A Pleuropneumonia Contagiosa Bovina (PPCB) é uma enfermidade respiratória grave, causada pelo Mycoplasma mycoides subsp mycoides Small Colony (MmmSC), sendo de primeira ordem no quadro nosológico em Angola, com alta prevalência e grandes repercussões econômicas na pecuária angolana. Objetivando caracterizar os achados anatomopatológicos e a resposta inflamatória na ocorrência natural da PPCB, realizou-se a coleta de amostras de pulmão e linfonodos regionais em 50 bovinos com lesões macroscópicas compatíveis com a doença e abatidos na cidade de Huambo, Angola. As amostras teciduais foram fixadas em formol 10% e incluídas em parafina para avaliação histopatológica, pela técnica de hematoxilina-eosina, e detecção do agente infeccioso por PCR a partir do DNA total extraído do tecido pulmonar parafinado. A caracterização da resposta inflamatória foi avaliada por imunohistoquímica, utilizando-se o método de detecção por polímero para os marcadores de linfócitos T (CD3) e B (Pax5), assim como as interleucinas (IL)-1β e 4. Após avaliação molecular, 7 amostras foram excluídas por não apresentarem DNA viável e 16/43 (37,2%) animais foram positivos para o MmmSC pelo PCR. Dos animais positivos, a lesão macroscópica mais frequente foi a aderência (7/16, 43,8%); enquanto a lesão microscópica mais frequente foi a hiperplasia linfoide (9/16, 56,2%). Houve associação significativa entre a positividade dos animais e a presença da alteração nas lesões de aderência (p=0,047), pulmão marmoreado (p=0,012), sequestro (p=0,001) e fibrose pulmonares (p<0,05). O pulmão esquerdo e lobo diafragmático esquerdo foram frequentemente afetados na doença, porém sem diferença significativa. Os resultados imunohistoquímicos demonstraram marcação focal para linfócitos B e T, assim como imunoexpressão de IL-1β e IL-4 em intensidade fraca a moderada na maioria dos casos analisados. Os resultados demostraram que a PPCB está presente em bovinos na região estudada, porém com lesões anatomopatológicas e resposta inflamatória inespecíficas. Estes resultados ressaltam a importância da avaliação integrada de dados epidemiológicos, anatomopatológicos, imunológicos e moleculares para estabelecer um diagnóstico preciso e definitivo da PPCB nestes animais. / Bovine Contagious Pleuropneumonia (PPCB) is a serious respiratory disease caused by Mycoplasma mycoides subsp mycoides Small Colony (MmmSC) , being of first order in the nosological framework in Angola, with high prevalence and great economic repercussions in Angolan livestock. Aiming to characterize the anatomopathological findings and the inflammatory response in the natural occurrence of PPCB, samples of lung and regional lymph nodes were collected in 50 cattle with macroscopic lesions compatible with the disease and slaughtered in the city of Huambo, Angola. Tissue samples were fixed in 10% formalin and embedded in paraffin for histopathological evaluation by the hematoxylin-eosin technique and detection of the infectious agent by PCR from the total DNA extracted from the paraffin-shaped lung tissue. The characterization of the inflammatory response was evaluated by immunohistochemistry, using the polymer detection method for T (CD3) and B (Pax5) lymphocytes, as well as interleukins (IL) -1β and 4. After molecular evaluation, 7 samples were excluded because they did not present viable DNA and 16/43 (37.2%) animals were positive for MmmSC by PCR. Of the positive animals, the most frequent macroscopic lesion was adhesion (7/16, 43.8%); while the most frequent microscopic lesion was lymphoid hyperplasia (9/16, 56.2%). There was a significant association between the positivity of the animals and the presence of changes in adhesion lesions (p = 0.047), marbled lung (p = 0.012), pulmonary sequestra (p = 0.001) and fibrosis (p <0.05). The left lung and left diaphragmatic lobe were frequently affected in the disease, but without significant difference. Immunohistochemical results demonstrated focal labeling for B and T lymphocytes as well as weak to moderate intensity of IL-1β and IL-4 immunoexpression in most of the analyzed cases. The results showed that CPBP is present in cattle in the studied region, but with nonspecific anatomopathological lesions and inflammatory response. These results highlight the importance of the integrated evaluation of epidemiological, anatomopathological, immunological and molecular data to establish an accurate and definitive diagnosis of CBPP in these animals.

As doenças entéricas associadas com diarreia, desidratação e perda de peso constituem um dos principais problemas da bovinocultura mundial, contribuindo para expressivos índices de morbidade e mortalidade, principalmente entre neonatos. Nesse contexto, exigências para o aprimoramento do diagnóstico laboratorial de doenças entéricas de natureza infectocontagiosa, tornaram-se constantes, face às perdas econômicas vinculadas. Entre os principais enteropatógenos virais, de distribuição mundial e história recente, destaca-se o astrovírus bovino (BoAstV), cuja primeira identificação data de 1978. BoAstV entérico induz diarreia principalmente entre neonatos e imunocomprometidos, tendo uma prevalência acima de 60% nas 5 primeiras semanas de vida dos animais. Devido à carência de dados a respeito da prevalência desse vírus no Brasil, o presente estudo foi realizado objetivando a detecção e caracterização moleculares de cepas de BoAstV em amostras fecais de bovinos com e sem diarreia de diferentes idades. O estudo foi conduzido a partir de 272 animais em diferentes estados do Brasil, obtendo-se 14,3% de positividade através de RT-PCR, sendo que 11 amostras, provenientes dos estados de São Paulo, Minas Gerais e Rio Grande do Sul, foram submetidas ao sequenciamento nucleotídico. A similaridade entre as sequências deduzidas de aminoácidos das amostras obtidas foi maior do que 86,8%, quando comparadas umas com as outras, e situou-se entre 86,2 a 94,8% quando comparadas com sequências de outros BoAstV descritos. Nas reconstruções filogenéticas, 9 amostras agruparam-se conjuntamente em clado distinto de outros BoAstV, uma amostra (BoAstV-155-BRA) formou ramo parafilético a clado que agrupa tanto outros BoAstV como astrovírus de cervídeos e a amostra restante (BoAstV-267-BRA) formou ramo basal aos astrovírus bovinos e suínos. No entanto, o posicionamento das 2 amostras divergentes (BoAstV-155-BRA e BoAstV-267-BRA) não derivou de episódios de seleção positiva ou recombinação. Em síntese, os resultados obtidos indicam, de maneira inédita, a circulação de variantes de BoAstV entérico em rebanhos de estados da região centro-sul do Brasil. / Enteric diseases associated with diarrhea, dehydration and weight loss is one of the major problems of cattle worldwide, contributing to significant morbidity and mortality, especially among newborns. In this context, demands for improving the laboratory diagnosis of infectious enteric diseases, became constant, given the economic losses involved. Among the major viral enteropathogens of worldwide distribution and recent history, highlight the bovine astrovirus (BoAstV), whose first mention dates from 1978. Enteric BoAstV induce diarrhea especially among newborns and immunocompromised animals, having a prevalence above 60% in the first 5 weeks of life. Due to lack of data concerning the occurrence of this virus in Brazil, the present study was aimed at molecular detection and characterization of BoAstV strains in fecal samples from cattle with and without diarrhea of different ages. The study was conducted on 272 animals from different states of Brazil, obtaining positivity of 14.3% by RT-PCR, and 11 samples from the states of São Paulo, Minas Gerais and Rio Grande do Sul were subjected to nucleotide sequencing. The similarity between the deduced amino acid sequences of the samples was greater than 86.8% when compared with each other and was between 86.2 to 94.8% compared with sequences of other BoAstV described. In phylogenetic reconstructions, 9 samples were grouped together in a separate clade from other BoAstV, a sample (BoAstV-155-BRA) formed a paraphyletic branch to a clade that groups both other BoAstV and deer astrovirus and the remaining sample (267-BRA-BoAstV ) formed a basal branch to bovine and porcine astrovirus. However, the positioning of the two different samples (BoAstV-155-BRA and BoAstV-267-BRA) did not derive from episodes of positive selection or recombination. In summary, the results indicate, in an unprecedented manner, the circulation of enteric BoAstV variants in herds of cattle form states of the south-central region of Brazil.

As doenças entéricas associadas com diarreia, desidratação e perda de peso constituem um dos principais problemas da bovinocultura mundial, contribuindo para expressivos índices de morbidade e mortalidade, principalmente entre neonatos. Nesse contexto, exigências para o aprimoramento do diagnóstico laboratorial de doenças entéricas de natureza infectocontagiosa, tornaram-se constantes, face às perdas econômicas vinculadas. Entre os principais enteropatógenos virais, de distribuição mundial e história recente, destaca-se o astrovírus bovino (BoAstV), cuja primeira identificação data de 1978. BoAstV entérico induz diarreia principalmente entre neonatos e imunocomprometidos, tendo uma prevalência acima de 60% nas 5 primeiras semanas de vida dos animais. Devido à carência de dados a respeito da prevalência desse vírus no Brasil, o presente estudo foi realizado objetivando a detecção e caracterização moleculares de cepas de BoAstV em amostras fecais de bovinos com e sem diarreia de diferentes idades. O estudo foi conduzido a partir de 272 animais em diferentes estados do Brasil, obtendo-se 14,3% de positividade através de RT-PCR, sendo que 11 amostras, provenientes dos estados de São Paulo, Minas Gerais e Rio Grande do Sul, foram submetidas ao sequenciamento nucleotídico. A similaridade entre as sequências deduzidas de aminoácidos das amostras obtidas foi maior do que 86,8%, quando comparadas umas com as outras, e situou-se entre 86,2 a 94,8% quando comparadas com sequências de outros BoAstV descritos. Nas reconstruções filogenéticas, 9 amostras agruparam-se conjuntamente em clado distinto de outros BoAstV, uma amostra (BoAstV-155-BRA) formou ramo parafilético a clado que agrupa tanto outros BoAstV como astrovírus de cervídeos e a amostra restante (BoAstV-267-BRA) formou ramo basal aos astrovírus bovinos e suínos. No entanto, o posicionamento das 2 amostras divergentes (BoAstV-155-BRA e BoAstV-267-BRA) não derivou de episódios de seleção positiva ou recombinação. Em síntese, os resultados obtidos indicam, de maneira inédita, a circulação de variantes de BoAstV entérico em rebanhos de estados da região centro-sul do Brasil. / Enteric diseases associated with diarrhea, dehydration and weight loss is one of the major problems of cattle worldwide, contributing to significant morbidity and mortality, especially among newborns. In this context, demands for improving the laboratory diagnosis of infectious enteric diseases, became constant, given the economic losses involved. Among the major viral enteropathogens of worldwide distribution and recent history, highlight the bovine astrovirus (BoAstV), whose first mention dates from 1978. Enteric BoAstV induce diarrhea especially among newborns and immunocompromised animals, having a prevalence above 60% in the first 5 weeks of life. Due to lack of data concerning the occurrence of this virus in Brazil, the present study was aimed at molecular detection and characterization of BoAstV strains in fecal samples from cattle with and without diarrhea of different ages. The study was conducted on 272 animals from different states of Brazil, obtaining positivity of 14.3% by RT-PCR, and 11 samples from the states of São Paulo, Minas Gerais and Rio Grande do Sul were subjected to nucleotide sequencing. The similarity between the deduced amino acid sequences of the samples was greater than 86.8% when compared with each other and was between 86.2 to 94.8% compared with sequences of other BoAstV described. In phylogenetic reconstructions, 9 samples were grouped together in a separate clade from other BoAstV, a sample (BoAstV-155-BRA) formed a paraphyletic branch to a clade that groups both other BoAstV and deer astrovirus and the remaining sample (267-BRA-BoAstV ) formed a basal branch to bovine and porcine astrovirus. However, the positioning of the two different samples (BoAstV-155-BRA and BoAstV-267-BRA) did not derive from episodes of positive selection or recombination. In summary, the results indicate, in an unprecedented manner, the circulation of enteric BoAstV variants in herds of cattle form states of the south-central region of Brazil.

The purpose of this research was to define the effects of various growth-promoting factors on in vitro embryonic development of in vitro matured and in vitro fertilized bovine embryos. The control medium was a chemically defined medium which improves the possibility of closely determining the in vivo conditions the embryo is actually exposed to. The growth-promoting factors tested in this experiment included transferrin, IGF-I (insulin-like growth factor-one), IGF-II (insulin-like growth factor-two), TGF-a (transforming growth factor-alpha) , TGF-B1 (transforming growth factor-beta1) , PDGF (platelet derived growth factor), EGF (epidermal growth factor), NGF (nerve growth factor), and bFGF (basic fibroblast growth factor). Transferrin was included at 10 micrograms/milliliter , while all other factors were utilized at 10 nanograms/milliliter in the control medium. Bovine cumulus-oocytes were retrieved from slaughterhouse ovaries and were matured i n Medium-199 containing 10% feta l bovine serum for 24 hours at 39°C in a 5% C02 atmosphere. Frozen-thawed bull spe r m were s wim-up separated and capacitated in medium containing heparin for 3 hours prior to insemination. Gametes were co- incubated fo r 18 hours and then cumulus cells were stripped from the ova. Ova which did not cleave were removed from culture 36 hours after insemi nati on and were stained for evidence of fertilization. Embryos were cultured in one of the 10 conditions (including control) described above. A total of 150 total oocy.t.es were cultured per treatment for a tota l of 10 days. EGF improved embryo development, while TGF-Bl and TGF-a only slightly improved embryo development compared to the control. All other factors tested did not have a beneficial effect on embryo development in this culture medium. In summary, EGF improved in vitro development of bovine embryos obtained from in vitro maturated and in vitro fertilized bovine oocytes. Other factors which were t est ed did not significantly improve in vitro bovine embryo development. Further experiments are necessary fo r determining the requirements of bovine embryos in vitro.

An analytical method was developed for the determination of the â-agonists clenbuterol, cimaterol and salbutamol in bovine retina. The method involved extraction into a pH 8.5 tris-HCl buffer, followed by protease enzyme digestion and immunoaffinity column cleanup before analysis by liquid chromatography with tandem mass spectrometry detection LC/MS/MS. The LOD for clenbuterol, salbutamol and cimaterol were 0.64, 1.20 and 1.92 ng/g respectively. The identities of the analytes were able to be confirmed to an acceptable standard. An analytical method was also developed for the analysis of the â-agonists clenbuterol, salbutamol, cimaterol, ractopamine, and mabuterol in bovine urine and emu muscle. The urine and muscle samples were digested with â-glcuronidase enzyme and cleaned up using a Bond Elute Certify SPE. The extracts were analysed by LC/MS/MS. Deuterated internal standards were used for quantitation. The LOD for urine [less than] 1ng g and for emu muscle it was [less than] 0.3 ng/g. The last part of the work describes the simultaneous gas chromatography-mass spectrometric analysis of diethylstilbestrol DES, hexestrol HEX, dienestrol DIEN, zeranol ZER, taleranol TAL and zeralenone ZON in fresh full cream and fresh skim milk. The analytes were analysed as their trimethyl silane (TMS) derivatives. A three phase solvent system was used for extraction and the extract was cleaned up using a combination of the anion exchange and hydrophobic properties of an anion exchange SPE. The detection limits for DES, DIEN, HEX, ZER, TAL and ZON were 9.6, 9.6, 16.8 , 7.2 , 13.5 and 34.8 ng/L respectively. / Doctor of Philosophy(PhD)

This study was performed to test the viability of administering a single 40 mg dose of Folltropin-V® (FSH, Bioniche Animal Health) diluted in SRF (MAP-5 50, Sodium Hyaluronate, Bioniche Animal Health) on day 5 of a recipient synchronization protocol to beef cows to evaluate its effect on recipient fertility. All recipients were administered an estradiol 17beta (2.5 mg, IM) and progesterone (50 mg, IM) combination injection on day 0 and a CIDR® (progesterone 1.34 g, Pfizer Animal Health) was inserted. Lutalyse® (dinoprost tromethamine, Pfizer Animal Health, 25 mg, IM) was administered at the time of CIDR removal on day 7, and estradiol 17beta (1 mg, IM) was administered on day 8. On day 16, the presence of at least one corpus luteum (CL), detected via ultrasound, resulted in the recipient receiving an embryo (both fresh and frozen-thawed embryos were used). Embryos were not transferred into cows that did not show the presence of a CL. Dependent variables for which data were collected included circulating progesterone levels at the time of transfer, number of CLs and CL diameter, circumference, and area; measured in millimeters. The study (n=572) consisted of a treatment group (n=268) and a control group (n=304), and included both Bos indicus (Brahman influenced) crossbred (n=115) and Bos taurus (Angus based) cows (n=457). Pregnancy rates for Treated recipients (40.67%A) and Control recipients (52.96%B) differed (P<.05). There was no difference in the mean number of CLs per recipient for Treated (1.14 +/- .03) and Control (1.10 +/- .02) cows, nor was there a difference in progesterone (P4) at the time of transfer for Treated (3.14 +/- .40 ng/mL) and Control (3.23 +/- .18 ng/mL) recipients. Overall, the inclusion of Folltropin-V® diluted in hyaluronan in a FTET synchronization protocol did not improve the fertility of beef recipients.

Thesis (Ph. D.)--Carleton University, 1999. / Also available in electronic format on the Internet.

La variabilité non maîtrisée de la tendreté de la viande bovine est un problème majeur pour la filière industrielle, cette qualité étant très recherchée par les consommateurs. Depuis de nombreuses années, différents programmes de recherche ont identifié des marqueurs ADN, ARN et protéines de la tendreté de la viande bovine, dans des contextes différents. Aux Etats-Unis et en Australie, des recherches ont abouti à la conception de tests efficaces de prédiction de la qualité de la viande. Ces tests ne fonctionnent cependant pas sur les élevages français. La filière française est donc demandeuse de tests de prédiction simples permettant de mesurer la tendreté sur l’animal vivant et la carcasse. De tels tests sont inexistants à l’heure actuelle. L’objectif de la thèse est de valider ou non une liste de potentiels marqueurs protéiques de la tendreté, et de développer des équations de prédiction de cette qualité, afin d’envisager la conception de tests immunologiques de prédiction à destination de la filière sur l’animal vivant et la carcasse, dans un avenir proche. Une nouvelle technique pour quantifier les protéines d’un échantillon de muscle bovin a été développée, le Dot-Blot, et permet de disposer de prototype pour de futurs tests de phénotypage de la tendreté. L’utilisation de cette technique a permis de quantifier 24 protéines sur 111 échantillons de deux muscles de boeufs et de taurillons de race Charolaise. Ces travaux ont identifié en premier lieux des effets biologiques liés au type de muscle et d’animal sur l’abondance des protéines. Puis, trois analyses différentes, dont les résultats sont concordants, ont validé des marqueurs de tendreté et mis en lumière les principaux mécanismes cellulaires impliqués dans la tendreté, générant des équations de prédiction de la tendreté. Des outils de bioinformatique ont été construits à partir de données expérimentales de la thèse sur les protéines de la tendreté et de bases de données humaines, afin de mieux comprendre les mécanismes biologiques impliqués dans la mise en place de la tendreté. En conclusion, ce travail de thèse a développé un nouvel outil d’analyse et les premières équations de prédiction de la tendreté de la viande bovine fiables sur un système centré sur les mâles, supports indispensables au développement de tests de prédiction de la tendreté sur l’animal vivant et la carcasse. De plus, ce travail a permis de mieux comprendre et caractériser les mécanismes moléculaires impliqués dans la mise en place de la tendreté. / The uncontrolled variability of beef tenderness is a major problem for industry. This quality is very important for consumers. For many years, different research programs have identified DNA, RNA and proteins markers of beef tenderness, in different contexts. In United States and Australia, these tests reached to the conception of efficient meat quality prediction tests. However, these tests do not work in France. So, French industry asks for simple tenderness prediction tests, which measure tenderness on living animals and carcasses. This type of tests is currently missing. The objective of the PhD is to validate or not a list a proteins tenderness potential markers, and to develop quality prediction equation, to engage the conception of immunological tests for industry, usable on living animals and carcasses. A new technique for quantify proteins in bovine muscle samples have been developed, the Dot-Blot, and allow to have prototypes of the future tenderness tests. Utilization of Dot-Blot provides phenotypic data of 224 proteins on 111 muscle samples of Charolais beefs and young bulls. This work first identified biological effects due to animal and muscle types on proteins abundance. Then, three different analyses, with corroborating results, validated tenderness markers and revealed main cellular pathways implied in tenderness, and generated tenderness prediction equations. Bioinformatics tools have been built upon experimental data from this work on tenderness proteins and human databases, to better understand the main mechanisms implied intenderization processes. In conclusion, this work have developed a new analyzing tool and the first beef tenderness prediction equation, trusty on a male-based system, essential supports for the development of beef tenderness prediction tests on living animals and carcasses. Moreover, this work enabled to better understand and characterize the molecula rmechanisms involved in tenderization processes.

Realizou-se um estudo para caracterizar a situação epidemiológica da brucelose e tuberculose bovinas na região de Campinas, Piracicaba, Bragança Paulista, Limeira, Mogi Mirim e São João da Boa Vista. Foram aleatoriamente amostradas 251 propriedades com criação de bovinos, sendo que nestas, eram sorteadas 40 ou 20 fêmeas acima de 24 meses para diagnóstico de tuberculose e 15 ou 10 fêmeas acima de 24 meses para diagnóstico sorológico de brucelose, dependendo do total de fêmeas existentes. O rebanho foi considerado positivo para brucelose quando havia pelo menos um animal com diagnóstico positivo. Para tuberculose, em propriedades com mais de 99 fêmeas acima de 24 meses, era necessário pelo menos dois animais positivos para a propriedade ser classificada como positiva, enquanto que em propriedades com até 99 fêmeas acima de 24 meses, um animal positivo classificava a propriedade como positiva. Foi aplicado questionário epidemiológico e com base nesse documento, foram feitas as análises univariada e regressão logística para identificar os fatores de risco associados à condição de foco e calculado o valor de odds ratio para quantificar o risco. A prevalência aparente de focos de brucelose foi 11,2% (IC95% = 7,8;15,8) e de tuberculose foi 14,1% (IC95% = 10,2%; 19%). A prevalência aparente de animais positivos para brucelose foi 2,5% (IC95% = 1,5%; 4,1%) e para tuberculose foi 2,7% (IC95% = 1,6;4,5%). Como fatores de risco associados à brucelose ter mais de 57 bovinos no rebanho, apresentou valor de OR = 4,2 (IC95% = 1,9; 9,5). Para tuberculose, exploração leite apresentou valor de OR = 2,6 (IC95% = 1,3; 5,3), ajustado por aquisição de bovinos, OR = 2,4 (IC95% = 1,2; 5,0). Os esforços para redução da prevalência de brucelose não surtiram efeito mesmo após 10 anos de aplicação de medidas de controle preconizadas pelo PNCEBT, sugerindo-se a sua reformulação na área de estudo. / A study was conducted to characterize the epidemiological situation of bovine brucellosis and tuberculosis in Campinas, Piracicaba, Bragança Paulista, Limeira, Mogi Mirim and São João da Boa Vista área, State of São Paulo. A total of 251 farms were randomly selected and in each of them 40 or 20 cows over 24 months were selected for tuberculosis diagnosis and 15 or 10 females over 24 months for brucellosis diagnosis depending on the existing total females. The herd was considered positive for brucellosis when there was at least one positive animal. For tuberculosis in herds above 99 females over 24 months at least two positive animals to classify the herd as positive. As long as in farms with up to 99 females over 24 months, a positive animal classified the property as positive. An epidemiological questionnaire was applied and based on this document, univariate and multivariate logistic regression were performed to identify risk factors associated with the diseases, based on odds ratio calculation. The apparent prevalence of brucellosis positive herds was 11.2% (95% CI = 7.8, 15.8) and tuberculosis positive herds was 14.1% (95% CI = 10.2%, 19%). The apparent prevalence of positive animals for brucellosis was 2.5% (95% CI = 1.5%, 4.1%) and tuberculosis was 2.7% (95% CI = 1.6, 4.5%). Having more than 57 cattle in the herd, with an OR = 4.2 (95% CI = 1.9, 9.5) was associated with brucellosis. Milk farm type with an OR = 2.6 (95% CI = 1.3, 5.3), and cattle purchase, with an OR = 2.4 (95% CI = 1.2, 5.0) were associated with tubercullosis. Efforts to reduce the prevalence of brucellosis do not reached the expected effects even after 10 years of implementation of control measures recommended by the PNCEBT. Therefore, the reformulation of the Program is recommended in the study area.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-10T16:24:10Z No. of bitstreams: 1 Hamilton Pereira Santos.pdf: 661343 bytes, checksum: 002eebd8b1e7c7b5293fde2164fb6317 (MD5) / Made available in DSpace on 2016-10-10T16:24:10Z (GMT). No. of bitstreams: 1 Hamilton Pereira Santos.pdf: 661343 bytes, checksum: 002eebd8b1e7c7b5293fde2164fb6317 (MD5) Previous issue date: 2010-02-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Bovine Enzootic Leukosis (BL) is a disease caused by bovine leukemia virus (BLV), a Deltaretrovirus of the Retroviridae family, characterized by lymphocyte proliferation and lymphosarcoma, mainly distributed in dairy cattle. Based on national and international literature was found that all continents are affected by BLV, including Brazil. The state of Maranhão is characterized by intense trading of cattle from various regions of the country, where the presence of the EBL was found. This work aimed to study the prevalence and risk factors associated with EBL in the dairy herd of the state of Maranhao and the evaluation of a microimmunediffusion technique (micro-AGID) in agar gel for the diagnosis of EBL. In order to know the seroprevalence and risk factors associated with EBL, a total of 920 blood samples were collected from 92 dairy HZ breed cattle distributed in 23 municipalities of the five that make up the regional dairy farming country. For the diagnosis of EBL, the AGID was used to test (kit produced by the Technological Institute of Parana - TECPAR). The estimated prevalence was of 53.80% of seropositive animals, distributed in98.91% (91/92) of herds, mainly affecting animals over the age of 48 months (P <0.05). The Bacabal, Sao Luis and Pedreiras regionals presented higher seropositive frequencies (63.50%, 61.87, and 60.62%%) respectively; Imperatriz, intermediate (41.18%), and Açailândia, the lowest (30.83%) (P<0.05). All municipalities had seropositive animals, with frequencies ranging from 22.50% (San Francisco Brejao) to 75.00% (Bernardo do Mearim). When analyzing the variables studied as potential risk factors for EBL a statistically significant association (P <0.05) was found between seropositive for EBL and repeated use of the same needle for blood sampling or vaccination (Odds Ratio - OR=2.76; IC – 1,73 - 4,93), repeated use of the same obstetric glove (OR=1.74; IC – 1.2 a 2.49), animal housing (OR=1.97; IC – 1.28 – 3.02 ), and lack of veterinary care (OR=1.42; IC – 1.06 – 1.88), was found. Knowledge of BEL by farmers and the purchase of animals from other farms for breeding did not interfere in seroreactivity to BEL (OR=1.09; IC – 0.82 – 1.44 and OR=0.88; IC – 0.57 – 1.36, respectively) (P> 0.05). In order to improve the development of a diagnostic test for EBL,micro-gel immuno-Agarose (micro-AGID) was used with simple protocol for obtaining the antigen compared to a macro-AGID. A total of 450 serum samples from 92 properties in 23 counties that make up the dairy state of Maranhao were used. The antigen used in micro-AGID was obtained by dialysis of supernatant of FLK cells infected by BLV against the polyethylenogricol. In micro-AGID 10 μl of antigen and positive serum control was used and 30 μl of test serum, in the macro-AGID 25 μl of all reagents wereused. This produced by the Technological Institute of Parana (TECPAR). Of the sera compared, 259 (57.56%) and 245 (54.44%) showed positive results in micro-AGID and macro-AGID, respectively. There was a very good agreement between both techniques (K = 0.91), with sensitivity and specificity of the macro-AGID for micro-AGID of 93.43% and 98.43% with an accuracy of 95.96%. Micro-AGID showed clearer lines than those observed in the macro-AGID and reading can be made 24 hours before the macro-AGID. It is concluded that micro-AGID can be used successfully in the serological diagnosis of EBL, with the advantage of greater speed in issuing the results and obtaining the antigen with a simple technique. / A Leucose Enzoótica Bovina (LEB) é uma doença causada por um Deltaretrovirus da família Retroviridae, caracterizada por proliferação linfocitária e/ou formação de linfosarcomas, principalmente distribuída em bovinos leiteiros. O vírus da LEB está presente em todos os continentes. No Brasil foram encontrados 27,6% do rebanho leiteiro, infectado. O Estado do Maranhão se caracteriza por intensa comercialização de bovinos de várias regiões do país, onde foi constatada a presença da LEB. Neste trabalho se teve como objetivos, estudar a soroprevalência e fatores de riscos associado à LEB na Bacia Leiteira do Estado do Maranhão e aperfeiçoar técnica de imunodifusão em gel de agar (IDGA) para diagnóstico da LEB. Assim foram coletadas 920 amostras sanguíneas de 92 rebanhos leiteiros da raça Girolanda distribuídos em 23 municípios das cinco Regionais que compõem a bacia leiteira do Estado. Para o diagnóstico da LEB foi utilizada a prova de Imunodifusão em Gel deÁgar (IDGA). A prevalência estimada foi de 53,80% de animais sororeagentes, distribuídos em98,91% (91/92) dos rebanhos estudados, afetando principalmente animais de idade superior aos 48 meses (P<0,05). As Regionais Bacabal, São Luís e Pedreira apresentaram as freqüências de sororeatividade mais elevadas (63,50%, 61,87,% e 60,62%, respectivamente); Imperatriz, intermediária (41,18%); e Açailândia, a mais baixa (30,83%) (P<0,05). Todos os municípios apresentaram animais sororeagentes, com freqüências variando de 22,50% (São Francisco do Brejão) a 75,00% (Bernardo do Mearim). Ao se analisar as variáveis estudadas como potenciais fatores de risco para LEB foi verificada associação estatisticamente significativa (P<0,05) entre sororeagentes para LEB e uso repetido da mesma agulha para colheita de sangue ou vacinação (Odds Ratio – OR = 2,76; IC – 1,73 - 4,93), uso repetido da mesma luva obstétrica (OR=1,74; IC - 1,2 a 2,49), estabulação dos animais (OR=1,97; IC – 1,28 – 3,02) e ausência de assistência Veterinária (OR=1,42; IC – 1,06 – 1,88). O conhecimento da LEB pelos criadores e a aquisição de animais de outras criações parareprodução não interferiu na sororeatividade para LEB (OR=1,09; IC – 0,82 – 1,44 e OR=0,88; IC – 0,57 – 1,36, respectivamente) (P>0,05). Para aperfeiçoamento de uma prova de diagnóstico para LEB, utilizou-se a micro-imunodifusão em gel de agarose (micro-IDGA) usou-se protocolo simples para obtenção do antígeno comparativamente a uma macro-IDGA. Foram utilizadas 450 amostras de soro bovino provenientes de 92 propriedades dos 23 municípios que compõem a bacia leiteira do estado do Maranhão. O antígeno usado na micro-IDGA foi obtido por diálise frente ao polietilenogricol de sobrenadante de células FLK infectadas pelo VLEB. Na micro-IDGA utilizou-se 10 μl de antígeno e soro controle positivoe 30 μl do soro teste; na macro-IDGA 25 μl de todos os reagentes, produzidos pelo Instituto Tecnológico do Paraná (TECPAR). Dos soros comparados, 259 (57,56%) e 245 (54,44%) apresentaram resultados positivos na micro-IDGA e macro-IDGA, respectivamente. Houve ótima concordância entre as duas técnicas (K=0,91), com sensibilidade e especificidade da macro-IDGA em relação a micro-IDGA de 93,43% e 98,43%. A micro-IDGA apresentou linhas mais claras do que as observadas na macro-IDGA e a leitura pode ser feita 24 horas antes da macro-IDGA. Conclui-se que a micro-IDGA pode substituir a macro-IDGA no diagnóstico sorológico da LEB, com a vantagem de maior rapidez na emissão dos resultados e da obtenção do antígeno com técnica simples.