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

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.

Bacteriophage cocktail application for Campylobacter mitigation - from in vitro to in vivo.

BACKGROUND: Effective strategies are urgently needed to control Campylobacteriosis, one of the most important foodborne gastrointestinal diseases worldwide. Administering bacteriophages (phages) is under evaluation as a possible intervention strategy in primary poultry production to reduce the public health risk of human infection. A major challenge is the translation of results from small-scale animal studies to large broiler flocks. In this study, the in vitro lytic activity of 18 Campylobacter-specific group II phages and 19 group III phages were examined singly, and in different combinations from the same group and from both groups using a planktonic killing assay. Based on these results, a combination of phage NCTC 12,673 (group III) and vB_CcM-LmqsCPL1/1 (group II) was selected for in vivo application in a seeder bird model to study its effectiveness under conditions as close as possible to field conditions. One hundred eighty Ross 308 broiler chickens were divided into a control and a treatment group. Ten days post hatch, seeder birds were orally inoculated with the C. jejuni target strain. Phages were administered via drinking water at a total concentration of 107 PFU/mL four, three, and two days before necropsy. RESULTS: Combining group II and group III phages resulted in significantly higher in vitro growth inhibition against the C. jejuni target strain BfR-CA-14,430 than single application or combinations of phages from the same group. The results of the animal trial showed that the application of the two phages significantly reduced Campylobacter counts in cloacal swabs. At necropsy, Campylobacter counts in colonic content of the treatment group were significantly reduced by 2 log10 units compared to the control group. CONCLUSIONS: We demonstrated that combining phages of groups II and III results in significantly increased lytic activities. The in vitro results were successfully translated into practical application in a study design close to field conditions, providing new data to apply phages in conventional broiler flocks in the future. Phage application reduced the fecal Campylobacter excretion and Campylobacter concentrations in the colon of broilers.

Antimicrobial effect of a drinking water additive comprising four organic acids on Campylobacter load in broilers and monitoring of bacterial susceptibility.

Application of organic acids via feed or drinking water is under discussion as a possible intervention strategy to reduce Campylobacter (C.) load in primary poultry production. A previous in vitro study showed that reduced concentrations of sorbic acid, benzoic acid, propionic acid, and acetic acid were required for antibacterial activity against Campylobacter when using a mixture of these 4 acids compared to when using the single acids. The present study aimed at determining the antibacterial efficiency of this combination in vivo as a drinking water additive for reducing shedding and intestinal C. jejuni colonization in broilers. Furthermore, we assessed whether the inoculated C. jejuni strain BfR-CA-14430 adapted in vivo to the applied organic acids. Results of this study showed that adding the organic acids consistently reduced Campylobacter loads in cloacal swabs. While significant reductions were observed within the entire study period, a maximum 2 log reduction occurred at an age of 18 d. However, after dissection at the end of the trial, no significant differences were detected in Campylobacter loads of cecal and colon contents compared to the control group. Susceptibility testing of re-isolates from cloacal swabs and cecal content revealed equal minimum inhibitory concentration (MIC) values compared to the inoculated test strain, suggesting that C. jejuni remained susceptible throughout the trial.

Transmission pathways of campylobacter spp. at broiler farms and their environment in Brandenburg, Germany.

Broiler meat is widely known as an important source of foodborne Campylobacter jejuni and Campylobacter coli infections in humans. In this study, we thoroughly investigated transmission pathways that may contribute to possible Campylobacter contamination inside and outside broiler houses. For this purpose we carried out a comprehensive longitudinal sampling approach, using a semi-quantitative cultivation method to identify and quantify transmissions and reservoirs of Campylobacter spp.. Three german broiler farms in Brandenburg and their surrounding areas were intensively sampled, from April 2018 until September 2020. Consecutive fattening cycles and intervening downtimes after cleaning and disinfection were systematically sampled in summer and winter. To display the potential phylogeny of barn and environmental isolates, whole genome sequencing (WGS) and bioinformatic analyses were performed. Results obtained in this study showed very high Campylobacter prevalence in 51/76 pooled feces (67.1%) and 49/76 boot swabs (64.5%). Average counts between 6.4 to 8.36 log10MPN/g were detected in pooled feces. In addition, levels of 4.7 and 4.1 log10MPN/g were detected in boot swabs and litter, respectively. Samples from the barn interior showed mean Campyloacter values in swabs from drinkers 2.6 log10MPN/g, walls 2.0 log10MPN/g, troughs 1.7 log10MPN/g, boards 1.6 log10MPN/g, ventilations 0.9 log10MPN/g and 0.7 log10MPN/g for air samples. However, Campylobacter was detected only in 7/456 (1.5%) of the environmental samples (water bodies, puddles or water-filled wheel tracks; average of 0.6 log10MPN/g). Furthermore, WGS showed recurring Campylobacter genotypes over several consecutive fattening periods, indicating that Campylobacter genotypes persist in the environment during downtime periods. However, after cleaning and disinfection of the barns, we were unable to identify potential sources in the broiler houses. Interestingly, alternating Campylobacter genotypes were observed after each fattening period, also indicating sources of contamination from the wider environment outside the farm. Therefore, the results of this study suggest that a potential risk of Campylobacter transmission may originate from present environmental sources (litter and water reservoirs). However, the sources of Campylobacter transmission may vary depending on the operation and farm environmental conditions.

A Complex Competitive Exclusion Culture Reduces Campylobacter jejuni Colonization in Broiler Chickens at Slaughter Age In Vivo.

Diminishing Campylobacter prevalence in poultry flocks has proven to be extremely challenging. To date, efficacious control measures to reduce Campylobacter prevalence are still missing. A potential approach to control Campylobacter in modern poultry productions is to occupy its niche in the mucosal layer by administering live intestinal microbiota from adult chickens to dayold-chicks (competitive exclusion (CE)). Therefore, this in vivo study investigates the efficacy of a complex CE culture to reduce Campylobacter (C.) jejuni colonization in broiler chickens. For this purpose, the complex CE culture was applied twice: once by spray application to day-old chicks immediately after hatching (on the 1st day of life) and subsequently by an additional application via drinking water on the 25th day of life. We observed a consistent and statistically significant reduction of C. jejuni counts in cloacal swabs throughout the entire fattening period. At the end of the trial after necropsy (at 33 days of age), C. jejuni cecal counts also showed a statistically significant decrease of 1 log10 MPN/g compared to the control group. Likewise, colon counts were reduced by 2.0 log10 MPN/g. These results suggest that CE cultures can be considered a practically relevant control strategy to reduce C. jejuni colonization in broiler chickens on poultry farms.

Emission Sources of Campylobacter from Agricultural Farms, Impact on Environmental Contamination and Intervention Strategies.

Although extensive research has been carried out to describe the transmission pathways of Campylobacter entering livestock farms, the role of livestock farms as source of Campylobacter contamination of the environment is still poorly investigated. It is assumed that Campylobacter-positive livestock farms contribute to an environmental contamination, depending on the animal species on the farm, their Campylobacter status, the housing system, manure management as well as their general farm hygienic and biosecurity management. Different emission sources, like manure, air, water, insects and rodents as well as personnel, including equipment and vehicles, contribute to Campylobacter emission into the environment. Even though Campylobacter are rather fastidious bacteria, they are able to survive in the environment for even a longer period of time, when environmental conditions enable survival in specific niches. We conclude that a significant reduction of Campylobacter emission in the environment can be successfully achieved if various intervention strategies, depending on the farm type, are applied simultaneously, including proper general and personal hygiene, establishing of hygienic barriers, insect controls, manure management and hygienization of stables, barns and exhaust air.