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Article

Assessment of Immunological Interference Between Live Infectious Bursal Disease Virus and Avian Reovirus Vaccines in SPF Chickens

by
Jiaolong Wen
1,2,†,
Mingwei Li
1,†,
Yuecheng Long
1,
Shenghua Yang
2,3,
Chuang Lyu
4,
Junxian Li
1,
Guanming Huo
1,
Ermin Xie
2,
Yiming Liu
2,
Yanhua Xu
2,
Xuesong Li
1,2,
Jianping Qin
2,
Lijuan Yin
2,3,* and
Wencheng Lin
1,2,*
1
College of Veterinary Medicine & College of Animal Science, South China Agricultural University, Guangzhou 510642, China
2
Wen’s Foodstuffs Group Co., Ltd., Yunfu 527400, China
3
Yunfu Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Yunfu 527400, China
4
Qingdao Jiazhi Biotechnology Co., Ltd., Qingdao 266100, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Animals 2026, 16(4), 690; https://doi.org/10.3390/ani16040690
Submission received: 14 January 2026 / Revised: 6 February 2026 / Accepted: 15 February 2026 / Published: 23 February 2026
(This article belongs to the Special Issue Common Infectious Diseases in Poultry)
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Simple Summary

Vaccination is a key tool for preventing infectious diseases in poultry, but the timing of different vaccines can influence how well they work. In this study, we investigated whether two widely used live vaccines, one targeting infectious bursal disease and the other targeting viral arthritis, affect each other when they are given at the same time or within a short period. Using chickens raised without prior exposure to pathogens, we evaluated immune responses and disease protection under different vaccination schedules. The results showed that the infectious bursal disease vaccine always provided full protection and was not influenced by the second vaccine. In contrast, the viral arthritis vaccine was less effective when both vaccines were administered on the same day or only three days apart, resulting in weaker immune responses and lower protection against disease. When the interval between the two vaccinations was extended to five days or longer, this negative effect disappeared and strong protection was restored. These findings demonstrate that vaccine timing is critical for achieving optimal protection. Adjusting vaccination schedules to include a sufficient interval can improve disease prevention, reduce production losses, and support better health and welfare in poultry flocks, contributing to more efficient and sustainable poultry production.

Abstract

Infectious bursal disease virus (IBDV) and avian reovirus (ARV) are major immunosuppressive pathogens controlled through the widespread use of live attenuated vaccines. Concerns persist regarding potential immune interference when these vaccines are co-administered, though comprehensive in vivo data are lacking. Here, we reported the immunogenicity and protective efficacy of a live IBDV vaccine (W2512G-61) and a live ARV vaccine (ZJS) administered simultaneously or sequentially at 3-, 5-, and 7-day intervals in specific-pathogen-free (SPF) chickens. The IBDV live vaccine elicits strong, interval-independent humoral immunity and conferred 100% protection, demonstrating no compromise from ARV co-administration. Conversely, ARV-specific immunity was severely impaired by close temporal vaccination. ARV protection rates fell from 86.7% (ARV-only) to 46.7% with simultaneous administration and from 93.3% to 66.7% with a 3-day interval. Extending the interval to five or seven days eliminated this interference, restoring ARV antibody titers and protection to levels equivalent to ARV-only control vaccinated groups. This study provides the first definitive evidence of asymmetric immune interference between live IBDV and ARV vaccines. The results establish a minimum safe interval of five days to prevent interference and ensure robust ARV vaccine efficacy. These findings offer critical, evidence-based guidance for optimizing vaccination schedules to improve disease control in commercial poultry production.

1. Introduction

Infectious bursal disease virus (IBDV) and avian reovirus (ARV) are major immunosuppressive pathogens that inflict substantial economic losses on global poultry production [1]. Both viruses exhibit a pronounced tropism for immunologically critical tissues in young chickens, disrupting normal immune development. This results in heightened susceptibility to secondary infections, poor vaccine responsiveness, and increased mortality [2,3]. Their early-age infectivity, environmental persistence, and capacity to induce prolonged immunosuppression make their effective control a persistent challenge in modern poultry operations [4].
IBDV, a member of the genus Avibirnavirus within the family Birnaviridae, is a non-enveloped virus with a double-stranded RNA genome [5]. The genome comprises two segments: segment A (~3.2 kb) encodes a polyprotein cleaved into the structural proteins VP2, VP3, VP4, and the non-structural VP5, while segment B (~2.8 kb) encodes the viral RNA-dependent RNA polymerase, VP1 [6,7]. VP2, the major capsid protein, carries the principal neutralizing epitopes and is the primary determinant of antigenicity [8,9].
IBDV exhibits a marked tropism for immature B lymphocytes in the bursa of Fabricius. Viral infection results in lymphocytolysis and varying degrees of immunosuppression, the severity of which depends on viral virulence [10]. Infection with very virulent IBDV (vvIBDV) strains causes high mortality, severe bursal atrophy, and profound lymphoid necrosis, leading to long-term humoral immunosuppression [11,12]. Surviving birds usually display reduced vaccine responsiveness and increased susceptibility to opportunistic infections [13]. Live-attenuated IBDV vaccines—classified as mild, intermediate, or intermediate-plus—are widely used. However, their replication within the bursa and the associated innate immune activation can transiently modulate the host’s immune status, potentially interfering with the efficacy of other live vaccines administered concurrently [14,15].
ARV, a member of the genus Orthoorthoreovirus within the family Reoviridae, is a non-enveloped virus with a segmented double-stranded RNA genome comprising 10 segments [16]. The σC protein, encoded by the S1 genome segment, functions as the primary attachment protein and harbors both type-specific and cross-reactive epitopes, making it the key molecular target for strain differentiation [17].
Globally, ARV strains cluster into at least six genotypes [18,19]. This genetic diversity underlies the broad spectrum of clinical diseases, ranging from primary manifestations like viral arthritis and tenosynovitis—characterized by synovitis, hock swelling, lameness, and growth retardation—to involvement in enteric disease, malabsorption syndromes, and immunosuppression [19,20]. ARV replicates in tendons, synovium, the intestinal tract, and lymphoid organs (e.g., spleen, bursa of Fabricius), amplifying pathology and increasing susceptibility to secondary infections [21]. Although live attenuated ARV vaccines are widely used in breeder and broilers, their efficacy is contingent upon adequate early replication. This can be compromised by several factors, including interference from maternal antibodies, the immature state of the neonatal immune system, and concurrent viral infections [20,22].
In commercial poultry production, live attenuated vaccines against both IBDV and ARV are routinely administered during the first two weeks of life to establish early protection [23]. However, both viruses share significant biological similarities: they replicate in lymphoid tissues, induce potent innate immune responses, and require robust early replication to elicit protective immunity [3,23]. These overlapping characteristics raise concerns about potential immune interference, especially when vaccines are administered simultaneously or in close succession.
Immune interference among live poultry vaccines is a well-documented phenomenon. A primary mechanism involves the strong innate antiviral response triggered by one vaccine virus, which can suppress the replication of a concurrently administered live attenuated virus [1,24,25]. Notably, IBDV infection is known to transiently alter host innate signaling and antigen-presenting cell (APC) function, which could theoretically impair ARV vaccine take. From a vaccination practice perspective, live ARV vaccines are designed for early administration to ensure sufficient viral replication and induction of protective immunity prior to field exposure. Commercial live ARV vaccines based on the S1133 or ZJS strain, are recommended to be administered from 7 days of age, reflecting the requirement for early vaccine virus replication to establish effective immunity [26]. In contrast, attenuated live IBDV vaccines, including the W2512 strain, are typically administered around 10 days of age and have been demonstrated to efficiently replicate in the bursa of Fabricius, providing effective protection against both classical and novel variant IBDV strains while inducing strong early immune activation and transient bursal immune modulation [27]. Therefore, the close temporal proximity between the recommended immunization windows for live ARV (7 days of age) and live IBDV (approximately 10 days of age) vaccines creates a biologically plausible scenario for immune interference, particularly if the innate immune responses elicited by IBDV vaccination overlap with the critical replication phase of the ARV vaccine virus. Despite the widespread industry practice of co-administering or closely spacing these vaccines, no comprehensive in vivo study has systematically quantified the degree of interference between live IBDV and live ARV vaccines or established a safe, non-interfering immunization interval.
To address this critical knowledge gap, this study investigated the effects of different immunization intervals on the immunogenicity and protective efficacy of live attenuated IBDV (W2512G-61 strain) and ARV (ZJS strain) vaccines. In this study, we systematically evaluated humoral response, protection against virulent challenge, clinical and pathological outcomes, and tissue viral loads to determine whether vaccine interference occurs and, if so, to establish the minimum safe interval required to eliminate its detrimental effects. The findings provide crucial, evidence-based data to optimize vaccination schedules and enhance the control of these economically significant immunosuppressive diseases in commercial poultry production.

2. Materials and Methods

2.1. Ethics Statement

All animal procedures were reviewed and approved by the Animal Care Committee of South China Agricultural University (approval ID: SYXK-2024-0136). Experimental operations complied with the Guidelines for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China. To minimize pain and distress, birds were euthanized under isoflurane anesthesia followed by administration of pentobarbitone, as previously described [28].

2.2. Animals, Vaccines and Viruses

A total of 300 one-day-old specific pathogen-free (SPF) White Leghorn chickens and the live ARV vaccine (ZJS strain) were purchased from Dahuanong Animal Health Products Co., Ltd. (Zhaoqing, China). The live IBDV vaccine (W2512G-61 strain) was purchased from Huadu Shihua Biological Co., Ltd. (Beijing, China). The IBDV strain YYB and ARV strain S1133 were stored in our lab. The hypervirulent IBDV YYB strain used for challenge has been previously characterized as causing severe bursal lesions, high mortality, and marked immunosuppression in SPF chickens, and is routinely used as a standard virulence challenge strain in vaccine efficacy studies [27]. The ARV S1133 strain is a well-established virulent reference strain that induces typical viral arthritis and tenosynovitis following footpad inoculation, and has been widely applied for evaluating the protective efficacy of live ARV vaccines [22].

2.3. Animals Experimental Design

A total of 300 one-day-old SPF chickens were randomly assigned into 20 groups (15 birds per group) and reared under standard controlled environmental conditions with ad libitum access to feed and water. The study evaluated four primary immunization schedules relative to a subsequent homologous challenge, alongside essential controls. The complete schedule and group allocation are detailed in Table 1. The IBDV live vaccine and ARV live vaccine were administered via eye drop and subcutaneous injection, respectively. Briefly, chickens in groups 1 and 2 received the ARV vaccine only (on day 7 or 10), and birds in groups 3, 4, and 5 were immunized with IBDV vaccine (on day 10, 12, or 14). In the combination vaccine groups, sequential administration of ARV was performed on day 7 followed by IBDV on day 10 (3-day interval, groups 6 & 7), day 12 (5-day interval, groups 8 & 9), or day 14 (7-day interval, groups 10 & 11). Birds in groups 12 & 13 were vaccinated with both vaccines simultaneously on day 10. Birds in groups 14, 15, and 16 served as IBDV challenge controls, and birds in groups 17 and 18 served as ARV challenge controls, while birds in groups 19 and 20 served as blank controls. Blood samples were collected via the wing vein at 7, 14, and 21 days post IBDV vaccination, or at 7, 14, 21, and 28 days post ARV vaccination.
Birds designated for ARV challenge were inoculated at 28 days post-vaccination (35 or 38 days of age, see Table 1). Each bird received 0.1 mL containing 107.0 TCID50 of the virulent ARV strain S1133 via injection into the left footpad, a standard model for viral arthritis. Following challenge, birds were monitored daily for 10 days for clinical signs of arthritis, including lameness, hock swelling, footpad discoloration, and reluctance to move. Clinical disease was defined as the presence of characteristic signs for ≥5 consecutive days. Detailed clinical examinations of the footpad and joints were performed at 3, 5, 7, and 10 days post-challenge. At 10 days post-challenge, all birds were euthanized and necropsied. Gross lesions in the hock joint and associated tendons were observed and recorded, including synovial thickening, tendon edema, fibrinous adhesions, and hemorrhage, according to established criteria [29]. Protection was defined as the absence of both clinical arthritis and ARV-associated gross lesions. Tissue including tendon tissue, cecal tonsils, and cloacal swabs were collected to measure viral loads by a TaqMan-based RT-qPCR assay [30].
Birds designated for IBDV challenge were inoculated at 21 days post-vaccination (31, 33, or 35 days of age, see Table 1) with 104.5EID50 of the virulent IBDV YYB strain via ocular route. Birds were monitored daily for 4 days for clinical signs, including depression, huddling, ruffled feathers, diarrhea, and mortality. At 4 days post-challenge, all surviving birds were humanely euthanized; birds that died during the observation period were necropsied immediately. The bursa of Fabricius was collected, and examined for gross lesions typical of IBD (e.g., swelling, edema, hemorrhage, gelatinous transudate). Total RNA was extracted from bursal tissue homogenates using TRIzol reagent, and the vp2 gene was amplified [31], and sequenced to confirm the identity of the challenge virus.

2.4. Serological Assessment

IBDV-specific antibodies were measured using a commercial indirect ELISA kit (ID Screen® IBD Indirect, IDvet, Grabels, France). A titer ≥ 875 was defined as seropositive per the manufacturer’s instructions. ARV-specific antibodies were quantified with a commercial indirect ELISA kit (ID Screen® ARV Indirect, IDvet, Grabels, France). A titer ≥ 853 was considered seropositive.

2.5. Cytokine Analyses

The production of interferon-gamma (IFN-γ) in the serum were determined to assess the cellular immune response using a commercial chicken IFN-γ ELISA kit (Cat. No. [SP15253], Wuhan Saipei Biotechnology Co., Ltd., Wuhan, China), according to the manufacturer’s protocol. Serum samples collected at the indicated time points were diluted 1:5 and assayed in duplicate alongside a recombinant chicken IFN-γ standard curve (range: 100–1600 ng/L). Absorbance was measured at 450 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific, Waltham, MA, USA), and IFN-γ concentrations were calculated by interpolation from the standard curve. All samples were analyzed in the same assay batch to minimize inter-assay variation.

2.6. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Antibody titers, IFN-γ concentrations and viral load data were analyzed using two-way analysis of variance (ANOVA) to assess the effects of vaccination group and time, followed by appropriate post hoc multiple comparison tests. Data are presented as mean ± standard error of the mean (SEM). A value of p < 0.05 was considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

3. Results

3.1. IBDV Antibody Responses Are Unaffected by Co-Administration with ARV

IBDV-specific antibody titers measured at 7, 14, and 21 days post-vaccination demonstrated robust and comparable humoral immunity across all groups, irrespective of the vaccination schedule. As shown in Figure 1, the antibody titers in the simultaneous group (both vaccines on day 10) and in all sequential interval groups (3, 5, or 7 days) did not differ significantly from those of the IBDV vaccinated groups (p > 0.05).
All immunized groups achieved 100% seropositivity by 21 days post-vaccination, with titers consistently within the protective range. Specifically, the IBDV antibody titers in the IBDV (10 d) group and the ARV (10 d) + IBDV (10 d) group were 6129.59 ± 826.53 and 5609.86 ± 773.75, respectively (Figure 1A). In the ARV (7 d) + IBDV (10 d) group, the IBDV antibody titer reached 6726.14 ± 585.82 (Figure 1B). Furthermore, the antibody titers in the IBDV (12 d) group and ARV (7 d) + IBDV (12 d) group reached 9025.45 ± 478.18 and 7746.22 ± 970.33, respectively (Figure 1C), while the corresponding titers in the IBDV (14 d) group and the ARV (7 d) + IBDV (14 d) group were 8296.59 ± 648.28 and 8033.94 ± 551.65, respectively (Figure 1D). These results demonstrate that the immunogenicity of the live IBDV vaccine is stable and remains uncompromised by the concurrent or closely timed administration of the live ARV vaccine.

3.2. ARV Antibody Responses Are Significantly Reduced by Simultaneous or 3-Day Interval Vaccination

ARV-specific antibody titers were critically dependent on the vaccination schedule. ARV antibody titers measured at 7, 14, 21, and 28 days post vaccination differed significantly among immunization groups (Figure 2). Pronounced interference occurred when vaccinations were administered in close succession. In the simultaneous group (both vaccines on day 10), the ARV antibody titer at 28 days post vaccination was significantly reduced to 1172.33 ± 377.15, compared with that of the ARV vaccinated group (4653.43 ± 975.86) (Figure 2A). Similarly, in the 3-day interval group, ARV antibody titers at 28 days post-vaccination remained significantly lower (3470.33 ± 843.99) than those observed in the corresponding ARV vaccinated group (5243.06 ± 1141.64) (Figure 2B). In contrast, extending the interval between ARV and IBDV vaccination to five or seven days effectively eliminated this interference. In the 5- and 7-day interval groups, the ARV antibody titers at 28 days post-vaccination reached 4512.40 ± 616.63 and 4938.93 ± 1036.47, respectively, and did not differ significantly from those of the ARV vaccinated group (Figure 2C,D). These data demonstrate that a minimum interval of five days is required to prevent interference and ensure robust ARV-specific humoral immunity.

3.3. IFN-γ Production Following Single or Combined Vaccination

To evaluate the cellular immune responses induced by different vaccination schedules, IFN-γ concentrations in the serum were measured at 7, 14, 21, and 28 days post-vaccination in chickens receiving single or combined ARV and IBDV immunization. As shown in Figure 3, IFN-γ levels remained low at 7 and 14 days post-vaccination in all groups, with no significant differences between immunized and non-immunized chickens. A pronounced but transient increase in IFN-γ production was consistently observed at 21 days post-vaccination in chickens receiving live IBDV vaccination, either alone or in combination with ARV. At this time point, IBDV immunized groups exhibited significantly higher IFN-γ levels than the non-immunized control group across multiple vaccination schedules. Combined ARV and IBDV vaccination did not result in additive enhancement of IFN-γ production compared with IBDV vaccination alone. By 28 days, IFN-γ concentrations declined in all groups and approached baseline levels, indicating that IFN-γ induction was transient and temporally associated with IBDV vaccination.

3.4. Protective Efficacy of the IBDV Vaccine

As shown in Table 2 and Figure 4, all vaccinated groups, including those that received IBDV and ARV vaccines simultaneously or sequentially, achieved 100% survival. RT-PCR analysis of bursal tissues confirmed this protection, with 100% detection of the vaccine virus and 0% detection of the wild-type challenge virus across all immunized groups. In contrast, the unvaccinated challenge control groups exhibited mortality rates of 20–80%, with 0% vaccine virus detection and 100% wild-type virus detection, confirming successful infection (Table 2, Figure 4). Gross lesion assessment was consistent with these results. All birds in the challenge control groups developed characteristic severe lesions, including bursal congestion, hemorrhage, and exudation (Figure 5H). In contrast, bursae from all vaccinated groups exhibited normal morphology with preserved architecture (Figure 5A–G). No clinical signs or pathological changes were observed in any vaccinated bird. These results demonstrate that the live attenuated IBDV vaccine confers complete protection against a hypervirulent challenge. Crucially, its protective efficacy remained uncompromised regardless of co-administration or timing with the live ARV vaccine, highlighting its robust immunodominance.

3.5. Protective Efficacy of the ARV Vaccine

The ARV challenge results corroborated the serological findings, confirming the presence of immune interference when vaccination intervals were insufficient (Table 3). Administration of the ARV vaccine alone at 10 days of age conferred 86.7% protection. In contrast, simultaneous co-administration of the ARV and IBDV vaccines on day 10 resulted in a significant reduction in protection to 46.7%. Likewise, when ARV was given on day 7 followed by IBDV on day 10 (a 3-day interval), the protection rate declined from 93.3% (ARV vaccinated group) to 66.7%, indicating that short intervals significantly compromise vaccine efficacy. Extending the vaccination interval effectively eliminated this interference. Birds receiving ARV on day 7 followed by IBDV on day 12 (5-day interval) or day 14 (7-day interval) achieved protection rates of 93.3% and 86.7%, respectively—both statistically equivalent to the ARV vaccinated groups. This establishes a minimum 5-day interval between vaccines to maintain optimal protection against ARV challenge. Clinical lesion scoring supported these results. Birds in the simultaneous and 3-day interval groups exhibited pronounced arthritic lesions, including synovial thickening, tendon exudation, hock joint edema, and adhesion formation (Figure 6). In contrast, birds in the 5- and 7-day interval groups displayed minimal to no lesions. Viral load measurements in tissues and swabs further corroborated the protection data (Figure 7). In the ARV vaccinated group, viral loads in tendon tissue were significantly lower than in challenge controls and the simultaneous vaccination group (Figure 7A). All sequential vaccination groups (ARV on day 7; IBDV on days 10, 12, or 14) exhibited significantly reduced ARV loads in tendons, cecal tonsils, and cloacal swabs compared to challenge controls, consistent with effective vaccine-mediated protection (Figure 7B). These results demonstrate that simultaneous or closely spaced (≤3 days) vaccination impairs ARV-induced humoral immunity and protection, whereas an interval of 5 days or more prevents interference, ensuring robust ARV-specific antibody responses and full protective efficacy.

4. Discussion

This study provides the first systematic in vivo evidence that co-administration or close sequential administration of live attenuated IBDV and ARV vaccines induces significantly asymmetric immune interference. The live IBDV vaccine (W2512 G-61) consistently elicited strong seroconversion and complete protection, regardless of vaccination interval. In contrast, the live attenuated ARV vaccine (ZJS) exhibited significantly reduced antibody responses and compromised protection when administered simultaneously with IBDV or within a 3-day interval. Extending the interval between vaccines to at least 5 days completely eliminated this interference. These results offer clear, evidence-based guidance for optimizing vaccination schedules in commercial poultry operations to ensure robust protection against both immunosuppressive diseases.
The differential susceptibility of both vaccines can be explained by their distinct immunobiological characteristics. Live attenuated IBDV rapidly targets the bursa of Fabricius and replicates efficiently in B lymphocytes, inducing transient lymphoid depletion, apoptosis, and potent early innate immune activation, including the induction of antiviral cytokines and inflammatory mediators [32,33,34]. Previous studies have shown that even attenuated IBDV vaccine strains can generate substantial antiviral signaling capable of suppressing the replication of other viruses during the early post-vaccination period [27,35,36]. In contrast, effective ARV immunization depends on sufficient early viral replication and antigen availability to initiate adaptive immune responses, rendering it inherently more sensitive to such transient immune modulation. The interference observed here was further supported by multiple functional endpoints, including reduced ARV-specific antibody titers, decreased protection rates, and increased viral loads following challenge. These findings are consistent with previous reports demonstrating that early innate immune activation induced by IBDV vaccination can transiently alter antigen-presenting cell function and suppress the replication of co-administered live attenuated viruses [27]. Collectively, these data delineate a biologically meaningful “interference window” during the early post-IBDV vaccination period.
IFN-γ is a key cytokine associated with Th1-type immune responses and antiviral defense [37]. In the present study, IFN-γ analysis revealed that vaccination-induced cellular immune activation was predominantly associated with live IBDV administration. IFN-γ elevation was transient and peaked at 21 days post vaccination, temporally coinciding with the period of reduced ARV-specific antibody responses and protection in co-administration and short-interval sequential groups. Actually, we also tried to detect the expression of IFN-β and IL-6 in the serum of vaccinated chickens. However, no typical change in both cytokines were observed. More efforts will be required to evaluate cytokine expression in the future.
To explain the biological basis of this defined interference window, several immunological mechanisms may contribute to the observed phenomenon. First, the type I interferon response induced by live IBDV vaccination can establish a transient antiviral state, which has been reported to suppress the replication of other live attenuated viruses, including ARV [27,38,39]. Second, IBDV infection is known to trigger rapid recruitment and functional modulation of antigen-presenting cells (APCs), such as dendritic cells and macrophages, which may temporarily limit their capacity to process and present heterologous viral antigens [3,32,40]. Third, IBDV-associated cytokine responses may bias the immune environment toward a Th1-dominant or regulatory profile, potentially constraining Th2-associated humoral responses that are critical for optimal ARV-specific antibody production [41,42]. These mechanisms align with previous studies reporting interactions among live poultry vaccines, particularly those involving highly immunomodulatory viruses [1,24,43]. Collectively, these studies indicate that live vaccines against IBDV, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and chicken infectious anemia virus (CIAV) can induce transient immune modulation or deviation, affecting responses to co-administered antigens [1,24,25]. However, the extent to which these interactions occur is likely dependent on vaccine type, timing, and host immune status.
From a practical perspective, the implementation of a minimum five-day interval between live IBDV and ARV vaccinations is operationally feasible within commercial broiler production systems. Live IBDV vaccination is routinely administered at an early age, and adjusting the timing of subsequent ARV vaccination generally does not require additional handling events or labor input. On the contrary, separating vaccinations may reduce cumulative physiological stress associated with simultaneous or closely spaced immunizations, potentially improving overall immune responsiveness. Given the substantial improvement in ARV vaccine efficacy observed in this study, the benefits of adopting a five-day interval are likely to outweigh the minimal logistical adjustments required at the farm level.
Although this study focused on ARV and IBDV, the findings also highlight a broader concept of “immunization priority” when highly immunomodulatory live vaccines are administered in combination. Live IBDV vaccination induces rapid and dominant immune activation, which may similarly affect other replication-dependent live vaccines commonly used in poultry production, such as Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) vaccines [24,44,45]. While direct extrapolation is beyond the scope of the present study, these results suggest that interval-based scheduling strategies may represent a generalizable framework for optimizing multi-vaccine immunization programs. Future studies should systematically evaluate optimal vaccination interval matrices and immune prioritization when IBDV is co-administered with multiple live vaccines under both experimental and field conditions.
In conclusion, this study demonstrates a clear and asymmetric immune interference between live attenuated IBDV and ARV vaccines in SPF chickens. While IBDV vaccination remained fully effective regardless of timing, ARV vaccine immunogenicity and protection were significantly compromised when vaccines were administered simultaneously or within a 3-day interval. Establishing a minimum 5-day interval effectively eliminated this interference and restored robust ARV protection. These findings provide practical, evidence-based guidance for optimizing early vaccination schedules and improving disease control in commercial poultry production.

5. Conclusions

This study provides clear in vivo evidence that live attenuated vaccines against infectious bursal disease and avian viral arthritis can interact in an asymmetric manner when administered simultaneously or within a short interval. While the infectious bursal disease vaccine consistently induced strong immune responses and complete protection regardless of vaccination timing, the efficacy of the viral arthritis vaccine was markedly reduced when both vaccines were given on the same day or within a three-day interval. This interference was reflected by decreased antibody responses, higher viral loads after challenge, and reduced protection against clinical disease.
Importantly, extending the interval between the two vaccinations to at least five days effectively eliminated immune interference and fully restored the immunogenicity and protective efficacy of the viral arthritis vaccine. These findings demonstrate that vaccination timing is a critical determinant of vaccine performance when multiple live vaccines are used early in life. The results provide practical, evidence-based guidance for optimizing vaccination schedules in poultry production, enabling improved disease control, enhanced flock health, and more efficient use of live vaccines under field conditions.

Author Contributions

J.W.: Writing—original draft, Writing—review and editing, Methodology, Investigation. M.L.: Writing—original draft, Methodology, Data curation. Y.L. (Yuecheng Long): Methodology, Software, Validation. S.Y.: Software, Formal analysis, Investigation. C.L.: Project administration, Software, Resources. J.L.: Validation, Project administration, Conceptualization. G.H.: Investigation, Methodology, Software. E.X.: Methodology, Investigation, Data curation. Y.L. (Yiming Liu): Software, Project administration, Conceptualization. Y.X.: Methodology, Software, Data curation. X.L.: Investigation, Methodology, Data curation. J.Q.: Supervision, Methodology, Investigation, Conceptualization. L.Y.: Writing—original draft, Writing—review and editing, Formal analysis. W.L.: Writing—review and editing, Validation, Supervision, Resources, Project administration. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Science and Technology Plan Program of Yunfu city (2024090301 & YF2025NYRC03 & 2024020101), the Natural Science Foundation of Guangdong Province, China (Grant No. 2025A1515012150), the Science and Technology Plan Program of Guangdong Province (2023B1212070018), the Key R&D Program of Shandong Province (2023CXPT059), and the Fourth Round of Guangdong Provincial Modern Agricultural Industry Technology System Innovation Team Construction Project (2024CXTD15).

Institutional Review Board Statement

All animal procedures were reviewed and approved by the Animal Care Committee of South China Agricultural University (approval ID: SYXK-2024-0136, approval date: 30 June 2024).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflict of interest. The authors affiliated with Wen’s Foodstuffs Group Co., Ltd. (Jiaolong Wen, Shenghua Yang, Ermin Xie, Yiming Liu, Yanhua Xu, Xuesong Li, Jianping Qin, Lijuan Yin, Wencheng Lin), the Yunfu Branch of Guangdong Laboratory for Lingnan Modern Agriculture (Shenghua Yang, Lijuan Yin), and Qingdao Jiazhi Biotechnology Co., Ltd. (Chuang Lyu) were involved in this study as part of their employment and confirmed that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. IBDV-specific humoral immune responses following different vaccination schedules. IBDV-specific antibody titers in the serum were measured by ELISA at 0, 7, 14, and 21 days post vaccination. (A) Simultaneous vaccination with ARV and IBDV administered at 10-day-old age. (B–D) Sequential vaccination with intervals of 3 days: (B) ARV vaccination at 7-day-old age, IBDV vaccination at 10-day-old age, 5 days. (C) ARV vaccination at 7-day-old age, IBDV vaccination at 12-day-old age, and 7 days. (D) ARV vaccination at 7-day-old age, IBDV vaccination at 14-day-old age. Data are presented as mean ± SEM. No statistically significant differences were observed among groups at any time point (p > 0.05).
Figure 1. IBDV-specific humoral immune responses following different vaccination schedules. IBDV-specific antibody titers in the serum were measured by ELISA at 0, 7, 14, and 21 days post vaccination. (A) Simultaneous vaccination with ARV and IBDV administered at 10-day-old age. (B–D) Sequential vaccination with intervals of 3 days: (B) ARV vaccination at 7-day-old age, IBDV vaccination at 10-day-old age, 5 days. (C) ARV vaccination at 7-day-old age, IBDV vaccination at 12-day-old age, and 7 days. (D) ARV vaccination at 7-day-old age, IBDV vaccination at 14-day-old age. Data are presented as mean ± SEM. No statistically significant differences were observed among groups at any time point (p > 0.05).
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Figure 2. ARV-specific humoral immune responses following different intervals of IBDV co-administration. ARV-specific antibody titers in the serum were determined by ELISA at 0, 7, 14, 21, and 28 days post vaccination. (A) Simultaneous vaccination with ARV and IBDV administered at 10-day-old age. (B–D) Sequential vaccination with intervals of 3, 5, and 7 days, respectively: (B) ARV vaccination at 7-day-old age, IBDV vaccination at 10-day-old age; (C) ARV vaccination at 7-day-old age, IBDV vaccination at 12-day-old age; (D) ARV vaccination at 7-day-old age, IBDV vaccination at 14-day-old age. Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01.
Figure 2. ARV-specific humoral immune responses following different intervals of IBDV co-administration. ARV-specific antibody titers in the serum were determined by ELISA at 0, 7, 14, 21, and 28 days post vaccination. (A) Simultaneous vaccination with ARV and IBDV administered at 10-day-old age. (B–D) Sequential vaccination with intervals of 3, 5, and 7 days, respectively: (B) ARV vaccination at 7-day-old age, IBDV vaccination at 10-day-old age; (C) ARV vaccination at 7-day-old age, IBDV vaccination at 12-day-old age; (D) ARV vaccination at 7-day-old age, IBDV vaccination at 14-day-old age. Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01.
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Figure 3. IFN-γ responses following ARV and IBDV vaccination under different immunization schedules. IFN-γ concentrations (ng/L) in the serum were measured by ELISA at indicated time points after vaccination in chickens immunized with ARV alone, IBDV alone, sequential ARV + IBDV vaccination, or unvaccinated as controls. (A) Simultaneous vaccination with ARV and IBDV administered at 10-day-old age. (B–D) Sequential vaccination with intervals of 3, 5, and 7 days, respectively: (B) ARV vaccination at 7-day-old age, IBDV vaccination at 10-day-old age; (C) ARV vaccination at 7-day-old age, IBDV vaccination at 12-day-old age; (D) ARV vaccination at 7-day-old age, IBDV vaccination at 14-day-old age. Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01.
Figure 3. IFN-γ responses following ARV and IBDV vaccination under different immunization schedules. IFN-γ concentrations (ng/L) in the serum were measured by ELISA at indicated time points after vaccination in chickens immunized with ARV alone, IBDV alone, sequential ARV + IBDV vaccination, or unvaccinated as controls. (A) Simultaneous vaccination with ARV and IBDV administered at 10-day-old age. (B–D) Sequential vaccination with intervals of 3, 5, and 7 days, respectively: (B) ARV vaccination at 7-day-old age, IBDV vaccination at 10-day-old age; (C) ARV vaccination at 7-day-old age, IBDV vaccination at 12-day-old age; (D) ARV vaccination at 7-day-old age, IBDV vaccination at 14-day-old age. Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01.
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Figure 4. Survival of chickens following challenge with hypervirulent IBDV (YYB strain). Survival was monitored to assess protective efficacy. (A–C) Groups vaccinated with IBDV alone or co-administered with ARV at simultaneous (day 10), 3-day, 5-day, or 7-day intervals were challenged at 31, 33, or 35 days of age, according to schedule. All vaccinated groups showed 100% survival, regardless of ARV co-administration or vaccination interval, demonstrating complete protection. In contrast, three challenge control groups showed low and variable survival (20–80%), confirming challenge virulence.
Figure 4. Survival of chickens following challenge with hypervirulent IBDV (YYB strain). Survival was monitored to assess protective efficacy. (A–C) Groups vaccinated with IBDV alone or co-administered with ARV at simultaneous (day 10), 3-day, 5-day, or 7-day intervals were challenged at 31, 33, or 35 days of age, according to schedule. All vaccinated groups showed 100% survival, regardless of ARV co-administration or vaccination interval, demonstrating complete protection. In contrast, three challenge control groups showed low and variable survival (20–80%), confirming challenge virulence.
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Figure 5. Gross bursal pathology following IBDV challenge. Representative bursae photographed at necropsy: (A–G) Vaccinated groups showed normal bursal morphology with preserved architecture and absence of congestion, edema, or hemorrhage. (H) Challenge control group showed severe bursal lesions, including congestion, hemorrhage, and fibrinous exudation. Red arrow indicate the bursal lesion area. (I) Blank control group displayed normal bursae.
Figure 5. Gross bursal pathology following IBDV challenge. Representative bursae photographed at necropsy: (A–G) Vaccinated groups showed normal bursal morphology with preserved architecture and absence of congestion, edema, or hemorrhage. (H) Challenge control group showed severe bursal lesions, including congestion, hemorrhage, and fibrinous exudation. Red arrow indicate the bursal lesion area. (I) Blank control group displayed normal bursae.
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Figure 6. Clinical evaluation of footpad swelling following ARV challenge. Representative footpad photographs collected at the end of the observation period. (A) ARV (10 d) only. (B) Simultaneous ARV (10 d) + IBDV (10 d). (C) ARV (7 d) only. (D) ARV (7 d) + IBDV (10 d) (3-day interval). (E) ARV (7 d) + IBDV (12 d) (5-day interval). (F) ARV (7 d) + IBDV (14 d) (7-day interval). (G) ARV challenge control. (H) Blank control. ARV-only groups (A,C) and sequential vaccination groups with 5- or 7-day intervals (E,F) showed minimal swelling, indicating effective protection. Pronounced swelling was evident in the simultaneous (B) and 3-day interval (D) groups, consistent with insufficient immunity. Severe swelling in challenge controls (G) confirmed challenge virulence. Red arrows in panels B, D, and G indicate tibiotarsal joint swelling.
Figure 6. Clinical evaluation of footpad swelling following ARV challenge. Representative footpad photographs collected at the end of the observation period. (A) ARV (10 d) only. (B) Simultaneous ARV (10 d) + IBDV (10 d). (C) ARV (7 d) only. (D) ARV (7 d) + IBDV (10 d) (3-day interval). (E) ARV (7 d) + IBDV (12 d) (5-day interval). (F) ARV (7 d) + IBDV (14 d) (7-day interval). (G) ARV challenge control. (H) Blank control. ARV-only groups (A,C) and sequential vaccination groups with 5- or 7-day intervals (E,F) showed minimal swelling, indicating effective protection. Pronounced swelling was evident in the simultaneous (B) and 3-day interval (D) groups, consistent with insufficient immunity. Severe swelling in challenge controls (G) confirmed challenge virulence. Red arrows in panels B, D, and G indicate tibiotarsal joint swelling.
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Figure 7. ARV viral load and shedding following challenge. ARV viral loads were quantified in tendon tissue, cecal tonsils, and cloacal swabs. (A) ARV (10 d) only, simultaneous (ARV 10 d + IBDV 10 d), and challenge control groups. The ARV vaccinated group showed significantly lower viral loads in tendon tissue compared with the simultaneous and challenge controls. Both vaccinated groups showed reduced viral loads in cecal tonsils and cloacal swabs compared with challenge controls. (B) Sequential groups: ARV (7 d) followed by IBDV at 10, 12, or 14 days (3-, 5-, or 7-day intervals). All sequential groups demonstrated significantly lower viral loads across all sample types compared with challenge controls, indicating effective suppression of viral replication and shedding. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.
Figure 7. ARV viral load and shedding following challenge. ARV viral loads were quantified in tendon tissue, cecal tonsils, and cloacal swabs. (A) ARV (10 d) only, simultaneous (ARV 10 d + IBDV 10 d), and challenge control groups. The ARV vaccinated group showed significantly lower viral loads in tendon tissue compared with the simultaneous and challenge controls. Both vaccinated groups showed reduced viral loads in cecal tonsils and cloacal swabs compared with challenge controls. (B) Sequential groups: ARV (7 d) followed by IBDV at 10, 12, or 14 days (3-, 5-, or 7-day intervals). All sequential groups demonstrated significantly lower viral loads across all sample types compared with challenge controls, indicating effective suppression of viral replication and shedding. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.
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Table 1. Experimental group allocation and vaccination schedule.
Table 1. Experimental group allocation and vaccination schedule.
GroupDesignationARV
Vaccination Age		IBDV
Vaccination Age		IntervalChallenge Age, StrainNo. of Birds
1ARV (7 d) group (ARV challenge)7 d //35 d, ARV S1133 strain15
2ARV (10 d) group (ARV challenge)10 d //38 d, ARV S1133 strain15
3IBDV (10 d) group (IBDV challenge)/10 d/31 d, IBDV YYB strain15
4IBDV (12 d) group (IBDV challenge)/12 d/33 d, IBDV YYB strain15
5IBDV (14 d) group (IBDV challenge)/14 d/35 d, IBDV YYB strain15
6ARV (7 d) + IBDV (10 d) group (ARV challenge)7 d 10 d3 d35 d, ARV S1133 strain15
7ARV (7 d) + IBDV (10 d) group (IBDV challenge)7 d 10 d3 d31 d, IBDV YYB strain15
8ARV (7 d) + IBDV (12 d) group (ARV challenge)7 d 12 d5 d35 d, ARV S1133 strain15
9ARV (7 d) + IBDV (12 d) group (IBDV challenge)7 d 12 d5 d33 d, IBDV YYB strain15
10ARV (7 d) + IBDV (14 d) group (ARV challenge)7 d 14 d7 d35 d, ARV S1133 strain15
11ARV (7 d) + IBDV (14 d) group (IBDV challenge)7 d 14 d7 d35 d, IBDV YYB strain15
12ARV (10 d) + IBDV (10 d) group (ARV challenge)10 d 10 d0 d38 d, ARV S1133 strain15
13ARV (10 d) + IBDV (10 d) group (IBDV challenge)10 d 10 d0 d31 d, IBDV YYB strain15
14IBDV challenge control group 1///31 d, IBDV YYB strain15
15IBDV challenge control group 2///33 d, IBDV YYB strain15
16IBDV challenge control group 3///35 d, IBDV YYB strain15
17ARV challenge control group 1///35 d, ARV S1133 strain15
18ARV challenge control group 2///38 d, ARV S1133 strain15
19Blank control group 1////15
20Blank control group 2////15
Table 2. Protective efficacy of IBDV live vaccine following hypervirulent IBDV YYB challenge.
Table 2. Protective efficacy of IBDV live vaccine following hypervirulent IBDV YYB challenge.
GroupSurvival RateLesion RateIBDV Vaccine
Virus Positivity		IBDV Wild
Virus Positivity		Protection Rate
IBDV (10 d) group100% (15/15)0% (0/15)100% (15/15)0% (0/15)100% (15/15)
ARV (10 d) + IBDV (10 d) group (IBDV challenge)100% (15/15)0% (0/15)100% (15/15)0% (0/15)100% (15/15)
ARV (7 d) + IBDV (10 d) group
(IBDV challenge)		100% (15/15)0% (0/15)100% (15/15)0% (0/15)100% (15/15)
IBDV challenge control group 120% (3/15)100% (15/15)0% (0/15)100% (15/15)0% (0/15)
IBDV (12 d) group100% (15/15)0% (0/15)100% (15/15)0% (0/15)100% (15/15)
ARV (7 d) + IBDV (12 d) group
(IBDV challenge)		100% (15/15)0% (0/15)100% (15/15)0% (0/15)100% (15/15)
IBDV challenge control group 240% (6/15)100% (15/15)0% (0/15)100% (15/15)0% (0/15)
IBDV (14 d) group100% (15/15)0% (0/15)100% (15/15)0% (0/15)100% (15/15)
ARV (7 d) + IBDV (14 d) group
(IBDV challenge)		100% (15/15)0% (0/15)100% (15/15)0% (0/15)100% (15/15)
IBDV challenge control group 380% (12/15)100% (15/15)0% (0/15)100% (15/15)0% (0/15)
Blank control group 1100% (15/15)0% (0/15)0% (0/15)0% (0/15)100% (15/15)
Table 3. Protective efficacy of ARV live vaccine following virulent ARV S1133 challenge.
Table 3. Protective efficacy of ARV live vaccine following virulent ARV S1133 challenge.
GroupClinical
Morbidity		Lesion RateTendon ARV PositivityCecal Tonsil ARV PositivityCloacal Swab ARV
Positivity		Protection Rate
ARV (10 d) group (ARV challenge)13.3% (2/15)13.3% (2/15)13.3% (2/15)6.7% (1/15)0% (0/15)86.7% (13/15)
ARV (10 d) + IBDV (10 d) group (ARV challenge)33.3% (5/15)53.3% (8/15)53.3% (8/15)40% (6/15)26.7% (4/15)46.7% (7/15)
ARV challenge control group 186.7% (13/15)86.7% (13/15)86.7% (13/15)80% (12/15)80% (12/15)13.3% (2/15)
ARV (7 d) group (ARV challenge)0% (0/15)6.7% (1/15)6.7% (1/15)0% (0/15)0% (0/15)93.3% (14/15)
ARV (7 d) + IBDV (10 d) group (ARV challenge)26.7% (4/15)33.3% (5/15)33.3% (5/15)26.7% (4/15)13.3% (2/15)66.7% (10/15)
ARV (7 d) + IBDV (12 d) group (ARV challenge)13.3% (2/15)6.7% (1/15)6.7% (1/15)0% (0/15)0% (0/15)93.3% (14/15)
ARV (7 d) + IBDV (14 d) group (ARV challenge)13.3% (2/15)13.3% (2/15)13.3% (2/15)6.7% (1/15)6.7% (1/15)86.7% (13/15)
ARV challenge control group 286.7% (13/15)86.7% (13/15)86.7% (13/15)80% (12/15)80% (12/15)13.3% (2/15)
Blank control group 20% (0/15)0% (0/15)0% (0/15)0% (0/15)0% (0/15)100% (15/15)
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Wen, J.; Li, M.; Long, Y.; Yang, S.; Lyu, C.; Li, J.; Huo, G.; Xie, E.; Liu, Y.; Xu, Y.; et al. Assessment of Immunological Interference Between Live Infectious Bursal Disease Virus and Avian Reovirus Vaccines in SPF Chickens. Animals 2026, 16, 690. https://doi.org/10.3390/ani16040690

AMA Style

Wen J, Li M, Long Y, Yang S, Lyu C, Li J, Huo G, Xie E, Liu Y, Xu Y, et al. Assessment of Immunological Interference Between Live Infectious Bursal Disease Virus and Avian Reovirus Vaccines in SPF Chickens. Animals. 2026; 16(4):690. https://doi.org/10.3390/ani16040690

Chicago/Turabian Style

Wen, Jiaolong, Mingwei Li, Yuecheng Long, Shenghua Yang, Chuang Lyu, Junxian Li, Guanming Huo, Ermin Xie, Yiming Liu, Yanhua Xu, and et al. 2026. "Assessment of Immunological Interference Between Live Infectious Bursal Disease Virus and Avian Reovirus Vaccines in SPF Chickens" Animals 16, no. 4: 690. https://doi.org/10.3390/ani16040690

APA Style

Wen, J., Li, M., Long, Y., Yang, S., Lyu, C., Li, J., Huo, G., Xie, E., Liu, Y., Xu, Y., Li, X., Qin, J., Yin, L., & Lin, W. (2026). Assessment of Immunological Interference Between Live Infectious Bursal Disease Virus and Avian Reovirus Vaccines in SPF Chickens. Animals, 16(4), 690. https://doi.org/10.3390/ani16040690

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