Protection of Penaeus monodon against white spot syndrome by continuous oral administration of a low concentration of Bacillus subtilis spores expressing the VP28 antigen.

In this study, Bacillus subtilis spores expressing a chimeric protein, CotB-VP28, were used as a probiotic vaccine to protect black tiger shrimps (Penaeus monodon) against white spot syndrome virus (WSSV) infection. Oral administration of pellets coated with CotB-VP28 spores (at ≥1 × 109  CFU per g pellet) to shrimps induced immune-relating phenoloxydase activity (PO) in shrimps after 14 days of feeding (prior challenge) and at day 3 post challenge (1·26 and 1·70 fold increase respectively). A 75% protection rate was obtained by continuous feeding of the spore-coated pellets at ≥1 × 109  CFU per g for 14 days prior to WSSV challenge and during all the postchallenge period. Even when the amount of CotB-VP28 spores in feed pellets was reduced down to ≥5 × 107  CFU per g and ≥1 × 106  CFU per g, relatively high protection rates of 70 and 67·5%, respectively, were still obtained. By contrast, feeding pellets without spores (untreated group) and with naked spores (PY79 group) at ≥1 × 109  CFU per g could not protect shrimps against WSSV. These data suggest that supplementation of CotB-VP28 spores at low dose of ≥1 × 106  CFU per g could be effective as a prophylactic treatment of WSS for black tiger shrimps. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the protective efficacy of Bacillus subtilis CotB-VP28 spores on black tiger shrimps (Penaeus monodon) against white spot syndrome virus infection. Oral administration of pellets coated with CotB-VP28 spores (≥1 × 109  CFU per g) conferred 75% protection after white spot syndrome virus challenge. Even after reducing CotB-VP28 spores in feed pellets to ≥1 × 106  CFU per g, 67·5% protections was still obtained. These data indicate that supplementation of CotB-VP28 spores at a low dose of ≥1 × 106  CFU per g could be effective in prophylaxis against white spot syndrome in black tiger shrimps.

Investigating the Efficiency of Recombinant FliC-Loaded Bacillus subtilis Spores in Mice Immunization against Salmonella enterica Serovar Typhi.

BACKGROUND: Bacterial spores are among the most efficient vaccine delivery vehicles. Because of their safety and efficacy, Bacillus subtilis spores are increasingly used in this regard. The negatively charged surfaces of the spores allow antigens to be adsorbed onto these structures. In this study, a candidate vaccine against Salmonella enterica serovar Typhi was adsorbed onto B. subtilis spores and the immunogenicity of the formulation was investigated in BALB/c mice. METHODS: This work was performed during 2018-2019 in Islamic Azad University of Lahijan. FliC protein was recombinantly expressed in E. coli BL21 (DE3) cells and purified by affinity chromatography. On the other hand, B. subtilis strain PY79 (ATCC1609) was cultured in DSM medium and after the sporulation, FliC protein was adsorbed onto the spores in three different pH values (4, 7 and 10) and the adsorption was verified using dot-blot assay. FliC-adsorbed spores were then administered to BALB/c mice through the subcutaneous route. Mice immunization was evaluated by serum IgG assessment and challenge study. RESULTS: FliC protein was successfully expressed and purified. Sporulation was controlled by phase-contrast microscopy. Serum IgG assay showed significant stimulation of the mice's humoral immune system. Immunized mice were able to resist bacterial infection. CONCLUSION: The results showed the efficiency of spores as natural adjuvants for the stimulation of mice immune system. The formulation can be exploited for the delivery of recombinant vaccines against bacterial pathogens.

Genetically Intact Bioengineered Spores of Bacillussubtilis.

Developments in genome editing offer potential solutions to challenges in agriculture, industry, medicine, and the environment. However, many technologies remain unexploited due to limitations in the use of genetically altered organisms. In this study, we use B. subtilis spores to explore the possibility of bioengineering organisms while leaving their genome intact. Taking advantage of the differential expression between the mother cell and the fore-spore compartments during sporulation, we created plasmids programmed to modify the spore phenotype from the mother cell compartment, but to "self-digest" in the fore-spore. At the end of sporulation, the mother cell undergoes lysis and releases the phenotypically engineered, genetically unaltered spores. Using this approach, we demonstrated the potential to express foreign proteins in B. subtilis spores without genome alterations by producing spores expressing GFP in their protective coats, where approximately 90% of the spore population had no detectable plasmid or chromosome alterations. In a separate demonstration, we programmed KinA overexpression during vegetative growth to artificially induce sporulation, and also obtained spores with nearly 90% of them free of detectable plasmid. Artificial induction of sporulation could potentially simplify the bioprocess for industrial spore production, as it reduces the number of steps involved. Overall, these findings demonstrate the potential to create genetically intact bioengineered organisms.

Integration of Membrane Distillation with solar photo-Fenton for purification of water contaminated with Bacillus sp. and Clostridium sp. spores.

Although Membrane Distillation (MD) has been extensively studied for desalination, it has other applications like removing all kinds of solutes from water and concentrating non-volatile substances. MD offers the possibility of producing a clean stream while concentrating valuable compounds from waste streams towards their recovery, or emerging contaminants and pathogens present in wastewater in order to facilitate their chemical elimination. This paper analyses the elimination of bacterial spores from contaminated water with MD and the role of MD in the subsequent treatment of the concentrate with photo-Fenton process. The experiments were performed at Plataforma Solar de Almería (PSA) using a plate and frame bench module with a Permeate Gap Membrane Distillation (PGMD) configuration. Tests were done for two different kinds of spores in two different water matrixes: distilled water with 3.5wt% of sea salts contaminated with spores of Bacillus subtilis (B. subtilis) and wastewater after a secondary treatment and still contaminated with Clostridium sp. spores. An analysis of the permeate was performed in all cases to determine its purity, as well as the concentrated stream and its further treatment in order to assess the benefits of using MD. Results showed a permeate free of spores in all the cases, demonstrating the viability of MD to treat biological contaminated wastewater for further use in agriculture. Moreover, the results obtained after treating the concentrate with photo-Fenton showed a shorter treatment time for the reduction of the spore concentration in the water than that when only photo-Fenton was used.