Fig 1.
Preparation of different starting inoculum sizes.
To determine the effect of starting inoculum size on the growth of different Brachyspira spp. in both agar and liquid media, serial dilutions (10−1 to 10−9) of starting inoculum sizes were prepared. Then broth cultures with different inoculum sizes were inoculated by adding 100 μl of starting inoculum to brain heart infusion (BHIS) vial and by adding 2 μl to the agar media.
Fig 2.
The effect of optical density on determination of bacterial density (CFU/ml) in a broth culture of Brachyspira spp.
Relationship between optical density and colony forming unit of: (A) B. pilosicoli (OD), (B) B. hampsonii, (C) B. hyodysenteriae. (D) Plotted equations for all three species determined from experimental data and averaged (red line) into have an equation which can predict the bacterial density (CFU/ml) in a broth culture.
Fig 3.
The growth of B. pilosicoli in brain heart infusion (BHI) broth with different starting inoculum sizes.
Visible growth (turbidity = (+) growth, no turbidity = (-) growth) in brain heart infusion broth (supplemented with 1% defibrinated serum) with decreasing starting inoculum size (from left to right: uninoculated control (blank), (A) 8.7×108 growth, (B) 8.7×107 growth, (C) 8.7×106 growth, (D) 8.7×105 no-growth, (E) 8.7×104 no-growth, (F) 8.7×103 no-growth, (G) 8.7×102 no-growth, (H) 8.7×101 no-growth, (I) ~8.7 CFU/ml) no-growth.
Fig 4.
Visible growth of B. hampsonii on trypticase soy agar (TSA) with different starting inoculum sizes.
Three replicates were included for each inoculum and a total volume of 2 μl were spotted onto each plate. (1a-1c) 8,000 CFU/spot, (2a-2c) 800 CFU/spot, (3a-3c) 80 CFU/spot, (4a-4c) 8 CFU/spot, (5a-5c) >1 CFU/spot, (6a) >1 CFU/spot. Haemolysis was used as an indicator of visible growth.
Table 1.
Minimum inoculum of three Brachyspira species required for growth on liquid and solid media.
Table 2.
Effect of inoculum size on observed minimum inhibitory concentration.
Table 3.
Reproducibility of the standard agar dilution method developed.
Fig 5.
Phylogenetic tree of Brachyspira spp. tested.
Phylogenetic tree of partial nox sequences of diagnostic isolates and types strains of B. hyodysenteriae, B. pilosicoli, B. innocens and B. murdochii based on a 765 base pair alignment. Sequences associated with B. hamponii genomevar I (B. hampsonii_30599; AOMM00000000), and B. hampsonii genomevar II (B. hampsonii_30446; IDAC No 161111–01, ALNZ00000000) are also included.
Table 4.
The number of isolates from each production system.
Table 5.
Antimicrobial minimum inhibitory concentration distribution.