Detection of Viable but Non-Culturable (VBNC)-Campylobacter in the Environment of Broiler Farms: Innovative Insights Delivered by Propidium Monoazide (PMA)-v-qPCR Analysis

Abstract

Campylobacteriosis cases in humans are of global concern, with high prevalence rates in the poultry reservoir considered the most important source of infection. Research findings show Campylobacters’ ability to enter a viable but non-culturable (VBNC) state, remaining “viable” but unable to grow on culture media. We explored the persistence of VBNC states in specific environments, particularly at broiler farms, as this state may lead to an underestimation of the present Campylobacter prevalence. For VBNC detection, a propidium monoazide PMA-dye viability qPCR (v-qPCR) was used in combination with cultivation methods. We examined samples collected from broiler farm barns and their surroundings, as well as chicken manure from experimental pens. In addition, the tenacity of culturable and VBNC-Campylobacter was studied in vitro in soil and water. In a total of three visits, Campylobacter was not detected either culturally or by v-qPCR (no Campylobacter DNA) in the environment of the broiler farms. In four visits, however, VBNC-Campylobacter were detected both inside and outside the barns. The overall prevalence in environmental samples was 15.9% for VBNC-Campylobacter, 62.2% for Campylobacter DNA, and 1.2% for culturable C. jejuni. In the experimental pens, no cultivable C. jejuni was detected in chicken manure after 24 h. Strikingly, “VBNC-Campylobacter” persisted even after 72 h. “VBNC-Campylobacter” were confirmed in barn surroundings and naturally contaminated chicken manure. Laboratory studies revealed that VBNC-Campylobacter can remain intact in soil for up to 28 days and in water for at least 63 days, depending on environmental conditions.