Abordagens distintas à Interação entre Anopheles spp. e Plasmodium spp: estabelecendo um modelo murino de laboratório, estudando o escape dos esporozoítos e a microbiota

Submitted by Nuzia Santos ([email protected]) on 2017-07-07T18:12:30Z No. of bitstreams: 1 Tese_BCM_AlessandradaSilvaOrfanó.pdf: 14649502 bytes, checksum: 8b8f69f21d98530d030988780ffb840b (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2017-07-07T18:22:19Z (GMT) No. of bitstreams: 1 Tese_BCM_AlessandradaSilvaOrfanó.pdf: 14649502 bytes, checksum: 8b8f69f21d98530d030988780ffb840b (MD5)Made available in DSpace on 2017-07-07T18:22:19Z (GMT). No. of bitstreams: 1 Tese_BCM_AlessandradaSilvaOrfanó.pdf: 14649502 bytes, checksum: 8b8f69f21d98530d030988780ffb840b (MD5) Previous issue date: 2016CAPESFundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brasil.Nas Américas, a região Amazônica é a área com o maior risco de transmissão de malária. No entanto, a falta de modelos experimentais adequados de anofelinos das Américas tem limitado o progresso para compreender a biologia de transmissão de malária nesta região. Entretanto, com colonização de vetores naturais da Amazônia, como o A. aquasalis, abriu pela primeira vez a possibilidade de estudar sua interação com o Plasmodium sp. No presente trabalho, questões básicas dessa interação foram investigadas tais como: 1) a susceptibilidade do A. aquasalis à diferentes espécies de Plasmodium sp. 2) o processo de escape dos esporozoítos dentro do vetor 3) a dinâmica microbiana do A. aquasalis em diferentes estágios de vida e fonte alimentar. Como resultados, (1) O A. aquasalis se mostrou refratário às infecções com P. falciparum (NF54 e 7G8), P. berghei e P. yoelii 17xnl, e altamente susceptível ao P. yoelii N67 tornando a utilização do P. yoelii N67/ A. aquasalis um bom modelo de estudo de laboratório. (2) Nesse trabalho foi descrita a microanatomia do escape dos esporozoítos de oocistos de quatro espécies de Plasmodium P. gallinaceum e P. berghei, e as duas espécies principais que causam a malária em humanos, P. vivax e P. falciparum. Verificou-se que os esporozoítos possuem mecanismos específicos de escape do oocisto. O P. berghei e o P. gallinaceum têm um mecanismo comum, na qual a parede do cisto se decompõe antes dos esporozoítos escaparem. Em contraste, os esporozoítos de P. vivax e P. falciparum possuem um mecanismo de escape ativo, através de propulsão polarizada. (3) A diversidade microbiana descrita e monstrou que as pupas possuem uma riqueza maior de fOTUs comparado aos outros grupos estudados. Foi visto que, a abundância das fOTUs de Enterobacteriacea e aumenta nos mosquitos pós infectados com P. vivax comparado aos outros grupos, sugerindo uma possível relação com o parasito. Acreditamos que com estes conhecimentos, poderemos abrir novas fronteiras para estudos de controle da malária focado no contexto epidemiológico brasileiro, como, por exemplo, produção de insetos geneticamente modificados potencialmente resistentes ao patógeno, ou mesmo indicar candidatos para estudo de vacinas de bloqueio de transmissão.In the Americas, the Amazon region is the área with highest risk of malária transmission. However, the lack of adequate experimental models of malaria vectors in the Americas to infect has limited the progress in understanding the biology of transmission in this region. Nevertheless, the colonization of natural vectors of the Amazon as the A. aquasalis has been achieved, and for the first opened the possibility of studying their interaction with the Plasmodium sp. In this present study, basic questions were investigated such as: 1) the susceptibility of A. aquasalis the different species of Plasmodium sp. 2) how does the escape process of sporozoites in the mosquito vector occurs using different species of Plasmodium and vectors 3) how does the microbial dynamics of A. aquasalis varies depending on the life stage and food source. As results: (1)The A. aquasalis proved refractory to infection with P. falciparum (NF54 and 7G8), P. berghei and P. yoelii 17xnl, and high susceptibility to P. yoelii N67. This shows that the P. yoelii N67 and A. aquasalis is a functional study model of malaria transmission outside the endemic areas. (2)We also investigated the microanatomy of escape of sporozoites from oocysts from four Plasmodium species: P. berghei and P. gallinaceum, and the two major species that cause malaria in humans, P. vivax and P. falciparum. It was found that the sporozoites have specific mechanisms for the oocyst escape. The P. berghei and P. gallinaceum have a common mechanism in which the oocyst wall decomposes before sporozoites escape. In contrast, the sporozoites of P. vivax and P. falciparum show an active escape mechanism from the oocyst through polarized propulsion. (3)We also saw that microbial diversity of pupae presented a greater wealth of fOTUs compared to the other groups. However, it was seen that the Enterobacteriaceae family increases in mosquitoes post infected with P. vivax. We believe that this knowledge can open new frontiers for malaria control studies focused on the epidemiological context, such as, production of genetically modified insect that are potentially resistant to the pathogen, or to indicate candidates for the study of transmission blocking vaccine

Avaliação do papel da microbiota intestinal do Aedes aegypti no desenvolvimento esporogônico do Plasmodium gallinaceum

Submitted by Nuzia Santos ([email protected]) on 2013-01-07T15:58:08Z No. of bitstreams: 1 Dissertação Alessandra Orfanó.pdf: 10376056 bytes, checksum: 69183dcae9646089fc702958e90fc51d (MD5)Made available in DSpace on 2013-01-07T15:58:08Z (GMT). No. of bitstreams: 1 Dissertação Alessandra Orfanó.pdf: 10376056 bytes, checksum: 69183dcae9646089fc702958e90fc51d (MD5)Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.A malária é responsável por cerca de 350 a 500 milhões de casos clínicos anuais, permanecendo como um dos maiores problemas de saúde mundial. Estudos sobre a influência da microbiota intestinal do vetor no desenvolvimento do ciclo de vida dos parasitos vêm sendo desenvolvidos. Em mosquitos, as pesquisas vêm mostrando que infecções por bactérias do intestino podem inibir o desenvolvimento esporogônico dos parasitas da malária. No presente trabalho, o papel da microbiota intestinal do Aedes aegytpi infectado com Plasmodium gallinaceum foi avaliado. Os insetos foram submetidos a cinco diferentes antibióticos (canamicina, carbenicilina, espectinomicina, gentamicina e tetraciclina). Somente canamicina e carbenicilina tiveram efeito sob a infecção com o parasito. Para esses dois antibióticos e o controle, técnicas de cultivo bacteriano de isolamento e sequenciamento do DNA ribossomal 16S foram feitas. Asaia bogorensis, Asaia Krungthepensis, Asaia siamensis, Bacillus licheniformis e Chryseobacterium meningosepticum foram identificadas para o grupo controle. No grupo tratado com carbenicilina foram identificadas Chryseobacterium meningosepticum e uma bactéria da família Microbacteriaceae, e para o grupo com canamicina Microbacterium lacticum. Este trabalho sugere que a interação entre espécies de Asaia e C. meningosepticum são relevantes na resistência/suscetibilidade do A. aegypti ao P. gallinaceum. Para observar o efeito do tratamento com os dois antibióticos que mostraram efeito na infecção na expressão de peptídeos antimicrobianos, os níveis de gambicina e defensina foram medidos utilizando-se a técnica de Real-time PCR. Os níveis de defensina se mostraram super-expressos quando os mosquitos foram submetidos aos tratamentos com canamicina e carbenicilina, enquanto gambicina se mostrou super-expressa nos mosquitos tratados com canamicina e sub-expressa naqueles com carbenicilina. A expressão desses dois AMPs também foi medida quando esses grupos de insetos se alimentaram com sangue e com sangue infectado. Nossos resultados mostraram que a expressão de defensina e gambicina no grupo tratado com carbenicilina alimentado com sangue apresentou um aumento de 24h para 36h, e uma queda no grupo controle alimentado com sangue. Insetos tratados com canamicina infectados aumentaram a expressão de defensina 24h e 36h após a alimentação, diminuídas nos tratados com carbenicilina. É observada uma inibição do mRNA de gambicina no grupo carbenicilina infectado sugerindo assim que a ativação desse peptídeo não ocorre somente pelo parasita e necessita da ação da microbiota. Estes resultados indicam que as bactérias associadas ao intestino do A. aegypti tem um papel importante na infecção e resposta imune ao parasito.Malaria is responsible for about 350 to 500 million clinical cases annually, remaining as one of the largest health problems worldwide. Studies on the influence of the vector gut microbiota in the life cycle of the parasites are being performed. In mosquitoes, studies have shown that infection with midgut microbiota may inhibit the sporogonic development of malaria parasites. Here, the role of intestinal microbiota of Aedes aegytpi infected with Plasmodium gallinaceum was assessed. The insects were subjected to five different antibiotics (kanamycin, carbenicillin, spectinomycin, gentamicin and tetracycline). Only kanamycin and carbenicillin had effect over the infection process. The response of treated groups and control was analyzed by: bacterial culture techniques of isolation and sequencing of 16S ribosomal DNA. Asaia bogorensis, Asaia krungthepensis, Asaia siamensis, Bacillus licheniformis and Chryseobacterium meningosepticum were identified for the control group. In the carbenicillin treated group Chryseobacterium meningosepticum and Microbacteriaceae family bacterium were identified. In the kanamycin treated group Lacticum microbacterium was found. This work suggests that the interaction between C. meningosepticum and Asaia species is relevant in the resistance/susceptibility balance in the A. aegypti/P. gallinaceum model. To observe the effect of treatment over the expression of antimicrobial peptides, gambicin and defensin levels were measured by Real-time PCR. Levels of defensin proved to be up-regulated when the mosquitoes were subjected to treatment with kanamycin and carbenicillin. Gambicin levels were be up-regulated in mosquitoes treated with kanamycin and down-regulated in those treated with carbenicillin. The expression of these two AMPs was also measured when these groups of insects were fed with blood and infected blood. Our results showed that the expression of defensin and gambicin in carbenicillin treated blood fed group increased between 24 to 36 hours, and decreased in the blood fed control group. Infected insects treated with kanamycin increased the expression of defensin between 24 to 36 hours after feeding, while it decreased in those treated with carbenicillin. An inhibition in the expression of gambicin in the infected group treated with carbenicillin was observed, suggesting that activation of these peptides does not occur only by the parasite, or microbial action, but by both. These results indicate that the microbiota associated with A. aegypti has an important role in infection and immune response to the parasite

Plasmodium yoelii nigeriensis (N67) Is a Robust Animal Model to Study Malaria Transmission by South American Anopheline Mosquitoes

Submitted by Nuzia Santos ([email protected]) on 2017-02-23T18:24:28Z No. of bitstreams: 1 ve_Orfano_Alessandra_ Plasmodium yoelii_CPqRR_2016.pdf: 6369959 bytes, checksum: d459b9a02443550b0378396bc879eedd (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2017-02-23T18:27:19Z (GMT) No. of bitstreams: 1 ve_Orfano_Alessandra_ Plasmodium yoelii_CPqRR_2016.pdf: 6369959 bytes, checksum: d459b9a02443550b0378396bc879eedd (MD5)Made available in DSpace on 2017-02-23T18:27:19Z (GMT). No. of bitstreams: 1 ve_Orfano_Alessandra_ Plasmodium yoelii_CPqRR_2016.pdf: 6369959 bytes, checksum: d459b9a02443550b0378396bc879eedd (MD5) Previous issue date: 2016National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Rockville, MA, United States of America/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Entomologia Médica. Belo Horizonte, MG, BrasilNational Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Rockville, MA, United States of America/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Entomologia Médica. Belo Horizonte, MG, Brasil/Fundação de Medicina Tropical Dr. Heitor Vieira Dourado. Manaus, AM, BrasilNational Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Rockville, MA, United States of America.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Entomologia Médica. Belo Horizonte, MG, Brasil/Fundação de Medicina Tropical Dr. Heitor Vieira Dourado. Manaus, AM, Brasil.National Institutes of Health. National Institute of Allergy and Infectious Diseases. Laboratory of Malaria and Vector Research. Rockville, MA, United States of America.Malaria is endemic in the American continent and the Amazonian rainforest is the region with the highest risk of transmission. However, the lack of suitable experimental models to infect malaria vectors from the Americas has limited the progress to understand the biology of transmission in this region. Anopheles aquasalis, a major vector in coastal areas of South America, was found to be highly refractory to infection with two strains of Plasmodium falciparum (NF54 and 7G8) and with Plasmodium berghei (mouse malaria), even when the microbiota was eliminated with antibiotics and oxidative stress was reduced with uric acid. In contrast, An. aquasalis females treated with antibiotics and uric acid are susceptible to infection with a second murine parasite, Plasmodium yoelii nigeriensis N67 (PyN67). Anopheles albimanus, one of the main malaria vectors in Central America, Southern Mexico and the Caribbean, was more susceptible to infection with PyN67 than An. aquasalis, even in the absence of any pre-treatment, but was still less susceptible than Anopheles stephensi. Disruption of the complement-like system in An. albimanus significantly enhanced PyN67 infection, indicating that the mosquito immune system is mounting effective antiplasmodial responses. PyN67 has the ability to infect a broad range of anophelines and is an excellent model to study malaria transmission by South American vectors

Schistosoma mansoni in susceptible and resistant snail strains Biomphalaria tenagophila: in vivo tissue response and in vitro hemocyte interactions

Submitted by Nuzia Santos ([email protected]) on 2014-06-06T16:44:11Z No. of bitstreams: 1 Schistosoma mansoni in susceptible and resistant snail strains Biomphalaria tenagophila.pdf: 2901781 bytes, checksum: 96c4b0f3ef87d7b200bb246bbdd16ad7 (MD5)Made available in DSpace on 2014-06-06T16:44:12Z (GMT). No. of bitstreams: 1 Schistosoma mansoni in susceptible and resistant snail strains Biomphalaria tenagophila.pdf: 2901781 bytes, checksum: 96c4b0f3ef87d7b200bb246bbdd16ad7 (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Entomologia Médica. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Esquistossomose. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Entomologia Médica. Belo Horizonte, MG, BrasilUniversidade Federal de Sergipe. Laboratório de Entomologia e Parasitologia Tropical. Aracajú, SE, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Entomologia Médica. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Esquistossomose. Belo Horizonte, MG, BrasilSchistosomiasis is a parasitic disease that is highly prevalent, especially in developing countries. Biomphalaria tenagophila is an important invertebrate host of Schistosoma mansoni in Brazil, with some strains (e.g. Cabo Frio) being highly susceptible to the parasite, whereas others (e.g. Taim) are completely resistant to infection. Therefore, B. tenagophila is an important research model for studying immune defense mechanisms against S. mansoni. The internal defense system (IDS) of the snail comprises hemocytes and hemolymph factors acting together to recognize self from non-self molecular patterns to eliminate the threat of infection. We performed experiments to understand the cellular defenses related to the resistance and/or susceptibility of B. tenagophila to S. mansoni. During the early stages of infection, fibrous host cells of both snail strains were arranged as a thin layer surrounding the sporocysts. However, at later stages of infection, the cellular reactions in resistant snails were increasingly more intense, with thicker layers surrounding the parasites, in contrast to susceptible strains. All parasites were damaged or destroyed inside resistant snails after 10 h of infection. By contrast, parasites inside susceptible snails appeared to be morphologically healthy. We also performed experiments using isolated hemocytes from the two strains interacting with sporocysts. Hemocyte attachment started as early as 1 h after initial infection in both strains, but the killing of sporocysts was exclusive to hemocytes from the resistant strain and was time course dependent. The resistant strain was able to kill all sporocysts. In conclusion, our study revealed important aspects of the initial process of infection related to immune defense responses of strains of B. tenagophila that were resistant to S. mansoni compared with strains that were susceptible. Such information is relevant for the survival or death of the parasites and so is important in the development of control measures against this parasite

Schistosoma mansoni in susceptible and resistant snail strains Biomphalaria tenagophila: in vivo tissue response and in vitro hemocyte interactions.

Schistosomiasis is a parasitic disease that is highly prevalent, especially in developing countries. Biomphalaria tenagophila is an important invertebrate host of Schistosoma mansoni in Brazil, with some strains (e.g. Cabo Frio) being highly susceptible to the parasite, whereas others (e.g. Taim) are completely resistant to infection. Therefore, B. tenagophila is an important research model for studying immune defense mechanisms against S. mansoni. The internal defense system (IDS) of the snail comprises hemocytes and hemolymph factors acting together to recognize self from non-self molecular patterns to eliminate the threat of infection. We performed experiments to understand the cellular defenses related to the resistance and/or susceptibility of B. tenagophila to S. mansoni. During the early stages of infection, fibrous host cells of both snail strains were arranged as a thin layer surrounding the sporocysts. However, at later stages of infection, the cellular reactions in resistant snails were increasingly more intense, with thicker layers surrounding the parasites, in contrast to susceptible strains. All parasites were damaged or destroyed inside resistant snails after 10 h of infection. By contrast, parasites inside susceptible snails appeared to be morphologically healthy. We also performed experiments using isolated hemocytes from the two strains interacting with sporocysts. Hemocyte attachment started as early as 1 h after initial infection in both strains, but the killing of sporocysts was exclusive to hemocytes from the resistant strain and was time course dependent. The resistant strain was able to kill all sporocysts. In conclusion, our study revealed important aspects of the initial process of infection related to immune defense responses of strains of B. tenagophila that were resistant to S. mansoni compared with strains that were susceptible. Such information is relevant for the survival or death of the parasites and so is important in the development of control measures against this parasite

Bacterial diversity of wild-caught Lutzomyia longipalpis (a vector of zoonotic visceral leishmaniasis in Brazil) under distinct physiological conditions by metagenomics analysis

Abstract Background The leishmaniases are a group of diseases caused by protozoans of the genus Leishmania, which are transmitted by the bite of phlebotomine sand flies. In the New World, Lutzomyia longipalpis is the most important vector of visceral leishmaniasis and is a proven vector for Leishmania infantum chagasi in Brazil. During development within the vector, Leishmania can interact with a variety of microorganisms such as fungi and bacteria. The presence of bacteria in the midgut of sand flies can influence the development and survival of the parasite. Results The bacteria-targeted metagenomic analysis revealed different community compositions between the distinct physiological stages of those tested. The amplicon-oriented metagenomic profiling revealed 64 bacterial genera and 46 families. By crossing the taxa indices from each experimental condition a core composed of 6 genera was identified (Enterobacter, Serratia, Stenotrophomonas, Enhydrobacter, Pseudomonas and Chryseobacterium). Conclusions The observed dynamic nature of the bacterial community expands the knowledge pertaining to the tripartite host-microbiota-pathogen interactions. Further studies addressing how laboratory and field collected communities differ are critical to successfully develop control strategies based on bacterial symbionts and paratransgenesis, as already tested in other arthropod vectors

Interaction between hemocytes from the <i>B. tenagophila</i> Taim strain and <i>S. mansoni</i> sporocysts.

<p>(<b>A</b>) After 1 h of interaction, showing a sporocyst with normal morphology and no attached hemocyte. Various germinal cells are present (white arrows). Magnification 40X. (<b>B,C</b>) After 1 h of interaction, showing sporocysts with a few attached hemocytes (arrowheads). ‘h’ indicates free hemocytes. Magnification 40X. (<b>D,E</b>) After 5 h of interaction, showing porocysts with several attached (arrowheads) and free h. There is also a great quantity of cellular debris (arrows). Magnification 40X. (<b>F,G</b>) After 10 h of interaction, several attached hemocytes (arrowheads) are seen on the sporocyst. The parasite morphology is showing disturbance of the internal organelles (asterisks). Cellular debris can also be seen (arrows). Magnification 40X. (<b>H</b>) After 10 h of interaction detailing the anterior region of the sporocyst. There is a hemocyte adhered to the surface (arrowheads) and the disarrangement of the syncytial layer on the surface of the parasite. Magnification 63X. (<b>I</b>,<b>J</b>) After 10 h of interaction. sporocysts with a few attached hemocytes (arrowheads) can be seen, as well as a large amount of cellular debris around the parasite (arrows). The sporocyst in (J) is completely destroyed and is now in several parts (asterisks). Magnification 40X.</p

Histological sections showing distinct views of <i>S. mansoni</i> sporocysts.

<p>(<b>A</b>) Longitudinal section of the sporocyst medial plane showing the main internal organelles: germinal cells (gc), neural mass (nm), terebratorium (t) and several vesicles (v). The two dotted lines delimit the anterior (A) and posterior (P) regions of the parasite. (<b>B</b>) Transversal section of the anterior region of the parasite showing the gc. (<b>C</b>) Transversal section of the posterior region of the parasite, showing several types of v. (<b>D</b>) Oblique section of the anterior region of the parasite, showing the t, gc and nm. (<b>E</b>) Oblique sequential section of the anterior region of the parasite showing the large area of the t, as well as the apical glands (ag) and gc. Magnification 40X.</p

Histological sections of uninfected host <i>B. tenagophila</i> (control).

<p>(<b>A</b>) General view of the heat-foot region of <i>B. tenagophila</i>. Observe the mouth (Mo) near the anterior region (A), posterior region (P) and the mantle (m) that covers the heat-foot region (Cr). Magnification 2.5X. (<b>B</b>) Details of the region between the cr and the m. The lateral m region shows several types of vesicle (v) with distinct stain densities. Magnification 40X. (<b>C</b>) Details of the heat-foot region. Observe the ciliar cylindrical epithelium (Ep) and just below it the dense connective tissue (Ct) that is supported by the muscular connective tissue (Mt) comprising a network of muscle fibers (white arrow). A large crypt is seen that is similar to a large epithelial fold (large arrow). Small arrows indicate the cilia. Magnification 100X. (<b>D</b>) Detail of the heat-foot region, showing the Ep with several crypts (arrows) and moat-like invaginations. Key: Ct, connective tissue; Mt, muscle tissue; arrows, muscle fibers; arrowheads, crypts. Magnification: 40X. (<b>E</b>) Large magnification showing details of one crypt (large arrow) deep into the Ep. Details of the cilia on the epithelium are shown (arrowheads) Magnification: 100X. (<b>F</b>) Posterior area of the heat-foot region. Notice the presence of numerous v in the Ct supported by Mt. The Ep shows several crypts (arrowheads) Magnification 40X.</p