https://orcid.org/0009-0007-3866-7520
Figures
Abstract
Spotty Liver Disease (SLD) is an acute bacterial infection of layer chickens in production, caused by Campylobacter hepaticus, and occurs most frequently in barn-housed and free-range systems. The disease is characterized by a sharp decline in egg production and increased mortality. The hallmark pathological feature is 1–2 mm white to grey necrotic foci distributed across the liver surface. Despite its growing economic impact on commercial poultry, the molecular mechanisms underlying host responses to C. hepaticus infection remain poorly understood. To address this gap, we performed a comprehensive transcriptome analysis of liver tissue from chickens naturally infected with SLD compared to uninfected controls. Illumina NovaSeq-based RNA sequencing was performed on liver tissues collected from three SLD-infected and three uninfected control chickens, with three biological samples for each sample type (n = 6), yielding 9,277 differentially expressed genes (DEGs), of which 3,063 were upregulated and 6,214 were downregulated. Functional pathway enrichment analysis revealed significant alterations in immune and metabolic processes associated with SLD pathophysiology. Infected chickens exhibited significant activation of immune response pathways, particularly cytokine-cytokine receptor interactions involving interleukins IL-22, IL-21, and IL-6, along with enhanced cell signaling, and cell adhesion. Among the individual genes, C1QTNF1 and the adhesion molecule gene ADGRD1 were notably overexpressed, indicating enhanced inflammatory activity. In contrast, core hepatic metabolic functions were profoundly reduced (adjusted p < 0.05) as evidenced by downregulation of oxidative phosphorylation, fatty acid metabolism, iron ion binding, and heme binding pathways. A marked increase in serum amyloid A (SAA) gene expression further indicated robust acute-phase responses and compromised liver function during infection. Together, these findings demonstrate a complex interplay between inflammatory activation and metabolic dysregulation during SLD. The strong upregulation of acute-phase proteins and pro-inflammatory cytokines demonstrates the host’s vigorous attempt to combat bacterial infection, whereas the concurrent suppression of essential metabolic pathways reflects the pathological consequences of SLD. This study provides a transcriptomic characterization of host responses to C. hepaticus infection, offering insights into SLD pathogenesis and potential avenues for targeted intervention.
Citation: Bommineni V, Edison LK, Gottapu C, Butcher GD, Kariyawasam S (2026) Hepatic transcriptomic landscape of chicken reveals host responses to spotty liver disease. PLoS One 21(9): e0346466. https://doi.org/10.1371/journal.pone.0346466
Editor: Birendra Mishra, University of Hawai’i at Manoa, UNITED STATES OF AMERICA
Received: March 18, 2026; Accepted: August 31, 2026; Published: September 16, 2026
Copyright: © 2026 Bommineni et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files. The transcriptomic profile data (raw and processed) described in this study were deposited in the Gene Expression Omnibus (GEO) database in NCBI under accession number GSE325026. (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE325026).
Funding: This research was funded by Agriculture and Food Research Initiative Competitive Grant no. 2023-67015-40780 from the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) and the USDA NIFA Animal Health and Disease Grant no. FLA-VME-005986. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Spotty Liver Disease (SLD) is an emerging bacterial disease of layer chickens in production that has become a major economic concern for the global egg industry. The global expansion of cage-free and free-range production systems has increased the burden of SLD with more than 350 million laying hens across major egg-producing countries estimated to be at risk. Seroprevalence surveys conducted across 12 free-range layer flocks detected anti-Campylobacter hepaticus antibodies in an average of 20% of birds, increasing to 27% in flocks with a previous history of SLD compared with 11% in flocks with no prior history. Furthermore, SLD has been associated with substantial economic losses, with farm-level costs ranging from USD 0.32 to 2.71 per bird and an estimated annual industry-wide impact of up to USD 489 million. Despite increasing research efforts and recent progress in vaccine development, SLD continues to pose a major challenge to the commercial layer industry [1–3]. While Campylobacter hepaticus remains the primary and better-characterized causative agent of SLD [4,5], Campylobacter bilis was more recently isolated from bile samples of clinical SLD-affected birds and confirmed as a second causative species following fulfilment of Koch’s postulates in experimental infection trials, where oral challenge reproduced characteristic hepatic lesions within six days. The pathogenic mechanisms of C. bilis remain poorly understood and require further investigation to elucidate its exact role in SLD pathogenesis and associated liver pathology in affected layer hens [6].
Outbreaks have been reported in the United States, United Kingdom, New Zealand, Jordan, and across Europe [7–9]. Outbreaks typically occur during the peak laying period, between 22 and 35 weeks of age, mostly during warm conditions [10] and are associated with flock mortalities of up to 10% and production losses of 10–35% [11] resulting reduction in egg production and survivability makes SLD one of the most important health challenges in layer operations.
SLD is characterized by a variable clinical presentation, ranging from subclinical infection, in which birds show no apparent clinical signs but may exhibit characteristic hepatic lesions at postmortem examination, to acute disease manifested by reduced egg production, lethargy, pyrexia, icterus, or sudden death [11]. This disease is more prevalent in layers, which are more vulnerable than broilers or non-laying chickens, perhaps as a result of physiological or hormonal factors and increased circulating iron levels, however, the precise mechanisms underlying this increased susceptibility remain unclear and require further investigation [10,11]. The characteristic postmortem lesion of SLD is in the liver, where 1–2 mm grey-white lesions are distributed across the surface of the liver parenchyma [12]. Histologically, these foci represent multifocal hepatocellular necrosis, often accompanied by fibrin deposition and infiltration of heterophils and other inflammatory cells [13]. Notably, lesions are not restricted to specific hepatic structures but appear randomly distributed, suggesting a systemic process. Although such damage is expected to affect liver function, it is unclear how exactly it contributes to mortality and loss of production. These unresolved questions highlight the need for molecular approaches to uncover the host–pathogen interactions driving hepatic pathology [14]. In chickens, the liver is the major site for de novo lipid synthesis and yolk precursor production. The continuous assembly of egg yolk relies entirely on the liver’s ability to synthesize vitellogenin and specialized very-low-density lipoproteins (VLDLy) [15] Consequently, hepatic health directly dictates chicken egg production, as the liver is essential for nutrient metabolism, yolk precursor synthesis, and hormonal regulation of laying. Pathologies like SLD, caused by C. hepaticus, damage liver tissue and are consistently associated with marked reductions in egg output and increased flock mortality in affected layer hens [16]. Also, the liver serves as a unique immune organ that contributes to pathogen defense. It eliminates pathogens through innate immune cells such as Kupffer cells, dendritic cells, and natural killer cells, while adaptive immunity is supported by resident T and B lymphocytes [16,17]. Previous work on immune responses of SLD has demonstrated that infected hens develop protective antibodies and can resist reinfection, with seroprevalence studies revealing variable antibody responses (2–64%) across affected flocks [1]. Limited studies suggest that C. hepaticus infection triggers proinflammatory responses, including elevated levels of IL-1β, IL-6, and IL-8 [18,19]. However, the hepatic immune response, the key site of pathology in SLD, remains poorly characterized.
To bridge this knowledge gap, our hypothesis is that the progression of Spotty Liver Disease (SLD) involves differential expression of host genes associated with inflammation and tissue injury and metabolic pathways, thereby contributing to liver pathology. We conducted high-throughput RNA sequencing to analyze the liver transcriptomes of hens naturally infected with SLD and exhibiting lesions characteristic of the disease. We further expected that transcriptomic analysis would identify differentially expressed genes and biological pathways associated with upregulation of immune and inflammatory genes, tissue remodeling, and downregulation of metabolic dysfunction in the livers of naturally infected hens. These findings contribute to a deeper understanding of liver-specific host–pathogen interactions and may highlight targets for improved diagnostics, prevention, and control strategies.
2. Materials and methods
2.1. Sample collection and study design
Infected livers were obtained from 31-week-old White Leghorn commercial layer chickens (n = 3) during an active SLD outbreak at a commercial egg production facility that was experiencing elevated mortality and decreased egg production and was free from other diseases. Postmortem examination of deceased birds revealed characteristic SLD lesions, including multiple white-to-tan foci distributed across the liver surface. The presence of C. hepaticus was confirmed through microbiological isolation and PCR-based identification of affected tissues [20]. PCR assays also excluded the presence of C. billis [6]. Negative control liver samples (n = 3) were collected from age-matched laying hens (31-week-old) sourced from a SLD-free commercial facility with no history of C. hepaticus infection and exhibited no gross liver lesions at necropsy. All liver samples were collected during necropsy procedures, immediately stabilized in RNAlater (Thermo Fisher Scientific, Lenexa, KS, USA) solution, transported on ice, and stored at −80°C until RNA extraction. For transcriptomic analysis, three independent biological replicates from each infected and control liver were selected for downstream processing comprising six biological samples (n = 6) in total. Three independent biological replicates per group were used, consistent with RNA-seq experimental design recommendations that consider three biological replicates as the minimum acceptable number for differential gene expression studies. All samples were obtained as part of routine disease surveillance activities conducted under veterinary supervision.
2.2. RNA extraction and rRNA depletion
Total RNA was extracted from liver tissue samples using the RiboPure RNA Purification Kit (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol to ensure optimal yield and purity. Ribosomal RNA (rRNA) was depleted from the total RNA preparations using the RiboMinus Eukaryote Kit v2 (Invitrogen, Waltham, MA, USA) and the enriched messenger RNA (mRNA) fraction was concentrated using the RiboMinus Concentration Module (Invitrogen, Waltham, MA, USA) to achieve the optimal input concentration for high-throughput sequencing library preparation. RNA quality and quantity were determined at each step using a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Wilmington, DE, USA) to ensure that all samples required standards for downstream transcriptomic analysis.
2.3. RNA sequencing library construction and transcriptomic analysis
Purified chicken liver mRNA (100 ng per sample) was processed for sequencing library construction using the TruSeq Stranded mRNA Library Prep Kit (Illumina InC., San Diego, CA, USA). The enriched mRNA was enzymatically fragmented and used for double-stranded cDNA synthesis. The resulting cDNA fragments were subjected to 3′ end adenylation and ligated with TruSeq RNA Combinatorial Dual Index Adapters (Illumina InC., San Diego, CA, USA), followed by PCR enrichment to amplify the indexed libraries. The prepared cDNA libraries were pooled, distributed across two flow-cell lanes, and sequenced on the Illumina NovaSeq X Series platform (Illumina InC., San Diego, CA, USA). Raw sequencing data were subjected to quality control processing using CLC Genomics Workbench v.24.0.04 (Qiagen, Redwood City, CA, USA), including adapter trimming and removal of poor-quality reads (Phred score < 30). Quality-filtered reads were aligned to the White Leghorn layer chicken reference genome (NCBI Ref Seq Assembly GCF_016700215.2 & Gen Bank Assembly GCA_016700215.2) for transcript quantification. Differential gene expression analysis between SLD-affected and control liver samples was performed using CLC Genomics Workbench v.24.0.04 to identify differentially expressed genes (DEGs). Statistical significance thresholds were established at p-value < 0.01, false discovery rate (FDR) < 0.05, and fold change (FC) > 2 [21,22]. Each experimental group consisted of three independent biological replicates to ensure statistical robustness.
2.4. Quantitative real-time PCR verification of RNA-seq data
Total RNA from chicken liver samples was isolated using the RiboPure RNA Purification Kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. A total of 100 ng of RNA from liver control and infected liver samples was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). To validate the expression trends observed in the RNA-seq dataset, selected chicken genes were examined by reverse transcription quantitative PCR (RT-qPCR) with gene-specific primers listed in S1 File. Reactions were prepared using SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and run on a QuantStudio™ 5 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The thermal cycling program included an initial denaturation at 95 °C for 3 minutes, followed by 40 cycles at 95 °C for 15 seconds and 60 °C for 30 seconds. Transcript levels were normalized against the housekeeping gene β-actin, which served as the internal control. The results of RT-qPCR presented as means ± standard deviation (SD). Statistical significance was evaluated using unpaired two-tailed Student’s t-test in GraphPad Prism version 10.0.1, with p < 0.05 considered statistically significant.
3. Results
3.1. Transcriptomic profiling reveals extensive gene expression changes
Quality trimmed, high-throughput RNA sequencing reads were mapped to the White Leghorn chicken reference genome (NCBI RefSeq Assembly, GCF_016700215.2) with an average global mapping rate of 70.7% (range: 65.0–80.5%). After quality control and normalization, 25,462 genes were retained for differential gene expression analysis. The complete, unfiltered differential gene expression dataset is provided in detail in S2 File. The analysis identified 9,277 significantly DEGs in the liver samples, comprising 3,063 upregulated and 6,214 downregulated genes. The volcano plot illustrates the transcriptomic changes contributing to SLD pathogenesis, with significant DEGs showing fold changes in both directions (Fig 1A). Expression density plots showed apparent transcriptional differences between control and SLD-affected samples (Fig 1B), whereas a heatmap analysis demonstrated clear clustering of genes and expression pattern differences between infected and control livers (Fig 1C).
(A) Volcano plot depicting the differential transcriptomic profile of C. hepaticus in infected chicken liver. In the volcano plot, the x-axis represents log2 fold change and the y-axis represents –log10 FDR p-value. Each dot represents a gene, with red indicating significantly upregulated genes and blue indicating significantly downregulated genes. (B) Density plot showing the distribution of normalized gene expression values in infected and control liver samples. (C) Hierarchical clustering heatmap of differentially expressed genes (DEGs) in infected and control chicken liver samples.
3.2. Gene ontology (GO) enrichment analysis
GO enrichment analysis was performed using g:Profiler. GO terms were classified into three standard categories: Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). For upregulated genes, g:Profiler identified 291 significantly enriched GO terms (49 MF, 196 BP, and 46 CC; provided in detail in S3 File as upregulated GO terms), whereas downregulated genes yielded 172 significant GO terms (33 MF, 89 BP, and 50 CC; provided in detail in S4 File as downregulated GO terms). To simplify interpretation, the top 30 GO terms in the downregulated and upregulated categories are presented in (Fig 2A and 2B), respectively. Statistical significance was determined using the Benjamini–Hochberg false discovery rate (FDR) with a threshold of p < 0.05. Upregulated GO pathways were predominantly enriched for processes related to cell surface signaling receptor activity, cell migration, cell adhesion, calcium ion binding, and developmental processes. In contrast, downregulated GO pathways were significantly associated with small molecule metabolic process, fatty acid metabolic process, Iron ion binding, heme binding, and oxidoreductase activity, consistent with broad suppression of core hepatic metabolic functions in hens with SLD.
(A) downregulated genes and (B) upregulated genes. Bar plot showing the significantly enriched GO terms categorized under BP, MF, and CC. The x-axis represents the number of upregulated genes associated with each GO term (gene count), while the y-axis lists the GO terms. The color of each bar indicates the GO category.
3.3. Kyoto encyclopedia of genes and genomes (KEGG) analysis
KEGG pathway enrichment analysis (g:Profiler) identified nine and 20 significantly enriched pathways among the upregulated and downregulated DEGs, respectively; provided in detail in S3 File and S4 File as upregulated and downregulated KEGG terms. To highlight the most biologically relevant processes, the top pathways are shown in (Fig 3A and 3B). Upregulated pathways were dominated by cytokine–cytokine receptor interaction, cell adhesion molecules, extracellular matrix (ECM)–receptor interaction, and focal adhesion, consistent with immune activation and tissue remodeling. In contrast, downregulated pathways were primarily associated with metabolic pathways, oxidative phosphorylation, carbon metabolism, and fatty acid degradation, reflecting marked suppression of mitochondrial and core metabolic functions in SLD-affected hens.
(A) Upregulated genes and (B) downregulated genes. The dot plot illustrates the top significantly enriched KEGG pathways derived from the differentially expressed genes. The x-axis represents the number of genes involved in each pathway, and the y-axis lists the pathway names. The color gradient corresponds to the − log10(p-value), where darker red indicates higher statistical significance, and the dot size reflects the number of genes contributing to each pathway.
3.4. Patterns of highly expressed gene modules
Immune and inflammatory pathways.
Elevated expressions were observed in immune mediator genes such as IL2RB, IL21, IL22, IL8L2, IL-6, IL17REL, IL17RE, IL18BP and the chemokine gene CXCL13, accompanied by up upregulation of multiple immunoglobulin variable region genes (LOC121112463, LOC107049423, LOC121108126, LOC124418263, LOC121108139, LOC121112462, LOC121112468, LOC112531091, LOC124418264, LOC121108133, LOC124418261, LOC121112461, LOC124418262, LOC121112469, LOC121111827, LOC112531092) suggesting B-cell activation. Increased expression was also noted in immune receptor and regulator genes, including IGSF6, FCMAR, LRRC70, and ZNF467. The LYGL (lysozyme G-like) was also upregulated, indicating the activation of innate antibacterial defense mechanisms. Additionally, the avian beta-defensin (AVBD1) and CATH2 genes were upregulated, indicating the activation of antimicrobial peptide defenses against bacterial infection. Together, these findings indicate robust innate inflammatory and humoral immune responses in SLD-affected livers, further supported by the marked upregulation of the serum amyloid A (SAA) gene. In contrast, transcripts associated with NK and T cell activation (e.g., LOC121106526 and loci annotated to immune response-regulating pathways LOC121107862, LOC112531339, LOC107050230) were downregulated (Fig 4).
Infected1, Infected 2, and Infected 3 represent the three biological replicates of the liver-infected group; and Control1, Control 2, and Control 3 represent the three biological replicates of control groups.
Structural integrity.
Genes related to the ECM and cytoskeletal structures were broadly suppressed, including collagen-associated genes (e.g., LOXL4, collagen alpha-1(I)-like gene), intermediate filaments (KRT75), junctophilin-2 like, outer dense fiber protein 3–like, and coiled-coil domain proteins, consistent with matrix degradation and cytoskeletal disruption. In contrast, TUBB2A (tubulin beta 2A class IIa), which is essential for microtubule-based cell migration, was upregulated. SKA1 (spindle and kinetochore-associated complex subunit 1), which regulates chromosome segregation and mitotic cell cycle progression, was also upregulated. Additionally, PERPA (periplakin A), a cell-cell adhesion protein, was upregulated, suggesting the activation of cellular adhesion mechanisms during infection.
Metabolic dysfunction.
Core hepatic pathways were markedly downregulated, including those involved in carbohydrate metabolism (FBP2, HKDC1, ALDOB, PDHA2, GLUL), lipid metabolism (APOB, APOA2, PLIN1), amino acid and urea cycle processes (OTC, PYCR1), detoxification (CYP2C18, CYP2AC1), and mitochondrial/energy homeostasis and transport (ACAA1, SLC30A2), collectively indicating metabolic dysfunction. Several genes involved in iron homeostasis, heme biosynthesis, and iron-dependent metabolic processes (FTH1, FECH, MELTF, ISCU, ACO2, and RRM2) were also downregulated.
Cell signaling and ion transport.
Signaling receptor genes ACKR4 (chemokine scavenger) and ADGRD1 (adhesion GPCR) were upregulated, along with ion/transport regulator genes (ANO9, GABRA5, CAP2), suggesting altered chemokine gradients, G protein-coupled receptor (GPCR) mediated signaling, and ionic homeostasis in the infected livers. KCNJ13, which mediates potassium ion transmembrane transport, was upregulated. TRPV4 (transient receptor potential cation channel subfamily V member 4), a calcium channel that regulates the production of pro-inflammatory chemokines, was upregulated in SLD-affected livers.
DNA repair and transport.
Genes associated with genome maintenance and transport functions were downregulated, including RAD51C (homologous recombination), SLC18A1 (serotonin uptake), and SLC28A2 (nucleoside transport), indicating compromised cellular integrity in the terminal SLD.
Apoptotic regulation.
Genes encoding key regulators of apoptosis, including ITPRIP (extrinsic apoptotic signaling via death domain receptors), PLEKHO2 (macrophage apoptotic processes), and C1QTNF1, were significantly upregulated, consistent with the activation of programmed cell death pathways during hepatic damage.
3.5. Confirmation of RNA-seq results with RT-qPCR
To validate the transcriptional profiles identified by RNA-seq, four representative genes (IL-22, IL-6, RAD51C, and FBP2) were analyzed using RT-qPCR. As shown in Fig 5, the expression trends observed by RT-qPCR were consistent with the RNA-seq data in liver samples, with both methods showing similar patterns of upregulation or downregulation. These results confirm the reliability of the RNA-seq expression profiles.
Relative expression levels of four representative chicken genes, IL-6 (A), IL-22 (B), RAD51C (C), and FBP2 (D) in liver samples were compared between RNA-seq (blue bars) and RT-qPCR (red bars). Statistical significance was determined using an unpaired two-tailed Student’s t-test; p < 0.05 was considered significant. Data are presented as means ± SD.
4. Discussion
Transcriptome analysis of chicken liver affected by SLD revealed a complex host-pathogen relationship marked by significant immunological dysregulation, metabolic disruption, and cellular failure. These findings shed light on the molecular mechanisms underlying the severe pathology of SLD, distinguishing it from other Campylobacter infections that are often asymptomatic in chickens.
Previous poultry transcriptomic studies have demonstrated that Salmonella Enteritidis infection, downregulated genes were associated with metabolic pathways, inflammation, and mitochondrial apoptosis [23], while recent APEC liver transcriptome studies reported enrichment of cytokine–cytokine receptor interaction, Toll-like receptor, MAPK, PPAR signaling, and lipid metabolism-related pathways [17]. Campylobacter transcriptomic studies in chicken intestinal tissues have also reported differential expression of genes involved in cytokine signaling [24]. In the recent SLD biomarker study upregulation of inflammation genes together with downregulation of yolk precursor genes [25], whereas our pathway enrichment analysis further demonstrated coordinated suppression of oxidative phosphorylation, fatty-acid metabolism, and heme/iron-related pathways. The pathway enrichment analysis revealed robust activation of immune-related processes, cytokine-mediated signaling, intestinal immune network for IgA production, MAPK signaling pathway, and cell surface receptor signaling pathway among the most significantly enriched pathways associated with upregulated genes. Upregulation of key cytokine genes, including IL2RB, IL-21, IL-22, IL8L2, IL-6, IL17REL, and IL17RE, reflects activation of distinct immune cell populations and a coordinated response to C. hepaticus invasion. IL-22, a member of the IL-10 cytokine family that directly targets hepatocytes to stimulate antimicrobial peptide production and pro-inflammatory cytokine release [26] was markedly upregulated in SLD-affected livers [27]. The IL-6 gene was also upregulated in infected liver tissue. Previous studies have consistently identified IL-6 as a key inflammatory mediator in both C. jejuni and C. hepaticus infections, where it serves as an immediate marker for inflammation alongside other proinflammatory cytokines marking the onset of inflammation [18]. Acute-phase proteins (APPs), largely synthesized by hepatocytes in response to pro-inflammatory cytokines, are released after infection or inflammation. SAA is the major and most sensitive APP in chickens, and was upregulated in SLD-affected liver tissues, confirming a severe hepatic inflammatory response and extensive hepatocyte activation during C. hepaticus infection [28]. IL-21 plays a dual immunomodulatory role by enhancing T cell activation while simultaneously blocking dendritic cell maturation, which may contribute to the dysregulated immune response observed in terminal SLD. Thus, the observed immune activation likely reflects an active host attempt to control infection, whereas continued bacterial persistence may indicate that this response is insufficiently coordinated. The concurrent upregulation of the IL-21 gene alongside its ability to inhibit dendritic cell maturation may paradoxically impair antigen presentation and contribute to the unsuccessful pathogen clearance observed in fatal SLD cases [29]. Additionally, the strong upregulation of CXCL13 in SLD-affected liver tissues indicates activation of B cell recruitment pathways, as chicken CXCL13 variants function as the primary B cell-attracting chemokines through binding to CXCR5 receptors on B cells and T cell subsets. Similarly, the upregulation of ACKR4 (atypical chemokine receptor 4) in C. hepaticus-infected liver tissues indicates activation of chemokine scavenging mechanisms. In chickens, scavenger receptors are known to play important roles in innate immune defenses and to stimulate dose-dependent inflammatory responses in macrophages [30]. The upregulation of LYGL (lysozyme G-like) and avian β-defensin (AVBD) genes indicates activation of innate antimicrobial defense mechanisms in SLD-affected livers. Lysozyme G-like proteins degrade bacterial peptidoglycan cell walls, providing direct bactericidal activity. Avian β-defensins are antimicrobial peptides with potent activity against multiple bacterial species, including Campylobacter [27]. Previous studies have demonstrated that C. jejuni infection induces dynamic expression of avian host defense peptides, with significant upregulation of multiple AVBD genes in infected chickens [31]. The marked upregulation of AVBD1 in C. hepaticus-infected livers suggests hepatocytes are mounting a vigorous antimicrobial response to bacterial challenge. However, the persistence of severe hepatic lesions and high mortality in SLD despite robust antimicrobial gene upregulation indicate that C. hepaticus may have evolved mechanisms to evade these defenses in the hepatic microenvironment. Previous transcriptomic studies of C. hepaticus have reported reduced expression of stress-associated genes, including groL and dnaK, during infection, suggesting that decreased production of these highly immunogenic proteins may limit bacterial recognition by the host immune system. In addition, the presence of the conserved N-linked protein glycosylation (pgl) system and glycosyltransferase genes indicates that C. hepaticus may remodel its surface glycoconjugates, potentially influencing host–pathogen interactions, while the upregulation of cmeA, napA, and nrfA reflects adaptation to the oxidative and nutrient-limited hepatic environment. However, the precise contribution of these mechanisms to immune evasion and chronic colonization remains to be experimentally validated, and further functional studies are required to confirm their roles in the pathogenesis of SLD [32,33]. Conversely, the downregulation of NK and T cell activation [34] and multiple immune-regulatory loci reflect suppression of adaptive immune control mechanisms during terminal SLD. These findings suggest that C. hepaticus establishes an immunosuppressive microenvironment, enabling bacterial persistence despite the robust inflammatory response observed.
The pathway enrichment analysis of downregulated genes revealed profound suppression of core metabolic functions. The genes involved in oxidative phosphorylation, fatty acid metabolic processes, carbon metabolism, fatty acid degradation, iron ion binding, heme binding, Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway were significantly downregulated, reflecting a profound impairment of mitochondrial and cellular core metabolic processes. Collectively, these alterations suggest that C. hepaticus infection disrupts metabolic processes in hepatocytes, contributing to hepatic failure and the characteristic pathology observed in SLD. This pattern of metabolic suppression is consistent with findings from other avian bacterial liver infections [23] where metabolic genes are typically downregulated during the acute phase response. However, the extent of metabolic dysfunction in SLD exceeds what has been reported for other avian pathogens, with the downregulation of genes encoding key glucose metabolism genes FBP2, ALDOB and HKDC1 indicating comprehensive disruption of glucose homeostasis [35]. The suppression of lipid metabolism genes, including APOB, APOA2, and PLIN1, reflects severe disruption of hepatic lipid transport and storage functions [36]. The downregulation of OTC (ornithine transcarbamylase gene) indicates a compromised ammonia detoxification capacity, which is particularly problematic in chickens, given their differential ammonia metabolism compared to mammals. The concurrent suppression of cytochrome P450 enzyme genes CYP2C18 and CYP2AC1 indicates severely compromised xenobiotic metabolism [37], while the downregulation of ACAA1 reflects disrupted mitochondrial fatty acid β-oxidation and energy production. This metabolic and energy failure not only threatens hepatocyte survival but also compromises the liver’s role as the primary site for yolk precursor synthesis in laying hens. The suppression of fatty acid synthesis pathways is expected to decrease triglyceride and phospholipid production [38] resulting in reduced egg production and inferior egg quality, clinical observations consistent with field reports of SLD-affected flocks. C.hepaticus genome exhibits a reduced repertoire of genes associated with iron acquisition and metabolism [39], indicating adaptation to iron-rich host environments like the liver. In chickens, the liver functions as the primary site for iron storage and regulation, maintaining systemic iron balance. Infected livers exhibited significant downregulation of pathways associated with iron ion, heme, and tetrapyrrole binding pathways, suggesting a localized modulation of hepatic iron homeostasis, potentially increasing iron bioavailability within the hepatic microenvironment, thereby favoring bacterial persistence and exacerbating disease progression. Notably, our previous studies on bacterial transcriptomic analysis of C. hepaticus during infection LMH cell line demonstrated upregulation of the siderophore transport gene ceuB [32]. When considered together, the host downregulation of iron-binding pathways and the bacterial upregulation of ceuB support a model in which C. hepaticus exploits infection-induced disruption of hepatic iron homeostasis to secure iron within the hepatic microenvironment, thereby promoting persistence and disease progression. This pattern is consistent with host–pathogen competition for iron, whereby the host attempts to limit iron availability while pathogens activate iron acquisition systems to overcome this nutritional barrier [40]. The dramatic upregulation of C1QTNF1 (C1q and TNF-related protein 1) and ADGRD1 genes in SLD-infected liver tissues represents a critical finding with implications for both inflammatory responses and cellular stress adaptation. C1QTNF1 functions as both a complement-related immune effector and metabolic regulator, with its upregulation suggesting activation of complement-mediated inflammatory pathways consistent with the severe hepatic lesions characteristic of terminal-stage disease [41]. Similarly, ADGRD1, although not well characterized in chickens, is an adhesion G protein-coupled receptor known in mammals to mediate hypoxia and inflammatory-signaling. This pronounced upregulation during C. hepaticus infection [42] suggesting that hepatocytes are undergoing substantial stress and attempting to cope with the combined metabolic and inflammatory burden imposed by infection.
5. Conclusions
In summary, transcriptomic profiling of SLD-affected chicken livers revealed marked immune dysregulation accompanied by profound suppression of core metabolic pathways. The identification of key cytokines, acute-phase proteins, and metabolic genes may uncover promising biomarkers for disease surveillance and therapeutic intervention. Although this study provides transcriptomic evidence of host responses to SLD, further functional studies are needed to validate the biological roles of the identified genes and pathways. Together, these findings establish a molecular framework for developing targeted diagnostic tools and control strategies to reduce the economic and welfare burden of SLD in commercial layer operations.
Supporting information
S2 File. Overall differential gene expression profile data control liver and infected liver.
https://doi.org/10.1371/journal.pone.0346466.s002
(CSV)
S3 File. GO and KEGG analysis list of upregulated genes.
https://doi.org/10.1371/journal.pone.0346466.s003
(NUMBERS)
S4 File. GO and KEGG analysis list of downregulated genes.
https://doi.org/10.1371/journal.pone.0346466.s004
(NUMBERS)
S5 File. Immune and metabolic downregulated genes.
https://doi.org/10.1371/journal.pone.0346466.s005
(PDF)
References
- 1. Muralidharan C, Huang J, Anwar A, Scott PC, Moore RJ, Van TTH. Prevalence of Campylobacter hepaticus specific antibodies among commercial free-range layers in Australia. Front Vet Sci. 2022;9:1058110. pmid:36452145
- 2. Wade B, Rautenschlein S, Hao Van TT, Moore RJ. Campylobacter hepaticus and spotty liver disease in poultry. Avian Dis. 2025;68:481–9.
- 3. Gottapu C, Edison LK, Neelawala RN, Bommineni V, Butcher GD, Sahay B, et al. Identification and expression of immunogenic mimotopes of C. hepaticus Using an E. coli-based surface display system. Vaccines (Basel). 2026;14(4):298. pmid:42042774
- 4. Courtice JM, Ahmad TB, Wei C, Mahdi LK, Palmieri C, Juma S, et al. Detection, characterization, and persistence of Campylobacter hepaticus, the cause of spotty liver disease in layer hens. Poult Sci. 2023;102(7):102462. pmid:37209651
- 5. Gregory M, Klein B, Sahin O, Girgis G. Isolation and characterization of Campylobacter hepaticus from layer chickens with spotty liver disease in the United States. Avian Dis. 2018;62(1):79–85. pmid:29620465
- 6. Van TTH, Phung C, Anwar A, Wilson TB, Scott PC, Moore RJ. Campylobacter bilis, the second novel Campylobacter species isolated from chickens with spotty liver disease, can cause the disease. Vet Microbiol. 2023;276:109603. pmid:36423482
- 7. Arukha A, Denagamage TN, Butcher G, Kariyawasam S. Complete genome sequence of Campylobacter hepaticus strain UF2019SK1, isolated from a commercial layer flock in the United States. Microbiol Resour Announc. 2021;10(12):e01446–20. pmid:33766908
- 8. Gottapu C, Sahin O, K Edison L, Srednik ME, Kariyawasam S. Complete genome sequences of Campylobacter hepaticus strains USA1 and USA5 isolated from a commercial layer flock in the United States. Microbiol Resour Announc. 2025;14(5):e0091924. pmid:40243316
- 9. Hananeh W, Ababneh M. Spotty liver disease in Jordan: an emerging disease. Vet Med (Praha). 2021;66(3):94–8. pmid:40201433
- 10.
Grimes T, Reece R. Spotty liver disease – an emerging disease in free-range egg layers in Australia. In: Proceedings of the Sixtieth Western Poultry Disease Conference. Sacramento, CA; 2011. pp. 53–6.
- 11. Crawshaw T. A review of the novel thermophilic Campylobacter, Campylobacter hepaticus, a pathogen of poultry. Transbound Emerg Dis. 2019;66(4):1481–92. pmid:31081981
- 12. Crawshaw TR, Chanter JI, Young SCL, Cawthraw S, Whatmore AM, Koylass MS, et al. Isolation of a novel thermophilic Campylobacter from cases of spotty liver disease in laying hens and experimental reproduction of infection and microscopic pathology. Vet Microbiol. 2015;179(3–4):315–21. pmid:26130518
- 13. Courtice JM, Mahdi LK, Groves PJ, Kotiw M. Spotty liver disease: a review of an ongoing challenge in commercial free-range egg production. Vet Microbiol. 2018;227:112–8. pmid:30473340
- 14. Moore RJ, Scott PC, Van TTH. Spotlight on avian pathology: Campylobacter hepaticus, the cause of Spotty Liver Disease in layers. Avian Pathol. 2019;48(4):285–7. pmid:30942612
- 15. Li J, Xu Y, Ren P, Xiong X, Qiu M, Yu C, et al. Integrative metabolomic and transcriptomic analysis reveals stage-specific shifts in hepatic lipid metabolism of broiler chickens. Anim Biotechnol. 2026;37(1):2622124. pmid:41634535
- 16. Zaefarian F, Abdollahi MR, Cowieson A, Ravindran V. Avian liver: the forgotten organ. Animals. 2019;9(2):63.
- 17. Chen M, He Y, Jia Y, Wu L, Zhao R. Liver transcriptome response to avian pathogenic Escherichia coli infection in broilers with corticosterone treatment. Poult Sci. 2025;104(5):105020. pmid:40088534
- 18. Eastwood S, Wilson TB, Huang J, Campbell BE, Scott PC, Moore RJ, et al. Immune responses and recovery from spotty liver disease in layer birds. Poult Sci. 2025;104(8):105351. pmid:40446677
- 19. Quinteros JA, Scott PC, Wilson TB, Anwar AM, Scott T, Muralidharan C, et al. Isoquinoline alkaloids induce partial protection of laying hens from the impact of Campylobacter hepaticus (spotty liver disease) challenge. Poult Sci. 2021;100(11):101423. pmid:34534853
- 20. Van TTH, Gor M-C, Anwar A, Scott PC, Moore RJ. Campylobacter hepaticus, the cause of spotty liver disease in chickens, is present throughout the small intestine and caeca of infected birds. Vet Microbiol. 2017;207:226–30. pmid:28757028
- 21. Edison LK, Kudva IT, Kariyawasam S. Comparative transcriptome analysis of Shiga Toxin-Producing Escherichia coli O157:H7 on Bovine rectoanal junction cells and human colonic epithelial cells during initial adherence. Microorganisms. 2023;11(10):2562.
- 22. Edison LK, Kariyawasam S. From the gut to the brain: transcriptomic insights into neonatal meningitis Escherichia coli across diverse host niches. Pathogens. 2025;14.
- 23. Coble DJ, Sandford EE, Ji T, Abernathy J, Fleming D, Zhao H, et al. Impacts of Salmonella enteritidis infection on liver transcriptome in broilers. Genesis. 2013;51(5):357–64. pmid:23097340
- 24. Connell S, Meade KG, Allan B, Lloyd AT, Kenny E, Cormican P, et al. Avian resistance to Campylobacter jejuni colonization is associated with an intestinal immunogene expression signature identified by mRNA sequencing. PLoS One. 2012;7(8):e40409. pmid:22870198
- 25. James H, Gan E, Moore RJ, Andrews DM, Van TTH. Infection dynamics and host biomarker identification for spotty liver disease in chickens. Vet Sci. 2025;12(12):1136. pmid:41472116
- 26. Kim S, Faris L, Cox CM, Sumners LH, Jenkins MC, Fetterer RH, et al. Molecular characterization and immunological roles of avian IL-22 and its soluble receptor IL-22 binding protein. Cytokine. 2012;60(3):815–27. pmid:22980486
- 27. Cuperus T, Coorens M, van Dijk A, Haagsman HP. Avian host defense peptides. Dev Comp Immunol. 2013;41(3):352–69. pmid:23644014
- 28. Chagneau S, Gaucher M-L, Fravalo P, P. Thériault W, Thibodeau A. Intestinal colonization of Campylobacter jejuni and its hepatic dissemination are associated with local and systemic immune responses in broiler chickens. Microorganisms. 2023;11(7):1677.
- 29. Rothwell L, Hu T, Wu Z, Kaiser P. Chicken interleukin-21 is costimulatory for T cells and blocks maturation of dendritic cells. Dev Comp Immunol. 2012;36(2):475–82. pmid:21911004
- 30. He H, MacKinnon KM, Genovese KJ, Nerren JR, Swaggerty CL, Nisbet DJ, et al. Chicken scavenger receptors and their ligand-induced cellular immune responses. Mol Immunol. 2009;46(11–12):2218–25. pmid:19446336
- 31. Garcia JS, Byrd JA, Wong EA. Expression of nutrient transporters and host defense peptides in Campylobacter challenged broilers. Poult Sci. 2018;97(10):3671–80. pmid:29931274
- 32. Bommineni V, Edison LK, Gottapu C, Butcher GD, Kariyawasam S. Campylobacter hepaticus transcriptomics identified genes involved in spotty liver disease (SLD) Pathogenesis. Pathogens. 2025;14(10):1048.
- 33. McDonald JB, Scott NE, Underwood GJ, Andrews DM, Van TTH, Moore RJ. Characterisation of N-linked protein glycosylation in the bacterial pathogen Campylobacter hepaticus. Sci Rep. 2023;13.
- 34. Woo SJ, Kim J, Lee HJ, Lee KY, Park KJ, Kim J-K, et al. Conserved functional features of natural killer cell subsets in chicken, human, and murine immune systems. iScience. 2025;28(8):113144. pmid:40792038
- 35. Pilkis SJ, El-Maghrabi MR, Claus TH. Fructose-2,6-bisphosphate in control of hepatic gluconeogenesis from metabolites to molecular genetics. Diabetes Care. 1990;13:582–99.
- 36. Zhai G, Pang Y, Zou Y, Wang X, Liu J, Zhang Q, et al. Effects of PLIN1 gene knockout on the proliferation, apoptosis, differentiation and lipolysis of chicken preadipocytes. Animals (Basel). 2022;13(1):92. pmid:36611701
- 37. Ren J, Yang L, Li Q, Zhang Q, Sun C, Liu X, et al. Global investigation of cytochrome P450 genes in the chicken genome. Genes (Basel). 2019;10(8):617. pmid:31416226
- 38. Liu X-T, Lin X, Mi Y-L, Zeng W-D, Zhang C-Q. Age-related changes of yolk precursor formation in the liver of laying hens. J Zhejiang Univ Sci B. 2018;19(5):390–9. pmid:29732750
- 39. Petrovska L, Tang Y, Jansen van Rensburg MJ, Cawthraw S, Nunez J, Sheppard SK, et al. Genome reduction for niche association in Campylobacter Hepaticus, a cause of spotty liver disease in poultry. Front Cell Infect Microbiol. 2017;7:354. pmid:28848714
- 40. Marchetti M, De Bei O, Bettati S, Campanini B, Kovachka S, Gianquinto E, et al. Iron metabolism at the interface between host and pathogen: from nutritional immunity to antibacterial development. Int J Mol Sci. 2020;21(6):2145. pmid:32245010
- 41. Yu H, Yan X, Chen G, Li R, Yang Z, Liang Z, et al. Dynamic network biomarker C1QTNF1 regulates tumor formation at the tipping point of hepatocellular carcinoma. Biomol Biomed. 2024;24(4):939–51. pmid:38498315
- 42. Žáčková S, Pávová M, Trylčová J, Chalupová J, Priss A, Lukšan O. Upregulation of mRNA expression of ADGRD1/GPR133 and ADGRG7/GPR128 in SARS-CoV-2-infected lung adenocarcinoma Calu-3 Cells. Cells. 2024;13.