Selection of Bacillus subtilis for animal and chicken embryo supplementation
Authors: Reis Thais Fernanda Martins , Hoepers Patricia Giovana , Carvalho Vasco Ariston , Silva Gabriela Ribeiro , Notário Fabiana Oliveira , Soares Maria Cecília , Schlemper André Eduardo , Costa Ícaro Mendonça , Coelho-Rocha Nina Dias , Costa Mylla Spirandelli , Sommerfeld Simone , Ronchi Alessandra Aparecida Medeiros , Rossi Daise Aparecida , Castro Igor Paula , Fonseca Belchiolina Beatriz 
Published: October 2022

Bacillus subtilis (B.S.) has been used as an excellent probiotic; however, some B.S. strains seem to be opportunist pathogens or do not present inhibitory effects in the pathogenic bacterium, so the characterization of B.S. strains for use in animals is mandatory. This study aimed to select nonpathogenic strains of B.S., which have beneficial effects on birds and can inhibit Salmonella spp., avian pathogenic Escherichia coli coli (APEC) and Campylobacter jejuni (C.J.). We tested nine (9) strains of B.S. isolated from several sources (named A to I) in in vitro by tests of mucin degradation activity, haemolytic activity, apoptosis, and necrosis in fibroblasts from chickens. After the in vitro test, we tested the remaining seven (7) strains (strains A to G) in a chicken embryo (C.E.) as an in vivo model and target animal. We inoculated 3 log CFU/CE of each strain via allantoic fluid at the 10th day postincubation (DPI). Each treatment group consisted of eight C.Es. At the 17th DPI. We checked C.E. mortality, gross lesions, C.E. weight, and whether B.S. strains were still viable. To perform the cytokine, total protein, albumin, and reactive C protein analysis, we collected the C.E. blood from the allantoic vessel and intestine fragments in the duodenum portion for histomorphometric analysis. After the results in C.Es., we tested the inhibition capacity of the selected B.S. strains for diverse strains of Salmonella Heidelberg (S.H.), S. Typhimurium (S.T.), S. Enteritidis (S.E.), S. Minnesota (S.M.), S. Infantis (S.I.), Salmonella var. monophasic (S.V.M) and C. jejuni. After the in vitro trial (mucin degradation activity, haemolytic activity, apoptosis, and necrosis), we removed two (2) strains (H and I) that showed β-haemolysis, mucin degradation, and/or high apoptosis and necrosis effects. Although all strains of B.S. were viable in C.Es. at the 17th DPI, we removed four (4) strains (A, B, D, F) once they led to the highest mortality in C.Es. or a high albumin/protein ratio. C. jejuni inoculated with strain G had greater weight than the commercial strain, which could be further used for egg inoculation with benefits to the C.E. Moreover, the cytokine analysis indicated that strains E and G have immunomodulatory effects on C.Es. From the tests in C.Es., we selected the strains C, E, and G for their ability to inhibit pathogenic strains of relevant foodborne pathogens. We found that the inhibition effect was strain dependent. In general, strains E and/or G presented better or similar results than commercial control strains in the inhibition of S.H., S.T., S.I., APEC and two (2) strains of C.J. In this study, we selected B.S. strains C, E and G due to their in vitro and in vivo safety and beneficial effects. In addition, we emphasize the value of C.E. as an in vivo experimental model for assessing B.S.'s safety and possible benefits for poultry and other animals.