Efeito antimicrobiano e citolítico da trialisina recombinante da saliva de Triatoma infestans

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, 2002.Insetos hematófagos têm grande interesse médico-biológico devido a transmissão de doenças endêmicas de alta prevalência, morbidade e mortalidade em populações humanas e de animais, em todos os continentes. Por exemplo, a doença de Chagas transmitida pelo triatomineo Triatoma. infestans está amplamente distribuída no continente americano, onde cerca de 18 milhões de pessoas já estariam infectadas com o Trypanossoma. cruzi, o agente causai da doença. Esses insetos produzem e armazenam proteínas farmacologicamente ativas em suas glândulas salivares, e as inoculam no hospedeiro durante o repasto. Ao inibir os mecanismos homeostásicos da sua presa, essas bioaminas facilitam o repasto do inseto e a transmissão do agente patógeno. O estudo de proteínas salivares com notável efeito farmacológico pode ser útil no estudo da filogenia e diversidade genética de insetos, mas elas também poderiam ser usadas como ferramentas para a caracterização dos processos hematológicos, vasculares, e teciduais relacionados às modificações que ocorrem no sítio da picada. Contudo, nós achamos que, igualmente importante, seria a produção de uma vacina contra a picada do inseto hematófago, criando imunoprofilaxia contra a transmissão do T. cruzi. A produção de vacina com proteínas recombinantes expressas na glândula salivar do triatomineo é um objetivo em longo prazo no Laboratório Multidisciplinar de Pesquisa em Doença de Chagas. O trabalho aqui apresentado faz parte dessa estratégia visando á identificação de genes e expressão de proteínas recombinantes com funções importantes no repasto do inseto, Nós analisamos 20 clones oriundos de uma biblioteca de cDNA de glândula salivar de T. infestans. As seqüências gênicas desses clones mostraram similaridade, e não descartamos a possibilidade de se tratar de uma família mulíigênica. A proteína recombinaníe resultante da expressão do clone 30/LMPDC recebeu o nome de rTrialisina. Ainda que a seqüência do clone 30/LMPDC tenha 97% de similaridade com o gene da Trialisina (Amino e cols., 2002), nós obtivemos a expressão da rTrialisina e descrevemos suas características funcionais ainda desconhecidas. Ademais, a modelagem molecular permitiu-nos conhecer parte da estrutura terciária da proteína e seu sítio ativo foi predito, o que também eram desconhecidos. O cDNA completo do gene da Triaiisina que codifica a pró-proteína foi inserido em um vetor de expressão de proteínas de fusão pGEX-5x-3. A proteína de fusão foi expressa sob a indução de 1PTG em E. coli BL21(DE3) e purificada em cromatografia de afinidade com agarose ligada à glutationa. A proteína recombinante (rTrialisina) ativada pela saliva de T. infestans mostrou efeito citolítico sobre E. coli, T. cruzi, L donovani e células murinas da linhagem L6. A atividade fosfoiipásica A2 da ririalisina estaria relacionada com a sua atividade lítica formadora de poros. A rTrialisina foi expressa em E. coli e a proteína recombinante foi utilizada para a imunização de coelhos. Os anticorpos policlonais obtidos localizaram a expressão da proteína no primeiro par de glândulas salivares do inseto. De grande interesse, esses anticorpos inibiram parcialmente a atividade citolííica da rTrialisina in vitro. Em resumo, os resultados desse trabalho adicionam informações e insumos para a avaliação da estratégia montada pela equipe de pesquisadores do LMPDC. Nós postulamos que juntamente com outras proteínas da saliva do triatomíneo, a rTrialisina deve ser testada como fração de imunógenos na formulação de vacina polivalente visando a imposição de dificuldades ao repasto do inseto hematófago transmissor da doença de Chagas. Uma possível atividade imunobiológica ou biotecnológica da rTrialisina deverá ser avaliada após sua expressão em vetor indicado para produção em larga escala. Diante desse potencial, a aplicabilidade da rTrialisina e de outras proteínas recombinantes produzidas no LMPDC deverão ser submetidos ao processo de patente.Blood-sucking insects have been studied for their bio-medical importance associated with the transmission of prevalent endemic diseases showing high morbidity and mortality ali over the world. For exampie, Chagas disease transmitted by the reduviid bug {Hemíptera: Reduviidae) T. infestans is broadly distributed in the American Continent, where 18 millíon people became infected with T. cruzi, the causai agent of the disease These insects store and secreíe in their salivary gland pharmacologically active proíeins, and insert these into the host during feeding. The host homeostatic inhibition mechanisms help insectfeeding and the pathogen transfer. The study of salivary proteins study with notable pharmacologicai effect may be useful in understanding the genetic diversity and biogenetics of the insects, but it can be used as a tool for characterization of the hematological, vascular and tissue specific hematological processes. Therefore we see the importance of vaccine production against the hemathophagous insect bite, as prophylaxis against T. cruzi transmission. The vaccine production with recombinant protein expressed in the triatomine salivary gland is a future project in the LMPDC. The work we have presented here is part of this strategy that iníends to identify genes and express recombinant proteins with important functions for insect feeding. We have analyzed 20 clones from the T. infestans salivary gland cDNA library. These clones sequences showed high similarity, and we donl discard the possibility of a multigene family. The recombinant protein that resulted from expression of the clone 30/LMPDC was named rThalysin. The rThalysin sequence showing 97% homology with Triaiysin's gene (Amino et al., 2002) was further subjected to expression and the still unknown recombinant protein functionat characterization was described. In addition, the molecular modeling hetped to know part of the tertiary structure and the active site could be predicted. The complete Trialysin cDNA that codes for a pre-protein was inserted mto a fusion protein expressiion vector pGEX-5x-3. The fusion protein was expressed with IPTG induction in E. coli BL21(DE3) and purified by affinity chromatography with glutathione agarose. The recombinant protein processed by T. infestans saliva-.showed cytolytic effect against E. coli. T. cruzi, L. donovani and murine L6 cells. The rTrialysin phospholipase A2 activity could be related to pore-forming activity of the rTrialysin. The rTrialysin was expressed in E. coli and the recombinant protein was used to immunize rabbits. The polyctonal antibodies recognized the native Trialysin in the first pair of salivary glands in the insect. These antibodies also inhibited partially the rTrialysin cytolyses in vitro. In summary, the results of this work add information and evaluate the strategy set forth by the LMPDC staff researchers. We have planned that together with oíher proteins from the Triatomine salivary gland the rTrialysin should be tested with immunogenic fractions in the polyvalent vaccine preparation, creating difficulty for the haematophagous insect feeding vector of Chagas disease. Hence the immunobiological and biothecnological activities or the rTrialysin should be evaluated after expression on large scale. The rTrialysin and the other recombinant proteins expressed exclusively in the Triatomine salivary glands is promising for the future and therefore, its applicability needs Paíent protection

Testicular biometric characteristics in Santa Inês sheep submitted to protein supplementation

Para avaliar as características biométricas testiculares de carneiros da raça Santa Inês, 24 cordeiros machos, inteiros, com peso médio inicial de 24,5 kg ± 2,88, aos quatro meses de idade, foram distribuídos em quatro tratamentos: APv (animais vermifugados + concentrado com alta proteína), APn (animais não-vermifugados + concentrado com alta proteína), BPv (animais vermifugados + concentrado com baixa proteína), BPn (animais não vermifugados + concentrado com baixa proteína) durante 8 meses e meio. Antes do abate, realizaram-se as medições testiculares in situ: perímetro escrotal (PE), comprimento (COMP), largura testicular (LARG) e a forma dos testículos, bem como o volume testicular (cilindro e prolato esferóide). Após o abate, determinaram- se o volume testicular real (VOLR), o comprimento (COMPV) e a largura testicular (LARGV). Observou-se que os testículos dos animais eram simétricos e que o uso do paquímetro é uma forma altamente confiável para se realizar as medições testiculares in situ. O formato dos testículos afetou diretamente seu volume. O valor médio das equações do prolato esferóide e cilíndrica é a forma mais acurada para se estimar o volume testicular real em ovinos Santa Inês. O alto teor de proteína da dieta e o tratamento antiparasitário possibilitaram uma melhoria do peso corporal dos ovinos, sendo o principal fator de variação para as características biométricas testiculares. _____________________________________________________________________________________ ABSTRACTBiometric testicular traits were evaluated on 24 entire male Santa Inês lambs, with mean initial weight of 24.5 kg ± 2.88. These were distributed in four treatments (n=6): HPv (animals drenched + high protein concentrate), HPn: (animals not drenched + high protein concentrate), LPv: (animals drenched + low protein concentrate), LPn: (animals not drenched + low protein concentrate) for eight and a half months. Before slaughter testicular measurements were taken in situ. Scrotal perimeter (SC) was measured using tape as well as length (LENG) and width (WID) using calipers. Testicular shape and volume (using prolate and cylinder equation) were calculated. After slaughter the measurements were taken in vitro. Real testicle volume (VOLR) was measured using water displacement. Length (LENGV) and width (WIDV) were measured with calipers after skin removal. The testicles were symmetrical and calipers were an accurate means of in situ measurement of the testicle size. The form of the testicles affected the volume. The average of the prolate sphere and cylinder equations gave the best estimate for real testicle volume. High protein levels in the diet accompanied by antihelminth treatment led to higher body weight in the sheep, this being the main factor for variation in testicle biometric traits

Phosphorus deficiency diagnosis in sheep using labeled phosphorus uptake by erythrocytes

Este trabalho teve como objetivo avaliar a técnica de incorporação de 32P pelos eritrócitos de ovinos jovens, mantidos em pastejo de Andropogon gayanus, como método de diagnóstico da deficiência de fósforo. Vinte ovinos, com peso vivo inicial de 13,88±2,51 kg, foram divididos em dois tratamentos de dez animais cada; num dos tratamentos, os animais foram suplementados com 3 g de P por animal por dia e, no outro, os animais não receberam suplementação de P. Foram realizadas cinco pesagens dos animais, coletas de sangue e fezes nos 8º, 29º, 43º, 57º e 71º dias do experimento, para avaliar a incorporação de 32P pelos eritrócitos, determinar as concentrações de Ca, glicose e P no soro e a porcentagem de P nas fezes. Foi encontrada diferença significativa na concentração de Ca no 57º dia. Na concentração de glicose, porcentagem de P nas fezes e peso vivo não foram observadas diferenças significativas entre os tratamentos. A partir da segunda coleta, a concentração de P no soro foi mais elevada e a incorporação de 32P foi menor no grupo de animais suplementados com P. A incorporação de 32P pelos eritrócitos é uma técnica adicional para avaliar o status de P e identificar sua deficiência subclínica em ovinos jovens.This work had the objective of evaluating the method of 32P uptake by the erythrocytes of young sheep, supplemented with phosphorus and kept at pasture of Andropogon gayanus, as a tool for phosphorus deficiency diagnosis. Twenty lambs, initially weighing 13.88±2.51 kg, were divided in two treatments of ten animals each. In one of them, animals were supplemented with 3 g of phosphorus per animal per day and, in the other, animals did not receive supplementation of phosphorus. Weighing, and blood and faeces collection were carried out in the 8th, 29th, 43rd, 57th e 71st days of the experiment, to evaluate the incorporation of 32P by the erythrocytes, to determine calcium, glucose and phosphorus concentrations in the serum and percentage of P in the faeces. Significant difference for the concentration of Ca was found in the 57th day. For the glucose concentration, the percentage of phosphorus in faeces, and the weight, significant differences between the treatments were not observed. From the 29th to the 71st day, the concentration of phosphorus in the serum was higher, and the incorporation of 32P was lower in the group of animals supplemented with phosphorus. The analysis of 32P incorporation by the erythrocytes is an additional tool to evaluate the status of P, with identification of the subclinic deficiency in lambs

Dry matter intake, performance and carcass characteristics of hair sheep reared under different grazing systems

The aim of this study was to evaluate the effect of three different grazing systems: isolated, alternate and simultaneous, on feed intake, performance and carcass characteristics of sheep. About 5.2 ha area of Tanzania grass (Panicum maximun Jacq cultivate Tanzania) was divided into 13 paddocks. This area was used as a stocking rate of two animal units (AU) per ha for 7 days’ occupation and 21 days rest. A total number of 58 animals were used consisting of 12 heifers and 30 Santa Ines lambs with the addition of 16 adult ewes that were used to stabilize grazing pressure in the isolated system. The sheep were fed on 200 g per head per day of concentrate and cattle 2 kg per head per day. The parameters determined were the following: weekly weight (WW), total live weight gain (LWG) and mean daily weight gain (MDW). Also dry matter intake was estimated 84 days after the start of the experiment using external indicators (Purified and Enriched Lignin) in addition to carcass traits and composition which were also estimated. The result obtained for carcass composition revealed that the muscle:bone ratio and bone percentage were better in the alternate system. Moreover, the simultaneous and isolated systems showed higher lamb performance than the alternate system, while there was no effect on dry matter intake. Furthermore, there was no difference of the different systems on carcass traits and feed intake of sheep. Nevertheless, the simultaneous grazing system showed better sheep performance than the alternate grazing system

Dry matter intake, performance and carcass characteristics of hair sheep reared under different grazing systems

ABSTRACT The aim of this study was to evaluate the effect of three different grazing systems: isolated, alternate and simultaneous, on feed intake, performance and carcass characteristics of sheep. About 5.2 ha area of Tanzania grass (Panicum maximun Jacq cultivate Tanzania) was divided into 13 paddocks. This area was used as a stocking rate of two animal units (AU) per ha for 7 days’ occupation and 21 days rest. A total number of 58 animals were used consisting of 12 heifers and 30 Santa Ines lambs with the addition of 16 adult ewes that were used to stabilize grazing pressure in the isolated system. The sheep were fed on 200 g per head per day of concentrate and cattle 2 kg per head per day. The parameters determined were the following: weekly weight (WW), total live weight gain (LWG) and mean daily weight gain (MDW). Also dry matter intake was estimated 84 days after the start of the experiment using external indicators (Purified and Enriched Lignin) in addition to carcass traits and composition which were also estimated. The result obtained for carcass composition revealed that the muscle:bone ratio and bone percentage were better in the alternate system. Moreover, the simultaneous and isolated systems showed higher lamb performance than the alternate system, while there was no effect on dry matter intake. Furthermore, there was no difference of the different systems on carcass traits and feed intake of sheep. Nevertheless, the simultaneous grazing system showed better sheep performance than the alternate grazing system