Buffered peptone water (bpw)

On 7 dpi (d 21), one chicken per cage was euthanized, and approximately 20 g of jejunal contents (lower jejunal to upper ileal) were collected in sterile Whirl-Pak filter bags (Nasco, Fort Atkinson, WI). To each filter bag containing the jejunal content, 10 mL of buffered peptone water (BPW; Himedia, Mumbai, India) was added, and the samples were homogenized using a Masticator Silver Panoramic (Neutec Group Inc., Farmingdale, NY) for 1 min. Following homogenization, 1 mL of the mixed jejunal contents was transferred to a sterile glass dilution tube and diluted to 10⁻⁸ by serial dilution with BPW. Subsequently, 100 µL of the diluted contents (10⁻², 10⁻⁴, 10⁻⁶, and 10⁻⁸) were plated on Tryptose Sulfite Cycloserine and Shahadi Ferguson Perfringens (TSC/SFP; Oxoid Ltd., Hampshire, United Kingdom) agar. The plates were incubated at 37°C for 24 h under anaerobic conditions created using an anaerobic gas-pack system (AnaeroPack™, Thermo Scientific, MA).

Using appropriate hygiene procedures, samples were collected in separate, sterile, ziplocked bags. After collection, the samples were transported at refrigeration temperature to the Department of Microbiology and Veterinary Public Health (DMVPH), CVASU, for further investigation. The samples were diced and transferred to separate sterile test tubes containing buffered peptone water (BPW; HIMEDIA, Mumbai, India) and incubated at 37 °C overnight for primary enrichment.

Blood samples (n = 9; 3 chicks from each replicate in each treatment) were collected early in the morning from wing vein and centrifuged (2661× g for 5 min at 4 °C) at 21 and 35 days of age to extract serum, which was used for the hemagglutination inhibition (HI) test. The clear sera were obtained from the collected samples for the HI test for the NDV. Additionally, at 21 and 35 days of age, fecal samples (n = 9; 3 chicks from each replicate in each treatment) were collected and transferred to buffered peptone water (BPW, HiMedia Laboratories, Mumbai, India), which was immediately used for counting the intestinal microbes. On day 35, nine birds (3 chicks from each replicate in each group) were killed under anesthesia with an intravenous injection of sodium pentobarbital (50 mg/kg) (CAMEO Chemicals, Tampa,FL, USA) and necropsied for carcass traits. Additionally, 3 cm of duodenal samples were collected, washed with saline, and maintained in neutral buffered formalin (10%) (Algomhoria Co. Cairo, Egypt) for histological examination. The fixed samples were processed with the conventional paraffin-embedding technique. Histological sections of 3 μM were prepared from the paraffin blocks of samples. These sections were stained with the Hematoxylin and Eosin (H&E) technique [23 ].

Previously flamed organ samples were cut into small pieces and were transferred to test tubes with 10 ml of Buffered Peptone Water (BPW) (HiMedia, India) for general enrichment. Cultivation was carried out at 37 °C for 18–24 h. The loopful of BPW was then streaked onto Xylose–Lysine Deoxycholate Agar (XLD Agar) (HiMedia, India) and incubated for 18–24 h at 37 °C. The separate pink and yellow colonies were replated on Nutrient agar (NA) (HiMedia, India) to obtain a pure culture for further research. All isolated cultures were stored in Nutrient broth (NB) (HiMedia, India) with 15% glycerol at − 80 °C.