Meconium

All animal work was reviewed and approved by the Laboratory Animal Care Committee of Shandong Agricultural University (permit number SDAUA-2021-034) on 10 March 2022. Informed verbal consent was obtained from farmers to allow samples to be collected from their farms.
In July 2020, a total of 693 samples (458 dead embryos, 100 cloacal swabs, 20 water samples, 15 feed samples, 30 environmental swabs, and 70 meconium samples of newly hatched chicks) were selected from four different chicken breeds in Tai’an (White feather broiler), Jinan (Bairi), Rizhao (Langya), and Jining (Luhua) in Shandong Province, China, for Salmonella isolation and identification. The isolation and identification of Salmonella species were performed according to ISO standard 6579:2017 and the Chinese national standard (GB 4789.4-2016) with minor modifications [35 ,36 ]. Each sample was added to 4.5 mL of buffered peptone water (BPW, Qingdao Hope Bio-technology Co., LTD., China) and the BPW mixture was incubated at 37 °C for 12 h for pre-enrichment. Approximately 0.5 mL of pre-enriched culture was inoculated into 4.5 mL of Tetrathionate Broth Base (TTB, Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) and Selenite Cystine Broth (SC, Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China). Cultures of each TTB and SC broth were inoculated on a Xylose Lysine Desoxycholate Agar base (XLD, Qingdao Hope Bio-technology Co., Ltd., Qingdao, China) and incubated at 37 °C for 48 h [37 ]. Smooth and round without a black center or large with a black center colonies were confirmed by polymerase chain reaction (PCR) assays with primers designed for the FimW gene (Table S1) [10 (link),38 (link)]. Bacterial DNA was extracted using a TIANamp Bacterial DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions.
Amplification reactions were carried out at a final volume of 25 µL containing 1 µL of each primer (10 µM), 12.5 µL of 2 × Taq Master Mix (Vazyme Biotech, Nanjing, China), 1 μL of genomic DNA, and 9.5 μL of distilled deionized water. PCR of the FimW gene was performed with the following program: 94 °C for 4 min, followed by 24 cycles of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C, and a final extension at 72 °C for 10 min. The PCR products were separated through 1% agarose gel electrophoresis and visualized under UV light after staining with ethidium bromide. To confirm the presence of Salmonella, the standard strain of S. enterica (CVCC 3377) purchased from the China Center for the preservation and management of veterinary microorganisms was used as a positive control, and Escherichia coli (ATCC 25922) was used as a negative control.

The data were retrieved from the electronic medical records of the parturient and the neonate [32 (link)]. The data collected included maternal age, gestational age at delivery, gravidity, parity, smoking, BMI (kg/m²), hypertensive disorders (chronic hypertension, gestational hypertension, and preeclampsia), pre-gestational diabetes mellitus (DM), and gestational DM. Birth and delivery outcomes included onset of delivery (spontaneous vs. induced labor), use of epidural anesthesia, duration of the second stage of labor, intrapartum fever (defined as 38 °C during labor), trial of labor after cesarean delivery (CD) (TOLAC) and mode of labor as CD due to non-reassuring fetal heart rate (NRFHR), or instrumental delivery. Preterm delivery was defined as spontaneous labor at <37 weeks of gestation.
Neonatal outcomes included neonatal birth weight: small for gestational age ([SGA] defined as birthweight <10th percentile according to local growth charts [33 (link)]). Additional data collected included 1- and 5-min Apgar scores, cord pH, neonatal intensive care unit (NICU) admissions, neonatal hypoglycemia, respiratory distress with mechanical ventilation, and meconium aspiration syndrome (MAS). The clinical diagnosis of respiratory distress syndrome (RDS) with mechanical ventilation was made for preterm infants with typical respiratory difficulties. MAS was diagnosed when respiratory distress occurred soon after birth, with radiological findings that could not be explained otherwise, in the presence of meconium during labor.