Publicação
Use of bacteriophages to control Campylobacter in poultry
| Resumo: | Recent publications indicate that Campylobacter is the leading cause of foodborne diseases worldwide, clearly surpassing other foodborne pathogens such as Salmonella. Poultry meat is regarded as the most common source of human campylobacteriosis, being Campylobacter jejuni and Campylobacter coli the most prevalent species. Measures commonly used to control foodborne pathogens have had little success for Campylobacter, which is a reflection of differences in the physiology, epidemiology and ecology of these organisms. Moreover, recent legislation restricting the use of antibiotics as growth promoters in animal production, together with the risk of antibiotic-resistant bacteria entering the human food chain, highlight the importance of finding alternative ways to control and treat animal infection. Bacterio(phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and capacity to evolve to overcome bacterial resistance. These characteristics make phages an appealing option to control bacterial pathogens. The present study exploits phages as biocontrol agents to reduce the levels of Campylobacter jejuni and Campylobacter coli in broilers and thus preventing this pathogen for entering the human food chain. In order to accomplish this aim, Campylobacter phages were isolated and characterized and three phages considered the best candidates for therapy were selected for composing a phage cocktail that was further tested in animal trials. This work presented several challenges mostly related to the fastidious growth of Campylobacter and to unique features that Campylobacter phages exhibit, hindering the use of conventional methods of phage isolation, propagation and characterization. Therefore these methods had to be adapted, optimized and implemented. One of the main achievements was the development of a novel method to isolate Campylobacter phages that allowed the isolation of 43 phages from poultry intestines. From this collection, three phages (phiCcoIBB12, phiCcoIBB35, phiCcoIBB37) were selected based on the best performance in vitro and the broadest lytic spectra against both Campylobacter jejuni and Campylobacter coli strains. The selected phages were further characterized in terms of morphology and physiology, showing to belong to the Myoviridae family of lytic phages. The DNA of one of the selected phages (phiCcoIBB35) was sequenced and analyzed, contributing to deepen the knowledge of Campylobacter phage genomes, since only two Campylobacter phage genomes have been reported so far. The DNA sequence data of phiCcoIBB35 consists of five DNA contigs in a total of 172,065 bp, being most of the open reading frames (ORFs) unique. It was possible to identify the genes encoding for tail fibers and for the lytic cassette, this later expressing enzymes involved in bacterial capsular polysaccharides (CPS) degradation, which has never been reported before for Campylobacter phages. The three selected and characterized phages were used to compose a cocktail that was administered by oral gavage or on feed to one-day old Campylobacter infected chicks. The results of the in-vivo phage performance evaluation revealed that both administration routes enabled the same levels of Campylobacter reduction by approximately 2 log10 cfu/g, however, feed incorporating phages resulted in an earlier Campylobacter reduction. This can be considered a promising, effective and innovative approach of phage administration to be used under commercial condition in a poultry unit. Overall this work contributed for the improvement of knowledge of Campylobacter phages biology, physiology, genomics and proteomics and established the proof-of-principle that the administration of Campylobacter phages in living poultry can be an effective measure to reduce the levels of Campylobacter colonization. |
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| Autores principais: | Carvalho, Carla A. O. C. M. |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Recent publications indicate that Campylobacter is the leading cause of foodborne diseases worldwide, clearly surpassing other foodborne pathogens such as Salmonella. Poultry meat is regarded as the most common source of human campylobacteriosis, being Campylobacter jejuni and Campylobacter coli the most prevalent species. Measures commonly used to control foodborne pathogens have had little success for Campylobacter, which is a reflection of differences in the physiology, epidemiology and ecology of these organisms. Moreover, recent legislation restricting the use of antibiotics as growth promoters in animal production, together with the risk of antibiotic-resistant bacteria entering the human food chain, highlight the importance of finding alternative ways to control and treat animal infection. Bacterio(phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and capacity to evolve to overcome bacterial resistance. These characteristics make phages an appealing option to control bacterial pathogens. The present study exploits phages as biocontrol agents to reduce the levels of Campylobacter jejuni and Campylobacter coli in broilers and thus preventing this pathogen for entering the human food chain. In order to accomplish this aim, Campylobacter phages were isolated and characterized and three phages considered the best candidates for therapy were selected for composing a phage cocktail that was further tested in animal trials. This work presented several challenges mostly related to the fastidious growth of Campylobacter and to unique features that Campylobacter phages exhibit, hindering the use of conventional methods of phage isolation, propagation and characterization. Therefore these methods had to be adapted, optimized and implemented. One of the main achievements was the development of a novel method to isolate Campylobacter phages that allowed the isolation of 43 phages from poultry intestines. From this collection, three phages (phiCcoIBB12, phiCcoIBB35, phiCcoIBB37) were selected based on the best performance in vitro and the broadest lytic spectra against both Campylobacter jejuni and Campylobacter coli strains. The selected phages were further characterized in terms of morphology and physiology, showing to belong to the Myoviridae family of lytic phages. The DNA of one of the selected phages (phiCcoIBB35) was sequenced and analyzed, contributing to deepen the knowledge of Campylobacter phage genomes, since only two Campylobacter phage genomes have been reported so far. The DNA sequence data of phiCcoIBB35 consists of five DNA contigs in a total of 172,065 bp, being most of the open reading frames (ORFs) unique. It was possible to identify the genes encoding for tail fibers and for the lytic cassette, this later expressing enzymes involved in bacterial capsular polysaccharides (CPS) degradation, which has never been reported before for Campylobacter phages. The three selected and characterized phages were used to compose a cocktail that was administered by oral gavage or on feed to one-day old Campylobacter infected chicks. The results of the in-vivo phage performance evaluation revealed that both administration routes enabled the same levels of Campylobacter reduction by approximately 2 log10 cfu/g, however, feed incorporating phages resulted in an earlier Campylobacter reduction. This can be considered a promising, effective and innovative approach of phage administration to be used under commercial condition in a poultry unit. Overall this work contributed for the improvement of knowledge of Campylobacter phages biology, physiology, genomics and proteomics and established the proof-of-principle that the administration of Campylobacter phages in living poultry can be an effective measure to reduce the levels of Campylobacter colonization. |
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