Submitted:
08 June 2026
Posted:
10 June 2026
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Abstract
Nicholson et al. (2026) have established the first adult stem cell-derived chicken lung organoid system and validated it for in vitro modelling of avian influenza virus replication, representing a landmark advance in respiratory avian biology. The model is derived exclusively from White Leghorn chickens, a single commercially selected genotype that sits at one extreme of the phenotypic and genomic diversity of Gallus gallus domesticus. Indigenous African chicken breeds, which constitute 70–80% of sub-Saharan poultry production and represent the primary interface between endemic avian influenza viruses and unvaccinated smallholder flocks, are entirely unrepresented. This short communication argues that extension of the organoid platform to indigenous African breeds is scientifically urgent for One Health surveillance, outlines the molecular basis for anticipating meaningful breed-level transcriptomic divergence particularly at the ANP32A locus and proposes three tractable protocol adaptations required for indigenous breed organoid derivation: biosafety characterisation of non-SPF donor animals, variant-aware transcriptomic alignment to account for indigenous breed allelic diversity, and experimental designs that accommodate within-breed genetic heterogeneity.
Keywords:
chicken lung organoids
; indigenous African chickens
; avian influenza
; ANP32A
; breed-specific transcriptomics
; One Health
; sub-Saharan Africa
INTRODUCTION
Nicholson et al. (2026) describe the first chicken lung organoids derived from adult stem cells a landmark in the advancement of avian respiratory biology. The model, derived from three-week-old specific pathogen-free (SPF) White Leghorn chickens, characterized by bulk and single-nuclei RNA sequencing, and validated through successful replication of both low and high pathogenic avian influenza A viruses, fills a genuine gap in the in vitro toolkit for studying respiratory host–pathogen interactions in poultry. The authors themselves note that the successful establishment of cultures from domestic chickens may aid in producing and expanding organoids from other avian species. However, this extension is not merely desirable but scientifically urgent, and the current model carries a structural limitation whose derivation from a single, commercially selected genotype prevents it from addressing the most consequential questions in African avian influenza surveillance.
A Model Built on a Single, Commercially Derived Genotype
The White Leghorn is the product of more than a century of directional selection for egg production under high-biosecurity, temperature-controlled, high-input industrial conditions. It sits at one extreme of the phenotypic and genetic diversity of Gallus gallus domesticus, and its immune gene expression landscape reflects this selection history in ways that are well documented at the transcriptomic level. The Iowa State University Leghorn line has been used for decades as the susceptible comparator in NDV and avian influenza resistance studies, consistently showing greater transcriptomic disruption, higher viral loads, and more severe immunopathology than breeds adapted to lower-input or tropical environments (Deist et al., 2017; Vanamamalai et al., 2024).
This matters for the Nicholson et al. model for a specific molecular reason. The paper correctly identifies ANP32A and ANP32B expression in both organoid and tissue samples as evidence that the model recapitulates the cellular context relevant to influenza A virus replication, and notes that the antiviral restriction factor PLSCR1 was more highly expressed in organoids than in tissues. These are significant findings. However, ANP32A expression level and splicing isoform distribution are not fixed properties of Gallus gallus they are subject to breed-level variation with direct functional consequences for viral replication efficiency. Avian ANP32A carries a characteristic 33-amino-acid insertion between its leucine-rich repeats and carboxy-terminal low-complexity acidic region ,the structural feature that distinguishes it from mammalian ANP32A and enables efficient support of avian influenza polymerase activity (Sugiyama et al., 2019; Long et al., 2019). Whether this insertion is differentially expressed, alternatively spliced, or subject to distinct regulatory controls across genetically divergent chicken populations remains entirely unknown. The Nicholson et al. dataset, restricted to a single commercial genotype, cannot address this question.
Transcriptomic Divergence Between Chicken Breeds Is Detectable at Organoid Resolution
The feasibility of detecting breed-specific gene expression differences in organoid systems was directly demonstrated by Sun et al. (2025), who performed single-cell RNA sequencing on intestinal organoids derived from broiler and layer chicken embryos under identical baseline culture conditions. Layer and broiler organoids showed significant differences in cell-specific transcriptomes, most pronounced in epithelial cells, pointing to divergent selection having acted on gastrointestinal physiology in ways detectable at cellular resolution in vitro. Broiler organoids comprised approximately 77% mesenchymal cells compared to 65% in layer organoids, while layer organoids showed a proportionally higher epithelial cell fraction ,a compositional difference with direct functional implications for pathogen encounter, mucosal immunity, and barrier integrity (Sun et al., 2025).
This finding establishes the foundation for the present argument. If transcriptomic and compositional differences between two commercial lines ,broiler and layer, separated by selective breeding within a relatively constrained genetic bottleneck are robustly detectable in organoid systems, the signals from indigenous African breeds will be substantially more pronounced. The genomic distance between an SPF White Leghorn and a Kenyan or Ethiopian village breed is vastly larger than the distance between industrial broiler and layer lines. Genomic studies of indigenous African breeds have identified strong selection signatures at heat resistance and immune loci including HSF1, TSHR, HIF3A, SLC44A2, and cytokine cluster genes on chromosomes 1 and 5 (Gheyas et al., 2024; Fleming et al., 2017) that predict qualitatively distinct baseline and induced gene expression programmes in the respiratory epithelial and innate immune cell compartments most directly profiled by the Nicholson et al. model.
The Indigenous Breed Gap: Why It Matters for Avian Influenza Surveillance
Indigenous chickens represent 70–80% of poultry production in sub-Saharan Africa and constitute the primary protein and income source for hundreds of millions of smallholder households. They are also the interface at which endemic Newcastle disease, infectious bursal disease, and high pathogenicity avian influenza H5N1 clade 2.3.4.4b viruses encounter the largest unvaccinated poultry population on the continent. The epidemiology and evolutionary trajectory of avian influenza in Africa are actively shaped by the mucosal immunologies and environmental adaptations of these indigenous breeds biological realities that a White Leghorn-derived organoid model cannot represent.
The key question for regional One Health surveillance is not whether a commercial White Leghorn organoid supports viral replication; Nicholson et al. have established that it does. The critical unanswered question is whether indigenous African breeds which have co-existed with diverse pathogen communities under continuous natural selection possess distinct ANP32A isoform distributions, altered receptor expression profiles, or unique co-factor regulations that modulate viral replication kinetics. This question has direct implications for understanding spillover risk, viral adaptation, and the design of breed-appropriate vaccination strategies.
A directly relevant parallel exists in NDV resistance research. The Fayoumi line, adapted to Egyptian arid conditions and long used as an indigenous breed proxy, shows constitutively higher baseline expression of interferon-stimulated genes (ISGs) and mounts a more rapid, coordinated innate immune transcriptomic response to viral challenge than the Leghorn comparator (Deist et al., 2017; Vanamamalai et al., 2024). These differences are transcriptomic before they are virological ,they reflect baseline gene expression architecture, not merely post-infection regulation. Whether indigenous East African breeds possess analogous constitutional differences in respiratory epithelial immune gene expression is precisely the question that breed-specific lung organoids could resolve.
Protocol Adaptations Required for Indigenous Breed Organoid Derivation
Three categories of breed-specific adaptation are required before the Nicholson et al. protocol can be applied to indigenous African chicken breeds, each scientifically tractable with current methods.
First, source tissue logistics. Nicholson et al. used three-week-old SPF White Leghorns from a commercial supplier, ensuring standardized tissue quality and a defined health status. Indigenous breed donors sourced from village populations or conservation flocks carry complex histories of natural pathogen exposure and are not SPF. This necessitates pre-derivation biosafety characterisation of donor animals, systematic serology for endemic pathogens, and antibiotic and antifungal supplementation protocols calibrated to an unmanaged microflora baseline. The growth factor composition of the organoid culture medium developed and validated for White Leghorn tissue may also require optimisation, since growth factor receptor expression profiles and signalling pathway activity vary across breeds and are subject to selection pressure.
Second, the reference transcriptome. Nicholson et al. aligned to the GalGal5.0 assembly. The current reference (GRCg7b) provides improved annotation but remains predominantly derived from commercial line sequences. Indigenous African breeds carry extensive allelic diversity at critical immune loci ,including the highly polymorphic MHC-B region on chromosome 16 that is deeply underrepresented in the reference genome. Variant-aware alignment strategies, such as STAR two-pass mode incorporating population-specific SNP data from published genomic studies on East African breeds (Walugembe et al., 2020; Gheyas et al., 2024), are necessary to minimize reference bias-driven false negatives in differential expression analysis at precisely the immune loci most relevant to the biological questions at hand.
Third, experimental design for within-breed genetic heterogeneity. Unlike inbred commercial lines or SPF stocks, indigenous village chickens are genetically heterogeneous. This diversity is an adaptive asset rather than an experimental flaw ,it is the biological resource the research seeks to characterize ,but it demands expanded biological replication (a minimum of six independent donors) and appropriate statistical modelling, donor identity treated as a random effect, and factorial breed-by-condition interaction terms deployed within DESeq2 or edgeR to separate true breed-level transcriptomic signals from within-breed background variation.
Conclusion
The Nicholson et al. paper establishes that chicken lung organoids can be derived, maintained, and infected in vitro while faithfully recapitulating the cellular heterogeneity and gene expression landscape of the avian respiratory tract. This is the necessary first step. The field must now take the second ,extending the platform to the breeds that matter most for the One Health questions that motivated its development.
Two upstream challenges remain unresolved. First, no validated iPSC reprogramming protocol exists for indigenous African chicken breeds, and the conventional mammalian OSKM cocktail is frequently insufficient to drive full reprogramming in avian somatic cells (Zahoor et al., 2024); breed-specific iPSC lines must therefore be developed in parallel with organoid work.
Second, transcriptomic data generated from indigenous genetic resources carries Digital Sequence Information (DSI) governance obligations under the CBD Nagoya Protocol and the COP15 multilateral benefit-sharing mechanism, making institutional anchoring at centres with established in-country data infrastructure both ethically and legally necessary.
Research programmes with direct access to indigenous breed populations including ILRI in Nairobi, which maintains conservation flocks of multiple East African breeds, operates BSL-2 certified containment facilities, and has established genomic characterisation and DSI governance infrastructure are uniquely positioned to lead this work. The White Leghorn organoid is an invaluable tool. But the chickens most at risk from avian influenza and most critical to the food security of populations least able to absorb production losses are not White Leghorns. The platform Nicholson et al. have built should be extended to represent them.
Funding
No funding was received for this work.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
No new data were created or analyzed in this study. Data sharing is not applicable to this article.
Generative AI Disclosure
Claude (Anthropic, version Sonnet 4.6) was used to assist with the manuscript formatting only. All scientific content, arguments and conclusions are solely author’s own.
Conflicts of Interest
The author declares no conflict of interest.
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