BACILLUS LICHENIFORMIS STRAIN WITH PROBIOTIC ACTIVITY

Abstract

The current invention concerns a new B. licheniformis strain with strong inhibition of C. perfringens and its use as probiotic.

Claims

1-19. (canceled)

20. A Bacillus licheniformis strain or preparation thereof selected from the group consisting of: a) a B. licheniformis strain as deposited under DSM 32314 at the DSMZ; b) a mutant of the B. licheniformis strain as deposited under DSM 32314 having all identifying characteristics of the strain DSM 32314; c) a preparation of (a) or (b); and d) a preparation comprising an effective mixture of the strains or preparations of paragraphs (a), (b) or (c).

21. The Bacillus licheniformis strain or preparation of claim 20, wherein said strain is a mutant having all identifying characteristics of DSM 32314 and a DNA sequence identity to DSM 32314 of at least 95%.

22. The Bacillus licheniformis strain or preparation of claim 20, wherein the B. licheniformis strain further comprises: a) a 16S rDNA sequence with a sequence identity of at least 99% to the polynucleotide sequence of SEQ ID NO: 1; b) a yqfD sequence with a sequence identity of at least 99% to the polynucleotide sequence of SEQ ID NO: 2; and/or c) a gyrB sequence with a sequence identity of at least 99% to the polynucleotide sequence of SEQ ID NO: 3.

23. The Bacillus licheniformis strain or preparation of claim 20, wherein the B. licheniformis strain further comprises: a) an rpoB sequence with a sequence identity of at least 99% to the polynucleotide sequence of SEQ ID NO: 4; and/or b) a groEL sequence with a sequence identity of at least 99% to the polynucleotide sequence of SEQ ID NO: 5.

24. The B. licheniformis strain or preparation of claim 20, wherein said strain is able to grow anaerobically.

25. The B. licheniformis strain or preparation of claim 20, wherein said strain inhibits the growth of C. perfringens.

26. The B. licheniformis strain or preparation of claim 20, wherein said strain is able to grow in the presence of 0.05 wt. % acetic acid, 0.05 wt. % propionic acid and/or 0.2 wt. % lactic acid.

27. The B. licheniformis strain or preparation of claim 20, wherein said strain comprises a cellulase activity of at least 200 mU/mL and/or a xylanase activity of at least 10 mU/mL.

28. The B. licheniformis strain or preparation of claim 20, wherein said strain is able to grow in presence of 2 mM bile.

29. A composition comprising the B. licheniformis strain or preparation of claim 20, and further comprising at least one other ingredient or compound.

30. The composition of claim 29, wherein said composition is a feed or foodstuff and said at least one other ingredient or compound is selected from the group consisting of: proteins; carbohydrates; fats; further probiotics; prebiotics; enzymes; vitamins; immune modulators; milk replacers; minerals; amino acids; coccidiostats; acid-based products; medicines; and combinations thereof.

31. The composition of claim 29, wherein said composition is a pharmaceutical composition and said at least one other ingredient or compound is a pharmaceutically acceptable carrier.

32. A method comprising administering the Bacillus licheniformis strain or preparation of claim 20 to animals or humans or applying the strain or preparation to plants, liquids or solids in the environment.

33. The method of claim 32, wherein the Bacillus licheniformis strain or preparation is administered to an animal or human either alone, or as part of a feed or pharmaceutical composition, to improve gut health status.

34. The method of claim 32, wherein the Bacillus licheniformis strain or preparation is administered to animals either alone, or as part of a feed, to improve the health of the animals; improve the feed conversion rate of the animals; decrease the mortality rate of the animals; increase the survival rate of the animals; improve the weight gain of the animals; increase the disease resistance of the animals; increase the immune response of the animals; establish or maintain a healthy gut microflora in the animals; and/or reduce pathogen shedding through the feces of the animals.

35. The method of claim 32, wherein the Bacillus licheniformis strain or preparation is applied to manure, contaminated liquids, litter, a pit, or a manure pond to control or avoid detrimental environmental effects.

36. The method of claim 32, wherein the Bacillus licheniformis strain or preparation is applied to drinking, rearing water or to aqueous solutions to control or improve the quality of these fluids.

37. The method of claim 32, wherein the Bacillus licheniformis strain or preparation is applied to cultivated plants to treat or prevent microbial diseases.

Description

WORKING EXAMPLES

Example 1. Strain Characteristics Relevant to Survival in the Gastrointestinal Tract

[0116] Bacillus licheniformis strains were screened from various environmental samples in order to obtain a superior strain as animal direct-fed microbial (DFM)/probiotic. As the strain is intended to reach its full potential in the intestine of the target animal, the strain was pre-screened to withstand various environmental and gut related conditions. Strain spores were generated (Nicholson and Setlow 1990), washed and incubated at 80° C. for 20 minutes (pasteurization), then titrated in logarithmic/1 in 10 dilutions using veal infusion broth agar (VI, Difco™, no. 234420, Becton Dickinson GmbH, Heidelberg, Germany). The second highest dilution prior to no growth was stored at −80° C. and used as standardized starting point for all further assessments from spore state. To simulate gastric passage (Argenzio 2004a; Trampel and Duke 2004), survival of acid exposure was assessed based on Larsen et al. (2014). Growth of vegetative cells was furthermore assessed at low pH indicating growth under stomach/proventriculus and gizzard conditions, as well as in presence of up to 4 mM bile (B8631, CAS 8008-63-8, Sigma-Aldrich) at pH 7 in order to confirm strain growth at the proximal part of the small intestine right after clearance of the stomach or gizzard (Argenzio 2004b; Trampel and Duke 2004).) Strain fitness in the anaerobe intestine (Argenzio 2004b; Trampel and Duke 2004) was assessed by inoculating standardized spore solutions under anaerobic conditions (AnaeroPak™, Thermo Fisher Scientific) in VI medium supplemented with 2.5 mM KNO.sub.3 (Glaser et al. 1995). Furthermore was the anaerobe proteolytic and cellulytic activity of strains assessed on VI agar plates supplemented with 1% skim milk powder (70166, Sigma-Aldrich) or 0.1% water insoluble AZCL-HE cellulose (I-AZCEL, Megazyme International, Bray, Ireland). Osmotic stress, as also found in the gut (Argenzio 2004b; Trampel and Duke 2004), was assessed by determining growth on VI agar with addition of 5% NaCl (den Besten et al. 2009). Finally, spore heat stability was assessed to determine pelleting stability by exposing spores to 99° C. for 20 min (Palop et al. 1996) and subsequent inoculation on VI agar. Bacillus licheniformis strain DSM 32314 survived simulated gastric passage, growth of strain DSM 32314 was observed starting at pH 6. Strain DSM 32314 grew anaerobically and was able to degrade water-insoluble cellulose under anaerobic conditions. Strain DSM 32314 was able to grow in presence of 2 and 4 mM bile, as well as in presence of 10% NaCl. Strain DSM 32314 reached an average spore count of 4.35×10.sup.9 CFU/mL, and spores of strain DSM 32314 were viable after exposure to 99° C. for 20 min. Furthermore, B. licheniformis wildtype strains WT1 and WT2 were assessed to grow anaerobically and to withstand pH 6, but they were unable to anaerobically degrade cellulose and were therefore disqualified as DFM/probiotic candidates. However, they were used as wildtype comparison in the following examples.

REFERENCES

[0117] Argenzio, R. A. (2004a). Secretion of the Stomach and Accessory Glands, p. 405-418. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 25; Cornell University Press, Ithaca, N.Y., USA.

[0118] Argenzio, R. A. (2004b). Digestive and Absorptive Functions of the Intestines, p. 419-437. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 26; Cornell University Press, Ithaca, N.Y., USA.

[0119] Dawson, R. M. C.; Elliot, D. C.; Elliot, W. H.; Jones, K. M. (1986). Data for Biochemical Research; 3.sup.rd edition, Oxford Science Publishing, United Kingdom.

[0120] Den Besten HMW, Mols M, Moezelaar R, Zwietering MH, Abee T. (2009). Phenotypic and transcriptomic analyses of mildly and severely salt-stressed Bacillus cereus ATCC 14579 cells. Appl Environ Microbiol. 75:4111-9.

[0121] Glaser, P., A. Danchin, F. Kunst, P. Zuber, and M. M. Nakano. (1995). Identification and isolation of a gene required for nitrate assimilation and anaerobic growth of Bacillus subtilis. J. Bacteriol. 177:1112-1115

[0122] Nicholson W. L., Setlow P. Sporulation, germination and outgrowth. In: Harwood C R, Cutting S M, editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons Ltd.; 1990. pp. 27-74.

[0123] Palop, A., Raso, J., Pagan, R., Condon, S. and Sala, F. J. (1996). Influence of pH on heat resistance of Bacillus licheniformis in buffer and homogenized foods. International Journal of Food Microbiology 29, 1-10.

[0124] Trampel, D. W. and Duke, G. E. (2004). Avian Digestion, p. 488-500. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 29; Cornell University Press, Ithaca, N.Y., USA.

Example 2. Comparative Strain Performance Relative to Wild-Type Bacillus licheniformis—Quantitative Assessment of Bile Tolerance

[0125] In order to assess the competitiveness of the Bacillus licheniformis strain DSM 32314 selected from example 1, analysis was performed comparing to representative Bacillus licheniformis wildtype 1 (WT1) and wildtype 2 (WT2) and readiness of strains to perform in the proximal small intestine in presence of bile at neutral pH right after gastric passage (Argenzio 2004b; Trampel and Duke 2004) was determined by strain growth in VIB media with addition 2 mM bile. Overnight culture with 50 uL candidate strain cell suspension and 10 mL VIB in 100 mL conical flask was incubated at 37° C. and 200 rpm, then approximately 50 uL of overnight culture was transferred to 100 well honeycomb plates (Oy Growth Curves Ab Ltd, former Thermo Labsystems, Helsinky, Finland) with 1 mL VIB at pH 7 with 2 mM bile in order to obtain OD 0.2 per mL. Strain specific growth at 37° C. and 200 rpm was observed for 48 h with OD determined every 15 mM using Bioscreeen C MBR with BioLink software package (Oy Growth Curves Ab Ltd). Averaged triplicate blank OD read of broth with bile only (blanks) were subtracted per culture at each time point before area under the curve (AUC) was calculated. Quantitative assessment for each strain was compared as area under the curve between 0-5 h (AUC5, in OD×time in h), area under the curve between 0-10 h (AUC10 in OD×time in h), and time until strains reached its maximum optical density (Tmax in h). Statistical analysis was performed using one-way ANOVA procedure of MiniTab ® 16 Statistical Software (Minitab Inc., State College, Pa., USA). Results can be found in Table 2.1.

TABLE-US-00001 TABLE 2.1 Growth of Bacillus licheniformis strains DSM 32314 and wildtype strains WT1 and WT2 in presence of 2 mM bile. Strain ID AUC5 AUC10 Tmax DSM 32314 0.539 .sup.A 2.144 .sup.A 18.2 .sup.C WT1 0.266 .sup.B 0.714 .sup.B 30.3 .sup.B WT2 0.378 .sup.B 1.177 .sup.B 36.4 .sup.A P-value P < 0.01 P = 0.001 P < 0.001 SEM 0.018   0.076   0.5 

[0126] AUC5, area under the curve between time point 0 and 5 h in optical density×h; AUC10, area under the curve between time point 0 and 10 h in optical density×h; Tmax, time in h until maximum optical density was reached; SEM, pooled standard error of the mean;.sup.A, B means that do not share a letter are significantly different.

[0127] In direct comparison, strain DSM 32314 reached its maximum OD in presence of 2 mM bile 10 h faster than the wildtype Bacillus licheniformis strains WT1 and WT2. In addition, strain DSM 32314 grew 1.4-2.0 fold faster during the first 5 hours, and 1.8-3.0 fold faster during the first 10 h of the test, compared to growth of wildtype B. licheniformis strains, respectively.

REFERENCES

[0128] Argenzio, R. A. (2004b). Digestive and Absorptive Functions of the Intestines, p. 419-437. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 26; Cornell University Press, Ithaca, N.Y., USA.

[0129] Trampel, D. W. and Duke, G. E. (2004). Avian Digestion, p. 488-500. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 29; Cornell University Press, Ithaca, N.Y., USA.

Example 3. Comparative Strain Performance Relative to State of the Art Direct-Fed Microbial (DFM)/Probiotic for Animal Nutrition—Growth in Presence of Short Chain Fatty Acids (SCFA)

[0130] Comparative growth of strains DSM 32314 and WT1 was assessed in presence of short chain fatty acids as those are observed in the gut with increasing importance towards the large intestine (Argenzio 2004b; Trampel and Duke 2004). Tests were initiated using standardized spore solution as described in example 1 testing aerobe growth in VI medium at 37° C. and pH 6, read-out parameter was growth versus no growth. For this test, VI medium was adjusted to pH 6 using Mcllvaine buffer (Palop et al. 1996) and subsequently supplemented with 0.05% acetic acid (HA, 537020, CAS 64-19-7, Sigma-Aldrich), 0.05% propionic acid (HP, P1386, CAS 79-09-4, Sigma-Aldrich) or 0.2% lactic acid (HL, W261106, CAS 50-21-5, Sigma-Aldrich). Results can be found in Table 3.1.

TABLE-US-00002 TABLE 3.1 Assessment of growth of Bacillus licheniformis strain DSM 32314 and wildtype strain WT1 in presence of short chain fatty acids at pH 6. Strain ID Acetic acid Propionic acid Lactic acid DSM 32314 Yes Yes Yes WT1 No growth No growth No growth

[0131] Bacillus licheniformis strain DSM 32314 was able to grow at pH 6 in the presence of acetic, propionic and lactic acid, whereas WT1 strain was unable to grow from spore stage under these conditions.

REFERENCES

[0132] Argenzio, R. A. (2004b). Digestive and Absorptive Functions of the Intestines, p. 419-437. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 26; Cornell University Press, Ithaca, N.Y., USA.

[0133] Palop, A., Raso, J., Pagan, R., Condon, S. and Sala, F.J. (1996). Influence of pH on heat resistance of Bacillus licheniformis in buffer and homogenized foods. International Journal of Food Microbiology 29, 1-10.

[0134] Trampel, D. W. and Duke, G. E. (2004). Avian Digestion, p. 488-500. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 29; Cornell University Press, Ithaca, N.Y., USA.

Example 4. Comparative Strain Performance Relative to State of the Art Direct-Fed Microbial (DFM)/Probiotic for Animal Nutrition—Quantitative Assessment of Enzymatic Activity

[0135] Similar to test conducted in Example 2, strains DSM 32314, WT1 and WT2 were compared evaluating the respective aerobe cellulytic, xylanolytic and proteolytic activity. Cellulase and Xylanase activity were determined as described in Larsen et al. (2014). For proteolytic activity analysis, starter and main culture of strains were grown in VIB at 37° C. as previously described. From main culture, 10 uL were added to 20 uL 0.5% Fluorescein Isothiocyanate Casein (FITC; C3777, Sigma-Aldrich) solution with 20 uL buffer consisting of 20mM sodium phospate (dibasic, anhydrous) with 150 mM sodium chloride (all components from Sigma-Aldrich), then incubated for 1 h at 37° C. After addition of 150 uL of 10% (v/v) trichlor acetic acid (Sigma-Aldrich) and another 30 min incubation at 37° C., samples were centrifuged at 19,000 rpm for 15 min, then 2 uL of supernatant transferred to 200 uL 500 mM TRIS HCl Solution (Trizma BaseTRIS, Sigma-Aldrich). Fluorescence of soluble peptides due to proteolytic release were determined (TECAN GENios Microplate Reader, Tecan Group Ltd., Männedorf, Switzerland) at excitation 494 nm, emission 518 nm. Analysis was performed in three independent runs, then averaged as milliunits per microliter solution, statistical analysis was performed using one-way ANOVA procedure of MiniTab ® 16 Statistical Software (Minitab Inc., State College, Pa., USA). Results can be found in Table 4.1.

TABLE-US-00003 TABLE 4.1 Cellulase, protease and xylanase activity of Bacillus licheniformis strain DSM 32314 compared to wildtype strains WT1 and WT2. Cellulase activity Protease activity Xylanase activity Strain ID (mU/mL) (mU/mL) (mU/mL) DSM 32314 250.3 A  9.8 A 20.5 A WT1 n/d 3.3 B 14.3 B WT2 56.6 B 2.7 B 14.6 B P-value P < 0.001 P < 0.005 P < 0.001 SEM 15.3  1.8  0.8  SEM, pooled standard error of the mean; A, B means that do not share a letter are significantly different. In direct comparison, strain DSM 32314 demonstrated significant 4.4 fold increased cellulase activity comparing to WT2. In addition, DSM 32314 demonstrated significant 3.0 fold increased protease activity comparing to WT1 and 3.6 fold increased protease activity comparing to WT2. Moreover, DSM 32314 demonstrated significant 1.4 fold increased xylanase activity comparing to WT1 and WT2.

REFERENCE

[0136] Larsen, N., Thorsen, L., Kpikpi, E. N., Stuer-Lauridsen, B., Cantor, M. D., Nielsen, B., Brockmann, E., Derkx, E. M. F. and Jespersen, L. (2014). Characterization of Bacillus spp. strains for use as probiotic additives in pig feed. Applied microbiology and biotechnology, 98(3), 1105-1118.

Example 5. Comparative Strain Performance Relative to State of the Art Direct-Fed Microbial (DFM)/Probiotic for Animal Nutrition—Expression of Metabolites and Pathogen Inhibition

[0137] Similar to tests conducted in Example 2, B. licheniformis strains DSM 32314, WT1 and WT2 were compared evaluating the respective number of metabolites expressed and pathogens inhibited in the respective media. For metabolite expression analysis, starter cultures were grown and tests performed as described in Scholz et al. (2011). From 10 mL Luria Bertami broth (LB, Thermo Fisher Scientific) culture grown for 24 h at 37° C. and 160 rpm in 100 mL flask, 100 uL were transferred to main culture. Main culture was grown either in 10 mL LB containing 0.2 mL/L KellyT trace metal solution (LBKelly, Scholz et al. 2011), or 10 mL Trypticase Soy Broth (Oxoid, Thermo Fisher Scientific) with 0.6% yeast extract (Y1625, CAS 8013-1-2, Sigma-Aldrich; resulting broth abbreviated TSBYE), both for 24 hat 37° C. at 160 rpm in 100 mL flask. Of the main culture, 4 mL were combined with 2 mL n-Butanol in 15 mL test tube, vortexed for 3 min, then sonicated for 15 min. After centrifugation for 1 min at 5000 rpm, organic phase was transferred, vacuum dried and analyzed using High-performance liquid chromatography—electrospray ionization mass spectrometry (HPLC-ESI-MS; Chen et al. 2006). Every sample was measured in two different modes, negative and positive mode, and mass spectra were acquired. Resulting peaks as similarly reported in Teo and Tan (2005) were converted to molecular mass in Da. Results for comparison can be found in Table 5.1.

TABLE-US-00004 TABLE 5.1 Comparison of metabolites expressed by strains DSM 32314, WT1 and WT2, in L B Kelly, respectively. Molecular Mass 496 389 855 865 943 969 1008 1022 1036 1050 1420 1423 2471 Da Da Da Da Da Da Da Da Da Da Da Da Da DSM 32143 yes n/d yes n/d n/d yes yes yes yes yes yes yes yes WT1 n/d yes n/d yes yes n/d n/d n/d n/d n/d n/d n/d n/d WT2 n/d n/d n/d n/d n/d n/d yes yes traces n/d n/d n/d n/d

[0138] In addition, Clostridium perfringens inhibition via Bacillus licheniformis bacteriocin production, as part of metabolites from table 5.1. but not closer investigated, was assessed using well diffusion antagonismus test (Parente et al. 1995). Four pathogenic C. perfringens candidates were tested being C. perfringens type strain ATCC 13124 from Teo and Tan (2005), as well as three pathogenic C. perfringens field isolates from poultry and swine, obtained from University of Leipzig, Faculty of Veterinary Medicine, Department of Bacteriology and Mycology, Prof. Dr. Christoph Baums, Potsdam, Germany. The C. perfringens type A-strains from Leipzig describe as follows: Strains 2300-1-17 and 2300-1-18 were isolated from necrotic enteritis positive chicken digestive tract. Both strains produce α-toxin, strain 2300-1-17 also expressing NetB toxin (Savva et al. 2013; Uzal et al. 2014,) whereas strain 2300-1-18 tested positive for β2-toxin (Allaart et al. 2012). Strain 2300-1-19 was isolated from digestive tract of a scouring piglet exhibiting symptoms of Clostridial type A enteritis (Songer and Uzal 2005). Growth conditions and media were described by Teo and Tan (2005). In brief, Bacillus strains were grown in 10 mL TSBYE and LBKelly starter culture for 24 hat 37° C. and 160 rpm in 100 mL flask in 5% CO.sub.2 atmosphere, respectively. Clostridium perfringens starter cultures were cultivated anaerobically (AnaeroPak™, Thermo Fischer Scientific) in fluid thioglycolate broth (FTB, Becton Dickenson) for 24 h at 37° C. and 160 rpm in 100 mL flask, then spread with sterile cotton swab agar plates (TSBYE with 1% agar, short TSAYE). Inoculated Inoculated TSAYE plates were then incubated anaerobically overnight at 37° C. in order to obtain lawn of C. perfringens. After overnight growth, three 9 mm diameter wells were cut into the agar with lawn, 1S.sup.t well was used as non-inoculated control without culture, 2.sup.nd well was inoculated with 100 uL not—C. perfringens-inhibiting Bacillus strain (B. cereus var. toyoi, NCIMB 40112), the 3.sup.rd well was inoculated with 100 uL of Bacillus licheniformis DSM 32314 or DSM 17299 starter culture. After 24 h incubation at 37° C., zone of clearance in mm was determined measuring from the edge of the cut well to the border of the cleared lawn, each colony was measured twice (horizontally, vertically), then averaged. For each Bacillus licheniformis antagonism test and media, analysis was performed in duplicate plate runs. Statistical analysis was performed using one-way ANOVA procedure of MiniTab ® 16 Statistical Software (Minitab Inc., State College, Pa., USA) using Fisher LSD for mean separation, results can be found Table 5.2. for pathogen inhibition (strains grown in LBKelly), in Table 5.3 for pathogen inhibition (strains grown in TSBYE).

TABLE-US-00005 TABLE 5.2 Comparison of Bacillus licheniformis DSM 32314 and DSM 17299 inhibitory capacity of pathogenic C. perfringens well diffusion assay (strains grown in LBKelly), values in mm clearance of pathogen. Pathogenic C. perfringens Poultry Poultry Swine Necrotic Necrotic Clostridial Type Enteritis Enteritis Enteritis strain strain strain strain ATCC Average Bacillus 2300-1-17 2300-1-18 2300-1-19 13124 inhibition DSM 32314 15.50 11.50 14.50 14.00 13.88 .sup.A DSM 17299 2.00 0.00 6.00 2.00  2.50 .sup.B P-value <0.001.sup.  SEM 2.15  SEM, pooled standard error of the mean; .sup.A, B means that do not share a letter are significantly different.

TABLE-US-00006 TABLE 5.3 Comparison of Bacillus licheniformis DSM 32314 and DSM 17299 inhibitory capacity of pathogenic C. perfringens well diffusion antagonism (strains grown in TSBYE), values in mm clearance of pathogen. Pathogenic C. perfringens Poultry Poultry Swine Necrotic Necrotic Clostridial Type Enteritis Enteritis Enteritis strain Bacillus strain strain strain ATCC Average strains 2300-1-17 2300-1-18 2300-1-19 13124 inhibition DSM 32314 21.00 17.00 21.00 18.00 19.25 .sup.A DSM 17299 0.00 0.00 0.00 0.00  0.00 .sup.B P-value <0.001.sup.  SEM 1.46  SEM, pooled standard error of the mean; .sup.A, B means that do not share a letter are significantly different.

REFERENCES

[0139] Allaart, J. G., de Bruijn, N. D., van Asten, A. J., Fabri, T. H., and Gröne, A. (2012). NetB-producing and beta2-producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands. Avian Pathol., 41:541-546

[0140] Chen, X. H., Vater, J., Piel, J., Franke, P., Scholz, R., Schneider, K., Koumoutsi, A., Hitzeroth, G., Grammel, N., Strittmatter, A. W., Gottschalk, G., Süssmuth, R. and Borriss, R. (2006). Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42. Journal of bacteriology, 188(11), 4024-4036.

[0141] Parente, E., Brienza, C., Moles, M., & Ricciardi, A. (1995). A comparison of methods for the measurement of bacteriocin activity. Journal of microbiological methods, 22(1), 95-108.

[0142] Sawa, G. S., Fernandes da Costa, S. P., Bokori-Brown, M., Naylor, C. E., Cole, A. R., Moss, D. S., Titball, R-W., and Basak, A. 2013. Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens. J Biol. Chem., 288: 3512-3522.

[0143] Scholz, R., Molohon, K. J., Nachtigall, J., Vater, J., Markley, A. L., Süssmuth, R. D., Mitchell, D. A. and Borriss, R. (2011). Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42. Journal of bacteriology, 193(1), 215-224.

[0144] Songer, J. G., and Uzal, F. A. (2005). Clostridial enteric infections in pigs. Journal of veterinary diagnostic investigation, 17(6), 528-536.

[0145] Teo, A. Y.-L. and Tan, H.-M. (2005). Inhibition of Clostridium perfringens by a novel strain of Bacillus licheniformis from the gastrointestinal tracts of healthy chickens. Appl. Environm. Microbiol., 71:4185-90.

[0146] Uzal, F. A., Freedman, J. C., Shrestha, A., Theoret, J. R., Garcia J., Awad, M. M., Adams, V., Moore, R. J., Rood, J. I., and McClane, B. A. (2014). Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiol., 9:361-377

Example 6. Comparison of Performance of Broilers Reared in India Fed a Novel Bacillus licheniformis or an Antibiotic Growth Promoter

[0147] A broiler growth performance study was conducted with day-old, male, Vencobb 400 (Venkateshwara Hatcheries Pvt. Ltd, India) chicks placed in floor pens with used litter. Three dietary treatments were randomly assigned, with 18 replicate pens per treatment containing 25 birds per pen. Birds were fed with one of the dietary treatments in three phases consisting of starter (1-14 days), grower (15-28 days) and finisher (29-42 days) phases. The basal diet was mainly based on corn-soybean meal (Table 6.1) containing 500g/MT of dinotolmide to control coccidiosis. The basal diet also included 4% meat and bone meal (MBM), as additional challenge as MBM is a predisposing factor for development of necrotic enteritis caused by Clostridium perfringens in broiler chickens (M'Sadeqa et al. 2015). Three dietary treatments were mainly based on corn-soybean meal (Table 6.1) and included; 1. Basal control (Control), 2. Control+50 g of Bacitracin Methylene Disalicyclate/MT of feed (BMD), 3. Control+a Bacillus licheniforomis strain DSM 32314 at 250 g/MT containing 2.0*109 cfu/g (DSM 32314). Experimental treatments were fed ad libitum in mash form from 1-42 days of age. Statistical analysis was performed using one-way ANOVA procedure and LSD post-test analysis with SAS vs9.4 (SAS Institute Inc., USA). The results of the treatments on body weight, feed conversion ratio and mortality are reported in Table 6.2.

TABLE-US-00007 TABLE 6.1 Basal diet and diet nutrient composition for starter grower and finisher phases. Starter Grower Finisher (1-14 d) (15-28 d) (29-42 d) Ingredients, % Corn 53.98 62.27 64.54 Soybean meal, 48% CP 36.09 27.64 24.63 Meat and bone meal 4.00 4.00 4.00 Soybean oil 2.46 2.60 3.60 Dicalcium phoslphate 1.48 1.51 1.53 Limestone 0.39 0.45 0.16 Premix 0.65 0.65 0.65 (including vit-min mix) Sodium chloride 0.28 0.29 0.29 Sodium bicarbonate 0.10 0.10 0.10 Choline chloride 50 0.10 0.10 0.10 DL-Methionine 0.29 0.23 0.22 L-Lysine HCl 0.12 0.13 0.14 L-Threonine 0.05 0.04 0.05 Nutrient composition ME, kcal/kg 2950 3050 3150 CP, % 23.50 20.08 18.83 Ca 1.00 1.00 1.00 Available P 0.45 0.45 0.45 Lys 1.36 1.14 1.06 Met 0.63 0.53 0.50 M + C 0.99 0.85 0.80 Thr 0.92 0.78 0.74 Trp 0.27 0.22 0.20 Arg 1.59 1.33 1.23 Ile 0.97 0.81 0.75 Leu 1.93 1.70 1.61 Val 1.08 0.92 0.86

TABLE-US-00008 TABLE 6.2 Animal performance between days 0 and 42 with and without diet supplementation with B. licheniformis based feed additive or antibiotic growth promoter. 1-21 d 1-42 d Treatment BW, g FCR, g/g BW, g FCR, g/g 1. Control 880.7 1.43 2757.4 1.76 2. BMD 899.0 1.42 2781.6 1.74 3. DSM 32314 905.2 1.42 2824.2 1.74 Difference 24.5 −0.01 66.8 −0.02 Relative % 2.8 −0.70 2.4 −1.14 BW, average bird body weight in specified time period; FCR, feed conversion ratio calculated as feed to gain in specified time period; Control, no additives in basal diet; BMD, treatment with addition of bacitracin methylene disalicylate to basal diet; DSM 32314, treatment with addition of strain DSM 32314 to basal diet; Difference, the numeric difference observed when DSM 32314was compared to control; Relative %, the difference between DSM 32314and control as a percent change from control.

[0148] The data from this broiler indicates that both products, BMD and DSM 32314, improved the body weight compared to the control. However, the pre-product group D treated group had the highest average weights at both 21 and 42 days with a 24.5g and 66.8g increase compared to the control, respectively. Likewise the, FCR was reduced in both the BMD and DSM 32314 treated groups at D21 and D42. This study indicates that in these conditions the DSM 32314 was able to improve FCR of broilers at least as well as the potent antibiotic growth promoter BMD compared to the control, and was even responsible for achieving the highest average weights of all the groups.

REFERENCES

[0149] M'Sadeqa, S., Wua S., Swicka. R. A. and M. Chocta (2015). Towards the control of necrotic enteritis in broiler chickens with in-feed antibiotics phasing-out worldwide. Animal Nutrition. 1:1-11.

Example 7: Well Diffusion Antagonism Tests with Respect to Different Pathogenic Strains

[0150] A well diffusion antagonism test with 3 different pathogens, Enterococcus cecorum DSM 20683, Streptococcus gallinaceus DSM 15349 and Streptococcus suis ATCC 43765 was performed.

[0151] E. cecorum is known to cause lameness, arthritis and osteomyelitis in broilers usually caused by an inflammation of a joint and/or bone tissue. Additional E. cecorum can cause an inflammation of the pericardium [Kense et al. 2011]. DSM 20683 was isolated from caecum of a chicken.

[0152] S. gallinaceus can cause septicaemia in poultry. The gross lesions included splenomegaly, hepatomegaly, renomegaly and congestion. Multiple areas of necrosis and/or infarction in the liver and spleen associated with valvular endocarditis were also observed [Collins et al. 2002].

[0153] S. suis is an important pathogen in pigs and one of the most important causes of bacterial mortality in piglets after weaning causing septicemia, meningitis and many other infections [Goyette-Desjardins et al. 2014]. ATCC 43765 belongs to Serological group: R; serovar 2 and was isolated from pigs.

[0154] Bacillus strains were grown in 10 mL TSBYE (30 g/l TSB+6 g/l Yeast extract) or LB-Kelly (LB-Media supplemented with trace elements solution of DSMZ media 1032) for 16 hat 37° C. and 200 rpm in 100 mL shaking flask. The pathogenic strains were grown under suitable conditions as liquid culture to an optical density of 595 nm of at least 1, then 100 μl were spread with sterile spatula on the surface of agar plates. For S. gallinaceus BHI agar plates, for E. cecorum and S. suis TSBYE agar plates are used. Three 9 mm diameter wells were cut into the dried plates. 1.sup.st well was used as non-inoculated media control without culture, 2.sup.nd well was inoculated with 100 uL not-inhibiting Bacillus strain (B. cereus var. toyoi, NCIMB 40112), the 3.sup.rd well was inoculated with 100 uL of Bacillus subtilis DSM 32315 or DSM 17299 culture. After 24 h incubation under suitable conditions at 37° C., the zone of clearance in mm was determined measuring from the edge of the cut well to the border of the cleared lawn. Each colony was measured twice (horizontally, vertically), then averaged. The results can be found in table 7.1.

TABLE-US-00009 TABLE 7.1 Comparison of Bacillus licheniformis DSM 32314 and Bacillus subtilis DSM 17299 inhibitory capacity on pathogenic strains in well diffusion antagonism assays, values in mm clearance of pathogen. Pathogen E. cecorum S. gallinaceus S. suis Probiotic DSM 20683 DSM 15349 ATCC 43765 DSM 32314 5.7 16.1 18.0 DSM 17299 0.0 0.0 5.5 The data show that DSM 32314 is able to inhibit E. cecorum, S. gallinaceus and S. suis very effectively, in particular in comparison to DSM 17299.

REFERENCES

[0155] M J Kense, W J M Landman (2011). Enterococcus cecorum infections in broiler breeders and their offspring: molecular epidemiology. Avian Pathology Vol. 40 , Iss. 6.

[0156] M D Collins, R A Hutson, E Falsen, E Ingana, M Bisgaard (2002). Streptococcus gallinaceus sp. nov., from chickens. International Journal of Systematic and Evolutionary Microbiology. 52: 1161-1164.

[0157] G Goyette-Desjardins, J-P Auger, J Xu, M Segura, M Gottschalk (2014). Streptococcus suis, an important pig pathogen and emerging zoonotic agent—an update on the worldwide distribution based on serotyping and sequence typing. Emerg Microbes Infect. 3(6):e45.