Supplementary Material for: Effects of highly leukotoxic Aggregatibacter actinomycetemcomitans associated with ligature-induced periodontitis on oral and gut tissues in male rats

This study evaluates the effects of gastric administration of the highly leukotoxic Aggregatibacter actinomycetemcomitans JP2 strain (AaJP2), both with and without ligature-induced periodontitis, on periodontal and small intestinal tissues using an in vivo animal model comprising 40 male Wistar rats (8 weeks old) assigned to four experimental groups (n=10/group) over a 42-day period: a control group (CG) receiving phosphate-buffered saline (PBS) gavage only; a periodontitis group (PG) with ligature-induced periodontitis and PBS gavage; a combined periodontitis and AaJP2 group (PAaG) with both interventions; and an AaJP2-only group (AaG). Periodontitis was induced through cotton ligatures placed around the cervical region of lower first molars bilaterally and maintained throughout the experimental period, while the A. actinomycetemcomitans JP2 strain (HK921) at a concentration of 1×10⁹ CFU/mL in PBS was administered twice weekly via gastric gavage (200 µL) for 6 weeks. Comprehensive morphological and histological analyses were performed, including stereomicroscopic quantification of alveolar bone loss; micro-computed tomography (µ-CT) assessment of bone volume density (%BV/TV), total bone porosity ratio, and trabecular thickness; and histopathological evaluation of inflammatory infiltrate scoring, vascular congestion quantification, and goblet cell density in small intestine segments (duodenum, jejunum, ileum), with measurements of villus height and width. Statistical analysis employed the Kolmogorov-Smirnov test for normality assessment, Kruskal-Wallis and Mann-Whitney U tests for intergroup comparisons, chi-square test for categorical variables, Wilcoxon signed-rank test for weight gain analysis, and Spearman's rank correlation coefficient for correlation studies, with significance set at p<0.05 using SPSS version 22.0.