Development and Functional Characterization of Egg-Yolk-Derived Polyclonal IgY Induced by Lumpy Skin Disease Virus Vaccination

1. Introduction

1.1. Background

Lumpy skin disease (LSD) is caused by the lumpy skin disease virus (LSDV) from the family Poxviridae, genus Capripoxvirus (CapV), the Neethling strain prototype [1]. It is characterized by fever; nodules on the skin, mucous membranes and internal organs; emaciation; enlarged lymph nodes; edema of the skin; and sometimes death [2]. The disease is a highly contagious transboundary infection that causes significant economic losses in the livestock industry as it can cause a temporary reduction in milk production, temporary or permanent sterility in bulls, damage to hides, and, occasionally, death [3].

Lumpy skin disease virus is endemic to Ethiopia, first reported in 1983 in the northwestern part of Ethiopia in the Amhara National Regional State, surrounding the southwest part of Lake Tana [4]. Because of the wide distribution of the disease and the size and structure of the cattle population in Ethiopia, LSDV is one of the most economically important livestock diseases in the country [5]. A study conducted in central and northwestern Ethiopia estimated the median total economic loss of an LSD outbreak at USD 1,176 per affected herd, with losses of USD 489 and USD 2,735 reported for subsistence and commercial farms, respectively[6]

Mammalian polyclonal antibodies (pAbs), monoclonal antibodies (mAbs), and avian polyclonal antibodies (IgY) have been extensively adopted as valuable tools for assay-specific target development and detection in basic research, diagnostics, and biomarker discovery [7].

Avian polyclonal antibodies (IgY) can be produced from chickens using non-invasive protocols, offering several advantages over mammalian antibodies. IgY antibodies enable the detection of conserved epitopes across species, making them particularly suitable for targeting proteins with high sequence similarity [8,9].

Egg-yolk-derived antibodies provide a high yield per animal, and the harvesting process is simple, ethical, and non-invasive [10]. Purified IgY antibodies exhibit higher avidity and sensitivity, broader epitope recognition, and reduced interference in immunoassays compared with mammalian IgG antibodies. [11].

Despite these advantages, the application of IgY technology to lumpy skin disease virus (LSDV) remains limited. In this context, the present study was designed as a proof-of-concept to investigate the production, purification, and functional characterization of egg-yolk-derived IgY antibodies against the LSDV vaccinal strain. Specifically, this study aimed to evaluate the kinetics of IgY production following immunization and to assess the functional activity of the generated antibodies using in vitro assays. The findings of this study provide preliminary evidence supporting the feasibility of IgY-based approaches and highlight their potential application in the future development of diagnostic tools for LSDV.

Despite the potential advantages of IgY technology, its application for generating antibodies against LSDV has not been investigated in Ethiopia. Therefore, this study aimed to produce and functionally characterize egg-yolk-derived IgY antibodies against the LSDV vaccinal strain as a proof-of-concept for their potential use in the development of serological diagnostic assays.

1.2. Objectives

1.2.1. General Objective

To generate and functionally characterize the polyclonal IgY antibodies produced in chickens immunized with the lumpy skin disease virus (LSDV) vaccinal strain, and to evaluate their potential for future application in LSDV-related research and serological assay development.

1.2.2. Specific Objectives

To isolate and purify IgY antibodies from chicken egg yolk following immunization with the LSDV vaccinal strain.

To evaluate the temporal antibody response and production kinetics of IgY following immunization.

To assess the virus-neutralizing activity of egg-yolk-derived IgY against the LSDV vaccinal strain.

2. Materials and Methods

2.1. Study Site

The experimental work was conducted at the Animal Health Institute (AHI), Sebeta, Oromia, Ethiopiaand the AU-PANVAC Laboratory in Bishoftu, Ethiopia.

2.2. Study Design

The experimental study was conducted over 42 days between January and May 2024 to produce and functionally characterize egg-yolk-derived IgY antibodies against the LSDV vaccinal strain as a proof-of-concept for future serological assay development. Following virus adaptation and titration, the chickens were randomly allocated into an experimental group and a control group. The experimental group received two doses of the LSDV vaccinal strain, while the control group received phosphate-buffered saline (PBS) as a placebo. The injections were administered on days 0 and 15 into the pectoral muscles [12] (Supplementary Figure S2).

2.3. Experimental Chickens

Ten healthy 52-week-old Bovans Brown layer chicks with no history of vaccination, from the same hatch batch were bought from a local farm in Sebeta town and managed in controlled environments for housing, inoculation, and sample collection. The chickens were allocated into two groups and housed separately in mesh wire enclosures. For a successful recovery from the stress caused by transportation, feed, and environmental alterations, the chickens were given a week to acclimate before the trial commenced. Chickens were managed in a good state of welfare: they were healthy, comfortable, well-nourished, safe, able to express natural behaviors, and free from pain, fear, and distress The study was approved by the College of Veterinary Medicine and Animal Science, University of Gondar Research Ethics Review Committee (CVMASC-RERC; Ref. No. CVMASC/UoG/RERC/26/04/2023), and all procedures involving experimental animals were conducted in accordance with the approved protocol.

2.4. Sample Size

The sample size was determined based on feasibility and alignment with previous exploratory IgY immunization and kinetic studies, which commonly employ small experimental groups due to the ethical and logistical considerations associated with animal-based research. A total of ten (n = 10) egg-laying chickens were included and randomly allocated into an experimental group (n = 6) and a control group (n = 4), and housed under controlled conditions to minimize bias.

2.5. Preparation, Adaptation, and Titration of Lumpy Skin Disease Virus Vaccine

Preparation of Vero Cell Culture for LSDV Adaptation

Vero cells (obtained from the Virus Isolation and Cell Culture Laboratory of Animal Health Institute; Sebeta, Oromia, Ethiopia) were cultured in Minimum Essential Medium (MEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2% (v/v) streptomycin sulfate, and Amphotericin B (250 µg/mL) [14]. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂ and allowed to reach approximately 70% confluence within 3 days before viral inoculation.

2.6. Titration of Lumpy Skin Disease Virus Vaccinal Strain

A live lumpy skin disease virus (LSDV) vaccine was obtained from the National Veterinary Institute (NVI). The virus was therefore harvested and passaged into fresh Vero cells. In the subsequent passage, characteristic CPEs were observed within 7 days [2], after which the virus was harvested and used for titration and downstream applications (see Supplementary Figure S1).

The viral titer was determined by tenfold serial dilution in 96-well microtitration plates, and the median tissue culture infectious dose (TCID₅₀) was calculated using the Reed–Muench method [15]. The final viral stock used for immunization was standardized to 10³ TCID₅₀/mL.

2.7. Immunization of Chickens, Sample Collection, and IgY Extraction

Immunization and Sample Collection

The chickens in the experimental group were immunized intramuscularly with 10³ TCID₅₀/mL of LSDV into both pectoral muscles [12], while the control birds received 1 mL of PBS. A booster dose was administered on day 15 using the same virus dose and placebo.

Eggs and blood samples were collected weekly from day 7 to day 42 (Table 1). The eggs were stored at 4 °C, and approximately 3 mL of blood was collected from the brachial vein of each chicken (see Supplementary Figure S3).

The serum was separated after clotting at room temperature and stored in cryovials. The IgY from egg yolk and corresponding sera were later evaluated by the virus neutralization test (VNT) [16,17].

2.8. Extraction of Chicken IgY

Fresh, dark-brown-shelled eggs were processed using a modified extraction protocol as described in [17].The egg yolks were separated from the egg whites using an egg strainer and transferred into 50 mL Falcon tubes. Each egg yielded approximately 15 mL of yolk (V1).

The yolk was mixed with PBS at a 1:2 ratio, yielding a final volume of approximately 45 mL. The mixture was vortexed, followed by the addition of 3.5% (w/v) PEG 6000 to the total volume (V2), and vortexed again. The solution was then agitated in a thermoshaker incubator at 300 rpm for 10 min, at room temperature followed by centrifugation at 4 °C and 3500 rpm for 15 min. The supernatant was carefully decanted and filtered through folded filter paper into a sterile tube, producing a new volume (V3).

Afterward, 8.5% (w/v) PEG 6000 was added to the volume (V3), and the mixture was vortexed and incubated on a thermoshaker at 300 rpm. The tubes were then centrifuged at 4 °C and 3500 rpm for 15 min, and the supernatant was discarded. The resulting pellet was resuspended and homogenized in 1 mL of PBS using a glass rod, and PBS was added to adjust the total volume to 10 mL (V4).

The solution was further treated with 12% (w/v) PEG 6000, mixed on a thermoshaker at 300 rpm for 10 min, and centrifuged again at 4 °C and 3500 rpm for 15 min. The supernatant was discarded, and the pellet was resuspended in 1.8 mL of PBS, mixed thoroughly, and transferred into a 2 mL cryovial for storage (see Supplementary Figure S4).

Finally, the extracts were purified using a Membra-Cel^® (Serva Electrophoresis GmbH, Heidelberg, Germany) dialysis membrane with a 14 kDa molecular weight cut-off (MWCO) to remove PEG 6000 and further refine the IgY preparation against PBS.

2.9. Purification of the IgY Extract

The IgY extracts were purified using a Membra-Cel^® dialysis membrane with a 14 kDa molecular weight cut-off (MWCO). The tubing was pretreated with 5 mM EDTA and thoroughly rinsed with distilled water prior to use. The IgY extract was loaded into the membrane and dialyzed overnight against 0.1% saline under continuous stirring, followed by an additional 3 h of dialysis against phosphate-buffered saline (PBS). The purified IgY was then recovered and stored at −20 °C until further analysis.

2.10. Detection and Quantification of Antibodies

Virus Neutralization Test

The virus neutralization test (VNT) was performed to evaluate the functional activity of IgY antibodies against LSDV by assessing their ability to inhibit viral infection in Vero cells. This assay enabled the detection and comparison of neutralizing polyclonal antibodies (pAbs) in both serum- and egg-yolk-derived IgY samples.

The serially diluted IgY samples were incubated with a standardized viral dose prior to inoculation onto the Vero cells. Each dilution was tested in duplicate (two wells per dilution), and the presence or absence of cytopathic effects (CPEs) was recorded following incubation. Neutralization endpoints were determined based on the proportion of wells protected from CPEs across serial dilutions. When partial neutralization was observed between consecutive dilutions, the endpoint titer was estimated using an interpolation approach.

2.11. Protein Concentration and Purity

The total protein concentration and purity of the preparationwere determined using a NeoDot microvolume spectrophotometer (NeoBiotech Co., Ltd., South Korea) by measuring absorbance at 280 nm. Phosphate-buffered saline (PBS) was used as the blank. For each measurement, 2 µL of egg-yolk-derived protein preparationsample was loaded onto the pedestal, and readings were recorded according to the manufacturer’s instructions. Proteinpurity was assessed based on the A260/A280 absorbance ratio and UV spectral profile (see Supplementary Figure S5).

2.12. SDS-PAGE Analysis

Reducing SDS-PAGE was performed to determine the purity and molecular weight of purified IgY. The samples (10 µg) were separated on NuPAGE™ Novex^® Bis-Tris Mini Gels using the XCell SureLock™ Mini-Cell system, Invitrogen (Thermo Fisher Scientific: Carlsbad, CA, USA). A prestained protein molecular weight marker (BenchMark™ Protein Ladder, Invitrogen (Thermo Fisher Scientific: Carlsbad, CA, USA)) was included for size estimation. Electrophoresis was conducted at 200 V for approximately 2 h, and protein bands were visualized by Coomassie-based staining according to the manufacturer’s instructions.2.13. Data Management and Analysis

Data were analyzed using R version 4.4.1. Changes in IgY concentration over time were evaluated using linear regression and repeated-measures ANOVA, with week treated as the within-subject factor. When significant differences were detected, post hoc comparisons were performed. For the virus neutralization analysis, log-transformed neutralization titers were treated as the dependent variable and analyzed using a linear mixed-effects model, with week and antibody source (egg yolk vs. serum) included as fixed effects and individual chickens included as random effects to account for repeated measurements. Assumptions of normality and sphericity were assessed and met prior to analysis. A p-value of <0.05 was considered statistically significant (see Supplementary Table S1).

3. Results

3.1. Virus Titration

The viral stock had a calculated titer of 10⁵ TCID₅₀/mL. For immunization, the virus was diluted 1:100 to obtain a final concentration of 10³ TCID₅₀/mL.

3.2. Quantification of IgY Concentration

IgY concentrations were determined weekly using a microvolume spectrophotometer at 280 nm. In the experimental group, mean IgY levels increased markedly from 0.023 ± 0.004 mg/mL at Week 1 to a peak of 0.961 ± 0.059 mg/mL at Week 5, followed by a slight decline to 0.837 ± 0.055 mg/mL at Week 6 (Figure 1) (see Supplementary Table S3). In contrast, the control group maintained consistently low IgY concentrations throughout the study period (range: 0.027–0.043 mg/mL) (see Supplementary Table S4).

A linear regression analysis demonstrated a significant increase in IgY concentration over time in the experimental group (slope = 0.162, p = 0.024), whereas no significant trend was observed in the control group (slope = 0.001, p = 0.301) (Table 2, Figure 1).

A repeated-measures ANOVA was conducted to assess changes in IgY concentration over time in the experimental group. A significant effect of time was observed (p < 0.0001). Assumptions of normality (Shapiro–Wilk test) and sphericity (Mauchly’s test) were satisfied (p > 0.05); therefore, no corrections were applied. Post hoc comparisons indicated significant differences between weeks (see Supplementary Table S2). These findings demonstrate a time-dependent IgY response following immunization, consistent with an active humoral immune response to the LSDV vaccinal strain.

3.3. Confirmation of IgY Identity

The reduced SDS-PAGE analysis of the egg-yolk-derived protein preparation showed two prominent protein bands at approximately 65 kDa and 25 kDa, with molecular weights consistent with the expected heavy and light chains of IgY, respectively (Figure 2 and Figure S5).

3.4. Evaluating the Virus Neutralization Efficacy of IgY

Virus neutralization testing demonstrated that IgY exhibited effective antiviral activity, with the endpoint titer estimated at approximately 1:125 (log₁₀ ≈ 2.10), based on interpolation between adjacent dilution levels showing partial neutralization. No neutralizing activity was detected in the serum or egg-yolk preparations from the control group at any of the tested dilutions.

Neutralization titers were expressed as log₁₀ reciprocal dilution values. Egg-yolk-derived IgY exhibited a progressive increase in neutralizing activity over time, reaching peak levels at Week 5, which corresponded to the highest recorded IgY concentration, followed by a modest decline thereafter.

In contrast, serum-derived antibodies showed lower neutralization titers, with a maximum of approximately 1:50 (log₁₀ ≈ 1.70) and an earlier decline over time. The linear mixed-effects model analysis confirmed a statistically significant difference in neutralization responses between the egg-yolk-derived and serum-derived antibody preparations across the study period (p < 0.0001).

Overall, egg-yolk-derived IgY demonstrated stronger and more sustained neutralizing activity against lumpy skin disease virus compared to serum-derived antibodies (Figure 3).

4. Discussion

Immunoglobulin Y technology is a scalable production technology that started its foundation in 1980 [18] and was rapidly adopted in developing countries and regions such as South America and India, as well as Japan, Germany, and other developed countries [19]. It is highly relevant in immunodiagnostics, playing a crucial role in the development of immunoassays such as ELISA due to its specificity and sensitivity [20]. Its relevance extends to disease control and serosurveillance, where IgY is potentially used for the detection, prevention, and treatment of human and animal infections [21].

In this study, we demonstrated the feasibility of producing and functionally characterizing IgY antibodies against the LSDV vaccine strain using a polyethylene glycol (PEG)-based extraction method [22] and dialysis purification protocols [23]. The generated egg-yolk-derived antibody preparationswere characterized by SDS-PAGE, quantified spectrophotometrically, and evaluated for functional activity using virus neutralization tests. The results provide proof-of-concept evidence that the immunization of chickens with the LSDV antigen can induce the production of biologically active IgY antibodies capable of neutralizing the virus in vitro. This expands the application of IgY technology to an economically important transboundary livestock disease and highlights its potential as a scalable platform for antibody production.

The SDS-PAGE analysis was suggestive of the characteristic IgY subunit profile, with distinct bands corresponding to the heavy (~65 kDa) and light (~25 kDa) chains, consistent with the expected molecular structure of avian IgY. Comparable molecular weights have been reported in previous IgY studies targeting Nosema ceranae [24], avian influenza viruses (H5/H9) [25], and rotaviruses [26], demonstrating structural consistency across antigen systems. Similar IgY isolation and profiling patterns were also described in comparative studies of immunized chicken breeds [27]. Progressive increases in band intensity from Week 2 onward paralleled the quantified rise in IgY concentration, reflecting active humoral immune maturation following primary and booster immunization [26]. The absence of detectable bands during the first week is consistent with the known kinetics of primary antibody responses in chickens, where IgY becomes measurable after the initial lag phase [28]. The marked enhancement in band intensity following booster administration further supports effective secondary immune stimulation and amplified polyclonal antibody production [29]. Collectively, these findings confirm the structural integrity and time-dependent accumulation of functional IgY in egg yolk.

IgY quantification demonstrated a progressive increase in mean egg yolk antibody concentration over the study period, with peak levels observed following booster immunization and a subsequent decline toward the final week. Linear regression confirmed a significant time-dependent increase in IgY production across six weeks, consistent with the expected kinetics of primary and secondary humoral responses. These findings align with previous reports describing time-dependent increases in IgY production against ricin and Clostridium botulinum neurotoxins [30], as well as studies documenting the cyclical patterns of antibody titer elevation and decline following immunization [31]. Together, these results confirm that immunization with the LSDV vaccine strain induces a robust and dynamic IgY response in chickens.

The functional virus neutralization analysis demonstrated that the increased IgY concentrations observed in egg yolk translated into superior neutralizing activity against LSDV compared with serum-derived antibodies. The mixed-effects modeling of dilution endpoints further confirmed significantly higher neutralization titers in yolk IgY. This functional advantage is biologically consistent with the preferential accumulation of IgY in egg yolk, where antibody levels are typically higher than in serum. Similar observations have been reported in previous IgY-based studies targeting viral pathogens [32], supporting egg yolk as a reliable source of high-potency polyclonal antibodies [8].

Notably, direct within-animal comparisons between serum-derived antibodies and egg-yolk-derived IgY remain limited in the literature. In this study, the inclusion of paired serum and egg yolk samples from the same immunized chickens enabled a controlled evaluation of their relative functional performance. The comparatively lower and delayed neutralization response observed in serum further supports the preferential accumulation and functional enrichment of virus-specific antibodies in egg yolk. These findings provide additional insight into the differential distribution and activity of IgY across biological compartments, although further studies are needed to validate these observations across larger sample sizes and different viral systems.

Although this study demonstrated the production and functional neutralization capacity of egg-yolk-derived IgY against lumpy skin disease virus, several important limitations should be acknowledged.

First, IgY quantification was based on absorbance at 280 nm, which measures total protein concentration rather than IgY specifically. As a result, the reported values may include non-IgY proteins and should be interpreted as approximate estimates rather than precise antibody concentrations.

Second, functional characterization relied primarily on virus neutralization testing without complementary antigen-specific assays such as ELISA. Therefore, while neutralizing activity was demonstrated, the specificity and binding characteristics of the antibodies were not directly evaluated.

Third, purification was performed using polyethylene glycol precipitation followed by dialysis, which may provide lower purity compared to chromatographic methods.

Finally, the relatively small sample size, although consistent with exploratory IgY studies and supported by a repeated-measures design, may limit the generalizability of the findings.

Additionally, total protein also increased during the later time points; therefore, the observed increase in bands consistent with the expected IgY profile cannot be attributed exclusively to IgY production. Furthermore, SDS-PAGE was not performed on the control-group samples; therefore, the observed changes in bands consistent with the expected IgY profile cannot be attributed specifically to IgY.

These limitations indicate that the present study should be considered a preliminary proof-of-concept. Future studies incorporating antigen-specific immunoassays, advanced purification techniques, and larger sample sizes are required to further validate and optimize the application of IgY for diagnostic purposes.5. Conclusions and Recommendations

This study demonstrates the production, purification, and functional characterization of egg-yolk-derived IgY against the LSDV vaccinal strain. IgY-associated neutralizing activity was detectable from the second week post-immunization, with peak activity observed at Week 5. The SDS-PAGE analysis indicated the structural integrity of the purified IgY, while virus neutralization testing demonstrated its functional activity. Egg-yolk-derived IgY exhibited stronger and more sustained neutralizing activity compared to serum-derived antibodies, consistent with its accumulation in egg yolk.

Collectively, these findings provide a proof-of-concept foundation for the potential use of IgY in future LSDV serological assay development. However, additional studies incorporating antigen-specific immunoassays, improved purification strategies, and broader immunological characterization are required to further validate and optimize this approach.