Host immune dysregulation during streptococcosis and bacterial/viral co-infections in cultured fish: synthesis of experimental transcriptomic, proteomic, qPCR, histopathological and intervention studies (2015–2026)
Introduction
Research question and scope
This report synthesizes experimental evidence (2015–2026) on mechanisms of host immune dysregulation in fish during streptococcosis—principally infections caused by Streptococcus iniae and Streptococcus agalactiae—and interactions with common co-pathogens (Aeromonas hydrophila, Vibrio spp., and viral agents such as iridoviruses and tilapia lake virus). The focal host species are Nile tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss), and Japanese yellowtail (Seriola quinqueradiata) when available; the review emphasizes studies that applied transcriptomics (bulk RNA‑seq), single‑cell RNA sequencing (scRNA‑seq), proteomics or qPCR profiling of immune genes (notably pro‑ and anti‑inflammatory cytokines IL‑1β, TNF‑α, IL‑10; Toll‑like receptors; antimicrobial peptides; immunoglobulins), in vivo challenge models (mono‑ vs. co‑infection), and histopathology of primary lymphoid/immune tissues (spleen, head kidney/anterior kidney, gills). Environmental modulators (temperature, stocking density, hypoxia) and aquaculture interventions (polyvalent vaccines, probiotics, immunostimulants, antimicrobial stewardship) are evaluated.
Summary of key findings (high level)
- Co‑infections with Streptococcus spp. and other bacterial or viral agents commonly produce synergistically stronger inflammatory responses and more severe pathology than mono‑infections, with higher mortality reported in field outbreaks and experimental co‑challenge models
. - Transcriptomic profiling (bulk RNA‑seq) indicates that coinfected fish often show larger numbers of differentially expressed genes (DEGs) and stronger upregulation of cytokine/chemokine, proteasome, antigen processing, and oxidative phosphorylation pathways than mono‑infected fish; for example, a tilapia intestine RNA‑seq study reported 6,185 DEGs and markedly higher expression of IL‑1β, CXCL10, and proteasome/ubiquitin genes in co‑infected groups
. - Single‑cell RNA‑seq studies of head kidney leukocytes reveal cell‑type specific trajectories: non‑classical cytotoxic cells (NCCs) and monocytes/macrophages (Mos/Mφs) are dominant responders; Mos/Mφs may follow distinct differentiation paths to Gram‑positive versus Gram‑negative bacteria, associated with differential cytokine signatures (e.g., IL‑1β/TNF‑α dominated responses with Aeromonas)
. - Histopathology across species and challenge models consistently documents intense inflammatory infiltrates (macrophages, lymphocytes), splenitis and anterior kidney lesions, and tissue necrosis in susceptible fish; selectively bred resistant lines show markedly reduced pathological changes
. - Mechanistic themes of dysregulation include exaggerated pro‑inflammatory cytokine production (IL‑1β, TNF‑α, chemokines such as CXCL10/CCL19), proteasome/ubiquitin pathway activation, suppression or perturbation of energy metabolism/oxidative phosphorylation (ATP synthase downregulation), and immune evasion by bacterial virulence factors (nucleases, 5′‑nucleotidases) that degrade neutrophil extracellular traps (NETs) and generate immunosuppressive adenosine
. - Epigenetic correlates (DNA methylation) of resistance to S. agalactiae have been reported in tilapia, indicating promoter methylation changes for immune‑relevant genes (e.g., Arnt2, Nhr38) that correlate with differential expression and histopathology between resistant and susceptible lines; however, histone modification data and single‑cell epigenomics in co‑infection contexts are lacking
.
The remainder of the report integrates extracted primary experimental evidence, compares species and infection‑route specific patterns, analyzes environmental modulators, evaluates implications for aquaculture interventions, documents research gaps, and proposes testable hypotheses and priorities for future immunotherapeutic and omics‑driven studies.
Experimental evidence: transcriptomics, proteomics, qPCR and histopathology
Overview of selected experimental studies (citations after each claim)
- Bulk transcriptomics of intestinal responses in tilapia to mono‑ and co‑infection (Cui et al., 2022)
- Study design and methods: Intraperitoneal injection challenge of Nile tilapia with S. agalactiae (AG), S. iniae (IG) or a 1:1 co‑infection (MG); intestine sampled at 6, 12, 24 h and 7 d; RNA‑seq (Illumina HiSeq), DEGs (|FC| ≥ 2, FDR < 0.01), RT‑qPCR validation for 8 genes
. - Key quantitative results: 6,185 total DEGs (3,876 up; 2,309 down); peak DEG numbers at 24 h post‑infection; 136 immune‑related DEGs; strongest upregulation in the MG co‑infection group for genes such as IL‑1β, CXCL10, CCL19, PSMB8, TRIM39, IgM VH and IgG Fc‑binding
. - Mechanistic interpretation: Coinfection produced a amplified inflammatory/chemokine signal and B‑cell/immunoglobulin responses in intestine relative to mono‑infections; proteasome/ubiquitin pathway activation and decreased oxidative phosphorylation genes (ATP synthase subunit B1 down) suggest heightened antigen processing and metabolic stress during coinfection—mechanisms that may both reflect and exacerbate immune dysregulation
. - Supporting visualization: comparative fold‑change patterns for selected genes (PSMB8, ATP‑B1, CXCL10, IL‑1β, CCL19, IgG‑Fc) show orders‑of‑magnitude increases in some immune genes in co‑infection vs mono‑infection (figure referenced below). The underlying data and fold changes were numerically extracted and visualized (chart files produced and embedded as figures). See figure:
.
- scRNA‑seq and bulk RNA‑seq of head kidney leukocytes in tilapia responding to S. agalactiae and A. hydrophila (Li et al., 2025 — extract of a 2025 Animals paper included in searched results)
- Study design and methods: Intraperitoneal mono‑infection challenges with S. agalactiae or A. hydrophila; head kidney lymphocytes isolated at 24 hpi; bulk RNA‑seq (DESeq2) plus single‑cell RNA‑seq (10x Genomics) with clustering and pseudotime trajectory analyses
. - Key quantitative results: Bulk RNA‑seq DEGs: 5,555 (S. agalactiae), 6,596 (A. hydrophila), with 4,077 common DEGs. scRNA‑seq: 20,191 cells (6,731 control, 8,133 S. agalactiae, 5,327 A. hydrophila); NCCs and Mos/Mφs had the highest DEG burdens (NCC >4,000 DEGs; Mos/Mφ >2,000 DEGs). Pseudotime analyses: NCCs followed a unified differentiation trajectory across both pathogens (2,134 trajectory DEGs), while Mos/Mφs diverged by pathogen—S. agalactiae‑associated states enriched for second‑messenger pathways and chemokines; A. hydrophila‑associated states enriched for proinflammatory cytokines (TNF‑α, IL‑1β)
. - Mechanistic interpretation: The single‑cell resolution demonstrates that different leukocyte lineages and subclusters mediate distinct responses to Gram‑positive vs Gram‑negative bacteria. Mos/Mφ plasticity and divergent differentiation likely underpin the pathogen‑specific cytokine milieus and downstream immune regulation or dysregulation.
- miRNA profiling and immune regulation in head kidney (tilapia) after S. iniae challenge (Qiang et al., 2017)
- Methods: Small RNA‑seq of head kidney 24 h post S. iniae injection; 19 differentially expressed miRNAs validated by qRT‑PCR; predicted targets include complement C3, MAPK1 and other immune regulators
. - Mechanistic interpretation: Infection‑responsive miRNAs may modulate innate immune signaling (complement, TLR‑NF‑κB pathways) and contribute to dynamic up‑/down‑regulation of inflammatory mediators; miRNA‑driven regulation is a plausible mechanism of early immune modulation and dysregulation.
- Epigenetic correlates of resistance to S. agalactiae (Hu et al., 2020; follow‑ups summarized)
- Methods and main results: Whole‑genome bisulfite sequencing (WGBS) and RNA‑seq compared resistant vs susceptible GIFT tilapia 5 h post S. agalactiae challenge; identified thousands of DMRs and DEGs with 337 overlapping genes; validated promoter hypomethylation correlating with higher expression of immune‑related genes (e.g., Arnt2, Nhr38) in resistant fish
. - Mechanistic interpretation: Epigenetic reprogramming (DNA methylation patterns) is associated with differential immune gene expression and histopathology, suggesting that host epigenetic state contributes to resistance or susceptibility and can shape dysregulated inflammatory responses.
- Histopathological evidence from experimental infection and selective breeding (multiple sources)
- Pathology: Experimental S. iniae infection in tilapia produces lymphocyte and macrophage infiltrations in gill, liver, kidney, and other organs; susceptible lines show severe splenitis, nephritis, necrotizing vasculitis and meningitis with bacterial presence, while resistant lines show minimal acute lesions
. - Co‑infection field pathology: Natural triple co‑infection (TiLV + A. hydrophila + S. agalactiae) on farmed red hybrid tilapia produced severe histological lesions (syncytial giant cells, intracytoplasmic inclusions from TiLV; inflammatory cell infiltrates, necrosis, haemorrhages) and high mortality (70%), with suggestions of synergistic exacerbation of pathology
.
Synthesis of immune markers quantified across studies
A cross‑study extraction of immune markers (cytokines, chemokines, proteasome genes, immunoglobulins, lysozyme, phagocytosis measures, antimicrobial peptides, TLRs) reveals recurring patterns:
- Proinflammatory cytokines: IL‑1β and TNF‑α are consistently upregulated in bacterial infection contexts, with stronger expression in Gram‑negative A. hydrophila challenges for TNF‑α/IL‑1β in scRNA‑seq data and in vaccinated/immunostimulant contexts
. - Chemokines: CXCL10 and CCL19 are elevated in co‑infection (tilapia intestine), indicating enhanced chemotactic signaling and immune cell recruitment
. - Antigen presentation/proteasome and ubiquitin pathways: PSMB8 and E3 ubiquitin ligases (TRIM39) are markedly upregulated in co‑infection, signifying increased antigen processing and turnover
. - Metabolic perturbation: ATP synthase subunit (ATP‑B1) downregulation in co‑infection indicates impaired oxidative phosphorylation and potential metabolic exhaustion or ROS dysregulation
. - Immunoglobulins: IgM heavy chain variable regions and IgG Fc‑binding proteins show dynamic induction, sometimes elevated more in co‑infection, reflecting heightened humoral activation
. - Lysozyme and phagocytic activity: Common endpoints in vaccine and probiotic studies; increases correlate with improved survival or RPS in vaccination/co‑challenge contexts
.
Table: Key immune markers dysregulated in mono‑ vs co‑infection (synthesized)
| Marker / pathway | Direction in mono‑infection | Direction in co‑infection | Representative citations |
|---|---|---|---|
| IL‑1β | ↑ (early) | ↑↑ (greater fold in co‑infection) | |
| TNF‑α | ↑ (noted strongly with Gram‑negative A. hydrophila) | ↑↑ (depending on pathogen mix) | |
| CXCL10, CCL19 (chemokines) | ↑ (mono) | ↑↑ (co‑infection shows higher expression) | |
| PSMB8, TRIM39 (proteasome/ubiquitin) | ↑ | ↑↑ (co‑infection markedly) | |
| ATP synthase (ATP‑B1) | variable | ↓ (co‑infection—metabolic stress) | |
| IgM, IgT, IgG‑Fc markers | ↑ (adaptive activation) | ↑ (often higher or earlier in co‑infection) | |
| Lysozyme activity, phagocytic index | ↑ (vaccination/probiotic induced) | — |
(See table caption: markers synthesized from transcriptomic and functional vaccine/probiotic studies cited above.)
Visual summary of reported transcriptome DEG counts across studies
- A horizontal bar chart of reported DEG counts from selected studies illustrates that (i) bulk RNA‑seq of intestinal tissue in coinfection contexts reports several thousand DEGs (Cui et al., 2022 reported 6,185 total DEGs), (ii) scRNA‑seq/bulk RNA studies in head kidney report thousands of DEGs per pathogen (Li et al., 2025 reports >5,000 DEGs per pathogen), and (iii) tissue‑specific DEGs (head kidney vs spleen) can differ in magnitude (e.g., Maekawa et al., 2023 for Eleutheronema tetradactylum reported 1,584 DEGs in head kidney, 1,981 in spleen) (chart file produced and embedded). See figure:
.
Mechanistic patterns of immune dysregulation and cellular processes
Synthesis of mechanisms supported by experimental evidence
- Exaggerated innate inflammation and chemokine signaling
- Coinfections amplify cytokine and chemokine expression: IL‑1β, CXCL10, CCL19 among the most frequently reported. Elevated chemokines promote increased leukocyte recruitment and may drive tissue‑damaging inflammation when regulatory mechanisms are inadequate
. - Single‑cell data show Mos/Mφ subclusters are principal cytokine producers; Gram‑negative infections (A. hydrophila) provoke stronger TNF‑α/IL‑1β signatures while Gram‑positive (S. agalactiae) stimulate second messenger/chemokine responses—this dichotomy suggests pathogen‑pattern recognition differences (e.g., distinct TLR ligand engagement) that shape dysregulated immune phenotypes
.
- Antigen processing/proteasome activation and ubiquitination
- Strong upregulation of proteasome subunits (e.g., PSMB8) and E3 ubiquitin ligases (TRIM family) in coinfection contexts indicates heightened antigen processing and protein turnover—processes that accompany increased presentation but can also reflect proteostasis stress and inflammation‑associated tissue damage
.
- Metabolic exhaustion—oxidative phosphorylation perturbation
- Downregulation of mitochondrial ATP synthase subunits and enrichment of oxidative phosphorylation pathways in transcriptome data point to mitochondrial dysfunction or reprogramming during coinfection (ATP‑B1 reduction in coinfected tilapia intestine). This metabolic shift may impair cellular energetics for phagocytosis and adaptive responses, and increase ROS production, contributing to immunopathology
.
- Suppression or impairment of innate effector functions (phagocytosis, NETs)
- Functional assays and mechanistic bacterial virulence studies indicate that Streptococcus nucleases (SpnAi) and 5′‑nucleotidases (S5nAi) degrade NETs and produce adenosine, respectively; these activities reduce neutrophil‑mediated clearance and phagocyte activation and thereby facilitate immune evasion and persistent infection
.
- Adaptive responses and regulatory imbalance
- Coinfections often accelerate and elevate humoral markers (IgM, IgG‑Fc binding protein, IgT), yet this adaptive activation may be dysregulated in timing or quality. The literature reports Th1/Th2 differentiation pathway enrichment in some spleen studies (e.g., Maekawa et al. 2023), but clear, consistent Th1/Th2 skewing patterns across species and models are not yet fully resolved by available bulk transcriptomics
. - Regulatory T cell (Treg) expansion is not comprehensively profiled in the surveyed transcriptome or single‑cell studies; where anti‑inflammatory cytokines (TGF‑β, IL‑10) were measured (mostly in vaccination studies), they tended to rise in vaccinated fish but direct quantification of Treg populations in infection/co‑infection remains a gap
.
- Apoptosis and lymphoid depletion
- Histopathology in susceptible lines indicates lymphoid depletion in anterior kidney and spleen, consistent with apoptosis or cell loss; several transcriptome studies reported DEGs related to apoptosis and hematopoietic lineage disruption, but direct quantitation of apoptotic immune cells (TUNEL, flow cytometry) in coinfection models is lacking in the literature reviewed
.
- Microbiome perturbation and barrier breach
- Field reports and reviews emphasize that co‑occurring parasites, poor water quality, or viral lesions that damage epithelia (gills, skin) can enable bacterial invasion (e.g., Gyrodactylus enabling S. iniae invasion), and that dysbiosis can increase susceptibility; direct high‑resolution microbiome studies in coinfected streptococcosis remain limited in number
.
Species‑specific comparisons and infection routes
- Tilapia (Oreochromis niloticus): Extensive transcriptomic and single‑cell studies show robust innate activation (NCCs, Mos/Mφs), cytokine/chemokine surges, and metabolic perturbations in intestine and head kidney. Co‑infection studies (S. agalactiae + S. iniae) show amplified intestinal inflammation and proteasome activation
. - Rainbow trout (Oncorhynchus mykiss): The literature includes histopathology and vaccine/immune studies but fewer scRNA‑seq co‑infection data; trout mount classic proinflammatory innate responses to Streptococcus spp., with environmental temperature strongly influencing outcomes (colder temperatures slow immune responses but may favor certain bacterial growths). Specific transcriptomic co‑infection studies in trout with Streptococcus + Aeromonas/Vibrio were not prominent in the extracted corpus (gap).
- Yellowtail (Seriola quinqueradiata): Experimental data specific to streptococcosis co‑infections (with Vibrio or Streptococcus spp.) using modern omics are limited in the extracted literature; microarray studies of kidney cells stimulated by LPS/ConA (earlier work) indicate robust innate/adaptive gene expression programs but do not model coinfection or in vivo disease dynamics
.
Infection routes
- Intraperitoneal/intramuscular injection is the dominant infection route in controlled experimental challenge models used for transcriptomics and scRNA‑seq, producing reproducible systemic infection and permitting tissue sampling at defined time points. However, injection bypasses mucosal barriers and may alter early innate signaling compared with immersion or natural exposure. Several field/natural outbreak studies document immersion/waterborne transmission and show more complex pathology and co‑pathogen profiles, but lack transcriptomic resolution
.
Environmental stressors modulating immune dysregulation
- Temperature: Most experiments standardize temperature (e.g., 27–28°C for tropical tilapia studies). Temperature modulates immune kinetics and pathogen replication; low temperatures slow immune responses in coldwater species like trout and may alter Th1/Th2 balance, but detailed temperature‑interaction transcriptomics for streptococcosis co‑infections are scarce in the surveyed set
. - Hypoxia and stocking density: Reviews and field studies identify hypoxia and crowding as predisposing factors that increase mortality in co‑infection outbreaks (e.g., Francisella + S. agalactiae under chronic hypoxia leading to 100% mortality in one experiment summarized in a review)
. However, mechanistic transcriptomic data linking specific stressors to immune gene dysregulation in co‑infections are limited.
Implications for aquaculture disease control and therapeutics
Vaccine efficacy and polyvalent approaches
- Feed‑based formalin‑killed bivalent vaccines combining S. iniae and A. hydrophila demonstrate significant upregulation of innate and adaptive immune measures (IL‑1β, TNF‑α, TGF‑β, lysozyme activity, phagocytic index, IgT/IgM) and confer meaningful protection in co‑challenge studies (relative percent survival RPS ~76–80% in bivalent groups vs. lower for monovalent or controls)
. - Immunological profiling after vaccination illustrates that bivalent vaccines can stimulate mucosal and systemic immunity (gene induction in spleen, head kidney, gut), increase lysozyme and phagocytosis, and elevate antibody titers—features plausibly protective against both Gram‑positive and Gram‑negative pathogens when used preventively.
- Limitations: Many vaccine studies measure immune proxies (lysozyme, phagocytosis, gene expression) and RPS under controlled challenge; field efficacy varies with strain variation, dosing, route of administration (oral vs injection), and co‑infection pressures. Long‑term durability, breadth across diverse strains/serotypes, and the impact on microbial ecology of fish mucosa under vaccine pressure require further study.
Probiotics and immunostimulants
- Meta‑analyses and controlled trials indicate dietary probiotics (Bacillus spp., Lactobacillus spp., mixed consortia) enhance growth, serum lysozyme, and phagocytic activity and can increase survival after pathogen challenge, positioning probiotics as antibiotic‑sparing measures for disease prevention
. - Evidence indicates that probiotics can reduce pathogen colonization and modulate innate immunity, but robust multi‑site trials assessing efficacy against real‑world coinfections (e.g., TiLV + bacterial opportunists) are limited.
Antimicrobial stewardship and resistance
- Field outbreak studies document mixed antibiotic susceptibility patterns for co‑pathogens and instances of multiple antibiotic resistance indices (MAR) in A. hydrophila and S. agalactiae isolates, underscoring the need for judicious antibiotic use; vaccines and probiotics offer alternatives that may reduce selective pressure for resistance
.
Immunotherapeutic concepts and practical considerations
- Strategies informed by omics findings could include: (i) vaccines that drive balanced Th1/Th2 and mucosal immunity to limit early bacterial colonization; (ii) adjuvants or feed‑additives that boost phagocytic function and NET stability; (iii) probiotics/synbiotics to stabilize mucosal microbiota and competitive exclusion; and (iv) selective breeding guided by epigenetic markers or methylation QTLs for enhanced resistance.
- Practical constraints: delivery (oral vaccine uptake, antigen stability), heterologous protection across serotypes, regulatory hurdles, and farm‑level variability in water quality and pathogen pressure will influence effectiveness.
Research gaps and priorities
- Single‑cell multi‑omics in co‑infection models
- While scRNA‑seq of single‑pathogen challenges has clarified cell‑type specific responses, there is an absence of single‑cell transcriptomic or multi‑omic (scATAC‑seq, single‑cell methylome) studies in controlled co‑infection models (e.g., S. agalactiae + A. hydrophila, or Streptococcus spp. + TiLV). Single‑cell profiling across multiple time points would resolve cell‑level decision points (e.g., Mos/Mφ branch choice) underlying dysregulation.
- Functional validation of candidate dysregulatory pathways
- Proteasome/ubiquitin activation, ATP/mitochondrial dysfunction, and NET degradation by bacterial nucleases are implicated but require targeted functional experiments (e.g., proteasome inhibition, mitochondrial rescue, nuclease knockouts) in fish models to establish causality for immune impairment and mortality.
- Epigenetic mechanisms and heritable resistance
- DNA methylation studies indicate regulatory control of resistance, but the dynamics of methylation during coinfection, potential transgenerational inheritance, and the role of histone modifications remain unexplored.
- Microbiome dynamics under coinfection
- High‑resolution longitudinal microbiome profiling in controlled coinfection experiments is largely missing; such studies could clarify whether dysbiosis precedes or follows systemic immune dysregulation and whether probiotic interventions restore protective community structures.
- Standardized, multi‑stress experimental designs
- Integrative experiments combining environmental stressors (temperature, hypoxia, stocking density), coinfection, and omics readouts are required to model realistic aquaculture scenarios; existing studies often isolate single variables.
- Functional Treg and Th‑cell subset profiling
- Few studies directly quantify regulatory T cells, Th1/Th2 polarization, or apoptosis in immune compartments during coinfection. Flow cytometry panels and scRNA‑seq markers for Treg/Th subsets in fish should be developed and applied.
Proposed hypotheses and experimental approaches for future studies
Hypotheses
H1: Coinfection with Streptococcus spp. and Gram‑negative bacteria (Aeromonas/Vibrio) causes divergent macrophage differentiation trajectories that amplify proinflammatory mediators and reduce effective phagocytic clearance, driving tissue damage and increased mortality.
H2: Streptococcal nucleases and 5′‑nucleotidases directly impair neutrophil function (NET stability and ROS‑mediated killing) and shift local purinergic signaling toward immunosuppression, facilitating secondary bacterial overgrowth in coinfection contexts.
H3: Host epigenetic states (DNA methylation of promoter regions in T‑cell differentiation and antigen presentation genes) modulate the speed and balance of adaptive responses; methylation profiles predict resistance and can be selected for in breeding programs.
Experimental approaches (suggested designs)
- Controlled co‑infection experiment (tilapia): factorial design crossing pathogen pair (S. agalactiae, S. iniae, A. hydrophila; mono vs S1+S2 coinfections) × environmental stressor (normoxia vs chronic hypoxia) × time points (6, 12, 24, 72 h, 7 d). Collect anterior kidney, spleen, gill mucosa, and intestine for bulk RNA‑seq, scRNA‑seq (10x), proteomics (label‑free LC‑MS/MS), WGBS (subset) and microbiome (16S rRNA/shotgun). Functional assays: phagocytosis, lysozyme, ROS, NET quantitation, and survival.
- Functional bacterial genetics: generate Streptococcus nuclease and 5′‑nucleotidase mutants, test NET degradation, phagocytic inhibition, and virulence in mono‑ and co‑infection settings; complement with host rescue experiments (e.g., adenosine receptor antagonists).
- Single‑cell epigenomics: apply scATAC‑seq and targeted single‑cell methylation to immune cell populations isolated during coinfection to link chromatin accessibility/methylation with gene expression trajectories.
- Vaccine challenge trials: evaluate polyvalent vaccines with adjuvants targeting both innate boosting (e.g., TLR agonists that avoid excessive IL‑1β/TNF storm) and balanced adaptive responses; measure scRNA‑seq profiles pre‑ and post‑challenge to map protective cell states.
Conclusion
The assembled experimental corpus (2015–2026) indicates that streptococcal infections in cultured fish, particularly when combined with co‑pathogens such as Aeromonas spp., Vibrio spp., or viruses like TiLV, produce amplified and sometimes maladaptive immune responses characterized by elevated proinflammatory cytokines (IL‑1β, TNF‑α), chemokine surges (CXCL10, CCL19), proteasome/ubiquitin pathway activation, metabolic perturbations (oxidative phosphorylation downregulation), and virulence‑mediated impairment of innate effectors (NET degradation, phagocytosis inhibition). Single‑cell transcriptomics has begun to reveal the cellular heterogeneity and divergent macrophage/NCC trajectories underlying pathogen‑specific responses. Vaccination (polyvalent killed vaccines) and dietary probiotics show promise to bolster innate measures (lysozyme, phagocytosis) and reduce mortality, but broader validation in co‑infection and field settings is needed. Epigenetic evidence suggests that DNA methylation contributes to resistance phenotypes, opening avenues for selective breeding and epigenetic biomarker development.
Limitations of the reviewed evidence
- Evidence base skewed toward bulk transcriptomics and single‑pathogen challenges; few controlled co‑infection experiments employ single‑cell resolution or proteomics.
- Many field outbreak studies provide histopathology and pathogen detection but lack molecular profiling; conversely, molecular studies often use injection challenges that bypass mucosal defenses and may not fully recapitulate natural exposure.
- Epigenetic and multi‑omic integration is nascent; histone modification studies and single‑cell epigenomics are largely missing.
- Comparative data for rainbow trout and yellowtail are sparser in contemporary transcriptomic scRNA‑seq co‑infection studies compared with tilapia.
TLDR (concise takeaways)
- Coinfection involving Streptococcus spp. commonly provokes amplified inflammatory and chemotactic responses (IL‑1β, CXCL10, CCL19) and proteasome activation, with metabolic stress (ATP synthase downregulation) that may exacerbate immunopathology; single‑cell analyses identify macrophage and NCC subclusters as central drivers
. - Vaccination with bivalent formulations and dietary probiotics increase lysozyme, phagocytosis and immunoglobulin responses and can significantly reduce mortality in experimental co‑challenges; however, field heterogeneity, strain variation, and environmental stressors complicate generalizability
. - Key research needs: (i) multi‑timepoint single‑cell multi‑omic co‑infection studies, (ii) functional validation of proteasome/mitochondrial/NET pathways, (iii) epigenetic dynamics during coinfection, and (iv) standardized trials of immunotherapeutics under farm‑relevant stressors.
Appendix: Selected citations and data sources (representative)
- Cui M, Wang Z, Yang Y, et al. Comparative Transcriptomic Analysis Reveals the Regulated Expression Profiles in Oreochromis niloticus in Response to Coinfection of Streptococcus agalactiae and Streptococcus iniae. Front Genet. 2022;13:782957.
- Li Q, Fang Z, Li Z, Wei X, Wei Y. RNA‑Seq and Single‑Cell RNA‑Seq Analyses of Tilapia Head Kidney in Response to Streptococcus agalactiae and Aeromonas hydrophila. Animals. 2025;15(20):2951.
- Hu Q, Ao Q, Tan Y, et al. Genome‑Wide DNA Methylation and RNA Analysis Reveal Potential Mechanism of Resistance to Streptococcus agalactiae in GIFT Strain of Nile Tilapia (Oreochromis niloticus). J Immunol. 2020;204(12):3182–3190.
- Monir MS, Yusoff SBM, Zulperi ZBM, et al. Haemato‑immunological responses and effectiveness of feed‑based bivalent vaccine against Streptococcus iniae and Aeromonas hydrophila in hybrid red tilapia. BMC Vet Res. 2020;16:226.
- Basri A, Othman R, Abdullah MF, et al. Co‑Infections of Tilapia Lake Virus, Aeromonas hydrophila and Streptococcus agalactiae in Farmed Red Hybrid Tilapia. Animals. 2020;10(11):2141.
- Maekawa S, Wang P‑C, Chen S‑C. Differential Expression Genes of the Head Kidney and Spleen in Streptococcus iniae‑Infected East Asian Fourfinger Threadfin Fish (Eleutheronema tetradactylum). Int J Mol Sci. 2023;24:3832.
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