In vivo effect of minocycline alone and in combination with immunotherapy against pythium insidiosum.

This study examined the effect of minocycline alone and in combination with immunotherapy against pythiosis. Twenty rabbits, aged three months old and subcutaneously inoculated with Pythium insidiosum zoospores were divided into four groups (n = 5): treated with minocycline (10 mg/kg/day twice daily), treated with immunotherapy (34 mg subcutaneously every 14 days), treated with minocycline plus immunotherapy, and untreated (control group). The treatments were started 30 days after inoculation and continued for 70 days. The subcutaneous nodular injury areas in infected groups were measured every seven days after the beginning of treatment. Only the rabbits that developed lesions were selected for this study. When compared with the control group over 70 days, the minocycline and minocycline plus immunotherapy groups of rabbits with pythiosis showed significantly reduced injuries. The histopathology showed the presence of inflammation, macrophages and eosinophils. Grocott's staining revealed irregular hyphae-like structures that were ramified and occasionally septate. Our results suggest that minocycline has fungistatic activity and that the combination of minocycline and immunotherapy is more effective than the individual therapies tested.

Efeitos in vitro de ocratoxina A, deoxinivalenol e zearalenona sobre a viabilidade celular e atividade de E-ADA em linfócitos de frangos de corte / In vitro effects of ochratoxin A, deoxynivalenol and zearalenone on cell viability and E-ADA activity in broiler chickens lymphocytes

Mycotoxins are a group of chemically diverse naturally occurring substances resulting from the secondary metabolism of pathogenic filamentous fungi. They are produced mainly by the genera Fusarium, Alternaria, Aspergillus and Penicillium which can contaminate grains and cereals such as wheat, corn and soy. According to the nature and the concentration levels, mycotoxins can induce toxic effects in food-production animals and humans. An in vitro study was conducted to evaluate the susceptibility of broiler chickens lymphocytes to different concentrations of ochratoxin A, deoxynivalenol and zearalenone. Each toxin was added to the cell medium at different concentrations (0.001, 0.01, 0.1 and 1μg/mL). Cell viability and ecto-adenosine deaminase activity were assessed at 24, 48 and 72 hours by colorimetric assays. Thus, it were used 0.7x10(5) lymphocytes/mL in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 2.5 IU of penicillin/streptomycin per mL, incubated at 37°C in a 5% CO2 atmosphere. All the experiments were carried out in triplicate and the results were expressed as mean ± standard error of the mean. The results showed that OTA and DON induced lymphocyte proliferation and reduced enzymatic activity in vitro (P<0,05), whereas ZEA also promoted proliferation (P<0,05), but neither alteration on enzymatic activity (P>0,05). It was possible to correlate the results about viability cell and ecto-adenosine deaminase activity, suggesting that, at minimal concentrations, the evaluated mycotoxins do not stimulated the enzymatic activity, which has proinflammatory action and contributes for the immunosuppression process, thus, avoiding a decrease on the viability cell. This is the first in vitro study conducted with OTA, DON and ZON in broiler chickens lymphocytes evaluating these parameters.

Micotoxinas representam um vasto grupo de contaminantes químicos naturais originados a partir do metabolismo secundário de fungos filamentosos patogênicos. Elas são produzidas, principalmente, pelos gêneros Fusarium, Alternaria, Aspergillus e Penicillium, os quais podem contaminar grãos e cereais, como trigo, milho e soja. Conforme sua natureza e níveis de concentração, micotoxinas podem induzir efeitos tóxicos em animais de produção e humanos. Um estudo in vitro foi realizado para avaliar a susceptibilidade das células linfocitárias de frangos de corte a diferentes concentrações de ocratoxina A, deoxinivalenol e zearalenona. Cada micotoxina foi adicionada ao meio celular em diferentes concentrações (0,001; 0,01; 0,1 e 1μg/mL). A viabilidade celular e atividade de ecto-adenosina desaminase foram analisadas em 24, 48 e 72 horas através de ensaios colorimétricos. Para isso, foram utilizados 0,7x10(5) linfócitos/mL em meio RPMI 1640, suplementado com 10% de soro fetal bovino e 2,5 UI de penicilina/estreptomicina por mL, incubados em atmosfera de 5% de CO2 a 37 °C. Todos os experimentos foram realizados em triplicata e os resultados foram expressos como média e erro padrão da média. Os resultados obtidos demonstraram que tanto ocratoxina A como deoxinivalenol induziram proliferação linfocitária e baixa atividade enzimática in vitro (P<0,05), enquanto zearalenona também induziu proliferação (P<0,05), mas nenhuma alteração na atividade enzimática (P>0,05). Foi possível correlacionar os dados referentes à viabilidade celular e atividade de ecto-adenosina desaminase, sugerindo que, em concentrações mínimas, as micotoxinas testadas não estimularam a atividade da enzima, que possui ação pró-inflamatória e contribui para o processo de imunossupressão e, portanto, evitando um decréscimo na viabilidade celular. Este é o primeiro estudo feito com OCRA, DON e ZEA sobre linfócitos de frangos de corte em cultivos in vitro na avaliação desses parâmetros.

E-NTPDase and E-ADA activities in rats experimental infected by Cryptococcus neoformans.

Cryptococcus neoformans, the etiological agent of cryptococcosis, is an opportunistic fungal pathogen of immunocompromised individuals. The aim of this study was to evaluate the activities of E-NTPDase and E-ADA in rats experimentally infected by C. neoformans var. grubii. Adult rats (35) were divided in two groups: 18 for the control group (uninfected) (A), and 17 for the infected group (B). Each group was separated into three sub-groups (A1, A2, A3-B1, B2, B3), and samples were collected on 10, 20, and 30 days post-infection (PI). Leukocyte counts, IFN-γ, TNF-α, IgM, IgG levels, and E-NTPDase and E-ADA activities were analyzed. It was possible to observe that IgG and IgM seric levels of infected rats were significantly elevated (P<0.01) on days 10, 20 and 30 PI, as well as the levels of TNF-α and INF-γ when compared to uninfected rodents. Regarding E-NTPDase activity in lymphocytes, it was possible to observe that the ATP hydrolysis was significantly decreased on days 20 (P<0.01) and 30 PI (P<0.05), while ADP hydrolysis was significantly reduced only on day 20 PI (P<0.01) when compared with uninfected group. Seric E-ADA activity had a significant reduction (P<0.01) during all three evaluated periods when compared to the control group, while E-ADA activity in lymphocytes increased significantly (P<0.01) when compared to the group A on day 10 PI; however on days 20 and 30 PI, its activity was considerable reduced in lymphocytes of infected animals (P<0.01). Therefore, it is possible to conclude that the infection caused by C. neoformans in immunocompetent rats leads to changes in the purinergic signaling (NTPDase and E-ADA), concomitantly with an inflammatory response (increased levels of cytokines and immunoglobulins) associated with inflammatory infiltrates and histological lesions in the lung.

In vitro trypanocidal activity of macela (Achyrocline satureioides) extracts against Trypanosoma evansi.

The aim of this study was to verify the trypanocidal effectiveness of aqueous, methanolic, and ethanolic extracts of Achyrocline satureioides against Trypanosoma evansi in vitro. A. satureioides extracts, known as macela, were used on trypomastigotes at different concentrations (1, 5, 10, 50, 100, 500, and 1,000 µg/ml) and exposure times (0, 1, 3, 6, and 9 hr). A dose-dependent effect was observed when the 3 extracts were tested. The concentrations of 1, 5, and 10 µg/ml were not able to kill trypomastigotes until 3 hr after exposure, and the highest concentrations (500 and 1,000 µg/ml) were able to kill all trypomastigotes after 1 hr. When the time of exposure was increased up to 9 hr, the concentrations at 50 and 100 µg/ml were 100% effective to 3 extracts. The chemical analysis of the extracts revealed the presence of flavonoids, a trypanocidal compound already described. Based on the results, we can conclude that the A. satureioides extracts exhibit trypanocidal effects.

Trypanocidal activity of the essential oils in their conventional and nanoemulsion forms: in vitro tests.

The aim of this study was to investigate the susceptibility in vitro of Trypanosoma evansi to the essential oils of andiroba (Carapa guaianensis) and aroeira (Schinus molle), in their conventional and nanostructured forms. For that, pure oils at concentrations of 0.5%, 1.0% and 2.0% were used. A negative control (untreated) and a positive control (diminazene aceturate 0.5%) were used as comparative parameters. Later, the same tests were performed, using nanoemulsions oils at concentrations of 0.5% and 1.0%. The tests were carried out in triplicates and the numbers of parasites were quantified on 1, 3 and 6 h from onset of the study. A dose-dependent reduction in the number of parasites to the forms of two oils tested was observed after 1 h. The concentration of parasites was significantly reduced at low concentrations after 3 h, as well as at 6 h no alive parasites were observed for the essential oils tested. Ours findings indicate, for the first time, that oils of andiroba and aroeira (in their conventional and nanoemulsion forms) have high activity against T. evansi in vitro, leading to the suggestion that these oils may be applied as an alternative treatment for this disease.