Effect of Plant Derived Antimicrobials on Salmonella Enteritidis Adhesion to and Invasion of Primary Chicken Oviduct Epithelial Cells in vitro and Virulence Gene Expression

Abstract

Salmonella Enteritidis (SE) is a major foodborne pathogen in the United States and one of the most frequently reported Salmonella serotypes globally. Eggs are the most common food product associated with SE infections in humans. The pathogen colonizes the intestinal tract in layers, and migrates to reproductive organs systemically. Since adhesion to and invasion of chicken oviduct epithelial cells (COEC) is critical for SE colonization in reproductive tract, reducing these virulence factors could potentially decrease egg yolk contamination. This study investigated the efficacy of sub-inhibitory concentrations of three plant-derived antimicrobials (PDAs), namely carvacrol, thymol and eugenol in reducing SE adhesion to and invasion of COEC, and survival in chicken macrophages. In addition, the effect of PDAs on SE genes critical for oviduct colonization and macrophage survival was determined using real-time quantitative PCR (RT-qPCR). All PDAs significantly reduced SE adhesion to and invasion of COEC (p < 0.001). The PDAs, except thymol consistently decreased SE survival in macrophages (p < 0.001). RT-qPCR results revealed down-regulation in the expression of genes involved in SE colonization and macrophage survival (p < 0.001). The results indicate that PDAs could potentially be used to control SE colonization in chicken reproductive tract; however, in vivo studies validating these results are warranted.

In-Feed Supplementation of trans -Cinnamaldehyde Reduces Layer-Chicken Egg-Borne Transmission of Salmonella enterica Serovar Enteritidis

“…The pelleted epithelial cells were resuspended in whole medium and allowed to grow until a confluent monolayer was formed. After four successive passages, the cells were seeded onto 24-well cell culture plates (ϳ2 ϫ 10 5 cells per well) and grown at 39°C under 5% CO 2 for 24 to 36 h. The identity of COEC was confirmed by determining the constitutive expression of the avian beta defensin (Av␤D) genes by realtime quantitative PCR (RT-qPCR) as described previously (42).…”

“…Primary chicken oviduct epithelial cells (COEC) were isolated as described previously (41,42). The oviduct tissues of 25-to 28-week-old Salmonella-free layer hens (single comb, White Leghorn) were obtained from the University of Connecticut poultry farm.…”

“…The adhesive and invasive abilities of three S. Enteritidis isolates on COEC were investigated as previously described (42). S. Enteritidis was cultured to mid-log phase in either the absence (control) or presence of TC at the SICs before inoculation onto COEC.…”

“…Chicken macrophages (HTC; chicken monocyte cell line) were cultivated in RPMI 1640 with 10% FBS. The cells were activated and plated as described previously (42,43). Twenty-four hours prior to infection, the cells were seeded in 24-well tissue culture plates and incubated at 39°C under 5% CO 2 to form a monolayer.…”

“…The infected COEC were lysed with 0.1% Triton X-100 (Invitrogen), and the number of viable adherent S. Enteritidis organisms was determined by serial dilution and plating on TSA and XLD plates. For the invasion assay, a gentamicin protection assay was performed as described previously (38,42). The S. Enteritidis-inoculated monolayer was incubated for 1 h, followed by three washings in specific minimal medium (MEM).…”