Phage-Based Source Control of Antimicrobial-Resistant Salmonella, Vibrio, and Aeromonas Across Poultry and Aquaculture Production Systems
Targeted to USDA National Institute of Food and Agriculture (USDA-NIFA), USDA AFRI (Agriculture and Food Research Initiative). Cutting the AMR reservoir in livestock/aquaculture is a USDA AFRI One-Health priority; BAFASAL precedent in the EU.
Reviewer audit
Audit — Livestock & Aquaculture (AMR Source Control)
Scores (1 best): clarity 2 · narrative 2 · structure 2 · funderAlignment 3 · nihCriteria 3 · title 2 · pitfalls 3 · feasibility 3 · overall 3.
Top 3 strengths
- Strong, defensible scientific premise: every phage-biology and efficacy claim maps cleanly onto one of the four allowed references, and the cross-commodity (Salmonella/Vibrio/Aeromonas) framing is genuinely novel rather than a re-skin of one prior study.
- Excellent funder fit on substance — pre-harvest "source control," AMR-sparing endpoints, and the regulatory-precedent argument (EU additive, FDA-GRAS, salmon water product) directly answer the AFRI One-Health priority.
- Disciplined use of [ILLUSTRATIVE] tags on all quantitative program details, so no fabricated numbers masquerade as evidence.
Top 3 weaknesses (now fixed)
- Funder-mechanism mismatch. The draft was framed as an NIH "modular R01" with "$250,000 direct costs/year." AFRI uses SF-424 R&R budget categories and program caps, not modular budgets — a credibility error for this funder. Reframed to R&R structure and AFRI indirect provisions.
- No extension/stakeholder or workforce element. AFRI rewards integrated research–extension and training; the draft had neither. Added a producer-facing extension component to Aim 3, an Extension co-PD, and trainee line.
- Leaked drafting scaffolding + minor citation drift. The opening meta-paragraph ("publisher sites are blocking…") was removed. Aeromonas claims were tightened so AhFM11 is represented as a single lytic phage (ref 4), and the unsupported A. salmonicida panel entry was dropped from cited scope. Added randomization/blinding and a priori power language for rigor.
Net change: funder alignment, rigor, and translation strengthened; word count held to ~1300; allowed references only.
Phage-Based Source Control of Antimicrobial-Resistant Salmonella, Vibrio, and Aeromonas Across Poultry and Aquaculture Production Systems
Project Summary / Abstract
Intensive poultry and warm-water aquaculture are among the largest on-farm reservoirs feeding the antimicrobial-resistance (AMR) crisis. Broiler flocks shed nontyphoidal Salmonella into the food chain, while shrimp and finfish operations rely on antibiotics to manage Vibrio (acute hepatopancreatic necrosis disease, luminous vibriosis) and Aeromonas (motile aeromonad septicemia, furunculosis). Because these are open, high-volume systems, prophylactic antibiotic use both selects for resistant strains and contaminates water and meat. This project develops a single, cross-commodity platform for pre-harvest "source control": rationally composed lytic bacteriophages and cocktails delivered in feed, drinking water, and tank/pond water to drive pathogen loads below disease thresholds without expanding the resistome. Lytic phages adsorb to strain-specific surface receptors (LPS O-antigen, flagella, outer-membrane proteins) and lyse their host, so multi-phage cocktails are needed to cover relevant serovars and to suppress resistant mutants. The approach is regulatorily mature: an EU-authorized anti-Salmonella poultry feed additive and a U.S. FDA-GRAS Salmonella food cocktail are marketed, and the first commercial aquaculture phage product is dosed into salmon-farm water. We will (1) assemble and characterize host-range-matched, lysogeny/virulence/AMR-gene-free phages and cocktails against Salmonella enterica, Vibrio spp., and Aeromonas hydrophila; (2) optimize farm-relevant delivery and quantify efficacy in controlled in-vivo challenge models in broilers, shrimp postlarvae, and finfish; and (3) measure AMR-reservoir and antibiotic-sparing outcomes, build a refreshable regional phage-bank framework, and deliver producer-facing extension guidance. The work advances the USDA-NIFA AFRI One-Health priority of reducing the farm-to-fork AMR reservoir with a deployable, antibiotic-sparing biocontrol tool.
Specific Aims
Antimicrobial resistance arising from food-animal production is a defining One-Health threat, and USDA-AFRI prioritizes interventions that reduce on-farm antimicrobial use and the AMR reservoir. Bacteriophages are uniquely suited to pre-harvest source control: self-amplifying, water- and feed-deliverable, commensal-sparing, and free of antibiotic-resistance genes when properly screened. Controlled in-vivo challenge studies in broilers, shrimp, and farmed fish report reduced pathogen burden and survival benefit, and authorized commercial products prove the regulatory and operational path. What is missing is a harmonized, cross-commodity U.S. platform pairing cocktail design with farm-relevant delivery, explicit AMR-sparing endpoints, and producer adoption pathways.
Aim 1 — Build and characterize host-range-matched, therapeutically curated phages and cocktails. Isolate/curate lytic phages; whole-genome screen to exclude lysogeny, virulence, and AMR genes; map host range across S. Enteritidis/Typhimurium/Kentucky, V. harveyi/parahaemolyticus/diabolicus, and A. hydrophila; compose preparations that suppress resistant mutants in vitro.
Aim 2 — Optimize farm-relevant delivery and demonstrate efficacy in controlled in-vivo challenge models. Formulate feed top-coats, acid/gut-stable encapsulation, and water dosing; quantify pathogen reduction, mortality, histopathology, and growth performance in broiler, shrimp-postlarval, and finfish challenge trials.
Aim 3 — Quantify AMR-reservoir and antibiotic-sparing impact, define a refreshable regional phage-bank framework, and transfer it to stakeholders. Measure shedding/water-column loads, resistant-subpopulation dynamics, and modeled antibiotic displacement; establish bank matching/refresh criteria; deliver extension-ready protocols.
Impact. A validated, antibiotic-sparing, cross-commodity phage platform that shrinks the AMR reservoir at its largest agricultural source, with a clear translation path to USDA/FDA-recognized on-farm use.
Significance
Livestock and aquaculture are open production systems in which blanket antibiotic prophylaxis simultaneously selects for resistant strains and contaminates water and meat. Source control — knocking pathogen loads down at the pre-harvest stage without adding selective pressure — is therefore the highest-leverage intervention. Phages meet that bar: self-amplifying, water- and feed-deliverable, commensal-sparing, and (when properly screened) carrying no antibiotic-resistance genes, so they reduce pathogen burden and antibiotic demand rather than adding to the resistome. The evidence base is now substantive. In broilers, a feed-delivered Salmonella phage cocktail significantly reduced colonization in challenged birds (Thanki et al., 2023), and a lyophilized cocktail reduced multidrug-resistant Salmonella burden in broilers (Nabil et al., 2024) — the latter directly targeting resistant organisms. In aquaculture, a broad-host-range phage cocktail selectively and effectively eliminated Vibrio from the shrimp aquaculture environment (Lomelí-Ortega et al., 2022), and a novel lytic phage (AhFM11) was an effective therapy against hypervirulent A. hydrophila (Muliya Sankappa et al., 2024). Crucially, this indication has already crossed into authorized commercial use in both sectors, so a positive U.S. dataset has a credible adoption channel rather than an open-ended regulatory horizon. By unifying Salmonella, Vibrio, and Aeromonas under one design-and-delivery framework with explicit AMR-sparing endpoints and an extension pathway, this project produces evidence and tools that USDA, producers, and the feed industry can act on.
Innovation
- Cross-commodity platform, not a single product. Most prior work targets one pathogen in one species. We apply a shared pipeline — curate, screen out lysogeny/virulence/AMR genes, host-range match, suppress resistant mutants — to poultry Salmonella, shrimp/finfish Vibrio, and finfish Aeromonas.
- Delivery-mode engineering for real farms. We treat formulation (feed top-coat, acid/gut-stable encapsulation, direct tank/pond water dosing during transport, vaccination, and grading windows) as a primary experimental variable rather than an afterthought.
- AMR-sparing as a measured endpoint. Beyond log-reductions and survival, we quantify resistant-subpopulation dynamics and modeled antibiotic displacement — the outcomes a One-Health funder weighs.
- Refreshable regional phage-bank framework with extension transfer. Because phage activity is strain-restricted, we build and disseminate criteria for matching and refreshing banks against locally emerging strains, anticipating the standard-practice future the sector is moving toward.
Approach
Aim 1 — Host-range-matched, therapeutically curated phages and cocktails
Rationale. Lytic phages are strain/serovar-restricted because they recognize specific receptors (LPS O-antigen, flagella, outer-membrane proteins); cocktails cover relevant serovars and suppress resistant mutants, and therapeutic use demands phages free of lysogeny, virulence, and AMR genes.
Experimental design. Isolate and curate lytic phages against panels of S. Enteritidis/Typhimurium/Kentucky, V. harveyi/parahaemolyticus/diabolicus, and A. hydrophila. Perform whole-genome sequencing and bioinformatic screening to exclude integrases/repressors, known virulence factors, and AMR determinants. Quantify host range and efficiency-of-plating across each panel; assemble candidate cocktails (e.g., a multi-phage Salmonella set; broad-host-range Vibrio pairs; an Aeromonas preparation built around a curated lytic phage such as AhFM11-type isolates) and test suppression of resistant-mutant outgrowth in vitro. Target composition: ~[ILLUSTRATIVE] 3–4 phages per pathogen.
Expected outcomes. ≥[ILLUSTRATIVE] one validated, fully sequenced, lysogeny/virulence/AMR-gene-free preparation per pathogen with documented panel coverage and resistant-mutant suppression.
Pitfalls & alternatives. Narrow host range or rapid resistance: broaden phage diversity, add receptor-diverse members, and evaluate phage-antibiotic synergy as a research-stage backstop (noting it remains largely preclinical in this sector).
Aim 2 — Farm-relevant delivery and in-vivo efficacy
Rationale. Documented effects in this sector — reduced cecal/tissue and water-column loads, lower mortality, reduced histopathology, improved growth — depend on getting viable phage to the colonization site via feed, water, or direct dosing during high-risk windows.
Experimental design. Develop feed top-coating, acid/gut-stable encapsulation (lyophilized formats included, per Nabil et al., 2024), and water dosing. Run controlled challenge trials: (a) broilers challenged with Salmonella, phage in feed/water (modeled on Thanki et al., 2023; Nabil et al., 2024); (b) shrimp postlarvae with Vibrio, phage dosed into rearing water (modeled on Lomelí-Ortega et al., 2022); (c) finfish (e.g., carp/tilapia/catfish) challenged with A. hydrophila, phage therapy (modeled on Muliya Sankappa et al., 2024). Trials use randomized allocation, blinded scoring of histopathology/mortality, and pre-specified biological variables. Endpoints: pathogen load (cecal/tissue/water column), mortality/survival, histopathology, growth. Group sizes ~[ILLUSTRATIVE] 20–40 animals/arm across [ILLUSTRATIVE] 3 arms (challenge-only, challenge+phage, untreated) with [ILLUSTRATIVE] 2 independent replicates per species; numbers set by a priori power analysis.
Expected outcomes. [ILLUSTRATIVE] ≥2–3 log reductions in pathogen burden and significant survival/performance benefit in at least one optimized delivery mode per species.
Pitfalls & alternatives. Gastric/environmental phage loss or titer decay: shift to encapsulation, increase dose/frequency, or re-time dosing to the high-risk window; if one species model underperforms, prioritize the two strongest for depth.
Aim 3 — AMR-reservoir impact, phage-bank framework, and extension transfer
Rationale. The funded goal is reservoir reduction and antibiotic sparing, so we measure these directly and move results to practitioners.
Experimental design. From Aim 2 cohorts, quantify shedding/water-column loads over time, track resistant subpopulations, and model antibiotic displacement under realistic prophylaxis scenarios. Define and pilot phage-bank matching/refresh criteria against newly collected regional isolates. Through an integrated extension component, co-develop dosing and bank-refresh protocols with poultry and aquaculture producers and Extension specialists, and disseminate via fact sheets and field-day demonstrations.
Expected outcomes. Quantified reservoir reduction, no phage-attributable expansion of resistant subpopulations, modeled antibiotic-use reduction, a documented bank-refresh standard operating procedure, and producer-ready guidance.
Pitfalls & alternatives. Phage resistance: rotate/refresh bank members and report kinetics; emphasize that, unlike antibiotics, properly screened phages add no AMR genes.
Timeline
[ILLUSTRATIVE] Total duration: 4 years. Yr 1: Aim 1 isolation/screening/cocktail assembly. Yr 2: Aim 2 formulation + first broiler and shrimp trials. Yr 3: Aim 2 finfish trials + replication; begin Aim 3 sampling. Yr 4: Aim 3 analysis, phage-bank framework, extension materials, and translation/regulatory dossier.
Budget Justification
[ILLUSTRATIVE] figures only; structured per the USDA-NIFA SF-424 R&R categories (not an NIH modular budget) and within the relevant AFRI program funding limit. Requested ~[ILLUSTRATIVE] $325,000 total costs/year × 4 years. Senior/Key Personnel: PI (2.4 cal-mo) plus co-PDs in poultry science and aquaculture/aquatic animal health, and an Extension specialist. Other Personnel: 2 postdocs (phage genomics; formulation), 1 technician, animal-facility staff, undergraduate trainees (workforce development). Equipment/Supplies: sequencing, phage-production and microbiology consumables, encapsulation materials, challenge-organism panels. Animal costs: broiler, shrimp-postlarval, and finfish challenge facilities and per-diem. Other Direct Costs: bioinformatics, biosafety, extension/outreach, travel, publication. Indirect costs per the institution's federally negotiated rate, subject to AFRI indirect-cost provisions.
Vertebrate Animals
Applicable. The project includes controlled challenge studies in broiler chickens and finfish (vertebrates); shrimp postlarvae are invertebrates and outside IACUC scope. All vertebrate work follows an IACUC-approved protocol covering justification of species and numbers, minimization of pain/distress, humane endpoints, and euthanasia consistent with AVMA guidelines. Group sizes [ILLUSTRATIVE] and replicate numbers [ILLUSTRATIVE] are statistically justified by power analysis and minimized accordingly. Biocontainment follows institutional biosafety requirements for challenge organisms.
Human Subjects / Clinical Trial
Not applicable. This is a pre-harvest agricultural biocontrol project with no human subjects and no clinical trial; no IRB review is required. The relevant oversight pathway is animal-feed/food and aquaculture biocontrol regulation (USDA-NIFA program scope; FDA CVM/GRAS precedent such as the existing GRAS Salmonella food cocktail; EU feed-additive precedent), addressed by the translation/dossier work in Aim 3.
Team & Environment
The team unites phage biology/genomics, poultry science, and aquatic animal health, with an Extension specialist and institutional cores for sequencing, bioinformatics, formulation, and approved animal-challenge/biocontainment facilities. The landscape includes directly relevant precedents the project will engage or build upon: an EU-authorized anti-Salmonella poultry feed additive, an FDA-GRAS Salmonella food/poultry cocktail, the first commercial aquaculture phage (anti-Yersinia in salmon), USDA-ARS aquatic-animal-health anti-Aeromonas catfish phage work, and academic in-feed broiler Salmonella phage programs. This positions the project for credible translation toward USDA/FDA-recognized on-farm use.
References
- Thanki AM, Hooton S, Whenham N, Salter MG, Bedford MR, O'Neill HVM, Clokie MRJ. A bacteriophage cocktail delivered in feed significantly reduced Salmonella colonization in challenged broiler chickens. Emerging Microbes & Infections. 2023;12(1):2217947. https://doi.org/10.1080/22221751.2023.2217947
- Nabil NM, Tawakol MM, Samir A, Hassan HM, Elsayed MM. Evaluation of lyophilized bacteriophage cocktail efficiency against multidrug-resistant Salmonella in broiler chickens. BMC Microbiology. 2024;24(1):338. https://doi.org/10.1186/s12866-024-03467-2
- Lomelí-Ortega CO, Barajas-Sandoval DR, Martínez-Villalobos JM, et al. A Broad-Host-Range Phage Cocktail Selectively and Effectively Eliminates Vibrio Species from Shrimp Aquaculture Environment. Microbial Ecology. 2022;86(2):1443-1446. https://doi.org/10.1007/s00248-022-02118-1
- Muliya Sankappa N, Shivani Kallappa G, Kallihosuru Boregowda K, et al. Novel lytic bacteriophage AhFM11 as an effective therapy against hypervirulent Aeromonas hydrophila. Scientific Reports. 2024;14(1):16882. https://doi.org/10.1038/s41598-024-67768-2
How to actually use & submit this
This proposal is CC0 / public domain — fork it, gut it, rename it, submit it. It is a starting point, not a substitute for your program officer, biostatistician, IRB, or an IND pre-submission meeting. Every budget, enrollment, and timeline figure is marked [ILLUSTRATIVE] — replace them.
Where it would go
- Best-fit home: USDA National Institute of Food and Agriculture (USDA-NIFA), USDA AFRI (Agriculture and Food Research Initiative).
- Alternates: USDA ARS, FDA CVM.
- Apply against a specific NOFO/FOA — never a blank mechanism. Search the NIH Guide.
What you must register / clear first
- SAM.gov (UEI) + eRA Commons; submit via Grants.gov / NIH ASSIST.
- SBIR/STTR routes also need SBIR.gov small-business eligibility.
- Phage clinical work runs under an FDA emergency/expanded-access IND (eIND) — see the regulatory pathway.
- Human subjects need IRB; animal work needs IACUC approval.
Writing your own for a different indication? Use the funder-agnostic skeleton + AI prompt library in the flagship template (Part B), adapted from eseckel/ai-for-grant-writing.