In order to verify that the mice were actively infected with Leptospira, bladders were cultured up to 14 days post infection in EMJH and examined for the presence of motile Leptospira by dark field At 7 d.p.i., no leptospires were detected in the bladders of Trif−/− mice and only one WT bladder was positive for bacteria. In contrast, Leptospires were detected in bladder cultures from both groups at 14 d.p.i., indicating a failure to clear the bacteria and establishment of persistent infection in mice (Supplemental Table 1). Even though the burden was elevated in the kidneys of Trif−/− mice at 1 and 3 d.p.i. compared to WT mice, measurement of creatinine in serum and observation of kidneys did not indicate any significant physiological differences in either infected group at 1 or 3 d.p.i. (Supplemental Table 1, Data not shown, Supplemental Fig. 1). Serum Nitrite levels did not differ between WT and infected groups at 7 or 14 d.p.i., further suggesting that there were no physiological differences between WT and Trif−/− mice at 7 or 14 d.p.i. (Supplemental Fig. 1). These results suggest that chronic leptospiral infection is ultimately established in both WT and Trif−/− mice, but significant differences in weight gain and spirochete burden exhibited by the Trif−/− mice compared to WT mice at 1 and 3 d.p.i. indicate that TRIF contributes to murine host defense during leptospiral infection.