Abstract

We successfully established fluorescence in situ hybridization (FISH) method for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific FISH probes were designed based on the L. thermotolerans16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNAgene, 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. The assay was then applied to two individual chicken trials. In trial 1, the cell population of L. thermotolerans ranged from 1.6 ´ 10⁶ to 3.4 ´ 10⁸ cells/g feces and from 2.6 ´ 10⁷ to 3.6 ´ 10⁸ cells/g cecal content. In trial 2, L. thermotolerans had also almost similar concentration (2.0 ´ 10⁶ to 3.4 ´ 10⁸ cells/g feces and 2.7 ´ 10⁷ to 2.9 ´10⁸ cells/g cecal content). We were not able to detect any bacterial cells at day one in both the trials. The results suggest that the newly developed FISH technique might be used for monitoring L. thermotolerans in the chicken intestine despite of its low sensitivity.

Key words: Lactobacillus thermotolerans, fluorescence in situ hybridization (FISH), probiotic, chickens.

Abbreviation

FISH, Fluorescence in situ hybridization; DAPI, 4', 6'-diamidino-2- phenylindole.