MATERIALS AND METHODS

Ethical approval

All experimental procedures involving animals were reviewed and approved by the Chulalongkorn University Institutional Animal Care and Use Committee (IACUC Approval No. 1531069) and the Chulalongkorn University Institutional Biosafety Committee (IBC Approval No. 1531003) before commencement of the study. The research was conducted in accordance with the institutional guidelines for the ethical care and use of laboratory animals, the applicable national regulations governing animal experimentation in Thailand, and internationally accepted principles for the ethical use of animals in scientific research.

The approved protocol covered all experimental procedures, including procurement and housing of broiler chickens, administration of live-attenuated IBV vaccines, experimental challenge with the Thai QX-like IBV strain, clinical monitoring, blood and tracheal sample collection, laboratory investigations involving infectious materials, and humane euthanasia. All procedures involving the challenge virus were performed under the approved institutional biosafety regulations to ensure the safety of personnel, animals, and the surrounding environment.

Throughout the study, birds were housed in separate biosecure isolation rooms equipped with independent ventilation systems to prevent cross-contamination among experimental groups. Standard husbandry practices were maintained, and birds had unrestricted access to commercial feed and clean drinking water. Animal health and welfare were monitored at least twice daily by trained personnel, and birds exhibiting signs of severe distress, pain, or illness beyond the expected effects of the experimental infection would have received immediate veterinary attention and been humanely euthanized according to the approved humane endpoint criteria.

Vaccination, viral challenge, blood collection, and tissue sampling were performed by experienced veterinarians or trained research personnel using standardized procedures designed to minimize handling stress and discomfort. At the completion of the challenge experiment (5 days post-challenge [dpc]), all birds were humanely euthanized by carbon dioxide (CO₂) asphyxiation in accordance with the approved IACUC protocol before postmortem tissue collection. Every effort was made throughout the study to minimize animal suffering, reduce the number of animals used, and adhere to the principles of Replacement, Reduction, and Refinement (3Rs).

Study period and location

The study was conducted from March to September 2021. The animal challenge experiment was performed at the experimental animal facility of the Faculty of Veterinary Science, Chulalongkorn University, Nakhon Pathom Campus, Thailand. Laboratory analyses, including virological and serological assays, were carried out at the Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.

Study design

A randomized controlled challenge study was performed to evaluate the protective efficacy of concomitant hatch-day vaccination with two live-attenuated IBV vaccines against a Thai QX-like IBV strain. Seventy commercial day-old Cobb500 broiler chickens were randomly allocated to vaccinated, positive control, and negative control groups. Birds in the vaccinated group received concomitant ocular vaccination on the day of hatch, whereas birds in the control groups remained unvaccinated. At 21 days of age, vaccinated and positive control birds were challenged with a Thai QX-like IBV strain, while the negative control birds remained unchallenged. Protective efficacy was assessed by evaluating tracheal ciliostasis, viral detection by RT-PCR, serological responses, and body weight.

Viruses and vaccines

Challenge virus: The challenge virus was IBV strain THA80151 (GenBank accession number FJ156075) [11]. The virus was propagated in 9- to 11-day-old SPF embryonated chicken eggs and used at passage 4 in the challenge experiment. Following incubation, allantoic fluids were harvested from embryos exhibiting characteristic IBV lesions and clarified by centrifugation. Virus stocks were aliquoted and stored at −80°C until use. Viral infectivity was determined by endpoint titration in SPF embryonated chicken eggs using the Reed and Muench method and expressed as the median embryo infectious dose (EID₅₀)/mL. Based on the calculated titer, the virus stock was diluted in sterile phosphate-buffered saline (pH 7.2) to obtain a final challenge dose of 10⁴ EID₅₀ per 0.1 mL.

Vaccines: Two commercial live-attenuated vaccines were used in this study: Cevac IBird (strain 1/96; Batch No. 0604D4U2; Ceva Animal Health, Libourne, France) at a dose of 0.03 mL (10³.⁸ EID₅₀/bird) and Cevac Vitabron L (strains H120 + PHY.LMV.42; Batch No. 0603D3SKF; Ceva Animal Health, Libourne, France) at a dose of 0.03 mL (10⁰.³ EID₅₀/bird for IBV and 10³.⁰ EID₅₀/bird for Newcastle disease virus). Vaccines were transported and stored under the manufacturer's recommended cold-chain conditions until reconstitution with sterile diluent immediately before administration. All vaccine doses were administered within 1 h after reconstitution to preserve vaccine viability.

Chickens and experimental design

Seventy commercial day-old Cobb500 broiler chickens, which more closely represent commercial production systems than SPF chickens, were obtained from a commercial hatchery (GFPT Public Company Limited, Samut Prakan, Thailand) and transported to the experimental animal facility at Chulalongkorn University, Nakhon Pathom Campus.

Birds were randomly assigned to three experimental groups and housed separately in accordance with the experimental design (Table 1). The mean maternally derived IBV antibody titer at 1 day of age was 2,565.5 ± 712.55 enzyme-linked immunosorbent assay units (BioChek B.V., Reeuwijk, the Netherlands).

On the day of hatch, birds in Group A received concomitant ocular vaccination with Cevac IBird (strain 1/96) and Cevac Vitabron L (strains H120 + PHY.LMV.42) according to the manufacturer's recommendations. Birds in Groups B and C remained unvaccinated. This concomitant vaccination strategy differs from most previous studies, which evaluated either single vaccine strains or sequential vaccination schedules.

At 21 days of age, birds in Groups A and B were challenged with the Thai QX-like IBV strain by the intraocular and intranasal routes, whereas birds in Group C served as the unchallenged negative control. At 5 dpc, all birds were humanely euthanized using CO₂ asphyxiation in accordance with the approved IACUC protocol. Tracheal tissues were collected for assessment of ciliary activity and RT-PCR analysis.

Vaccination at 1 day of age | Group | Number of broilers | Challenge age ( days ) | Birds for ciliostasis ( 5 dpc ) | Birds for RT - PCR ( 5 dpc )
Cevac IBird (1/96) + Cevac Vitabron L (H120 + PHY.LMV.42) | A | 20 | 21 | 10 | 10
None | B | 20 | 21 | 10 | 10
None | C | 20 | NA | 10 | 10

Housing of the animals

Each experimental group was housed in an independent isolation room within the Livestock Hospital, Faculty of Veterinary Science, Chulalongkorn University, Nakhon Pathom, Thailand. Each room had an independent ventilation system to prevent cross-contamination among treatment groups.

Strict biosecurity procedures were maintained throughout the study. Personnel followed a one-way movement protocol from non-challenged to challenged rooms and changed dedicated protective clothing, gloves, boots, and disposable equipment before entering each room.

Throughout the experiment, birds had unrestricted access to commercial feed (Betagro Public Company Limited, Bangkok, Thailand) and clean drinking water.

Vaccination

Cevac IBird and Cevac Vitabron L were reconstituted in chilled, sterile demineralized water to obtain a single commercial dose per bird. The reconstituted vaccines were maintained at 3-5°C until administration. Vaccines were administered by the ocular route, with each bird receiving two drops (0.03 mL/drop), equivalent to one commercial dose.

IBV challenge

Frozen allantoic fluid containing the Thai QX-like IBV strain was thawed and diluted in chilled, sterile, demineralized water to a final concentration of 10⁴ EID₅₀/0.1 mL. The virus suspension was maintained under refrigerated conditions until use. Each bird received two ocular drops (0.05 mL/drop), providing a total challenge volume of 0.1 mL.

Ciliary activity test

The ciliary activity test was performed as previously described by Cook et al_._ [19] with minor modifications. Each trachea was divided into proximal, middle, and distal regions and sectioned into five rings (two proximal, one middle, and two distal), each measuring <2 mm in thickness. Individual rings were placed into separate wells of a 24-well culture plate containing minimum essential medium/Earle's balanced salt solution medium (Merck KGaA, Darmstadt, Germany).

Ciliary activity was evaluated microscopically by an investigator blinded to treatment allocation. Each ring was scored using a 0-4 scale:

1. 0 = all cilia beating

2. 1 = approximately 75% ciliary activity

3. 2 = approximately 50% ciliary activity

4. 3 = approximately 25% ciliary activity

5. 4 = complete ciliostasis

The maximum ciliostasis score for each trachea was 20 (4 × 5 rings).

Birds were considered protected when the total ciliostasis score was <10.

The ciliostasis protection score (CPS) was calculated as:

\documentclass{article} \usepackage{amsmath} \usepackage{amssymb} \begin{document} \[ CPS=100-\frac{100\times \sum _{}^{}{individual ciliostasis scores}}{n\times 20} \] \end{document}

where n represents the number of birds examined.

The binomial protection score (BPS) was calculated as the percentage of birds exhibiting >50% ciliary activity (ciliostasis scores of 0-2), as described by de Wit et al_._ [20].

IBV detection by RT-PCR

At 5 dpc, tracheal samples were collected from 10 randomly selected birds in each group for RT-PCR detection of IBV.

Samples were homogenized in 10% PBS and centrifuged at 1,800 × g for 10 min. Viral RNA was extracted from the clarified supernatant using the HiYield™ Viral Nucleic Acid Extraction Kit (RBC Bioscience Corp., New Taipei City, Taiwan).

Primers previously described by Sarueng et al_._ [15] were used to differentiate the challenge strain (QX-like THA80151) from the vaccine strains (H120 and 793/B). Primer sequences were:

1. F1547: 5′-TAATGAAACTGGTTCTCAGCC-3′

2. R1691: 5′-GCGGTACTATTTGCTTAATAA-3′

One-step RT-PCR consisted of reverse transcription at 48°C for 45 min, initial denaturation at 94°C for 5 min, followed by 35 amplification cycles comprising denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min, with a final extension at 72°C for 10 min [15].

Amplified products were separated on 1.2% agarose gels stained with ethidium bromide (0.5 μg/mL) and visualized using an ultraviolet transilluminator.

Serological evaluation

Blood samples were collected at 1, 7, 14, 21, and 26 days of age. Serum samples were separated and analyzed for IBV-specific antibodies using a commercial enzyme-linked immunosorbent assay kit (BioChek B.V., Reeuwijk, the Netherlands) to evaluate humoral immune responses following vaccination and challenge.

Statistical analysis

Continuous data were evaluated for normality using the Shapiro-Wilk test and visual inspection of Q-Q plots. Body weight and antibody titers were analyzed using one-way analysis of variance, followed by Duncan's multiple range test for pairwise comparisons. Ciliostasis scores and RT-PCR detection data were analyzed using the Kruskal–Wallis test.

All statistical analyses were performed using IBM SPSS Statistics version 29 (IBM Corp., Armonk, NY, USA). Statistical significance was established at p < 0.05.