Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-03-19T20:01:22Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ArbovirusMorumbiPhlebovirus.pdf: 62905996 bytes, checksum: a5b43d27d591f2456824a2b93e0fe46c (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-03-21T13:33:10Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ArbovirusMorumbiPhlebovirus.pdf: 62905996 bytes, checksum: a5b43d27d591f2456824a2b93e0fe46c (MD5) / Made available in DSpace on 2013-03-21T13:33:10Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ArbovirusMorumbiPhlebovirus.pdf: 62905996 bytes, checksum: a5b43d27d591f2456824a2b93e0fe46c (MD5) Previous issue date: 2000 / O vírus Morumbi é membro do sorogrupo Phlebotomus fever (família Bunyavírídae: gênero Phlebovírus) nativo da Região Amazônica. Seu vetor é desconhecido, mas supõem-se ser transmitido por flebotomíneos. Foi isolado em 1988 de ser humano apresentando quadro febril agudo. Este arbovírus, quando inoculado em camundongo por via cerebral, demonstrou viscerotropismo, induzindo inclusive lesões no fígado do animal inoculado. Com os objetivos de: i) estabelecer as características anátomo-patológicas e imuno-histoquímicas em fígado de camundongos albinos Swíss recém-nascidos experimentalmente infectados pelo vírus Morumbi; ii) verificar se o vírus apresenta hepatotropismo diferenciado na dependência de inoculação pelas vias cerebral, peritoneal ou subcutânea; iii) caracterizar detalhadamente os padrões anátomo-patológicos sequenciais no fígado; iv) demonstrar a localização do antígeno viral no tecido hepático ao longo da infecção experimental; v) estudar possíveis inter-relações entre os achados anátomo-patológicos e os imuno-histoquímicos. Foram estudados experimentalmente 71 camundongos Swíss recém-nascidos (dois e três dias), distribuídos ao final do experimento como segue: 21 animais inoculados por via intracerebral (IC), 21 por via intraperitoneal (IP) e 29 animais inoculados por via subcutânea (SC). Utilizou-se a dose infectante 5,0DL 50 /0,02ml de suspensão de vírus. Outros trinta, animais que não receberam inóculos, foram utilizados como grupo controle. Subgrupos de oito animais (seis inoculados e dois do grupo controle) foram sacrificados diariamente a intervalos de 24 em 24 horas, até 96 horas para os grupos IC e IP e até 120 horas para o grupo SC. Fragmentos de fígado de todos os animais foram fixados em solução de formalina neutra a 10%, incluídos em parafina, de onde foram obtidos cortes de 5 mm que foram corados pela técnica de hematoxilina-eosina para análise morfológica e, cortes adicionais, foram submetidos à técnica de imuno-histoquímica (Sistema Envision, DAKO, USA), utilizando a fosfatase alcalina e soro hiperimune do vírus Morumbi preparado em camundongos jovens, para detecção de antígeno viral. Foram estudados seis parâmetros de lesão em áreas portais e nove outros nos lóbulos, que foram semiquantificados numa escala que variou de zero (0) a três cruzes (+++), onde zero significou ausência de lesão e três cruzes lesão intensa. À microscopia óptica, ficou evidente que o vírus Morumbi inoculado em camundongos por três diferentes vias induz lesões em áreas portais e lobulares, caracterizando uma hepatite aguda com presença de corpúsculos acidófilos, semelhantes aos corpúsculos de Councilman -Rocha Lima, de distribuição irregular nos lóbulos, cujo aparecimento foi observado 24 horas pós-inoculação (p.i.) e atingiu o máximo de intensidade às 72 horas p.i. em animais inoculados por via IP. O exame imuno-histoquímico mostrou presença leve de antígeno viral a partir de 24 horas p.i. no grupo IC e a partir de 48 horas p.i. nos grupos IP e SC, havendo certo paralelismo em relação a intensidade de lesão morfológica, tendo- se observado o máximo de detecção de antígeno viral em animais inoculados por via IP e sacrificados às 72 horas p.i. A distribuição geral de antígeno foi observada especificamente nos lóbulos hepáticos, no citoplasma de hepatócitos íntegros e necrosados e no interior de células de Kupffer, não havendo preferência por nenhuma das três zonas do lóbulo. Concluiu-se que: i) o modelo de infecção experimental em camundongos foi excelente para o estudo das lesões causadas pelo vírus Morumbi, podendo ser selecionada a via IP como referencial; ii) em todas as vias utilizadas (IP, IC e SC) se confirmou a infecção pelo vírus Morumbi com marcante detecção de seu antígeno, no tecido hepático de camundongos Swiss; iii) a presença de antígeno do vírus Morumbi no fígado desses camundongos associou-se ao aparecimento de hepatite aguda, com necrose focal; iv)hepatite intensa pôde ser observada em fígado de camundongos sacrificados 72 h p.i. com o vírus Morumbi por via IP, o que não foi verificado com as outras duas vias; v) a hepatite aguda mostrou-se limitada, neste experimento, tendendo a desaparecer na maioria dos camundongos inoculados, com avançar das horas; vi) colestase não alteração freqüente na hepatite experimental pelo vírus Morumbi, quando inoculada por via IC, IP e SC; vii) o antígeno do vírus Morumbi teve predominância pela localização intracitoplasmática, padrão granular, nos hepatócitos e células de Kupffer; viii) antígeno viral foi detectado em fragmento hepático de animais experimentalmente inoculados com o vírus Morumbi, a partir das 24 horas via IC e a partir de 48 horas nas vias IP e SC. / Morumbi virus is a member of Phlebotomus Fever serogroup (Bunyaviridae family, Phlebovirus genus), native from the Brazilian Amazon region. It'svector is unknown, but is supposed to be, transmitted by phlebotomine sandflies. It was isolated in 1988 from a human presenting an acute febrile illness. When inoculated in the brain of Newborn Swiss mice, this arbovirus showed visceral tropism, including liver damage. In order to establish the anatomopathological and immunohistochemical characteristics in the liver of albino Swiss suckling mice experimentally infected with Morumbi virus; to assess the differences of tropism of the virus in the liver following the intra cerebral, intra peritoneal or subcutaneous routes of inoculation; to detail the sequence of anatomical and pathological events in the liver; to demonstrate the location of the viral antigen in the hepatic tissue during the experimental assay; and to study the possible relationship between the anatomopathological and immunohistochemical findings, an experimental study was conducted with 71 Newborn Swiss mice (two or three days of age), distributed as follows: 21 received intra cerebral inoculation (IC), 21 received intraperitoneal inoculation (IP) and 29 received subcutaneous inoculation (SC). 0.02ml of virus suspension was used as the infectious dose. The control was comprised of 30 mice that were not inoculated. Subgroups of 8 mice (6 inoculated, 2 controls) were euthanized daily, at intervals of 24 - 96 hours in the IC and IP groups, and until 120 hours in the SC group. Liver specimens from all mice were fixed in a 10% neutral formalin solution, then embedded in paraffin wax to obtain 5f.lm sections that were stained with haematoxylin/eosin for morphological analysis. Immunohistochemical technique (Envision System, DAKO, USA) was employed in additional sections, using alkaline phosphatase and hyper-immune anti-Morumbi virus serum prepared into suckling mice, to detect the viral antigen. 6 patterns of portal lesion and 9 patterns of lobule lesion were studied in a scale from zero (0) to three (+++), where zero represented absence of lesion and three represented severe lesion. Light microscopy examination revealed that Morumbi virus was able to produce hepatic lesions in the portal and lobular areas when inoculated in mice in three different routes, raising an acute hepatitis, in which bodies similar to Councilman - Rocha Lima bodies were observed, irregularly distributed in the lobules. The appearance of those bodies occurred 24 hours post-inoculation (pi), with a peak at 72 hours pi in miceis inoculated. The immunohistochemical technique showed mild presence of viral antigen from 24 hours after inoculation in IC group and from 48 hours after in IP and SC groups, showing a certain parallelism between the detection of viral antigens and the morphological lesions. Maximum detection of the viral antigen was observed in IP rout, specially those mice euthanized at 72 hours pi. General distribution of the antigen was concentrated in the hepatic lobules, in the cytoplasm of normal and necrotic hepatocytes, as well as inside the Kupffer cells, without any preference for the three lobule areas. The following conclusions are made: i) experimentally infected mice model was excellent to study the Morumbi virus-induced lesions, with preference to IP rout; ii) in all inoculated routes (IP, IC and SC) there were evidence of Morumbi virus infection, with a remarkable detection of its antigen in the hepatic tissue of Swiss mice; iii) Morumbi virus antigen detected in the liver of Swiss mice was associated with acute hepatitis and with focal necrosis; iv) an intense acute hepatitis occurred in the liver of euthanized mice 72 hours pi with Morumbi virus by the intraperitoneal route but was not observed in the other two used routes; v) in this experiment, acute hepatitis was limited, showing a clear tendency to disappear in the follow up in the majority of inoculated animals; vi) cholestasis was not very often observed in the experimental hepatitis due to Morumbi virus; vii) Morumbi virus antigen was detected predominantly in the cytoplasma and was exhibiting a granular pattern in hepatocytes and Kupffer cells; viii) Morumbi viral antigen was detected as early as 24 hours in hepatic tissue in IC route and 48 hours after IP and SC routes.

The main aim of this thesis was to prove the hypothesis that the intake of blood with specific antibodies against sand fly saliva affects sand fly fecundity and mortality. Phlebotomus argentipes and Mesocricetus auratus were used for most experiments. ELISA revealed high levels of specific antibodies in repeatedly exposed hosts. However, a five-day study of mortality and fecundity of bloodfed females demonstrated that feeding on repeatedly bitten hamsters has no effect on number of eggs or survival of females. Salivary antigens of P. argentipes recognized by sera of repeatedly bitten hamsters were characterized by immunoblotting and mass spectrometry. Immunoblotting showed that antibodies in the hamster sera recognize salivary proteins with molecular weight from 25 to 60 kDa. Mass spectrometry revealed that the antigens correspond to D7, apyrases, antigen 5-related proteins and yellow-related proteins. In addition, Phlebotomus females were fed through a chicken membrane on rabbit blood with high concentrations of histamine, serotonin or prostaglandin E2 to find out if they influence fecundity or mortality of sandfly females. While the approximate number of eggs layed by one female did not significantly differ from controls in any experimental group studied, the total mortality of females was lower...

The Trypanosoma theileri group are blood parasites of ungulates. Confirmed vectors of these protists are horseflies or sheep keds for T. melophagium, but these trypanosomes were also detected in deer keds, mosquitoes and phlebotomus. We targeted on the investigation of possibility mosquitoes acting as vectors and additionally we targeted on the prevalence of T. theileri in mosquitoes at selected locations in the Czech Republic due to previous positive detection there. The measured prevalence has reached 6,4 % in mosquitoes, 4 % in deer keds and 16,7 % in sheep keds. The aim of experimental infections was to discover a potential of mosquitoes and phlebotomus acting as vectors of the T. theileri. Phlebotomus perniciosus and three species of mosquitoes Culex pipiens molestus, Culex pipiens quinquefasciatus and Aedes vexans were used for those experiments. Furthermore, isolates of T. theileri and T. melophagium from different sources were used. The divergence among these isolates and among used species of insect was found. The best results were achieved with Ae. aegypti and isolates from mosquitoes, where prevalence had reached 90,8 %. The infected mosquitoes were used for prediuresis research which had shown infection forms of the trypanosomes in the urine liquid. The phylogenetic analyse of T....

Sand flies (Diptera, Phlebotominae) are small biting insects and vectors of Leishmania spp. which cause medically and veterinary important disease - leishmaniasis. During the piercing of the host skin, sand fly females inject saliva to facilitate the blood feeding. The sand fly saliva is composed of many bioactive molecules which were shown to possess anti-inflammatory and anti-haemostatic functions. The saliva affects host's immunity in the bite site and consequently enhances the survival and development of transmitted pathogens. Most of the studies focus on salivary proteins and enzymes of sand flies belonging to Phlebotomus and Lutzomyia genera, while salivary proteins from sand flies of the third genus Sergentomyia were neglected so far. In this thesis we focused on comparison of salivary proteins from two Phlebotomus species, namely Phlebotomus perniciosus and Phlebotomus orientalis, and Sergentomyia schwetzi. These sand fly species differ not only by the ecology and geographical distribution but also by host preferences. Both Phlebotomus species prefer large or medium-size mammals as the bloodmeal source, particularly rabbits, hares and dogs for P. perniciosus and cattle, goats, sheep and humans for P. orientalis. Contrarily, Sergentomyia sand flies are known for preferred feeding on reptiles...