Identification of lactate-utilisers bacteria isolated from the rumen of Sanjabi sheep

Lactic acidosis, characterized by excessive accumulation of lactic acid in the rumen, is a major ruminal disorder that reduces productivity and the welfare of animals (Hutton et al., 2010). Normal concentrations of lactate in the rumen are maintained by the microbial balance between the major lactate-producers, and major lactate-utilisers(Klieve et al., 1999). Consequently, due to the accumulation of some organic acids pH decreases followedincreases lactic acid bacteria (LAB) and rumen acidosis occurs. Therefore, isolation and identification of LAB from the rumen should bring about to our knowledge and controlling of fermentative acidosis. In order to characterization of lactate-utilisers bacteria, 48 isolates were collected from ruminal fluid of three Sanjabi sheepfed with diet based on 40 percent roughage and 60 percent concentrate twice a day cultured in specific mediadescribed by Al-Jassim (2003). After total DNA was extracted, 16S rRNA was amplified by PCR from the genomic DNA of each isolate. The primers 27F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and 1512R (5ʹ- ACGGCTACCTTGTTACGACTT-3ʹ) were used to amplify the 16S rRNA (Lane, 1991). To test the genetichomogeneity of isolates, RFLP technique was used with the restriction enzymes Alu1 and Dde1. Four purified PCR products were sequenced in both directions using an automated sequencer via Bioneer laboratory (Bioneer Co., South Korea). DNA sequence data sets were edited using BIOEDIT and characterized by the online BLAST analysis for the most closely related sequences. One of the notable results of this experiment, was Close genetically (99% identity) one of the 16S rRNA gene sequences to the Selenomonus ruminantium. This is maybe the first report of S. ruminantium from ruminal fluid of ruminants

Keywords:

lactate-utilisers bacteria , 16s rRNA gene , Selenomonus ruminantium