In vitro selection and characterization of ssDNA aptamers by cross-over SELEX and its application for detection of S. Typhimurium.

S. Typhimurium is the most common food-borne pathogen frequently encountered in contaminated food and poses a major threat to public health, animals, and the food industry worldwide. Early and sensitive detection is the prime step to ensuring the microbiological quality of fresh and processed food. We used the cross-over SELEX strategy to select ssDNA aptamers against OM usher protein, FimD that could be employed in the S. Typhimurium detection assay. Altogether, 7 rounds of protein-SELEX, 3 rounds of intermittent counter-SELEX, and 4 rounds of whole bacterial cell-SELEX were undertaken for specific aptamer selection. The aptamer specificity was achieved by performing counter-SELEX with non-target bacterial cells. The enriched pool of aptamers obtained post SELEX process was cloned, sequenced, and further the specificity and affinity were characterized by flow cytometry. The aptamers DFRM-3 and DFRM-10 were specific and showed relatively high binding affinity towards S. Typhimurium with an apparent dissociation constant (Kd value) of 76 nM and 90.01 nM respectively. A dual aptamer-based sandwich assay was developed using biotinylated DFRM-3 aptamer for magnetic capture and AlexaFluor-488 labeled DFRM-10 aptamer for detection of S. Typhimurium. The limit of detection (LOD) of S. Typhimurium by the developed assay was found to be 102 CFU/ml in pure culture and 103 CFU/ml in the artificially contaminated chicken meat samples.

Oral co-administration of bivalent protein r-BL with U-Omp19 elicits mucosal immune responses and reduces S. Typhimurium shedding in BALB/c mice.

The increase in international food trade and travel has dramatically increased the global incidences of Salmonellosis. In the light of widespread resistance to frontline antibiotics, oral vaccines remain the most reliable alternative. In this study, the fusion protein, r-BL was rationally constructed by splicing the Salmonella Typhimurium sseB and ompL genes through G4S linker by over-lap extension PCR. The oral coadministration of r-BL with B. abortus U-Omp19 protein with known protease inhibitor activity resulted in significant increase of mucosal IgA titres to antilog 4.5051 (p < 0.0001) and 4.806 (p < 0.0001) in the fecal samples and intestinal washes respectively. Antibody isotyping of the intestinal washes demonstrated increase in mucosal IgM, IgG1 and IgG2a isotypes also and demonstrated a significant reduction in fecal shedding of S. Typhimurium in challenge study. The r-BL + U-Omp19 treated mice demonstrated a complete termination of Salmonella fecal shedding by the 12th day of challenge as compared to other study groups. In summary, the bivalent protein r-BL when administered with the mucosal adjuvant U-Omp19 was successful in triggering mucosal arm of the immune system which forms the first line of defence in combating the infections caused by the enteric pathogen like Salmonella.