Colonization patterns of Enterococcus cecorum in two different broiler production cycles detected with a newly developed quantitative real-time PCR

Arne Jung, Henning Petersen, Lydia Teske, Silke Rautenschlein

Although Enterococcus cecorum (EC) infection is one of the most important bacterial diseases in modern broiler chickens today, many aspects of epidemiology and pathogenesis are still unknown. There is a need for better detection methods for EC than classical cultivation. In the present study, we describe the validation and application of a newly developed quantitative TaqMan real-time PCR (qPCR) assay based on the 16S-rRNA-gene for the detection of EC. Fifty EC strains isolated from 12 different animal species were detected with the assay, while none of the other 26 examined bacterial species were tested positive during validation procedure. The detection limit of the PCR was 6.25 CFU/ml PBS. The qPCR assay was also considerably more sensitive using intestine and organ samples than the classical cultivation method. Field application of the PCR setup was tested comparing two different broiler production cycles on one farm: in cycle I broilers showed signs of enterococcal spondylitis (ES) from day 24 post hatch onwards while broilers in cycle II developed no ES. Two totally different colonization patterns were found in the two cycles with the qPCR using cloacal swabs. Animals in cycle I showed significantly (P ≤ 0.05) higher detection rates of EC at the day of placement and throughout the cycle than broilers of cycle II. Additionally, significantly higher detection rates were found in the cecum compared to duodenum, jejunum and ileum within one cycle. The new qPCR for EC is highly specific, more sensitive than classical cultivation and was able to show differences in colonization in a broiler cycle with later EC disease outbreak compared to a healthy cycle. These findings may be explained by infection with different strains, pathogenic EC isolates are probably more effective in colonization than commensal isolates. A high correlation was found between qPCR results from cecum and cloacal swabs in this study, indicating that cloacal swabs can be used to examine intestinal colonization of broilers with EC. The new qPCR significantly improves the diagnostic of EC infections and may help to answer open questions concerning epidemiology and pathogenesis.