DISCUSSION

Overall protective efficacy of concomitant vaccination

To our knowledge, this is the first study to demonstrate protective efficacy against a Thai QX-like IBV strain following concomitant hatch-day administration of two commercially available live-attenuated IBV vaccines in commercial broilers. This vaccination strategy addresses the practical limitations associated with sequential vaccination programs in modern hatcheries while providing early protection during a critical period of susceptibility.

The present study evaluated the protective efficacy of concomitant administration of Cevac IBird (strain 1/96) and Cevac Vitabron L (strains H120 + PHY.LMV.42) in broilers challenged with a Thai QX-like IBV strain at 21 days of age. The results demonstrated that this vaccination strategy provided measurable protection against heterologous challenge, particularly by preserving tracheal ciliary function, enhancing humoral immune responses, and partially reducing viral detection.

Tracheal protection and preservation of ciliary function

One of the principal findings of this study was that CPS was significantly higher in vaccinated birds than in the positive control group. Vaccinated birds achieved a CPS of 58.5%, whereas the unvaccinated challenged birds exhibited complete ciliostasis (CPS = 0%), indicating severe tracheal damage. The mucociliary apparatus constitutes one of the primary defense mechanisms of the avian respiratory tract against inhaled pathogens, including IBV [21, 22]. Preservation of ciliary activity facilitates mucociliary clearance, thereby limiting viral persistence and reducing tissue damage.

The markedly higher CPS observed in vaccinated birds indicates that concomitant vaccination effectively preserved tracheal integrity following heterologous challenge. In contrast, the complete loss of ciliary activity in unvaccinated challenged birds highlights the susceptibility of unprotected birds to severe respiratory injury following infection with Thai QX-like IBV.

Similarly, vaccinated birds achieved a BPS of 100%, whereas the positive control group showed no protected birds. These findings indicate that concomitant vaccination maintained functional tracheal cilia in a substantially greater proportion of birds, which may reduce disease severity and limit within-flock virus transmission [23].

Viral detection following challenge

RT-PCR analysis demonstrated a lower viral detection rate in the vaccinated group than in the positive control group, although the difference was not statistically significant. These findings suggest that concomitant vaccination did not completely prevent infection but partially suppressed viral replication following heterologous challenge.

Previous studies have similarly demonstrated that heterologous vaccination strategies may reduce viral replication and disease severity without completely preventing infection [9, 18]. Partial suppression of viral replication may nevertheless reduce environmental virus contamination, improve flock health, and lessen production losses under commercial conditions. However, because RT-PCR was used for qualitative detection, the present findings should be interpreted as reduced viral detection rather than a confirmed reduction in viral load or shedding.

Humoral immune response

The serological findings further supported the immunogenic effect of concomitant vaccination. Vaccinated birds developed significantly higher antibody titers than the negative control group following challenge, indicating effective stimulation of humoral immunity. Although maternally derived antibody titers declined with age across all groups, vaccinated birds maintained numerically higher antibody responses than control birds at 14 and 21 days of age.

The significantly higher antibody titers in vaccinated birds at 5 dpc suggest that vaccination primed the immune system for a rapid anamnestic response following challenge. However, the positive control group also developed significantly higher titers than the negative control group at this time point, indicating that challenge infection contributed to the observed serological response. Therefore, antibody titers alone cannot fully distinguish vaccine-induced protection from the immune response to infection.

Growth performance following challenge

No significant differences in body weight were detected among the groups before challenge, indicating that hatch-day vaccination did not adversely affect early growth performance. However, at 5 dpc, birds in both the vaccinated and positive control groups had significantly lower body weights than those in the negative control group.

These findings indicate that the Thai QX-like IBV challenge adversely affected short-term growth performance regardless of vaccination status. The absence of a significant difference between the vaccinated and positive control groups suggests that vaccination did not prevent challenge-associated growth depression during the short observation period. Therefore, although vaccination preserved tracheal ciliary activity and enhanced antibody responses, its benefit was not reflected in improved body weight by 5 dpc.

Comparison with previous studies and possible protective mechanisms

The emergence of genetically diverse IBV variants continues to complicate disease control. The extensive genetic variability of IBV, driven by mutation and recombination, may reduce vaccine-induced cross-protection and necessitates continuous evaluation of vaccination programs.

The combination of Cevac IBird and Cevac Vitabron L represents a practical application of the protectotype concept, in which antigenically distinct vaccine strains are combined to broaden heterologous protection [15]. The present findings are consistent with those reported by Sarueng et al_._ [15] and Thomrongsuwannakij et al_._ [24], who demonstrated improved heterologous protection following administration of two IBV vaccine strains. Whereas those studies evaluated vaccines administered at different ages, the present investigation demonstrates that concomitant hatch-day administration can also provide measurable protection in commercial broilers.

The previous studies were conducted in commercial broilers and SPF chickens, respectively, suggesting that the benefits of dual-strain vaccination may extend across different experimental and production settings. Simultaneous priming with Massachusetts-type H120 and 1/96 (793/B) strains may broaden heterologous immunity by stimulating responses to a wider range of IBV antigens, consistent with the protectotype concept described by Cook et al_._ [19] and further supported by de Wit et al_._ [20].

Exposure to antigenically distinct vaccine strains may increase immune recognition of conserved IBV epitopes and promote cross-reactive CD4⁺ and CD8⁺ T-cell responses against relatively conserved viral proteins, including the nucleocapsid and membrane proteins [25, 26]. Replication of live vaccine viruses in the respiratory tract may also induce local mucosal immunity, including secretory immunoglobulin A production, which can interfere with viral attachment and replication at mucosal surfaces [27–29]. In addition, local memory T-cell responses generated after vaccination may facilitate more rapid viral clearance following heterologous challenge [29, 30]. Collectively, these mechanisms may complement systemic humoral immunity and contribute to the protection observed after combined Massachusetts and 793/B vaccination programs [19, 21, 31].

Vaccine strain selection and IBV evolution

Recent studies evaluating live-attenuated IBV vaccines further emphasize the importance of vaccine strain selection in optimizing protection against emerging variants. Kilany et al_._ [32] demonstrated that a live-attenuated GI-23 vaccine was safe, did not revert to virulence, and provided effective protection against homologous challenge. Although the genotype-matched strategy evaluated by Kilany et al_._ [32] differs from the protectotype-based strategy used in the present study, both approaches highlight the importance of antigenic compatibility between vaccine strains and circulating field viruses.

Elfeil et al_._ [33] reported differences in protection among commercially available vaccination programs against QX challenge, indicating that vaccine strain selection and vaccination strategy can substantially influence heterologous protection. Similarly, Shosha et al_._ [34] demonstrated extensive genetic diversity and recombination among circulating IBV strains, particularly within the GI-19 and GI-23 lineages. These findings reinforce the need for continuous molecular surveillance and periodic reassessment of vaccination programs.

Emerging evidence also indicates that recombination involving vaccine-related lineages may contribute to the evolution of novel IBV variants. Huang et al_._ [35] identified a naturally occurring recombinant virus containing genomic material derived from a 4/91-like vaccine lineage, which showed greater virulence than its parental field strain. These observations emphasize the need to balance the benefits of live-attenuated vaccines against the ongoing monitoring of vaccine-derived and field virus evolution.

Concurrent IBV and Newcastle disease virus (NDV) vaccination

In the present study, concomitant administration of live IBV and NDV vaccine components at 1 day of age was associated with improved protection against subsequent challenge with a Thai QX-like IBV strain. These findings suggest that hatchery vaccination may provide a practical foundation for early protection during the period when broilers are highly susceptible to respiratory infection.

Nevertheless, potential interference between live IBV and NDV vaccines administered concurrently should be considered. The magnitude of vaccine interaction may depend on vaccine strain, route of administration, maternally derived antibody levels, and replication kinetics. Competition for respiratory epithelial cells and the induction of innate antiviral responses could potentially alter the replication of one or both vaccine viruses.

However, concurrent administration does not necessarily compromise protective immunity. Ball et al_._ [36] demonstrated that day-old broilers receiving NDV VG/GA-Avinew concurrently with IBV H120 and CR88 developed protective NDV antibody responses while maintaining high levels of ciliary protection against both M41 and QX challenge strains. Collectively, these findings suggest that concurrent administration of appropriately selected IBV and NDV vaccine strains can provide satisfactory protection, although the interactions between vaccine viruses require further evaluation under commercial conditions.

Practical implications for commercial poultry production

The present findings have important implications for commercial poultry production in Thailand, where QX-like IBV strains continue to circulate. Because IBV infection commonly occurs during the first weeks of life, establishing early protective immunity is critical for reducing respiratory disease, secondary bacterial infections, impaired growth, and production losses.

Hatchery-based concomitant vaccination offers several operational advantages over farm-level administration, including standardized vaccine delivery, improved dose uniformity, reduced labor requirements, lower handling stress, and a reduced risk of administration errors. These advantages are particularly relevant to large integrated poultry operations in which large numbers of chicks must be vaccinated efficiently.

The protective outcomes observed in this study support the potential inclusion of the evaluated vaccine combination in an integrated IBV control program. However, vaccine selection should be guided by the genetic and antigenic characteristics of locally circulating field strains. Continuous surveillance of IBV genotypes in Thailand remains essential to determine whether existing vaccination programs provide adequate cross-protection against emerging variants.

Successful field implementation also depends on hatchery management practices, including vaccine storage and handling, reconstitution procedures, equipment calibration, dose delivery, chick quality, and biosecurity. Routine monitoring of vaccine uptake, serological responses, respiratory health, and flock performance should therefore be incorporated into commercial vaccination programs to optimize protection and minimize the economic impact of IB.

Study strengths, limitations, and future perspectives

A major strength of this study was the use of commercial broilers with maternally derived antibodies, which provides greater practical relevance than studies conducted exclusively in SPF chickens. The concomitant hatch-day vaccination schedule also closely reflects the operational requirements of modern commercial hatcheries. In addition, protection was evaluated using complementary outcomes, including body weight, ciliostasis, CPS, BPS, RT-PCR detection, and serological responses.

Several limitations should nevertheless be acknowledged. First, the relatively small sample size may limit the generalizability of the findings to commercial field conditions. Second, the short experimental period restricted assessment to the early post-challenge phase and did not permit evaluation of long-term protection, prolonged virus persistence, later clinical progression, or sustained production performance. Third, only one Thai QX-like challenge strain was evaluated; therefore, the findings may not represent protection against other circulating genotypes or emerging recombinant variants. Fourth, RT-PCR detection was qualitative, and quantitative viral load and shedding kinetics were not determined. Finally, production performance was assessed mainly through body weight, without evaluating feed intake, feed conversion ratio, carcass characteristics, or economic outcomes.

Future studies should include larger controlled and commercial field trials, quantitative assessment of viral load and shedding duration, and challenges with multiple circulating Thai IBV genotypes. Longer follow-up periods are also required to determine the durability of protection and its effects on production performance. Comparative studies of concomitant, sequential, genotype-matched, and protectotype-based vaccination programs would further clarify the most effective strategies for controlling QX-like IBV under commercial conditions.

Although this study was performed under controlled experimental conditions, its findings may also be relevant to other regions where QX-like or related IBV variants circulate, including parts of the Middle East, Europe, and Latin America. Field validation will be essential to determine whether the observed tracheal protection and partial reduction in viral detection translate into meaningful improvements in flock health and economic performance.