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.

Made available in DSpace on 2014-07-29T15:13:43Z (GMT). No. of bitstreams: 1 Dissertacao Vanessa Silvestre Ferreira de Oliveira.pdf: 1341350 bytes, checksum: 9ce3345b7f150b232b983b427448e976 (MD5) Previous issue date: 2011-03-16 / Este trabalho teve como objetivos identificar os protozoários encontrados em fezes de cães, observar a ocorrência da infecção por Neospora caninum e Toxoplasma gondii em cães pela Reação de Imunofluorescência Indireta (RIFI), desenvolver e validar técnicas de ELISA para o diagnóstico sorológico de N. caninum em cães, determinar a utilidade do Western Blot para esse diagnóstico e observar a ocorrência de DNA de N. caninum, T. gondii e Hammondia heydorni em tecido cerebral de cães. Para isso, foram colhidas amostras de cérebro e sangue de 251 cães, e fezes de 106 cães, provenientes do Centro de Zoonoses de Goiânia, Goiás. Foram utilizados oito soros de infecção experimental por N. caninum e quatro por T. gondii para comprovação dos resultados sorológicos. As amostras de fezes foram concentradas pelo método de Teleman seguido da flutuação com solução de Sheather, para visualização dos oocistos. Foi realizada a extração de DNA de todas as amostras, posteriormente analisadas por nested-PCR específicas para detecção de DNA de N. caninum, H. heydorni, T. gondii, Cryptosporidium spp. e Giardia spp. Uma nested-PCR foi ainda realizada nas amostras em que foram visualizados oocistos menores de 20 µm, para a detecção de DNA de coccídios e posterior seqüenciamento, para correta identificação dos mesmos. A técnica de RIFI foi realizada em todas as amostras de sangue, e técnicas de ELISA foram padronizadas, utilizando-se extrato solúvel de taquizoítos e as proteínas recombinantes rNcGRA7, rNcSAG4, rNcBSR4 e rNcSRS9 de N. caninum. Para a detecção de DNA dos parasitos nos cérebros, estes foram divididos em três partes, e de cada parte foi realizada a extração de DNA. Após a extração, foram realizadas nested-PCR específicas para cada parasito. Nas fezes dos cães, foram encontradas doze amostras contendo oocistos. Em seis, os oocistos eram maiores que 20 µm, e foram identificados como de Cystoisospora canis, e nas outras seis, oocistos menores que 20 µm foram visualizados e identificados como sendo quatro de H. heydorni, um de Cystoisospora spp. e uma amostra apresentava DNA tanto de H. heydorni quanto de Cystoisospora spp. DNA de C. canis foi identificado em quatro amostras, uma amostra foi identificada como G. duodenalis genótipo C e quatro como G. duodenalis genótipo D. Nenhuma amostra de fezes foi positiva para N. caninum e T. gondii, e quatro foram positivas para H. heydorni na PCR específica para este parasito. Nos cérebros analisados, foram encontradas seis amostras (2,4%) positivas a N. caninum e 23 amostras (9,1%) foram positivas a T. gondii. Nenhuma amostra foi positiva a H. heydorni. Anticorpos anti-N. caninum foram encontrados em 18,32% dos cães analisados, e 56,57% dos cães apresentaram sorologia positiva a T. gondii pela técnica de RIFI. Técnicas de ELISA foram padronizadas, utilizando-se extrato solúvel de taquizoítos e as proteínas recombinantes rNcGRA7, rNcSAG4, rNcBSR4 e rNcSRS9 de N. caninum. Foram encontrados valores de sensibilidade e especificidade menores que 84%, além do reconhecimento das proteínas recombinantes pelos dois parasitos, nos soros de infecções naturais e experimentais. Uma baixa concordância entre as técnicas foi observada, com valores de Kappa inferiores a 0,4. Os resultados encontrados indicam que N. caninum está menos presente em fezes de cães, enquanto H. heydorni é mais facilmente encontrada. As amostras que continham Cryptosporidium e Giardia foram identificadas como espécies-específicas. A pesquisa de DNA de parasitos em órgãos de cães pode ser utilizada como uma ferramenta para o diagnóstico post-mortem de infecções por protozoários, porém as técnicas de PCR podem apresentar baixa sensibilidade devido à pequena quantidade de tecido utilizado. Os resultados encontrados no WB indicam não haver um padrão de reconhecimento das proteínas imunodominantes, e que as técnicas sorológicas na neosporose canina têm limitações, devido aos baixos títulos encontrados e à reação cruzada com T. gondii, além da alta prevalência da infecção por T. gondii em cães, e presença de infecções mistas.

Um dos estudos observacionais mais difundidos e usados em epidemiologia veterinária é o estudo do tipo transversal. Sua popularidade ocorre por fatores como baixo custo e rapidez comparados com outros tipos de estudos, além de ajudar a estimar a prevalência de uma doença (desfecho) e postular fatores associados com o desfecho, que poderão ser confirmados como fatores causais em outros tipos de estudos epidemiológicos. Porém, este tipo de estudo apresenta dois importantes desafios: a dependência dos dados, muito frequente dada a típica estrutura populacional de animais dentro do mesmo rebanho ou fazenda e a escolha da medida de associação para desfechos binários, tão frequentes neste modelo de estudo. Com o objetivo de contribuir com a compreensão global da epidemiologia do aborto bovino associado à Neospora caninum tendo em conta a estrutura populacional, construiu-se um modelo misto com os dados de um estudo transversal realizado em duas regiões do Rio Grande do Sul. Usaram-se dados de 60 propriedades amostradas em duas regiões (noroeste e sudeste) e 1256 bovinos. A percentagem de aborto dentro de cada rebanho variou entre 1% e 30%. Vacas soropositivas tiveram 6,63 vezes mais chances de ter histórico de aborto (IC 95%: 4,41-13,20). As chances de uma vaca ter histórico de aborto foram 5,18 vezes maiores na região noroeste em relação à região sudeste (IC 95%: 1,83-20,80). Um coeficiente de correlação intraclasse de 16% foi estimado, indicando que 16% da variação da ocorrência de abortamentos não explicados pelos efeitos fixos foram devido as fazendas. Já na segunda parte deste trabalho, uma revisão sistemática foi realizada considerando um conjunto diverso de revistas e jornais com o objetivo de verificar os métodos estatísticos usados e a adequação das interpretações das medidas de associação estimadas em estudos transversais na área de medicina veterinária. Um total de 62 artigos foi avaliado. A revisão mostrou que, independentemente do nível de prevalência relatado no artigo, 96% deles empregou regressão logística e, portanto, estimaram razão de chances (RC). Nos artigos com prevalência superior a 10%, 23 deles fizeram uma interpretação adequada da RC como uma “razão de chances” ou simplesmente não fizeram uma interpretação direta da RC, enquanto 23 artigos interpretaram de forma inadequada a RC, considerando-a como risco ou probabilidade. Entre os artigos com prevalência inferior a 10%, apenas três interpretaram a RC como uma “razão de chances”, cinco interpretaram como risco ou probabilidade e em um, apesar de ter estimado a razão de prevalências (RP), foi interpretado de forma inadequada. Paralelamente, com o objetivo de exemplificar o uso de métodos estatísticos que estimam diretamente a razão de prevalências (RP), medida mais adequada para os estudos transversais, um conjunto de dados obtidos a partir de um estudo transversal sobre a ocorrência de anticorpos (AC) contra o vírus da diarreia viral bovina (BVDV) foi usado. Os AC foram medidos em amostras de tanque de leite de rebanhos leiteiros localizados no estado do Rio Grande do Sul, em que os possíveis fatores associados puderam ser avaliados. Entre os métodos utilizados, as maiores discrepâncias nas medidas de associação estimadas foram observadas com a regressão logística tomando-se como referência a regressão log-binomial. Finalmente, é importante que este tipo de desafio seja atendido pelos pesquisadores que realizam estudos transversais, ou seja, considerar a estrutura das populações nas análises, cuidado ao escolher o tipo de modelo estatístico empregado para desfecho binário e interpretação dos estimadores. / The commonest study design used in veterinary epidemiology is the cross-sectional study. Its popularity lies on the fact of the short time needed and low costs compared with other types of studies; moreover, this type of study estimates prevalence and associated factors, which may be elucidated as causal in another type of epidemiological studies. However, this type of study presents two major challenges: a very common dependence between data given the typical structure of the animal population, i.e., animals within herds or farms and the choice of measure of association for binary outcomes, frequently used in this type of study. In order to contribute to the understanding of the epidemiology of bovine abortion associated with Neospora caninum, a mixed model accounting for the hierarchical structure of cattle population using data from a cross-sectional study conducted in two regions (northwest and southeast) of Rio Grande do Sul was made. Data from 60 dairy herds and 1256 sampled cattle were used. The percentage of abortions in each herd ranged between 1% and 30%. Seropositive cows were 6.63 times more likely to have a history of abortion (95% CI: 4.41 to 13.20). The chances of a cow have a history of abortion were 5.18 times higher in the northwest comparing with the southeast region (95% CI: 1.83 to 20.80). An intraclass correlation coefficient (ICC) of 16% was estimated which means that 16% of the variation in abortion occurrence not explained by the fixed effects is due to farms. In the second part of this work, a systematic review was conducted considering a range of journals and newspapers in order to verify the statistical methods used and the adequacy of the interpretations of the measures of association estimated in cross-sectional studies from the veterinary medicine field. A total of 62 articles were revised. The review showed that, regardless of the reported prevalence, 96% of them employed logistic regression, therefore estimating odds ratio (OR). From the articles that reported prevalence rates above 10%, 23 of them did a proper interpretation of OR as an odds ratio, or simply did not make a direct interpretation of the OR, while 23 articles interpreted improperly the OR as a risk or probability. Among the articles that reported prevalence rates lower than 10%, only three interpreted the OR as an odds ratio, five interpreted as a risk or probability and only one, despite the estimated prevalence ratio (PR), it was improperly interpreted. Meanwhile, in order to exemplify the use of statistical methods to estimate directly the PR, the more appropriate measure of association in cross-sectional studies, a data set obtained from a cross-sectional study to estimate the occurrence of antibodies (AB) against bovine viral diarrhea virus (BVDV) in milk was used; AB were measured in bulk tank samples from dairy herds located in the state of Rio Grande do Sul, Brazil, and also possible associated factors were estimated. Among the methods used, major discrepancies in the measures of association estimated were observed with the logistic regression, comparing with the log-binomial regression. Finally, it is important that such challenges are met by the researchers that undertake cross-sectional studies.

Um dos estudos observacionais mais difundidos e usados em epidemiologia veterinária é o estudo do tipo transversal. Sua popularidade ocorre por fatores como baixo custo e rapidez comparados com outros tipos de estudos, além de ajudar a estimar a prevalência de uma doença (desfecho) e postular fatores associados com o desfecho, que poderão ser confirmados como fatores causais em outros tipos de estudos epidemiológicos. Porém, este tipo de estudo apresenta dois importantes desafios: a dependência dos dados, muito frequente dada a típica estrutura populacional de animais dentro do mesmo rebanho ou fazenda e a escolha da medida de associação para desfechos binários, tão frequentes neste modelo de estudo. Com o objetivo de contribuir com a compreensão global da epidemiologia do aborto bovino associado à Neospora caninum tendo em conta a estrutura populacional, construiu-se um modelo misto com os dados de um estudo transversal realizado em duas regiões do Rio Grande do Sul. Usaram-se dados de 60 propriedades amostradas em duas regiões (noroeste e sudeste) e 1256 bovinos. A percentagem de aborto dentro de cada rebanho variou entre 1% e 30%. Vacas soropositivas tiveram 6,63 vezes mais chances de ter histórico de aborto (IC 95%: 4,41-13,20). As chances de uma vaca ter histórico de aborto foram 5,18 vezes maiores na região noroeste em relação à região sudeste (IC 95%: 1,83-20,80). Um coeficiente de correlação intraclasse de 16% foi estimado, indicando que 16% da variação da ocorrência de abortamentos não explicados pelos efeitos fixos foram devido as fazendas. Já na segunda parte deste trabalho, uma revisão sistemática foi realizada considerando um conjunto diverso de revistas e jornais com o objetivo de verificar os métodos estatísticos usados e a adequação das interpretações das medidas de associação estimadas em estudos transversais na área de medicina veterinária. Um total de 62 artigos foi avaliado. A revisão mostrou que, independentemente do nível de prevalência relatado no artigo, 96% deles empregou regressão logística e, portanto, estimaram razão de chances (RC). Nos artigos com prevalência superior a 10%, 23 deles fizeram uma interpretação adequada da RC como uma “razão de chances” ou simplesmente não fizeram uma interpretação direta da RC, enquanto 23 artigos interpretaram de forma inadequada a RC, considerando-a como risco ou probabilidade. Entre os artigos com prevalência inferior a 10%, apenas três interpretaram a RC como uma “razão de chances”, cinco interpretaram como risco ou probabilidade e em um, apesar de ter estimado a razão de prevalências (RP), foi interpretado de forma inadequada. Paralelamente, com o objetivo de exemplificar o uso de métodos estatísticos que estimam diretamente a razão de prevalências (RP), medida mais adequada para os estudos transversais, um conjunto de dados obtidos a partir de um estudo transversal sobre a ocorrência de anticorpos (AC) contra o vírus da diarreia viral bovina (BVDV) foi usado. Os AC foram medidos em amostras de tanque de leite de rebanhos leiteiros localizados no estado do Rio Grande do Sul, em que os possíveis fatores associados puderam ser avaliados. Entre os métodos utilizados, as maiores discrepâncias nas medidas de associação estimadas foram observadas com a regressão logística tomando-se como referência a regressão log-binomial. Finalmente, é importante que este tipo de desafio seja atendido pelos pesquisadores que realizam estudos transversais, ou seja, considerar a estrutura das populações nas análises, cuidado ao escolher o tipo de modelo estatístico empregado para desfecho binário e interpretação dos estimadores. / The commonest study design used in veterinary epidemiology is the cross-sectional study. Its popularity lies on the fact of the short time needed and low costs compared with other types of studies; moreover, this type of study estimates prevalence and associated factors, which may be elucidated as causal in another type of epidemiological studies. However, this type of study presents two major challenges: a very common dependence between data given the typical structure of the animal population, i.e., animals within herds or farms and the choice of measure of association for binary outcomes, frequently used in this type of study. In order to contribute to the understanding of the epidemiology of bovine abortion associated with Neospora caninum, a mixed model accounting for the hierarchical structure of cattle population using data from a cross-sectional study conducted in two regions (northwest and southeast) of Rio Grande do Sul was made. Data from 60 dairy herds and 1256 sampled cattle were used. The percentage of abortions in each herd ranged between 1% and 30%. Seropositive cows were 6.63 times more likely to have a history of abortion (95% CI: 4.41 to 13.20). The chances of a cow have a history of abortion were 5.18 times higher in the northwest comparing with the southeast region (95% CI: 1.83 to 20.80). An intraclass correlation coefficient (ICC) of 16% was estimated which means that 16% of the variation in abortion occurrence not explained by the fixed effects is due to farms. In the second part of this work, a systematic review was conducted considering a range of journals and newspapers in order to verify the statistical methods used and the adequacy of the interpretations of the measures of association estimated in cross-sectional studies from the veterinary medicine field. A total of 62 articles were revised. The review showed that, regardless of the reported prevalence, 96% of them employed logistic regression, therefore estimating odds ratio (OR). From the articles that reported prevalence rates above 10%, 23 of them did a proper interpretation of OR as an odds ratio, or simply did not make a direct interpretation of the OR, while 23 articles interpreted improperly the OR as a risk or probability. Among the articles that reported prevalence rates lower than 10%, only three interpreted the OR as an odds ratio, five interpreted as a risk or probability and only one, despite the estimated prevalence ratio (PR), it was improperly interpreted. Meanwhile, in order to exemplify the use of statistical methods to estimate directly the PR, the more appropriate measure of association in cross-sectional studies, a data set obtained from a cross-sectional study to estimate the occurrence of antibodies (AB) against bovine viral diarrhea virus (BVDV) in milk was used; AB were measured in bulk tank samples from dairy herds located in the state of Rio Grande do Sul, Brazil, and also possible associated factors were estimated. Among the methods used, major discrepancies in the measures of association estimated were observed with the logistic regression, comparing with the log-binomial regression. Finally, it is important that such challenges are met by the researchers that undertake cross-sectional studies.

Um dos estudos observacionais mais difundidos e usados em epidemiologia veterinária é o estudo do tipo transversal. Sua popularidade ocorre por fatores como baixo custo e rapidez comparados com outros tipos de estudos, além de ajudar a estimar a prevalência de uma doença (desfecho) e postular fatores associados com o desfecho, que poderão ser confirmados como fatores causais em outros tipos de estudos epidemiológicos. Porém, este tipo de estudo apresenta dois importantes desafios: a dependência dos dados, muito frequente dada a típica estrutura populacional de animais dentro do mesmo rebanho ou fazenda e a escolha da medida de associação para desfechos binários, tão frequentes neste modelo de estudo. Com o objetivo de contribuir com a compreensão global da epidemiologia do aborto bovino associado à Neospora caninum tendo em conta a estrutura populacional, construiu-se um modelo misto com os dados de um estudo transversal realizado em duas regiões do Rio Grande do Sul. Usaram-se dados de 60 propriedades amostradas em duas regiões (noroeste e sudeste) e 1256 bovinos. A percentagem de aborto dentro de cada rebanho variou entre 1% e 30%. Vacas soropositivas tiveram 6,63 vezes mais chances de ter histórico de aborto (IC 95%: 4,41-13,20). As chances de uma vaca ter histórico de aborto foram 5,18 vezes maiores na região noroeste em relação à região sudeste (IC 95%: 1,83-20,80). Um coeficiente de correlação intraclasse de 16% foi estimado, indicando que 16% da variação da ocorrência de abortamentos não explicados pelos efeitos fixos foram devido as fazendas. Já na segunda parte deste trabalho, uma revisão sistemática foi realizada considerando um conjunto diverso de revistas e jornais com o objetivo de verificar os métodos estatísticos usados e a adequação das interpretações das medidas de associação estimadas em estudos transversais na área de medicina veterinária. Um total de 62 artigos foi avaliado. A revisão mostrou que, independentemente do nível de prevalência relatado no artigo, 96% deles empregou regressão logística e, portanto, estimaram razão de chances (RC). Nos artigos com prevalência superior a 10%, 23 deles fizeram uma interpretação adequada da RC como uma “razão de chances” ou simplesmente não fizeram uma interpretação direta da RC, enquanto 23 artigos interpretaram de forma inadequada a RC, considerando-a como risco ou probabilidade. Entre os artigos com prevalência inferior a 10%, apenas três interpretaram a RC como uma “razão de chances”, cinco interpretaram como risco ou probabilidade e em um, apesar de ter estimado a razão de prevalências (RP), foi interpretado de forma inadequada. Paralelamente, com o objetivo de exemplificar o uso de métodos estatísticos que estimam diretamente a razão de prevalências (RP), medida mais adequada para os estudos transversais, um conjunto de dados obtidos a partir de um estudo transversal sobre a ocorrência de anticorpos (AC) contra o vírus da diarreia viral bovina (BVDV) foi usado. Os AC foram medidos em amostras de tanque de leite de rebanhos leiteiros localizados no estado do Rio Grande do Sul, em que os possíveis fatores associados puderam ser avaliados. Entre os métodos utilizados, as maiores discrepâncias nas medidas de associação estimadas foram observadas com a regressão logística tomando-se como referência a regressão log-binomial. Finalmente, é importante que este tipo de desafio seja atendido pelos pesquisadores que realizam estudos transversais, ou seja, considerar a estrutura das populações nas análises, cuidado ao escolher o tipo de modelo estatístico empregado para desfecho binário e interpretação dos estimadores. / The commonest study design used in veterinary epidemiology is the cross-sectional study. Its popularity lies on the fact of the short time needed and low costs compared with other types of studies; moreover, this type of study estimates prevalence and associated factors, which may be elucidated as causal in another type of epidemiological studies. However, this type of study presents two major challenges: a very common dependence between data given the typical structure of the animal population, i.e., animals within herds or farms and the choice of measure of association for binary outcomes, frequently used in this type of study. In order to contribute to the understanding of the epidemiology of bovine abortion associated with Neospora caninum, a mixed model accounting for the hierarchical structure of cattle population using data from a cross-sectional study conducted in two regions (northwest and southeast) of Rio Grande do Sul was made. Data from 60 dairy herds and 1256 sampled cattle were used. The percentage of abortions in each herd ranged between 1% and 30%. Seropositive cows were 6.63 times more likely to have a history of abortion (95% CI: 4.41 to 13.20). The chances of a cow have a history of abortion were 5.18 times higher in the northwest comparing with the southeast region (95% CI: 1.83 to 20.80). An intraclass correlation coefficient (ICC) of 16% was estimated which means that 16% of the variation in abortion occurrence not explained by the fixed effects is due to farms. In the second part of this work, a systematic review was conducted considering a range of journals and newspapers in order to verify the statistical methods used and the adequacy of the interpretations of the measures of association estimated in cross-sectional studies from the veterinary medicine field. A total of 62 articles were revised. The review showed that, regardless of the reported prevalence, 96% of them employed logistic regression, therefore estimating odds ratio (OR). From the articles that reported prevalence rates above 10%, 23 of them did a proper interpretation of OR as an odds ratio, or simply did not make a direct interpretation of the OR, while 23 articles interpreted improperly the OR as a risk or probability. Among the articles that reported prevalence rates lower than 10%, only three interpreted the OR as an odds ratio, five interpreted as a risk or probability and only one, despite the estimated prevalence ratio (PR), it was improperly interpreted. Meanwhile, in order to exemplify the use of statistical methods to estimate directly the PR, the more appropriate measure of association in cross-sectional studies, a data set obtained from a cross-sectional study to estimate the occurrence of antibodies (AB) against bovine viral diarrhea virus (BVDV) in milk was used; AB were measured in bulk tank samples from dairy herds located in the state of Rio Grande do Sul, Brazil, and also possible associated factors were estimated. Among the methods used, major discrepancies in the measures of association estimated were observed with the logistic regression, comparing with the log-binomial regression. Finally, it is important that such challenges are met by the researchers that undertake cross-sectional studies.