Made available in DSpace on 2014-06-11T19:27:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-25Bitstream added on 2014-06-13T18:56:53Z : No. of bitstreams: 1 andre_mr_me_jabo.pdf: 427316 bytes, checksum: 366b68e3f9cae8ab0938b9ab1032ffb9 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Poucos relatos têm sido feitos sobre o diagnóstico da erliquiose e babesiose em felinos domésticos e selvagens brasileiros, os quais são baseados diretamente pela presença de mórulas em leucócitos e piroplasmas em eritrócitos, e indiretamente pela detecção de anticorpos anti-Ehrlichia canis. O presente estudo teve como objetivo realizar a detecção molecular de E. canis e B. canis e a presença de anticorpos da classe IgG contra esses hemoparasitas em amostras de sangue e soro, respectivamente. Neste utilizamos 72 felídeos selvagens brasileiros mantidos em cativeiro em algumas instituições e zoológicos. Pela Reação de Imunofluorescência Indireta (RIFI), dezoito (25%) e cinqüenta e três (73,6%) dos 72 animais amostrados foram sororeagentes frente aos antígenos de E. canis e B. canis, respectivamente. Na PCR para E. canis, onze (15,3%) dos 72 animais amostrados foram positivos. Os amplicons foram confirmados por seqüenciamento e o DNA de E. canis encontrado mostrou grande similaridade genética com amostras de E. canis isoladas no Brasil, México, Portugal, Grécia e Taiwan, com 98% de similaridade. Nenhuma das amostras foi positiva na PCR para B. canis. Destaca-se a importância e a primeira detecção molecular de E. canis e presença de anticorpos anti-E. canis e anti-B. canis em felídeos selvagens brasileiros mantidos em cativeiro. / Few are the reports that have been carried out on ehrlichiosis and babesiosis diagnostic in Brazilian domestic and wild felids, which are based directly on the presence of morulae in leucocytes and piroplasms in erythrocytes, and indirectly by detection of antibodies against E. canis. The aim of this study was to detect molecularly E. canis and B. canis and the presence of anti-E. canis and B. canis IgG antibodies in the blood and sera samples, respectively, from 72 Brazilian wild captive felids maintained in some instituitions and zoos. Eighteen (25.0%) and fifty-three (73.6%) out of 72 animals were seroreagent for E. canis and B. canis antigen, respectively, by IFA (Indirect Immunofluorescent Assay). Eleven (15.3%) of the 72 samples were positive for nPCR E. canis. The amplicons were confirmed by sequencing and the E. canis DNA found appeared to be closely related to E. canis samples from Brazil, Mexico, Portugal, Greece and Taiwan with 98% percent identity. None of the 72 samples were positive for B. canis by PCR. This is the first molecular detection of E. canis and presence of seroreactivity for both B. canis and E. canis in Brazilian wild captive felids.

Orientador: Mirela Tinucci Costa / Banca: Aguemi Kohayagawa / Banca: Tiago Wilson Patriarca Mineo / Banca: Rosangela Zacarias Machado / Banca: Aureo Evangelista Santana / Resumo: A infecção aguda experimental pela amostra Jaboticabal de Ehrlichia canis provoca alterações clínicas severas no hospedeiro, com graves distúrbios sanguíneos e imunológicos, que podem comprometer a vida do animal. Com este estudo buscou-se avaliar a expressão gênica de TNF-α, IFN-γ e IL-10, pesquisar a presença de mórulas no baço e avaliar o imunofenótipo das células esplênicas antes, nos dias 6, 18 e 30 após a inoculação e 25 dias após o tratamento com cloridrato de doxicilina, em cinco cães sem definição racial inoculados com a amostra Jaboticabal de Ehrlichia canis. Nas condições experimentais desta pesquisa, o início do desenvolvimento dos sinais clínicos, seis dias após a inoculação (D6) foi acompanhado pela expressão de TNF-α e aumento de células MHC II+ (P<0,05) na citologia esplênica em relação ao controle. Com o desenvolvimento da infecção experimental (D18) ocorreu o agravamento dos sinais clínicos, os cães apresentaram febre, linfadenomegalia e esplenomegalia acompanhados de trombocitopenia, leucopenia e anemia, mórulas na citologia esplênica, aumento significativo da expressão de TNF-α em leucócitos e células esplênicas, detecção de IL-10, tanto em leucócitos como em células esplênicas e redução de células CD4+ (P<0,05), em relação ao momento anterior e ao grupo controle, macrófagos (P<0,05) em relação ao controle, e aumento de células B (P<0,05) em relação a D-1 e ao grupo controle. Aos 30 dias os cães já não apresentavam sinais clínicos da infecção, porém persistia a trombocitopenia. Além disso, persistência do aumento das células B+ esplênicas (P<0,05), diminuição significativa das células CD4+ e dos macrófagos em relação ao D18 e ao controle. O TNF-α atingiu sua maior taxa de expressão e ocorreu a detecção de IFN-γ. / Abstract: The experimental acute infection by Ehrlichia canis Jaboticabal sample provokes severe clinical alterations in the host with serious blood and immunological disorders that may compromise the animal's life. The present study aimed to evaluate the gene expression of TNF-α, IFN-γ and IL-10, search morulae presence in the spleen and evaluate the splenic cells' immunophenotype before, at days 6, 18 and 30 days post inoculation and 25 days after the treatment with doxycycline cloridrate, in five cross-bred dogs inoculated with Ehrlichia canis Jaboticabal sample. At the experimental conditions of this research, the beginning of the development of the clinical signs, six days after the inoculation (D6) was accompanied by expression of TNF-α and increase of MHC II+ cells (P<0,05) at the splenic cytology when compared to the control group. As long as the experimental infection was developed (D18) the clinical signs were becoming worse, the dogs presented fever, lymphadenomegalia and spleenomegalia accompanied by thrombocytopenia, leucopenia and anemia, morulae in the splenic cytology, significant increase of the expression of TNF-α in leukocytes and splenic cells, detection of IL-10 both in leukocytes and in splenic cells and the decrease of CD4+ cells (P<0,05) in comparison to the previous moment and to the control group, macrophages (P<0,05) compared to the control group, and increase of cells B (P<0,05) in comparison to the control group and D-1. At day 30 the dogs no more presented the infection clinical signs, although the thrombocytopenia persisted. Besides, persistent splenic cells B+ increase (P<0,05), significant reduction of CD4+ cells and macrophages compared to D18 and to the control group were observed. The TNF-α reached its highest expression rate and the detection of IFN-γ ocurred. / Doutor

Made available in DSpace on 2014-08-20T14:31:33Z (GMT). No. of bitstreams: 1 dissertacao_fernanda_antunes_brum.pdf: 139752 bytes, checksum: 989f0c94f1c779f13cc72237d9ab8552 (MD5) Previous issue date: 2010-12-23 / The Ehrlichia canis is a canine monocytic ehrlichiosis responsible for (EMC). The incubation period of EMC is 8 to 20 days, the disease has three phases: acute, subclinical and chronic. The species E. canis is transmitted to the dog and the man by the tick Rhipicephalus sanguineus. It is diagnosed by blood smear, serological or polymerase chain reaction (PCR), with the immunofluorescence method used. Recently E. canis was described as being capable of causing severe disease in humans, with death cases, mainly children and elderly. The gp 19 protein is an important immunodominant antigen, it induces rapid immune response in dogs. The similarity between the geographically distinct samples suggests that the gp19 protein can be used for testing the diagnostic immunoassays, as well as vaccination programs, because this protein is specific for E. canis and thus not have cross reactions with other genera of Ehrlichia.Este study aimed to clone and express the glycoprotein 19 of Ehrlichia canis in Escherichia coli for use as immunobiological, rapid and accurate detection of this disease. The gp 19 gene was amplified by polymerase chain reaction (PCR) using specific primers containing sites for restriction enzymes. The PCR product after digestion, was purified and cloned into a vector and inserted into E. pAE coli TOP 10 competent by electroporation. Of the identified clones was extracted plasmid, which was digested with restriction enzymes to confirm the presence of the insert. After selection of recombinant clones containing the gene linked to the vector, this was purified by heat shock and inserted in expression strain E. coli Star, cultured and induced to express the protein gp19. / A Ehrlichia canis é a responsável pela erliquiose monocitica canina (EMC). O período de incubação da EMC é de 8 a 20 dias; a doença apresenta três fases: aguda, subclínica e crônica. As espécies de E. canis são transmitidas para o cão e para o homem pelo carrapato da espécie Rhipicephalus sanguineus. O diagnóstico é realizado através de esfregaços sanguíneos, métodos sorológicos ou reação em cadeia da polimerase (PCR), sendo a imunofluorescencia o método mais utilizado. Recentemente E. canis, foi descrita como sendo capaz de causar doença grave em humanos, com casos de óbito principalmente em crianças e idosos. A proteína gp 19 é um importante antígeno imunodominante, pois induz rápida resposta imunológica nos cães. A similaridade entre as amostras geograficamente distintas sugere que a proteína gp19 possa ser usada para ensaios de imunoenzimáticos de diagnóstico, bem como em programas vacinais, pois esta proteína é especifica para E. canis não tendo assim reações cruzadas com outros gêneros de Ehrlichia. O gene gp 19 foi amplificado pela reação da polimerase em cadeia (PCR) utilizando oligonucleotídeos iniciadores específicos contendo sítios para enzimas de restrição. O produto da PCR, após digestão, foi purificado e clonado no vetor pAE e inserido em E. coli TOP 10 competente por eletroporação. Dos clones identificados extraiu-se o plasmídio, o qual foi digerido com as enzimas de restrição para comprovar a presença do inserto. Após seleção dos clones recombinantes contendo o gene ligado ao vetor, este foi purificado e inserido por choque térmico na cepa de expressão E. coli Star cultivado e induzido para expressar a proteína gp19. Esta expressão foi verificada por gel de SDS-Page 12% e confirmada por Dot-Blot.

Ehrlichia ruminantium, the causative organism of heartwater infections, places severe economic constraint on the livestock industry wherever Amblyomma tick vectors are present. Angora goats are particularly susceptible to this disease and the current live blood vaccine cannot safely be used to protect these animals. An attenuated E. ruminantium (Welgevonden) experimental vaccine has previously shown promising results in Merino sheep and Boer goats. The vaccine was administered by intravenous route (i/v). The general objective of this study was to test the efficacy and safety of the attenuated heartwater vaccine E. ruminantium (Welgevonden) in Angora goats. The specific objectives were, firstly to assess the intra-muscular route of administration of the attenuated vaccine as compared to the standard i/v route and, secondly, to study the haematological changes in Angora goats before, during and after vaccination under controlled conditions at the Onderstepoort Veterinary Institute tick-free stables. A total of 55 Angora goats were used in this trial. They were purchased from an area in South Africa which is known to be Amblyomma-free and heartwater-free. Furthermore, on arrival, the goats were screened for E. ruminantium infection by the immunofluorescent antibody (IFA) test to confirm their disease-free status. The Angora goats were divided into 3 groups: In Group 1, ten were vaccinated by the standard i/v route, in Group 2, 31 received the vaccine by i/m route and 10 served as untreated controls for Group 3. Five of the 10 i/v vaccinated group, 20/31 of the i/m vaccinated and 5 controls were challenged by feeding of known infected adult A hebreaum. The other remaining animals within the three groups were challenged using a known infected blood stabilate administered by the standard i/v route (dose 5xLD50). All animals were challenged 42 days after vaccination. The vaccine did not produce any inflammatory reactions at the site of injection. However, 3/31 (9.7%) of i/m and 7/10 (70%) of i/v vaccinated goats developed febrile reactions starting on Day 11 post-immunisation and were treated. All vaccinated goats were fully protected against either needle i/v or tick challenge, while the control non-vaccinated goats reacted severely to the challenge materials and required oxytetracycline treatment. Despite treatment, two of the unvaccinated goats died from the challenge material. 9 Haematological values (packed cell volume, differential blood cells count) were obtained on blood samples taken from the treatment and control groups at different times during the course of the trial. Wide within group variations as shown by the high standard deviation values were found. As no significant changes were found between vaccinated and control animals, it is likely that the attenuated vaccine does not cause significant clinical haematological changes. This study has demonstrated that the attenuated E. ruminantium (Welgevonden) vaccine is safe in 90.3% and efficacious (100% efficacy) for intramuscular administration in Angora goats. However, further laboratory and on-farms studies are needed in order to establish the lowest effective and safety dose, duration of immunity, and the vaccine’s safety in young and pregnant animals. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted

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Made available in DSpace on 2016-08-17T18:39:33Z (GMT). No. of bitstreams: 1 3168.pdf: 2139042 bytes, checksum: 58468f87414d6567ebe9a007d0fc5966 (MD5) Previous issue date: 2010-07-30 / Financiadora de Estudos e Projetos / Because the close relationship between men and dogs, possibly some ectoparasites of dogs can be observed parasitizing man. The tick Rhipicecephalus sanguineus is an ectoparasite that has the ability to carry and transmit pathogens to humans. This arthropod, that is blood-feeding, is the main biological vector of the bacteria Ehrlichia canis. Currently, the diagnosis of this disease ehrlichiasis is based on blood, biochemical and serological tests, although they are unreliable for diagnosing the disease, since their clinical and clinicopathological features are largely nonspecific. Tetracyclines are commonly used in the treatment of ehrlichiosis, but studies show that some dogs remain positive for E. canis after treatment or after the loss of spontaneous infection. The chronic ehrlichiosis, having high prognosis fails, can result in high mortality. Therefore, more sensitive and reliable tests may help in the selection of dog carrying the disease. The PCR (polymerase chain reaction) has been used successfully in the diagnosis of E. canis. However, this molecular technology requires expensive equipment and specialized personnel to handle, which limits its use in laboratories routines. A more sensitive, specific, and simple to detect the microorganism method is desirable. In this work we develop the technique for detection of the bacterium Ehrlichia canis using the LAMP (Loop- Mediated Isothermal Amplification), which proved being very effective. Specific sequences of the E. canis dsb gene (disulfide bond) were used as target for the tested techniques. Dsb gene was highly specific in order to detect E. canis, since it is divergent of phylogenetically similar bacteria. Performed molecular tests showed a disease incidence greater than that indicated by authors using traditional diagnostic techniques. In the public kennel, 80% of the samples were infected, while in private clinics and veterinary hospital 40% had the disease. The developed diagnostic technique using LAMP is more sensitive than PCR, highly specific and does not require the prior DNA extraction to amplification. In addition, the product of amplification can be seen with the naked eye. We conclude that the diagnosis by the LAMP method enables the specific and high sensitivity identification of infected animals with minimal laboratory settings. These factors make possible the use of this diagnostic methodology for veterinary clinics, laboratories and educational institutions. / Devido à estreita relação entre o homem e o cão, eventualmente alguns ectoparasitas de cães podem ser observados parasitando o homem. O carrapato Rhipicecephalus sanguineus é um ectoparasita que possui a capacidade de carregar e transmitir patógenos aos seres humanos. Esse artrópode, por exercer hematofagia, é o principal vetor biológico e reservatório da bactéria Ehrlichia canis. Atualmente, o diagnóstico da erliquiose é baseado em testes hematológicos, bioquímicos e sorológicos, embora sejam pouco confiáveis para diagnostica-lá, uma vez que suas características clínicas e clinicopatológicas são amplamente inespecíficas. As tetraciclinas são comumente utilizadas no tratamento da Erliquiose, mas estudos mostram que alguns cães permanecem soropositivos para E. canis após o tratamento ou após a perda da infecção espontânea. A Erliquiose crônica por ter falhas de prognóstico, pode resultar em alta mortalidade. Portanto, testes mais sensíveis e confiáveis poderão auxiliar na seleção de cães portadores. A PCR (reação em cadeia da polimerase) tem sido usada com sucesso no diagnóstico da bactéria E. canis. Entretanto, esta tecnologia molecular necessita de equipamentos caros e pessoal especializado para sua execução, o que limita o seu uso na rotina dos laboratórios. Assim uma metodologia mais sensível, específica, e simples de detectar o microorganismo é desejável. Neste trabalho desenvolvemos a detecção da bactéria Ehrlichia canis pena da técnica de LAMP (Loop-Mediated Isothermal Amplification), que se mostrou muito eficaz. Utilizamos sequências específicas do gene dsb (disulfide bond) de E. canis como alvo para as técnicas testadas. O gene dsb mostrou-se altamente específico para a detecção de E. canis, já que é divergente até mesmo das bactérias filogeneticamente próximas. Os testes moleculares realizados mostram uma incidência maior da doença do que aquela preconizada por autores que utilizam técnicas diagnósticas tradicionais. No canil municipal, 80% das amostras estavam infectadas; das clínicas particulares e do hospital veterinário 40% apresentaram a doença pela técnica de PCR. A técnica de diagnóstico por LAMP que foi desenvolvida é mais sensível do que o PCR, altamente específica e não é necessária a extração do DNA para a sua amplificação. Além disso, o produto da amplificação pode ser visualizado a olho nu. Concluímos que o diagnóstico pela metodologia LAMP possibilita a identificação específica e com alta sensibilidade de animais infectados, com mínima estrutura laboratorial. Tais fatores viabilizam a utilização dessa metodologia diagnóstica para clínicas veterinárias, laboratórios e instituições de ensino.

Ehrlichia ruminantium est l'agent causal de la cowdriose, une maladie tropicale mortelle des ruminantstransmis par les tiques Amblyomma. Jusqu'à présent, il n'existe pas de vaccin efficace dû à la faible protection croisée des souches vaccinales vis-à-vis des isolats de terrain. Ceci est principalement lié àdiversité génétique d'E. ruminantium au sein les zones géographiques. Par conséquent, la caractérisation de lastructure génétique de la population d'E. ruminantium à l'échelle mondiale et régionale est importante pour définir les meilleures stratégies de contrôle et améliorer les stratégies de surveillance de la cowdriose. / Ehrlichia ruminantium is the causal agent of heartwater, a ruminant tropical fatal diseasetransmitted by Amblyomma ticks. Up to now, no effective vaccine is available due to a limitedcross protection of vaccinal strains on field isolates mainly associated to a high geneticdiversity of E. ruminantium within geographical locations. Thus, both characterization of E.ruminantium genetic population structure at worldwide and regional scale and estimation of E.ruminantium tick prevalence are important to delimitate better control strategies and improveheartwater monitoring strategies

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia chaffeensis, a Gram-negative organism belonging to the order Rickettsiales, is responsible for an emerging infectious disease in humans, the human monocytic ehrlichiosis. E. chaffeensis also infects several other vertebrate hosts including dogs, goats, coyotes and white tailed deers. This organism is transmitted by an infected tick, Amblyomma americanum. The exact pathogenic mechanisms involved for the persistence of the pathogen in vertebrate hosts are still unclear. E. chaffeensis protein expression varies significantly in vertebrate and tick hosts. Differentially expressed proteins include the immunodominant outer membrane proteins encoded by the p28-Omp multigene locus. The p28-Omp 14 is expressed primarily in tick cells and the p28-Omp 19 is the major expressed protein in macrophages both under in vitro and in vivo conditions. The objective of this study is to prepare recombinant proteins and use them to assess the secondary structures and protein functions. The protein sequences were analyzed with the aid of bioinformatics programs to make structural predictions. The analysis suggested the presence of eight β barrel structures for both the p28-Omp proteins. The coding sequence of the p28-Omp genes were cloned and over expressions of proteins in in E. coli was accomplished by using the plasmid expression construct, pET28. The proteins were purified to near homogeneity and used to refold using detergents to mimic native protein structure in the bacterial outer membrane. Refolding of proteins was analyzed by two methods; SDS-PAGE and Circular Dichroism. The Circular dichroism spectroscopy analysis suggested the formation of β-sheet structures of proteins in micelles formed with the detergents. β-sheet structures may have been formed with the hydrophobic domains of the protein imbedded in the micelles. The hydrophilic segments (predicted by bio informatics analysis) may be exposed to the aqueous phase. The recombinant proteins were also iii used to prepare proteoliposomes and tested for the porin activity. The analysis demonstrated the porin activity for both p28-Omp 14 and 19 recombinant proteins by using mono-, di- and tetra- saccharides as well as for amino acid L-glutamine. This study forms the basis for initiating studies to compare the structural difference between the two differentially expressed proteins of E. chaffeensis.