A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345 T and Lactobacillus fermentum JCM 1173 T , and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 ؋ 10 3 cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10 4 cells/g feces on day 4 and 10 5 cells/g feces on days 11 to 18. However, cell populations of 10 6 to 10 7 cells/g feces were detected in the second trial. The total bacterial count, measured by 4,6-diamidino-2-phenylindole (DAPI) staining, was approximately 10 11 cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.Previously, we isolated Lactobacillus thermotolerans, a novel species, from chicken feces collected in Thailand (9). The preference of this bacterium for the chicken intestine may be due to the body temperature of chickens, 42°C (2), which corresponds to the optimum temperature for growth of this bacterium (42°C), as determined by measurement of the specific growth rate (9).Our current interest in L. thermotolerans is to characterize this bacterium ecologically in the chicken intestine, since no studies have been conducted to date on the ecology of this new organism. Studies on the distribution and colonization of L. thermotolerans in different organs of the gastrointestinal tract should provide new insights into chicken intestinal microbiology. For this an effective method for enumeration of this bacterium is required. Development of a molecular ecological enumeration method appears to be particularly valuable in the case of L. thermotolerans, since conventional culture methods are insufficient due to the relatively high temperature required for culturing this bacterium. Real-time PCR offers significant advantages over other molecular enumeration techniques in terms of the speed at which assays are performed and the ability to quantify the target microbial population. Real-time PCR has already been established as a promising tool for studies of the composition of microbial communities in the gastrointestinal tract or feces of humans (1,4,5,12), as well as chickens (13). However, most studies that have focused on the chicken microbiota have been conducted using conventional culture methods (6,8...
