Immunogenicity and invasiveness of the genus Bacillus microorganisms with oral administration

Immunomodulatory effects of genus Bacillus -based probiotics result from the ability of Bacillus to penetrate through the mucous membranes of the gastrointestinal tract into the bloodstream. Creation of recombinant Bacillus -based vaccines with antigens of the most significant and antigenically variable viral and bacterial agents of farm animals (poultry, pigs and cattle) is prospective. The problem of oral vaccination is the nutritional immunotolerance which is due to the involvement of CD4+ and CD8+/Treg lymphocytes in adaptive events that suppress the immune response to the administered antigen. The article for the first time indicates that the invasiveness of the genera Bacillus and Geobacillus strains is important but not the only factor of their immunogenicity. It was found that both B subtillis and B. atrophaeus could be vectors to deliver recombinant antigens. Our objective was to search for Bacillus strains with optimal invasiveness and immunogenicity when used per or and without effects of food immunotolerance. Bacillus strains were provided by the Institute of Chemical Biology and Fundamental Medicine SB RAS (ICBFM SB RAS) (22 cultures) and LLC SibAF (Berdsk) (7 cultures). Bacteria were identified by Sanger sequencing of 16S ribosomal RNA. The strains were incubated in 3 % tryptic-soybean broth (TSB), 0.5 % yeast extract (YE) and on 1.5% Bacto agar (BD Biosciences, USA) at 37 °C for 16 h. Bacterial cells were inactivation by heating at 65 °C for 3 h and furacilin adding to a concentration of 0.1 %. Cultures of 29 strains were tested for in vivo immunogenicity in F1 hybrid mice (A/Sn × Balb/C). Animals were orally fed 200 μl of culture fluid of microorganisms of Bacillus and Geobacillus genera at a concentration of 0.7 McF, three times every 7 days. After 3 weeks, blood was sampled for serologic studies. Immunogenicity and invasiveness were evaluated in agglutination test of blood sera with fluorescently labeled O-antigens. Invasiveness upon oral administration was evaluated by a bioassay on 10-day-old Shaver cross chickens (Gallus gallus L.). Every day for 10 days chickens were administered orally a 1-day culture of the tested microorganism at a dose of 200 μl (1 billion CFU/ml). The chickens were slaughtered on day 11, and the bacterial insemination of muscle, liver and heart tissues was assed using meat-peptone agar and meat-peptone broth. Invasiveness of Bacillus strains were tested on C57Black mice. The animals were orally administered 100 μl of bacterial culture once a day for two consecutive days. On day 3, the animals were slaughtered and liver and heart tissue samples were tested using Eugonic agar, meat-peptone agar and meat-peptone broth. For Oil spreading assay of biosurfactant activity, the culture fluid was diluted with distilled water and added with 20 µl of oil. Distilled water was a negative control and SDS (1 % v/v) was a positive control. Destruction of the oil slick was considered a positive test. Only 27.5 % of Bacillus cultures generated production of antibody to their own corpuscular antigens during oral administration to F1 (A/Sn × Balb/C) mice. Among the B. subtilis cultures studied, 71.42 % induced antibody synthesis. Other species which stimulated antibody production were B. atrophaeus B8, isolate B23 attributed to B. zhangzhoulensis / B. pumilus / B. safensis , and B. licheniformis B10. Most tested microorganisms did not cause production of antibodies to their own antigens. B. mucoides / B. weihenstephanensis B31, B. simplex B32, B. thuringiensis B20 , B. subtillis, B. amyloliquefaciens 2-15, Bacillus spp. 1'-37-7 had a pathogenic effect with 25 to 50 % death of mice. In all chicks administered orally with the Bacillus cultures, 100 % of breast and thigh muscles, 96 and 52 % of liver and heart, respectively, were contaminated. Invasiveness for mice was assessed by detection of microorganisms in liver and heart samples 18 h after the last oral administration. Bacillus spp. B56, Bacillus spp. B50, B. atrophaeus B8, and B. safensis B23 consistently showed invasiveness. However, antibodies were detected only to B. atropheus B8 and B. safensis B23. B23 and B24 isolates of B. zhangzhoulensis / B. safensis / B. pumilus , and B. atrophaeus B8 had biosurfactant activity. No statistically significant association between biosurfactant propertied and invasiveness is found in the analyzed sample. Thus, the potential of probiotic B. subtilis strains as carriers of recombinant vaccine antigens seems to us the most promising.