Despite Neospora caninum being recognised as a major cause of bovine abortion, its pathogenesis is only partially understood. Evidence of immune mediated placental pathology has been reported as being responsible for compromising pregnancy probably due to an exacerbated Th1 immune response at the maternal-foetal interface. Different clinical outcomes are known to follow experimental infections at different stages of gestation, with foetal death being the most common finding during early gestation infections, and the birth of live congenitally infected calves following infection in mid or late gestation. The aim of the current study was to characterise the placental cellular immune responses and cytokine expression following experimental Neospora infection during pregnancy. Placentomes were collected from cattle experimentally inoculated with the tachyzoites of the Nc-1 strain during early, mid and late gestation. Inflammation in early gestation was generally moderate to severe. Differently in mid gestation, inflammation was mild to moderate and minimal to mild in late gestation. Generally cellular infiltrates were mainly characterised by the presence of CD3+, CD4+ and γδ T-cells; whereas CD8+ and NK cells were less numerous. Macrophages were detected in larger numbers during later time-points after infection. A moderate to severe infiltration of IL-12, IFN-γ and TNF-α expressing cells was observed in the placentas collected in early gestation. This infiltration was more pronounced in the samples of placentome collected from dams carrying a dead foetus or in those that had aborted, compared with mothers carrying live foetuses at the time of sampling. The distribution of the cellular subsets observed in the three studies was similar. However, cellular infiltrates were more severe following infection during the first trimester in comparison to the second and third trimester. Similarly, the infiltration of Th1 cytokine expressing-cells was more severe in early gestation compared with the milder and more minimal infiltrations observed following N. caninum infection in mid and late gestation, respectively. These results may explain the milder clinical outcome observed when animals are infected in later stages of pregnancy.

The papers included in this thesis examine the host–parasite relationship in small and large animals following experimental challenges with Neospora caninum. This apicomplexan parasite is a major cause of abortion and reproductive losses in cattle worldwide. Economic and welfare issues make the development of a vaccine against the transplacental transmission of Neospora highly desirable. This thesis evaluates the host-parasite interactions in a non-pregnant mouse model examining whether the actively multiplying stage of the parasite (tachyzoite) could be attenuated through prolonged in vitro cultivation (passage) and used as a live vaccine. We show that continued maintenance of tachyzoites in tissue culture produced significantly reduced levels of morbidity and mortality in the mice following challenge, compared to mice receiving virulent parasites. Inoculation with a sub-lethal dose of tachyzoites was shown to protect against a subsequent lethal challenge of virulent parasites. Mice showing higher levels of cell mediated immunity (CMI) (antigen-specific splenocyte proliferation and interferon-γ (IFN-γ) production) had lower parasite burdens compared to mice with less pronounced CMI responses. Combined, these works show that it is possible to protect against a lethal challenge using attenuated tachyzoites and that a strong T-helper Type-1 CMI response is involved in protection and in reducing clinical disease severity. As the most commonly known route of infection with N. caninum is transplacental, from dam to foetus, we also wanted to examine the host-parasite relationship in pregnant cattle. This was done through the serial examination of the maternal and foetal immune responses of experimentally challenged cattle under controlled conditions at different stages throughout pregnancy. These works show the importance of the timing, location and magnitude of multiple components of the host immune response in determining foetal survival and also whether vertical transmission occurs. We show that both the maternal and foetal immune responses are critical in determining the clinical outcome of infection. A strong maternal CMI response was shown to aid foetal survival by reducing the numbers of parasites reaching and thus damaging the placenta. Due to the syndesmychorial nature of the ruminant placenta, any foetal responses observed are as a result of foetal infection. These experiments show that as pregnancy progresses the foetus goes from being immunologically immature and incapable of mounting a protective immune response (70 days of gestation (dg)) to becoming capable of mounting parasite-specific humoral, innate and CMI responses from around 140dg onwards. The experiments in pregnant cattle confirm the importance of parasite-specific proliferation and IFN-γ production, in reducing the magnitude of the parasite challenge reaching the maternal–foetal interface and aiding foetal survival. We also examined the immunodominant parasite peptides expressed in HPLC fractionated tachyzoite antigen, which are recognised by the cellular immune response of experimentally challenged cattle. Through LC-ESI-MS/MS, 6 Neospora proteins (including SAG1, SRS2 and GRA2) and a number of Toxoplasma gondii orthologues were identified and found to be recognised by CD4+ T-cells. These works collectively demonstrate the complexity of the host-parasite interaction in Neospora infections and show the importance of a CMI response in protection against the parasite.

The 16 publications presented in this thesis summarise the author’s contribution to seroepidemiological approaches for the diagnosis and monitoring of animal diseases of importance to New Zealand. The first four publications not only contribute to the above in relation to three important animal pathogens, namely Brucella ovis, Mycobacterium avium spp. paratuberculosis and Bovine Leukaemia Virus but also give an insight into more general consideration associated with the optimisation and validation of serological assays, namely regarding the definition and choice of gold standard reference sera, the determination of the cutoff threshold and discrimination between negative and positive reference populations. Two further publications deal with the establishment and validation of serological assays for the diagnosis of Neospora caninum infection and abortion in New Zealand. Then, baseline data were obtained for the sero-prevalence of the infection in dog and cattle populations in New Zealand. Three case studies provided initially information about the kinetics of serological responses after a N caninum abortion outbreak, and information about the usefulness of herd-based techniques rather than individual cowbased abortion diagnoses. A further study provided some early information about the mode of transmission seemingly predominating in New Zealand, which tends to be mainly via post-natal infection, in contrast to evidence provided by overseas researchers. A final case study, a longitudinal study of serological and other responses over a period of three years also provided data on the production effects of N caninum. The dissertation is completed by a number of reviews on sero-diagnosis of N caninum infection, its presence in Australasia and suggests finally control options, based on the present state of knowledge.

The 16 publications presented in this thesis summarise the author’s contribution to seroepidemiological approaches for the diagnosis and monitoring of animal diseases of importance to New Zealand. The first four publications not only contribute to the above in relation to three important animal pathogens, namely Brucella ovis, Mycobacterium avium spp. paratuberculosis and Bovine Leukaemia Virus but also give an insight into more general consideration associated with the optimisation and validation of serological assays, namely regarding the definition and choice of gold standard reference sera, the determination of the cutoff threshold and discrimination between negative and positive reference populations. Two further publications deal with the establishment and validation of serological assays for the diagnosis of Neospora caninum infection and abortion in New Zealand. Then, baseline data were obtained for the sero-prevalence of the infection in dog and cattle populations in New Zealand. Three case studies provided initially information about the kinetics of serological responses after a N caninum abortion outbreak, and information about the usefulness of herd-based techniques rather than individual cowbased abortion diagnoses. A further study provided some early information about the mode of transmission seemingly predominating in New Zealand, which tends to be mainly via post-natal infection, in contrast to evidence provided by overseas researchers. A final case study, a longitudinal study of serological and other responses over a period of three years also provided data on the production effects of N caninum. The dissertation is completed by a number of reviews on sero-diagnosis of N caninum infection, its presence in Australasia and suggests finally control options, based on the present state of knowledge.

The 16 publications presented in this thesis summarise the author’s contribution to seroepidemiological approaches for the diagnosis and monitoring of animal diseases of importance to New Zealand. The first four publications not only contribute to the above in relation to three important animal pathogens, namely Brucella ovis, Mycobacterium avium spp. paratuberculosis and Bovine Leukaemia Virus but also give an insight into more general consideration associated with the optimisation and validation of serological assays, namely regarding the definition and choice of gold standard reference sera, the determination of the cutoff threshold and discrimination between negative and positive reference populations. Two further publications deal with the establishment and validation of serological assays for the diagnosis of Neospora caninum infection and abortion in New Zealand. Then, baseline data were obtained for the sero-prevalence of the infection in dog and cattle populations in New Zealand. Three case studies provided initially information about the kinetics of serological responses after a N caninum abortion outbreak, and information about the usefulness of herd-based techniques rather than individual cowbased abortion diagnoses. A further study provided some early information about the mode of transmission seemingly predominating in New Zealand, which tends to be mainly via post-natal infection, in contrast to evidence provided by overseas researchers. A final case study, a longitudinal study of serological and other responses over a period of three years also provided data on the production effects of N caninum. The dissertation is completed by a number of reviews on sero-diagnosis of N caninum infection, its presence in Australasia and suggests finally control options, based on the present state of knowledge.

This study represents an analysis of serological and risk factor data collected previously in Alberta, Canada, involving neosporosis in beef and dairy cattle. The causative agent of neosporosis, Neospora caninum (NC), is a single-celled, apicomplexan protozoan parasite in which domesticated dogs have been identified as the definitive host. The primary economic impact involves beef and dairy cattle due to associated abortions and neonatal mortality. The data used in this study were collected for cattle in both dairy and beef herds in an identical manner permitting a direct comparison of host-, herd-, and environmental risk factors for neosporosis among beef and dairy cattle using descriptive statistical methods and the construction of multivariable models. The outcome assessed in the multivariable models was cow-level seropositivity for antibodies to N. caninum. Individual-level fixed, herd-level fixed, and random effects were evaluated with respect to the outcome. In the final multivariable models, there were few statistically significant potential risk factors identified. In the beef multivariable model, the significant explanatory factors were related to acreage of farm, site of calving, and pH of soil. Among the potential risk factors identified in the three multivariable models it appeared seropositivity to NC among beef cattle is more related to environmental conditions; on the other hand, it seems that seropositivity to NC in dairy cattle pertains to associated management factors. In the future, longitudinal studies are needed to explore the validity of the current knowledge regarding N. caninum by investigating potential risk factors that have been identified due to the fact that crosssectional studies can not prove association.

Los problemas reproductivos en el ganado bovino lechero, producidos por el parásito Neospora caninum, son de gran impacto económico en el ámbito mundial. El objetivo del presente estudio fue determinar la seroprevalencia de Neospora caninum en bovinos lecheros del valle de Lima en el año 2000. Con este fin se evaluaron 304 sueros de vacas lecheras adultas provenientes de 19 establos lecheros ubicados en la zona norte (n = 12) y en la zona sur (n = 7) del valle de Lima para la detección de anticuerpos contra Neospora caninum mediante la prueba de inmunofluorescencia indirecta (IFI). El 29.61% ± 5.13% (90/304) presentó anticuerpos contra el parásito en una dilución de 1:200. En la zona norte el 40.83% ± 8.79% (49/120) y en la zona sur 22.28% ± 6.01% (41/184). Con estos resultados se confirma la presencia del parásito Neospora caninum en bovinos lecheros del valle de Lima. / The reproductive problems in dairy cattle produced for Neospora caninum have a great economic impact and world-wide distribution. The aim of this study was found the seroprevalence of Neospora caninum in dairy bovines from the valley of Lima in 2000. was evaluated with IFAT 304 serum samples of 19 dairy herds from north (n = 12) and south (n = 7) Lima. 29.61± 5.13% (90/304) presented antibodies against this parasite in 1:200 dilution. 40.83% ± 8.79% (49/120) in north zone and 22.28% ± 6.01% (41/184) in the south zone. Which confirm the presence of Neospora caninum in dairy bovines in the valley of Lima.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2015-03-27T12:50:18Z No. of bitstreams: 1 Dissertação_ICS_ Gregory Alves Ferraz.pdf: 1703623 bytes, checksum: 42fd6e6502aa336c756f4be148d6d919 (MD5) / Made available in DSpace on 2015-03-27T12:50:18Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Gregory Alves Ferraz.pdf: 1703623 bytes, checksum: 42fd6e6502aa336c756f4be148d6d919 (MD5) / neosporose acomete muitas espécies animais, causando-lhes manifestações clínicas de natureza neuro muscular e tem sido apontada como um dos fatores que acarreta prejuízos econômicos na pecuária mundial. Seu agente etiológico, o N. caninum, é controlado por mecanismos de imunidade mediada por células, com destaque de papel protetor para os linfócitos T, que tem variação na sua resposta a depender do tecido parasitado. No sistema nervoso central ocorre um balanço imunológico muito preciso onde a forma infectante do parasito - os taquizoítos - estimula a produção de citocinas do tipo TH1, que acarretariam no controle da sua proliferação. Neste tecido, as células neurogliais parecem responder com a produção de citocinas TH2, com intuito de evitar ou controlar lesões causadas pela resposta pró-inflamatória. O presente estudo investigou o papel imunomodulatório das culturas primárias de microglia durante infecção com taquizoítos de N. caninum. As células, obtidas de cérebro de ratos neonatos, foram infectadas por 72h com o parasito e, para obtenção de controle positivo, foram tratadas com LPS pelo mesmo período. Ensaios de Azul de Trypan e MTT foram realizados para testar a viabilidade celular. Foi observado que as células infectadas e aquelas tratadas com LPS apresentaram significativa perda de viabilidade, cuja causa pode ter sido o aumento significativo dos níveis de nitrito, contudo este evento não contribuiu para o controle da proliferação parasitária. A resposta dessas células ao estímulo parasitário não acarretou tampouco liberação de citocinas inflamatórias (TNF e IFN-γ) e somente quando pré-tratadas com LPS, tiveram níveis de TNF aumentados. Esses dados são compatíveis com a possível modulação negativa da citocina IL-10, que foi encontrada aumentada em ambos tratamentos. Os resultados apontam para a incapacidade das células microgliais isoladas em controlar a proliferação de taquizoítos, ainda que estas, em interação com demais tipos celulares no contexto do tecido cerebral, tenham papel destacado na modulação da resposta ao N. caninum.

No description available.

Neospora caninum é um parasita Apicomplexa que causa doença neuromuscular em cães e abortamento em bovinos. Cães e coiotes são as únicas espécies reconhecidas como hospedeiros definitivos, nas quais ocorre a fase sexuada do ciclo evolutivo do N. caninum, com eliminação de oocistos através das fezes, os quais esporulam no ambiente e tornam-se infectantes. Apesar da importância dos oocistos como fonte de infecção para várias espécies de hospedeiros, a viabilidade e resistência desses oocistos a tratamentos físicos e químicos ainda são desconhecidas. Este estudo teve por objetivo avaliar a viabilidade de oocistos esporulados de N. caninum após tratamentos com diferentes desinfetantes, temperaturas e tempos de exposição. Três cães foram alimentados com tecido cerebral de búfalos soropositivos para anticorpos anti-N.caninum pela reação de imunofluorescência indireta (RIFI 100) para a obtenção de oocistos. Desses, somente um cão eliminou oocistos tipo Neospora-Hammondia sendo confirmado serem de N. caninum por bioensaio em gerbilos e por métodos moleculares (PCR-RFLP). Os oocistos esporulados foram purificados e 11 alíquotas contendo aproximadamente 3000 oocistos por alíquota constituíram cada um dos tratamentos, sendo estes: Formol 10% por 1h; Amônia 10% por 1h; Álcool 70% por 1h; Álcool absoluto por 1h; Iodo 2% por 1h; Hipoclorito de sódio 10% por 1h; Temperatura ambiente, controle; -20ºC por 6h; 4ºC por 6h; 60ºC por 1m e 100ºC por 1m. Os tratamentos químicos foram todos realizados à temperatura ambiente. Após tratamento os oocistos foram divididos em alíquotas com 1000 oocistos cada e estas foram administradas, via oral, a gerbilos (1000 oocistos por gerbilo) sendo cada grupo experimental constituídos por três animais. Depois de 63 dias os gerbilos foram sacrificados e colheu-se sangue para a pesquisa de anticorpos anti-N. caninum (RIFI e western blotting) e tecidos para a pesquisa de cistos em esfregaço direto de cérebro e DNA do parasito (PCR em tempo real), além de pesquisa do agente por técnicas histopatológicas e imuno-histoquímica. Considerou-se eficaz o tratamento que confirmou a inviabilidade dos oocistos por resultados negativos em todas as cinco provas realizadas. Dos tratamentos realizados mostrou-se eficaz o uso de calor a 100ºC por 1 minuto e do hipoclorito de sódio a 10% por 1 hora, podendo ser estes indicados para o controle dessas formas no ambiente. / Neospora caninum is an Apicomplexan parasite that causes neuromuscular disorders in dogs and abortion in cattle. Dogs and coyotes are the only species identified as definitive hosts. The sexual phase of the N. caninum life cycle occurs within the host, and results in the shedding of oocysts in the feces that will sporulate in the environment and become infective. Despite their relevance as a source of infection for a number of different hosts, the resistance and viability of such oocysts to physical and chemical treatments are yet to be known. The purpose of this study was to assess the viability of N. caninum sporulated oocysts after exposure to treatments using different disinfectants, temperatures and periods of time. For acquisition of the oocysts, three dogs were fed brain tissue from buffaloes positive for antibodies to N. caninum by an indirect fluorescent antibody test (IFAT 100). Only one of the dogs excreted Neospora-Hammondia type oocysts. Such oocysts were confirmed to be N. caninum by bioassay in gerbils and molecular methods (PCR-RFLP). The sporulated oocysts were purified and 11 doses with approximately 3,000 oocysts each were treated as follows: 10% formaldehyde (formol) for 1 h; 10% ammonia for 1 h; 70% alcohol for 1 h; absolute alcohol for 1 h; 2% iodine for 1 h; 10% sodium hypochlorite for 1 h; Room temperature, control; -20ºC for 6 h; 4ºC for 6 h; 60ºC for 1 min, and 100ºC for 1 min. All chemical treatments were performed at room temperature. After treatment, the oocysts were divided into doses of 1,000 oocysts each and administered into three gerbils (Meriones unguiculatus) orally (1,000 oocysts per gerbil) per treatment. The gerbils were euthanized after 63 days. Blood samples were taken to be tested for the presence of N. caninum antibodies (IFAT and Western blotting analysis), and tissues samples to be tested for the presence of cysts by brain smear technique and detection of the parasite DNA (real-time PCR), and the identification of the parasite by immunohistochemical and histopathological examinations. In order to be considered an effective treatment, negative results should be observed in the gerbils of all five evaluations conducted. Out of the treatments carried out in this study, exposures to a temperature of 100ºC for 1 min and to a 10% sodium hypochlorite solution for 1 h were effective.