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.

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.

Paracoccidioidomycosis-related mortality trend, state of Sao Paulo, Brazil: a study using multiple causes of death

Objective. To investigate mortality in which paracoccidioidomycosis appears on any line or part of the death certificate. Method. Mortality data for 1985-2005 were obtained from the multiple cause-of-death database maintained by the Sao Paulo State Data Analysis System (SEADE). Standardized mortality coefficients were calculated for paracoccidioidomycosis as the underlying cause-of-death and as an associated cause-of-death, as well as for the total number of times paracoccidioidomycosis was mentioned on the death certificates. Results. During this 21-year period, there were 1950 deaths related to paracoccidioidomycosis; the disease was the underlying cause-of-death in 1 164 cases (59.69%) and an associated cause-of-death in 786 (40.31%). Between 1985 and 2005 records show a 59.8% decline in the mortality coefficient due to paracoccidioidomycosis as the underlying cause and a 53.0% decline in the mortality as associated cause. The largest number of deaths occurred among men, in the older age groups, and among rural workers, with an upward trend in winter months. The main causes associated with paracoccidioidomycosis as the underlying cause-of-death were pulmonary fibrosis, chronic lower respiratory tract diseases, and pneumonias. Malignant neoplasms and AIDS were the main underlying causes when paracoccidioidomycosis was an associated cause-of-death. The decision tables had to be adapted for the automated processing of causes of death in death certificates where paracoccidioidomycosis was mentioned. Conclusions. Using the multiple cause-of-death method together with the traditional underlying cause-of-death approach provides a new angle on research aimed at broadening our understanding of the natural history of paracoccidioidomycosis.

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.

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar-gel immunodiffusion (ACID) test for canine brucellosis, were used as the control panel for B. cants infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies. (C) 2010 Elsevier Ltd. All rights reserved.

Immunohistochemical Characterization of Canine Prostatic Intraepithelial Neoplasia

The development of prostate cancer is believed to be a multistep process, progressing sequentially from normal epithelium, to prostatic intraepithelial neoplasia (PIN) and, finally, to invasive neoplasia. Malignant stem cells within the basal cell layer of the prostatic epithelium are believed to play an important role in the failure of androgen-ablation therapy that occurs in the most advanced form of prostate cancer. The aim of the present study was to immunohistochemically characterize the lesions of canine PIN. Prostatic tissue from five dogs with PIN was compared with normal prostate tissue from nine further dogs. There was an increase in the number of basal epithelial cells in lesions consistent with PIN as defined by expression of the nuclear protein p63. These lesions had elevated expression of proliferating cell nuclear antigen (PCNA) and heterogeneous labelling for the nuclear androgen receptor (AR). These findings suggest that the basal cells present in PIN may play a role in canine prostate carcinogenesis and that the proliferation of these cells occurs despite the heterogeneous expression of the AR. (C) 2009 Elsevier Ltd. All rights reserved.

Expression of Connexins in Normal and Neoplastic Canine Bone Tissue

Previous studies showed that intercellular communication by gap junctions has a role in bone formation. The main connexin involved in the development, differentiation, and regulation of bone tissue is connexin (Cx) 43. In addition, Cx46 is also expressed, mostly localized within the trans-Golgi region. Alterations in the expression pattern and aberrant location of these connexins are associated with oncogenesis, demonstrating a deficient gap junctional intercellular communication (GJIC) capacity in neoplastic tissues. In this study, we evaluated normal and neoplastic bone tissues regarding the expression of Cx43 and Cx46 by immunofluorescence, gene expression of these connexins by real-time PCR, and their correlation with cell proliferation index and deposition of collagen. Fourteen neoplastic bone lesions, including 13 osteosarcomas and I multilobular tumor of bone, were studied. The mRNA levels of Cx43 were similar between normal and neoplastic bone tissue. In normal bone tissue, the Cx43 protein was found mainly in the intercellular membranes. However, in all bone tumors studied here, the Cx43 was present in both cell membranes and also aberrantly in the cytoplasm. Regarding only tumor samples, we determined a possible inverse correlation between Cx43 expression and cellular proliferation, although a positive correlation between Cx43 expression and collagen deposition was also noted. In contrast, Cx46 had lower levels of expression in neoplastic bone tissues when compared with normal bone and was found retained in the perinuclear region. Even though there are differences between these two connexins regarding expression in neoplastic versus normal tissues, we concluded that there are differences regarding the subcellular location of these connexins in normal and neoplastic dog bone tissues and suggest a possible correlation between these findings and some aspects of cellular proliferation and possibly differentiation.

Isolation, Characterization, and Differentiation Potential of Canine Adipose-Derived Stem Cells

Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in veterinary medicine. It can be obtained in large quantities, under local anesthesia, and with minimal discomfort. In this study, canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by suction-assisted lipectomy (i.e., liposuction). Adipose tissue was processed to obtain a fibroblast-like population of cells similar to human adipose-derived stem cells (hASCs). These canine adipose-derived stem cells (cASCs) can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of cASCs are of mesodermal or mesenchymal origin. cASCs are able to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirate, canine adipose tissue may also contain multipotent cells and represent an important stem cell source both for veterinary cell therapy as well as preclinical studies.

CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28(-/-)) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28(-/-) mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro-and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.

Attempts at a peptide vaccine against paracoccidioidomycosis, adjuvant to chemotherapy

Chemotherapy is the basis of treatment of paracoccidioidomycosis in its various forms. Depending on the Paracoccidioides brasiliensis virulence, the status of host immunity, the degree of tissue involvement and fungal dissemination, treatment can be extended for long periods with an alarming frequency of relapses. Association of chemotherapy with a vaccine to boost the cellular immune response seemed a relevant project not only to reduce the time of treatment but also to prevent relapses and improve the prognosis of anergic cases. The candidate immunogen is the gp43 major diagnostic antigen of P. brasiliensis and more specifically its derived peptide P10, carrying the CD4(+) T-cell epitope. Both gp43 and P10 protected Balb/c mice against intratracheal infections with virulent P. brasiliensis strain. P10 as single peptide or in a multiple-antigen-peptide (MAP) tetravalent construction was protective without adjuvant either by preimmunization and intratracheal challenge or as a therapeutic agent in mice with installed infection. P10 showed additive protective effects in drug-treated mice stimulating a Th-1 type immune response with high IFN-gamma and IL-12. P10 and few other peptides in the gp43 were selected by Tepitope algorithm and actually shown to promiscuously bind several prominent HLA-DR molecules suggesting that a peptide vaccine could be devised for a genetically heterogenous population. P10 was protective in animals turned anergic, was effective in a DNA minigene vaccine, and increased the protection by monoclonal antibodies in Balb/c mice. DNA vaccines and peptide vaccines are promising therapeutic tools to be explored in the control of systemic mycoses.

Experimental paracoccidioidomycosis: alternative therapy with ajoene, compound from Allium sativum, associated with sulfamethoxazole/trimethoprim

Ajoene has been described as an antithrombotic, anti-tumour, antifungal, antiparasitic and antibacterial agent. This study deals with the efficacy of ajoene to treat mice intratracheally infected with Paracoccidioides brasiliensis. The results indicate that ajoene therapy is effective in association with antifungal drugs (sulfametoxazol/trimethoprim), showing a positive additive effect. Ajoene-treated mice developed Th1-type cytokine responses producing higher levels of IFN-gamma and IL-12 when compared to the infected but untreated members of the control group. Antifungal activity of ajoene involves a direct effect on fungi and a protective pro-inflammatory immune response. Reduction of fungal load is additive to chemotherapy and therefore the combined treatment is mostly effective against experimental paracoccidioidomycosis.

Poly(lactic acid-glycolic acid) nanoparticles markedly improve immunological protection provided by peptide P10 against murine paracoccidioidomycosis

Background and purpose: The present study reports on the preparation and testing of a sustained delivery system for the immunomodulatory peptide P10 aimed at reducing the in vivo degradation of the peptide and the amount required to elicit a protective immune response against paracoccidioidomycosis. Experimental approach: BALB/c mice were infected with the yeast Paracoccidioides brasiliensis to mimic the chronic form of paracoccidioidomycosis. The animals were treated daily with sulfamethoxazole/trimethoprim alone or combined with peptide P10, either emulsified in Freund`s adjuvant or entrapped in poly(lactic acid-glycolic acid) (PLGA) nanoparticles at different concentrations (1 mu g, 5 mu g, 10 mu g, 20 mu g or 40 mu g center dot 50 mu L-1). Therapeutic efficacy was assessed as fungal burden in tissues and the immune response by quantitative determination of cytokines. Key results: Animals given combined chemotherapy and P10 nanotherapy presented a marked reduction of fungal load in the lungs, compared with the non-treated animals. After 30 days of treatment, P10 entrapped within PLGA (1 mu g center dot 50 mu L-1) was more effective than `free` P10 emulsified in Freund`s adjuvant (20 mu g center dot 50 mu L-1), as an adjuvant to chemotherapy. After treatment for 90 days, the higher doses of P10 entrapped within PLGA (5 or 10 mu g center dot 50 mu L-1) were most effective. Treatment with P10 emulsified in Freund`s adjuvant (20 mu g center dot 50 mu L-1) or P10 entrapped within PLGA (1 mu g center dot 50 mu L-1) were accompanied by high levels of interferon-gamma in lung. Conclusions and implications: Combination of sulfamethoxazole/trimethoprim with the P10 peptide entrapped within PLGA demonstrated increased therapeutic efficacy against paracoccidioidomycosis. P10 incorporation into PLGA nanoparticles dramatically reduced the peptide amount necessary to elicit a protective effect.

Reactivity of p53 protein in canine transmissible venereal tumor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)