Poster Presentation The 2nd Prato Conference on the Pathogenesis of Bacterial Diseases of Animals 2012

Screening of phytogenics against Lawsonia intracellularis with a flow cytometric viability assay (#58)

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

Lawsonia intracellularis (LI) are Gram-negative intracellular pathogens that cause diseases in a wide range of animals. In pigs, LI are of economic importance as the cause of proliferative enteropathy (ileitis). While the acute form of ileitis can cause sudden mortality in finishing pigs, the chronic and subclinical forms can drastically impair the growth performance of grower pigs. For the assay described here, LI were obtained either from a live vaccine or purified from infected cell cultures, diluted in PBS and seeded in U-bottom 96 well plates. Either dilution series of samples or PBS (negative control) were added to the plates, which were then incubated for 30 min at 37 °C, 8% O2 and 8.8% CO2. Afterwards, the bacteria were stained with the BacLight viability kit (Invitrogen), and analyzed with an ACCURI C6 flow cytometer. Bacteria were distinguished from debris in a FSC/SSC-plot. Live and dead bacteria were distinguished by their fluorescence and gated in a green/red fluorescence plot to determine viability rates. The viability rates of sample-treated LI were compared with the mean viability of the control LI to calculate viability rates. The results of samples that showed a dose-dependent anti-bacterial effect were used to calculate EC50 values with a probit regression. Several phytogenic samples showed antibacterial activity. Among them silymarin and a chestnut extract performed best. In vitro screening assays like the one presented in this study shall be the basis for the development of a phytogenic feed additive for LI control.