Made available in DSpace on 2014-06-11T19:33:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-31Bitstream added on 2014-06-13T21:06:05Z : No. of bitstreams: 1 furuta_pi_dr_jabo.pdf: 1073038 bytes, checksum: 5e772861c92b1e24fce5e65feea5cf02 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A neosporose é uma doença causada pelo protozoário Neospora caninum, e causa um grande impacto econômico na pecuária mundial. Sabe-se que muitos mamíferos são considerados hospedeiros intermediários, e que recentemente, foi confirmado o papel das aves no ciclo de vida de N. caninum como hospedeiros intermediários. O presente trabalho teve como finalidade estudar o ciclo evolutivo e patogenia de N. caninum em cães alimentados com ovos embrionados de Gallus gallus domesticus previamente inoculados com taquizoítas. Realizou-se diariamente o exame de fezes dos animais, pela técnica de flutuação, constatando a presença de oocistos nas fezes a partir de 7 dia apósinfecção. Anticorpos anti- N. caninum foram pesquisados semanalmente nos cães pela RIFI, sendo observados anticorpos a partir dos 14dai. Semanalmente um cão foi eutanasiado e durante a necropsia observou-se em todos os cinco animais, aumento dos linfonodos mesentéricos e das tonsilas, presença de muco no duodeno e jejuno e hepatoesplenomegalia, com hiperplasia de polpa branca do baço. Pelo exame histopatológico detectou-se reatividade dos linfonodos, lesões no fígado, coração, pulmão, baço e rim. Pelas técnicas de imunoistoquímica, imunofluorescência indireta dos tecidos e no Real-time PCR, observou-se a presença do parasito. Os resultados obtidos apontam que, os ovos embrionados infectados pelo parasito mostraram eficiência como modelo experimental para infectar cães, que eliminaram oocistos nas fezes, mesmo não apresentando sinais clínicos da neosporose. Ademais, os cães infectados apresentaram prematura soroconversão pela prévia administração de bicarbonato de sódio e o uso das técnicas citadas tornou possível a detecção do parasito nos tecidos analisados. / Neosporosis is a protozoan disease caused by Neospora caninum and affects the reproductive system in cattle worldwide. Infection by the parasite has been widely described in mammals, and recently the role of birds in its lifecycle was confirmed as intermediate hosts. This work aimed to study the life cycle of N. caninum and the pathogeny in dogs that were fed with embryonated eggs inoculated with tachyzoites. The tested dogs were followed up for oocyst shedding, by flotation technique, and it was detected at 7 days after infection. Antibodies anti- N. caninum were detected at 14dai. Once a week, one dog was euthanized and at the necropsy alterations was observed on the mesenteric lymph nodes and tonsils, presence of mucus at duodenum and jejunum portions, and hepatosplenomegaly. At the histopathological examination lymph nodes alterations were noticed ranging from moderate to intense. In addition, there were alterations in liver, heart, lung, spleen and kidney. The parasite was detected by immunochemistry and indirect immunofluorescense tests as well as by Real-time PCR. The results herein presented suggest that inoculated embryonated eggs showed to be an efficient model to infect dogs, because of shedding oocysts, and no clinical alterations. Moreover, the infected dogs presented earlier seroconvertion because of sodium bicarbonate solution administration and the parasite detection in the analyzed tissues was successful by the techniques used.

Orientador: Rosangela Zacarias Machado / Banca: Solange Maria Gennari / Banca: Aramis Augusto Pinto / Banca: Deise Aparecida de Oliveira Silva / Banca: Ana Patricia Yatsuda Natsui / Resumo: Neospora caninum é um protozoário do Filo Apicomplexa, que foi primeiramente descrito como causa de encefalomielite em filhotes caninos sorologicamente negativos para Toxoplasma gondii. Estudos anteriores neste importante hospedeiro da cadeia epidemiológica de N. caninum demonstram que as respostas de anticorpos IgG são tardiamente detectadas e que a infecção clínica é de difícil indução. Desta forma, este trabalho objetivou o estudo da imunidade de cães frente à infecção oral por N. caninum. Os resultados obtidos a partir de análises de diversos animais experimentalmente infectados indicam que os cães apresentam uma prolongada fase aguda da infecção, com eliminação de oocistos associado à queda nos níveis de linfócitos T CD4+ e CD8+ e diminuição de MHC de classe II por células apresentadoras de antígeno. Adicionalmente, os animais apresentam soroconversão instável durante o mesmo período, sendo que somente IgG1 e IgG3 foram detectados em adultos e filhotes, respectivamente, entre o 2o e 3o mês de infecção. De forma concomitante, observa-se uma predominância da expressão de citocinas imunomoduladoras como TGF 1, IL-4 e IL-10. Após dois meses de infecção, o perfil da resposta se inverte, sendo observado picos de produção dos marcadores CD4 e CD8 de linfócitos T e citocinas próinflamatórias como IFN , IL-6 e IL-12, além do aumento nos títulos de anticorpos, principalmente IgG1 e IgG4 nos cães jovens. Com base nestes resultados, conclui-se que os cães apresentam uma relação de equilíbrio com N. caninum, a qual induz nesta espécie uma modulação da resposta imunológica durante a fase de merogonia. / Abstract: Neospora caninum is an Apicomplexan parasite firstly described as the cause of encephalomyelitis in puppies serologically negative to Toxoplasma gondii. Previous reports on the parasite’s definitive host indicate a late IgG antibody response and that clinical disease is difficult to be induced. The aim of this study was to investigate canine immunity during N. caninum oral infection. The results obtained from the analysis of infected animal’s samples indicate that dogs present a protracted acute phase, with oocyst shedding correlated to a drop in CD4+ and CD8+ T cell levels, and low MHC class II expression by antigen presenting cells. Additionally, the dogs presented an unstable seroconversion pattern in the same period, with only IgG1 and IgG3 being detected in adult dogs and puppies, respectively, between the second and third months of infection. Concomitantly, dominant Th2 cytokine expression was observed, with peak expression levels of TGF 1, IL-4 and IL-10. After 2 months of infection, the immunity profile shifts towards a Th1 response, with high levels of CD4 and CD8 lymphocytary marker production and pro-inflammatory cytokine expression (IFN , IL-6 and IL-12), besides of the raise in antibody levels, especially IgG1 and IgG4 in puppies. Based in the results presented herein, we may conclude that dogs present a balanced host-parasite relationship, modulating the host immune response during N. caninum merogony. / Doutor

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-04-25T12:16:44Z No. of bitstreams: 1 Carlos Andre Barbosa de Franca.pdf: 2030226 bytes, checksum: 965977096ae399d28e6c310614ac0961 (MD5) / Made available in DSpace on 2017-04-25T12:16:44Z (GMT). No. of bitstreams: 1 Carlos Andre Barbosa de Franca.pdf: 2030226 bytes, checksum: 965977096ae399d28e6c310614ac0961 (MD5) Previous issue date: 2016-05-16 / This study aimed to carry out a sero-epidemiological investigation associated with the infection by Neospora caninum in dairy cattle herds deriving from family-based farming in the microregion of Vale do Ipojuca, in the Brazilian State of Pernambuco. Blood serum samples were collected from 341 bovines derived from 25 farms in 16 municipalities of the microregion. The samples were processed by the Indirect Immunofluorescent Assay Technique (IFAT). An investigative questionnaire was applied for the analysis of the risk factors, with objective questions about the hygiene-sanitary and reproductive management. Subsequently, the data were analyzed by logistic regression, considering as a dependent variable the serological results. The prevalence of anti-Neospora caninum antibodies was 18.2% (62/341) in cattle herds, with 80% (20/25) of foci. No risk factors associated with the infection have been confirmed in this study. These results show that the infection by N. caninum is present in cattle herds of the region of Vale do Ipojuca, and can cause loss to family farmers. / Objetivou-se com este estudo realizar um inquérito soroepidemiológico associado à infecção por Neospora caninum em rebanhos bovinos leiteiros da agricultura familiar na microrregião do Vale do Ipojuca, Estado de Pernambuco. Foram coletadas amostras de soro sanguíneo de 341 bovinos, procedentes de 25 propriedades rurais, em 16 municípios da microrregião. As amostras obtidas foram processadas pela técnica de Reação de Imunofluorescência Indireta (RIFI). Para a análise dos fatores de risco foi aplicado um questionário investigativo, com perguntas objetivas sobre o manejo higiênico-sanitário e reprodutivo. Posteriormente, os dados foram analisados por regressão logística, considerando como variável dependente o resultado sorológico. A prevalência de anticorpos anti-Neospora caninum foi 18,2% (62/341) no rebanho bovino, com 80% (20/25) de focos. Neste estudo não foi confirmado nenhum fator de risco associado à infecção. Esses resultados evidenciam que a infecção pelo N. caninum encontra-se presente nos bovinos da região do Vale do Ipojuca e pode ocasionar prejuízos aos produtores da agricultura familiar.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2014-01-28T18:04:42Z No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_InqueritoSorologicoNeospora.pdf: 2233716 bytes, checksum: f4635514f7396cea5559b87600b8bcd0 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-01-31T13:46:39Z (GMT) No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_InqueritoSorologicoNeospora.pdf: 2233716 bytes, checksum: f4635514f7396cea5559b87600b8bcd0 (MD5) / Made available in DSpace on 2014-01-31T13:46:39Z (GMT). No. of bitstreams: 2 license_rdf: 22974 bytes, checksum: 99c771d9f0b9c46790009b9874d49253 (MD5) Dissertacao_InqueritoSorologicoNeospora.pdf: 2233716 bytes, checksum: f4635514f7396cea5559b87600b8bcd0 (MD5) Previous issue date: 2010 / Neospora caninum é um protozoário coccídio que tem ampla difusão e provoca consideráveis prejuízos aos criatórios dos animais. Para levantar a freqüência de ocorrência de anticorpos anti-N. caninum em bovinos leiteiros do município de Parauapebas, Mesorregião Sudeste do Estado do Pará, 465 amostras de soros de bovinos leiteiros de 45 propriedades rurais foram submetidos à reação de imunofluorescência indireta (RIFI), adotando-se como ponto de corte a diluição 1:100; 62 (13,33 %) amostras de soros demonstraram a presença de anticorpos anti-N. caninum, distribuídos pelos seguintes percentuais e títulos 27(43,55 %) na titulação 1:100; 14 (22,58 %) na 1:200; 16 (25,80) na 1:400 e 5 (8,07 %) com título de 1:800. Nenhum animal apresentou título superior a 1:800; não foi observada diferença significativa entre os sexos dos animais, mas, animais com idade igual ou superior a cinco anos apresentaram percentagem maior de soros positivos, proporcionalmente à quantidade coletada em relação aos animais mais jovens. Os resultados demonstram que os bovinos leiteiros no município de Parauapebas tinham com uma considerável freqüência anticorpos anti-N. caninum. Medidas e ações de vigilância sanitária são recomendadas para prevenir a entrada de novas fontes de N. caninum nos rebanhos e controlar a difusão desse agente no criatório regional. / Neospora caninum is a world-wide coccidian protozoa which causes considerable damages at animals breeding. To survey the frequence of anti-N. caninum antibodies in bovine milkmen in Parauapebas county, Southeast Mesoregion of Pará State, Northern Brazil, 465 serum samples coming from 45 farmers were submitted to indirect immunofluorescence reaction (cutting point 1:100); 62 (13,33%) samples have demonstrated the presence of anti-N. caninum antibodies, with the following distribution: 27 (43,55%) in 1:100 dilution; 14 (22,58%) in 1:200 dilution; 16 (25,80%) in 1:400; and 5 (8,07%) in 1:800 dilution. None serum had tittle in over 1:800 dilution. In a high percentage there was only an animal with anti-N. caninum antibodies; the major frequence was observed between females, but was’nt observed significative difference between animal sexes; animals with equal or over five years old presented major percentage of positive serum proporcionaly to number of positives serum of younger animals. The results have demonstrated that bovine milkmen of Parauapebas county had a considerable frequence of anti-N. caninum antibodies. Sanitary surveillance measures are necessary to prevent the entrance of N. caninum and to control the difusion of this protozoa at the regional bovine milkmen.

Aquesta tesi (mitjançant cinc estudis) va ser destinada a investigar factors que tenen un efecte potencial sobre la salut materna durant la gestació i el postpart temprà, tenint en compte la melatonina com una possible millora de la salut i el rendiment de les vaques. En el primer estudi, els factors que afecten els leucòcits perifèrics de la mare, com un indicador d'estat immune, entre els dies 90-210 de gestació van ser, la interacció entre l'edat de la vaca i Neospora caninum, l'estació, la gestació gemelar, la producció de llet i el temps de mostreig. Seguint el sistema immune matern més enllà en la seva fase més crítica, el segon i el tercer estudis resumeixen els diferents factors que afecten el sistema immunològic de la mare des del dia 220 de gestació fins a 30 dies postpart. El toro d'IA, les glicoproteïnes associades a la gestació (PAG), la interacció N. caninum-C. burnetii, l'estació, l'edat, la gestació gemelar i el temps de mostreig afectaven significativament els leucòcits materns. L'objectiu del quart estudi va ser avaluar la dosi adequada de la melatonina, per provar els seus possibles efectes en la millora de la salut de la mare durant el peripart i la dosi de 332 mg / kg era la més adequada. Finalment, els possibles efectes de la melatonina en millorar la salut materna durant el peripart van ser avaluats en el cinquè estudi. Les vaques tractades amb melatonina tenien menys probabilitat de ser repetidores i de perdre la gestació, i, per tant, tenien menys dies oberts. / Esta tesis (mediante cinco estudios) fue destinada a investigar factores que tienen un efecto potencial sobre la salud materna durante la gestación y el posparto temprano, teniendo en cuenta la melatonina como una posible mejora de la salud y el rendimiento de las vacas. En el primer estudio, los factores que afectan a los leucocitos periféricos de la madre, como un indicador de estado inmune, entre los días 90-210 de gestaion fueron, la interacción entre la edad de la vaca y Neospora caninum, la estación, la gestación gemelar, la producción de leche y el tiempo de muestreo. Siguiendo el sistema inmune materno más allá en su fase más crítica, el segundo y el tercer estudios resumen los diferentes factores que afectan al sistema inmunológico de la madre desde el día 220 de gestación hasta 30 días posparto. El toro de IA, las glicoproteínas asociadas a la gestación (PAG), la interacción N. caninum-C. burnetii, la estación, la edad, la gestación gemelar y el tiempo de muestreo afectaban significativamente los leucocitos maternos. El objetivo del cuarto estudio fue evaluar la dosis adecuada de la melatonina, para probar sus posibles efectos en la mejora de la salud de la madre durante el periparto y la dosis de 332 mg/kg era la más adecuada. Por último, los posibles efectos de la melatonina en mejorar la salud materna durante el periparto fueron evaluados en el quinto estudio. Las vacas tratadas con melatonina tenian menos probabilidad de ser repetidoras y de perder la gestación, y, por lo tanto, tenian menos días abiertos. / This thesis (by means of five studies) aimed at investigating factors that have potential effect on the maternal health during gestation and the early postpartum, plus considering melatonin for improving cows’ health and performance. In the first study, factors affecting maternal peripheral leukocytes, as an indicator to immune status, between Days 90-210 of gestaion were, the interaction between cows’ age and Neospora caninum-seropositivity, season, twin-pregnancy, milk production and time. Following the maternal immune system further in its most critical phase, the second and third studies summarize different factors affecting maternal immune system from gestation Day 220 till 30 days postpartum. Artificial inseminating bull, plasma pregnancy associated glycoproteins (PAGs), N. caninum-C. burnetii interaction, season, age, twin-pregnancy and time were found to significantly affect maternal peripheral leukocytes during that stage. The aim of the forth study was to evaluate proper melatonin dose as subcutaneous implants, for testing its possible effects in enhancing maternal health during the peripartum. Melatonin dose of 332 μg/kg was the most adequate. Finally, possible melatonin effects on enhancing maternal health during the peripartum were evaluated in the fifth study. Melatonin treated cows were found to have less likelihood of repeat breeding syndrome and pregnancy loss, and, therefore, less days open.

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.

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.

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.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-06-16T11:25:33Z No. of bitstreams: 1 texto completo.pdf: 659479 bytes, checksum: a72044bcb96556303124778af280b069 (MD5) / Made available in DSpace on 2016-06-16T11:25:33Z (GMT). No. of bitstreams: 1 texto completo.pdf: 659479 bytes, checksum: a72044bcb96556303124778af280b069 (MD5) Previous issue date: 2005-11-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Neospora caninum é um protozoário importante por causar abortamento em bovinos em todo o mundo. O seu ciclo biológico completo foi descrito em 1998, tendo como hospedeiros definitivos os canídeos e entre os hospedeiros intermediários os herbívoros. A principal forma de permanência da infecção nos rebanhos é pela via transplacentária. Ovelhas infectadas experimentalmente são utilizadas como modelo da neosporose bovina e para se estudar a transmissão vertical e a patologia da infecção nas várias gestações subseqüentes, sendo utilizadas sete ovelhas adultas e seus respectivos filhotes de segunda, terceira, quarta e quinta gestações com diferentes idades. Lesões características de neosporose foram encontradas nos animais estudados, compreendendo focos inflamatórios não-supurativos no fígado, cérebro e pulmão, além de infiltrado inflamatório mononuclear perivascular nos mesmos, principalmente nos filhotes da segunda gestação. Além disso, observaram-se cistos teciduais de Neospora caninum na língua e coração de ovelhas adultas. Comprovou-se através de imunofluorescência indireta que todos os animais apresentaram-se positivos à infecção. Estes dados demonstram que o parasita é transmitido verticalmente durante várias gestações, causando lesões características, o que pode em condições naturais manter a infecção no rebanho sem, no entanto, causar a morte fetal. / Neospora caninum is an important protozoan cause of abortion in cattle worldwide. The whole life cycle was described in 1998, had canids as definitive hosts and herbivores as intermediate hosts. The main pattern of persistent infection in herds it is by transplacental route. Sheep experimentally infected are used as a model of bovine neosporosis and to study vertical transmission and the pathology of infection at several subsequent gestations, being used seven ewes and their respective offspring at second, third, fourth and fifth pregnancies with different ages. Typical neosporosis lesions were found in the studied animals, consisting of non- suppurative inflammatory focuses on liver, brain and lungs, besides mononuclear perivascular infiltrate, principally on lambs of second gestation. Moreover, Neospora caninum tissues cysts were observed on the tongue and heart of adult sheep. It was proved by indirect immunofluorescence that all the animals were positives to the infection. These data demonstrate the parasite is vertically transmitted during several pregnancies, causing characteristic lesions, which, in natural conditions, keep the infection in herd without, nevertheless, to induce fetal death. / Não foi encontrado o CPF do autor.

Made available in DSpace on 2014-06-11T19:33:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-03-15Bitstream added on 2014-06-13T19:23:20Z : No. of bitstreams: 1 mineo_twp_dr_jabo.pdf: 3433348 bytes, checksum: fac62f81f1b33434ac64c0e811ae8d5a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Neospora caninum é um protozoário do Filo Apicomplexa, que foi primeiramente descrito como causa de encefalomielite em filhotes caninos sorologicamente negativos para Toxoplasma gondii. Estudos anteriores neste importante hospedeiro da cadeia epidemiológica de N. caninum demonstram que as respostas de anticorpos IgG são tardiamente detectadas e que a infecção clínica é de difícil indução. Desta forma, este trabalho objetivou o estudo da imunidade de cães frente à infecção oral por N. caninum. Os resultados obtidos a partir de análises de diversos animais experimentalmente infectados indicam que os cães apresentam uma prolongada fase aguda da infecção, com eliminação de oocistos associado à queda nos níveis de linfócitos T CD4+ e CD8+ e diminuição de MHC de classe II por células apresentadoras de antígeno. Adicionalmente, os animais apresentam soroconversão instável durante o mesmo período, sendo que somente IgG1 e IgG3 foram detectados em adultos e filhotes, respectivamente, entre o 2o e 3o mês de infecção. De forma concomitante, observa-se uma predominância da expressão de citocinas imunomoduladoras como TGF 1, IL-4 e IL-10. Após dois meses de infecção, o perfil da resposta se inverte, sendo observado picos de produção dos marcadores CD4 e CD8 de linfócitos T e citocinas próinflamatórias como IFN , IL-6 e IL-12, além do aumento nos títulos de anticorpos, principalmente IgG1 e IgG4 nos cães jovens. Com base nestes resultados, conclui-se que os cães apresentam uma relação de equilíbrio com N. caninum, a qual induz nesta espécie uma modulação da resposta imunológica durante a fase de merogonia. / Neospora caninum is an Apicomplexan parasite firstly described as the cause of encephalomyelitis in puppies serologically negative to Toxoplasma gondii. Previous reports on the parasite s definitive host indicate a late IgG antibody response and that clinical disease is difficult to be induced. The aim of this study was to investigate canine immunity during N. caninum oral infection. The results obtained from the analysis of infected animal s samples indicate that dogs present a protracted acute phase, with oocyst shedding correlated to a drop in CD4+ and CD8+ T cell levels, and low MHC class II expression by antigen presenting cells. Additionally, the dogs presented an unstable seroconversion pattern in the same period, with only IgG1 and IgG3 being detected in adult dogs and puppies, respectively, between the second and third months of infection. Concomitantly, dominant Th2 cytokine expression was observed, with peak expression levels of TGF 1, IL-4 and IL-10. After 2 months of infection, the immunity profile shifts towards a Th1 response, with high levels of CD4 and CD8 lymphocytary marker production and pro-inflammatory cytokine expression (IFN , IL-6 and IL-12), besides of the raise in antibody levels, especially IgG1 and IgG4 in puppies. Based in the results presented herein, we may conclude that dogs present a balanced host-parasite relationship, modulating the host immune response during N. caninum merogony.