Orientador: Antonio Carlos Paes / Resumo: A Ehrlichiose Monocítica Canina (EMC), causada por Ehrlichia canis, destaca-se dentre as hemoparasitoses de cães. A EMC causa doença multissistêmica e pode ser classificada em fase aguda, subclínica ou crônica. A fase crônica caracteriza-se por hipoplasia/aplasia medular e muitos cães vêm a óbito por falência medular, embora as causas da supressão medular ainda sejam indefinidas. Neste estudo, foi investigado se a fase crônica resulta de alterações medulares iniciadas na fase aguda. Em particular, verificar se, durante a fase aguda, células precursoras podem ser parasitadas por E. canis e se processos imunomediados podem ser responsáveis por alterações hematológicas durante o curso da doença. Dezoito cães com EMC na fase aguda foram submetidos a exames hematológicos (hemograma, bioquímica sérica e mielograma) e sorológicos. A presença de E. canis foi confirmada mediante PCR e cultivo celular. A citologia de medula óssea revelou alterações principalmente na série eritróide que comumente estão associadas a doenças imunomediadas, inclusive em animais não anêmicos. Também confirmou-se a presença de E. canis na medula óssea de 11/18 cães e certa tendência entre a positividade das amostras medulares e a série eritróide, embora sem significância estatística. Os títulos sorológicos de 2560 em 9/18 e 10240 nos demais confirmou a resposta humoral exacerbada descrita pela literatura, contudo também sem relação significativa com a presença de E. canis na medula e algumas variáveis medula... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Submitted by Walter Bertequini Nagata null (walter.bn@hotmail.com) on 2017-01-04T19:05:41Z No. of bitstreams: 1 DISSERTAÇÃO DE MESTRADO - Walter Bertequini Nagata.pdf: 726100 bytes, checksum: 44016cb83ccb52d2853fbd5e43a37790 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-01-06T13:34:43Z (GMT) No. of bitstreams: 1 nagata_wb_me_araca.pdf: 726100 bytes, checksum: 44016cb83ccb52d2853fbd5e43a37790 (MD5) / Made available in DSpace on 2017-01-06T13:34:43Z (GMT). No. of bitstreams: 1 nagata_wb_me_araca.pdf: 726100 bytes, checksum: 44016cb83ccb52d2853fbd5e43a37790 (MD5) Previous issue date: 2017-01-03 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do presente estudo foi investigar, por métodos estatísticos, a ocorrência de reações cruzadas entre Leishmania spp. e Ehrlichia spp. com o uso de técnicas sorológicas e moleculares. Um total de 100 amostras sanguíneas de cães foram colhidas e processadas por meio do ELISA (Ensaio Imunoenzimático Indireto) e PCR (Reação em Cadeia da Polimerase). A análise estatística consistiu nos testes de McNemar e Coeficiente de concordância Kappa. As estatísticas foram consideradas significativas quando p<0,05. A sensibilidade e especificidade dos testes foram determinadas pela curva ROC (Receiver Operator Characteristic), considerando o PCR como teste padrão ouro. A partir das análises pode-se verificar que 48,0% dos animais foram reativos na ELISA e 58,0% foram positivos na PCR, no caso da Leishmania spp. Para Ehrlichia spp., a ocorrência de anticorpos pelo ELISA foi de 54,0% e, pela PCR, 48,0% dos cães foram positivos. Nota-se, também, que 37,0% dos animais foram positivos para os dois patógenos por meio do teste molecular, e quatro cães foram reativos no teste ELISA para ambas as doenças e tiveram resultado negativo apenas no teste molecular de Ehrlichia spp. Estes resultados, na sorologia, podem sugerir possíveis casos de reatividade cruzada entre a Leishmania spp. e Ehrlichia spp.. Entretanto, é mais provável que estes resultados estejam relacionados com a coinfecção desses agentes. Por meio dos resultados obtidos, há mais evidências de coinfecção por estes dois agentes patogênicos no cão, do que reatividade cruzada entre eles. / The aim of this study is to investigate, by statistical methods, the occurrence of cross-reactivity between Leishmania spp. and Ehrlichia spp. using serological and molecular techniques. A total of 100 blood samples from dogs were collected and processed by ELISA (indirect Enzyme-Linked Immunosorbent Assay) and PCR (polymerase chain reaction). Statistical analysis consists of McNemar tests and agreement coefficient Kappa. The statistics were considered significant when p <0.05. The sensitivity and specificity of the tests were determined by ROC curve (Receiver Operator Characteristic), considering the PCR as gold standard. From the analysis it can be seen that 48,0% of the animals were reactive in ELISA and 58,0% were positive in PCR in the case of Leishmania spp. For Ehrlichia spp., the occurrence of antibodies by ELISA was 54,0% and, by PCR, 48,0% of the dogs were positive. It can be noted also that 37,0% of the animals were positive for both parasites through molecular test and four dogs were reactive in the ELISA test for both diseases and were negative only in molecular test for Ehrlichia spp. These results in serology can suggest possible cases of cross-reactivity between Leishmania spp. and Ehrlichia spp.. However, it is more likely that these results are related to coinfection of these agents. Through the results obtained, there are more evidences of coinfection by these two pathogens in dogs than cross-reactivity between them. / FAPESP: 2014/26413-2

A erliquiose monocitotrópica canina é caracterizada como uma infecção persistente, podendo evoluir para doença fatal. Mecanismos imunopatogênicos estão implicados no desenvolvimento da doença, porém não é completamente compreendido o papel da resposta imune celular nas infecções causadas por Ehrlichia canis transmitida pelo carrapato Rhipicephalus sanguineus. A infecção do vetor ocorre somente durante o repasto em cães riquetsêmicos, porém não é reconhecido o nível de riquetsemia infectante durante o curso da infecção nos cães. O objetivo desse estudo foi verificar os níveis de riquetsemia relativa capazes de infectar o vetor (R. sanguineus) durante o curso da erliquiose canina; e avaliar a resposta imune celular induzida por exposição de cães a carrapatos infectados com Ehrlichia canis. Um cão foi utilizado para produção do inoculo e infecção de ninfas de R. sanguineus. Subseqüentemente, os carrapatos adultos infectados, oriundos das ninfas que se alimentaram nos cães infectados, foram utilizados para infecção dos cães dos grupos I (n=3) e II (n=3). Cães do grupo III (n=3) foram inoculados com sangue infectado com E. canis, por via intravenosa. Os grupos IV (n=3) e V (n=3) foram compostos por cães não infectados. Os grupos II e IV foram reinfestados periodicamente com ninfas não infectadas de R. sanguineus. Foram observadas, alterações clínicas como febre, apatia e disorexia nos cães infectados, durante a fase aguda da infecção, porém de forma mais intensa e precoce nos cães infectados por inoculação intravenosa (grupo III). As alterações hematológicas mais evidentes foram redução do número de plaquetas, leve redução na série vermelha e branca. Todos os animais infectados soroconverteram aos 14 dias após inoculação ou infestação com carrapatos infectados (dpi), mantendo títulos entre 10240 e 81920. Os níveis de riquetsemia foram variáveis durante o curso da infecção, persistindo até 364 dpi, porém mais constante na fase aguda da doença. Um pequeno número de carrapatos alimentados nos cães infectados apresentou amplificação de DNA de E. canis, porém foram demonstrados até 308 dpi. Foi observada uma redução na relação CD4:CD8 nos animais infectados, no 28° dpi, mantendo-se de forma mais branda até 252 dpi. Uma maior proporção de linfócitos T CD4+ produtores de IFN-&gamma; e IL-4 foi observada no tempo zero de cães que não adquiriram infecção após infestação com carrapatos infectados. Os níveis séricos de citocinas mostraram de forma mais evidente a elevação de IL-10 e TNF-&alpha; em um cão que desenvolveu doença fatal, podendo indicar a participação de mecanismos imunes na apresentação clínica e na persistência do agente no organismo hospedeiro. / The canine monocytic ehrlichiosis is characterized as a persistent infection, which can develop fatal disease. Immunopathogenic mechanisms are implicated in the development of the disease, however it is not completely understood the role of cellular immune in the infection caused by Ehrlichia canis transmitted by the tick Rhipicephalus sanguineus. The vector becomes infected only while feeding on rickettsemic dogs, however the level of rickettsemia is not recognized during the course of the infection in the dogs. The objective of this study was to verify the level of relative rickettsemia capable to infect the vector (R. sanguineus) during the course of the canine ehrlichiosis; and to evaluate the cellular immune induced in dogs exposed to Ehrlichia canis-infected ticks. A dog was used for production of the inoculum and infection of nymphs of R. sanguineus. Infected adult ticks that had fed as nymphs on the infected dog were used to feed on and transmit the infection to dogs of groups I (n=3) and II (n=3). Group III dogs (n=3) were intravenously inoculated with E. canis-infected dogs. Group IV (n=3) and V (n=3) dogs were the control groups, never exposed to E. canis. Dogs of groups II and IV were reinfested periodically with uninfected R. sanguineus nymphs. Clinical alterations such as fever, apathy and dysorexia were observed in the infected dogs during the acute phase of the infection, however in a more intense and precocious form in the dogs infected by intravenous inoculation (group III). The more evident hematological alterations were reduction of the number of platelets, mild reduction in the red and white cells series. All infected animals soroconverted by 14 days after inoculation or infestation by infected ticks (dpi), showing titers between 10240 and 81920 during the study. The rickettsemia levels were variable during the course of the infection, persisting up to 364 dpi, however more constant in the acute phase of the disease. A small number of ticks that fed on infected dogs presented amplification of E. canis DNA, however they were demonstrated up to 308 dpi. A reduction in the relationship CD4:CD8 in the infected animals was observed in 28th dpi, keeping in a mild form up to 252 dpi. A larger proportion of lymphocytes T CD4+ producing IFN-&gamma; and IL-4 it was observed in the zero time of dogs that did not acquire infection after infestation with infected ticks. The cytokine seric levels showed the elevation in a more evident form of IL-10 and TNF-&alpha; in a dog that developed fatal disease, what could indicate the participation of immune mechanisms in the clinical presentation and in the persistence of the agent in the organism host.

Made available in DSpace on 2016-04-28T20:18:30Z (GMT). No. of bitstreams: 1 2008 - Erica Mateus Toledo Accetta.pdf: 809163 bytes, checksum: 04d79d0b9854d1e99fd77cff6ae68a4f (MD5) Previous issue date: 2009-08-27 / Canine ehrlichiosis is an important infectious disease whose prevalence has been increasead in most areas of Brazil. Clinical signals and the laboratorial findings are variable. The present work had as objective to determine the frequency of the infection for E. canis and Anaplasma platys in dogs with trombocytopenia at Regi?o dos Lagos - State of Rio de Janeiro. It has been evaluated the CBC of 1127 dogs with trombocytopenia, a total of 3019 laboratorial tests carried through in the period of June 2006 and July 2007, at CEVET Lagos Lab, providing atendence to several clinics of Araruama, Iguaba Grande, S?o Pedro d Aldeia, Cabo Frio, Arraial do Cabo and B?zios. Erliquiosis was diagnosised through haemoparasit in smears of total blood s research stained with Panoptic kit. Eighty-four dogs (7.45%) were considered infected by the discovery of morulae of Ehrlichia spp. e Anaplasma platys. Normocytic normochromic anaemia and monocytosis were the most hematological alterations found. / A erliquiose canina ? uma importante doen?a infecciosa cuja preval?ncia tem aumentado significativamente em v?rias regi?es do Brasil. Os sinais cl?nicos e os achados laboratoriais s?o vari?veis. O presente trabalho teve como objetivo determinar a freq??ncia da infec??o por E. canis e A. platys em c?es com trombocitopenia na Regi?o dos Lagos do Estado do Rio de Janeiro. Foram avaliados os exames hematol?gicos de 1127 c?es com trombocitopenia de um total de 3019 exames realizados no per?odo de junho de 2006 a julho de 2007, no laborat?rio CEVET Lagos, que presta atendimento ?s diversas cl?nicas dos munic?pios de Araruama, Iguaba Grande, S?o Pedro d Aldeia, Cabo Frio, Arraial do Cabo e B?zios. A erliquiose foi diagnosticada atrav?s da pesquisa de hemoparasitos em esfrega?os de sangue total corados com kit pan?ptico. O diagn?stico baseou-se no achado de m?rulas de Ehrlichia spp. e Anaplasma platys, sendo considerados infectados 84 (7,45%) c?es. Anemia normoc?tica normocr?mica e monocitose foram as altera??es hematol?gicas mais freq?entes.

Made available in DSpace on 2016-12-23T13:53:37Z (GMT). No. of bitstreams: 1 Mara Rubia Rocha Pereira Sales.pdf: 980226 bytes, checksum: 42a133fd63917e32301d6df4c9b4fca5 (MD5) Previous issue date: 2012-06-26 / The aim of this study was determined by nested-PCR the presence of Ehrlichia canis in dogs located in the municipality of Alegre-ES and evaluate its correlation with morulae and thrombocytopenia. For this purpose blood samples were collected from 85 dogs, regardless of race, age, sex or health status. With these, slides were obtained for the detection of morulae, thrombocytopenia, and execution of nested-PCR technique. We verified a prevalence of 1.17% to investigated the presence of morulae, 5.6% when using the technique of nested-PCR, and was verified that 17.64% of the CBCs were thrombocytopenia. However, only 40% of positive samples by nested-PCR showed thrombocytopenia. The results presented in this study demonstrate that the introduction of molecular diagnostic techniques such as nested-PCR is an important method to aid in the early diagnosis of diseases / Objetivou-se com este estudo determinar por meio da nested-PCR a prevalência da Ehrlichia canis em cães da região de Alegre-ES, e avaliar sua correlação com a presença de mórulas e trombocitopenia. Para isso, foram colhidas amostras sanguíneas de 85 cães, independente de raça, sexo, idade e estado de saúde. Com estas, foram confeccionadas lâminas para a pesquisa de mórulas, trombocitopenia, e execução da técnica de nested-PCR. Foi verificada uma prevalência de 1,17% ao pesquisar a presença de mórulas, 5,6% ao utilizar a técnica da nested-PCR, e foi verificada que 17,64% dos hemogramas apresentavam trombocitopenia. No entanto, somente 40% das amostras positivas pela nested-PCR, apresentaram trombocitopenia. Os resultados apresentados neste estudo demonstram que a introdução de técnicas de diagnóstico molecular como a nested-PCR é um método importante para o auxílio no diagnóstico precoce de patologias

A erliquiose monocitotrópica canina é caracterizada como uma infecção persistente, podendo evoluir para doença fatal. Mecanismos imunopatogênicos estão implicados no desenvolvimento da doença, porém não é completamente compreendido o papel da resposta imune celular nas infecções causadas por Ehrlichia canis transmitida pelo carrapato Rhipicephalus sanguineus. A infecção do vetor ocorre somente durante o repasto em cães riquetsêmicos, porém não é reconhecido o nível de riquetsemia infectante durante o curso da infecção nos cães. O objetivo desse estudo foi verificar os níveis de riquetsemia relativa capazes de infectar o vetor (R. sanguineus) durante o curso da erliquiose canina; e avaliar a resposta imune celular induzida por exposição de cães a carrapatos infectados com Ehrlichia canis. Um cão foi utilizado para produção do inoculo e infecção de ninfas de R. sanguineus. Subseqüentemente, os carrapatos adultos infectados, oriundos das ninfas que se alimentaram nos cães infectados, foram utilizados para infecção dos cães dos grupos I (n=3) e II (n=3). Cães do grupo III (n=3) foram inoculados com sangue infectado com E. canis, por via intravenosa. Os grupos IV (n=3) e V (n=3) foram compostos por cães não infectados. Os grupos II e IV foram reinfestados periodicamente com ninfas não infectadas de R. sanguineus. Foram observadas, alterações clínicas como febre, apatia e disorexia nos cães infectados, durante a fase aguda da infecção, porém de forma mais intensa e precoce nos cães infectados por inoculação intravenosa (grupo III). As alterações hematológicas mais evidentes foram redução do número de plaquetas, leve redução na série vermelha e branca. Todos os animais infectados soroconverteram aos 14 dias após inoculação ou infestação com carrapatos infectados (dpi), mantendo títulos entre 10240 e 81920. Os níveis de riquetsemia foram variáveis durante o curso da infecção, persistindo até 364 dpi, porém mais constante na fase aguda da doença. Um pequeno número de carrapatos alimentados nos cães infectados apresentou amplificação de DNA de E. canis, porém foram demonstrados até 308 dpi. Foi observada uma redução na relação CD4:CD8 nos animais infectados, no 28° dpi, mantendo-se de forma mais branda até 252 dpi. Uma maior proporção de linfócitos T CD4+ produtores de IFN-&gamma; e IL-4 foi observada no tempo zero de cães que não adquiriram infecção após infestação com carrapatos infectados. Os níveis séricos de citocinas mostraram de forma mais evidente a elevação de IL-10 e TNF-&alpha; em um cão que desenvolveu doença fatal, podendo indicar a participação de mecanismos imunes na apresentação clínica e na persistência do agente no organismo hospedeiro. / The canine monocytic ehrlichiosis is characterized as a persistent infection, which can develop fatal disease. Immunopathogenic mechanisms are implicated in the development of the disease, however it is not completely understood the role of cellular immune in the infection caused by Ehrlichia canis transmitted by the tick Rhipicephalus sanguineus. The vector becomes infected only while feeding on rickettsemic dogs, however the level of rickettsemia is not recognized during the course of the infection in the dogs. The objective of this study was to verify the level of relative rickettsemia capable to infect the vector (R. sanguineus) during the course of the canine ehrlichiosis; and to evaluate the cellular immune induced in dogs exposed to Ehrlichia canis-infected ticks. A dog was used for production of the inoculum and infection of nymphs of R. sanguineus. Infected adult ticks that had fed as nymphs on the infected dog were used to feed on and transmit the infection to dogs of groups I (n=3) and II (n=3). Group III dogs (n=3) were intravenously inoculated with E. canis-infected dogs. Group IV (n=3) and V (n=3) dogs were the control groups, never exposed to E. canis. Dogs of groups II and IV were reinfested periodically with uninfected R. sanguineus nymphs. Clinical alterations such as fever, apathy and dysorexia were observed in the infected dogs during the acute phase of the infection, however in a more intense and precocious form in the dogs infected by intravenous inoculation (group III). The more evident hematological alterations were reduction of the number of platelets, mild reduction in the red and white cells series. All infected animals soroconverted by 14 days after inoculation or infestation by infected ticks (dpi), showing titers between 10240 and 81920 during the study. The rickettsemia levels were variable during the course of the infection, persisting up to 364 dpi, however more constant in the acute phase of the disease. A small number of ticks that fed on infected dogs presented amplification of E. canis DNA, however they were demonstrated up to 308 dpi. A reduction in the relationship CD4:CD8 in the infected animals was observed in 28th dpi, keeping in a mild form up to 252 dpi. A larger proportion of lymphocytes T CD4+ producing IFN-&gamma; and IL-4 it was observed in the zero time of dogs that did not acquire infection after infestation with infected ticks. The cytokine seric levels showed the elevation in a more evident form of IL-10 and TNF-&alpha; in a dog that developed fatal disease, what could indicate the participation of immune mechanisms in the clinical presentation and in the persistence of the agent in the organism host.

No Brasil, carrapatos do gênero Rhipicephalus são representados pelas espécies Rhipicephalus sanguineuss.l. (Latreille) e Rhipicephalus (Boophilus) microplus (Canestrini), sendo que a primeira espécie possui especificidade com cães domésticos e a segunda com bovinos. Para o cão, R. sanguineus s.l. é o principal vetor de agentes causadores da babesiose canina e da erliquiose, enquanto que nos bovinos, R. microplus é responsável, principalmente, pela transmissão de agentes causadores da babesiose e anaplasmose. Alguns dos patógenos causadores dessas doenças são difíceis de serem cultivados in vitro, e não crescem em meios artificiais, especialmente Anaplasma spp. Assim, muitas linhagens celulares de carrapatos foram desenvolvidas nos últimos 40 anos e têm sido amplamente utilizadas para isolamento e propagação de microrganismos patogênicos. Cultivos celulares obtidos de células embrionárias de carrapatos oferecem um sistema de vetor in vitro, que é útil principalmente para estudos de agentes intracelulares. Dessa forma, a obtenção de culturas de células embrionárias de R. sanguineus (origens tropical e temperada) e de R. (B.) microplus, bem como, sua infecção com Ehrlichia canis e Anaplasma marginale, respectivamente, são objetivos do presente estudo. Os cultivos celulares dessas espécies de carrapatos foram preparados com massas de ovos de diferentes idades e mantidos à 30 &deg;C em meio de cultura L15-B, suplementado com 10% de Triptose Fosfato e 20% de Soro Fetal Bovino. Subcultivos foram realizados após a formação da monocamada celular confluente. As identidades celulares foram confirmadas pela PCR e sequenciamento, utilizando um fragmento do gene mitocondrial 16S rDNA. As sequências das culturas de células de R. sanguineus (tropical e temperada) e de R. microplus foram depositadas no GenBank. Essas culturas de células foram infectadas com E. canis e A. marginale (cepas Jaboticabal), respectivamente, e mantidas em meio L-15B (Vitrocell) suplementado com Soro Fetal Bovino enriquecido com ferro (Hyclone) nas concentrações de 2% e 5%. As células infectadas foram mantidas em propagações sucessivas até a terceira passagem, para novas culturas de células não infectadas, sendo então criopreservadas. O DNA extraído das células infectadas e não infectadas de R. sanguineus (de ambas as origens) e de R. microplus, foi analisado pela PCR quantitativa em tempo real (qPCR), utilizando os genes E. canis-dsb e msp1&#946;, respectivamente. Propagações de células de carrapatos infectadas para as novas culturas de R. sanguineus (origem tropical) e R. (B) microplus, foram bem sucedidas. Por outro lado, as células de R. sanguineus (origem temperada) não se tornaram infectadas no presente estudo. / In Brazil, ticks of the genus Rhipicephalus are represented by the species Rhipicephalus sanguineus s.l. (Latreille) and Rhipicephalus (Boophilus) microplus (Canestrini), being that the first one has specificity with domestic dogs and the second with cattle. For dogs, R. sanguineus s.l. is the main vector of causative agents of canine babesiosis and ehrlichiosis, whereas in cattle, R. microplus is responsible, mainly, for the transmission of agents that cause babesiosis and anaplasmosis. Some of the pathogens that cause these diseases are difficult to grow in vitro, and do not grow on artificial media, especially Anaplasma spp. Therefore, many tick cell lines were developed in the last 40 years and have been used for the isolation and propagation of pathogenic microorganisms. Cell cultures obtained from embryonic tick cells offer an in vitro vector system, which is mainly useful for studies of intracellular agents. The aim of the present study was to obtain cultures of R. sanguineus (tropical and temperate lineages) and R. microplus embryonic cells and their infection with Ehrlichia canis and Anaplasma marginale. Cell cultures from the tick species were prepared with egg masses of different ages and kept at 30 &deg; C in L15-B culture medium supplemented with 10% Tryptose Broth and 20% Fetal Bovine Serum. Subcultures were done after confluent cell monolayer formation. Cellular identities were confirmed by PCR and sequencing using a 16S rDNA mitochondrial gene fragment. Sequences of R. sanguineus (tropical and temperate) and R. microplus cell cultures were deposited on GenBank. These cell cultures were infected with E. canis and A. marginale (Jaboticabal strains), respectively, and maintained in L-15B medium and Fetal Bovine Serum with iron (Hyclone) supplemented at 2% and 5% concentrations. The infected cells were maintained in successive propagations until the third passage, to new cultures of uninfected cells, and then were cryopreserved. DNA extracted from infected and uninfected R. sanguineus (both origin) and R. microplus cells was analyzed by quantitative real-time PCR (qPCR) using the E. canis-dsb and msp1&#946; genes, respectively. Propagations of infected tick cells to the new cultures of R. sanguineus (tropical origin) and R. microplus, were successful. On the other hand, the R. sanguineus cells (temperate origin) did become infected in the present study.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is a Gram negative, rickettsial organism responsible for human monocytic ehrlichiosis, an emerging disease in people. E. chaffeensis infection to a vertebrate host occurs when the pathogen is inoculated by an infected tick, Amblyomma americanum. White-tailed deer is a reservoir host for this pathogen. The strategies employed by E. chaffeensis in support of its dual host adaptation and persistence are not clear. One of the possible mechanisms by which the pathogen adapts and persists, is by altering its gene expression in response to its host cell environments. Recently, we reported that E. chaffeensis protein expression including that from a 28 kDa outer membrane protein multigene locus (p28-Omp), is influenced by macrophage and tick cell environments. E. chaffeensis expresses p28-Omp gene 14 product predominantly when it is grown in tick cells and p28-Omp gene 19 protein in macrophages. We hypothesize that E. chaffeensis achieves its host-specific gene expression by employing transcriptional regulation by sensing the host cell signals. In support of this hypothesis, transcriptional analysis of 14 and 19 genes was performed utilizing several RNA analysis methods. The results supported our hypothesis that the gene regulation occurs at mRNA level in a host cell-specific manner. This analysis also identified transcription start sites and located putative promoters for the p28-Omp genes 14 and 19. Promoter regions of genes 14 and 19 were mapped to identify gene-specific differences, RNA polymerase binding sequences and the putative regulatory elements that may influence the promoter activities. Electrophoretic mobility shift assays revealed interaction of E. chaffeensis proteins with gene 14 and 19 promoters. Several E. chaffeensis putative regulatory proteins were expressed as recombinants and their effects on a p28-Omp gene promoter activity were evaluated. In summary, we demonstrated that the differences in the E. chaffeensis p28-Omp genes 14 and 19 are the result of their regulation at transcriptional level in response to the host cell environment. We also identified RNA polymerase binding regions and several DNA sequences that influenced promoter activity. This is the first description of a transcriptional machinery of E. chaffeensis. The data from these studies provide important insights about molecular mechanisms of gene regulation in E. chaffeensis.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen that causes infection in people and several vertebrate animals. One of the striking features of E. chaffeensis infection is the prolonged persistence in its vertebrate and tick hosts. The mechanism of persistent infection and the reasons for the host immune system failure to clear the infection are not well understood. One hypothesis is that differential gene expression serves as an important adaptive mechanism used by E. chaffeensis in support of its continued survival in both tick and vertebrate hosts. One way to test this hypothesis is by performing mutational analysis. However, the methods for introducing mutations in this pathogen have not yet been documented and are challenging, possibly due to its obligate, intraphagosomal growth requirement. Recently, a novel gene mutation method called ‘TargeTron Gene Knockout System’ that is based on the modified group II intron insertion strategy has been developed. This method appears to be effective in creating mutations in a wide range of gram positive and gram negative bacterial organisms. The group II intron can be programmed for insertion into virtually any desired DNA target with possibly high frequency and specificity. In this study, I focus on creating mutations in E. chaffeensis using the TargeTron gene knockout system. I prepared modified group II intron constructs retargeting for insertion into three E. chaffeensis genes: Ech_0126 (a transcriptionally silent gene), macrophage-specific expressed gene (p28-Omp 19, Ech_1143) and tick cell-specific expressed gene (p28-Omp 14, Ech_1136). In support of driving the expression of the modified group II introns in E. chaffeensis, the pathogen- specific high-expressing gene promoter (tuf) was inserted upstream to the transcription start site. In addition, a chloramphenicol acetyltransferase gene with E. chaffeensis rpsl promoter was introduced for use as a selection marker. The constructs were then evaluated by transforming into E. chaffeensis. Transformants with mutations, introduced in two of the three genes (Ech_0126 and Ech_1143), were identified by PCR and Southern blot methods. Although the mutants are detectable for up to 48 hours, establishment of stable transformants remains to be challenging. The outcomes of this project will have important implications in defining the pathogenesis of E. chaffeensis, particularly to assess the differences in the organism in tick and vertebrate hosts.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause the emerging tick-transmitted disease, human granulocytic anaplasmosis (HGA). Following entry, the pathogen replicates within a host cell-derived vacuole that fails to mature along the endocytic pathway, does not acidify, and does not fuse with lysosomes. Selective fusogenicity is prototypical of many vacuole-adapted pathogens and has been attributed, at least in part, to pathogen modification of the vacuolar inclusion membrane and/or to selective recruitment or exclusion of host trafficking regulators. As a result, the A. phagocytophilum-occupied vacuolar membrane (AVM) provides a unique interface to study the host-pathogen interactions critical to A. phagocytophilum intracellular survival. Diverse vacuole-adapted pathogens; including Chlamydia, Legionella, and Salmonella; selectively recruit host Rab GTPases to their vacuolar membranes to establish replicative permissive niches within their host cells. Rab GTPases coordinate many aspects of endocytic and exocytic cargo delivery. We determined that the A. phagocytophilum-occupied vacuole (ApV) selectively recruits a subset of fluorescently-tagged Rabs that are predominantly associated with recycling endosomes. Another emerging theme among vacuole-adapted pathogens is the ability to hijack ubiquitin machinery to modulate host cellular processes. Mono- and polyubiquitination differentially dictate the subcellular localization, activity, and fate of protein substrates. Monoubiquitination directs membrane traffic from the plasma membrane to the endosome and has been shown to promote autophagy. We show that monoubiquitinated proteins decorate the AVM during infection of promyelocytic HL-60 cells, endothelial RF/6A cells, and to a lesser extent, embryonic tick ISE6 cells. Importantly, tetracycline treatment concomitantly promotes loss of the recycling endosome-associated GFP-Rabs and ubiquitinated proteins and acquisition of the late endosomal marker, Rab7, and lysosomal marker, LAMP-1, implicating bacterial-derived proteins in the ApV's altered fusogenicity. Therefore, we rationalized that A. phagocytophilum-encoded proteins that associate with the AVM may establish interactions with the host cell that are important for intracellular survival. By focusing on A. phagocytophilum proteins that are induced during host infection, we identified the first two bacterial-encoded proteins -- APH_1387 and APH_0032 -- that modify the AVM. Although functional studies are hindered by the lack of a system to genetically manipulate Anaplasma, the pathobiological roles of APH_1387 and APH_0032 are likely unique, as both proteins exhibit very little or no homology with any previously described protein. APH_1387 and APH_0032 are present at the cytoplasmic face of the AVM, therefore they likely interact with host proteins. We demonstrate that ectopic expression of APH_1387 and APH_0032 inhibits the ApV development in A. phagocytophilum infected cells. The results presented in this dissertation contribute to our understanding of how A. phagocytophilum modifies the vacuolar membrane in which it resides to establish a safe haven and evade lysosomal degradation.