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Abstract:

Avian pathogenic Escherichia coli (APEC) cause yearly a huge economic damage to the poultry industry due to mortality, slower growth, a lower feed conversion efficiency and condemnation of carcasses at the abattoir. Inhalation of dust contaminated with high concentrations of Escherichia coli can infect the air sacs and lungs, whereafter bacteria can enter the bloodstream, causing systemic disease (known as colibacillosis). The disease is traditionally treated with antibiotics, but due to frequent use of antibiotics many APEC strains have become multidrug resistant. The use of antibiotics raises a public health concern, due to transfer of resistance genes/resistant bacteria from animals to humans. Therefore in this study the use of disinfectants and bacteriophage therapy were tested as alternative prophylaxis/treatment for colibacillosis in chickens. First, antibiotic resistance of a selection of APEC strains (n = 97), representative for the Belgian laying hen industry, was determined since APEC isolates from laying hens are less studied than isolates from broilers. Furthermore, it was determined if these APEC isolates were resistant against the most included active ingredients (formaldehyde, glutaraldehyde, glyoxal, hydrogen peroxide (H2O2), and a quaternary ammonium compound (QAC)) in routinely used disinfectants in the poultry industry. The biofilm-forming capacity of the strains was determined on materials frequently used in poultry barns, since biofilm formation can have an effect on the efficacy of disinfectants. High resistance percentages were found against antibiotics frequently used in the poultry industry (ampicillin, nalidixic acid, sulfonamides and trimethoprim). However, no phenotypically detectable acquired resistance against routinely used disinfectants could be found, while resistance genes were present. Most APEC strains were only moderate biofilm producers on polystyrene, while on polypropylene and polyvinyl chloride they were strong biofilm producers. Hence, the efficacy of two of the five selected active ingredients in disinfectants (H2O2 and QAC) was tested to eliminate sessile cells. H2O2 and QAC were able to eliminate all adhering cells at a concentration of 1% and 0.01%, respectively. Normally, disinfectants are used in poultry houses to prevent disease carry-over between sequential flocks, whereby the number of specific pathogens is lowered as much as possible. However, during this study the efficacy of nebulization of low concentrations of H2O2 to reduce infection pressure in the presence of chickens was determined. Chickens respiratorily exposed to APEC and subsequently to 2% H2O2 had nonetheless a higher chance of developing a high lesion score associated with APEC, than chickens that were exposed to APEC and subsequently to 1% H2O2 or distilled water, contraindicating H2O2 nebulization in the presence of chickens. During the second part of the study, a bioluminescent APEC strain (CH2-lux) was developed by transposition of the lux operon of Photorhabdus luminescens in the strain used for infection studies (CH2). The transposition of the lux operon into the chromosome of the APEC isolate did not affect the virulence of the latter strain, nor its sensitivity for lytic bacteriophages (phages). Ex vivo correlations were found between the luminescence and bacterial numbers in the homogenized heart, liver and lung of four-week-old CH2-lux infected chickens. The marked strain can also be used to increase knowledge on the pathogenesis of the disease, in order to find targeted treatments against colibacillosis. A bacteriophage cocktail composed of four phages was developed after an in vitro selection of the phages. They were administered to experimentally infected chickens intratracheally, intra-esophageally or via the drinking water. However, treated groups did not show a significant decrease in mortality, lesion scores or weight loss compared to infected/untreated groups. The results indicate that even though in vitro the phage cocktail efficiently lysed the infecting strain, the phage cocktail could not cure experimentally infected chickens. More research is needed to analyze the in vivo therapeutic and prophylactic effect of phages. The developed luminescent APEC strain can thereby possibly provide solutions. During this study various experiments were performed to evaluate the application of disinfectants and bacteriophages to reduce avian colibacillosis, even though many valuable results were found, still much research is needed to expand knowledge on the use of disinfectants and bacteriophages in the battle against colibacillosis.