Em bovinos, o desenvolvimento embrionário pré implantacional depende das funções do endométrio bovino que tem suas funções mediadas por uma complexa interação da ação e dos efeitos dos hormônios esteroides ovarianos E2 e P4. Estes hormônios regulam a expressão gênica e controlam o ambiente uterino modulando, entre outros, a expressão de microRNAs e a rede de citocinas relacionadas ao sistema imune. Os objetivos do presente trabalho foram abordados em dois capítulos, sendo (I) comparar os efeitos dos distintos ambientes endócrinos peri-ovulatórios sobre a expressão de microRNAs (II) e na modulação do sistema IL1/TLR no endométrio bovino nos dias 4 e 7 após a indução da ovulação. Para isso, controlou-se farmacologicamente o crescimento do folículo objetivando induzir a ovulação de folículos de maior diâmetro (grupo folículo grande-CL grande, FG-CLG) ou de menor diâmetro (grupo folículo pequeno-CL Pequeno, FP-CLP). Vinte e duas vacas multíparas nelore, foram pré-sincronizadas, metade destes animais foram destinados para o grupo FG-CLG e receberam uma dose de prostaglandina F2&#945; (PGF) e um dispositivo de progesterona, juntamente com benzoato de estradiol no D10. No momento da retirada dos dispositivos de progesterona (entre D1,75 e D2,5) todos os animas receberam uma dose de PGF. A ovulação foi induzida com acetato de buserelina (D0). O que diferiu entre os tratamentos foi que os animais do grupo FP-CLP não receberam uma dose de PGF no D10 e o momento da retirada dos dispositivos foi entre D1,25 e o D1,5. No capítulo I, o a expressão de microRNAs foi determinada por qPCR nos dias 4 e 7. Dos 90 microRNAs testados, 21 apresentaram se up-regulated e dois down-regulated no grupo FG-CLG (P<0.1) no D4. No D7, quatro microRNAs foram diferentemente expressos, sendo um up-regulated e três down-regulated no grupo FG-CLG (P<0.1) no D7. Para os microRNAs diferentemente expressos determinou-se mRNA-alvos preditos. Uma análise de ontologia demonstrou que os mRNAs-alvos apresentaram enriquecimento funcional na via dos receptores de hormônios esteroides, entre outras. No capítulo II, o sistema IL1/TLR foi avaliado quanto a abundância de transcriptos envolvidos neste sistema, do microRNA bta-mir-155 e das proteínas IL1&#946; e IL1R1. A abundância relativa de mRNA apresentou diferença (P<0.1) na abundância dos mRNAs de IL1R1, TAB1 e FOXP3, das proteínas IL1&#946; e IL1R1, sendo essas moléculas up-regulated no grupo FG-CLG. O microRNA bta-mir-155 foi down-regulated no grupo FG-CLG (P<0.1). Diante disto, pode-se concluir que o ambiente endócrino peri-ovulatório determina o perfil de expressão de microRNAs e modula o sistema IL1/TLR no endométrio bovino / In cattle, the pre implantation embryo development depends on the functions of the bovine endometrium that has its functions mediated by a complex interaction of action and the effects of ovarian steroid hormones E2 and P4. These hormones regulate gene expression and control the modulating uterine environment among others, the expression of microRNAs and the network of cytokines related to the immune system. The objectives of this study were discussed in two chapters, (I) to compare the effects of different peri-ovulatory endocrine environment on the expression of microRNAs (II) and modulation of the IL-1 system / TLR in bovine endometrium on days 4 and 7 after induction of ovulation. For this, it was controlled pharmacologically follicle growth aiming to induce ovulation of follicles larger diameter (great grand-CL follicle group, FG-CLG) or smaller in diameter (small-CL Small follicle group, FP-PLC). Twenty two nelore multiparous cows were pre-sync, half of these animals were used for the FG-NCG group and received a dose of F2á prostaglandin (PGF) and progesterone device along with oestradiol benzoate in D-10. Upon withdrawal of progesterone devices (between 1.75 and D-D-2,5) all animas received a dose of PGF. Ovulation was induced with buserelin acetate (D0). What differed between treatments was that animals FP-CLP group did not receive a dose of PGF in the D-10 and the time of removal of the devices was between D-1,25 and D-1.5. In Chapter I, the expression of microRNAs was determined by qPCR on 4 and 7. Of the 90 microRNAs tested, 21 showed was up-regulated and down-regulated in two FG-CLG group (P <0.1) in the D4. In D7 four microRNAs were differently expressed, one up-regulated and down-regulated in three FG-CLG group (P <0.1) at D7. For differently expressed microRNAs was determined predicted mRNA-target. An ontology analysis showed that the mRNA-targets had functional enrichment in via the steroid hormone receptors, among others. In Chapter II, the IL-1 / TLR system was evaluated as the abundance of transcripts involved in this system, the bta-mir-155 microRNA and IL1&#946; and IL1R1 proteins. The relative abundance of mRNA was different (P <0.1) in the abundance of mRNAs IL1R1, TAB1 and FOXP3, the IL1&#946; and IL1R1 proteins, and these up-regulated molecules in the FG-CLG group. The bta-mir-155 microRNA was down-regulated in the FG-CLG group (P <0.1). Given this, we can conclude that the peri-ovulatory endocrine milieu determines the profile of microRNA expression and modulates the IL1 / TLR system in bovine endometrium

A utilização de uréia na alimentação de fêmeas bovinas pode prejudicar o desempenho reprodutivo destes animais, provavelmente decorrente do aumento do teor de nitrogênio uréico plasmático (NUP). O objetivo deste estudo foi avaliar se alimentação com uréia por curto prazo oferecida a novilhas, e conseqüente aumento de NUP, exerce influência sobre a quantidade, qualidade e competência dos complexos cumulus-oócito (CCO). O experimento teve duração de 62 dias, dividido em oito semanas e dois períodos. Foram utilizadas 40 novilhas mestiças mantidas a pasto, alocadas a dois tratamentos, utilizando-se delineamento \"cross-over\": dieta sem uréia (SU): 5 kg (matéria original) de silagem de milho e 1 kg de concentrado/animal/dia ou dieta com uréia (U): 5 kg de silagem de milho e 1 kg de concentrado contendo 75 g de uréia/animal/dia. Os animais selecionados a cada semana receberam as dietas por seis dias, uma única vez ao dia. No sexto dia de oferecimento das dietas, foram realizadas aspiração folicular (OPU) e colheita de sangue, em jejum e 3 horas após a alimentação. Imediatamente após a OPU, foi realizada contagem total de CCO recuperados e classificação dos mesmos em viáveis e inviáveis. Apenas os viáveis foram destinados à produção In vitro de embriões. Em relação ao teor de NUP, houve efeito de interação entre tratamento e tempo de colheita (P=0,04) e dentro de cada tempo foi observado aumento significativo (P<0,01) para os animais do tratamento U. Não foi observado efeito de tratamento em relação ao número total de CCO recuperados por animal (média ± EPM: SU=9,15 ± 0,82 vs. U=8,82 ± 0,95), nem sobre a porcentagem de CCO viáveis sobre total de CCO recuperados por animal (SU=73,61% ± 2,47 vs. U=70,26% ± 2,31). A taxa de clivagem obtida no dia 3 após a inseminação In vitro (IIV) e as taxas de blastocisto nos dias 6, 7 e 9 após a IIV, não foram influenciadas pelo tratamento. Porém, no 11º pós IIV, houve diminuição (P=0,04) da taxa de blastocistos eclodidos para o tratamento U (SU=82,50% ± 0,05 vs. U=64,33% ± 0,07). Apesar do aumento do teor de NUP observado nas novilhas do tratamento U, a quantidade, qualidade e competência dos CCO durante as primeiras clivagens até atingirem o estádio de blastocisto In vitro não foram influenciadas pelo oferecimento de 75 g de uréia na dieta, durante seis dias. Porém, foi observada redução da taxa de eclosão dos blastocistos das novilhas do tratamento U. / Urea feeding offered to bovine dams may damage their reproductive performance, probably due to an increase in levels of plasmatic urea nitrogen (PUN). The aim of this study was evaluate if short-term urea feeding of heifers, following high PUN levels, would have an influence on the quantity, quality and competence of cumulus-oocyte complexes (COC). Trial lasted 62 days, divided into eight weeks and two periods. Forty crossbred grazing heifers were allocated to one of two treatments, using a cross-over design: diet without urea (WU): 5 kg (as fed) of corn silage and 1 kg of concentrated/animal/day, or diet with urea (U): 5 kg of corn silage and 1 kg of concentrated with 75 g of urea/animal/day. Heifers selected in each week received these diets once a day, for six days. On the sixth day of diets? offer, ovum pick-up (OPU) and blood sampling at fasting and 3 hours after feeding were carried out. Immediately after OPU, total COC recovered was counted and classified as either viable or unviable. Only viable were destined to In vitro embryo production. In relation to PUN level, there was a significant interaction between treatment and sampling time (P=0.04) and a significant (P<0.01) increase in animals undergoing U treatment for each of the test times. No significant effect was observed relative to either the total number of recovered COC per animal (mean ± SEM: WU=9.15 ± 0.82 vs. U=8.82 ± 0.95), or the viable COC as a percentage of total recovered COC per animal (WU=73.61% ± 2.47 vs. U=70.26% ± 2.31). Cleavage ratio assessed on day 3 post In vitro insemination (IVI) and blastocyst ratio on days 6, 7 and 9 post IVI were not influenced by treatments. However, there was a significant treatment effect (P=0.04) in relation to hatched blastocysts on day 11 after IVI (WU=83% ± 0.05 vs. U=64% ± 0.07). Even though higher PUN levels were observed in animals from treatment U, quantity, quality and competence of the COC during first cleavages until reaching the blastocyst stage In vitro were not influenced by adding 75 g of urea on the diet offered to heifers, during six days. Neverthless, a decline in hatched blastocysts rate was observed in heifers of treatment U.

A pele dos vertebrados é alvo da maioria das 15.000 espécies de artrópodes hematófagos existentes e pouco se sabe sobre as estratégias imunológicas utilizadas pelos hospedeiros para expulsar esse tipo de ectoparasitos. É fato que carrapatos, como artrópodes hematófagos, são capazes também de induzi-las. Entre a grande variedade de hospedeiros, os bovinos, que apresentam fenótipos variáveis de resistência ao Ripicephalus (Boophilus) microplus, constituem o único modelo no qual é possível correlacionar as respostas imunológicas entre os fenótipos contrastantes na mesma espécie hospedeira. Para tal, as populações celulares do infiltrado induzido na pele pelo carrapato foram quantificadas nos bovinos resistentes, Bos taurus indicus e nos suscetíveis, Bos taurus taurus. Como esperado, o carrapato induziu inflamação cutânea local nos bovinos estudados e a composição celular do infiltrado apresentou diferenças que variaram entre os fenótipos contrastantes de infestação. A pele resistente apresentou maior número de basófilos em comparação a pele suscetível infestada pelo carrapato adulto (P < 0,05) o que sugere a participação desse tipo de granulócito na resposta imune, prejudicando a hematofagia do carrapato. Eosinófilos não foram observados na pele naïve, mas sim na pele normal e infestada, apresentando maiores quantidades (P < 0,05) na pele resistente infestada por ninfa e por adulto. Esse granulócito também se mostrou importante para a aquisição de resistência a carrapatos, a cinética observada na pele dos animais sugere um efeito sistêmico de eosinófilos na infestação. Já mastócitos se mostraram reduzidos de forma semelhante na pele resistente e suscetível infestada por ninfa e adulto em comparação a pele não infestada das mesmas (P < 0,05), sugerindo desgranulação induzida pela saliva do carrapato. As citocinas e mediadores inflamatórios liberados por mastócitos poderiam desencadear e até modular as respostas imunes de contra o carrapato. Neutrófilos estavam em 9 quantidades semelhantes na pele infestada de ambas as raças, apresentando maior quantidade na fase de adulto em relação à fase de ninfa (P < 0,05). Esse fato sugere que a saliva da ninfa expressa desintegrinas contendo RGD, que possivelmente são específicas para esse granulócito. Em contrapartida, as células mononucleares foram mais abundantes na pele resistente e suscetível infestadas por ninfa em relação às infestadas por adulto (P = 0,001). Entre as populações mononucleares fenotipadas, as células T CD3+ foram recrutadas em maior número na pele resistente infestada por ninfa e adulto que na pele suscetível nas mesmas fases (P < 0,05), indicando sua importância na regulação da resposta imune de resistência ao ectoparasito. Células CD4+ foram mais numerosas na pele resistente infestada por adulto que a pele suscetível infestada pela mesma fase (P < 0,05); já as células CD8+ estavam em maior número na pele resistente infestada por ninfa do que na pele suscetível na mesma fase de infestação (P < 0,05). Células T gd/WC1+ foram mais abundantes (P < 0,05) na pele resistente infestada por adulto que a mesma não infestada, indicando que esses linfócitos podem desempenhar papel importante na aquisição de resistência. Já os linfócitos B estiveram em número reduzido na pele suscetível infestada por ninfa e por adulto em comparação à mesma não infestada (P < 0,05). Entretanto, na pele resistente infestada por adulto, o número desses linfócitos foi maior (P < 0,05) em comparado ao encontrado na pele suscetível infestada pela mesma fase, sugerindo a participação de anticorpos na aquisição de resistência a carrapatos. Embora não significativa, o maior número de células Natural Killers na pele resistente, sugere seu envolvimento na proteção contra doenças transmitidas por carrapatos. Em suma, os resultados obtidos sugerem que a resposta imune local envolve células residentes e recrutam outras que, em conjunto, atuam na imunorregulação e na elaboração de respostas imunes efetoras e de memória eficazes contra carrapatos. / The skin of vertebrates is the largest for over 15,000 species of hematophagous arthropods. Among them are ticks, which are long-term feeders and interact with host defenses for days to weeks. Little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in host. Bovines present variable and heritable levels of resistance to the Ripicephalus (Boophilus) microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. In order to obtain some of the immune correlates of these outcomes, we quantified populations of leukocytes and lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of resistant and susceptible bovine breeds, respectively, Bos taurus indicus and Bos Taurus taurus. As expected, ticks induce cutaneous inflammatory infiltrates around their mouthparts. However the composition of infiltrate presented with significant differences that varied according to the phenotype of infestation. Inflammation of resistant breed contained significantly more basophils than in susceptible adult-infested skin (P<0.05). Eosinophils were absent in skin from naïve animals, but were present in normal and infested skin. They were present in infested skin of both breeds and more significantly so in nymph and adult-infested skin of resistant breed (P<0.05). However, mast cell numbers were equally diminished in nymph and adult-infested skin of both breeds when compared with non-infested skin (P<0.05). The neutrophils were equally present in infested skin of both breeds, but they are more numerous in skin infested with adults than with nymphs (P<0.05). Conversely, mononuclear cells were more abundant in skin infested with nymphs than with adults (P=0.001). Bites by nymphs and adults recruit more CD3+ T cells in skin of resistant breeds than in that of susceptible ones (P<0.05). The CD4+ populations of cells 11 were more numerous in adult-infested skin of resistant than in that of susceptible (P<0.05); CD8+ cells were increased in nymph-infested skin of resistants relative to that of susceptibles (P<0.05). The population of gd/WC1+ T cells were more abundant (P<0.05) in adult-infested skin of resistant when compared with those found in control skin of the same animals. The numbers of B lymphocytes were diminished in nymph and adult-infested skin of susceptibles when compared with those found in control skin (P<0.05); in adult-infested skin of resistants, B lymphocytes were more numerous (P < 0,05) than in skin of susceptibles. Bites by nymph and adult tends to recruited more Natural Killers cells to skin in resistant breed than in susceptible one (P > 0,05). The results suggest that mast cells are source of cytokines and inflammatory mediators that play effectors and modulator roles in immune responses, their reduction possibly due to degranulation by tick saliva. Amount of neutrophils in infested skin may reflect the fact that only nymphs express RGD-containing disintegrins, which are possibly neutrophils-specific. Eosinophils, as well as basophils, are important to ticks resistance, their skin kinetic suggesting a systemic effect of tick infestations. Basophils resistant host increasing suggesting that they are the pivotal cells that impair haematophagy. The observed increase of CD3+ T cells in nymph and adult-infested skin of resistants suggests their importance to regulate anti-tick immune responses. The diminished numbers of B cells in susceptible breed indicate that antibodies are important in acquired resistance to ticks. On the other hand, the reduction of gd/WC1+ T cells seen in the infested skin of susceptible bovines indicates that these cells may play a role in resistance to ticks. Natural Killers cells could help the development of efficacious immune responses to ticks-borne diseases. In conclusion, these results reflect the fact that local responses involve resident and infiltrating leukocytes and lymphocytes that are sources of immunoregulatory, effectors and memory responses elicited against ticks.

A raiva bovina continua sendo uma importante zoonose. A ocorrência de casos confirmados da doença no meio rural é um problema atual de saúde pública, sendo mais relevante quando se consideram os casos de raiva huma na que ocorreram nos últimos anos no país. Este projeto teve por objetivo estudar a situação da raiva bovina no município de Guaxupé - MG, consideran do: a presença do morcego hematófago no município; o nível de conhecimento dos produtores/empregados rurais sobre a raiva; a vacinação contra a raiva bovina; a notificação de casos da doença junto ao Instituto Mineiro Agro pecuário (IMA); os casos confirmados de raiva animal. Quanto à metodologia, fo ram realizados: um levantamento de dados secundários junto ao IMA e junto à Vigilância Epidemiológica da Gerência Regional de Alfenas - MG, da qual faz parte o município de Guaxupé; a apli cação de um questionário sobre a raiva bovina junto a uma amostra de produtores/empregados rurais do município. Foi feita uma estratificação da amostra em dois grupos, sendo o questionário aplicado em 35 pequenas e médias propriedades e em cinco grandes propriedades. De acordo com os dados do IMA, durante o período de 1999 a 2003, foram realizados 34 exames laboratoriais para diagnóstico da raiva, entre eles, 10 foram conside rados positivos, sendo oito da espécie bovina e dois da espécie eqüina. Com relação ao conhecimento de produto res/empregados rurais sobre a raiva, constatou-se que 74,3% consideram o veterinário como uma importante fonte de informação; 85% procuram o veterinário se houver um animal doente; 97,5% conhecem a importância da vacinação; 72,5% indicaram a salivação aumentada do animal como sintoma característico da doença; 58,3% indicaram o carbúnculo como doença que se confunde com a raiva; 90,0% afirmaram ser o morcego hematófago o transmissor da doença; 57,1% afirma ram aplicar algum tipo de produto em mordeduras por morcegos; 92,5% afirmaram ter conhecimento da presen ça do morcego em sua propriedade; 17,5% afirmaram ter tido caso confirmado de raiva na propriedade; 53% apontaram galpões de secador de café como abrigo para os morcegos; 90% afirmaram vacinar o rebanho durante as campanhas de vacinação. Com relação aos dados da Vigilância Epidemiológica, entre os anos de 2000 e 2006, ocorreram 587 casos de agressão por animais no município de Guaxupé. Desses casos, 24,3% ocorreram com crianças de até nove anos de idade; 91,5% ocorreram na zona urbana; 82,9% dos ferimentos foram por mordedura; 49,0% foram nas mãos; em 78,2% dos casos a espécie agressora era a canina; ocorreu apenas um caso de raiva canina com diagnós tico laboratorial e quatro casos de raiva animal com diagnóstico clínico. Esses estudos permitiram concluir que ainda é atual a necessidade de manter todas as ações que visam o controle da raiva, especialmente a vacinação; é importante a realização de um trabalho de consci entização com relação aos casos de agressão por animais, especialmente os cães. / Bovine rabies continues to be an important zoonosis. The occurrence of confirmed cases of the disease in the rural environment is a current public health problem, which is even more relevant when the cases of human rabies that occurred in the last few years are considered. The objective of the present project was to study the situation of bovine rabies in the municipality of Guaxupé - MG, on the basis of the presence of blood-sucking bats in the municipality, the level of knowledge of rural producers/employe es about rabies, the vaccination against bovine rabies, the notification of cases of the disease to the \"Instituto Mineiro Agropecuário\" (IMA), and the confirmed cases of animal rabies. The methodology consisted of a survey of secondary data at IMA and at the Epidemiological Surveillance of the Regional Management of Alfenas - MG, to which the municipality of Guaxupé belongs, and the application of a questionnaire about bovine rabies to a sample of rural producers/employees of the municipality. The sample was stratified into two groups, with the questionnaire being applied to 35 small and medium-sized properties and to 05 large properties. According to IMA data, 34 laboratory exams for the diagnosis of rabies were performed during the period from 1999 to 2003. Of these, 10 were considered to be positive, 08 of them involving cattle and 02 involving horses. Regarding the knowledge of rural producers/employe es about rabies, 74.3% were found to consider the veterinary to be an impor tant source of information; 85% called a veterinary when an animal was sick, 97.5% were aware of the importance of vaccination, 72.5% indicated that incre ased animal salivation is a characteristic symptom of the disease, 58.3% indica ted that carbuncle is a disease often confused with rabies, 90.0% stated that blood-sucking bats are the transmitters of the disease, 57.1% stated that they applied some type of product to bat bites, 92.5% stated that they were aware of the presence of bats on their property, 17.5% stated that they had a confirmed case of rabies on their property, 53% pointed out that coffee-drying barns are a shelter for bats, and 90% stated that they vaccinated the herd during vaccination campaigns. Regarding the data of Epidemiological Surveillance, 587 cases of aggression by animals occurred between 2000 and 2006 in the municipality of Guaxupé. Of these, 24.3% involved children up to 09 years of age, 91.5% occurred in the urban zone, 82.9% of the wounds were bites, 49.0% were in the hands, in 78.2% of cases the aggressing species was the canine one, and there was only one case of canine rabies with a laboratory diagnosis, plus 04 cases of animal rabies with a clinical diagnosis. These studies led to the conclusion that there still is the need to maintain all actions aiming at the control of rabies, especially vaccination, and that it is important to conduct awareness-raising work regarding cases of aggression by animals, especially dogs.

Made available in DSpace on 2016-04-28T20:16:24Z (GMT). No. of bitstreams: 1 2007- Flavio Augusto Soares Graca01.pdf: 782491 bytes, checksum: aca3b139eb03d06af06c5dd1c95ec3d7 (MD5) Previous issue date: 2007-04-26 / Crotalus poisoning was experimentally reproduced by inoculation of Crotalus durissus terrificus (South American Rattlesnake) venom by subcutaneous via in ten clinically healthy mixed breed bovine, weighing between 125 and 449 kg and aged 12 to 36 months. Two bovine of the same age range and breed standard were used as controls. The bovine that received a 0.03 mg/kg p.v. dose died 7h40min after inoculation. A 0.015 mg/kg p.v. dose provoked death in four out of seven inoculated bovine, while two bovine experimentally poisoned with 0.0075 mg/kg p.v. became mildly sick and recovered. Onset of symptoms occurred between 1h30min and 13h45min after inoculation. Evolution oscillated between 5h25min and 44h59min for bovine deaths and between 33h15min and 17 days for bovine that recovered. The principal nervous signs observed were diminished response to external stimuli, hypotonic reflexes, dragging of hooves along the ground, apparent lethargy, difficulties in moving around obstacles, ocular globe paralysis, lateral and sternal decubitus and tongue paralysis. Constant adypsia and petechiae in the conjunctival and vaginal mucosa was also verified. A discrete to moderate increase in bleeding time was verified in six bovine and a moderate increase in partial thromboplastin time was activated in seven bovine. Moderate leukocytosis with neutrophilia, relative lymphopenia, eosinopenia and monocytosis occurred and a discrete increase in the number of rods. A significant increase in creatine kinase serum levels was observed, of a ten-fold order. No significant alterations were observed at urinalysis. At necropsy there were minimal edema at the inoculation site, discrete petechiae and suffusions in the epicardium, omentum, biliary vesicle and bladder mucosa. Histopathological examination revealed necrosis (hyalinization) of groups or isolated myocytes in ten different muscles examined, both close and distant from the inoculation site. Faced with the clinic-pathological picture, observations were made regarding the diagnosis of crotalus envenomation and its differentiation from deseases which course with paralysis and muscular necrosis in cattle in Brazil. / Reproduziu-se experimentalmente o envenenamento crot?lico, atrav?s da inocula??o, por via subcut?nea, do veneno de Crotalus durissus terrificus (Cascavel sul-americana) em dez bovinos mesti?os, com peso variando entre os 125 e 449 quilogramas e idade entre 12 e 36 meses. Dois animais na mesma faixa et?ria e padr?o racial foram utilizados como controle. O animal que recebeu dose de 0,03 mg/kg p.v. foi a ?bito 7h40min ap?s a inocula??o. A dose de 0,015 mg/kg p.v. induziu o ?bito em quatro de sete bovinos inoculados, enquanto os dois animais que receberam 0,0075 mg/Kg, adoeceram discretamente e se recuperaram. Os sintomas tiveram in?cio entre 1h30min e 13h45min ap?s a inocula??o. A evolu??o oscilou entre 5h25min e 44h59min para os animais que morreram e entre 33h15min e 17 dias entre os animais que se recuperaram. Os principais sinais nervosos observados foram diminui??o da resposta aos est?mulos externos, reflexos hipot?nicos, arrastar dos cascos no solo, aparente letargia, paralisia do globo ocular e da l?ngua, dec?bito esternal e lateral. Verificou-se tamb?m adipsia constante e pet?quias nas mucosas vaginal e conjuntival. Observaram-se discreto a moderado aumento do tempo de sangramento em seis animais e moderado aumento do tempo de tromboplastina parcial ativada em sete bovinos. Houve moderada leucocitose com neutrofilia, linfopenia relativa, eosinopenia, monocitose e discreto aumento do n?mero de bast?es. Foi evidenciado significativo aumento dos n?veis s?ricos de creatinaquinase, da ordem de dez vezes. N?o foram observadas altera??es significativas atrav?s da urin?lise. ? necropsia constataram-se edema quase impercept?vel no local da inocula??o, discretas pet?quias e sufus?es no epic?rdio, omento, ves?cula biliar e mucosa da bexiga. Os exames histopatol?gicos revelaram necrose (hialiniza??o) de grupos de mi?citos ou em mi?citos isolados em dez diferentes m?sculos esquel?ticos examinados, pr?ximos ou distantes do local de inocula??o. Diante do quadro cl?nico-patol?gico foram feitas observa??es sobre o diagn?stico do envenenamento crot?lico e sua diferencia??o com enfermidades que cursam com paralisia e necrose muscular em bovinos do Brasil.

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O objetivo geral desta pesquisa foi avaliar a transferência de imunidade passiva e a sua influência na resposta vacinal para as viroses envolvidas na Doença Respiratória Bovina (DRB). Os dados obtidos nesta pesquisa estão apresentados em dois capítulos. Capítulo 1 - Objetivou-se avaliar a dinâmica de anticorpos (Acs) específicos para as viroses respiratórias e subpopulações de linfócitos em bezerros do nascimento aos 240 dias (d) de vida. Para tanto, acompanhou-se a transferência de imunidade passiva de Acs específicos para as viroses respiratórias em 19 bezerros, destes cinco foram selecionados para acompanhamento da dinâmica de Acs neutralizantes e subpopulações de linfócitos dos 14 aos 240d. O colostro fornecido era proveniente de vacas doadoras vacinadas. A análise da qualidade individual do colostro revelou índice Brix &ge;21%, observando-se forte correlação desses valores com proteína total (r&#61; 0,942 e P&#61; 0,001). Após 48 horas da ingestão do colostro, pôde-se observar soroconversão dos 19 animais (100%) para os agentes virais envolvidos na DBR. As medianas (Log2) encontradas foram de 12,3, 9,0, 5,0 e 8,5 para BVDV, BoHV-1, BRSV e BPIV-3. Dos 14 aos 240d os 5 bezerros avaliados, demonstraram declínio gradual dos títulos de Acs (Log2) para BVDV (12,8-3,3), BoHV-1 (10,0-3,3) e BPIV-3 (10,0-2,0), embora o BVDV não tenha apresentado soronegatividade até os 240d. Manifestações clínicas de broncopneumonia foram observadas em 4/5 (80%) bezerros dos 80 aos 135 d. BRSV (11,3-2,0) apresentou perfil diferenciado na dinâmica de anticorpos em relação às demais viroses. Não foram detectadas diferenças estatísticas entre os momentos para as viroses apesar das variações detectadas (P&gt;0,05). A meia-vida e tempo para soronegatividade foram de 36,2&#177;6,1 e 367,01&#177;68,7d para BVDV, 50,7&#177;18,0 e 239,67&#177;66,88d para BoHV-1 e, 46,8&#177;21,1 e 303,36&#177;60,15d para BPIV- 3. BRSV não respeitou modelo de regressão para cálculos de meia-vida e soronegatividade. Valores absolutos e relativos das populações de linfócitos não revelaram diferenças estatísticas entre os momentos (P&gt;0,05). Contudo, a dinâmica das subpopulações de linfócitos revelou aumento de células B CD21+ até 150d; aumento nos valores relativos das subpopulações CD4+, CD8+ e CD3+CD4-CD8- principalmente aos 74-90d. Assim, pôde-se determinar uma janela de susceptibilidade a partir dos 74d especialmente ao BRSV e BoHV-1, momento que precede o aumento dos títulos de Acs após exposição natural. Capítulo 2 Objetivou-se avaliar a influência dos Acs maternos na resposta imune para DRB induzida pela vacinação. Foram selecionados 23 bezerros recém-nascidos, distribuídos aleatoriamente entre 4 grupos experimentais: G1 vacinado aos 14d e booster aos 44d; G2 aos 90d e aos 120d; G3 aos 180d e aos 210d. Além disso manteve-se um grupo controle não vacinado CG1, CG2 e CG3. Os bezerros foram vacinados com a mesma vacina comercial empregada para vacinação das doadoras de colostro. Observou-se: (1) a vacina com BVDV inativado não promoveu aumento dos títulos de Acs para nenhum dos grupos avaliados; (2) G1 não demonstrou soroconversão para nenhuma das viroses, enquanto controle CG1 exibiu decréscimo dos títulos para BVDV, BoHV-1 e BPIV-3; (3) o BRSV apresentou baixa soroconversão no G2, enquanto o controle demonstrou altos títulos dos 44-120d; (4) não foi possível distinguir entre quais tempos ocorreram diferenças entre títulos (P&gt;0,0167); (5) linfócitos B (CD21+) aumentaram do T0-T2 para G1, diminuíram para G2, e aumentaram no G3; (6) linfócitos T CD3+ diminuíram ao longo do tempo para todos os grupos, exceto CG3; (7) apesar de oscilações, linfócitos T CD4+, CD8+ e WC1+ se mantiveram praticamente constantes até 240d, exibindo maiores proporções nos grupos vacinados; (8) a expressão do marcador CD25+foi mantida pelo grupo G1 até o T2, mas apresentou aumento no G2 e G3; (9) manifestações de broncopneumonias foram identificadas nos bezerros do grupo controle (4/5 - 80%) e podem ter exercido influência nas diferenças encontradas para as células entre os grupos. Em geral, a vacinação dos bezerros aos 90 (G2) e 180d (G3) manteve ou estimulou a produção de Acs para o BoHV-1, BRSV e BPIV-3, e a ativação das células T expressas pelo marcador CD25+ pode ter sido responsável pela proteção dos bezerros frente à DRB. Assim, com base nos resultados, concluiu-se que a intensidade da imunidade dos bezerros induzida pela vacinação aumentou de acordo com o desenvolvimento etário e diminuição dos títulos de Acs maternos. A conclusão geral desta dissertação aponta para a necessidade precoce de imunização dos bezerros, especialmente pela susceptibilidade observada para BRSV e BPIV-3 aos 74-90d de vida. Entretanto, esta pesquisa não encontrou resposta humoral induzida pela vacinação no grupo de bezerros vacinados aos 14 e 44 dias, apesar dos indícios de resposta imune celular. Assim, estudos futuros devem ser elaborados considerando estratégias para amplificar a resposta imune precoce dos bezerros para os agentes virais envolvidos na DRB / The main purpose of this research was to evaluate the transfer of passive immunity and its influence on vaccine response to the viruses involved in bovine respiratory disease (BRD). The data obtained in this study are presented in two chapters. Chapter 1 - The objective was to evaluate the dynamics of specific antibodies to the respiratory viruses and lymphocyte subpopulations in the calves from birth to 240 days (d) of life. Thus, the transfer of passive immunity of specific antibodies to the respiratory viruses were assessed in 19 calves; from these, five were selected for monitoring the dynamic of neutralizing Abs and lymphocytes subpopulations from 14 to 240d. The colostrum was provided from donor vaccinated cows. The analysis of individual quality of the colostrum revealed Brix &ge;21%, observing strong correlation of these values with total protein (r &#61; 0.942 and P &#61; 0.001). After 48 hours of colostrum intake, was observed seroconversion of the 19 animals (100%) for the viral agents involved in the DBR. The median (Log2) ratio found was 12.3, 9.0, 5.0 and 8.5 for BVDV, BoHV-1, BRSV and BPIV-3. The 5 calves followed from 14 to 240d showed gradual decline in antibody titers (Log2) to BVDV (12.8 to 3.3), BoHV-1 (10.0 to 3.3) and BPIV-3 (10, 0-2.0), although could not be detected seronegative calves for BVDV up to eight months of age. Clinical manifestations of bronchopneumonia were observed in 4/5 (80%) calves from 80 to 135 days of life. BRSV (11.3 to 2.0) showed a distinct profile in the dynamics of antibodies compared to other viruses. There were no statistical differences between times for viruses despite variations detected (P&gt; 0.05). The half-life and time to become seronegative were 36.2&#177;6.1 and 367.01&#177;68.7d for BVDV, 50.7&#177;18.0 and 239.67&#177; 66.88d for BoHV-1 and, 46.8&#177;21.1 and 303.36&#177;60.15d for BPIV-3. BRSV did not respect regression model to perform half-life and seronegative calculations. Absolute and relative values of lymphocyte populations revealed no statistical differences between times (P&gt; 0.05). However, the dynamics of lymphocyte subpopulations showed increase in B cells CD21+ up to 150d; increase in the relative values of CD4+, CD8+ and CD3+CD4-CD8- subpopulations, mainly to 74-90d. Thus, it was possible to determine a window of susceptibility since 74d especially for BRSV and BoHV-1, moment that precede the increase in antibody titers after natural exposure. Chapter 2 - The objective was to evaluate the influence of maternal antibodies in the immune response to respiratory viruses induced by vaccination. Was selected 23 newborn calves that were randomly distributed in four groups: G1 - vaccinated at 14d and booster at 44d; G2 - vaccinated at 90d and booster at 120d; G3 - vaccinated at 180d and booster at 210d. Furthermore were kept a non-vaccinated control group - CG1, CG2 and CG3. Calves were vaccinated with the same commercial vaccine in the colostrum donors. From these, could be observed: (1) the vaccine with inactivated BVDV did not promote increase of antibodies titers in any of the assessed groups; (2) G1 did not demonstrated seroconversion for any of the viruses while CG1 control exhibited decrease in the titers for BVDV, BoHV-1 and BPIV-3; (3) BRSV had low seroconversion in G2, while the control showed high titers from 44 to 120d; (4) where the differences between times occurred could not be distinguished (P &lt;0.0167); (5) B cells (CD21+) increased from T0 to T2 for G1, decreased for G2, and increased for G3; (6) T lymphocytes CD3+ decreased over time for all groups except for CG3; (7) despite variations, T lymphocytes CD4+, CD8+ and WC1+ remained almost constant until 240d, displaying greater proportions in the vaccinated groups; (8) the expression of the CD25+ marker was maintained in the vaccinated group G1 up to T2, whereas vaccination promoted an increase of this expression in G2 and G3; (9) clinical manifestations of bronchopneumonia were identified in the control group (4/5 calves - 80%) and may have influenceon the differences found for the cells between the groups. In general, vaccination of calves at 90 (G2) and 180d (G3) maintained or stimulated the production of Abs to BoHV-1, BRSV and BPIV-3, and the activation of T cells expressed by the CD25+ marker may have been responsible for the protection of calves from BRD. Thus, based on the results, it was concluded that the intensity of the immunity induced by vaccination of calves increased according to the age of development and decay of maternal antibody titers. The general conclusion of this research, points to the need for early immunization of calves, especially by the susceptibility observed for BRSV and BPIV-3 from 74-90d of life. However, this research didnot found humoral response induced by vaccination in the group of calves vaccinated at 14 and 44 days, despite the evidence of cellular immune response. Thus, future studies should be designed considering strategies to amplify the early immune response of calves to the viral agents involved in the BRD

Somatic cell nuclear transfer (SCNT), or cloning, is a form of artificial reproductive technology that can be used to improve economic traits of domestic animals. However, extreme inefficiency of producing viable offspring via this method is a major limitation. An aggressive immune response at the maternal-fetal interface is an important reason for SCNT pregnancy loss. The goal of this project was to investigate the molecular mechanisms of immune-mediated miscarriage in cloned cattle pregnancies. Many publications hint that immune-mediated miscarriage is associated with abnormal MHC-I expression in the placenta. The regulation of bovine MHC-I genes was systematically studied to identify the cause of abnormal MHC-I expression during immune-mediated miscarriage. We also produced cloned pregnancies to study immune- mediated pregnancy loss. MHC-I and cytokines involved in proinflammatory responses were highly expressed in the placental trophoblast cells of cloned fetuses and in the uterine endometrium of recipients carrying MHC-I incompatible fetuses, respectively, suggesting that MHC-I compatibility between fetus and surrogate mother is important for the success of animal cloning. The results from this research not only reveal the cause of high pregnancy loss in cloned animals but also provide molecular clues to prevent immune-mediated miscarriage in cattle and potentially in human clinics.

Bovine reproductive performance is one of the most important factors influencing dairy farm profitability. Present-day techniques for oestrus- and pregnancy-detection are unreliable and labour-intensive. Although measuring milk-progesterone at regular intervals allows the fertility status of a cow to be determined reliably, the labour cost of collecting and analysing samples is prohibitive. This project aimed to develop a progesterone sensing system that could be automated and integrated with the milking unit, thus minimising labour costs. The proposed system involved mixing the milk sample with an enzyme-antibody conjugate and then passing the sample through a column containing immobilised progesterone. Any progesterone in the milk would inhibit conjugate binding to the column. An enzyme substrate would then flow through the column and bound conjugate would be detected as a colour change at the column's outlet. Periodate-coupling was used to attach horseradish peroxidase enzyme to anti-progesterone antibody, and progesterone-3-carboxymethyloxime was immobilised on the polystyrene bead surface using amine-coupling. Both techniques are widely used. Initial experiments attempted to verify the success of these two reactions simultaneously, whereas later experiments focused on the bead-coating. Beads were suspended in a specially-constructed syringe and the antibody activity of the eluted solution was measured by SPR. However, a combination of non-specific binding and antibody stability and activity issues meant neither reaction was conclusively verified. Many trials were done to investigate how to overcome the problems encountered but a suitable, workable procedure was not developed. Despite poor progress, the problems encountered did not undermine the project's potential. There remains optimism of developing an on-line method if research were to continue.

Consumers' risk perceptions are examined to explain the underlying reasons for consumer concern associated with milk from dairy herds treated with recombinant bovine growth hormone (rbGH). A focus group study was employed as an initial step in exploring the primary influences of consumer apprehension toward rbGH's use. The information obtained through the focus group sessions was invaluable in strengthening empirical measures of the factors affecting risk perception, and in formulating concise survey questions for a national study. Data from a nationwide survey of 1,910 primary household food purchasers were used in understanding the influence of risk characteristics on consumers' risk perceptions toward rbGH treated herd milk, as well as investigating consumer risk perception profiles. One conclusion is evident from the data, consumers remain concerned about the rbGH product despite FDA approval for commercial use. Results suggest that particular characteristics of the rbGH product hypothesized as being more risky and less tolerable elicit consumer outrage perceptions. Results also showed systematic differences between consumers, producing a range of risk perception profiles. Overall, the results support the idea that consumers' risk perceptions are multi-dimensional and differ in emphasis compared to the risk assessments by scientific experts. Consumers' risk perceptions warrant recognition as playing a vital role in product acceptance. A recommendation proposed for those involved in risk assessment is to integrate consumer beliefs and perceptions into assessments of risk, perhaps increasing consumer trust and reducing product apprehension. Additionally, the range of risk perceptions among consumers imply that one public policy strategy is unlikely to satisfy all consumers. Risk communicators can design more effective risk communication strategies by understanding the ways consumers differ in their behavioral response to a particular perceived concern. / Graduation date: 1997