Publicação
Development of a bacteriophage based product to control colibacillosis in poultry
| Resumo: | Escherichia coli (E. coli) is part of the commensal microflora of the chicken intestinal tract, being commonly an opportunistic bacteria that causes disease in immunologic deprived chickens. However, there are extra-intestinal E. coli strains, the avian pathogenic Escherichia coli (APEC) that are able to cause colibacillosis by itself, due to its invasive ability. The colibacillosis and colisepticemia are responsible for significant economic losses in poultry industries worldwide, mainly due to the low feed conversion rate with consequent weight loss, high cost of treatments during production, poor carcass quality with consequent rejection at slaughter and high mortalities rates. The increasing high patterns of antibiotic resistance acquired by these bacteria, as well as the restriction to the antibiotic usage implemented by the European Union, have encouraged the search of new solutions to control severe infections ensuring good meat quality and minimizing environmental impact. Bacteriophages (phages), virus infecting exclusively bacteria, have been proposed as valuable alternatives to antibiotics based on their capacity to infect and destroy the bacteria, releasing in few minutes progeny that will infect the surrounding hosts. The presented work aimed at developing an efficient, safe and competitive phage based product to control colibacillosis in poultry. The work encompassed five different stages: firstly, different bacteriophages active for a wide range of APEC strains were isolated and characterized; secondly, an in vivo evaluation of the toxicity of the phage suspensions were performed, thirdly the effect of the route of administration and phage titre on phage dissemination in the chickens’ organisms was assessed; fourthly the efficacy of the phages presenting the wider lytic spectrum was evaluated through in vivo efficiency trials; finally, large animal trials were performed to validate the efficacy of the phage product. Phages were isolated from poultry sewage and tested against 148 O-serotyped APEC strains. The results showed that 70.5% of the tested strains were sensitive to a combination of three of the five isolated phages. Taxonomically, two of these three phages, phi F61E and phi F78E look like 16-19, T4-like phages (Myoviridae) and the other, phi F258E is a T1-like phage and belongs to the Syphoviridae family. All belong to the Caudovirales order. Restriction fragment length polymorphism (RFLP) patterns demonstrate that all phages were genetically different. The in vivo evaluation of the toxicity of the phage lysate revealed that the phage suspensions did not promote any decrease in feed and water intake, or body weight lost during the in vivo trial and the post mortem necropsies did not show any macroscopic lesions in the internal organs. These observations supported that the lysate was not toxic for chickens. The in vivo assessment of the effect of the route of administration and the dosage in the dissemination of the phages in the chicken’s organs, revealed that when administered orally and by spray, all the phages reached the respiratory tract, as well as the bloodstream. Intramuscular administration enabled the phages to reach all chickens’ internal organs. Results suggested that, besides the intramuscular administration (not feasible to use in flocks), the oral and spray administration can be considered promising administration routes to treat respiratory E. coli infections in poultry. The in vivo evaluation of the efficacy of phi F78E to control severe E. coli infections revealed that phage performance is dosage dependant and only a high concentration of 109 PFU/ml allowed a decrease in 25% and 43% in chickens’ mortality and morbidity, respectively. Interestingly, the phage cocktail (of phi F61E, phi F78E and phi F258E) administered in the water drinking and by spray in a single application, and composed by 5×107 PFU/ml of each bacteriophage, was able to control the mortality rate in naturally infected chicken flocks, refractive to antibiotherapy. The mortality felt from 2.2% in average, to under 0.5% in no more than three weeks, with no recidivism. In conclusion, with this work it was possible to obtain an antimicrobial product, comprised by a combination of three different lytic phages, which demonstrated to be safe for chickens and efficient against colibacillosis in the poultry industry. |
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| Autores principais: | Oliveira, Ana Cristina Afonso |
| Ano: | 2009 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Escherichia coli (E. coli) is part of the commensal microflora of the chicken intestinal tract, being commonly an opportunistic bacteria that causes disease in immunologic deprived chickens. However, there are extra-intestinal E. coli strains, the avian pathogenic Escherichia coli (APEC) that are able to cause colibacillosis by itself, due to its invasive ability. The colibacillosis and colisepticemia are responsible for significant economic losses in poultry industries worldwide, mainly due to the low feed conversion rate with consequent weight loss, high cost of treatments during production, poor carcass quality with consequent rejection at slaughter and high mortalities rates. The increasing high patterns of antibiotic resistance acquired by these bacteria, as well as the restriction to the antibiotic usage implemented by the European Union, have encouraged the search of new solutions to control severe infections ensuring good meat quality and minimizing environmental impact. Bacteriophages (phages), virus infecting exclusively bacteria, have been proposed as valuable alternatives to antibiotics based on their capacity to infect and destroy the bacteria, releasing in few minutes progeny that will infect the surrounding hosts. The presented work aimed at developing an efficient, safe and competitive phage based product to control colibacillosis in poultry. The work encompassed five different stages: firstly, different bacteriophages active for a wide range of APEC strains were isolated and characterized; secondly, an in vivo evaluation of the toxicity of the phage suspensions were performed, thirdly the effect of the route of administration and phage titre on phage dissemination in the chickens’ organisms was assessed; fourthly the efficacy of the phages presenting the wider lytic spectrum was evaluated through in vivo efficiency trials; finally, large animal trials were performed to validate the efficacy of the phage product. Phages were isolated from poultry sewage and tested against 148 O-serotyped APEC strains. The results showed that 70.5% of the tested strains were sensitive to a combination of three of the five isolated phages. Taxonomically, two of these three phages, phi F61E and phi F78E look like 16-19, T4-like phages (Myoviridae) and the other, phi F258E is a T1-like phage and belongs to the Syphoviridae family. All belong to the Caudovirales order. Restriction fragment length polymorphism (RFLP) patterns demonstrate that all phages were genetically different. The in vivo evaluation of the toxicity of the phage lysate revealed that the phage suspensions did not promote any decrease in feed and water intake, or body weight lost during the in vivo trial and the post mortem necropsies did not show any macroscopic lesions in the internal organs. These observations supported that the lysate was not toxic for chickens. The in vivo assessment of the effect of the route of administration and the dosage in the dissemination of the phages in the chicken’s organs, revealed that when administered orally and by spray, all the phages reached the respiratory tract, as well as the bloodstream. Intramuscular administration enabled the phages to reach all chickens’ internal organs. Results suggested that, besides the intramuscular administration (not feasible to use in flocks), the oral and spray administration can be considered promising administration routes to treat respiratory E. coli infections in poultry. The in vivo evaluation of the efficacy of phi F78E to control severe E. coli infections revealed that phage performance is dosage dependant and only a high concentration of 109 PFU/ml allowed a decrease in 25% and 43% in chickens’ mortality and morbidity, respectively. Interestingly, the phage cocktail (of phi F61E, phi F78E and phi F258E) administered in the water drinking and by spray in a single application, and composed by 5×107 PFU/ml of each bacteriophage, was able to control the mortality rate in naturally infected chicken flocks, refractive to antibiotherapy. The mortality felt from 2.2% in average, to under 0.5% in no more than three weeks, with no recidivism. In conclusion, with this work it was possible to obtain an antimicrobial product, comprised by a combination of three different lytic phages, which demonstrated to be safe for chickens and efficient against colibacillosis in the poultry industry. |
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