predominantly humoral, associated with progression of the disease and mediated by anti-inflammatory cytokines such as interleukin 10 (IL-10). Although seemly important in the dynamics of leishmaniosis, other gene products such as toll-like receptor 2 (TRL-2) and inducible nitric oxide synthase (iNOS) exert unclear roles in the determination of the type of immune response. Given that the dog skin serves as a micro-environment for the multiplication of Leishmania spp., we investigated the parasite load and the expression of TLR-2, iNOS, IL-10 and TNF-α in the skin of 29 infected and 8 control dogs. We found that increased parasite load leads to upregulation of TLR-2, IL-10 and TNF-α, indicating that abundance of these transcripts is associated with infection. We also performed a xenodiagnosis to demonstrate that increased parasitism is a risk factor for infectiousness to sandflies.

Matrix metalloproteinases (MMPs) are a group of calcium- and zinc-dependent endopeptidases that are involved in maintaining the extracellular matrix. MMP-2 and MMP-9 are thought to be related to the disruption of the blood-brain-barrier (BBB) by their ability to cleave type IV collagen, the main component of the basal membrane. To establish the presence of MMP-2 and MMP-9 in the pathogenesis of canine cerebral leishmaniasis, we examined the levels of these metalloproteinases in the cerebrospinal fluid (CSF) and serum of dogs with visceral leishmaniasis and neurological symptoms (n=16) and in the CSF and serum of uninfected healthy dogs (n=10) using zymography. In the CSF of dogs with cerebral leishmaniasis there was a massive presence of active MMP-2, whereas only the levels of both proMMP-2 and proMMP-9 were elevated in the serum. Although the detected MMP activity in the CSF might merely be related to CNS inflammation, these enzymes may also play a collaborative role in the disease progression. Both MMP-2 and MMP-9 are known to target critical constituents of the BBB, and once activated, they may promote cerebral barrier breakdown, allowing the entrance of inflammatory cells and proteins within the nervous system milieu.

Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. Because glial cells are extensively associated with the immune response within the brain, we evaluated the morphology of astrocytes and microglia of dogs naturally infected with Leishmania chagasi. We used immunohistochemical and lectin-histochemical techniques for morphological analyses and we also examined the glial correlation with lymphocyte infiltration of the brain and with the presence of anti-Leishmania antibodies within the cerebrospinal fluid of the dogs. Although we did not detect a shared morphological pattern in the astrocytes or microglia in the brain tissue, these cells were more intensely labelled in infected dogs than in the control group. The density of microglia was increased in the ependymal/subependymal area, thus demonstrating a strong correlation with the presence of T lymphocytes and with cerebrospinal fluid antibody titres. Thus, our results indicate a pro-inflammatory state in the brains of dogs naturally infected with L. chagasi and strongly suggest that microglia and astrocytes are involved in the pathogenesis of the neurological disorders of visceral leishmaniasis in dogs.

Inflammation causes increases in the level of matrix metalloproteinases (MMPs), which, in central nervous system (CNS), are associated with neuroinflammation and disruption of blood-brain barrier. Analysis of cerebrospinal fluid (CSF) is pivotal for detecting diseases in CNS and, although a specific diagnosis may not be achieved, this analysis is helpful to confirm the diagnosis or to rule out relevant differential diagnoses. This study examined the levels of MMP-2 and MMP-9 in the CSF of dogs using gelatin zymography to verify possible alterations in these enzymes during natural systemic infection with Leishmania chagasi. Latent and active forms of MMP-2 were detected in some dogs of both groups, with high levels in the control group. In contrast, latent and active forms of MMP-9 were detected only in some animals with leishmaniasis. These results clearly demonstrate that MMP-9 is elevated in CSF of dogs with visceral leishmaniasis (VL). Although these results are preliminary, they suggest that MMP-9 might play a role in disruption of blood-brain barrier and/or blood-CSF barrier. While the presence of MMPs in CSF is not a condition exclusive to VL, their presence and persistence in CSF supports the hypothesis of an inflammatory state within CNS of dogs with VL.

Summary An 18-year-old Quarter Horse mare presented with an 18 week history of dermatological lesions characterised by recurrent urticaria and recent signs of enlarged mammary glands, pyrexia, alopecia and intense pruritus. Haematological and serum biochemical analyses revealed anaemia, leucocytosis, thrombocytopenia and hyperfibrinogenaemia. Mammary discharge was evaluated and revealed neutrophilia. Cytological examinations of fine needle aspirates from the parotid lymph nodes were normal. Common causes of anaemia were ruled out by specific tests. The initial diagnosis was mastitis and 2 weeks later the animal returned to the hospital with worsening of clinical signs, intermittent fever, intense pruritus and generalised alopecia. Blood samples were collected for haematological and serum biochemical analyses, and the mammary gland and parotid lymph nodes were biopsied. Rectal palpation revealed a large irregular mass near the left kidney with adhesions of surrounding structures. The animal died before the laboratory results were ready. This report details the clinical, histological and immunophenotypic findings of a case of large lymphoma in a mare.

The present study aimed to estimate the cell response and parasite load in the popliteal lymph nodes of dogs with visceral leishmaniasis (VL), comparing these findings with the clinical staging of the disease. From the necropsy, 33 dogs were classified as symptomatic (S), asymptomatic (A), or oligosymptomatic (O). Cytology and histopathology were used to determine any presence of microscopic lesions and immunohistochemistry, for parasite load. Dog hyperimmune serum was used as the primary antibody. The inflammatory infiltrate in lymph nodes consisted of macrophages and plasmocytes. The granulomas invaded the trabecular and sinusoid regions and sometimes compressed the lymphocytes of the cortical region (atrophy) and medullary cord cells. Parasite load intensity was unrelated to the density of the macrophages infiltrating the lymph node. Significant differences in parasite load (P < 0.05) were observed between the three groups of infected dogs. Follicular hyperplasia of the cortical region occurred among A and O, while follicular atrophy predominated among S. The parasite load was the greatest among S, followed by O. It can be concluded that, regardless of clinical condition, the most evident cell response consisted of macrophages and plasmocytes. Lymphoid atrophy was observed among animals with intense granulomatous reaction and high parasite load, such as among the symptomatic dogs (P < 0.05). Likewise, the oligosymptomatic dogs also presented high density of parasites in the lymph nodes. Thus, we can confirm that dogs with clinical manifestations of VL have an immune system that is less effective for controlling infection by Leishmania chagasi, thereby favoring parasite multiplication.

Made available in DSpace on 2018-12-11T16:40:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-15 Visceral leishmaniasis is a chronic disease caused by Leishmania infantum. We aimed to detect the parasite in the brain of fifteen naturally-infected dogs using in situ hybridization and immunohistochemistry, and the gene expression of selected chemokines by RT-qPCR. We detected no parasite in the brain, but perivascular deposition of parasite DNA and IgG in the choroid plexus. We noticed up-regulation of CCL-3, CCL-4 and CCL-5, coherent with T lymphocyte accumulation, stating the brain as a pro-inflammatory environment. Indeed, not necessarily the parasite itself, but rather its DNA seems to act as a trigger to promote brain inflammation during visceral leishmaniasis. Faculdade de Medicina Veterinária UNESP - Univ Estadual Paulista Laboratório de Patologia Aplicada (LApap) Faculdade de Medicina Veterinária UNESP - Univ Estadual Paulista Laboratoire des Processus Infectieux à Trypanosomatidés Département Infection et Epidemiologie Institut Pasteur Faculdade de Medicina Veterinária UNESP - Univ Estadual Paulista Laboratório de Patologia Aplicada (LApap) Faculdade de Medicina Veterinária UNESP - Univ Estadual Paulista

Visceral leishmaniasis is a complex disease caused by Leishmania infantum, and in dogs, besides the classical symptoms, there are descriptions of inflammatory alterations in the brain. Brain inflammation is a strictly controlled process, and as the brain counts on the efficiency of the blood-brain barrier (BBB), we aimed to assess BBB integrity in dogs with spontaneous visceral leishmaniasis. Therefore, we evaluated markers in the cerebrospinal fluid (CSF) and in brain tissue related to BBB disruption and brain inflammation. Elevated albumin quota revealed BBB breakdown, corroborated by increased concentrations of anti-Leishmania antibodies in the CSF. In the brain, albumin and IgG staining formed halos around blood vessels, a classical indicator of BBB leakage. Soluble IgG was also detected in the choroid plexus and ependyma, and in these structures, IgG stained random resident cells. IgG(+) cells and Fcγ-RI(+) cells were identified in the choroid plexus, ependyma and perivascular in the brain parenchyma. The data support the occurrence of BBB disruption in dogs with spontaneous visceral leishmaniasis, and IgG as a key molecule that is capable of initiating and/or maintaining the inflammatory stimuli in the nervous milieu and the CSF as an important disseminator of inflammatory stimuli within the CNS.

Made available in DSpace on 2015-10-21T13:10:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-01-01. Added 1 bitstream(s) on 2015-10-22T09:53:04Z : No. of bitstreams: 1 S1984-29612015000100006.pdf: 2287154 bytes, checksum: 39b358e024b9b99871c03aef16177dcd (MD5) Para avaliar a prevalência de hipertensão arterial e sua correlação com a severidade da lesão renal e proteinúria em cães com leishmaniose, 66 cães foram divididos em dois grupos. O grupo 1 (G1), composto por 54 cães em estágio 1 de doença renal crônica (DRC), e o grupo 2 (G2) por 12 cães em estágios 2 e 3 de DRC. A prevalência de hipertensão foi de 28,8%, compreendendo 22,2% dos cães de G1 e 58,3% dos cães de G2 (p = 0,011). A pressão arterial média (PA) de G1 (135,7 ± 20,5) foi inferior a de G2 (170,0 ± 26,3) (P

The hallmark microscopic lesion in canine visceral leishmaniosis is the accumulation of lymphocytes and macrophages parasitized by amastigotes of Leishmania in the lymphoid organs. Matrix metalloproteinase (MMP)-2 and MMP-9 are important for leukocyte migration as they degrade the type IV collagen in the basal lamina. Sera from 65 dogs, 53 with serological diagnosis of visceral leishmaniosis and 12 healthy ones, were analysed by gelatin-zymography to detect MMP-2 and MMP-9 activity. The infected dogs presented higher levels of serum mature MMP-9, proMMP-9 and proMMP-2 than control dogs. No mature MMP-2 activity was observed. The levels of mature and proMMP-9 were highly correlated. These findings suggest that the multi-systemic inflammatory lesions observed in visceral leishmaniosis are associated with an increase in serum MMPs, especially MMP-9. In concert with other clinical data, quantification of serum MMP-9 in infected dogs may lead to a better understanding of the pathogenesis of visceral leishmaniosis.

Made available in DSpace on 2015-10-21T13:10:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-03-15. Added 1 bitstream(s) on 2015-10-22T09:53:05Z : No. of bitstreams: 1 WOS000351267000001.pdf: 2449473 bytes, checksum: b225c3d24561829c859fb56354c7405b (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação para o Desenvolvimento da UNESP (FUNDUNESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: Recent studies have demonstrated numerous biological properties of mesenchymal stem cells and their potential application in treating complex diseases or injuries to tissues that have difficulty regenerating, such as those affecting the central and peripheral nervous system. Thus, therapies that use mesenchymal stem cells are promising because of their high capacity for self-regeneration, their low immunogenicity, and their paracrine, anti-inflammatory, immunomodulatory, anti-apoptotic and neuroprotective effects. In this context, the purpose of this study was to evaluate the feasibility and safety of intrathecal transplantation of bone marrow-derived mesenchymal stem cells in horses, for future application in the treatment of neurological diseases.Results: During the neurological evaluations, no clinical signs were observed that were related to brain and/or spinal cord injury of the animals from the control group or the treated group. The hematological and cerebrospinal fluid results from day 1 and day 6 showed no significant differences (P > 0.05) between the treated group and the control group. Additionally, analysis of the expression of matrix metalloproteinase (MMP) -2 and -9 in the cerebrospinal fluid revealed only the presence of pro-MMP-2 (latent), with no significant difference (P > 0.05) between the studied groups.Conclusions: The results of the present study support the hypothesis of the feasibility and safety of intrathecal transplantation of autologous bone marrow-derived mesenchymal stem cells, indicating that it is a promising pathway for cell delivery for the treatment of neurological disorders in horses. Universidade Estadual de Maringá, Departamento de Medicina Veterinária Universidade Estadual Paulista, Departamento de Reprodução Animal e Radiologia Veterinária, Faculdade de Medicina Veterinária e Zootecnia de Botucatu Universidade Estadual Paulista, Departamento de Clínica, Cirurgia e Reprodução Animal, Faculdade de Medicina Veterinária de Araçatuba Universidade Estadual Paulista, Departamento de Clínica Veterinária, Faculdade de Medicina Veterinária e Zootecnia de Botucatu FAPESP: 2009/51431-6 FUNDUNESP: 00571/09 CNPq: 481350/2009-8

Summary Visceral leishmaniasis is an important parasitic disease that affects humans and animals. The response against the protozoan involves the interaction of both innate and adaptive branches of the immune system, and an important immune sensor is represented by the toll-like receptor (TLR) family. Here, we investigated the pattern of TLR-2, TLR-4 and TLR-9 gene expression in different compartments (brain, choroid plexus, spleen and lymph node) of dogs naturally infected with Leishmania infantum. Gene expression of the TLRs varied according to the compartment evaluated. In the brain, there was only an upregulation of TLR-2, whereas in the choroid plexus, TLR-2 and TLR-9 were both upregulated. Further, the peripheral lymphoid organs (spleen and lymph nodes) showed increased TLR-2 and TLR-4 expression. This study provides the first insight about TLR expression in the central nervous system of infected dogs, and gives additional evidence of the compartmentalization of the immune response during visceral leishmaniasis.

Visceral leishmaniasis is an anthropozoonosis caused by the protozoan Leishmania infantum (L. chagasi). In dogs, the disease presents with systemic manifestations, including neurological disorders. There are rare reports of the presence of the parasite in the central nervous system of infected dogs, and some evidences of inflammatory lesions and the breakdown of cerebral barriers have been described. The aim of this study was to investigate the presence of L. infantum DNA in five specific areas of the brains of 20 naturally infected dogs by real-time PCR. For the first time, the presence of parasite DNA was detected and quantified in the brains of naturally infected dogs, in all evaluated regions. These data provide strong evidence of the presence of the Leishmania parasite in the nervous milieu and contribute to a new perspective of the pathogenesis of visceral leishmaniasis.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:29:40Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:32:37Z : No. of bitstreams: 1 2-s2.0-84878646777.pdf: 1927931 bytes, checksum: 20fb74309fe7de3e76cb2016bfd195cf (MD5) Made available in DSpace on 2014-05-27T11:29:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-11 Background: Visceral leishmaniasis is a disease with great variability regarding the clinical manifestations in humans and dogs. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterize the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis. Concomitant with Leishmania chagasi, dogs may be infected by opportunistic pathogens, such as Toxoplasma gondii and Neospora caninum, which may contribute to the occurrence of lesions in the central nervous system. Hence, we aimed to compare the T and B lymphocytes population in the brains of infected dogs with seropositivity to L. chagasi, T. gondii and N. caninum concurrently (n = 24), seropositivity only to L. chagasi (n = 31), and seropositivity to T. gondii and N. caninum (n = 16). Uninfected dogs were used as control (n = 10). Results: Inflammatory lesions, characterised by mononuclear cell accumulation, composed mainly of CD3+ T lymphocytes predominated in several encephalic regions of the dogs from all the three infected groups, with no difference among them (P = 0.0004), whereas CD79α+ B lymphocytes were detected in very small intensity and presented no difference among groups (P = 0.5313). Furthermore, no association among diseases was detected at the serological enquire. Conclusions: We demonstrate that the peripheral infection by L. chagasi per se can promote the influx of lymphocytes within the nervous milieu as occurs during Toxoplasma and Neospora infections, and the concomitant seropositivity against these pathogens does not exacerbate the inflammatory brain lesions. Therefore, these findings give additional support that the brain should be included in the list of organs affected by visceral leishmaniasis and that even asymptomatic infected dogs may develop brain lesions. © 2013 Sakamoto et al.; licensee BioMed Central Ltd. Laboratory of Applied Pathology (LAPAP) College of Veterinary Medicine Universidade Estadual Paulista (UNESP), Rua Clóvis Pestana 739, CEP 16050-680, Araçatuba, São Paulo Laboratory of Applied Pathology (LAPAP) College of Veterinary Medicine Universidade Estadual Paulista (UNESP), Rua Clóvis Pestana 739, CEP 16050-680, Araçatuba, São Paulo

The hearts of 30 dogs naturally infected with Leishmania infantum chagasi were evaluated histologically and immunohistochemically. Myocardial lesions were detected in all dogs, including lymphoplasmacytic myocarditis (27/30), myonecrosis (24/30), increased interstitial collagen (22/30), lepromatous-type granulomatous myocarditis (7/30), fibrinoid vascular change (3/30), and vasculitis (1/30). The parasite was detected in the hearts of 20 of 30 dogs. The number of parasitized cells correlated with the intensity of the inflammation and with the number of granulomas. The results indicate that cardiac lesions are prevalent in dogs with naturally occurring leishmaniasis even in the absence of clinical signs of cardiac disease.