Objectives: “Broken mouth” periodontitis (BMP) is a condition of sheep grazed on rough pasture which involves periodontal infection of the incisor teeth and progressive tooth loss. This can reduce the efficiency of grazing of the sheep, which contributes to malnutrition, weight loss and systemic health problems. Consequently, this condition is a major economic problem to sheep farmers. However, there are no treatment or control methods available. The aim of this preliminary study was to use both culture-dependent (aerobic and anaerobic culture) and culture-independent (bacterial 16S rRNA gene cloning and sequencing) methods to identify the bacteria associated with BMP, including cultivable, uncultivable and potentially novel species.
Methods: Swabs were collected from the gingival pockets of two sheep with BMP. Bacterial 16S rRNA genes were amplified from samples using consensus PCR primers and PCR products cloned into a plasmid vector. For each cloned sample, 16S rRNA gene inserts from 50 recombinant clones were digested with RsaI and MnlI and one clone from each RFLP group was partially sequenced. Bacteria were identified by comparing sequence data with 16S rRNA gene sequences from the GenBank and EMBL sequence databases using the BLAST algorithm. Bacteria were also identified by aerobic and anaerobic culture.
Results: Culture-independent methods identified 14 different phylotypes from 36 sequenced clones. Eight clones represented potentially novel and previously undetected phylotypes and nine clones represented uncultivable phylotypes. The most prevalent known species identified were Virgibacillus marismortui, Bacterium ‘New Zealand C', Salibacillus spp., Lysobacter spp. and Streptococcus entericus. Conventional microbiological culture methods identified a much more limited microflora. The species isolated by culture included Mannheimia ruminalis, Veillonella ratti and various streptococci.
Conclusions: A wide range of bacteria may be associated with BMP. This study demonstrates the importance of culture-independent methods in determining the total bacterial flora associated with BMP in sheep.