Nigeria recently became the leading economy in Sub-Saharan Africa with a total GDP of 522.64 billion of US dollars (Tradingeconomics.com). As GDP increases, population rises and food demand intensifies. Within this context it is of critical importance to achieve food security. However, Nigeria heavily relies in exportations in order to meet the growing food demand, especially of meat products, a situation which is not desirable. The livestock industry, although one of the largest in Sub-Saharan Africa, still constrained by several endemic livestock diseases which result in annual economic loses for value of 140 million of US dollars (Fadiga et al., 2013). Within this group, bovine and porcine trypasosomiasis alone has been estimated to be responsible for 50 million of US dollars in economic loses in Nigeria (Fadiga et al., 2013). However, the real epidemiological situation, and hence the possibility of developing a rational control programme, remains largely unknown across the country due to the absence of large epidemiological studies. Majority of the trypanosomiasis research studies in Nigeria employ the Haematocrit technique or the Buffy coat technique and Giemsa stain as a diagnostic method. These techniques possess a high specificity but a much lower sensitivity than the molecular method employed in this research study. In fact, better epidemiological studies employing molecular techniques have been conducted in recent times such (Majekodumni et al., 2013a; Takeet et al., 2013) and results displayed much higher trypanosomiasis prevalence than previously detected by microscopy. In many sub-Saharan countries the majority of national livestock herds are owned by mobile communities; however, the trypanosome status of cattle owned by mobile pastoralist communities have been less thoroughly studied when compared to those of sedentary livestock keepers. In this doctoral work, the epidemiology of trypanosomiasis was studied, in transhumant herds located in two different Nigerian enclaves: the Kachia grazing reserve and the Jos Plateau, both located in North-central Nigeria. Within Kachia, the ecology appears to determine the presence of infection with a spatially differentiated distribution of the detected trypanosome species being observed across the reserve that appears not to be related to the migration of livestock. While upon the Jos Plateau, the current reduction in trypanosome prevalence suggests an abrupt change in the trypanosome infection rates in this part of the country. The hypothesis established in this doctoral work is that these epidemiologically different scenarios are the result of land pressures that have ultimately resulted in the habitat destruction of the vector. Longitudinal data was also collected in order to assess the effectivity of different formulations of synthetic pyrethroids for the combined control of trypanosomiasis and tick-borne diseases. Insecticide treated cattle represents at the moment the best long-term and cost-effective method for the control of the vector responsible for the transmission of trypanosomiasis, the tsetse fly. Since no data exist about the efficacy of the insecticide or the compliance of the pastoralist population with its application under migratory conditions, its performance was assessed in this doctoral work. In addition, animal health outcomes were monitored to stablish the possible relationship between clinical symptoms and disease outcome and socio-economic data relevant for the dynamics of disease such as migration trends, husbandry practices, awareness and administered treatment has been also analysed. The compiled information of this data will establish the risk associated with contracting the disease and provide further indications for the control of African bovine trypanosomiasis in the specific context of transhumant pastoral systems of sub-humid sub-Saharan African.

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Orientador: Simon Biagio Chiacchio / Banca: Roberto Calderon Gonçalves / Banca: Rogério Martins Amorim / Banca: José Giometti / Banca: Laurenil Gaste / Resumo: O objetivo do experimento foi avaliar o efeito de diferentes suplementações com selênio (Se) sobre a resposta imune humoral anti-rábica, na concentração sérica de selênio e cortisol em bovinos. Utilizou-se sessenta bovinos machos não castrados da raça Nelore, com 10 a 12 meses de idade, alimentados com pastagem de Brachiaria decumbens. Os animais foram divididos em 4 grupos (N=15) sendo que o primeiro deles não receberam suplementação (Gc) e os outros três suplementados diariamente e individualmente com concentrações de Se de 3,6 mg (G3,6), 5,4 mg (G5,4) ou 6,4 mg (G6,4). Os animais foram imunizados no dia zero com uma dose de vacina anti-rábica comercial, líquida e inativada. Nos dias zero, 15, 30, 60, 90 e 120 durante os quais submetidos ao estresse de manejo no curral e colheita de sangue. As amostras de sangue foram colhidas depois da vacinação e depois de serem submetidos ao estresse para determinação da concentração sérica de selênio, títulos de anticorpos anti-rábicos e cortisol sérico. A concentração de selênio também foi determinada nas amostras das forrageiras colhidas dos piquetes utilizados pelos bovinos. A concentração de Se na B. decumbens foi de 0,04 mg de Se/Kg de matéria seca. A concentração sérica de Se obtida no dia zero no grupo Gc era mais elevada que nos grupos G5,4 e G6,4 (P= 0,005). A concentração sérica de Se diminuiu ao longo do experimento no Gc (P<0,004), aumentou no G3,6 (P<0,000) e no grupo G5,4 (P<0,000), entretanto, no grupo G6,4 aumentou no dia 60 (P<0,002). Os títulos de anticorpos anti-rábicos não diferiram entre os grupos tratados ou não com selênio. Entretanto, 120 dias após a vacinação os títulos de anticorpos no grupo G3,6 permaneceram acima do mínimo considerado protetor (=0,5 UI/mL) (P<0,000), enquanto que nos outros grupos permaneceram baixos (P<0,05). A concentração sérica de cortisol dos bovinos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study evaluated the effect of different concentrations of selenium (Se) supplementation on cattle antirabies humoral immune response, serum Se concentrations and cortisol levels. Sixty uncastrated male Nelore calves from 10 to 12 months grazing on Brachiaria decumbens forage were studied. The animals were assigned to one of four groups (N=15 each), which received nonsupplemented diets (Gc) or supplemented with daily and individual Se concentrations of 3.6 mg (G3.6), 5.4 mg (G5.4) or 6.4 mg (G6.4). The animals were immunized on day 0 with one dose of commercial liquid inactivated rabies vaccination. On days 15, 30, 60, 90 and 120, the cattle underwent the same stressing procedures used for vaccination in the corral. Cattle blood samples were collected after vaccination and stressing procedures to determine serum Se levels, rabies antibody titers and serum cortisol. Se levels were also determined in forage samples collected from the paddocks in which the cattle were held. Se concentration in B. decumbens was 0.04 mg of Se/Kg dry matter. Baseline Se levels obtained on day 0 were higher in Gc than in G5.4 and G6.4 (P= 0.005). Serum Se levels decreased in Gc throughout the experiment (P<0.004), increased in G3.6 (P<0.000) and G5.4 (P<0.000) and were kept high from day 60 on in group G6.4 (P<0.002). Rabies antibody titers did not differ between control and supplemented groups. However, 120 post-vaccination rabies antibody titers were kept above the protecti ve levels (=0.5 UI/mL) only in group G3.6 (P<0.00002), whereas they dropped in the other groups (P<0.05). Serum cortisol levels did not differ among the experimental groups (P=0.79), reached peak levels on day 90 and returned close to baseline levels on day 120. Se and cortisol levels were not markedly correlated. Serum cortisol and rabies antibody titers were correlated only in group G6.4, on day 60 (R=0.513; P=0.05)... (Complete abstract click electronic access below) / Doutor

Studying the dynamics of pathogen transmission within wildlife populations presents an array of challenges. Where populations are socially structured, this can influence parasite transmission, impacting on the effectiveness of disease management strategies. In this thesis, I focus on a well-studied social mammal, the European badger (Meles meles) which is a key wildlife reservoir of a disease of economic importance; bovine TB (caused by infection with Mycobacterium bovis). The social structuring, characteristic of high density badger populations, is of well-established importance in the transmission of bovine TB and has resulted in unexpected management outcomes. However, little is known about the role of kin structure or host genotype on transmission dynamics. In this thesis, I combine traditional spatial epidemiology and ecological analysis of a well-studied badger population with more novel genetic and genomic approaches. Firstly, I investigate the role of kin structure within badger social groups in determining early life infection risk (Chapter 3). Using host genotype data, I demonstrate that cubs who are related to infected adults experience enhanced infection risks. I then explore the role of badger genotype on outcomes of M. bovis exposure and demonstrate that inbred badgers are more likely to show evidence of progressive infection (Chapter 4). Where the social structure of badgers is stable and unmanaged, this is predicted to result in a stable spatial distribution of M. bovis infection. Motivated by an observation of change in the spatial distribution of M. bovis infection in the study population, in the absence of management, I characterise the attrition of a spatially stable infection distribution (Chapter 5). To explore the drivers of this, I detect changes in the genetic population structure (Chapter 6) and present evidence that the population has experienced a period of demographic flux. Finally, I use a novel dataset generated by whole genome sequencing of M. bovis isolates and present evidence of spatial spread of M. bovis infection across the study population (Chapter 7). To conclude, I discuss how my findings demonstrate how genetic and genomic approaches can complement traditional wildlife epidemiology approaches, how they contribute to our understanding of heterogeneity in transmission dynamics and discuss their implications for wildlife disease management.

Poor reproductive performance is a major challenge for the bovine dairy industry, with implications for profitability and animal welfare. Early pregnancy diagnosis and accurate oestrus detection can improve reproductive performance through efficient herd management. However currently available methods do not allow this. Circulating microRNAs (miRNAs) have been proposed as non-invasive biomarkers of reproductive status in humans. The hypothesis for this work was that differentially expressed miRNAs in plasma will be detectable during early pregnancy / oestrus, which may provide novel potential biomarkers. Using sequencing and PCR-based platforms I successfully identified and validated increases in miR-26a and the miR-26a / miR-205 ratio as early as Day 8 of pregnancy (max. 7.5-fold) in the plasma of pregnant compared to non-pregnant heifers. These miRNAs are known regulators of immunity, angiogenesis and metabolism, however their specific roles in early pregnancy remain to be investigated. I also identified small but significant increases in the levels of miR-125b, let-7f, miR-145 and miR-99a-5p at oestrus, when compared with the luteal phase of the cycle. These miRNAs have been previously shown to regulate the follicular to luteal transition in the bovine ovary. Finally, I provide a validated high-throughput resource which can help identify potential global biomarkers of tissue function, as shown for the liver-enriched miR-802 in the present results. The findings of this work may be useful in the development of diagnostic methods for early pregnancy and oestrus, and pave the way for the functional characterisation of these miRNAs in bovine reproduction.

Orientador: Cáris Maroni Nunes / Banca: Valéria Marçal Félix de Lima / Banca: Hélio José Montassier / Resumo: A cisticercose bovina, causada pela forma metacestóide de Taenia saginata, é responsável por prejuízos econômicos devido à desvalorização de carcaças infectadas. A inspeção sanitária tem sido a principal forma de diagnóstico desta zoonose, porém apresenta baixa sensibilidade em identificar animais com infecção menos intensa. Ensaios imunoenzimáticos para detecção de anticorpos e de antígenos representam importante alternativa de diagnóstico ante mortem para a identificação de animais infectados, pois apresentam maior sensibilidade. Neste trabalho foram avaliadas a detecção de anticorpos anti-Taenia saginata e de antígenos do mesmo parasita em amostras de soro de bovinos naturalmente infectados, por meio dos testes ELISA indireto e ELISA sanduíche, respectivamente. Além das condições de realização dos testes, como o preparo das soluções e o tipo de extrato antigênico utilizados, a intensidade da infecção é um dos fatores que mais interferem nos resultados diagnósticos. Dessa forma, as características diagnósticas dos testes ELISA indireto, realizado com três diferentes extratos antigênicos, e ELISA sanduíche, realizado com o anticorpo monoclonal HP10, foram também relacionadas com a intensidade de infecção dos bovinos por Taenia saginata / Abstract: Bovine cysticercosis caused by Taenia saginata metacestodes is responsible for economic losses due to depreciation of the infected carcasses. Sanitary inspection has been the main diagnostic tool but has low sensitivity in animals light infected. Immunoassays for antibodies and antigens detection are important ante mortem diagnosis, because present higher sensitivity. Antibodies anti-T. saginata and T. saginata antigens detection were evaluated in naturally infected bovine serum samples by indirect ELISA and sandwich ELISA. Besides reagents and test conditions the intensity of animal's infection represents one of the most important interfering factors on the diagnostic results. Thus, diagnostic characteristics of indirect ELISA performed with three different antigenic extracts and sandwich ELISA, with monoclonal antibody HP10, were related to the intensity of infection in samples from Taenia saginata infected bovines / Mestre

Proteomic technology has been increasingly incorporated into agricultural research, as characterization of proteomes can provide valuable information for potential biomarkers of health and physiological status of an animal. As dairy cattle are a dominant production animal in the USA, their biofluids such as milk, blood, urine, and rumen fluid have been examined by proteomic analysis. The research outlined herein was performed to further characterize the dynamics of specific proteomes and relate them to dairy cattle physiology. The first experiment evaluated the diurnal dynamicity of the rumen metaproteome in Holstein dairy cattle. Rumen fluid was collected from three mid to late lactation multiparous dairy cattle (207 ± 53.5 days in milk) at three time points relative to their first morning offering of a total mixed ration (TMR) (0 h, 4 h, and 6 h after feeding). Samples were processed and labeled using Tandem Mass tagging before being further fractionated with a high pH reversed-phase peptide fractionation kit. Samples were analyzed by LC-MS/MS and statistically analyzed for variations across hour of sampling using the MIXED procedure of SAS with orthogonal contrasts. A total of 242 proteins were characterized across 12 microbial species, with 35 proteins identified from a variety of 9 species affected by time of collection. Translation-related proteins were correlated positively with increasing hour of sampling while more specific metabolic proteins were negatively correlated with increasing hour of sampling. Results suggest that as nutrients become more readily available, microbes shift from conversion-focused biosynthetic routes to more encompassing DNA-driven pathways. The second experiment aimed to characterize the milk fat globule membrane (MFGM) proteomes of colostrum and transition milk for comparison from multi- (n = 10) and primiparous (n = 10) Holstein dairy cattle. Samples were collected at four timepoints post-partum (milkings 1, 2, 4, and 14). After isolation of the protein lysates from the MFGM, proteins were labeled using Tandem Mass tagging and analyzed using LC-MS/MS techniques. Protein identification was completed using MASCOT and Sequest in Proteome Discoverer 2.2. Protein abundance values were scaled and analyzed using the MIXED procedure in SAS to determine the effect of parity, milking number, and parity x milking number, and the adaptive false-discovery rate (FDR)-adjusted P values were determined using the MULTTEST procedure of SAS. There were 104 proteins identified within the MFGM. Statistical analysis revealed that 44.2% of proteins were affected by parity, 70.2% by milking number, and 32.7% by the variable of parity x milking number. There was a two-fold difference in calcium sensing S100 proteins in cows differing in parity possibly due to the multiparous mammary gland being more adapted to the physiological demand of lactation or the lesser requirement of calcium in primiparous cows because of a lower production rate.

Orientador: Vera Fernanda Martins Hossepian de Lima / Resumo: O processo transcricional em embriões extremamente complexo, nosso trabalho estimou o impacto de perturbações nos processos de transcricionais, durante as fases de ativação do genoma embrionário sobre o desenvolvimento embrionário in vitro de embriões; analisamos dados de sequenciamento de rna (RNA-seq) depositados nos bancos públicos (GEO) desde o estágio de oóocito até o dia 19 do desenvolvimento embrionário; Isolamos e caracterizamos a massa celular interna (ICM) e a trofectoderma (TE) do sexo masculino e feminino, oriundos de um mesmo blastocisto produzido in vitro com espermatozoides sexados (X e Y) e com sêmen convencional e caracterizamos e exploramos o transcriptoma desses isolados celulares. Concluímos então que a EGA menor é essencial para o desenvolvimento embrionário bovino, blastocistos possuem a maior atividade transcricional de um total de 6457 genes diferentemente expressos entre os contrastes avaliado encontramos; 2065 genes diferencialmente expressos entre a ICM e a TE, enquanto a ICM está voltada para a manutenção da pluripotência, a TE está voltada ao metabolismo energético. Os nossos dados sugerem que os embriões fêmeas são mais sensíveis ao cultivo in vitro. / Abstract: Transcription process in embryos is a complex process, our work estimated the impact of perturbations in the transcriptional processes during genome activation of vitro produced bovine embryos on their development; we analyzed public data (GEO) from rna sequencing data (RNA-seq) of oocyte up to the 19th day of embryonic development; We’d performed isolation and characterization of male and female inner cell mass (ICM) and trofectoderma (TE) from the same blastocyst produced in vitro with sorted semen (X and Y) and with conventional semen. We did the characterization and exploratory analysis of the transcriptome of these cells. We conclude that minor EGA is essential for bovine embryonic development. Blastocysts possess the highest transcriptional activity of 6457 differentially expressed genes among analyzed contrasts. We found 2065 genes differentially expressed between ICM and TE, while ICM is maintaining pluripotency, TE is focused on energy metabolism. Our data suggest that female embryos are more sensitive to in vitro culture. / Doutor

Two microarray designs were developed and produced to screen for multiple bovine pathogens commonly found in the cattle industry today. The first microarray was designed, built, and processed in-house using conventional material and equipment and targeted Pasteurella multocida, Manheimia haemolytica, Histophilus somni, and Arcanobacterium pyogenes. For each pathogen, 12 perfect-match oligonucleotide probes, which were also designed in-house, targeted different sections of the respective 16S ribosomal genes, and were coupled with 12 corresponding mismatched probes for background. These arrays were able to produce distinct hybridization patterns for each pathogen that were easily visible without the need for computer analysis. However, the need for PCR amplification of the 16S gene prior to hybridization motivated us to explore more efficient array options. The second designed microarray, a custom Affymetrix GeneChip, targeted Escherichia coli, Salmonella typhimurium, and Salmonella dublin in addition to the previously mentioned pathogens and was more successful in overall performance than the "in-house" arrays. In addition to the 16S gene, oligonucleotide probes targeted other genes (from 2 to >4500, depending on whether the genome was sequenced) that were unique to each pathogen. This array also differed from the "in-house" arrays in that mismatched probes were not designed. The different probe sets performed at different detection limits as P. multocida, A. pyogenes, S. typhimurium, and S. dublin were detected with as little as 250ng of hybridized genomic DNA (gDNA), while M. haemolytica, H. somni, and E. coli required as much as 1μg gDNA. These pathogens were also spiked into bovine tissue to simulate multiorgan infections in which they were individually detected with the microarray design.

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