Salmonella enterica serovars in absence of ttrA and pduA genes enhance the cell immune response during chick infections.

Salmonella spp. is one of the major foodborne pathogens responsible for causing economic losses to the poultry industry and bringing consequences for public health as well. Both the pathogen survival ability in the intestinal environment during inflammation as well as their relationship with the host immune system, play a key role during infections in poultry. The objective of this study was to quantify the presence of the macrophages and CD4+/CD8+ cells populations using the immunohistochemistry technique, in commercial lineages of chickens experimentally infected by wild-type and mutant strains of Salmonella Enteritidis and Salmonella Typhimurium lacking ttrA and pduA genes. Salmonella Enteritidis ∆ttrA∆pduA triggered a higher percentage of the stained area than the wild-type, with exception of light laying hens. Salmonella Typhimurium wild-type strain and Salmonella Typhimurium ∆ttrA∆pduA infections lead to a similar pattern in which, at 1 and 14 dpi, the caecal tonsils and ileum of birds showed a more expressive stained area compared to 3 and 7 dpi. In all lineages studied, prominent infiltration of macrophages in comparison with CD4+ and CD8+ cells was observed. Overall, animals infected by the mutant strain displayed a positively stained area higher than the wild-type. Deletions in both ttrA and pduA genes resulted in a more intense infiltration of macrophages and CD4+ and CD8+ cells in the host birds, suggesting no pathogen attenuation, even in different strains of Salmonella.

Mesenteric metastasis of granulosa cell tumor in a bitch: a case report / Metástasis mesentérica de tumor de células de granulosa en una perra: reporte de caso / Metástase mesentérica de tumor das células da granulosa em uma cadela: relato de caso

Abstract Anamnesis: A 5-year-old mixed breed entire bitch was presented with bloody vaginal discharge and abdominal distension. Clinical and laboratory findings: Physical examination and abdominal ultrasonography suggested the presence of a left ovarian tumor. At histopathological examination it was diagnosed as granulosa cell tumor. Treatment approach: An exploratory laparotomy was performed followed by ovariohysterectomy to remove the intra-abdominal mass. Conclusion: The clinical, ultrasonographic and histologic signs in this case support other literature reports. Ultrasonography, in combination with histopathologic analysis is a fundamental tool for detection of ovarian tumors, especially tumors of the granulosa cells.

Resumen Anamnesis: Una perra entera sin raza definida, con cinco años de edad, presentando secreción vaginal sanguinolenta con una distensión abdominal acentuada. Hallazgos clínicos y de laboratorio: El examen clínico y la ultrasonografía abdominal sugirieron la presencia de la neoformación ovárica izquierda. En el examen histopatológico fue diagnosticado como tumor de células de la granulosa. Aproximación terapéutica: Se realizó una laparotomía exploratoria seguida de ovariohisterectomía para eliminar la masa intra-abdominal. Conclusión: Los signos clínicos, ecográficos e histológicos en este caso respaldan otros informes de la literatura. La ecografía, en combinación con el análisis histopatológico, es una herramienta fundamental para la detección de tumores ováricos, especialmente tumores de las células de la granulosa.

Resumo Anamnese: Uma cadela sem raça definida, com cinco anos de idade, não castrada, apresentando secreção vaginal sanguinolenta com acentuada distensão abdominal. Achados clínicos e laboratoriais: O exame clínico e ultrassonografia abdominal sugeriram a presença de neoformação ovariana esquerda. No exame histopatológico foi diagnosticado como tumor de células da granulosa. Abordagem terapêutica: Foi realizada laparotomia exploratória seguida de ovariohisterectomia para remoção da massa intra-abdominal. Conclusão: Os sinais clínicos, ultrassonográficos e histológicos corroboram a literatura, concluindo que a modalidade ultrassonográfica, em associação a análise histopatológica é uma ferramenta fundamental na detecção de neoformações ovarianas, contribuindo significativamente no diagnóstico final.

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.

p-mTOR, p-4EBP-1 and eIF4E expression in canine prostatic carcinoma.

The mTOR/4E-BP1/eIF4E pathway plays important roles in the neoplastic transformation process and in tumour growth. In men, the mTOR/4E-BP1/eIF4E pathway was described as altered in different tumours, including prostate cancer (PC). Apart from humans, the dog is the only species that develops PC with high frequency and is considered a good model for comparative oncology initiatives. Due to limited information on this pathway in canine tumours, this study aimed to investigate mTOR, 4E-BP1 and eIF4E gene and protein expression in canine PC, as well as in metastatic and normal prostatic tissues, and to evaluate the correlations between gene/protein expression and Gleason score (GS) in PC. A total of 35 formalin-fixed paraffin-embedded (FFPE) samples, including 13 of normal prostatic tissue, 17 PC samples and 5 metastasis samples, were evaluated by immunohistochemistry and qPCR. mTOR gene mutation in the kinase domain was also investigated. We identified higher p-mTOR and eIF4E protein levels in canine PC with higher GS values (≥ 8) and a significant positive correlation in expression between these proteins. eIF4E overexpression was observed in metastasis relative to expression in normal samples. Our data suggest that p-mTOR and eIF4E expression is positively correlated with GS in canine PC, similar to the pattern in humans. More studies of the mTOR/4EBP1/eIF4E pathway should be performed to identify possible correlations of the proteins involved with clinical and pathologic findings in canine PC and the roles of these proteins as therapeutic targets for the treatment of canine PC.

Immunodetection of myeloid and plasmacytoid dendritic cells in mammary carcinomas of female dogs / Imunodetecção de células dendríticas mieloides e plasmocitoides nos carcinomas mamários em cadelas

Dendritic cells have attracted great interest from researchers as they may be used as targets of tumor immune evasion mechanisms. The main objective of this study was to evaluate the relationship between the dendritic cells (DCs) subpopulation in simple type mammary carcinomas in female dogs. Two groups of samples were used: the control group consisted of 18 samples of mammary tissue without changes and the tumor group with 26 simple type mammary carcinomas. In these groups, we evaluated the immunodetection of immature and mature myeloid DCs, plasmacytoid DCs and MHC-II. In mammary tumor, mature myeloid DCs predominated in the peritumoral region, while immature myeloid DCs and plasmacytoid DCs were evident in the intratumoral region. Immunostaining of MHC-II was visualized in mammary acini (control group), in tumor cells and inflammatory infiltration associated with tumors. The comparison between the control and tumor groups showed a statistically significant difference between immature myeloid DCs, mature myeloid DCs and plasmacytoid DCs. The immunodetection of MHC-II was not significant when comparing the groups. The predominance of immature DCs in the tumor group is possibly related to an inefficient immune response, promoting the development and survival of tumor cells. The presence of plasmacytoid DCs in the same group suggests a worse prognosis for female dogs with mammary tumors. Therefore, the ability of differentiation of canine dendritic cells could be influenced by neoplastic cells and by the tumor microenvironment...

As células dendríticas têm despertado grande interesse dos pesquisadores, pois podem ser alvo dos mecanismos de evasão imune do tumor. O objetivo principal deste estudo foi avaliar a relação entre as subpopulações de células dendríticas (DCs) nos carcinomas mamários do tipo simples em cadelas. Dois grupos de amostras foram utilizados, o grupo controle composto por 18 amostras de tecido mamário sem alterações e o grupo tumor com 26 carcinomas mamários do tipo simples. Nestes grupos foram avaliadas a imunodetecção de DCs mieloides imaturas e maduras, DCs plasmocitoides e de MHC-II. Nas mamas com tumor, as DCs mieloides maduras predominaram na região peritumoral, enquanto que as DCs mieloides imaturas e as DCs plasmocitoides foram evidentes na região intratumoral. A imunomarcação do MHC-II foi visualizada nos ácinos mamários (grupo controle), nas células tumorais e no infiltrado inflamatório associado aos tumores. Na comparação entre os grupos controle e tumor houve diferença estatística significativa entre as DCs mieloides imaturas, DCs mieloides maduras e DCs plasmocitoides. A imunodetecção de MHC-II não foi significativa na comparação entre os grupos. A predominância de DCs imaturas no grupo tumor, possivelmente, está relacionada com uma resposta imune ineficiente, favorecendo o desenvolvimento e a sobrevivência das células tumorais. A presença das DCs plasmocitoides no mesmo grupo sugere um prognóstico pior para cadelas com tumores de mama. Portanto, a capacidade de diferenciação das células dendríticas caninas poderia ser influenciada pelas células neoplásicas e pelo microambiente tumoral...

Increased expression of interleukin-6 related to nephritis in chickens challenged with an avian infectious bronchitis virus variant / Aumento da expressão de interleucina-6 relacionada com nefrite in galinhas desafiadas com uma variante do vírus da bronquite infecciosa

A Brazilian field isolate (IBV/Brazil/PR05) of avian infectious bronchitis virus (IBV), associated with development of nephritis in chickens, was previously genotyped as IBV variant after S1 gene sequencing. The aim of this study was to evaluate the levels of IL-6 in kidneys and trachea of birds vaccinated and challenged with IBV/Brazil/PR05 strain, correlating these results with scores of microscopic lesions, specific IBV antigen detection and viral load. The up-regulation of IL-6 and the increased levels of viral load on renal and tracheal samples were significantly correlated with scores of microscopic lesions. Reduced levels of viral load were detected in kidneys of birds previously vaccinated and challenged, compared to non-vaccinated challenged group, although markedly microscopic lesions were observed for both groups. The expression of IL-6, present both in the kidney and in the tracheas, was dependent on the load of the virus present in the tissue, and the development of lesions was related with IL-6 present in the tissues. These data suggest that variant IBV/Brazil/PR05 can induce the expression of proinflammatory cytokines in a manner correlated with viral load and increased IL-6 is involved in the tissue with the influx of inflammatory cells and subsequent nephritis. This may contribute with a model to the development of immunosuppressive agents of IL-6 to prevent acute inflammatory processes against infection with IBV and perhaps other coronaviruses, as well as contribute to the understanding of the immunopathogenesis of IBV nephropatogenic strains.

Uma estirpe variante do vírus da bronquite infecciosa (VBI) associada com o desenvolvimento de nefrite em galinhas, foi isolado e identificado como variante por análise do gene S1. A estirpe IBV/Brazil/PR05 foi testada quanto à sua capacidade de induzir a expressão de interleucina-6 (IL-6) nos tecidos renais e traqueais. Galinhas vacinadas com a estirpe Massachusetts H120 e não vacinadas foram desafiadas com a estirpe IBV/Brazil/PR05. Cinco dias após a infecção, traquéias e rins foram coletados para análise por RT-qPCR, imunohistoquímica e histopatologia. Foi determinada a expressão relativa de IL-6 e da carga viral. A expressão de IL-6 e carga viral foram correlacionadas com o desenvolvimento de nefrite e lesão traqueal. A expressão de IL-6 foi maior quando houve aumento da carga viral na traqueia e nos rins. A carga viral presente nos rins foi inferior quando as aves foram vacinadas, entretanto foi observada nefrite acentuada. Houve alta correlação entre o desenvolvimento de nefrite e o nível de expressão de IL-6, bem como a expressão de IL-6 e a carga viral. A expressão de IL-6, presente tanto nos rins e nas traqueias, foi relacionada a carga viral presente nestes tecidos, e o desenvolvimento das lesões foi relacionado com a expressão de IL-6. Estes dados sugerem que a variante IBV/Brazil/PR05 pode induzir a expressão de citocinas pró-inflamatórias de forma correlacionada com a carga viral, e o aumento de IL-6 está envolvido com o influxo de células inflamatórias no tecido, o que evolui para o desenvolvimento de nefrite. Isto pode contribuir como um modelo para o desenvolvimento de agentes imunossupressores da IL-6 para evitar processos inflamatórios agudos contra infecção com o VBI e talvez outros coronavírus, bem como contribuir para o entendimento da imunopatogênese das estirpes nefropatogênicas deste vírus.

CD4+FOXP3+ cells produce IL-10 in the spleens of dogs with visceral leishmaniasis.

Visceral Leishmaniasis (VL) is caused by intracellular parasites of the genus Leishmania that affect humans and several animal species. Dogs are one of the main urban reservoirs of the parasite and play a central role in the transmission cycle to humans via sandflies. Studies concerning the immune response in dogs with VL have demonstrated that protective immunity is associated with cellular immune response, while disease progression is associated with humoral response and IL-10 and TGF-ß production. The study aimed to evaluate IL-10 and TGF-ß production by regulatory T (Treg) cells in the blood and spleen of dogs naturally infected by Leishmania spp. and correlate this with parasite load. Five healthy dogs and 29 dogs with proven infection were selected for the study group. Real-time PCR was used to quantify parasite load and confirm infection by Leishmania spp. Treg cells producing IL-10 and TGF-ß were quantified using flow cytometry. An increase in IL-10 production by Treg cells was verified in the spleen of dogs naturally infected by Leishmania spp. Concurrently, a decrease in the total number of T cells in these dogs was verified compared with healthy dogs. No association was determined between parasite load and the percentage of spleen Treg cells producing IL-10 and TGF-ß. These findings suggest that Treg cells are an important source of IL-10 in the spleen, participating in immune response modulation, while the reduced percentage of these cells in infected dogs could be attributed to persistent immune activation.

Doxorubicin induced dilated cardiomyopathy in a rabbit model: an update.

Dilated cardiomyopathy (DCM) is characterized by chamber dilation and cardiac dysfunction. Because of the poor prognosis, models are needed for the investigation of and development of new therapeutic approaches, as well as stem cell therapy. Doxorubicin (DOX), used as chemotherapeutic agent, is reported to be cumulative cardiotoxic causing DCM. The aim of the study was to investigate the onset of systolic dysfunction using echocardiography in rabbits receiving two different doses of DOX (1mg/kg twice a week and 2 mg/kg once a week). Twenty rabbits were treated with doxorubicin in two different doses for 6 weeks and compared with a control group treated with NaCl 0.9%. The effect of doxorubicin on the myocardium was investigated with histological analysis and scanning electron microscopy of left ventricle (LV), as well as in the interventricular septum (IVS) and right ventricle (RV). The results showed a high mortality rate for rabbits receiving 2 mg/kg once a week. A significant reduction in systolic function was present in animals treated with DOX after 6 weeks, with decreased ejection fraction and shortening fraction. Histology and electron microscopy revealed vacuolization, intracytoplasmic granulation, necrosis and interstitial fibrosis in LV, as well as in the IVS and RV. Doxorubicin induced changes are present in the LV, RV and IVS, and the administration at the dose of 1 mg/kg twice a week for only 6 weeks is safe and sufficient to induce DCM in rabbits.

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.

Alterações morfológicas de tecido laminar do casco e parâmetros clínicos e laboratoriais de equinos com síndrome cólica letal / Morphologic alterations of the hoof lamellar tissue, and clinic and laboratorial analyses of horses with lethal colic syndrome

As afecções gastrintestinais dos cavalos são agravadas por complicações como a laminite, cuja etiopatogenia está relacionada à degradação da membrana basal do tecido laminar por metaloproteinases (MMPs). A ativação das MMPs pode ocorrer devido à liberação local de citocinas inflamatórias ou enzimas provenientes de leucócitos infiltrados no tecido laminar. O objetivo deste trabalho foi avaliar as alterações morfológicas do tecido laminar de equinos com síndrome cólica letal e sua provável associação com parâmetros clínicos e laboratoriais. Observou-se intensa destruição da arquitetura laminar, principalmente nos animais com alterações físicas e laboratoriais mais acentuadas, como tempo de preenchimento capilar prolongado (TPC), membranas mucosas congestas, taquicardia, hemoconcentração e redução nas contagens de plaquetas e leucócitos. Os resultados sinalizam o provável momento do desenvolvimento de lesões do tecido laminar em equinos com síndrome cólica, no qual é possível adotar medidas preventivas contra a laminite.

The gastrointestinal diseases of horses are aggravated by complications such as laminitis. The laminitis etiopathogeny are connected with lamellar basement membrane degradation by matrix metalloproteinases (MMPs). Inflammatory cytokines and leukocytes enzymes can active MMPs. The object of this study was to evaluate morphological changes on lamellar tissue of horses with colic syndrome and its association with clinical and laboratorial parameters. It was observed intensive destruction of lamellar architecture, mainly on animals with severe physical and laboratorial alterations, such as delayed capillary refill time, congested mucous membrane, tachycardia, hemoconcentration and low count of platelet and leukocytes. The results sign to the most likely moment of development of lamellar tissue injuries in horses with colic syndrome, which can be adopted preventive measures against laminitis.

An investigation of Leishmania spp. in Didelphis spp. from urban and peri-urban areas in Bauru (São Paulo, Brazil).

Blood and bone marrow samples were taken from 112 Didelphis spp., collected between March 2005 and February 2006, from urban and peri-urban areas of Bauru, São Paulo State, Brazil, to evaluate the hypothesis that these animals might constitute a reservoir of Leishmania spp. Anti-Leishmania ssp. antibodies were screened in the serum samples using an enzyme-linked immuno-sorbent assay (ELISA) and the polymerase chain reaction (PCR). PCR was performed on fragments of DNA samples from Leishmania spp. using primers 13A and 13B, and showed a positive outcome in 91.6% of the 112 samples tested. Of the 107 samples analyzed by ELISA, 71% were positive. Evidence of epidemiological risk factors such as a circulating parasite and freely moving vectors suggests that Didelphis spp. may participate in the transmission cycle of Leishmania spp. in Bauru.