Fig 1.
Stages of proximal femoral head degeneration (Upper row) or tibial head necrosis (Low row) leading progressively to bacterial chondronecrosis with osteomyelitis (BCO).
1. Normal proximal femoral head; 2. Femoral head separation (FHS: epiphyseolysis); 3. Femoral head necrosis, FHN. 4. Normal proximal tibial head with struts of trabecular bone in the metaphyseal zone fully supporting the growth plate; 5. Tibial head necrosis (THN). Lytic channels (small arrows) penetrate from the necrotic voids into the growth plate. 6. Tibial head necrosis (THNsc). Bacterial infiltration and sequestrae (open arrows) provide macroscopic evidence of osteomyelitis.
Fig 2.
Design of the PCR primers and the amplicon library.
V6 rRNA region were amplified using the two primers with barcode tags and Illumina adaptors attached at 5’ ends.
Fig 3.
The incidence of lameness in two lines (line B and line D) developed on wire flooring and on wood-shavings litter flooring from days 14 through 49.
Fig 4.
Taxonomic composition of the bone samples at phylum level.
Fig 5.
Comparison of the α-diversity in different groups of bone samples.
Diversity levels were estimated using Chao1 index with rarefied 180 V6 rRNA reads per sample. Means with the same letters in red color within the same row are not different significantly (p>0.05). Means with the same letters in blue within the same column are not different significantly (p>0.05).
Fig 6.
Hierarchical clustering analysis of the bacterial communities.
Phylogenetic tree shows that most of FHS (dark blue), THN (green), FHN (pink) and THNsc (red) samples can be clustered. Femur Normal (black), and Tibia Normal (light blue) were largely mixed together in two different clusters.
Table 1.
Analysis of similarities (ANOSIM).
Fig 7.
Principal Coordinate analysis.
Unweighted PCoA on Qualitative measures and weighted PCoA on Quantitative measures illustrates the differences between Normal and BCO communities. Normal communities are tightly clustered as compared to the scattered BCO communities.
Fig 8.
Comparison between Normal and BCO samples using LEfSe analysis.
Taxa abundance analysis determined 7 features were differentially abundant in BCO group (LDA score >2.4) and 21 features (LDA >2.4) in Normal cases. The genus Staphylococcus, which has been frequently isolated from BCO samples by previous researchers, is an abundant feature in BCO case.
Fig 9.
Taxonomic composition of the bone samples based at genus level.
Only 15 major genera of significant importance were selected and shown in the graphs.
Fig 10.
Comparison of samples using unweighted UniFrac.
Unweighted UniFrac was used to generate a matrix of pairwise distances between communities, then a scatterplot was generated from the matrix of distances using Principal Coordinate Analysis. Number = chicken ID, R = right, L = left, F = femur, T = tibia. A. Comparison of 4 samples from the same chicken, which has FHS on both femurs and THN on both tibias. B. Comparison of the samples from different individual chickens. Paired samples from same individual chickens have FHS on femurs and THN on tibias.