Six-Herb Drinking Water Formula Protects Broilers Against Clostridium Necrotic Enteritis

A six-herb traditional botanical formula administered via drinking water significantly mitigated necrotic enteritis in broiler chickens challenged with Clostridium perfringens, enhancing daily weight gain, restoring feed conversion efficiency, and strengthening intestinal structural integrity without damaging bacterial cellular membranes. Published in Frontiers in Veterinary Science, the study led by researchers at the Hebei Province Key Laboratory of Preventive Veterinary Medicine explored practical non-antibiotic interventions for intensive poultry production. Necrotic enteritis represents a major bacterial threat globally, occurring in acute forms with flock mortality reaching fifty percent or in subclinical forms that erode digestive efficiency and induce heavy economic losses estimated to exceed six billion US dollars annually. Historically managed with antibiotic growth promoters, the disease has rebounded following regulatory restrictions in jurisdictions including China and the European Union. The investigative team formulated a multi-target botanical mixture designed specifically to attenuate pathogen virulence phenotypes and support epithelial barrier maintenance under disease challenge.

To establish an evidence-based formulation, the researchers screened forty individual traditional Chinese herbal medicines using agar well diffusion and broth microdilution assays against Clostridium perfringens serotype A strain ATCC 13124. Single-herb aqueous decoctions exhibited inhibition zone diameters spanning 6.00 to 29.00 millimeters and minimum inhibitory concentrations ranging from 7.81 to 500.00 milligrams per milliliter. Mume Fructus, Schisandrae Fructus, and Coptidis Rhizoma produced the largest clearing zones at 29.00, 27.33, and 27.00 millimeters, while Chebulae Fructus and Scutellariae Radix demonstrated the lowest minimum inhibitory concentrations at 7.81 milligrams per milliliter. Pairwise checkerboard testing across twenty-one herbal combinations quantified interactions using the fractional inhibitory concentration index. Synergistic interactions emerged between Lignum Sappan and Phellodendri Cortex with an index of 0.38, as well as Chebulae Fructus and Lignum Sappan at 0.50. Guided by synergy metrics and traditional Jun-Chen-Zuo-Shi compatibility principles, the authors combined Phellodendri Cortex, Berberidis Radix, Lignum Sappan, Chebulae Fructus, Caryophylli Flos, and Glycyrrhizae Radix et Rhizoma in a defined 2.5:2.5:1.5:1.5:1:1 ratio.

Chemical characterization of the composite herbal formula was conducted using high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Following raw spectral processing, noise filtering, and alignment, forty-five high-confidence chemical constituents were identified through spectral matching against the mzCloud database. Peak area normalization demonstrated that the ten most abundant constituents comprised more than seventy-two percent of the identified chemical profile. Isoliquiritigenin constituted the predominant compound at 16.56 percent relative content, followed by citric acid at 12.70 percent and 3,4,5-trihydroxycyclohex-1-ene-1-carboxylic acid at 9.85 percent. Additional prominent molecules included 1,6-bis-O-(3,4,5-trihydroxybenzoyl) hexopyranose at 8.94 percent, miquelianin at 7.66 percent, trigonelline at 4.28 percent, quercetin at 3.67 percent, arjungenin at 3.36 percent, ononin at 3.02 percent, and D-(-)-quinic acid at 2.65 percent. The remaining thirty-five identified compounds collectively accounted for 27.30 percent of total peak area. The authors noted that this rich composition combined alkaloid-rich components known for antibacterial properties with polyphenol and flavonoid fractions associated with antioxidant, mucosal protective, and anti-inflammatory activities.

Evaluating the crude aqueous decoction against the test pathogen revealed robust in vitro antibacterial activity, generating an inhibition zone diameter of 22.17 ± 0.29 millimeters, a minimum inhibitory concentration of 15.63 milligrams per milliliter, and a minimum bactericidal concentration of 31.25 milligrams per milliliter. Crucially, the compound formula displayed a non-membranolytic mode of action rather than inducing rapid bacterial envelope destruction. Measurement of alkaline phosphatase release into culture supernatants, an established indicator of outer membrane permeability alterations, showed only minor time-dependent increases over a three-hour incubation window that did not differ significantly from untreated control cultures at either inhibitory or bactericidal concentrations. Transmission electron microscopy further corroborated the preservation of bacterial envelope structure following eight hours of exposure at the minimum inhibitory concentration. Treated cells maintained regular rod-shaped contours, well-defined cellular perimeters, and intact surface architectures indistinguishable from untreated controls, with no discernible signs of membrane rupture, cell wall disintegration, or intracellular cytoplasmic extravasation under the experimental parameters evaluated.

Rather than acting through physical lysis, the botanical preparation suppressed multiple virulence-associated phenotypes in Clostridium perfringens across a series of phenotypic assays. Biofilm biomass, quantified through crystal violet staining after seventy-two hours of anaerobic incubation at thirty degrees Celsius, was significantly reduced at concentrations of one-half the minimum inhibitory concentration and higher. Hemolytic activity against sheep erythrocytes was significantly curtailed by exposure to bactericidal and bacteriostatic concentrations, falling substantially below positive control lysis levels. Surface motility on semi-solid agar plates was completely abolished at the minimum inhibitory and bactericidal concentrations, while sub-inhibitory treatment at one-half the minimum inhibitory concentration significantly restricted swarming migration radii. Cellular biosafety assays using human colorectal adenocarcinoma Caco-2 monolayers confirmed cell viability above eighty-four percent at sub-inhibitory thresholds, though viability fell sharply at inhibitory levels. While bacterial adhesion to Caco-2 monolayers declined significantly at bacteriostatic concentrations, the concurrent reduction in epithelial viability precluded concluding that direct anti-adhesion occurred independently of cytotoxic effects.

The therapeutic utility of the botanical formula was subsequently examined in vivo using one hundred and fifty one-day-old specific-pathogen-free white-feathered broiler chicks. Chicks were allocated into six experimental groups across five replicate cages containing five birds each: an uninfected blank control, an untreated necrotic enteritis model, an amoxicillin-treated positive control receiving sixty milligrams per liter, and three herbal treatment cohorts receiving 1.5, 2.5, or 3.5 grams per liter in drinking water. Disease induction occurred from day seventeen to day twenty through daily oral gavage of one milliliter containing 1 × 10^8 colony-forming units of Clostridium perfringens. Water-mediated treatments were administered from day twenty-one to day twenty-five, following temporary water withdrawal, by providing freshly prepared medicated solutions for two hours daily at an estimated daily intake of approximately 0.15, 0.25, and 0.35 grams per bird. Birds were continuously monitored until the experimental endpoint on day twenty-eight to quantify systemic and intestinal responses.

Bacterial challenge substantially impaired broiler growth performance between days seventeen and twenty-eight, but supplementation with the herbal formula counteracted these production losses in a dose-dependent manner. In untreated infected birds, average daily weight gain plummeted to 8.42 grams compared with 10.97 grams in unchallenged controls, while the feed conversion ratio deteriorated from 1.53 to 1.96. Providing the high-dose botanical preparation at 3.5 grams per liter elevated average daily gain to 10.32 grams and lowered the feed conversion ratio to 1.63, reflecting a statistically significant gain increase of 1.90 grams and an efficiency improvement of 0.33 relative to diseased models. The intermediate dosage of 2.5 grams per liter also produced marked improvements, reaching an average daily gain of 9.96 grams and a feed conversion ratio of 1.69. Performance parameters in the high-dose group did not differ significantly from birds treated with amoxicillin or healthy uninfected controls, indicating that the botanical water additive restored growth trajectories to baseline levels observed in healthy flocks.

Pathological evaluation of intestinal tissues demonstrated pronounced mitigation of structural mucosal injury following herbal administration. Gross lesion scoring of the jejunum revealed severe focal and extensive necrotic damage in untreated infected birds, which averaged a lesion score of 3.36 ± 0.70 on a four-point scale. Administration of 3.5 grams per liter herbal decoction significantly attenuated tissue destruction, lowering the mean lesion score to 1.48 ± 0.96, an effect statistically indistinguishable from the amoxicillin group mean of 1.60 ± 1.12. Histomorphological analysis of hematoxylin and eosin-stained jejunal cross-sections documented severe blunting of intestinal architecture in diseased models, with villus height dropping to 597.48 ± 92.31 micrometers and crypt depth expanding to 235.46 ± 10.49 micrometers. High-dose herbal treatment restored villus height to 1185.86 ± 60.16 micrometers and reduced crypt depth to 188.20 ± 9.49 micrometers, normalizing the villus height-to-crypt depth ratio to 6.32 ± 0.58, which closely mirrored uninfected control values.

Systemic immune organ monitoring and serum cytokine profiling confirmed that the herbal formula modulated systemic inflammatory cascades triggered by the enteric infection. Pathogen challenge induced significant enlargement of the spleen and bursa of Fabricius relative to body weight, whereas thymus indices remained unchanged across all experimental groups. High-dose herbal intervention and amoxicillin treatment normalized spleen and bursa indices back to levels observed in healthy birds. Serum enzyme-linked immunosorbent assays revealed marked elevations of pro-inflammatory tumor necrosis factor-alpha and interleukin-6 alongside sharp reductions in anti-inflammatory interleukin-10 in diseased chickens. Water supplementation with the 3.5 grams per liter formula significantly suppressed circulating tumor necrosis factor-alpha and interleukin-6 while elevating interleukin-10 concentrations. Concurrently, jejunal tissue homogenate analyses demonstrated substantial upregulation of the mucosal barrier proteins mucin-2, occludin, claudin-1, and zonula occludens-1, which had been severely depleted during infection, with high-dose herbal treatment restoring mucin-2 and zonula occludens-1 to levels comparable to uninfected controls.

Despite these favorable experimental findings, the investigators identified several experimental constraints and interpretive boundaries that must guide translational application. A notable divergence existed between the in vitro minimum inhibitory concentration of 15.63 milligrams per milliliter and the effective in vivo drinking water concentration of 3.5 grams per liter, indicating that therapeutic protection in live animals relied on gut physiological modulation rather than direct luminal bactericidal eradication alone. In addition, tight junction and mucin protein quantities in jejunal homogenates were quantified as concentration values in nanograms per milliliter without normalization to total soluble protein content, potentially constraining absolute quantitative comparisons. Furthermore, the study did not include high-throughput sequencing of the intestinal microbiome, leaving the extent of microfloral community restructuring unresolved. The authors emphasized that future research must delineate exact molecular targets within quorum-sensing systems, evaluate pathogen-adhesion dynamics under non-cytotoxic sub-inhibitory conditions, and confirm both product stability and clinical efficacy across commercial, field-scale poultry production environments.

Subject of Research: Drinking-water Chinese herbal formula as an antibiotic alternative for broiler necrotic enteritis

Article Title: Drinking-water Chinese herbal formula as an antibiotic alternative for broiler necrotic enteritis: efficacy and mechanism

Article References: Liu, Y., Li, S., Han, Z., Wang, Z., Chen, L., Wang, X., Li, Q., Zhang, Z., Wu, T., & Jia, Q. (2026). Drinking-water Chinese herbal formula as an antibiotic alternative for broiler necrotic enteritis: efficacy and mechanism. Frontiers in Veterinary Science, 13, Article 1899410. https://doi.org/10.3389/fvets.2026.1899410

Image Credits: AI Generated

DOI: 10.3389/fvets.2026.1899410

Keywords: necrotic enteritis, Clostridium perfringens, broiler chickens, Chinese herbal formula, antibiotic alternatives, Phellodendri Cortex, Berberidis Radix, Lignum Sappan, Chebulae Fructus, isoliquiritigenin, feed conversion ratio, average daily gain, intestinal barrier integrity, tight junction proteins, bacterial biofilm inhibition, swarming motility, jejunal morphology, poultry health