Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 90-98). Also available via the Internet.

Thesis--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 67-77).

Thesis (MSc. (Veterinary Science))--University of Pretoria, 2005. / Includes bibliographical references.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (p. 169-200). Also available via the World Wide Web.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-03-28T13:58:36Z No. of bitstreams: 1 texto completo.pdf: 1220110 bytes, checksum: 803118ec7eccfad2cba1645f25679903 (MD5) / Made available in DSpace on 2017-03-28T13:58:36Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1220110 bytes, checksum: 803118ec7eccfad2cba1645f25679903 (MD5) Previous issue date: 2016-12-19 / A carne de frango consiste em uma matriz ideal para a multiplicação de micro- organismos, e, portanto, é associada a numerosas infecções em humanos. Entre as bactérias que mais geram infecções intestinais, a partir do consumo de produtos avícolas, encontra-se a espécie Campylobacter jejuni. O uso indiscriminado de antibióticos na produção animal, pode contribuir na seleção de bactérias resistentes, que podem ser disseminadas durante a produção ou processamento dos alimentos. Desta forma, são necessárias novas alternativas para o controle desse patógeno, como a utilização de bacteriófagos. O objetivo deste trabalho foi isolar, caracterizar e avaliar o efeito de um coquetel de bacteriófagos de forma individual ou associado ao antibiótico enrofloxacina, no controle de C. jejuni, em frangos de corte. Foram isolados seis bacteriófagos (BC2, BC7, BC10, BC14, BC18 e BC19) a partir de fezes de frango coletadas em duas granjas avícolas na região de Viçosa, no Estado de Minas Gerais. Os bacteriófagos foram avaliados quanto à especificidade frente a diferentes cepas bacterianas. Cinco bacteriófagos foram específicos para C. jejuni, e um (bacteriófago BC14) além de apresentar ação lítico nas cepas de C. jejuni, apresentou atividade lítica frente a Salmonella Typhimuium (ATCC 14028) e Escherichia coli (CDC0111ab). A caracterização morfológica de dois bacteriófagos (BC7 e BC14) por meio de Microscopia Eletrônica de Transmissão (MET), evidenciou que os bacteriófagos apresentam cabeça icosáedrica e cauda de tamanho médio, indicando que provavelmente eles são vírus da ordem Caudovirales, família Myoviridae, e subfamília Eucampyvirinae. O coquetel de bacteriófagos foi afetado maiormente quando exposto a pH 2,5 (redução de 6,90) ciclos logarítmicos, e aos sais biliares nas concentrações de (0,3 e 1,0) %, com redução de (0,37 e 0,51) ciclos logarítmicos, respectivamente. Portanto, os animais devem ser previamente tratados com um antiácido como CaCO 3 , a fim de manter a viabilidade dos bacteriófagos no trato gastrointestinal das aves. A sinergia entre os bacteriófagos e a enrofloxacina, levou a redução de 1,08 ciclos logarítmicos de C. jejuni (B12313), e 1,48 ciclos logarítmicos de C. jejuni (IOC/ATCC 33560) nas fezes dos frangos após cinco dias de tratamento. No conteúdo cecal, a redução de C. jejuni (B12313) com tratamento de coquetel de bacteriófagos foi de 2,58 ciclos logarítmicos, e o tratamento com o antibiótico foi de 1,67 ciclos logarítmicos. Nos frangos infectados com C. jejuni (IOC/ATCC 33560), a redução foi de 2,42 ciclos logarítmicos com o uso de coquetel de bacteriófagos, e de 2,51 ciclos logarítmicos com o antibiótico enrofloxacina. Os bacteriófagos são apresentados como uma ferramenta útil na redução do patógeno C. jejuni, antes e após o abate. Mais investigações devem ser feitas com outros antibióticos, com o objetivo de aumentar o conhecimento de sinergia entre bacteriófagos e antibióticos como terapêutico dentro da granja. Em conteúdo cecal, as análises indicaram atuação mais eficiente do coquetel de bacteriófagos isoladamente. Isso indica que o tratamento com fagos seria eficiente no controle da contaminação da carne durante o abate. / Poultry meat, consist of an ideal matrix for the multiplication of microorganisms and thus is a source of numerous infections in humans. Among the bacteria, which generates more intestinal infection from the consumption of poultry products, is the species Campylobacter jejuni. The indiscriminate use of antibiotics in animal production may contribute to the selection of resistant bacteria, which may be disseminated during food production or processing. Therefore, new alternatives are needed to control this pathogen, such as the use of bacteriophages. The objective of this study was to isolate, characterize and evaluate the effect of a cocktail of bacteriophages individually or associated with the antibiotic enrofloxacin, in the control of C. jejuni, in broilers. Six bacteriophages were isolated (BC2, BC7, BC10, BC14, BC18 and BC19) from chicken feces collected from two poultry farms in the region of Viçosa, in the State of Minas Gerais. The bacteriophages were evaluated for specificity against different bacterial strains. The bacteriophage BC14 besides presenting lytic power over the strains of C. jejuni presented lithic activity against Salmonella Typhimuium (ATCC 14028) and Escherichia coli (CDC0111ab). The morphological characterization of two bacteriophages (BC7 e BC14) by transmission electron microscopy (TEM) showed that bacteriophages had icosahedral head and medium-sized tail indicating that they are probably viruses of the order Caudovirales order and Myoviridae family. The bacteriophage cocktail was mostly affected when exposed to pH 2.5 (reduction of 6.90) log 10 units and bile salt in the concentrations of (0.3 and 1.0) %, with a reduction of (0.37 and 0.51) log 10 units respectively. Therefore, animals should be pretreated with an antacid such as CaCO 3 , in order to maintain the viability of bacteriophages in the gastrointestinal tract of birds. A synergy was observed between bacteriophage and enrofloxacin with a reduction of 1,08 log 10 units of C. jejuni (B12313) and 1.48 log 10 units of C. jejuni (IOC/ATCC 33560) in the feces of the chickens after five days of treatment. In the cecal content, the reduction of C. jejuni (B12313) with treatment of bacteriophage cocktail was 2.58 log 10 units, and treatment with the antibiotic was 1.67 log 10 units. In chickens infected with C. jejuni (IOC / ATCC 33560), the reduction was 2.42 log 10 units with the use of bacteriophage cocktail, and 2.51 log 10 units with the antibiotic enrofloxacin. Bacteriophages are presented as a useful tool in the reduction of the C. jejuni pathogen, before and after slaughter. Further investigations should be made with other antibiotics, with the aim of increasing the knowledge of synergy between bacteriophages and antibiotics as a therapeutic process within the farm. In cecal content, the analyzes indicated a more efficient performance of the bacteriophage cocktail alone. This indicates that phage treatment would be effective in controlling meat contamination during slaughter.

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias . / Campylobacter jejuni (C. jejuni) y Campylobacter coli (C. coli) son microorganismos comensales en animales productivos y constituyen una de las principales causas de enteritis de transmisión alimentaria. C. jejuni es responsable del 90% de las campylobacteriosis humanas y C. coli cerca del 10%. Ambas especies son aisladas en proporciones similares de la carne de pollo, la principal fuente de infección del ser humano, representando cada una de ellas cerca del 50% de los Campylobacter spp. aislados. Para estas bacterias, la adherencia e invasión a células intestinales son mecanismos fundamentales de patogenicidad. Se han identificado diversos factores de virulencia asociados a estos mecanismos, como también diferencias entre C. jejuni y C. coli en las prevalencias y tamaños de algunos de los genes que los codifican. El propósito del presente trabajo fue caracterizar la capacidad de adherencia e invasión a células intestinales humanas de cepas de C. jejuni y C. coli aisladas de personas y animales productores de alimentos y relacionar esta capacidad con la presencia de siete genes de virulencia (cadF, flaA, racR, dnaJ, virB11, ciaB y pldA). La hipótesis fue que las cepas de C. jejuni tendrían mayor capacidad de adherir e invadir células intestinales humanas que las cepas de C. coli, y que esta capacidad se relacionaría positivamente con la presencia de genes de virulencia. Se emplearon 15 cepas de C. jejuni y 17 de C. coli aisladas desde pacientes humanos, cerdos, bovinos y pollos broiler. La presencia de los genes de virulencia de cada una de las cepas fue caracterizada en un estudio previo mediante la técnica de PCR convencional. Para evaluar la capacidad de adherencia e invasión a células intestinales humanas se realizaron estudios in vitro empleando la línea celular T84 de epitelio colónico T84 y midiendo el número de bacterias adheridas luego de una h de infección y las bacterias internalizadas luego de tres. La asociación con los genes de virulencia se valoró mediante análisis de regresión logística, el que se complementó con el test Kruskal-Wallis para evaluar diferencias en adherencia e invasión entre cepas portadoras y no portadoras de los genes. Los resultados demostraron que tanto las cepas humanas como las aisladas desde animales productores de alimento tienen la capacidad de adherir e invadir células intestinales in vitro y que esta capacidad varía entre las diferentes cepas. Estadísticamente, no se encontraron diferencias en la capacidad de adherencia e invasión entre C. jejuni y C. coli. El análisis Kruskal- Wallis (y test post-hoc Dunn) reveló que las cepas de C. coli portadoras del gen dnaJ tenían una mayor capacidad de invasión que las cepas de C. coli no portadoras del gen y que las cepas C. jejuni portadoras del mismo. Asimismo, con el análisis de regresión logística se encontró una asociación significativa entre la presencia del gen dnaJ y una mayor capacidad invasora en la especie C. coli. Los resultados de este trabajo sugieren que C. jejuni y C. coli tendrían la misma capacidad de adherir e invadir células intestinales humanas y que solo existiría una asociación positiva entre el gen dnaJ y la invasión de cepas de C. coli. / Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are commensals microorganisms of food-producing animals, and they are considered one of the major causes of food-borne enteritis. 90% of human campylobacteriosis is caused by C. jejuni and most of the rest by C. coli. Both species are isolated in similar proportions from chicken meat, the main source of human infection, representing each of them almost the 50% of Campylobacter spp. isolated. The adherence to and invasion of human intestinal epithelial cells are essential mechanisms in Campylobacter pathogenesis. There have been identified several virulence factors related to these mechanisms, besides differences between C. jejuni and C. coli in the prevalence and size of some of the genes that encode them. The aim of this work was studied the adherence to and invasion of human intestinal epithelial cells by C. jejuni and C. coli isolated from humans and foodproducing animals, and to relate those abilities to the presence of seven virulence genes (cadF, flaA, racR, dnaJ, virB11, ciaB y pldA). The hypothesis was that C. jejuni strains would have more ability to adhere to and invade human intestinal epithelial cells than C. coli strains, and those abilities would be associated with the presence of virulence genes. We used 15 C. jejuni strains and 17 C. coli strains isolated from human patients, broiler chickens, swine, and bovines. The presence of virulence genes of each of the strains was determined using PCR before this work. We employed the human colonic epithelial cell line T84 to test in vitro the adherence and invasion abilities, and we checked them after one and three hours of infection to determine the number adhered and internalized bacteria, respectively. To test the association with the virulence genes we used logistic regression, and to evaluate differences of adherence and invasion between strains carrying and non-carrying virulence genes we used the Kruskal-Wallis test. We observed that both Campylobacter isolates from humans and from food-producing animals are capable of adhering to and of invading intestinal epithelial cells in vitro, and there are variations between strains in those abilities. Statistically, no significant differences were detected between C. jejuni and C. coli in their abilities to adhere to and to invade T84 cells. The Kruskal-Wallis test (and post-hoc Dunn test) showed that C. coli strains carrying dnaJ gene invaded more than C. coli strains non-carrying the gene, and more than C. jejuni strains carrying the same gene. Besides, a significant association was detected between the presence of the dnaJ gene and a higher invasion in C. coli strains by logistic regression. Our results suggest that C. jejuni and C. coli would have the same adherence and invasion abilities, and that, statistically, it would exist only a positive association between the dnaJ gene and the invasion ability of C. coli strains. / Financiamiento: Programa de Apoyo Económico de Actividades de Investigación para estudiantes de Magíster en Ciencias Animales Veterinarias.

Magister Scientiae - MSc / South Africa

Campylobacter jejuni infection is one of the leading causes of gastroenteritis in humans worldwide. During colonization of the gastrointestinal tract, C. jejuni will be unavoidably exposed to reactive oxygen species (ROS) produced by the host immune system and other intestinal microbiota. Identification of defenses against ROS is therefore important for understanding how Campylobacter survives this environmental stress during infection. Construction of isogenic deletion mutants into genes encoding potential oxidative stress defense systems followed by phenotypic screening revealed genes important for oxidant defense within C. jejuni. Surprisingly, genes involved in motility were found to play an indirect role in resistance to oxidative stress. Deletion of the flagellar motor apparatus genes, motAB, resulted in increased sensitivity towards superoxide which could be restored by fumarate supplementation or tandem deletion of motAB with ccoQ (cytochrome c oxidase). This finding suggested that disruption of the proton gradient across the inner membrane resulted in increased superoxide production in non-motile flagellar mutants. Phenotypic screening of the mutant library also identified a novel gene (cj1386) specifically involved in hydrogen peroxide defense within the cell. Hydrogen peroxide detoxification within living organisms is predominantly carried out by catalase enzymes. Interestingly, cj1386 is located directly downstream from katA (catalase) in the C. jejuni genome and it was found that a ∆cj1386 mutant had reduced catalase activity relative to wild-type C. jejuni. Immunoprecipitation of KatA from ∆cj1386 revealed a significant reduction in hemin content associated with KatA suggesting a role for cj1386 in hemin trafficking to KatA. Hemin binding experiments with purified Cj1386 demonstrated the ability of Cj1386 to bind hemin with a 1:1 hemin-to-protein binding ratio. Furthermore, co-immunoprecipitation experiments revealed an interaction between KatA and Cj1386. Mutagenesis of conserved amino acids in Cj1386 demonstrated that tyrosine 57 plays an important role in hemin affinity and is required for proper hemin content of KatA within the cell. Overall, this work provides a global characterization of key oxidant defenses within C. jejuni and provides one of the first studies investigating hemin trafficking to KatA.

Campylobacter organisms are the most commonly reported bacterial causes of foodborne infection in the world, with Campylobacter jejuni and Campylobacter coli responsible for over 99% of reported infections. Traditionally, Campylobacter species detection is an arduous process, requiring a special incubation environment as well as specific growth media for an extended growth period. The development of a rapid and reliable diagnostic tool for the detection of Campylobacter species would be a valuable aid to the medical diagnostic decision process, especially to rule out Campylobacter infection during the enteric pre-surgical time period. Improved patient outcomes would result if this rapid assay could reduce the number of enteric surgeries. Assays performed during this dissertation project have demonstrated that both SYBR® green and hydrolysis probe assays targeting an 84 nucleotide portion of cadF, a fibronectin-binding gene of Campylobacter jejuni and Campylobacter coli, were able to detect from 101 to 108 copies of organism from stool specimens, did not detect nonspecific targets, and exhibited a coefficient of variation (CV) of 1.1% or less. Analytical validation of sensitivity, specificity and precision, successfully performed in these studies, warrants additional clinical validation of these assays.

Mononuclear molybdenum enzymes catalyze a variety of reactions that are essential in the cycling of nitrogen, carbon, arsenic, and sulfur. For decades, the structure and function of these crucial enzymes have been investigated to develop a fundamental knowledge for this vast family of enzymes and the chemistries they catalyze. The dimethyl sulfoxide reductase (DMSOR) family is the most diverse family of molybdoenzymes and, the members of this family catalyze a myriad of reactions that are important in microbial life processes. Periplasmic nitrate reductase (Nap) is an important member of the DMSO reductase family that catalyzes the reduction of nitrate to nitrite, and yet the physiological role of Nap is not completely clear. Enzymes in this family can transform multiple substrates; however, quantitative information about the substrate preference is sparse and more importantly, the reasons for the substrate selectivity are not clear. Substrate specificity is proposed to be tuned by the ligands coordinating the molybdenum atom in the active site. As such, periplasmic nitrate reductase is utilized as a vehicle to understand the substrate preference and delineate the mechanistic underpinning of these differences. To this end, NapA from <i>Campylobacter jejuni </i>has been heterologously overexpressed, and a series of variants, where the molybdenum-coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic and biochemical properties of these variants will be discussed and compared with those of the native enzyme, providing quantitative information to understand the function.