Performance of IFAT, ELISA, direct parasitological examination and PCR on lymph node aspirates for canine visceral leishmaniasis diagnosis

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

Antibacterial activity of propolis collected in different regions of Brazil

The aim of this study was to investigate the antibacterial activity of propolis samples from Goiás, Paraná and São Paulo States, Brazil, and their flavonoids content. Ethanolic extracts of propolis (EEP) were prepared (30g of propolis in 70% ethanol), and the microorganisms Staphylococcus aureus and Escherichia coli were tested. The methodology employed was agar diffusion using filter paper discs. Ampicillin and tetracycline were used as controls. Antibacterial activity was determined by the reading of inhibition zone diameters (mm) after 24 hours incubation at 37°C. Results demonstrated that EEP inhibited the growth of Staphylococcus aureus but not that of Escherichia coli. Tetracycline and ampicillin showed an efficient action against both bacteria. Flavonoids content was variable, depending on the propolis sample. According to the results, it may be concluded that EEP showed effective action against Gram-positive bacteria, independently on their geographic origin, and a positive correlation between antibacterial activity and flavonoids content.

Association between handling stress in the corral and rabies antibody titers in selenium-supplemented cattle

This study determined the correlation between serum cortisol levels and rabies antibody titers in cattle primo-vaccinated against rabies and supplemented with dietary selenium (Se). Sixty Nelore male calves (10 to 12 months old) received daily and individual dietary supplementation with 0, 3.6, 5.4 and 6.4 mg Se (groups Gc, G(3.6), G(5.4) and G(6.4), respectively). The animals were vaccinated against rabies (day 0) and subjected to handling stress in the corral for 120 days. Blood sampling procedures were performed on days 0, 15, 30, 60, 90 and 120. Cortisol levels increased until day 90, but had dropped significantly by day 120 (P < 0.01). Rabies antibody titers on days 30 and 90 were similar among Se-supplemented groups; in the control group, rabies antibodies decreased significantly from day 30 to 60, and 90 to 120. Serum cortisol levels and antibody titers were not correlated in most of the groups or blood sampling days. A positive correlation among these variables was found only in G(6.4) on days 60 (R = 0.513; P = 0.05) and 120 (R = 0.644; P = 0.009). In conclusion, repeated handling in the corral stresses cattle, but without compromising rabies humoral immune response.

Propolis effect on streptozotocin-induced diabetic rats

Propolis is one of the hive products that has been used extensively in folk medicine, due to its several biological and pharmacological properties. Besides, propolis-containing products have been intensely marketed by the pharmaceutical industry and health-food stores. This work was carried out in order to investigate whether propolis treatment could revert the metabolic alterations of streptozotocin-induced diabetic rats. Animals were kept in metabolic cages and diabetes was induced by a single dose of streptozotocin (35 mg/kg, IV). After a week, rats with glycemia higher than 230 mg/dL were divided into two groups and treated with ethanolic extract of propolis (10 and 90 mg/kg, PO) for seven days. Glycemia and free fatty acids were determined, as well as food and water intake, body weight and urine volume were registered weekly. Data showed no significant differences in the analyzed variables. Based on these results, one may conclude that propolis had no effects after diabetes establishment, in our conditions assays. Further assays with different concentrations of propolis and periods of administration should be carried out in order to evaluate its therapeutic potential in this disease.

Antimicrobial activity of propolis and essential oils and synergism between these natural products

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

Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin B and itraconazole

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

Ballast water: a review of the impact on the world public health

Since the nineteenth century ships have been using ballast water (BW) for safety, stability, propulsion and maneuverability, as well as to redress loss of fuel weight and water consumption, and to maintain structural stress at acceptable levels. Ballast water has been spreading many non-native species around the globe, but little is known about the extent and potential significance of ship-mediated transfer of microorganisms. The global movements of ballast water by ships create a long-distance dispersal mechanism for human pathogens that may be important in the worldwide distribution of microorganisms, as well as for the epidemiology of waterborne diseases. Only a few studies have been carried out on this subject, most of them involving ballast water containing crustacean larvae and phytoplankton. Specialized microbiological studies on these waters are necessary to avoid a repeat of what happened in 1991, when epidemic cholera was reported in Peru and rapidly spread through Latin America and Mexico. In July of 1992, Vibrio cholerae was found in the USA and the Food and Drug Administration (FDA) determined that it came from ballast water of ships whose last port of call was in South America. In Brazil, just a few studies about the subject have been performed. An exploratory study by the Brazilian National Health Surveillance Agency (Agencia Nacional de Vigilancia Sanitaria - ANVISA) found in ballast water different microorganisms, such as fecal coliforms, Escherichia coli, Enterococcus faecalis, Clostridium perfringens, coliphages, Vibrio cholerae O1 and Vibrio cholerae non-O1. Until now, Brazil has been focusing only on organisms transported to its territory from other countries by ballast water, to avoid their establishment and dissemination in Brazilian areas. Studies that can assess the probability that water ballast carries pathogenic microorganisms are extremely important, as is the examination of ships that arrive in the country. Treatment of the human infections caused by BW exists but none is completely safe and efficient.

Modulatory effects of tuberculosis vaccines on experimental autoimmune encephalomyelitis

Although BCG is the only accepted vaccine against tuberculosis (TB), its protective ability is very limited. Therefore, many new vaccines are being evaluated. Our group has been working on DNAhsp65 - a genetic construction containing the hsp65 gene from Mycobacterium leprae. In previous experimental works, we demonstrated that both DNAhsp65 alone or associated with BCG, in a prime-boost regimen, were effective to control TB. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 alone or associated with BCG in a rat model of multiple sclerosis. Female Lewis rats were immunized with three doses of DNAhsp65 or primed with BCG followed by two DNAhsp65 boosters. The animals were, then, immunized with myelin associated with complete Freund's adjuvant to develop experimental autoimmune encephalomyelitis (EAE). The following parameters were evaluated: weight loss, clinical score, central nervous system (CNS) inflammation and anti-myelin immune response. No deleterious effect was associated with these immunizations schedules. Immunized animals equally lost weight, the clinical scores were similar and CNS inflammation did not increase. Interestingly, both procedures determined decreased inflammation in the brain and lumbar spinal cord. This was concurrent with a modulatory effect over cytokine production by peripheral lymphoid organs. Cell cultures from spleen and lymph nodes in vitro stimulated with myelin produced less IFN-gamma and IL-10, respectively. This phenomenon was more clear in rats immunized with the genetic vaccine alone than with the prime-boost strategy. Together the results suggest that these strategies for TB prophylaxis would not accelerate or aggravate multiple sclerosis, being therefore, safe in this aspect. In addition, they indicate that these vaccination regimens have a potential anti-inflammatory activity that could be better explored in the future.

The inhibitory effect of MK886 on the inflammatory process caused by Paracoccidioidomycosis brasiliensis infection in genetically selected mice

Leukotrienes are classic inflammatory response mediators considered chemotactic agents and microbicidal activity regulators in cells of the innate immune system, playing a protective role against different infectious agents. In this study, we investigated the involvement of leukotrienes in the course of murine paracoccidioidomycosis based on the following immunologic parameters: cell influx, mieloperoxydase activity, NO production, cytokine production, and fungal recovery in lungs of mice selected according to the intensity of their low (AIRmin) and high (AIRmax) acute inflammatory response. Infection by P. brasiliensis induced considerable production of IL-6, IL-10, IFN-gamma and TNF-alpha cytokines, and led to cell recruitment, as well as NO production in lungs at different study periods. In animals treated with MK886, a leukotriene biosynthesis inhibitor, IFN-gamma, IL-6 and TNF-alpha production was lower, while neutrophil influx and NO production decreased. These results may explain the higher fungal load in lungs of animals in which leukotriene synthesis was inhibited, suggesting that leukotrienes have a possible protective role in experimental paracoccidioidomycosis. AIRmax animals had lower fungal load in comparison with AIRmin ones, which can be related to the AIR phenotype regarding neutrophil migration, besides lower production of NO and pro-inflammatory cytokines. Thus, mice presenting AIRmax background are more resistant to infection by P. brasiliensis.

Bacterial biofilms with emphasis on coagulase-negative staphylococci

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

Cytokine production in experimental paracoccidioidomycosis of murine selected lineages for acute inflammatory response (air)

Paracoccidioidomycosis is a systemic human mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), an imperfect dimorphic fungus whose conidia are its infective form. Mice genetically selected for maximum (AIRmax) and minimum (AIRmin) acute inflammatory response were used as experimental paracoccidioidomycosis models. The animals were intraperitoneally inoculated with P. brasiliensis (strain 18) and killed 6, 12 and 24 hours or 3, 7 and 14 days after infection. In these periods, fragments from their spleen, liver and lung were removed for evaluation of the infection level by fungal cells, assessment of macrophagic activity by peritoneal and splenic macrophages - through the determination of nitric oxide (NO) concentrations and production of pro- and anti-inflammatory cytokines of lung and spleen homogenate supernatants. In the present study, it was observed that AIRmax lineages presented greater control of the infectious process than the AIRmin ones. Regarding NO production, AIRmax animals produced more metabolites in late periods, what may help control the infectious process. Concerning cytokine production, it was observed that the production of INF-gamma, TNF-alpha, IL-1, IL-6, IL-8 and IL-12 were increased in AIRmax lineages in most analyzed organs and periods, thus contributing to the greater resistance exhibited by such lineages against infection, except for IL-4 and IL-10 that showed decreased production in AIRmax lineage, reproducing its suppressive biological effect. From these results, it was observed that the AIRmax lineage was more effective in controlling the infectious process, with an important involvement of the analyzed cytokines. These findings are probably related to the genetically selected factors involved in the acute inflammatory response.

ROLE of TLR2 and TLR4 on human neutrophil functions against Paracoccidioides brasiliensis

In paracoccidioidomycosis, a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb), studies have focused on the role of neutrophils that are involved in the primary response to the fungus. Neutrophil functions are regulated by pro- and anti-inflammatory cytokines. Molecular mechanisms involved in this process are not fully understood, but there are strong evidences about the involvement of toll-like receptors (TLRs). We aimed at evaluating TLR2 and TLR4 expression on human neutrophils activated by GM-CSF, IL-15, TNF-alpha or IFNgamma and challenged with a virulent strain of P. brasiliensis (Pb18). Moreover, we asked if these receptors have a role on fungicidal activity, H(2)O(2) and IL-6, IL-8, TNFalpha and IL-10 production, by activating and challenging cells. All cytokines increased TLR2 and TLR4 expression. Pb18 also increased TLR2 expression, inducing an additional cytokine effect. on the contrary, it inhibited TLR4 expression. All cytokines increased neutrophil fungicidal activity and H(2)O(2) production; however, this process was not associated with TLR2 or TLR4. Neutrophil activation by GMCSF and TNF-alpha resulted in a significant increase of IL-8 production, while IL-15 and IFN-alpha have no effect. Pb18 also augmented IL-8 expression, inducing an additional effect to that of cytokines. None of the cytokines activated neutrophils by releasing IL-10. This cytokine was only detected after Pb18 challenge. Interestingly, IL-8 and IL-10 production involved TLR2 and mainly TLR4 modulation. The present results suggest that Pb18 interaction with neutrophils through TLR2 and TLR4 with consequent IL-8 and IL-10 production may be considered a pathogenic mechanism in paracoccidioidomycosis.

Biological properties of medicinal plants: a review of their antimicrobial activity

Plants have been used for thousands of years to flavor and conserve food, to treat health disorders and to prevent diseases including epidemics. The knowledge of their healing properties has been transmitted over the centuries within and among human communities. Active compounds produced during secondary vegetal metabolism are usually responsible for the biological properties of some plant species used throughout the globe for various purposes, including treatment of infectious diseases. Currently, data on the antimicrobial activity of numerous plants, so far considered empirical, have been scientifically confirmed, concomitantly with the increasing number of reports on pathogenic microorganisms resistant to antimicrobials. Products derived from plants may potentially control microbial growth in diverse situations and in the specific case of disease treatment, numerous studies have aimed to describe the chemical composition of these plant antimicrobials and the mechanisms involved in microbial growth inhibition, either separately or associated with conventional antimicrobials. Thus, in the present work, medicinal plants with emphasis on their antimicrobial properties are reviewed.

Anti-coagulase-negative Staphylococcus activity of ethanolic extracts of propolis from two Brazilian regions and synergism with antimicrobial drugs by the E-test method

Propolis is a natural resinous substance collected by bees from vegetal sources and its therapeutic properties have been investigated. In this work, we evaluated the inhibitory activity of ethanolic extracts of propolis (EEP) from the Southeast and South of Brazil on coagulase-negative Staphylococcus (CNS) growth as well as the EEP in vitro synergism with antimicrobial drugs by using the diffusion method (E-test). The EEP chemical characteristics (dry weight, pH, flavonoid and phenolic compounds) were determined. Seven drugs were tested, and synergism was observed between three drugs and Southeast EEP, six drugs and South EEP, and one drug and ethanol control. Ethanolic extracts of propolis from the South of Brazil presented the greatest flavonoid content and synergism rate, while EEP from the Southeast presented the greatest anti-CNS activity and phenolic compound content. Results showed the correlation among anti-CNS activity, synergism rate and chemical characteristics of propolis.

Evalution of methods for the detection of MRSA in patients of Botucatu Medical School Hospital, São Paulo, Brazil

Oxacillin is the main drug of choice for the treatment of S. aureus infections. However, S. aureus resistance to oxacillin has become a major problem in the recent decades. The study aimed assess the rates of oxacillin resistance in S. aureus samples obtained at the Botucatu Medical School Hospital, UNESP, and to compare phenotypic techniques for the detection of MRSA against the gold standard method (mecA gene detection) in these samples. A total of 102 samples, previously isolated between 2002 and 2006, and kept at the Culture Collection of the Department of Microbiology and Immunology, in the Botucatu Biosciences Institute, UNESP, were included. Oxacillin resistance was assessed by oxacillin and cefoxitin disk diffusion and agar dilution tests, screening tests using Mueller-Hinton agar with 6 mu g/mL of oxacillin and 4% NaCl, E-test, and mecA gene detection. of the samples analyzed, 46 (45.1%) were mecA-positive. Oxacillin disk sensitivity and specificity were 86.9% and 91.1%, respectively. Cefoxitin disk sensitivity and specificity were respectively 91.3% and 91.1%. The screening test with the cefoxitin disk showed almost the same level of sensitivity (91.3%) and specificity (91.1%). With E-test strips, sensitivity was higher (97.8%) and specificity was comparable to that found with the other methods (91.1%). Ninety-three percent of the samples produced beta-lactamase and five of them were mecA-negative. There was a gradual increase in the number of oxacillin-resistant S. aureus samples between 2002 and 2004. However, from 2004 to 2006, the number of resistant samples dropped from 55% of MRSA in 2004, to 45% in 2005 and 34.6% in 2006. The data obtained reveal that, among phenotypic methods, the E-test yielded the best results, with higher sensitivity levels when compared to the other methods. The decreased resistance rate observed over the most recent years may be explained by the rational use of antimicrobial agents associated with good practices in the control of hospital infection, or may be related to the diminished use of oxacillin as a treatment option.