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Bacillus species have demonstrated beneficial effects on intestinal health, production parameters, and immune function in poultry under both standard and disease-challenged conditions. Previously, we found that several novel ingredients (beta-glucan, vegetable protein hydrolysate, and liquid seaweed extract) demonstrated growth stimulation effects on Bifidobacterium lactis and Lactobacillus plantarum , Here, we extended this approach to examine in vitro synbiotic combinations of five novel marine-derived candidate probiotic Bacillus strains to assess their potential for in ovo applications. Beta-glucan enhanced the growth of all candidate Bacillus probiotic strains compared to a glucose control ( p  ≤ 0.05), suggesting a broad-spectrum modulatory role over a 24-h period, with variable magnitudes of response observed between strains. Species specificity was also observed, with lentinus stimulating the Bacillus pumilus but not the Bacillus altitudinis strains. A seaweed extract consistently stimulated the growth of one of the B. altitudinis strains ( p  ≤ 0.05), which, like all of the strains evaluated here, is seaweed-derived. This suggests potential ecological adaptation in substrate utilization. The shared environmental origin may influence substrate specificity and metabolic complementarity between strains and prebiotic candidates. Both B. altitudinis strains also exhibited enhanced growth at almost all time points ( p  ≤ 0.05) when cultured with vegetable protein hydrolysate. Based on these findings, we evaluated the effect of a potential synbiotic formulation comprising one of the B. altitudinis strains and vegetable protein hydrolysate in chickens, in ovo. The components were administered intra-amniotically at embryonic development day 18.5, utilizing a standard vaccination protocol. The hatchability of the chickens was not affected, thereby demonstrating the established dose as safe and applicable for further investigation. Key points • Shared origin of bioactive compounds may enhance probiotic-prebiotic compatibility in vitro • Protein hydrolysate offers a novel alternative to carbohydrate prebiotics • In ovo delivery of Bacillus-based synbiotic formulations offers potential as an early microbiome programming strategy

A stable gut microbiota promotes a healthy gut and enhances immune function, antioxidant status, and metabolic activities in chickens. The present research work aimed to investigate the modulatory impacts of in ovo delivery of prebiotic and probiotic on oxidative stress, the intestinal transcriptome, and various plasma metabolites in chickens. Fertilized Ross 308 eggs were administered in ovo either with galactooligosaccharide (GOS) (3.5 mg/egg or Lactiplantibacillus plantarum (LP) 1 × 10 6 /egg on the 12th day of egg incubation. Three hundred viable Ross 308 broiler hatching eggs in total were randomly assigned to four groups, namely, the negative control not injected group, the group receiving physiological saline injections as the positive control, GOS, and LP. The analysis of genes associated with immune functions, antioxidants, barrier functions, and free fatty acid receptors were determined via qPCR. The analysis of the selected plasma blood metabolites was performed automatically with Pentra C 400. The antioxidant capacity of the chickens’ liver, breast muscle, and spleen was enhanced by the in ovo injection of GOS and LP. The immune-related gene expression levels were upregulated after in ovo stimulation with either GOS or LP which improved the gut health of broiler chickens. In addition, several genes related to gut barrier functions were upregulated, thus ensuring epithelial integrity. As for blood plasma metabolites, no adverse effects were observed. In summary, we report that in ovo stimulation with either GOS or LP stimulates the immune system and improves the antioxidant status and gut health of chickens with no negative impact on plasma blood metabolite indices.

Wastewater surveillance has proven to be a useful tool to monitor pathogens such as SARS-CoV-2 as it is a nonintrusive way to survey the potential disease burden of the population contributing to a sewershed. With the expansion of this field since the beginning of the COVID-19 pandemic, laboratory methods to process wastewater and quantify pathogen nucleic acid levels have improved as technologies changed, efforts expanded in size and scope, and supply chain issues were resolved. Maintaining data continuity is crucial for labs undergoing method transitions to accurately assess infectious disease levels over time and compare measured RNA concentrations to public health data. Despite the dynamic nature of laboratory methods and the necessity to ensure uninterrupted data, to our knowledge there has not been a study that unites two datasets from different lab methods for pathogen quantification from environmental samples. Here, we describe a lab transition from SARS-CoV-2 RNA quantification using a low-throughput, manual filtration-based wastewater concentration and RNA extraction followed by qPCR to a high-throughput, automated magnetic bead-based concentration and extraction followed by dPCR. During the two-month transition period, wastewater samples from across the Chicago metropolitan area were processed with both methods in parallel. We evaluated a variety of regression models to relate the RNA measurements from both methods and found a log-log model was most appropriate after removing outliers and discrepancy points to improve model performance. We also evaluated the consequences of assigning values to samples that were below the detection limit. Our study demonstrates that data continuity can be maintained throughout a transition of laboratory methods if there is a sufficient period of overlap between the methods for an appropriate model to be constructed to relate the datasets.