Method to reduce enteric colonization by hatchery-associated opportunistic pathogens and improve performance of chickens

Abstract

The present invention provides compositions comprising specific isolates of bifidobacterium bacteria. The composition may further comprise lactic acid bacteria. Methods of administering the compositions to a subject in ovo are also provided. The methods can be used, for example, to reduce the colonization of pathogens in the gastrointestinal tract of hatching chickens and thereby improve their growth performance.

Description

[0001] METHOD TO REDUCE ENTERIC COLONIZATION BY HATCHERY-ASSOCIATED OPPORTUNISTIC PATHOGENS AND IMPROVE PERFORMANCE OF CHICKENS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 600,459, which was filed on November 17, 2023, the contents of which are incorporated by reference in their entireties.

[0004] BACKGROUND

[0005] In commercial poultry farms, eggs are promptly removed from the hen and transferred to a hatchery. As a result, the eggs have limited exposure to the hen’s microbiota and the gastrointestinal tracts of neonatal chicks are instead colonized by the first microorganisms that they encounter in the hatch cabinet. Hatch cabinets provide optimal conditions for microbial proliferation, and opportunistic pathogens flourish in this environment. Accordingly, there is a need in the art for methods for controlling the colonization of the chick gastrointestinal tract during hatching.

[0006] SUMMARY

[0007] In a first aspect, the present invention provides compositions comprising a Bifidobacterium bacterium selected from: (a) a bacterium designated as B2-2 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B- 68468, (b) a bacterium designated as B3-4 and deposited at the NRRL under accession number NRRL B-68467, and (c) a combination of (a) and (b). In some embodiments, the compositions further comprise a lactic acid bacterium selected from (a) a bacterium designated as LAB 18 and deposited at the NRRL under accession number NRRL B-50964, and (b) a bacterium designated as LAB46 and deposited at the NRRL under accession number NRRL B-50961.

[0008] In a second aspect, the present invention provides methods for administering the bacterial compositions described herein to a subject. In preferred embodiments, administration is in ovo.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 (Example 1, Trial 2) shows Bifidobacterium bacteria recovery from the gastrointestinal tract of chicks at hatch. On day 18 of embryogenesis, eggs were injected with either saline or a Bifidobacterium isolate (i.e., Bl-2, Bl-3, B2-1, B2-2, B3-1, or B3-4). On day 21, whole gut samples were collected from hatched chicks and were enriched in trypticase phytone yeast extract (TPY) broth overnight in anaerobic boxes and plated on TPY agar to assess total Bifidobacterium recovery.

[0011] FIG. 2 (Example 1, Trial 3) shows Bifidobacterium bacteria recovery from the gastrointestinal tract of chicks at hatch. On day 18 of embryogenesis, eggs were injected with either saline or a Bifidobacterium isolate (i.e., Bl-2, Bl-3, B2-2, B2-4, B3-1, B3-2, or B3-4). On day 21, whole gut samples were collected from hatched chicks and were enriched in TPY broth overnight in anaerobic boxes and plated on TO S -propionate agar with mupirocin (TOS MUP agar) to assess total Bifidobacterium recovery.

[0012] FIG. 3 (Example 1, Trial 4) shows the effect of in ovo inoculation on hatchability. On day 18 of embryogenesis, eggs (30 eggs per isolate) were injected with either saline or a bacterial treatment. The treatments included either one or two Bifidobacterium isolates (i.e., B2-2, B3-4, or a combination thereof). On day 21, hatched chicks were pulled from hatch cabinets and hatchability was determined.

[0013] FIG. 4 (Example 1, Trial 4) shows Bifidobacterium bacteria recovery from the gastrointestinal tract of chicks at hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included either one or two Bifidobacterium isolates (i.e., B2-2, B3-4, or a combination thereof). On day 21, the whole gut was removed from hatched chicks and samples were plated on TOS MUP agar to evaluate total Bifidobacterium recovery.

[0014] FIG. 5 (Example 1, Trial 5) shows the effect of in ovo inoculation on hatchability. On day 18 of embryogenesis, eggs (30 eggs per isolate) were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, either alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). On day 21, chicks were pulled from hatch cabinets and hatchability was determined.

[0015] FIG. 6 (Example 1, Trial 5) shows lactic acid bacteria recovery from the gastrointestinal tract of chicks at hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, either alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). On day 21, the whole gut was removed from hatched chicks and samples were plated on Man Rogosa Sharpe (MRS) agar to evaluate lactic acid bacteria recovery. FIG. 7 (Example 1, Trial 5) shows Bifidobacterium bacteria recovery from the gastrointestinal tract of chicks at hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included combinations of the lactic acid bacterium LAB 18 with either one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). On day 21, the whole gut was removed from hatched chicks and samples were plated on TOS MUP agar to evaluate total Bifidobacterium recovery.

[0016] FIG. 8 (Example 1, Trial 6) shows the effect of in ovo inoculation on hatchability. On day 18 of embryogenesis, eggs (60 eggs per isolate) were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, a combination two Bifidobacterium bacteria (i.e., B2-2 and B3-4), and combinations of LAB18 with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). On day 21, chicks were pulled from hatch cabinets and hatchability was determined. Notably, the hatchability reported in this figure is lower than expected because it was not adjusted to account for mortalities and infertile eggs as it was in FIG. 3 and FIG. 5.

[0017] FIG. 9 (Example 1, Trial 6) shows lactic acid bacteria recovery from the gastrointestinal tract of chicks at hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, a combination two Bifidobacterium bacteria (i.e., B2-2 and B3-4), and combinations of LAB18 with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). On day 21, the whole gut was removed from hatched chicks and samples were plated on MRS agar to evaluate lactic acid bacteria recovery.

[0018] FIG. 10 (Example 1, Trial 6) shows Bifidobacterium bacteria recovery from the gastrointestinal tract of chicks at hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, a combination two Bifidobacterium bacteria (i.e., B2-2 and B3-4), and combinations of LAB18 with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). On day 21, the whole gut was removed from hatched chicks and samples were plated on TOS MUP agar to evaluate total Bifidobacterium recovery.

[0019] FIG. 11 (Example 1, Trial 6) shows Gram-negative bacteria recovery from the gastrointestinal tract of chicks at hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, a combination two Bifidobacterium bacteria (i.e., B2-2 and B3-4), and combinations of LAB18 with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). On day 21 , the whole gut was removed from hatched chicks and samples were plated on MacConkey’s agar to evaluate total Gram-negative bacteria recovery.

[0020] FIG. 12 (Example 1, Trial 6) shows the average body weight gain of chicks from day 0-7 post hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, either alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). Chicks were challenged with Salmonella enterica serovar Enteritidis on the day of hatch and individual bodyweights were recorded on day 1 and day 7 post hatch. Data are expressed as average bodyweight gain per bird from day 0 to day 7. Outliers more than 1.5 standard deviations from the mean were removed from each treatment.

[0021] FIG. 13 (Example 1, Trial 6) shows the average body weight gain of chicks from day 0- 10 post hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, either alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). Chicks were challenged with Salmonella enterica serovar Enteritidis on the day of hatch and individual body weights were recorded on day 1, day 7, and day 10 post hatch. Data are expressed as average body weight gain per bird from day 0 to day 10. Outliers more than 1.5 standard deviations from the mean were removed from each treatment.

[0022] FIG. 14 (Example 1, Trial 6) shows lactic acid bacteria recovery from the gastrointestinal tract of chicks 10 days after hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, either alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). Chicks were challenged with Salmonella enterica serovar Enteritidis on the day of hatch. On day 10 post hatch, the whole gut was removed from hatched chicks and samples were plated on MRS agar to evaluate lactic acid bacteria recovery.

[0023] FIG. 15 (Example 1, Trial 6) shows Bifidobacterium bacteria recovery from the gastrointestinal tract of chicks 10 days after hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB18, either alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). Chicks were challenged with Salmonella enterica serovar Enteritidis on the day of hatch. On day 10 post hatch, the whole gut was removed from hatched chicks and samples were plated on TOS MUP agar to evaluate total Bifidobacterium recovery.

[0024] FIG. 16 (Example 1, Trial 6) shows Gram-negative bacteria recovery from the gastrointestinal tract of chicks 10 days after hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB 18, either alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). Chicks were challenged with Salmonella enterica serovar Enteritidis on the day of hatch. On day 10 post hatch, the whole gut was removed from hatched chicks and samples were plated on MacConkey’s agar to evaluate total Gram-negative bacteria recovery.

[0025] FIG. 17 (Example 1, Trial 6) shows Salmonella bacteria recovery from the gastrointestinal tract of chicks 10 days after hatch. On day 18 of embryogenesis, eggs were injected with either saline or a bacterial treatment. The treatments included the lactic acid bacterium LAB18, either alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4). Chicks were challenged with Salmonella enterica serovar Enteritidis on the day of hatch. On day 10 post hatch, the whole gut was removed from hatched chicks and samples were plated on brilliant green agar (BGA) supplemented with novobiocin (NO) and nalidixic acid (NA) to evaluate Salmonella recovery. Notably, the Salmonella used as the challenge organism is resistant to NO and NA.

[0026] FIG. 18 (Example 2, Experiment 1) shows Enterococcus bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included one lactic acid bacterium (i.e., LAB 18 or LAB46), one Bifidobacterium bacterium (i.e., B2-2 or B3-4), or a combination of one lactic acid bacterium and one Bifidobacterium bacterium and are compared to a non-challenged, non-treated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at <0.05.

[0027] FIG. 19 (Example 2, Experiment 1) shows Gram-negative bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included one lactic acid bacterium (i.e., LAB 18 or LAB46), one Bifidobacterium bacterium (i.e., B2-2 or B3-4), or a combination of one lactic acid bacterium and one Bifidobacterium bacterium and are compared to a non-challenged, non-treated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at P<0.05.

[0028] FIG. 20 (Example 2, Experiment 1) shows Bifidobacterium bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included one lactic acid bacterium (i.e., LAB 18 or LAB46), one Bifidobacterium bacterium (i.e., B2-2 or B3-4), or a combination of one lactic acid bacterium and one Bifidobacterium bacterium and are compared to a non-challenged, non-treated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at P<0.05.

[0029] FIG. 21 (Example 2, Experiment 1) shows lactic acid bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included one lactic acid bacterium (i.e., LAB 18 or LAB46), one Bifidobacterium bacterium (i.e., B2-2 or B3-4), or a combination of one lactic acid bacterium and one Bifidobacterium bacterium and are compared to a non-challenged, non-treated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at P<Q.Q5.

[0030] FIG. 22 (Example 2, Experiment 2) shows Enterococcus bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included the lactic acid bacterium LAB46 alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) and are compared to a non-challenged, nontreated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at f 0.05.

[0031] FIG. 23 (Example 2, Experiment 2) shows Gram-negative bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included the lactic acid bacterium LAB46 alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) and are compared to a non-challenged, non- treated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at / ?0.05.

[0032] FIG. 24 (Example 2, Experiment 2) shows Bifidobacterium bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included the lactic acid bacterium LAB46 alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) and are compared to a non-challenged, nontreated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at <0.05.

[0033] FIG. 25 (Example 2, Experiment 2) shows lactic acid bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included the lactic acid bacterium LAB46 alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) and are compared to a non-challenged, nontreated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at C 0.05.

[0034] FIG. 26 (Example 2, Experiment 2) shows the average body weight gain from day 0-7, day 7-14, and day 0-14 after hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included the lactic acid bacterium LAB46 alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) and are compared to a non-challenged, nontreated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at f 0.05.

[0035] FIG. 27 (Example 2, Experiment 3) shows Enterococcus bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included the lactic acid bacterium LAB46 alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) and are compared to a non-challenged, nontreated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at P 0.05. FIG. 28 (Example 2, Experiment 3) shows Gram-negative bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included the lactic acid bacterium LAB46 alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) and are compared to a non-challenged, nontreated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at f 0.05.

[0036] FIG. 29 (Example 2, Experiment 3) shows Bifidobacterium bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included the lactic acid bacterium LAB46 alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) and are compared to a non-challenged, nontreated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at 7’<0.05.

[0037] FIG. 30 (Example 2, Experiment 3) shows lactic acid bacteria recovery from the gastrointestinal tract of chicks at hatch. Eggs were injected with various bacterial treatments on day 18 of embryogenesis and were challenged with a pathogen mix (PM) on day 19. The treatments included the lactic acid bacterium LAB46 alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) and are compared to a non-challenged, nontreated control (NC) and a challenged, non-treated control (PM). Differing superscripts indicate significant differences between treatment groups at / ?0.05.

[0038] FIG. 31 (Example 3) shows a meta-analysis of Gram-negative bacterial recovery from DOH GIT samples combined from trials 2, 3, and 4. Data reported as ‘mean ± SE’ LoglO CFU / g of sample. n=36 samples per media per treatment. ANOVA (JMP Pro 17) was used to determine significant differences at E < 0.05. Means further separated by Student’s T test. Differing letters indicate significant differences across treatment groups. Treatment abbreviations: NC (negative control), PM (pathogen mix), LAB46 (lactic acid bacteria isolate 46), LAB46 + B2-2 (LAB46 + Bifidobacterium saeculare isolate 2-2), LAB46 + B3-4 (LAB46 + Bifidobacterium saeculare isolate 3-4), LAB46 + B-Combo (LAB46 + both Bifidobacterium saeculare isolates).

[0039] FIG. 32 (Example 3) shows a meta-analysis of Enterococcus spp. recovery from DOH GIT samples combined from trials 2, 3, and 4. Data reported as ‘mean ± SE’ LoglO CFU / g of sample. n=36 samples per media per treatment. ANOVA (JMP Pro 17) used to determine significant differences at < 0.05. Means further separated by Student’s T test. Differing letters indicate significant differences across treatment groups. Treatment abbreviations: NC (negative control), PM (pathogen mix), LAB46 (lactic acid bacteria isolate 46), LAB46 + B2-2 (LAB46 + Bifidobacterium saeculare isolate 2-2), LAB46 + B3-4 (LAB46 + Bifidobacterium saeculare isolate 3-4), LAB46 + B-Combo (LAB46 + both Bifidobacterium saeculare isolates).

[0040] FIG. 33 (Example 3) shows a meta-analysis of lactic acid bacterial recovery from DOH GIT samples combined from trials 2, 3, and 4. Data reported as ‘mean ± SE’ LoglO CFU / g of sample. n=36 samples per media per treatment. ANOVA (JMP Pro 17) used to determine significant differences at E < 0.05. Means further separated by Student’s T test. Differing letters indicate significant differences across treatment groups. Treatment abbreviations: NC (negative control), PM (pathogen mix), LAB46 (lactic acid bacteria isolate 46), LAB46 + B2-2 (LAB46 + Bifidobacterium saeculare isolate 2-2), LAB46 + B3-4 (LAB46 + Bifidobacterium saeculare isolate 3-4), LAB46 + B-Combo (LAB46 + both Bifidobacterium saeculare isolates).

[0041] FIG. 34 (Example 3) shows a meta-analysis of Bifidobacterium spp. recovery from DOH GIT samples combined from trials 2, 3, and 4. Data reported as ‘mean ± SE’ LoglO CFU / g of sample. n=36 samples per media per treatment. ANOVA (JMP Pro 17) used to determine significant differences at < 0.05. Means further separated by Student’s T test. Differing letters indicate significant differences across treatment groups. Treatment abbreviations: NC (negative control), PM (pathogen mix), LAB46 (lactic acid bacteria isolate 46), LAB46 + B2-2 (LAB46 + Bifidobacterium saeculare isolate 2-2), LAB46 + B3-4 (LAB46 + Bifidobacterium saeculare isolate 3-4), LAB46 + B-Combo (LAB46 + both Bifidobacterium saeculare isolates).

[0042] DETAILED DESCRIPTION

[0043] The present invention provides compositions comprising specific isolates of Bifidobacterium bacteria. The compositions may further comprise lactic acid bacteria. Methods of administering the compositions to a subject in ovo are also provided.

[0044] In poultry, the initial colonization of the gastrointestinal tract typically occurs at hatch. However, the gastrointestinal tract of chicks can be pre-colonized with microorganisms via in ovo injection during the late stage of embryogenesis. Pre-colonization with beneficial microorganisms helps to protect chicks from colonization by pathogenic microorganisms present in the hatching environment. Advantageously, in ovo injection allows one to expose chicks to higher concentrations of beneficial bacteria than can feasibly be achieved in a natural setting.

[0045] The present inventors have identified several beneficial bacteria that can be used to precolonize the gastrointestinal tract of chicks. These beneficial bacteria include two isolates of Bifidobacterium bacteria, i.e., B2-2 and B3-4, and two isolates of lactic acid bacteria, i.e., LAB 18 and LAB46. As is demonstrated in the Examples, injecting specific combinations of these bacteria into chicken eggs results in increased colonization by the injected bacteria, reduced colonization by pathogenic bacteria, and improved growth performance in newly hatched chicks.

[0046] Bacterial compositions:

[0047] In a first aspect, the present invention provides compositions comprising a Bifidobacterium bacterium selected from: (a) a bacterium designated as B2-2 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B- 68468, (b) a bacterium designated as B3-4 and deposited at the NRRL under accession number NRRL B-68467, and (c) a combination of (a) and (b).

[0048] The terms “Bifidobacterium bacteria” and “bifidobacteria” are used interchangeably herein to refer to bacteria of the genus Bifidobacterium . Bifidobacteria are Gram-positive, nonmotile, anaerobic bacteria that are ubiquitous inhabitants of the gastrointestinal tracts of animals. As is described in Example 1, the inventors isolated the bifidobacteria isolates B2-2 and B3-4 from turkey fecal samples. Thus, the bifidobacteria isolates included in the compositions of the present invention are poultry derived. B2-2 and B3-4 were both classified as belonging to the species Bifidobacterium saeculare based on 16S DNA sequencing data.

[0049] In the Examples, the inventors tested bifidobacteria isolates in combination with the lactic acid bacteria LAB 18 and LAB46. Thus, in some embodiments, the compositions further comprise a lactic acid bacterium selected from (a) a bacterium designated as LAB 18 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B-50964, and (b) a bacterium designated as LAB46 and deposited at the NRRL under accession number NRRL B-50961. The term “lactic acid bacteria” is used herein to refer to bacteria of the order Lactobacillales, which are Gram-positive bacteria that produce lactic acid via carbohydrate fermentation. Lactic acid bacteria are present in high numbers in fermented foods and are widely used as probiotics. The lactic acid bacteria used with the present invention, i.e., LABI 8 and LAB46, are the bacteria used in the poultry probiotic product FloraMax®-Bl 1 . FloraMax®-Bl 1 has been shown to increase resistance to Salmonella infections, reduce idiopathic diarrhea in commercial turkey brooding houses, and increase poultry performance. LAB 18 (previously referred to as FM18) is an isolate of the species Pediococcus acidilactici, and LAB46 (previously referred to as FM46) is an isolate of the species Lactobacillus salivarius. Both of these lactic acid bacteria were isolated from the poultry gastrointestinal tract. The inventors have previously used these lactic acid bacteria as a poultry probiotic, as is described in U.S. Patent No. 10,786,565, which is hereby incorporated by reference in its entirety.

[0050] The compositions of the present invention may comprise various combinations of the bifidobacteria B2-2 and B3-4 and the lactic acid bacteria LAB 18 and LAB46. In some embodiments, the compositions comprise a combination of the lactic acid bacterium LAB46 and one or two bifidobacteria. Specifically, these compositions may comprise: (a) LAB46 and B2-2, (b) LAB46 and B3-4, or (c) LAB46, B2-2, and B3-4. In other embodiments, the compositions comprise a combination of the lactic acid bacterium LAB 18 and one or two bifidobacteria. These compositions may comprise: (a) LAB 18 and B2-2, (b) LAB 18 and B3-4, or (c) LAB 18, B2-2, and B3-4.

[0051] In some embodiments, the bacterial compositions are dried or stabilized in some other manner. As used herein, a “dried” composition is a composition from which some amount of water has been removed. Drying can be accomplished using any method known in the art including, without limitation, heat drying, dielectric drying, freeze drying (lyophilization), supercritical drying, and air drying.

[0052] The bacteria may be included in the compositions at any concentration that is appropriate for the intended application. For example, in a dried composition, the concentration of a bacterium may be at least IxlO7CFU per gram of composition, at least IxlO8CFU per gram of composition, at least IxlO9CFU per gram of composition, at least IxlO10CFU per gram of composition, or higher.

[0053] The compositions of the present invention may further comprise additional reagents. In some embodiments, the compositions further comprise a carrier. As used herein, the term “carrier” refers to a diluent, excipient, or propellant. In embodiments in which the composition is designed to be administered to a subject, the carrier may be a pharmaceutically acceptable carrier, i.e., a carrier that is not deleterious to a recipient to which it is administered. Examples of pharmaceutically acceptable carriers include diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., thimerosal, benzyl alcohol, parabens), solubilizing agents (e.g., glycerol, polyethylene glycerol), emulsifiers, liposomes, nanoparticles, and adjuvants. In embodiments in which the composition is dried, the carrier may be a dry substance, such as a powder.

[0054] The compositions may further comprise additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), antioxidants (e.g., ascorbic acid, sodium metabisulfite), bulking substances or tonicity modifiers (e g., lactose, mannitol). Components of the compositions may be covalently attached to polymers (e.g., polyethylene glycol), complexed with metal ions, or incorporated into or onto particulate preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels) or onto liposomes, microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. The compositions may also be formulated in lipophilic depots (e.g., fatty acids, waxes, oils) for controlled or sustained release.

[0055] The compositions of the present invention were initially designed to be injected into poultry eggs to serve as an in ovo probiotic. In large-scale commercial poultry operations, eggs are commonly injected with vaccines to vaccinate developing chicks prior to hatching. Thus, in some embodiments, the compositions of the present invention are formulated to be administered in combination with a vaccine. For example, the compositions may further comprise a vaccine and / or a vaccine carrier. Examples of poultry vaccines that may be combined with the compositions of the present invention include vaccines against Marek's disease, avian influenza, Newcastle disease, fowlpox, infectious bursal disease (Gumboro disease), infectious laryngotracheitis, coccidiosis, infection by coronaviruses, and infection by reoviruses.

[0056] The active ingredient in all vaccines is either an antigen or a nucleic acid encoding an antigen. Thus, in some embodiments, the compositions further comprise an antigen. As used herein, the term “antigen” refers to a substance that induces an immune response in a recipient. Antigens can be any type of biologic molecule including, for example, simple intermediary metabolites, sugars, lipids, and hormones as well as macromolecules such as complex carbohydrates, phospholipids, nucleic acids, and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa antigens, parasitic antigens, and tumor antigens. In other embodiments, the compositions further comprise a nucleic acid (i.e., a polymer of DNA or RNA) encoding an antigen. Methods:

[0057] In a second aspect, the present invention provides methods for administering the bacterial compositions described herein to a subject.

[0058] The bacterial compositions utilized in the methods of the present invention comprise a Bifidobacterium bacterium selected from: (a) a bacterium designated as B2-2 and deposited at the NRRL under accession number NRRL B-68468, (b) a bacterium designated as B3-4 and deposited at the NRRL under accession number NRRL B-68467, and (c) a combination of (a) and (b). In the Examples, the inventors demonstrate that injecting bifidobacteria into chicken eggs at a concentration of IxlO8CFU per embryo is sufficient to allow the bifidobacteria to colonize the chick gastrointestinal tract. Thus, in some embodiments, the Bifidobacterium bacterium is administered at a concentration of IxlO7- IxlO9CFU per embryo.

[0059] In some embodiments, the compositions utilized in the methods further comprise a lactic acid bacterium selected from (a) a bacterium designated as LAB 18 and deposited at the NRRL under accession number NRRL B-50964, and (b) a bacterium designated as LAB46 and deposited at the NRRL under accession number NRRL B-50961. In the Examples, the inventors demonstrate that including LAB 18 or LAB46 in their probiotic compositions at a concentration of IxlO4CFU per embryo can increase bifidobacteria colonization, reduce colonization by pathogenic bacteria, and increase early chicken performance. Thus, in some embodiments, the lactic acid bacterium may be administered at a concentration of IxlO3- IxlO3CFU per embryo.

[0060] “Administering” refers to the introduction of a substance into a subject's body. Methods of administration are well known in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraoral administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, intrathecal administration, and subcutaneous administration. Administration can be continuous or intermittent.

[0061] As is noted above, the compositions of the present invention were initially designed to be injected into poultry eggs to serve as an in ovo probiotic. Thus, in preferred embodiments, administration is in ovo. In ovo administration involves puncturing the eggshell with a fine needle and injecting a composition into the egg. This is commonly accomplished using an in ovo machine. Within the egg, the composition may be injected into the amnion or into the air cell (i.e., a pocket of air between the outer and inner membranes that typically rests at the large end of the egg). In the Examples, the inventors injected their compositions into the amnion, as this route of administration has been shown to be more efficacious for delivering beneficial bacteria to developing embryos in late embryogenesis. Thus, in preferred embodiments the compositions are injected into the amniotic fluid (i.e., the fluid contained within the amnion).

[0062] In ovo administration of bacteria during the early stages of embryogenesis could potentially be detrimental to the developing embryo. Thus, in some embodiments in which the composition is administered in ovo, the composition is administered in the late stage of embryogenesis. In chickens, the late state of embryogenesis is from day 17 to day 19 of embryogenesis whereas, in turkeys, the late state of embryogenesis is from day 24 to day 26 of embryogenesis. In the Examples, the inventors injected bacterial compositions into chicken eggs on day 18 of embryogenesis. Thus, in certain embodiments, the compositions are administered on day 18 of embryogenesis. However, the age of the eggs into which the compositions are injected should be selected in view of the developmental timing of the subject.

[0063] The “subject” to which the methods are applied may an animal of any species. For example, in some embodiments, the subject is a livestock animal, such as a cow, horse, pig, rabbit, goat, sheep, or chicken. However, in preferred embodiments, the subject is a poultry animal. Poultry are domesticated birds that are raised for the production useful animal products, such as eggs and meat. Examples of poultry birds include, chickens, turkeys, quails, ducks, geese, guinea fowl, and pigeons. In the Examples, the inventors administered bacterial compositions to broiler chicken eggs. Thus, in certain specific embodiments, the subject is a chicken.

[0064] Development of a healthy gut microbiome is a crucial for the health and performance of livestock animals. In the Examples, the inventors demonstrate that in ovo administration of the bacterial compositions described herein alters enteric colonization in newly hatched chicks. Specifically, they show that their compositions can reduce colonization by pathogens and / or increase colonization by the beneficial bacteria provided in the composition. Thus, in some embodiments, the method reduces the colonization of a pathogen in the gastrointestinal tract of the subject as compared to an untreated control and / or increases the colonization of Bifidobacterium bacteria and / or lactic acid bacteria in the gastrointestinal tract of the subject as compared to an untreated control.

[0065] As used herein, an “untreated control” is a comparable animal to the subject (e.g., of the same species, breed, sex, and age) that was hatched or birthed in a similar environment but that was not treated with a bacterial composition described herein.

[0066] As used herein, “colonization” refers to the introduction of microorganisms into the gastrointestinal tract of an animal. The microorganisms present in the gastrointestinal tract of a chick can be assessed by collecting chick droppings or gastrointestinal tract samples and enumerating the bacteria present therein on various culture media. Alternatively, the microorganisms present in the gastrointestinal tract of a chick can be assessed via metagenomic sequencing. Colonization may be assessed immediately after hatch / birth or may be assessed at a later stage of development.

[0067] As used herein the term “pathogen” refers to an organism that is capable of causing disease in an animal. Exemplary pathogens include bacteria, fungi, viruses, algae, arachnids, insects, nematodes, parasitic plants, and protozoans. In Example 1, the inventors tested the ability of bacterial compositions to prevent colonization by an isolate of Salmonella enterica serovar Enteritidis, bacteriophage type 13 A, that was isolated from poultry. Thus, in some embodiments, the pathogen is a Salmonella bacterium. In Example 2, the inventors tested the ability of bacterial compositions to prevent colonization by a mixture of pathogens comprising Enterococcus cecorum, Enterococcus faecalis, and Escherichia coli. Thus, in some embodiments, the pathogen is an Enterococcus bacterium or an Escherichia bacterium. In some embodiments, the pathogen is a mixture of several pathogenic bacteria.

[0068] Additionally, the inventors have demonstrated that in ovo administration of their bacterial compositions increases the growth performance of chickens after hatch. Thus, in some embodiments, the method increases the growth performance of the subject as compared to an untreated control. Growth performance may be assessed in terms of body weight gain, i.e., the difference between an animal’s final weight and initial weight. As is described in Example 2, the inventors observed an 8.01-10.47% increase in body weight gain from hatch to day 14 post-hatch in response to their probiotic treatments. Thus, in some embodiments, the body weight gain is increased by 14 days post-hatch. In some embodiments, body weight gain is increased at least 5- 15%, at least 7-12%, or at least 8-11% as compared to an untreated control. In the disclosed methods, the composition may be administered to the subject once or multiple times. For example, the composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

[0069] The methods of the present invention can be used in combination with other methods for controlling enteric colonization. For example, the methods may further comprise administering an additional probiotic composition to the subject to pre-colonize the subject with beneficial microorganisms or may further comprise applying an antimicrobial composition to the environment in which the subject is born or hatched to inhibit the growth of pathogens therein. The additional composition may comprise one or more living microorganisms or may comprise a cell-free supernatant produced using a microorganism. The additional composition may be applied or administered using the same method used for the bacterial composition described herein or a different method. For example, to control a microorganism in a poultry hatching environment, the additional composition may be sprayed into the hatch cabinet, applied to the surface of eggs, injected into eggs, or administered directly to chicks after they have hatched.

[0070] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0071] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0072] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents form part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or descriptions found in the cited references.

[0073] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0074] EXAMPLES

[0075] Example 1:

[0076] The following example describes the isolation of novel Bifidobacterium isolates from poultry and a preliminary assessment of their potential for use as in ovo probiotics. The primary goals for performing the experiments described in this example were: (1) to determine whether Bifidobacterium bacteria (bifidobacteria) are present in poultry, (2) to determine whether bifidobacteria could be isolated from poultry, and (3) to determine whether poultry-derived bifidobacteria are useful as in ovo probiotics.

[0077] While little has been published on the benefits of bifidobacteria probiotics in poultry, they have been shown to have many beneficial effects in humans. For example, bifidobacteria have been shown to have antimicrobial activity, including against pathogenic bacteria such as C. difficile and H. pylori (Lee, 2003). Bifidobacteria also positively impact the immune system by secreting lysosomal enzymes and enhancing phagocytosis (Gill, 1998), and certain isolates of bifidobacteria promote downregulation of the pro-inflammatory cytokines IL-6 and IL-12 and upregulation of the anti-inflammatory cytokine IL- 10 (Citar, 2015). Further, bifidobacteria have beneficial properties for use as probiotics. For example, bifidobacteria are resistant to bile and have traits that that allow them to rapidly colonize the human get, such as high cell surface hydrophobicity (which improves adhesion to host cells) and strong adherence to human mucus (Ljungh & Wadstrom, 2006).

[0078] Bifidobacteria are prominent constituents of the gut microbiota of various animals. However, the particular strains of bifidobacteria that are found in the gut appear to be host specific. For example, infant-type bifidobacteria are never found in children or adults, and animal -type bifidobacteria are never found in humans (Mitsuoka, 1990). This high specificity may be indicative of the important role that these bacteria plat in the gut microbiome.

[0079] Results:

[0080] Ten Bifidobacterium isolates were obtained from fecal samples from broody turkeys. The species of each isolate was determined via 16S DNA sequencing (Table 1). The viability of five of these isolates following exposure to air (for 0-120 minutes) was tested, and the results demonstrate that these bacteria can be exposed to air for the duration of time required for in ovo injection without affecting their viability (Table 2). Table 1. Bifidobacterium isolate species identity

[0081] Table 2. Bifidobacterium isolate viability following exposure to air

[0082] Numbers expressed as Logio CFU.

[0083] TO: Bacteria were plated and immediately placed in an anaerobic box. T120: Bacteria were exposed to air for 120 minutes before being placed in an anaerobic box.

[0084] 3 days: Bacteria were incubated at 2-8°C for 3 days in an air-tight injection port tube.

[0085] Six trials were performed to test the effect of in ovo inoculation with the Bifidobacterium isolates on hatchability and gastrointestinal colonization. Eighteen-day-old embryos were obtained from Cobb-Vantress (Siloam Springs, AR). In each trial, eggs were candled and inoculated with 200 pl of either saline or a bacterial treatment via in ovo injection into the amnion. The bacterial treatments comprised various combinations of the Bifidobacterum isolates (approximately 108CFU) and the lactic acid bacterium LAB 18 (approximately 104CFU). The different treatment groups were placed in separate hatchers to avoid cross contamination. On day 21 of embryogenesis (i.e., day of hatch), chicks were pulled from hatch cabinets and the hatchability was determined (Table 3 (trial 1-3), FIG. 3 (trial 4), FIG. 5 (trial 5), and FIG. 8 (trial 6)).

[0086] Table 3. Bifidobacterium isolate effect on hatchability (trial 1) In trials 2-6, 12 chickens from each treatment group were humanely euthanized to evaluate their gastrointestinal compositions. To this end, the whole gut (i.e., ventriculus to cecum) was aseptically removed on day 21 of embryogenesis. Sections were collected into sterile bags and homogenized. Samples were weighed and 1 :4 wt / vol dilutions were made with sterile 0.9% saline.

[0087] To assess Bifidobacterium bacteria recovery, 10-fold dilutions of each sample were made in a sterile 96 well Bacti flat bottom plate and the diluted samples were plated on trypticase phytone yeast extract (TPY) agar (FIG. 1 (trial 2)) or TOS-propionate agar with mupirocin (TOS MUP agar) (FIG. 2 (trial 3), FIG. 4 (trial 4), FIG. 7 (trial 5), and FIG. 10 (trial 6)). Note: In trials 2 and 3, samples were enriched overnight in media at 37°C prior to plating. To assess lactic acid bacteria recovery, the samples were plated on Man Rogosa Sharpe (MRS) agar (FIG. 6 (trial 5) and FIG. 9 (trial 6)). To assess total Gram-negative bacteria recovery, samples were plated on MacConkey’s agar (FIG. 11 (trial 6)).

[0088] Additionally, in trial 6, hatched chicks were challenged with a poultry isolate of Salmonella enterica serovar Enteritidis, bacteriophage type 13A (obtained from the USDA National Veterinary Services Laboratory). This isolate was resistant to 25 pg / mL of novobiocin (NO) (catalog no. N-1628, Sigma, St. Louis, MO 63103) and was selected for resistance to 20 pg / mL of nalidixic acid (NA) (catalog no. N-4382, Sigma). Chicks were challenged with this isolate at approximately 104CFU / chick on the day-of-hatch (day 0) after being pulled from the hatch cabinets. Individual body weights were recorded on days 1, 7, and 10 (FIG. 12 and FIG. 13). On day 10, whole gut samples were collected from 12 birds per group and were processed as described above. Diluted samples were plated on various media to select for the growth of particular bacteria. Specifically, lactic acid bacteria recovery was assessed using MRS agar (FIG. 14), Bifidobacterium bacteria recovery was assessed using TOS MUP agar (FIG. 15), total Gram-negative bacteria recovery was assessed using MacConkey’s agar (FIG. 16), and Salmonella bacteria recovery was assessed using brilliant green agar (BGA) supplemented with novobiocin (NO) and nalidixic acid (NA) (FIG. 17).

[0089] Discussion:

[0090] The studies described in this example demonstrate that bifidobacteria are present in poultry and can be isolated therefrom. Further, these studies demonstrate that in ovo injection of poultry-derived bifidobacteria does not negatively affect hatchability and that the injected bacteria can colonize the gastrointestinal tract of chickens, as approximately 5 to 6 logs of bifidobacteria were recovered from the whole gut at hatch. When bifidobacteria isolates were combined with the lactic acid bacterium LAB 18 for in ovo treatment, there was no adverse effect on hatchability and a significant increase in the recovery of both lactic acid bacteria and bifidobacteria from the gastrointestinal tract of hatched chicks was observed.

[0091] A Salmonella challenge experiment was conducted to evaluate the effect of the in ovo probiotics on the growth of Salmonella and 10-day performance parameters. At hatch, all probiotic treated groups showed significantly lower recovery of Gram-negative bacteria as compared to a saline control. However, 10 days later, the recovery of both Salmonella and total Gram-negative bacteria was higher in the probiotic treated groups. From day 0 to day 7, body weight gain was elevated in groups treated with specific combinations of LAB 18 and Bifidobacterium isolates (i.e., LAB18+B2-2 and LAB18+B2-2+B3-4), but not when body weight The gut microbiome is established rapidly post hatch in chickens (i.e., within the first 72 hours) and early colonization is known to be important for postnatal development. For example, in ovo injection with beneficial bacteria has been shown to impact gut maturation and immune system development (Wilson, 2020). Thus, while early exposure to beneficial bacteria may not shift the population of recoverable bacteria in the gut long term, it may nonetheless have lasting effects on host development.

[0092] Materials and Methods:

[0093] Isolation of Bifidobacteria

[0094] Bifidobacterium isolates were obtained from broody turkeys via intraperitoneal (IP) injection of pilocarpine, which causes cecal evacuation within minutes. Fecal samples were acquired and cultured in trypticase phytone yeast extract (TPY) broth in anaerobic chambers on a culture shaker at 37°C for 48 hours. After enrichment, samples were struck for isolation on TOS- propionate agar with mupirocin (TOS MUP agar) and the plates were grown anaerobically for 48 hours at 37°C. Next, individual colonies were picked from the plates and enriched again in TPY broth. After 48 hours of growth, the samples were struck for isolation on TOS MUP agar, which yielded plates with similar colony morphologies. A single colony from each plate was observed under a bright-field microscope to confirm bifidobacterial morphology. Ten Bifidobacterium isolates were obtained using this process.

[0095] Viability following exposure to air An in vitro test was performed to assess the resilience of five of the Bifidobacterium isolates following exposure to oxygen. A 200 pl sample of each tested isolate was placed in a 96 well plate. Each sample was diluted and plated on TPY and TOS MUP agar at four timepoints: after 0 minutes, 30 minutes, 60 minutes, and 120 minutes. The samples were then placed in the cold room at 2°C for 3 days to determine if the exposure to air impacted viability. No difference in growth was observed at any time point or after the 3-day incubation in the cold room (Table 2).

[0096] Hatchability

[0097] Hatchability was calculated as the number of hatched eggs divided by the total number of eggs and is reported as a percentage. In trials 1-5, hatchability was adjusted by subtracting the number of infertile eggs, early dead embryos, and late dead embryos from the total number of eggs, such that these results reflect the hatchability of only the viable embryos. Hatchability was not adjusted in trial 6.

[0098] Bacterial recovery

[0099] To assess bacterial recovery from the gut, whole gut samples (i.e., ventriculus to cecum) were aseptically removed from chicks and collected into sterile bags. The samples were weighed, homogenized, and diluted 1 :4 wt / vol with sterile 0.9% saline. Ten-fold dilutions of each sample were made in sterile 96-well Bacti flat bottom plates. Diluted samples were drop plated onto various media to select for the growth of specific bacteria. The plates were incubated overnight and the bacteria that grew on each plate were counted. Bacterial counts are expressed as LoglO CFU / g of sample.

[0100] To evaluate bifidobacteria recovery, samples were plated on trypticase phytone yeast extract (TPY) agar in trial 2 and were plated on TOS-propionate agar with mupirocin (TOS MUP agar) in trials 3, 4, 5, and 6. In trials 2 and 3, samples were also enriched overnight in 2X TPY media at 37°C and streaked onto TOS MUP agar. Note: TOS MUP agar proved to be superior to TPY agar for selective recovery of bifidobacteria from gut samples. Each of these plated samples was incubated anaerobically at 37° C overnight (~18 hours).

[0101] To evaluate lactic acid bacteria recovery, samples were plated on Man Rogosa Sharpe (MRS) agar in trials 5 and 6. To evaluate Gram negative bacteria recovery, samples were plated on MacConkey’s agar in trial 6. To evaluate Salmonella recovery, samples were plated on brilliant green agar (BGA) supplemented with novobiocin (NO) and nalidixic acid (NA) in trial 6. Each of these plated samples was incubated aerobically at 37°C overnight (~18 hours).

[0102] Body weight gain

[0103] In trial 6, chicks were neck tagged and placed into floor pens on fresh pine shavings for 10 days. The chicks were individually weighed at hatch, on day 7 post-hatch, and on day 10 posthatch. Body weight gain was calculated by subtracting the body weight at hatch from the body weight at day 7 or day 10. The average 7-day body weight gain and average 10-day body weight gain were calculated for each treatment group except for the B2-2+B3-4 group, for which body weight was not evaluated.

[0104] References:

[0105] Citar M, Hacin B, Tompa G, Stempelj M, Rogelj I, Dolinsek J, et al. Human intestinal mucosa- associated Lactobacillus and Bifidobacterium strains with probiotic properties modulate IL-10, IL-6 and IL-12 gene expression in THP-1 cells. Benef Microbes Wageningen Academic Publishers. 2015;6:325-36.

[0106] Lee, Y.-J., Yu, W.-K. and Heo, T.-R. (2003). Identification and screening for antimicrobial activity against Clostridium difficile of Bifidobacterium and Lactobacillus species isolated from healthy infant faeces. Int. J. Antimicrob. Ag. 21, 340-346.

[0107] Ljungh, A. and T. Wadstrom. 2006. Lactic acid bacteria as probiotics. Curr. Issues Intest.

[0108] Microbiol. 7(2):73-89.

[0109] Gill, H.S. (1998). Stimulation of the immune system by lactic cultures. Int. Dairy J. 8, 535-544. Mitsuoka, Tomotari. 1990. Bifidobacteria and their Role in Human Health. J. Industrial

[0110] Microbiology. Vol. 6: 263-267.

[0111] Wilson, K. M., Rodrigues, D. R., Briggs, W. N., Duff, A. F., Chasser, K. M., Bottje, W. G., & Bielke, L. R. (2020). Impact of in ovo administered pioneer colonizers on intestinal proteome on day of hatch. Poultry science, 99(3), 1254-1266.

[0112] Example 2:

[0113] In the following example, the inventors demonstrate that in ovo administration of specific isolates of lactic acid bacteria and poultry-derived Bifidobacterium bacteria (bifidobacteria) results in enteric colonization of hatched chicks and improves their growth performance in the presence of opportunistic pathogens. Early establishment of beneficial microbes is crucial for proper gut maturation and development. In previous work, the inventors have demonstrated the ability of probiotics introduced during hatch (Graham et al., 2018) or via in ovo injection (Teague et al., 2017) to prevent colonization by opportunistic pathogens and to improve performance parameters posthatch. Pioneer colonization by lactic acid bacteria has been shown to result in a favorable shift in the gut microbiota composition post-hatch (Rodrigues et al., 2019), and in ovo administration of lactic acid bacteria has been shown to improve early performance and significantly lower gut colonization by Gram-negative bacteria in a virulent E. coli horizontal challenge model (Arreguin-Nava et al., 2020). Further, in ovo injection of Bifidobacterium spp. at 17 days of embryogenesis has been shown to increase body weight gain and reduce Gram-negative bacterial colonization (Abdel -Moneim et al., 2020). Accordingly, the inventors conducted a series of three experiments to assess the effects of in ovo treatment with specific isolates of lactic acid bacteria and bifidobacteria.

[0114] Results:

[0115] Experiment 1

[0116] Experiment 1 was conducted to evaluate the effects of in ovo administration of a lactic acid bacterium (i.e., LAB 18 or LAB46), either alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4), on enteric colonization of chicks at hatch. On day 18 of embryogenesis, eggs were candled and inoculated with a bacterial treatment via in ovo injection into the amnion. Then, on day 19 of embryogenesis, a pathogen mix (PM) consisting of Enterococcus cecorum, Enterococcus faecalis, and Escherichia coli was applied to a 28mm surface of the eggshell. This pathogen mix was adapted from a previously described challenge model (Graham et al., 2022) and was used to simulate the environmental contamination that occurs in commercial poultry hatch cabinets. FIG. 18 shows the effects of the various bacterial treatments on Enterococcus spp. recovery at hatch. Of the treatments evaluated, 5 / 8 significantly (P<0.05) reduced recovery of Enterococcus spp. at hatch. This suggests that certain strains or combinations of strains more effectively prevent gut colonization by Enterococcus spp.. The effects of the bacterial treatments on the recovery of Gram-negative bacteria (FIG. 19), Bifidobacteria bacteria (FIG. 20), and lactic acid bacteria (FIG. 21) at hatch were variable. There were no differences in hatchability across the treatment groups (data not shown). Experiment 2 Experiment 2 was performed to further assess the effects of in ovo administration of the lactic acid bacteria LAB46, alone or in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4), following challenge with the pathogen mix. Specifically, the effects of these treatments on enteric colonization at hatch were reevaluated and the effects on 14-day performance (i.e., body weight gain) were newly evaluated. The results of this experiment show that Enterococcus spp. recovery from the gut at hatch was significantly (P<0.05) reduced in chicks treated with LAB46 alone, a combination of LAB46 with B2-2 (LAB46+B2-2), or a combination of LAB46 with both B2-2 and B3-4 (LAB46+combo) as compared to the challenge control group, while recovery from chicks treated with a combination of LAB46 with B3-4 (LAB46+B3-4) was not statistically different than the challenge control group (FIG. 22). No differences in Gram-negative bacteria recovery were observed across all challenged groups (FIG. 23). Bifidobacteria recovery was markedly increased for the LAB46+B2-2 group and the LAB46+combo group and was moderately increased for the LAB46+B3-4 group (FIG. 24). All the in ovo treatments significantly increased lactic acid bacteria recovery as compared to the control groups (FIG. 25). The pathogen mix utilized in this study contained a strain of Enterococcus cecorum, referred to as EC7, that was previously shown to be pathogenic (Arango et al., 2023). EC7 was not included in the “standard” pathogen mix that was previously described by Graham et al. (2022). Its inclusion in the pathogen mix appears to negatively impact chick performance (FIG. 26), as negative effects on performance were not observed with the standard pathogen mix in previous studies (Graham et al., 2022; Selby et al., 2023). However, this effect was alleviated by all tested in ovo treatments (FIG. 26). Together, these data suggest that precolonizing the gut microbiome of chicks with beneficial bacteria improves 14-day body weight gain.

[0117] Experiment 3

[0118] In Experiment 3, enteric colonization at hatch was reassessed to confirm the results of Experiment 2. The results of Experiment 3 demonstrate that in ovo treatment with LAB46+B3-4 or LAB46+combo markedly reduced enteric Enterococcus spp. recovery from the gut compared to the challenge control group (FIG. 27). Additionally, treatment with LAB46+B2-2 significantly reduced Gram-negative bacteria recovery compared to the challenge control group (FIG. 28). As in Experiment 2, in ovo treatment with LAB46 in combination with B2-2 and / or B3-3 increased bifidobacteria recovery at hatch (FIG. 29) and LAB46 treatment, alone or in combination with B2-2 and / or B3-3, increased lactic acid bacteria recovery compared to the control groups (FIG. 30).

[0119] Materials and Methods:

[0120] Challenge experiment

[0121] Lactic acid bacteria (i.e., LAB 18 or LAB46), both alone and in combination with one or two Bifidobacterium bacteria (i.e., B2-2 and / or B3-4) were injected into chicken eggs at 18 days of embryogenesis. The lactic acid bacteria were diluted to achieve a concentration of IxlO4CFU / embryo and the bifidobacteria were concentrated to achieve a concentration of IxlO8CFU / embryo. Then, on day 19 of embryogenesis, a pathogen mix (PM) consisting of Enterococcus cecorum, Enterococcus faecalis, and Escherichia coli was applied to a 28mm surface of the eggshell. The effects of the probiotic treatments were assessed following hatch using the assays described below.

[0122] Bacterial recovery

[0123] To assess bacterial recovery from the gut, whole gut samples (i.e., ventriculus to cecum) were aseptically removed from chicks and collected into sterile bags. The samples were weighed, homogenized, and diluted 1:4 wt / vol with sterile 0.9% saline. Ten-fold dilutions of each sample were made in sterile 96-well Bacti flat bottom plates. Diluted samples were drop plated onto various media to select for the growth of specific bacteria. The plates were incubated overnight and the bacteria that grew on each plate were counted. Bacterial counts are expressed as Log 10 CFU / g of sample.

[0124] TOS-propionate agar with mupirocin (TOS MUP agar) was used to evaluate bifidobacteria recovery, MacConkey’s agar was used to evaluate Gram negative bacteria recovery, Chromagar Orientation (CO) agar was used to evaluate Enterococcus spp. recovery, and Man Rogosa Sharpe (MRS) agar was used to evaluate lactic acid bacteria recovery. Samples plated on TOS MUP agar were incubated anaerobically at 37°C overnight (~18 hours), whereas samples plated on MacConkey’s agar or CO agar were incubated aerobically at 37°C for 18 hours and samples plated on MRS agar were incubated at 37°C for 48 hours.

[0125] Body weight gain

[0126] Pen weights were recorded at hatch, on day 7 post-hatch, and on day 14 post-hatch. Body weight gain was calculated by subtracting the pen weight at hatch from the pen weight at day 7 or day 14. The average 7-day body weight gain and average 14-day body weight gain were calculated for each treatment group.

[0127] References:

[0128] Abdel-Moneim, AM. E., Elbaz, A.M., Khidr, R.ES. 2020. Effect of in Ovo Inoculation of Bifidobacterium spp. on Growth Performance, Thyroid Activity, Ileum Histomorphometry, and Microbial Enumeration of Broilers. Probiotics & Antimicro. Prot. 12, 873-882.

[0129] Arango, M., Forga, A., Liu, J., Zhang, G., Gray, L., Moore, R., Coles, M., Atencio, A., Trujillo, C., Latorre, J. D., Tellez-Isaias, G., Hargis, B., & Graham, D. (2023). Characterizing the impact of Enterococcus cecorum infection during late embryogenesis on disease progression, cecal microbiome composition, and early performance in broiler chickens. Poultry science, 102(11), 103059.

[0130] Arreguin-Nava, M.A., Graham, B.D., Adhikari, B., Agnello, M., Selby, C M., Hernandez- Velasco, X., Vuong, C.N., Solis-Cruz, B., Hernandez-Patlan, D., Latorre, J.D. and Tellez Jr, G., 2020. In ovo Administration of Defined Lactic Acid Bacteria Previously Isolated From Adult Hens Induced Variations in the Cecae Microbiota Structure and

[0131] Enterob act eriaceae Colonization on a Virulent Escherichia coli Horizontal Infection Model in Broiler Chickens. Frontiers in Veterinary Science, 7.

[0132] Graham, B.D., Selby, C M., Forga, A.J., Coles, M.E., Beer, L.C., Graham, L.E., Teague, K.D., Tellez-Isaias, G., Hargis, B.M. and Vuong, C.N., 2022. Development of an environmental contamination model to simulate the microbial bloom that occurs in commercial hatch cabinets. Poultry Science, 101(6), p.101890.

[0133] Graham, L., K. Teague, J. Latorre, Y. Yang, M. Baxter, B. Mahaffey, X. Hernandez-Velasco, L. Bielke, B. Hargis, and G. Tellez. 2018. Use of probiotics as an alternative to formaldehyde 280 fumigation in commercial broiler chicken hatch cabinets. J. of Appl. Poult. Res. 27:371-379.

[0134] Teague, K.D., Graham, L.E., Dunn, J.R., Cheng, H.H., Anthony, N., Latorre, J.D., Menconi, A., Wolfenden, R.E., Wolfenden, A.D., Mahaffey, B.D. and Baxter, M., 2017. In ovo evaluation of FloraMax®-Bl l on Marek’s disease HVT vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis infection in broiler chickens. Poultry science, 96(7), pp.2074-2082. Rodrigues, D. R., E. Winson, K. M. Wilson, W. N. Briggs, A. F. Duff, K. M. Chasser, and L. R.

[0135] Bielke. 2020. Intestinal pioneer colonizers as drivers of ileal microbial composition and diversity of broiler chickens. Frontiers in microbiology 10:2858.

[0136] Selby, C M., Beer, L.C., Forga, A.J., Coles, M.E., Graham, L.E., Teague, K.D., Tellez-Isaias, G., Hargis, B.M., Vuong, C.N. and Graham, B.D., 2023. Evaluation of the impact of formaldehyde fumigation during the hatching phase on contamination in the hatch cabinet and early performance in broiler chickens. Poultry Science, 102(5), p.102584.

[0137] Example 3:

[0138] In the following example, the inventors demonstrate that in ovo administration of specific isolates of lactic acid bacteria and poultry-derived Bifidobacterium bacteria (bifidobacteria) results in enteric colonization of hatched chicks and improves their growth performance in the presence of opportunistic pathogens. Meta-analyses of three trials are presented.

[0139] Pioneer colonization by beneficial microbes has been shown to improve performance in broiler chickens. In the present study, one screening trial followed by three additional trials were conducted to evaluate the effect of in ovo administration of lactic acid bacteria (LAB; LAB 18 or LAB46), a Bifidobacterium saeculare (B2-2 or B3-4), or combinations thereof on hatchability, enteric colonization, and 14-day performance in broiler chickens. Briefly, on day 18 of embryogenesis (DOE 18) beneficial bacteria were administered via in ovo injection into the amniotic cavity and embryos were transferred to treatment specific hatch cabinets (trials 1-3, n=195 embryos / hatcher, n=2 hatchers / group or n=225 embryos / hatcher, n=2 hatchers / group for NC and PM and n=l hatcher / group LAB46, LAB46 + B2-2, LAB46 + B3-4, and LAB46 + B- Combo, trial 4). On DOE19, a PM consisting of two Escherichia coli strains (021 and 2016 LG), Enterococcus faecalis (03 IB), and an Enterococcus cecorum (EC7) was applied to the blunt end of the eggshell. In all trials, gastrointestinal (GIT) samples (n=12 / group) were collected at hatch to enumerate LAB, Bifidobacterium spp., Enterococcus spp., and Gram-negative bacteria. Performance was only evaluated in trials 2 and 4. At hatch, chicks were randomized within treatment groups and allocated into floor pens (trial 2, n=20 chicks / pen; n=8 pens / group; trial 4 n=22 chicks / pen; n=12 pens / group). Pen and feed weights were recorded at day 0, day 7, and day 14 to calculate average body weight (BW), body weight gain (BWG), and feed conversion ratio (FCR). GIT sample data were combined and analyzed as a single data set (n=36 / group) for trials 2-4. ANOVA was used to determine significant differences at P<0.05. Student’s T test was used to further separate means. Groups containing LAB showed a significant increase in GIT LAB recovery compared to NC and PM. Similarly, there was a marked increase in Bifidobacterium from groups that received B. saeculare. Groups LAB46, LAB46 + B2-2, LAB46 + B3-4, and LAB46 + B-Combo experienced a significant reduction in Enterococcus spp. recovery compared to PM while only LAB46 + B2-2 resulted in a significant reduction in Gram-negative bacterial recovery compared to PM. In trial 2, a significant increase in 14-day BWG was observed for LAB46, LAB46 + B2-2, and LAB46 + B-Combo compared to PM. For trial 4, statistical differences in BW were observed on day 7 where LAB46 + Combo was significantly higher compared to PM. While no significant differences were observed for FCR in either trial 2 or 4, LAB 46 + B3-4 and LAB46 + B-Combo showed a 16-point numerical improvement on day 14 compared to PM in trial 2 and LAB46 + B2-2 showed a 13-point numerical improvement compared to PM in trial 4. These results indicate that pioneer colonization by probiotic bacteria during late embryogenesis may positively impact the composition of GIT in chicks hatched under challenge conditions.

[0140] Pioneer colonization is a term used to describe the process of organisms populating a sterile or near sterile environment (Dalling, 2008). More specifically, in poultry, pioneer colonization may be used to define the microorganisms which colonize the presumably sterile gastrointestinal (GIT) tract of neonatal chicks during the hatching phase. Pioneer colonization by beneficial microorganisms has been shown to have positive effects on development and maturation of the GIT (Awad et al., 2008; Abdel-Moneim et al. 2019; Castaneda et al. 2020; Lee et al., 2023), establishment of the GIT microbiome (Wilson et al. 2020; Rodrigues et al. 2020; Lee et al., 2023), and regulation of the immune system (Karaffova et al., 2017; Alizadeh et al. 2020; Alizadeh et al., 2021; Sharma et al. 2024). However, pathogenic microorganisms may also serve as pioneer colonizers of the GIT and consequently exclude colonization by beneficial microorganisms (Litvak et al. 2019). Additionally, early colonization by pathogenic microorganisms can negatively affect flock performance which may have financial and animal welfare implications. Thus, initial colonization by beneficial microorganisms is critical for optimizing GIT function and flock performance in commercial poultry.

[0141] In nature, the avian maternal microbiota may be initially transmitted to the embryo during oviposition (Lee et al., 2019). After oviposition, the hen will incubate the developing embryonated eggs and remain close to the chicks post-hatch (Li et al., 2022). Through this natural hatching and rearing process, the chicks become exposed to the maternal microflora directly and indirectly via the environment. Additionally, coprophagic behavior by the chick may also play a role in the transfer of maternal microbiota (Dunbar et al., 2024). However, natural rearing conditions are vastly different that those of commercial hatcheries. In a commercial setting, fertile eggs are promptly removed from the hen and transported to a hatchery. Fertile eggs may be held in a cooler for up to 5 days (Ozlu, 2021) followed by setting in an incubator for the next 17-19 days of embryogenesis. After incubation (~18 days of embryogenesis), embryonated eggs will be candled, vaccinated via hi ovo injection, and transferred to a hatch cabinet until day 21 of embryogenesis (Proudfoot et al., 1980; Tona et al., 2001). Temperature and humidity in the incubator and hatch cabinet are maintained to provide ideal embryogenic conditions (French 1997; Cobb-Vantress 2008; Tullett 2009) but these conditions also promote extensive microbial proliferation. Given the contrast between natural and commercial hatching conditions, the microorganisms present in the environment during the hatching phase also differ. As a result, there is a need to replace the lost transfer of maternal microbiota post-oviposition in commercial hatcheries.

[0142] Early administration of beneficial bacteria during late embryogenesis has been investigated as a means to promote pioneer colonization by beneficial microorganisms in commercial settings (Abdel-Moneim et al., 2019; Duan et al., 2021). In ovo administration of probiotics, such as Lactobacillus spp., Bifidobacterium spp., and Enterococcus faecium have been shown to reduce colonization by opportunistic pathogens, improve histological properties of the GIT, and positively impact GIT microbiome (Abdel-Moneim et al., 2019; Wilson et al., 2020; Castaneda et al., 2020; Rodrigues et al., 2020; Duan et al., 2021; Lee et al., 2023). While the findings from these studies are relevant to the poultry industry, investigation of similar probiotic isolates under a simulated hatchery challenge has yet to be explored. The purpose of the present study was to evaluate the effect of in ovo administration of lactic acid bacteria and / or Bifidobacterium saeculare on enteric colonization at hatch and impact on performance in broiler chickens. For this, a multi-species challenge model was adapted from Graham et al. (2022) to better assess the efficacy of the in ovo treatments under simulated commercial hatching conditions.

[0143] Materials and Methods:

[0144] Experimental Design Four trials were conducted to evaluate the effect of administration of Pediococcus acidilactici or Lactobacillus salivarius (lactic acid bacteria, LAB) and / or two Bifidobacterium saeculare during late embryogenesis on hatchability, enteric colonization at hatch, and early performance in broiler chicks. To identify top performing treatments that reduced enteric colonization by Gram-negative bacteria and / or Enterococcus spp. at hatch in Trial 1, individual LAB and bifidobacteria were administered alone or in combination by in ovo injection into the amniotic cavity at D0E18. The treatment groups included in the screening trial (Trial 1) were: (1) non-challenged control (NC), (2) pathogen mix challenged control (PM), (3) PM + LAB isolate 18 (PM + LAB 18), (4) PM + LAB isolate 46 (PM + LAB 46), (5) PM + B. saeculare isolate 2-2 (PM + B2-2), (6) PM + B. saeculare isolate 3-4 (PM + B3-4), (7) PM + LAB 18 + B2- 2, (8) PM + LAB46 + B3-4, (9) PM + LAB18 + B3-4, and (10) PM + LAB46 + B2-2. The treatment groups included in the subsequent trials (Trials 2-4) were: (1) NC, (2) PM, (3) PM + LAB46, (4) PM + LAB46 + B2-2, (5) PM + LAB46 + B3-4, and (6) PM + LAB46 + both B. saeculare isolates (PM + LAB46 + B-Combo).

[0145] One hatcher (GQF 1550) was utilized per treatment (n=195 embryos / hatcher) in Trial 1 and 4. For Trials 2 and 3, two hatchers were used per treatment (n=195 embryos / hatcher; n=390 embryos / treatment). In all experiments, in ovo treatments were administered on day of embryogenesis 18 (DOE18) and challenge was administered to the eggshell at DOE19 as described in detail below. At hatch, hatchability was recorded, and GIT samples were collected to evaluate enteric colonization of relevant bacteria for all trials. Performance was only evaluated in Trials 2 and 4, where chicks were weighed individually by treatment, allocated by weight and placed on fresh pine shavings in treatment specific pens (n=8 pens / treatment; n=22 chicks / pen). The allocation process was performed to normalize and prevent treatment effects on body weight (BW) by providing similar (within 10g) starting pen weights. Pen BW was recorded at day of hatch (DOH), day 7, and day 14 to evaluate average body weight (BW) and body weight gain (BWG). Feed was also weighed at day 7 and day 14 to evaluate feed conversion ratio (FCR). Chicks were provided ad libitum access to water and a balanced, unmedicated corn and soybean meal diet meeting nutritional requirements for broilers recommended by Aviagen (Aviagen, 2018).

[0146] Lactic acid bacteria LAB (LABI 8 and LAB46) used in these trials have been shown to reduce Salmonella colonization and improve performance in broiler chickens when administered by in ovo injection into the amniotic cavity at D0E18 (Teague et al., 2017). LAB18 and LAB46 have been identified as Pediococcus acidilactici and Lactobacillus salivarius, respectively (Teague et al., 2017). To prepare each isolate for in ovo injection, lOOmL of Man Rogosa Sharpe (MRS Agar, Hardy Diagnostics C5932) broth was inoculated with a ImL stock aliquot and incubated aerobically at 37°C for 24h. After incubation, a subsample of the turbid culture was removed to enumerate the stock concentration (CFU / mL). Stock concentration was determined by drop plating onto MRS agar followed by incubation at 37°C for 24h. The remaining stock was stored at 4°C overnight. Final LAB concentration for in ovo treatments was IxlO4CFU / embryo and was confirmed by drop plate enumeration on MRS agar incubated at 37°C for 24h under aerobic conditions.

[0147] Bifidobacteria

[0148] The two B. saeculare isolates used in these trials were obtained from broody turkeys by intraperitoneal injection of pilocarpine, which within minutes causes cecal evacuation. These samples were enriched in Trypticase Phytone Yeast Extract (TPY; Scardovi, 1986) broth and incubated anaerobically on an orbital shaker at 37°C for 48h (Matto et al., 2006). After enrichment, samples were struck for isolation on TOS-propionate (TOS Agar, EMD Millipore VM795943) agar containing 440 mg / L mupirocin (MUP, Glemark Pharmaceuticals NDC 68462- 180-22, Vlkova et al., 2015 & Thitaram et al., 2005). TOS + MUP agar plates were grown anaerobically using a BD GasPak EZ Anaerobe Container System (BD 260678) at 37°C for 48h. Next, distinct individual colonies were removed and used to inoculate fresh TPY broth for a secondary enrichment. Samples were incubated anaerobically on an orbital shaker at 37°C for 48h. Like above, turbid TPY broth cultures were streaked onto TOS + MUP following the secondary enrichment and incubated anaerobically at 37°C for 48h. An individual colony from each plate was observed microscopically using a Bright-field microscope to confirm Bifidobacterium morphology. Ten candidates were isolated from this process which consisted of five B. saeculare and five B. pseudoIongum. Identification was confirmed by 16S sequencing. Since bifidobacteria are obligate anaerobes, each candidate was evaluated to determine the resilience of the bacteria when exposed to oxygen in vitro. A 200 pl sample of a turbid culture was placed in a 96 well plate, diluted, and plated on TPY and TOS agar at times 0, 30, 60, and 120 minutes. Two B. saeculare (B2-2 and B3-4) isolates were selected as top candidates based on their ability to persist when exposed to oxygen for 120 minutes. Further evaluation of B2-2 and B3-4 indicated that these candidates readily colonized the GIT of chicks at hatch and had no impact on hatchability when administered by in ovo injection (IxlO8CFU / 200uL / embryo) at DOE18 (see Example 1).

[0149] For the current study, a stock aliquot of B2-2 and B3-4 was thawed and swabbed onto two TOS + MUP agar to generate a Tawn-of-growth.’ Plates were incubated anaerobically at 37°C for 48h. Following incubation, sterile cotton swabs were used to inoculate an additional 20 TOS + MUP agar plates. After anaerobic incubation at 37°C for 48h, 5 mL 0.9% sterile saline was added to a single plate and sterile cell plate scrapers were used to gently scrape the colonies from the surface of the agar. A serological pipet was used to collect the cell suspension and transfer it to a 50 mL conical tube. This process was repeated for all plates for each isolate. The cell suspension was washed by centrifugation at 1000 x g for 15min. Following centrifugation, the supernatant was decanted, and the pellet was resuspended in 20 mL of sterile saline. This was done to ensure no residual MUP was remaining in the suspension. Final bifidobacteria concentration for in ovo treatments was IxlO8CFU / embryo. Treatments were plated onto TOS + MUP plates and incubated anaerobically at 37°C for 48h to confirm final concentration for each trial.

[0150] Application of probiotic treatments

[0151] To administer the probiotic treatments to DOE18 embryos in each trial, an 18-gauge needle was used to puncture the eggshell where the air cell was located followed by a 200 pL injection of the respective treatment into the amniotic cavity using a 21 -gauge blunt tip needle and 1 mL syringe. The embryos were then placed into the respective hatchers but remained in the transport flats until challenged on DOE19. Treatment concentrations were confirmed as described above.

[0152] Challenge preparation and application

[0153] Preparation and application of the pathogen mix (PM) followed the procedures described by Graham et al. (2022). However, in the current study, modifications were made to the composition of the PM challenge. Specifically, the PM challenge consisted of presumptive pathogens commonly recovered from commercial poultry production, including two wild-type Escherichia coli isolates (field isolate and hatchery isolate), Enterococcus faecalis (hatchery isolate), and Enterococcus cecorum (field isolate, Arango et al., 2023). E. cecorum had not been included in the PM challenge in previous studies (Graham et al., 2022; Selby et al., 2023). E. cecorum used in the current study has been shown to reduce early performance in broiler chickens exposed during late embryogenesis (Arango et al., 2023). All challenge isolates were grown at 37°C for ~18h. The E. coli isolates were grown aerobically, and the Enterococcus spp. isolates were grown in microaerophilic conditions (5% CO2). Following incubation, each challenge isolate was washed 3x and enumerated on respective media to determine CFU / mL. Washed and enumerated challenge isolates were resuspended in 2x tryptic soy broth (TSB, cat. no. 90000-378, VWR, Suwanee, GA) containing 0.01% xanthan gum serving as a nutrient source and thickening agent (Graham et al., 2022). After mixing, a subsample of the PM was removed and enumerated via drop plate to confirm CFU / mL on MacConkey Agar (BD 212122) for recovery of Gram-negative bacteria or CHROMagar Orientation (RT412, CO agar) for Enterococcus spp.

[0154] As described in Graham et al. (2022), embryos were removed from the hatchers by treatment at DOE19 in the present study and 100 uL of the PM challenge inoculum was applied to the air cell end of each embryo (excluding NC) using a pipette followed by even distribution over a 28 mm area with a disposable loop. Attention was given to not spread the challenge inoculum directly over the in ovo hole. The embryos were then removed from the transport trays and placed into the hatcher trays. Gastrointestinal Sampling

[0155] For all trials, the GIT samples (n=12 chicks / treatment) were aseptically removed from chicks on day 21 of embryogenesis (DOH) after a whole-body rinse. The GIT (duodenum to cloaca) was collected into sterile bags. GIT samples were weighed and homogenized, and 1 :4 wt / vol dilutions were made using sterile 0.9% saline. Ten-fold dilutions of each sample were made in a sterile 96-well plates followed by drop plating of each dilution to evaluate presumptive Gram-negative bacteria, LAB, Bifidobacterium spp., and Enterococcus spp. on the media described above. MacConkey, MRS, and CO agar plates were incubated at 37°C for 18-24h. MacConkey and MRS plates were incubated under aerobic conditions and CO plates were incubated under microaerophilic conditions. TOS + MUP plates were incubated at 37°C for 48h under anaerobic conditions. Colonies were counted for each plate and recovery is expressed as Log 10 CFU / g of sample. Animal Source

[0156] All trials utilized candled, unvaccinated eighteen-day-old Ross 308 embryos obtained from a local commercial hatchery. Embryos were obtained at DOE18 and were randomly allocated and placed into separate hatchers based on treatment group. All experiments and animal handling procedures complied with the requirements set forth by the Institutional Animal Care and Use Committee at the University of Arkansas, protocol #23028. Statistical Analysis

[0157] Microbial recovery from DOH GIT samples and performance data were subjected to oneway ANOVA using JMP Pro 17. Means were further separated using Student’s t test. Significance was reported as P < 0.05 unless otherwise noted. DOH GIT recovery is expressed as mean Log 10 CFU / g.

[0158] Results:

[0159] DOH GIT Recovery

[0160] Table 4 summarizes the results for GIT samples collected at DOH by trial and type of bacteria. In trial 1, LAB46 (0.99 ± 0.69) and LAB46 + B3-4 (0.24 ± 0.24) had the lowest recovery of Gram-negative bacteria from the GIT compared to all groups. Conversely, LAB 18 (3.82 ± 0.68) and LAB18 + B2-2 (5.93 ± 0.63) had markedly more Gram-negative bacterial recovery from the GIT compared to PM. However, none of the groups were significantly (P<0.05) different from PM (1.32 ± 0.75). All other treatments resulted in numerically higher, and in some cases significantly higher, Gram-negative bacterial recovery from the GIT. In trial 2, all groups were statistically similar to PM (2.77 ± 0.84) for Gram-negative bacterial recovery from the GIT, excluding NC (0.22 ± 0.22), which was not significantly different from LAB46 + B2-2 (2.28 ± 0.98). In trial 3, Gram-negative bacterial recovery for PM (3.51 ± 0.83), LAB46 (3.96 ± 0.89), and LAB46 + B-Combo (2.57 ± 1.11) were similar and all three were significantly higher than LAB46 + B2-2 (0.38 ± 0.38) and NC (0.00 ± 0.00) while LAB46 + B3-4 (6.11 ± 0.69) was significantly higher than all groups. In trial 4, LAB46 + B2-2 (2.60 ± 0.77) had significantly lower Gram-negative bacterial recovery compared to PM (7.05 ± 0.42) while LAB46 (6.26 ± 0.57), LAB46 + B3-4 (5.64 ± 1.21), and LAB46 + B-Combo (6.55 ± 0.56) were not significantly different from PM.

[0161] Table 4. Bacterial recovery (LoglO CFU / g) from GIT samples at hatch by trial

[0162] Data reported as mean ± SE, n=12 / treatment. ANOVA (JMP Pro 17) used to determine significant differences at P < 0.05. Means further separated by Student’s T test.a'K c_ dsuperscripts indicate significance across treatment groups. 'Treatment abbreviations: NC (negative control), PM (pathogen mix), LAB18 (LAB isolate 18), LAB46 (LAB isolate 46), B2-2 (Bifidobacterium saeculare isolate 2-2), B3-4 (Bifidobacterium saeculare isolate 3-4), LAB46 + B-Combo (LAB46 + both Bifidobacterium saeculare isolates).

[0163] Bifidobacterium spp. recovery from the GIT at DOH was elevated overall, in some cases significantly, in groups treated with / t. saeculare in trial 1 with the exception of LAB 18 + B3-4 (0.00 ± 0.00). In trial 2, LAB46 + B2-2 (2.81 ± 0.92) and LAB46 + B-Combo (5.79 ± 0.58) were significantly higher than all other groups while LAB46 + B3-4 (1.16 ± 0.64), although numerically higher, was not significantly different for Bifidobacterium spp. recovery compared to NC (0.00 ± 0.00), PM (0.00 ± 0.00), and LAB46 (0.00 ± 0.00). In trial 3, LAB46 + B2-2 (1 .92 ± 0.74), LAB46 + B3-4 (3.01 ± 0.81), and LAB46 + B-Combo (2.67 ± 0.76) were similar but there was a significant increase in Bifidobacterium spp. recovery compared to NC (0.00 ± 0.00), PM (0.00 ± 0.00), and LAB46 (0.00 ± 0.00). A similar trend also observed in trial 4 where LAB46 + B2-2 (5.10 ± 0.84), LAB46 + B3-4 (3.50 ± 1.08), and LAB46 + B-Combo (5.26 ± 0.97) were similar but had a significant increase Bifidobacterium spp. recovery compared to NC (1.04 ± 0.70), PM (0.31 ± 0.31), and LAB46 (0.00 ± 0.00).

[0164] In trial 1, all treatment groups except LAB46 + B3-4 (3.07 ± 0.70), LAB18 + B3-4 (3.08 ± 0.85), and LAB46 + B2-2 (2.39 ± 0.80) had a significant increase in lactic acid bacterial recovery compared to NC (0.98 ± 0.70) and PM (2.61 ± 0.89). In trials 2 and 3, LAB46 (5.57 ± 0.58; 5.45 ± 0.58), LAB46 + B2-2 (3.49 ± 0.88; 5.14 ± 0.54), LAB46 + B3-4 (5.75 ± 0.63; 4.97 ± 0.71), and LAB46 + B-Combo (4.60 ± 0.26; 5.82 ± 0.37) had significantly higher lactic acid bacterial recovery compared to PM (0.00 ± 0.00) and NC (0.84 ± 0.45). In trial 4, LAB46 (5.52 ± 0.68), LAB46 + B2-2 (6.26 ± 0.38), LAB46 + B3-4 (7.08 ± 0.70), and LAB46 + B-Combo (5.32 ± 0.60) had significantly increased lactic acid bacterial recovery compared to NC (0.31 ± 0.31). However, only LAB46 + B2-2 and LAB46 + B3-4 had a significant increase in lactic acid bacterial recovery compared to PM (4.38 ± 0.99).

[0165] In trial 1, there were no significant decreases in Enterococcus spp. recovery from DOH GIT samples for any groups compared to PM (0.64 ± 0.64). However, B2-2 (5.38 ± 0.81) and B3-4 (6.05 ± 0.66) had significantly more Enterococcus spp. recovered from the GIT compared to PM in trial 1. In trial 2, NC (0.00 ± 0.00), LAB46 (1.86 ± 0.69), LAB46 + B2-2 (1.81 ± 0.84), and LAB46 + B-Combo (1.43 ± 0.57) had a significant decrease in Enterococcus spp. recovery compared to PM (4.11 ± 0.86). Although LAB46 + B3-4 (3.57 ± 0.86) had numerically lower Enterococcus spp. recovery compared to PM in trial 2, this difference was not significant. Enterococcus spp. recovery in trial 3 was significantly lower in NC (0.00 ± 0.00), LAB46 + B3-4 (1.10 ± 0.75), and LAB46 + B-combo (2.16 ± 0.84) compared to PM (4.75 ± 0.69), while LAB46 (2.76 ± 0.89) and LAB46 + B2-2 (4.24 ± 0.72) only had a numerical reduction compared to PM. In trial 4, NC (0.87 ± 0.61), LAB46 (3.24 ± 0.91), and LAB 46 + B2-2 (4.17 ± 1.01) resulted in a significant reduction of Enterococcus spp. recovery compared to PM (6.45 ± 0.49), while LAB46 + B3-4 (5.87 ± 0.92) and LAB46 + Combo (6.27 ± 0.67) only had a numerical reduction compared to PM. Meta-analysis of DOH GIT Trials 2-4

[0166] A meta-analysis of bacterial recovery from the GIT at DOH for trials 2, 3, and 4 is presented in in FIG. 31-34. NC and LAB46 + B2-2 were the only treatments that resulted in a significant reduction in Gram-negative bacterial recovery from DOH GIT samples compared to PM (FIG. 31). LAB46, LAB46 + B2-2, LAB46 + B3-4, and LAB46 + B-Combo showed a significant reduction in Enterococcus spp. recovery compared to PM while recovery from NC was significantly lower than all treatments (FIG. 32). Groups containing LAB resulted in a significant increase in LAB compared to NC and PM (FIG. 33). Likewise, groups containing B. saeculare had a significant increase in Bifidobacterium spp. recovered from the GIT collected at hatch compared to NC and PM (FIG. 34).

[0167] Performance

[0168] Hatchability for trials 2, 3, and 4 is reported in Table 5. Trial 1 hatchability was not collected, as evaluating DOH GIT recovery was the sole objective for that trial. No significant differences were observed in any of the three trials where hatchability data was collected. Average BW and mortality for trials 2 and 4 have been presented in Table 6. While significant differences in DOH average BW were observed across groups in both trials, the range was 0.8g or less across groups. No significant differences were observed for D7 or D14 average BW in trial 2. However, LAB46 + B-Combo (144.3 ± 4.44) had a significantly higher average BW at day 7 compared to PM (134.0 ± 3.26) in trial 4. No significant differences were observed for D14 average BW in trial 4. Also, no significant differences were observed for day 0-7 day or 0- 14 day mortality for either trial.

[0169] Table 5. Hatchability (Trials 2, 3, and 4),

[0170] Treatment1Trial 2 Trial 3 Trial 4

[0171] NC 237 / 239 (99.16) 357 / 354 (99.16) 432 / 441 (97.96)

[0172] PM 230 / 236 (97.46) 356 / 351 (98.60) 439 / 446 (98.43)

[0173] LAB46 237 / 238 (99.58) 358 / 357 (99.71) 221 / 223 (99.10)

[0174] LAB46 + B3-4 215 / 219 (98.17) 359 / 352 (98.06) 208 / 209 (99.52)

[0175] LAB46 + B2-2 230 / 230 (100) 357 / 353 (98.88) 206 / 207 (99.52)

[0176] LAB46 + B-Combo 224 / 225 (99.56) 354 / 352 (99.42) 206 / 208 (99.04) treatment abbreviations: NC (negative control), PM (pathogen mix), LAB46 (Lactic acid bacteria isolate 46), LAB46 + B2-2 (LAB46 + Bifidobacterium saeculare isolate 2-2), LAB46 + B3-4 (LAB46 + Bifidobacterium saeculare isolate 3-4), LAB46 + B-Combo (LAB46 + both Bifidobacterium saeculare isolates).2Adjusted hatchability excluded embryos not viable prior to D0E18 and infertile embryos. No embryos were killed due to in ovo injection. Trial 2 n=l hatcher / treatment. Trial 3 n=2 hatchers per treatment. Trial 4 n=2 hatchers per treatment for NC and PM, n=l hatcher per treatment for LAB46, LAB46 + B2-2, LAB46 + B3-4, and LAB46 + B-Combo.

[0177] Table 6. Average BW and mortality (Trial 2 and 4)

[0178] NC 38.7 ± 0.04ab133.4 ± 3.21ab346.5 ± 9.31 4 / 264 (1.52) 4 / 264 (1.52)

[0179] PM 38.6 ± 0.06b134.0 ± 3.26b344.9 ± 12.73 1 / 242 (0.41) 4 / 242 (1.65) ri lLAB46 38.5 ± 0.06b140.1 ± 3.62ab360.6 ± 9.42 1 / 198 (0.51) 4 / 198 (2.02)

[0180] 4 LAB46 ± B2-2 38.5 ± 0.06b142.1 ± 5.14ab361.8 ± 15.88 0 / 154 (0) 0 / 154 (0)

[0181] LAB46 ± B3-4 38.5 ± 0.04b140.1 ± 1.51ab363.3 ± 1.18 3 / 132 (2.27) 5 / 132 (3.79)

[0182] LAB46 ± B- 38.9 ± 0.25a144.3 ± 4.44a365.2 ± 11.38 4 / 154 (2.60) 5 / 154 (3.25)

[0183] Combo

[0184] Data reported as mean ± SEM. ANOVA (JMP Pro 17) used to determine significant differences at / 5< 0.05. Means further separated by Student’s T test.a’b, c’dsuperscripts indicate significant difference across treatment groups by column and trial.

[0185] 'Treatment abbreviations: NC (negative control), PM (pathogen mix), LAB46 (Lactic acid bacteria isolate 46), LAB46 + B2-2 (LAB46 + Bifidobacterium saeculare isolate 2-2), LAB46 + B3-4 (LAB46 + Bifidobacterium saecidare isolate 3-4), LAB46 + B-Combo (LAB46 + both Bifidobacterium saeculare isolates).

[0186] 2Trial 2 n=4-8 pens / treatment.

[0187] 3Trial 4 n=5-12 pens / treatment.

[0188] Average BWG and FCR for trials 2 and 4 have been summarized in Table 7. No significant differences were observed for 0-7 day BWG or 0-14 day FCR for either trial. No significant differences were observed in 0-7 day FCR except between LAB46 + B2-2 (1.53 ± 0.15) and NC (1.27 ± 0.04) in trial 2. LAB46 (417.22 ± 3.70), LAB46 + B2-2 (415.97 ± 17.60), and LAB46 + B-Combo (405.76 ± 13.29) had significantly higher 0-14 day BWG compared to PM (375.83 ± 10.27) in trial 2. In trial 4, although a 20g improvement in 0-14 day BWG over PM (296.80 ± 12.58) was observed for LAB46 + B-Combo (316.80 ± 11.15), there were no significant differences between any groups. Table 7. Average BWG and FCR (Trial 2 and 4)

[0189] Day 0-7 Day 0-14

[0190] Trial Treatment1BWG (g) FCR BWG (g) FCR

[0191] NC 103.85 ± 1.85 1.27 ± 0.04b398.83 ± 5.63ab1.37 ± 0.03

[0192] PM 105.46 ± 2.65 1.41 ± 0.10ab375.83 ± 10.27b1.55 ± 0.11

[0193] Trial LAB46 110.34 ± 4.44 1.30 ± 0.03ab417.22 ± 3.70a1.32 ± 0.02

[0194] 22LAB46 + B2-2 102.54 ± 5.85 1.53 ± 0.15a415.97 ± 17.60a1.53 ± 0.21

[0195] LAB46 ± B3-4 107.99 ± 1.56 1.33 ± 0.06ab404.39 ± 12.27ab1.39 ± 0.03

[0196] LAB46 + B-Combo 108.94 ± 2.69 1.38 ± 0.04ab405.76 ± 13.29a1.39 ± 0.04

[0197] NC 90.86 ± 3.21 1.56 ± 0.10 299.00 ± 9.39 1.74 ± 0.10

[0198] PM 91.37 ± 3.21 1.54 ± 0.09 296.80 ± 12.58 1.64 ± 0.06

[0199] Trial LAB46 97.70 ± 3.59 1.49 ± 0.11 312.40 ± 9.64 1.62 ± 0.09

[0200] 43LAB46 ± B2-2 99.81 ± 5.12 1.30 ± 0.07 314.80 ± 15.86 1.51 ± 0.09

[0201] LAB46 ± B3-4 95.36 ± 2.49 1.50 ± 0.11 314.60 ± 1.74 1.63 ± 0.09

[0202] LAB46 + B-Combo 100.10 ± 4.60 1.46 ± 0.10 316.80 ± 11.15 1.63 ± 0.09

[0203] Data reported as ‘mean ± SEM’ Log 10 CFU / g of sample. ANOVA (JMP Pro 17) used to determine significant differences atP < 0.05. Means further separated by Student’s T test.a bSuperscripts indicate significant difference across treatment groups by column and trial, treatment abbreviations: Non-challenged control (NC), pathogen mix (PM), lactic acid bacteria (LAB), Bifidobacterium saeculare (B) 2-2, 3-4, or -Combo.

[0204] 2Trial 2 n=4-8 pens / treatment

[0205] 3Trial 4 n=5-12 pens / treatment

[0206] Discussion:

[0207] Natural and commercial hatching conditions differ in terms of the microbial communities present at hatch. While the transfer of maternal microbiota through oviposition (Lee et al., 2019), close physical contact (Li et al., 2022), and coprophagic behaviors (Dunbar et al., 2024) results in microaerophilic and anaerobic GIT associated microbial communities resembling those of the hen, microbial communities found in hatching cabinets can often favor aerobic bacteria which are generally not associated with gastrointestinal colonization. Lactobacillus spp. and Bifidobacterium spp. are commonly associated with the maternal microbiota of mature healthy hens. While fertilized eggs produced in commercial operations are exposed to the maternal microbiota during oviposition, they lack the ability to receive further transfer of these microbial communities once they are promptly removed from the hen. As the maternal microbiota contains a multitude of beneficial microorganisms which act as pioneer colonizers of the neonate gastrointestinal tract, there is a need in commercial hatcheries to find an alternative to the natural transfer of maternal microbiota. Recent publications have shown that in ovo application of probiotic bacteria such as Lactobacillus spp., Bifidobacterium spp., and Enterococcus faecalis can reduce colonization by opportunistic pathogens, improve GIT maturation, and positively shift GIT microbiome (Abdel-Moneim et al., 2019; Wilson et al., 2019; Castaneda et al., 2020; Rodrigues et al., 2020; Duan et al., 2021; Lee et al., 2023).

[0208] An established hatchery challenge model was used in this study to evaluate the effects of pioneer colonization by lactic acid bacteria and / or Bifidobacterium saeculare administered via in ovo injection on microbial recovery in DOH GIT samples and subsequent 7- and 14-day performance. The natural challenge model established by Graham et al. (2022) included isolates of E. coli, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus chromogens, and Aspergillus fumigatus, which were isolated from a homogenate of non-viable and infected embryos obtained from a commercial hatchery. For the present study, the same E. coli and Enterococcus faecalis isolates described in Graham et al. (2022) were used while the Enterococcus faecium, both Staphylococcus spp., and A. fumigatus isolates were replaced with an additional E. coli isolate as well as an Enterococcus cecorum isolate described by Arango et al. (2023). Although Staphylococcus spp. and A. fumigatus are hatchery associated opportunistic pathogens, they are more associated with septicemia and respiratory diseases, respectively. Historically, E. cecorum has caused disease in longer lived broilers and broiler breeders. However, more recently, it has been isolated from diseased younger broilers at as early as two weeks of age (Borst et al., 2017) and has been associated with reduced performance (Arango et al., 2023). Thus, it was selected for the current study.

[0209] In the current study, a reduction in Gram-negative bacteria in the GIT at hatch was observed in chicks that received LAB46 + B2-2 at DOE18. In ovo application of a probiotic containing Lactobacillus plantarum (Duan et al., 2021) or Bifidobacterium spp. (Abdel-Moneim et al., 2019) also lowered Gram-negative bacteria, although evaluation for these studies was done on day 14 and 28, respectively. This indicates that introducing beneficial bacteria during the prehatch phase may prevent or perturb colonization by Gram-negative bacteria in laboratory and commercial settings. Performance improvements, such as improved body weight gain, was associated with exposure to LAB46, LAB46 + B2-2, and LAB46 + B-Combo pre-hatch in trial 2 as well as improved day 7 BW for LAB46 + B-Combo in trial 4 compared to PM. Teague et al. (2017) observed similar effects associated with in ovo administration of lactic acid bacteria in broiler chickens. Thus, application of probiotics via in ovo injection into the amnion not only alters the gut microflora at hatch but may considerably improve growth performance. Sustainable alternatives to antibiotics are needed to enhance gut health and mitigate performance losses associated with opportunistic pathogens related to commercial hatcheries in a reduced antibiotic or antibiotic-free era.

[0210] A meta-analysis of bacterial recovery from DOH GIT samples revealed significantly lower Gram-negative bacteria recovery for LAB46 + B2-2 compared to PM, while LAB46, LAB46 + B3-4, and LAB46 + B-Combo were not significantly different compered to PM. Additionally, a significant decrease in Enterococcus spp. recovery was observed in all probiotic treated groups compared to PM. Additionally, improved BW on day 7 was observed in LAB46, LAB46 + B2-2, and LAB46 + B-Combo in trial 2 as well as improved BWG on day 14 in LAB46 + B2-2 in trial 4. The results of this study suggest that pre-hatch colonization of beneficial pioneer colonizers may reduce colonization by opportunistic pathogens at hatch which may lead to subsequent performance improvements.

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[0246] DEPOSIT INFORMATION

[0247] A deposit of the University of Arkansas Division of Agriculture proprietary Bifidobacterium saeculare strain designated as B2-2, which is disclosed above and recited in the appended claims, has been made with the Agricultural Research Service Culture Collection (NRRL), 1815 N. University Street, Peoria, IL 61604, and has been accepted under the terms of the Budapest Treaty. The date of deposit was November 6, 2024. The deposit comprises agar plates, which were found viable on November 13, 2024. All restrictions will be irrevocably removed upon granting of a patent. This deposit is intended to meet all the requirements of 37 C.F.R. §§1.801-1.809. The NRRL accession number is NRRL B-68468. The deposit will be maintained in the depository for a period of thirty years, or five years after the last request, or for the enforceable life of the patent, whichever is longer, and will be replaced as necessary during that period.

[0248] A deposit of the University of Arkansas Division of Agriculture proprietary Bifidobacterium saeculare strain designated as B3-4, disclosed above and recited in the appended claims, has been made with the NRRL, 1815 N. University Street, Peoria, IL 61604, and has been accepted under the terms of the Budapest Treaty. The date of deposit was November 6, 2024. The deposit comprises agar plates, which were found viable on November 13, 2024. All restrictions will be irrevocably removed upon granting of a patent. This deposit is intended to meet all the requirements of 37 C.F.R. §§1.801-1.809. The NRRL accession number is NRRL B-68467. The deposit will be maintained in the depository for a period of thirty years, or five years after the last request, or for the enforceable life of the patent, whichever is longer, and will be replaced as necessary during that period.

[0249] A deposit of the University of Arkansas Division of Agriculture proprietary Pediococcus acidilactaci strain previously designated as FM18, but which is referred to herein as LAB 18, has been made with the NRRL, 1815 N. University Street, Peoria, IL 61604, and has been accepted under the terms of the Budapest Treaty. The date of deposit was April 1, 2014. This deposit is intended to meet all the requirements of 37 C.F.R. §§1.801-1.809. The NRRL accession number is NRRL B-50964. A deposit of the University of Arkansas Division of Agriculture proprietary Weissella confiisa strain previously designated as FM46, but which is referred to herein as LAB46, has been made with the NRRL, 1815 N. University Street, Peoria, IL 61604, and has been accepted under the terms of the Budapest Treaty. The date of deposit was April 1, 2014. The NRRL accession number is NRRL B-50961. These deposits are intended to meet all the requirements of 37 C.F.R. §§1.801-1.809. The deposits will be maintained in the depository for a period of thirty years, or five years after the last request, or for the enforceable life of the patent, whichever is longer, and will be replaced as necessary during that period.

Claims

CLAIMSWhat is claimed:

1. A composition comprising a Bifidobacterium bacterium, wherein the Bifidobacterium bacterium is selected from: a) a bacterium designated as B2-2 and deposited at the Agricultural Research Service Culture Collection (NRRL) under accession number NRRL B-68468, b) a bacterium designated as B3-4 and deposited at the NRRL under accession number NRRL B-68467, and c) a combination of (a) and (b).

2. The compositions of claim 1 further comprising a lactic acid bacterium, wherein the lactic acid bacterium is selected from: a) a bacterium designated as LAB 18 and deposited at the NRRL under accession number NRRL B-50964, and b) a bacterium designated as LAB46 and deposited at the NRRL under accession number NRRL B-50961.

3. The composition of claim 2, wherein the composition comprises: a) LAB46 and B2-2, b) LAB46 and B3-4, or c) LAB46, B2-2, and B3-4.

4. The composition of claim 2, wherein the composition comprises: a) LAB 18 and B2-2, b) LAB 18 and B3-4, or c) LAB 18, B2-2, and B3-4.

5. The composition of any one of the preceding claims, wherein the composition is dried.

6. The composition of any one of the preceding claims, further comprising a carrier.

7. The composition of any one of the preceding claims, further comprising an antigen.

8. A method comprising administering the composition of any one of the preceding claims to a subject.

9. The method of claim 8, wherein administration is in ovo.

10. A method comprising administering a composition to a subject in ovo, wherein the composition comprises a Bifidobacterium bacterium selected from: a) a bacterium designated as B2-2 and deposited at the NRRL under accession number NRRL B-68468, b) a bacterium designated as B3-4 and deposited at the NRRL under accession number NRRL B-68467, and c) a combination of (a) and (b).

11. The method of claim 10, wherein the composition further comprises a lactic acid bacterium selected from: a) a bacterium designated as LAB 18 and deposited at the NRRL under accession number NRRL B-50964, and b) a bacterium designated as LAB46 and deposited at the NRRL under accession number NRRL B-50961.

12. The method of claim 11, wherein the lactic acid bacterium is administered at a concentration of IxlO3- IxlO5CFU per embryo.

13. The method of any one of claims 9-11, wherein the Bifidobacterium bacterium is administered at a concentration of IxlO7- IxlO9CFU per embryo.

14. The method any one of claims 9-13, wherein the composition is administered in the late stage of embryogenesis.

15. The method of any one of claims 9-14, wherein the subject is a chicken.

16. The method of any one of claims 8-15, wherein the method reduces the colonization of a pathogen in the gastrointestinal tract of the subject as compared to an untreated control.

17. The method of claim 16, wherein the pathogen is an Enterococcus bacterium, an Escherichia bacterium, a Salmonella bacterium, or a combination thereof.

18. The method of claim 17, wherein the Enterococcus bacterium is selected from Enterococcus cecorum, Enterococcus faecalis, and a combination thereof.

19. The method of claim 17, wherein the Escherichia bacterium is Escherichia coll.

20. The method of claim 17, wherein the Salmonella bacterium is Salmonella enteritidis.

21. The method of any one of claims 8-20, wherein the method increases the colonization of Bifidobacterium bacteria and / or lactic acid bacteria in the gastrointestinal tract of the subject as compared to an untreated control.

22. The method of any one of claims 8-21, wherein the method increases the body weight gain of the subject as compared to an untreated control.

23. The method of claim 22, wherein the body weight gain is increased by 14 days post-hatch.

24. The method of claim 22 or 23, wherein the body weight gain is increased at least 5-15% as compared to an untreated control.

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