Made available in DSpace on 2016-04-28T20:16:24Z (GMT). No. of bitstreams: 1 2007- Katia Roberta Fernandes.pdf: 2143619 bytes, checksum: 6ff7127f4642317cf1f53d7aceb93f1f (MD5) Previous issue date: 2007-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The aim of the present study was to evaluate the natural and experimental infection of B. equi in nymphs and adult Boophilus microplus using optical microscopy and molecular analysis. The experimental infection was observed in nymphs, males and females of B. microplus fed on equines chronically infected with B. equi and in non fed larvae and eggs. For the experiments two equines, of not defined breed, proven to be infected with B. equi were used. The animals were infested with B. microplus larvae of generation F4 obtained from a laboratory maintained population, known to be free of Babesia spp. infection. Daily collections of ticks were realized using as starting point the ecdisys to nymph state. After the collection the hemolymph was examined and the intestine and salivary glands were dissected, smashed on glass slides for microscopy, methanol treated and stained with Giemsa. There were dissected 860 specimens (432 nymphs, 280 females and 148 males). The dissected salivary glands were divided into two parts. The first one was smashed on microscopy glass slides, stained and examined by optical microscopy for morphologic analyses. From the second part was realized DNA extraction and PCR with specific primers for the 18S rRNA. On optical microscopy of nymph hemolymph was possible to be observed the presence of kinetes with claviform aspect characteristic for the genus Babesia. In the salivary glands of B. microplus nymphs the morphology and the sequence of developmental stages of B. equi were observed initiating on day 4 after ecdysis, being possible to see in acinary cells the formation of sporoblasts and ovoid sporozoites measuring 1.5 μm of diameter and elongated sporozoites with 3.8 to 5.2μm of length and 0.8 to 1.5 μm of width. The PCR confirmed the presence of B. equi in DNA samples extracted from salivary glands of nymphs, male and female ticks as well as in larvae and eggs. To evaluate the natural infection were collected nymphs and adult B. microplus from two equines naturally infested by these ticks and naturally infected with B. equi. These horses were originated from Seropedica city in the state of Rio de Janeiro. There were dissected 324 specimens (145 nymphs, 138 females and 41 males). The proceedings with the salivary glands were identical to the previously described for the experimental infection. Of the salivary glands submitted to PCR, 70% showed to be infected with B. equi. Microscopical analysis of the salivary glands revealed the presence of sporoblast stages and the development of elongated sporozoites in acinary glands. The morphologic, morphometric and molecular analysis confirmed the experimental and natural infection of the salivary glands of nymphs and adult B. microplus with B. equi. The results of the present study show the ability of B. equi to develop in this tick species. The detection of B. equi DNA in eggs and larvae also suggests the possibility of transovarian transmission in B. microplus. The results allow to consider the tick B. microplus as a potential biologic vector of B. equi in horses from the studied region. / Este trabalho teve como objetivo avaliar a infec??o natural e experimental de Babesia equi em ninfas e adultos de Boophilus microplus por meio de microscopia ?ptica e an?lise molecular. A infec??o experimental foi observada a partir de ninfas, machos, f?meas, ovos e larvas n?o alimentadas de B. microplus alimentados em equinos com infec??o cr?nica por B. equi. Para a realiza??o do experimento foram utilizados dois eq?inos, mesti?os, com infec??o por B. equi. Os animais foram infestados com larvas de B. microplus de gera??o F4 obtidas de col?nia mantida em laborat?rio e livres de infec??o por Babesia spp. A partir da ecdise para ninfa foram realizadas coletas di?rias. Ap?s a coleta foram realizados os exames de hemolinfa e extra??o do intestino e das gl?ndulas salivares os quais foram macerados em l?minas de vidro para microscopia, fixados em metanol e corados com Giemsa. As gl?ndulas salivares dissecadas foram divididas em duas partes. A primeira parte foi macerada em l?minas de vidro para microscopia, corada com corante Giemsa e observada em microsc?pio ?tico para an?lise morfol?gica. A segunda parte foi realizada a extra??o de DNA, sendo submetida a PCR com primers especif?cos para o gene 18S rRNA para B. equi. Foram dissecados 860 esp?cimes (ninfas= 432; f?meas= 280 e machos= 148). Na microscopia ?ptica foi poss?vel observar nas hemolinfas das ninfas a presen?a de cinetos com aspecto claviforme t?picos do g?nero Babesia. Nas gl?ndulas salivares, a morfologia e a seq??ncia dos est?gios de desenvolvimento de B. equi das ninfa s de B. microplus, foram observadas a partir do 4? dia ap?s ecdise, sendo poss?vel observar nos ?cinos celulares a forma??o de esporoblastos e de esporozo?tas ov?ides medindo 1,5 μm de di?metro e alongados medindo 3,8 a 5,2μm de comprimento e 0,8 a 1,5μm de largura. A rea??o em cadeia de polimerase (PCR) confirmou a presen?a de B. equi em DNA de gl?ndulas salivares extra?das de ninfas, machos, f?meas, assim como dos ovos e larva. A infec??o natural foi observada a partir de ninfas e adultos de B. microplus coletados de dois eq?inos naturalmente infestados e comprovadamente infectados por B. equi, procedentes do munic?pio de Serop?dica, Rio de Janeiro. Foram dissecados 324 esp?cimes (ninfas= 145, f?meas= 138 e machos= 41). O processamento das gl?ndulas salivares dissecadas foi semelhante ao descrito para infec??o experimental. Das gl?ndulas salivares submetidas a PCR, 70% apresentaram resultados positivos para B. equi. As an?lises por microscopia ?ptica das gl?ndulas salivares das ninfas e dos adultos revelaram a presen?a nos ?cinos celulares os est?gios de esporoblastos e o desenvolvimento de esporozo?tas alongados. As an?lises morfol?gicas, morfom?tricas e moleculares confirmaram a infec??o experimental e natural das gl?ndulas salivares de ninfas e adultos de B. microplus por B. equi. Os resultados deste estudo demonstram a capacidade de multiplica??o de B. equi em gl?ndulas salivares de ninfas e adultos de B. microplus. A detec??o de DNA de B. equi em ovos e larvas de B. microplus tamb?m sugere a possibilidade da transmiss?o transovariana nesta esp?cie de carrapato. Estes resultados sugerem que o carrapato B. microplus ? vetor biol?gico de B. equi na regi?o estudada.

Made available in DSpace on 2014-12-17T15:32:33Z (GMT). No. of bitstreams: 1 MarciaCCMiguel_tese.pdf: 1534160 bytes, checksum: 1a3ec415bfaf10c088ec1ae5f6224189 (MD5) Previous issue date: 2005-05-27 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Pleomorphic adenoma and adenoid cystic carcinoma (ACC) consist benign and malignant neoplasm from salivary gland, respectively. These neoplasms share some characteristics, such as cellular origin and considerable production of extracellular matrix, however, with distinct biological behavior. The aim of the present study was to compare the expression of D2E1, D3E1 e D5E1 integrins in pleomorphic adenoma from minor and major salivary glands and ACCs. Furthermore, it was investigated possible differences in the expression of these integrins according to histological subtypes of ACC. Fourteen cases of pleomorphic adenoma from major salivary gland, fourteen cases from minor salivary gland and ten cases of ACC were selected. It was taken into consideration the presence or absence, localization and intensity of integrin immunoexpression. The cases of pleomorphic adenoma were grouped in order to compare the expression between the distinct neoplasms. It was observed a highly significant difference (p<0,0001) in relation to D2E1 integrin between the neoplasms since pleomorphic adenoma showed a pronounced immunostaining. It was not possible to perform statistical tests considering the D2E1 integrin expression; nevertheless, it could be observed a tendency of higher staining in pleomorphic adenoma. For comparative reasons the cases ACCs were divided in two groups: solid and tubular/cribriform. It was not detected significant differences in regard to D2E1 integrin; and statistical analysis could not be realized in relation to D3E1 and D5E integrin expression. However, it was also verified a tendency of absence or reduced expression in the solid subtype. It can be concluded that the reduced D2E1 integrin expression observed in CACs may be related to a lesser degree of cell differentiation in this neoplasm and the reduced D5E1 integrin expression can be associated with aggressive biological behavior. Moreover, the absence and/or reduced expression of the studied integrins in solid ACC suggests a role in pathogenesis and more aggressive biological behavior of this histological subtype / O adenoma pleom?rfico e o carcinoma aden?ide c?stico (CAC) representam neoplasias de gl?ndula salivar benigna e maligna, respectivamente, as quais compartilham algumas caracter?sticas como a mesma origem celular e uma marcante presen?a de matriz extracelular, apresentando, por?m, comportamentos biol?gicos distintos. O prop?sito desta pesquisa consistiu em comparar a express?o das integrinas D2E1, D3E1 e D5E1 em adenomas pleom?rficos de gl?ndula salivar menor e maior e CACs. Al?m disso, procurou investigar se havia diferen?as na express?o destas integrinas entre os subtipos histopatol?gicos do CAC. Foram selecionados 14 casos de adenoma pleom?rfico de gl?ndula salivar maior, 14 casos de gl?ndula salivar menor e 10 casos de CACs. Analisou-se a presen?a ou aus?ncia, localiza??o e intensidade de marca??o das integrinas. Os dois grupos de adenomas pleom?rficos foram reunidos em um s? para fazer a compara??o entre os dois tumores. Verificou-se que houve diferen?a estat?stica altamente significativa (p<0,0001) para a integrina D2E1 entre os dois tumores, apresentando o adenoma pleom?rfico, uma marca??o mais intensa para esta integrina. Em rela??o ? integrina D5E1 n?o foi poss?vel a realiza??o de testes estat?sticos, ficando patente, por?m, que houve uma tend?ncia da referida integrina ser mais intensamente expressa no adenoma pleom?rfico. Para an?lise comparativa, os CACs foram subdivididos em 2 grupos: s?lido e tubular/cribriforme. Para a integrina D2E1 observou-se que n?o houve diferen?a estatisticamente significativa e em rela??o ? D3E1 e D5E1 n?o foi poss?vel a realiza??o do teste estat?stico; no entanto, tamb?m foi verificada uma clara tend?ncia para os casos do subtipo s?lido apresentarem express?o ausente ou reduzida das integrinas avaliadas. Concluiu-se que a reduzida express?o da integrina D2E1 observada nos CACs, pode estar relacionada com a menor diferencia??o das c?lulas deste tumor e ? poss?vel que a reduzida express?o da D5E1, possa estar implicada em seu comportamento mais agressivo. Al?m disso, sugere-se que a aus?ncia e/ou redu??o da express?o das integrinas pesquisadas nos casos do subtipo s?lido, pode desempenhar algum papel na patog?nese e no comportamento biol?gico mais agressivo deste subtipo tumoral