A self-designed Chinese herbal formula alleviates GAstV infection-induced gout in goslings by inhibiting xanthine oxidase (XOD) and regulating intestinal microbiota

Abstract

Goose gout, a metabolic disorder characterized by systemic urate deposition in visceral organs (e.g., liver, kidneys) and joints of goslings, is strongly associated with goose astrovirus (GAstV) infection and environmental stressors such as high humidity. This disease severely compromises growth performance and survival rates, yet no licensed prophylactic or therapeuti interventions are currently available. Here, we investigated the efficacy of SIA, a Chinese herbal formula comprising Saposhnikovia divaricata, Isatis indigotica and Astragalus membranaceus against GAstV-induced gout in goslings. An experimental model was established by combining oral GAstV challenge with high-humidity environmental exposure. We systematically evaluated the protective effects of SIA by monitoring mortality rates, growth parameters (body weight, feed conversion ratio), serum biomarkers (uric acid, creatinine, inflammatory cytokines), and histopathological alterations in target organs. Furthermore, 16S rRNA sequencing was employed to analyze shifts in intestinal microbiota composition. Notably, medium-dose SIA administration significantly reduced mortality (p<0.05), restored growth performance, and attenuated hepatic and renal pathologies. Serum analysis revealed marked decreases in serum uric acid and creatinine levels, alongside suppression of pro-inflammatory cytokines. Mechanistically, SIA treatment modulated gut dysbiosis by reducing the relative abundances of pathogenic taxa, including Oceanobacillus picturae, Veillonellales, Streptococcus equinus and Synechococcus while promoting commensal microbiota associated with intestinal homeostasis.These findings highlight the dual prophylactic and therapeutic potential of SIA against goose gout, likely mediated through multi-target mechanisms involving renal urate excretion, anti-inflammatory activity, and gut microbiota regulation. This study provides a scientific foundation for developing SIA as a phytotherapeutic agent for goose gout management.

Introduction

Goose gout is a metabolic disorder characterized by the dysregulated protein metabolism, leading to hyperuricemia due to either overproduction of uric acid or impaired renal excretion (Chen et al., 2020). This pathological condition results in the deposition of monosodium urate crystals in joints and visceral organs. The etiology goose gout is multifactorial, with primary triggers including goose astrovirus (GAstV) infection, dietary calcium-phosphorus imbalance, and suboptimal husbandry practices..As the largest global producer of geese, China has faced escalating GAstV outbreaks since its first detection in 2017. A recent epidemiological survey of 15 goose farms across multiple provinces revealed a 35 % GAstV-positive rate (300 clinical samples analyzed), highlighting its nationwide prevalence. These outbreaks have precipitated substantial economic losses in the poultry industry (Yin et al., 2021; Zhang et al., 2017), underscoring the urgent need for effective control strategies.

GAstV is a non-enveloped, icosahedral virus with a diameter of 28-30 nm,. exhibiting distinctive star-like surface projections under electron microscopy. The virus's genome (6.9 −7.9 kb) consists of a 5′ untranslated region (UTR), three open reading frames (ORF1a, ORF1b, and ORF2), a 3′ UTR, and a polyadenylic acid tail (poly A) (Liu et al., 2018). Functional analysis indicates that ORF2 encodes the capsid protein VP90, essential for viral particles assembly and host cell entry. ORF1a and ORF1b, conversely, encode non-structural proteins including a proteases and RNA-dependent RNA polymerase (RdRp), which orchestrate viral genome replication and transcription regulation (Arias and DuBois, 2017; Zhang et al., 2023; Zhu et al., 2024). GAstV spreads primarily via the fecal-oral route, with highest susceptibility observed in goslings under three weeks of age. Clinical signs include gross pathological lesions such as urate deposition on cardiac surfaces, renal tubules, and ureters, accompanied by synovial swelling. Notably, field outbreaks demonstrate alarming morbidity and mortality rates of up to 80 % and 50 %, respectively, positioning GAstV as a critical pathogen threatening goose production sustainability.

Current gout management relies on small-molecule drugs including Allopurinol, Benzbromarone, and Colchicine. Although effective for hyperuricemia control, prolonged administration induces adverse effects such as hepatotoxicity (elevated ALT/AST), gastrointestinal disturbances, and renal impairment (Fontana et al., 2021). These safety concerns contraindicate their use in livestock production. Traditional Chinese medicine (TCM) offers multi-target therapeutic advantages with favorable safety profiles, aligning with antibiotic-free farming initiatives (Liang et al., 2021). While classic formulas (e.g., Simiao San, Xuanbi Decoction, and Duhuo Jisheng Decoction) demonstrate efficacy in human gout management through anti-inflammatory and urate-lowering mechanisms (Fan et al., 2021; Wang et al., 2020; Xu et al., 2022), their application in avian species remains unexplored.In this study, Sini Tang (SNT) was selected as the positive control, mainly for the following core reason: first, derived from Treatise on Febrile Diseases, it is a classic TCM formula for "warming yang to rescue adverse qi and warming the middle-jiao to dispel cold".It can target the characteristic of "gosling gout induced by cold and humid environment" in this study (both modeling and clinical observations showed that low temperature and high humidity significantly increased the incidence rate). Through the synergistic effect of Aconitum carmichaelii (warming the whole body's yang qi) and Zingiber officinale (warming the spleen to dispel cold), it matches the core pathogenesis of "cold bi syndrome" in gosling gout (Lu et, al., 2023).Second, preliminary experiments confirmed that 5 mL/kg SNT could stably reduce serum uric acid levels and alleviate urate deposition in the liver and kidneys of gouty goslings, meeting the requirement for effective intervention as a positive control.Third, it possesses the characteristics of multi-component and multi-pathway regulation, whose action mode is similar to that of the tested compound SIA. This can avoid the interference of "drug category differences" on the interpretation of experimental results and ensure the comparability of the control.

Saposhnikovia divaricata (Fangfeng) demonstrates efficacy in wind-dispelling and dampness-resolving, alleviating pain, and suppressing convulsions. Experimental evidence confirms its ability to reduce joint swelling and inhibit the inflammatory cell infiltration, corresponding pathologically with gout manifestations (Chun et al., 2016); Isatis indigotica (Banlangen) exhibits heat-clearing and detoxifying properties, with documented antiviral activity against respiratory syncytial virus (RSV), porcine reproductive and respiratory syndrome virus (PRRSV), and influenza virus. Its bioactive components impede viral infection through three mechanisms: adsorption inhibition, invasion blockade, and suppression of viral nucleic acid/protein synthesis (D. Jiang et al., 2022; Wang et al., 2018; Zhang et al., 2023). Astragalus membranaceus (Huangqi) enhances DNA synthesis in bone marrow hematopoietic cells, accelerates nucleated cell mitotic, and promotes nucleic acid metabolism. These actions facilitate efficient conversion of purines into readily excretable metabolites, thereby reducing uric acid accumulation. Additionally, it improves immune function and microcirculation, enhancing livestock growth performance and economic benefits (Chen et al., 2023; Han et al., 2024; Liu et al., 2017). Based on this rational, we hypothesize that the SIA formulation - comprising Saposhnikovia divaricata (Apiaceae), Isatis indigotica (Brassicaceae), and Astragalus membranaceus (Fabaceae) - may inhibit GAstV infection and attenuate organ inflammation. To test this hypothesis, a goose gout model was established through oral GAstV challenge under high-humidity conditions., Model goslings were subsequently treated with SIA. The prophylactic and therapeutic efficay of SIA was evaluated by assessing: mortality rate, growth performance, serum biochemical parameters, and histopathological alterations in target organs. Furthermore, intestinal microbiota composition was analyzed using 16S rRNA sequencing to investigate potential mechanisms of action.

Materials and methods

Rejuvenation of GAstV on Goose Embryos and Determination of 50 % Egg Infectious Dose (EID₅₀) of Goose Embryos

The GAstV strain CH—HNXX-2021 (provided by Prof. Wang Zeng) was thawed from −80°C and inoculated (0.2 mL/embryo) onto the chorioallantoic membranes of 11-day-old goose embryos under aseptic conditions. After 7 days of incubation at 37°C, early mortalities (<24 h post-inoculation) were discarded. Surviving or dead embryos were chilled at 4°C before samples collection. Harvested tissues and allantoic fluid were collected, homogenized, and centrifuged at 8,000 rpm for 5 minutes. The supernatant was then filtered through a 0.22 µm membrane filter and stored at −80°C. For viral titration, 10-fold serial dilutions (10¹ to 10⁶) of the stock were inoculated into six 11-day-old goose embryos per dilution, with parallel saline-inoculated control. Following 7-day incubation at 37°C, samples were collected, and EID₅₀ was determined by RT-PCR and the Reed-Muench method.

Preparation of experimental drugs

Saposhnikovia divaricata, Isatis indigotica and Astragalus membranaceus (Henan Runhong Bencao Pharmaceutical Co., Ltd., Batch No. Z41020627) were weighed in the selected ratio. The herbs were soaked in 5 - fold water for 30 min, decocted twice for 60 min each, and the combined filtrates were concentrated under reduced pressure to 1 g crude drug/mL, aliquoted, and stored for feed mixing.Sini Tang, a classic TCM formula derived from Shang Han Lun (Treatise on Febrile Diseases), consists of three herbs: Aconitum carmichaelii (Fuzi), Zingiber officinale (Ganjiang), and Glycyrrhiza uralensis (Zhigancao). All herbs were purchased from Hebei Anguo TCM Decoction Piece Co., Ltd. (Batch No.: 20240315) and authenticated by the TCM Identification Laboratory of Henan Agricultural University, conforming to the quality standards of the Chinese Pharmacopoeia (2020 Edition, Volume I). The herbs were weighed at a ratio of 3:2:1 (Fuzi:Ganjiang:Zhigancao), soaked in 8-fold purified water for 40 min, boiled with strong fire then simmered for 90 min (to reduce the toxicity of Aconitum carmichaelii), and the filtrate was concentrated under reduced pressure to 1 g crude drug/mL. The prepared SNT was aliquoted and stored at 4°C for feed mixing, consistent with the administration route of SIA.

Animal experiments

Forty 1-day-old goslings (purchased from Zhengzhou Ruixiang Poultry Co., Ltd., unvaccinated, and negative for goose astrovirus) were randomly divided into four groups: three GAstV-inoculated groups with high (0.4 mL), medium (0.2 mL), and low (0.1 mL) doses, and a control group. The 50 % egg infectious dose (EID₅₀) of the virus was 1 × 10^1.03/mL, and the viral load was 1 × 10^9.28 copies/mL. Each group contained 10 goslings. The virus challenge was administered via oral gavage at 1 day of age, the experimental period lasted for 14 days, and all goslings were reared in an environment with a relative humidity of 80 ± 5 %.Additionally, 40 one-day-old goslings were randomly divided into 4 groups, including 3 goose astrovirus (GAstV)-inoculated groups (high dose: 0.4 mL, medium dose: 0.2 mL, low dose: 0.1 mL) and 1 control group. The experimental procedure was consistent with that of the aforementioned group, with the only difference being that the goslings were housed in an environment with a relative humidity of 50 ± 5 %.

To screen the optimal dosage of SIA for prophylactic and therapeutic purposes in gosling gout, 300 one - day - old goslings were randomly assigned to ten groups.The control group received an oral administration of 0.2 mL physiological saline as the negative control. The model group was orally inoculated with 0.2 mL of goose astrovirus (GAstV) allantoic fluid (1 × 10^9.28 copies/mL) on day 1. The SIA prevention groups (P-LSIA, P-MSIA, P-HSIA) and therapy groups (T-LSIA, T-MSIA, T-HSIA) were fed diets supplemented with low (5 mL/kg), medium (10 mL/kg), and high (20 mL/kg) doses of SIA before and after the onset of symptoms, respectively. The positive control groups (P-SNT, T-SNT) were treated with 5 mL/kg Sini Tang (SNT) via feed at the same time points as the prevention and therapy groups. The environmental humidity for all goslings was measured using a hygrometer. Cardboard boxes were used for feeding, and sterile PBS water was sprayed on the boxes to maintain the humidity within the desired range. If the humidity was found to be outside the range, the boxes were replaced immediately.And the goslings had free access to feed and water. This experimental protocol has been approved by the Scientific Ethics Committee of Henan Agricultural University (Approval No.: HNND202400347).

Observation of the overall status of goslings and determination of growth performance

Record the daily feed intake and body weight of goslings in each group, then calculate the Average Daily Feed Intake (ADFI), Average Daily Gain (ADG), and the Feed-to-Gain ratio (F/G). The ADG is calculated using the formula: ADG = (Final body weight - Initial body weight) / Number of experimental days. The ADFI is calculated using: ADFI = Total feed intake / (Number of goslings in each group × Number of experimental days). The F/G ratio is calculated using: F/G = ADFI / ADG.

Determination of serum biochemical indicators and liver XOD activity

After the experiment, 5 goslings per group were randomly selected. Intravenous injection of pentobarbital sodium injection was performed at an anesthetic dose of 25 mg/kg. Cardiac blood collection was conducted, and the blood samples were allowed to stand at room temperature for 30 minutes. Serum was then separated via centrifugation at 3000 rpm for 15 minutes. Serum creatinine (CRE), blood urea nitrogen (BUN), uric acid (UA), xanthine oxidase (XOD), alanine transaminase (ALT), and aspartate transaminase (AST) were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute).The activity of XOD in liver tissue was also determined using a commercial kit from Nanjing Jiancheng Bioengineering Institute (catalog number: A002-1-1).

Detection of serum inflammatory factors

After separating the serum, measure the contents of AST,ALT,ALB,TP,GLB,Cre,BUN,XOD,IFN - γ, IL - 1β, IL - 6, and iNOS using an ELISA kit (Jiangsu Enzyme Immunoassay Industry Co., Ltd.). The operation steps should be strictly carried out in accordance with the instructions.

HistoPathological changes

The tissue samples were fixed in a 4 % paraformaldehyde solution. The samples were dehydrated in a series of graded alcohols, cleared with xylene, and embedded in paraffin. Subsequently, the samples were serially cut into 4 - micrometer - thick sections and stained with hematoxylin - eosin using the conventional method. The stained sections were observed under an optical microscope (Carl Zeiss, Germany).

Collection of cecal contents and 16S sequencing

Following the experiment, the cecal contents of five randomly selected goslings from each group were collected and immediately stored at −80°C for subsequent analysis. The extraction of microbial DNA was conducted in accordance with the instructions provided by the DNeasy PowerSoil Pro Kit from Qiagen (Hilden, Germany). After quality control checks using a Qubit 2.0 fluorometer, agarose gel electrophoresis, and an Agilent 2100 Bioanalyzer, the DNA was utilized to construct a DNA library. The V4 region of the bacterial 16S rRNA was amplified, and the sequencing was carried out on the Illumina platform by Personal Biotechnology Co., Ltd. (Shanghai, China).

Processing of 16S rDNA sequence data

During preprocessing, the demux plugin of QIIME 2 was initially used to demultiplex the pooled sequences, followed by primer removal using cutadapt. Two distinct analysis paths were then pursued: one was DADA2-based denoising, setting a maximum expected error threshold to remove low-quality sequences, merge the remaining ones, eliminate chimeras, and generate an ASV table. The second path utilized Vsearch-based clustering, where Vsearch functions were employed for sequence merging, filtering, and deduplication. After two rounds of clustering at different similarity levels, OTU representative sequences and a table were produced. Subsequently, mafft was employed for multiple sequence alignment, and fasttree2 was used to construct a phylogenetic tree based on the aligned sequences. For diversity analysis, the diversity plugin was used to calculate alpha and beta diversity indices, with sample sequences being rarefied to a uniform depth before the analysis. Finally, the feature classifier's classifier was utilized to compare the sequences with a reference database to complete taxonomic assignment.

Statistical analysis

A one-way analysis of variance (one-way ANOVA) was employed to analyze the data of various parameters across different experimental groups. The outcomes were represented as mean ± standard deviation. A difference was deemed statistically significant when P < 0.05, and highly statistically significant when P < 0.01.(Table.1).

Table 1.

Key nutritional indicators of diet for goslings within 14 days of age (based on NYT 4641-2025).

Items	%	Nutrient levels	%
Ingredients Maize Wheat flour Soybean meal (46 % CP) Fish meal (60 % CP) Whey powder Vegetable oil Limestone Calcium hydrogen phosphate Sodium chloride L-Lysine HCl DL-Methionine Threonine Choline chloride (50 %) Mineral and vitamin premix¹ Total	40.0 15.0 28.0 5.0 4.0 2.0 1.2 1.0 0.3 0.6 0.3 0.2 0.1 0.3 100.0	Metabolizable energy (MJ/kg) Crude protein Calcium Total phosphorus Lysine Methionine	12.15 21.0 0.95 0.65 1.15 0.55

All values are expressed as content per kilogram of diet.

Results

Quantitative evaluation of EID₅₀ in the rejuvenation of GAstV virus

The cytotoxic CH—HNXX-2021 was inoculated and cultured in goose embryos. Following the cultivation, the goose embryos were dissected. Thickening of the allantoic membranes of the goose embryos (Fig. 1B), embryonic hemorrhage with stunted growth (Fig. 1D) were observed. After virus inoculation, the allantoic fluid, membranes, and embryo bodies of the goose embryos were collected. Total RNA was extracted, and RT-PCR detection was performed using specific primers for goose astrovirus. The measured viral load was 1 × 10^9.28 copies/mL. The test results for determining the median infectious dose of the embryo bodies are presented in Table 2. Based on these results, the Reed-Muench method was employed to calculate the median infectious dose (EID₅₀) of the goose embryos, which was 1 × 10^1.03/mL.

Fig. 1.

Pathological conditions of goose embryos. The allantoic membrane of goose embryos in the control group is normal (A), while that in the GAstV group is thickened (B). The embryo body of goose embryos in the control group is normal (C), and Goose embryos in the GAstV-infected group exhibited hyperemic changes in embryonic tissues (D).

Table 2.

Determination results of the median infective dose of goose embryos.

Inoculation concentration	Number of deaths	Number of infections	The number of effective inoculations	Infection rate
virulent stock	2	6	6	100 %
10-1	1	5	6	83.33 %
10-2	0	2	6	33.33 %
10-3	0	0	6	0
10-4	0	0	6	0
Control	0	0	0	0

Under humid conditions, oral administration of GAstV can better simulate goose gout

During the experiment, goslings in the control groups under both humidity conditions maintained normal mental status, diet, water intake, and defecation, with no mortality observed. In contrast, under the rearing humidity of 80 ± 5%, goslings in the high-dose GAstV group (GAstV-H group) and medium-dose GAstV group (GAstV-M group) began to show clinical symptoms from the 3rd day of the experiment, which were characterized by depression, pale and loose feces, and accompanied by mortality.Post-mortem examination results showed that under both humidity conditions, no obvious lesions were observed in the heart, liver, or kidneys of goslings in the control groups. In contrast, compared with the condition of rearing humidity at 50 ± 5%, obvious urate deposition was visible on the surfaces of the heart and liver in goslings of the GAstV-H and GAstV-M groups under the environmental humidity of 80 ± 5%. Goslings in the low-dose GAstV group (GAstV-L group) exhibited pale livers with more severe damage (Figure S1A and Figure 2A). Under both environmental conditions, the kidneys of goslings in the high-dose and medium-dose groups showed swelling with bleeding spots, and white urate deposition was observed in the bilateral ureters.Under the rearing humidity of 80 ± 5%, the mortality rates of goslings in each group were as follows: 70% in the GAstV-H group, 50% in the GAstV-M group, and 10% in the GAstV-L group (Figure 2B). Among these, the mortality rate of the GAstV-M group was highly close to that of clinical gosling gout. Under the rearing humidity of 50 ± 5%, the mortality rates of goslings in each group were 30% in the D-GAstV-H group, 10% in the D-GAstV-M group, and 0% in the D-GAstV-L group (Figure S1B). After GAstV infection, compared with the control group, the serum uric acid levels of goslings in the GAstV-H and GAstV-M groups increased significantly (P < 0.01), and were higher than those in the D-GAstV-H and D-GAstV-M groups (Figure S1C). In addition, the weight growth of goslings in the GAstV-H, GAstV-M, and GAstV-L groups was significantly inhibited, with a substantial decrease in body weight compared with the control group (P < 0.01) (Figure 2D). Based on the aforementioned observations, the GAstV-M group (medium-dose group) was selected as the optimal viral dose for establishing the gosling gout model, due to its relatively high mortality rate of 50% and significantly elevated serum uric acid levels.

Fig. 2.

Changes in geese with or without GAstV infection. Changes in visceral organs in the control group and the GAstV group (A), changes in body weight (B), and changes in mortality rate (C).Values are expressed as mean ± standard deviation, n = 4,***P < 0.001.

SIA can reduce the death rate and improve the growth performance of goslings with gout

Goslings' mortality rate in the model group was great, reaching 50 % compared to the control group. The feed-to-gain ratio (F/G) was significantly increased (P < 0.01), and the final weight and average daily gain were significantly reduced (P < 0.01). In the gout prophylaxis study, the average daily weight gain in the P-HSIA group was significantly improved (P < 0.01), F/G was significantly reduced (P < 0.01), and mortality rate was 30 %. The average daily intake and F/G were significantly reduced in the P-MSIA group (P < 0.05), and the average daily weight gain was significantly improved (P < 0.01), and the mortality rate reached 10 %. The mean daily weight gain was significantly improved in the P-LSIA group (P < 0.01), the F/G was significantly reduced (P < 0.01), and the mortality rate was 20 %. It can be seen that medium doses of SIA effectively reduce the effect of goose astrovirus infection on Goslings' growth performance and may have a positive impact on Goslings' growth.

Interestingly, in the gout therapeutic experiment, the average daily gain was significantly increased in the T-HSIA group and the F/G was significantly decreased compared with the model group (P < 0.01). The mortality rate was 20 %. The mean daily feed intake and F/G were significantly reduced in the T-MSIA group (P < 0.01) with a mortality rate of 6.7 %. However, the weight of this group significantly increased at the end of the experiment (P < 0.01). The final weight and mean daily gain in the T-LSIA group significantly increased (P < 0.01), with a significant decrease in the F/G (P < 0.01), and a mortality rate of 13.3 %. This suggests that the medium dose of SIA can significantly improve the growth performance of goslings during therapy, surpassing the effects of SNT (Table 3). Therefore, medium-dose of SIA was used in this study.

Table 3.

The influence of SIA on the mortality rate and growth performance of goslings.

Group	initial weight(g)	final weight(g)	ADFI(g/d)	ADG(g/d)	F/G	death rate (%)
Control	80.95±7.0	212.76±25.1*	20.11±1.0	9.71±0.5***	2.07±0.1***	0
Model	80.26±6.5	164.54±51.1	21.60±0.2	6.51±0.4	3.33±0.2	50.0
P-SNT	79.60±5.7	192.49±21.6	20.87±0.5	7.78±0.4*	2.69±0.2***	23.3
P-HSIA	79.75±8.0	195.33±36.8	20.68±0.5	8.32±0.2***	2.49±0.1***	30.0
P-MSIA	79.67±7.1	209.20±37.8*	19.51±0.8*	9.61±0.6***	2.03±0.1***	10.0
P-LSIA	79.50±8.1	198.04±21.2	20.36±0.7	8.37±0.1***	2.43±0.1***	20.0
T-SNT	79.44±9.2	201.92±39.5	20.26±0.5	7.96±0.4**	2.55±0.2***	23.3
T-HSIA	79.55±7.4	204.73±30.6	20.78±0.4	8.91±0.1***	2.33±0.1***	20.0
T-MSIA	80.13±6.9	220.47±29.6**	19.24±0.6**	10.41±0.4***	1.85±0.1***	6.7
T-LSIA	80.82±6.3	213.37±39.4**	19.87±0.6	9.45±0.1***	2.10±0.1***	13.3

Values are expressed as mean ± standard deviation,n = 30,Compared with Group Model,Values are expressed as mean ± standard deviation, n = 30, *P <0.05; **P <0.01; ***P <0.001.

SIA can enhance the immunity of goslings with gout and reduce the load of GAstV in the livers and kidneys of goslings

The effects of SIA against GAstV infection and on the organ index of goslings are shown in Fig. 3. Compared to the control group, the model group exhibited a marked elevation in the spleen index (P < 0.01) but displayed a significant reduction in the bursa of Fabricius index (P < 0.01), indicating distinct organ-specific immune responses. Concurrently, GAstV viral loads in the kidneys, liver, and feces samples of the model group were substantially upregulated (P < 0.01), suggesting enhanced viral replication following immune dysregulation. In the prophylactic experiment, both P-SNT and P-MSIA therapies induced a downward trend in liver indices compared to the model, albeit without statistical significance (P > 0.05). Notably, these groups exhibited a significant reduction in spleen indices (P < 0.01), accompanied by elevated bursa of Fabricius indices. Specifically, the P-MSIA group demonstrated a pronounced increase in the bursa of Fabricius index(P < 0.01), indicating its potential to enhance the immune capability of goslings. Following viral challenge at 14 days post-infection (dpi), both P-SNT and P-MSIA therapies significantly suppressed GAstV replication in the kidneys and liver (P < 0.01). Although a decline in viral loads was observed in fecal samples, the reduction did not reach statistical significance (P > 0.05).

Fig. 3.

Effects of the SIA on the immune organ indices of geese with gout and the viral load of GAstV. Liver index (A), Spleen index (B), Bursa of Fabricius index (C), Viral load of GAstV (D).Values are expressed as mean ± standard deviation, n = 5,*P < 0.05; **P < 0.01; ***P < 0.001.

In the therapeutic experiment, both T-SNT and T-MSIA therapies induced a significant reduction in spleen indices (P < 0.01). Concurrently, the bursa of Fabricius indices were elevated across all therapy groups, with the T-MSIA group exhibiting a markedly higher increase (P < 0.01). These findings collectively demonstrate that T-MSIA enhances adaptive immunity in goslings, potentially through promoting B-lymphocyte maturation in the bursa of Fabricius. At 14 days post-challenge (dpc), T-SNT and T-MSIA therapies significantly attenuated renal viral loads (P < 0.05), while T-MSIA further achieved a robust suppression of hepatic viral replication (P < 0.01). Although a downward trend in viral loads was observed in the fecal samples, the reduction lacked statistical significance (P > 0.05).

SIA can alleviate the liver damage of goslings with gout

SIA significantly attenuated gout - associated hepatotoxicity in goslings, as evidenced by histopathological and gross morphological analyses (Fig. 4). Gross examination revealed that control group livers exhibited a uniform light - red coloration with distinct and regular edges, whereas the model group exhibited hepatic atrophy, surface dullness, and macroscopic urate deposition on the hepatic and cardiac surfaces. Notably, both SNT and SIA interventions abolished visible urate crystal accumulation (Fig. 4A).

Fig. 4.

Effects of SIA on the livers of goslings infected with GAstV. (A) Liver injury during necropsy; (B) Liver pathological sections. In the control group, hepatocytes showed no obvious lesions with clear hepatic cord structures. The model group exhibited marked vacuolar degeneration, along with extensive aggregation of inflammatory cells and erythrocytes. The P - SNT group had minimal inflammatory cell infiltration, while the P - MSIA group showed milder vacuolar degeneration compared to the model group. The T - SNT group had severe inflammatory cell infiltration, and the T - MSIA group showed only slight vacuolar degeneration. Blue arrows denote erythrocyte aggregation, green arrows indicate inflammatory cell aggregation, and orange arrows signify vacuolar degeneration.

Histologically, HE - stained liver sections from the control group showed intact hepatic cords and absence of pathological alterations. In contrast, livers in the model group demonstrated marked vacuolar degeneration, disrupted parenchymal architecture, and extensive inflammatory infiltrates admixed with erythrocyte aggregation. Prophylactic administration of P - SNT partially mitigated inflammation (minor inflammatory cell infiltration), while P - MSIA further reduced vacuolar degeneration severity compared to the model group (P < 0.05) (Fig. 4B).

In the therapeutic experiment, T - MSIA - treated goslings maintained near - normal hepatocyte morphology with minimal vacuolation, whereas T - SNT group livers showed persistent severe inflammatory infiltration. (Fig. 4B). These findings demonstrate that SIA outperforms SNT in ameliorating GAstV - induced hepatic injury.

SIA ameliorat GAstV-induced renal pathology in goslings

SIA significantly ameliorated GAstV-induced renal pathology in goslings, as demonstrated by gross morphological and histopathological evaluations (Fig. 5). Gross examination revealed control group kidneys maintained well-defined contours with smooth surfaces, devoid of distortion or atrophy. In stark contrast, GAstV-infected goslings exhibited renal enlargement, ureteral urate crystal accumulation, and mottled parenchyma. Prophylactic P-MSIA therapy preserved normal renal architecture, while P-SNT group kidneys displayed mild subcapsular hemorrhage and edema (Fig. 5A).Hematoxylin-eosin (HE) staining of kidney tissues from different therapy groups revealed distinct morphological differences. In the control group, renal tubules maintained their structural integrity with no discernible pathological alterations. Conversely, the model group exhibited characteristic signs of injury, including infiltration of inflammatory cells, necrosis and exfoliation of renal tubular epithelial cells, as well as cellular swelling. The P-MSIA group showed evidence of epithelial cell degeneration, although the extent of necrosis was markedly reduced compared to the model group (Fig. 5B). Notably, the T-MSIA group demonstrated a striking recovery phenotype, with clearly defined tubular structures, well-organized epithelial cell layers, and a lax interstitium devoid of fibrotic changes. Collectively, these data indicate that SIA confers greater renoprotective effects than SNT, effectively mitigating renal damage and promoting overall kidney health.

Fig. 5.

Effects of SIA on gosling gout - related kidney damage. (A) Gross examination: The control group had clear - contoured, smoothly - edged kidneys; GAstV group kidneys were swollen, mottled, with bilateral ureteral urate deposits; P - SNT group kidneys showed mild hemorrhage and edema, while P - MSIA group kidneys were normal. (B) Histopathology: Control group had intact renal tubules; the model group showed inflammatory cell infiltration, necrotic and shed epithelial cells, and cell swelling; T - MSIA group had clear - structured tubules, orderly - arranged epithelium, and non - fibrotic loose interstitium. The blue arrow denotes inflammatory cell infiltration; the red arrow, necrosis and shedding of renal tubular epithelial cells; the yellow arrow, swelling of these cells.

SIA can alleviate the abnormalities in the physiological functions of the liver and kidneys of goslings with gout and inhibit the production of xanthine oxidase (XOD)

The influence of SIA on the liver function of goslings is shown in Fig. 6. The serum liver function indicators showed a significant increase in the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the model group relative to the control group (P < 0.01). This trend reversed following therapy with SNT and SIA. In the prophylactic experiment, the levels of ALT were notably reduced in P-SNT and P-MSIA groups compared to the model group (P < 0.05). Similarly, both P-SNT and P-MSIA significantly decreased the AST levels (P < 0.05) (Fig. 6A - B). In the therapeutic experiment, the ALT content in T-SNT and T-MSIA groups showed a remarkably significant decrease (P < 0.01), and the AST levels were significantly reduced (P < 0.05). Notably, there was no significant difference in the serum levels of albumin (ALB), total protein (TP), and globulin (GLB) across all groups (P > 0.05) (Fig. 6C - E).

Fig. 6.

The Influence of the SIA on the Liver Function of Geese with Gout.ALT(A),AST(B),ALB(C),TP(D),GLB(E),Values are expressed as mean ± standard deviation, n = 5,*P < 0.05; **P < 0.01; ***P < 0.001.

The influence of SIA on the liver function of goslings is shown in Fig. 7. The results of the measurement of renal function indicators in serum indicated significant increases in blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), and xanthine oxidase (XOD) levels in the model group compared to the control group(P < 0.05). In addition, the hepatic XOD activity results showed that, compared with the control group, the hepatic XOD activity level was also significantly increased (P < 0.01).In the prophylactic experiment, both UA and XOD levels decreased significantly (P < 0.01) under the intervention of SNT and SIA, with P-MSIA showing the most promising results, nearly matching those of the control group (Fig. 7A-D). In the therapeutic experiment, the CRE level in the T-MSIA group significantly decreased compared to the model group (P < 0.05). Both SNT and SIA therapies could significantly reduce UA and XOD levels, with the T - MSIA group exhibiting the most effective results (Fig. 7A - E).

Figure 7.

Effects of the SIA on kidney function and XOD in goslings with gout. BUN (A), CRE (B), UA (C), XOD in serum (D), Liver XOD activity(E),Values are expressed as mean ± standard deviation, n = 5,*P < 0.05; **P < 0.01; ***P < 0.001.

SIA can improve the pathological damage of the small intestine in goslings with gout

SIA significantly ameliorates gout-induced pathological lesions in the gosling small intestine. Histopathological examination (Fig. 8A) demonstrated that the model group exhibited intestinal wall thinning, villus atrophy (↓ villus height), and crypt hyperplasia (↑ crypt depth) compared to controls. While the P - SNT group showed partial improvement in intestinal wall thickness and crypt depth (P < 0.01), villi height remained reduced. The T - SNT group displayed significant reduction in crypt depth (P < 0.001). In contrast, T-SIA administration substantially increased villus height (P < 0.001) and reduced crypt depth (P < 0.01). Quantitative analysis (Fig. 8B-D) confirmed that both prophylactic (P-MSIA) and therapeutic (T-MSIA) SIA interventions: Increased villus height by 45.79 % and 91.91 % vs. model (P < 0.01), Decreased crypt depth (T-MSIA: decreased by 28.6 %, P < 0.01), Elevated villus/crypt (V/C) ratio (T-MSIA:1.5-fold vs. T-SNT, P < 0.01). These results indicate SIA’s superior efficacy over SNT in mitigating intestinal mucosal damage, particularly during therapeutic application.

Fig. 8.

Effects of the SIA on the intestinal tissues of geese with gout. Sections of pathological damage of intestinal tissues (A), intestinal villus height (B), crypt depth (C), the ratio of intestinal villus height to crypt depth (D), Values are expressed as mean ± standard deviation, n = 5,*P < 0.05; **P < 0.01; ***P < 0.001.

SIA can reduce the levels of pro - inflammatory factors in goslings with gout

The effect of SIA on the inflammatory factors in goslings is shown in Fig. 9. Serum inflammatory factors TNF-α, IFN-γ, IL-1β, iNOS, and IL-6 were significantly elevated in the model group versus controls (P < 0.001). Substantial reductions in TNF-α, IFN-γ, IL-1β, iNOS and IL-6 in SNT and SIA groups versus model (P < 0.01; Fig. 9A-E). Critically, SIA exhibited superior anti-inflammatory efficacy relative to SNT, with more robust cytokine suppression across prophylactic and therapeutic regimens.

Fig. 9.

Effects of the SIA on the serum inflammatory factors of geese with gout. Tumor necrosis factor-α (TNF-α) (A), interferon-γ (IFN-γ) (B), interleukin-1β (IL-1β) (C), inducible nitric oxide synthase (iNOS) (D), interleukin-6 (IL-6) (E), Values are expressed as mean ± standard deviation, n = 5,*P < 0.05; **P < 0.01; ***P < 0.001.

SIA can ameliorate gosling gout through modulation of gut microbiota composition

SIA modulates the gut microbiota composition in gout-afflicted goslings (Fig. 10). Operational taxonomic unit (OTU) clustering revealed significant OTU reduction in all treatment groups versus controls (P < 0.05, Fig.10A), though α-diversity (Shannon/Simpson indices) showed no intergroup differences (P > 0.05). Non-metric multidimensional scaling analysis (NMDS) based on Bray-Curtis distance (stress value = 0.0707, indicating a good fit) showed clear separation between the model and the control groups, while convergence of T-MSIA samples toward the control cluster (Fig. 10C). At the phylum level, model group increased Bacillota (+71.79 %) with decreased Bacteroidota (−96 %) and Pseudomonadota (−75 %) versus controls, while partial reversal of these trends in T-SIA/T-SNT, especially microbial profile of T-MSIA were closest to controls (Fig. 10D). At the species level, model group increased Enterococcus cecorum(+1.5-fold), while decreased Phocaeicola vulgatus, Corynebacterium bovis, Akkermansia muciniphila, Phocaeicola dorei, Bacteroides thetaiotaomicron, Blautia wexlerae, and Niallia nealsonii versus controls,. In contrast, the T-SNT group increased Ligilactobacillus aviarius and Akkermansia muciniphila, while decreased Enterococcus cecorum, Corynebacterium bovis, and Phocaeicola dorei versus model group. The T-MSIA group increased Phocaeicola vulgatus(+30-fold) with overall composition resembling controls (Fig. 10E).

Fig. 10.

Effects of SIA on cecal microbiota.Operational Taxonomic Unit (OTU) analysis (A); α-diversity analysis (B); β-diversity analysis (C); species composition at the phylum level (D); species composition at the species level (E); Linear discriminant analysis Effect Size (LEfSe) analysis of differential microbiota (F); correlation analysis between microbiota and protection rate, liver/kidney function indices, inflammatory factor indices, and relative abundance of species (G-H); relationship between Akkermansia muciniphila and IL-1β (I); Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (J-K); relationship between Ko00281 (pathway enriched in the T-MSIA group) and serum XOD (L);Values are expressed as mean ± standard deviation, n = 5, **P < 0.01.

Based on the Lefse differential analysis, Corynebacterium amycolatum and Blautia marasmi were remarkably enriched in the control group; Oceanobacillus picturae, Veillonellales, Streptococcus equinus and Synechococcus were substantially enriched in the model group; Corynebacterium freneyi and Globicatella sanguinis were highly significantly enriched in the T-MSIA group; Lactobacills crispatvs, Lactobacills helveticus and Romboutsia timonensis were remarkably enriched in the T-SNT group (Fig. 10F). In this experiment, except for some unclassified and unnamed bacterial genera, Shigella, Acinetobacter, Escherichia, Corynebacterium freneyi and Globicatella sanguinis exhibited a statistical correlation trend of negative association with the levels of ALT, AST, CRE, BUN, and UA in the serum of goslings (P < 0.05); correspondingly, these genera showed a trend of positive association with the protection rate.In contrast, Oceanobacillus and Sporolactobacillus showed a trend of positive association with the contents of ALT, AST, CRE, BUN, and UA in the serum, and a trend of negative association with the protection rate (P < 0.05) (Fig. 10G). Further analysis demonstrated that Oceanobacillus and Sporolactobacillus were highly significantly enriched in the GAstV group, while Shigella, Escherichia, Corynebacterium freneyi and Globicatella sanguinis showed higher abundances in the T-MSIA group (Fig. 10H).Furthermore, correlation analysis revealed a negative correlation between Akkermansia muciniphila and IL-1β (Fig. 10I).The differential analysis of KEGG metabolic pathways indicated that, compared with the control group, the model group was upregulated in ko00523 (nitrogen metabolism pathway), ko00460 (cytokine - cytokine receptor interaction pathway) and ko00253 (tetracycline biosynthesis pathway); compared with the model group, the T-MSIA group was upregulated in the ko00281 pathway (Fig. 10J and K).In contrast, the ko00281 pathway exhibited a negative correlation with serum xanthine oxidase (XOD) (Fig. 10L).

Discussion

Goose Astrovirus (GAstV) induces highly lethal visceral gout in goslings, with mortality rates reaching 50 %, posing a substantial threat to waterfowl production due to the absence of licensed prophylactics or therapeutic (Hou et al., 2024; Zhu et al., 2022). Our study demonstrates that the herbal formulation SIA significantly: reduced mortality by 43.3 %, suppressed viral load (P < 0.001), attenuated systemic inflammation (TNF-α/IL-1β /IL-6 decreased by32-38 %, P < 0.001), ameliorated hepatorenal pathology , improved growth performance (FCR decreased by44 %, P < 0.05). These multi-target effects position SIA as a promising candidate for clinical management of GAstV-induced gout, potentially addressing critical gaps in veterinary therapeutics.

To establish a goose gout model, GAstV-infected goslings were administered three viral doses. The 0.2 mL GAstV-M dose was determined as optimal for consistent pathology induction. High-humidity exposure exacerbated inflammatory response and hyperuricemia, facilitating gout development. This finding aligns with prior research (Bi et al., 2024). Oral inoculation precisely replicated natural infection routes (Ding et al., 2021), providing a reliable platform for evaluating SIA’s prophylactic and therapeutic efficacu against goose gout.

The GAstV-challenged model group exhibited 50 % mortality following oral inoculation. During the 14-day trial with SIA administered via feed mixture: the therapeutic group’mortality was 6.7 %, lower than the prophylactic group of 10 %. The protection efficacy reached 93.3 %, indicating a significant protective effect of SIA against goose astrovirus. Compared with the control group, the average daily gain and final body weight of goslings in the model group decreased significantly (P < 0.05), and the feed - to - gain ratio increased significantly (P < 0.05), SIA could reverse this trend. Based on the data from this experiment, we hypothesize that the growth-promoting effect of SIA on goslings infected with GAstV may be attributed to its synergistic effects on intestinal morphology repair, hepatic and renal function optimization, and immune regulation.Compared with the model group, the increased villus height in the T-MSIA group directly expands the intestinal absorption area, while the reduced crypt depth decreases the energy consumption required for intestinal epithelial renewal (Bai et al., 2024; Tang and Xiong, 2022). The liver serves as the core organ for glucose and lipid metabolism. Compared with the model group, the decreased ALT/AST levels in the SIA group indicate the restored activity of metabolic enzymes in hepatocytes (e.g., pyruvate kinase, fatty acid synthase). This restoration promotes carbohydrate oxidative energy supply and fat storage. (Chen et al., 2025; Wang et al., 2022) Improved renal function reduces the energy expended on uric acid crystal clearance, diverting more energy to protein synthesis (e.g., muscle growth). This is consistent with the result that the final body weight of the T-MSIA group was higher than that of the model group (Table 3). SIA avoids excessive energy consumption in inflammatory responses by inhibiting pro-inflammatory cytokines, which may ultimately redirect immune-related energy expenditure toward growth metabolism. This is consistent with previous reports that Astragalus membranaceus polysaccharides can promote DNA synthesis in bone marrow hematopoietic cells and improve the immune-growth negative feedback. (Yao et al., 2023)

SIA significantly ameliorated hepatic and renal dysfunction in gout-afflicted goslings through multi-target mechanisms. Liver function restoration was evidenced by reduced ALT (↓55 %, P < 0.001) and AST (↓46 %, P < 0.01) versus the model group, indicating reversal of transaminase elevation and attenuated hepatocellular damage. Kidney function improvement manifested as:Kidney function improvement manifested as:decreased BUN (↓36 %, P < 0.01) and CRE (↓57 %, P < 0.01), suppressed XOD activity (↓45 %, P < 0.001) and UA levels (↓52 %, P < 0.001), inhibiting purine-to-urate conversion.. BUN, or Blood Urea Nitrogen, is the principal end product of human protein metabolism. Primarily filtered through the glomerulus, it is then excreted in urine. In the event of kidney function damage, the glomerular filtration rate diminishes, consequently reducing BUN excretion and leading to an increased concentration of BUN in the blood (Liu et al., 2016).The blood level of Creatinine (CRE) is an important indicator of glomerular filtration function. When this function deteriorates to a certain degree, the blood CRE levels significantly increase, potentially aggravating kidney damage. Xanthine Oxidase Dehydrogenase (XOD) is an essential enzyme in purine metabolism, catalyzing the conversion of hypoxanthine into xanthine, which subsequently produces uric acid - a byproduct of purine metabolism (L. Jiang et al., 2022; Lu et al., 2024). Elevated activity of XOD will promote higher uric acid production, thereby increasing uric acid levels in the blood. This elevated uric acid level constitutes a crucial biochemical foundation for gout. When the blood's uric acid concentration exceeds its solubility, monosodium urate crystals precipitate as uric acid precipitates. These crystals deposit in tissues such as joints and kidneys, potentially inducing a gout attack (Gao et al., 2022; Zhou et al., 2024). Concomitantly, SIA exerted potent anti-inflammatory effects via reduction of TNF-α (↓38 %), IFN-γ (↓39 %), IL-1β (↓36 %), and iNOS (↓37 %) (P < 0.001),validating Astragalus membranaceus-mediated immunomodulation. These biochemical improvements correlated with hepatic and renal histopathological organ protection. Concerning kidney protection, SIA maintains the kidneys' normal functions via various pathways. These include promoting the excretion of uric acid, inhibiting uric acid production, and mitigating inflammatory damage to the kidneys.

The gut microbiota of GAstV-infected goslings showed a dysbiotic pattern, with a notable increase in the relative abundance of Oceanobacillus picturae; this change showed a positive correlation trend with serum uric acid levels (P < 0.05, adjusted for small sample size). As a moderate halophile, O. picturae has the potential to secrete antimicrobial substances (e.g., lipopeptide antibiotics), which may affect microbial equilibrium. O. picturae was significantly enriched in the model group and exhibited a positive correlation with uric acid levels, suggesting its potential contribution to gout pathogenesis under GAstV infection. Concurrently, enhanced nitrogen metabolism (ko00523) promoted excessive catabolism of nitrogenous compounds → amino acids → purine nucleotides → uric acid overproduction. This metabolic dysregulation, combined with monosodium urate crystal-induced activation of the cytokine-cytokine receptor pathway (ko00460) and IL-1β release, exacerbated systemic inflammation. After SIA treatment, the branched-chain amino acid degradation pathway (ko00281) is enriched. Correlation analysis shows a negative correlation between this pathway and serum xanthine oxidase (XOD) activity. We speculate that the enrichment of the ko00281 pathway will exert a positive effect on the treatment of GAstV infection, but its specific mechanisms remain to be explored. (Abe and Yamamoto, 2021; Muraoka et al., 2020).

Overall, SIA demonstrates significant therapeutic efficacy against goose gout, reducing mortality, mitigating hepatorenal damage, and modulating systemic inflammation. Its multi-target actions—encompassing intestinal microbiota restoration, uric acid homeostasis regulation, and immune modulation—support its potential as a clinically viable prophylactic and therapeutic agent for GAstV-induced gout. Further studies should elucidate precise molecular mechanisms underlying these effects.

CRediT authorship contribution statement

Xue Zhang: Data curation, Conceptualization. Miaoge Zhu: Investigation, Formal analysis. Hongyan Cui: Project administration, Methodology. Junyang Fan: Supervision, Software. Mingfan Yang: Validation. Yue Jin: Visualization. Sai Mao: Writing – original draft. Hongying Zhang: Resources, Funding acquisition.

Disclosures

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.