This research used classical and molecular epidemiology tools to assess the potential importance of feedlot cattle as Campylobacter reservoirs. The project was conducted from November 2004 to September 2005 in southern Alberta.<p> Fresh pen-floor fecal samples were collected from commercial feedlot cattle near slaughter weight in seven feedlots. Overall, 87% of 2,776 fecal samples were culture positive for Campylobacter species (86% of 1,400 in winter, 88% of 1,376 in summer), and 69% of 1,486 Campylobacter positive isolates were identified as <i>Campylobacter jejuni</i>. After accounting for clustering within pen and feedlot, the number of days-on-feed and feedlot size were associated (p ¡Ü 0.05) with Campylobacter species isolation rates.<p> Retail ground beef was collected from 60 grocery stores (four chains, three cities). None of the 1,200 packages were culture positive for Campylobacter species. Polymerase chain reaction (PCR) results from a subset of samples (n=142) indicated that 48% of packages were positive for Campylobacter DNA. By species, 14.8% (21/142), 26.8% (38/142) and 1.4% (2/142) of packages were PCR positive for <i>C. jejuni</i>, <i>C. coli</i> and <i>C. hyointestinalis</i> DNA, respectively. The collection period (1, 2, 3 or 4) was associated (p ¡Ü 0.05) with the odds of detecting Campylobacter species DNA using PCR.<p> Oligonucleotide DNA microarrays were used as a platform for comparative genomic hybridization (CGH) analysis of 87 C. jejuni isolates (46 bovine, 41 human) obtained within the same geographical regions and time frame. Of the 13 CGH clusters identified based on overall comparative genomic profile similarity, nine contained human and cattle isolates, three contained only human isolates, and one contained only cattle isolates. In addition, human clinical and feedlot cattle C. jejuni isolates were compared on a gene-by-gene basis and only a small number of the 1,399 genes tested were unequally distributed between the two groups (p ¡Ü 0.05).<p> The high isolation rates of Campylobacter species and <i>C. jejuni</i> reported here may have implications for food safety, public health and environmental contamination. Our findings suggest that feedlot cattle and human <i>C. jejuni</i>strains are very similar and may be endemic within southern Alberta.

This research used classical and molecular epidemiology tools to assess the potential importance of feedlot cattle as Campylobacter reservoirs. The project was conducted from November 2004 to September 2005 in southern Alberta.<p> Fresh pen-floor fecal samples were collected from commercial feedlot cattle near slaughter weight in seven feedlots. Overall, 87% of 2,776 fecal samples were culture positive for Campylobacter species (86% of 1,400 in winter, 88% of 1,376 in summer), and 69% of 1,486 Campylobacter positive isolates were identified as <i>Campylobacter jejuni</i>. After accounting for clustering within pen and feedlot, the number of days-on-feed and feedlot size were associated (p ¡Ü 0.05) with Campylobacter species isolation rates.<p> Retail ground beef was collected from 60 grocery stores (four chains, three cities). None of the 1,200 packages were culture positive for Campylobacter species. Polymerase chain reaction (PCR) results from a subset of samples (n=142) indicated that 48% of packages were positive for Campylobacter DNA. By species, 14.8% (21/142), 26.8% (38/142) and 1.4% (2/142) of packages were PCR positive for <i>C. jejuni</i>, <i>C. coli</i> and <i>C. hyointestinalis</i> DNA, respectively. The collection period (1, 2, 3 or 4) was associated (p ¡Ü 0.05) with the odds of detecting Campylobacter species DNA using PCR.<p> Oligonucleotide DNA microarrays were used as a platform for comparative genomic hybridization (CGH) analysis of 87 C. jejuni isolates (46 bovine, 41 human) obtained within the same geographical regions and time frame. Of the 13 CGH clusters identified based on overall comparative genomic profile similarity, nine contained human and cattle isolates, three contained only human isolates, and one contained only cattle isolates. In addition, human clinical and feedlot cattle C. jejuni isolates were compared on a gene-by-gene basis and only a small number of the 1,399 genes tested were unequally distributed between the two groups (p ¡Ü 0.05).<p> The high isolation rates of Campylobacter species and <i>C. jejuni</i> reported here may have implications for food safety, public health and environmental contamination. Our findings suggest that feedlot cattle and human <i>C. jejuni</i>strains are very similar and may be endemic within southern Alberta.

C. jejuni, a pathogenic gram-negative bacterium infecting the human gastrointestinal tract has lately been shown to cause bacteraemia to a wider extent than previously known. In some genotypes, this is thought to be related to GDP-Mannose 4,6 dehydratase encoded by the gene wcbK in the capsule polysaccharide operon and its potential phase variated regulated nature mediated by a homopolymeric guanine tract. This potential regulatory tract has been reported to be controlling the survival in serum by switching expression of wcbK “ON” or “OFF”. This master thesis report evaluates C. jejuni’s ability to survive human serum and low pH, as proxies for the conditions that bacteria meet in human blood or the stomach, respectively. By next generation sequencing, I evaluated the correlation between survival in human serum and the wcbK gene’s “ON” or “OFF” state. Furthermore, the temporal stability of the serum resistant phenotype was assessed over multiple generations. I found that a serum resistant fraction of the C. jejuni population could be enriched by selection in normal human serum. The serum resistant part of the population did not decrease during repeated subculture for 10 generations in bacterial culture medium. However, there was no correlation between the extent of serum resistance in the population and the “ON” or “OFF” state of the wcbK gene.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2017-04-24T11:45:18Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação Tassiana Ramires.pdf: 1728606 bytes, checksum: a63d0903f11cb3f858c5a7fd4e32ad97 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-05-04T18:58:35Z (GMT) No. of bitstreams: 2 Dissertação Tassiana Ramires.pdf: 1728606 bytes, checksum: a63d0903f11cb3f858c5a7fd4e32ad97 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-04T18:58:35Z (GMT). No. of bitstreams: 2 Dissertação Tassiana Ramires.pdf: 1728606 bytes, checksum: a63d0903f11cb3f858c5a7fd4e32ad97 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-20 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Campylobacter termofílicos são, atualmente, as principais bactérias causadoras de doenças gastrointestinais em todo o mundo. Esse grupo é assim denominado devido a sua temperatura ótima de multiplicação oscilar entre 42 °C e 43 °C, sendo Campylobacter jejuni, C. coli, C. lari e C. upsaliensis, as principais espécies envolvidas nos casos de campilobacteriose em humanos. Dentre essas espécies, a mais relacionada à essa doença é C. jejuni, seguida por C. coli. O principal reservatório desses micro-organismos são as aves, principalmente os frangos, possivelmente pela temperatura corporal desses animais ser similar à temperatura ótima para Campylobacter termofílicos. Com isso, o objetivo desse estudo foi avaliar a ocorrência, a diversidade genética, o perfil de resistência a antimicrobianos e a presença de genes associados à virulência em isolados de Campylobacter termofílicos provenientes de frangos de corte e na cama de aviário em granjas aviárias da região sul do Rio Grande do Sul, Brasil. Um total de 48 amostras foram coletadas em três diferentes granjas (A, B e C), incluindo uma amostra de swab de arrasto da cama do aviário e 15 pools de amostras de swab de cloaca em cada granja. Das três granjas amostradas, apenas a granja C apresentou contaminação por Campylobacter termofílicos, sendo todos os isolados identificados por técnicas fenotípicas e moleculares como C. jejuni. Dessas 16 amostras positivas, obtiveram-se 28 isolados, sendo 16 pelo isolamento em ágar Preston e 12 do ágar mCCD. A diversidade genética entre os isolados foi avaliada por PFGE, verificando-se que todos os isolados apresentaram um único padrão de macrorestrição, sugerindo clonalidade entre os isolados e a presença de apenas uma fonte de infecção por Campylobacter nessa granja. O perfil de resistência a antimicrobianos foi avaliado pelo teste de disco difusão em ágar, utilizando-se oito antimicrobianos distintos, de três classes diferentes: tetraciclina, quinolonas e macrolídeos. Os isolados apresentaram perfil similar de resistência a antimicrobianos, sendo resistentes às quinolonas e tetraciclinas e sensíveis aos macrolídeos. Devido a relação clonal e ao perfil de resistência similar, um isolado representativo foi selecionado para detecção dos genes de virulência. A técnica de PCR foi utilizada para detectar a presença dos genes ciaB, cadF, cdtA, cdtB e cdtC, sendo o isolado selecionado positivo todos os genes pesquisados. Dessa forma, a presença de C. jejuni resistente a antimicrobianos e com potencial de virulência em frangos de corte prontos para o abate e na cama de aviário durante o período de produção é um risco à saúde pública, pois esses micro-organismos podem ser introduzidos no ambiente do abatedouro e contaminar as carcaças durante o abate. / Thermophilic Campylobacter are currently the leading bacteria causing of gastrointestinal diseases worldwide. This group is so named because its optimal multiplication temperature oscillates between 42 °C and 43 °C, being Campylobacter jejuni, C. coli, C. lari and C. upsaliensis, the main species involved in cases of human campylobacteriosis. Among these species, the most related to this disease is C. jejuni, followed by C. coli. The main reservoir of these microorganisms are birds, especially chickens, possibly because the body temperature of these animals coincides with the optimal temperature for thermophilic Campylobacter. Therefore, the aim of this study was to verify the occurrence, genetic relationship, antimicrobial susceptibility, and the presence of virulence genes in thermophilic Campylobacter from broilers and broiler bedding from the southern region of Rio Grande do Sul, Brazil. A total of 48 samples were collected in three different farms (A, B and C), which comprising one sample of drag swab and 15 pools of cloacal swabs in each farm. From the three farms sampled, only the farm C showed thermophilic Campylobacter contamination. All isolates were identified by phenotypic and molecular techniques such as C. jejuni. Of these 16 positive samples, 28 isolates were obtained, 16 being isolated by Preston agar and 12 by mCCD agar. The genetic diversity among the isolates was evaluated by PFGE, and it was observed that all the isolates belonged to the same macrorestriction pattern, suggesting clonality among the isolates and the presence of only one source of Campylobacter infection in this farm. The antimicrobial resistance profile was evaluated by the agar disc diffusion test using eight distinct antimicrobial agents from three different classes: tetracyclines, quinolones and macrolides. The isolates presented a similar antimicrobial resistance profile, being resistant to quinolones and tetracyclines and susceptible to macrolides. As the isolates shared the same PFGE pattern and similar resistance profile, a representative isolate was chosed for investigation of virulence genes. A PCR assay was carried out aiming to identify the presence of ciaB, cadF, cdtA, cdtB and cdtC virulence genes and all the genes evaluated were found. Thus, the presence of C. jejuni resistant to antimicrobial agents and harboring virulence genes in broilers and broiler farm during the broiler production period may represents a potential risk to public health, because these microorganisms can be introduced into the abattoir environment and may contaminate the carcasses during slaughter.

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