Poster Presentation The 3rd Prato Conference on the Pathogenesis of Bacterial Diseases of Animals 2014

Two assays for in vitro screening of phytogenics against Lawsonia intracellularis (#27)

Andreas Koestelbauer 1 , Klaus Teichmann 1 , Gerd Schatzmayr 1
  1. BIOMIN Research Center, Tulln, Austria

Lawsonia intracellularis (LI) are Gram-negative bacteria that are of economic importance in pig husbandry as the cause of proliferative enteropathy (ileitis). The acute form of ileitis can cause sudden mortality close to slaughter age, while chronic and subclinical forms can drastically impair growth performance in younger pigs. LI do not proliferate in a cell-free medium. This means that conventional inhibition assays like agar plate or broth micro dilution assays cannot be used for research. Our objective was to develop an assay to screen phytogenic samples for their activity against LI. Two methods were established: A viability assay, evaluated by flow cytometry, and a cell culture assay, evaluated by a microplate reader. For both, LI were reconstituted from a live vaccine (Boehringer Ingelheim, Germany), filtered and washed to remove debris. For the viability assay, LI were incubated with phytogenic samples, stained with fluorescent dyes and analyzed with a flow cytometer (ACCURI C6). Viable and damaged bacteria were distinguished via fluorescence. For the cell culture assay, LI were co-cultured with McCoy mouse fibroblast cells in 96 well plates in the presence of phytogenic samples. After 5 d the cells were fixed and intracellular bacteria were stained with a primary anti-LI and a secondary fluorescein-conjugated antibody. The fluorescence of intracellular bacteria was measured with a microplate reader. Several substances (phytogenics with known antibacterial activity, and antibiotics) were tested with both assays. Several phytogenic samples showed antibacterial activity. Among the used antibiotics tylosin tartrate was able to inhibit LI in the cell culture assay, but showed no activity in the viability assay. In vitro screening assays, like those presented in this study, are the first step for the development of a phytogenic feed additive for LI control.