Preparation of experimental treatments and diets
CR treatment consisted of a solid dispersion of curcumin with polyvinylpyrrolidone in a 1:9 ratio previously described [22,23], CA treatment was copper (II) acetate hydrate (98%, catalog no. 341746, Sigma), and CA-CR treatment consisted of a mixture of the previous treatments. Solid dispersion of curcumin was prepared by dissolving 1 part of curcumin in 9 parts of a polyvinylpyrrolidone (PVP) K30 solution, followed by water evaporation at 40°C and sieving. Mash corn-soybean-based broiler starter basal diet was formulated to approximate the nutritional requirements of broiler chickens, as recommended by the National Research Council [24] and then adjusted to breeder’s recommendations [25]. No antibiotics, coccidiostats or enzymes were added to the feed (Table 1). All animal handling procedures complied with the Institutional Animal Care and Use Committee (IACUC) at the University of Arkansas, Fayetteville (protocol #18029).
Table 1, Ingredient composition and nutrient content of a basal starter diet used in the experiment on as-fed basis
Item
|
Corn soybean-based diet
|
Ingredients (%)
|
|
Corn
|
57.34
|
Soybean meal
|
34.66
|
Poultry fat
|
3.45
|
Dicalcium phosphate
|
1.86
|
Calcium carbonate
|
0.99
|
Salt
|
0.38
|
DL-Methionine
|
0.33
|
L-Lysine HCl
|
0.31
|
Threonine
|
0.16
|
Vitamin premix1
|
0.20
|
Mineral premix2
|
0.10
|
Choline chloride 60%
|
0.20
|
Calculated analysis
|
|
Metabolizable energy (kcal/ kg)
|
3,035
|
Crude protein (%)
|
22.16
|
Ether extract (%)
|
5.68
|
Lysine (%)
|
1.35
|
Methionine (%)
|
0.64
|
Methionine + cystine (%)
|
0.99
|
Threonine (%)
|
0.92
|
Tryptophan (%)
|
0.28
|
Total calcium (%)
|
0.90
|
Available phosphorus (%)
|
0.45
|
Determined analysis
|
|
Crude protein (%)
|
21.15
|
Ether extract (%)
|
6.05
|
Calcium (%)
|
0.94
|
Phosphorus (%)
|
0.73
|
1Vitamin premix supplied per kg of diet: Retinol, 6 mg; cholecalciferol, 150 µg; dl-α-tocopherol, 67.5 mg; menadione, 9 mg; thiamine, 3 mg; riboflavin, 12 mg; pantothenic acid, 18 mg; niacin, 60 mg; pyridoxine, 5 mg; folic acid, 2 mg; biotin, 0.3 mg; cyanocobalamin, 0.4 mg.
. 2Mineral premix supplied per kg of diet: Mn, 120 mg; Zn, 100 mg; Fe, 120 mg; copper, 10 to 15 mg; iodine, 0.7 mg; selenium, 0.2 mg; and cobalt, 0.2 mg.
Salmonella strain and culture conditions
The poultry strain of Salmonella Typhimurium (PHL-2020) was obtained from the USDA National Veterinary Services Laboratory (Ames, IA, United States). This strain was selected for resistance to 25 µg/mL of novobiocin (NO, catalog no. N-1628, Sigma) and 20 µg/mL of nalidixic acid (NA, catalog no. N-4382, Sigma) in our laboratory. In the present study, 100 mL of S. Typhimurium from a frozen aliquot was added to 10 mL of tryptic soy broth (TSB, Catalog No. 22092, Sigma, St. Louis, MO, USA), incubated at 37°C for 8 h, and passed three times every 8 h to ensure that all bacteria were in log phase as previously described [26]. Post-incubation, bacteria were washed three times with sterile 0.9% saline by centrifugation at 1864 ´ g for 10 min, reconstituted in saline, quantified by densitometry with a spectrophotometer (Spectronic 20DC, Spectronic Instruments Thermo Scientific, Rochester, NY, USA) and finally diluted to an approximate concentration of 104 cfu/mL. Levels of S. Typhimurium were further verified by serial dilutions and plated on brilliant green agar (BGA, Catalog No. 70134, Sigma, St. Louis, MO, USA) with NO and NA for enumeration of actual cfu used in the experiment.
Animal source and experimental design
In the present study, two independent trials with 75 day-of-hatch male Cobb-Vantress broiler chickens (Fayetteville, AR, USA) were conducted. Chicks were individually weighed and randomly assigned to one of five groups (n = 15 chickens/group): 1) Negative control (NC, basal diet); 2) Positive control (PC, basal diet + challenged with 104 cfu of S. Typhimurium per bird on hatching day); 3) CA (basal diet supplemented with 250 mg/Kg of Copper (II) acetate hydrate + challenged with 104 cfu of S. Typhimurium per bird on hatching day); 4) CR (basal diet supplemented with 0.2% curcumin + challenged with 104 cfu of S. Typhimurium per bird on hatching day); and 5) CA-CR (basal diet supplemented with 250 mg/kg of Copper (II) acetate hydrate and 0.2% curcumin + challenged with 104 cfu of S. Typhimurium per bird on hatching day). In both trials, chicks were raised in floor pens (118 × 59 in), provided with their diet, water ad libitum, and maintained at an age-appropriate temperature during all experiments. Body weight (BW) and body weight gain (BWG) were evaluated at 10-days of age. On day ten post-S. Typhimurium challenge, chickens were given an appropriate dose of fluorescein isothiocyanate dextran (FITC-d) by oral gavage one hour before the chickens were euthanized by CO2 inhalation and only the right cecal tonsil (CT) from 12 broilers per group were aseptically collected for S. Typhimurium recovery. Furthermore, blood samples were also collected from the femoral vein for the determination of FITC-d. The concentration of FITC-d administered was calculated based on group body weight at day nine post-S. Typhimurium challenge. For microbiota analysis, the content of the left CT was collected aseptically and stored at −20°C until analysis. The number of broilers chosen per group for each determination was based on reproducible results from experiments previously described and published by our laboratory [22,23].
Salmonella recovery
In both independent trials, the right CT samples from 12 chickens per group were individually homogenized and diluted with saline (1:4 w/v), and 10-fold dilutions were plated on Xylose Lysine Tergitol-4 (XLT-4, Catalog No. 223410, BD DifcoTM) with NO and NA for S. Typhimurium recovery. Plates were incubated at 37°C for 24 h to enumerate total S. Typhimurium colony-forming units. Subsequently, the CT samples were enriched in 2× concentrated tetrathionate enrichment broth and further incubated at 37°C for 24 h. Enrichment samples were streaked onto XLT-4 with NO and NA selective media for confirmation of Salmonella presence. Samples that were negative in the plate dilution method but positive after enrichment with tetrathionate received an arbitrary value of 500 CFU / g (Lower limit of detection).
Serum determination of FITC-d leakage
FITC-d (MW 3–5 kDa; Sigma-Aldrich Co., St. Louis, MO, USA) was provided by oral gavage to 12 broiler chickens from each group at a dose of 8.32 mg/kg of body weight one hour before the chicks were euthanized by CO2 inhalation with the purpose of evaluating the paracellular transport and mucosal barrier dysfunction [27,28]. Three remaining broiler chickens of each group were used as controls. The blood samples were centrifuged (1000×g for 15 min) to separate the serum. Then, serum samples obtained were diluted (1:5) and measured fluorometrically at an excitation wavelength of 485 nm and an emission wavelength of 528 nm (Synergy HT, Multi-mode microplate reader, BioTek Instruments, Inc., VT, USA) to determine the serum FITC-d levels [29].
Microbiota analysis
DNA extraction, PCR, and library preparation for sequencing
V4 region of 16S rRNA gene from the genomic DNA of each of the 25 samples of cecal content (5 samples per group x 5 groups) was amplified using the primers 515F [30] and 806R [31]. The library of amplicons for DNA sequencing was prepared according to the 16S Illumina PCR protocol described in the Earth Microbiome Project (http://www.earthmicrobiome.org) with slight modifications [32]. In brief, Q5® High-Fidelity DNA Polymerase user guide protocol (New England Biolabs, Catalog No. M0491S) was used to conduct PCR in a 25 μl final reaction volume via 30 amplification cycles. The length of the amplified product was confirmed with 1% agarose gel electrophoresis, and equal amount (~300 ng) of the amplicons from each sample as measured by Qubit dsDNA BR Assay Kit (ThermoFisher Scientific, Catalog No. Q32850) were pooled together. The pooled amplicons were finally run on 1% agarose gel electrophoresis, purified using Zymoclean Gel DNA Recovery Kit (Zymo Research, Catalog No. D4007), and sequenced with Illumina MiSeq paired-end 300 cycle options at Admera Health, LLC (New Jersey, United States). Despite the small number of samples analyzed, previous studies using 6 samples have shown reliable results since even omitting outliers the trend is the same [33,34].
Amplicons sequence analysis
Nebula cloud computing platform of the University of Arkansas was used to process raw sequencing reads in QIIME 2 version 2018.8 utilizing the pipelines developed for paired-end data types [35]. In sum, “demux emp-paired” method of q2-demux plugin was used to demultiplex sequencing reads, followed by quality filtering and denoising with “dada2 denoise-paired” method of q2-dada2 [36] plugin available at QIIME 2. The truncation length of forward and reverse reads were set at 220 and 200 bp, respectively, which was based on the quality score criteria (≥30). Taxonomic assignment was performed using a Naive Bayes classifier pre-trained with Greengenes (Version 13.8) 99% OTUs [37] and q2-feature-classifier plugin, where the sequences have been trimmed to include only the V4 region of the 16S rRNA gene region which is defined by the 515F/806R primer pair. We detected the sequence reads assigned to Chloroplast and Mitochondria, which were subsequencingtly removed using taxonomy-based filtering option in QIIME2. The core-metrics-phylogenetic method at a sampling depth of 69,566 was used to analyze Alpha and Beta diversity. Observed OTUs were used to calculate alpha diversity, while weighted UniFrac distance and unweighted UniFrac distance metrics were used for beta diversity analysis. All figures were created using ggplot2 packages of R [38].
Data and statistical analysis
After demonstrating that data from S. Typhimurium counts (log10 cfu/g), serum determination of FITC-d leakage, BW and BWG presented a normal distribution and homogeneity of the variances using the Levene and Ryan-Joiner procedures, respectively, these data were subjected to analysis of variance (ANOVA) as a completely randomized design using the General Linear Models procedure of Statistical Analysis System (SAS®) [39]. Significant differences among the means were determined by Duncan's multiple range test at p < 0.05. Enrichment data were expressed as positive/total chickens (%), and the percentage of S. Typhimurium positive samples were compared by a chi-square test of independence [40], testing all possible combinations to determine the significance (p < 0.05).
Statistical differences of bacterial taxa at different levels (family and genus) among treatment groups were determined using Linear discriminant analysis effect size (LEfSe) using all against all comparison mode, where the level of significance was set at LDA score > 2.0 and P<0.05 [41]. The significant differences in alpha diversity were calculated using an alpha-group-significance command of QIIME2, which is based on the Kruskal-Wallis test. Statistical differences in beta diversity among groups were calculated by PERMANOVA [41] test using a beta-group-significance command of QIIME2 with a pairwise option. For both diversities analysis, the corrected p values for multiple comparisons (q) were used to report a significant difference between the two groups, where the level of significance was set at q < 0.05.