Ana Paula Madureira

Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

BackgroundPlasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBPII), which is the most variable segment of the protein.MethodsTo test whether diversifying natural selection has shaped the nucleotide diversity of PvDBPII in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBPII, and T- and B-cell epitopes were localized on the 3-D structure.ResultsThe results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBPII, and (ii) PvDBPII appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium.ConclusionsThis study shows that some polymorphisms of PvDBPII are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion.

Effects of Anterior Thalamic Nucleus Deep Brain Stimulation in Chronic Epileptic Rats

Deep brain stimulation (DBS) has been investigated for the treatment of epilepsy. In rodents, an increase in the latency for the development of seizures and status epilepticus (SE) has been reported in different animal models but the consequences of delivering stimulation to chronic epileptic animals have not been extensively addressed. We study the effects of anterior thalamic nucleus (AN) stimulation at different current intensities in rats rendered epileptic following pilocarpine (Pilo) administration. Four months after Pilo-induced SE, chronic epileptic rats were bilaterally implanted with AN electrodes or had sham-surgery. Stimulation was delivered for 6 h/day, 5 days/week at 130 Hz, 90 µsec. and either 100 µA or 500 µA. The frequency of spontaneous recurrent seizures in animals receiving stimulation was compared to that recorded in the preoperative period and in rats given sham treatment. To investigate the effects of DBS on hippocampal excitability, brain slices from animals receiving AN DBS or sham surgery were studied with electrophysiology. We found that rats treated with AN DBS at 100 µA had a 52% non-significant reduction in the frequency of seizures as compared to sham-treated controls and 61% less seizures than at baseline. Animals given DBS at 500 µA had 5.1 times more seizures than controls and a 2.8 fold increase in seizure rate as compared to preoperative values. In non-stimulated controls, the average frequency of seizures before and after surgery remained unaltered. In vitro recordings have shown that slices from animals previously given DBS at 100 µA had a longer latency for the development of epileptiform activity, shorter and smaller DC shifts, and a smaller spike amplitude compared to non-stimulated controls. In contrast, a higher spike amplitude was recorded in slices from animals given AN DBS at 500 µA.

Role of adenosine in the antiepileptic effects of deep brain stimulation

Despite the effectiveness of anterior thalamic nucleus (AN) deep brain stimulation (DBS) for the treatment of epilepsy, mechanisms responsible for the antiepileptic effects of this therapy remain elusive. As adenosine modulates neuronal excitability and seizure activity in animal models, we hypothesized that this nucleoside could be one of the substrates involved in the effects of AN DBS. We applied 5 days of stimulation to rats rendered chronically epileptic by pilocarpine injections and recorded epileptiform activity in hippocampal slices. We found that slices from animals given DBS had reduced hippocampal excitability and were less susceptible to develop ictal activity. In live animals, AN DBS significantly increased adenosine levels in the hippocampus as measured by microdialysis. The reduced excitability of DBS in vitro was completely abolished in animals pre-treated with A1 receptor antagonists and was strongly potentiated by A1 receptor agonists. We conclude that some of the antiepileptic effects of DBS may be mediated by adenosine.

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.

Public Knowledge about and Detection of Canine Visceral Leishmaniasis in Urban Divinópolis, Brazil.

Background. Leishmaniases are diseases with a wide spectrum of clinical manifestations including cutaneous (CL) and visceral (VL) forms. Many factors may affect their occurrence and expansion including environmental, geographic, and social conditions. In the past two decades, Divinópolis, Minas Gerais State, Brazil, has exhibited the potential for a disease outbreak, with the appearance of CL, and VL cases (human and canine). Hence, this study was initiated to monitor public knowledge of the disease. Questionnaires were administered in four neighborhoods (Jardim Belvedere, Esplanada, Danilo Passos I and II) where most of the human and canine cases have been reported. The analyses demonstrated that public knowledge of the disease is sparse and fragmented. A strong perception of the dog as the main reservoir was observed. Five veterinary clinics were evaluated for the presence of canine VL using serological (RIFI and ELISA) and molecular (PCR-RFLP) techniques. This is the first study demonstrating the occurrence of Leishmania infantum in Divinópolis, suggesting a possible urbanization of VL.

The management of bee communities by intercropping with flowering basil (Ocimum basilicum) enhances pollination and yield of bell pepper (Capsicum annuum)

Enhancements of floral resources in surrounding landscape and in local crop fields are expected to mitigate adverse human impacts on pollinator diversity and pollination services. We evaluated whether intercropping with flowering basil (Ocimum basilicum) increases the local abundance and richness of bees and improves fruit and seed production of bell pepper (Capsicum annuum). Fields of bell pepper were divided into intercropping (basil mixed with pepper) and single-cropping plots (only pepper). We also grew bell peppers in protected greenhouse in the presence and absence of beehives. The richness and abundance of visiting bees to pepper flowers were greater in the intercropped plots than the single-cropping plots. The increase in the number of bees visiting the peppers was due to the stronger attraction of Paratrigona lineata, Apis mellifera and Tetragonisca angustula to basil in the intercropping plots. The pepper fruits produced in the intercropping were wider, longer, and heavier and developed more seeds than the fruits produced by single-cropping. The same differences were observed between the fruits grown in greenhouse with and without beehives of P. lineata. We demonstrate that agricultural systems designed to attract and retain pollinators are more productive than growing pure stands. Our results also highlight the role of provisioning floral resources to enhance bee conservation and management in anthropogenic ecosystems.

Clinical treatment of cutaneous leishmaniasis in dogs with furazolidone and domperidone

Cutaneous leishmaniasis (CL) is a zoonosis and a public health problem in countries of subtropical America. The aim of this study was to investigate the efficacy of furazolidone and domperidone treatment of dogs naturally infected with Leishmania (Viannia) braziliensis. Infection was confirmed by PCR and parasite culture of tissue collected from skin scrapings of the lesion borders of dogs. Naturally infected animals were divided into control (n=4) and treatment (n=8) groups. The treatment group was administered furazolidone for 21 days interspersed with domperidone for 10 days by oral gavage. Dogs that showed no lesion healing during this period were administered the same treatment cycle for up to 93 days. Among the eight treated animals, seven were clinically cured without recurrence of skin lesions during the 12-month study period. However, during lesion healing, skin scrapings were positive for L. (V.) braziliensis by PCR; no growth of the protozoan in NNN-LIT medium occurred until the end of follow-up. These results suggest that treatment with furazolidone and domperidone is effective for epithelialisation and lesion healing of dogs with clinical CL caused by L. (V.) braziliensis.

Alcohol Abuse Promotes Changes in Non-Synaptic Epileptiform Activity with Concomitant Expression Changes in Cotransporters and Glial Cells

Non-synaptic mechanisms are being considered the common factor of brain damage in status epilepticus and alcohol intoxication. The present work reports the influence of the chronic use of ethanol on epileptic processes sustained by non-synaptic mechanisms. Adult male Wistar rats administered with ethanol (1, 2 e 3 g/kg/d) during 28 days were compared with Control. Non-synaptic epileptiform activities (NEAs) were induced by means of the zero-calcium and high-potassium model using hippocampal slices. The observed involvement of the dentate gyrus (DG) on the neurodegeneration promoted by ethanol motivated the monitoring of the electrophysiological activity in this region. The DG regions were analyzed for the presence of NKCC1, KCC2, GFAP and CD11b immunoreactivity and cell density. The treated groups showed extracellular potential measured at the granular layer with increased DC shift and population spikes (PS), which was remarkable for the group E1. The latencies to the NEAs onset were more prominent also for the treated groups, being correlated with the neuronal loss. In line with these findings were the predispositions of the treated slices for neuronal edema after NEAs induction, suggesting that restrict inter-cell space counteracts the neuronal loss and subsists the hyper-synchronism. The significant increase of the expressions of NKCC1 and CD11b for the treated groups confirms the existence of conditions favorable to the observed edematous necrosis. The data suggest that the ethanol consumption promotes changes on the non-synaptic mechanisms modulating the NEAs. For the lower ethanol dosage the neurophysiological changes were more effective suggesting to be due to the less intense neurodegenertation.

An Integrated Approach Using Spatial Analysis to Study the Risk Factors for Leishmaniasis in Area of Recent Transmission

Some epidemiological aspects of leishmaniasis in the municipality of Formiga, Brazil, an important touristic site, were evaluated. Those included phlebotomine sand fly vectors, canine infection, and geoprocessing analysis for determining critical transmission areas. Sand flies (224 insects) belonging to ten different species were captured. The most captured species included Lutzomyia longipalpis (35.3%), Lutzomyia cortelezzii (33.5%), and Lutzomyia whitmani (18.3%). A significant correlation between sand fly densities and climatic conditions was detected. Serological diagnosis (DPP and ELISA) was performed in 570 dogs indicating a prevalence of 5.8%. After sequencing the main species circulating in the area were Leishmania infantum and Leishmania braziliensis. Spatial analysis demonstrated that vegetation and hydrography may be related to sand fly distribution and infected dogs. The municipality of Formiga has proven leishmaniasis vectors and infected dogs indicating the circulation of the parasite in the city. Correlation of those data with environmental and human cases has identified the critical areas for control interventions (south, northeast, and northwest). In conclusion, there is current transmission of visceral and canine human cases and the city is on the risk for the appearance of cutaneous cases.

Phenotypic correlations among meat quality traits in broilers

The goals of this research were to estimate the phenotypic correlations among various meat quality traits from a male broiler line and to describe the relation among these variables. Phenotypical correlations were determined among quality traits, isolating the effects of slaughter date, the age of the mother and sex. The evaluated traits were pH measurements taken at time 0 and at 6 and 24 hours after slaughtering, color parameters, water loss due to exudation, thawing and cooking of the meat, and shear force. Important associations (P 0.05) to the other traits of the meat, whereas the pH at 24 hours after slaughter was able of directly interfering with the attributes of the meat, since this trait was inversely related with lightness and water losses, which indicates an effect of pH fall along 24h after slaughtering on protein denaturation. This study demonstrates that the variables of poultry meat quality are related and that there is a phenotypical association between lightness and cooking losses and the other attributes of the meat. The pH at 24 hours after slaughtering, lightness and cooking losses could be efficient meat quality indicators in this broiler line.