Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-09-28T14:27:47Z No. of bitstreams: 1 2011 - Erica Cristina Rocha Roier.pdf: 982721 bytes, checksum: 24c0654d64de8a5b21a0eea5376f828c (MD5) / Made available in DSpace on 2016-09-28T14:27:47Z (GMT). No. of bitstreams: 1 2011 - Erica Cristina Rocha Roier.pdf: 982721 bytes, checksum: 24c0654d64de8a5b21a0eea5376f828c (MD5) Previous issue date: 2011-02-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / This study aimed to evaluate the prevalence of antibodies against Neorickettsia risticii in horses in the microregion of Itagua? RJ, show possible associated factors and identify the agent by molecular techniques. The study was conducted in the districts of Serop?dica, Itagua? and Mangaratiba, Rio de Janeiro. The 350 blood samples were obtained by convenience from horse properties belonging to the region. Serologic testing was performed by Immunofluorescence Assay (IFAI) for N. risticii, being considered positive samples titer 1:50. To evaluate risk factors associated with the presence of antibodies to N. risticii an epidemiological questionnaire was applied to the owners or guardians of animals, which aimed especially farm characteristics and management of animals. Hematological and clinical evaluation was performed for all animals. Molecular analysis by a Real Time PCR was performed from leukocyte cover. The prevalence of antibodies anti-N.risticii in horses of the region of Itagua? was 26.3% (92/350). The frequencies of IgG anti-N.rristicii were 52.2% (48/92) for titers of 1:50, zero for titers of 1:100, 13% (12/92) for titles of 1:200, 28.3% (26/92) for evidence of 1:400 and 6.5% (6 / 92) for titles of 1:800. The age and quality level of the properties were associated (p <0.05) with horses seropositivity for N. risticii. DNA from N. risticii was not detectable in samples of this study. Also no significant changes in the clinical evaluation of animals was found in relation to seropositivity for the agent. Despite showing association (p <0.05) between the seropositivity of the animals and changes in white blood cell of negative animals, these changes were not outside reference limits for the species evaluated. The existence of horses seropositive for N. risticii indicates the presence of this agent in the microregion of Itagua?. / O presente estudo teve por objetivo avaliar a preval?ncia de anticorpos contra Neorickettsia risticii em equinos na microrregi?o de Itagua?-RJ, demonstrar os poss?veis fatores associados e identificar o agente atrav?s de t?cnicas moleculares. O estudo foi conduzido nos munic?pios de Serop?dica, Itagua? e Mangaratiba, estado do Rio de Janeiro. As 350 amostras de sangue foram obtidas por conveni?ncia, das propriedades de cria??o de equinos pertencentes ? regi?o. O teste sorol?gico foi realizado atrav?s da Rea??o de Imunofluoresc?ncia Indireta (RIFI) para N. risticii, sendo consideradas positivas amostras com t?tulo 1:50. Para avalia??o dos fatores de risco associados ? presen?a de anticorpos de N. risticii, aplicou-se um question?rio epidemiol?gico com os propriet?rios e/ou respons?veis dos animais, destacando caracter?sticas da propriedade e manejo dos animais. Realizou-se hemograma e avalia??o cl?nica de todos os animais. As an?lises moleculares atrav?s da t?cnica de Real Time PCR foram realizadas a partir de capa leucocit?ria. A preval?ncia de anticorpos anti-N. risticii em equinos da microrregi?o de Itagua? foi de 26,3% (92/350) pela RIFI. As frequ?ncias de anticorpos IgG anti-N. risticii foram 52,2% (48/92) para titula??es de 1:50, zero para titula??es de 1:100, 13% (12/92) para t?tulos de 1:200, 28,3% (26/92) para t?tulos de 1:400 e 6,5% (6/92) para t?tulos de 1:800. A idade e o n?vel de qualidade das propriedades apresentaram associa??o (p<0,05) com a soropositividade dos equinos para N. risticii. N?o foi detectado o DNA de N. risticii nas amostras do presente estudo. Tamb?m n?o foram evidenciadas altera??es significativas na avalia??o clinica dos animais, em rela??o ? soropositividade para o agente. Apesar de haver associa??o (p<0,05) entre a soropositividade dos animais e altera??es no leucograma dos animais negativos, estas altera??es n?o estiveram fora dos limites de refer?ncia para a esp?cie animal avaliada. A exist?ncia de equinos soropositivos para N. risticii indica a circula??o desse agente na microrregi?o de Itagua?

Tick-borne diseases are caused by a group of pathogenic microorganisms which are transmitted between animal and human population by vector which is most frequently tick. Evaluation of danger and perils of these diseases is important due to prevention and monitoring of current situation development. The most common tick-borne diseases in the Czech Republic are borreliosis, tick-borne encephalitis and ehrlichiosis. Rarely can we encounter bartonellosis, babesiosis, rickettsiosis and tularemia. All these diseases can have serious consequences and in critical cases they can result in death of the infected person. One of the basic and the most important of preventive measures which lower the risk of the infection by any tick-borne disease is prevention of tick encounter and eventually vaccination. Due to continual global warming we can expect that in near future ticks will spread even to locations which have been so far not suitable for their development. We can expect that we will even more frequently encounter tick-borne diseases - including diseases which were formerly not found in our territory or were very rare. As a part of health education it will be needed more to get known basic information of tick-borne diseases and their prevention. Different educational centres are an ideal place for...

Made available in DSpace on 2014-06-11T19:31:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-24Bitstream added on 2014-06-13T18:06:52Z : No. of bitstreams: 1 nakaghi_ach_dr_jabo.pdf: 627442 bytes, checksum: 53f339a01b25f98c06e48eed8f59cc13 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente estudo objetivou clonar e analisar a expressão do gene p28 da amostra Jaboticabal de Ehrlichia canis e avaliar sua utilização no diagnóstico molecular e sorológico da erliquiose canina. A PCR, baseada no gene p28 de E. canis, foi capaz de detectar o DNA de um único parasita entre um bilhão de células. Amostras sangüíneas de cães com suspeita clínica de erliquiose foram testadas pela PCR, baseada no gene p28 e pela nested PCR para detecção do gene 16S rRNA. O fragmento amplificado do gene p28 foi observado em 51,25% (n=41) das 80 amostras examinadas, e todas elas foram positivas para o gene 16S rRNA de E. canis, entretanto, em outros 21,25% (n=17) das amostras apenas o gene 16S rRNA foi detectado. A caracterização molecular do gene p28 mostrou similaridade com outras amostras de E. canis. Para a expressão da proteína recombinante P28, foram testados dois sistemas diferentes com linhagens de Escherichia coli BL21(DE3), BL21 Star(DE3)pLysS, BL21(DE3) pLysS e BL21-CodonPlus(DE3)-RIL. A análise pelo Western-blotting revelou reatividade de várias frações protéicas com anticorpo policlonal anti-E. canis. Porém, quando a membrana foi incubada com anticorpo monoclonal para detecção de moléculas de histidina, não houve reatividade, mostrando a ausência de expressão da proteína P28. / The aim of this study was to clone and express the p28 gene of Ehrlichia canis Jaboticabal strain and evaluate its application in molecular and serological diagnosis of canine ehrlichiosis. The p28-based PCR was able to detect E. canis DNA of one parasite among one billion cells. Blood samples from dogs for which a diagnosis of canine ehrlichiosis was suspected were tested by p28-based PCR and by 16S rRNA-based nested PCR. Amplified fragment from p28 gene were observed in 51.25% (n=41) among 80 assayed samples and all of this positive samples were also positives for 16S rRNA gene of E. canis, however in 21.25% (n=17) only 16S rRNA gene was detected. The molecular characterization of p28 gene showed similarity with others strains of E. canis. Expression of P28 recombinant protein was tested with two different systems and in Escherichia coli BL21(DE3), BL21 Star(DE3)pLysS, BL21(DE3) pLysS and BL21-CodonPlus(DE3)-RIL strains. After expression induction, Westernblotting showed reactivity of some protein fractions against anti-E. canis antibodies. However, when samples were incubated with anti-histidine monoclonal antibodies, they did not react, demonstrating non-expression of P28 protein.

Orientadora: Rosangela Zacarias Machado / Banca: Fernando Luiz de Lucca / Banca: Maria Célia Bertolini / Banca: Aramis Augusto Pinto / Banca: Jesus Aparecido Ferro / Resumo: O presente estudo objetivou clonar e analisar a expressão do gene p28 da amostra Jaboticabal de Ehrlichia canis e avaliar sua utilização no diagnóstico molecular e sorológico da erliquiose canina. A PCR, baseada no gene p28 de E. canis, foi capaz de detectar o DNA de um único parasita entre um bilhão de células. Amostras sangüíneas de cães com suspeita clínica de erliquiose foram testadas pela PCR, baseada no gene p28 e pela nested PCR para detecção do gene 16S rRNA. O fragmento amplificado do gene p28 foi observado em 51,25% (n=41) das 80 amostras examinadas, e todas elas foram positivas para o gene 16S rRNA de E. canis, entretanto, em outros 21,25% (n=17) das amostras apenas o gene 16S rRNA foi detectado. A caracterização molecular do gene p28 mostrou similaridade com outras amostras de E. canis. Para a expressão da proteína recombinante P28, foram testados dois sistemas diferentes com linhagens de Escherichia coli BL21(DE3), BL21 Star(DE3)pLysS, BL21(DE3) pLysS e BL21-CodonPlus(DE3)-RIL. A análise pelo Western-blotting revelou reatividade de várias frações protéicas com anticorpo policlonal anti-E. canis. Porém, quando a membrana foi incubada com anticorpo monoclonal para detecção de moléculas de histidina, não houve reatividade, mostrando a ausência de expressão da proteína P28. / Abstract: The aim of this study was to clone and express the p28 gene of Ehrlichia canis Jaboticabal strain and evaluate its application in molecular and serological diagnosis of canine ehrlichiosis. The p28-based PCR was able to detect E. canis DNA of one parasite among one billion cells. Blood samples from dogs for which a diagnosis of canine ehrlichiosis was suspected were tested by p28-based PCR and by 16S rRNA-based nested PCR. Amplified fragment from p28 gene were observed in 51.25% (n=41) among 80 assayed samples and all of this positive samples were also positives for 16S rRNA gene of E. canis, however in 21.25% (n=17) only 16S rRNA gene was detected. The molecular characterization of p28 gene showed similarity with others strains of E. canis. Expression of P28 recombinant protein was tested with two different systems and in Escherichia coli BL21(DE3), BL21 Star(DE3)pLysS, BL21(DE3) pLysS and BL21-CodonPlus(DE3)-RIL strains. After expression induction, Westernblotting showed reactivity of some protein fractions against anti-E. canis antibodies. However, when samples were incubated with anti-histidine monoclonal antibodies, they did not react, demonstrating non-expression of P28 protein. / Doutor

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In impoverished and resource limited communities such as townships, and welfare organizations, areas such as living and sleeping spaces are sometimes shared with animals, and occasionally humans. Dogs play an integral role in our lives and have become part of the family. Therefore, it is probable that ectoparasites, such as ticks, that feed on dogs also feed on other vertebrates, thereby, transmitting pathogens. The primary aim of this study was to screen for the presence of tick-borne pathogens in dogs from welfare organisations and townships in Cape Town, with special focus on Ehrlichia and Babesia spp. The reason for this choice of subject is due to the fact that very few tick-borne infection studies have focused on resource limited communities. Furthermore, welfare organisations have continuously attracted awareness due to the amount of unrestricted work performed by veterinarians in communities with limited resources. Consequently, the topic was borne. A total of 126 blood samples and 509 ticks (adults and nymphs) were collected directly from dogs from four welfare organisations and two townships in Cape Town. Samples were collected from April to July 2014. The four welfare organisations where samples were collected included the Animal Anti Cruelty League welfare organisations in Epping and Bellville, the Lucky Lucy Foundation in Joostenberg Vlakte and The Emma Animal Rescue Society (TEARS), located in the Sunnydale area. Samples were also collected from the Asanda village and Nomzamo, two townships located just outside the Cape Town suburb, the Strand. DNA was extracted from blood and ectoparasites and screened for the presence of Ehrlichia, Anaplasma, Theileria and Babesia species infections using touchdown PCR and RLB hybridization assays. Genus and species-specific probes were used during hybridization in order to identify specific parasite infections in the blood samples and the tick samples pooled according to geographical origin and species. Forty six (36.5%) of the blood samples tested positive for tick-borne pathogen DNA. Of the positive blood samples, 17 (13.5%) were infected with Ehrlichia canis; 16 (12.7%) with Babesia rossi and four (3.2%) samples were infected with Babesia vogeli. Incidental infections were also detected, these included Ehrlichia ruminantium (n=6, [4.7%]), Theileria taurotragi (n=2, [1.6%]) and Anaplasma sp. Omatjenne (n=1, [0.8%]) infections. DNA detected from 10 samples (7.94%) hybridized only to the Ehrlichia/Anaplasma genus-specific probes and four samples (3.17%) hybridized only to the Theileria/Babesia genus-specific probes. None of these 14 samples hybridized to any of the species-specific probes. Collected Rhipicephalus sanguineus (n=457) and Haemaphysalis elliptica (n=52) ticks were grouped into 15 pools, representing both tick species according to specific collection locations. Since only two H. elliptica from Asanda and one R. sanguineus from TEARS were collected, these ticks were mixed in pools of the dominant species as they were too few for DNA extraction. Ticks were collected from the Nomzamo Township (R. sanguineus n=400), Asanda village (H. elliptica n=2; R. sanguineus n=42), TEARS (H. elliptica n=21; R. sanguineus n=1), and the Animal Anti Cruelty League in both Epping (R. sanguineus n=14), and Bellville (H. elliptica n=29), in Cape Town. Analysis by the RLB assay showed that 11 (73.3%) of the 15 tick pools representing both tick species were positive for at least one parasite species. All positive samples hybridized with the Ehrlichia/Anaplasma genus-specific probe. Three (20%) tick pools containing both tick species tested positive for Ehrlichia canis infection, two (13.3%) tested positive for Babesia rossi and Babesia vogeli DNA was identified in one (6.6%) tick pool. The Theileria/Babesia genus-specific probe hybridised in three (20%) tick pools. These three pools were comprised of both R. sanguineus and H. elliptica tick species. These tick pools also tested positive for a specific Babesia tick-borne pathogen. Tick-borne pathogen DNA could not be detected in four (26.6%) tick pools. The fore-mentioned tick-borne pathogen DNA detected in the dog blood samples, and the ectoparasites collected from the same dogs during this study, suggests that dogs play a large role in the endemicity of these pathogens / Environmental Sciences / M. Sc. (Life Science)