Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--Murdoch University, 2003. / Title from t.p. of PDF document (viewed on Apr. 17, 2005). Includes bibliographical references (p. 195-239).

Title from title page of PDF (University of Missouri--St. Louis, viewed March 23, 2010). Includes bibliographical references.

DISSERTATION (PH.D.)--THE UNIVERSITY OF MICHIGAN

Made available in DSpace on 2014-06-11T19:25:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-14Bitstream added on 2014-06-13T19:53:32Z : No. of bitstreams: 1 schwardt_tf_me_araca.pdf: 288540 bytes, checksum: f6b713d25bd498b2a46d22b12adc7738 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Leishmaniose visceral em cães é descrita como uma doença de caráter crônico na qual os principais sintomas são perda progressiva de peso, caquexia, lesões dermatológicas, e recentemente, a doença tem sido relacionada com quadros neurológicos. Um total de 41 cães portadores de leishmaniose visceral, foram divididos em dois grupos. O primeiro composto por cães sem sintomas neurológicos (n=31) e o segundo grupo composto por cães com sintomas neurológicos (n=10). Amostras de encéfalo foram coletadas e armazenadas em formalina tamponada, para realização de imunoistoquímica para a pesquisa de formas amastigotas da Leishmania sp., linfócitos T CD3+, CD4+ e CD8+ e macrófagos. Não foram observadas formas amastigotas de Leishmania sp. Linfócitos T CD3+ estavam presentes em 24/31 (77,4%) dos cães sem sintomas neurológicos e em todos os cães do segundo grupo (p=0,0011). Linfócitos T CD4+ e CD8+ raramente foram observados, apresentando imunomarcação para CD4+ em 10/41 (24,3%) dos cães e em metade dos animais do grupo neurológico (p=0,0090). A presença de CD8+ foi detectada em 4/10 (40%) dos cães com doenças neurológicas (p=0,0021). Macrófagos foram observados em 38/41 (92,6%) cães, sem diferença estatística significante entre os dois grupos (p= 0,7664) / Visceral leishmaniasis in dogs is described as a chronic disease whose main symptoms are progressive weigth loss, dermatologic lesions and, recently, the disease has been associated to neurologic disorders in dogs. A total of 41 Leishmania chagasi infected dogs were divided into two groups. The first composed of dogs without neurological signs (n=31) and the second by dogs with neurological disorders (n=10). Brain samples were collected, stored in 10% buffered formalin and subjected to immunohistochemical examination for amastigotes forms of Leishmania sp, CD3+, CD4+ and CD8+ T lymphocytes and macrophages. Imunnohistochemistry not revealed amastigote any forms of Leishmania sp. CD3+ T lymphocytes were present in 24/31 (77,4%) dogs without neurological signs and in all dogs from the second group (p=0.0011). CD4+ and CD8+ were rarely observed, with CD4+ immunostaining in 10/41 (24,3%) dogs, from which half of them had neurological disease (p=0.0090). The presence of CD8+ was detected only in 4/10 (40%) dogs from neurological group (p=0.0021). Macrophages were detected in 38/40 (92,6%) dogs, without significant differences between groups (p=0.7664)

<p>Development of serological assays for the diagnosis of Baylisascaris larva migrans in birds and non-human primates</p>

O estudo dos teores protéicos oferecidos aos animais possui crucial importância, pois estes exercem influência determinante no desenvolvimento da infecção parasitária. Investigou-se o efeito da utilização de dois diferentes teores protéicos (18,5% e 8,6% de PB na MS) sobre as alterações fisiopatológicas desenvolvidas por bezerros primo infectados com o nematóide intestinal Cooperia punctata. Objetivando determinar se a suplementação possibilitaria aos animais infectados responderem mais prontamente ao desafio parasitário, realizaram-se estudos de composição corporal, retenção de compostos nitrogenados, acompanhamento clínico completo, avaliação de peso e carcaça, avaliação quantitativa e qualitativa da carga parasitária e exames histopatológicos. Os animais que receberam suplementação apresentaram um quadro clínico menos severo, perderam menos peso, tiveram um maior rendimento de carcaça, apresentaram lesões teciduais mais exuberantes, maiores taxas de excreção urinária de nitrogênio, expulsaram os parasitos para porções distais do trato gastrointestinal e demonstraram uma eficiência de retenção de nitrogênio 3,6 vezes maior que os animais mantidos na dieta basal. Demonstrou-se que a suplementação protéica resultou em efeitos positivos para os animais infectados. / The study of protein levels offered to cattle is very important when one aims at nematode control, since they can effectively affect the development of parasitic infection. The effect of two different levels - 18.5% and 8.6% CP on DM basis on the pathophysiology of Cooperia punctata infection on naive calves was examined. Aiming at determine if protein supplementation allow animals to better withstand parasitic challenge, several parameters were evaluated: body composition, nitrogen retention, clinical evolution, weight gain, carcass yeld, quantitative and qualitative assessment of parasitic burden and histopathology. Supplemented animal presented a milder clinical signs, lost less weight, had higher carcass yeld, greater tecidual lesions, higher urinary nitrogen excretion, expelled parasites to distal parts of gastrointestinal tract and showed a 3.6 greater efficiency for nitrogen retention than animal kept on basal diet. It became clear that protein supplementation resulted in benefits to infected animals.

Made available in DSpace on 2015-03-26T13:46:47Z (GMT). No. of bitstreams: 1 texto completo.pdf: 644915 bytes, checksum: d059fb831fbd9168fde56d0f1948a13a (MD5) Previous issue date: 2005-05-25 / During the past ten years, the protozoan Neospora caninum has been described and associated as an ethyological agent of infected abortions in cattle. This disease in dogs also has an abortive character, but since dogs are definitive hosts, they have relevant importance in the life cycle of this parasite, acting as a source of the disease. Other diseases such as leptospirosis and brucelosis are also disseminated in Brazil and are associated to reproductive disturbances. The objective of this study was to evaluate the presence of infection for N. caninum in cattle and dogs of rural properties in two municipalities, Viçosa and Mercês, located in the macro-region of Zona da Mata in the state of Minas Gerais, as well as to analyze the existing risk factors for the infection. Serum samples of female cattle and dogs were collected for research of anti-N. caninum antibodies, using the indirect immunofluorescence technique; Brucella abortus selective test (Buffered Acidified Antigen - BAA) and corroborative tests (2-ME and SAT) and Leptospira sp., Microscopic Agglutination Technique (MAT) without and with sera dilution. The bovine sera were tested for the three pathogenic bioagents and the canine sera for N. caninum and Leptospira sp. In addition, information was collected by applying a structured questionnaire on type of exploration, production system and reproductive problem history in cattle and habits, type of food, demographic characteristics in dogs. The prevalence of positive properties was of 54.3% and 18.5% for N. caninum in cattle and dogs, respectively; 15.2% for Brucella abortus and 6.5% for Leptospira sp. in cattle and 7.4% for Leptospira sp.in dogs. The prevalence of positive animals presented lower results: 23.3% of N. caninum in cattle and dogs, respectively; 15.2% for Brucella abortus and 6.5% for Leptospira sp. in cattle and 7.4% for Leptospira. However, it should be emphasized that the prevalence for N. caninum in cattle was significantly higher, compared to Brucella abortus (p < 0.01) and Leptospira sp. (p < 0.01). Positive properties for bovine neosporosis were associated to the presence of reproductive disturbances in the animals (p < 0.01), when the analysis considered the type of disturbance, abortion (OR = 5.2; IC 95% = 1.32-20.54) and prenatal mortality (OR = 6.0; IC 95% = 1.22 - 29.95) presented significant association. The occurrence of positive serology for N. caninum in cattle was associated to the presence of positive dogs in the property (p<0.01); however, reproductive problem in cattle was not associated with canine neosporosis (p = 0.1046). Significant association was also verified between bovine osteoporosis and type of exploration, serum positive cattle having, approximately, three times more chance (OR = 2.82 and IC 95% = 1.05 - 9.52) of belonging to a milk production system rather than mixed. The characterization of canine neosporosis did not show significant association with the variables sex (p = 0.55), age (p = 0.34), animal habit (p = 0.03; OR = 0.00; IC 95% = 0.0-1.06) and type of food (p = 0.11). The results suggest that sanitation in neosporosis has become of utmost importance in bovine herds, especially when reproductive problems resulting from the infection are considered. Therefore, it becomes evident the need for animal health authorities and institutions to fight this disease. / Durante os últimos dez anos o protozoário Neospora caninum tem sido descrito e associado como agente etiológico de abortamentos infecciosos em bovinos. Esta doença em cães também tem caráter abortivo, mas como os caninos são hospedeiros definitivos, possuem relevante importância no ciclo de vida deste parasita, funcionando como reservatório da doença. Outras doenças como a leptospirose e a brucelose também são disseminadas em nosso país e também estão associadas a transtornos reprodutivos. O objetivo deste estudo foi avaliar a presença de infecção para N. caninum em bovinos e cães existentes em propriedades rurais de dois municípios, Viçosa e Mercês, pertencentes à Macrorregião Zona da Mata do Estado de Minas Gerais, bem como analisar a existência de fatores de risco para a infecção. Foram coletadas amostras de soro de bovinos fêmeas e cães para pesquisa de anticorpos anti-N. caninum, pela técnica de imunofluorescência indireta; Brucella abortus prova seletiva (Antígeno Acidificado Tamponado - AAT) e provas confirmatórias (2-ME e SAT) e Leptospira sp., Técnica de Aglutinação Microscópica (MAT) sem e com diluição dos soros. Os soros bovinos foram testados para os três bio-agentes patogênicos e os de cães para N. caninum e Leptospira sp. Adicionalmente, foram coletadas informações, utilizando-se questionário estruturado, relativas ao tipo de exploração, sistema de produção e histórico de problemas reprodutivos em bovinos e hábitos, tipo de alimentação, características demográficas dos cães. As prevalências de propriedades positivas foram de 54,3% e 18,5% para N. caninum em bovinos e cães, respectivamente; 15,2% para Brucella abortus e 6,5% para Leptospira sp. em bovinos e 7,4% para Leptospira sp. em cães. As prevalências de animais positivos apresentaram resultados menores: 23,3% de N. caninum em bovinos e cães, respectivamente; 15,2% para Brucella abortus e 6,5% para Leptospira sp. em bovinos e 7,4% para Leptospira, entretanto, é de se destacar que, para os bovinos, a prevalência para N. caninum, significativamente superior quando comparada a Brucella abortus (p < 0,01) e Leptospira sp. (p < 0,01). Propriedades positivas para neosporose bovina estiveram associadas à presença de distúrbios reprodutivos nos animais (p < 0,01), quando a análise considerou o tipo de distúrbio, abortamento (OR = 5,2; IC 95% = 1,32-20,54) e mortalidade perinatal (OR = 6,0; IC95% = 1,22 - 29,95) apresentaram associação significativa. A ocorrência de sorologia positiva para N. caninum em bovinos esteve associada à presença de cães positivos na propriedade (p < 0,01), entretanto, problema reprodutivo em bovinos não esteve associado à neosporose canina (p = 0,1046). Associação significativa também foi verificada entre a neosporose bovina e o tipo de exploração, sendo que os bovinos soropositivos tiveram, aproximadamente, três vezes mais chance (OR = 2,82 e IC 95% = 1,05 - 9,52) de pertencerem a sistema de produção tipo leite do que tipo misto. A caracterização da neosporose canina não revelou associação significativa com as variáveis sexo (p = 0,55), idade (p = 0,34), hábito do animal (p = 0,03; OR = 0,00; IC 95% = 0,0-1,06) e tipo de alimentação (p = 0,11). Os resultados sugerem o significado sanitário que a neosporose vem assumindo em rebanhos bovinos, notadamente quando se considera a ocorrência de problemas reprodutivos decorrentes da infecção. Nesse sentido, evidencia-se a atenção necessária das autoridades e instituições de saúde animal, no combate a esta enfermidade.

O estudo dos teores protéicos oferecidos aos animais possui crucial importância, pois estes exercem influência determinante no desenvolvimento da infecção parasitária. Investigou-se o efeito da utilização de dois diferentes teores protéicos (18,5% e 8,6% de PB na MS) sobre as alterações fisiopatológicas desenvolvidas por bezerros primo infectados com o nematóide intestinal Cooperia punctata. Objetivando determinar se a suplementação possibilitaria aos animais infectados responderem mais prontamente ao desafio parasitário, realizaram-se estudos de composição corporal, retenção de compostos nitrogenados, acompanhamento clínico completo, avaliação de peso e carcaça, avaliação quantitativa e qualitativa da carga parasitária e exames histopatológicos. Os animais que receberam suplementação apresentaram um quadro clínico menos severo, perderam menos peso, tiveram um maior rendimento de carcaça, apresentaram lesões teciduais mais exuberantes, maiores taxas de excreção urinária de nitrogênio, expulsaram os parasitos para porções distais do trato gastrointestinal e demonstraram uma eficiência de retenção de nitrogênio 3,6 vezes maior que os animais mantidos na dieta basal. Demonstrou-se que a suplementação protéica resultou em efeitos positivos para os animais infectados. / The study of protein levels offered to cattle is very important when one aims at nematode control, since they can effectively affect the development of parasitic infection. The effect of two different levels - 18.5% and 8.6% CP on DM basis on the pathophysiology of Cooperia punctata infection on naive calves was examined. Aiming at determine if protein supplementation allow animals to better withstand parasitic challenge, several parameters were evaluated: body composition, nitrogen retention, clinical evolution, weight gain, carcass yeld, quantitative and qualitative assessment of parasitic burden and histopathology. Supplemented animal presented a milder clinical signs, lost less weight, had higher carcass yeld, greater tecidual lesions, higher urinary nitrogen excretion, expelled parasites to distal parts of gastrointestinal tract and showed a 3.6 greater efficiency for nitrogen retention than animal kept on basal diet. It became clear that protein supplementation resulted in benefits to infected animals.

Background: Neospora caninum is a parasite that causes disease, largely in cattle and dogs. It is a disease of significant interest within New Zealand due to its association with bovine abortion. The economic impact of bovine abortion justifies the development of a bovine vaccine against N. caninum. Aim: To develop and optimise diagnostic procedures for the detection of Neospora from a variety of blood and tissue samples and to isolate a New Zealand strain of Neospora caninum. Methods: A local strain of Toxoplasma gondii and an imported Neospora caninum strain, Nc-Liverpool, were used to optimise tachyzoite growing conditions in bovine endothelial (BE) cells and Vero host cell cultures. A serum study using 112 tissue culture flasks was performed to determine whether foetal bovine serum or horse serum supplemented media provided the optimal growing conditions for Nc-Liverpool tachyzoites. Nc-Liverpool tachyzoites were also used to determine the optimal growth period between passage, and harvest for cryopreservation and cryopreservation conditions. Percoll gradients were also tested using Nc-Liverpool tachyzoites. A known Neospora positive canine sample and murine tissues infected with Toxoplasma, were used during the development of the immunohistochemical diagnostic technique. Antibody concentrations and incubation temperatures were tested to reduce cross-reactivity and increase specific stain intensity. Immunohistochemistry was performed on sections of all tissue samples used for N. caninum isolation and experimentally infected murine tissue. Several PCR techniques were developed, the final PCR used being a combination of the different techniques, which produced a 250kb band. PCR-3 used the NF6/GA1 primer combination for Neospora detection and TF6/GA1 for Toxoplasma detection, additional Mg2+ and an annealing temperature of 55°C were required. Whole tissue was processed via DNA elution whereas cell culture and Percoll purified tachyzoites were used following crude lysis techniques. All bovine and canine tissues used for parasite isolation as well as all experimentally infected mouse tissues were tested for N. caninum using PCR. An immunoblot technique was developed for the detection of N. caninum antibodies in murine blood samples. Lysed Nc-Liverpool tachyzoites were used as antigen with varied results. The primary and secondary antibodies were commercially available and used at concentrations of 1:1,000 and 1:25,000 respectively. BALB/c and CF1 mice were experimentally infected with Toxoplasma gondii and Nc-Liverpool. Forty female BALB/c and 40 female CF1 mice were used in 2 studies to determine the optimal Nc-Liverpool inoculation dose and immunosuppression requirements. Mice were immunosuppressed with 2.5mg of methylprednisolone acetate (MPA) and Nc-Liverpool inoculation ranged from 1.3x106 to 5x103 tachyzoites. Upon death, the brain and blood was harvested from the mouse carcases. Attempts were made to isolate a New Zealand strain of N. caninum from bovine and canine central nervous system (CNS) tissue, and to maintain the parasites in cell culture and by small animal passage, in order to attenuate the parasite strain for use as a live large animal vaccine. Twenty one bovine tissue samples were used for N. caninum isolation attempts, 18 of which were positive for Neospora antibodies using a commercial IFAT. Isolation tissues were purified using a 30% Percoll gradient and inoculated onto 8 cell culture flasks and into 8 immunosuppressed mice (BALB/c and CF1). Results: Nc-Liverpool tachyzoites were found to be viable when grown at 37°C in antibiotic-MEM supplemented with either FBS or ES and grew optimally in FBS despite Neospora antibodies being detected using an IFAT. Passaging cultures at approx. 4 day intervals resulted in the greatest parasite growth. However, cryopreserved parasites should be harvested 2 days post inoculation (PI) for optimal viability. Viable parasites could be isolated using a 30% Percoll gradient and centrifuged at 2,700 x g (3,400 rpm) in a bucket centrifuge for 10 minutes. Tissue cysts could be detected using immunohistochemistry but some degree of cross reaction remained despite optimisation. Cysts were not found in tissues used for isolation attempts or in mouse brains following inoculation with Nc-Liverpool, however cysts were commonly found in mice experimentally infected with T. gondii tachyzoites. PCR-3 was successfully used to detect N. caninum and T. gondii infected tissue and tachyzoites from tissue culture. PCR-3 could detect N. caninum DNA in the brain tissue of 9/24 mice experimentally infected with Nc-Liverpool, even though most mice were culled within 1 week. Although production of N. caninum antigen was only moderately successful, N. caninum antibody detection in mouse blood using one specific antigen batch was reliable and specific. The immunoblot could only detect N. caninum antibody approximately 14 days PI, but was sensitive enough to detect 100% of mice experimentally infected with Nc-Liverpool tachyzoites. PCR-3 strongly correlated with the immunoblot results from 14 days PI. BALB/c mice were found to be far more sensitive to Nc-Liverpool than CF1 mice and developed severe disease at concentrations of approximately 1x106 Nc-Liverpool tachyzoites. Neither BALB/c nor CF1 mice developed peritoneal exudate, irrespective of the parasite inoculation concentration. Despite Neospora DNA being present in the brains of experimentally infected mice, re-isolation and continuous parasite passage from the brains could not be achieved. No mice experimentally infected with either Nc-Liverpool or isolation attempts were found to have brain cysts when tested using immunohistochemistry. Only 1 mouse inoculated with bovine isolation material was found to have a Neospora positive PCR. Through the detection of DNA, antigens and antibodies, parasites were determined to have been present in 10 of the 18 IFAT positive bovine isolation samples, indicating that 55% of calves born to seropositive dams were infected with N. caninum. However, despite numerous attempts to isolate Neospora parasites from naturally infected canine and bovine tissue and culturing using the optimised Nc-Liverpool technique, maintenance of a live culture of a New Zealand strain of N. caninum could not be established. Conclusions: Findings from this study could be used to assist in the maintenance of Neospora caninum and Toxoplasma gondii parasite strains and for detection or diagnosis of these parasites in host tissues.