Efficacy of hyperimmunized egg yolk antibodies (IgY) against Campylobacter jejuni: In Vitro and In Vivo evaluations

Abstract

Campylobacter infections are a prevalent cause of diarrheal disease in humans and are the most significant zoonotic pathogens worldwide. Human campylobacteriosis is generally via ingestion of contaminated poultry products. However, based on recent studies chicken egg yolk antibody (IgY) powder has great potential to reduce the cecum load of Campylobacter jejuni (C. jejuni) in broilers. To understand the effective and economically feasible dosage, two immunization and challenge studies were conducted using 30 layer hens and 250 broiler chickens and found a scientific approach, starting with in vitro evaluations and progressing with in vivo studies confirmed. In this study it was demonstrated that specific IgY powder (SIgY), produced by immunized hens via bacterin, was highly effective in inhibiting bacterial growth and adhesion, as well as exhibiting bactericidal and agglutination properties (P < 0.05). Notably, doses of 0.5 % and 1 % SIgY significantly enhanced both the height and width of intestinal villi, along with improving the villus height-to-crypt depth ratio when compared to the positive control group (P < 0.05). Furthermore, medium and high doses of SIgY were effective in preserving the integrity of the intestinal epithelium, as evidenced by a reduction in crypt depth and the number of goblet cells, which serve as important markers in the immune system (P < 0.01). Additionally, analyses of cecal and liver bacterial counts in response to the 0.5 % SIgY treatment revealed a significant reduction in C. jejuni counts compared to other challenged groups throughout the 28 d experiment (P < 0.01). Based on these results, it may be concluded that specific antibodies play a crucial role in maintaining the integrity of intestinal villi, support the health of the intestinal epithelium, and reduce the colonization of C. jejuni. These findings could form the basis for developing an economical and effective strategy to enhance poultry and human health in the context of C. jejuni infection.

Graphical abstract

Introduction

Campylobacter jejuni (C. jejuni) is a gram-negative pathogen that can cause gastroenteritis in humans, is a prominent source of zoonotic infections, and is responsible for more than 62 % of confirmed cases in the European Union in 2021 (EFSA and ECDC, 2022). In contrast to other foodborne illnesses such as salmonellosis, campylobacteriosis has the potential to give rise to severe autoimmune disorders that can be fatal to humans (Feye et al., al.,2020). Consequently, the presence of Campylobacter poses a substantial threat to human health, emphasizing the implementation of measures aimed at controlling or eliminating C. jejuni infection in food sources.

Exploring alternative approaches to ameliorate Campylobacter infections is crucial. Chicken egg yolks are rich in antibody, particularly immunoglobulin Y (IgY), which have emerged as promising candidates for immunological supplementation in food products and can provide rapid protection against diseases that do not respond to conventional antibiotic therapies (El-Kafrawy et al., 2023). Incorporating IgY as a feed additive has proven effective in controlling diseases of the alimentary tract (Rehan et al., 2020). Avian species have evolved a mechanism to transfer passive immunity to their offspring through the deposition of immunoglobulins in egg yolks. During the formation of an egg, immunoglobulins from the hen's serum are actively transported into the egg yolk resulting in higher concentrations of IgY in the yolk (Thibodeau et al., 2017; Kowalczyk et al., 2019). These transferred antibodies in the egg yolk provide passive immunity to the developing chick protecting it against potential pathogens (Wang et al., 2021). Typically, the generation of specific IgY takes place via the inoculation of laying avian with the particular antigen or whole cells, which may encompass simple molecules or complex structures such as viruses, bacteria, and other pathogens (Paul et al., 2014).

The application of IgY for the treatment and control of infectious and non-communicable diseases in both human and animal populations has been investigated (Diraviyam et al., 2014; Pereira et al., 2019; Leiva et al., 2020). In these studies, conflicting results were observed due to variations in the dosage of egg yolk powders, vaccines, period, and different bacterial strains used across experiments. For instance, regarding C. jejuni bacteria, Vandeputte et al. (2019) demonstrated a significant decrease in C. jejuni counts per infected broiler using 5 % hyperimmune egg yolk powder. However, Paul et al. (2014) reported no differences in C. jejuni counts when using 10 % hyperimmune egg yolk powder. This discrepancy underscores the critical importance of systematically assessing egg yolk powder conditions in both in vivo and in vitro experiments to ensure reliable and reproducible research findings.

Accordingly, In vitro analysis (agglutination test, bactericidal, and mobility assay) of IgY derived from egg yolk powder provided evidence of its beneficial effects in repressing C. jejuni. However, based on the in vivo data protected egg yolk powder containing anti-C. jejuni IgY does not provide evidence for the ability to impede C. jejuni colonization in the chicken cecum (Garba et al., 2019). Furthermore, in order to evaluate the effectiveness of chicken IgY in addressing diarrhea in diverse animal classes, such as piglets, mice, poultry, and calves, a comprehensive review of 374 research reports was undertaken. Following a rigorous selection process, 61 studies were identified for meta-analysis. The findings derived from the meta-analysis demonstrated that oral passive immunization with IgY yields favorable outcomes in the control and prevention of diarrhea in domestic animals. Nonetheless, further research is indispensable to optimize the appropriate dosage of IgY (Diraviyam et al., 2014).

The objective of the study was to evaluate the presence and efficacy of specific and non-specific IgY through in vitro testing, as well as in vivo experiments with broiler chickens. Additionally, a scond objective was to determine the optimal dose of egg yolk powder enriched with anti‐C. jejuni IgY to effectively reduce C. jejuni colonization within a farm environment.

Materials and methods

Experimental design

There were two steps in this study. In the first step, the goal was to prepare hyperimmune egg yolk powder, extract IgY, and determine the effectiveness of IgY in vitro. In the second step, experiments were conducted on broiler chickens to determine the effective dose of hyperimmune egg yolk powder. All experimental procedures (sampling, immunization of hens, and euthanasia methods), and animal husbandry were conducted following the guidelines approved by the Institutional Animal Care and Use Committee of Tarbiat Modares University (REC.1400.134).

Bacterial strain and culture conditions

C. jejuni ATCC 29428 was provided by Razi Vaccine and Serum Production Research Institute (Karaj, Iran) and was inoculated onto the Campylobacter selective agar (Oxoid, UK) with 10 % sheep blood agar. The plates were incubated for 48 h at 42°C under microaerophilic consisting of 5 % oxygen (O₂), 7.5 % carbon dioxide (CO₂), 2.5 % hydrogen (H₂), and 85 % nitrogen (N₂).

Formalin-killed campylobacter

A formalin-killed C. jejuni whole cell (bacterin) was prepared as follows: Freshly grown bacterial cultures were washed three times with sterile Phosphate-Buffered Saline (PBS, pH 7.2, Sigma-Aldrich). The bacterial cells were then harvested by centrifugation at 4000 × g for 10 min at 4°C. The resulting pellet was adjusted with sterile PBS containing 0.4 % formalin to a final concentration of 1.5 × 10⁸ CFU/mL, equivalent to a 0.5 McFarland Standard. To confirm the cells were completely inactivated, the suspension was plated on modified charcoal cefoperazone deoxycholate agar (mCCDA) (Oxoid, UK) and incubated overnight at 42°C. Once cell death was verified, the formalin-killed bacterial suspension was stored at 4°C for later use.

Immunization of laying hens

Thirty 30-week-old Single Comb White Layer hens, confirmed negative for C. jejuni through cloacal swab testing, were individually housed in sloping wire-floored cages measuring 40 cm × 50 cm × 45 cm (height × width × depth), until they reached 52 weeks of age. The room temperature was maintained at 20 ± 4°C, following a light/dark cycle of 16/8 h (h). Drinking water and a commercial diet were provided ad libitum, and the birds were randomly assigned to two groups. The first group (control = 10 hens) was not exposed to any bacterin while the second group was injected with 10⁸ CFU/mL bacterin at two sites of the breast muscles, with 0.5 mL per site (bacterin = 20 hens). Each immunization dose consisted of a 1:1 mixture of the inoculum with Freund's Complete Adjuvant (FCA) for the first dose at the age of 32 weeks. The subsequent four booster doses, a 1:1 mixture of the inoculum and Freund's Incomplete Adjuvant (FIA), were administered at 14 d intervals following the initial immunization. For sham immunization, 1 mL of PBS was used for each immunization in the negative control.

Antibody extraction and preparation of IgY powder

In the experiment conducted for the IgY powder production, approximately 129 eggs were obtained per bird, and stored at 4°C. The collected eggs were subsequently utilized in the powder manufacturing process every week from the sixth week onwards. The preparation of hyperimmune egg yolk powder involved isolating by the water-soluble fraction (WSF) method of egg yolk which contains IgY. This was achieved by utilizing cold acidified distilled water with a pH of 2.5 adjusted using 0.1 M HCl, following the protocols of Akita and Nakai (1992) and Mahdavi et al., 2010a, Mahdavi et al., 2010b. The WSF containing IgY was frozen at −60°C then lyophilized by a freeze dryer (Martin-Christ, Osterode am Harz, Germany) for 48 h to achieve a dry IgY Powder. Specific IgY Powder (SIgY) derived from hyperimmune egg yolks and non-specific IgY Powder (NSIgY) from non-immunized hen eggs were utilized as controls. Furthermore, the extraction of chicken IgY antibodies from egg yolk was by the polyethylene glycol (PEG) 6000 method (Pauly et al., 2011). The concentration of SIgY and NSIgY protein powder obtained from all collected eggs was determined using the Bradford method, with Bovine Serum Albumin (BSA) and purified chicken IgY (1 mg of protein/mL, Sigma Immunochemicals, Saint Louis, MO, USA) serving as standard solutions (Bradford, 1976). These standards were prepared in concentrations ranging from 0.0625 to 0.5 mg/mL to generate a standard curve.

SDS-page and ELISA

Both sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA) were performed to assess the characteristics of the IgY antibodies. The molecular weight of SIgY using the WSF and PEG-6000 methods was determined by SDS-PAGE under reducing conditions, as outlined by Laemmli (1970) and Rosen et al. (2010). The titers of anti-Campylobacter IgY, extracted by the WSF method, in serum and egg yolks were determined using the ELISA protocol previously described by Helmy et al. (2022) with minor changes to the ELISA protocol. Microtiter plates were coated with 100 µL of bacterin in equal concentration carbonate bicarbonate buffer (1/1; vol/vol) (2.16 g sodium carbonate decahydrate (Na₂CO₃.10H₂O), 1.935 g sodium bicarbonate (NaHCO₃) in 500 mL water (H₂O), pH 9.6), and incubated overnight at 4 °C. After washing three times with PBST (0.05 % Tween-20 in PBS), the plates were blocked (90 min at 37°C) with 100 µl blocking buffer (3 % nonfat dry (skim) milk in PBS). The plates were then washed (3 ×) with PBST. Next, 100 µL of three dilutions of samples (diluted 1:1000 in PBS) were added to duplicate wells and incubated at 37°C for 2 h, then washed again (3 ×) with PBST. After washing 100 µL of goat anti-chicken IgY conjugated with horse-radish peroxidase (HRP) (diluted 1:10000 in PBS, Sigma) were added to each well and incubated at 37°C for 90 min. After washing as described above, the plates were incubated with 100 µl 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma) for 20 min at room temperature in the dark. The reaction was stopped with 50 µL 0.5 M sulfuric acid (H₂SO₄) and absorbance was read at 450 nm in a microplate ELISA reader (Anthos 2020, Salzburg, Austria).

Agglutination test

The cultured C. jejuni cells were suspended in PBS until the absorbance at 600 nm reached approximately 1.0. Then, 200 µL of the suspension was transferred to an agglutination plate, followed by adding WSF extracts in the same volume ratio. The suspension was then agitated and agglutination was recorded within 2 min incubation at room temperature.

Growth inhibition assay

The C. jejuni inhibition assay was performed in Mueller Hinton Broth (MHB) (Oxoid, UK), prepared by suspending the microorganisms in normal saline and adjusting the density to 0.5 McFarland standard tube (∼1.5 × 10⁸ CFU/mL). SIgY and NSIgY were reconstituted to concentrations of 50, 100, and 150 mg/mL. For another treatment, MHB medium containing 25 µL/mL Enrofloxacin antibiotic (Rooyan Darou, Iran) was used. The mixture was subsequently filtered using a 0.22 µm filter. MHB medium without IgY and antibiotic was used as the negative control. These mixtures were incubated under microaerobic conditions (37°C) and their optical density at 600 nm (OD600) was measured in three biological replicates. Then IgY growth inhibition curves were measured by plotting the OD600 value as a function of time.

Collection of broiler chicken intestinal mucus

Campylobacter-free 21 d broiler small intestinal mucus was isolated as described by Hermans et al. (2014) with some modifications. The intestine was transported on an ice pack to the laboratory where sections of jejunum and ileum were gently rinsed with ice-cold PBS to remove any solids. The mucosal layer was then scraped from the intestinal segment using glass slides and collected into 5-mL tubes. The mucus was diluted 1:3 with PBS, then centrifuged at 2000 × g for 10 min at 4 °C (repeated three times) to remove solids. After filtering the supernatant through a 0.45-µm, the protein concentration was measured using the Bradford protein assay kit (Sigma-Aldrich).

Mucus adhesion test

The adherence of bacteria to IgY, was investigated with C. jejuni (∼1.5 × 10⁸ CFU/mL) mixed with 100 µL PBS, different concentrations of SIgY 50 and 100 mg/mL (SIgY-50 and SIgY-100), along with a NSIgY 100 mg/mL, as described by Hermans et al. (2014) and Wang et al. (2019). Briefly, intestinal mucus was diluted with buffer to a final concentration of 250 µg protein/mL, 100 µL of this mixture incubated overnight at 4°C. After washing, plates were incubated with 100 µL blocking buffer for 1 h at room temperature. The wells were then washed and 100 µL of the mixtures of IgY and C. jejuni were added to a plate coated with mucus and incubated at 42 °C for another 1 h. The wells were then washed and adherent bacteria were collected by adding 200 µL of 0.5 % Triton X100 to each well. The mixture was incubated for 30 min at room temperature while shaking. Next, 10-fold dilutions of the wells in PBS were titrated on mCCDA plates.

Bactericidal assay

The bactericidal assay was conducted in sterile microcentrifuge tubes using PBS, specific WSF, and non-specific WSF. The complement source consisted of serum collected from C. jejuni-free chickens, which had been sterilized with a 0.45-μm filter and inactivated by heating the serum to 56°C (referred to as inactive complement). Each test tube contained 50 µL of the bacterial suspension (OD₆₀₀ = 1), 50 µL of a 1:5 complement (in PBS), and 10 µL of WSF. Control tubes included (i) bacteria plus complement only, (ii) bacteria plus WSF only, and (iii) bacteria plus PBS only. The assays were incubated for 1 h at 37°C. Following the incubation period, 100 µL of the mixture was carefully cultured on mCCDA plates, which were incubated for 2 d at 42°C under microaerophilic conditions. Colonies were counted, and bactericidal activity was determined as the percentage reduction of live C. jejuni compared to the control. The percent reduction was calculated using the following formula: % reduction = [CFU (bacteria + complement only) − CFU (bacteria + antibody + complement)]/CFU (bacteria + complement only) × 100. The experiment was conducted thrice (Sahin et al., 2001).

Oral inoculating suspension

For the in vivo trial, freshly grown bacterial cultures were harvested and diluted in sterile PBS to achieve a specific viable concentration of 1.5 × 10⁸ CFU/mL. The inoculum concentration was determined using McFarland standard tubes. This concentration falls within the physiological range typically encountered by gut bacteria (Lamb-Rosteski et al., 2008). All groups, except the negative control group (NC), were orally gavaged with C. jejuni (1 mL containing 10⁸ CFU/mL) on d 7 and 8 after hatching. The goal was to achieve a persistent infection in the chicks and maintaining stable C. jejuni counts over time. In contrast, the negative control group was orally gavaged with 1 mL of sterile PBS and received no additional treatments.

Chicks and diets

A total of 250 1 d old chicks (Ross 308) were randomly divided into five groups with five replicates (n = 10/replicate) in a completely randomized design: Negative Control (NC); C. jejuni-infected chicks (Positive Control (PC)); PC + 0.25 % (SIgY-0.25 %); PC + 0.5 % (SIgY-0.5 %); and PC + 1.0 % (SIgY-1 %). Chicks were obtained from a commercial hatchery (Kosar Company, Karaj, Alborz Province, Iran) and were reared in floor pens (2 × 1 × 1.2 m³), and were given ad libitum access to water while being fed a commercial diet, with one feeder and drinker available for every 10 birds. The room temperature was maintained at 32°C for the first three days of the trial and gradually decreased by 2°C per week until it reached 24°C at four weeks of age. The negative control was housed in a separate isolating room. Prior to experimental infection, all birds were confirmed to be Campylobacter-free by taking cloacal swabs, which were streaked onto mCCDA plates and incubated for 48 h in microaerobic conditions at 42°C. SIgY mixed manually through the feed (wt/wt). At 7 days and 8 days of age, all chicks in each group, except for the negative control group, were orally gavaged with approximately 1 × 10⁸ CFU/mL of C. jejuni. One chicken from each replicate was humanely euthanized by CO₂ asphyxiation at 14, 21, and 28 days of age.

Histomorphological traits of ileum

At the end of the feeding trial (d 28), one bird from each replicate was euthanized (as described above) and 2 cm intestinal segments from proximal to the ileocecal junction (from Meckel's diverticulum to the ileocecal colonic junction) were collected. This trimmed segment was referred to as the ileum. After sampling, the ileum fragments were processed (serial dehydration, clearing, and embedding in paraffin), sectioned to 5 µm thickness by a microtome, and then placed on a glass slide and stained for microscopic evaluation. Further, for each intestinal cross-section, ten intact, well-oriented crypt-villus units were selected for experiments conducted in five replicates (resulting in 50 measurements for each sample). The morphometric indices evaluated were villus height (from the tip of the villus to the crypt, 100X the magnification), villus width (from one side to the other in the midpoint of the villus, 100X the magnification), crypt depth (from the base of the villi to the submucosa, 100X the magnification), the villus height to crypt depth ratio, and goblet cells were counted as the number of goblet cells per 100 μm villus height (400X the magnification) (Giannenas et al., 2010). All the measurements were performed under an Olympus light microscope (CX 21, Olympus, Stuttgart, Germany).

Liver and cecal campylobacter jejuni enumeration

The right lobe of the liver was sterilely harvested before removing the cecum to ensure an accurate count of C. jejuni and avoid the risk of false negatives. The liver surface was seared with a hot spatula, cut with a sterile scalpel, and then homogenized. Subsequently, the cecum was ligated at the ileocecal junction and aseptically excised. One gram of the cecal contents and homogenized liver from each bird were serially diluted in a 0.9 % sterile saline solution and used for the C. jejuni assay.

Statistics

The in vitro experiment was performed in triplicate on three separate occasions and microbial counts were converted to log₁₀ CFU/mL. The data were fitted by one-way ANOVA using the generalized linear model (GLM) procedure of SAS software version 9.4 (SAS Institute Inc., 2014). Moreover, significant differences (P < 0.05) between means were separated using Duncan's multiple range test. The following statistical model was used:

$${\mathrm{Y}}_{\text{ij}}=\mu +{\mathrm{T}}_{\mathrm{i}}+{\mathrm{e}}_{\text{ij}}$$

Where Y_ij is the dependent variable, μ is the sample mean, T_i is the fixed treatment effect, and e_ij is the effect of the error.

Results

Concentrations of protein in the WSF powder

The average protein and total IgY concentrations in the IgY powder obtained from all collected eggs over the 20 wk experimental period are presented in Table 1. As expected, the protein concentration, total IgY concentrations, and purity of IgY in the NSIgY from non-immunized chicken egg yolk were significantly lower than those in the SIgY (P < 0.05).

Table 1.

Characteristics of the IgY powder containing specific and non-specific IgY powder.

IgY Powder	Protein (mg/g)	Total IgY (mg/g)	Purity (%)
SIgY	498a	96.16a	19.32a
NSIgY	473.6b	79.5b	16.79b
SEM	5.055	1.354	0.395
P-Value	0.02	0.001	0.016

^a,bMeans within a column with no common superscript differ: P < 0.01; P < 0.05, SEM: Standard Error of the Mean. (n: 3 samples/treatment per group). SIgY: Specific IgY Powder; NSIgY: Non-Specific IgY Powder.

SDS page

The use of PEG-6000 and WSF methods, as demonstrated by the results obtained from SDS-PAGE analysis, effectively isolates antibodies from egg yolks (Fig. 1). Nevertheless, the gel displayed no significant differences in purity between the PEG-6000 and WSF methods. The antibody exhibited two bands under reducing conditions, as shown in Fig. 1, which corresponded to approximately $\approx$65-67 kDa and$\approx$25-27 kDa.

Fig. 1.

SDS-PAGE analysis of IgY, extract under the reducing conditions, from hyperimmune egg yolks. (1): The molecular weight marker (Sinaclon, Tehran, Iran), (2): IgY by water-soluble fraction method, (3): IgY by polyethylene glycol-6000 method. Two protein bands with a$\approx$ 65-67-kDa heavy chain and a$\approx$ 25-27-kDa light chain were observed in WSF and IgY.

Campylobacter-specific antibody activity

Mean ELISA OD values (Fig. 2) illustrate the ability of IgY from immunized hens to bind with C. jejuni and showed the pattern of the immune response by the hens over 26 wk in serum and egg. Based on the ELISA results, the level of IgY-binding activity in blood serum and egg increased one wk after the primary injection of bacterin with FCA and constantly elevated after the first and second booster injections. The binding activity of anti-C. jejuni IgY reached a peak (OD of 1.29 in blood serum and OD of 1.44 in Egg) in the 8th wk. The IgY titer increased from initial immunization and the high titer persisted for 10 wk (from 8 to 18 wk after the first immunization). However, the mean OD for the non-immunized group was significantly lower (OD = 0.44), compared with the immunized group (P = 0.0001).

Fig. 2.

The change of specific activity of IgY in the serum and egg yolk from broiler chickens immunized with C. jejuni bacterin and Freund's Complete Adjuvant (FCA) and C. jejuni bacterin and Freund's Incomplete Adjuvant (FIA). The arrows indicate immunization times (bacterin + FCA ; bacterin + FIA ). ^{a, g} Means bearing different superscripts during the immunization period are significantly different. Data are mean ± standard deviation (n = 3 samples/treatment per group).

Agglutination

Anti-C. jejuni IgY was assessed utilizing the agglutination technique. As anticipated, a discernible positive response was observed about the agglutination of C. jejuni in the presence of IgY.

Growth inhibition

The growth inhibition assay resulted in concentration-dependent outcomes for SIgY and NSIgY antibodies (Fig. 3). Antibiotic treatment significantly suppressed bacterial growth (P < 0.01). Notably, there was no significant difference in bacterial growth inhibition between the high dose of SIgY (SIgY-150) and the medium dose of SIgY (SIgY-100), both of which exhibited high efficacy (P < 0.01). While the low-dose SIgY (SIgY-50) had a comparatively reduced impact compared to the SIgY-150 and SIgY-100 groups. When incubated with a high dose of NSIgY (NSIgY-150) during the initial 24 h period, the bacteria exhibited significant inhibitory effectiveness. Upon subsequent observations, both the high and low dosages of NSIgY resulted in a lower level of inhibition, similar to that of the negative control group. The medium concentration of NSIgY (NSIgY-100) resulted in better inhibition of C. jejuni growth compared to the other two non-specific treatment concentrations. However, this difference between medium and high doses in the last hours of the period was not statistically significant. (P > 0.01).

Fig. 3.

Growth inhibition curve of C. jejuni treated with different concentrations of specific IgY Powder (SIgY), non-specific IgY Powder (NSIgY), and antibiotic. ^{a - e} Means bearing different superscripts within the 36, 38, and 48 h of the Growth curve are significantly different: P < 0.01. Data are mean ± standard deviation (n = 3 samples/treatment per group).

Mucus adhesion test

The comprehensive results from the colony count of C. jejuni adhered to broiler intestinal (jejunal or ileal) mucus in vitro by pretreatment SIgY groups significantly decreased compared to the NSIgY and control group (P < 0.01). Among the treatments, 50 mg/mL of SIgY was more effective in inhibiting adhesion than the other groups (P < 0.01) (Fig. 4).

Fig. 4.

Inhibition of adhesion of C. jejuni to intestinal mucus of broiler by non-specific IgY (NSIgY) and different concentrations of specific IgY (SIgY) in vitro. ^a-c Columns with no common letters differ: P < 0.01. Data are mean ± standard deviation (n = 3 samples/treatment per group). Data are mean ± standard deviation.

Bactericidal assay

The bactericidal efficacy was assessed in the presence of serum with an inactivated complement system and IgY by examining their impact on the cultivability of C. jejuni. Based on these results, there was a significant difference in the reduction of the mean percentage of C. jejuni cultivability between the specific and non-specific IgY groups at the P < 0.05 level (Table 2). Specific IgY induced a higher bactericidal effect (40.49 %) than the non-specific IgY (26.16 %) in CFU counts.

Table 2.

Bactericidal efficiency (%) of specific and non-specific IgY extracts on C. jejuni.

	Bacteria
Treatments	Mean % reduction C. jejuni (CFU)
Serum + Specific IgY	40.49a
Serum + non- Specific IgY	26.16b
SEM	2.25
P-Value	0.01

^a,bMeans within a row with no common superscript differ: P < 0.05. SEM: Standard Error of the Mean.

Histomorphological measurements

The effects of dietary Campylobacter-Specific antibody on morphological characters of the ileum at 28 d of age are presented in Table 3. The highest mean value of villus height was observed in broilers fed the 0.5 % IgY and NC groups (872 and 914 μm, respectively, P < 0.01). Moreover, villus width was higher in the SIgY-0.5 % (130 μm, P < 0.05). Notably, the crypt depth was significantly higher in the PC group compared to the other groups (P < 0.01). Furthermore, the SIgY-0.5 %, followed by the SIgY-1 %, significantly increased the villus height to crypt depth ratio (P < 0.01) when compared to the positive control. Regarding goblet cells, their number was significantly higher in the PC group (11.28; Table 3) than in the experimental NC and SIgY-0.5 % groups (8.32 and 9.68, respectively). After the PC group, the 0.25 % and 1 % treatment groups exhibited the highest number of goblet cells.

Table 3.

Effect of different doses of specific IgY powder on histomorphological parameters of the ileum in broiler (28 d of age).

			Parameters
Treatments	Villus height (μm)	Villus width (μm)	Crypt depth (μm)	Villus height to crypt depth	Goblet Cell Number
NC	914a	114ab	146b	6.33a	8.32b
PC	648b	70c	198a	3.29b	11.28a
SIgY-0.25 %	784ab	86bc	156b	5.09a	10.16a
SIgY-0.5 %	872a	130a	134b	6.71a	9.68ab
SIgY-1 %	766ab	80bc	120b	6.50a	10.08a
SEM	34.39	12.50	9.50	0.44	0.54
P-Value	0.0002	0.015	0.0002	0.0001	0.017

^a-cMeans within each column with no common superscript differ: P < 0.01; P < 0.05. SEM: Standard Error of the Mean. (n: 5 samples/treatment per group). NC: Negative Control; PC: Positive Control; SIgY-0.25 %: Positive Control + 0.25 % of specific IgY powder; SIgY-0.5 %: Positive Control + 0.5 % of specific IgY powder; SIgY-1 %: Positive Control + 1 % of specific IgY powder.

Campylobacter jejuni enumeration

C. jejuni counts per gram cecal content and liver after euthanasia (as described above) of the chickens, prophylactically fed different doses of specific IgY powder, at 14, 21, and 28 days are presented in Fig. 5. In infected broilers, average cecal C. jejuni counts were similar for the PC and SIgY-0.25 % groups until 21 d after which there was a significant reduction in C. jejuni counts in the SIgY-0.25 % group (respectively 7.43, 8.71 log₁₀ cfu/g cecal content; P < 0.01). Mean cecal C. jejuni counts were significantly reduced in groups receiving SIgY-0.5 % and SIgY-1 % compared to PC and SIgY-0.25 % groups (P < 0.01). In addition, it was observed that for the SIgY-0.5 % group, there was a general decreasing trend in bacteria count compared to other groups (respectively 6.79, 3.46, 2.53 log₁₀ cfu/g cecal content; P < 0.01). In all three periods, the numbers of C. jejuni in the ceca and livers of inoculated broilers receiving SIgY-0.5 % were reduced by at least five log₁₀ cfu (P < 0.0001) compared to the challenged groups.

Fig. 5.

Cecal (A) and liver (B) C. jejuni counts in broiler chickens fed different doses of specific IgY powder (n = 5 birds/treatment per slaughter). ^a-c Columns with no common letters differ: P < 0.01. Data are mean ± standard deviation (n = 5 birds/treatment per group). NC: Negative Control; PC: Positive Control; SIgY-0.25 %: Positive Control + 0.25 % of specific IgY powder; SIgY-0.5 %: Positive Control + 0.5 % of specific IgY powder; SIgY-1 %: Positive Control + 1 % of specific IgY powder.

C. jejuni was recovered from the liver of the PC group at all processing times (respectively, 5.60, 4.29, and 2.80 log₁₀ cfu/g, P > 0.01). On d 14, the SIgY-0.5 % group had the lowest level of C. jejuni colonization (3.49 log₁₀ cfu/g), and there were no differences in the level of colonization between the SIgY-0.25 % and SIgY-1 % groups (respectively, 4.08, 3.99 log₁₀ cfu/g, P > 0.01). On d 21 and 28, the groups treated with specific IgY powders exhibited a significant reduction in bacterial levels compared to the PC group, and C. jejuni was not isolated in the liver (0 vs. 2.8 log₁₀ cfu/g liver contents).

Discussion

Hyperimmune egg yolk has potential as a feed additive to significantly reduce the cecum load of C. jejuni in broilers. This can help decrease the risk of zoonotic infections (Hermans et al., 2014; Vandeputte et al., 2019). However, it is important to note that the results of these studies may not always be comparable due to variations in the immunizing antigen used for IgY production, as well as differences in the period and dosage of IgY administration for passive immunity (Paul et al., 2014). The main objective of the study herein was to assess the anti-Campylobacter activity of hyperimmune egg yolk through both in vitro and in vivo experiments. The ultimate goal was to develop a step-by-step scientific approach, starting with in vitro evaluations and progressing to in vivo studies, in order to determine an effective and economically feasible dosage.

Campylobacter is known to contain immunogenic outer membrane proteins (OMP) such as multidrug efflux pump component (CmeC), Campylobacter adhesion protein A (CapA), fibrin-like peptide A (FlpA), and Campylobacter adhesion protein to fibronectin (CadF) (Annamalai et al., 2013; Kreling et al., 2020). These specific proteins easily detected by the immune system lead to stimulation and increased specific antibodies in chicken serum (Kreling et al., 2020; Mortada et al., 2021). Antibodies obtained from hyperimmune egg yolks were compared as PEG-600 and WSF methods in Fig. 1. The analysis of WSF from hyperimmune egg yolk protein using SDS-PAGE confirmed the presence of two main IgY bands, along with minor bands of other proteins. These minor bands decreased after purification using the PEG-6000 method, indicating that these proteins can be omitted through further purification of IgY from the fraction. However, for the purpose of this study, the WSF was sufficient for subsequent experiments on the anti-C. jejuni effect of IgY. Moreover, this technique allows for the production of antibodies in substantial quantities while maintaining high quality, through simple and cost-effective methods (Mahdavi et al., 2010a, Mahdavi et al., 2010b; Kassim et al., 2012). The WSF method is one of the best methods for recovering and purifying total proteins (Siriya et al., 2013; Verdoliva et al., 2000). The WSF powder collected from the fourth immunization until the end of the immunization period (i.e., 26 wk) was pooled for protein and total IgY concentration analysis (Table 1). Based on this result, the concentrations of protein (Sunwoo et al., 2010) and total IgY and IgY purity (Sunwoo et al., 2010; Mahdavi et al., 2010a, Mahdavi et al., 2010b) in the SIgY were significantly higher than NSIgY during the immunization period. The total IgY concentration in the SIgY immunized with bacterin in this study matched the range of total IgY per yolk reported by Thibodeau et al. (2017), who identified the highest concentration of total IgY in two groups immunized with injections of C. jejuni OMP and bacterin.

The graph depicted in Fig. 2 illustrates a robust and enduring immune response in hens following the administration of bacterin. The WSF exhibits substantially elevated levels of binding in ELISA with IgY specific to C. jejuni, indicating high efficacy in immunization. These results corroborate other reports that demonstrated similar trends in the antibody response in serum and eggs of hens immunized with whole cell lysates or recombinant proteins (Sunwoo et al., 2010; Mahdavi et al., 2010a, Mahdavi et al., 2010b; Al-Adwani et al., 2013; Thibodeau et al., 2017; Hatamzade Isfahani et al., 2020). The peak specific antibody titer in immunized chickens typically occurs between 4 and 7 wk post-immunization (Sunwoo et al., 2010; Hatamzade Isfahani et al., 2020; Lyu et al., 2021). The study herein consistently demonstrated a significant increase in specific WSF titers one wk following the third booster immunization (i.e., 6th wk), followed by a stable platform between the 8th ∼ 26th wk. Enhancing the levels of anti-C. jejuni IgY following bacterin injection and ensuring its long-term stability is critical for optimizing the efficacy and utilization of specific immunoglobulins (Thibodeau et al., 2017). The consistent IgY level and value depend on the number and intervals between each injection. Based on these results, the immunization method utilizing bacterin emulsified with adjuvant, in combination with an appropriate immunization schedule, has proven to effectively stimulate the immune response of hens. The administration of booster vaccines is crucial in maintaining a heightened antibody concentration, with a notable increase in antibody levels observed after each bacterin injection (Thibodeau et al., 2017; Lyu et al., 2021). In the study herein, the high sensitivity of WSF containing IgY specific to the corresponding antigen was demonstrated through a dilution of 1:1000 on ELISA.

The protective role of antibodies obtained from hyperimmune egg yolks against certain foodborne pathogens has been studied in vitro. The inhibitory effects of antibacterial IgY on Escherichia coli (E. coli), Salmonella spp., Candida albicans, and C. jejuni have also been documented in vitro (Sunwoo et al., 2002; Mahdavi et al., 2010a, Mahdavi et al., 2010b; Garba et al., 2019; El-Ghany, 2021). The exact mechanisms through which IgY counteracts C. jejuni activity are not clearly understood. However, several mechanisms have been suggested for utilizing specific IgY in immunotherapy against bacteria. These include agglutination, inhibition of bacterial replication and enzyme activity, adherence-blockade, neutralization of bacterial toxins, and direct bacterial killing probably through opsonization followed by phagocytosis (Xu et al., 2011; Yang et al., 2020).

In the study herein, investigating the antibacterial properties of IgY against C. jejuni, there was successful agglutination induced by antibacterial IgY. Similarly, Garba et al. (2019) reported that antibodies extracted from hyperimmune egg yolk powders promoted agglutination in both homologous and heterologous C. jejuni strains. In contrast, Thibodeau et al. (2017) reported that IgY extracts failed to agglutinate most heterologous C. jejuni strains; however, the extracts recognized and agglutinated live homologous C. jejuni strains. Although agglutination may be one mediator of growth inhibition, it is unlikely to be the most important mediator. Therefore, other in vitro methods were employed to analyze the inhibitory effect of IgY extracts on live bacteria.

Based on the in vitro characterization assay, anti-C. jejuni IgY exerts a potent, dose-dependent inhibitory effect on the growth of live C.jejuni in broth culture medium. Notably, concentrations of 150 mg/mL and 100 mg/mL of SIgY proved particularly effective when compared with other treatments. These findings concur with those of Li et al. (2024), who also observed that SIgY suppresses C. jejuni growth in a dose-responsive manner, with higher concentrations yielding greater inhibition. In contrast, NSIgY did not significantly inhibit bacterial growth. In the context of immunized IgY, both dosage and specificity play critical roles in inhibiting and preventing bacterial proliferation (Mahdavi et al., 2010a, Mahdavi et al., 2010b). The proposed inhibitory mechanism involves antibodies binding to specific components on the bacterial surface, effectively blocking their function. For instance, this mechanism has been observed in E. coli O78:K80 (Mahdavi et al., 2010a, Mahdavi et al., 2010b) and S. Infantis (Hatamzade Isfahani et al., 2020). Furthermore, morphological alterations were noted on the surface of S. Typhimurium (Lee et al., 2002), E. coli O111 (Zhen et al., 2008), and E. coli O157:H7 (Sunwoo et al., 2010) due to binding with specific IgY. Consequently, the binding activities of IgY against bacterial surface components effectively inhibit bacterial proliferation (Sunwoo et al., 2010).

In consideration of the structural interactions between the host cell and bacteria, the type of immunization should encompass a broad spectrum of targets for pathogen attenuation. Since the specific antibody produced by bacterin is not limited to a set number of specific adhesions, it may emerge as one of the effective factors in inhibiting Campylobacter adhesion. In continuation of these research endeavors, the present study undertook the evaluation of SIgY concerning the inhibition of the adhesion ability of C. jejuni to the mucosal lining of the small intestine. The motivation for this research was derived from the well-documented significance of bacterial adhesion to the gastrointestinal (GI) mucosa in the mechanisms of colonization and persistence of pathogens within the GI tract (Froebel et al., 2020). These findings elucidate that the SIgY was found to significantly reduce the adhesion properties of C. jejuni to the small intestinal mucosa of broiler chickens. This outcome is further corroborated by the observed experimental evidence pertaining to agglutination and growth inhibition. The data presented in this study agree with earlier research where IgY antibodies directed against Helicobacter pylori (H. pylori) were effective in combating H. pylori through mechanisms that include agglutination, growth inhibition, and inhibition of Helicobacter adhesion to human gastric epithelial cells in vitro (Zhang et al., 2021). Additionally, Wang et al. (2019) reported that specific IgY antibodies were efficacious in significantly reducing the adhesion of E. coli K88 to the mucosal lining of the porcine small intestine. Therefore, the inhibition of bacterial adhesion to epithelial tissues may significantly contribute to the maintenance of gastrointestinal health. This adhesion is considered essential for pathogens to withstand the physical forces imparted by gastrointestinal peristalsis and to successfully establish colonization (Froebel et al., 2020). Specific IgY antibodies against target pathogens have lead to functional impairment in vitro. These IgYs, by coating the whole bacterial cell wall, which was recorded by the immunoelectron microscopy, lead to structural alterations which suppresses the biological functions of the pathogen itself (Sunwoo et al., 2010; Lee et al., 2002). It is also possible that specific IgY binding to bacteria could alter cellular signaling cascades that could result in decreased toxin production and release (Xu et al., 2011). Based on these results and observations, IgY may exhibit protective efficacy against bacterial adhesion to the mucosal surface since adhesion to host cell is an important step in the pathogenesis of C. jejuni. In particular, the adherence-blockade mechanism with SIgY seems to be a promising concept for reducing Campylobacter bacterial load in poultry production.

Another mechanism of the body against pathogens is phagocytosis along with direct pathogen neutralization (Yang et al., 2020). The complement system plays an important role in phagocytosis helping to defend the host against microbial infections (Kang et al., 2013). The active complement system defends the host by inducing inflammation, attracting phagocytes to the site of infection, promoting opsonization, lysing Gram-negative bacteria, and participating in B-cell activation (Juul-Madsen et al., 2014). In light of the response of the complement system against invading microbes, it is not surprising that pathogens have used strategies to counter the attack of the complement system (Kang et al., 2013). In the assessment of the bactericidal efficacy of specific WSF against Campylobacter in the presence of heated serum at 56°C (inactive complement), a significant difference was observed in the capacity of the specific immunoglobulin to diminish the count of Campylobacter colonies than the nonspecific group. The observed impact of the nonspecific powder in the bactericidal test can be attributed to the presence of chicken C3, a significant constituent of the serum complement. The presence of chicken C3 in egg yolk has been reported (Recheis et al., 2005). This finding is consistent with the results reported by Thibodeau et al. (2017), who demonstrated that IgY extract obtained from OMP antigen and bacterin exhibits a potent bactericidal effect against pathogenic C. jejuni strains. Additionally, other studies have shown that C. jejuni is susceptible to killing by maternal antibodies, with both complement and specific antibody playing a role in this process (Sahin et al., 2001). Therefore, the complement system alone is inadequate for the elimination of C. jejuni; the presence of specific antibodies is required to bind to the pathogen's surface antigens (Sahin et al., 2001). However, Garba et al. (2019) reported that egg yolk powder, even when containing the highest concentrations of antibodies (OMP and bacterin), exhibited no bactericidal effect in the presence of complement. The in vitro efficacy of SIgY against critical functions of C. jejuni, including agglutination, growth inhibition, and bactericidal activity, prompted the exploration of its potential as a feed additive for reducing chicken colonization by C. jejuni.

One of the primary mechanisms facilitating the survival and colonization of Campylobacter in the digestive tract is its aggressive adhesion to intestinal epithelial cells (Awad et al., 2015). C. jejuni-inoculated animals have been observed to have significant decreases in villus height, increases in crypt depth, and a reduction in the villus height-to-crypt depth ratio (Rahimi et al., 2020; Rzeznitzeck et al., 2022; Mantzios et al., 2024). Therefore, C. jejuni infection may disrupt the detrimental effects on villus integrity and function of the intestinal epithelium (Munoz et al., 2023). One characteristic associated with a healthy digestive system in broiler chickens is the presence of long and healthy villi coupled with shallow crypts (Ferket et al., 2002). In contrast, in the study herein the positive control group exhibited a decrease in both the height and width of the villi, as well as an increase in the maximum depth of the crypts in the ileum of broiler chickens (Table 3). These findings highlight the importance of maintaining intestinal health and the potential negative impacts of Campylobacter infection on the digestive system. Decreased villus height, villus-crypt ratio, and increased crypt depths were observed in C. jejuni-infected chickens compared to non-infected ones at 42 d in the ileum, as reported by Munoz et al. (2023) and Gharib Naseri et al. (2012). The histomorphometry examination of the intestinal ileum in broiler chickens infected with C. jejuni at 23 d confirmed the aforementioned results regarding the increase in crypt depth and the decrease in the villus-crypt ratio (Mantzios et al., 2024). Given the susceptibility of newly hatched chicks to infections due to limited maternal antibodies and immature immune and digestive systems, a well-developed mucus layer in the GI tract assumes a pivotal role in an active immunity system (Yang et al., 2021; Duangnumsawang et al., 2021). The hypothesis considered herin is that SIgY, obtained through immunization with bacterin, can prevent damage to intestinal villi. Interestingly, the treatment of chickens with 0.5 % and 1 % SIgY for 4 wk moderated the effect of C. jejuni infection on the intestine by increasing the villus height and villus-crypt ratio in the ileum. These results are in agreement with other reports concerning the effect of feeding chickens with anti-Clostridium perfringens IgY (Abadeen et al., 2022), mice with high and medium doses of anti-enterotoxigenic E. coli IgY (Han et al., 2021), and the duodenum of weaned pigs with hyperimmunized hen egg yolk powder (Han et al., 2019). The dietary intervention may have improved the intestinal damage repair capacity when facing infections, potentially leading to a reduction in the effects of Campylobacter on the epithelial surface of the ileum. Although the gut is not a distinct immune organ, it is often regarded as the largest immune organ and significantly impacts host immunity (Sterling et al., 2022). Alternatively, Campylobacter might utilize the mucous layer of intestinal crypts as a site for colonization and protection from additives and antibiotics in poultry (Cole et al., 2006). In the study herein, comparing the crypt depth results to the positive control group, the lowest crypt depth is observed first in the 1 % group and then in the 0.5 % group. These treatments restrict the environment for Campylobacter colonization within the crypt space by reducing the depth of the crypt. Hence, a shallower crypt depth may enhance bacterial exposure to the intestinal lumen and dietary or chemical environment variations. Furthermore, smaller crypts may impede the colonization of Campylobacter by fostering competition among Campylobacter populations and microflora (Cole et al., 2006). In addition to the role of crypt depth and surface villi morphology in the immune system, the intestinal epithelium typically serves its protective function through goblet cells. These specialized cells produce and secrete mucus, which serves as a barrier safeguarding against physical and chemical damage caused by ingested food, microbes, and microbial products (Kim and Ho, 2010; Sterling et al., 2022). Although the mucus layer serves a protective function in the intestinal tract, some pathogenic bacteria have developed strategies to exploit goblet cells and mucus secretion (Pelaseyed et al., 2014). In the study herein, after the bacterial challenge, the increase in goblet cell numbers was observed. However, the use of 0.5 % SIgY resulted in a remarkable decrease in the goblet cell numbers (Table 3). These findings are in agreement with other observations of a reduction in goblet cells after exposure to pathogenic microbes and subsequent treatment with specific IgY (Mahdavi et al., 2010a, Mahdavi et al., 2010b). Therefore, the effectiveness of the antibody in maintaining the integrity of the villi and the health of the intestinal epithelium in broilers may contribute to protection against colonization by C. jejuni. The high affinity of oral IgY to bind antigens of zoonotic bacteria indicates that orally administered IgYs are effective in providing passive immunity (Table 4). The antigens can capture C. jejuni ingested by birds, thereby preventing the colonization and transmission of these pathogens (Hermans et al., 2014; DuBourdieu, 2019).

Table 4.

Efficacy of varying doses of hyperimmune egg yolk powders against bacterial infections in broiler chickens.

Pathogen	vaccine	Dose (%)	Treatment protocol	Outcome	References
C. jejuni	Bacterin of strains C. jejuni and C. coli	0.5	Inoculated 0.5 IgY 14 days before infection (109 CFU)	Reduction in the feces	Tsubokura et al. (1997)
E. coli	Bacterin of E. coli O78:K80	0.2 0.4	In feed before infection (109 CFU)	Reduction in the ileal	Mahdavi et al., 2010a, Mahdavi et al., 2010b
C. jejuni	Whole-cell lysate or hydrophobic protein	5	In feed 10 days before infection and evaluating the efficacy 3 days after infection (3 × 104 CFU)	Reduction in the cecal	Hermans et al. (2014)
C. jejuni	Recombinant proteins	10	In feed before or after infection (105 CFU)	No reduction in the caecal	Paul et al. (2014)
C. jejuni	Bacterin or Outer membrane proteins of the same C. jejuni strains	5	In feed before infection (2 × 103 or 3 × 103 CFU)	No reduction in the caecal	Garba et al. (2019)
C. jejuni	Bacterin or subunit vaccine	5	In feed before infection (105 CFU)	Reduction in the cecal	Vandeputte et al. (2019)
S. Infantis	Bacterin of S. Infantis	0.5	In feed and water before infection (107 CFU)	Reduction in the cecal and liver	Hatamzade Isfahani et al. (2020)
C. jejuni	Bacterin of C. jejuni	0.5	In feed before infection (108 CFU)	Reduction in the cecal	This work

In their first in vivo experiment, Tsubokura et al. (1997) reported that the C. jejuni strain is susceptible to specific antibodies, which can kill it. The results of this study demonstrated 7 d after inoculation, that prophylactic administration of feed supplemented with 0.5 % or 1 % SIgY for 4 wk significantly reduced the CFU counts in both the cecum and liver (Fig. 5). The reduction of C. jejuni colonization induced by bacterin-specific antibodies is comparable to the results obtained by Vandeputte et al. (2019) and Tsubokura et al. (1997). Previous passive immunization studies utilizing a diet containing 0.4 % anti-E. coli WSF powder and 0.5 % anti-Salmonella Infantis hyperimmunized egg yolk powder, derived from a previously reported bacterin vaccine, resulted in a significant effect on the colonization of cecal pathogenic bacteria in broilers (Mahdavi et al., 2010a, Mahdavi et al., 2010b; Hatamzade Isfahani et al., 2020). Furthermore, The positive effects of specific antibodies derived from two novel vaccines—a bacterin of Campylobacter strains (Hermans et al., 2014; Vandeputte et al., 2019) and a subunit vaccine (Vandeputte et al., 2019)—were confirmed to reduce C. jejuni levels in broiler chickens. These hyperimmune yolks were administered at a concentration of 5 % (wt/wt) in the broiler diet. Moreover, Vandeputte et al. (2019) reported that hyperimmune yolk, particularly when derived from bacterin, effectively controlled Campylobacter colonization in poultry through passive immunization. In contrast, cecal C. jejuni counts in birds receiving feed supplemented with 10 % (wt/wt) hyperimmunized egg yolk powder were not significantly reduced compared to control chickens (Paul et al., 2014). Similar results were reported by Garba et al. (2019), who examined egg yolk powder against the C. jejuni OMP antigen and bacterin (5 % wt/wt) and observed no reduction in colonization of the chicken cecum. In the study herein, a positive effect of SIgY levels in reducing Campylobacter colonization in the liver was observed, which aligns with the role of encapsulated immune yolk in reducing Salmonella Infantis (Hatamzade Isfahani et al., 2020). Therefore, considering the connection between poultry livers and human campylobacteriosis, controlling the levels of this bacterium is an effective measure for protecting consumer health (Sylte et al., 2020). The inconsistency in the dose-response relationship for C. jejuni growth inhibition in the cecum of broilers, compared to previous studies, may be attributed to the following factors: (1) vaccine-specific differences in C. jejuni, (2) variations in immunization protocols, (3) differences in experimental setups and the duration of preventive treatment, and (4) the effectiveness of the IgY produced in this study at lower concentrations. The divergence between the results herein and other reports regarding the dose-response relationship for the inhibition of bacterial growth using egg yolk powder as an additive is not novel. For example, Holt et al. (1996) and Gürtler and Fehlhaber (2004) observed this divergence in vitro with the effect of hyperimmune yolk on Salmonella Enteritidis. Additionally, under in vivo conditions, Vandeputte et al. (2019) reported that hyperimmune yolk exhibited more pronounced inhibitory effects on Campylobacter at lower doses. However, in the study by Paul et al. (2014), this inhibitory effect was not present when the hyperimmune yolk was administered at double the dose.

Conclusion

The antagonistic activity of antibodies derived from bacterin under in vitro conditions and optimized for dosage for in vivo applications was studied. The anti-C. jejuni antibodies induced agglutination, effectively inhibited bacterial growth, prevented adhesion of bacteria to intestinal mucusa, and exhibited bactericidal activity in vitro. Furthermore, oral administration of broiler chickens with 0.5 % (wt/wt) anti-C. jejuni WSF powder significantly reduced C. jejuni colonization compared to the positive control without IgY. Therefore, the production of polyclonal antibodies through the immunization of hens can yield large amounts of high-quality antibodies at an economical dose using simple methods. However, to confirm the protective role of this dose of bacterin vaccine-induced antibodies, addition comprehensive experiments using chickens with different challenge strains are required.

Declaration of competing interest

The authors declare that they have no conflict of interest in this study.