Dietary peptide-specific antibodies against interleukin-4 differentially alter systemic immune cell responses during Eimeria challenge with minimal impacts on the cecal microbiota

Abstract

Eimeria spp. induce host interleukin (IL)-4 production, a potent immune regulator, during coccidiosis to evade immune responses. Dietary anti-IL-4 may preserve bird performance during challenge; however, specific mechanisms have not been investigated. Study objectives were to develop peptide-specific anti-IL-4 antibodies and evaluate immune cell profiles and the cecal microbiota during Eimeria challenge. Four candidate IL-4 peptides were selected based on antigenicity and location. Hens were injected with conjugated peptide or carrier-only control (3/injection), eggs were collected post-vaccination and yolks were pooled by peptide before freeze-drying. On d 0, 300 Ross 708 broilers were placed in floor pens (10/pen) and assigned to 5 diets consisting of basal diet + 2% egg yolk powder containing antibodies against 1 of 4 target peptides or carrier-only control for 14-d starter and grower periods (28 d total). Baseline blood and cecal contents were collected on d 14 (6 birds/diet) before half the remainder were inoculated with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ). Body weight (BW) and feed intake (FI) were recorded weekly and blood and cecal samples were collected at 3, 7, and 14 d post-inoculation (pi; 3/treatment). Immune cell profiles in peripheral blood mononuclear cells (PBMC) were evaluated flow cytometrically and cecal microbial communities determined by 16S/18S rRNA gene amplicon sequencing. Data were log-transformed when necessary and analyzed with diet, Eimeria, and timepoint fixed effects plus associated interactions (SAS 9.4; P ≤ 0.05). Anti-IL-4 did not alter baseline performance but generally increased PBMC Bu-1⁺ B cells 38.0 to 55.4% (P < 0.0001). Eimeria challenge reduced FI and BWG 16.1 and 30.3%, respectively, regardless of diet (P < 0.0001) with only birds fed peptide 4 antibodies not recovering feed conversion by d 28. Minimal diet-associated cecal microbiota changes were observed, indicating that anti-IL-4 effects were likely host-specific. Eimeria-challenged birds fed peptide 3 antibodies displayed minimal immune cell fluctuations compared to unchallenged counterparts, suggesting these antibodies potentially modulated intestinal immune responses to minimize systemic requirements, making them good candidates for further research.

INTRODUCTION

The rise in antibiotic-free (ABF) poultry production has been associated with the increased prevalence of damaging intestinal diseases like coccidiosis due to Eimeria spp. infection and downstream necrotic enteritis, as available antibacterial drugs no longer align with these ABF production practices (Noack et al., 2019; López-Osorio et al., 2020). This, combined with growing anticoccidial resistance by Eimeria has contributed to reduced production performance and persistent coccidiosis-associated economic losses for the global poultry industry amounting to an estimated $12.5 billion USD annually (Arabkhazaeli et al., 2013; Blake et al., 2020). Eimeria vaccination remains a viable strategy for coccidiosis control; however, vaccine efficacy is limited due to limited cross-protection between different Eimeria spp. and strains (Martin et al., 1997; Dalloul and Lillehoj, 2006). In turn, this has created opportunities for research into novel dietary coccidiosis mitigation strategies such as probiotics and phytochemicals supplemented with or without vaccination, all with promising, but variable, impacts on poultry health, immune responses, and the intestinal microbiota during Eimeria challenge (Alfaro et al., 2007; Bozkurt et al., 2013; Ritzi et al., 2014; Behnamifar et al., 2019). As a result, compounds that provide the benefit of being easy to administer to large flocks of birds like feed additives, with targeted effects against Eimeria similar to vaccination emerge as optimal candidates for coccidiosis control.

Dietary immunoglobulin (Ig)-Y antibodies derived from egg yolk present an intriguing approach to dietary coccidiosis mitigation. As feed additives, the neutralizing activity of IgY antibodies can survive freeze-drying and low pH environments in the upper gastrointestinal tract enabling effective incorporation into poultry rations (Hatta et al., 1993; Cook and Trott, 2010; Bobeck et al., 2016). These antibodies can be targeted against specific components of the Eimeria parasite or the host immune responses to confer passive immunity and specific pathogen targeting, much like vaccination but without the need for additional labor. When targeting the parasite specifically, spray- or freeze-dried egg yolk from hens hyperimmunized against Eimeria included in diets as low as 0.05% had protective effects on broiler body weight gain (BWG) during E. acervulina or E. tenella challenge (Lee et al., 2009; Xu et al., 2013). In addition to specifically targeting the parasite, IgY antibodies can be developed to target components of the immune response that may be modulated by Eimeria to gain a competitive advantage during infection. For example, Eimeria induces host production of anti-inflammatory interleukin (IL)-10 to evade host immune responses and dietary anti-IL-10 egg yolk antibodies have shown a protective effect on BWG during challenge with high-dose attenuated Eimeria spp. (Arendt et al., 2016, 2019a,b; Sand et al., 2016).

In poultry exposed to live or attenuated coccidia that is allowed to cycle fecal-oral, such as a vaccine strain or in the environment, immunity can be developed over a period of weeks. Pathogens, in turn, have developed mechanisms of immune evasion to gain a foothold in the complex intestinal environment. The helper T cell (T_H)1-T_H2 paradigm describes the polarization of effector lymphocytes that favor responses against intracellular pathogens and produce cytokines like interferon (IFN)-γ and tumor necrosis factor-α coordinated by T_H1 cells over those dominated by T_H2 cells that produce IL-4 and IL-13 which are effective against extracellular pathogens and vice versa (Muraille et al., 2014). In addition to T_H2 cells, interleukin-4 is produced by mast cells, and basophils with characterized functions in mammals to promote T_H2 polarization, B cell differentiation, and Ig class switching (Silva-Filho et al., 2014). While functions in chickens are still uncertain, IL-4 has been shown to induce expression of genes in cultured chicken macrophages associated with an anti-inflammatory, alternatively activated macrophage phenotype associated with T_H2 responses (Chaudhari et al., 2018).

As an intracellular parasite, Eimeria induces host IL-4 expression, to potentially shift immune responses away from anti-Eimeria T_H1 and IFN-γ production toward less-effective T_H2 phenotypes (Lillehoj and Choi, 1998; Hong et al., 2006). In mammalian models, unactivated B and T cells in vitro display low-level IL-4 receptor expression (<100 molecules/cell) with IL-4′s ability to exert biological function at low levels an indicator of its potency (Park et al., 1987). Similarly, in unchallenged chickens average serum IL-4 concentration was ∼5 pg/mL but increased 6-fold within 24 h of lipopolysaccharide challenge, suggesting similar potency in avian models (Chaudhari et al., 2018). This potency makes Eimeria-induced IL-4 production an intriguing strategy for evading host immune responses as small changes by the parasite can produce favorable conditions for replication. Theoretically, dietary anti-IL-4 egg yolk antibodies could diminish the effect of Eimeria-induced IL-4 and promote the initiation of more effective immune responses to preserve bird health during challenge; however, this has not been investigated previously. Additionally, the impacts of these dietary antibodies on systemic immune cell profiles and potential indirect action through modulation of the intestinal microbiota is uncertain. As such, the objectives of this study were to develop and evaluate egg yolk antibodies targeting different regions of the IL-4 protein on broiler performance, systemic immune cell profiles, and cecal microbiota during Eimeria challenge.

MATERIALS AND METHODS

Peptide Selection and Antibody Production

Animal protocols in this study were approved by the Iowa State University Institutional Animal Care and Use Committee. Chicken and mouse IL-4 sequences were accessed from the Universal Protein Resource (Uniprot) database (Uniprot ID: Q5W4U1 and 907750), aligned to identify taxonomically conserved regions, and copied into the BepiPred 2.0 server to identify potentially antigenic regions (epitopes; Jespersen et al., 2017). Candidate peptide sequences were mapped onto the human IL-4 protein structure obtained from Protein Data Bank (PDB; ID: 5FHX) to visualize their location within the molecule and evaluate potential antibody binding sites (Figure 1). Four candidate peptide regions were selected (Table 1) with peptides 1 and 2 representing areas identified as epitopes but located within alpha helices. Peptides 3 and 4 represented epitopes that were potentially accessible regions not exclusively composed of alpha helices and located toward the periphery of the IL-4 structure (Figure 1).

Figure 1.

Location of 4 selected peptide regions identified within chicken interleukin-4 and visualized within the human interleukin-4 protein structure (Protein Data Bank ID: 5FHX) to estimate potential accessibility of antiinterleukin-4 antibodies. Different color highlighting denotes peptide regions selected to prepare peptide conjugate for antibody production in laying hens. Peptide 4 is shown in contact with a ligand molecule included in the reference structure.

Table 1.

Candidate peptide sequences for generating anti-chicken interleukin-4 antibodies. Sequences were aligned to mouse interleukin-4 in the Universal Protein Resource (Uniprot) Knowledgebase to identify taxonomically conserved protein regions.

Peptide	Species	Sequence
1	Chicken1	PCPTAAGN
	Mouse2	SCTMNESK
2	Chicken	QGEVSCVK
	Mouse	TGETPCTE
3	Chicken	FADNKTNN
	Mouse	LTATKNTT
4	Chicken	TELLCKAS
	Mouse	SELVCRAS

¹Chicken Uniprot ID: Q5WU1.

²Mouse Uniprot ID: 907750.

Peptides were synthesized in 5 mg quantities by Genscript (Piscataway, NJ) and individually conjugated to bovine gamma globulin by glutaraldehyde conjugation as described previously (Bobeck et al., 2012). Briefly, 2 mg bovine gamma globulin were dissolved in acetate buffer (0.1 M), 2 mg of peptide was added, then 0.23 mL 2 M glutaraldehyde. The mixture was conjugated for 3 h at room temperature and the reaction was stopped by the addition of 10 mg glycine for 1 h. Each conjugated peptide was dialyzed overnight in phosphate buffered saline (PBS) using 6,000 to 8,000 molecular weight dialysis tubing. Peptide injections were prepared by adding 0.33 mg of each peptide conjugate to 1.5 mL PBS and emulsifying in an equal volume of Freund's complete adjuvant (Trott et al., 2009). Carrier-only control injections were prepared in a similar manner using unconjugated bovine gamma globulin in place of conjugated peptide. Three hens per carrier control or peptide were injected across 4 sites in the breast and leg muscle (0.25 mL/site). Hens received a booster vaccination prepared as described but emulsified in Freund's incomplete adjuvant 14 d after initial injection. Eggs were collected 21 d after initial injection and separated yolks were pooled by peptide before being lyophilized.

Animals and Diets

A total of 300 as-hatched Ross 708 broilers were placed in thirty 1 m² floor pens (10 birds/pen) and randomly assigned to 1 of 5 experimental diets (6 pens/diet) with ad libitum access to water and mash feed. The 28-d study was divided into 14 d starter and grower periods. Diets consisted of a corn-soybean meal basal diet + 2% egg yolk powder from hens injected with the carrier-only control (control) or 1 of 4 target peptides and formulated to meet NRC and breeder recommendations (NRC, 1994; Aviagen, 2019; Table 2). Body weight (BW) and feed intake (FI) were recorded weekly and used to calculate BWG and feed conversion ratio (FCR).

Table 2.

Starter (d 0–14) and grower (d 14–28) diets containing egg yolk ± peptide-specific antiinterleukin-4 antibodies fed to Ross 708 broilers for 28 d.

	Experimental diet
Ingredient, %	Basal starter					Basal grower
Corn	54.1					62.1
Soybean meal, 48% CP	36.3					30.2
Soybean oil	1.4					1.3
Salt	0.4					0.4
DL-Met	0.4					0.3
L-Lys*HCL	0.3					0.2
L-Thr	0.1					0.2
Limestone	2.0					0.9
Dicalcium phosphate	2.1					1.8
Choline chloride-60	0.4					0.0
Vitamin-mineral premix1	0.6					0.6
Egg yolk powder2	2.0					2.0
Calculated values, %
Crude fat	5.2					5.3
CP	23.2					20.9
Digestible Lys	1.3					1.2
Digestible Met	0.7					0.6
ME, kcal/Kg	3000					3100
Analyzed values (%)
	Control	Peptide 1	Peptide 2	Peptide 3	Peptide 4	Control	Peptide 1	Peptide 2	Peptide 3	Peptide 4
Moisture	8.8	8.9	8.8	8.9	9.0	8.4	8.4	8.4	8.4	8.5
DM	91.2	91.1	91.2	91.1	91.0	91.6	91.6	91.6	91.6	91.5
Crude fat	5.6	5.4	5.5	5.5	5.0	4.7	5.0	5.2	5.4	5.5
CP	19.7	19.9	19.3	19.5	19.0	18.4	18.2	17.4	18.0	17.7
GE, cal/g	3,898	3,906	3,838	3,884	3,869	3,903	3,909	3,911	3,923	3,924

¹Vitamin and mineral premix provided per kg of diet: selenium 250 μg; vitamin A (retinyl acetate) 8,250 IU; cholecalciferol (vitamin D₃) 2,750 IU; α-tocopherol acetate (vitamin E) 17.9 IU; menadione 1.1 mg; vitamin B₁₂ 12 μg; biotin 41 μg; choline 447 mg; folic acid 1.4 mg; niacin 41.3 mg; pantothenic acid 11 mg; pyridoxine 1.1 mg; riboflavin 5.5 mg; thiamine 1.4 mg; iron 282 mg; magnesium 125 mg; manganese 275 mg; zinc 275 mg; copper 27.5 mg; iodine 844 μg.

²Egg yolk powder contained either control antibody or antiinterleukin-4 antibody.

On d 14, blood was collected from 6 birds/diet by cardiac puncture under CO₂ anesthesia into heparinized syringes and blood tubes before euthanasia by cervical dislocation. Cecal contents were collected into sterile cryotubes and snap frozen in liquid nitrogen before being transferred to −80°C storage until analysis. Half the remaining birds were sham-inoculated with sterile PBS while the other half were administered 10X Coccivac-B52 by oral gavage (Merck Animal Health, Kenilworth, NJ). Diet and Eimeria challenge conditions contributed to 10 total groups arranged in a 5 × 2 factorial treatment design. Following inoculation, blood and cecal samples were collected at 3, 7, and 14 d post-inoculation (pi) from 3 birds/treatment. Fresh excreta samples were collected at 7 and 14 dpi and submitted to the Iowa State University Veterinary Diagnostic Laboratory (Ames, IA) for oocyst enumeration by the McMaster chamber method. Briefly, 2 g of excreta was homogenized in 28 mL of sucrose float solution (1.2–1.25 specific gravity), pipetted into both reservoirs of a McMaster chamber, and incubated undisturbed at room temperature for 5 min. Oocysts within each counting grid were enumerated using a microscope (10×) and the sum of both grids was multiplied by 50 to calculate oocysts/g excreta. Coccivac-B52 contains oocysts from E. acervulina, E. maxima, E. mivati, and E. tenella and lesion scores in the duodenum, jejunum, and ceca were evaluated using published criteria for each species at 14 dpi (Johnson and Reid, 1970). With this system, scores = 0 indicated no evidence of disease whereas scores = 4 denoted severe intestinal lesions.

Peripheral Blood Mononuclear Cell Isolation and Extracellular Staining

Blood was diluted 1:1 in sterile PBS, layered onto a Histopaque 1119-1077 density gradient (Sigma Aldrich, St. Louis, MO) and centrifuged at 600 × g for 35 min at the lowest acceleration setting and no brakes. Peripheral blood mononuclear cells (PBMC) were collected from the PBS-Histopaque 1119 and 1119-1077 interfaces into a clean 15 mL tube, washed twice in sterile PBS, and enumerated by hemocytometer. Cells were resuspended in RPMI supplemented with 42.5% heat-inactivated chicken serum and 7.5% DMSO before being frozen at −80°C.

For extracellular staining, PBMC were thawed in RPMI, washed once, and resuspended in 0.5 mL sterile PBS. Cells were enumerated and aliquoted into 7 polystyrene flow cytometry tubes/PBMC sample. Fluorochrome-conjugated antibodies against extracellular markers associated with innate immune cells and lymphocyte populations included monocyte/macrophage biotin (clone KUL01; mouse IgG₁κ), Bu-1 Alexa Fluor 647 (clone AV20; mouse IgG₁κ), cluster of differentiation (CD) 3 Alexa Fluor 700 (clone CT-3; mouse IgG₁κ), CD4 PE-Cy7 (clone CT-4; mouse IgG₁κ), CD8α Pacific Blue (clone CT-8; mouse IgG₁κ), and T cell receptor (TCR) γδ PE (clone TCR-1; mouse IgG₁κ; Southern Biotech, Birmingham, AL). Antibody-specific isotype controls for each marker and fluorescence-minus-one staining were used to account for nonspecific binding and applied to each PBMC sample. Master mixes for each stain were prepared by diluting antibodies 1:125 in PBS and 50 μL was added to corresponding flow cytometry tubes. Cells were incubated with primary antibody for 30 min in the dark at 4°C, washed in PBS, and 50 μL BrilliantViolet785-conjugated streptavidin (Biolegend, San Diego, CA; 1:250) was added to each flow cytometry tube for secondary staining of biotin-conjugated antibodies. After 30 min incubation at 4°C in the dark, cells were washed, resuspended in PBS, and analyzed by BD FACSCanto cytometer (Franklin Lakes, NJ). Cell population data were analyzed by FlowJo 10.5.0 software (BD Biosciences, San Jose, CA).

DNA Extraction, Sequencing, and Analysis

DNA was extracted from 0.2 g of thawed cecal contents collected at baseline, 7, and 14 dpi using the DNeasy PowerSoil kit according to manufacturer recommendations (Qiagen, Hilden, Germany) and quantified by a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA) before being stored at −20°C. The extraction process was also performed on sterile water using kit reagents as a process control. Two DNA aliquots designated for 16S and 18S rRNA gene amplicon sequencing to characterize bacterial/archaeal and eukaryotic communities, respectively, were diluted to approximately 30 ng/μL before submission to the Iowa State University DNA Facility (Ames, IA). PCR amplification of the 16S rRNA variable region V4 (515F, 806R; Caporaso et al., 2011, 2012) or 18S rRNA variable region V9 (1391f, EukBr; Stoeck et al., 2010) was performed prior to 250 bp paired-end sequencing on the Illumina MiSeq Platform.

Mothur V1.43.0 (Schloss et al., 2009) software was used to quality screen sequencing data and cluster reads into de novo operational taxonomic units (OTU) according to the MiSeq standard operating procedure (Kozich et al., 2013). Using this protocol, paired-end sequences were merged, quality-screened, and filtered to a minimum read length of 497 bp or 275 bp for 16S and 18S rRNA gene sequences, respectively, with thresholds of 0 ambiguous bp and ≤8 bp homopolymers. The SILVA reference database (v132; Quast et al., 2013) within Mothur was used to identify and remove sequences aligning outside the target 16S or 18S hypervariable region while possible chimeric regions were removed using “chimera.uchime” or “chimera.vsearch” commands for 16S and 18S sequences, respectively. Remaining sequences for 16S rRNA (4,227,653) and 18S rRNA (6,278,376) datasets were then classified using the SILVA database and clustered into OTUs at 99% similarity. Vertebrate, plant, and arthropod-associated OTUs were removed from the 18S rRNA dataset. 16S and 18S rRNA gene sequences were submitted to the NCBI sequence Archive (SRA) and are available under BioProject IDs PRJNA992535 (16S) and PRJNA992548 (18S).

Data were exported to R (version 4.2.2) for further analysis using the Phyloseq package (version 1.42.0). DNA extraction process controls were used to identify and remove potential contaminants and OTUs represented by fewer than 10 reads were removed from analysis. Within 16S and 18S rRNA data, 2 additional datasets were generated for baseline and post-inoculation timepoints to account for Eimeria absence at d 14 and samples were removed if they contained <5,000 sequences. Bray-Curtis distances were generated to evaluate community differences within baseline and post-inoculation datasets. Within R, alpha diversity measures were subsampled to the lowest read number (≥ 14,319), relative abundances were calculated, and log-transformed sequence counts for the 40 most abundant genera and 100 most abundant OTUs were exported as .csv files for statistical analysis.

Statistical Analysis

Data were evaluated using 3 different statistical models depending on timepoints (pre- or post-inoculation) or parameter. For outcomes in unchallenged birds (wk 1 and 2), data were analyzed using the following model:

$$y_{ij}=\mu +D_i+\text{iB}{\mathrm{W}}_{ijk}+e_{ij}$$

In this notation, y_ij is the dependent variable, μ is the overall mean, D_i is the effect of diet at the ith level, iBW_ijk is the initial BW covariate, and e_ij is the random error. Initial BW covariate was not used in the analysis of baseline immune cell populations or 16S/18S rRNA OTU relative abundance data; however, flow cytometry tube or individual bird were included as random effects for immune cell population and amplicon sequencing data, respectively.

Performance and immune cell population data within each timepoint following Eimeria-inoculation were analyzed using the following model:

$$y_{ijk}=\mu +D_i+E_j+{\left(D\times E\right)}_{ij}+\text{iB}{\mathrm{W}}_{ijk}+e_{ijk}$$

Much of the notation is retained from the previous model with additional terms for the diet main effect (D_i), Eimeria main effect (E_j), and interaction between diet and Eimeria ((D × E)_ij).

Postinoculation immune cell population data over time and relative abundances were analyzed using the following model:

$$y_{ijkl}=\mu +D_i+E_j+T_k+{\left(D\times E\right)}_{ij}+{\left(D\times T\right)}_{ik}+{\left(E\times T\right)}_{jk}+{\left(D\times E\times T\right)}_{ijk}+e_{ijkl}$$

This model contains additional terms for the timepoint main effect (T_k) and associated interactions of diet and timepoint ((D × T)_ik), Eimeria and timepoint ((E × T)_jk), and the 3-way diet, Eimeria, and timepoint interaction ((D × E × T)_ijk). Data analysis for immune cell population data included flow cytometry tube as the random effect while the random effect for 16S/18S analysis was the individual bird.

Performance and immune cell population data were analyzed using these models within the mixed procedure with Tukey's post hoc test to account for multiple comparisons (SAS 9.4, SAS Institute, Cary, NC). Relative abundances were normalized using the trimmed mean of the M value determined by log-transformed total sequences within individual samples and analyzed by generalized linear mixed model (PROC GLIMMIX) following a negative binomial distribution (Robinson and Oshlack, 2010). The false discovery rate within 16S/18S data was controlled using q values and outcomes were considered significant at P and q value thresholds ≤0.05.

Ordinal logistic regression in R (version 4.2.2) was used to analyze lesion scoring outcomes with the generalized linear model (vglm) package. Unchallenged birds did not have observable lesions (scores = 0) and were excluded from analysis. Using this method, comparisons were best interpreted as the likelihood of assigning a higher/lower lesion score when feeding anti-IL-4 peptide-specific antibodies vs. control during Eimeria challenge. The “link=logit” and “reverse=true” options were used to specify logistic regression and facilitate interpretation. With these options, negative estimates denote an increased likelihood of assigning a lower lesion score while positive estimates suggest increased likelihood of assigning a higher score. Changes in likelihood were considered significant at P ≤ 0.05.

RESULTS

Bird Performance, Lesion Scoring, and Oocyst Shedding

Feeding diets containing peptide-specific egg yolk antibodies against IL-4 did not affect bird performance in the first 14 d of the study prior to Eimeria inoculation (Table 3; Supplemental Table 1). The Eimeria main effect reduced d 21 BW and wk 3 BWG, and FI by 15.2, 30.4, and 16.2% in challenged vs. unchallenged birds, culminating in a 29.9-point reduction in efficiency (P < 0.00001). Day 28 BW was reduced 7.0% by the Eimeria main effect (P = 0.03); however, only challenged birds fed diets containing antibodies against IL-4 peptide 1 displayed a 24-point lower efficiency in wk 4 (P = 0.01; Table 3). For the entire grower period (d 14–d 28), the Eimeria challenge main effect reduced BWG 10.1% in challenged vs. unchallenged birds (P = 0.01), while Eimeria-challenged chicks fed antibodies against peptide 4 had a 16-point less efficient grower FCR (P = 0.03; Supplemental Table 1). For the entire 28-day study, Eimeria-challenged birds gained 7.3% less BW than their unchallenged counterparts, regardless of diet (P = 0.03) while Eimeria-challenged birds fed antibodies against IL-4 peptide 4 had a 9-point less efficient overall FCR compared to their unchallenged counterparts (P = 0.05; Table 3). Feeding diets containing anti-IL-4 antibodies against any of the 4 target peptides did not significantly affect the likelihood of assigning higher or lower lesion scores compared to feeding the control (Table 4). No oocysts were detectable in excreta from unchallenged birds (Table 5).

Table 3.

Weekly performance objectives in Ross 708 broilers fed diets containing egg yolks ± peptide-specific antiinterleukin-4 antibodies before and after Eimeria challenge¹.

	Treatment											P values
Measure	Control		Peptide 1		Peptide 2		Peptide 3		Peptide 4		SEM	Diet	Eimeria	Diet × Eimeria
D 0 BW2, g	44.00		43.67		44.00		44.17		44.33		0.0006	0.85	N/A	N/A
Wk 1
D 7 BW, g	155.40		150.00		150.40		152.20		151.30		4.20	0.72	N/A	N/A
BWG, g	111.20		105.90		106.20		108.10		107.10		4.20	0.72	N/A	N/A
FI, g	133.50		126.90		129.90		129.40		129.30		3.60	0.48	N/A	N/A
FCR	1.20		1.20		1.22		1.20		1.21		0.04	0.97	N/A	N/A
Wk 2
D 14 BW, g	376.6		343.00		342.60		353.10		358.90		13.26	0.10	N/A	N/A
BWG, g	221.40		193.20		192.50		200.90		207.80		10.75	0.07	N/A	N/A
FI, g	285.10		263.30		266.40		276.50		269.80		11.53	0.36	N/A	N/A
FCR	1.29		1.36		1.40		1.39		1.30		0.05	0.09	N/A	N/A
	Treatment											P values
	Control	Control + Eimeria	P1	P1 + Eimeria	P2	P2 + Eimeria	P3	P3 + Eimeria	P4	P4 + Eimeria	SEM	Diet	Eimeria	Diet × Eimeria
Wk 3
D 21 BW, kg	0.77	0.69	0.74	0.59	0.64	0.60	0.73	0.65	0.80	0.59	0.05	0.06	<0.0001	0.18
BWG, kg	0.40	0.31	0.39	0.26	0.32	0.25	0.38	0.29	0.44	0.23	0.03	0.09	<0.0001	0.12
FI, kg	0.56	0.52	0.56	0.44	0.50	0.45	0.54	0.46	0.59	0.44	0.04	0.19	<0.0001	0.29
FCR	1.41	1.68	1.44	1.68	1.58	1.82	1.43	1.62	1.37	1.92	0.09	0.07	<0.0001	0.09
Wk 4
D 28 BW, kg	1.27	1.18	1.17	1.13	1.12	1.09	1.20	1.18	1.32	1.06	0.09	0.30	0.03	0.27
BWG, kg	0.49	0.49	0.43	0.54	0.46	0.49	0.47	0.53	0.52	0.48	0.05	0.92	0.15	0.23
FI, kg	0.76	0.75	0.70	0.74	0.67	0.73	0.72	0.79	0.79	0.70	0.06	0.60	0.57	0.34
FCR3	1.54ab	1.54ab	1.62a	1.38b	1.47ab	1.49ab	1.54ab	1.49ab	1.52ab	1.47ab	0.05	0.50	0.01	0.01
D 0–28
BWG, kg	1.22	1.14	1.13	1.09	1.07	1.05	1.15	1.13	1.28	1.02	0.09	0.30	0.03	0.27
FI, kg	1.74	1.69	1.66	1.55	1.57	1.58	1.66	1.67	1.80	1.53	0.11	0.37	0.08	0.36
FCR	1.42ab	1.49ab	1.48ab	1.43ab	1.47ab	1.51a	1.44ab	1.47ab	1.41b	1.50a	0.03	0.32	0.02	0.05

¹Birds were challenged with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ) or PBS on d 14. Data represent the average of 6 pens/diet (wk 1 and 2) or 3 pens/diet × Eimeria challenge group (wk 3, 4, and overall) on a per bird basis. Eimeria effects not included in the statistical model for wk 1 and 2 as birds were not challenged until d 14.

²Abbreviations: BW, body weight; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio (intake/BWG).

³Values with different letter superscripts are significantly different, P ≤ 0.05.

Table 4.

Lesion scores recorded 14-day postinoculation with 10X Coccivac-B52¹ in Ross 708 broilers fed diets containing egg yolks ± peptide-specific antiinterleukin-4 antibodies from 3 birds/treatment.

	Lesion score2						Estimate3	P value
Treatment	0	1	2	3	4	Average
Duodenum	3	0	0	0	0	0.0	NA4	NA
Control	3	0	0	0	0	0.0	NA	NA
Peptide 1	3	0	0	0	0	0.0	NA	NA
Peptide 2	3	0	0	0	0	0.0	NA	NA
Peptide 3	3	0	0	0	0	0.0	NA	NA
Peptide 4	3	0	0	0	0	0.0	NA	NA
Control + Eimeria	0	2	1	0	0	1.3	NA4	NA
Peptide 1 + Eimeria	0	2	1	0	0	1.3	<0.0001	1.0
Peptide 2 + Eimeria	0	1	2	0	0	1.7	1.1	0.5
Peptide 3 + Eimeria	0	1	1	1	0	2.0	1.9	0.3
Peptide 4 + Eimeria	0	1	1	1	0	2.0	1.9	0.3
Jejunum
Control	3	0	0	0	0	0.0	NA	NA
Peptide 1	3	0	0	0	0	0.0	NA	NA
Peptide 2	3	0	0	0	0	0.0	NA	NA
Peptide 3	3	0	0	0	0	0.0	NA	NA
Peptide 4	3	0	0	0	0	0.0	NA	NA
Control + Eimeria	0	3	0	0	0	1.0	NA	NA
Peptide 1 + Eimeria	0	3	0	0	0	1.0	<0.0001	1.0
Peptide 2 + Eimeria	0	2	1	0	0	1.3	18.1	1.0
Peptide 3 + Eimeria	0	2	1	0	0	1.3	18.1	1.0
Peptide 4 + Eimeria	0	2	1	0	0	1.3	18.1	1.0
Cecum
Control	3	0	0	0	0	0.0	NA	NA
Peptide 1	3	0	0	0	0	0.0	NA	NA
Peptide 2	3	0	0	0	0	0.0	NA	NA
Peptide 3	3	0	0	0	0	0.0	NA	NA
Peptide 4	3	0	0	0	0	0.0	NA	NA
Control + Eimeria	0	2	1	0	0	1.3	NA	NA
Peptide 1 + Eimeria	0	0	2	1	0	2.3	19.0	1.0
Peptide 2 + Eimeria	0	0	2	1	0	2.3	19.0	1.0
Peptide 3 + Eimeria	0	2	1	0	0	1.3	<0.0001	1.0
Peptide 4 + Eimeria	0	1	2	0	0	1.7	1.4	0.4

¹Merck Animal Health, Kenilworth, NJ.

²Lesion scores were observed by a single observer within 3 birds/treatment in accordance with criteria published by Johnson and Reid (1970) for Eimeria acervulina, E. maxima, E. mivati, and E. tenella where score = 0 indicates no observable lesions and 4 indicates severe intestinal damage. Data represent the number of birds assigned each score within a treatment.

³Data were analyzed by ordinal logistic regression in R with reverse = true option in the vglm package. Positive estimates indicate an increased likelihood of assigning a higher lesion score whereas negative estimates indicate an increased likelihood of assigning a lower score.

⁴NA = not analyzed. No lesions were observed in unchallenged birds and were removed from the dataset to avoid zero vs. nonzero comparisons. All other comparisons were made based on feeding the diets containing 1 of 4 peptide-specific IL-4 antibodies during Eimeria challenge vs. the control.

Table 5.

Oocyst shedding per gram of excreta¹ in Ross 708 broilers fed diets containing egg yolks ± peptide-specific antiinterleukin-4 antibodies at 7- and 14-day postinoculation (pi) with 10X Coccivac-B52².

	Treatment
Study timepoint	Control	Peptide 1	Peptide 2	Peptide 3	Peptide 4	Control + Eimeria	Peptide 1 + Eimeria	Peptide 2 + Eimeria	Peptide 3 + Eimeria	Peptide 4 + Eimeria
7 dpi	ND	ND	ND	ND	ND	150,700	39,900	164,500	298,800	163,000
14 dpi	ND	ND	ND	ND	ND	400	4,750	65,500	100,600	800

¹Oocysts in pooled fresh excreta from 3 pens/diet × Eimeria challenge group were enumerated by McMaster chamber. Briefly, 2 g of fresh excreta were homogenized in 28 mL 1.2 to 1.25 specific gravity sucrose solution, the total oocysts within both counting grids were multiplied by 50 to determine oocysts/g excreta.

Immune Cell Populations

The panel implemented to evaluate immune cell populations within isolated PBMC was designed to include monocyte/macrophage⁺ cells as representatives of innate immunity with T and B lymphocytes as representatives of the adaptive immune response. Prior to Eimeria challenge, birds fed egg yolk powder containing antibodies against peptide 4 had 44.5 to 60.4% greater monocytes/macrophages than all other treatment groups (P < 0.0001; Figure 2A). While feeding egg yolk with peptide-specific anti-IL-4 antibodies resulted in 45.0 to 74.4% greater Bu-1⁺ B cells compared to control, regardless of peptide target, antibodies against peptide 4 resulted in 38.0 to 55.4% greater B cell populations than birds fed antibodies against peptides 1, 2, and 3 (P < 0.0001). Overall CD3⁺ T cell populations were unaffected by dietary anti-IL-4 antibodies; however, feeding antibodies against the different target peptides contributed to differential underlying T cell profiles (Figure 2). In birds fed antibodies against peptide 1, baseline CD3⁺CD4⁺ T_H and CD3⁺TCRγδ (γδ) T cell populations were 52.3% greater than the control (P ≤ 0.0005) without impacting CD3⁺CD8α⁺ cytotoxic T cell (T_C) populations (Figure 2B). In birds fed antibodies against peptides 2, 3, and 4, T_H cells were 65.9, 55.1, and 66.4% greater at baseline compared to the control, respectively (P < 0.0001) without affecting other measured T cell subpopulations. While dietary anti-IL-4 antibodies generally contributed to greater baseline PBMC T_H populations, birds fed antibodies against peptides 2 and 4 had 23.9 to 29.5% greater T_H populations compared to those fed antibodies against peptides 1 and 3 (P < 0.0001; Figure 2B).

Figure 2.

Baseline immune cell populations within peripheral blood mononuclear cells (PBMC) isolated from 14-day-old Ross 708 broilers fed diets containing egg yolk powder ± peptide-specific antiinterleukin-4 antibodies. Data represent the mean immune cell population positive for each marker within the (A) live cell gate or (B) CD3⁺ T cell gate from 6 birds/diet ± SEM. Bars within each population with different letter labels are significantly different (P ≤ 0.05).

During Eimeria challenge, a number of significant diet × Eimeria × timepoint interactions were observed within immune cell populations and demonstrate alterations in immune response timelines due to dietary anti-IL-4. To facilitate interpretation, immune cell population differences within each post-inoculation timepoint are presented in Figure 3. As innate immune responders, circulating monocytes as macrophage precursors were expected to show the most changes during earlier post-inouclation timepoints. At 3 dpi, birds fed the control and diets containing antibody against peptide 1 demonstrated 65.9 and 70.4% greater monocyte/macrophage⁺ cells, respectively, compared to their unchallenged counterparts (P ≤ 0.006; Figure 3A). From 3 to 7 dpi, PBMC monocyte/macrophages increased 63.9% in Eimeria-challenged birds fed antibodies against peptide 4, resulting in 64.0% greater populations compared to their unchallenged counterparts (P ≤ 0.02). In control-fed birds, PBMC monocyte/macrophage populations returned to similar levels between challenged and unchallenged birds at 7 dpi; however, in birds fed antibodies against peptide 1 these populations remained 41.8 and 67.7% greater in Eimeria-challenged vs. unchallenged birds at 7 and 14 dpi, respectively (P < 0.0001). Eimeria-challenged birds fed antibodies against peptide 2 had 57.0% greater monocytes/macrophages than their unchallenged counterparts only at 7 dpi with populations returning to similar levels at 14 dpi (P < 0.0001). No differences in monocytes/macrophages were observed over the course of Eimeria challenge in birds fed antibodies against peptide 3 (Figure 3A).

Figure 3.

Immune cell populations within peripheral blood mononuclear cells (PBMC) isolated from Ross 708 broilers fed diets containing egg yolk powder ± peptide-specific antiinterleukin-4 antibodies at different timepoints postinoculation (pi) with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ). Data represent the mean immune cell population positive for each marker within the (A) monocyte/macrophage⁺, (B) Bu-1⁺ B cells, and (C) CD3⁺ T cells the within live cell gate and (D) CD4⁺ helper, (E) CD8α⁺ cytotoxic, and (F) TCRγδ⁺ T cells within CD3⁺ populations from 3 birds/diet × Eimeria group ± SEM. Bars within each population and timepoint with different letter labels denote significant diet × Eimeria interactions (P ≤ 0.05).

As part of the adaptive immune response and contributing to long-term immunity through antibody production, Bu-1⁺ B cells were expected to respond at post-inoculation timepoints ≥7 dpi. At 3 dpi, Eimeria-challenged birds fed diets containing antibodies against peptides 1, 2, and 4 had 37.9, 49.1, and 63.6% greater B cells, respectively, than their unchallenged counterparts (P = 0.01; Figure 3B). Eimeria-challenged birds fed the control diet displayed stable PBMC B cell populations throughout the post-inoculation period while their unchallenged counterparts demonstrated a 58.7% increase from 3 to 7 dpi (P < 0.0001). From 3 to 7 dpi, Eimeria-challenged birds fed antibodies against peptides 1 and 4 displayed 38.9 and 47.7% increases in Bu-1⁺ cells, culminating in 46.6 and 55.2% greater populations compared to their unchallenged counterparts, respectively (P ≤ 0.005), followed by 64.2 and 56.2% decreases between 7 and 14 dpi (P < 0.00001). In Eimeria-challenged birds fed antibodies against peptide 1, this 7 to 14 dpi decrease in B cells resulted in populations 57.3% lower than their unchallenged counterparts (P < 0.0001) whereas B cell levels were recovered at 14 dpi in birds fed antibodies against peptide 4. While B cell populations in Eimeria-challenged birds fed antibodies against peptides 2 and 3 remained stable from 7 to 14 dpi, B cells increased in unchallenged birds fed these diets 66.4 and 47.7% from 7 to 14 dpi to levels 64.6 and 50.1% greater than their Eimeria-challenged counterparts, respectively (P ≤ 0.02; Figure 3B).

In addition to B cells, T lymphocytes initiate and coordinate adaptive immune responses and were expected to respond later during Eimeria challenge. At 3 dpi, unchallenged birds fed antibodies against peptide 1 had 35.0% greater CD3⁺ T cell populations than their Eimeria-challenged counterparts (P < 0.0001); however, cell populations were within the 6 to 10% range observed at baseline (Figures 2 and 3C). From 3 to 7 dpi PBMC T cell populations increased 40.3 and 41.7% in Eimeria-challenged birds fed control and peptide 1 antibodies, respectively, culminating in 36.3 and 382% greater T cell populations than their unchallenged counterparts at 7 dpi (P < 0.0001; Figure 3C). While T cells in Eimeria-challenged birds fed antibodies against IL-4 peptides 2 and 4 also increased 33.0 and 26.9% from 3 to 7 dpi (P ≤ 0.04), these changes were mirrored by their unchallenged counterparts resulting in similar 7 dpi T cell populations between Eimeria-challenged and unchallenged birds fed either diet. From 7 to 14 dpi, CD3⁺ T cell populations decreased 35.7 and 48.3% in Eimeria-challenged birds fed the control and antibodies against peptide 1 (P < 0.0001), recovering to levels similar to their unchallenged counterparts. At 14 dpi, Eimeria-challenged birds fed antibodies against peptide 2 had 27.7% greater T cells than their unchallenged counterparts (P < 0.0001); however, the change in populations from 7 to 14 dpi was not significant. Between 7 and 14 dpi, T cell populations also decreased 43.9% in Eimeria-challenged birds fed antibodies against peptide 4 to levels 41.2% lower than their unchallenged counterparts, potentially indicating T cell recruitment from systemic compartments to sites of infection in the intestine (P < 0.0001; Figure 3C).

Within overall T cell populations, subtypes analyzed in this flow cytometry panel included effector subtypes responsible for coordinating responses and activating B and other T cell subtypes (T_H), those with direct cytotoxic effects (T_C), or subtypes with proposed cytotoxic and immunoregulatory properties (γδ; Mombaerts et al., 1993; Vantourout and Hayday, 2013; Fenzl et al., 2017). At 3 dpi, Eimeria-challenged birds fed antibodies against peptides 2 and 4 had 38.4 and 42.4% greater CD3⁺CD4⁺ T_H populations than their unchallenged counterparts but only challenged birds fed peptide 4 antibodies simultaneously displayed 52.8% greater CD3⁺CD8α⁺ than their unchallenged counterparts (P < 0.0001; Figure 3D and E). Eimeria-challenged, control-fed birds also had 28.9% increased T_C cells at 3 dpi, whereas challenged birds fed antibodies against peptide 1 had 32.7% lower T_C cells at the same time (P ≤ 0.02; Figure 3D). From 3 to 7 dpi when overall CD3⁺ T cells increased in Eimeria-challenged birds fed control and peptide 1 antibodies, no alterations in T cell subtypes were observed in challenged control-fed birds. Eimeria-challenged birds fed peptide 1 antibodies displayed increased T_C and γδ T cell populations by 39.7 and 49.8%, respectively, from 3 to 7 dpi (P < 0.0001); however, only γδ T cell populations were 49.9% greater at 7 dpi between challenged and unchallenged birds fed antibodies against peptide 1 (P = 0.0004; Figure 3D–F). When Eimeria-challenged birds fed peptide 2 antibodies displayed greater overall CD3+ T cell populations at 14 dpi, T_H populations were simultaneously reduced 27.2% compared to their unchallenged counterparts (P = 0.0003) without additional changes observed within the other analyzed T cell subtypes (Figure 3D). In Eimeria-challenged birds fed antibodies against peptide 4, γδ T cells decreased 72.1% to levels 68.9% lower than their unchallenged counterparts (P < 0.0001), suggesting that this subtype may have been preferentially recruited when overall CD3⁺ populations decreased at the same time (Figure 3C–F).

Cecal Bacterial Communities: Alpha and Beta Diversity Measures

No differences in alpha diversity measures were observed at baseline (Figure 4). At 7 dpi, Eimeria-challenged birds had 44.8% fewer overall observed species in the cecum (P = 0.02) and 28.8% lower Shannon diversity indices (P = 0.006) compared to unchallenged birds, regardless of diet (Figure 4A and C). All alpha diversity measures in cecal microbial communities were recovered at 14 dpi. Principal coordinates analysis of Bray-Curtis distances for both baseline and post-inoculation datasets are presented in Figure 5. None of the diets displayed differential clustering patterns in either dataset.

Figure 4.

Alpha diversity measures of bacterial/archaeal communities determined by 16S rRNA gene amplicon sequencing within the cecum of Ross 708 broilers fed diets containing 2% egg yolk ± peptide-specific anti-IL-4 antibodies before and after inoculation with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ). Data represent the mean (A) observed species, (B) Chao1 species richness, (C) Shannon diversity, and (D) Simpson diversity indices from 6 birds/diet (baseline) or 3 birds/diet × Eimeria group/timepoint ± 95% confidence limit.

Figure 5.

Principal coordinate analysis of bacterial/archaeal communities in the cecum of Ross 708 broilers fed diets containing 2% egg yolk powder ± peptide-specific anti-IL-4 antibodies (A) before (d 14 baseline) and (B) after inoculation with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ). Each point represents a community isolated from an individual bird (6/diet at baseline; 3/diet × Eimeria challenge after inoculation) and ellipses represent the 95% confidence limit for each diet.

Cecal Microbial Communities: Genus-Level Changes

After removal of vertebrate, plant, and arthropod sequencing reads, 72 of the 90 original 18S rRNA gene amplicon sequencing samples were removed from the dataset due to inadequate sequence depth (<5,000 reads remaining), and eukaryotic community comparisons could not be analyzed. Within bacterial communities assessed by 16S rRNA gene amplicon sequencing, dietary anti-IL-4 antibodies did not affect relative abundance of the 20 most abundant genera prior to inoculation while Eimeria challenge caused microbial shifts favoring Bacteroides and Escherichia-Shigella at 7 dpi, which resolved by 14 dpi (Figure 6). Baseline shifts in microbial communities at the genus level were limited to detectable Bilophila relative abundance (mean = 0.40–0.47%) in the cecum of birds fed antibodies against peptides 2 and 4 while Bilophila was not present in birds fed the control or diets with antibodies against peptides 1 and 3 (P < 0.0001; Figure 7). Diet had minimal effect on cecal communities following Eimeria challenge and most changes were associated with the Eimeria and timepoint interaction. At 7 dpi, Eimeria-challenged birds had 7.2-, 17.5-, 2.1-, and 6-fold increased relative abundance of Escherichia-Shigella, Tyzzerella, Merdibacter, and Enterococcus, respectively, compared to unchallenged birds with 3.5-, 3.9-, 6.6-, 7.8-, 3.9-, and 8.5-fold reductions in Eisenbergiella, Lachnospiraceae GCA-900066575, Bacilli RF39, Shuttleworthia, Oscillospiraceae, and Clostridia UCG-014, respectively (P ≤ 0.04). At 14 dpi, these Eimeria-induced shifts in bacterial genera resolved to similar abundances between Eimeria-challenged and unchallenged birds (Figure 8).

Figure 6.

The 20 most abundant bacterial genera within the cecum of Ross 708 broilers fed diets containing 2% egg yolk powder ± peptide-specific antiinterleukin-4 antibodies at (A) baseline (d 14), (B) 7 d, and (C) 14-day postinoculation (pi) with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ). Color density represents the mean relative abundance from 6 birds/diet at baseline and 3 birds/diet × Eimeria challenge at 7 and 14 dpi.

Figure 7.

Relative abundance of the genus Bilophila in the cecum of 14-day-old Ross 708 broilers fed diets containing 2% egg yolk powder ± peptide-specific antiinterleukin-4 antibodies. Each point represents an individual bird and bars represent the mean relative abundance in cecal contents collected from 6 birds/diet ± SEM. Bars with different letter labels are significantly different (P ≤ 0.05).

Figure 8.

Genera affected by the Eimeria challenge × timepoint interaction within the cecal bacterial/archaeal communities of Ross 708 broilers fed diets containing 2% egg yolk powder ± peptide-specific antiinterleukin-4 antibodies following inoculation with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ). Data represent the mean relative abundance of each genus (15 birds/Eimeria group/timepoint) ± SEM. Bars with different letter labels within each genus are significant different (P ≤ 0.05).

DISCUSSION

This study evaluated the potential performance and physiological responses to feeding egg yolk powder containing anti-IL-4 antibodies against various peptide regions within the IL-4 protein structure. To simplify findings, comparing outcomes for each peptide antibody to the control provided more insight into how each antibody affected outcomes in unchallenged and Eimeria-challenged birds rather than comparing peptides to each other. During Eimeria challenge, control-fed birds ate 8.3% less feed, experienced a 10.9% BW reduction at d 21 and 23.3% BWG reduction in the first 7 dpi (wk 3), numerically, compared to their unchallenged counterparts. The magnitude of wk 3 performance reductions between Eimeria-challenged and unchallenged birds fed antibodies against peptides 2 and 3 were similar to those observed in control-fed birds. In contrast, Eimeria-challenged birds fed antibodies against peptides 1 and 4 displayed numerically greater magnitude performance losses at 19.5 to 26.8% reduced d 21 BW, 21.5 to 25.3% reduced FI, and 31.8 to 46.5% reduced wk 3 BWG compared to their unchallenged counterparts (Table 3). Performance recovery was apparent in birds fed the control diet with 6.7% reduced d 28 BW and <1.0% wk 4 BWG and FI reductions between Eimeria-challenged and unchallenged birds. Eimeria-challenged birds fed antibodies against peptides 1, 2, and 3 demonstrated compensatory BWG and FI in wk 4 compared to their unchallenged counterparts while Eimeria-challenged birds fed antibodies against peptide 4 had prolonged 19.3, 7.7, and 10.9% numerically reduced d 28 BW and wk 4 BWG and FI, respectively, compared to their unchallenged counterparts (Table 3). For the entire 28 d study, BWG between Eimeria-challenged and unchallenged birds fed the control, peptide 1, peptide 2, and peptide 3 antibody diets was reduced 7.0, 3.7, 2.3, and 1.6% while a 20.0% separation in BWG for the entire study was observed in birds fed antibodies against peptide 4 (Table 3).

Collectively, these outcomes demonstrate that feeding antibodies against peptides 2 and 3 do not protect bird performance at the height of Eimeria challenge but may contribute to compensatory responses during the recovery period that culminate in unchanged overall performance. Antibodies against peptide 4 contributed to prolonged, higher magnitude performance reductions whereas antibodies against peptide 1 demonstrated short-term high magnitude performance losses that were recoverable by the end of the study. While intriguing, the availability of egg yolk powder for each peptide antibody was a limiting factor in the current study, restricting the number of birds that could be housed for a 28 d preliminary challenge trial and euthanized for sample collection. Additionally, excreta samples were pooled by treatment to confirm Eimeria challenge and to account for unsynchronized Eimeria cycling in floor pens. Eimeria-challenged birds fed antibodies against peptide 1 displayed numerically reduced fecal oocysts at both 7 and 14 dpi compared to their control-fed counterparts while birds fed antibodies against peptides 2 and 3 displayed numerically greater fecal oocysts than other treatments at 14 dpi (Table 5). This could indicate intestinal environment changes due to anti-IL-4 that favored Eimeria clearance or persistence that could not be clarified by 18S rRNA gene sequencing to determine Eimeria relative abundance due to high presence of host and feed sequences. As such, future research is needed to better elucidate the long-term effects of anti-IL-4 peptide antibodies on Eimeria challenge recovery and oocyst cycling during a longer grow-out period and with higher stocking densities more reflective of commercial practice.

Despite sample size limitations, outcomes from flow cytometric analysis demonstrate differential immune cell responses that could provide insight into mechanistic responses to dietary peptide-specific anti-IL-4 antibodies before and after Eimeria challenge. Prior to Eimeria inoculation, antibodies against peptide 4 had the greatest impact on baseline immune cell populations, increasing PBMC monocytes/macrophages and inducing a higher magnitude of increased B cell populations compared to antibodies against other peptides (Figure 2A). Chicken Bu-1, the B cell marker used in this study, is expressed on B cells at all developmental stages but is absent following differentiation into antibody-producing plasma cells (Houssaint et al., 1987; Tregaskes et al., 1996). While the role of IL-4 in chicken B cell development is unknown, in mammals it plays a role in B cell maturation, activation, and antibody production (Nelms et al., 1999; Granato et al., 2014). In chickens, B cell development occurs within the bursa which connects to the cloaca through a duct that allows interaction with intestinal contents (Schaffner et al., 1974). While this association is explored in the context of host-microbiota interactions affecting B cell development, it is possible that anti-IL-4 reduced or diminished the activity of intestinal IL-4 and limited maturation of bursal Bu-1⁺ naïve B cells into Bu-1⁻ plasma cells, creating an enriched population of Bu-1⁺ populations that entered systemic circulation. As such, this could explain the systemic response in B cell populations by dietary anti-IL-4 despite the inability of egg yolk IgY antibodies to translocate the intestinal barrier (Cook and Trott, 2010). Though it is an intriguing possibility, further research is needed to elucidate the exact role of chicken IL-4 on B cell development, its concentration in intestinal contents, and whether intestinal IL-4 meaningfully contributes to B cell profiles entering systemic circulation from the bursa.

In addition to altered B cells, antibodies against IL-4 generally increased underlying CD3⁺CD4⁺ T_H populations in PBMC with the greatest magnitude of change observed in birds fed antibodies against peptides 2 and 4 (Figure 2B). Interluekin-4 is an important cytokine for the development of T_H cell differentiation into effector subtypes, particularly T_H2 cells associated with antihelminthic functions (Silva-Filho et al., 2014). Unlike B cells, chicken T cell development occurs in the thymus and is not located in close proximity to intestinal contents; however, research has shown that the intestinal microbiota can alter chicken thymic structures and T cell development (Lee et al., 2018; Cheng et al., 2021). In this study, peptide-specific anti-IL-4 antibodies did not alter baseline cecal microbial communities, but this may be a function of small sample size or the effects occurring in intestinal segments other than the ceca (Figure 4, Figure 5, Figure 6). As such, further research is needed to better characterize microbiota changes due to anti-IL-4 throughout the gastrointestinal tract and include immune cell functional assays to better determine the relationship between anti-IL-4, the microbiota, and altered T_H populations in unchallenged broilers.

Immune cell population changes in PBMC following Eimeria challenge can be better understood in the context of the interplay between systemic immunity and local tissues during disease challenge. Monocytes as macrophage precursors associated with innate immune responses were expected to respond at early timepoints during the challenge (Yang et al., 2014), which was observed as increased monocytes/macrophages in the PBMC of Eimeria-challenged birds fed control diets at 3 dpi. Monocyte/macrophage⁺ responses in Eimeria-challenged birds fed antibodies against IL-4 peptide 1 mirrored those seen in control-fed birds in the first 3 dpi but stayed elevated relative to their unchallenged counterparts for the duration of the study, suggesting a sustained monocyte/macrophage response. Eimeria-challenged birds fed antibodies against peptide 4 displayed a significant monocyte/macrophage increase from 3 to 7 dpi while increases at the same time were numeric, but not significant, in birds fed antibodies against peptide 2. In contrast, Eimeria-challenged birds fed peptide 3 antibodies displayed minimal monocyte/macrophage responses during Eimeria challenge (Figure 3A). This suggests that antibodies against peptide 2 and 4 antibodies may have delayed systemic monocyte/macrophage responses while peptide 3 antibodies may have altered intestinal immune responses in a way that eliminated the need to expand and recruit these cell types from systemic compartments; however, further research into intestinal immune responses to anti-IL-4 antibodies is needed.

Throughout the course of Eimeria challenge, control-fed birds did not display PBMC B cell population shifts with lymphocyte responsiveness limited to CD3⁺ T cell expansion between 3 and 7 dpi (Figure 3B). In contrast, lymphocyte populations in birds fed peptide-specific anti-IL-4 antibodies were not limited to T cell responses. Eimeria-challenged birds fed peptide 1 antibodies demonstrated PBMC B cell expansion from 3 to 7 dpi with potential recruitment from systemic circulation to intestinal sites of infection from 7 to 14 dpi as part of an adaptive response, suggesting a requirement to systemically expand and recruit this cell population during challenge. In contrast, birds fed peptide 4 antibodies demonstrated B cell increases as early as 3 dpi that persisted from 3 to 7 dpi before recovering to levels similar to their unchallenged counterparts as early as 14 dpi, potentially indicating that an earlier B cell response was initiated. Eimeria-challenged birds fed peptide 2 and 3 antibodies displayed stable B cell populations throughout the study while B cell expansion was observed in their unchallenged counterparts from 7 to 14 dpi with a much greater increase observed in birds fed peptide 2 antibodies (Figure 3B). In this case, the increased Bu-1⁺ populations in unchallenged birds fed peptide 2 and 3 antibodies could indicate alteration of systemic B cell profiles due to potential luminal IL-4 depletion that may be attenuated by intestinal B cell requirements during Eimeria challenge.

Control-fed birds displayed a 3 to 7 dpi increase in overall CD3⁺ T cells during Eimeria challenge that was mirrored in challenged birds fed peptide 1 antibodies (Figure 3C). Where the 2 responses differentiated was in the underlying T cell types. No changes in T cell subpopulations were observed in Eimeria-challenged control-fed birds while underlying T_C and γδ T cell populations were simultaneously increased in birds fed peptide 1 antibodies (Figure 3E and F). Both T_C and γδ T cells exhibit cytotoxic functions that may be favorable for the clearance of intracellular pathogens like Eimeria (Mombaerts et al., 1993; Fenzl et al., 2017; Kim et al., 2019), and while no evidence of T cell recruitment was observed in Eimeria-challenged birds fed peptide 1 antibodies, it is possible that recruitment occurred at timepoints not measured in the current study. Keeping with a general trend, Eimeria-challenged birds fed peptide 2 and 3 antibodies did not display significantly different T cell shifts from their unchallenged counterparts, suggesting that systemic T cell populations may not have been required to mount an adaptive immune response within the intestine. Birds fed peptide 4 antibodies during Eimeria challenge did not display T cell expansion different from their unchallenged counterparts; however, there was evidence of T cell recruitment from 7 to 14 dpi (Figure 3C). Examination of T cell subtypes at the same time indicate that γδ T cells may have been preferentially recruited (Figure 3F). As γδ T cell populations have poorly defined functions that encompass immune regulation or cytotoxic activity, it is uncertain if this late preferential recruitment represents immune response resolution or delayed recruitment of T cell subtypes suitable for intracellular pathogen clearance.

Generally, systemic immune system changes provide some insight into how these anti-IL-4 antibodies differentially altered immune responses but also indicated that intestinal immunity was altered to the point of minimal systemic immune input being necessary in the case of peptide 2 and 3 antibodies. Further research is needed to evaluate intestinal immune responses; however, the microbiota could provide insight into some of the intestinal changes due to anti-IL-4. At the genus level, changes were limited to the Eimeria and timepoint main effect with Eimeria-associated increases in the relative abundance of genera like Escherichia-Shigella and Tyzzerella corresponding with previous Eimeria challenge research (Macdonald et al., 2017; Jebessa et al., 2022; Figure 8). Eimeria challenge is not typically associated with altered alpha diversity measures, which contradicts findings in this study; however, previous work has implemented challenges with a single Eimeria species and the reduced cecal alpha diversity herein may be partially due to challenge with multiple Eimeria species or an artifact of low sample size (Madlala et al., 2021; Jebessa et al., 2022). As a result, the current study indicates that the effects of anti-IL-4 antibodies during Eimeria challenge may be host-specific, but further research on select peptide antibodies with greater sample sizes may better elucidate the relationship between anti-IL-4 and the intestinal microbiota.

Outcomes from this study serve as important preliminary results for further investigation into the role of anti-IL-4 antibodies on broiler immune development and responses to Eimeria challenge. When considering the immune and performance outcomes, prolonged systemic monocyte/macrophage responses in Eimeria-challenged birds fed peptide 1 antibodies coupled with relatively unaltered adaptive immune responses may have contributed to the numerically greater performance reductions in the first 7 dpi of Eimeria challenge that numerically recovered by wk 4. As a more extreme case, the delayed systemic monocyte/macrophage expansion and T cell recruitment coupled with early B cell responses in Eimeria-challenged birds fed peptide 4 antibodies could have created a prolonged, energetically expensive adaptive immune response that contributed to the increased magnitude of performance reductions observed in these birds (Klasing and Leschinsky, 1998). Instead, the minimal alterations in systemic immunity during Eimeria challenge in birds fed antibodies against peptides 2 and 3 indicate that local immune responses were favorably altered to reduce systemic immune requirements, resulting in potentially diminished immune-related energy expenditure and performance reductions that were recoverable during Eimeria challenge. As such, further research to evaluate the exact functions of anti-IL-4 antibodies should emphasize antibodies against peptides 2 and 3 for further refinement as a potential Eimeria mitigation strategy.