Made available in DSpace on 2014-06-11T19:31:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-01-19Bitstream added on 2014-06-13T20:22:02Z : No. of bitstreams: 1 faria_jlm_dr_jabo.pdf: 2769905 bytes, checksum: 15c4be53d67c11751ac4644728993d4f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / 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. / 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.

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.

Die vorliegende epidemiologische Studie umfaßte 153 Hunde aus der Nähe von Athen, Griechenland. Um die Prävalenz arthropogener Infektionen abschätzen zu können, wurden Serumproben auf Antikörper gegen Leishmania infantum, Ehrlichia canis und Borrelia burgdorferi sowie auf Antigene von Dirofilaria immitis überprüft. Blutausstriche wurden auf das Vorkommen von Babesia canis und Hepatozoon canis untersucht. Außerdem wurden von den Hunden abgesammelte Zecken bestimmt. Bei 126 Hunden erfolgte eine klinische Allgemeinuntersuchung. Des weiteren wurden die serologischen Testverfahren ELISA und IFAT für den Nachweis von Antikörpern gegen Borrelia burgdorferi miteinander verglichen. Insgesamt waren 94 (61,4 %) der 153 untersuchten Hunde infiziert, 63 (41,2 %) Hunde wiesen Antikörper gegen Ehrlichia canis auf. Infektionen mit Borrelia burgdorferi wurden im ELISA insgesamt bei 43 (28,1 %), im IFAT bei 35 (22,9 %) Tieren nachgewiesen. Außerdem konnte bei 28 (18,3 %) Tieren eine Infektion mit Leishmania infantum und bei 20 (13,1 %) mit Dirofilaria immitis gefunden werden. Ein Nachweis von Babesia canis im Blutausstrich gelang bei vier (2,6 %) Hunden, von Hepatozoon canis nur bei einem (0,7 %) Hund. Für keinen der untersuchten Infektionserreger konnten signifikante Alters-, Geschlechts- oder Rasseprädispositionen festgestellt werden. Die Infektionsraten mit Leishmanien, Babesien, Dirofilarien und Borrelien waren bei den im Tierheim lebenden Hunden geringer als bei den Streunern. Vierundvierzig (28,8 %) der 153 getesteten Hunde waren gleichzeitig mit zwei, drei oder vier durch Arthropoden übertragene Erregerarten infiziert. Zweifachinfektionen kamen bei 29 (19,0 %), Dreifachinfektionen bei sieben (4,6 %) und Vierfachinfektionen bei acht (5,2 %) Tieren vor. In der Regel war das Risiko für Hunde, einen Erreger zu beherbergen, höher, wenn bereits eine Infektion mit einem anderen Erreger vorhanden war. Insgesamt waren 28 (18,3 %) Streuner und 15 (9,8 %) Tierheimhunde von Mehrfachinfektionen betroffen. Von den 94 mit mindestens einem der berücksichtigten Erreger infizierten Hunden wurden 75 (79,8 %) einer klinischen Untersuchung unterzogen. Dreiunddreißig (44,0 %) dieser Tiere zeigten Krankheitserscheinungen. Bei 21 (67,7 %) der 31 klinisch untersuchten und mit mehreren Erregern gleichzeitig infizierten Hunde konnten Krankheitssymptome diagnostiziert werden. Von 44 klinisch untersuchten Tieren, die nur mit einem Erreger infiziert waren, zeigten 12 (27,3 %) klinische Symptome. Die diagnostizierten Symptome waren bei allen untersuchten Erregern von einer starken Variabilität geprägt. Die Übereinstimmung der Testsysteme IFAT und ELISA für den Nachweis von Antikörpern gegen Borrelia burgdorferi lag bei 92,2 %. Die Testverfahren sind damit als nahezu gleichwertig anzusehen, wobei der ELISA etwas sensitiver zu sein scheint. Eine Infektionsgefahr besteht demnach in Griechenland vor allem für Ehrlichia canis, Borrelia burgdorferi und Leishmania infantum, aber auch für Infektionen mit Dirofilaria immitis besteht ein Risiko. Infektionen mit Babesia canis kommen in dieser Region seltener vor, es muss jedoch insbesondere bei geschwächten oder in Deutschland geborenen und somit hochempfänglichen Tieren mit Erkrankungen gerechnet werden. Hepatozoon canis ist nach den vorliegenden Ergebnissen in der Umgebung von Athen von geringer Verbreitung. Ein Rückschluß vom klinischen Bild auf das Vorliegen einer Infektion mit einem bestimmten Erreger kann aufgrund der Variabilität der Symptome in Verbindung mit häufig auftretenden Mehrfachinfektionen nicht gezogen werden. Die Möglichkeit von gleichzeitig vorliegenden Infektionen sollte in der Diagnostik und Therapie unbedingt berücksichtigt werden. Gute Haltungsbedingungen und eine tierärztliche Überwachung und Prophylaxe reduzieren offensichtlich die Inzidenz von Infektionen mit arthropodenübertragenen Erregern. Die überwiegende Anzahl (449 von 457 Exemplaren) der bestimmten Zecken gehörte der Art Rhipicephalus sanguineus an. Es wurden sechs Nymphen und 443 adulte Tiere gefunden. Die adulten Tiere verteilten sich auf 243 männliche und 200 weibliche Zecken. Bei acht Zecken handelte es sich um weibliche Ixodes ricinus. Bei aus Griechenland stammenden Hunden wie auch bei Tieren, die sich reisebegleitend dort aufgehalten haben, muss mit einer starken Infestation mit Rhipicephalus sanguineus gerechnet werden. Da Rhipicephalus sanguineus der Vektor für Ehrlichia canis, Babesia canis vogeli und Hepatozoon canis ist, sollten Prophylaxemaßnahmen eine geeignete Zeckenbekämpfung einbeziehen. / 153 dogs from the environs of Athens, Greece, were surveyed for tick infestation and arthropod borne infections. Serology was performed for Leishmania infantum, Dirofilaria immitis, Ehrlichia canis and Borrelia burgdorferi and bloodfilms were microscopically examined for Babesia canis and Hepatozoon canis. Ticks collected from the dogs were differentiated. 126 dogs underwent clinical examination. Suitability of an indirect immunofluorescent antibody assay (IFAT) and an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of antibodies to Borrelia burgdorferi was compared. Altogether 94 (61.4 %) dogs were infected with an arthropod borne pathogen, 63 (41.2 %) produced antibodies to Ehrlicha canis. ELISA detected Borrelia burgdorferi infection in 43 (28.1 %) dogs while IFAT was positive in 35 (22.9 %). 28 (18.3 %) dogs were infected with Leishmania infantum and 20 (13.1 %) with Dirofilaria immitis. Babesia canis was found in blood smears of four (2.6 %) dogs, Hepatozoon canis was detected only in one case (0,7 %). No association was found between the breed, age or sex of the dogs and any of the tested pathogens. The rate of infection with Leishmania, Babesia, Dirofilaria or Borrelia was lower in dogs living in the animal shelter than in those living as strays. 44 (28.8 %) of the 153 dogs examined were infected concurrently with two (n = 29, 19,0 %), three (n = 7, 4,6 %) or four (n = 8, 5,2 %) arthropod borne pathogens. In general the risk of infection was higher in dogs that were already infected with another pathogen. Altogether 28 (18.3 %) of the strays and 15 (9.8 %) of the dogs living in the animal shelter had a multiple infection. 75 of the 94 dogs infected with at least one of the tested pathogens were examined clinically. 33 (44.0 %) showed clinical symptoms. In 21 (67,7 %) of the 31 clinically examined dogs with multiple infection symptoms of disease were diagnosed. Twelve (27.3 %) of the 44 clinically examined dogs that were only infected with one pathogen had symptoms of some kind. Clinical symptoms varied considerably irrespective of the causative agent. The concordance of the IFAT and the ELISA for the diagnosis of Borrelia burgdorferi was 92.2 %. The serologic diagnosis of Borrelia burgdorferi with IFAT or ELISA can be regarded similarly suitable although ELISA seems to be somewhat more sensitive. In conclusion the risk of infection in Greece is particularly high for Ehrlichia canis, Borrelia burgdorferi and Leishmania infantum and Dirofilaria immitis as well. Babesia canis is obviously transmitted less. However dogs in poor condition or born in non endemic areas may acquire babesiosis in Greece. According to the results of this study Hepatozoon canis seems to be of negligible relevance in Greece. Due to the variability of the clinical symptoms and the frequent occurrence of multiple infections diagnosis based on the clinical picture only is not possible. Multiple infections should also be considered with respect to treatment. Infections with arthropod borne pathogens seem to occur less often in prophylactically treated dogs living under good conditions. Most of the examined ticks were identified as Rhipicephalus sanguineus (449 out of 457 specimens). Six nymphs and 419 adults were found. Of the adult Rhipicephalus sanguineus ticks 243 were males and 200 were females. Eight of the ticks were female Ixodes ricinus. In dogs originating from Greece as well as in dogs having accompanied tourists into Greece infestation with Rhipicephalus sanguineus is probable. As Rhipicephalus sanguineus is known to transmit Ehrlicha canis, Babesia canis vogeli and Hepatozoon canis prophylactic measures should include a suitable tick-control.

Orientadora: Rosangela Zacarias Machado / Banca: Jose Mauricio Barbante Duarte / Banca: Natalino Hajime Yoshinari / Resumo: 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. / Abstract: 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. / Mestre

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.

Made available in DSpace on 2014-06-11T19:23:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-19Bitstream added on 2014-06-13T20:30:45Z : No. of bitstreams: 1 munhoz_td_me_jabo.pdf: 517533 bytes, checksum: 9d7063c670aa086ee9a1d333248d04b8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A erliquiose canina é uma doença de alta incidência na região nordeste do Estado de São Paulo, sendo responsável pela morte de muitos cães. O diagnóstico precoce favorece a pronta instituição do tratamento e melhora o prognóstico do animal. Estudos têm apontado que a determinação da concentração de proteínas de fase aguda (PFA) pode contribuir para detecção precoce de doenças e auxiliar na predição do prognóstico. O presente estudo objetivou avaliar o perfil de proteínas de fase aguda (PFA) em cães experimentalmente infectados com Ehrlichia canis, amostra Jaboticabal, no início da infecção e após o tratamento. Para tanto, foram utilizados 10 cães sem contato prévio com hemoparasitas. Deste, cinco foram infectados e cinco serviram de controle da infecção. Hemogramas, nested PCR, detecção de anticorpos anti-E. canis e eletroforetograma de proteínas séricas foram realizados em períodos pré-determinados. Os resultados mostraram que a proteína-C reativa, ceruplasmina e a α1-glicoproteína ácida tiveram suas concentrações aumentadas antes do aparecimento dos sinais clínicos e das alterações de hemograma nos cães infectados. Os sinais clínicos da doença tornaram-se evidentes por volta do 17º dia de infecção. Trombocitopenia foi registrada a partir do 3º dia de infecção. Mórulas intracitoplasmáticas foram detectadas a partir do 15º dia. Títulos sorológicos anti-E. canis, variando de 1:2560 a 1:5120, e nPCR positiva foram evidenciados no 18º dia. Nas avaliações após o tratamento os cães infectados estavam assintomáticos, com nPCR negativo e acentuada redução dos títulos de anticorpos específicos em quatro dos cinco cães. Todas as PFA estudadas reduziram suas concentrações a títulos próximos aos iniciais. Este estudo mostrou que as mensurações das PFA contribuem para o diagnóstico precoce e predição de cura... / Canine ehrlichiosis is an endemic disease, with high incidence in the Northwest area of Sao Paulo State – Brazil and it is responsible for the death of numerous dogs. The rapid and accurate diagnosis helps the prompt establishment of treatment and improves the prognosis of the animal. Evidence has shown that the measurement of acute phase proteins (APP) can contribute for the early detection of the disease and help to predict its prognosis. This study evaluated the profile of APP in dogs experimentally infected with Ehrlichia canis, Jaboticabal – SP – Brazil sample, at the onset of the infection and after its treatment. Ten dogs, with no previous hemoparasitosis, were randomly assigned in either the infected group (5 animals), which received the bacteria, or the control group (5 animals). In pre-determined intervals, their blood was colected and hemogram, nested PCR, anti-E. canis antibodies detection and tritation and eletrophoresis of serum proteins were performed. We showed that, in infected dogs, the concentration of C-reactive protein, ceruplasmin and α-1 acid glycoprotein were increased previous to the clinical signs forthcoming and hemogram alterations. The clinical signs were evident around the 17th day of infection. Thrombocytopenia was found on the 3th day of infection. Intra-cytosolic morules were detected on the 15th day of infection. Sorologic tritation of anti-E. canis, ranging from 1:2560 to 1:5120, and positive nPCR were found on the 18th day of infection. In the post-treatment evaluation, the infected dogs were asymptomatic with negative nPCR and decreased tritation of specific antibodies in four out of five dogs. All APP analyzed were similar to basal levels. This study showed that the measurement of APP can, indeed, contribute for the early diagnosis and prediction of cure, after treatment, of the experimental acute phase of canine Ehrlichiosis.

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

Die vorliegende epidemiologische Studie umfaßte 153 Hunde aus der Nähe von Athen, Griechenland. Um die Prävalenz arthropogener Infektionen abschätzen zu können, wurden Serumproben auf Antikörper gegen Leishmania infantum, Ehrlichia canis und Borrelia burgdorferi sowie auf Antigene von Dirofilaria immitis überprüft. Blutausstriche wurden auf das Vorkommen von Babesia canis und Hepatozoon canis untersucht. Außerdem wurden von den Hunden abgesammelte Zecken bestimmt. Bei 126 Hunden erfolgte eine klinische Allgemeinuntersuchung. Des weiteren wurden die serologischen Testverfahren ELISA und IFAT für den Nachweis von Antikörpern gegen Borrelia burgdorferi miteinander verglichen. Insgesamt waren 94 (61,4 %) der 153 untersuchten Hunde infiziert, 63 (41,2 %) Hunde wiesen Antikörper gegen Ehrlichia canis auf. Infektionen mit Borrelia burgdorferi wurden im ELISA insgesamt bei 43 (28,1 %), im IFAT bei 35 (22,9 %) Tieren nachgewiesen. Außerdem konnte bei 28 (18,3 %) Tieren eine Infektion mit Leishmania infantum und bei 20 (13,1 %) mit Dirofilaria immitis gefunden werden. Ein Nachweis von Babesia canis im Blutausstrich gelang bei vier (2,6 %) Hunden, von Hepatozoon canis nur bei einem (0,7 %) Hund. Für keinen der untersuchten Infektionserreger konnten signifikante Alters-, Geschlechts- oder Rasseprädispositionen festgestellt werden. Die Infektionsraten mit Leishmanien, Babesien, Dirofilarien und Borrelien waren bei den im Tierheim lebenden Hunden geringer als bei den Streunern. Vierundvierzig (28,8 %) der 153 getesteten Hunde waren gleichzeitig mit zwei, drei oder vier durch Arthropoden übertragene Erregerarten infiziert. Zweifachinfektionen kamen bei 29 (19,0 %), Dreifachinfektionen bei sieben (4,6 %) und Vierfachinfektionen bei acht (5,2 %) Tieren vor. In der Regel war das Risiko für Hunde, einen Erreger zu beherbergen, höher, wenn bereits eine Infektion mit einem anderen Erreger vorhanden war. Insgesamt waren 28 (18,3 %) Streuner und 15 (9,8 %) Tierheimhunde von Mehrfachinfektionen betroffen. Von den 94 mit mindestens einem der berücksichtigten Erreger infizierten Hunden wurden 75 (79,8 %) einer klinischen Untersuchung unterzogen. Dreiunddreißig (44,0 %) dieser Tiere zeigten Krankheitserscheinungen. Bei 21 (67,7 %) der 31 klinisch untersuchten und mit mehreren Erregern gleichzeitig infizierten Hunde konnten Krankheitssymptome diagnostiziert werden. Von 44 klinisch untersuchten Tieren, die nur mit einem Erreger infiziert waren, zeigten 12 (27,3 %) klinische Symptome. Die diagnostizierten Symptome waren bei allen untersuchten Erregern von einer starken Variabilität geprägt. Die Übereinstimmung der Testsysteme IFAT und ELISA für den Nachweis von Antikörpern gegen Borrelia burgdorferi lag bei 92,2 %. Die Testverfahren sind damit als nahezu gleichwertig anzusehen, wobei der ELISA etwas sensitiver zu sein scheint. Eine Infektionsgefahr besteht demnach in Griechenland vor allem für Ehrlichia canis, Borrelia burgdorferi und Leishmania infantum, aber auch für Infektionen mit Dirofilaria immitis besteht ein Risiko. Infektionen mit Babesia canis kommen in dieser Region seltener vor, es muss jedoch insbesondere bei geschwächten oder in Deutschland geborenen und somit hochempfänglichen Tieren mit Erkrankungen gerechnet werden. Hepatozoon canis ist nach den vorliegenden Ergebnissen in der Umgebung von Athen von geringer Verbreitung. Ein Rückschluß vom klinischen Bild auf das Vorliegen einer Infektion mit einem bestimmten Erreger kann aufgrund der Variabilität der Symptome in Verbindung mit häufig auftretenden Mehrfachinfektionen nicht gezogen werden. Die Möglichkeit von gleichzeitig vorliegenden Infektionen sollte in der Diagnostik und Therapie unbedingt berücksichtigt werden. Gute Haltungsbedingungen und eine tierärztliche Überwachung und Prophylaxe reduzieren offensichtlich die Inzidenz von Infektionen mit arthropodenübertragenen Erregern. Die überwiegende Anzahl (449 von 457 Exemplaren) der bestimmten Zecken gehörte der Art Rhipicephalus sanguineus an. Es wurden sechs Nymphen und 443 adulte Tiere gefunden. Die adulten Tiere verteilten sich auf 243 männliche und 200 weibliche Zecken. Bei acht Zecken handelte es sich um weibliche Ixodes ricinus. Bei aus Griechenland stammenden Hunden wie auch bei Tieren, die sich reisebegleitend dort aufgehalten haben, muss mit einer starken Infestation mit Rhipicephalus sanguineus gerechnet werden. Da Rhipicephalus sanguineus der Vektor für Ehrlichia canis, Babesia canis vogeli und Hepatozoon canis ist, sollten Prophylaxemaßnahmen eine geeignete Zeckenbekämpfung einbeziehen. / 153 dogs from the environs of Athens, Greece, were surveyed for tick infestation and arthropod borne infections. Serology was performed for Leishmania infantum, Dirofilaria immitis, Ehrlichia canis and Borrelia burgdorferi and bloodfilms were microscopically examined for Babesia canis and Hepatozoon canis. Ticks collected from the dogs were differentiated. 126 dogs underwent clinical examination. Suitability of an indirect immunofluorescent antibody assay (IFAT) and an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of antibodies to Borrelia burgdorferi was compared. Altogether 94 (61.4 %) dogs were infected with an arthropod borne pathogen, 63 (41.2 %) produced antibodies to Ehrlicha canis. ELISA detected Borrelia burgdorferi infection in 43 (28.1 %) dogs while IFAT was positive in 35 (22.9 %). 28 (18.3 %) dogs were infected with Leishmania infantum and 20 (13.1 %) with Dirofilaria immitis. Babesia canis was found in blood smears of four (2.6 %) dogs, Hepatozoon canis was detected only in one case (0,7 %). No association was found between the breed, age or sex of the dogs and any of the tested pathogens. The rate of infection with Leishmania, Babesia, Dirofilaria or Borrelia was lower in dogs living in the animal shelter than in those living as strays. 44 (28.8 %) of the 153 dogs examined were infected concurrently with two (n = 29, 19,0 %), three (n = 7, 4,6 %) or four (n = 8, 5,2 %) arthropod borne pathogens. In general the risk of infection was higher in dogs that were already infected with another pathogen. Altogether 28 (18.3 %) of the strays and 15 (9.8 %) of the dogs living in the animal shelter had a multiple infection. 75 of the 94 dogs infected with at least one of the tested pathogens were examined clinically. 33 (44.0 %) showed clinical symptoms. In 21 (67,7 %) of the 31 clinically examined dogs with multiple infection symptoms of disease were diagnosed. Twelve (27.3 %) of the 44 clinically examined dogs that were only infected with one pathogen had symptoms of some kind. Clinical symptoms varied considerably irrespective of the causative agent. The concordance of the IFAT and the ELISA for the diagnosis of Borrelia burgdorferi was 92.2 %. The serologic diagnosis of Borrelia burgdorferi with IFAT or ELISA can be regarded similarly suitable although ELISA seems to be somewhat more sensitive. In conclusion the risk of infection in Greece is particularly high for Ehrlichia canis, Borrelia burgdorferi and Leishmania infantum and Dirofilaria immitis as well. Babesia canis is obviously transmitted less. However dogs in poor condition or born in non endemic areas may acquire babesiosis in Greece. According to the results of this study Hepatozoon canis seems to be of negligible relevance in Greece. Due to the variability of the clinical symptoms and the frequent occurrence of multiple infections diagnosis based on the clinical picture only is not possible. Multiple infections should also be considered with respect to treatment. Infections with arthropod borne pathogens seem to occur less often in prophylactically treated dogs living under good conditions. Most of the examined ticks were identified as Rhipicephalus sanguineus (449 out of 457 specimens). Six nymphs and 419 adults were found. Of the adult Rhipicephalus sanguineus ticks 243 were males and 200 were females. Eight of the ticks were female Ixodes ricinus. In dogs originating from Greece as well as in dogs having accompanied tourists into Greece infestation with Rhipicephalus sanguineus is probable. As Rhipicephalus sanguineus is known to transmit Ehrlicha canis, Babesia canis vogeli and Hepatozoon canis prophylactic measures should include a suitable tick-control.