Haemoccult

IL-10KO mice did not develop significant spontaneous colitis in our animal facility. Therefore, colitis was induced by DSS (2 % DSS wt/vol, 36,00–50,00 kDa; MP Biomedicals, Eschwede, Germany) administered in drinking water ad libitum for 5 days at the age of 6 weeks. Cyclooxygenase inhibitor 2 celecoxib (500 μg per mouse per day) was applied by gastric gavage on day 7, 10, and 13 to trigger colon carcinogenesis as described (25 (link), 29 (link)) (Supplementary Figure 1). Clinical severity of colitis (stool consistency, weight loss, and fecal blood) were recorded daily for the first 15 days and three times weekly thereafter. Percent weight changes in relation to baseline were scored as follows: 0, no weight loss; 1, 1–5%; 2, 5–10%; 3, 10–15%; 4, >15%. Stool consistency was scored as follows: 0, well-formed pellets; 2, pasty and semi-formed stools; and 4, liquid stools that adhered to the anus. Rectal bleeding was scored as follows: 0, no blood using haemoccult (Beckman Coulter, Palo Alto, CA, USA); 2, positive haemoccult; and 4, gross bleeding. Deceased mice did not fulfill the criteria for premature exclusion from the experiments but died unexpectedly overnight.

Mice were administered DSS (3% w/vol; molecular weight: 36 to 50 kDa; MP Biomedicals) in drinking water for one week to generate a DSS-induced colitis model, followed by regular drinking water until the end of the study. When the antibiotic cocktails were administered for gut microbiota depletion, mice were administered 2% DSS to prevent preantibiotic-treated mice or KO mice from experiencing enhanced sensitivity^{64 (link)}. Mice were sacrificed by CO₂ inhalation after DSS treatment, and colonic tissues were collected for further examination.
Clinical signs of colitis were evaluated based on the DAI scores, including body weight loss, occult blood, and stool consistency. Mice were scored blindly for the colitis experiments. In brief, the weight loss score was determined as follows: 0, no weight loss; 1, loss of 1–5% original weight; 2, loss of 6–10% original weight; 3, loss of 11–20% original weight; and 4, loss of >20% original weight. The bleeding score was determined as follows: 0, no blood by using Haemoccult (Beckman Coulter) analysis; 1, positive Haemoccult; 2, visible blood traces in stool; and 3, gross rectal bleeding. The stool score was graded as follows: 0, well-formed pellets; 1, semiformed stools that did not adhere to the anus; 2, pasty semiformed stool that attached to the anus; and 3, liquid stools that attached to the anus.

To assess clinical signs of A. butzleri infection on a daily basis, a standardized cumulative clinical score (maximum 12 points), addressing the occurrence of blood in feces (0: no blood; 2: microscopic detection of blood by the Guajac method using Haemoccult, Beckman Coulter/PCD, Krefeld, Germany; 4: overt blood visible), diarrhea (0: formed feces; 2: pasty feces; 4: liquid feces), and the clinical aspect (0: normal; 2: ruffled fur, less locomotion; 4: isolation, severely compromised locomotion, pre-final aspect) was used [31 (link)].