Thesis (Ph. D.)--University of Sydney, 2009. / Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Pathology, Faculty of Medicine. Title from title screen (viewed Sept. 30, 2009) Includes bibliography. Also available in print form.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 139 pages. Includes vita. Includes bibliographical references.

Orientador: Mary Marcondes Feitosa / Banca: Márcia Dalastra Laurenti / Banca: Aparecido Antonio Camacho / Resumo: A importância do diagnóstico da leishmaniose visceral canina no Brasil reside no fato de que, dentre as estratégias de controle da doença indicadas pela Fundação Nacional de Saúde, encontra-se a eliminação do cão doméstico sorologicamente positivo. Desta forma, torna-se necessário o conhecimento da sensibilidade e da especificidade das provas sorológicas utilizadas para a correta identificação destes animais. Para avaliar o desempenho dos métodos sorológicos no diagnóstico da leishmaniose visceral canina, foram determinadas e comparadas as características dos métodos de ELISA, reação de imunofluorescência indireta (RIFI) e imunocromatografia, adotando-se como padrão o método parasitológico. Para tanto, foram utilizados 50 cães naturalmente acometidos por leishmaniose visceral e 45 cães sadios provenientes de área não endêmica para a doença. A RIFI revelou sensibilidade de 98% e especificidade de 91,1%, além de ótima concordância com o método parasitológico (Kappa = 0,893). Os métodos de ELISA e de imunocromatografia apresentaram boa concordância com o método parasitológico (coeficientes Kappa de 0,788 e 0,769, respectivamente) e valores de sensibilidade de 94% e 86% e especificidade de 84,4% e 91,1%, respectivamente. Com relação à ocorrência de reações cruzadas com leishmaniose visceral, das 14 amostras de soro positivas para doença de Chagas, nove (64,3%) foram consideradas positivas pela técnica de ELISA e seis (42,9%) pela RIFI, não se observando resultados positivos por imunocromatografia. Das 13 amostras de soros de animais portadores de erliquiose, uma (7,7%) apresentou resultado positivo por meio dos métodos de ELISA e de imunocromatografia. / Abstract: The importance of canine visceral leishmaniasis diagnosis in Brazil is based on the fact that, according to the Ministry of Health, a seropositive dog must be culled. Thus, itþs important to know the sensitivity and specificity values of the methods employed for the diagnosis of the disease. To evaluate the performance of the available sorological tests for the diagnosis of canine leishmaniasis it was determined and compared the sensitivity and specificity of ELISA, indirect immunofluorescent antibody test (IFAT) and immunochromatographic test using the parasitological test as the gold standard. For this purposal, the study was carried out with a group of 50 naturally infected dogs from an endemic area and 45 healthy dogs from a non endemic area for visceral leishmaniasis. The IFAT was 98% sensitive and 91,1% specific, and showed a very good concordance with the parasitiological test (k = 0,893). The ELISA showed a sensitivity of 94% and specificity of 84,4% and when kappa index was analysed, a good concordance was obseved (k = 0,788). The immunochromatographic test was 86% sensisitive and 91,1% specific and showed a good concordance with the parasitiological test (k = 0,769). About the occurrence of cross reaction with visceral leishmaniasis, nine (64,3%) out of 14 positive samples for Chagas’ disease were positive by ELISA and six (42,9%) out of 14 were positive by IFAT, while the immunocromatographic test didn’t yield any positive result. One (7,7%), out of 13 positive samples for ehrlichiosis, was positive by ELISA and immunocromatografic test. Five (83,3%) out of six positive samples for ehrlichiosis and babesiosis were positive by ELISA and three (50%) by immunocromatography, while IFAT didnþt yield any positive result. Five (50%) out of 10 positive samples for toxoplasmosis were positive by IFAT and one (10%) by immunocromatography. / Mestre

Orientador: Henrique Nunes de Oliveira / Coorientador: Márcia Cristina de Sena Oliveira / Banca: Jesus Aparecido Ferro / Banca: Teresa Cristina Goulart de Oliveira Sequeira / Banca: Anderson Ferreira da Cunha / Banca: Marcos Rogério André / Resumo: A babesiose bovina é uma hemoparasitose transmitida pelo carrapato Rhipicephalus (Boophilus) microplus que causa grandes prejuízos à bovinocultura do Brasil. Entre as espécies que causam as babesioses bovinas, B. bovis se destaca como a mais patogênica. É amplamente conhecida a resistência natural dos bovinos do grupo genético Bos taurus indicus frente a B. bovis. Entretanto, a relação quantitativa do nível de infecção em animais Bos taurus taurus e seus cruzamentos é desconhecida. O método clássico de detecção e quantificação da parasitemia por B. bovis apresenta baixa sensibilidade e falha no diagnostico em animais portadores. Recentemente, a técnica de qPCR tem sido utilizada com grande sucesso para este propósito. Assim, o presente estudo teve como objetivo verificar a associação entre o nível de infestação por R. (B.) microplus, a soroprevalência e a parasitemia por B. bovis, estimada através dos métodos do ELISA-teste e qPCR em bovinos de corte naturalmente infestados, de diferentes locais e grupos genéticos (grupos-locais).Verificou-se que a infestação por carrapatos foi maior nos grupos locais de taurinos, com diferenças significativas entre os grupos. Nos ensaios do ELISA-teste, 74,39% dos animais avaliados foram positivos, encontrando-se os maiores níveis de anticorpos em animais taurinos quando comparados aos cruzados. Nas quantificações do DNA através da qPCR, a técnica demonstrou alta sensibilidade analítica e todos os animais foram positivos. Foram verificadas diferenças significativas (P<0,05) para o numero de copias (NC) de DNA de B. bovis entre os grupos locais, onde o grupo Angus de José Bonifácio, SP (AJB) apresentou o maior nível de infecção. As repetibilidades encontradas para as contagens de R. (B.) microplus e os NC de DNA de B. bovis foram baixas, enquanto que para os níveis de anticorpos obtidos pelo ELISA-teste foram ... / Abstract: Bovine babesiosis is a tick-borne disease, transmitted by Rhiphicephalus (Boophilus) microplus tick, that imposes substantial economic losses in to Brazilian livestock, among the species that cause bovine babesiosis B. bovis is the most virulent. Is widely known the natural resistance of the Bos taurus indicus genetic group to B. bovis, however, the quantitative relationship of the infection levels when compared to Bos taurus taurus and their crosses is unknown. The classical detection and quantitation method is of low sensitivity and usually fails to diagnose carrier animals. Recently, the real-time PCR (qPCR) techniques have been used successfully for these purposes. The present study aimed to determine the association among infestations by R. (B.) microplus, infection levels by B. bovis, estimated by qPCR, and the antibody levels in beef cattle naturally infested, proceeding of different locations and genetic groups (local groups). It was found greater tick infestations in taurine local groups, with significant differences between groups. In the ELISA test, 74.39% of animals were positive, meeting the highest antibodies levels in taurine animals when compared to the crossbred. In the DNA quantification, the analytical sensitivity of qPCR technique was high and all animals were positive. There were significant differences (P <0.05) for the DNA copy number (NC) among local groups, where Angus-Jose Bonifácio group (AJB) showed the highest infection levels. R. (B.) microplus scores and DNA copy number of B. bovis show low repeatability, whereas the antibodies levels were higher. No correlation among the examined measures were found. It is concluded that there is variation in the seroprevalence and DNA copy number of B. bovis among different local groups and the antibodies levels in the animals is not directly associated with the DNA NC of B. bovis, and both are independent of tick infestation / Doutor

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-10-04Bitstream added on 2014-06-13T18:53:46Z : No. of bitstreams: 1 zanette_mf_me_araca.pdf: 477117 bytes, checksum: 33b9b6dcc94d610c9edaafc75071b1a8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A importância do diagnóstico da leishmaniose visceral canina no Brasil reside no fato de que, dentre as estratégias de controle da doença indicadas pela Fundação Nacional de Saúde, encontra-se a eliminação do cão doméstico sorologicamente positivo. Desta forma, torna-se necessário o conhecimento da sensibilidade e da especificidade das provas sorológicas utilizadas para a correta identificação destes animais. Para avaliar o desempenho dos métodos sorológicos no diagnóstico da leishmaniose visceral canina, foram determinadas e comparadas as características dos métodos de ELISA, reação de imunofluorescência indireta (RIFI) e imunocromatografia, adotando-se como padrão o método parasitológico. Para tanto, foram utilizados 50 cães naturalmente acometidos por leishmaniose visceral e 45 cães sadios provenientes de área não endêmica para a doença. A RIFI revelou sensibilidade de 98% e especificidade de 91,1%, além de ótima concordância com o método parasitológico (Kappa = 0,893). Os métodos de ELISA e de imunocromatografia apresentaram boa concordância com o método parasitológico (coeficientes Kappa de 0,788 e 0,769, respectivamente) e valores de sensibilidade de 94% e 86% e especificidade de 84,4% e 91,1%, respectivamente. Com relação à ocorrência de reações cruzadas com leishmaniose visceral, das 14 amostras de soro positivas para doença de Chagas, nove (64,3%) foram consideradas positivas pela técnica de ELISA e seis (42,9%) pela RIFI, não se observando resultados positivos por imunocromatografia. Das 13 amostras de soros de animais portadores de erliquiose, uma (7,7%) apresentou resultado positivo por meio dos métodos de ELISA e de imunocromatografia. / The importance of canine visceral leishmaniasis diagnosis in Brazil is based on the fact that, according to the Ministry of Health, a seropositive dog must be culled. Thus, itþs important to know the sensitivity and specificity values of the methods employed for the diagnosis of the disease. To evaluate the performance of the available sorological tests for the diagnosis of canine leishmaniasis it was determined and compared the sensitivity and specificity of ELISA, indirect immunofluorescent antibody test (IFAT) and immunochromatographic test using the parasitological test as the gold standard. For this purposal, the study was carried out with a group of 50 naturally infected dogs from an endemic area and 45 healthy dogs from a non endemic area for visceral leishmaniasis. The IFAT was 98% sensitive and 91,1% specific, and showed a very good concordance with the parasitiological test (k = 0,893). The ELISA showed a sensitivity of 94% and specificity of 84,4% and when kappa index was analysed, a good concordance was obseved (k = 0,788). The immunochromatographic test was 86% sensisitive and 91,1% specific and showed a good concordance with the parasitiological test (k = 0,769). About the occurrence of cross reaction with visceral leishmaniasis, nine (64,3%) out of 14 positive samples for Chagas disease were positive by ELISA and six (42,9%) out of 14 were positive by IFAT, while the immunocromatographic test didn t yield any positive result. One (7,7%), out of 13 positive samples for ehrlichiosis, was positive by ELISA and immunocromatografic test. Five (83,3%) out of six positive samples for ehrlichiosis and babesiosis were positive by ELISA and three (50%) by immunocromatography, while IFAT didnþt yield any positive result. Five (50%) out of 10 positive samples for toxoplasmosis were positive by IFAT and one (10%) by immunocromatography.

Made available in DSpace on 2014-06-11T19:32:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-11-25Bitstream added on 2014-06-13T19:42:44Z : No. of bitstreams: 1 000749443.pdf: 1003234 bytes, checksum: 782cda03d91059640e363de67ccc1519 (MD5) / A babesiose bovina é uma hemoparasitose transmitida pelo carrapato Rhipicephalus (Boophilus) microplus que causa grandes prejuízos à bovinocultura do Brasil. Entre as espécies que causam as babesioses bovinas, B. bovis se destaca como a mais patogênica. É amplamente conhecida a resistência natural dos bovinos do grupo genético Bos taurus indicus frente a B. bovis. Entretanto, a relação quantitativa do nível de infecção em animais Bos taurus taurus e seus cruzamentos é desconhecida. O método clássico de detecção e quantificação da parasitemia por B. bovis apresenta baixa sensibilidade e falha no diagnostico em animais portadores. Recentemente, a técnica de qPCR tem sido utilizada com grande sucesso para este propósito. Assim, o presente estudo teve como objetivo verificar a associação entre o nível de infestação por R. (B.) microplus, a soroprevalência e a parasitemia por B. bovis, estimada através dos métodos do ELISA-teste e qPCR em bovinos de corte naturalmente infestados, de diferentes locais e grupos genéticos (grupos-locais).Verificou-se que a infestação por carrapatos foi maior nos grupos locais de taurinos, com diferenças significativas entre os grupos. Nos ensaios do ELISA-teste, 74,39% dos animais avaliados foram positivos, encontrando-se os maiores níveis de anticorpos em animais taurinos quando comparados aos cruzados. Nas quantificações do DNA através da qPCR, a técnica demonstrou alta sensibilidade analítica e todos os animais foram positivos. Foram verificadas diferenças significativas (P<0,05) para o numero de copias (NC) de DNA de B. bovis entre os grupos locais, onde o grupo Angus de José Bonifácio, SP (AJB) apresentou o maior nível de infecção. As repetibilidades encontradas para as contagens de R. (B.) microplus e os NC de DNA de B. bovis foram baixas, enquanto que para os níveis de anticorpos obtidos pelo ELISA-teste foram... / Bovine babesiosis is a tick-borne disease, transmitted by Rhiphicephalus (Boophilus) microplus tick, that imposes substantial economic losses in to Brazilian livestock, among the species that cause bovine babesiosis B. bovis is the most virulent. Is widely known the natural resistance of the Bos taurus indicus genetic group to B. bovis, however, the quantitative relationship of the infection levels when compared to Bos taurus taurus and their crosses is unknown. The classical detection and quantitation method is of low sensitivity and usually fails to diagnose carrier animals. Recently, the real-time PCR (qPCR) techniques have been used successfully for these purposes. The present study aimed to determine the association among infestations by R. (B.) microplus, infection levels by B. bovis, estimated by qPCR, and the antibody levels in beef cattle naturally infested, proceeding of different locations and genetic groups (local groups). It was found greater tick infestations in taurine local groups, with significant differences between groups. In the ELISA test, 74.39% of animals were positive, meeting the highest antibodies levels in taurine animals when compared to the crossbred. In the DNA quantification, the analytical sensitivity of qPCR technique was high and all animals were positive. There were significant differences (P <0.05) for the DNA copy number (NC) among local groups, where Angus-Jose Bonifácio group (AJB) showed the highest infection levels. R. (B.) microplus scores and DNA copy number of B. bovis show low repeatability, whereas the antibodies levels were higher. No correlation among the examined measures were found. It is concluded that there is variation in the seroprevalence and DNA copy number of B. bovis among different local groups and the antibodies levels in the animals is not directly associated with the DNA NC of B. bovis, and both are independent of tick infestation

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-14Bitstream added on 2014-06-13T19:35:11Z : No. of bitstreams: 1 coelho_nmd_me_araca.pdf: 406925 bytes, checksum: b4d17da85b8093f899383793bd8d1c81 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Fundação para o Desenvolvimento da UNESP (FUNDUNESP) / O objetivo deste estudo foi avaliar a ocorrência de Cryptosporidium spp. em crianças e seus respectivos cães e gatos de estimação residentes no Município de Andradina, SP, por meio do Teste de Imunoadsorção Enzimática (ELISA). Durante o período de janeiro a agosto de 2009, foram analisadas 188 amostras fecais de crianças, bem como de seus respectivos cães e gatos de estimação para pesquisa de Cryptosporidium spp. O teste quiquadrado (χ2) foi utilizado para verificar a associação entre as variáveis: sexo, idade, raça e consistência fecal, com nível de significância de 5%. Pelo ELISA foi detectado Cryptosporidium spp. em em amostras fecais de 2,1% (4/188) das crianças com idade inferior a sete anos. Entre os animais examinados, 6,8% (9/132) dos cães e 5,4% (3/56) felinos apresentaram amostras positivas para este coccídeo. Tanto nas crianças como nos animais não houve influencia do sexo e da idade na detecção do Cryptosporidium spp. sendo que nestes últimos a raça também não influenciou (P> 0,05). Neste trabalho foi evidenciada uma baixa ocorrência deste parasito nas crianças, assim só será possível determinar com precisão a relação entre a infecção das crianças com dos animais, com a posterior caracterização molecular deste protozoário. / The objective this study was to evaluate the occurrence of Cryptosporidium spp. in children and their dogs and pet cats in the city of Andradina, SP. through Test-linked immunosorbent assay (ELISA), during the period from January to August 2009, we analyzed 188 fecal samples from children, as well as their dogs and pet cats for detection of Cryptosporidium spp. The chi-square test (χ2) was used to verify the association between gender, age, race and fecal consistency, with a significance level of 5%. Using ELISA was detected Cryptosporidium spp. in stool samples in 2.1% (4/188) of children under the age of seven years. Among the animals examined, 6.8% (9/132) of dogs and 5.4% (3/56) cats had positive samples for this coccidium. Both children and animals there was no influence of sex and age on the detection of Cryptosporidium spp. being that in these last race did not influence (P> 0.05). This study demonstrated a low occurrence of this parasite in children, so only you can accurately determine the relationship between the infection of children with the animal with the molecular characterization of this parasite.

Orientador: Tereza Cristina Cardoso Silva / Banca: Maria Cecília Rui Luvizotto / Banca: Edviges Maristela Pituco / Resumo: A língua azul é uma doença viral, cujo agente etiológico pertence à família Reoviridae, gênero Orbivírus, transmitida por um vetor (artrópode) hematófago, do gênero Culicoides. Por se tratar de doença confundível com Febre Aftosa está incluída na lista de diagnóstico diferencial juntamente com Estomatite Vesicular, Varíola Ovina, Ectima Contagioso. O estudo teve como objetivo detectar a presença de anticorpos para língua azul em ovinos da região de Araçatuba, por ser esta uma região com rebanho expressivo e condições climáticas favoráveis à multiplicação de insetos. As amostras foram colhidas ao longo do ano de 2006, de ovinos adultos, em fase reprodutiva, (acima de 12 meses e com pelo menos uma parição, ou em caso de machos sexualmente maduros) e sem sintomas característicos da doença, de ambos os sexos, cadastrados no Núcleo de Produtores de Ovinos da Região de Araçatuba, distribuídas nos municípios da Região administrativa de Araçatuba. O tamanho da amostras foi calculado, considerando-se distribuição normal, prevalência de 50%, nível de confiança de 95% e precisão absoluta de 3%. Foram analisadas 1002 amostras de soros ovinos adultos, provenientes de 31 cabanhas, pelas provas de imunodifusão dupla em gel de agar (IDGA) e ELISA (Enzyme Linked immunosorbent Assay) de competição da fase sólida (ELISA CFS), provenientes do Centro Panamericano de Febre aftosa. Dessas amostras, 744 (74,3%) foram reagentes ao vírus da língua azul , pelo teste de ELISA-CFS e 651 (65,1%), pela técnica de IDGA. Não houve associação significante entre prevalência da doença e o sexo dos animais. Esses resultados revelam que o Vírus da Língua Azul encontra-se disseminado nessas regiões, sugerindo a ocorrência de infecções inaparente. / Abstract: Bluetongue (BT) is an infectious, non-contagious, insect-born viral disease of Ruminants. The causative agent of BT is bluetongue virus (BTV) that belongs to the family Reoviridae genus Orbivirus. Insect vectors in the genus Culicoides transmit this virus. BT affects domestic and wild ruminants, however small ruminants are considered the most affected specie. BT is included in the OIE list based on the differential diagnosis with Foot and Mouth Disease (FMDV), Vesicular Stomatitis Virus (VSV) and Sheep Pox Virus (SPV). The aim of the study was to detect antibodies against BTV in commercial sheep farms, of the Northeastern region of Sao Paulo State, Brazil. The size of the samples was calculated, considering normal distribution, prevalence of 50%, confidence level of 95% and absolute precision of 3%.A total of 1002 sera samples collected from adult sheep (above 1 year-old), comprising a total of 31 farms, were screened for the presence of BTV antibodies, by agar gel immunodiffusion test (AGID) and ELISA-CFS (Enzyme Linked Immunosorbent Assay - competitive solid phase), both produced by Pan American Center of FMDV. From a total of 744 samples 74,3% were positive by ELISA - CFS and 651 (65,1%) were positive by AGID. There was no significant association between prevalence of the disease and sex of animals.These results suggest that the BTV is widespread among farms, probably causing subclinical infections, with high level of antibodies. / Mestre

Philosophiae Doctor - PhD / More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother’s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to PLBW. / South Africa

Philosophiae Doctor - PhD / More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother’s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to PLBW.