DNA amounts were quantified by using a standard curve obtained with results of tenfold serial dilutions of lysates of 1 to 106 bacteria. All measurements were done in duplicate. Guinea pig infection All experiments on animals were performed with the approval of the Animal Care and Use Committee of Gamaleya Institute of Epidemiology and Microbiology. T. pyriformis and L. monocytogenes EGDe strain were co-cultured for 7 days in 100 ml LB broth at 28°C. On day 7 cyst C59 wnt research buy concentration exceeded that of trophozoites.

After that https://www.selleckchem.com/products/BIBF1120.html in the remaining vegetative cells the encystment was promoted by their incubation at +4°C overnight. This was followed by the removal of extracellular bacteria with gentamycin treatment (100 μg/ml) for 2 h at room temperature. Control bacteria were grown overnight on LB plates, suspended in 1 ml of PBS, diluted with PBS to a concentration of 109 CFU/ml and kept frozen in 10% glycerin. Groups VX-680 mw of three female 350 g guinea pigs were infected intraconjunctivally

by applying a cotton wool tampon saturated with the T. pyriformis cyst water suspension at concentration 8.9 x104 cyst/ml, which contained 1 × 106 L. monocytogenes CFU/ml or with L. monocytogenes suspension at concentration 1 × 106 CFU/ml. Bacterial loads were equalized using qPCR as described above. Three guinea pigs were infected with 1 × 105 axenic T. pyriformis cysts as a control. For oral inoculation, 1 ml of water suspension containing L. monocytogenes in concentration 1 × 106 CFU/ml (clogged in cysts or from the culture) was introduced to the back of oral cavity of three animals. The animals were not fed for 12 h before triclocarban infection. The concentration of L. monocytogenes in faeces was determined daily by plating serial dilutions on the selective medium (PALCAM agar, HiMedia, India). On day 3 (72 h after infection) animals were anaesthetized by chloroform and sacrificed. The liver and the spleen were homogenized in PBS and serial dilutions of homogenate material were plated on LB agar. Microscopic studies Transmission electron microscopic investigations were performed in general

as described in [44]. In short, microorganisms were fixed with phosphate-buffered osmium tertraoxide according to [45], dehydrated in alcohols of increasing concentrations, and embedded in araldite M. Ultrathin sections were produced on an LKB-3 ultratome, and studied in a GEM 100B electron microscope. Up to six sections for one sample were studied. Light microscopic studies were performed with Olympus IX-71 microscope. Acknowledgements Authors are grateful to Prof. J.A. Vazquez-Boland, Univ. Bristol, UK, for a gift of the L. monocytogenes strains EGDe, EGDeΔhly, NCTC5105 and the L. innocua strain NCTC11288, and to Prof. T.R. Klaenhammer, North Carolina State University, for a gift of the vector pTRKL2. Authors highly appreciate Dr. L. Didenko and Dr. N. Konstantinova for the help with electron microscopy.