Quantification of FITC-labelled probiotic Lactobacillus salivarius DSPV 001P during gastrointestinal transit in broilers

FUSARI, M.L.; SEQUEIRA, J.G.; BLAJMAN, J.E.; ROSSLER, E.; OLIVERO, C.R.; SIGNORINI, M.L.; ROSSI, L.; FRIZZO, L.S.

San Miguel de Tucumán

Simposio; V Simposio Internacional de Bacterias Lácticas; 2016

Centro de Referencia para Lactobacilos

A good method to investigate the mechanisms by which probiotics mediate their effects is to mark probiotic bacteria and trace them. The aim of this research was to develop a new method to estimate in vivo fluorescein isothiocyanate (FITC)-labelled Lactobacillus salivarius DSPV 001P counts during passage through the gastrointestinal tract of broilers. The indigenous bacterium L. salivarius DSPV 001P with in vitro probiotic properties was made resistant to rifampicin, labelled with FITC, and administered to broilers through gavage. Forty five, 1 day old Cobb broilers were used in this trial. Programmed necropsies were performed 30 min, 6 h, and 12 h after administration of probiotic bacterium. Samples of 0.1 g of liver, crop, duodenum, and caecum were collected, homogenized in 1/4 Ringer solution, and spread in De Man, Rogosa, Sharpe with rifampicin agar plates to recover only the supplemented strain. Petri dishes were incubated at 37°C for 72 h in anaerobic conditions, and the characteristic colonies were counted. Also, 0.1 g of content and tissue samples were added to the appropriate wells of a black 96 well microtiter microplate and fluorescence was read in a multimode microplate reader. To determine spatial and temporal transit of L. salivarius DSPV 001P in broilers, the number of bacteria as well as its respective fluorescent signal produced by FITC were measured. In order to compare the standard plate count technique and the fluorescence technique, a logistic regression analysis was applied. The evaluation of logistic parameters was performed and the coefficients of determination (R2) were estimated. The amount of fluorescence could be used as an indicator of fluorescent probiotic bacteria in crop and duodenum 30 min after probiotic supplementation. In addition, fluorescent signal could be used to estimate bacterial counts in caecum 6 and 12 h after L. salivarius DSPV 001P administration. To the best of our knowledge, this research is the first in vivo trial to employ the bacterial FITC-labelling technique in order to enumerate probiotic bacteria during gastrointestinal transit in broilers. This model may be useful to monitor the fate of a strain when administered to broilers, simplifying and automating analysis of bacterial probiotic counts through gastrointestinal tract.