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. 2023 Apr 15;102(8):102723. doi: 10.1016/j.psj.2023.102723

Red osier dogwood extract versus Trimethoprim-sulfadiazine (Part 1). Effects on the growth performance, blood parameters, gut histomorphometry, and Salmonella excretion of broiler chickens orally challenged with Salmonella Enteritidis

Taiwo J Erinle *, Martine Boulianne †, Deborah I Adewole *,1
PMCID: PMC10404697  PMID: 37406598

Abstract

The poultry industry has not been spared from the prevalent incidence of diseases caused by invasive pathogens, especially Salmonella. Due to the pressing need to identify a suitable antibiotic alternative for use in poultry production, this study investigated the efficacy of red osier dogwood (ROD) extract on the growth, blood parameters, gut morphology, and Salmonella excretion in broiler chickens orally challenged with Salmonella Enteritidis (SE). A 4 × 2 factorial experiment was conducted based on 2 main factors, namely dietary treatments, and SE challenge. A total of 404, one-day-old male Ross broiler chicks were randomly assigned to 4 dietary treatments; 1) Negative control (NC), 2) NC + 0.075 ppm of Trimethoprim-sulfadiazine (TMP/SDZ)/kg of diet, 3) NC + 0.3% ROD extract, and 4) NC + 0.5% ROD extract. The absence of SE in the fecal samples obtained from chick delivery boxes was confirmed on d 0. On d 1, half of the birds were orally gavaged with 0.5 mL of phosphate-buffered saline each (noninfected group) and the remaining with 0.5 mL of 3.1 × 105 CFU/mL SE (infected group) in all treatment groups. Dietary treatments were randomly assigned to 8 replicate cages at 6 birds/cage. On 1-, 5-, 12-, and 18-day postinfection (DPI), cloacal fecal samples were collected on the 6 birds/cage to assess SE excretion. Average weight gain (AWG), average feed intake (AFI), feed conversion ratio (FCR), and mortality were determined weekly. On d 21, 10 chickens/treatment were euthanized to perform hematology, gut histomorphometry, serum immunoglobulins G and M (IgG and IgM), and superoxide dismutase measurements. Both ROD extract levels did not affect (P > 0.05) growth performance; however, the SE-infected birds showed increased (P < 0.05) AFI and FCR throughout the experimental period. Regardless of the SE-infection, both ROD extract levels improved (P < 0.05) duodenal villus height: crypt depth compared to other treatments. 0.5% ROD extract improved (P < 0.05) ileal villus width (VW) of noninfected birds and ileal crypt depth of infected birds, but it decreased (P < 0.05) the ileal VW of infected birds, compared to other treatments. The SE-infected birds showed lower (P < 0.05) lymphocytes (L) but increased (P < 0.05) heterophils (H), H:L, and monocytes (MON). Both ROD extract levels did not affect (P > 0.05) white blood cell differential, while dietary 0.3% ROD extract increased (P < 0.05) MON of the birds, regardless of infection model. Regardless of infection model, both TMP/SDZ and 0.5% ROD extract reduced the concentration of IgM in the serum, compared to the control and 0.3% ROD (P = 0.006). Conclusively, both ROD extract levels improved duodenal histomorphology and body defense against SE infection in broiler chickens; however, the 0.3% ROD extract was better.

Key words: red osier dogwood extract, antibiotic-replacement, Salmonella Enteritidis, oral gavage, white blood cell differentials

INTRODUCTION

Salmonella—a gram-negative, facultative anaerobic bacteria, is unarguably one of the most economically important causes of foodborne diseases following the consumption of Salmonella-contaminated food materials. In Canada, human nontyphoidal salmonellosis is notifiable, with an average case of approximately 6,500 reported annually between 2009 and 2013 (Public Health Agency of Canada, 2008, Public Health Agency of Canada, 2009; Government of Canada, 2007, Government of Canada, 2016). The United States seems to be most impacted by Salmonellosis incidence and has recorded approximately 1.4 million gastroenteritis cases, 26,500 hospitalizations, and 420 deaths caused by the nontyphoidal Salmonella (Cao, 2014; Centers for Disease Control and Prevention (CDC), 2018); resulting in an annual loss of $1.4 to 3.0 billion (McGruder, 1995; WHO, 2018). On a global scale, nontyphoidal Salmonella causes a yearly estimated illness and death of over 93.8 million and 39,000 to 303,000 deaths, respectively (Majowicz et al., 2010). While there are over 2,600 unique nontyphoidal Salmonella serovars, Salmonella enterica ssp. Enteritidis is the most typical culprit among human and nonhuman isolates in Europe, Africa, Latin America, and the Caribbean (Galanis et al., 2006; Afshari et al., 2018; Castro-Vargas et al., 2020).

It is notable that the epidemiological prevalence of Salmonella Enteritidis (SE) has been closely associated with edible poultry products and, consequently, has exclusively been reported to contribute to the increased incidence of salmonellosis (Antunes et al., 2016; Centers for Disease Control and Prevention, 2020; Boubendir et al., 2021). The menace of Salmonella infections in poultry is not limited to the host, as vertical transmission of the bacteria from the maternal into their eggs has been reported (Berchieri Jr. et al., 2001). For decades, antibiotics, including chloramphenicol, neomycin, ampicillin, ciprofloxacin, ceftriaxone, fluoroquinolones, and trimethoprim/sulfadiazine (TMP/SDZ) have been heavily relied upon in the combat of salmonellosis in animals. Despite the remarkable collaborative efforts, including critical biosecurity measures, medications, and vaccination, to control SE infection in poultry and prevent contamination of poultry products, the poultry industry is still constantly embattled by antibiotic-resistant SE. According to the Centre for Disease Control (2020) and White et al. (2001), Salmonella strains resistant to medically important antibiotics, including ceftriaxone, ciprofloxacin, and ceftiofur, have been isolated in chickens and turkeys. Thus, identifying a more suitable antibiotic alternative has become a desirable goal to address animal health and improve economic return for poultry farmers, and assure food safety concerns for the public.

Some phytogenic additives have undoubtedly proven worthy antibiotic substitutes due to their high concentrations of diverse polyphenols. Among the identified numerous phytogenic additives, red osier dogwood (ROD; Cornus stolonifera) has been reckoned to contain a high concentration of total polyphenol, with ranges between 220 and 234 mg gallic acid equivalence (GAE)/g dry weight (Isaak et al., 2013; Scales, 2015; Erinle et al., 2022), values higher than the 40.27 mg GAE/g dry weight obtained in an olive plant (Isaak et al., 2013). In addition, ROD extract boasts of a considerable antioxidant capacity given its higher oxygen reactive absorbance capacity when compared to that of methanol extract of tea, parsley, basil, and olive leaves (Isaak et al., 2013). Some virulent pathogens like Salmonella when invading gut epithelial tissue and barrier provoke inflammatory cytokines production (Ferenczi et al., 2016; Song et al., 2020; Thiam et al., 2021). In vitro studies by Jiang et al. (2019) and Yang et al. (2019) reported that polyphenols in ROD extracts exert their antioxidant prowess and prevent proinflammatory responses in Caco-2 cells by repressing gene expression of inflammatory cytokines.

Incorporation of ROD plant material into the diets of Escherichia coli-challenged pigs (Jayaraman et al., 2018; Koo et al., 2021) was reported to improve gut morphology, which has been associated with optimized nutrient digestion and absorption. Furthermore, dietary supplementation of ROD was also reported to suppress blood thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA) in weaned piglets infected with E. coli (Amarakoon, 2017), thereby reducing bacteria-induced oxidative stress. In the absence of antibiotics, dietary supplementation of ROD extract has been demonstrated to sustain growth performance and intestinal health of unchallenged broiler chickens (Mogire et al., 2021) and when challenged with SE lipopolysaccharide (LPS) (Erinle et al., 2022). Because the 2 studies lacked an actual Salmonella infection challenge it was deemed necessary to investigate the impact of ROD in poultry birds under disease-challenged conditions. In addition to the LPS, pathogenic bacteria produce several highly toxic exotoxins to attack their host. Until now, no in vivo study has been conducted to examine the effects of dietary ROD extract as a potential antibiotic alternative on broiler chickens infected with SE. This study investigates the effects of ROD extract on the growth performance, blood parameters, gut morphology, and Salmonella excretion of broiler chickens challenged with SE.

MATERIALS AND METHODS

The experimental protocol was approved by the University of Montreal Animal Care and Use Committee (Project 20-Rech-2063). The birds were handled following the guidelines established by the Canadian Council on Animal Care (2009).

Birds and Housing

A total of 404, one-day-old, male Ross 308 broiler chicks were obtained from a commercial hatchery in Quebec. They were raised in a 4Tier Poultry Super Brooder (from Alternate design website): (38” wide × 31” deep × 15” high for 21 d). Upon arrival, the birds were weighed into a group of 6 birds and allocated to cages in 2 different rooms to house the noninfected and about-to-be infected birds separately. The room temperature was monitored daily and was gradually reduced from 32°C to 24°C from d 0 to 21. The lighting program was set to produce 24 h, 16 h, and 10 h of light during d 1 to 3, d 4 to 14, and d 15 to 21, respectively, and illumination was gradually reduced from 20 lx on d 0 to 5 lx on d 21.

Preparation of Salmonella Enteritidis Inoculum

The SE (SHY 04 1540) used in the current study was isolated in 2004 from a clinical case and used in previous infection studies by Dr. Martine Boulianne's Avicole Research Laboratory, University of Montreal, Quebec, Canada. The strain was grown overnight on blood agar at 37°C. Three colonies of SE strain were transferred into 10 mL of Luria-Bertani (LB) broth culture. Following this, 1 mL from the SE-LB broth mixture was added to another 100 mL of prewarmed LB and shook at 150 rpm in an incubator at 37°C for about 3 h. The inoculum concentration was periodically confirmed using a spectrophotometer at a wavelength 600 nm. The inoculum was further serially diluted to achieve the appropriate bacteria coliform-forming unit (CFU). The SE concentration in this study was 3.1 × 105 CFU/mL.

Diets and Experimental Design

The ROD extract used in this study was obtained from Red Dogwood Enterprise Ltd., Swan River, Manitoba. The extract was in a powdery form, and it was obtained by using the spray drying method of extraction. The birds were randomly assigned to 8 treatment groups containing 8 replicate pens of 6 birds each. The experiment was designed as a 4 × 2 factorial arrangement based on 2 main factors, as shown in Table 1. The main factors were: 1) 4 dietary treatments: corn-wheat-soybean meal-based diet negative control (NC), negative control with 0.075 ppm of TMP/SDZ per kilogram of diet; and negative control supplemented with 0.3 or 0.5% ROD extract and 2) 2 infection model groups: the noninfected groups (N) were challenged orally with 0.5 mL of sterile 1 × phosphate buffered saline (PBS) per bird (AVL82762, HyClone Laboratories, Inc., Logan, UT), and the infected groups (I) were challenged orally with 0.5 mL/bird of 3.1 × 105 CFU/mL of SE. The individual oral gavage challenge was done on d 1. The basal diet was formulated as isocaloric and isonitrogenous to meet the nutrient requirements of broiler chickens as recommended by the Ross 308 feeding guide. The gross and proximate compositions of the experimental diets are presented in Table 2. The experimental diets containing TMP/SDZ and ROD extract were mixed from the negative control diets, and as a result, only the nutrient content of the negative control diet was reported. The proximate composition of the experimental diets was determined following the procedure of AOAC (1990). The polyphenols profile of ROD extract and the total polyphenols in the starter and grower ROD and control treatments have been reported in our previous study (Erinle et al., 2022).

Table 1.

Experimental design.

Challenge model
Dietary treatment (number of replicates; n) Unchallenged (U) Challenged (C)
Negative control 1) Basal (NC)1 + PBS (n = 8) 2) NC + SE (n = 8)
+ Antibiotics 3) NC + 0.075 ppm TMP/SDZ + PBS (n = 8) 4) NC + 0.075 ppm TMP/SDZ + SE (n = 8)
+0.3% ROD extract (5) NC + 0.3% ROD + PBS (n = 8) 6) NC + 0.3% ROD + SE (n = 8)
+0.5% ROD extract 7) NC + 0.5% ROD + PBS (n = 8) 8) NC + 0.5% ROD + SE (n = 8)
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N = noninfected group; I = infected group; PBS = phosphate buffered saline; SE = Salmonella Enteritidis.

1

Basal, negative control diet, TMP/SDZ (Trimethoprim-sulfadiazine) antibiotic diet, 0.3% ROD, diet containing 0.3% red osier dogwood extract, 0.5% ROD, diet containing 0.5% red osier dogwood extract.

Table 2.

Gross and nutrient compositions of experimental diets (as-fed basis, %, unless otherwise stated).1

Ingredients Starter phase (1–14 d)
 Grower phase (14–21 d)
Basal TMP/SDZ 0.3% ROD 0.5% ROD Basal TMP/SDZ 0.3% ROD 0.5% ROD
Corn 42.37 42.27 41.83 41.48 45.99 45.65 45.22 44.86
Soybean meal 40.13 40.15 40.17 40.2 36.15 36.21 36.24 36.26
Wheat 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00
Vegetable oil 2.82 2.85 3.01 3.14 3.74 3.85 4.01 4.14
Dicalcium phosphate 1.57 1.57 1.57 1.57 1.39 1.39 1.39 1.39
Limestone 1.45 1.45 1.45 1.45 1.32 1.32 1.32 1.32
DL methionine premix2 0.61 0.61 0.61 0.61 0.53 0.53 0.53 0.53
Starter vitamin/mineral premix3 0.50 0.50 0.50 0.50 - - - -
Grower/finisher vitamin/mineral premix 4 - - - - 0.50 0.50 0.50 0.50
Salt 0.40 0.40 0.40 0.40 0.38 0.38 0.38 0.38
Red dogwood extract - - 0.30 0.50 - - 0.30 0.50
TMP/SDZ 110 G5 - 0.05 - - - 0.05 - -
Lysine HCL 0.16 0.16 0.16 0.16 - 0.12 0.12 0.12
Calculated analysis
Crude protein 23 23 23 23 21.5 21.5 21.5 21.5
Metabolizable Energy (kcal/kg) 3,000 3,000 3,000 3,000 3,100 3,100 3,100 3,100
Calcium 0.96 0.96 0.96 0.96 0.87 0.87 0.87 0.87
Available phosphorus 0.48 0.48 0.48 0.48 0.44 0.44 0.44 0.44
Digestible lysine 1.28 1.28 1.28 1.28 1.15 1.15 1.15 1.15
Digestible methionine + cystine 0.95 0.95 0.95 0.95 0.87 0.87 0.87 0.87
Sodium 0.19 0.19 0.19 0.19 0.18 0.18 0.18 0.18
Analyzed composition
Crude protein 24.1 22.1
Calcium 0.81 0.75
Total phosphorus 0.68 0.62
Sodium 0.15 0.12
Crude fat 3.22 4.40
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1

Basal, negative control diet, TMP/SDZ (Trimethoprim-sulfadiazine) antibiotic diet, 0.3% ROD, diet containing 0.3% red osier dogwood extract, 0.5% ROD, diet containing 0.5% red osier dogwood extract.

2

Supplied/kg premix: DL-Methionine, 0.5 kg; wheat middlings, 0.5 kg.

3

Starter vitamin-mineral premix contained the following per kg of diet: 9,750 IU vitamin A; 2,000 IU vitamin D3; 25 IU vitamin E; 2.97 mg vitamin K; 7.6 mg riboflavin; 13.5 mg Dl Ca-pantothenate; 0.012 mg vitamin B12; 29.7 mg niacin; 1.0 mg folic acid, 801 mg choline; 0.3 mg biotin; 4.9 mg pyridoxine; 2.9 mg thiamine; 70.2 mg manganese; 80.0 mg zinc; 25 mg copper; 0.15 mg selenium; 50 mg ethoxyquin; 1,543 mg wheat middlings; 500 mg ground limestone.

4

Grower and finisher vitamin-mineral premix contained the following per kg of diet: 9,750 IU vitamin A; 2,000 IU vitamin D3; 25 IU vitamin E; 2.97 mg vitamin K; 7.6 mg riboflavin; 13.5 mg Dl Ca-pantothenate; 0.012 mg vitamin B12; 29.7 mg niacin; 1.0 mg folic acid, 801 mg choline; 0.3 mg biotin; 4.9 mg pyridoxine; 2.9 mg thiamine; 70.2 mg manganese; 80.0 mg zinc; 25 mg copper; 0.15 mg selenium; 50 mg ethoxyquin; 1,543 mg wheat middlings; 500 mg ground limestone.

5

Bacitracin methylene disalicylate (providing 55 mg/kg mixed feed); Alpharma, Inc., Fort Lee, NJ.

Fecal Excretion of Salmonella Enteritidis

To confirm for the absence of SE in purchased day-old chicks 4 papers containing fecal samples collected from chick delivery boxes bottom were put into 4 separate whirl-paks to which was added a LB broth (Fisher BP1426-500) at a 1:10 ratio depending on the weight of paper + fecal samples. LB-enriched fecal samples were incubated overnight at 37°C. Hundred microliters of LB-enriched fecal samples were plated onto xylose-lysine-tergitol 4 (XLT4) agar (Item No:223420, Bacto BBL Difco Microbiology, Franklin Lakes, New Jersey) then incubated for 24 h at 37°C. To assess excreted Salmonella, cloacal swabs were collected individually on all birds (n = 404) per cage at 1-, 5-, 12-, and 18-day postinfection (DPI) and diluted in 6 ml of buffered peptone water before streaking onto XLT4 agar.

Growth Performance

Individual body weight and feed intake (on a pen basis) were determined weekly. Feed intake and body weight were used to estimate average feed intake (AFI) and average weight gain (AWG). Mortality was recorded daily to correct for AFI and feed conversion ratio (FCR). Birds that died were necropsied by a poultry pathologist to determine cause of death.

Hematology, Serum Immunoglobulins, and Superoxide Dismutase

On d 21, 80 birds (10 birds from each treatment) were randomly selected for blood sampling and individually weighed. Approximately 5 mL of blood was collected from the brachial vein and divided into i) EDTA tubes for hematology and ii) serum tubes for serum immunoglobulins and superoxide dismutase analyses. The hematological parameters including hematocrit (HCT), total protein (TP), leukocytes (LEU), heterophils (HET), lymphocytes (LYM), HET:LYM (H:L), monocytes (MON), eosinophils (EP), and basophils (BP) were assayed at the Centre de diagnostic veterinaire de l'Université de Montréal. Samples for serum immunoglobulins G (IgG) and immunoglobulin M (IgM) were assayed using an enzyme-link immunosorbent assay (ELISA) kit from Bethyl Laboratories Inc. (catalog number E33-104-200218 and E33-102-180410, respectively) following manufacturer protocols. Superoxide dismutase (SOD) was analyzed using a SOD assay kit (Item Number 706002; Cayman Chemical, Ann Arbor, MI), following the manufacturer's protocol.

Gut Histomorphometry

A 1.5 cm segment at the mid-length of the duodenum, jejunum, and ileum was collected and preserved in 10% buffered formalin (Sigma-Aldrich, St. Louis, MO) for 3 d. The formalin-preserved intestinal segments were cross-sectioned and placed in cassettes and were then immersed in 4% paraformaldehyde tissue fixation solution and later into paraffin. Each of the cross-sectioned segments was mounted on a glass slide (n = 10 per treatment) and stained with HPS (COLHPS1). The morphological slides were examined under a microscope coupled with a digital camera. Ten well-oriented and distinct villi on each slide were identified and measured for villus height (VH), villus width (VW), and crypt depth (CD) as described by Shang et al. (2020). The villus height:crypt depth ratio (VH:CD) was subsequently estimated.

Statistical Analysis

Datasets were subjected to 4 × 2 factorial analysis of variance (ANOVA) using the General Linear Model of Minitab software. Error terms of individual response variables were confirmed for the validity of 3 basic assumptions, including normality, constant variance, and independence. A normal probability plot of residuals was done to verify the normality of error terms using the Anderson Darling test in the same statistical package. Where error terms of datasets were found to be non-normal or nonconstant, the respective original datasets were subjected to various transformation functions. If the normality and homoscedasticity of error terms were still violated upon transformations, then such datasets were analyzed using the Kruskal-Wallis test. Following ANOVA, differences between significant means were separated using Tukey's honest significant difference (HSD) test and Mann Whitney for the parametric and nonparametric datasets, respectively, in the same statistical package. Analyzed datasets were presented as means, standard error of the mean (SEM), and probability values. Statistically different values were considered at P < 0.05.

RESULTS

Fecal Excretion of Salmonella Enteritidis

The effect of ROD extract on the fecal excretion of SE is shown in Supplementary Table 1. There was no presence of SE in all the cloacal swab samples collected on d 0, 1, 5, 12, and 18 DPI.

Growth Performance

The effect of ROD extract as an antibiotic alternative on the growth performance of broiler chickens infected or uninfected with SE is presented in Table 3. There were no interactions; thus, the results are reported based on dietary treatments and the challenge model's main effects. Throughout the experimental period, dietary supplementation of 0.3 and 0.5% ROD extract did not affect the growth performance of birds with or without SE infection. AFI and AWG at wk 1, and AFI and FCR at wk 2, 3 and on overall were higher (P < 0.05) among the infected birds compared to the noninfected group of birds.

Table 3.

Effect of red osier dogwood extract on growth performance of broiler chickens challenged orally with Salmonella Enteritidis.

Treatment1
 Infection model2
 P value
Weeks Parameters Infection model2 Basal TMP/SDZ 0.3% ROD 0.5% ROD SE mean N I SE mean Treatment effect Model effect Interaction effect
Wk 1 Average feed intake (g/bird) N 121.9 122.4 122.5 117.2 1.77 119.9b 125.0a 1.20 0.305 0.025 0.305
I 127.0 126.9 124.2 122.0 1.53
Average weight gain (g/bird) N 96.81 97.54 101.6 90.16 2.68 96.53b 104.7a 1.96 0.238 0.035 0.238
I 111.6 109.7 98.89 98.50 2.70
FCR N 1.26 1.25 1.20 1.28 0.02 1.24 1.19 0.02 0.487 0.103 0.487
I 1.14 1.16 1.28 1.25 0.02
Mortality N 0.00 0.00 0.00 0.00 0.03 0.00 0.00 0.03 0.793 0.557 -
I 0.00 0.00 0.00 0.00 0.04
Wk 2 Average feed intake (g/bird) N 298.7 304.6 304.9 298.7 4.29 301.7b 315.0a 3.13 0.252 0.032 0.252
I 322.0 328.6 309.0 300.3 4.32
Average weight gain (g/bird) N 243.4 240.3 248.3 229.0 6.09 240.3 240.6 4.13 0.167 0.963 0.167
I 254.2 259.4 222.2 226.7 5.67
FCR N 1.23 1.27 1.23 1.33 0.03 1.25b 1.30a 0.02 0.393 0.031 0.393
I 1.25 1.28 1.34 1.27 0.02
Mortality N 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.107 0.154 -
I 0.00 0.00 0.00 0.00 0.00
Wk 3 Average feed intake (g/bird) N 539.4 533.4 538.0 508.9 10.5 529.9b 666.5a 11.0 0.592 <0.001 0.592
I 640.7 669.3 687.6 668.5 9.07
Average weight gain (g/bird) N 397.1 377.1 405.1 395.3 9.02 393.6 373.7 5.90 0.979 0.102 0.979
I 370.2 384.8 370.6 369.2 7.32
FCR N 1.37 1.43 1.33 1.29 0.04 1.34b 1.74a 0.04 0.916 <0.05 0.916
I 1.77 1.75 1.88 1.84 0.02
Mortality N 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.107 0.154
I 0.00 0.00 0.00 0.00 0.00
Overall Average feed intake (g/bird) N 959.0 959.7 964.9 922.9 12.9 951.6b 1106a 13.6 0.513 <0.05 0.513
I 1090 1125 1121 1091 13.9
Average weight gain (g/bird) N 737.3 714.9 755.1 714.5 15.7 730.4 719.0 10.3 0.690 0.588 0.690
I 736.0 754.0 691.8 694.4 13.6
FCR N 1.31 1.35 1.28 1.29 0.03 1.29b 1.50a 0.02 0.899 <0.05 0.899
I 1.50 1.50 1.64 1.59 0.01
Mortality N 0.00 0.00 0.00 0.00 0.05 0.00 0.00 0.03 0.255 0.644
I 0.00 0.00 0.00 0.00 0.04
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Basal, negative control diet, TMP/SDZ (Trimethoprim-sulfadiazine) antibiotic diet, 0.3% ROD, diet containing 0.3% red osier dogwood extract, 0.5% ROD, diet containing 0.5% red osier dogwood extract.

2

N = noninfected group; I = infected group.

In a row, means assigned different lowercase letters (a, b) are significantly different, P < 0.05 (Tukey's procedure).

Gut Morphology

The effect of ROD extract on gut histomorphometry of broiler chickens challenged orally with SE is presented in Table 4. In the duodenum, the VH of birds were significantly increased (P < 0.05) with 0.3% extract compared to the other treatments. Compared to birds in the TMP/SDZ antibiotic treatment, CD of 0.5% ROD extract treated birds was significantly depressed (P < 0.05). Comparing between infection model, duodenal VH and CD were significantly higher (P < 0.05) among the infected group of birds. Regardless of the challenge, duodenal VH:CD was significantly increased (P < 0.05) among fed 0.3 and 0.5% ROD extract compared to other treatments. There was a significant interaction effect (P < 0.05) on VW. In the jejunum, VH and VW were significantly influenced (P < 0.05) by the interaction between dietary treatment and infection model. In the ileum, VH was not influenced by the dietary treatments. In the ileum, there were significant interactive effects between infection model and diet on VW and CD. While VW was significantly increased by 0.5% ROD extract among the noninfected group, it was significantly reduced by 0.5% ROD extract among the infected group, compared to the control and antibiotic treatments. However, 0.3% ROD extract significantly increased VW among the infected group, compared to the control. In addition, while there were no effects of treatment on CD among the noninfected group, 0.3 and 0.5% ROD extracts significantly increased CD (P < 0.005) among the infected group, compared the TMP/SDZ antibiotic group.

Table 4.

Effect of red osier dogwood extract on gut morphology of broiler chickens challenged orally with Salmonella Enteritidis.

Treatment1
 Infection model2
 P value
Parameters Infection model2 Basal TMP/SDZ 0.3% ROD 0.5% ROD SE mean N I SE mean Treatment effect Mode effect Interaction effect
Duodenum
Villus height (mm) N 1.58 1.47 1.69 1.79 0.036 1.63b 1.79a 0.034 0.002 0.001 -
I 1.67 1.77 2.08 1.78 0.030
Average 1.52b 1.52b 1.85a 1.67b
Villus width (mm) N 0.15b 0.20a 0.14b 0.15b 0.007 0.16 0.17 0.005 0.067 0.204 0.003
I 0.16bc 0.17b 0.16bc 0.18ab 0.004
Crypt depth (mm) N 0.19 0.21 0.18 0.16 0.005 0.19b 0.20a 0.005 <0.005 0.036 0.345
I 0.21 0.21 0.21 0.18 0.010
Average 0.20ab 0.21a 0.19ab 0.17b
VH:CD3 N 6.97 7.26 10.2 9.92 0.398 8.37 8.76 0.346 <0.005 0.346 0.690
I 8.13 7.48 9.89 9.95 0.290
Average 7.55b 7.37b 9.95a 9.93a
Jejunum
Villus height (mm) N 1.08 0.82 0.76 0.85 0.058 0.90 0.89 0.046 0.728 0.703 0.019
I 0.86 0.96 0.92 0.82 0.034
Villus width (mm) N 0.22 0.18 0.18 0.21 0.011 0.20 0.20 0.008 0.844 0.998 0.001
I 0.19 0.22 0.22 0.19 0.005
Crypt depth (mm) N 0.15 0.14 0.13 0.14 0.004 0.13 0.13 0.004 0.955 0.368 0.282
I 0.12 0.14 0.14 0.13 0.004
VH:CD3 N 7.49 6.16 6.62 8.30 0.340 7.12 6.95 0.284 0.486 0.661 0.089
I 7.00 7.13 6.92 6.47 0.228
Ileum
Villus height (mm) N 0.39 0.34 0.38 0.38 0.010 0.37 0.38 0.010 0.165 0.399 0.786
I 0.38 0.36 0.39 0.41 0.011
Villus width (mm) N 0.15b 0.15b 0.17ab 0.18a 0.004 0.16 0.16 0.005 0.042 0.742 0.018
I 0.15c 0.17ab 0.18a 0.14cd 0.006
Crypt depth (mm) N 0.10a 0.09b 0.09b 0.10a 0.003 0.09 0.10 0.003 <0.005 0.273 0.047
I 0.09bc 0.08c 0.11ab 0.12a 0.004
VH:CD3 N 4.19 3.98 5.03 3.82 0.144 4.10 4.30 0.139 0.245 0.218 0.098
I 4.31 4.35 4.22 4.32 0.133
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1

Basal, negative control diet, TMP/SDZ (Trimethoprim-sulfamethoxazole) antibiotic diet, 0.3% ROD, diet containing 0.3% red osier dogwood extract, 0.5% ROD, diet containing 0.5% red osier dogwood extract.

2

N = noninfected group; I = infected group.

3

VH:CD = villus height: crypt depth ratio.

In a row, means assigned different lowercase letters (a–d) are significantly different, P < 0.05 (Tukey's procedure).

Hematology Parameters

The effect of ROD extract on hematology, serum immunoglobulins and superoxide dismutase of broiler chickens infected with SE is shown in Table 5. The dietary treatments did not significantly affect (P > 0.05) HCT, TP, HET, LYM, H:L, EP, and BP of birds with or without SE challenge. Regardless of infection model, 0.3% ROD extract increased the percentage of monocytes (P = 0.003) but it reduced the percentage of eosinophils (P = 0.047), compared to the control and antibiotic groups. Comparing the infection model, HET, H:L, and MON were significantly higher (P < 0.05) among the infected birds, while LYM was higher (P < 0.05) among the noninfected birds compared to their counterpart.

Table 5.

Effect of red osier dogwood extract on differential white blood cell count of broiler chickens challenged orally with Salmonella Enteritidis.

Treatment1
 Infection model2
 P value
Parameters Infection model2 Basal TMP/SDZ 0.3% ROD 0.5% ROD SE mean N I SE mean Treatment effect Model effect Interaction effect
Hematocrit (L/L) N 0.26 0.27 0.26 0.25 0.004 0.26 0.26 0.003 0.228 0.896 0.440
I 0.25 0.27 0.25 0.27 0.005
Total protein (g/L) N 40.0 40.0 37.0 39.6 0.614 39.15 39.65 0.455 0.200 0.604 0.536
I 40.3 41.4 39.0 37.9 0.688
Leukocytes (× s 109 /L) N 14.6 14.3 14.8 13.0 0.832 14.44 15.54 0.775 0.236 0.115 -
I 15.3 15.0 20.4 14.9 1.350
Heterophils (%) N 35.1 40.9 34.5 34.4 2.060 35.1b 44.0a 1.560 0.929 0.008 0.866
I 41.0 43.2 47.8 43.9 2.060
Lymphocytes (%) N 55.4 48.3 56.8 50.6 2.200 52.8a 43.6b 1.860 0.748 0.022 0.600
I 43.6 40.8 36.7 44.3 2.890
H:L3 N 0.66 0.78 0.61 0.52 0.090 0.64b 1.12a 0.063 0.599 <0.001 0.700
I 1.01 1.12 1.33 1.01 0.061
Monocytes (%) N 2.57 2.00 6.25 3.25 0.581 3.52b 5.68a 0.434 0.003 0.007 0.596
I 4.71 5.40 8.80 3.80 0.547
Average 3.64b 3.70b 7.53a 3.53b
Eosinophils (%) N 3.57 2.89 1.00 1.80 0.450 2.00 2.00 0.291 0.047 0.623 -
I 2.00 4.00 1.00 2.00 0.376
Average 2.00b 3.50a 1.00c 2.00b
Basophils (%) N 1.00 2.00 2.00 2.00 0.671 2.00 2.00 0.613 0.950 0.941 -
I 3.00 5.00 3.00 2.00 1.060
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1

Basal, negative control diet, TMP/SDZ (Trimethoprim-sulfamethoxazole) antibiotic diet, 0.3% ROD, diet containing 0.3% red osier dogwood extract, 0.5% ROD, diet containing 0.5% red osier dogwood extract.

2

N = noninfected group; I = infected group.

3

H:L = heterophils:lymphocytes ratio.

In a row, means assigned different lowercase letters (a–c) are significantly different, P < 0.05 (Tukey's procedure).

Serum Immunoglobulins and Superoxide Dismutase

The effect of ROD extract on serum immunoglobulins and superoxide dismutase of broiler chickens infected with SE is shown in Table 6. No interactions were observed. However, both the serum IgG and SOD were not affected by the dietary treatments and the challenge model. Regardless of infection model, both antibiotic and 0.5% ROD extract reduced the concentration of IgM in the serum, compared to the control and 0.3% ROD (P = 0.006). Serum IgM was higher (P = 0.024) in the noninfected group than the infected group.

Table 6.

Effect of red osier dogwood extract on serum immunoglobulins and superoxide dismutase of broiler chickens challenged orally with Salmonella Enteritidis.

Treatment1
 Infection model2
 P value
Parameters Infection model2 Basal TMP/SDZ 0.3% ROD 0.5% ROD SE mean N I SE mean Treatment effect Model effect Interaction effect
IgG (mg/mL)3 N 4.12 3.60 3.66 4.32 0.416 3.62 3.61 0.240 0.928 0.972 0.842
I 3.66 3.55 3.75 3.48 0.243
IgM (mg/mL)4 N 1.84 1.27 1.52 1.47 0.074 1.51a 1.36b 0.061 0.006 0.024 0.570
I 1.53 1.10 1.59 1.15 0.094
Average 1.69a 1.19cd 1.56ab 1.31c 0.08
SOD (U/mL)5 N 1.71 1.91 1.96 1.73 0.103 3.48 3.94 0.071 0.576 0.300 0.783
I 1.77 1.75 1.96 2.04 0.097
Open in a new tab
1

Basal, negative control diet, TMP/SDZ (Trimethoprim-sulfamethoxazole) antibiotic diet, 0.3% ROD, diet containing 0.3% red osier dogwood extract, 0.5% ROD, diet containing 0.5% red osier dogwood extract.

2

N = noninfected group; I = infected group.

3

IgG = immunoglobulin G.

4

IgM = immunoglobulin M.

5

SOD = superoxide dismutase.

In a row, means assigned different lowercase letters (a–d) are significantly different, P < 0.05 (Tukey's procedure).

DISCUSSION

Chicks are the most susceptible to Salmonella infection. This is partly attributable to their new but developing immune system. In the current study, oral inoculation of day-old broiler chicks with 0.5 mL/bird of 3.1 × 105 CFU/mL of SE did not result in the presence of SE in their cloacal fecal samples at 1 DPI. However, this does not suggest the systemic absence of SE among the SE-infected birds. According to the report of Van Immerseel et al. (2004), it is possible that birds could be carriers of Salmonella regardless of its failed detection in their fecal sample. In the research demonstration by Borsoi et al. (2011), SE was detected in the fecal samples of broiler chickens following the oral gavage of 1 × 105 CFU/mL of SE. This conflicts with the current research finding. Such conflict could be due to the variation in the SE inoculum strain and volume, which was not reported by Borsoi et al. (2011). Furthermore, in some SE-challenge studies using a lower concentration of Salmonella inoculum (usually <105 CFU/mL), the presence of Salmonella infection was confirmed using a nonfecal assessment method, particularly with the use cecal tonsil (Higgins et al., 2008, 2010; Prado-Rebolledo et al., 2017). This suggests that the challenged birds in the current study could be SE carriers even though SE was not detected by cloacal fecal samples. On 5, 12, and 18 DPI, no dietary treatment effect was observed on the SE shedding in the cloacal fecal samples of birds. This is not surprising as SE presence was not confirmed 24 h after the SE challenge.

No interaction effects were observed on all the evaluated growth performance parameters. As a result, the results discussed are based on the main effects of dietary treatments and the challenge model. Antibiotics have a proven capacity to reliably improve feed conversion efficiency and, consequently, accelerate the growth performance of birds (Mehdi et al., 2018; Ali et al., 2019; Saleh et al., 2020; Shang et al., 2020). According to Gaucher et al. (2015), absence of antibiotics use in poultry production has been associated with consequential production losses. It would therefore be interesting to verify if ROD extract could match up the positive impact of antibiotics on the growth performance indices of broiler chickens. In the present study, neither the supplementation of ROD extract (0.3 and 0.5%) nor TMP/SDZ have influenced AFI, AWG, FCR, and mortality of birds with or without SE infection. This corroborates the findings of Mogire et al. (2021), who reported an insignificant impact of 0.1 and 0.3% ROD extract on the growth performance of birds, although the study lacked a disease-challenged model. In contrast to the present findings, Erinle et al. (2022) demonstrated that supplementation of 0.3 and 0.5% ROD extract significantly sustained average feed intake, feed efficiency, and mortality of birds challenged with SE lipopolysaccharides compared to the birds fed in-feed bacitracin. Unlike the impact of antibiotic-free diet on poultry production as reported by Gaucher et al. (2015), our present results suggest that dietary supplementation of ROD extract at either 0.3 or 0.5% does not have negative impact on the growth response of broiler chickens and as a result, will not cause consequential production losses. Comparing the infection model, AFI and AWG at wk 1 were significantly higher among the infected birds, whereas AFI and FCR at wk 2 and 3 and overall were significantly higher among the noninfected group of birds. Salmonella infection has a dynamic impact on birds’ growth performance. The severity of the infection largely depends on the age and strain of birds and the concentration of Salmonella inoculum. In some studies where Salmonella infection was established in poultry birds older than 21 d, poorer growth performance indices of birds were reported (Marcq et al., 2011; El-Shall et al., 2020). Meanwhile, at d 9 to 11 posthatch, Zhen et al. (2018) reported that oral challenge of birds with 1 mL of 1 × 109 CFU of SE did not significantly affect feed intake, body weight gain, FCR of chicks at different periods. In theory, given its virulence, Salmonella is expected to have a drastic draconian impact on the growth of poultry birds. However, in the current study, the unexpected significant discrepancies which partly favored AFI in SE-challenged birds but with a depressed FCR could be due to the elongated time elapse during the weekly weighing of left-over feed and bodyweight of birds in the pen containing the noninfected birds before moving to the infected birds. Mortality was not affected by the dietary treatments and infection model. Ribeiro et al. (2007) reported a no significant difference in the livability of birds challenged with SE.

The gut is involved in the digestion and absorption of nutrients and serves as a selective barrier which permits the transportation of nutrients while blocking intestinal pathogens and their metabolic products. As a result, the gut structure, including the height and width of villi, and crypt depth, has been considered one of the critical indicators of a healthy gut (Laudadio et al., 2012). In the presence or absence of disease or immune stressors, it is pertinent that the gut architectural integrity must be maintained. In the duodenum, there was a significant increase in VH of noninfected birds fed either 0.3 or 0.5% ROD extract diets, while the VH of infected birds were significantly increased among those fed only 0.3% ROD extract. Both ROD levels were also observed to substantially increase duodenal VH:CD compared to control and antibiotic treatments. This is deviance from Mogire et al. (2021) report, where 0.1 and 0.3% ROD extract did not influence the duodenal and ileal histomorphometry. In addition, the present study was also different from our previous findings (Erinle et al., 2022) in which 0.3 and 0.5% ROD extract did not affect duodenal and jejunal morphology, which could be due to the different challenge models. Laudadio et al. (2012) reported that intestinal morphology from healthy and well performing birds is characterized by higher VH and VW, suggesting a large nutrient absorption surface area. Thus, the 2 ROD levels improved duodenal morphology. The duodenum interacts with accessory organs, including the pancreas and liver, which are critical in the digestion process of feed material. With the exception to VW, dietary TMP/SDZ antibiotic decreases duodenal morphology. Still, in the duodenum, the SE infection model presents a significantly higher VH and deeper CD among the infected birds than the noninfected birds. The higher VH is quite surprising and could reflect the increase in AFI throughout the experimental period. In the jejunum, there was a significant interaction effect between the dietary treatment and infection model on VH and VW. In the ileum, there was a significant interaction effect on CD; however, 0.3 and 0.5% ROD extract significantly deepened CD of infected birds compared to the antibiotic and control-fed infected birds. This was similar to previous findings where 0.3 and 0.5% ROD extract was reported to increase ileal CD of birds challenged with SE lipopolysaccharides (Erinle et al., 2022). Salmonella Typhimurium was reported to increase the incidence of intestinal epithelial exfoliation and, consequently, increase the goblet cell density (Fasina et al., 2010). According to Conrad and Stocker (2013), the number of goblet cells is highest in the crypts. Thus, a higher CD among the infected birds could be due to the detrimental effect of SE on gut morphology. Besides the possible detrimental effects of SE in the gut, we suspect that the increased CD could also be due to the gut architectural modulation by ROD extract in response to disease or immune stressors. However, there was a significant interaction effect on the ileal VW. An increase in VW has also been associated with increased villus surface area for nutrient absorption.

The reliability of blood as a predictable barometer of the physiological and health status of animals cannot be contested. White blood cells (WBC) component of the blood serves as the military defense system of the body against infections. WBC components, including HET, LYM, and H:L are reliable signals that indicate the severity of stressors (Zulkifli et al., 2000; Ghareeb et al., 2008). For example, an increased HET, decreased LYM, and consequently increased H:L have been reported in Salmonella infection (Jazi et al., 2019). Heterophils are the preponderant granulated leukocytes which increase birds’ response to acute inflammation caused by pathogens. Therefore, in the presence of SE, the body defense system deploys HET as the first counter defensive mechanism. Such heterophil response increases HET counts and is considered a worthy indicator of infection severity. In addition to HET, LYM, on the other hand, constitute the B immune cells which are responsible for synthesis of antibodies (Blumenreich, 1990). A reduction in the LYM components of the WBC is an indication of immunosuppression in poultry birds. Thiam et al. (2021) correlated a low H:L with an improved gut barrier and immune response and could be used as a biomarker for Salmonella resistance in chickens. As expected, the SE infection in the current study significantly increased HET, MON, and H:L and decreased LYM of SE-challenged birds. Although ROD extract did not affect the differential WBC in the present study, 0.3% ROD extract inclusion level stimulated the production of LEU and MON in SE-infected birds. Unfortunately, a similar feat was not achieved at 0.5% ROD extract inclusion level as it lowered LEU and MON but was comparable to antibiotic and control treatments. In humans, antibiotics like beta-lactam and vancomycin have been reported to decrease WBC and MON count (Shuman et al., 2012). A lower count of LEU is a common indicator of immunosuppression and consequently increases susceptibility to infections. As a part of the crucial component of the bodily immune system, MON travels around the body to scavenge pathogenic microbes and dead and damaged cells and contribute to immune responses during infection (Yáñez et al., 2017). This suggests that 0.3% ROD extract possesses the capacity to stimulate the inflammatory response by promoting the production of LEU and MON. Compared to the 0.3% ROD extract, 0.5% extract had a depressive effect on the concentration of monocytes. The reason for this is not clear from the current study but it demands further investigation.

Serum immunoglobulins, including IgM and IgG, are products of the WBC sought as indicators of humoral immunity. The IgM antibodies have been reported to exhibit a greater affinity for the antigen and, as a result, are potent pathogen neutralizers better than the IgG's (Keyt et al., 2020). In the present study, IgM was significantly highest among SE-infected birds fed 0.3% ROD extract and lowest among those fed TMP/SDZ treatment. This is different from our previous reports where both 0.3 and 0.5% ROD extract did not influence the IgG and IgM of birds challenged with SE-LPS (Erinle et al., 2022). This could be due to variation in the challenge models. According to Heyman and Shulman (2016), more than 80% of human patients with recurrent infections have a deficiency in IgM. In MOPC 104E plasma cells, antibiotics was reported to inhibit the secretion of serum immunoglobulins, notably IgM, by inhibiting the glycosylation of carbohydrate component of immunoglobulins (Hickman and Kornfeld, 1978). The desirable impacts 0.3% ROD extract on the WBC and IgM over the TMP/SDZ antibiotics suggest that it promotes antibody production and the amount of circulating leukocytes in the blood. Comparing the infection model groups, SE infection had significant effects on IgM and was significantly lower among the infected group of birds. The serum IgM is reported to be the first antibody produced during the early period postinfection (Larsson et al., 1993; Rathnapraba et al., 2007) and declines gradually with age (Khare et al., 1976; Holodick et al., 2016). Regardless of the SE infection, the range of IgM concentrations obtained in the current study was 1.1 to 1.84 mg/mL, however, was within the range of mean concentrations 0.71 to 2.55 mg/mL reported by Lebacq-Verheyden et al. (1974), Chhabra and Goel (1980), and Davis (1985).

CONCLUSIONS

SE-infected birds had higher VH and deeper CD. Regardless of the presence of SE infection, both 0.3 and 0.5% ROD extract improved duodenal VH:CD and marginally lowered CD compared to the antibiotic-fed broiler chickens. Meanwhile, the dietary treatment and infection model had an interaction effect on the ileal VW and CD. Furthermore, regardless of SE infection, MON and IgM were increased among chickens fed 0.3% ROD extract, compared to those fed antibiotic. However, SE infection adversely affected the hematology of infected birds compared to the noninfected birds. Given the sustained growth performance, improved duodenal histomorphology, monocyte percentage, and IgM concentration, both 0.3 and 0.5% ROD extract could be a suitable antibiotic replacement; however, 0.3% ROD extract had the best experimental outcome over the antibiotics.

ACKNOWLEDGMENTS

The authors thank Red Dogwood Enterprise Ltd. (Swan River, MB) for providing the red dogwood extract, Janice MacIsaac and Janie Fraser for diet formulation manufacturing, and transportation to Montreal. Appreciations go to Lila Maduro for the Salmonella Enteritidis inoculum preparation and some other microbiological assays, and the entire research team member of Dr. Martine Boulianne, including Karine Lamarre, Maude Lanoie, Laura Alejandra Guerrero, Anne-Marie Cardinal, Nicholas Deslauriers for all their assistance. The authors thank Shima Borzouie for helping with the final revision of the manuscript. Funding for this project was provided by the Canadian Poultry Research Council (38335), Mitacs (38335), and Natural Sciences and Engineering Council of Canada (NSERC) discovery grant (34288).

DISCLOSURES

The authors declare no conflicts of interest(s).

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2023.102723.

Appendix. Supplementary materials

mmc1.docx (16.2KB, docx)

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