Thesis (Ph. D.)--University of North Texas, May, 2009. / Title from title page display. Includes bibliographical references.

Thesis (M.Sc.)--Victoria University of Wellington, 2008. / Includes bibliographical references.

Con el objetivo de caracterizar la fosfolipasa A de la membrana externa (OMPLA E.C. 3.1.1.32) de Campylobacter jejuni aislado de pollos de carne, se estandarizaron métodos para el aislamiento e identificación de la bacteria a partir de hisopados rectales de aves comerciales. Para ello, primero se analizó la presencia de C. jejuni en hisopados cloacales mediante técnicas bacteriológicas y la reacción en cadena de la polimerasa anidada en base a los genes ribosómicos 16S e hipO, las cuales permitierón detectar Campylobacter jejuni en 29/50 (58%) y 48/50 (96%) de las muestras respectivamente. Después, se determinó la secuencia nucleotídica de los genes ribosómicos 16S y flaA del aislado MP4 con la finalidad de tener una cepa nativa de C. jejuni bien caracterizada que se utilice como modelo de estudio para la fosfolipasa A, las secuencias nucleotídicas de estos dos genes mostraron 98% y 92% de similiitud nucleotídica con las de Campylobacter jejuni subs. jejuni ATCC 33560. Las actividades hemolítica y de fosfolipasa A de C. jejuni MP4 fue dependiente de calcio, cuando este ión se reemplazó con EDTA, las actividades disminuyeron de 4119.4 a 44.78 U/mg de proteína. Utilizando cebadores específicos se amplificó y secuenció la parte central del gen pldA de C. jejuni MP4, se obtuvo 225 nucleótidos y 75 aminoácidos. La comparación de la secuencia aminoácidica por el ClustalW mostró 99% y 98% de identidad con las proteínas OMPLA de las cepas de Campylobacter jejuni RM1221 y C.jejuni subsp. jejuni ATCC 33560 respectivamente; esta alta conservación de secuencia aminoacídica de la fosfolipasa A la convierte en una candidata potencial para el diseño de vacunas y pruebas diagnósticas. / In order to characterize the Campylobacter jejuni’s outer membrane phospholipase A (OMPLA E.C. 3.1.1.32) from broiler chickens, it was standarized suitable methods for isolation and identification of this bacterium from cloacal swabs of commercial chickens. To that end, first it was analized the presence of C. jejuni in cloacal swabs by using bacteriological techniques and nested-PCR based on 16S ribosomal and hipO genes. The approaches used detected C. jejuni in the 29/50 (58%) and 48/50 (96%) of the samples respectively. Then, it was determined the nucleotidic sequence of 16S ribosomal gene to have a well-characterized native strain for using it as a model in the study of phospholipase A. The nucleotidic sequence of both genes showed 98% and 92% of similarity with Campylobacter jejuni subs. jejuni ATCC 33560. The haemolytic and phospholipase A enzymatic activities of the C. jejuni MP4 strain was calcium-depended, when calcium was replaced by EDTA both activities significantly decreased of 4119.4 to 44.78 U/mg protein. It was amplified and sequenced the central side from pldA gen of C. jejuni MP4 using specific primers. By means of that, it was obtained 225 nucleotides and 75 aminoacids. The comparison of aminoacidic sequences by CLUSTALW showed 99% and 98% of identity with OMPLA proteins from C. jejuni RM1221 and C. jejuni subsp. jejuni ATCC 33560. This high conservation of the aminoacidic sequence of phospholipase A become it into a potential target to design vaccines and diagnostic tests.

Broiler flocks become frequently asymptomatically contaminated by the enteric bacteria Salmonella sp. and Campylobacter sp. which are human pathogens. Among the strategies developed at farm level to reduce the incidence of these pathogens, some lactic acid bacteria have been shown to be interesting because of their antimicrobial activity and their stimulatory properties on the immune system of poultry. The aim of this thesis was to select bacteria with antagonistic activity against Salmonella or Campylobacter, and to improve their inhibitory effect by the combination with enzymes of polysaccharidase type. The first step of the thesis was an epidemiological study carried out in the Walloon region in order to determine the contamination way of broilers by Campylobacter in free range production. Results showed that the major way of contamination is the open-air range to which the animals have access during the rearing period. A preventive treatment of the open-air range and the straw litter with an antagonistic strain in combination with an enzyme seems thus to be suitable in this case. The second step of the work aimed at the selection of a xylanase for using as a dietary additive in combination with an antagonistic bacterial strain against Salmonella. Four xylanases were studied in vivo for their effect on growth performances of broiler chickens. Diet supplementation with enzyme led to an increased final body weight and daily weight gain (P < 0.05), without difference according to the bacterial or fungal origin of the xylanase. The Belfeed B1100MP xylanase, which is commercialized in he Walloon region, was selected in order to develop a probiotic-xylanase feed additive. The purpose of the third part was to select a bacterial strain with antagonistic activity against Campylobacter for applying on open-air range and broiler litter. An in vitro screening of 12 lactic acid bacteria was realised using a co-culture assay with a growth medium based on straw and dehydrated poultry excreta, supplemented with different cellulase concentrations. Lactobacillus pentosus and Enterococcus faecium showed inhibitory effect against Campylobacter without enzyme which was intensified by cellulose from 200 ppm. Finally, the effect of dietary supplementation with a L. plantarum strain combined with the Belfeed B1100MP (PE treatment) on growth performance, microflora, and faecal Salmonella Typhimurium concentrations, was studied with experimentally infected broiler chickens. The PE diet allowed to partially overcome the negative effects associated with the infection on growth performance and microflora, and to significantly reduce faecal Salmonella concentration.