In vitro effects of synbiotic fermentation on the canine faecal microbiota.

Stirred, pH-controlled anaerobic batch cultures were used to investigate the in vitro effects of galacto-oligosaccharides (GOS) alone or combined with the probiotic Bifidobacterium bifidum 02 450B on the canine faecal microbiota of three different donors. GOS supported the growth of B. bifidum 02 450B throughout the fermentation. Quantitative analysis of bacterial populations by FISH revealed significant increases in Bifidobacterium spp. counts (Bif164) and a concomitant decrease in Clostridium histolyticum counts (Chis150) in the synbiotic-containing vessels compared with the controls and GOS vessels. Vessels containing probiotic alone displayed a transient increase in Bifidobacterium spp. and a transient decrease in Bacteroides spp. Denaturing gradient gel electrophoresis analysis showed that GOS elicited similar alterations in the microbial profiles of the three in vitro runs. However, the synbiotic did not alter the microbial diversity of the three runs to the same extent as GOS alone. Nested PCR using universal primers, followed by bifidobacterial-specific primers illustrated low bifidobacterial diversity in dogs, which did not change drastically during the in vitro fermentation. This study illustrates that the canine faecal microbiota can be modulated in vitro by GOS supplementation and that GOS can sustain the growth of B. bifidum 02 450B in a synbiotic combination.

Investigation of the faecal microbiota associated with canine chronic diarrhoea.

Diarrhoea is a common problem in dogs and can result in disturbance of the normal intestinal microbiota. However, little is known about the gastrointestinal microbiota of dogs with chronic diarrhoea and controlled canine studies of dietary management are scarce. The aims of this study were to investigate the predominant faecal microbiota of chronic diarrhoea dogs and to examine the effect(s) of a fibre blend on the canine faecal microbiota. A 3-week fibre supplementation feeding study was performed in nine chronic diarrhoea and eight control dogs. Atopobium cluster, Lactobacillus-Enterococcus group and Clostridium cluster XIV were the predominant bacterial groups in all dogs. Chronic diarrhoea dogs had significantly higher Bacteroides counts at baseline and significantly lower Atopobium cluster counts following fibre supplementation compared with control dogs. Atopobium cluster levels increased significantly in control dogs, while counts of sulphate-reducing bacteria decreased significantly and Clostridium clusters I and II counts increased significantly in chronic diarrhoea dogs during fibre supplementation. Microbial profiles (detected by denaturing gradient gel electrophoresis) demonstrated interindividual variation, with greater similarity seen between the chronic diarrhoea and control dogs' profiles after fibre supplementation compared with baseline. In conclusion, fibre supplementation induced changes in the canine faecal microbiota, with greater resemblance between the microbiota of chronic diarrhoea and control dogs after this dietary modulation.

Análise das regiões polimórficas do gene HASPB (K26) de Leishmania infantum em amostras clínicas positivas para leishmaniose visceral e coinfecção LV/HIV

A leishmaniose visceral (LV) é uma doença grave que afeta a população de vários países, onde o Brasil apresenta a maior prevalência da infecção nas Américas. Com o estudo do gene codificante da proteína B de superfície (HASPB ou K26) de Leishmania infantum é possível identificar as variações polimórficas intraespecíficas e, assim, será possível consolidar a descrição de um perfil polimórfico presente no Estado de Pernambuco. O objetivo do trabalho foi analisar as regiões polimórficas do gene HASPB (K26) de Leishmania infantum em amostras clínicas positivas para leishmaniose visceral e coinfecção LV/HIV. O sistema K26 PCR foi otimizado utilizando concentrações variadas de DNA genômico de L. infantum. Foi realizado o screening de amostras clínicas de DNA através de dois sistemas de PCR simples, kDNA e ITS1/RFLP, para ensaios posteriores com a K26 PCR nas amostras positivas. A curva de dissociação de alta definição (qPCR-HRM) foi empregada na localização de temperaturas de melting específicas para L. infantum. Os amplicons do gene K26 foram sequenciados e alinhados as sequencias selecionadas em base de dados. A K26 PCR apresentou limiar de detecção de 1 pg para amplicon de 700 pb. A especificidade dos primers foi avaliada experimentalmente e in silico, apresentando anelamento inespecífico com DNA humano. Em paralelo, foram selecionadas 78 amostras de DNA através dos dois sistemas screening, sendo 17 caracterizadas como L. infantum. Os ensaios com DNA das amostras clínicas para o sistema K26 PCR revelaram bandas espúrias. A análise através qPCR-HRM em DNA genômico do parasita resultou em amplificação com Tm de 88,2 °C, já o ensaio com amostra clínica revelou duas amplificações com distintas temperaturas de melting, 84,6 e 88,2 °C. Três amplicons do gene K26 foram sequenciados e alinhados a cinco sequencias da base de dados, indicando 38,2 % de similaridade. Pode-se concluir que o sistema K26 PCR é recomendável para análise dos polimorfismos genéticos, contanto que o DNA seja extraído diretamente de espécies isoladas em meio de cultura.

Estratégias para o aprimoramento do diagnóstico molecular na leishmaniose visceral

A leishmaniose visceral (LV) é uma doença infecto-parasitária causada por protozoários do gênero Leishmania. O trabalho teve como objetivo avaliar estratégias para o aprimoramento do diagnóstico molecular na LV, que compreenderam a avaliação da eficiência de diferentes métodos de extração de DNA em amostras de urina; a análise do uso da Reação em Cadeia da Polimerase quantitativa em tempo real (qPCR) como ferramenta para a detecção do DNA de Leishmania infantum na referida amostra; e a padronização de uma reação duplex qPCR para a detecção simultânea do DNA de L. infantum e do gene G3PD (controle endógeno). Depois da escolha do protocolo de extração de DNA mais apropriado, e após a otimização e a análise de reprodutibilidade, uma qPCR em urina foi padronizada. Em paralelo, após o desenho e a síntese de sondas TaqMan® compatíveis com os sistemas LINF 1B e G3PD1, após otimização e análise de reprodutibilidade, uma duplex qPCR em sangue também foi padronizada. Para avaliação dos protocolos desenvolvidos foram utilizadas técnicas de estatística descritiva. Para análise comparativa com técnicas clássicas de diagnóstico da LV utilizou-se Teste Qui Quadrado de independência ou Teste Exato de Fisher (p<0,05 e p<0,01, respectivamente). Como resultados, após otimização, o limite de detecção alcançado pela qPCR em urina utilizando o protocolo de extração selecionado (kit comercial) foi de 5 fg/microlitros de amostra 0,034 parasitos) A duplex qPCR em sangue alcançou um limite de detecção de 2x102 fg/microlitros de amostra 1,4 (ou ~ 1,4 parasito), após a otimização. A partir dos dados estatísticos obtidos, pôde-se analisar alta concordância percentual entre a qPCR e urina e o conjunto de critérios diagnósticos (sorologia rK39 + qPCR em sangue), bem como entre a duplex qPCR em sangue e a qPCR em sangue, para os Grupos 01 (pacientes com suspeita de LV) e 02 (pacientes HIV positivos co-infectados ou não). Como um conjunto de critérios, os dois novos ensaios obtiveram excelentes concordâncias com o conjunto de técnicas clássicas: 88,89 por cento e 94,74 por cento para os Grupos 01 e 02, respectivamente. Não houve diferenças estatísticas significativas entre os testes. Pôde-se concluir que ambos os ensaios mostraram bom potencial para a incorporação, após validação, ao diagnóstico da LV; em conjunto ou individualmente (quando necessário), trazendo mais conforto, praticidade, confiabilidade e rapidez ao diagnóstico definitivo da patologia

Implementação da tecnologia de IgY para o diagnóstico da leishmaniose visceral canina

Os métodos de diagnóstico sorológico são ainda as ferramentas mais realistas e aplicáveis para identificação de cães com leishmaniose visceral. A pesquisa por métodos sorológicos mais eficientes e de produção simplificada é importante para a identificação do reservatório canino, ação primordial para o controle da doença. Este estudo teve por objetivo o desenvolvimento e a avaliação do desempenho de um ELISA para leishmaniose visceral canina, utilizando como conjugado enzimático uma IgY anti-IgG canina. Para isto, galinhas poedeiras foram imunizadas com IgG canina purificada e a IgY anti-IgG foi isolada das gemas dos ovos por precipitação com polietilenoglicol e purificada por meio de adsorção tiofílica. A especificidade frente ao antígeno imunizante foi verificada por imunodifusão radial dupla, ELISA e western-blot. O ELISA foi desenvolvido utilizando IgY conjugada a enzima peroxidase, tendo seus parâmetros de acurácia avaliados e comparados com o ELISA utilizando IgG de mamíferos conjugada a peroxidase. A concentração total de IgY isolada nas gemas foi constante, sem diferença significativa nos meses analisados (...), com média de 97,55 mg de IgY/ gema. A IgY produzida reconheceu a IgG canina, demonstrando uma forte sensibilidade e especificidade no ELISA, porém na imunodifusão não foi detectada ligação da IgY produzida a IgG canina. Após a imunização, houve um aumento significante da produção de IgY específica do primeiro para o segundo mês (...) onde ocorreu um pico estável sem queda de produção até o final do período analisado (...). A IgY demonstrou ainda, forte reatividade no western-Blot frente a IgG canina purificada e a presente no soro de cão clinicamente saudável, não sendo capaz de reconhecer IgG de outras espécies animais.

Supranutritional selenium induces alterations in molecular targets related to energy metabolism in skeletal muscle and visceral adipose tissue of pigs

While selenium (Se) is an essential micronutrient for humans, epidemiological studies have raised concern that supranutritional Se intake may increase the risk to develop Type 2 diabetes mellitus (T2DM). We aimed to determine the impact of Se at a dose and source frequently ingested by humans on markers of insulin sensitivity and signalling. Male pigs were fed either a Se-adequate (0.17 mg Se/kg) or a Se-supranutritional (0.50 mg Se/kg; high-Se) diet. After 16 weeks of intervention, fasting plasma insulin and cholesterol levels were non-significantly increased in the high-Se pigs, whereas fasting glucose concentrations did not differ between the two groups. In skeletal muscle of high-Se pigs, glutathione peroxidase activity was increased, gene expression of forkhead box O1 transcription factor and peroxisomal proliferator-activated receptor- coactivator 1 were increased and gene expression of the glycolytic enzyme pyruvate kinase was decreased. In visceral adipose tissue of high-Se pigs, mRNA levels of sterol regulatory element-binding transcription factor 1 were increased, and the phosphorylation of Akt, AMP-activated kinase and mitogen-activated protein kinases was affected. In conclusion, dietary Se oversupply may affect expression and activity of proteins involved in energy metabolism in major insulin target tissues, though this is probably not sufficient to induce diabetes.

Role for protease activity in visceral pain in irritable bowel syndrome

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.

Visceral and central abdominal fat and anthropometry in relation to diabetes in Asian Indians

OBJECTIVE: The objective of the study was to examine body fat distribution using computed tomography (CT), dual-energy X-ray absorptiometry (DEXA), and anthropometry in relation to type 2 diabetes in urban Asian Indians. RESEARCH DESIGN AND METHODS: This is a case-control study of 82 type 2 diabetic and 82 age- and sex-matched nondiabetic subjects from the Chennai Urban Rural Epidemiology Study, an ongoing epidemiological study in southern India. Visceral, subcutaneous, and total abdominal fat were measured using CT, while DEXA was used to measure central abdominal and total body fat. Anthropometric measures included BMI, waist circumference, sagittal abdominal diameter (SAD), and waist-to-hip ratio. RESULTS: Visceral and central abdominal fat showed a strong correlation with each other (P <0.0001), and kappa analysis revealed a fairly good agreement between tertiles of visceral and central abdominal fat (kappa=0.44, P <0.0001). Diabetic subjects had significantly higher visceral (P=0.005) and central abdominal (P=0.011) fat compared with nondiabetic subjects. Waist circumference and SAD showed a strong correlation with visceral (P <0.01) and central abdominal (P <0.0001) fat in both diabetic and nondiabetic subjects. Logistic regression analysis revealed visceral (odds ratio [OR] 1.011, P=0.004) and central abdominal (OR 1.001, P=0.013) fat to be associated with diabetes, even after adjusting for age and sex. CONCLUSIONS: Visceral and central abdominal fat showed a strong association with type 2 diabetes. Both measures correlated well with each other and with waist circumference and SAD in diabetic and nondiabetic urban Asian Indians.

An assessment of the prebiotic potential of single and blended substrates in anaerobic in vitro batch culture fermentations using canine faecal samples as inocula

Previously, using an in vitro static batch culture system, it was found that rice bran (RB), inulin, fibersol, mannanoligosaccharides (MOS), larch arabinogalactan and citrus pectin elicited prebiotic effects (in terms of increased numbers of bifidobacteria and lactic acid bacteria) on the faecal microbiota of a dog. The aim of the present study was to confirm the prebiotic potential of each individual substrate using multiple faecal donors, as well as assessing the prebiotic potential of 15 substrate blends made from them. Anaerobic static and stirred, pH-controlled batch culture systems inoculated with faecal samples from healthy dogs were used for this purpose. Fluorescence in situ hybridization (FISH) analysis using seven oligonucleotide probes targeting selected bacterial groups and DAPI (total bacteria) was used to monitor bacterial populations during fermentation runs. High-performance liquid chromatography was used to measure butyrate produced as a result of bacterial fermentation of the substrates. RB and a MOS/RB blend (1:1, w/w) were shown to elicit prebiotic and butyrogenic effects on the canine microbiota in static batch culture fermentations. Further testing of these substrates in stirred, pH-controlled batch culture fermentation systems confirmed the prebiotic and butyrogenic effects of MOS/RB, with no enhancement of Clostridium clusters I and II and Escherichia coli populations.

Visceral behaviours and leadership: a dark side of boardroom life?

Purpose – Characteristics of leaders whose behaviour is visceral include taking action based on instinct rather than intellect and exhibiting coarse, base and often negative emotions. Despite the challenge of precisely defining the nature of visceral behaviour, the purpose of this paper is to provide insight into this less attractive side of boardroom life. Design/methodology/approach – Following a literature review of the research into the negative behaviour leaders exhibit, the paper highlights four forms of visceral behaviour based on focused and intimate qualitative case studies involving the experiences of those on the receiving end of that behaviour within a boardroom context. Findings – Based on interviews with an international sample of five chief executive officers (CEOs), plus three subordinates with substantial profit and loss responsibility, the study reveals a distinctly human experience from which no one is exempt. The idiosyncratic nature of the visceral behaviour experienced resulted in each study participant's unique experience. The authors conclude that leaders need to adopt specific measures in order to control and reduce the darker human tendencies. Research limitations/implications – The experiences of study participants are presented in four case studies, providing insight into their experiences whilst also protecting their identity. The study participants were drawn from a sample of companies operating globally within a single sector of the manufacturing industry. The concepts the authors present require validating in other organisations with different demographic profiles. Originality/value – The paper presents a model based on two dimensions – choice and level of mastery – that provides the reader with insight into the forms of visceral behaviour to which leaders succumb. Insight enables us to offer managers strategic suggestions to guard against visceral behaviour and assist them in mitigating its worst aspects, in both those with whom they work and themselves.

Observations on the feeding habits of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Campo Grande, an endemic area of visceral leishmaniasis in Mato Grosso do Sul, Brazil

Sand flies were captured weekly with CDC light traps from December 2003 to November 2005 in three areas of Campo Grande, in the Brazilian state of Mato Grosso do Sul. These areas incorporated two patches of remnant forest and five houses. The blood meals of engorged female sand flies were identified using the avidin-biotin system of immunoenzymatic ELISA capture. Most (327/355) of the females analysed were Lutzomyia longipalpis, of which 66.4% reacted with human blood, 64.8% with that of birds and 8.9% with that of dogs. Females that had taken human blood predominated in the residential areas and two forest patches. The following combinations of blood were also detected for L. longipalpis in some of the samples analysed: bird + human (43.4%), bird + human + dog (6.1%). The combination bird + human + dog + pig was also found for Nyssomyia whitmani. Dogs and pigs appear to have little attractiveness for L. longipalpis. The results obtained demonstrate the eclecticism and high anthropophily of L. longipalpis and raise new questions with regard to the importance of dogs in VL epidemiology and the possible role of man as a source of infection for sand flies. (C) 2008 Elsevier B.V. All rights reserved.

Seasonal variation of Lutzomyia longipalpis (Lutz & neiva, 1912) (Diptera : Psychodidae : Phlebotominae) in endemic area of visceral leishmaniasis, Campo Grande, state of Mato Grosso do Sul, Brazil

The seasonal distribution of Lutzomyia longipalpis was studied in two forested and five domiciliary areas of the urban area of Campo Grande; MS, from December 2003 to November 2005. Weekly captures were carried out with CDC light traps positioned on ground and in the canopy inside a residual forest and on the edge (ground) of a woodland and in at least one of the following ecotopes in peridomiciles-a cultivated area, a chicken coop, a pigsty, a kennel, a goat and sheep shelter and an intradomicile. A total of 9519 sand flies were collected, 2666 during the first year and 6853 during the second. L. longipalpis was found throughout the 2-year period, presenting smaller peaks at intervals of 2-3 months and two greater peaks, the first in February and the second in April 2005, soon after periods of heavy rain. Only In one of the woodlands was a significant negative correlation (p < 0.05) between the number of insects and temperature during the first year and the climatic factors (temperature, RHA and rain) was observed. In the domiciliary areas in four domiciles some positive correlations (p < 0.05) occurred in relation to one or more climatic factors; however, the species shows a clear tendency to greater frequency (72%) in the rainy season than in the dry (28%). Thus, we recommend an intensification of the VL control measures applied in Campo Grande, MS, during the rainy season with a view to reducing the risk of the transmission of the disease. (C) 2007 Elsevier B.V. All fights reserved.

In vitro inhibitory effect of trichostatin A on canine grade 3 mast cell tumor

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M -phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1). B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis. (c) 2008 Elsevier Ltd. All rights reserved.