This thesis explores the genetic characterization of the mechanisms underlying complex traits in chicken through the use and development of bioinformatics tools. The characterization of quantitative trait loci controlling complex traits has proven to be very challenging. This thesis comprises the study of experimental designs, annotation procedures and functional analyses. These represent some of the main ‘bottlenecks’ involved in the integration of QTLs with the biological interpretation of high-throughput technologies. The thesis begins with an investigation of the bioinformatics tools and procedures available for genome research, briefly reviewing microarray technology and commonly applied experimental designs. A targeted experimental design based on the concept of genetical genomics is then presented and applied in order to study a known functional QTL responsible for chicken body weight. This approach contrasts the gene expression levels of two alternative QTL genotypes, hence narrowing the QTL-phenotype gap, and, giving a direct quantification of the link between the genotypes and the genetic responses. Potential candidate genes responsible for the chicken body weight QTL are identified by using the location of the genes, their expression and biological significance. In order to deal with the multiple sources of information and exploit the data effectively, a systematic approach and a relational database were developed to improve the annotation of the probes of the ARK-Genomics G. gallus 13K v4.0 cDNA array utilized on the experiment. To follow up the investigation of the targeted genetical genomics study, a detailed functional analysis is performed on the dataset. The aim is to identify the downstream effects through the identification of functional variation found in pathways, and secondly to achieve a further characterization of potential candidate genes by using comparative genomics and sequence analyses. Finally the investigation of the body weight QTL syntenic regions and their reported QTLs are presented.

As bactérias do gênero Salmonella, especialmente S. Enteritidis e S. Typhimurium, são responsáveis por imensos prejuízos econômicos ao setor avícola, uma vez que produtos como carne de frango e ovos são apontados como a principal fonte desse patógeno em surtos de salmonelose humana. Perdas econômicas na avicultura também estão relacionadas à infecção em aves jovens devido ao desenvolvimento de doença clínica e ao aumento da mortalidade. A patogenicidade de Salmonella é considerada complexa e multifatorial necessitando de estudos que possam esclarecer a interação entre patógeno e hospedeiro. O presente trabalho teve por objetivo avaliar a virulência de 130 isolados de S. Enteritidis e 70 de S. Typhimurium, inoculadas em pintos de um dia de idade através do estabelecimento de um índice de patogenicidade. Para cada cepa, foram utilizados 10 pintos comerciais da linhagem Cobb provenientes de matrizes de corte com idade superior a 58 semanas. As aves foram inoculadas com 0,2 mL de uma solução contendo aproximadamente 2x108 UFC de S. Enteritidis ou S. Typhimurium, através da via intraperitoneal. A mortalidade e a presença de lesões associadas à septicemia foram observadas diariamente durante sete dias. As aves encontradas mortas durante esse período foram necropsiadas e observadas quanto à presença de aerossaculite (A), perihepatite (Ph), pericardite (Pc), peritonite (Pt), onfalite (O) e celulite (C). Para cada lesão foi atribuído o valor de “0,833” se presente e o valor “0” quando ausente. Animais mortos nas primeiras 24 horas pósdesafio receberam a pontuação 10, o que representa o índice máximo. Do segundo ao sétimo dia, houve uma diminuição proporcional da pontuação no tempo de morte (TM) a cada dia em que o animal sobrevivia. O cálculo do índice de patogenicidade de cada pintinho inoculado (IP) obedeceu a seguinte fórmula: IP = (TMx5) + A + Ph + Pc + Pt + O + C. Para obtenção do IP de cada amostra foi realizada a média do IP obtido com as 10 aves inoculadas. A partir dos resultados do trabalho, foi possível atribuir um valor de patogenicidade a cada uma das cepas permitindo classificá-las em grupos de baixa, intermediária e de alta patogenicidade. Os sorotipos analisados neste estudo não diferiram na capacidade de induzir a formação de lesões septicêmicas e elevada mortalidade. Desta forma conclui-se que, tanto S. Enteritidis quanto S. Typhimurium, são potencialmente capazes de causar doença clínica e mortalidade de forma semelhante aos sorovares adaptados às aves. / Bacterials of the Salmonella genus, particularly Salmonella (S.) Enteritidis and S. Typhimurium, are responsible for serious economic loss on the poultry industry, once eggs and meat chicken are the principle sources of the pathogens on outbreak of salmonellosis in human. Economic loss on the poultry industry, also is related infection in young chicks due to development clinical disease and increased mortality. The pathogenicity of Salmonella is complex and multifactorial which requires studies that elucidate the interaction between pathogen and host. The present study, was conducted to evaluate the virulence of 130 isolate of S. Enteritidis and 70 of S. Typhimurium, inoculated in 01 day old chicks, through establishment of one index of pathogenicity. For each strain was used 10 commercial lineage Cobb chicks older than 58 weeks old. Chickens were inoculated with 0,2 mL containing approximately 2 x 108 CFU of S. Enteritidis or S. Typhimurium by route intraperitoneal. Mortality and lesions associated septicemia were observed daily for seven days. Dead chicks during this period were necropsied and observed as the presence of airsaculitis (A), perihepatitis (Ph), pericarditis (Pc), peritonitis (Pt), omphalitis (O) and cellulitis. For each lesion was attributed the value “0,833” when present and “0”when absent. Dead animals on first 24 hours post-challenge received the punctuation 10. The second to seventh day, there was a proporcional decrease of punctuation on time of death (TM) each day that the chickens survived. The calculation of the index of pathogenicity obeyed the following formula: IP = (TMx5) + A + Ph + Pc + Pt + O + C. For obtainment IP of each strain was realized the average IP obtained with 10 inculated chickens. Based on results this study, was possible to assign a value of pathogenicity for each isolated allowing to classify them on groups of low, intermediate and high pathogenicity. The sorovars analyzed did not differ on ability to induce septicemic lesions and high mortality. Therefore it is concluded that, such S. Enteritidis as S. Typhimurium, are potencially capable to cause clinical disease and mortality similar to serotypes poultry adapted.

Thesis (M.Sc. (Agriculture)) --University of Limpopo, 2010 / Ten experiments were conducted to determine the effect of dietary energy to protein ratio level on growth and productivity of indigenous Venda chickens raised in closed confinement from day-old up to thirteen weeks of age. The ten experiments were based on five different energy levels of 12.2, 13, 13.2, 13.4 and 14 MJ ME/kg DM. Each dietary energy level had five different levels of protein concentrations of 220, 190, 180, 170 and 160 g/kg DM, thus ending up with twenty five different dietary energy to protein ratio levels. Experiments 1 to 5 examined the effect of dietary energy to protein ratio on productivity of unsexed Venda chickens aged one to six weeks. Experiments 1 to 5 each commenced with 160 unsexed day-old indigenous Venda chicks with an initial weight of 25 ± 2 g per bird and each lasted for a period of six weeks. In each experiment, the chicks were randomly assigned to five treatments with four replications, each having eight birds. Thus, 20 floor pens (1.5 m2 /pen) were used in total for each experiment. All the five experiments were carried out around the same time. A complete randomized design was used for each experiment. A quadratic regression model was used to determine the ratios for optimum feed intake, growth rate, feed conversion ratio, live weight, metabolisable energy and nitrogen retention in each experiment while a linear model was used to determine the relationship between optimal responses in the above variables and dietary energy to protein ratio levels. Dietary energy to protein ratio level for optimal response for any variable was relative and depended on the energy to protein ratio values of the diet. Energy to protein ratios of 63, 67, 70, 74 and 78 MJ ME/kg protein promoted optimal live weights of 415, 408, 370, 365 and 344 g at six weeks of age for diet energy levels of 12.2, 13, 13.2, 13.4 and 14 MJ ME/kg DM, respectively. However, optimal response trends for different variables were also influenced by the dietary energy to crude protein ratio. Optimal feed intake increased linearly with increasing dietary energy to protein ratio levels. Similarly, optimal metabolisable energy level increased linearly with increasing dietary energy to protein ratio levels without any increase in live weight. On the other hand, increasing dietary energy to protein ratio level decreased growth rate with a linear deteriorating feed conversion ratio,thus, negatively affecting live weight of the chickens at six weeks of age. However, increasing dietary energy to protein ratio levels improved efficiency of protein utilization with a linear deteriorating metabolisable energy utilization. In experiments 1 to 5, correlation analysis indicated that optimal feed intake, feed conversion ratio and metabolisable energy level were positively and strongly correlated with dietary energy to protein ratio levels while optimal live weight, growth rate and nitrogen retention were negatively and strongly correlated with dietary energy to protein ratio levels. The study indicated that a diet containing a crude protein content level of 178 g/kg DM and an energy level of 14 MJ ME/kg DM allowed for optimal utilization of absorbed protein and energy for growth in unsexed indigenous Venda chickens aged between one and six weeks. Experiments 6 to 10 examined the effect of dietary energy to protein ratio level on productivity of male Indigenous Venda chickens raised in closed confinement from seven up to 13 weeks of age. Each experiment commenced with 100 seven-week old male Venda chickens with an initial weight of 320 ± 2 g per bird. In each experiment, the chickens were randomly assigned to five treatments with four replications, each having five birds. Thus, 20 floor pens (1.5 m2/pen) were used in total for each experiment. All the five experiments were carried out around the same time and for a period of seven weeks. A complete randomized design wasused for each experiment. A quadratic regression model was used to determine energy to protein ratios for optimum feed intake, growth rate, feed conversion ratio,live weight, metabolisable energy, nitrogen retention, carcass yield, breast meatyield and fat pad deposition in each experiment while a linear model was used to determine the relationship between optimal responses of the above variables and dietary energy to protein ratio levels. Energy to protein ratios of 60, 71, 66, 72 and 71 MJ ME/kg protein promoted optimal live weights of 1167, 950, 983, 1235 and 1172 g at thirteen weeks of age for diet energy levels of 12.2, 13, 13.2, 13.4 and 14 MJ ME/kg DM, respectively. Optimal response trends in feed intake, growth rate, feed conversion ratio, nitrogen retention, and metabolisable energy intakes in male Venda chickens tended to increase with increase in dietary energy to protein ratio level. In contrast, optimal carcass and breast meat yield tended to decrease with increasing dietary energy to protein ratio level. However, optimal live weight and fat pad remained unchanged with increase in dietary energy to protein ratio value. Additionally, increasing dietary energy to protein ratio level improved efficiency of protein utilization with deteriorating metabolisable energy utilization.On the other hand, experiments 6 to 10 showed that optimal feed intake, growth rate, feed conversion ratio, live weight, metabolisable energy level, nitrogen retention and fat pad were positively and poorly correlated with dietary energy to protein ratio levels while optimal carcass weight and breast meat yield were negatively and poorly correlated with dietary energy to protein ratio levels This study indicated that a diet containing 193 g CP/kg DM and an energy level of 14 MJ ME/kg DM allowed for optimal utilization of absorbed protein and energy for growth in male indigenous Venda chickens aged between seven and thirteen weeks.It is, therefore, concluded that optimal response trends for different variables in growing unsexed Venda chickens offered diets differing in optimal dietary energy to protein ratio levels are influenced by the diet crude protein content rather than the feed energy level per se. Diets containing decreased dietary energy to protein ratio levels may promote improved growth rate and efficient feed conversion ratio while naturally restricting feed intake. Diets containing increased dietary energy to protein ratio levels favoured efficient utilization of consumed dietary protein. However, with such diets, protein became limiting and birds increased their feed intake attempting thereby to obtain more of the limiting protein in order to meet their protein requirement regardless of the energy value of the diet.It is, also, concluded that growing male Venda chickens increased their feed intake with increase in dietary energy to protein ratio. This is contrary to what has been observed in broiler chickens which decrease their intake with increase in diet energy value. It is suggested that this might reflect the differences between indigenous and broiler chickens in terms of their genetic and physiological abilities to regulate their feed intakes according to dietary energy levels. / National Research Foundation (NRF)

Corrigenda inserted behind title page. Copies of author's previously published articles inserted. Bibliography: leaves 193-210. This study investigated whether estrogen could modulate the proteins associated with calcium transport (eg calcium binding protein, calbindin D28k) in the intestinal tissue during sexual maturation of laying hens. The first study investigated the temporal and spatial expression of estrogen receptors (ER) and calcium binding protein in jejunal tissue. Findings indicated the potential of estrogen to induce calbindin D28K in the intestinal tissue, from which it can be concluded that estrogen may increase calcium absorption and consequently calcium retention. It was shown that the amount of estrogen normally present in the reproductive system is sufficient for the normal capacity of egg and eggshell production. Egg production and shell quality were significantly reduced by phytoestrogen. It is recommended that the concentration of phytoestrogen in poultry diets be investigated in more detail as it may act as an anti-nutritive factor and affect metabolism and productivity.

Monoclonal antibodies (MCA) produced against infectious laryngotracheitis virus (ILTV) of chickens reacted in western blotting experiments with several different ILTV protein bands in the absence of tunicamycin which inhibits carbohydrate synthesis. Most of the MCA lost their reactivity in western blotting experiments when extracts of tunicamycin-treated ILTV CELC were used, suggesting their specificity for carbohydrate-based epitopes. In an indirect immunofluorescence test most of the MCA bound primarily to cytoplasmic antigens except some MCA which bound primarily to nuclear antigens. Additivity ELISA was also performed to study whether MCA are against the same epitope or different epitopes. The polymerase chain reaction (PCR) was developed as a diagnostic technique for detection of ILTV using primers made from a portion of the ILTV thymidine kinase gene. The 647-basepair amplified ILTV PCR product was labeled to create a non-radioactive, biotinylated DNA probe. Hybridization was performed using the probe to detect ILTV. Both PCR and hybridization detected ILTV, and neither hybridization nor PCR gave positive results with any other pathogen. Hybridization was specific for ILTV, However, slight hybridization occurred with CELC DNA when relatively relaxed conditions were used. In another experiment, diagnostic tests to detect ILTV in tracheas of experimentally-infected chickens, including the indirect fluorescent antibody test (IFAT), immunoperoxidase (IP), virus isolation (VI), histopathology, PCR, and hybridization, were performed and compared. Using virus isolation as a reference, the sensitivity and specificity of the tests were calculated. The IP test and IFAT performed better than any other test used in this study. / Graduation date: 1995

Marek?s disease virus serotype-1 (MDV-1) causes T cell lymphomas in chickens. Vaccines prepared from attenuated CVI988/Rispens MDV-1 strain currently offer the best protection. Although attenuated CVI988 is non-oncogenic, it codes for two forms of the MDV-1 oncoprotein Meq (CVI-Meq and CVI-L Meq). In this study, both CVI-Meq proteins, like the Meq protein of Md5 (a very virulent oncogenic strain), transformed Rat-2 and NIH3T3 cells. Both CVI-Meq and CVI-L Meq proteins activated the meq promoter only in the presence of chicken c-Jun (CK-Jun) whereas Md5-Meq activated the same promoter irrespective of CK-Jun co-expression. However, all three Meq proteins bound the meq promoter regardless of whether CK-Jun was co-expressed. We constructed three chimeric Meq proteins, namely, Md5-CVI-Meq, CVI-Md5-Meq, and Md5-CVI-L by exchanging domains between Md5 meq and CVI meq genes. Although these chimeric Meq proteins transactivated the meq promoter, the activation was significantly less than Md5-Meq. The current study indicated amino acid residues at positions 71 and 320 were important for Md5-Meq increase transcription of its own promoter. All three Meq proteins activated the MDV gB, MMP-3 and Bcl-2 promoters and suppressed transcription from the MDV pp38/pp14 bidirectional promoter. CVI-Meq protein in the context of other Md5 genes caused tumors only in 6% of chickens when compared to parental rMd5 (a very virulent strain), which induced lymphomas in 100% of chickens, (Reddy and Lupiani, unpublished data). Taking advantage of these two different phenotypes, we constructed two chimeric Meq proteins, Md5/CVI-Meq and CVI/Md5-Meq, by exchanging DNA binding and transactivation domains between Md5-Meq and CVI-Meq to understand the role of the DNA binding and the transactivation domains of Meq in transformation. rMd5-Md5/CVI-Meq virus caused 100% mortality in chickens and T lymphomas were found at high frequency in the peripheral nerves and various organs such as the heart, spleen, kidney, and gonads. On the other hand, rMd5-CVI/Md5-Meq induced disease in 36% of chickens on average and lesions were primarily in the nerves. Very rarely, lesions were present in the spleen and heart and no tumors were present in the kidney or gonads. Our results suggest that both the DNA binding domain and transactivation domain of Meq could cooperatively determine the nature of lymphomas in chickens.

Thesis (Ph. D.)--Mississippi State University. Department of Biochemistry and Molecular Biology. / Title from title screen. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vii, 78 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 52-62).

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains ix, 109 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 88-104).

Thesis (Ph. D.)--Institute of Paper Chemistry, 1969. / Includes bibliographical references (p. 63-65).