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 %. 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 September 2018 to June 2021 and sampled 51 case flocks (flocks with high first week mortality; FWM), 22 control flocks, 21 parent flocks and 14 samples from hatcheries. 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 necropsy, 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 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). Based on the results we could conclude that show that E. coli within a bird usually were similar. Phylogenetic analysis also showed a high degree of similarity between isolates within a flock and we proposed to sequence one E. coli from at least three individuals. 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. 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. We have sequenced E. coli isolates from target and control flocks, 218 and 80 isolates respectively. Bioinformatic analysis showed high diversity within both groups. E. coli from the target flocks are published in a separate paper where two outbreaks have been described. E. coli ST429, O2/O50:H1 caused an outbreak in 2018-2019 including six of the sampled flocks. In spring 2021, there was a colibacillosis outbreak in Norway and E. coli ST23, O78:H4 was detected in 17 target flocks, but flocks with high mortality at a later stage was also affected. The outbreak was followed up closely in APEC-Seq with tracing of parent flocks as well as grandparent flocks and regularly WGS. Bioinformatic analysis indicate that the isolates are similar and probably originate from a common source. WGS and bioinformatics analysis, in combination with epidemiological data, indicate that this E. coli variant is transmitted from a higher level of the breeding pyramid, possible at grandparent level. All sampled data from the sampling 2018-2021 have been collected and transcribed. For each target flock, two control flocks were identified retrospectively, and not all data could be collected retrospectively. Collected data was used for further analysis and identification of risk factors associated with high FWM, where two variables came out as significant; "high FWM in the previous flock" and "flock size". The PhD student employed in APEC-Seq completed and defended her thesis in July 2023. Through APEC-Seq, we have build experience with WGS of E. coli and what opportunities it provides for the poultry industry. After the end of the project, the possibility of setting up a monitoring system to monitor and control APEC in poultry will be followed up by the poultry industry. It is difficult to define clear interventions for reducing high FWM in broilers based on the risk factors identified in the project. The project participants agree that management in a poultry flock is an important factor for success, and the first days after the broilers have arrived in the house are particularly important. In addition, our results, together with existing knowledge about E. coli, show that washing, disinfection and time without broilers in the house can be decisive factors. A webinar will be held for veterinarians and advisers in the poultry industry focusing on what is required to provide a good start, as well as routines and recommendations for washing and disinfection.

Kunnskapsoverføring til fjørfenæringen om helgenomsekvensering (WGS) og bruk av ny teknologi særlig med tanke på karakterisering av sykdomsfremkallende E. coli og smittesporing har vært fokus gjennom hele prosjektperioden og ble demonstrert for gjennom utbruddet av colibacillose våren 2021. APEC-Seq har bygget en basis for diagnostikk og overvåkning for APEC basert på detaljert karakterisering gjennom WGS. Etter prosjektets slutt er det et ønske om å teste ut muligheten for overvåkning av APEC basert på WGS for å monitorere utviklingen APEC og fange opp mulige utbrudd så tidlig som mulig, og dermed kunne redusere sykdom og tap av dyr. Gjennom APEC-Seq prosjektet har forskerne på Veterinærinstituttet og fjørfenæringen lært mye av hverandre gjennom kunnskapsdeling, som gjør at vi lettere kan diskutere/identifisere problemstillinger vi bør/kan studere nærmere. For forskningsfeltet har APEC-Seq gitt økt detaljkunnskap om APEC. Det har også gitt en økt forståelse for hvor like/ulike bakterieisolater innad i et individ eller en flokk er, og om utvikling/evolusjon av APEC. Vi har bygget opp en database av helgenomsekvensdata for sykdomsfremkallende E. coli fra fjørfe som er systematisk innsamlet, som vil ha forventet virkning og effekt for fremtidig overvåkning nasjonalt og eventuelt internasjonalt. Databasen av helgenomsekvenser vil være relevant for forståelse av evolusjon av bakterier og for hvordan man definerer utbrudd i store dyrepopulasjoner sammenliknet med utbrudd av zoonotiske agens på mennesker. Økt detaljkunnskap om APEC, norsk fjørfeproduksjon og en relevant database av helgenomsekvenser vil være relevant og fordelaktig for internasjonalt forskningssamarbeid. I videre forstand har APEC-Seq prosjektet gitt oss økt kunnskap og kompetanseutvikling med tanke på bruk av helgenomsekvensering i smittesporing og karakterisering av sykdomsfremkallende bakterier i dy generelt. Det vil også ha en effekt for utvikling av helgenomsekvensering, bioinformatikk og forståelse av bruken av ny teknologi i relevante problemstillinger for forskerne på Veterinærinstituttet. En annen viktig virkning i prosjektet er en stipendiat som har fullført PhD utdannelse og som bidrar til økt kompetanse innen fjørfehelse, fjørfesykdommer og sekvensering og bioinformatisk analyse.

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.