Avaliação da toxicidade de florações naturais e de cultura de cianobactérias: efeitos sobre Ceriodaphnia silvestri (Crustacea, Cladocera)

The incidence of toxic cyanobacterial blooms is one of the important consequences of eutrophication in aquatic ecosystems. It is a very common phenomenon in reservoirs and shrimp ponds in the State of Rio Grande do Norte (RN), Brazil. Cyanobacterias produce toxins which can affect aquatic organisms and men trough the food chain. Aiming to contribute to the studies of cyanobacterias in RN, we propose: a) to evaluate the toxicity of isolated cyanobacterias in important fresh-water environments; and b) to verify the effects of both natural and cultured blooms occurred in reservoirs for human supply and in the cladoceran Ceriodaphnia silvestrii. This study was carried out using samples of natural blooms occurred between March and October of 2004 in Gargalheiras Dam (08º L e 39º W), in July of 2004 in Armando Ribeiro Gonçalves Dam (06o S e 37o W) and in commercial shrimp ponds (Litopenaeus vannamei) located in fresh-water environments. The samples were collected with plankton net (20µm.) for identification, isolation and obtaining of phytoplanktonic biomass for liophilization and later toxicity bioassays. The toxicity of cultured samples and natural blooms was investigated through bioassays in Swiss mice. Quantification of cyanobacteria in samples was conducted following the Ütermol method, with 300mL samples fixed with lugol. The toxicity test with Ceriodaphnia silvestrii followed ABNT, 2001 recommendations, and were accomplished with natural hepatotoxic bloom s samples and cultured samples of both non-toxic and neurotoxic C. raciborskii. In this test, five newborns, aged between 6 and 24 hours, were exposed to different concentrations (0 a 800 mg.L-1) of crude cyanobacterial extracts during 24 and 48 hours. Three replicates were used per treatment. The pH, temperature and dissolved oxygen at the beginning and after 24 and 48hours from the test were measured. We estimated the CL50 through the Trimmed Spearman-Karber method. The blooms were constituted by Microcystis panniformis, M. aeruginosa, Anabaena circinalis, Cylindrospermopsis raciborskii and Planktothrix agardhii, producers of mycrocistin-LR confirmed with HPLC analysis. Samples of hepatotoxic blooms registered toxinogenic potential for C. silvestrii, with CL50-24h value of 47.48 mg.L-1 and CL5048h of 38.15 mg.L-1 for GARG samples in march/2005; CL50-24h of 113,13 mg.L-1 and CL5048h of 88,24 mg.L-1 for ARG July/2004; CL50-24h of 300.39 mg.L-1 and CL50-48h of 149.89 mg.L-1 for GARG October/2005. For cultured samples, values of CL50-24h and CL50-48h for C. raciborskii toxic strains were 228.05 and 120.28 mg.L-1, respectively. There was no mortality of C. silvestrii during the tests with non-toxic C. raciborskii strain. The toxicity test with C. silvestrii presented good sensitivity degree to cyanotoxins. The toxicity of natural hepatotoxic blooms samples (microcystins) and cultured neurotoxic saxitoxins producer samples analyzed in this study give us strong indications of that toxin s influence on the zooplanktonic community structure in tropical aquatic environments. Eleven cyanobacteria strains were isolated, representing 6 species: Anabaenopsis sp., Cylindrospermopsis raciborskii, Chroococcus sp., Microcystis panniformis, Geitlerinema unigranulatum e Planktothrix agardhii. None presented toxicity in Swiss mice. The strains were catalogued and deposited in the Laboratório de Ecologia e Toxicologia de Organismos Aquáticos (LETMA), in UFRN, and will be utilized in ecotoxicológical and ecophysiological studies, aiming to clarify the causes and control of cyanobacterial blooms in aquatic environments in RN. This state s reservoirs must receive broader attention from the authorities, considering the constant blooms occurring in waters used for human consumption

Análise do transcriptoma das glândulas venenosas do escorpião Tityus stigmurus

In Brazil, accidents with scorpions are considered of medical importance, not only by the high incidence, but also for the potentiality of the venom from some species in determining severe clinical conditions. Tityus stigmurus is a widely distributed scorpion species in Northeastern Brazil and known to cause severe human envenomations, inducing pain, hyposthesia, edema, erythema, paresthesia, headaches and vomiting. The present study uses a transcriptomic approach to characterize the molecular repertoire from the non-stimulated venom gland of Tityus stigmurus scorpion. A cDNA library was constructed and 540 clones were sequenced and grouped into 37 clusters, with more than one EST (expressed sequence tag) and 116 singlets. Forty-one percent of ESTs belong to recognized toxin-coding sequences, with antimicrobial toxins (AMP-like) the most abundant transcripts, followed by alfa KTx- like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Our analysis indicated that 34% include other possible venom molecules , whose transcripts correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases, cystein-rich peptides and lectins. Fifteen percent of ESTs are similar to cellular transcripts. Sequences without good matches corresponded to 11%. This investigation provides the first global view of cDNAs from Tityus stigmurus. This approach enables characterization of a large number of venom gland component molecules, which belong either to known or atypical types of venom peptides and proteins from the Buthidae family

Xylella fastidiosa: An in vivo system to study possible survival strategies within citrus xylem vessels based on global gene expression analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Methodology for fast evaluation of Bacillus thuringiensis crystal protein content

O desenvolvimento da produção e uso do Bacillus thuringiensis no Brasil em escala comercial enfrenta certas dificuldades, entre elas o estabelecimento de metodologias para a quantificação de produtos tóxicos a serem comercializados. Atualmente, a quantidade de toxinas é expressa como porcentagem do total de proteínas presentes em amostras em consideração. Tal metodologia, entretanto, não mede a quantidade real de uma determinada proteína presente em um produto qualquer, além do fato de diferentes linhagens bacterianas possuírem diferentes genes codificadores para endotoxinas e mesmo para b-toxina. Desde que os diferentes tipos de toxinas apresentam diferentes características antigências, este trabalho tem como objetivo a utilização de técnicas imunológicas para quantificar específicamente o conteúdo de proteína cristal presente em diferentes amostras. A proteína cristal produzida pela subespécie B. thuringiensis var. israelensis foi purificada por ultracentrifugação e utilizada para imunizar coelhas e produzir soros hiperimunes. Tais soros foram posteriormente usados para avaliar o nível de proteína cristal em bioinseticidas comerciais e em culturas de laboratório desta bactéria utilizando-se a técnica do imunodot. Os resultados foram obtidos por comparação de reações com concentrações conhecidas de proteína cristal permitindo assim avaliar com segurança os níveis desta proteína em várias preparações.

Pré-compostagem de cadáveres de bovinos acometidos pelo botulismo

Foram pré-compostados, em células individuais e isoladas, cinco cadáveres de bovinos acometidos pelo botulismo com a finalidade de monitorar a presença de esporos de Clostridium botulinum e de toxina botulínica antes e após o processo de decomposição em leira estática com fonte de carbono. O diagnóstico da intoxicação nos animais foi baseado nas características clínico-patológicas, epidemiológicas ou laboratoriais. Dos cinco bovinos com evolução clínica crônica de botulismo, cujos cadáveres foram pré-compostados, três foram acometidos pela toxina tipo D, um pelo complexo CD e um dos animais foi negativo na tentativa de detecção da toxina e de esporos da bactéria nas vísceras pelo bioensaio e neutralização em camundongo. O processo de pré-compostagem foi realizado em leira estática, com o uso de material carbonáceo umidificado como substrato e esquartejamento do animal, vedado individualmente com lona plástica e sem aeração por um período de 50 dias. A temperatura das leiras foi monitorada durante o período e oscilou de 40,5-52,4ºC. Após a abertura das leiras, pôde-se constatar a completa decomposição de todo material mole, com redução significativa do seu peso (de 26,5-44,5%), restando apenas os ossos. Não foi detectado esporo ou toxina botulínica no interior dos ossos (n=5 para cada cadáver). Nas 200 amostras examinadas do homogeneizado restante (n=40 para cada cadáver), em apenas duas amostras de uma leira foram detectados esporos de C. botulinum tipo C, enquanto que todas foram negativas para a tentativa de detecção da toxina botulínica pelo bioensaio em camundongo. da forma como foi avaliado o processo de pré-compostagem de bovinos mortos pela intoxicação botulínica não contribuiu para a proliferação de C. botulinum.

Produção de enterotoxinas e da toxina da síndrome do choque tóxico por cepas de Staphylococcus aureus isoladas na mastite bovina

A total of 72 strains of Staphylococcus aureus were examined for the production of staphylococcal enterotoxins (SE) A, B, C, D and toxic shock syndrome toxin (TSST-1). The strains were isolated from milk samples from cows with mastitis in dairy herds of São Paulo State, Brazil. Off 72 isolates, 38 (52.8%) produced SEA, 38 (52.8%) SEB, 32 (44.4%) SED, 28 (38.9%) SEC and 27 (37.5%) TSST-1. From the 72 strains, 66 (91.7%) produced, at least, one or more toxin, including TSST-1.

Ocorrência de Bacillus cereus em leite integral e capacidade enterotoxigênica das cepas isoladas

Pesquisaram-se a presença de Bacillus cereus e a produção de enterotoxinas produzidas por esses microrganismos em 120 amostras de diversos tipos de leite. Bacillus cereus foi isolado e identificado em 22 (73,3%), 15 (50,0%), 29 (96,7%) e quatro (13,3%) amostras de leite em pó, cru, pasteurizado e UAT (longa vida), respectivamente. Para a detecção de enterotoxinas pela técnica da alça ligada de coelho, foram positivos, respectivamente, três (13,6%), um (7,1%) e 10 (35,7%) microrganismos isolados das amostras de leite em pó, leite cru e leite pasteurizado. Pelo teste de aumento de permeabilidade vascular, dois (9,1%), um (7,1%), um (3,6%) e um (4,0%) microrganismos isolados de leite em pó, cru, pasteurizado e UAT apresentaram-se enterotoxigênicos, respectivamente. O uso da técnica de aglutinação passiva em látex demonstrou a produção da toxina diarréica por três (33,3%), sete (63,6%), quatro (30,8%) e oito (80,0%) microrganismos isolados, respectivamente, de leite em pó, cru, pasteurizado e UAT. Os resultados indicam um risco potencial, podendo colocar em risco a saúde dos consumidores desses produtos.

CONTROL of THE CENTRAL CHEMOREFLEX BY A5 NORADRENERGIC NEURONS IN RATS

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

Toxoplasma gondii: Evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in BALB/c mice

The purpose of this work was to evaluate protective activity against brain cyst formation in BALB/c mice intranasally vaccinated with recombinant proteins from Toxoplasma gondii. The recombinant proteins rROP2, rGRA5 and rGRA7 were used in vaccine preparation. Thirty-three female mice were divided into three groups, these animals received two doses by intranasal route at days 0 and 21 as follows; group 1 (G1, n = 11) received 12.5 mu g of each recombinant protein plus 0.5 mu g of cholera toxin, group 2 (G2, n = 11) received phosphate buffer saline (PBS) plus 0.5 mu g of cholera toxin, and group 3 (G3, n = 11) received PBS only. At challenge day (day 33) three animals from each group were euthanatized for IgA measure from intestine. Mice were infected orally with 50 cysts from the VEG strain at day 33. At challenge day the G1 animals had high immunoglobulin A levels, however, they only showed IgG antibody titers against rROP2 and rGRAT Animals from G1 also exhibited strong resistance to cyst formation compared with the control group (G3, P < 0.05). However, we did not observe differences in protection against brain cyst formation between G1 and G2 (P > 0.1). These results indicate that intranasal immunization in BALB/c mice with recombinant proteins rROP2, rGRA5 and rGRA7 associated with cholera toxin induced partial protection, when compared with G3, against tissue cyst formation after oral infection with tissue cysts from T gondii. (c) 2007 Elsevier B.V. All rights reserved.

Molecular Identification and Characterization of Ileal and Cecal Fungus Communities in Broilers Given Probiotics, Specific Essential Oil Blends, and Under Mixed Eimeria Infection

Broiler digestive tract fungal communities have gained far less scrutiny than that given corresponding bacterial communities. Attention given poultry-associated fungi have focused primarily on feed-associated toxin-producers, yeast, and yeast products. The current project focused on the use of pyrosequencing and denaturing gradient gel electrophoresis (DGGE) to identify and monitor broiler digestive fungal communities. Eight different treatments were included. Four controls were an Uninfected-Unmedicated Control, an Unmedicated-Infected Control, the antibiotic bacitracin methylene disalicylate plus the ionophore monensin as Positive Control, and the ionophore monensin alone as a Negative Control. Four treatments were two probiotics (BC-30 and Calsporin) and two specific essential oil blends (Crina Poultry Plus and Crina Poultry AF). All chickens except the Unmedicated-Uninfected Control were given, at 15 days of age, a standard oral Eimeria inoculum of sporulated oocysts. Ileal and cecal digesta were collected at pre-Eimeria infection at 14 days of age and at 7 days post-Eimeria infection at 22 days of age. Extracted cecal DNA was analyzed by pyrosequencing to examine the impact of diet supplements and Eimeria infection on individual constituents in the fungal community, while DGGE was used to compare more qualitative changes in ileal and cecal communities. Pyrosequencing identified three phyla, seven classes, eight orders, 13 families, 17 genera, and 23 fungal species. Ileal and cecal DGGE patterns showed fungal communities were clustered mainly into pre- and post-infection patterns. Post-infection Unmedicated-Uninfected patterns were clustered with pre-infection groups demonstrating a strong effect of Eimeria infection on digestive fungal populations. These combined techniques offered added versatility towards unraveling the effects of enteropathogen infection and performance enhancing feed additives on broiler digestive microflora.

Adaptabilidade de híbridos entre Gossypium barbadense E G. hirsutum N contendo o gene cry1Ac

Hybrids among transgenic plants and related species are expected to occur if they are sympatric and when there are not crossing barriers; as is the case, in Brazil, of cry1Ac transgenic cotton and Gossypium barbadense. This species has been maintained as dooryard plants, and should be preserved as a genetic resource. Hybrids were evaluated about traits related to fitness, leading to infer about its chances of survivor and selection. A barbadense genotype collected at the state of Mato Grosso was outcrossed to the variety DP 404, containing the gene cry1Ac, and to the isoline DP 404. All the F1 individuals and 122 among 170 F2 individuals expressed the toxin, and presented levels of resistance to pink bollworm (Pectinophora gossypiella) and cotton leafworm (Alabama argillacea) equivalent to the transgenic parent and superior to the isoline, barbadense or non transgenic hybrids. The percentage of germination and number of days to germinate did not differ among genotypes. Anthesis of the first flower and opening of the first cotton boll occurred earlier for herbaceous cotton and F1 hybrids than F2 population in average; all the populations presented a number of days to flower and opening of the first boll smaller then barbadense. The highest plants were barbadenses, and herbaceus the smallest, with F1 and F2 populations presenting intermediary heights. The number of seeds per plants were superior for F1 hybrids an herbaceous cotton, F2 populations were in average intermediary; the barbadense genotype produced the smallest number of seeds per plant. Pink bollworm, mainly, and also cotton leafworm, are important barbadense pests, so the transgene positive effect could favor the selection of hybrids, and hence G. hirsutum genome, against the maintenance of pure G. barbadense genome. The selection may be influenced by the plant uses: the smaller size of hybrids when compared to the barbadense may lead them to be differentiated from these parents to which medicinal properties are attributed; on the other hand, the greater boll production may favor hybrids maintenance with the purpose of producing lamp wicks, or use as an ornamental or swab

Efeitos da eliminação de neurônios infragranulares sobre a especificação de neurônios supragranulares do córtex cerebral

The cerebral cortex of mammals is histologically organized into different layers of excitatory neurons that have distinct patterns of connections with cortical or subcortical targets. During development, these cortical layers are established through an intricate combination of neuronal specification and migration in a radial pattern known as "insideout": deep-layer neurons are generated prior to upper-layer neurons. In the last few decades, several genes encoding transcription factors involved in the sequential specification of neurons destined to different cortical layers have been identified. However, the influence of early-generated neurons in the specification of subsequent neuronal cohorts remains unclear. To investigate this possible influence, we induced the selective death of cortical neurons from layer V and VI before the generation of layer II, III and IV neurons. Thus, we can evaluate the effects of ablation of early born neurons on the phenotype of late born neurons. Our data shows that one-day after ablation, layer VI neurons expressing the transcription factor TBR1 are newly generated while virtually no neuron expressing TBR1 was generated in the same age in control animals. This suggests that progenitors involved in the generation of neurons destined for superficial layers suffer interference from the selective death of neurons in deep layers, changing their specification. We also observed that while TBR1-positive neurons are located exclusively in deep cortical layers of control animals, many TBR1-positive neurons are misplaced in superficial layers of ablated animals, suggesting that the migration of cortical neurons could be controlled independently of neuronal phenotypes. Furthermore, we observed an increase in layer V neurons expressing CTIP2 and neurons expressing SATB2 and that these cells have changed their distributions. As a conclusion, our data indicate the existence of a mechanism of control exercised by the early-generated neurons in the cerebral cortex on the fate of the progenitors involved in the generation of the following cortical neurons. This mechanism could help to control the number of neurons in different layers and contribute to the establishment of different cortical areas

"O núcleo supraquiasmático e o folheto intergeniculado do mocó (Kerodon rupestris): Projeções retinianas e caracterização imuno-histoquímica

In this study, two circadian related centres, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) were evaluated in respect to their cytoarchitecture, retinal afferents and chemical content of major cells and axon terminals with a tract tracer and immunohistochemical techniques in the rock cavy (Kerodon rupestris), a Brazilian caviidae rodent species. The rock cavy SCN is innervated in its ventral portion by terminals from the predominantly contralateral retina. It also contains neurophisin and vasoactive intestinal polypeptide immunoreactive cell bodies and neuropeptide Y and enkephalin immunopositive fibres and terminals and is marked by intense GFAP immunoreactivity. The IGL receives a predominantly contralateral retinal projection, contains neuropeptide Y and nitric oxide synthase producing neurons and enkephalin immunopositive terminals and is characterized by dense GFAP immunoreactivity. This is the first report examining the neural circadian system in a crepuscular rodent species for which circadian properties have been described. The results are discussed comparing with what has been described for other species and in the context of the functional significance of these centres

Centros subcorticais dos sistemas visual primário e óptico acessório no mocó (kerodon rupestris): caracterização pela projeção retiniana e citoarquitetura.

The primary and accessory optic systems comprise two set of retinorecipient neural clusters. In this study, these visual related centers in the rock cavy were evaluated by using the retinal innervations pattern and Nissl staining cytoarchigtecture. After unilateral intraocular injection of cholera toxin B subunit and immunohistochemical reaction of coronal and sagittal sections from the diencephalon and midbrain region of rock cavy. Three subcortical centres of primary visual system were identified, superior colliculus, lateral geniculate complex and pretectal complex. The lateral geniculate complex is formed by a series of nuclei receiving direct visual information from the retina, dorsal lateral geniculate nucleus, intergeniculate leaflet and ventral lateral geniculate nucleus. The pretectal complex is formed by series of pretectal nuclei, medial pretectal nucleus, olivary pretectal nucleus, posterior pretectal nucleus, nucleus of the optic tract and anterior pretectal nucleus. In the accessory optic system, retinal terminals were observed in the dorsal terminal, lateral terminal and medial terminal nuclei as well as in the interstitial nucleus of the superior fasciculus, posterior fibres. All retinorecipient nuclei received bilateral input, with a contralateral predominance. This is the first study of this nature in the rock cavy and the results are compared with the data obtained for other species. The investigation represents a contribution to the knowledge regarding the organization of visual optic systems in relation to the biology of species.

O trato retino-hipotalâmico no mocó (Kerodon rupestris): Um estudo de traçado anterógrado com a subunidade b da toxina colérica

The light, besides the vision stimuli, controls other process completely independent of image formation, such as the synchronization of the organismic circadian rhythms to the enviromental light/dark cycle. In mammals, this adjust occurs through the retinohypothalamic tract, a direct retinal projection to the suprachiasmatic nucleus, considered to be the major circadian pacemaker. Early studies have identified only the suprachiasmatic nucleus as a retinal target in the hypothalamus. However, using more sensitive neuroanatomic tracers, other retinorecipient hypothalamic regions outside to suprachiasmatic nucleus were pointed in a great number of mammalian species. In this study, the retinohypothalamic tract was shown in the rock cavy (Kerodon rupestris), an endemic rodent of the semiarid region of the Brazilian Northeast, using unilateral intravitreal injections of cholera toxin subunit b as a neuronal tracer. The results reveal that in the rock cavy, besides the suprachiasmatic nucleus, several hypothalamic regions receive direct retinal projection, such as the ventrolateral preoptic nucleus, medial and lateral preoptic areas, the supraoptic nucleus and bordering areas, anterior, lateral and rectrochiasmatic hypothalamic areas, and the subparaventricular zone. The results are discussed by comparing with those of the literature, into a functional context