Immunoprophylaxis using polypeptide chimera vaccines plus adjuvant system promote Th1 response controlling the spleen parasitism in hamster model of visceral leishmaniasis.

In recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras inMesocricetus auratushamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-γ and TNF-α cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease.

Immunochemotherapy for visceral leishmaniasis: combinatorial action of Miltefosine plus LBSapMPL vaccine improves adaptative Th1 immune response with control of splenic parasitism in experimental hamster model.

The control of human visceral leishmaniasis (VL) is hard since there are no vaccines available as well as the treatment is hampered by toxicity and resistant parasites. Furthermore, as human, and canine VL causes immunosuppression, the combination of drugs with immunostimulatory agents is interesting to upregulate the immunity, reducing side-effects, improving treatment approaches against disease. Herein, we assessed the immunochemotherapy using miltefosine along with a vaccine formulated by Leishmania braziliensis antigens + saponin + monophosphoryl lipid-A (LBSapMPL) in L. infantum-infected hamsters. Two months after infection, the animals received treatments, and after 15 days they were evaluated for the treatment effect. The potential anti-Leishmania effect of miltefosine + LBSapMPL-vaccine was revealed by a specific immune response activation reflecting in control of spleen parasitism using half the miltefosine treatment time. The treated animals also showed an increase of total and T-CD4 splenocytes producing IFN-γ and TNF-α and a decrease of interleukin-10 and anti-Leishmania circulating IgG. In addition, it was demonstrated that the control of spleen parasitism is related to the generation of a protective Th1 immune response. Hence, due to the combinatorial action of miltefosine with LBSapMPL-vaccine in immunostimulating and controlling parasitism, this immunochemotherapy protocol can be an important alternative option against canine and human VL.

Establishment of monoclonal antibodies to evaluate the cellular immunity in a hamster model of L infantum infection.

Syrian hamsters (Mesocricetus auratus) are largely used as a model for infectious diseases because it is very susceptible to several pathogens, including Leishmania spp. parasites. However, the research community faces limitations in its use due to the lack of immunological reagents and tools to study the immune system in this model. In this context, we proposed the validation of some important commercially anti-mouse mAbs (CD4, TNF-α, IFN-γ and IL-10) and how this could be useful to evaluate a specific cellular immune response in Leishmania-infected hamster using flow cytometry experiments. Our data demonstrated a cross-reactivity between these anti-mouse mAbs and hamster molecules that were herein studied. Beyond that, it was able to characterize the development of a specific cellular immune response through cytokine production in L infantum-infected hamsters when compared to uninfected ones. These data not only aid the usage of hamsters as experimental model to investigate various infectious diseases, but they contribute to the design of novel approaches to further investigate the immunological mechanisms associated to pathogen infections.

Heterologous vaccine therapy associated with half course of Miltefosine promote activation of the proinflammatory response with control of splenic parasitism in a hamster model of visceral leishmaniasis.

Visceral leishmaniasis (VL) is a serious and neglected disease present worldwide. Chemotherapy using pentavalent antimony (SbV) is the most practical and inexpensive strategy available for the VL treatment today, however, it has high toxicity. Alternatively, other drugs are used as viable leishmanicidal therapeutic options. Miltefosine is the only anti-leishmanial agent administered orally, however, it has been reducing its effectiveness. In this sense, there is no ideal therapy for VL since the drugs currently used trigger severe side effects causing discontinuation of treatment, which carries an imminent risk for the emergence of parasite resistance. With that, other therapeutic strategies are gaining prominence. Among them, immunotherapy and/or immunochemotherapy, which the activation/modulation of the immune system can redirect the host's immune response to an effective therapeutic result. Therefore, this work was designed to assess an immunochemotherapy protocol composed of half course of Miltefosine associated with LBSap vaccine (Milt+LBSap) using the hamster Mesocricetus auratus as an experimental model for VL treatment. When evaluating the main hematobiochemical, immunological and therapeutic efficacy parameters, it was demonstrated that the treatment with Milt+LBSap showed restoration of hematobiochemical condition and reduced serum levels of IgG-anti-Leishmania compared to animals infected non treated (INT). Beyond that, an increase in the number of CD4+ lymphocytes producers of IFN-γ in relation to INT or to animals treated with miltefosine during 28 days, and TNF-α increased compared to INT were observed. Also, it was found a reduction of IL-10-production in relation to INT, or animals that received LBSap vaccine only, or miltefosine, following by a reduction in the splenic parasitic burden. These results demonstrate that the immunochemotherapy protocol used can stimulate the immune response, inducing an expressive cellular response sufficient to control spleen parasitism, standing out as a promising proposal for the VL treatment.

Histopathological changes in the gastrointestinal tract and systemic alterations triggered by experimental oral infection with Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in almost all countries of Latin America. In Brazil, oral infection is becoming the most important mechanism of transmission of the disease in several regions of the country. The gastrointestinal tract is the gateway for the parasite through this route of infection, however, little is known about the involvement of these organs related to oral route. In this sense, the present study evaluated the impact of oral infection on the digestive tract in mice infected by Berenice-78 (Be-78) T. cruzi strain, in comparison with the intraperitoneal route of infection. In this work, the intraperitoneal route group showed a peak of parasitemia similar to the oral route group, however the mortality rate among the orally infected animals was higher when compared to intraperitoneal route. By analyzing the frequency of blood cell populations, differences were mainly observed in CD4+ T lymphocytes, and not in CD8+, presenting an earlier reduction in the number of CD4+ T cells, which persisted for a longer period, in the animals of the oral group when compared with the intraperitoneal group. Animals infected by oral route presented a higher tissue parasitism and inflammatory infiltrate in stomach, duodenum and colon on the 28th day after infection. Therefore, these data suggest that oral infection has a different profile of parasitological and immune responses compared to intraperitoneal route, being the oral route more virulent and with greater tissue parasitism in organs of the gastrointestinal tract evaluated during the acute phase.

Liver infusion tryptose (LIT): the best choice for growth, viability, and infectivity of Leishmania infantum parasites.

Leishmania spp. parasites have a complex biological cycle presenting basically two different morphological stages, the amastigote and promastigote forms. In vitro cultivation allows a more complete study of the biological aspects of these parasites, indicating better conditions for infection, immunoassay tests, drug evaluations, and vaccines. Thus, we evaluated the three most used culture media for Leishmania spp., Grace's insect cell culture medium (Grace's), liver infusion tryptose (LIT), and Schneider's insect medium (Schneider's), without supplementation or supplemented with fetal calf serum (FCS) and bovine serum albumin (Albumin) to evaluate the growth, viability, and infectivity of the L. infantum promastigotes. It was observed that promastigote forms have a better growth in LIT and Schneider's with or without FCS when compared to that in Grace's. The supplementation with albumin promoted greater viability of the parasites independent of the medium. For in vitro infection of J774.A1 macrophages using light microscopy and flow cytometry analyses, FCS-supplemented LIT and Grace's promoted higher percentage of infected macrophages and parasite load compared with Schneider's media. Taken together, our results demonstrated that the supplementation of LIT culture medium with FCS is the most suitable strategy to cultivate Leishmania infantum parasites enabling the maintenance of growth and infective parasites for research uses.

Low-dose of benznidazole promotes therapeutic cure in experimental chronic Chagas' disease with absence of parasitism in blood, heart and colon.

Studies suggest that the dose of the standard benznidazole (BNZ) treatment regimen might be too high. We investigated the efficacy of BNZ 20 and 40 mg/kg/day compared with standard dose (100 mg/kg/day) to induce cure in mice infected with Trypanosoma cruzi Y strain in the acute and chronic phases of Chagas' disease. Our findings indicate that an experimental treatment with a BNZ low-dose (40 mg/kg/day) is similarly effective as the usual dose in the chronic mice model (100% of cure). In addition, the treatment in the chronic model of Chagas' disease presented better results than the acute model and colon appears to be a key tissue when it comes to evaluating treatment efficacy compared to blood and heart. Therefore, our data suggest the reconsideration of the current therapy, mainly in the chronic phase of the disease.

Manejo de pragas em cultivo de roseira de sistema de produção integrada e sistema convencional / Pest management in rose crop in integrated production system and conventional system

A cultura da roseira é suscetível ao ataque de várias pragas e o uso de práticas agrícolas menos agressivas à natureza têm sido incentivadas como alternativa ao controle químico. O objetivo do trabalho foi avaliar o uso de agentes de controle biológico e defensivos alternativos no manejo de pragas na produção integrada de rosas e comparálo com o uso de defensivos químicos no sistema convencional. O experimento foi conduzido de fevereiro de 2010 a janeiro de 2011 em cultivo de roseira em casa de vegetação (18 x 6 m) em São João Del Rei (MG). Foram utilizadas mudas de rosas da cultivar 'Carolla' (espaçamento de 1,20 m x 0,20 m). Foram separadas duas áreas na casa de vegetação, sendo uma com controle químico convencional e outra de produção integrada. Cada área continha quatro linhas com 50 plantas por linha. Foram feitas amostragens semanais através da contagem dos artrópodes fitófagos e inimigos naturais presentes nas plantas. Os principais artrópodes fitófagos encontrados foram ácaros, pulgões, moscas brancas e tripes. Não foi constatado dano nas plantas em ambas as áreas avaliadas. Não foram utilizados defensivos químicos na área de produção integrada. Na área de produção integrada foram encontrados 84,2% dos inimigos naturais quantificados. Os resultados obtidos nesse estudo permitem concluir que o cultivo de rosas em sistema de produção integrada possibilita a redução da aplicação de defensivos químicos e o aumento da população de inimigos naturais na área.

The culture of the rose is susceptible to attack by various pests and the use of agricultural practices less aggressive to nature has been encouraged as an alternative to chemical control. The objective was to evaluate the use of biological control agents and alternative pesticides in pest management in integrated production of roses and compare it with the use of pesticides in the conventional system. The experiment was conducted from february 2010 to january 2011 rose cultivation in greenhouse (18 x 6 m) in São João Del Rei (MG). Was used rose variety 'Carolla' (1.20 m x 0.20 m). Two areas were separated in a greenhouse, one with conventional chemical control and other integrated production. Each area contained four lines with 50 plants per row. Samples were collected weekly by counting the insects present in plants. The mains phytophagous arthropods were found mites, aphids, whiteflies and thrips. Damage was not observed in plants in both areas studied. No pesticides were used in the area of Integrated Production. In the area of integrated production found 84.2% of natural enemies quantified. The results of this study support the conclusion that the cultivation of roses in an integrated production system allows the reduction of the application of pesticides and increased population of natural enemies in the area.

Efeito da presa alternativa no desenvolvimento e consumo de Orius insidiosus (Say) (Hemiptera, Anthocoridae) e comportamento de oviposição em cultivares de crisântemo / Effect of alternative prey on development and consumption of Orius insidiosus (Say) (Hemiptera, Anthocoridae) and oviposition behavior on chrysantemum cultivars

Este trabalho teve como objetivo avaliar o desenvolvimento e o consumo de Orius insidiosus (Say, 1832) tendo Aphis gossypii Glover, 1877 como presa, bem como seu comportamento de oviposição em duas cultivares de crisântemo. O experimento foi conduzido em câmara climática a 25 ± 1ºC, UR 70 ± 10 por cento e fotofase de 12 horas. Ninfas do predador com até 24 horas de idade foram colocadas individualmente em placas de petri (5 cm) contendo 20 ninfas de A. gossypii (1º, 2º e 3º ínstares), as quais estavam posicionadas sobre disco foliar (4 cm) de cada cultivar ('White Reagan' e'Yellow Snowdon') em camada de ágar-água . Na avaliação da oviposição foram utilizados pecíolos de cada cultivar como substrato de oviposição e ovos de Anagasta kuehniella (Zeller, 1879) como alimento. O predador completou seu desenvolvimento alimentando-se somente de A. gossypii presente em ambas as cultivares. A duração da fase ninfal de O. insidiosus foi de 21,1 e 18,3 dias, em 'White Reagan' e 'Yellow Snowdon', respectivamente. O consumo de A. gossypii por fêmeas foi maior (P<0,01) em 'White Reagan' (2,63 ninfas), comparado a 'Yellow Snowdon' (0,7 ninfas). Fêmeas de O. insidiosus ovipositaram em pecíolos das cultivares, com 22,5 e 23,3 ovos/fêmea em 'White Reagan' e 'Yellow Snowdon', respectivamente. Liberações de O. insidiosus em cultivos de crisântemo podem auxiliar na diminuição da população de A. gossypii, uma vez que o predador completa o seu desenvolvimento tendo este inseto como presa e as cultivares de crisântemo oferecem condições para colonização e estabelecimento de O. insidiosus.

This work aimed to evaluate the development time and the consumption of Orius insidiosus (Say, 1832) feeding on Aphis gossypii Glover, 1877 as weel as its oviposition behavior on two crysanthemum cut cultivars. The trials were conducted in climatic chamber at 25±1ºC, RH 70±10 percent and 12h photophase. Nymphs of the predator, less than 24h old, were kept individualy in petri dishes (5cm) with 20 nymphs of A. gossypii (1st, 2nd and 3rt instars) on leaf disc (4cm) of each cultivar ("White Reagan" and "Yellow Snowdon") in a layer of agar-water (1 percent). Petiole of each crysanthemum cultivar as oviposition substrate was evaluated and the females were feeding on eggs of Anagasta kuehniella (Zeller, 1879). The predador complete its development feeding on A. gossypii kept in both cultivars. The duration of nymphal phase of O. insidiosus were 21.1 and 18.3 days on "White Reagan" and "Yellow Snowdon", respectively. The consumption of the females of O. insidiosus was higher (P<0.01) on A. gossypii in "White Reagan" (2.63 nymphs) compared to the consumption in "Yellow Snowdon" (0.7 nymphs). Females of O. insidiosus oviposited in petiole of both cultivars with 22.5 and 23.3 eggs/female on "White Reagan" and "Yellow Snowdon", respectively. Release of O. insidiosus on chrysanthemum crops could be important to decrease the A. gossypii population, as the predator completes its development having this aphid as prey, and the chrysanthemum cultivars offer conditions to colonization and establishment of O. insidiosus.

Desenvolvimento e exigências térmicas de Orius insidiosus ( Say ) ( Hemiptera, Anthocoridae ) / Development and thermal requirements of Orius insidiosus ( Say ) ( Hemiptera: Anthocoridae )

A temperatura exerce grande influência no desenvolvimento dos insetos e o conhecimento desse aspecto é essencial para subsidiar o uso de inimigos naturais como agentes de controle biológico, bem como para a sua criação massal. O presente trabalho teve como objetivo avaliar o efeito de diferentes temperaturas no desenvolvimento de Orius insidiosus (Say, 1832), bem como as suas exigências térmicas. O experimento foi conduzido em câmaras climáticas, a 16, 19, 22, 25, 28 e 31±1°C; UR de 70±10 por cento e fotofase de 12 horas. Como alimento foram utilizados ovos de Anagasta kuehniella (Zeller, 1879). O período embrionário foi de 14,0; 8,9; 6,6; 4,8; 3,9 e 3,3 dias nas temperaturas de 16, 19, 22, 25, 28 e 31°C, respectivamente. Ninfas de todos os instares (independente do sexo que deram origem) foram influenciadas pela temperatura quanto ao seu desenvolvimento, com redução nesse período com o aumento da temperatura. Machos e fêmeas, na temperatura de 25°C, apresentaram um período de desenvolvimento em torno de 12 dias. A temperatura base da fase de ovo foi de 11,78°C e a da fase ninfal foi de 12,27°C e de 13,03°C, para machos e fêmeas, respectivamente. A constante térmica para a fase de ovo foi de 63,75 e para a fase de ninfa de 161,97 e 157,24 graus-dia, para machos e fêmeas, respectivamente. A temperatura de 25°C foi a mais adequada para o desenvolvimento de O. insidiosus.

Influência da temperatura na reproducão e longevidade do predador Orius thyestes Herring (Hemiptera, Anthocoridae) / Influence of the temperature on reproduction and longevity of the predator Orius thyestes Herring (Hemiptera, Anthocoridae)

O desenvolvimento e a fecundidade das espécies de Orius são bastante influenciados por uma série de fatores, como as condicões ambientais, e em particular, pela temperatura. Esse trabalho teve como objetivo avaliar a reproducão e a longevidade de Orius thyestes Herring 1966 em diferentes temperaturas, tendo como alimento, ovos de Anagasta kuehniella (Zeller, 1879). O experimento foi conduzido em câmaras climatizadas com temperaturas de 16, 19, 22, 25, 28, 31 mais ou menos 1ºC, UR de 70 mais ou menos 10 por cento e fotofase de 12horas. Efeito deletério da temperatura em O. thyestes foi obtido a 16ºC, na qual apenas 40 por cento das ninfas atingiram a fase adulta, e destes apenas 19 por cento não apresentaram deformacões morfológicas. O maior período de pré-oviposicão foi observado a 19ºC (17,8 dias). Os maiores valores para a fecundidade média total foram registrados a 25 e 28ºC, com 109,2 e 128,2 ovos/fêmea, respectivamente, e o menor a 19ºC, com 22,8 ovos/fêmea. A 22 e 31ºC as fêmeas viveram mais que os machos, sendo que a 19ºC a longevidade foi maior, independente do sexo. As baixas temperaturas influenciaram a reproducão e longevidade de O. thyestes sugerindo que esta espécie poderá ter melhor performance reprodutiva em temperaturas mais elevadas, como aquelas de regiões tropicais e ou subtropicais.