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. 2024 Jul 10;103(10):104078. doi: 10.1016/j.psj.2024.104078

Oligodeoxynucleotides containing CpG motifs upregulate bactericidal activities of heterophils and enhance immunoprotection of neonatal broiler chickens against Salmonella Typhimurium septicemia

Iresha Subhasinghe *, Ayumi Matsuyama-Kato *, Khawaja Ashfaque Ahmed *, Lisanework E Ayalew †, Hemlata Gautam *, Shelly Popowich *, Betty Chow-Lockerbie *, Suresh K Tikoo ‡, Philip Griebel ‡,§, Susantha Gomis *,1
PMCID: PMC11345621  PMID: 39096829

Abstract

In the past, we demonstrated that oligodeoxynucleotides containing CpG motifs (CpG-ODN) mimicking bacterial DNA, stimulate the innate immune system of neonatal broiler chickens and protect them against Escherichia coli and Salmonella Typhimurium (S. Typhimurium) septicemia. The first line of innate immune defense mechanism is formed by heterophils and plays a critical protective role against bacterial septicemia in avian species. Therefore, the objectives of this study were 1) to explore the kinetics of CpG-ODN mediated antibacterial mechanisms of heterophils following single or twice administration of CpG-ODN in neonatal broiler chickens and 2) to investigate the kinetics of the immunoprotective efficacy of single versus twice administration of CpG-ODN against S. Typhimurium septicemia. In this study, we successfully developed and optimized flow cytometry-based assays to measure phagocytosis, oxidative burst, and degranulation activity of heterophils. Birds that received CpG-ODN had significantly increased (p < 0.05) phagocytosis, oxidative burst, and degranulation activity of heterophils as early as 24 h following CpG-ODN administration. Twice administration of CpG-ODN significantly increased the phagocytosis activity of heterophils. In addition, our newly developed CD107a based flow cytometry assay demonstrated a significantly higher degranulation activity of heterophils following twice than single administration of CpG-ODN. However, the oxidative burst activity of heterophils was not significantly different between birds that received CpG-ODN only once or twice. Furthermore, delivery of CpG-ODN twice increased immunoprotection against S. Typhimurium septicemia compared to once but the difference was not statistically significant. In conclusion, we demonstrated enhanced bactericidal activity of heterophils after administration of CpG-ODN to neonatal broiler chickens. Further investigations will be required to identify other activated innate immune cells and the specific molecular pathways associated with the CpG-ODN mediated activation of heterophils.

Key words: neonatal broiler chicken, CpG-ODN, antibacterial immunity, innate immunity, Salmonella Typhimurium

INTRODUCTION

Antimicrobials were used in the poultry industry frequently in the past to maintain animal health and welfare, however there are growing concerns about the emergence of antimicrobial resistant (AMR) pathogens. These bacteria could eventually cause adverse effects on both animal and human health and the environment (Hedman et al., 2020). Studies have shown that animal food sources (beef, pork, chicken, turkey meat, eggs, and raw milk) have mainly contributed to the development of antibiotic-resistant bacterial strains such as methicillin (oxacillin)-resistant Staphylococcus aureus (Lee, 2003; Spinelli et al., 2019). As a result, many countries have banned, restricted, or reduced antibiotic use for disease prevention and growth promotion in the food animal industry. One of the major problems in the broiler chicken industry is economic losses associated with bacterial infections such as Escherichia coli (E.coli) and Salmonella Typhimurium (S. Typhimurium) species associated with yolk sac infections in the neonatal period and subsequent chronic infections in the grow out period of broiler chickens (Swelum et al., 2021). Therefore, alternatives to antibiotics are a priority in broiler chicken production (Robinson et al., 2016; Aslam et al., 2021).

Toll-like receptors (TLRs) are capable of recognizing a varied range of pathogen-associated molecular patterns (PAMPs), including lipopeptides, glycerophosphatidylinositol, lipopolysaccharides (LPS), microbial nucleic acids (such as dsRNA, ssRNA, and unmethylated cytosine-phosphodiester-guanine (CpG) dinucleotides), and microbial proteins like flagellin and profilin. Upon binding to these PAMPs, TLRs initiate signaling pathways that cause the production of pro-inflammatory cytokines and other molecules necessary for mounting an effective immune response against the invading pathogens (Kawai and Akira, 2009). Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) trigger innate immune responses in vertebrate hosts (Klinman et al., 2004; Patel et al., 2008). The immunoprotective ability of CpG-ODN against pathogens has been shown in mice, humans, sheep, cattle, chickens, and fish (Harandi et al., 2003; Jørgensen et al., 2003; Nichani et al., 2004; Shirota et al., 2015; Lin et al., 2018). In chicken, it binds to TLR-21 which is a functional homolog to TLR-9 in mammalian species (Liu et al., 1998; Cross et al., 2015; Gursel and Gursel, 2016).

Polymorphonuclear leukocytes (PMNs), particularly heterophils in avian species, play an imperative role in the innate immune response. The ability of these cells to recognize, phagocytose, and subsequently kill pathogenic microbes is essential for protecting the bird from various infections. The unique characteristics and functions of heterophils make them a vital component of the innate immune system of chickens as they actively contribute to defense mechanisms against invading pathogens (Maxwell and Robertson, 1998; Kogut, 2022). As the first line of defense, heterophils are crucially important in chickens’ immune system (Wang et al., 2023). They constitutively express most of the TLRs such as TLR1, TLR2 type 1 and type 2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR10 (Kogut et al., 2005). The activation of oxidative burst and degranulation of heterophils as well as the upregulation of pro-inflammatory cytokines such as Interleukin (IL)-1β, IL-6 and inflammatory chemokines such as CXC chemokine ligand (CXCL)i2 and CCLi4 upon stimulation with specific TLR agonists underlines the active role of heterophils in the innate immune response of chickens (Kogut et al., 2005). We previously demonstrated that CpG-ODN can protect broiler chickens against E. coli and S. Typhimurium septicemia following a single injection by the in ovo, intramuscular (IM) or intrapulmonary (IPL) route (Gomis et al., 2003; Gunawardana et al., 2015; Goonewardene et al., 2017; Goonewardene et al., 2020; Gunawardana et al., 2020). The ability of CpG ODN to protect chickens against S. enteritidis challenge was reported by other researchers (He et al., 2007). We also reported the comparable effect of the prophylactic use of CpG-ODNs and select antimicrobials in protecting neonatal broiler chickens against lethal E. coli septicemia (Gunawardana et al., 2022) including the immunoprotective mechanisms (Gunawardana et al., 2019). Although we successfully established the imunoprotective efficacy of CpG-ODN against bacterial infections in chickens, the specific role of vital innate immune cells like heterophils in this process have not yet been explored. In addition, the effect of repeated administration of CpG-ODN in the immune mediated protection of neonatal broilers against bacterial infections has not been established. Therefore, the objectives of this study were to repeatedly administer CpG-ODN to neonatal broiler chickens and explore the kinetics of the bactericidal activities of heterophils and evaluate the degree of immunoprotection of birds against S. Typhimurium septicemia in comparison to birds challenged after receiving only single dose CpG-ODN.

MATERIALS AND METHODS

Ethics Statement

This animal experiment was approved by the University Committee on Animal Care and Supply Animal Research Ethics Board (Protocol number : 20070008) and conducted following the guidelines of the Canadian Council on Animal Care.

Housing and Maintenance of Broiler Chickens

Broiler chickens, at the day of hatch (1-day-old), were obtained from a broiler breeder flock (Ross 308) and maintained at the Animal Care Unit, Western College of Veterinary Medicine, University of Saskatchewan, Canada. A broiler chicken ration without antibiotics was provided ad libitum. Each room was ventilated with a HEPA filter and non-recirculated air at a rate of 10 to 12 changes/h. Strict sanitation and air pressure differentials were maintained during the entire experimental period. Birds were raised at 32°C for the first 7 d of life, after that, temperature conditions were reduced by 0.5°C per day until a temperature of 20°C. Lighting (30–40 lux) was provided continuously until 2 d post-hatch, thereafter lux and duration were decreased until 10 to 20 lux and 7 h of darkness were achieved.

CpG-ODN

CpG-ODN2007 (5′-TCGTCGTTGTCGTTTTGTCG TT-3′) with a modified phosphorothioate backbone was purchased from Operon Biotechnologies, Inc. (Huntsville, AL) and was dissolved in sterile pyrogen-free saline.

Phagocytosis assay

Blood samples were collected using heparin-coated syringes. Phagotest kit (Glycotope Biotechnology GmbH, Heidelberg, Germany) manufactured for use in mammalian species was modified and optimized to be used in chickens. Briefly, each blood sample was aliquoted into 2 tubes (200 µL/tube) as an experimental test (tube A) and experimental control (tube B). In each sample, tube A was mixed (vortexed) with 40 μL of fluorescein isothiocyanate (FITC) – labeled E. coli (2 × 109 bacteria/mL) supplied in the kit and incubated for 10 min at 37 °C in a water bath. Tube B was incubated on ice for 10 min after vortex mixing with 40 μL of FITC-labeled E. coli as a control. After incubation, all the samples were taken out and ice-cold quenching solution (supplied by the kit) was added to blood samples to stop the bacterial attachment and internalization by quenching the FITC fluorescence of surface bound bacteria leaving the fluorescence of internalized particles unaltered. Then, cells were washed 2 times with 2 mL of wash solution (supplied in the kit) and centrifuged for 5 min at 250 G. Cells were re-suspended in 2 mL of pre-warmed erythrocyte lysis solution (supplied by the kit) and kept for 10 min at room temperature. Then, samples were centrifuged for 5 min at 250 G and the supernatant was discarded. Samples were washed twice with 3 mL of wash solution and centrifuged for 5 min at 250 G. Cells (0.5×106 - 1×106 cells) from peripheral blood were stained with mouse anti-chicken CD45- Allophycocyanin (APC) (LT40) (Southern Biotech, Birmingham, AL) antibody and incubated for 30 min at 4°C in the dark. Cells were washed twice with ∼ 500 µL fluorescence-activated cell sorting (FACS) buffer [Phosphate buffered saline (PBS) containing 2% fetal bovine serum (Gibco, Grand Island, New York, NY) and 0.1% sodium azide] and centrifuged for 5 min at 250 G and re-suspended in 300 µL of FACS buffer and stored at 4°C. The percentage of heterophils was obtained by calculating differential cell counts twice using stained blood smears with Wright-Geimsa (Sigma Aldrich). Stained FITC+ cells were acquired by Cytoflex flow cytometer (Beckman Coulter, Carlsbad, CA) and results were analyzed using FlowJo Version 10.8.1 (Ashland, Becton, Dickinson and Company, 2021).

Oxidative Burst Assay

Phagoburst kit (Glycotope Biotechnology GmbH, Heidelberg, Germany) was used for cell preparation and antibody staining for flow cytometry (Parment et al., 2007). Blood samples were collected using heparin-coated syringes and kept on ice for 10 min before processing. Each sample was aliquoted into 2 tubes, 200 µL/tube and labeled as experimental test (tube A) and experimental control (tube B). In each sample, tube A was mixed with pre-warmed 20 µL of phorbol 12- myristate 13-acetate (PMA) solution (5 μL of PMA + 995 μL wash buffer solution), final concentration 1.62 µM, and incubated for 5 min at 37 °C. Tube B was mixed with wash buffer and use as experimental control blood samples. Fluorogenic substrate, 20 μL of dihydrorhodamine (DHR) 123, was added to each blood sample, vortexed thoroughly, and incubated at 37°C for 10 min. After incubation, 2 mL of lysis solution was mixed with samples and the mixtures were incubated at room temperature for 10 min. Samples were centrifuged for 5 min at 250 G to discard the supernatant. All the samples were washed twice with 2.5 mL of wash solution and centrifuged for 5 min at 250 G. Peripheral blood cells (0.5 × 106 – 1 × 106 cells) were stained with mouse anti-chicken CD45-APC antibody and incubated for 30 min at 4 °C in the dark. After washing cells twice with ∼ 500 µL of FACS buffer and centrifuging for 5 min at 250 G, cells were re-suspended in 300 µL of FACS buffer. Gating, percentage, staining and Cytoflex flow cytometer analysis of heterophils were performed as described above.

Isolation of Heterophils

Peripheral blood samples were collected from the brachial vein into BD Vacutainer® blood collection tubes (Becton, Dickinson and Company, Mississauga, ON). Heterophils were isolated as described previously (Kogut et al., 1995; Kogut et al., 2001; He et al., 2007) with some modifications. Briefly, collected blood was mixed with 1% methylcellulose (25 centiposes; Sigma Chemical Co., St. Louis, MO) at a ratio of 3: 2 and centrifuged at 250 G for 30 min. The serum and buffy coat layers were collected and suspended in sterile PBS (pH = 7.4). The suspension was overlaid on Histopaque (Sigma Chemical Co.) gradient (specific gravity 1.077 g/mL over 1.119 g/mL) and centrifuged at 250 G for 60 min. After centrifugation, the heterophil layer from the 1.077/ 1.119 interfaces and 1.119 band, was collected. Collected cells were washed 3 times with RPMI 1640 (Sigma Chemical Co.) supplemented with 10 % heat-inactivated fetal bovine serum, 1% penicillin/streptomycin (Gibco), and 10 µg/mL gentamicin sulfate (Gibco). Live cells were counted by the trypan blue exclusion method. Purity of heterophils was confirmed by staining cells with mouse anti-chicken Bu-1-FITC (AV20), anti CD3-APC (CT-3), anti-monocyte/macrophage (MoMa)-PE (KUL01) antibodies (Southern Biotech) and 7-Aminoactinomycin D dye (7-AAD, Invitrogen). Cells were prepared at a density of 1×107 cells/mL and stored on ice until use.

Degranulation Assay

Five hundred thousand isolated heterophils (purity > 98 % as determined by FACS analysis) were seeded in a round bottom 96-well tissue culture plate. Cells were stimulated with 50 ng/mL of PMA (Millipore-Sigma, St. Louis, MO) and 500 ng/mL of ionomycin (Ion) (Millipore-Sigma). A flow cytometry-based degranulation assay was developed and optimized using rabbit polyclonal anti-lysosomal membrane protein-1 (LAMP1) antibody (Abcam, Toronto, ON, Canada) conjugated with APC using Lightning-Link antibody conjugation kit (Abcam). The conjugated antibody was added into each well. Cells were incubated for 4 h at 41°C with 5% CO2. After incubation, cells were washed twice with 200 µL FACS buffer and stained with LIVE/DEAD Fixable Green Dead Cell Stain Kit (Invitrogen, Thermo fisher Scientific, MA) on ice in the dark for 20 min. Subsequently, cells were re-suspended in FACS buffer and data was acquired by Cytoflex flow cytometer and results were analyzed using FlowJo.

Gating Strategies for Phagocytosis, Oxidative Burst, and Degranulation of Heterophils From PBMC

Cells were gated based on the forward scatter area (FSC-A) vs. the side scatter area (SSC-A). Single cells were defined by forward scatter height (FSC-H) vs. FSC-A. The live cell population was confirmed using 7AAD. Heterophils with elevated phagocytosis and oxidative burst activities were identified as CD45+/FITC+ in 2 separate assays. The heterophil population with increased degranulation activity was characterized as CD107a+ population. Fluorescence minus one (FMO) controls were performed to identify the CD107a+ population.

S. Typhimurium Challenge Preparation

S. Typhimurium was isolated from a 25-wk-old broiler breeder chicken with septicemia and S. Typhimurium culture was prepared for challenge studies as previously described (Taghavi et al., 2008). Briefly, the bacteria was cultured on tryptic soy agar containing 5 % sheep blood (Thermo Scientific, Canada) and incubated aerobically for 18 to 24 h at 37 °C. Then, 2 to 3 colonies were added into 200 mL of Luria broth (Miller, BDH, Poole, United Kingdom) in a 500 mL Erlenmeyer flask and incubated at 37°C for 16 to 18 h on a shaker with shaking at 200 rpm. After incubation, the cultures contained approximately 1 × 109 colony-forming units (CFU) of stationary-phase bacteria. The S. Typhimurium inoculum was serially diluted with saline to achieve 1 × 107 CFU/mL and 1 × 108 CFU/mL in a total volume of 250 µL, and administered to broilers subcutaneously at 9 d of age.

Experimental Design

Effects of Delivery of CpG-ODN Once in Neonatal Broiler Chickens on Phagocytosis, Oxidative Burst and Degranulation of Heterophils

Broiler chicks at the day of hatch (d 1 of age), were randomly divided into 2 groups, and on the same day, Group 1 received CpG-ODN (50 µg/bird) and Group 2 received saline by the intramuscular (IM) route in the thigh. Peripheral blood samples were collected at 24, 48, and 72 h following CpG-ODN administration. Then, blood samples were processed to quantify phagocytosis, oxidative burst, and degranulation activities. Phagocytosis and oxidative burst assays were performed in duplicates with 4 biological replicates each time (Total number of birds/group/time point = 8), and the degranulation assay was conducted using 5 birds (n = 5).

Effects of Delivery of CpG-ODN Once or Twice in Neonatal Broiler Chickens on Phagocytosis, Oxidative Burst and Degranulation of Heterophils

Broiler chickens were randomly divided into 2 groups at the day of hatch (day 1 of age). Group 1 received CpG-ODN (50 µg/bird) by the IM route in the thigh at 1 and 4 d of age. The second group received saline by the IM route in the thigh at the day of hatch and then again at 4 d post-hatch. Peripheral blood samples were collected at 48 h following first and second CpG-ODN or saline administration. Phagocytosis and oxidative burst assays were repeated twice each time with 4 birds (Total number of birds/group/time point = 8). The degranulation assay was conducted using 5 birds (n = 5).

Delivery of CpG-ODN Once or Twice in Neonatal Broiler Chickens Against S. Typhimurium Septicemia

Broiler chickens were randomly allocated into 3 groups on the day of hatch (day 1 of age), each group containing 40 birds (n = 40/group). CpG-ODN (50 µg/bird) was injected by the IM route in the thigh muscle; Group 1 received CpG-ODN at 3 and 6 d of age; Group 2 received CpG-ODN at 6 d of age; Group 3 received saline and kept as a negative control. S. Typhimurium challenge was conducted at 9 d of age by the subcutaneous route. Half of the birds in each group (n = 20/group) were given the low dose (1 × 107 CFU/bird), while the other half received the higher dose (1 × 108 CFU/bird) as previously described (Taghavi et al., 2008). Birds were monitored 3 times per day until 10 d postchallenge. Clinical signs and daily cumulative clinical score (CCS)s were recorded for each bird as previously described: briefly, slow to move; 0 = normal; 0.5 = slightly abnormal appearance; 1 = birds are depressed, reluctant to move; 1.5 = reluctant to move, may drink and peck; 2 = not able to reach food or water or stand; and 3 = found dead (Taghavi et al., 2008). Chicks who received a clinical score of 2 were euthanized by cervical dislocation. A CCS was summarized at the end of the trial, with each bird given a sum of daily clinical scores. Dead or euthanized chicks were necropsied immediately. All remaining birds were euthanized at 10 d postchallenge. Swabs were taken from the air sacs, and a semi-quantitative estimate of bacteria isolation was conducted on 5% Columbia sheep blood agar by the quadrant streaking method. Bacterial growth on these cultures was recorded from 0 to 4 +, where 0 = no growth or few colonies (less than 5 colonies); 1 + = bacterial growth on quadrant 1; 2 + = bacterial growth on quadrants 1 and 2; 3 + = bacterial growth on quadrants 1, 2, and 3; and 4 + = bacterial growth on all quadrants as previously described (Gomis et al., 2004).

Statistical Analysis

Statistical analysis was conducted using GraphPad Prism ver.6 (GraphPad Software Inc., San Diego, CA). Survival rates and differences in CCS, and bacterial load were analyzed between the different groups. Log-rank (Mantel-cox) and the chi-square tests were used to compare survival patterns and median survival time. One way ANOVA and multiple comparison analysis were performed to compare more than 2 test groups. The unpaired 2-tailed Student's t-test was performed to compare phagocytosis, oxidative burst and degranulation functions of heterophils between the CpG-ODN and saline groups. P values ≤ 0.05 were considered statistically significant.

RESULTS

Effects of Delivery of CpG-ODN Once or Twice in Neonatal Broiler Chickens on Phagocytosis of Heterophils

We investigated the immunomodulatory effects of CpG-ODN on the phagocytic activity of heterophils at 24, 48, and 72 h after a single administration on the day of hatch using FITC-labeled E. coli. The percentage of granulocytes was ≥ 75% with heterotrophs representing ≥ 98% of the granulocytes in our assays. The gating strategy used to quantify phagocytosis of heterophils was demonstrated in pseudocolor plots. The phagocytized heterophil population was identified as FITC+CD45+ cells (Figure 1A). Phagocytosis by CpG-ODN-stimulated heterophils was significantly higher compared to the saline group at 24, 48, and 72 h (P = 0.0002, P = 0.0002, and P = 0.0002, respectively) (Figure 1B). Next, we examined the degree of stimulation of the phagocytic activity of heterophils after administration of CpG-ODN once or twice. A significantly higher phagocytic activity of heterophils was observed after administration of CpG ODN to neonatal broiler chickens once or twice (P < 0.0002 and P = 0.0002, respectively) compared to the saline control group. The phagocytic activity of heterophils was significantly higher in neonatal broiler chickens that received CpG-ODN twice compared to only once (P < 0.0001) (Figure 1C).

Figure 1.

Figure 1

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Phagocytic activity of heterophils in neonatal broiler chickens following CpG-ODN administration once or twice. Blood samples were collected from the groups of birds administered with CpG-ODN and incubated with FITC-labeled E. coli for 10 min at 37°C for phagocytosis assay. (A) The gating strategy to identify FITC+ heterophils. Pseudocolor plots show the percentage of FITC+ cells in birds administered with CpG-ODN or saline. (B) Significantly higher percentages of heterophils phagocytosis at 24, 48, and 72 h following administration of CpG-ODN once compared to the saline group. (C) The mean percentages of FITC+ labeled heterophils at 48 hr following administration of CpG-ODN once or twice. Phagocytic activity of heterophils was significantly higher in birds administered with CpG-ODN twice compared to once. Significant differences are indicated by ***P ≤ 0.001 and **** P ≤ 0.0001.

Effects of Delivery of CpG-ODN Once or Twice in Neonatal Broiler Chickens on Oxidative Burst of Heterophils

To examine immunostimulatory effects of CpG-ODN on oxidative burst of heterophils, blood samples were collected at 24, 48, and 72 h following CpG-ODN administration. Cells were then stimulated with PMA in-vitro. The percentage of granulocytes was ≥ 75% with heterophils accounting ≥ 98% of the granulocytes in our assays. The percentage of cellular uptake of fluorescent compound, Rhodamine 123 (oxidized DHR 123 by ROS), by heterophils was analyzed by flow cytometry. The gating strategy used to identify oxidative burst activity of heterophils was demonstrated in pseudocolor plots (Figure 2A). Oxidative burst of heterophils isolated from birds that received CpG-ODN was significantly higher than the saline-treated group at 24, 48, and 72 h (P < 0.0001, P < 0.0001, and P < 0.0001, respectively) (Figure 2B). Then, we examined the difference in the degree of oxidative burst activity of heterophils after once or twice administration of CpG ODN to neonatal broiler chickens. A significantly higher oxidative burst activity was observed in heterophils of broiler chickens that recieved CpG-ODN once or twice compared to the saline control group (P < 0.0001 and P < 0.0001, respectively). However, there was no significant difference in oxidative burst activity of between heterophils isolated from birds that received CpG-ODN once and twice (P = 0.2477). (Figure 2C).

Figure 2.

Figure 2

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Oxidative burst capacity of heterophils in neonatal broiler chickens following CpG-ODN administration once or twice. Blood samples were collected from the groups of birds following the administration of CpG-ODN to examine the effect of CpG-ODN on oxidative burst activity in heterophils. (A) The gating strategy to identify oxidative burst activity. Pseudocolor plots demonstrate the percentage of cells undergoing oxidative burst in the birds administered with CpG-ODN or saline. (B) Significantly higher oxidative burst percentages of heterophils at 24, 48, and 72 h following administration of CpG-ODN once compared to the saline administered group. (C) The mean percentages of heterophils undergoing oxidative burst at 48 h following administration of CpG-ODN once or twice. Significant differences are indicated by ****P ≤ 0.0001.

Effects of Delivery of CpG-ODN Once or Twice in Neonatal Broiler Chickens on Degranulation of Heterophils

This experiment was conducted to investigate the degranulation activity of heterophils following IM delivery of CpG-ODN to neonatal broiler chickens. Blood samples were collected at 24, 48, and 72 h following CpG-ODN administration and heterophils were isolated. Heterophils were stimulated with PMA/Ion and CD107a expression was used as a marker to assess the degranulation activity of the cells by flow cytometry. The gating strategy to quantify CD107a expression was demonstrated in pseudocolor plots (Figure 3A). Data were presented as a percentage of CD107a+ cells in the total live heterophil population. The degranulation activity of heterophils at 24, 48, and 72 h after CpG-ODN administration to neonatal broiler chickens was significantly higher compared to the saline group (P = 0.0100, P = 0.0127, and P = 0.0029, respectively) (Figure 3B). Degranulation of heterophils was also examined at 48 h following administration of CpG-ODN once or twice to birds. Birds that received CpG-ODN once or twice had significantly higher heterophil degranulation activity compared to the saline control group (P = 0.0019 and P = 0.0006, respectively). Furthermore, degranulation activity of heterophils was significantly higher in neonatal broiler chickens that received CpG-ODN twice compared to once (P = 0.0140). (Figure 3C).

Figure 3.

Figure 3

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Degranulation activity of heterophils in neonatal broiler chickens following CpG-ODN administration once or twice. Heterophils were isolated from the groups of birds following administration of CpG-ODN to examine effect of CpG-ODN on degranulation of heterophils. (A) The gating strategy to identify CD107a+ heterophils. Pseudocolor plots demonstrate percentage of CD107a+ heterophils in the groups of birds following administration of CpG-ODN or saline. (B) Significantly higher degranulation activity of heterophils in the groups of birds at 24, 48, and 72 h following administration of CpG-ODN once compared to saline group. (C) Degranulation activity of heterophils in the groups of birds at 48 hr following administration of CpG-ODN once or twice. Degranulation of heterophils was significantly higher in groups administered with CpG-ODN twice compared to once. Significant differences are indicated by * P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Delivery of CpG-ODN Once or Twice in Neonatal Broiler Chickens Against S. Typhimurium Septicemia

The objective of this experiment was to explore the immunoprotective effects of CpG-ODN against S. Typhimurium septicemia in neonatal broiler chickens following delivery of CpG-ODN once or twice. Neonatal broiler chickens that received CpG-ODN once or twice were significantly protected against S. Typhimurium challenge compared to the saline administered group (P < 0.0001). However, there was no significant difference of survival percentages between groups of birds who received CpG-ODN once and twice against S. Typhimurium (P = 0.1001) (Figure 4A). Birds that received CpG-ODN once or twice had significantly low cumulative clinical score (CCS) following S. Typhimurium challenge compared to the group that received saline (P < 0.0002 or P < 0.0001, respectively) (Figure 4B). S. Typhimurium was isolated from the air sacs and the bacterial load was quantified. Birds that received CpG-ODN twice had a statistically lower bacterial load in air sacs compared to birds that received saline (χ2 = 14.07, P = 0.0009) (Figures 4C and 4D). Birds that died due to S. Typhimurium septicemia had airsacculitis, perihepatitis, pericarditis, or a combination of airsacculitis, perihepatitis together with either pericarditis or polyserositis.

Figure 4.

Figure 4:

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Immunoprotective efficacy of CpG-ODN against S. Typhimurium septicemia in neonatal broiler chickens following administration of CpG-ODN once or twice. Following administration of CpG-ODN or saline at 3 and 6 d of age, birds were challenged with a lethal dose of S. Typhimurium.(A) Survival of the groups of birds administered with CpG-ODN once or twice against S. Typhimurium septicemia (n = 40/group). The CpG-ODN administered groups had a significantly increased survival compared to the saline group (P < 0.0001). Group administered with CpG-ODN twice had an increased survival compared to the group administered with CpG-ODN once, but difference was not significant. (B) Cumulative Clinical Score (CCS) of individual birds following S. Typhimurium challenge. A significantly lower CCS in the group administered with CpG-ODN once (P < 0.0002) and twice (P < 0.0001) than saline administered group. (C) Bacterial load (0, 1 +, 2 +, 3 +, & 4 +) in the air sacs of individual birds in the groups of birds administered with CpG-ODN once or twice. (D) Bacterial load in the air sacs as low (no growth & few colonies) and high (scores 1 +, 2 +, 3 + & 4 +) in the groups of birds administered with CpG-ODN once or twice. Groups administered with CpG-ODN had a significantly lower bacterial load (χ2 = 12.93, P = 0.0003) compared to the saline control group. Significant differences are indicated by **P ≤ 0.01, *** ≤ 0.001, ****P ≤ 0.0001.

DISCUSSION

As the prophylactic use of antibiotics is being discontinued in the poultry industry, infection rates with pathogenic bacteria like E. coli, Salmonella, Campylobacter jejuni, and Clostridium perfringens will likely increase in chickens. These infections not only adversely affect the health and welfare of birds, but also increase the likelihood of zoonotic disease transmission to humans (Immerseel et al., 2004; Pleydell et al., 2007; Kassem et al., 2017). The experience in the European poultry industry indicates that the withdrawal of prophylactic antibiotic use causes a substantial increase in the use of therapeutic antibiotics in the poultry industry (Casewell et al., 2003; Cogliani et al., 2011). Therefore, several alternatives, such as prebiotics and probiotics, are being developed as alternatives to antibiotics. The use of probiotics (Timmerman et al., 2006) and prebiotics (Huff et al., 2010) as growth promoters significantly improves feed conversion ratios and reduces stress, but does not provide significant protection against E. coli challenge (Gunawardana et al., 2022). We previously reported that CpG-ODN can be used as an immunostimulant to protect neonatal broiler chickens against lethal bacterial septicemias such as E. coli and S. Typhimurium (Gomis et al., 2003; Gomis et al., 2004; Gomis et al., 2007). However, no information is available in broiler chickens following CpG-ODN administration twice on bactericidal activities of heterophils. In this study, we specifically explored the bactericidal activities of heterophils following administration of CpG-ODN once or twice. The phagocytosis, oxidative burst, and degranulation activities of the cells were evaluated. In addition, the immunoprotective effect of CpG-ODN against challenge with S. Typhimurium was examined after single or twice administration of CpG-ODN to neonatal broiler chickens.

Immune cells of chicken, including heterophils and lymphocytes undergo a period of maturation following hatch. It has been reported that these immature immune cells are not fully functional until the third to fifth week of life. Hence, immaturity of immune cells increases the susceptibility of neonatal broiler chickens to bacterial infections (Cooper et al., 1966; Rodák et al., 1969; Song et al., 2021). Thisis associated with low production of cytokines such as IFN-γ and IL-1β, IL-4, IL-10 in peripheral blood and reduction in the expression of IL-2, IFN-γ, IL-4, and Lysozyme C gene in the ileum and the expression of IL-2 gene in the spleen during day 6 to 13 of neonatal chicks (He et al., 2011). In contrast, we demonstrated that CpG-ODN-mediated immunoprotective mechanisms are mediated through enhanced cytokine production such as IL-1, IL-4, IL-6, IL-8, IL-10, IL-18, IFN-γ, IFN-α, and LITAF in immune compartments like the spleen and lungs of neonatal broiler chickens (Gunawardana et al., 2019). The recruitment and activation of circulating granulocytes mainly heterophils play a crucial role in forming the first line of defense against bacterial invasions, such as salmonellosis in chickens. (Gast and Porter Jr, 2020).

In this study, we observed that neonatal broiler chickens administered with CpG-ODN once had significantly increased phagocytic heterophils as early as 24 h following CpG-ODN administration. Furthermore, heterophil phagocytic potential was significantly enhanced when birds received CpG-ODN for the second time compared to single administration. These results suggest that although immune cells are still in the process of maturity in neonatal chickens, they can be simulated with bacterial DNA or CpG-ODN very effectively to become mature and functional.

Following phagocytosis, granulocytes including heterophils kill ingested bacteria by generating reactive oxygen radicals through oxidative burst (Robinson, 2008). Although we did not observe a dose dependent oxidative burst activity of heterophils, our results demonstrated a significant elevation in the oxidative burst activity of heterophils in birds that received CpG ODN once or twice. In contrast, a dose dependent significant increase in oxidative burst activity was reported in neutrophils isolated from the bone marrow of mice following exposure to PMA (Ward et al., 2008). This indicates that full oxidative burst activity of heterophils can be achieved only by using a single dose of CpG-ODN. Reactive oxygen species and nitrogen species (ROS–RNS) and other redox active molecules have a balancing action (Wink et al., 2011). The main ROS are hydrogen peroxide (H2O2) and superoxide anion (O2•−) and the RNS are peroxynitrite (ONOO−), nitrogen dioxide (NO2•), and nitrogen trioxide produced by reaction of nitric oxide (NO•) with ROS. Imbalances between production and removal of ROS–RNS are linked with pathophysiological reactions during immune responses (Gostner et al., 2013). Therefore, it is important to concurrently evaluate nitric oxide and oxygen mediated killing mechanisms by innate immune cells following administration of CpG-ODN.

Our study established that CpG ODN stimulates significant degranulation activity of heterophils in a dose dependent manner. This is consistent with previous reports which indicated the in vitro and in-vivo stimulation of chicken heterophils with CpG-ODN with increased degranulation and significantly higher β-glucuronidase activity (He et al., 2005). Another study demonstrated that in ovo delivery of CpG-ODN to 18-days-old embryos increased degranulation of heterophils at day 2 post-hatch and subsequently reduced S. enteritidis invasion of the cecum when chickens were challenged at 10 d of age (Mackinnon et al., 2009). In our CpG-ODN dose dependent experiment, degranulation of heterophils increased as early as 24 h and continued to increase in all the time points examined. However, the molecular mechanisms associated with this phenomenon needs to be further investigated. As degranulation progresses, secretory lysosomes are released and the LAMP-1 or CD107a is transported to the surface of the cell rendering it accessible for antibody binding thus making it possible to identify cells with active degranulation. This event highlights the important role that LAMP-1 or CD107a plays in the degranulation process. CD107a is a crucial transmembrane protein that is heavily glycosylated and found on the membrane of lysosomes. Its glycosylated domain is located on the luminal side, while a short tail hangs in the cytoplasm . The expression of CD107a on the cell surface is a significant measure to explore degranulation activity of heterophils with its vital role in lysosomal membrane stability and vesicle trafficking, CD107a is an essential protein for cellular function (Parkinson-Lawrence et al., 2005).Therefore, detecting and analyzing its expression on the cell surface can provide valuable insights into cellular degranulation activity (Lorenzo-Herrero et al., 2019; Boodhoo and Behboudi, 2022). CD107a has been used as a marker to quantify degranulation activity in CD8 cytotoxic T cells, natural killer (NK) cells, and gamma delta T cells in chickens (Betts and Koup, 2004; Gostner et al., 2013; Voigt et al., 2014; Meijerink et al., 2021; Matsuyama-Kato et al., 2022). Here, our optimized flow cytometry-based degranulation assay which employed CD107a as a marker will be a very valuable tool to detect degranulation activity of chicken heterophils.

Our previous study demonstrated that CpG-ODNs protect neonatal broiler chickens against lethal E. coli septicemia comparable to the therapeutic use of tetracycline or sodium sulfamethazine which are commonly used in the broiler chicken industry (Gunawardana et al., 2022). Moreover, it was demonstrated that although therapeutic antibiotics can treat bacterial infections, the immunosuppressive effects induced by some antibiotics may negatively impact chicken health. In the current study, any adverse effects of CpG-ODN on the immune system and growth of boiler chickens were not observed over a 30 d period (data not shown) following administration of CpG-ODN twice in neonatal broiler chickens. Hence, CpG-ODNs are very effective and safe alternatives to antibiotics in the poultry industry.

Besides, we observed significant protection of birds that received CpG ODN (once or twice) against S. Typhimurium with significantly lower CCS and bacterial scores in air sacs. Birds that received CpG-ODN twice had better survival rate although it was not statistically significant. This might indicate that maintenance of the bioavailability of CpG-ODN may ensure a longer term protection of chickens against pathogens. Our previous studies demonstrated that a single administration of CpG-DN to neonatal broiler chickens by the IM, in ovo, and IPL routes protects against E. coli and S. Typhimurium septicemia at statistically significant levels. However, this immunoprotection declines by six days after CpG-ODN administration (Gomis et al., 2003; Gunawardana et al., 2015; Goonewardene et al., 2017; Gunawardana et al., 2019). The long term immunoportective effect of repeated administration of CpG ODN to neonatal broiler chickens against bacterial infections needs to be examined in the future.

In summary, we have demonstrated that the delivery of CpG-ODN twice significantly increased phagocytosis and degranulation activity of heterophils compared to a single administration. We have also demonstrated that the administration CpG-ODN twice to neonatal broiler chickens increased immunoprotection against S. Typhimurium septicemia, improved survival rates and clinical signs, and reduced bacterial load in the body compared to single dose administration. The exact molecular mechanism by which CpG ODN exerts its effects in heterophils, and other innate immune cells needs to be explored in the future. In addition, the long-term antibacterial prophylactic effects of CpG ODN should be studied in chickens after repeated dose administration.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

We thank the staff of the Animal Care Unit at the Western College of Veterinary Medicine, University of Saskatchewan. Financial support was provided by grants from the Chicken Farmers of Saskatchewan (424357), Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery program (420261) and Results Driven Agriculture Research program (RDAR) (425526).

Author Contributions: IS conducted the acquisition, analysis, and interpretation of the data and drafted the manuscript under the supervision of SG. IS, SP, HG, LEA, AM, and BL helped in acquiring the experimental material and supported the animal experiments. KAA, LEA, ST, and PG verified the underlying data and revised the manuscript. All authors read and approved the final manuscript.

Consent for Publication: Not applicable.

Availability of Data and Materials: The data used to support the findings of this study are available from the corresponding author upon request.

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