Made available in DSpace on 2014-06-11T19:23:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-10-05Bitstream added on 2014-06-13T19:50:59Z : No. of bitstreams: 1 baroza_pfj_me_jabo.pdf: 472300 bytes, checksum: 8c0c255232fc2e33839f99f65b3e5d49 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os objetivos do estudo foram: 1. avaliar as concentrações de proteína total, imunoglobulina G, enzimas e minerais na secreção láctea de cabras e vacas, nos primeiros 30 dias pós-parto, estabelecendo o limite máximo possível para se obter secreção láctea que permita adequada transferência de imunidade passiva aos neonatos; 2. avaliar a influência do congelamento nestes constituintes; e, disponibilizar informações que possam auxiliar na montagem de bancos de colostro. Foram examinadas amostras de secreções lácteas de 10 cabras da raça Saanen e 10 vacas da raça Holandesa recém-paridas e sadias, de terceira ou quarta lactação, coletadas nos momentos: dia do parto e 1, 3, 5, 9, 14 e 29 dias após o parto. As amostras foram obtidas após a ordenha completa das cabras e vacas. As amostras de colostro caprino foram submetidas aleatoriamente a seis tratamentos, de acordo com o período de congelamento; enquanto as amostras de colostro bovino foram submetidas a cinco tratamentos. As amostras do grupo G1 são colostros caprinos “in natura”; as amostras do grupo G2 são as mesmas amostras do G1, porém submetidas ao aquecimento de 56ºC durante 60 minutos; as amostras do grupo G3 foram submetidas a 30 dias de congelamento, as amostras do grupo G4, a 60 dias de congelamento, as amostras do grupo G5, a 90 dias de congelamento e as do grupo G6, a 180 dias de congelamento. As amostras de colostro bovino foram submetidas aos mesmos períodos de congelamento... / The goals of the study were: 1. to evaluate total protein, G immunoglobulin, enzymes and minerals concentrations in goat and cow milky secretion, during the first 30 days postparturition, establishing the possible maximum limit to obtain milky secretion that allows appropriate transfer of passive immunity to the newborns kids; 2. to evaluate the influence of freezing in these constituents; and, 3. to create information that can aid in the assembly of colostrum banks. It were used samples of milky secretions of 10 Saanen goats and 10 Holstein cows recently-given birth and healthy, of third or fourth nursing, collected in the moments: in the day of the parturition and 1, 3, 5, 9, 14 and 29 days after the parturition. The samples were obtained after goats and cows complete milks. The samples of goat colostrum were submitted to six treatments, in agreement with the freezing period; the samples of cow colostrum were submitted to five treatments. The samples of the group G1 are goat colostrums “in natura; the samples of the group G2 are the same samples of G1, however submitted to the heating of 56ºC for 60 minutes; the samples of the group G3 were submitted to 30 days of freezing, the samples of the group G4, to 60 days of freezing, the samples of the group G5, to 90 days of freezing and the one of the group G6, to 180 days of freezing. The samples of cow colostrum were submitted to the same freezing periods... (Complete abstract, click electronic access below)

Orientador: José Jurandir Fagliari / Banca: Mario Roberto Hatayde / Banca: Eduardo Harry Birgel Junior / Resumo: Os objetivos do estudo foram: 1. avaliar as concentrações de proteína total, imunoglobulina G, enzimas e minerais na secreção láctea de cabras e vacas, nos primeiros 30 dias pós-parto, estabelecendo o limite máximo possível para se obter secreção láctea que permita adequada transferência de imunidade passiva aos neonatos; 2. avaliar a influência do congelamento nestes constituintes; e, disponibilizar informações que possam auxiliar na montagem de bancos de colostro. Foram examinadas amostras de secreções lácteas de 10 cabras da raça Saanen e 10 vacas da raça Holandesa recém-paridas e sadias, de terceira ou quarta lactação, coletadas nos momentos: dia do parto e 1, 3, 5, 9, 14 e 29 dias após o parto. As amostras foram obtidas após a ordenha completa das cabras e vacas. As amostras de colostro caprino foram submetidas aleatoriamente a seis tratamentos, de acordo com o período de congelamento; enquanto as amostras de colostro bovino foram submetidas a cinco tratamentos. As amostras do grupo G1 são colostros caprinos "in natura"; as amostras do grupo G2 são as mesmas amostras do G1, porém submetidas ao aquecimento de 56ºC durante 60 minutos; as amostras do grupo G3 foram submetidas a 30 dias de congelamento, as amostras do grupo G4, a 60 dias de congelamento, as amostras do grupo G5, a 90 dias de congelamento e as do grupo G6, a 180 dias de congelamento. As amostras de colostro bovino foram submetidas aos mesmos períodos de congelamento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The goals of the study were: 1. to evaluate total protein, G immunoglobulin, enzymes and minerals concentrations in goat and cow milky secretion, during the first 30 days postparturition, establishing the possible maximum limit to obtain milky secretion that allows appropriate transfer of passive immunity to the newborns kids; 2. to evaluate the influence of freezing in these constituents; and, 3. to create information that can aid in the assembly of colostrum banks. It were used samples of milky secretions of 10 Saanen goats and 10 Holstein cows recently-given birth and healthy, of third or fourth nursing, collected in the moments: in the day of the parturition and 1, 3, 5, 9, 14 and 29 days after the parturition. The samples were obtained after goats and cows complete milks. The samples of goat colostrum were submitted to six treatments, in agreement with the freezing period; the samples of cow colostrum were submitted to five treatments. The samples of the group G1 are goat colostrums "in natura"; the samples of the group G2 are the same samples of G1, however submitted to the heating of 56ºC for 60 minutes; the samples of the group G3 were submitted to 30 days of freezing, the samples of the group G4, to 60 days of freezing, the samples of the group G5, to 90 days of freezing and the one of the group G6, to 180 days of freezing. The samples of cow colostrum were submitted to the same freezing periods... (Complete abstract, click electronic access below) / Mestre

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Passive immunity acquired by the newborn foal through the colostrum is essential to prevent perinatal infections. The aim of this study was to establish the sensitivity and specificity of the zinc sulfate turbidity test (ZST) 12 hours after first suckling for the detection of immunoglobulins (IgG) to indicate or not the failure of passive immunity transfer. There was no difference in the ZST results obtained from blood collected at 12 (n=110) and 18 (n=38) hours, confirming that the test may be performed as early as 12 hours after the first colostrum intake. When compared to the single radial immunodiffusion test, the ZST test showed 76.50%, 95.65% and 94.68% sensitivity and 93.75%, 93.94% and 75.92% specificity, respectively, for a standard of 400, 600 and 800 mg dL-1 of immunoglobulins (IgG). The results of the ZST test, i.e. according to label instructions, performed 12 hours after the first suckling at 37oC is a reliable diagnostic tool for detecting IgG levels in newborn foals. / A imunidade passiva adquirida pelo potro neonato através da ingestão do colostro é essencial para a prevenção das infecções neonatais. O objetivo deste trabalho foi determinar a sensibilidade e a especificidade do teste da turvação pelo sulfato de zinco (TSZ) 12 horas após a primeira ingestão de colostro para detecção de imunoglobulinas G (IgG) para indicar ou não a deficiência de transferência da imunidade passiva. Não houve diferença nos resultados do teste TSZ com amostras séricas de potros PSC coletadas 12 (n=110) ou 18h (n=38) após a primeira ingestão do colostro, confirmando que o teste pode ser conduzido 12h após a ingestão de colostro. Quando comparado com a imunodifusão radial simples, o teste TSZ apresentou sensibilidade de 76,50%, 95,65% e de 94,68% e especificidade 93,75%, 93,94% e de 75,92%, para padrões de 400, 600 e 800mg dL-1 de IgG, respectivamente. Assim, o TSZ é um valioso teste para o diagnóstico no campo, desde que sejam seguidas estritamente as indicações do fabricante, especialmente quanto à temperatura. O teste pode e deve ser realizado 12 horas após a primeira mamada, pois apresenta bom índice de precisão.

Resumo: O objetivo do estudo foi avaliar a transferência de imunidade passiva (TIP) de vacas Canchim aos seus bezerros. Um grupo experimental foi constituído por 13 vacas primíparas e seus bezerros, e o outro por 13 vacas pluríparas e seus bezerros. Foram coletadas amostras de sangue venoso dos bezerros e secreções lácteas das vacas até 1 hora e 1, 2, 7, 15 e 30 dias após o nascimento/parto. No hemograma, notaram-se alterações características do período neonatal, como redução na contagem de hemácias, teor de hemoglobina e volume globular após a ingestão do colostro, além de aumento na contagem de linfócitos e redução da contagem de neutrófilos segmentados. Os exames bioquímicos séricos revelaram aumento nas atividades de GGT (até 3.746 U/L) e ALP (até 1.030 U/L) e nos teores de proteína total (até 7,77 g/dL), globulinas (até 6,01 g/dL), IgA (até 322 mg/dL) e IgG (até 2.918 mg/dL) após a ingestão do colostro, seguidos por redução gradual nestes parâmetros até os 30 dias de idade. As avaliações bioquímicas das secreções lácteas revelaram alta concentração de todos os componentes colostrais analisados, com redução gradual nos seus teores no decorrer do período experimental, à exceção dos minerais, cujas concentrações oscilaram pouco entre os momentos. A TIP foi eficiente em ambos os grupos de bezerros, e a qualidade das secreções lácteas, embora diferentes entre vacas primíparas e pluríparas, não interferiu na passagem de imunoglobulinas da vaca para o bezerro. / Abstract: The aim of this study was to evaluate the passive immunity transfer from Canchim cows to its calves. One experimental group comprised 13 primiparous cows and its calves and the other group, 13 multiparous cows and its calves. Samples of calves' venous blood and cow's lacteal secretions were collected until 1 hour and 1, 2, 7, 15 and 30 days after birth/parturition. In the hemogram, characteristic alterations of this period, such as reduction in the erytocyte count, hemoglobin concentration and corpuscular volume after colostrum intake and rise in the lymphocyte count and reduction in the neutrophil count were noticed. The serum biochemistry revealed augmentation in the GGT (up to 3,746 U/L) and ALP (up to 1,030 U/L) activities and in the total protein (up to 7.77 g/dL), globulin (up to 6.01 g/dL), IgA (up to 322 mg/dL) and IgG (up to 2,918 mg/dL) concentrations after colostrum intake, followed by a gradual reduction in these parameters until 30 days of age. The biochemical evaluation of the lacteal secretions revealed high concentrations of all the colostral components analysed, with gradual reduction in their concentrations along the experimental period, except for the minerals, which showed little oscillation between the moments. The passive immunity transfer was efficient in both groups of calves, and the quality of the lacteal secretions, although different between primiparous and multiparous cows, did not interfere in the transference of immunoglobulins from the cows to the calves. / Orientador: José Jurandir Fagliari / Coorientador: Alexandre Amstalden Moraes Sampaio / Banca: Eduardo Harry Birgel Júnior / Banca: Daniela Gomes da Silva / Mestre

La grippe porcine est une infection enzootique touchant 50% du cheptel français. Elle passe parfois inaperçue, mais peut également induire une forte morbidité au sein des lots d’animaux touchés, entraînant une baisse des performances zootechniques et des pertes économiques importantes. La sévérité de l’infection à virus influenza A chez le porc peut dépendre de divers facteurs, comme les virus eux-mêmes, les pratiques d’élevage, le statut immunitaire des animaux, les infections concomitantes par d’autres pathogènes respiratoires, etc. De la même manière, diverses formes épidémiologiques de la grippe existent en élevage. Ainsi, des infections peuvent se répéter à un âge déterminé, sur toutes les bandes successives d’un élevage, notamment chez des jeunes présentant une immunité passive. Afin de mieux comprendre cette diversité clinique et épidémiologique de la grippe porcine, et aider à l’élaboration de stratégies d’intervention adéquates pour le contrôle de la maladie, nous avons cherché à apporter de nouvelles connaissances quant à certains facteurs pouvant favoriser l’exacerbation du syndrome grippal et/ou son caractère récurrent, et plus généralement aux mécanismes sous-jacents à la pathogenèse des virus influenza A chez le porc, en relation avec les réponses de l’hôte infecté. Nous avons d’abord comparé, suite à inoculations expérimentales de porcs exempts d’organismes pathogènes spécifiés, la pathogénicité des deux virus influenza porcins les plus fréquemment rencontrés chez le porc en France, l’un du lignage européen « avian-like swine H1N1 » (H1avN1), l’autre du lignage européen « human-like reassortant swine H1N2 » (H1huN2), seuls ou en association avec Mycoplasma hyopneumoniae (Mhp), autre pathogène respiratoire répandu en élevage. Nous avons montré que l’infection H1huN2 induit une pathologie plus marquée que l’infection H1avN1, et que la pré-infection des porcs par Mhp induit une exacerbation de l’infection H1avN1, mais pas H1huN2. Nous avons utilisé le modèle de co-infection Mhp/H1avN1 pour évaluer deux approches alternatives qui permettraient de diminuer l’impact des infections grippales et de leurs complications : l’apport de composés aux propriétés antioxydantes via l’alimentation ; et la restriction alimentaire de courte durée. Dans ces deux cas nous avons montré des effets bénéfiques sur les paramètres zootechniques pendant les jours suivant l’infection grippale. Ce travail a également apporté de nouvelles connaissances quant aux modifications des marqueurs plasmatiques de stress oxydant, ainsi que sur les modifications métaboliques faisant suite à la co-infection Mhp/H1avN1. La sévérité des manifestations cliniques de la grippe étant liée à la qualité de la réponse immunitaire mise en place chez l’hôte infecté, nous avons entrepris d‘étudier les réponses immunitaires du porc touché par la grippe et d’évaluer l’impact de facteurs tels que la présence de Mhp ou d’anticorps d’origine maternelle sur ces réponses. Nous avons ainsi montré que l’infection virale induit une inflammation et une réponse interféron. La pré-infection par Mhp exercerait un effet additif sur cette inflammation et pourrait favoriser la persistance du virus dans le poumon. Nous avons également montré que la présence d’immunité passive protège cliniquement le porcelet mais n’empêche pas l’excrétion du virus, retarde la réponse lymphocytaire T et inhibe la réponse humorale post-infectieuse. Malgré la réponse immunitaire humorale défaillante, les animaux étaient totalement protégés d’une seconde infection homologue lorsque les anticorps maternels avaient disparus. Ces travaux ont permis d’apporter de nouvelles connaissances sur les facteurs influençant l’infection grippale en élevage porcin ainsi que sur les mécanismes sous-jacents, ce qui est une prérequis pour l’amélioration des mesures de lutte et de maitrise de la maladie. Ils permettent, d’envisager d’améliorer la santé des animaux en agissant sur leur régime alimentaire. / Swine influenza is an enzootic infection affecting 50% of the French livestock. The infection can be unnoticed but can also induce high morbidity among batches of affected animals, resulting in lower production performance and significant economic losses. The severity of influenza A virus in pig is influenced by many factors such as the virus strain, husbandry practices, the immune status of animals, concomitant infections with other respiratory pathogens, etc. In the same way, various epidemiological forms of influenza exist in farms. Thus, infections can be repeated in all successive batches within a farm, especially among young animals with passive immunity. In order to better understand the clinical and epidemiological diversity of the swine flu, and help develop appropriate strategies to control the disease, we tried to bring new knowledge about factors that promote the exacerbation of the flu syndrome and/or its recurrence, and more generally to give new information about the mechanisms underlying the pathogenesis of influenza viruses in pigs, in relation to the response of the infected host. Firstly, we compared, through experimental infections of specific pathogen free pigs, the pathogenicity of the two swine influenza viruses mostly detected in pigs in France, i.e. one from the European “avian-like swine H1N1” lineage (H1avN1) and the other one from the European “human-like reassortant swine H1N2” lineage (H1huN2), each one alone or in co-infection with Mycoplasma hyopneumoniae (Mhp), another respiratory pathogen widespread in French farms. We showed that the H1huN2 infection induced a more marked pathology than the H1avN1 infection, and that Mhp pre-infection induced the exacerbation of the H1avN1, but not the H1huN2, infection. Then, we used the Mhp/H1avN1 co-infection model to evaluate alternative approaches that could reduce the impact of influenza infections and their complications: firstly, a supply of compounds with antioxidant properties in food; and secondly, a feed restriction of short duration. In both cases, we showed beneficial effects on zootechnical parameters the days following influenza infection. This work has also brought new knowledge on modulation of oxidative stress markers in plasma, as well as metabolic changes following the co-infection with Mhp and H1avN1 in pigs. The severity of flu clinical manifestations being related, among other, to the quality of the immune responses developed by the infected host, we studied these responses in pigs experimentally infected by H1avN1 and assessed the impact of factors such as the presence of Mhp or maternal derived antibodies on these responses. We showed that the viral infection induced inflammation and interferon response. The Mhp pre-infection exerted an additive effect on inflammation of lung tissue and may promote the virus persistence in the lung. Finally, we have shown that the presence of maternally-derived immunity protected the piglets clinically but did not prevent viral shedding, delayed the T cell response and strongly inhibited the post-infectious humoral response. However, despite the failed humoral immune response, animals were completely protected from a second infection occurring when maternal antibodies had disappeared. Therefore, this work have brought new knowledge on factors influencing influenza infection in pig as well as the underlying mechanisms, which is a prerequisite for improving disease control. They allow, between-other, to consider improving the health and welfare of animals by acting on their diet.

Bovine maternal antibodies are not transferred across the placenta during pregnancy and newborn calves are unable to produce their own antibodies within the first weeks after birth. As neonates are born agammaglobulinemic they need to acquire immunoglobulins (Ig) from their dam’s colostrum to acquire passive immunity. Colostrum fed to dairy calves, which is not timeous, of inadequate quantity and of unverified quality, might result in decreased neonate health. The aim of this study was to conduct a survey of the management of colostrum on commercial dairy farms, to estimate the quality of the Ig titre of colostrum fed to neonates and finally to recommend methods and techniques critical to the successful management of colostrum. The methods used included a questionnaire which was conducted as a structured interview on a one-on-one basis among 50 randomly selected commercial dairy farmers in the Eastern Cape Coastal Region of South Africa. The estimation of the colostrum Ig titre of colostrum fed to neonates was made by the on-farm measurement of specific gravity (SG) by making use of a commercially available KRUUSE colostrometer (Fleenor and Stott, 1980). A pooled colostrum sample, from each of the four quarters, from 90 randomly selected post-partum cows was collected on a leader commercial dairy farm. This method was used to compare colostrum samples from cows run under similar management practices. These samples were collected for analysis within 6 hrs of calving and were done over 3 seasons (autumn, winter and spring). Survey - The colostrum mass and timing of the initial feed are the most important factors when aiming to achieve adequate passive immunity. The results of the survey indicated that most of the farmers of this region feed an inadequate mass of colostrum (volume and Ig concentration) and only 52% of farmers surveyed feed colostrum less than 6 hrs post- partum. The majority (78%) of surveyed farmers did not follow up their initial colostrum feeding.Colostrum sampling - At the trial site only 10% (9 from 90 colostrum samples measured), were found to be of adequate SG quality. Cow age (parity), season of calving and colostrum temperature had an influence on the estimated colostrum SG. However, season of calving was found to have the greatest influence on SG values. These results were consistent with findings from previous studies that SG values from the cooler months were higher than those of the hotter months. Tables 4.7 (P=0.330), 4.8 (P=0.012) and 4.9 (P=0.005) showed that regression analysis confirmed that LS means across seasons were inadequately below the required 50 mg/ml Ig required for sufficient passive immunity. Tables 4.1 (P=0.164), 4.2 (P=0.011) and 4.3 (P=0.021) shows that season of calving had a much greater effect on CR than did parity, Table 4.5 (P=0.177). Table 4.4 shows that colostrum temperature has an significant effect on SG value. Recommendations for methods and techniques critical to the successful management of colostrum were made. These recommendations were based on the analysis of the data obtained from the questionnaire and the on-farm colostrum sampling study. The most important and critical management practices surveyed includes the timing of the cow and calf separation where it was found that only 30 from the 50 (60%) of the farms surveyed separate calves and dams at day (0), 19 from 50 farms (38%) separate at day (3-5) and 1 from 50 farms (2%) separate only at day 7 or later. Thus 40% of surveyed farms allow cows to nurse their calves. With regards to early exposure to pathogens this is a high risk management practice. Further to that, only 2 from 50 surveyed farms (4%) measure the colostrum quality fed to their calves and 48 from 50 farms (96%) feed colostrum of unmeasured quality. The mass of colostrum fed to calves is an important parameter for successful transmission of Ig. In the survey it was found that 28 from 50 farms (56%) feed 2L – 4L of colostrum and 11 from 50 farms (22%) feed 2L of colostrum. Thus 78% of farms feed approximately 50% of the amount of colostrum required for successful transmission of Ig. Finally only 1 from 50 farms (2%) freeze excess colostrum and 1 from 50 farms (2%) pool excess colostrum. Both these farms measure colostrum quality by colostrometer. / Agriculture Animal Health and Human Ecology / M. Sc. (Agriculture)

No description available.

Bovine maternal antibodies are not transferred across the placenta during pregnancy and newborn calves are unable to produce their own antibodies within the first weeks after birth. As neonates are born agammaglobulinemic they need to acquire immunoglobulins (Ig) from their dam’s colostrum to acquire passive immunity. Colostrum fed to dairy calves, which is not timeous, of inadequate quantity and of unverified quality, might result in decreased neonate health. The aim of this study was to conduct a survey of the management of colostrum on commercial dairy farms, to estimate the quality of the Ig titre of colostrum fed to neonates and finally to recommend methods and techniques critical to the successful management of colostrum. The methods used included a questionnaire which was conducted as a structured interview on a one-on-one basis among 50 randomly selected commercial dairy farmers in the Eastern Cape Coastal Region of South Africa. The estimation of the colostrum Ig titre of colostrum fed to neonates was made by the on-farm measurement of specific gravity (SG) by making use of a commercially available KRUUSE colostrometer (Fleenor and Stott, 1980). A pooled colostrum sample, from each of the four quarters, from 90 randomly selected post-partum cows was collected on a leader commercial dairy farm. This method was used to compare colostrum samples from cows run under similar management practices. These samples were collected for analysis within 6 hrs of calving and were done over 3 seasons (autumn, winter and spring). Survey - The colostrum mass and timing of the initial feed are the most important factors when aiming to achieve adequate passive immunity. The results of the survey indicated that most of the farmers of this region feed an inadequate mass of colostrum (volume and Ig concentration) and only 52% of farmers surveyed feed colostrum less than 6 hrs post- partum. The majority (78%) of surveyed farmers did not follow up their initial colostrum feeding.Colostrum sampling - At the trial site only 10% (9 from 90 colostrum samples measured), were found to be of adequate SG quality. Cow age (parity), season of calving and colostrum temperature had an influence on the estimated colostrum SG. However, season of calving was found to have the greatest influence on SG values. These results were consistent with findings from previous studies that SG values from the cooler months were higher than those of the hotter months. Tables 4.7 (P=0.330), 4.8 (P=0.012) and 4.9 (P=0.005) showed that regression analysis confirmed that LS means across seasons were inadequately below the required 50 mg/ml Ig required for sufficient passive immunity. Tables 4.1 (P=0.164), 4.2 (P=0.011) and 4.3 (P=0.021) shows that season of calving had a much greater effect on CR than did parity, Table 4.5 (P=0.177). Table 4.4 shows that colostrum temperature has an significant effect on SG value. Recommendations for methods and techniques critical to the successful management of colostrum were made. These recommendations were based on the analysis of the data obtained from the questionnaire and the on-farm colostrum sampling study. The most important and critical management practices surveyed includes the timing of the cow and calf separation where it was found that only 30 from the 50 (60%) of the farms surveyed separate calves and dams at day (0), 19 from 50 farms (38%) separate at day (3-5) and 1 from 50 farms (2%) separate only at day 7 or later. Thus 40% of surveyed farms allow cows to nurse their calves. With regards to early exposure to pathogens this is a high risk management practice. Further to that, only 2 from 50 surveyed farms (4%) measure the colostrum quality fed to their calves and 48 from 50 farms (96%) feed colostrum of unmeasured quality. The mass of colostrum fed to calves is an important parameter for successful transmission of Ig. In the survey it was found that 28 from 50 farms (56%) feed 2L – 4L of colostrum and 11 from 50 farms (22%) feed 2L of colostrum. Thus 78% of farms feed approximately 50% of the amount of colostrum required for successful transmission of Ig. Finally only 1 from 50 farms (2%) freeze excess colostrum and 1 from 50 farms (2%) pool excess colostrum. Both these farms measure colostrum quality by colostrometer. / Agriculture Animal Health and Human Ecology / M. Sc. (Agriculture)

Tesis por compendio / [ES] La producción en acuicultura se ha visto menguada por aparición de enfermedades en los sistemas de cría de peces. En concreto, en la dorada (Sparus aurata), hay dos parásitos destacados: Enteromyxum leei (Myxozoa) y Enterospora nucleophila (Microsporidia). Hasta la fecha, para ninguno de los dos se ha establecido un cultivo in vitro, y solo para E. leei se ha conseguido establecer un modelo de mantenimiento de la infección in vivo. La presente tesis pretende incrementar el conocimiento sobre estos parásitos y sus relaciones con el hospedador, sentando las bases para generar soluciones que puedan ser aplicadas en la acuicultura. El objetivo con E. leei fue estudiar la inmunidad adquirida inducida en la dorada y la posibilidad de generar herramientas de diagnóstico y vacunas frente a esta enfermedad. Para ello, primero se demostró la resistencia del pez al parásito tras una segunda exposición, la cual duró hasta 16 meses. Además, la resistencia parece estar correlacionada con altos niveles de inmunoglobulina (Ig) M específica en sangre, y una alta expresión de Igs, incluso antes de la re-exposición al parásito. El siguiente paso fue afinar el protocolo de infección con E. leei. Los resultados mostraron que una semana es suficiente para transmitir la infección de E. leei por efluente, independientemente de la temperatura. Tras la demostración de la respuesta adaptativa eficaz frente a E. leei, y al disponer de un modelo de infección refinado, se realizó un ensayo de inmunización pasiva. Aquí, los resultados mostraron que los anticuerpos especi'ficos efectivamente consigue ralentizar la invasión del intestino por el parásito y disminuir los síntomas de la enfermedad. Paralelamente, el resultado del análisis del repertorio de las regiones variables de la IgM e IgT del intestino peces resistentes mostró la inducción de una respuesta policlonal en las ce'lulas B. En base a estos resultados, se realizó una búsqueda de antígenos de E. leei que pudieran ser utilizados como candidatos para la producción de vacunas (análisis proteómico) o herramientas de diagnóstico (análisis in silico). Para ello, se ensambló un transcriptoma de novo utilizando una muestra mixta de intestino de dorada y parásito. Los resultados dieron lugar a 7 y 12 candidatos en la búsqueda in silico y proteómica, respectivamente. En los estudios de E. nucleophila, debido a que fue descrita muy recientemente, el punto de partida fue más básico. Las muestras de este parásito solo se pueden obtener de brotes naturales en piscifactorias. Por ello, primero se realizó un estudio de caracterización de la patología de la infección a partir de peces infectados naturalmente. En etapas tempranas de la infección, el parásito se localiza principalmente en el intestino, pero meses después, la prevalencia en intestino baja e incrementa en los órganos hematopoyéticos y el esto'mago. Los signos clínicos de la infección consistieron en una reducción significativa del crecimiento, emaciación, y palidez de las paredes intestinales. A nivel celular, en los casos ma's graves se observó hipercelularidad en el epitelio intestinal y proliferación de ce'lulas rodlet, un elevado número de linfocitos en la base del epitelio e infiltración de granulocitos acidófilos en el epitelio intestinal. Finalmente se probaron varias formas de transmisión horizontal de E. nucleophila (cohabitación, efluente, intubación oral y anal) con para desarrollar un modelo de mantenimiento in vivo. Se consiguió la transmisión el parásito por todas las vías, pero con una disminución de prevalencia a lo largo del tiempo. Variables como la temperatura, la dosis, y el estado de los peces donantes parecen ser más determinantes que la ruta seleccionada para la transmisión. Entre las rutas probadas, la intubación anal parece ser la más prometedora, pero ninguna de ellas fue capaz de reproducir los signos clínicos observados en las infecciones naturales. / [CA] La producció en aqüicultura s'ha vist minvada per aparició de malalties en els sistemes de cria de peixos. En concret, en l'orada (Sparus aurata), hi ha dos paràsits destacats: Enteromyxum leei (Myxozoa) i Enterospora nucleophila (Microsporidia). Fins avui, per a cap dels dos s'ha establert un cultiu in vitro, i només per a E. leei s'ha aconseguit establir un model de manteniment de la infecció in vivo. La present tesi pretén incrementar el coneixement sobre aquests paràsits i les seves relacions amb l'hoste, establint les bases per a generar solucions que puguin ser aplicades en l'aqüicultura. L'objectiu amb E. leei va ser estudiar la immunitat adquirida induïda en l'orada i la possibilitat de generar eines de diagnòstic i vacunes enfront d'aquesta malaltia. Per a això, primer es va demostrar la resistència del peix al paràsit després d'una segona exposició, la qual va durar fins a 16 mesos. A més, la resistència sembla estar correlacionada amb alts nivells d'immunoglobulina (Ig) M específica en sang, i una alta expressió de Igs, fins i tot abans de la re-exposició al paràsit. El següent pas va ser afinar el protocol d'infecció amb E. leei. Els resultats van mostrar que una setmana és suficient per a transmetre la infecció de E. leei per efluent, independentment de la temperatura. Després de la demostració de la resposta adaptativa eficaç enfront de E. leei, i en disposar d'un model d'infecció refinat, es va realitzar un assaig d'immunització passiva. Aquí, els resultats van mostrar que els anticossos específics efectivament aconsegueix alentir la invasió de l'intestí pel paràsit i disminuir els símptomes de la malaltia. Paral·lelament, el resultat de l'anàlisi del repertori de les regions variables de la IgM i IgT de l'intestí peixos resistents va mostrar la inducció d'una resposta policlonal en les cèl·lules B. Sobre la base d'aquests resultats, es va realitzar una cerca d'antígens de E. leei que poguessin ser utilitzats com a candidats per a la producció de vacunes (anàlisis proteómico) o eines de diagnòstic (anàlisi in silico). Per a això, es va assemblar un transcriptoma de novo utilitzant una mostra mixta d'intestí d'orada i paràsit. Els resultats van donar lloc a 7 i 12 candidats en la cerca in silico i proteòmica, respectivament. En els estudis de E. nucleophila, pel fet que va ser descrita molt recentment, el punt de partida va ser més bàsic. Les mostres d'aquest paràsit només es poden obtenir de brots naturals en piscifactorias. Per això, primer es va realitzar un estudi de caracterització de la patologia de la infecció a partir de peixos infectats naturalment. En etapes primerenques de la infecció, el paràsit es localitza principalment en l'intestí, però mesos després, la prevalença en intestí baixa i incrementa en els òrgans hematopoètics i l'estómac. Els signes clínics de la infecció van consistir en una reducció significativa del creixement, emaciació, i pal·lidesa de les parets intestinals. A nivell cel·lular, en els casos més greus es va observar hipercelularidad en l'epiteli intestinal i proliferació de cèl·lules rodlet, un elevat nombre de limfòcits en la base de l'epiteli i infiltració de granulòcits acidòfils en l'epiteli intestinal. Finalment es van provar diverses formes de transmissió horitzontal de E. nucleophila (cohabitació, efluent, intubació oral i anal) amb per a desenvolupar un model de manteniment in vivo. Es va aconseguir la transmissió el paràsit per totes les vies, però amb una disminució de prevalença al llarg del temps. Variables com la temperatura, la dosi, i l'estat dels peixos donants semblen ser més determinants que la ruta seleccionada per a la transmissió. Entre les rutes provades, la intubació anal sembla ser la més prometedora, però cap d'elles va ser capaç de reproduir els signes clínics observats en les infeccions naturals. / [EN] Aquaculture production is hampered by the emergence of parasite diseases in fish farming systems. Among them, in Sparus aurata, there are two important enteric parasites described: Enteromyxum leei (Myxozoa) Enterospora nucleophila (Microsporidia). To date, no in vitro culture has been established for either parasite, and only for E. leei was it possible to establish a model for maintaining the infection in vivo. The aim of this thesis is to gain new knowledge about these parasites and their relationship with the host, also the basic foundations for generating solutions that can be applied in aquaculture. The general objective for E. leei was to study the acquired immunity induced in gilthead bream and the possibility of generating diagnostic tools and vaccines against this disease. To this end, resistance against the parasite was assessed with a second exposure against the parasite, which showed a resistance for at least 16 months. Besides resistance seemed to be correlated with high levels of specific immunoglobulin (Ig) M in blood, and a high expression of Igs, in particular, the soluble forms, even before re-exposure to the parasite. The next step was refining the protocol for effluent infection with E. leei by studying infection at different exposure time points, temperatures and population densities. The results showed that one week of exposure is sufficient to spread E. leei infection by effluent, regardless of temperature. After demonstrating the resistance against E. leei, and with a refined infection model, a passive immunization assay was performed. The results showed that the serum with specific antibodies effectively slows down the invasion of the gut by the parasite and reduces the symptoms of the disease. At the same time, the analysis of the repertoire of the variable regions of intestinal IgM and IgT showed an induction of a polyclonal response in B cells. On the basis of these results, a research was carried out for E. leei antigens that could have use as candidates for the production of vaccines (proteomic study) or diagnostic tools (in silico study) using the parasite transcriptomic data. To do this, a de novo transcriptome was assembled using a mixed sample of gilthead sea bream and parasite, with a posterior filtrate of the sequences. The In silico and proteomic analysis search resulted in 7 and 12 transcripts, respectively, which are being used for diagnostic and vaccine production. The starting point was more basic in E. nucleophila studies, since this is a recently described disease. The samples of this parasite can only be obtained from natural outbreaks in fish farms. Therefore, first study was carried out to characterize the pathology of the infection of naturally infected fish. In the early stages of the infection, the parasite is mainly located in the intestine, but months later, the prevalence is lower in the intestine and increases in the hematopoietic organs and the stomach. Clinical signs of infection were significant reduction in growth, wasting, and intestinal walls paleness. At the cellular level, in the most severe cases hypercellularity in the intestinal epithelium, proliferation of rodlet cells, high number of lymphocytes at the base of the epithelium and infiltration of acidophilic granulocytes in the intestinal epithelium were observed. Finally, horizontal transmission of E. nucleophila was tried using different transmission methods: cohabitation, effluent, and oral and anal intubation. Transmission of the parasite was achieved with all routes, but there was a decrease in prevalence over time in all cases except for the anal route. Variables such as temperature, dose, and the status of the donor fish appear to be more important than the selected route. Among the routes tested, anal intubation seemed to be the most promising, as it was sustained over a longer period of time, but none of them was able to reproduce the same clinical signs of infection observed in natural infections. / The authors kindly acknowledge the collaboration of anonymous fish farming companies allowing access to the animals during the disease outbreaks. We thank J. Monfort and L. Rodríguez (IATS-CSIC) for the technical assistance on histological processing.This work has been carried out with financial support from the European Union and the Spanish Ministry of Economy and Competitiveness (MINECO) under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. APS was contracted under ParaFishControl project. Primer sequences and access to the gilthead sea bream transcriptomic database were kindly provided by Prof. J. Pérez-Sánchez of the IATS- Nutrigenomics group. The authors thank I. Vicente for fish maintenance and technical assistance during samplings. The authors thank P. Boudinot (INRAE) for his help in designing and interpreting the immunoglobulin repertoire study and results, J. Pérez-Sánchez (IATS-CSIC) for providing access to the gilthead sea bream genome sequences to perform the repertoire analysis.This work was funded by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM). / Picard Sánchez, MA. (2021). Control of enteric parasitic diseases of farmed gilthead sea bream: New insights into Enteromyxum leei (Myxozoa) and Enterospora nucleophila (Microsporidia) infections [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167035 / Compendio