Ascophyllum nodosum extract (ANE) preparation and chemical composition analyses
A. nodosum was harvested in February 2019 (Quality Sea Veg Ltd., Burtonport, Co. Donegal, Ireland). Whole seaweed biomass was oven-dried at 50°C for 9 days and milled to a 1 mm particle size (Christy and Norris Hammer Mill, Chelmsford, UK) and stored at room temperature. The ANE extract was obtained using a hydrothermal-assisted extraction method using the optimal conditions for best fucoidan yield (120°C, 62.1 min, 30 ml 0.1M HCl/g seaweed) as described previously [34].
The ANE composition as % w/w dry matter was as follows: 46.6% fucoidan, 18.6% laminarin, 10.7% mannitol, 4.6% alginate, 4.5% protein and 0.75% ash. The ANE was stored at -20°C. The concentration of fucoidan was estimated according to the method described by Usov et al. [35], with modifications as described by Garcia-Vaquero et al. [34]. The concentration of laminarin and mannitol was determined using standard kits (Megazyme, Bray, Ireland) according to the manufacturer’s instructions. The concentration of alginate was estimated according to the method described by Truus et al. [36]. The ash content was determined after ignition of a weighed sample in a muffle furnace (Nabertherm, Bremen, Germany) at 550°C for 6 h according to the AOAC.942.05 [37]. The nitrogen content was determined using the LECO FP 528 instrument (Leco Instruments UK Ltd., Cheshire, UK) according to the AOAC.990.03 [37]. The conversion factor 4.17 was used to calculate protein content, as described for brown macroalgae [38].
In vitro screening of ANE antibacterial activity
The revival and culture of the S. Typhimurium phage type (PT) 12 and Bifidobacterium thermophilum (DSMZ 20210) and the subsequent pure culture growth assays were carried out as described by Venardou et al. [39]. Briefly, S. Typhimurium and B. thermophilum were revived from cryoprotective beads (TS/71-MX, Protect Multi-purpose, Technical Service Consultants Ltd., Lancashire, UK) and sub-cultured following standard procedures to obtain 24 h cultures. The pure culture growth assays were carried out in 96-well microtiter plates (CELLSTAR, Greiner Bio-One, Kremsmünster, Austria). ANE was diluted appropriately in 10% de Man, Rogosa and Sharpe broth (MRS, Oxoid Ltd., Hampshire, UK) and 10% Tryptone Soya broth (TSB, Oxoid Ltd., Hampshire, UK) to obtain a final concentration of 5, 4, 3, 2 and 1 mg/ml prior to the assay. S. Typhimurium and B. thermophilum were diluted in 10% TSB and MRS, respectively, to obtain an inoculum of 106 − 107 CFU (colony-forming unit)/ml with initial bacterial enumeration performed each time. Equal quantities of each ANE concentration and inoculum were transferred to duplicate wells and control wells containing no ANE were also included. To evaluate the sterility, blank wells containing equal quantities of 10% medium and each ANE concentration were included. Plates were agitated gently for thorough mixing and incubated at 37°C for 18 h aerobically for S. Typhimurium or anaerobically for B. thermophilum. After incubation, a 10-fold serial dilution (10− 1 − 10− 8) followed by spread plating onto Tryptone Soya agar (Oxoid Ltd., Hampshire, UK) for S. Typhimurium and de Man, Rogosa and Sharpe agar (Oxoid Ltd., Hampshire, UK) for B. thermophilum were used to determine both the bacterial viability and counts at the increasing ANE concentrations. Plates were incubated aerobically at 37°C for 24 h for S. Typhimurium and anaerobically at 37°C for 48 h for B. thermophilum. Anaerobic conditions were established within sealed containers using AnaeroGen 2.5 and 3.5 L sachets (Thermo Fisher Scientific, Waltham, MA, USA). The dilution resulting in 5–50 colonies was selected for the calculation of CFU/ml using the formula CFU/ml = Average colony number * 50 * dilution factor. The bacterial counts were logarithmically transformed (logCFU/ml) for the subsequent statistical analysis. Zero counts at the neat dilution (100) were assigned the arbitrary value of 1.30 logCFU/ml which was considered the minimum detection limit using spread plating [40]. All experiments were carried out with technical replicates on three independent occasions (3 biological replicates).
Newly weaned pig experiment (day 0–21)
Experimental design and diets
The experiment had a randomised complete block design and consisted of the following dietary treatments: (T1) basal diet (control); (T2) basal diet + 3.1 g ZnO (pharmacological dose)/kg feed (ZnO); (T3) basal diet + 2 g ANE/kg feed (low ANE) and (T4) basal diet + 4 g ANE/kg feed (high ANE). The ANE inclusion levels were selected based on the in vitro anti-S. Typhimurium activity of the 2 and 4 mg/ml ANE. Ninety-six healthy pigs [progeny of meat-line boars x (large white x landrace sows)] with average weight 8.6 (standard deviation (SD) 1.12) kg were sourced from a commercial pig farm at weaning (28 days of age) and were penned in groups of two. At the time of weaning, the Salmonella seroprevalence for the herd in the farm of origin was estimated at 46.7% (weighted average of previous three months data). The pigs were blocked based on weaning weight, litter of origin and sex and within each block assigned to one of the four treatments (12 replicates/treatment). The basal diet contained 10.6 MJ/kg net energy and 14.0 g/kg standard ileal digestible lysine. All amino acid requirements were met relative to lysine [41]. The ingredient composition and the analysed and calculated chemical composition of the diet are presented in Table 1. All treatment diets were milled on site and fed in meal form for 21 days. The ZnO (Cargill, Naas, Ireland) was included at 3100 mg/kg feed and contained 80% Zn, resulting in an inclusion level of 2500 mg Zn per kg feed.
Table 1
Ingredient composition and chemical analysis of the basal dieta
Ingredient (g/kg) |
Wheat | 355.4 |
Full fat soya bean | 170.0 |
Soya bean meal | 105.0 |
Flaked wheat | 130.0 |
Flaked maize | 70.0 |
Soya oil | 30.0 |
Soya concentrate | 65.0 |
Whey powder (90%) | 50.0 |
Vitamins and mineralsb | 2.5 |
Sodium bicarbonate | 2.0 |
Monocalcium phosphate | 4.0 |
Calcium carbonate (Limestone) | 6.0 |
Salt | 2.0 |
Lysine HCl | 4.0 |
DL-methionine | 2.0 |
L-threonine | 1.8 |
Tryptophan | 0.3 |
Analysed and calculated chemical composition |
Dry matter | 899.0 |
Crude protein (N x 6.25) | 208.0 |
Gross energy (MJ/kg) | 16.9 |
Crude fat | 80.0 |
Crude fibre | 28.0 |
Ash | 46.0 |
Neutral detergent fibre | 99.0 |
Lysine %c | 1.43 |
Methionine %c | 0.50 |
Methionine and cysteine %c | 0.84 |
Threonine %c | 0.93 |
Tryptophan %c | 0.30 |
Valine % c | 0.98 |
Leucine % c | 1.45 |
Isoleucine % c | 0.87 |
aDietary treatments: (T1) basal diet (control); (T2) basal diet + 3.1 g ZnO/kg feed (ZnO); (T3) basal diet + 2 g ANE/kg feed (low ANE); (T4) basal diet + 4 g ANE/kg feed (high ANE) |
bProvided (mg/kg complete diet): Cu, 25; Fe, 140; Mn, 47; Zn, 120; I, 0.6; Se, 0.3; retinol, 1.8; cholecalciferol, 0.025; tocopherol, 67; menaquinone, 4; cyanocobalamin, 0.01; riboflavin, 2; nicotinic acid, 12; pantothenic acid, 10; choline chloride, 250; thiamine, 2; pyridoxine, 0.015 |
cCalculated for tabulated nutritional composition [42] |
Housing and animal management
The pigs were housed in fully slatted pens (1.7 m x 1.2 m) and weighed at the beginning of the experiment (day 0) and on days 7, 14 and 21. The ambient environmental temperature within the house was thermostatically controlled at 30°C for the first 7 days and reduced by 2°C per week for the remainder of the experiment. The humidity was maintained at 65%. Feed and water were available ad libitum from four-space feeders and nipple drinkers; precaution was taken to avoid feed wastage. Faecal scores (FS) were recorded twice daily in the individual pens by the same operator on a scale ranging from 1 to 5. The scoring system was as follows: 1 = hard, firm faeces; 2 = slightly soft faeces; 3 = soft, partially formed faeces; 4 = loose, semi-liquid faeces; and 5 = watery, mucous like faeces [43].
Sample collection for Salmonella presence and quantification
Faecal samples were collected after natural defaecation into sterile containers (Sarstedt, Nümbrecht, Germany) on arrival on day 0 from 19 pigs to determine the Salmonella status of the herd. Rectal faecal samples were collected into sterile containers from the same pig in each pen (n = 48 pigs) on days 14 and 21. Samples were obtained by natural defaecation with rectal stimulation employed only if necessary and solely on day 21. All faecal samples were immediately stored at -20°C.
Challenge experiment (day 25–34)
Experimental design and diets
On day 21, ZnO supplementation ceased and one pig from each pen from (T1), (T2) and (T4) of the newly weaned pig experiment (n = 36, 12 replicates/treatment) proceeded to the challenge experiment. The pigs from (T1) and (T4) were kept on their original diets as described in the newly weaned pig experiment with (T4) being renamed as ANE, whereas (T2) was renamed as ZnO-residual, whereby the animals were fed a basal diet upon ZnO removal. Between day 21 and 25, all pigs were on their respective diet, however, performance data was collected after the initiation of the challenge experiment on day 25. The challenge experiment had a randomised complete block design. The thirty-six pigs with an average weight of 18.3 (2.44 SD) kg on day 25 were blocked on weight basis and penned in pairs.
Housing and animal management
The pigs were weighed at the beginning (day 25) and end (day 34) of the experiment. The housing and animal management were as described in the newly weaned pig experiment apart from the ambient environmental temperature that was kept at 25°C during the nine-day experimental period in each house and the FS that was recorded once daily in the individual pens.
S. Typhimurium experimental infection
On day 25, each animal was manually restrained and orally challenged with 5 ml of a S. Typhimurium culture (infectious dose ≈ 4 x 107 CFU) using a syringe (no needle attached).
Sample collection
Rectal faecal samples were collected in sterile containers from all pigs on days 25 (prior to S. Typhimurium infection), 27 and 34 for Salmonella quantification and immediately stored at -20°C. Samples were obtained following natural defaecation or if necessary with rectal stimulation. On day 34, all 36 pigs were euthanised by pentobarbitone sodium (Euthatal Solution, 200 mg/ml; Merial Animal Health, Essex, UK) overdose (1 ml/kg body weight injected into the cranial vena cava). Euthanasia was completed by a competent person in a separate room away from sight and sound of the other pigs. The entire intestinal tract was immediately removed. Colonic and caecal digesta were collected in sterile containers, snap frozen on dry ice and stored at -20°C for bacterial quantification using quantitative real time polymerase chain reaction (QPCR). Additionally, 1 cm2 sections from the ileum (15 cm from ileocaecal junction) and colon were removed, emptied by dissection along the mesentery and rinsed using sterile phosphate buffered saline (Sigma-Aldrich, St. Louis, MO, USA). The tissue sections were stripped of the overlying smooth muscle before overnight storage in 5 ml RNAlater® solution (Sigma-Aldrich, St. Louis, MO, USA) at 4°C. The RNAlater® was removed before storing the samples at -80°C. These ileal and colonic tissue samples were used for gene expression analysis.
Feed analyses
The feed was milled through a 1 mm screen (Christy and Norris Hammer Mill, Chelmsford, England). The dry matter content was determined after drying overnight at 104°C. Ash content was determined after ignition of a weighted sample in a muffle furnace at 550°C for 6 h according to the AOAC.942.05 [37]. The gross energy content was determined using an adiabatic bomb calorimeter (Parr Instruments, Moline, IL, USA). Crude protein content was determined by measuring the nitrogen content of the feed samples using the LECO FP 528 instrument and the conversion factor of 6.25 according to the AOAC.990.03 [37]. The neutral detergent fibre content was determined according to the method of Van Soest et al. [44] and the crude fibre content according to the AOAC method [37]. The crude fat content of the diets was determined using light petroleum ether and Soxtec instrumentation (Tecator, Sweden) according to the AOAC.920.39 [37].
Salmonella isolation and serotyping
Faecal samples from day 0 were screened for the presence or absence of Salmonella in accordance with the protocol of the International Organisation for Standardization (ISO) 6579-1:2017. Salmonella serotyping, which involved agglutination tests with hyperimmune antisera specific for a range of somatic (O) and flagellar (H) antigens and comparison with the White-Kauffmann-Le Minor scheme [45], was also performed on Salmonella positive samples in accordance with ISO protocol 6579-3:2014. Isolates with a phenotypic partial serotyping were further analysed using a multiplex QPCR for differentiating S. Typhimurium and its monophasic variant S. 4,[5],12:i:- as described previously [46].
Quantification of selected bacterial groups using QPCR
DNA extraction
Microbial genomic DNA was extracted from faecal, colonic and caecal samples using QIAamp® PowerFecal® Pro DNA Kit (Qiagen, West Sussex, UK) according to the manufacturer’s instructions. The DNA quantity and quality were evaluated using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Bacterial primers: The domain-, function-, family- or genus-specific primers for the selected bacterial groups were available in the literature (with the exception of Salmonella enterica) and are provided in Table 2. The 16S rRNA gene was targeted for most bacterial groups except for Salmonella where the hilA gene, the transcriptional regulator of the Salmonella pathogenicity island 1 was selected [47] and also the butyrate-producing bacteria where the butyryl-CoA:acetate CoA-transferase (B-CoA) gene associated with this function was selected [48, 49]. Primers were designed using two tools, Primer3 (https://primer3.org/) for larger amplicons (> 150 bp) and Primer Express™ (Applied Biosystems, Foster City, CA, USA) for smaller amplicons optimised for QPCR (< 125 bp), and their specificity was verified using Primer Basic Local Alignment Search Tool (Primer-BLAST), https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi.
Plasmid preparation and QPCR for absolute quantification: The quantification of the selected bacterial groups using QPCR and the preparation of specific plasmids (total bacteria, Lactobacillus spp., Bifidobacterium spp., Prevotella spp., Enterobacteriaceae) to obtain the standard curves was carried out as described by Venardou et al. [39]. Additionally, plasmids containing the hilA and B-CoA genes were prepared from genomic DNA of S. Typhimurium extracted from pure cultures (DNeasy® Blood & Tissue kit, Qiagen, West Sussex, UK) and Faecalibacterium prausnitzii (DSMZ 17677) purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) respectively. The primers and genomic locations of all targeted genes that were incorporated into plasmids are outlined in Table S1 (Additional file 1). The plasmids were quantified spectrophotometrically and the copy number/µl was determined using an online tool which employs the formula mol/g * molecules/mol = molecules/g using Avogadro's constant, 6.022x1023 molecules/mole (http://cels.uri.edu/gsc/cndna.html). The QPCR reaction (20 µl) included 3 µl template DNA, 1 µl or 2 µl (for B-CoA) of each primer (10 µM), 5 µl or 3 µl (for B-CoA) nuclease-free water and 10 µl of GoTaq® qPCR Master Mix (Promega, Madison, WI, USA). All QPCR reactions were performed in duplicate on the 7500 ABI Prism Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with the following cycling conditions; a denaturation step of 95°C for 10 mins, 40 cycles of 95°C for 15 sec and 60°C for 1 min. Dissociation curves were generated to confirm the specificity of the amplicons. The efficiency of each QPCR assay was established from the slope of the curve derived from plotting the cycle threshold (Ct) obtained from 5-fold serial dilutions of the plasmid against their arbitrary quantities. Only assays exhibiting 90–110% efficiency and generating specific products were used in this study. Bacterial counts were determined from the standard curve derived from the mean Ct value and the log transformed gene copy number of the plasmid and expressed as log transformed gene copy number per gram of faeces or digesta (logGCN/g faeces or digesta).
Table 2
List of forward and reverse primers used for the bacterial quantification by QPCR
Target bacterial group | Forward primer (5'-3') Reverse primer (5'-3') | Amplicon length (bp) | Tm (°C) | References |
Salmonella enterica | F: TACTCAACATGGACGGCTCC R: TTTGCAAGAGAGAAGCGGGT | 630 | 59.3 57.3 | This study |
Total bacteria | F: GTGCCAGCMGCCGCGGTAA R: GACTACCAGGGTATCTAAT | 291 | 64.2 52.4 | [50] |
Lactobacillus spp. | F: AGCAGTAGGGAATCTTCCA R: CACCGCTACACATGGAG | 341 | 54.5 55.2 | [49] |
Bifidobacterium spp. | F: GCGTGCTTAACACATGCAAGTC R: CACCCGTTTCCAGGAGCTATT | 125 | 60.3 59.8 | [51] |
Enterobacteriaceae | F: ATGTTACAACCAAAGCGTACA R: TTACCYTGACGCTTAACTGC | 185 | 54.0 56.3 | [52] |
Butyryl-CoA:acetate CoA-transferase (B-CoA) | F: GCIGAICATTTCACITGGAAYWSITGGCAYATG R CCTGCCTTTGCAATRTCIACRAANGC | 530 | 67.0 64.0 | [48] |
Prevotella spp. | F: CACRGTAAACGATGGATGCC R: GGTCGGGTTGCAGACC | 514 | 58.3 56.9 | [53] |
bp, base pairs; Tm, melting temperature |
Gene expression
RNA extraction and cDNA synthesis
Total RNA was extracted from ileal and colonic tissue using TRI Reagent® (Sigma-Aldrich, St. Louis, MO, USA) and purified using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA) and a DNase removal step (On-Column DNase I Digestion Set, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturers’ instructions. The quantity and purity (260/280 nm absorbance ratio ≥ 2.0) of the total RNA was determined using a Nanodrop spectrophotometer. The complimentary DNA (cDNA) was synthesised from 2 µg total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. The total reaction volume (20 µl) was adjusted to 400 µl using nuclease-free water.
QPCR for relative quantification
The QPCR reaction mix (20 µl) contained 10 µl GoTaq® qPCR Master Mix, 1.2 µl forward and reverse primers (5 µM), 3.8 µl nuclease-free water and 5 µl cDNA. All QPCR reactions were carried out in duplicate on the 7500 ABI Prism Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with the following cycling conditions; a denaturation step of 95°C for 10 mins, 40 cycles of 95°C for 15 sec and 60°C for 1 min. All primers were designed using the Primer Express™ Software and synthesised by MWG Biotech UK Ltd (Milton Keynes, UK) and are presented in Table 3. The sequences of the forward and reverse primers have been described and validated previously for porcine gastrointestinal tissues [29, 54, 55] except for IL7, CCL20, TP53, STAT3, CHRM1, NOX1 and DUOX2 genes for which the primer pairs were newly designed, and their specificity was verified in silico using Primer-BLAST. Dissociation curves were generated to confirm the specificity of the resulting PCR products. The efficiency of each QPCR reaction was established by plotting the Ct derived from 4-fold serial dilutions of cDNA against their arbitrary quantities. Assays exhibiting 90–110% efficiency and single products were solely used in this study. Normalised relative quantities were obtained using the qbase™ PLUS software (Biogazelle, Ghent, Belgium) from two stable housekeeping reference genes, GAPDH and PPIA for the ileum and B2M and PPIA for the colon. These genes were selected as reference genes due to their lowest stability M value (< 1.5) generated by the geNorm application.
Table 3
Panel of target genes evaluated in the ileum and colon
Target gene | Accession No. | Forward primer (5'-3') Reverse primer (5'-3') | Amplicon length (bp) | Tm (°C) |
Immune response |
IL1A | NM_214029.1 | F: CAGCCAACGGGAAGATTCTG R: ATGGCTTCCAGGTCGTCAT | 76 | 63.0 60.5 |
IL6 | NM_214399.1 | F: GACAAAGCCACCACCCCTAA R: CTCGTTCTGTGACTGCAGCTTATC | 69 | 59.8 62.7 |
IL7 | NM_214135.2 | F: GAGTGACTATGGGCGGTGAGA R: GCGGGCGTGGTCATGA | 63 | 61.8 56.9 |
CXCL8 | NM_213867.1 | F: TGCACTTACTCTTGCCAGAACTG R: CAAACTGGCTGTTGCCTTCTT | 82 | 61.9 61.7 |
IL10 | NM_214041.1 | F: GCCTTCGGCCCAGTGAA R: AGAGACCCGGTCAGCAACAA | 71 | 63.4 63.1 |
IL17A | NM_001005729.1 | F: CCCTGTCACTGCTGCTTCTG R: TCATGATTCCCGCCTTCAC | 57 | 60.6 60.4 |
IL22 | XM_001926156.1 | F: GATGAGAGAGCGCTGCTACCTGG R: GAAGGACGCCACCTCCTGCATGT | 112 | 66.0 66.0 |
IFNG | NM_213948.1 | F: TCTAACCTAAGAAAGCGGAAGAGAA R: TTGCAGGCAGGATGACAATTA | 81 | 61.1 61.5 |
TNF | NM_214022.1 | F: TGGCCCCTTGAGCATCA R: CGGGCTTATCTGAGGTTTGAGA | 68 | 62.5 62.8 |
TGFB1 | NM_214015.1 | F: AGGGCTACCATGCCAATTTCT R: CGGGTTGTGCTGGTTGTACA | 101 | 60.6 61.7 |
FOXP3 | NM_001128438.1 | F: GTGGTGCAGTCTCTGGAACAAC R: AGGTGGGCCTGCATAGCA | 68 | 60.6 61.2 |
CCL20a | NM_001024589.1 | F: GCTCCTGGCTGCTTTGATG R: TTGCTTGCTGCTTCTGACTTG | 66 | 58.8 57.9 |
TLR4 | NM_001293317.1 | F: TGCATGGAGCTGAATTTCTACAA R: GATAAATCCAGCACCTGCAGTTC | 140 | 57·1 60·6 |
TP53a | NM_213824.3 | F: CCGGGTGGAAGGGAATTT R: CCACAACGCTGTGTCGAAAA | 68 | 56.0 57.3 |
STAT3a | NM_001044580 | F: TCTTGAGAAGCCAATGGAGATTG R: TGGAGGAGGCGGGACTCT | 69 | 58.9 60.5 |
Intestinal integrity |
MUC2 | AK231524 | F: CAACGGCCTCTCCTTCTCTGT R: GCCACACTGGCCCTTTGT | 70 | 63.1 62.1 |
TJP1/ZO-1 | XM_005659811.1 | F: TGAGAGCCAACCATGTCTTGAA R: CTCAGACCCGGCTCTCTGTCT | 76 | 59.9 60.0 |
Cholinergic receptor |
CHRM1a | NM_214034.1 | F: GCCATGGCCGCCTTCT R: GGTTCTCTGTCTCCCGGTAGATG | 76 | 56.9 64.2 |
NADPH oxidases |
NOX1a | XM_003484140.3 | F: CTTTGAAAGGATCCTCCGATTTT R: ATGGATACATGACCACCTTGGTAA | 71 | 57.1 59.3 |
DUOX2a | NM_213999.2 | F: CTGGGCCTTGACATAGATGAGAT R: GGCAAAAAGGTGTCTGAAGAAGA | 108 | 60.6 58.9 |
Reference genes |
PPIA | NM_214353.1 | F: CGGGTCCTGGCATCTTGT R: TGGCAGTGCAAATGAAAAACT | 75 | 62.1 60.7 |
B2M | NM_213978.1 | F: CGGAAAGCCAAATTACCTGAAC R: TCTCCCCGTTTTTCAGCAAAT | 83 | 58.2 58.4 |
GAPDH | AF017079.1 | F: CAGCAATGCCTCCTGTACCA R: ACGATGCCGAAGTTGTCATG | 72 | 62.2 62.1 |
bp, base pairs; Tm, melting temperature; IL1A, interleukin 1 alpha; IL6, interleukin 6; IL7, interleukin 7; CXCL8, C-X-C motif chemokine ligand 8; IL10, interleukin 10; IL17A, interleukin 17 alpha; IL22, interleukin 22; IFNG, interferon gamma; TNF, tumour necrosis factor; TGFB1, transforming growth factor beta 1; FOXP3, forkhead box P3; CCL20, C-C motif chemokine ligand 20; TLR4, toll-like receptor 4; TP53, tumour protein p53; STAT3, signal transducer and activator of transcription 3; MUC2, mucin 2; TJP1/ZO-1, tight junction protein 1/zona occludens 1; CHRM1, cholinergic receptor muscarinic 1; NOX1, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1; DUOX2, dual oxidase 2; PPIA, peptidylprolyl isomerase A; B2M, beta-2-microglobulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase |
aGenes encoding proteins with an established role in facilitating or inhibiting Salmonella infection. These proteins are associated with chemotaxis (CCL20), production of reactive oxygen species (NOX1, DUOX2) anti-inflammatory activity (STAT3, CHRM1) and cell survival and death (TP53) [56–61] |
Statistical analysis
All data was initially checked for normality using PROC UNIVARIATE procedure of Statistical Analysis Software (SAS) 9.4 (SAS Institute, Cary, NC, USA). The bacterial counts from the pure culture growth assays were analysed using PROC GLM procedure for the presence of linear and quadratic effects of ANE concentration. The biological replicate was the experimental unit. The LSMEANS statement was additionally used to calculate the least-square mean values and the standard error of the means (SEM). The performance data from the newly weaned pig experiment, FS data from both experiments and Salmonella shedding data from the challenge experiment were analysed by repeated measures analysis using PROC MIXED procedure of SAS [62]. The model included the fixed effects of treatment and time and their associated interaction. For the performance data, the initial weight was used as a covariate. Salmonella shedding data from the newly weaned pig experiment, performance data from the challenge experiment, bacterial populations data and gene expression data were analysed using PROC GLM procedure of SAS. The Bonferroni adjustment was used in the analysis of the gene expression data. The model assessed the effect of treatment with the experimental unit being the pen for the performance data and the animal within the pen for the bacterial populations and gene expression data. For the performance data, the body weight on day 25 was used as a covariate. Probability values of < 0.05 denote statistical significance. Results are presented as least-square mean values ± SEM.