Contribution of astrocytes and macrophage migration inhibitory factor to immune-mediated canine encephalitis caused by the distemper virus.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is produced by many cell types in situations of homeostasis or disease. One of its functions is to act as a proinflammatory molecule. In humans, several studies have shown that MIF levels become elevated in the serum, urine, cerebrospinal fluid and tissues of patients with chronic inflammatory diseases (systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, sepsis, atheromas, diabetes and cancer). In dogs, distemper is a viral infectious condition that may lead to demyelination and inflammation in the central nervous system (CNS). In addition to the action of the virus, the inflammatory process may give rise to lesions in the white matter. Therefore, the objectives of the present study were to evaluate the role of MIF in the encephalitis that the canine distemper virus causes and to compare this with immunodetection of major histocompatibility complex-II (MHC-II), CD3 T lymphocytes, MMP-9 and glial fibrillary acidic protein (GFAP; astrocytes) in demyelinated areas of the encephalon, in order to ascertain whether these findings might be related to the severity of the encephalic lesions. To this end, a retrospective study on archived paraffinized blocks was conducted, in which 21 encephala from dogs that had been naturally infected with the canine distemper virus (infected group) and five from dogs that had been free from systemic or CNS-affecting diseases (control group) were used. In the immunohistochemical analysis on the samples, the degree of marking by GFAP, MHC-II, MMP-9 and MIF was greater in the demyelinated areas and in the adjacent neuropil, and this was seen particularly in astrocytes. Detection of CD3 was limited to perivascular cuffs. In areas of liquefactive necrosis, Gitter cells were positive for MMP-9, MIF and MHC-II. Hence, it was concluded that activated astrocytes influenced the afflux of T lymphocytes to the encephalon (encephalitis). In the more advanced phases, activated phagocytes in the areas of liquefactive necrosis (Gitter cells) continued to produce inflammatory mediators even after the astrocytes in these localities had died, thereby worsening the encephalic lesions. Distemper virus-activated astrocytes and microglia produce MIF that results in proinflammatory stimulus on glial cells and brain-infiltrating leukocytes. Therefore, the effect of the inflammatory response is potentiated on the neuropil, resulting in neurological clinical signs.

Fatal hemothorax caused by pleural mesothelioma in a lion / Hemotórax fatal causado por mesotelioma pleural em um leão

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.(AU)

O mesotelioma é considerado um neoplasma maligna causada pela proliferação de células mesoteliais, principalmente da pleura, peritôneo e pericárdio. O presente caso descreve os achados macroscópicos, microscópicos e imuno-histoquímicos do hemotórax fatal causado por um mesotelioma pleural em um leão. Macroscopicamente, na cavidade torácica, foi observado cerca de 4 litros de sangue. Além disso, foram observadas numerosas massas macias, vermelho-rosa, irregulares, com até 1cm de diâmetro e distribuídas aleatoriamente pelas pleuras parietal e visceral. Microscopicamente, as massas torácicas apresentavam estruturas papilares, compostas por uma camada de células mesoteliais, que apresentavam citoplasma eosinofílico, núcleo central e nucléolo evidente, suportada por um estroma fibrovascular pouco celular. A imuno-histoquímica foi positiva para ambas citoqueratina e vimentina nas células neoplásicas. Este trabalho descreve o que parece ser o primeiro relato de um hemotórax fatal causado por um mesotelioma pleural em um leão.(AU)

Expression of matrix metalloproteinase-2 and metalloproteinase-9 in the skin of dogs with visceral leishmaniasis.

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.

Toll-like receptors and cytokines in the brain and in spleen of dogs with visceral leishmaniosis.

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-1ß 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.

Matrix metalloproteinases 2 and 9 in rabbits with doxorubicin-induced cardiomyopathy / Metaloproteinases de matriz 2 e 9 em coelhos com cardiomiopatia induzida pela doxorrubicina

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.(AU)

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.(AU)

Detection of natural occurrence of Tritrichomonas foetus in cats in Araçatuba, São Paulo, Brazil / Detecção da ocorrência natural de Tritrichomonas foetus em gatos em Araçatuba, São Paulo

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.(AU)

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.(AU)

Unveiling Cerebral Leishmaniasis: parasites and brain inflammation in Leishmania donovani infected mice.

Visceral leishmaniasis (VL) is a systemic disease with multifaceted clinical manifestations, including neurological signs, however, the involvement of the nervous system during VL is underestimated. Accordingly, we investigated both brain infection and inflammation in a mouse model of VL. Using bioluminescent Leishmania donovani and real-time 2D-3D imaging tools, we strikingly detected live parasites in the brain, where we observed a compartmentalized dual-phased inflammation pattern: an early phase during the first two weeks post-infection, with the prompt arrival of neutrophils and Ly6Chigh macrophages in an environment presenting a variety of pro-inflammatory mediators (IFN-γ, IL-1ß, CXCL-10/CXCR-3, CCL-7/CCR-2), but with an intense anti-inflammatory response, led by IL-10; and a re-inflammation phase three months later, extremely pro-inflammatory, with novel upregulation of mediators, including IL-1ß, TNF-α and MMP-9. These new data give support and corroborate previous studies connecting human and canine VL with neuroinflammation and blood-brain barrier disruption, and conclusively place the brain among the organs affected by this parasite. Altogether, our results provide convincing evidences that Leishmania donovani indeed infects and inflames the brain.

T lymphocyte immunophenotypes in the cerebrospinal fluid of dogs with visceral leishmaniasis.

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.

Blood pressure and renal injury in dogs with visceral leishmaniasis / Pressão sanguínea e lesão renal em cães com leishmaniose visceral

Systemic hypertension is known to be a common consequence of chronic renal disease, which is frequently diagnosed in dogs with visceral leishmaniasis. Although many veterinary investigations have looked at the renal injury caused by Leishmania spp., the role played by this complication in the development of arterial hypertension documented in some animals with visceral leishmaniasis is not completely understood. In this study, 18 adult dogs with naturally-occurring visceral leishmaniasis and varying clinical signs underwent an indirect blood pressure measurement. Also, sera and spot urine were used for laboratory tests. The median systolic blood pressure was 135.2mmHg (95% confidence interval: 128.5-147.7), median mean arterial pressure was 105.8mmHg (98.3-110.4), and median diastolic arterial pressure was 88.5mmHg (77.8-92.5). No differences existed between asymptomatic and symptomatic animals regarding arterial pressure, and no correlations were documented between blood pressure and serum creatinine, blood urea, urine protein-to-creatinine ratio, urine specific gravity, and the fractional excretion of sodium and potassium. Although an association between hypertension and the identification of inflammation on histopathology could not be demonstrated in hypertensive animals, the assessment of kidney samples from 12 dogs indicated mild inflammation with a lymphoplasmacytic infiltrate (6/12), moderate inflammation with multifocal lymphoplasmacytic and histiocytic infiltrates (3/12), and multifocal degeneration and protein casts (2/12). Anti-Leishmania spp. immunohistochemistry assays stained the renal epithelium in 2/12 of the animals. Even though mild systemic hypertension was documented in a small subset of animals, no relationship between the severity of clinical signs and hypertension could be anticipated.(AU)

A hipertensão sistêmica é reconhecida como uma consequência comum da doença renal crônica, cujo diagnóstico é frequente em cães com leishmaniose visceral. Embora muitas pesquisas veterinárias tenham investigado a lesão renal causada pela Leishmania spp., o papel dessa complicação no desenvolvimento da hipertensão arterial documentada em alguns animais com leishmaniose visceral ainda não é completamente compreendido. Neste estudo, 18 cães adultos com diagnóstico de leishmaniose visceral e sinais clínicos variáveis foram submetidos à avaliação indireta da pressão arterial. Além disso, foram coletados soro e urina para análises laboratoriais. As medianas das pressões arteriais sistólica, média e diastólica foram 135,2mmHg (intervalo de confiança de 95%: 128,5-147,7), 105,8mmHg (98,3-110,4) e 88,5mmHg (77,8-92,5), respectivamente. Não foram constatadas diferenças entre os cães assintomáticos e sintomáticos em relação à pressão arterial, assim como não houve correlação entre a pressão arterial e a creatinina e uréia séricas, relação proteína-creatinina urinária, densidade urinária e excreção fracionada de sódio e potássio. Embora não tenha sido evidenciada associação entre hipertensão arterial e inflamação do tecido renal à histopatologia, a avaliação das amostras oriundas de 12 cães indicou inflamação leve, com infiltrado linfoplasmocitário (6/12), inflamação moderada com infiltrados linfoplasmocitário e histiocítico multifocais (3/12), além de degeneração multifocal e cilindros protéicos (2/12). Ensaios imunoistoquímicos anti-Leishmania spp. marcaram o epitélio renal em 2/12 animais. Apesar de hipertensão leve ter sido documentada em uma pequena parcela dos cães estudados, não se evidenciou relação entre a severidade dos sinais clínicos e o desenvolvimento de hipertensão arterial.(AU)

Leishmania infection and neuroinflammation: Specific chemokine profile and absence of parasites in the brain of naturally-infected dogs.

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.

First detection of Leishmania infantum DNA within the brain of naturally infected dogs.

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.

Zymographic patterns of MMP-2 and MMP-9 in the CSF and cerebellum of dogs with subacute distemper leukoencephalitis.

Distemper leukoencephalitis is a disease caused by the canine distemper virus (CDV) infection. It is a demyelinating disease affecting mainly the white matter of the cerebellum and areas adjacent to the fourth ventricle; the enzymes of the matrix metalloproteinases (MMPs) group, especially MMP-2 and MMP-9 have a key role in the myelin basic protein fragmentation and in demyelination, as well as in leukocyte traffic into the nervous milieu. To evaluate the involvement of MMPs during subacute distemper leukoencephalitis, we measured the levels of MMP-2 and MMP-9 by zymography in the cerebrospinal fluid (CSF) and in the cerebellum of 14 dogs naturally infected with CDV and 10 uninfected dogs. The infected dogs presented high levels of pro-MMP-2 in the CSF and elevated levels of pro-MMP-2 and pro-MMP-9 in the cerebellar tissue. Active MMP-2 was detected in the CSF of some infected dogs. As active MMP-2 and MMP-9 are required for cellular migration across the blood-brain barrier and any interference between MMPs and their inhibitors may result in an amplification of demyelination, this study gives additional support to the involvement of MMPs during subacute distemper leukoencephalitis and suggests that MMP-2 and MMP-9 may take part in the brain inflammatory changes of this disease.

Bovine herpesvirus-5 infection in a rabbit experimental model: immunohistochemical study of the cellular response in the CNS.

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 µl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 10(7.5) TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways.

Pro-inflammatory cytokines predominate in the brains of dogs with visceral leishmaniasis: a natural model of neuroinflammation during systemic parasitic infection.

Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. To investigate the pathogenesis of brain alterations occurring during visceral leishmaniasis infection, the expression of the cytokines IL-1ß, IL-6, IL-10, IL-12p40, IFN-γ, TGF-ß and TNF-α and their correlations with peripheral parasite load were evaluated in the brains of dogs naturally infected with Leishmania infantum. IL-1ß, IFN-γ and TNF-α were noticeably up-regulated, and IL-10, TGF-ß and IL-12p40 were down-regulated in the brains of infected dogs. Expression levels did not correlate with parasite load suggestive that the brain alterations are due to the host's immune response regardless of the phase of the disease. These data indicate the presence of a pro-inflammatory status in the nervous milieu of dogs with visceral leishmaniasis especially because IL-1ß and TNF-α are considered key factors for the initiation, maintenance and persistence of inflammation.

Canine cerebral leishmaniasis: potential role of matrix metalloproteinase-2 in the development of neurological disease.

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.

Glial reactivity in dogs with visceral leishmaniasis: correlation with T lymphocyte infiltration and with cerebrospinal fluid anti-Leishmania antibody titres.

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.

High levels of serum matrix metalloproteinases in dogs with natural visceral leishmaniosis: a preliminary report.

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.

Differential alterations in the activity of matrix metalloproteinases within the nervous tissue of dogs in distinct manifestations of visceral leishmaniasis.

Canine visceral leishmaniasis is an important emerging disease with a multitude of clinical symptoms, including neurological alterations. Matrix metalloproteinases (MMP) are proteases implicated with the extracellular matrix remodelling and, within the central nervous system, these enzymes are involved with blood-brain-barrier disruption and inflammation. To establish the involvement of MMP-2 and -9 within the nervous tissue of dogs with spontaneous visceral leishmaniasis, fragments of nervous tissue from oligosymptomatic (n=9), symptomatic (n=8), neurological (n=12) and normal dogs (n=8) were subjected to zymographic and to immunohistochemical analysis. Immunohistochemistry evidenced MMP-2 staining in inflammatory cells inside and outside blood vessels. MMP-9 was found in endothelial cells and in the ependyma. Zymographic evaluation revealed only the latent forms of MMP-2 and -9 within the nervous tissue. ProMMP-9 activity in the infected animals was found higher than the normal dogs, but with no difference among the infected dogs. Oligosymptomatic dogs presented the highest proMMP-2 activity, followed by the symptomatic and then, by the neurological and the normal dogs. In summary, the nervous tissue compartment seems to be preserved in dogs with VL, due to the absence of active MMPs, even though the elevated levels of proMMP-2 and -9 would indicate a pro-inflammatory state in the brain.

Feline cholangitis/ chonlangiohepatitis complex secondary to Platynosomum fastosum infection in a cat.

Platynosomiasis has been associated with cholangitis/cholangiohepatitis complex in domestic cats. Despite being typically asymptomatic, some individuals may develop severe disease resulting in hepatic cirrhosis. Definitive diagnosis by means of parasite eggs direct visualization is not always possible and complementary tests should be associated. This paper reports a case of a three-year-old female cat with anorexia and jaundice. Ultrasound and biochemistry laboratory findings as well as liver biopsy were not conclusive. Definitive diagnosis of intense Platynosomum fastosum infestation was only possible through direct examination of biliary fluid at necropsy, stressing the importance of including platynosomiasis, commonly an underdiagnosed disease, among differential diagnoses of feline liver diseases.