Rodrigo de Almeida Vaucher

Antimicrobial activity of Amazonian oils against Paenibacillus species

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the primary bacterial pathogen of honeybee brood and the causative agent of American foulbrood disease (AFB). One of the feasible alternative treatments being used for their control of this disease is essential oils. In this study in vitro antimicrobial activity of Andiroba and Copaíba essential oils against Paenibacillus species, including P. larvae was evaluated. Minimal inhibitory concentration (MIC) in Mueller-Hinton broth by the microdilution method was assessed. Andiroba registered MIC values of 1.56-25%, while the MICs values obtained for Copaíba oil were of 1.56-12.5%. In order to determine the time-response effect of essential oils on P. larvae, this microorganism was exposed to the oils for up to 48 h. After 24 h treatment with Andiroba oil and after 48 h treatment with Copaíba oil no viable cells of P. larvae ATCC 9545 were observed. The possible toxic effect of essential oils were assessed by the spraying application method of the same concentrations of MICs. Bee mortality was evident only in treatment with Andiroba oil and the Copaíba oil shows no toxic effects after 10 days of observation. Taking together ours results showed for the first time that these oils presented a high activity against Paenibacillus species showing that Copaíba oil may be a candidate for the treatment or prevention of AFB.

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.

Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34

The in vitro cytotoxicity of the antimicrobial peptide P34 was evaluated in different eukaryotic cells. The food‐grade bacteriocin nisin was also analysed for comparison. Vero cells were treated with different concentrations (0.02–2.5 μg·ml−1) of antimicrobial peptide P34 and nisin. Cell viability and plasma membrane integrity were checked by MTT [3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide], NRU (Neutral Red dye uptake) and LDH (lactate dehydrogenase) assays. The EC50 values of the peptide P34 in MTT and NRU assays were 0.60 and 1.25 μg·ml−1 respectively, while values of nisin found were 0.50 and 1.04 μg·ml−1. In the LDH assay, the EC50 values were 0.65 and 0.62 μg·ml−1 for P34 and nisin, respectively. The peptide P34 revealed similar haemolytic activity on human erythrocytes (5.8%) when compared with nisin (4.9%). The effects on viability, motility and acrosomal exocytosis of human sperm were also evaluated. Nisin and P34 showed similar effects on sperm parameters. The evaluation of cytotoxicity of antimicrobial peptides is a critical step to guarantee their safe use.

Trypanocidal action of tea tree oil (Melaleuca alternifolia) against Trypanosoma evansi in vitro and in vivo used mice as experimental model

This study aimed to evaluate the Trypanosoma evansi susceptibility to tea tree oil (TTO - Melaleuca alternifolia) and tea tree oil nanocapsules (TTO nanocapsules) in vitro and in vivo tests. In vitro, we observed a mortality curve of trypomastigotes proportional to dose, i.e., the TTO and TTO nanocapsules have trypanocidal effect. Treatment with TTO in vivo was assessed in experiments (I and II). For Experiment I, T. evansi infected mice were treated with TTO and/or combinations of essential oil with chemotherapy (diminazene aceturate - D.A.). Treatment with TTO at a dose of 1mLkg(-1) was able to extend animal longevity, but had no curative efficacy. However, when TTO was combined with D.A. a disease curative efficacy of 100% for disease was observed, a much better result than the D.A. treatment (33.3%). In Experiment II, T. evansi infected mice were treated with TTO nanocapsules with doses of 0.3, 0.6 and 0.9mLkg(-1). Animals treated with 0.9mLkg(-1) showed higher longevity however without curative effect. Active compounds present in natural products, such as M. alternifolia, may potentiate the treatment of trypanosomosis when associated with other trypanocidal drugs.

Evaluation of the immunogenicity and in vivo toxicity of the antimicrobial peptide P34.

Immunogenicity and toxicity of antimicrobial peptide P34 were evaluated in vivo. BALB/c mice were inoculated intraperitoneally with peptide P34 alone and associated with Freunds adjuvant. For acute toxicity testing, different concentrations of the peptide P34 (82.5, 165.0, 247.5 and 330.0mg/kg) were orally administered. To evaluate the sub-chronic toxicity the tested dose of 0.825 mg/kg/day of the peptide P34 or nisin were administered for 21 days. There were no hypersensitivity reactions or significant increase in antibody titer during the immunogenicity experiment or death of animals during the acute or sub-chronic toxicity tests. The LD(50) was higher than 332.3 ± 0.76 mg/kg. No significant changes in serum biochemical parameters were observed in the animals treated with the peptide P34 unlike nisin-treated group showed a significant increase in alanine transaminase levels in comparison to controls. The group treated with 0.825 mg/kg/day of nisin showed histological changes in the spleen, skin and liver. In the group treated with peptide P34 histological changes in the spleen were observed, with the presence of megakaryocytes. Few studies report the use of animal models to evaluate the in vivo toxicity of antimicrobial peptides and such investigation is an essential step to ensure it safe use in foods.

Antimicrobial activity of Scutia buxifolia against the honeybee pathogen Paenibacillus larvae

The honeybee disease American foulbrood (AFB) is a serious problem since its causative agent (Paenibacillus larvae) has become increasingly resistant to conventional antibiotics. One of the feasible alternative treatments being used for control of this disease are plants extracts. The aim of the present work was to evaluate the effect of crude extract and fractions of Scutia buxifolia against six Paenibacillus species, including P. larvae, and its potential use for the control of AFB. In vitro activity of S. buxifolia samples against Paenibacillus species were evaluated by the disk diffusion and microdilution methods, and the minimal inhibitory concentration (MIC) were also determined. All Paenibacillus species were sensitive to crude extract and fractions of S. buxifolia. The dichloromethane (DC) fraction showed the better MIC (1.56 mg/mL), followed by ethyl acetate (EtAc) (6.25 mg/mL), n-butanol (BuOH) (25 mg/mL) and Crude extract (CE) (50 mg/mL). Toxic effect of S. buxifolia crude extracts and fractions against bees were also evaluated by the spraying application method of the same concentrations of MICs. The samples tested showed no toxic effects for the bees after 15 days of observation. These results are first time described for this species and showed that S. buxifolia presented a important activity against Paenibacillus species and proved to be a natural alternative for the prevention/control of AFB.

Physiological responses of Rhamdia quelen (Siluriformes: Heptapteridae) to anesthesia with essential oils from two different chemotypes of Lippia alba

The aim of this study was to evaluate if Lippia alba has different chemotypes according to the chemical composition of the essential oil (EO) considering collection site, and if the EO may have different effects on blood and plasma parameters in silver catfish, Rhamdia quelen, during and immediately after anesthesia. The citral (EO-C) and linalool (EO-L) chemotypes were identified, and both presented similar anesthetic effects for silver catfish. Fish were exposed to two concentrations of each EO, which induced slow and fast anesthesia (100 and 300 µL L-1, respectively). Blood ions did not change at any time of anesthesia induction and recovery and, therefore, the electrolyte balance was not altered. Blood gases oscillated through all exposure and recovery times, but there was an increase in pO2 after 10 min recovery in fish anesthetized with EO-C. Glucose increased in fish exposed to both EOs when compared with the control group. Overall, exposure to both EOs (except 100 µL L-1 EO-L at most times) reduced plasma cortisol levels compared to the control and/or ethanol groups. However, as plasma creatinine levels in fish anesthetized with EO-C were higher than control fish, the use of EO-L is preferable.

Insecticidal and repellent effects of tea tree and andiroba oils on flies associated with livestock

This study aimed to evaluate the insecticidal and repellent effects of tea tree, Melaleuca alternifolia (Myrtales: Myrtaceae), and andiroba, Carapa guianensis (Sapindales: Meliaceae), essential oils on two species of fly. For in vitro studies, free‐living adult flies were captured and reared in the laboratory. To evaluate the insecticidal effects of the oils, adult flies of Haematobia irritans (L.) and Musca domestica L. (both: Diptera: Muscidae) were separated by species in test cages (n = 10 per group), and subsequently tested with oils at concentrations of 1.0% and 5.0% using a negative control to validate the test. Both oils showed insecticidal activity. Tea tree oil at a concentration of 5.0% was able to kill M. domestica with 100.0% efficacy after 12 h of exposure. However, the effectiveness of andiroba oil at a concentration of 5.0% was only 67.0%. The insecticidal efficacy (100.0%) of both oils against H. irritans was observed at both concentrations for up to 4 h. The repellency effects of the oils at concentrations of 5.0% were tested in vivo on Holstein cows naturally infested by H. irritans. Both oils demonstrated repellency at 24 h, when the numbers of flies on cows treated with tea tree and andiroba oil were 61.6% and 57.7%, respectively, lower than the number of flies on control animals. It is possible to conclude that these essential oils have insecticidal and repellent effects against the species of fly used in this study.

Relationship between behavioral alterations and activities of adenylate kinase and creatine kinase in brain of rats infected by Trypanosoma evansi

The aim of this study was to investigate the behavioral assessment and activities of important enzymes involved in the phosphoryl transfer network in rat brains that were experimentally infected with Trypanosoma evansi. Behavioral assessment (cognitive performance), pro-inflammatory cytokines in serum and activities of adenylate kinase (AK), pyruvate kinase (PK), and creatine kinase (CK) in brain were evaluated at 5 and 15 days post-infection (PI). Here we demonstrate a cognitive impairment in the rats infected with T. evansi. At 5 and 15 days PI, a memory deficit and a depressant activity were demonstrated by an inhibition avoidance test and increase in the immobility time in a tail suspension test, respectively. On day 5 PI, a decrease in the CK activity and an increase in the AK activity were observed. On day 15 PI, an increase in the CK activity and a decrease in the AK activity were observed. Considering the importance of energy metabolism for brain functioning, it is possible that the changes in the activity of enzymes involved in the cerebral phosphotransfer network and an increase in the proinflammatory cytokines (TNF and IFN) may be involved at least in part in the cognitive impairment in infected rats with T. evansi.

Effect of tea tree oil (Melaleuca alternifolia) on the longevity and immune response of rats infected by Trypanosoma evansi

This study aimed to evaluate the effect of tea tree oil (TTO - Melaleuca alternifolia) on hepatic and renal functions, and the immune response of rats infected by Trypanosoma evansi. A pilot study has shown that rats treated with TTO orally (1 ml kg(-1)) had increased survival rate without curative effect. In order to verify if increased longevity was related to a better immune response against T. evansi when using tea tree oil, a second experiment was conducted. Thus, twenty-four rats were divided into four groups. The groups A and B were composed of uninfected animals, and the groups C and D had rats experimentally infected by T. evansi. Animals from the groups B and D were treated orally with TTO (1 ml kg(-1)) for three days. Blood samples were collected to verify humoral response analysis for immunoglobulins (IgA, IgM, IgE, and IgG) and cytokines (TNF-α, INF-γ, IL-1, IL-6, IL-4, and IL-10) at days 0, 3, 5 and 15 post-infection (PI). TTO treatment caused changes in the immunoglobulins and cytokines profile, as well as the course of T. evansi infection in rats. It was found that the TTO was not toxic, i.e., hepatic and renal functions were not affected. Therefore, it is possible to conclude that TTO influences the levels of inflammatory mediators and has trypanocidal effect, increasing life expectancy of rats infected by T. evansi.