Litter is a possible source of Campylobacter colonization for broilers as well as contamination of crops when used as fertilizer. A survey of Arkansas broiler litter indicated that Campylobacter recovery rates were higher in pine shavings and rice hulls than sand. Two experiments utilized three types of litter, which were artificially contaminated with Campylobacter. After 24 hours no Campylobacter could be recovered from any sample. Campylobacter growth was also examined for used pine shaving litter in varying conditions: aerobic atmosphere, micro aerobic atmosphere (6% O2), and moisture content. Campylobacter was recovered for all treatments at the initial sampling, and by the 12 hour sampling time, only the added moisture and micro aerobic atmosphere yielded recoverable Campylobacter. This research suggests that without birds present in the house to shed fresh Campylobacter cells onto the litter, that the litter itself is incapable of harboring the bacteria long enough to colonize sequential flocks.

Litter is a possible source of Campylobacter colonization for broilers as well as contamination of crops when used as fertilizer. A survey of Arkansas broiler litter indicated that Campylobacter recovery rates were higher in pine shavings and rice hulls than sand. Two experiments utilized three types of litter, which were artificially contaminated with Campylobacter. After 24 hours no Campylobacter could be recovered from any sample. Campylobacter growth was also examined for used pine shaving litter in varying conditions: aerobic atmosphere, micro aerobic atmosphere (6% O2), and moisture content. Campylobacter was recovered for all treatments at the initial sampling, and by the 12 hour sampling time, only the added moisture and micro aerobic atmosphere yielded recoverable Campylobacter. This research suggests that without birds present in the house to shed fresh Campylobacter cells onto the litter, that the litter itself is incapable of harboring the bacteria long enough to colonize sequential flocks.

Campylobacter is considered to be the leading cause of acute bacterial gastroenteritis in humans in the United States with Campylobacter jejuni being responsible for 80-90% of those infections. Many cases of Campylobacter gastroenteritis have been linked to the consumption of raw or undercooked chicken. The population of bacteria on the breast skin has been reported to be greater than on other edible portions of the chicken carcass making this an important site to control the organism and to study bacterial attachment properties. This research examined the efficacy of trisodium phosphate (TSP)(10%), cetylpyridinium chloride (CPC)(0.1% & 0.5%), acidified sodium chlorite (ASC)(0.1%), Tween 80 (polysorbate 80) (1%) and water (50°C) for reducing the number of viable Campylobacter jejuni on inoculated chicken breast skin. All chemicals were evaluated using contact times of 30 sec., 3 min. or 10 min. Statistically significant (p £ 0.05) differences in the reduction of C. jejuni populations were observed across chemical treatments and contact time. When bacteria were applied before treatment, a reduction of >1.0 log10 CFU/skin was achieved with 0.5% CPC (2.89), 10% TSP (1.63), 0.1% ASC (1.52), and 0.1% CPC (1.42). When bacteria were applied after treatment, a reduction of >1.0 log10 CFU/skin was achieved with 0.5% CPC (4.67) and 10% TSP (1.28). The main effects of contact time were statistically significant (p=0.02) only when bacteria were applied after treatment. / Master of Science

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.

A importância da espécie suína na transmissão de Campylobacter spp. assemelha-se aos demais grupos de animais que se destinam à produção de carne, incluindo aves, bovinos e ovinos. Os objetivos deste estudo foram isolar Campylobacter spp. a partir de fezes e carcaças de suínos abatidos no Estado de São Paulo; identificar as espécies de Campylobacter spp. presentes nos animais abatidos; caracterizar os isolados obtidos através do Polimorfismo do Comprimento de Fragmentos Amplificados (AFLP). Para tal, foram utilizadas 120 amostras de fezes e 120 suabes de carcaças de suínos, colhidas de quatro diferentes abatedouros do Estado de São Paulo. Das 120 amostras de fezes analisadas, 30 foram positivas para o isolamento de Campylobacter coli (25%) e duas foram positivas para isolamento de Campylobacter jejuni (1,6%). Todas as amostras analisadas de suabes de carcaça foram negativas para Campylobacter spp. As estirpes isoladas que apresentaram características bioquímicas sugestivas de Campylobacter spp. foram submetidas ao teste de susceptibilidade ao ácido nalidixico e cefalotina, destas 19,16% (23/120) apresentaram resistência ao ácido nalidixico apesar de todas as características bioquímicas indicarem se tratar de Campylobacter coli. Foram selecionadas para a análise genotípica 38 amostras isoladas, sendo 36 de C. coli e dois de C. jejuni. A análise dos isolados através do AFLP revelou a presença de 28 perfis que foram designados P1 a P28. A técnica discriminou as cepas de acordo com a espécie, porém, uma cepa previamente caracterizada como C. coli foi agrupada com isolados de C. jejuni. Não foi possível estabelecer a correlação entre os isolados e o abatedouro de origem, no entanto observa-se uma forte tendência dos isolados resistentes ao ácido nalidixico em formar grupamentos de maior similaridade. / The importance of swine species in the transmission of Campylobacter spp. resembles to the other groups of animals that are destined to the meat production, including bovine and ovine animals. The objectives of this study were to isolate Campylobacter spp. from swine feces and carcasses slaughtered in São Paulo State; to identify the Campylobacter spp. species presents in the slaughtered animals; to characterize the isolated samples by amplified fragment length polymorphism (AFLP), for this, 120 swine feces samples and the same number of carcasses swabs were collected of four different slaughterhouses in São Paulo State. From 120 feces samples, 30 (25%) were positive for Campylobacter coli isolation and two (1,6%) were positive for Campylobacter jejuni. All the analyzed carcass swabs samples were negatives for Campylobacter spp. The isolated samples that presented suggestive biochemical characteristics of Campylobacter spp. were submitted to the susceptibility test to the nalidixic acid and cefalotine, from that 19.16% (23/120) presented resistance to the nalidixic acid in spite of all the biochemical characteristics indicate that they were Campylobacter coli. They were selected for the genotypic analysis 38 isolated samples, being 36 of C. coli and two of C. jejuni. The analysis of the 38 tested samples by AFLP showed the presence of 28 profiles that had been assigned P1 to P28. The technique discriminated the samples in agreement with the species, however, one sample previously characterized as C. coli was clustered as C. jejuni. It was not possible to establish the correlation between the isolated samples and the origin slaughterhouse, however a strong tendency of the isolated samples acid nalidixic resistant to create clusters with more similarity.

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.

Renata Rakštelytė Marinuotų paukštienos pusgaminių užkrėstumas kampilobakterijomis ir jų rūšinė įvairovė Baigiamasis magistro darbas Darbo vadovas: prof. dr. Mindaugas Malakauskas Lietuvos sveikatos mokslų universitetas Veterinarijos akademija Veterinarijos fakultetas Maisto saugos ir kokybės katedra Darbo apimtis 36 puslapiai, 4 lentelės, 9 paveikslai. Darbo tikslas buvo įvertinti Lietuvoje parduodamų marinuotų paukštienos pusgaminių užkrėstumą dažniausiai žmonių susirgimus sukeliančiomis kampilobakterijų rūšimis. Tyrimų metu buvo ištirti 67 marinuotos paukštienos pusgaminiai. Trijų skirtingų paukštienos gamybos įmonių pusgaminiai tyrimui buvo perkami mažmeninės prekybos įmonėse be išankstinio įspėjimo. Kampilobakterijos buvo išskiriamos tiesiogiai sėjant ant mCCDA agaro ir atlikus pagausinimą Bolton sultinyje. Kampilobakterijos buvo identifikuojamos iki rūšies naudojant šiek tiek pakeistą Wang ir kt. (2002) aprašytą PGR metodą ir pradmenis. Tyrimai parodė, kad 22 mėginiai (32,8%)iš 67 tirtų marinuotų paukštienos pusgaminių, buvo užkrėsti kampilobakterijomis. Dažniausiai marinuoti paukštienos pusgaminiai buvo užkrėsti C. jejuni rūšies bakterijomis (16 mėginių), tuo tarpu C.coli bakterijos buvo aptiktos tik 2 mėginiuose, o vienas mėginys buvo užkrėstas nenustatytos rūšies kampilobakterijomis. Dar dviejuose mėginiuose rastos tiek C.jejuni, tiek ir Campylobacter spp. bakterijos ir viename - C.coli ir nenustatytos rūšies kampilobakterijos. Tyrimų laikotarpiu iš viso buvo... [toliau žr. visą tekstą] / Renata Rakštelytė CONTAMINATION OF MARINATED POULTRY MEAT WITH CAMPYLOBACTER SPP. AND THEIR SPECIES DIVERSITY Master thesis Thesis supervisor: prof. dr. Mindaugas Malakauskas Lithuanian University of Health Sciences Lithuanian Veterinary Academy Faculty of Veterinary medicine Departament of Food Safety and Quality Kaunas, 2014 m. The coverage of the work: 36 pages, 4 tables, 9 pictures. The aim of the study was to evaluate contamination of marinated poultry products with Campylobacter spp. sold at the retail level sale in Lithuania. It total 67 ready-to-cook marinated poultry products were examined. Detection of Campylobacter was carried out using direct plating on mCCDA agar and after enrichment in Bolton broth with further planting on mCCDA. Campylobacter species were identified by PCR method described by Wang et al. (2002 ) with minor changes. Only specific primers for identification of Campylobacter spp., C. jejuni and C. coli were used. The study showed that out of the 67 tested marinated poultry products 22 (32.8 %) were contaminated with Campylobacter spp. Most often marinated poultry preparations were contaminated with C. jejuni (16 samples), while C.coli species were isolated only from 2 samples. Other Campylobacter species then C. jejuni or C. coliwas found in one sample, C.jejuni and Campylobacter spp.bacteria were found in two samples and C.coli and unidentified Campylobacter species in one sample, respectively. Eleven C.jejuni strains isolated from marinated... [to full text]

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).

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.