Erysipelothrix spp. and other Erysipelotrichales detected by 16S rRNA microbial community profiling in samples from healthy conventionally reared chickens and their environment

Outbreaks of erysipelas, a disease caused by infection with Erysipelothrix rhusiopathiae (ER), is a re-emerging problem in cage-free laying hen flocks. The source of ER infection in hens is usually unknown and serological evidence has also indicated the presence of ER or other antigenically related bacteria in healthy flocks. The aim of the present study was to evaluate sample collection, culture methods and DNA-based methodology to detect ER and other Erysipelotrichales in samples from healthy chickens and their environment. We used samples from a research facility with conventionally reared chickens with no history of erysipelas outbreaks where hens with high titres of IgY recognising ER previously have been observed. Microbial DNA was extracted from samples either directly or after pre-culture in nonselective or ER-selective medium. Real-time PCR was used for detection of Erysipelothrix spp. and high-throughput amplicon sequencing of 16S rRNA sequencing was used for detection of Erysipelotrichales. A pilot serological analysis of some Erysipelotrichales members with IgY from unvaccinated and ER-vaccinated high-biosecurity chickens, as well as conventionally reared chickens, was also performed. All samples were negative for ER, E. tonsillarum and E. piscisicarius by PCR analysis. However, 16S rRNA community profiling indicated the presence of several Erysipelotrichales genera in both environmental samples and chicken intestinal samples, including Erysipelothrix spp. that were detected in environmental samples. Sequences from Erysipelothrix spp. were most frequently detected in samples pre-cultured in ER-selective medium. At species level the presence of Erysipelothrix anatis and/or Erysipelothrix aquatica was indicated. Serological results indicated that IgY raised to ER showed some cross-reactivity with E. anatis . Hence, environmental samples pre-cultured in selective medium and analysis by 16S rRNA sequencing proved a useful method for detection of Erysipelotrichales, including Erysipelothrix spp., in chicken flocks. The observation of such bacteria in environmental samples offers a possible explanation for the observation of high antibody titres to ER in flocks without a history of clinical erysipelas.