Enhancement of protective efficacy of innate immunostimulant based formulations against yolk sac infection in young chicks

This study was conducted to characterize and compare the protective effects of various innate immune stimulants against yolk sac infection (YSI) caused by an avian pathogenic Escherichia coli in young chicks. The immune stimulants were administered alone or in various combinations of unmethylated CpG oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (Poly I:C), and avian antimicrobial peptides (AMPs). Routes included in ovo or in ovo followed by a subcutaneous (S/C) injection. CpG alone and in combination with Poly I:C, truncated avian cathelicidin (CATH)-1(6-26), avian beta defensin (AvBD)1, and CATH-1(6-26) + AvBD1, were administered in ovo to 18-day-old embryonated eggs for gene expression and challenge studies. Next, CpG alone and the potentially effective formulation of CpG + Poly I:C, were administrated via the in ovo route using 40 embryonated eggs. At 1 day post-hatch, half of each group also received their respective treatments via the S/C route. Four hours later, all chicks were challenged using E. coli strain EC317 and mortalities were recorded for 14 d. The first challenge study revealed that amongst the single use and combinations of CpG with different innate immune stimulants, a higher protection and a lower clinical score were offered by the combination of CpG + Poly I:C. The second challenge study showed that this combination (CpG + Poly I:C) provides an even higher level of protection when a second dose is administered via the S/C route at 1 day post-hatch. The current research highlights the efficacy of a combination of CpG + Poly I:C administered either in ovo or in ovo along with a S/C injection and its potential use as an alternative to antibiotics against yolk sac infection in young chicks.

CpG, consisting of CpG motifs that are highly prevalent in bacterial DNA and recognized by TLR9 (Hemmi et al., 2000) and TLR21 (Brownlie et al., 2009) in mammals and chickens, respectively, has shown protection against some common avian pathogens . For instance, administration of CpG via subcutaneous (S/C), intramuscular (Gomis et al., 2003), or in ovo routes (Allan et al., 2018;Gunawardana et al., 2019;Nguyen et al., 2021) or through intrapulmonary delivery (Goonewardene et al., 2017) has shown protection against Escherichia coli infection in chickens. In addition, following intraperitoneal or in ovo administration of CpG, it has exhibited protection against Salmonella Enteritidis (He et al., 2005;Mackinnon et al., 2009) and Salmonella Typhimurium infections (Taghavi et al., 2008). Previous studies reported that in ovo delivery of CpG resulted in reduced mortality and morbidity and induced protection from viral infections including infectious laryngotracheitis virus (ILTV)  and reticuloendotheliosis virus (REV) infections (Yuan et al., 2017). Similarly, application of nanoparticle encapsulated CpG enhanced immune activity and increased the half-life and cellular uptake of CpG (Bavananthasivam et al., 2018). Notably, oral administration of poly D, L-lacticco-glycolic acid (PLGA)-encapsulated CpG reduced colonization of Campylobacter jejuni in cecal contents of chickens (Taha-Abdelaziz et al., 2018). Likewise, in ovo delivery of CpG formulated with carbon nanotubes or liposomes showed protection from E. coli infection in broiler chickens (Gunawardana et al., 2015). Taken together, CpG has potential as an alternative to antibiotics and as a vaccine adjuvant for induction of protective immunity against infectious diseases in poultry.
Poly I:C is a double-stranded RNA and a TLR3 ligand that exhibits antiviral and antibacterial activities. In a previous experiment, administration of Poly I:C enhanced E. coli phagocytosis in murine microglial cells through elevated cytokine induction (Ribes et al., 2010). In addition, enhanced expression of IL-6, IL-8, IL-1a, and monocyte chemoattractant protein-1 in murine lungs was reported following intranasal spray of Poly I: C solution leading to protection against Francisella tularensis infection (Pyles et al., 2010). Moreover, administration of Poly I:C in chicken-derived cells has led to rapid induction of pro-inflammatory cytokines and nitrite production in chicken spleenocytes and monocytes (Villanueva et al., 2011;He et al., 2012;St Paul et al., 2013). Notably, in ovo administration of Poly I:C protected against an E. coli challenge (Allan et al., 2018). In addition, Poly I:C has been used as a vaccine adjuvant for improving humoral and cellular immune responses. Administration of Poly I:C as an adjuvant in a H5N1 influenza vaccine significantly reduced virus shedding in mice (Ichinohe et al., 2009). Similarly, the combination of Poly I:C and H9N2 vaccine significantly induced higher anti-influenza antibody titers, reduced virus shedding, and elevated mRNA levels of IFN-a, IFN-g, IL-6, and MHC-II in ducks (Zhang et al., 2017). In addition, Poly I:C triggered production of pro-inflammatory cytokines and interferon-b, leading to restriction of Marek's disease virus infection in chicken embryo fibroblasts (CEFs) (Zou et al., 2017). In another study, Poly I:C combined with gp90 recombinant proteins significantly reduced the viremia and immunosuppressive effects caused by reticuloendotheliosis virus (REV) challenge in chickens (Yuan et al., 2017). Hence, Poly I:C is not only a potential anti-infection agent, but also a good adjuvant to enhance immune responses for its incorporated vaccines.
Antimicrobial peptides are important signaling molecules of the innate immune system and demonstrate high potency as immunostimulants and antibacterial agents in birds. Besides being directly bactericidal, AMPs can modulate host immunity through 1) activation or recruitment of immunocytes; 2) neutralization of bacterial products, including lipopolysaccharide (LPS) or lipoteichoic acid (LTA) to suppress inflammation; or 3) enhancement of nucleic acid recognition to promote autoinflammation (van Dijk et al., 2011;Zhang and Gallo, 2016;Nguyen et al., 2021). Regarding antibacterial activity, AMPs have shown in vitro killing effects against food-borne pathogens including E. coli, C. jejuni, Clostridium perfringens, S. Typhimurium, and other important pathogenic bacteria and fungi such as Staphylococcus aureus, Streptococcus pyogenes, and Candida albicans (Xiao et al., 2006;van Dijk et al., 2007van Dijk et al., , 2009Zhao et al., 2014;van Dijk et al., 2016;Nguyen et al., 2021). In ovo administration of Cathelicidin-2 (D-CATH-2) showed protection against E. coli infection in chickens by reducing mortality and respiratory bacterial load (Cuperus et al., 2016). Furthermore, recombinant avian CATH and b-defensin (AvBD) have been produced using an E. coli system and proven their antimicrobial activities against multiple microorganisms (Zhao et al., 2014;Yu et al., 2015;Tanhaiean et al., 2018). In our recent study, various peptides, including CATH-1(6-26), CATH-2(1-15), AvBD1, AvBD2, AvBD6, AvBD9 were tested in vitro. CATH-1 (6−26) and AvBD1 were determined to have the most potential as candidates against avian pathogens including E. coli strain EC317, Salmonella sp., C. jejuni, C. perfringens (Nguyen et al., 2021). Notably, in ovo administration of AvBD1 offered a comparable anti-E. coli effect to CpG in young chicks (Nguyen et al., 2021). Used alone, these immunostimulants have potential for prevention of bacterial infections in birds; however, the effects of their combinations are still not fully understood.
Yolk sac infection (YSI) or omphalitis has emerged as the most common bacterial infection that leads to high rates of early chick mortality (ECM) and economic losses in poultry production (Saskatchewan, 2021). Avian pathogenic E. coli (APEC) is a frequently isolated pathogen from poultry farms, followed by various bacteria such as Enterococcus, Staphylococcus, Salmonella, Pseudomonas, Proteus, Streptococcus, and Klebsiella (Olsen et al., 2012;Amare et al., 2013;Swelum et al., 2021). YSI induces mortality or low weight gains and leaky navels in survivor birds (Fasenko and O'Dea, 2008), and increases susceptibility to other infections (Allan et al., 2018). Currently, YSI is managed by heavy culling and administration of antibiotics via in ovo injection or post-hatch (Allan et al., 2018). However, consumer demand for antibiotic-free products and reduction of antibiotic-resistant bacteria has prompted the poultry industry to search for alternative products. Although multiple studies have been performed for development of vaccines as well as antimicrobial alternatives, there are no commercially available alternatives for prevention and control of mortalities due to YSI and other bacterial infections in young chicks (Swelum et al., 2021). The goal of the present investigation was to determine the immune response and protective effects of CpG alone and in combination with Poly I:C and AMPs when administered in ovo or in ovo followed by a S/C injection at one day of age for control of YSI.

Reagents
CpG ODNs 2007 was purchased from Biospring (Frankfurt, Germany) with >85% purity and endotoxin level below 1,000 EU/g. Poly I:C (P1530) was purchased from Sigma_Aldrich (MO). AMPs [AvBD1 and CATH-1(6-26)] were synthesized in linear forms by Genscript (NJ) using the Fmoc solid-phase peptide synthesis (SPPS) method with >95% purity assessed by mass spectrometry (MS). Sequences of CpG ODNs 2007, AvBD1, and CATH-1(6-26) used in this study are listed in Table 1. CpG, Poly I:C, and AMPs were suspended in sterile, endotoxin-free water (Millipore Sigma, MA) as stock solutions. Working solutions for in ovo experiments were prepared by mixing stock solutions with phosphate-buffered saline (PBS; Millipore Sigma, MA).

Animal Experiments
Experiment 1 For the investigation of gene expression following in ovo administration of innate immune stimulants, 185 eighteen-day-old embryonated eggs (from Lohmann LSL-lite layers) were obtained from the breeder operation of the Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. Yolk sacs and livers were collected from 5 embryos and cultured on MacConkey plates to ensure they were free from E. coli infection. The remaining 180 eggs were randomly divided into 6 equal groups as follows: Group A (CpG, 30 mg /embryo); Group B (CpG + AvBD1, 15 mg each/ embryo); Group C [CpG + CATH-1(6-26), 15 mg each/ embryo]; Group D [CpG + AvBD1 + CATH-1(6-26), 10 mg each/embryo]; Group E (CpG + Poly I:C, 15 mg each/embryo); Group F (PBS Control). Each embryo received 100 mL of treatment via the in ovo route into the amniotic sac. Following in ovo injection, spleen samples were collected from 6 embryos/group at 6, 24, 48, 72, and 96 h postadministration. Experiment 2 This experiment aimed at studying protective efficacy of the combinations of innate immune stimulants described in Experiment 1. A total of 240 eighteen-day-old embryonated eggs were randomly divided into 6 groups (A-F, n = 40 eggs/group) and received the same formulations as described in Experiment 1 via the in ovo route. The hatching rate was recorded, and yolk sacs and livers from 3 birds/group were collected for E. coli isolation using MacConkey plates. Twenty-four hours post-hatch all groups were subjected to E. coli 317 challenge. Experiment 3 In order to enhance the protection achieved by administration of the combination CpG + Poly I:C, we designed an experiment using a total of 145 eighteen-day-old embryonated eggs. Prior to innate immune stimulant administration, 5 eggs were Abbreviations: AMPs, avian antimicrobial peptides; CpG, unmethylated CpG oligodeoxynucleotides.
ENHANCED EFFICACY OF CPG+POLY I:C checked for E. coli contamination as described above.
The remaining 140 embryos were randomly divided into 3 groups (A, B, and C) with n = 40 embryos/group and group D with 20 embryos. Each in ovo treatment was 100 mL of the respective stimulant or sham solution/ embryo. Group A was treated with CpG, 30 mg/embryo, whereas group B received CpG + Poly I:C, 15 mg each/ embryo. Group C was given PBS in ovo as a control, while group D received no in ovo injection. One day post-hatch, all birds in groups A, B, and C were further divided into 2 subgroups with 20 birds per group. Birds in one of the subgroups from treatments A, B, and C received a second dose (same dosages as in ovo injection) via the S/C route. Birds in Group D were injected with the combination (CpG + Poly I:C, 15 mg each/embryo) via the S/C route only. Four hours after S/C injection, all birds were challenged with APEC E. coli strain EC317 as described below.

E. coli Challenge
Prior to challenge, birds were tagged individually and placed into an isolation room in the animal care unit of VIDO, University of Saskatchewan. Liver and yolk sac samples from 3 birds per group were collected for isolation of E. coli as described previously. Each bird was challenged with 35 CFU/100 mL of E. coli strain EC317 into the yolk sac via the intranavel route. For first 6 days postchallenge (dpc), the birds were checked and clinically scored 4 times daily while later clinical observations and scoring was done twice daily up to 14 dpc as reported previously (Allan et al., 2018;Nguyen et al., 2021). Dead and euthanatized birds were necropsied, and livers and yolk sacs were swabbed for E. coli isolation using MacConkey plates. The cumulative clinical scores (CCS) were determined as a sum of the clinical scores over the 14 dpc observational period. All animal experiments were approved by the University of Saskatchewan's Animal Research Ethics Board (proto-col#20160079) following the Canadian Council on Animal Care guidelines for animal use.

Gene Expression Study
RNA Isolation and cDNA Synthesis Spleen samples collected from embryos were directly placed into 1 mL Trizol tubes, homogenized and kept at À80°C until further processing for RNA isolation, which was performed as per manufacturer's instruction. The NanoDrop-ND1000 Spectrophotometer (Thermo Scientific, Canada) was used to determine RNA quality and quantity. Genomic DNA contamination was removed from the isolated RNA samples using DNase I (Thermo Scientific, Canada). cDNA synthesis was carried out using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.) as described previously (Nguyen et al., 2021). Quantitative Reverse Transcription Polymerase Chain Reaction Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using SsoAdvanced Universal SYBR Green Supermix (ThermoFisher scientific) and the primers listed in Table 2. The reaction was performed in a CFX96 System thermocycler (Bio-Rad Laboratories, Inc.) and conditions were as follows: 95°C for 3 min; then 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Target gene expression was normalized to the expression of b-actin using the 2 −ΔΔCt method as previously described (Livak and Schmittgen, 2001).

Statistical Analysis
Data was analyzed and graphs were generated using the Prism 8 software (GraphPad Software Inc., San Diego, CA). The survival proportions were compared between treated and control groups at all time-points using the Gehan-Breslow-Wilcoxon test. Cumulative clinical scoring (CCS) data was analyzed by Kruskal-Wallis nonparametric analysis of variance in Prism 8. Significant differences of gene expression in spleen samples at each time point among groups were analyzed using one-way ANOVA (and nonparametric or mixed) with Tukey's post-hoc test. P value < 0.05 represents significance difference.

Cytotoxicity of Immunostimulants and Their Combinations to HD11 Cells
The cellular toxicity of stand-alone and combinations of CpG, AvBD1, CATH-1(6-26), and Poly I:C with a dosage ranging from 1.56 to 30 mg/mL was tested in HD11 cells. Twenty-four hours post exposure, 2 to 36% of treated cells showed effects when treated with peptides, however, enhanced cytotoxicity to HD11 cells (32 to 81%) was observed in cells treated with CpG and its combinations. Stand-alone use of CpG showed a dose dependent increase in cell toxicity. For instance, CpG at a dose of 1.56 mg/mL caused »32% cell death, whereas, this percentage was increased to 74% at the highest dose of 30 mg/mL. The combinations of CpG with peptides or with Poly I:C also induced toxicity to HD11 cells with an average mortality of 50 to 81% with a dose ranging from 1.56 to 30 mg/mL (Figure 1).

Experiment 1: Cytokine Expression Following In Ovo Injection
To understand effects of innate immune stimulants on cytokine gene expression, spleen samples were collected at 6, 24, 48, 72, and 96 h post in ovo administration. Transcriptional changes in 3 pro-inflammatory cytokine genes, namely IL-1b, IL-8, and IFN-g were investigated ( Figure 2). In most of the treatment groups, upregulation of the IL-1b gene was noticed at various time points. Significantly higher up-regulation of IL-1b was observed in CpG treated birds at 72 h postinjection (PI) compared to the PBS group (1.06-fold vs. 3.6-fold respectively), whereas, CpG + AvBD1 + CATH-1(6-26) treated groups showed expression similar to the PBS group. At 96 h post administration of the immunostimulants, IL-1b gene expression was significantly higher in birds treated with CpG + AvBD1 (2.4-fold) compared to those treated with CpG + CATH-1(6-26) (1.24-fold), CpG + Poly I:C (1.27-fold), and PBS (1.03-fold). Upregulation of the IL8 gene occurred at the early time points post in ovo injection (PI) in birds treated with CpG (6-fold and 3.3-fold at 6 h and 24 h, respectively) and CpG + Poly I:C (2.1fold at 6h PI). The IFN-g gene showed higher gene expressions in CpG and CpG + Poly I:C treated groups at 6 h (6.9-fold and 2.96-fold, respectively), 48 h (9.1-fold and 3.1-fold, respectively), and 72 h PI (4.5-fold and 2.8fold, respectively). However, statistically significant expression of IFN-g compared to the control group was only noticed at 48 h PI.

Experiment 3: Enhancement of Protection Following In Ovo and a Subcutaneous Administration of CpG + Poly I:C Combination
The combination of CpG (15mg) + Poly I:C (15 mg) was chosen to be administered via different routes including in ovo only, in ovo plus S/C, and S/C only. Figure 1. In vitro toxicity of studied immunostimulants and their combinations to HD11 cells. Cytotoxicity to HD11 cells was determined by WST-1 assays. Cells were exposed to stimulants with dosages ranging from 1.56 to 30 mg/mL for 24 h. The graph represents data from three independent experiments on different days with each experiment carried out using three replicates for each group.

ENHANCED EFFICACY OF CPG+POLY I:C
In addition, administration of CpG alone (30 mg) in ovo and in ovo + S/C in two different groups was included as standard controls. Following 14 dpc with E. coli via the intra-navel route, birds in the group treated with CpG + Poly I:C via the in ovo plus S/C route showed the highest protection (100% survival rate), followed by CpG alone (93.33%) and CpG + Poly I:C (89.47%) injected in ovo only. Similar survival rates were found in birds receiving CpG via in ovo + S/C (67%), CpG + Poly I:C S/C only (65%) and those receiving PBS (53 and 64% for in ovo and in ovo + S/C, respectively; Figure 4).

DISCUSSION
Antibiotics have played a pivotal role in prevention and treatment of infectious diseases worldwide, however, their overuse and inappropriate application have led to progressively increasing antibiotic resistance. Emergence of various antibiotic-resistant microorganisms has resulted in multiple setbacks for disease treatment, leading to health and economic burdens on health care systems (Li and Webster, 2018). Recently, innate immune stimulants have been evaluated and proven efficacious as alternatives to antibiotics in several human and animal trials. Immunostimulants including CpG, Poly I:C, and AMPs have shown many benefits including antimicrobial activity, wound recovery efficacy, and ability to reduce sepsis (Ichinohe et al., 2009;Ribes et al., 2010;Allan et al., 2018;Nguyen et al., 2021). In chickens, stand-alone use of these stimulants has produced positive effects against various bacterial pathogens such as avian pathogen E. coli (APEC), Campylobacter sp., and Salmonella sp. and shown good adjuvant potential against viral diseases (Ichinohe et al., 2009;Ribes et al., 2010;van Dijk et al., 2016;Zhang et al., 2017;Allan et al., 2018;Nguyen et al., 2021). In this study, we aimed to characterize the protective efficacies of combinations of these immunostimulants using different administrated approaches for control of YSI in young chicks.
Although the antimicrobial and immunostimulatory effects of CpG, Poly I:C, and AMPs have been investigated in multiple in vitro as well as in vivo experiments, the potential toxicity of these immunostimulators has not been fully understood. In a previous study, we found that among 6 tested antimicrobial peptides, CATH-1(6-26) showed the highest in vitro antibacterial activities and the highest toxicity to chicken macrophage cells (HD11) at concentrations of 7.5 to 30 mg/mL (Nguyen et al., 2021). In the current study, AMPs showed lower Figure 2. Cytokine expression in spleen tissues at 6, 24, 48, 72, and 96 h post in ovo administration of immunostimulants. Six embryos from each group per time point were randomly collected for RNA isolation and qRT-PCR. Data represents mean § SEM. Asterisks (*), (**) mean significant differences with P < 0.05 and P < 0.01, respectively, using one-way ANOVA analysis for comparing data at each time point. toxic effects to HD11 cells compared to CpG and its combinations. HD11 cells treated with AvBD1, CATH-1(6-26), or AvBD1 + CATH-1(6-26) showed up to 36% cell mortality, however, cells receiving CpG and CpG combined with Poly I:C, AvBD1, CATH-1(6-26), or AvBD1 + CATH-1(6-26) had 32% to 81% cell death at 1.56 to 30 mg/mL. In contrast, in ovo administration of these immune stimulants indicated no or very low toxic effects to the chicken embryos as indicated by the improved hatching rates. For instance, the lowest hatching rate (82.05%) was found in the control chicks receiving PBS, whereas these rates were higher in the treated groups (84.62−94.87%). These results are in agreement with our previous report which showed non-significant differences in weights and hatchability of chicks treated with AMPs at a dose of 30 mg/embryo (Nguyen et al., 2021). Therefore, despite in vitro toxicity shown in an HD11 cell line, CpG, Poly I:C, AMPs, and their combinations were shown to be safe for in ovo administration in chicken embryos within the tested dose ranges in the current study.
Previous reports suggest that CpG could promote strong immune responses against pathogens due to the enhanced expression of cytokines and chemokines. CpG administrated via the in ovo route promoted inflammatory responses through enhanced expressions of proinflammatory cytokines such as IL-1b, IL-6, IL-18, and lipopolysaccharide induced tumor necrosis factor (LITAF) (mainly produced by macrophages when stimulated by bacterial products) in lungs and spleen and it was suggested that it could stimulate both Th1 and Th2 types of cytokines (Gunawardana et al., 2019). In addition, the induction of Th1 type responses following intramuscular administration of CpG ODNs in chicken was also reported (Patel et al., 2008). In our study, we evaluated the effects of in ovo administration Survival rates were compared at all time-points between treated and control groups using the Gehan-Breslow-Wilcoxon test. (*) means a significant difference with P < 0.05. (C) Cumulative clinical scores during 14-d post E. coli challenge. Each bird was assigned a daily clinical score as follows: 0 = normal; 0.5 = slow to move; 1 = ruffled feathers, sitting, reluctant to stand, and mouth breathing; 2 = unable to stand or walk, unable to reach feed or water, wings extended, and difficult breathing; and 3 = found dead. Birds with clinical score of 2 were euthanized for bacterial isolation and recorded lesion score. Clinical scoring was performed four times a day for the first 6 d postchallenge, and then twice a day until the experiment termination.
ENHANCED EFFICACY OF CPG+POLY I:C of CpG and its combinations on the expression of three important cytokines (IL-1b, IL-8, IFN-g) in spleen cells. In agreement with previous reports, our data showed that CpG alone could stimulate higher expression levels of all the examined genes than in combination with AMPs and Poly I:C. In addition, we found that the expression levels of these cytokines in the CpG group decreased from 6 h to 96 h postadministration (PA). Due to the variations in expression among the individual embryos, CpG injected birds showed only significant differences in expression of IL-1b at 72 h PA and IFN-g at 48 h PA. Co-administrations of CpG with other immune stimulants induced upregulation of these cytokines in a different manner. For instance, CpG + AvBD1 caused an increase in IL-1b gene expression at 6 h PA, however, a statistically significant difference was only noticed at 96 h PA. Two combinations including CpG + CATH-1 (6-26) and CpG + AvBD1 + CATH-1(6-26) showed increasing expressions of IL-1b and IFN-g, although no statistical difference was observed. The CpG + Poly I:C combination also elicited increased expression of these genes; however, relatively lower expression levels were observed compared to embryos that received CpG alone. This difference in gene expression might be a result of the distinct amounts of each delivered TLR agonists or the interaction between them. During a bacterial infection, multiple nonspecific immune responses are involved such as the recruitment of neutrophils, dendritic cells, and macrophages and the activation of complement and cytokine production to eliminate pathogens (Tosi, 2005). Pro-inflammatory cytokines and chemokines such as IL-1b and IL-8 rapidly induce pro-inflammatory mediators that play pivotal roles in the first line of host defenses (Tosi, 2005). IFN-g, a type II interferon, has both antiviral activity and immunoregulatory functions (Lee and Ashkar, 2018). Therefore, we assume that induction of these cytokines and chemokines following in ovo injection might have led to reduced E. coli infection in the experimentally challenged birds.
It is of note that the differences in administration routes and ages of animals may contribute to differences in antimicrobial responses of toll-like receptor ligands (TLR-Ls). In a previous study, antiviral effects of Poly I:C against avian influenza virus H4N6 LPAIV strain was observed following in ovo and in vitro administration, whereas this antiviral effect was absent when Poly I:C was administered in post-hatched chicks . Administration of CpG in chickens via a single route such as in ovo, S/C, intramuscular, or intrapulmonary was previously reported (Gomis et al., 2003;Goonewardene et al., 2017;Allan et al., 2018;Nguyen et al., 2021). However, the combination of these administration methods has not yet been studied. In the present study, we investigated the protective efficacies of these immune stimulants and their combinations administered in ovo and in ovo followed by a S/C injection at hatch. Our results from the second in ovo treatments demonstrated that administration of CpG + Poly I:C (15 mg each/embryo) provided the highest protective efficacy among various combinations of CpG with AMPs and Poly I:C. The CpG + Poly I:C treatment resulted in 72.41% survival rate compared to 50% survival rate in the control group (PBS) at 14 dpc with E. coli via the intra-navel route. In addition, the third experiment showed that CpG + Poly I:C injected in ovo and then again S/C at hatch induced a significantly higher protection (100% survival rate) than when given in ovo alone (89.47% survival proportion). Interestingly, we also found that CpG in ovo + S/C and CpG + Poly I:C only via the S/C route proved to be poorly protective in the treated chicks (67% and 65% survival rates, respectively). Thus, CpG treatment was more effective via in ovo injection than in ovo + S/C, whereas the combination of CpG + Poly I:C resulted in the highest protection when given both in ovo + S/C, and was only moderate efficacy when delivered by in ovo only.
Poly I:C has been well characterized as an antiviral agent by activation of both innate and adaptive immune responses in animals. However, Poly I:C has also shown protection against multiple bacterial infections. For instance, Ribes et al., 2020 demonstrated that administration of Poly I:C induced protection of immunocompromised mice against E. coli meningitis (Ribes et al., 2020). Poly I:C increased expression of RANTES (Regulated upon Activation, Normal T cell Expressed, and Secreted, also known as CCL5), IFN-g, recruitment of natural killer (NK) cells, and also increased microglial numbers, resulting in a more effective clearance of the pathogen (Ribes et al., 2020). Similarly, feeding turbot (Scophthalmus maximus) a low-dose of 0.00125% Poly I: C for 4 wk induced a stronger inflammatory response, long-term protection against Edwardsiella piscicida, and alleviated white feces syndrome after 3 to 7 wk resting period (He et al., 2021). In chickens, recent reports showed that chicks receiving Poly I:C via in ovo injection had higher survival rates than those in the control group after an E. coli infection (Allan et al., 2018;Sarfraz et al., 2022). A previous study in chickens showed that after Poly I:C treatment, AvBD2, 11, 12, 13, CATH2, CATHB1, and LEAP2 (liver-expressed antimicrobial peptide 2) significantly increased in bone marrow-derived cells (BMCs), and AvBD2, 3, 6, 9, 11, 12, 13, 14, CATH1, CATH3, CATHB1, GNLY (chicken NK-lysin), and LEAP2 increased in chicken embryonic fibroblasts (CEFs) (Jang et al., 2020). Moreover, Poly I:C stimulates the activity of various immune cells, including macrophages (Reimer et al., 2008), dendritic cells (DCs; Pulko et al., 2009), natural killer (NK) cells (Akazawa et al., 2007), and gdT lymphocytes (Shojaei et al., 2009). Therefore, a combination of Poly I:C and CpG could enhance the protection against E. coli challenge in day-old chicks. Moreover, we believe this is the first report showing that repeated CpG + Poly I:C injection after in ovo administration was more effective than a single dose. These results will facilitate further studies to evaluate protective efficacy of the combination of CpG + Poly I:C for control of other bacterial and viral pathogens in the poultry industry. In addition, this combination may be useful as a vaccine adjuvant for chickens as well as other animals.

ACKNOWLEDGMENTS
We acknowledge the Canadian Poultry Research Council (CPRC)/Agriculture-Agrifood Canada (AAFC) (Grant number #421319) and Chicken Farmers of Saskatchewan (SCIDF) (Grant number #421141) for their financial support. We are grateful for the assistance of Animal Care Services at the Vaccine and Infectious Disease Organization (VIDO). VIDO receives operational funding from the Government of Saskatchewan through Innovation Saskatchewan and the Ministry of Agriculture and from the Canada Foundation for Innovation through the Major Science Initiatives for its CL3 facility. This manuscript is published with the permission of the Director of VIDO as journal series no: 987.

DISCLOSURES
The authors declare no conflict of interest.


s2fieldsofstudy: Biology Medicine

text:
The Delta SARS-CoV-2 Variant of Concern Induces Distinct Pathogenic Patterns of Respiratory Disease in K18-hACE2 Transgenic Mice Compared to the Ancestral Strain from Wuhan

ABSTRACT Compared to the original ancestral strain of SARS-CoV-2, the Delta variant of concern has shown increased transmissibility and resistance toward COVID-19 vaccines and therapies. However, the pathogenesis of the disease associated with Delta is still not clear. In this study, using K18-hACE2 transgenic mice, we assessed the pathogenicity of the Delta variant by characterizing the immune response following infection. We found that Delta induced the same clinical disease manifestations as the ancestral SARS-CoV-2, but with significant dissemination to multiple tissues, such as brain, intestine, and kidney. Histopathological analysis showed that tissue pathology and cell infiltration in the lungs of Delta-infected mice were the same as in mice infected with the ancestral SARS-CoV-2. Delta infection caused perivascular inflammation in the brain and intestinal wall thinning in K18-hACE2 transgenic mice. Increased cell infiltration in the kidney was observed in both ancestral strain- and Delta-infected mice, with no clear visible tissue damage identified in either group. Interestingly, compared with mice infected with the ancestral strain, the numbers of CD45+ cells, T cells, B cells, inflammatory monocytes, and dendritic cells were all significantly lower in the lungs of the Delta-infected mice, although there was no significant difference in the levels of proinflammatory cytokines between the two groups. Our results showed distinct immune response patterns in the lungs of K18-hACE2 mice infected with either the ancestral SARS-CoV-2 or Delta variant of concern, which may help to guide therapeutic interventions for emerging SARS-CoV-2 variants.

RESULTS
Disease and weight loss following SARS-CoV-2 ancestral strain and Delta infection in K18-hACE2 mice. SARS-CoV-2-infected K18-hACE2 mice have been shown to recapitulate the clinical signs of severe COVID-19, including a decline in pulmonary function, upregulated innate immune responses in the lung, pulmonary inflammation, and respiratory failure, making them an ideal model to further investigate COVID-19 pathogenesis and compare the diseases caused by SARS-CoV-2 variants (25). To assess the disease induced by the SARS-CoV-2 ancestral strain and Delta infection, 8-week-old female K18-hACE2 mice were inoculated intranasally with 10 4 PFU of SARS-CoV-2 ancestral strain or SARS-CoV-2 Delta strain in a volume of 20 mL. Disease development and weight loss were monitored daily until 6 days postinfection (dpi). At 5 dpi, mice infected with both ancestral and Delta SARS-CoV-2 showed significant weight loss (approximately 6%) (Fig. 1A). Ancestral strain-and Delta-infected mice showed similar disease manifestations (P = 0.0578), including body weakness, restricted movement, labored breathing, and dry eyes (Fig. 1B). By 6 dpi, ancestral strain-and Delta-infected mice had lost approximately 10% to 13% of body weight and showed a disease score between 5 and 7.
Virus replication and tissue tropism of SARS-CoV-2 ancestral strain and Delta in infected K18-hACE2 mice. To determine virus load and dissemination of SARS-CoV-2 ancestral strain and Delta in K18-hACE2 mice, infected mice were euthanized, and tissues were dissected for analysis by plaque assay and qualitative real-time PCR (qRT- Eight-week-old female K18-hACE2 mice were intranasally inoculated with PBS (mock group) or 10 4 PFU of SARS-CoV-2 ancestral or Delta strain (n = 8). Mock-infected mice (n = 4) received 20 mL sterile DMEM with 2% FBS intranasally. Individual mice were monitored daily until reaching a clinical score of .3, when twice-daily monitoring was performed. (A) Weight change was monitored and compared with the initial weight on day 0. (B) Mice were given a disease score according to general health (eating habit, locomotion, and behavior), appearance, and weight loss. All values represent the means 6 standard errors of the mean from one experiment. Data were analyzed using two-way analysis of variance (ANOVA) with Bonferroni post hoc test (ns, not significant). SARS-CoV-2 Variants Induce Distinct Disease in Mice mBio PCR) at 6 dpi. High levels of infectious virus were detected by plaque assay in the lung tissue of mice infected with ancestral SARS-CoV-2 (;7 Â 10 5 PFU/g of tissue) and Delta (;4 Â 10 6 PFU/g of tissue) (P = 0.0556) ( Fig. 2A). Although the difference was not statistically significant, the higher average viral load in the lung tissue of Delta-infected mice than that of ancestral strain-infected mice was approaching statistical significance (P = 0.0556). We detected infectious virus in the brain (;2.3 Â 10 8 PFU/g tissue), intestine (;1.9 Â 10 5 PFU/g tissue), and kidney (;1.4 Â 10 5 PFU/g tissue) of all mice infected with Delta but none of the mice infected with the ancestral strain ( Fig. 2B to D). No infectious virus was detected in the serum or heart tissue of mice infected with either the ancestral or Delta strain at 6 dpi (data not shown). The copy number of viral genomes in tissues was quantified by qRT-PCR. There was no significant difference in the viral copy number in the lungs of mice infected with the ancestral or Delta strain (Fig. 2E). However, in brain, intestine, and kidney, the copy number of the Delta genome was significantly higher than that of ancestral strain-infected mice (Fig. 2F to H).
FIG 2 Viral burden in tissues of the mice infected by SARS-CoV-2 ancestral strain and Delta variant. Eight-week-old female K18-hACE2 mice were intranasally inoculated with PBS (mock group) or 10 4 PFU of SARS-CoV-2 ancestral or Delta strain (n = 5). At 6 dpi, the titers of infectious virus in the lung (A), brain (B), intestine (C), and kidney (D) were determined by plaque assay. The copy numbers of viral genome in lung (E), brain (F), intestine (G), kidney (H), heart (I), and serum (J) were determined by probe-based qRT-PCR. Each symbol represents the mean titer for one mouse. Data were analyzed by Mann-Whitney test (**, P , 0.01; ns, not significant).

SARS-CoV-2 Variants Induce Distinct Disease in Mice mBio
The viral genome copy numbers of both ancestral strain and Delta were at similarly low levels in heart tissue and serum ( Fig. 2I and J). Histopathological analysis of lung, brain, intestine, and kidney tissue following SARS-CoV-2 ancestral strain and Delta infection. To assess the histopathology of the disease associated with the ancestral and Delta strain infection in K18-hACE2 mice, lung, brain, intestine, and kidney of infected mice were collected at 6 dpi. Multiple areas of the lung in ancestral strain-and Delta-infected mice manifested moderate (1/5 in ancestral strain group; 2/5 in Delta group) to severe (4/5 in ancestral strain group; 3/5 in Delta group) pathological changes, typified by prominent immune cell infiltration surrounding blood vessels, thickened interalveolar septa, and consolidation with necrotic debris. Most of the pathological changes were located in the marginal areas of the lung or in proximity to apical or anterior basal regions. No significant difference in the number of cells infiltrating the lungs of ancestral strain-and Delta-infected mice was observed histologically (P = 0.0829) ( Fig. 3A and E). In the brain, localized vascular congestion, perivascular cuffing, and mononuclear cell infiltration in the cerebral cortex of the brain were observed in both ancestral strain-and Delta-infected mice ( Fig. 3B and F), with occasional meningeal or choroidal plexus infiltrates. Notably, this was more prominent in Delta-infected mice FIG 3 Histopathological analysis of lung, brain, kidney, and intestine from the mice infected by SARS-CoV-2 ancestral strain and Delta variant. Eight-weekold female K18-hACE2 mice were intranasally inoculated with PBS (mock group) or 10 4 PFU of SARS-CoV-2 ancestral or Delta strain (n = 5). At 6 dpi, lung (A and E), brain (B and F), kidney (C and G), and intestine (D, H, and I) tissues were collected and processed with H&E staining. Cell density of the wholetissue region was quantified using ImageScope. Arrows: green arrows, perivascular infiltration (B); blue arrows, cell infiltration (C); red arrows, intestine wall (D). Each image is representative of $3 mice. Statistical analysis performed using one-way ANOVA with Bonferroni post hoc test (ns, not significant; *, P , 0.05; **, P , 0.01; ***, P , 0.001; ****, P , 0.0001).
SARS-CoV-2 Variants Induce Distinct Disease in Mice mBio (3/5 mice showed perivascular infiltration) than in mice infected with ancestral strain (1/5 mice showed perivascular infiltration), consistent with our observation that live Delta and Delta viral RNA were detected in the brain at higher levels than ancestral strain. In the kidney, no clear pathology due to SARS-CoV-2 infection was identified by histological analysis (Fig. 3C). However, occasional enhanced glomerular infiltration by mononuclear cells was observed in both ancestral strain-and Delta-infected mice (Fig. 3G). In the small intestine of infected mice, no morphological changes were observed in the villi of the small intestine or any increase in general cellular infiltration ( Fig. 3D and H), although occasional small focal basal mononuclear cell infiltrates were noted in Delta-infected mice. However, the intestinal wall, which is composed of circular and longitudinal layers of smooth muscle, was significantly thinner in Delta-infected than in mock-and ancestral strain-infected mice (Fig. 3I). Inflammatory gene expression in the lungs of SARS-CoV-2 ancestral strain-and Delta-infected K18-hACE2 mice. Cytokine storm, characterized by excessive production of proinflammatory cytokines such as interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-a), and IL-1b, is reported to contribute to ARDS in patients with severe COVID-19 (26,27). We therefore measured the transcription levels of proinflammatory cytokines and chemokines in the lung tissue of the mice infected with SARS-CoV-2 ancestral strain or Delta at 6 dpi. Compared to mock-infected mice, the expression of IL-6, TNFa, IL-1b, CCL2, CXCL10, CCL5, gamma interferon (IFN-g), IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-13, IFN-b, and IL-12p35, in both infected groups, was all upregulated in lung homogenates (Fig. 4). No significant differences were identified in any of these factors between ancestral strain-and Delta-infected groups.
Leukocyte profile of the lungs of SARS-CoV-2 ancestral strain-and Delta-infected K18-hACE2 mice. Immune cell dysregulation induced by SARS-CoV-2 infection has been suggested to contribute to pulmonary pathology in COVID-19 patients (28). To assess the immune cell response in mice infected with SARS-CoV-2 ancestral strain or Delta, lung tissue was analyzed by flow cytometry at 6 dpi (Fig. 5A). Interestingly, the numbers of CD45 1 immune cells were significantly higher in the lungs of mice infected with ancestral strain, compared to mice infected with Delta (Fig. 5B). Moreover, the numbers of inflammatory monocytes (IM), alveolar macrophages (AM), dendritic cells, and CD4 1 and CD8 1 T cells and B cells were all found to be significantly higher in ancestral strain-infected mice than in mice infected with Delta ( Fig. 5E to K and M). No significant difference was found in the numbers of NK cells and Ly6G 1 neutrophils between the two infected groups ( Fig. 5C and L). Delta-infected mice had slightly more SSC hi neutrophils in lung tissue than mice infected with the ancestral strain; however, this was not statistically significant ( Fig. 5D).
Immunofluorescence analysis of lung tissue from SARS-CoV-2 ancestral strainand Delta-infected K18-hACE2 mice. To further characterize the leukocyte profile in infected lung tissue, immunofluorescence analysis of T cells, neutrophils, and CD169 1 alveolar macrophages was performed. Compared to the lung tissue of mock-infected mice, higher levels of CD3 1 T cells were present in the parenchyma of ancestral strainand Delta-infected mice. T cells were mainly distributed in the adventitia around bronchioles (Fig. 6). Gr-1 1 neutrophils in both ancestral strain-and Delta-infected mice were sparsely distributed in the lung parenchyma; however, neutrophils in the lungs of Deltainfected mice were more localized in proximity to bronchiole mucosal fold areas (Fig. 6). In the lungs of both ancestral strain-and Delta-infected mice, CD169 1 alveolar macrophages were sparingly distributed in the lung tissue, with no significant difference in the distribution observed between the two infected groups (Fig. 6).

DISCUSSION
Transmissibility, vaccine breakthrough, and disease severity are the three biggest concerns for human health when characterizing new emerging SARS-CoV-2 variants. The Delta variant of concern has been shown to be more transmissible (29), and vaccine effectiveness against Delta is reduced, compared to the ancestral strain (30,31). However, there is very limited information on the disease severity associated with Delta infection. There are a number of factors that make it difficult to compare the disease profile of Delta infection to that of the ancestral strain. For example, the Delta variant originated from different geographic regions than did the ancestral strain of SARS-CoV-2, making it prevalent in different patient populations with different genetic backgrounds and access to diverse health care resources. Additionally, as the COVID-19 pandemic has progressed, patients infected with Delta have been treated under    (32). Notably, the analysis shows that Delta has the highest risk of ICU admission and mortality compared to the Alpha, Beta, and Gamma variants. A recent study to determine pathogenicity of the Delta variant conducted by Lee et al. showed a higher disease score in K18-hACE2 mice infected with Delta compared with the Alpha variant (hCoV19/ England/204820464/2020, GISAID: EPI_ISL_683466) (33). We observed comparable disease severity and weight loss over 6 days in K18-hACE2 mice infected with ancestral or Delta SARS-CoV-2. Our observations are similar to a recent study in which Syrian hamsters infected with ancestral or Delta strain (at 10 5 PFU per animal) developed comparable levels of weight loss from 5 dpi and both peaked at 7 dpi (34). Because the hamster model of SARS-CoV-2 infection is a nonlethal model, infected hamsters recovered quickly after peak weight loss (34). Additional kinetic investigations based on more time points and bigger sample size would provide further details of disease development in K18-hACE2 mice. The disease observed in ancestral strain-and Delta-infected mice was associated with high viral load in lung tissue. Importantly, in K18-hACE2 mice, Delta infection spread beyond pulmonary tissues to various organs, including brain, kidney, and intestine, where live virus and viral RNA were detected. The viral RNA level in the brain of Deltainfected mice was ;10 5 -fold higher than that of ancestral strain-infected mice. However, our observations differ from the study by Lee et al. that showed low levels (below detection limit) of Delta viral RNA in mouse brain at 6 dpi (33). Further studies on the brain tropism of Delta are therefore recommended. Interestingly, compared to the difference in brain viral RNA, the difference in viral RNA load in the intestines and kidney between ancestral strain-and Delta-infected mice was only approximately 100-fold. The expression  level of ACE2 in organs is likely to affect virus tissue tropism, as Delta's enhanced ability to disseminate in K18-hACE2 mice may be due to the enhanced entry efficiency of its spike protein on recognizing the hACE2 receptor (17,35). However, the brain and kidney of K18-hACE2 mice express comparable levels of hACE2 (25,36). The mechanism of tissue tropism of Delta warrants further investigation. It is also worth noting that, compared to the recently emerged Omicron variant (B.1.1.529), the Delta strain showed no difference in the viral load in nasal turbinates but more profound infection in lung tissue of infected hamsters (34). Based on a meta-analysis involving 241 COVID-19 patients, diffuse alveolar damage (DAD), pulmonary hemorrhage and thromboembolism represented common lung-associated pathology (65.1%) (37). Histopathological analysis of ancestral strain-and Delta-infected lung tissue revealed moderate to severe alveolar damage and inflammation in the parenchyma. Similar pathology has been observed in smallanimal models (33,38). The brain has been shown to be another important organ involved in COVID-19 pathology in both clinical patients (37) and K18-hACE2 mice following infection with SARS-CoV-2 clinical isolate USA-WA1/2020 (39)(40)(41). SARS-CoV-2-induced lesions in the mouse brain were mainly found in the cerebral cortex. Pathological change in the cerebral cortex may be due to the proximity of the cerebral cortex to the olfactory bulb, as SARS-CoV-2 has been shown to invade the central nervous system through the neural-mucosal interface in olfactory mucosa (42). In line with previous reports, multiple sites of mononuclear cell infiltration were identified within the cerebral cortex region of the brain in both ancestral strain-and Delta-infected mice. More abundant tissue involvement was found in brains from Delta-infected mice than in those from ancestral strain-infected mice, suggestingat least in part-that, as more infectious virus and viral RNA were found in the brain of Delta-infected than of ancestral strain-infected mice, SARS-CoV-2 infection of brain tissue may have a role in brain pathology in K18-hACE2 mice. Although live ancestral SARS-CoV-2 was not detected at 6 dpi in the brain tissue, the lesions identified in the brain of ancestral strain-infected mice indicate that virus may be replicating at very low levels or being cleared earlier than the Delta strain. Previous studies have identified live virus in the brain of K18-hACE2 mice infected with the ancestral strain at similar disease time points as those analyzed here (43). Further assays to determine virus growth and histopathology of brain tissue at earlier and later time points would help clarify if virus replication correlates with brain pathology. Infectious virus in the brain of the Delta-infected mice did not cause overt cell infiltration at day 6 postinfection. We observed only localized vascular congestion, perivascular cuffing, and mononuclear cell infiltration sparsely distributed in the brain of 3 out of 5 Delta-infected mice and in 1 out of 5 ancestral strain-infected mice. From previous reports, based on either COVID-19 clinical patient samples (44,45) or animal data (46,47), microhemorrhages, perivascular neutrophilic/lymphocytic infiltrates, and fibrin microthrombi were detected at various levels in the brain. However, there was no evidence of overall increased cell infiltration in the wholebrain tissue, whether viral RNA genome was present in the tissue or not. Specifically, Song et al. (47) observed that, in the brain tissue of deceased COVID-19 patients, regions with positive viral staining did not show lymphocyte or leukocyte infiltration. Their results suggested that SARS-CoV-2 may not induce immune responses typical of other neurotropic viruses, which induce large numbers of immune cell infiltrates neighboring sites of virus infection (47). Brain damage in COVID-19 patients may result in long-term cognitive sequelae (48). It is unclear whether SARS-CoV-2 replication and associated pathology in the mouse brain will lead to any longterm consequence in mouse behavior. The brain tissue damage observed in this study may help to establish a disease model for future studies. Leukocyte infiltration and tissue damage in the kidneys due to SARS-CoV-2 infection, such as renal tubular necrosis or hemorrhage, have been reported in postmortem analyses (37), multiple small-animal models infected with SARS-CoV-2 Canada/ON/VIDO-01/2020 or a mouse-adapted strain, MASCp36 (49,50), and in clinical studies (51,52). In our study, glomerular mononuclear cell infiltration was observed to similar degrees in ancestral strain-and Delta-infected mice, but no clear tissue pathology was identified in the kidney. Further study is needed to elucidate the link between virus replication and cell infiltration in kidney tissue. SARS-CoV-2 has been shown to infect intestinal tissue and cause leukocyte infiltration and morphological changes in small-animal models infected with SARS-CoV-2 Canada/ON/VIDO-01/2020 or USA-WA1/2020 (36,49) and in humans (53,54). In this study, thinning of the intestine wall in response to SARS-CoV-2 infection was observed. The intestine wall in Delta-infected mice was found to be thinner than that in mock-and ancestral strain-infected mice, which again may be a result of higher levels of infectious Delta and Delta RNA genome present in the intestinal tissue. Thinning of the small intestine wall is thought to be associated with intestinal pneumatosis (55), which has been reported in COVID-19 patients (56). Thinning of the intestine wall may also be attributed to excessive gas in the intestine of infected mice (57) or the worsening nutritional status of SARS-CoV-2-infected mice. SARS-CoV-2 Canada/ON/VIDO-01/2020 and USA-WA1/2020, SARS-CoV-2 clinical isolates used in previous animal studies (36,39,40,49), have only three amino acid mutations (ORF1a [I1607V], E [S6L], and N [S194L]) and one mutation (L84S in ORF8), respectively, compared to the ancestral strain. The two isolates were therefore both genetically close to the original ancestral strain (Nextstrain). Thus, our comparison here of the tissue damage following ancestral strain and Delta infection is valuable in determining the difference in pathogenesis of COVID-19 caused by emerging SARS-CoV-2 variants. Future studies on the histopathology of other COVID-19-related organs, such as heart, liver, spleen, colon, and reproductive organs, would help to further decipher SARS-CoV-2 variant-induced pathology.
In agreement with previous clinical reports showing cytokine storm is correlated with severe COVID-19 (3), elevated levels of proinflammatory cytokines were detected in the lung tissue of ancestral strain-and Delta-infected mice. However, there was no significant difference in cytokine expression between the two infected groups. The comparable levels of cytokine profile we observed in ancestral strain-and Delta-infected mice concur with the similar disease manifestations of the two groups, as cytokine storm has been shown to correlate directly with COVID-19 severity (27). The study by Lee et al. detected higher levels of proinflammatory cytokines (TNF-a, IL-1b, CXCL13, IL-6, and CXCL10) which were associated with more severe disease in Delta-infected K18-hACE2 mice, compared to the Alpha variant (hCoV19/England/204820464/2020; GISAID: EPI_ISL_683466) (33). Future studies on cytokine/chemokine profile at both the protein level (e.g., multiplex Luminex assay) and the transcriptional level (e.g., RNA sequencing and NanoString) would provide a more comprehensive outlook on the role of cytokines in COVID-19.
Immature/total neutrophils were reported to be increased in severe COVID-19 patients (58). In our study, although not statistically significant, both the clinical disease score and the number of infiltrating SSC hi neutrophils in Delta-infected mice were higher than in mice infected with the ancestral strain, which is in line with the observation made in the work of Carissimo et al. (58). Moreover, IL-6 also appeared to be higher in Delta-infected mice than in mice infected with the ancestral strain, although not statistically significantly, supporting the correlation between immature neutrophil counts and IL-6 (58). As neutrophil-derived extracellular traps (NETs) have been reported to be induced by SARS-CoV-2 infection and associated with lung tissue damage (lung epithelial apoptosis) in COVID-19 patients (59), it would be interesting to investigate whether NETs contribute to the higher disease (P disease = 0.0578) and cell infiltration (P lung cell infiltration = 0.0829) in the lungs of Delta-infected K18-hACE2 mice. The numbers of CD45 1 cells, T cells, B cells, inflammatory monocytes, and dendritic cells (DCs) in the inflamed lung tissue of Delta-infected mice were significantly lower compared to mice infected with the ancestral strain. This was despite disease manifestations, lung virus titers, and proinflammatory cytokines/chemokines being at comparable levels between the two groups. Although leukocytes have been shown to be recruited into lung tissue following SARS-CoV-2 infection in K18-hACE2 mice (25) and COVID-19 patients (60), various reports regarding the correlation between total leukocytes and COVID-19 severity have been published (61). Several studies have shown that surviving patients usually present with lower total leukocyte counts, compared to nonsurviving patients (62). However, leukopenia has also been reported in COVID-19 patients in several studies (3,63). A clinical report based on 326 COVID-19 patients with pneumonia at normal, low, or high levels suggested that leukocyte numbers, at least in the blood, do not closely corelate with COVID-19 severity (64,65). Furthermore, a clinical study of 31 COVID-19 patients showed that the percentage of infection in the lung was negatively correlated with peripheral blood lymphocyte percentage and count but positively correlated with neutrophil percentage in the blood (66). In our study, the disease severity, virus replication, and pathology in lungs of Delta-infected mice were all slightly higher (close to significant) than with the ancestral strain, although the differences were not statistically significant (P disease = 0.0578; P lung virus titer = 0.0556; P lung cell infiltrates = 0.0829). Furthermore, the levels of IFN-g and IFN-b both trended lower in Delta-infected mice, compared to that of ancestral strain-infected mice (P IFN-g = 0.1324; P IFN-b = 0.5346). The low levels of leukocytes in the lungs observed in Delta strain-infected mice may therefore be due to as yet unknown immune evasion mechanisms. Related to this, the mechanism behind the differences in leukocyte recruitment between ancestral strain and Delta infections in mice warrants further investigation, such as exploring the determinants of disease severity by investigating the role of essential leukocytes (T cells, monocytes, alveolar macrophages, etc.) in the pathogenesis of COVID-19. Notwithstanding these differences, the severe histopathological changes in the lungs of both ancestral strain-and Delta-infected animals are consistent with the progressive compromise of pulmonary function, rather than the functional failure of other organs.
In summary, using a K18-hACE2 mouse model, the Delta variant disseminated to multiple tissues and caused more tissue damage than the ancestral strain of SARS-CoV-2. Numbers of leukocytes recruited to the lung tissue during Delta infection were lower than with the ancestral strain, suggesting Delta may have a novel invasion and evasion mechanism by which the host leukocyte response was suppressed, facilitating dissemination. As new SARS-CoV-2 variants, such as Omicron, continue to emerge, knowledge of the variant-specific pathogenesis of variants of concern will be valuable for the optimization and development of therapeutic strategies.

MATERIALS AND METHODS
Animal ethics statement. Animal experiments were approved by the Animal Ethics Committee of Griffith University (protocol no. MHIQ/07/20/AEC). All procedures conformed to the Australian Code for the Care and Use of Animals for Scientific Purposes (67).
Mouse infections and disease monitoring. K18-hACE2 C57BL/6 mice were obtained from the Animal Resource Centre (Perth, Australia). Eight-week-old female K18-hACE2 mice were anesthetized with ketamine-xylazine and inoculated intranasally with 10 4 PFU of SARS-CoV-2 ancestral strain or SARS-CoV-2 Delta strain in a volume of 20 mL. Mock-infected mice received 20 mL of sterile Dulbecco's modified Eagle's medium (DMEM) with 2% fetal bovine serum (FBS). The mice were monitored for disease manifestations daily. Mice were scored using a cumulative and progressive clinical disease matrix. Mice were given a score between 0 and 3 for each of the following health indicators: eating habit, locomotion, behavior and appearance, and weight loss, as well as general health (including breathing, body weakness, eye symptoms, etc.). A score of 0 was normal. After receiving an overall score of .3, mice were monitored twice daily. A score of 3 in any one of the 5 categories (e.g., .15% weight loss) or an overall score of .10 led to the mouse being euthanized.
Plaque assay. The titers of infectious virus were enumerated by plaque assay (69). Mouse tissues were collected in 1Â phosphate-buffered saline (PBS) and homogenized using the Bead Ruptor 24 Elite homogenizer according to the manufacturer's instructions. The supernatant was collected by centrifugation at 12,000 Â g, 4°C, for 10 min and stored at 280°C for longer preservation. Vero E6 cells were seeded in 12-well plates at 2.5 Â 10 5 cells per well and cultured in DMEM with 5% FBS overnight. The cells were infected with a dilution series of virus from samples and overlaid with 1.2% colloidal microcrystalline cellulose (Sigma-Aldrich) in DMEM supplemented with 2% FBS. The plates were incubated for 72 h at 37°C. Cells were fixed with 4% paraformaldehyde (PFA) and stained with 0.1% crystal violet. Viral titters were calculated using the following formula: PFU/mL = (average number of plaques/volume [mL] of virus added) Â the dilution factor.
Flow cytometry. The infected mice were culled and perfused with PBS. Lung tissues were collected for flow cytometry (fluorescence-activated cell sorting [FACS]) analysis (70). Lungs were minced and digested in type IV collagenase (0.2 mg/mL; Worthington) with DNase I (0.05 mg/mL; Sigma) and passed through 70-mm cell strainers. Cells were stained with antibodies for 45  Immunofluorescent histology and confocal microscopy. Lung tissues were harvested and fixed in 4% paraformaldehyde overnight. The tissues were subsequently washed in PBS and dehydrated in in 30% sucrose overnight. The tissues were embedded in optimal-cutting-temperature (OCT) compound (Sakura Finetek) and then frozen at 280°C. Tissue blocks were cut into 20-mm-thick sections and permeabilized in cold acetone. The sections were blocked with serum-free protein block (Dako) and immunolabeled with antibodies against mouse CD3 (2A7; BioLegend), Gr-1 (RB6-8C5; BD Biosciences), CD169 (3D6-1.12; AbD Serotec), and laminin (rabbit polyclonal; Abcam). The slides were mounted with ProLong Gold antifade agent (Thermo Fisher). Images were acquired using a confocal microscope (Olympus FV3000) using a 10Â and 20Â (0.95-numerical-aperture [NA]) objective and processed using Imaris (v9.5; Bitplane).
Qualitative real-time PCR assay for mouse cytokines. The infected mice were culled and perfused with PBS. Mouse tissues of interest were collected in TRIzol (Invitrogen) and homogenized using the Bead Ruptor 24 Elite homogenizer according to manufacturer's instructions. Total RNA was extracted from tissue homogenates using TRIzol (Invitrogen) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using reverse transcriptase (Sigma-Aldrich) with random primers. SYBR green real-time PCR was performed on a CFX96 Touch real-time PCR system (Bio-Rad) to measure the expression of inflammatory mediators. Primers for HTRP (housekeeping gene), IL-6, TNF-a, IL-1-b, CCL2, CXCL10, CCL5, IFN-g, IL-10, GM-CSF, IL-13, IFN-b, and IL-12p35 were purchased from Qiagen. SYBR green real-time PCR conditions were set as follows: (i) 1 cycle of 95°C for 15 min and (ii) 40 cycles of 94°C for 15 s, followed by 55°C for 30 s and 72°C for 30 s. The DNA amplification specificity was evaluated by melting curve analysis. The mRNA level of each target gene was expressed as fold change from the level in mock-infected samples.
Qualitative real-time PCR (qRT-PCR) assay for viral genomic RNA. The infected mice were culled and perfused with PBS. Lung, heart, brain, kidney, intestine, and serum were collected in TRIzol reagent (Invitrogen) and homogenized using the Bead Ruptor 24 Elite homogenizer according to manufacturer's instructions. Total RNA was extracted and reverse transcribed into cDNA using reverse transcriptase (Sigma-Aldrich) with random primers. QuantiTect probe real-time PCR was performed on a CFX96 Touch real-time PCR system (Bio-Rad) to measure the viral genome copy numbers. SARS-CoV-2 RdRp gene-targeting primer and probe sequences were 59-GTGAAATGGTCATGTGTGGCGG-39 (forward), 59-CAAATGTTAAAAACACTATTA GCATA-39 (reverse), and 6-carboxyfluorescein (FAM)-CAGGTGGAACCTCATCAGGAGATGC-black hole quencher (BHQ) (probe sequence). The QuantiTect probe real-time PCR conditions were set as follows: (i) 1 cycle of 95°C for 15 min and (ii) 40 cycles of 94°C for 15 s followed by 60°C for 1 min. The DNA amplification specificity was evaluated by melting curve analysis. Viral genome copy number was calculated against a standard curve by a viral infectious clone (GenBank accession no. MT926410).
H&E staining. The infected mice were culled and perfused with PBS. Lung, kidney, brain, and intestine tissues were collected and fixed in 4% PFA. Paraffin blocks of the tissues were cut into 5-mm-thick sections and stained with hematoxylin and eosin (H&E). Images of the tissues were taken by an Aperio AT2 digital whole-slide scanner (Leica). Tissue cell density was quantified using ImageScope software (Algorithm Nuclear v9) using the threshold settings: image zoom of 0.5, a minimum nuclear size (mm 2 ) of 10, a maximum nuclear size (mm 2 ) of 100, a minimum roundness of 0.4, a minimum compactness of 0.4, and a minimum elongation of 0.2.
Statistical analysis. Data for mouse disease scores and weight loss were analyzed using two-way analysis of variance (ANOVA) with a Bonferroni post hoc test. Flow cytometry results and cell density results were analyzed using one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. Plaque assays and viral genome copy number data were analyzed using the Mann-Whitney test. Cytokine RNA qRT-PCR data were analyzed using Student's t test. All data (except for mouse disease scores) were tested for normality using the D'Agostino-Pearson normality test prior to analysis. All statistical analyses were performed with GraphPad Prism software version 9.0.