57. Mitigating the Zoonotic Risk of Shiga-toxigenic Escherichia coli (STEC) O157:H7 in Cattle: Evaluation of Limosilactobacillus Reuteri as a Direct-fed Microbial.

Abstract Background Shiga toxin-producing Escherichia coli (STEC), particularly serotype O157:H7, remains a major food safety concern in North America. Cattle are the primary reservoir, shedding STEC in feces and contributing to contamination of beef products and farm environments. Direct-fed microbials (DFMs) are beneficial microorganisms administered to livestock to improve gut health and potentially reduce pathogen load. Among them, Lactobacillus spp. are widely used due to their probiotic properties and ability to inhibit enteric pathogens. Objective This study evaluated whether Limosilactobacillus reuteri R0444 could reduce STEC O157:H7 shedding in calves. Methods Two independent animal trials were conducted in 2025. After a two-week adaptation period, twelve Holstein male calves (13-14 weeks old) per trial were randomly assigned to a control group inoculated with a five-strain mixture of nalidixic acid-resistant STEC O157 or a treatment group receiving both STEC and L. reuteri R0444. All calves were orally inoculated with 1010 CFU of STEC O157 on Day 0, while calves supplemented with L. reuteri (1010 CFU) on d 0, 10, and 17. Fecal samples were collected on d 1, 3, 5, 7, 10, 15, 22, and 28 to quantify and detect STEC shedding using selective plating and immunomagnetic separation (IMS). The quantity of DFM strain in fece was monitored by qPCR throughout the experiments. Fecal microbiota was characterized by 16S rRNA gene sequencing processed in QIIME2 with DADA2. Data were analyzed using generalized linear mixed models (SAS PROC GLIMMIX). Results Across both trials, STEC shedding differed by treatment (p = 0.04) and time (p < 0.001), with DFM treatment reducing shedding by an average of 0.7 log10 CFU/g of feces. When the study period was stratified (Days 1-3, 5-7, and 10-28), a treatment-by-time interaction was detected (p = 0.03) along with a strong effect of time (p < 0.001). Differences between groups became evident after the initial post-inoculation phase. Treated calves shed fewer STEC (p < 0.05) during Days 5-7 and Days 10-28 compared with controls. The largest difference occurred on Day 22 (p = 0.003), when only 1-2 treated calves shed STEC (1-2.5 log10 CFU/g), whereas 3-5 control calves shed higher levels (1.4-5.6 log10 CFU/g). At Day 28, STEC O157 was not detected by IMS in 67% (Trial 1) and 83% (Trial 2) of DFM-treated calves. In contrast, STEC was recovered from 67% (Trial 1) and 100% (Trial 2) of control calves. qPCR detected L. reuteri R0444 shortly after administration, but levels declined to below detection at later sampling points. Conclusions Administration of L. reuteri R0444 reduced STEC O157 shedding in calves, supporting its potential as a DFM-based pre-harvest intervention to mitigate this pathogen in cattle.