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

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

Tucumán

Simposio; SIBAL 2016 V Simposio Internacional de Bacterias Lácticas; 2016

Centro de Referencia para Lactobacilos CERELA CONICET

A good method to investigate the mechanisms by whichprobiotics 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 duringpassage through the gastrointestinal tract of broilers. The indigenousbacterium L. salivarius DSPV 001Pwith in vitro probiotic properties wasmade resistant to rifampicin, labelled with FITC, and administered to broilers throughgavage. Forty five, 1 day old Cobb broilers were used in this trial. Programmednecropsies were performed 30 min, 6 h, and 12 h after administration ofprobiotic bacterium. Samples of 0.1 g of liver, crop, duodenum, and caecum werecollected, 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 thecharacteristic colonies were counted. Also, 0.1 g of content and tissue sampleswere added to the appropriate wells of a black 96 well microtiter microplate andfluorescence was read in a multimode microplate reader. To determine spatialand temporal transit of L. salivarius DSPV 001P in broilers, thenumber of bacteria as well as its respective fluorescent signal produced byFITC were measured. In order to compare the standard plate count technique andthe fluorescence technique, a logistic regression analysis was applied. Theevaluation of logistic parameters was performed and the coefficients of determination(R2) were estimated. The amount of fluorescence could be used as anindicator of fluorescent probiotic bacteria in crop and duodenum 30 min afterprobiotic supplementation. In addition, fluorescent signal could be used toestimate bacterial counts in caecum 6 and 12 h after L. salivarius DSPV 001P administration. To the best of ourknowledge, this research is the first invivo trial to employ the bacterial FITC-labelling technique in order toenumerate probiotic bacteria during gastrointestinal transit in broilers. Thismodel may be useful to monitor the fate of a strain when administered tobroilers, simplifying and automating analysis of bacterial probiotic countsthrough gastrointestinal tract.