Induction of Trained Immunity in Broiler Chickens by Combination of in Ovo Delivery of Oligodeoxynucleotides Containing CpG Motifs and Intrapulmonary Delivery of a Live Clostridium perfringens Vaccine at Hatch to Protect Against Escherichia coli Septicemia Later in the Grow-Out Period Open Access

Emergence of antimicrobial-resistant (AMR) strains of bacteria due to antimicrobial use (AMU) in food animal production has become a global threat on one health (1). The chicken industry is exploring AMU reduction strategies to control AMR development. Nevertheless, it has been shown that the reduction of prophylactic antibiotic use has led to a substantial increase in therapeutic antibiotic use in the poultry industry (2). Therefore, the poultry industry urgently needs alternatives to antibiotics to prevent bacterial diseases. Evolutionary conserved molecules or structures called pathogen-associated molecular patterns (PAMPs) presented by infectious microorganisms recognized by toll-like receptors (TLRs) are among the most broadly studied classes of pattern-recognition receptors (3). Oligodeoxynucleotides containing unmethylated cytosine-phosphodiester-guanine motifs (CpG-ODN) mimic bacterial DNA and act as a PAMP, which is recognized by TLR-9 in mammals (4) and TLR-21 in chickens (5). Upon internalization by target cells, synthetic CpG-ODNs can induce protective immunity against bacterial and viral agents (6,7). The immunostimulatory effect of CpG-ODN has been proven in many species, including mice (8), humans (9), and chickens (10). Our group has been pioneers in studying the immunomodulatory properties of CpG-ODN in chickens against Escherichia coli and Salmonella Typhimurium septicemia in neonatal chickens by different delivery routes such as in ovo, intramuscular (IM) and intrapulmonary (IPL). We have also demonstrated that administration of a single dose of CpG-ODN can protect chicks from lethal septicemia as an alternative to antibiotics (10,11,12,13,14,15). In ovo delivery of CpG-ODN induced T-helper (Th)1 cytokines (interferons), Th2 cytokine, interleukin (IL) -4, and regulatory cytokine IL-10 types of response, hence suggesting that CpG-ODN in chickens can be used as modulator of both Th1 and Th2 types of cytokines, particularly in lungs (16). It was demonstrated that enhanced expression of pro-inflammatory cytokines, IL-1β, IL-6, IL-18, and tumor necrosis factor (TNF) -α in lungs and spleen following in ovo delivery of CpG-ODN suggested that CpG-ODN promotes inflammatory responses in chickens. Therefore, in ovo delivery of CpG-ODN enriches immunological niches in the immature immune system of the chicken embryo so that pathogenic insults can be tolerated more efficiently at hatch (16). We have also demonstrated the enrichment and maturation of immune cells in different immune compartments, such as the lungs and spleens of neonatal chicks, by delivering CpG-ODN by the IPL route under laboratory and field conditions (14,15).

Among bacterial infections, E. coli infections in neonatal broiler chicks cause a variety of disease syndromes, including yolk-sac infection, omphalitis, respiratory tract infection, and septicemia (17). Escherichia coli infections in neonatal poultry are characterized by acute septicemia resulting in death, and subacute infection resulting in pericarditis, airsacculitis, and perihepatitis (17,18). Many E. coli isolates commonly associated with commercial broiler chickens belong to serogroups O1, O2, and O78 (19,20).

Necrotic enteritis (NE), caused by Clostridium perfringens (CP), is an economically important disease associated with the intestinal tract of broiler chickens (21). Mortality rates caused by CP range from 2% to 50%, leading to production and economic losses associated with poor weight gain and increased feed conversion ratio, in addition to increased mortality (21). Approximately $6 billion USD annual production losses are associated with NE globally (22). Different endotoxins produced during CP proliferation in the intestines are responsible for intestinal necrosis and peracute death of birds (23).

Although the respiratory and intestinal mucosal systems have considerable variations in terms of functional role, they do share anatomical similarities (24,25). Substantial evidence suggests the existence of a two-way interaction between the respiratory and intestinal mucosal immune systems. This crosstalk is referred to as the gut–lung axis (GLA) (26). The GLA results from a complex interaction between the different microbial products such as short chain fatty acids, antimicrobial peptides, lipopolysaccharides, migrating immune cells (lymphocytes, dendritic cells [DCs] and macrophages) and interactions of these molecules and immune cells with the respective microbiome of the intestine and lungs. It was demonstrated in a mouse model that DCs of the lung were capable of inducing migration of protective T cells to the gastrointestinal tract from the lungs and produce gut immunity against a highly pathogenic strain of Salmonella Typhimurium by intranasal immunization (27). We have recently demonstrated that delivery of a live CP vaccine at hatch with no booster vaccine by the IPL route protected broiler chickens against NE at a significant level against heterologous isolates of CP (28).

Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious and immunosuppressive disease of young broiler chickens caused by IBD virus (IBDV) which is responsible for major economic losses in the poultry industry worldwide (29). Most IBDV strains circulating in broiler chickens in Canada are variant IBDV (varIBDV) strains. Among them, varIBDV SK09 is the most common strain circulating in the broiler chicken industry in Canada (30).

Innate immunity is crucial as the initial line of defense against infectious pathogens. It actively restricts the early proliferation and spread of these pathogens within the body (31). Recent advancements in research have observed emerging evidence of innate immune memory or trained immunity after vaccination and infection in innate immune cells exhibiting long-term changes in their functional programs in mice (32,33), and humans (34,35). Innate immune cells, like monocytes, macrophages, and natural killer cells, undergo profound changes that lead to increased responsiveness upon secondary stimulation by microbial pathogens, enhanced production of inflammatory mediators, and the increased ability to eliminate infection without specificity (36). Trained immunity is based on nonpermeant genetic changes such as epigenetic reprogramming, histone modifications, DNA methylation, or modulation of microRNA and long noncoding RNA expression as well metabolic reprogramming (36). These changes lead to transcriptional programs that rewire the intracellular immune signaling of innate immune cells. Shift of cellular metabolism between oxidative phosphorylation (OXPHOS) and cellular glycolysis in order to enhance immune cell functions to defeat pathogens also has been recognized (37). IL-1 signaling plays an essential role in the induction of trained immunity. There is compelling evidence regarding trained immunity in mice (36,38,39) and humans (40,41). A recent study in mice demonstrated protection against pulmonary tuberculosis, Candida albicans, and Schistosoma mansoni infections following Bacillus Calmette-Guérin (BCG) vaccination (33). Recently, we have demonstrated the proof of concept that delivering CpG-ODN twice by the IM route in neonatal broiler chickens at Days 1 and 4 of age can induce trained immunity and protect against lethal E. coli infections later in life (42). Therefore, the objectives of this study were to explore the ability of CpG-ODN to induce trained immunity in broiler chickens (1) by administering CpG-ODN by the in ovo and IPL routes; and (2) by administering CpG-ODN by the in ovo route and IPL delivery of a CP vaccine at hatch to evaluate nonspecific protection of chickens against E. coli challenge.

Broiler hatching eggs were obtained from a Ross 308 broiler breeder flock and maintained at the Animal Care Unit (ACU), Western College of Veterinary Medicine (WCVM), University of Saskatchewan, Canada. Following hatch, water and commercial broiler starter ration were provided ad libitum. Air from each room was exhausted through a HEPA filter, and non-recirculated intake air was provided at a rate of 15–20 air changes/hour. Air pressure differentials and strict sanitation were maintained in this isolation facility. Broiler chicks were raised at 32 C for the first 7 days of life, after that, the temperature was decreased by 0.5 C per day until a room temperature reached 27.5 C. Light (30 lux) was provided for 24 hr/day during Days 0–2 posthatch. Darkness was introduced at 3 days posthatch with 1 hr of darkness added daily until 4 hr of darkness per day was achieved.

The sequence of Class B CpG-ODN²⁰⁰⁷ used in all animal experiments was 5′-TCGTCGTTGTCGTTTTGTCGTT-3′ (free of endotoxin) as previously described (11). CpG-ODN was synthesized with a modified phosphorothioate backbone (Operon Biotechnologies, Inc., Huntsville, AL, USA). CpG-ODN was diluted in sterile pyrogen-free phosphate-buffered saline (PBS). Each embryonating broiler hatching egg at Day 18 of incubation or neonatal broiler chicken received CpG-ODN (50 μg/egg) (11) in a total volume of 100 μl by the in ovo route, intramuscular route (IM; 50 μg/bird) (10) or IPL route (50 μg/bird) (15). The control group received saline by the IM, in ovo, and IPL routes.

The E. coli challenge preparation was conducted as previously described (11,43). The E. coli strain was isolated from a turkey with septicemia; the bacterium belongs to serogroup O2 and is a nonhemolytic, serum-resistant, produced aerobactin with a contained K1 capsule and type 1 pili. Briefly, E. coli was cultured for 18–24 hr at 37 C on 5% Columbia sheep blood agar (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). A single colony of bacteria from the agar plate was added to 100 ml of Luria broth in a 250-ml Erlenmeyer flask. The culture was grown at 37 C for 16–18 hr, shaking at 150 rpm. After incubation, the cultures contained approximately 1 × 10⁹ colony forming units (CFU)/ml of stationary phase of bacteria. These were further diluted in sterile saline to the concentration of bacteria required in the challenge experiments. The E. coli challenge was conducted with 1 × 10⁶ CFU/bird and 1 × 10⁷ CFU/bird of bacteria in a total volume of 250 μl of saline, subcutaneously (SC) in the neck (44,45). Mortality, cumulative clinical score (CCS), and bacterial load in the air sac were recorded over 7 days postchallenge. Clinical scoring and bacterial load from the air sac were measured as described above. Clinical signs and a daily CCS were assigned to each bird as previously described (36): 0 = normal; 0.5 = slightly abnormal appearance, slow to move; 1 = depressed, reluctant to move; 1.5 = reluctant to move, may take a drink and peck; 2 = unable to stand or reach for food or water; and 3 = found dead. Chicks with a clinical score of 2 were euthanized by cervical dislocation. A CCS was given at the end of the trial, with each bird given a sum of daily clinical scores, as previously described (13). Dead or euthanized chicks were necropsied immediately. All remaining birds were euthanized at 7 days postchallenge. Swabs were taken from the air sacs, and a semiquantitative 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 = less than 5 colonies; 1+ = growth of bacteria on quadrant 1; 2+ = growth of bacteria on quadrants 1 and 2; 3+ = growth of bacteria on quadrants 1, 2, and 3; and 4+ = growth of bacteria on all quadrants 1–4 as reported previously (46).

In this study, a well-established animal model of NE was used (47). Briefly, day-old broiler chickens were fed a commercial raised without antibiotics (RWA) broiler starter ration containing 20% protein (Farm Choice^TM RWA, Masterfeeds, Canada) until 18 days of age followed by feed withdrawal at 19 days of age. Then at 20 days of age, the diet was abruptly changed to a new feed ration containing 25% RWA turkey starter (MasterFeeds, Canada) and 38% poultry supplement (MasterFeeds, Canada) at a 1:3 ratio to obtain a ration containing 28% protein. In order to challenge broilers with CP, broilers were fed the 28% protein ration containing a fluid thioglycolate (FTG) -grown, clinical isolate of CP with cpa, netB, cpb2, and tpeL toxin genes. Briefly, a single colony was individually transferred to cooked meat broth media (CMM, Sigma-Aldrich, Canada) and incubated at 37 C for 24 hr, under anaerobic conditions. Ten milliliters of cultured CMM broth was diluted with 490 ml of CMM (dilution factor 50; v/v) and incubated for 24 hr at 37 C. Subsequently, the CMM culture was used to inoculate FTG (Sigma-Aldrich; 3% [v/v]) and incubated anaerobically at 37 C for 15–16 hr and 1 × 10⁹ CFU/bird was used to challenge broilers at 20, 21, and 22 days of age as previously described (47). Chickens were observed for clinical signs and mortality three times per day until the termination of the experiment at 23 days of age. At termination of the trial, birds were euthanized and the entire length of the intestine (duodenum, jejunum, and ileum) were examined for macroscopic lesions of NE. Samples for histopathology were collected in 10% neutral buffered formalin and stained with hematoxylin and eosin (47). Histopathological intestinal lesions were scored as 0 = no lesions/healthy mucosa; 1 = focal necrosis of intestinal villi, acute; 2 = necrosis of intestinal villi, multifocal to coalescing, acute; and 3 = diffuse, necrosis of intestinal villi, acute, severe in all the birds (47). To prepare the live CP vaccine for IPL delivery at the day of hatch, the bacterial culture was centrifuged at 4000 rpm for 20 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS (pH 7.4, 0.01M), suspended in 10% sucrose at a ratio of 1:1 and lyophilized overnight at −80 C (Labconco Freeze Dry System Freezone 2.5). After lyophilization, CP was stored at 4 C. At the time of IPL delivery, live lyophilized CP (1 × 10⁸ CFU/bird) was mixed in sterile 0.9% saline and aerosolized as 0.5-μm microdroplets using a compressor nebulizer (705-470; AMG Medical Inc., Montreal, QC, Canada) as previously described (15).

An inactivated CP booster was administered by the SC route at 10 days of age. Briefly, to prepare the inactivated CP booster vaccine, the CP (1 × 10⁸ CFU/bird) culture was mixed with 0.04% formaldehyde and incubated at 37 C aerobically overnight. To ensure inactivation of the bacteria, 100 μl of culture was plated on 5% Columbia sheep blood agar and incubated anaerobically for 18–24 hr. Then, the inactivated CP (1 × 10⁸ CFU/bird) was formulated with CpG-ODN²⁰⁰⁷ as an adjuvant (10 μg/bird; Operon Biotechnologies, Inc., Huntsville, AL).

Variant IBDV (varIBDV) SK09, the most prevalent varIBDV in the broiler chicken industry in Canada, was used as the challenge isolate (44,48). The vIBDV SK09 was propagated in specific-pathogen free (SPF) birds by oral administration as described previously (49). Briefly, the bursa of Fabricius (BF) from the SPF birds were collected 3 days following oral administration of varIBDV SK09. Then, BF was homogenized to obtain varIBDV stock containing 40% (w/v) suspension in sterile saline. The viral stocks were examined by qPCR to ensure that stocks are free from other avian pathogens. The varIBDV stocks were titrated in SPF embryos, and the egg infectious dose (EID₅₀) was determined according to the Reed–Muench method (50). Neonatal broiler chickens were infected with varIBDV SK09 at 6 days of age of by the oral route. Each broiler chicken received 3 × 10³ EID₅₀ of varIBDV SK09 as previously described (51).

Real-time live cell analysis of glycolysis based on extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation (OXPHOS) based on the oxygen consumption rate (OCR) were quantified using the Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, CA, USA) as previously described (42). Briefly, XFp culture plates were coated with 50 μl poly-d lysine in distilled water (1:8) and incubated at 4 C for 24 hr and sensor cartridges were hydrated overnight with 200 μl XF calibrant fluid in wells and 400 μl in each moat at 37 C without CO₂. XF calibrant fluid and Roswell Park Memorial Institute (RPMI) culture media were incubated overnight at 37 C without CO₂. On the day of the experiment, peripheral blood samples were collected into heparinized tubes from the wing vein using 21-gauge, 1-in. hypodermic needle. Heparinized blood was mixed with same volume of PBS (pH = 7.4) and layered on 3 ml Histopaque 1077® (Sigma Aldrich) and centrifuged for 20 min at 560 × g at 20 C to collect peripheral blood mononuclear cells (PBMC). The PBMC layer was collected gently and washed with 5 ml pre-warmed RPMI and spun at 300 × g for 5 min at 20 C. The supernatant was discarded, and the washing step was repeated. The PBMC pellet was re-suspended in 2–3 ml of RPMI and the live cells were counted using trypan blue. Cells from six birds (n = 6) at 1.5 × 10⁵ cells in 50 μl were added to each well. Then plates were spun for 2 min at 300 × g at 20 C. Then 130 μl of RPMI was added to each well, total volume 180 μl and the plate was incubated for 60 min at 37 C, without CO₂.

Mitochondrial stress assay was performed as previously described (42). Briefly, Seahorse XFp cell culture medium was prepared using RPMI 1640 XF RPMI Medium pH 7.4 (with 1 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), without phenol red, glucose, pyruvate, and l-glutamine), 1 mM pyruvate, 2 mM l-glutamine, and 10 mM glucose (Seahorse, Agilent). Four ports of the XFp cartridge were loaded with different reagents. (a) Twenty microliters of oligomycin (1.5 μM/well) was added into Port A, which inhibits mitochondrial OXPHOS based on the OCR (ATP synthase inhibitor) to shift cellular energy production toward glycolysis (i.e., ECAR values increase). (b) Twenty-two microliters of carbonyl cyanide p-(tri-fluromethoxy)phenyl-hydrazone (FCCP; 2.5 μM/well) was added to Port B to depolarize the mitochondrial membrane to increase oxygen consumption (i.e., OCR values increase, cells achieve maximal ECAR). (c) Twenty-five microliters of rotenone/antimycin A (Rot/AA) mixture (0.5 μM/well) was added in Port C to inhibit complex 1 and 3 of electron transport chain of the mitochondrial respiration. (d) Port D was loaded with culture media.

The assay procedure was similar to mitochondrial stress assay, except the cell culture media was prepared without glucose (only RPMI 1640 and phenol red-free) + pyruvate (1 mM) + (2 mM) l-glutamine solution: (a) 20 μl of glucose (10 mM/well) was added to Port A to stimulate aerobic glycolysis; (b) 22 μl of phorbol 12-myristate 13-acetate (2.5 ng/ml/well) was added specifically to activate protein kinase C; nuclear factor-kappa was added to Port B to reach the highest glycolytic capacity (ECAR); (c) 25 μl of Rot/AA (0.5 μM/well) was added to Port C; (d) 27 μl of 2-deoxy-d-glucose (2-DG; 50 mM/well) was added into Port D to inhibit cellular glycolysis competitively, as previously described (42).

This assay was conducted as previously described (52). Briefly, blood samples (3–4 ml/bird) were collected into heparinized tubes from the brachial vein using a 21-gauge, 1-in. hypodermic needle. Blood samples were mixed with the same volume (1:1) of 4 ml of PBS with 1% penicillin-streptomycin (Thermo Fisher Scientific Inc., Waltham, MA, USA) and layered on 3 ml Histopaque 1077® and centrifuged for 20 min at 560 × g at 20 C to collect the PBMC layer. The PBMC layer was collected in a new 15-ml centrifuge tube and washed three times with MACS buffer prepared using PBS supplemented with 0.5% bovine serum albumin (Sigma Aldrich) and 2 mM disodium ethylenediaminetetraacetate dehydrate (Sigma Aldrich). Live cells were counted using trypan blue. Monocytes at the concentration of 1 × 10⁵ cells were resuspended in MACS buffer. MACS was performed according to the manufacturer’s instructions (Miltenyl Biotec Inc., San Diego, CA). Monocytes were stained with mouse anti-chicken monocyte/macrophage-PE (KUL01) and incubated for 20 min on ice in the dark. Then, cells were washed twice with 10 ml of MACS buffer and centrifuged at 300 × g at 4 C for 10 min. After washing, the cell pellet was resuspended in the MACS buffer. Anti-PE microbeads (Miltenyi Biotec, Auburn, CA, USA) were added into tubes and incubated for 15 min on ice in the dark. The washing step was repeated with 10 ml of MACS buffer and cells were resuspended in MACS buffer. OctoMACS™ separator and MS columns (Miltenyi Biotec) were used to sort monocytes. The number and viability of sorted cells after sorting were determined using a hemacytometer and a trypan blue exclusion method. Live and dead cells were confirmed using 7-amino-actinomycin D dye, followed by incubation for 20 min at 4 C. Cells were washed three times and resuspended in ∼300 μl of flow cytometric buffer (PBS containing 2% fetal bovine serum). Samples were processed for flow cytometric analysis. Cells were gated based on a forward and side scatter. Fluorescence minus one control was used to identify and gate positive populations. Flow cytometry data were acquired by Cytoflex Flow Cytometer (Beckman Coulter, Carlsbad, CA). FlowJo (Tree Star, Ashland, OR, USA) software was used to analyze data.

Expression of mRNA of IL-1β and TNF-α cytokine genes in the isolated peripheral monocyte/macrophages were measured. RNA was extracted according to the manufacturer's instructions using RNeasy Kit (mini; QIAGEN, Toronto, Ontario, Canada,). The RNA concentration was calculated by NanoDrop® ND-1000 spectrophotometry. cDNA was synthesized from DNase-treated RNA (monocyte/macrophages 60 ng of RNA) using QuantiTect Reverse Transcription Kit (QIAGEN) and Quantiscript Reverse Transcriptase (QIAGEN) according to the manufacturer’s instructions.

Diluted cDNA at 1:2 (monocyte/macrophage samples) in nuclease-free water was used in a RT-PCR to evaluate host gene cytokine expressions. RT-qPCR was conducted using the BioRad CFX96 (Bio-Rad Laboratories Ltd, ON, Canada) and iTaq™ Universal SYBR® Green Supermix (Bio-Rad Laboratories). Primers mentioned in Table 1 were used for qPCR analysis. The primers were synthesized by Integrated DNA Technologies (IDT, Coralville, IA). The relative expression of target genes was calculated using BioRad CFXmaestro software in relation to chicken β-actin.

This experiment was conducted to investigate the effect of in ovo delivery of CpG-ODN (50 μg/egg) on cellular glycolysis and mitochondrial OXPHOS capacity of PBMC in broiler chickens vaccinated with live CP by the IPL route at hatch and boosted by the SC route at 10 days of age with inactivated CP adjuvanted with CpG-ODN (10 μg/bird). Metabolic output was measured at 1 (11 days of age) and 19 days (29 days of age) post-CP booster vaccination. Fertile broiler eggs were incubated at the ACU and randomly assigned (n = 35/group) to treatment groups: (1) in ovo CpG-ODN + live CP vaccine by the IPL route + booster, (2) in ovo saline + live CP by the IPL route + booster, (3) in ovo saline + no CP vaccine. CP challenge was conducted at 20 days of age for three consecutive days, and pathology of the intestines was scored at 23 days of age. Additional birds from each group (n = 6) were not challenged with CP were performed with cellular glycolysis and mitochondrial OXPHOS at 11 and 29 days of age as a longitudinal study.

The foregoing experiment was repeated with groups of birds to explore the effect of in ovo delivery of CpG-ODN (50 μg/egg) as a control group to compare groups vaccinated at hatch with live CP on cellular glycolysis and mitochondrial OXPHOS capacity of PBMC following booster vaccination at 10 days of age with inactivated CP adjuvanted with CpG-ODN (10 μg/bird). Fertile broiler eggs were incubated at the ACU and randomly assigned (n = 35/group): (1) in ovo CpG-ODN + live CP vaccine by the IPL route + booster; (2) in ovo saline + live CP vaccine by the IPL route + booster; (3) in ovo CpG-ODN + no CP vaccine. CP challenge was conducted at 20 days of age and pathology of the intestines was scored until 23 days of age. Additional birds from each group (n = 6) were not challenged with CP and conducted cellular glycolysis and mitochondrial OXPHOS at 13, 24, and 31 days of age or 3, 14, and 21 days postbooster vaccination as a longitudinal study.

We have previously demonstrated induction of trained immunity by two injections of CpG-ODN by the IM route at Days 1 and 4 of age leading to protect birds against E. coli infection at Day 27 of age (42). The current experiment was conducted to investigate the induction of trained immunity by administration of CpG-ODN (50 μg/bird) at 1 and 4 days of age to protect birds against NE at 20 days of age. At hatch, birds were randomly divided into two groups (n = 27/group). One group was administered with CpG-ODN by the IM route at 1 and 4 days of age and the other group was administered with saline as the control group. Before challenge, at 19 days of age, PBMCs were collected from each group (n = 6/group) and cellular metabolism (mitochondrial OXPHOS and cellular glycolysis) was quantified. Birds were challenged with CP at 20–22 days of age, as described previously (47). Morality and clinical signs were recorded. Gross and histopathological lesions of the intestines were scored at 23 days of age as described previously (47).

This experiment was conducted to explore the immunoprotective effects against E. coli septicemia at 27 days of age following administration of CpG-ODN by the in ovo and IPL routes or a combination of CpG-ODN by the in ovo route and IPL delivery of a live CP vaccine at hatch. Fertilized eggs were incubated in ACU and randomly divided into five groups (n = 30/group); (1) in ovo CpG-ODN + live CP vaccine by the IPL route + booster + E. coli challenge; (2) in ovo CpG-ODN + live CP vaccine by the IPL route + E. coli challenge; (3) in ovo CpG-ODN + IPL CpG-ODN + E. coli challenge; and (4) in ovo saline + E. coli challenge. Birds were challenged with E. coli at 27 days of age with low dose (1 × 10⁶ CFU/bird) and high dose (1 × 10⁷ CFU/bird) by the SC route in the neck as previously described (n = 30/group) (44). Birds were monitored three times per day for 7 days postchallenge.

Kinetics of immune cell metabolism, mitochondrial OXPHOS, and cellular glycolysis was quantified at 9 days of age (before booster) and 25 days of age (after booster and before challenge; n = 4/group) in Groups 1, 2, 3, and 4.

Following E. coli challenge, monocyte/macrophages were isolated from the PBMC layer from groups (1) in ovo CpG-ODN + live CP vaccine by the IPL route + booster + (with and without E. coli challenge); (4) in ovo saline, and (5) in ovo saline + E. coli challenge and quantified IL-1β and TNF-α gene expression (n = 5/group).

This experiment was conducted to investigate the effect of immunosuppression on cellular glycolysis and mitochondrial OXPHOS capacity of PBMC in broiler chickens vaccinated with live CP by the IPL route at hatch following in ovo delivery of CpG-ODN (50 μg/egg) and boosted at 10 days of age by the SC route with inactivated CP adjuvanted with CpG-ODN. One group was exposed to varIBDV SK09 with (1 × 10³ EID₅₀) at 6 days of age by the oral route as previously described (44). Fertile broiler eggs were incubated at the ACU and randomly assigned (n = 35/group): (1) in ovo CpG-ODN + live CP vaccine by the IPL route + booster; (2) in ovo CpG-ODN + live CP vaccine by the IPL route + varIBDV + booster; (3) in ovo saline + no CP vaccine. CP challenge was conducted at 20 days of age for three consecutive days, and pathology of the intestines was scored at 23 days of age. Additional birds from each group (n = 6) were not challenged with CP. Cellular glycolysis and mitochondrial OXPHOS quantification was conducted at 12 and 31 days of age or 2 and 21 days postbooster vaccination as a longitudinal study.

Survival, gross, and histopathologic data were analyzed using Prism (Prism 6.0; GraphPad Software Inc., San Diego, CA) with a significance level of P < 0.05. Survival and mortality data were compared using the log-rank (Mantel-cox) and chi-square tests. Microscopic lesions of the intestine were analyzed between groups using nonparametric one-way ANOVA and Tukey’s multiple comparisons. Cellular metabolism of each group's minimum, maximum, and spare respiratory capacity and compensatory glycolysis were analyzed using two-sample t-tests (nonparametric—Mann-Whitney test) when two groups were compared. A one-way ANOVA test (non-parametric—Kruskal-Wallis test) was used to compare more than two groups. Dunnett's multiple comparison was used as a post hoc test following ANOVA to assess significance among each treatment group compared to the control group.

The cellular glycolysis and mitochondrial OXPHOS capacity of PBMC was analyzed at 1 (11 days of age) and 19 days (29 days of age) post-IPL delivery of CP at hatch and CP booster vaccination at 10 days of age to investigate the effect of in ovo delivery of CpG-ODN on induction of immunity against NE. Kinetics of OCR representing mitochondrial OXPHOS of isolated PBMCs at 1 and 19 days postbooster showed an increasing pattern in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) compared to Group 2 (in ovo saline + live CP vaccine by the IPL route + booster) and Group 3 (in ovo saline + no CP vaccine; Fig. 1A,B). At 1 day postbooster, maximal and spare respiratory capacity of Group 1 were significantly higher (P = 0.0068 and P < 0.0001) compared to Group 2 (in ovo saline + live CP vaccine by the IPL route + booster) and Group 3 (in ovo saline + no CP vaccine; P < 0.0001 and P < 0.0001). Compared to Group 2 (in ovo saline + live CP by the IPL route + booster) and Group 3 (in ovo saline + no CP vaccine), Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) had significantly higher maximal and spare respiratory capacities (P = 0.0183 and P < 0.001) and (P = 0.0005 and P < 0.0001) at 19 days postbooster (Fig. 1C,D).

Kinetics of ECAR representing cellular glycolysis of isolated PBMCs at 1 day postbooster were significantly (P < 0.0443 and P < 0.0001) elevated in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) compared to Group 2 (in ovo saline + live CP vaccine by the IPL route + booster) and Group 3 (in ovo saline + no CP vaccine; Fig. 1E,G); however, no difference was observed at Day 19 postbooster (Fig. 1F,H).

After challenging birds with CP at 20 days of age, Group 3 (in ovo saline + no CP vaccine) had 20% mortality. In contrast, Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) and Group 2 (in ovo saline + live CP vaccine by the IPL route + booster) had no mortality following CP challenge. Of the birds in Group 3 (in ovo saline + no CP vaccine), 100% had NE lesions (70% of birds with macroscopic lesions [Score 3] and 30% of birds with microscopic lesions [score 2 in 20% birds and score 1 in 10% of birds]). Compared to Group 3, NE lesion scores were significantly (P < 0.0001) lower in Group 1 (in ovo CpG-ODN + live CP by the IPL route; Score 1 in 5% of birds and Score 2 in 5% of birds (P < 0.0001) and Group 2 (in ovo saline + live CP by the IPL route) NE lesion scores (Score 1 in 15% of birds).

To explore the effect of in ovo delivery of CpG-ODN on the cellular glycolysis and mitochondrial OXPHOS capacity of PBMC, glycolysis and mitochondrial OXPHOS was conducted at Day 3 (13 days of age), Day 14 (24 days of age), and Day 21 (31 days of age) following CP booster to investigate induction of immunity against NE. At Day 3 postbooster, Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) had significantly higher basal (P = 0.0365, P = 0.0116), maximal (P = 0.0036, P = 0.0003), and spare (P < 0.0001, P < 0.0001) respiratory capacities compared to Group 2 (in ovo saline + live CP vaccine by the IPL route + booster) or Group 3 (in ovo CpG-ODN + no CP vaccine), respectively. At Day 14 postbooster, mitochondrial OXPHOS was further increased in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) basal (P = 0.0093, P = 0.0201), maximal (P < 0.0001, P < 0.0001), and spare respiration (P < 0.0001, P < 0.0001) compared to Group 2 (in ovo saline + live CP vaccine by the IPL route + booster) or Group 3 (in ovo CpG-ODN, no CP vaccine), respectively. At Day 21 post-CP booster, mitochondrial OCR was significantly higher in Group 1 (in ovo saline + live CP vaccine by the IPL route + booster) basal (P = 0.0182), maximal (P = 0.0150, P < 0.0001), and spare respiration (P = 0.0035, P < 0.0001) compared to Group 2 (in ovo saline + live CP vaccine by the IPL route + booster) or Group 3 (in ovo CpG-ODN + no CP vaccine) respectively (Fig. 2A,B).

Kinetics of ECAR in Groups 1, 2, and 3 at 3, 14, and 21 days post-CP booster are shown in Fig. 2C,D. At 3 days postbooster, Group 1 (in ovo CpG-ODN+ live CP vaccine by the IPL route + booster) had significantly (P < 0.0001 and P < 0.0001) higher ECAR compared to Group 2 (in ovo saline + live CP vaccine by the IPL route + booster) or Group 3 (in ovo CpG-ODN + no CP vaccine). At 14 days post-CP booster, Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) had significantly (P = 0.0003 and P = 0.0005) higher ECAR compared to Group 2 (in ovo saline + live CP by the IPL route + booster) and Group 3 (in ovo CpG-ODN + no CP vaccine) . There was no significant difference in compensatory glycolysis among groups at Day 21 post-CP booster (Fig. 2D).

This experiment was conducted to investigate the induction of trained immunity by two IM injections of CpG-ODN at Days 1 and 4 of age against NE at Day 20 of age by measuring cellular glycolysis and mitochondrial OXPHOS capacity. Kinetics of OCR showed an increasing pattern in the CpG-ODN–treated group compared to the saline group (Fig. 3A). The group that was administered CpG-ODN had significantly higher maximal (P = 0.0006) and spare (P = 0.0242) respiratory capacity than the saline group prior to challenge with CP at 20 days of age (Fig. 3B). There was no difference between ECAR values between the two groups (Fig. 3C,D).

The group that received CpG-ODN had 11% mortality, and the group that received saline had 18.5% mortality (Fig. 3E). Gross lesions of NE were observed in 11% of birds in the group that received CpG-ODN (Fig. 3F). In contrast, 58% of birds in the saline group had gross lesions of NE (P < 0.0001). On histopathology, 14% of birds in the CpG-ODN group had NE lesions (Score 3 [11%], Score 2 [3%], Score 1 [0%], and Score 0 in [86%]). In contrast, 100% of birds in the saline group had NE lesions (Score 3 [60%], Score 2 [20%], and Score 1 [20%]; P < 0.0001; Fig. 3G).

This experiment was conducted to investigate the induction of trained immunity by in ovo CpG-ODN at 18 days of incubation and IPL delivery of a live CP vaccine at hatch against E. coli challenge at 27 days of age. Mitochondrial OXPHOS and cellular glycolysis were quantified at 9 days (before booster) and 25 days (after booster and before E. coli challenge) of age. OCR was increased (Groups 1, in ovo CpG-ODN + live CP vaccine by the IPL route + booster; Group 2, in ovo CpG-ODN + live CP vaccine by the IPL route; and Group 3 in ovo CpG-ODN + IPL CpG-ODN compared to Group 4 in ovo saline) at both time points (Fig. 4A,B). At 9 days of age, basal, maximal, and spare respiratory capacities were significantly higher in Groups 1 and 2 (in ovo CpG-ODN + live CP vaccine by the IPL route; P = 0.0014, P < 0.0001, P < 0.0001), and 3 (in ovo CpG-ODN + IPL CpG-ODN; P = 0.0067, P < 0.0001, P < 0.0001) compared to Group 4 (in ovo saline; Fig. 4C). OCR values (basal, maximal, and spare respiratory capacity) were significantly higher prior to challenge with E. coli at 25 days of age in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster; P = 0.0059, P < 0.0001, P = 0.0012), Group 2 (in ovo CpG-ODN + live CP vaccine by the IPL route; P = 0.0031, P < 0.0001, P = 0.0008), and Group 3 (in ovo CpG-ODN + IPL CpG-ODN; P = 0.0040, P < 0.0001, P = 0.0045; Fig. 4D). In contrast, cellular glycolysis was elevated only at 9 days of age (Fig. 4E,F). At 9 days of age, cellular glycolysis was also significantly elevated in Groups 1 and 2 (in ovo CpG-ODN + live CP vaccine by the IPL route; P = 0.0003) and Group 3 (in ovo CpG-ODN + IPL CpG-ODN; P = 0.0025; Fig. 4G). However, there was no difference in ECAR values among groups at 25 days of age (Fig. 4H).

Birds were significantly protected against E. coli challenge (P < 0.0001) in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster), Group 2 (in ovo CpG-ODN + live CP vaccine by the IPL route), and Group 3 (in ovo CpG-ODN + IPL CpG-ODN) with survival of 83%, 77%, and 77%, respectively, compared to Group 5 (in ovo saline + E. coli challenge) with 33% survival (Fig. 5A). CCS of Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster; P < 0.0001), Group 2 (in ovo CpG-ODN + live CP vaccine by the IPL route; P = 0.0003), and Group 3 (in ovo CpG-ODN + IPL CpG-ODN; P = 0.0016) were significantly lower compared to Group 5 (in ovo saline + E. coli challenge; Fig. 5B).

Bacterial loads were significantly lower in Group 1 (in ovo CpG-ODN + live CP vaccine + booster; P = 0.0040), Group 2 (in ovo CpG-ODN + live CP vaccine by the IPL route; P = 0.0045), and Group 3 (in ovo CpG-ODN + IPL CpG-ODN; P = 0.0050) compared to Group 5 (in ovo saline + E. coli challenge; Fig. 5C). IL-1β and TNF-α were significantly elevated (P = 0.0070 and P < 0.0001) in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) compared to Group 5 (in ovo saline + E. coli challenge; Fig. 5D,E).

This experiment was conducted to measure the immunosuppressive effects of varIBDV SK09 on cellular glycolysis and mitochondrial OXPHOS following in ovo delivery of CpG-ODN and subsequent IPL delivery of CP at hatch and a CP booster at 10 days of age against NE. OCR was significantly higher at both time points in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster; basal [P < 0.0003], maximal [P = 0.0003], and spare respiratory capacity [P = 0.0003]). In contrast, OCR did not increase in Group 2 (in ovo CpG-ODN + live CP vaccine by the IPL route + varIBDV + booster) compared to Group 3 (in ovo saline + no CP vaccine; Fig. 6A,B). At Day 21 postbooster, maximal and spare respiratory capacities were significantly higher (P < 0.01 and P < 0.01) in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) compared to Group 3 (in ovo saline + no CP vaccine). In contrast, no difference was observed for basal, maximal, and spare respiratory capacity of Group 2 (in ovo CpG-ODN + live CP vaccine by the IPL + varIBDV SK09 + booster) and Group 3 (Fig. 6C,D). Compensatory glycolysis was significantly (P = 0.0001) higher at 2 days postbooster in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) than in Group 3 (in ovo saline + no CP vaccine) or Group 2 (in ovo CpG-ODN + live CP vaccine by the IPL route + varIBDV SK09 + booster). There was no difference in ECAR among groups at Day 21 postbooster (Fig. 6G,H).

Following CP challenge, Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster) had no mortality. There was 20% mortality in Group 3 (in ovo saline + no CP vaccine) and 100% of birds had NE lesions (70% of birds with macroscopic lesions and 30% of birds with microscopic lesions). In contrast, 10% of birds in Group 1 (in ovo CpG-ODN + live CP vaccine by the IPL route + booster + CP challenge) had NE lesions (5% birds with macroscopic lesions and 5% of birds with microscopic lesions; P < 0.0001).

Trained immunity or innate immune memory describes the long-term functional reprogramming of innate immune cells. This is evoked by endogenous or exogenous insults leading to an enhanced response towards a secondary challenge after return to a nonactivated state. Nonpermanent genetic changes occur in innate immune cells during this training, such as epigenetic modifications to augment defense mechanisms like cytokine production and bactericidal activities (53). This concept has been studied widely in humans (36,54), mice (32,55), and plants (56). A study conducted in humans demonstrated protection of people against yellow fever virus infection 1 mo following BCG vaccination because of induction of epigenetic reprogramming of the PI3K/AKT/mTOR pathway and associated cytokine production (i.e., IL-1β and IL-6) by monocytes (54). Immunizing mice with BCG led to a T-cell–independent protection against secondary infections such as C. albicans and S. mansoni (57). It had been demonstrated that trained immunity protected mice against E. coli, S. aureus, and L. monocytogenes infections following injection of zymosan at 3 and 7 days prior (58). Furthermore, countries where people were vaccinated with BCG had reduced disease spread, severity, and death during the COVID-19 pandemic because of trained immunity induced by the BCG vaccine (59). Therefore, the objectives of this study were to explore the ability of CpG-ODN to induce trained immunity in broiler chickens (1) by administering CpG-ODN by the in ovo route and IPL route at hatch and (2) administration of CpG-ODN by the in ovo route and IPL delivery of a live CP vaccine at hatch to protect them against E. coli infections.

Recently, we were able to demonstrate protection of broiler chickens against NE by a novel technique of IPL delivery of live CP at hatch with no booster vaccine, utilizing the lung–gut-axis concept (28). During that study we found IPL delivery of live CP following in ovo delivery of CpG-ODN to enhance immune maturation and immune enrichment and provide protection against NE without utilizing lung–gut-axis concept (28). In order to study the mechanisms associated with IPL delivery of live CP vaccine following in ovo delivery of CpG-ODN, we conducted the first experiment (Experiment A). The group that received CpG-ODN by the in ovo route prior to IPL delivery of a CP vaccine at hatch had significantly higher maximal and spare respiratory capacity of OXPHOS in mitochondria. In contrast, birds that received a CP vaccine by the IPL route at hatch without in ovo CpG-ODN did not have increased mitochondrial OXPHOS and mitochondrial respiratory capacity, similar to the group that received saline by the in ovo route. This observation indicated that the group that received CpG-ODN by the in ovo route prior to IPL delivery of CP induced trained immunity and protected the animals against NE. Then, we compared the immunoprotective effects of the group that received CpG-ODN by the in ovo route prior to IPL delivery of CP at hatch against the group that only received CpG-ODN (Fig. 2). Here, we confirmed that the group that received CpG-ODN by the in ovo route prior to IPL delivery of CP at hatch had significantly higher mitochondrial basal, maximal, and spare respiratory OXPHOS capacity compared to the group that received a CP vaccine by the IPL route alone or CpG-ODN by the in ovo route without a CP vaccine. We have previously demonstrated that induction of trained immunity associated with shifting metabolic pathways of immune cells toward mitochondrial OXPHOS from glycolysis is likely caused by immune cell transitioning to trained immune cells or innate memory cells to increase the ability to survive and maintain energy production (42). In this experiment, we see the same phenomenon that glycolytic activity switched to mitochondrial OXPHOS following CpG-ODN delivery by the in ovo route and CP vaccine delivery by the IPL route. Because groups in Experiment A received a booster vaccine containing inactivated CP adjuvanted with CpG-ODN at Day 10 of age, we cannot confirm whether induction of trained immunity needs both CP vaccine at hatch and the booster vaccine.

We have previously demonstrated the utility of a single dose of CpG-ODN against yolk sac infections in neonatal broiler chickens; however, the immunoprotective ability of CpG-ODN declines by 6 days post administration (10,15,60) We have studied extensively to improve the duration of activity of CpG-ODN with different formulations such as carbon nanotubes and liposomes, but we were not successful in finding a formulation to lengthen the duration of the immunoprotective activity of CpG-ODN (11). Recently, we have demonstrated the proof of concepts that delivering CpG-ODN twice by the IM route in neonatal broiler chickens at Days 1 and 4 of age can induce trained immunity and protect birds against lethal E. coli septicemia later in life (42). The aim of Experiment B was to explore whether IM administration of two doses of CpG-ODN at Days 1 and 4 of age protected against NE at Day 20 of age. Birds that received two doses of CpG-ODN at Days 1 and 4 of age had significantly higher maximal and spare respiratory capacity of mitochondrial OXPHOS until Day 20 of age compared to the group that receive two doses of saline. In contrast, glycolytic activity was not elevated at Day 20 of age. The group that received two doses of CpG-ODN by the IM route had significantly lessened mortality and gross and histopathological lesions of NE following CP challenge at Day 20 of age compared to the group that received two IM injections of saline, because of induction of trained immunity. We have previously demonstrated the induction of trained immunity by IM delivery of two doses of CpG-ODN against a gram-negative bacterium, E. coli. Here, we demonstrated protection of birds against a Gram-positive bacterium, CP, following induction of trained immunity by CpG-ODN.

One of the aims in Experiment C was to study whether induction of trained immunity could be ensured by in ovo delivery of CpG-ODN and IPL delivery of CpG-ODN at hatch. Birds administered with CpG-ODN by the in ovo route and IPL route at hatch had increased mitochondrial OXPHOS; hence it was confirmed that induction of trained immunity can be accomplished by in ovo delivery of CpG-ODN and IPL delivery of CpG-ODN at hatch. Neonatal broiler chickens that received CpG-ODN by the in ovo and IPL routes had significantly low mortality, clinical score, and bacterial load in the air sacs at Day 27 of age following E. coli challenge compared to birds that did not receive CpG-ODN. Delivery of CpG-ODN by the in ovo route and IPL routes are industry feasible techniques, hence increasing the utility of CpG-ODN in the broiler chicken industry. We have demonstrated IPL delivery of CpG-ODN under field conditions previously as an industry feasible technique (61). The second aim in Experiment C was to study whether induction of trained immunity needed a combination of a CP vaccine at hatch and CP booster at 10 days of age following in ovo delivery of CpG-ODN. Birds administered with in ovo CpG-ODN and a live CP vaccine by the IPL route at hatch with or without a CP booster vaccine had increased mitochondrial OXPHOS; hence it is confirmed that only in ovo delivery of CpG-ODN and IPL delivery of CP vaccine at hatch was required to induced trained immunity. Furthermore, the same phenomenon of switching cellular energy demand from glycolytic activity to mitochondrial OXPHOS in birds induced with trained immunity was observed. This trained immunity protected them against E. coli septicemia following administration of CpG-ODN by the in ovo route and delivery of CP at hatch. Two CpG-ODN administrations are required to induce trained immunity in birds,. Here, we have only administered one dose of CpG-ODN by the in ovo route and a CP vaccine at hatch by the IPL route. We hypothesize that birds received a second dose of CpG from the live CP vaccine at hatch. Furthermore, we hypothesize that birds likely received a second dose of CpG-ODN from the dead CP in the lungs, since we could not culture a high number of CP at 24 hr post-IPL vaccination as we have seen previously (28). We have seen increased IL-1β and TNF-α gene expression in monocyte/macrophages from birds administered CpG-ODN by the in ovo route and CP delivery by the IPL route following E. coli challenge compared to birds challenged with E. coli with no CpG-ODN by the in ovo route and CP by the IPL route (P < 0.05). It has been demonstrated previously that individuals vaccinated with BCG had enhanced IL-1β following challenge with yellow fever virus and increased expression of IL-1β strongly correlated with reduced viremia associated with yellow fever (54).

The aim in Experiment D was to study whether the process of induction of trained immunity could be abolished by varIBDV SK09. Groups of chickens received both in ovo CpG-ODN, an IPL CP vaccine and booster then were exposed to varIBDV SK09 at 6 days of age. Birds that received in ovo CpG-ODN, IPL CP vaccine and a booster had significantly higher maximal and spare respiratory capacity of mitochondrial OXPHOS and glycolytic activity. In contrast, birds that received varIBDV SK09 in addition to in ovo CpG-ODN, IPL CP vaccine and a booster abolished higher basal, maximal and spare respiratory capacity of mitochondrial OXPHOS and glycolytic activities and OXPHOS and glycolytic activities, similar to birds that received only saline. IBDV depletes B cells and survive in macrophages. We hypothesize that IBDV abrogates induction of trained immunity by modulating functions of immune cells in broiler chickens (62,63). The scope of this study was not to examine the trained immunity mediated protection against varIBDV, but to observe the role of varIBDV SK09 during induction of trained immunity as birds were challenged soon after CpG-ODN administration (Day 6 of age). However, in future studies we will explore this aspect by analyzing transcriptomics and single cell RNA sequence. This observation illustrates the importance of varIBDV control in the broiler chicken industry as varIBDV associated immunosuppression leads not only to increase secondary bacterial infections but also abrogate induction of trained immunity and associated immunoprotection against other pathogens in broiler chickens.

In summary, we have demonstrated that delivery of CpG-ODN twice in neonatal broiler chickens can induce trained immunity to protect them against NE. Our results revealed that induction of trained immunity was associated with switching cellular energy metabolism from glycolysis to mitochondrial OXPHOS following two CpG-ODN administrations. Switching cellular energy demand from glycolysis to mitochondrial respiration likely associated with immune cells transitioning to trained immune cells or innate memory cells to survive longer. However, it would be important to explore epigenetic modifications such as DNA methylation and transcription of long noncoding RNAs associated with trained immunity to further elucidate mechanisms. We also demonstrated, for the first time, that birds administered with in ovo CpG-ODN and IPL delivery of a live CP vaccine at hatch were able to induce trained immunity and demonstrated protection against E. coli septicemia at 27 days of age. We have previously demonstrated that a single dose of CpG-ODN protected birds against yolk sac infections as an alternative to antibiotics, but here we demonstrated that two CpG-ODN administrations in neonatal broiler chickens induced trained immunity and protected them against bacterial infections later in life.

The authors are grateful to animal care technicians at the ACU, WCVM, 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), NSERC Alliance (427560), Canadian Poultry Research Council (427541) and Results Driven Agriculture Research Program (425526). Authors sincerely thank Dr. Ayumi Matsuyama-Kato (Department of Veterinary Pathology, University of Saskatchewan) for her commendable help for cytokine gene expression analysis. The animal use protocol was approved by the University of Saskatchewan’s Animal Care Committee Animal Research Ethics Board (Certificate of approval 20070008) and was conducted according to the Canadian Council on Animal Care guidelines.

2-DG =

2-deoxy-d-glucose;

ACU =

Animal Care Unit;

AMR =

antimicrobial resistant;

AMU =

antimicrobial use;

BCG =

Bacillus Calmette-Guérin (BCG);

BF =

bursa of Fabricius;

CCS =

cumulative clinical score;

CFU =

colony forming unit;

CMM =

cooked meat broth media;

CP =

Clostridium perfringens;

CpG-ODN =

oligodeoxynucleotides containing unmethylated cytosine-phosphodiester-guanine motifs;

DCs =

dendritic cells;

ECAR =

extracellular acidification;

EID₅₀ =

egg infectious dose rate;

FCCP =

carbonyl cyanide p-(tri-fluromethoxy)phenyl-hydrazone;

GLA =

gut–lung axis;

IBD(V) =

infectious bursal disease (virus);

IL =

interleukin;

IM =

intramuscular;

IPL =

intrapulmonary;

MACS =

magnetic activated cell sorting;

NE =

necrotic enteritis;

NSERC =

Natural Sciences and Engineering Research Council of Canada;

OCR =

oxygen consumption rate;

OXPHOS =

oxidative phosphorylation;

PAMPs =

pathogen-associated molecular patterns;

PBMC =

peripheral blood mononuclear cells;

PBS =

phosphate-buffered saline;

RPMI =

Roswell Park Memorial Institute;

Rot/AA =

rotenone/antimycin A;

rRT-qPCR =

real time quantitative PCR;

RWA =

raised without antibiotics;

SC =

subcutaneous;

SPF =

specific-pathogen free;

Th =

T-helper;

TLR =

toll-like receptors;

TNF =

tumor necrosis factor;

vIBDV =

variant infectious bursal disease virus;

WCVM =

Western College of Veterinary Medicine