Caco3

The collected samples were serially diluted with de Man, Rogosa and Sharpe (MRS) broth (Oxoid) and plated on MRS agar plates containing 0.5% (w/v) CaCO₃ (BDH, Poole, UK). These were then incubated under aerobic and anaerobic conditions at 37 °C for 48 h. For anaerobic incubations, liquid or solid MRS medium was supplemented with 0.3 g L⁻¹ L-cysteine-HCl (Merck, Darmstadt, Germany) giving rise to MRS-Cys. After incubation, individual colonies surrounded by clear acidification halos were randomly selected, purified on MRS agar with or without cysteine (depending on their aerobic or anaerobic isolation), and examined according to colony morphology, Gram staining, catalase reaction, and hemolytic activity using sheep blood agar plates (Oxoid). Catalase negative, γ-hemolytic, Gram-positive rods were deemed to be LAB and were stored at −80 °C in MRS broth supplemented with 15% (v/v) glycerol (Merck).

Vaginal swabs were suspended and serially diluted in de Man, Rogosa and Sharpe (MRS) broth (Oxoid), and the dilutions spread on MRS agar plates containing 0.5% (w/v) CaCO₃ (BDH, Poole, United Kingdom). Plates were incubated at 37°C for 48 h in aerobic or anaerobic (with Anaerocult; Merck, Darmstadt, Germany) conditions. Individual colonies surrounded by clear halos from both culture conditions were randomly selected and subcultured in MRS and incubated under the same conditions. Isolates were then screened by Gram staining, colony morphology, and the catalase test. Their hemolytic activity was assessed using sheep blood agar plates (Oxoid). Only Gram-positive, catalase negative, and γ-hemolytic cocci were selected. These isolates were routinely cultivated in MRS agar and incubated at 37°C for 48 h, and then stored in MRS broth supplemented with 15% (w/v) glycerol (Merck) at −80°C.

The collected cecal contents of HBW chickens were opened and processed under a super-clean bench. Then, cecal content samples were serially diluted in sterile saline (1 g of content was squeezed into 9 mL sterile saline) and spread on MRS agar plates containing 0.5% (wt/vol) CaCO₃ (BDH, Poole, United Kingdom) or LB agar plates. All plates were incubated at 37°C for 1–5 days. Well-separated colonies of different morphology were picked from each agar plate and purified by repeated streaking onto new plates. Each isolate was subjected to 16S rRNA gene amplicon sequencing analysis, and the amplification primers were 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGCTACCTTGTTACGACTT-3′). Using the taxonomically united database in EzBioCloud (http://www.ezbiocloud.net) (52 (link)), the full 16S rRNA gene nucleotide was definitively identified with high similarity. Then all isolates identified as Lactobacillus or Bacillus were resuspended in the corresponding growth medium with glycerol (20%, vol/vol) and stored at −80°C.