L and D lactic acid producing bacteria of the equine gastrointestinal tract : identification and molecular characterisation. (Abstract)

Abstract

24A L_and D_lactic acid producing bacteria of the equine gastrointestinal tract: identification and molecular characterisation P.T. Scott1, A.L. Trebbin 1,2 and R.A.M. Al Jassim 2 1 2 Agricultural Molecular Biotechnology Laboratory and School of Animal Studies, University of Queensland, Gatton Qld 4343 raj@sas.uq.edu.au The contribution of lactic acid producing bacteria (LAB) to acidosis and laminitis in horses is well established. Stomach ulceration in horses is also thought to be associated with increases in the production of VFA and lactic acid in the stomach from the relevant LAB. To date, the majority of studies of LAB in the horse have focussed on Streptococcus bovis and Streptococcus equinus (Al Jassim and Rowe 1999). Little is known about other members of the LAB population of the equine gastrointestinal tract. In this paper we report on the identification and molecular characterisation of LAB not previously described from the equine gastrointestinal tract. LAB were obtained from the stomach, small intestine, caecum, colon and rectum contents collected from both roughage fed and laminitisinduced horses. Isolates were initially cultured from serial dilutions (107 and 108) of the contents on modified MRS media. On the basis of fermentation end products, 25 isolates were selected as LAB from a total of 68 isolates. Volatile fatty acid production and L and Dlactate production were determined from pure cultures of each isolate. The 16S rDNA was amplified by PCR from genomic DNA of each isolate. The genetic diversity was initially determined by RFLP analysis with the restriction enzymes MboI, HhaI and HinfI. The PCR products were cloned into pGEMT and sequenced. DNA sequence data were then used to characterise in more detail the selected LAB population. All the selected isolates were able to produce L and/or Dlactate in pure culture. DNA sequence analysis (Figure 1) of the 16S rDNA indicated that most of the isolates were very closely related to species from the genus Lactobacillus, including L. salivarius, L. mucosae and L. delbrueckii. Some isolates were very closely related to Mitsuokella jalaludinii, an organism recently isolated and characterised from the rumen of cattle (Lan et al. 2002). Characterisation of LAB from the equine gastrointestinal tract should contribute to our understanding and management of acidosis, ulceration of the stomach and laminitis. Al Jassim, R.A.M. and Rowe, J.B. (1999). Better understanding of acidosis and its control. Recent Advances in Animal Nutrition in Australia 12, 9197. Lan, G.Q., Ho, Y.W. and Abdullah, N. (2002). Mitsuokella jalaludinii sp. nov., from the rumen of cattle in Malaysia. International Journal of Systematic and Evolutionary Microbiology 52, 713718. 97 100 RA2105 Lactobacillus salivarius 99 RA2116 T057 100 T059/2 RA2053 RA2071 100 100 RA2087 86 Lactobacillus mucosae RA2062 100 Lactobacillus delbrueckii Selenomonas ruminantium Mitsuokella jalaludinii RA2083 100 66 RA2074 58 100 0.02 Figure 1 Phylogenetic relationship of LAB isolates from the equine gastrointestinal tract, based on near complete 16S rDNA sequence data. Recent Advances in Animal Nutrition in Australia, Volume 14 (2003)