Avian pathogenic E. coli in Norwegian broiler Production - characterisation, identification of risk factors and prevention

The first week of life is a critical phase for broilers. The Norwegian poultry industry is aiming to reduce first-week mortality (FWM) in broilers to a level below 0.8 %. Nevertheless, Norway as well as other European countries have experienced periods of increased FWM due to E. coli infections (colibacillosis) during the previous years. The research project APEC-Seq aims to reduce the overall occurrence of colibacillosis in broilers through a close collaboration with participants from the poultry industry and scientists working with animal health, bacteriology and epidemiology. We have performed a systematic sampling from 54 target flocks (flocks with high first week mortality; FWM), 20 control flocks, 21 parent flocks and 7 samples from hatcheries from 2018-June 2021. Protocols for sampling and a submission form - questionnaire have been developed for systematic sampling from target flocks (flocks with high FWM), control flocks, parent flocks and hatcheries. The sampling includes autopsy, registration of pathological findings, sampling for bacteriological examination and registration of data from the flock and its environment and will be associated with the whole genome sequencing (WGS) data. In a pilot project, we have performed WGS on 47 E. coli isolates from three target flocks. We wanted to explore how similar E. coli isolates are within an animal and within a flock, and also investigate which organ was best suited for a harmonized sampling. We aimed to sequence 15 E. coli isolates per flock (five animals per flock and three organs per animal). Results show that E. coli within a bird usually have the same serotype and 7-gene MLST sequence type (ST). Phylogenetic analysis also show a high degree of similarity between isolates within a flock as well as in two of the flocks. E. coli isolates from liver seems to represent avian pathogenic E. coli (APEC), the main cause of disease, and can be a preferred organ for sampling. Paper 1 is submitted and we are waiting for the revision from the scientific journal. Based on the pilot project we selected one E. coli per bird (five in total per flock), mainly from liver, for WGS for further studies. At the moment we have focused on target ? and control flocks and have sequenced 286 and 78 isolates respectively. Initial bioinformatic analysis as quality control of data, assembly and identification of STs have been performed. The main aim is to compare APEC from target flocks within and between flocks as well as comparison to E. coli from control flocks. Further bioinformatics analyses will contain screening for virulence associated genes and serotype in addition to ST. Based on these results phylogenetic analysis will be performed on a selection of isolates. This work will end up in paper 2 for the PhD student. In spring 2021 there was a colibacillosis outbreak in Norway. Some of the samples in this project were part of the outbreak and was followed up closely with tracing of parent flocks as well as grandparent flocks. APEC was sequenced consecutively and identified as ST23. APEC-Seq has in collaboration with the industry and collaborators abroad got access to WGS data from the grandparent flock and poultry isolates from Finland with the same grandparent flock. Preliminary results indicate that the isolates are similar and probably a common source. However, the isolates are more different than we are used to see in outbreaks with zoonotic agents. We plan to write a scientific paper about the outbreak. All sampled data from the sampling 2018-2021 have been collected and transcribed. In addition, we plan to retrospectively sample data from control flocks in collaboration with the industry partners. The data will be used for further analysis and identification of risk factors associated with high FWM and are planned as paper 3 for the PhD student. Through the systematic sampling APEC-Seq aims to characterize and compare E. coli strains causing disease in poultry by WGS, a tool for determining the complete genetic material (DNA) from the bacteria. Combining these data with production-, management- and environmental data we want to identify potential risk factors for disease, as well as increase the research-based knowledge on E. coli causing disease in poultry; how similar are these bacteria really; can we isolate them from both parent and production animals (breeding pyramid); which parameters in the broiler environment are risk factors for disease. Results will be used to develop a framework for surveillance and monitoring of E. coli infections in broilers, i.e. identification of bacterial strains with strong disease-inducing properties in an early phase and implementation of best practice guidelines for prevention of colibacillosis in broiler production. APEC-Seq will have a significant impact on animal health and welfare, enable sustainable and cost-effective food production and increase consumer confidence in poultry meat.

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By whole genome sequencing (WGS) of avian pathogenic Escherichia coli (APEC) combined with advanced statistical modelling of complex data APEC-Seq aims to reduce the overall occurrence of colibacillosis in broilers. Important gaps of knowledge are clonality and dissemination of APEC strains through the breeding pyramid and significance of risk factors linked to management and environment. The Norwegian poultry industry has an aim of keeping first-week mortality (FWM) in broilers below 0.8 %. However, during the last couple of years broiler flocks in Norway, other Nordic countries and elsewhere in Europe have experienced periods of sudden and inexplicable increase in FWM due to colibacillosis. APEC-Seq joins actors from research and industry working with clinical bacteriology, epidemiology, poultry health management and broiler production in order to improve the evidence-based knowledge on APEC and predisposing factors. By systematic sampling in high mortality flocks, APEC-Seq will characterize and compare APEC isolates by WGS, identify potential risk factors from production, management and environment and quantify their relative contribution in advanced statistical modelling. Results will be used to develop a framework for monitoring APEC in broilers, i.e. identification of APEC with strong disease-inducing properties at an early phase and implementation of best practice guidelines for prevention of colibacillosis in broiler production. APEC-Seq will have a significant impact on animal health and welfare, enable sustainable and cost-effective food production and increase consumer confidence in poultry meat.