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Ingestion of Artemisia argyit essential oil combats Salmonella pullorum infections by altering gut microbiota composition in chicks

Veterinary Research volume 56, Article number: 98 (2025) Cite this article

Abstract

Pullorum disease, caused by Salmonella pullorum (S. pullorum), is a highly contagious illness affecting the poultry industry. Emerging evidence suggests that Artemisia argyit essential oil can influence the composition of gut microbes in the host, thereby promoting overall health. However, the specific mechanisms by which Artemisia argyit essential oil modulates gut microbiota to combat S. pullorum infection remains unclear. This study explored the effectiveness of various doses of Artemisia argyit essential oil in preventing S. pullorum infection in chicks. Our findings indicate that consuming this essential oil can mitigate the intestinal mucosal barrier damage and excessive inflammatory response caused by S. pullorum, as well as reverse the weight loss seen in infected chicks. Additionally, chicks that received faecal microbiota transplantation (FMT) from the gut microbiota of Artemisia argyit essential oil donors exhibited notable recovery from S. pullorum infections. This suggests that the observed protection may be linked to the modulation of gut microbiota. Furthermore, 16S rRNA sequencing revealed an increased abundance of Lactobacillus reuteri (L. reuteri), which along with the activation of Wnt/β-catenin pathways, played critical roles in the enhanced health of S. pullorum-infected chicks treated with Artemisia argyit essential oil. In summary, these findings highlight that the dietary inclusion of Artemisia argyit essential oil promotes the intestinal enrichment of L. reuteri, offering a promising strategy for the treatment and prevention of pullorum disease in chicks.

Introduction

The chick industry has experienced rapid growth over the past half-century, becoming the second-largest breeding industry in China [1]. This growth is largely due to the industry’s short feeding cycles, and its ability to provide energy, protein, and essential micronutrients to humans. However, high-density feeding may lead to immune stress or disease in chicks, which negatively impacts their growth performance and the quality of their meat [2].

Newly hatched chicks are particularly susceptible to Salmonella pullorum (S. pullorum). Infections can result in symptoms such as lethargy, loss of appetite, intestinal damage, and high mortality rates [3, 4]. Furthermore, S. pullorum can persist in the macrophages of the infected chicks throughout their lives, posing a risk of transmission to offspring and causing various health issues [5].

For decades, antibiotics have been used effectively to prevent and treat pathogenic infections in chicks. However, the long-term use of antibiotic growth promoters (AGP) disrupts the balance of gut microbiota, leads to antibiotic drug residues, and fosters the development of drug-resistant bacteria, which poses serious risks to public health and contributes to environmental pollution [6, 7].

In contrast, Traditional Chinese Medicine (TCM) offers promising alternatives with several advantages, including ready availability, low toxicity, minimal clinical side-effects, significant structural diversity, and biocompatibility, which solidifies its efficacious anti-infection effects [8, 9]. Growing evidence suggests that gut microbiota can serve as a reference for guiding the clinical application of TCM [10]. TCM helps establish microbiome homeostasis, control pathogen proliferation [11], and improves meat quality and immunity in poultry production [12].

Among the various natural products derived from TCM, essential oils have gained significant attention due to their effective properties. Studies have shown essential oils can significantly reduce colonisation of Clostridium perfringens and Campylobacter jejuni in chicks [13,14,15]. One notable example is Artemisia argyit, a traditional herbal medicine in China for over one thousand years. The essential oil extracted from Artemisia argyit (AAEO), commonly known as mugwort (Chinese name: aicao), contains a variety of aromatic compounds that offer pharmacological properties, including anti-inflammatory and antioxidant effects [16].

Collectively, essential oils present great potential in combating bacterial infections in chickens. However, there is still limited knowledge regarding the effectiveness of Artemisia argyit essential oil in modulating gut microbiota homeostasis to alleviate bacterial infections in chicks.

The intestinal microbiota, often referred to as the “invisible organ”, plays a crucial role in improving the digestive system and maintaining gastrointestinal homeostasis [17]. This population of microorganisms residing in the intestine significantly impacts both growth performance and host health [18]. It is well established that intestinal microorganisms influence host physiology in various ways. In cases of pathogen infection, an imbalance in gut microbiota can lead to decreased immune function, inflammatory diseases, and impaired intestinal function [19]. Therefore, establishing a stable gut microbiome in chicks is essential for combating bacterial infections [20].

Recent studies have focused on the effects of probiotics on chick intestinal development, microstructure and microbiological characteristics [21, 22]. Among the major probiotics found in the intestinal tracts of chickens, Lactobacillus reuteri (L. reuteri) has garnered much attention for its role in restoring growth performance and preventing intestinal damage in chicks [23,24,25]. Additionally, L. reuteri supplementation can help mitigate the adverse effects of Staphylococcus aureus-induced mastitis in mice by enhancing intestinal barrier function [26].

Encouragingly, previous research has shown that Artemisia argyi polyphenols can alleviate dextran sulfate sodium (DSS)-induced ulcerative colitis by altering gut microbiota [27]. In particular, Artemisia argyi polysaccharide treatment enhances the abundance of Lactobacillus in the gut microbiota of mice [28, 29]. These findings suggest that extracts from Artemisia argyi possess probiotic properties that influence the composition and metabolism of the microbiota, thereby improving intestinal homeostasis in the host.

However, it remains unclear whether Artemisia argyit essential oil can regulate gut microbiota to strengthen the intestinal barrier and provide therapeutic benefits against S. pullorum infection in chicks.

The present study aimed to investigate the effects of Artemisia argyit essential oil on the recovery of growth performance and the inhibition of intestinal damage in chicks affected with S. pullorum. Additionally, it sought to explore the possible mechanisms behind these effects. This research provides valuable insights into the pathogenesis of S. pullorum infection and offers practical guidance for developing targeted interventions.

Materials and methods

Bacteria culture conditions

Salmonella pullorum (SP), used in this study, was generously provided by Prof. Jingmin Gu from Jilin University. The bacteria were resuscitated in Luria–Bertani (LB, HopeBiol, China) broth culture medium at 37 ℃ and shaken at 200 rpm for 16 h.

L. reuteri, also used in this study, was kindly provided by Prof. Yunhe Fu from Jilin University. The bacteria were cultured in MRS (De Man, Rogosa and Sharpe) medium (Qingdao HopeBiol, China) for 24 h under anaerobic conditions at 37 °C.

Animals and experimental design

Animal experimentation protocols for this study received approval from the Ethics Committees of the Laboratory Animal Centre of Jilin University (No. SY202405028). White-feathered chicks were obtained from the Zhengda Group Breeding Base (Dewei, China). All chicks were randomly assigned in a completely randomised design and were exposed to 16 h of light each day. The room temperature was maintained at 34 °C during the first week and was gradually decreased to 30 °C by the 14th day. All chicks had free access to food and water [30]. The basal diet used in this experiment was formulated in accordance with the nutritional requirements outlined in the Chinese Feeding Standard for Chickens (NY/T33-2004) (Table 1). The animal experiments were conducted in the following three stages.

Table 1 The composition and nutrient levels of diets
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S. pullorum infections in chicks and the therapeutic effects of Artemisia argyit essential oil

Studies have demonstrated the negative effects of Salmonella challenges on chick growth performance [31,32,33]. To assess the protective effect of Artemisia argyit essential oil against S. pullorum-infections, a model using S. pullorum-infected chicks was established (Figure 1A). A total of 150 one-day-old chicks were randomly divided into five treatment groups, with five replicates of six chicks each:

  • NC group: chicks challenged with carboxymethyl cellulose (CMC-Na) solution

  • SP group: chicks challenged with S. pullorum

  • EOL, EOM and EOH groups: received 30, 60, and 100 mg/kg/day of Artemisia argyit essential oil via oral gavage from day 0 to day 14, respectively.

Figure 1
figure 1

Effect of Artemisia argyi essential oil on growth performance and internal organs bacteria colonization of chicks challenged by S. pullorum. A Experimental protocol of the Artemisia argyi essential oil treatment. Chicks were treated with Artemisia argyi essential oil for 14 days, and challenged with S. pullorum on days 8–10. B–D Effect of Artemisia argyi essential oil on GH (B), Ghrelin (C), and IGF-1(D) levels in serum challenged with S. pullorum (n = 6). E–G Evaluation of S. pullorum colonised in the liver (E), spleen (F) and ileum (G) of chicks (n = 6). Data are expressed as the mean ± SD. #P < 0.05, ##P < 0.01 vs the uninfected (NC) group; ns P > 0.05, *P < 0.05, **P < 0.01 vs the S. pullorum infection (SP) group.

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To ensure consistent and standardised infection, all inoculation experiments involved the oral administration of 500 μL of S. pullorum (4 × 108 CFUs/mL) on the 8th day [34], followed by inoculations for three consecutive days. The average daily gain (ADG), average feed intake (ADFI), and feed-to-gain ratio (F/G) were calculated on days 7 and 14 of the study, respectively. On the 14th day, caecal contents were collected and immediately transferred to liquid nitrogen for storage until microbial 16S rRNA analysis.

Faecal Microbiota Transplantation (FMT)

The faecal suspension was prepared as described previously [35, 36]. Fresh feces from chicks receiving 100 mg/kg/day of Artemisia argyit essential oil group for seven days were collected and mixed with sterile phosphate buffered saline (PBS) at a ratio of 1:6. This mixture was then placed in a tissue grinder (JingXin, China) and thoroughly ground. The supernatant was collected and stored for subsequent FMT experiments.

A total of 90 one-day-old chicks were selected as recipients for the FMT treatment and were randomly assigned into three groups: NC, SP and FMT. Each group consisted of five replicates, with six chicks per replicate. The chicks in the FMT group were orally administered 1 mL of the prepared faecal microbiota suspension daily for a consistent 14-day period. During the experiment, the body weights, ADG, ADFI, and F/G of the chicks were recorded on days 7 and day 14. The infection of S. pullorum was induced through oral gavage, following the method described previously [34].

Probiotic validation experiment

One-day-old chicks were randomly divided into three groups (n = 6 per group): the NC group, the SP group, and the SP + L. reuteri group. The SP + L. reuteri group received an administration of L. reuteri (1 × 108 CFUs/day/chick) to help re-establish the gut microbiota, before inducing the S. pullorum infection model [22]. The S. pullorum infection was conducted via oral gavage, following previously established methods [34].

Samples collection

In therapeutic experiments involving Artemisia argyit essential oil and FMT procedures, blood samples were collected from the wing veins on day 14, which marked the end of the experiment. Following this, euthanasia was performed on all chicks via jugular vein puncture. Under aseptic conditions, liver, spleen, and ileum tissues were collected from the subjects. All samples were immediately stored at − 80 °C until analysis. Additionally, ileum samples (approximately 1 cm) were collected and fixed in 4% paraformaldehyde.

Determination of growth factors in serum

ELISA kits (Jiangsu Meimian Industrial Co., Ltd., China) were used to measure levels of ghrelin, insulin-like growth factor-1 (IGF-1), and growth hormone (GH) serum samples. The serum naturally solidifies at room temperature, and the supernatant was collected by centrifugation at 12 000 rpm for 10 min.

Determination of bacteria in the internal organs

The bacterial load in Salmonella-infected chicks was enumerated following previously described methods [37]. Liver, spleen, and ileum samples were weighed and then homogenised in sterile PBS for 30 min. The homogenate was diluted to the appropriate volume with sterile PBS, and each sample was placed on Xylose Lysine Deoxycholate (XLD) agar plates for microorganism counting.

Histological analysis

Chick ileum tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and stained with haematoxylin and eosin (H&E) [38]. The intestinal morphology of various groups was observed using light microscopy. Villus height and crypt depth were measured using CaseViewer software, and the ratio of villus height to crypt depth (V/C) was calculated.

Total RNA extraction and quantitative RT-qPCR

As previously described [39], RNA was extracted from frozen ileum tissues using TransZol Up (TransGen, China). The extracted RNA was then reverse transcribed using a cDNA reverse transcription kit (Mei5bio, China). Real-time qPCR was conducted on a Step One Plus system (Applied Biosystems, Foster City, USA). β-actin served as the internal control, and the 2−△△Ct method was utilised to quantify the relative mRNA expression levels of the genes [40]. The primers used in this analysis are detailed in Additional File 1 [41].

Inflammatory cytokine and oxidative stress factor detection

ELISA kits (Jiangsu Meimian Industrial Co., Ltd., China) were used to measure the levels of key inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-1β (IL-1β), and nuclear factor kappa-B (NF-κB), as well as oxidative stress factors such as malondialdehyde (MDA), superoxide dismutase (SOD) in ileum tissue samples. To prepare a homogenate of the ileum tissue, PBS was used, and the supernatant was collected by centrifugation at 12 000 rpm for 10 min at 4 °C. The levels of corresponding cytokines were detected following the manufacturer's instructions, and absorbance was measured at 450 nm using a microplate reader (Tecan, CH).

16S ribosomal RNA (16S rRNA) sequencing and data analysis

Microbial DNA was extracted from the caecal contents of chicks (TianGen, China) following the manufacturer’s protocol. Spike-in sequences, along with bacterial 16S rRNA sequences from each DNA sample, were amplified across the variable regions V3-V4 using primers F (5′-GTGCCAGCMGCCGCGGTAA-3′) and R (5′-GGACTACHVGGGTWTCTAAT-3′). The amplifies sequences were then sequenced by Novogene Inc. (China) on the Novaseq 6000 platform (Illumina, San Diego, USA).

The raw data obtained from the sequencing process contained some interference data, which could affect the accuracy of the information analysis. To address this, the raw data was aggregated and filtered to obtain valid data. Noise reduction was then performed using DADA2 or denoising techniques, resulting in the final ASVs.

Statistical analysis

GraphPad Prism 9.3 (GraphPad software) was utilised for statistical analysis. All data are presented as the mean ± SD. Significant differences between the two groups were assessed using a t-test (parametric). For comparisons involving more than two groups, one-way ANOVA was employed. The P values are categorised as follows: ns P > 0.05; *P < 0.05; **P < 0.01.

For analysing caecal microbiota data, a t-test and Wilcoxom rank-sum test were applied for two subgroups, while Tukey and Kruskal–Wallis rank-sum tests were used for groups with more than two subgroups to calculate alpha diversity. The significance of segregation in PCoA was tested using R software version 4.0.3 with a weighted UniFrac distance measure. Linear discriminant analysis (LDA) combined with effect size measurements (LEfSe) was used to identify bacterial differences among groups. The functional contents of the intestinal metagenome was estimated using PICRUSt 2.

Results

Artemisia argyit essential oil improves the health of chicks affected by S. pullorum infection

The growth performance of the chicks at two stages (days 1 to 7 and days 7 to 14) was presented in Table 2. From days 1 to 7, before the S. pullorum challenge, Artemisia argyit essential oil supplementation (30, 60, and 100 mg/kg) exerted no significant effect on BW, ADG, ADFI and F/G of chicks compared with the NC group (P > 0.05). However, from day 7 to 14, the results showed that S. pullorum infection substantially reduced the BW, ADG, ADFI and potentiated the F/G of chicks in comparison with the NC group (P < 0.05). Compared with the infected group, the supplementation of 100 mg/kg Artemisia argyit essential oil significantly increased BW, ADG, ADFI and reduced the F/G of chicks (P < 0.05). Based on the above findings, Artemisia argyit essential oil addition alleviated growth retardation of S. pullorum-challenged chicks.

Table 2 Effect of Artemisia argyit essential oil on growth performance of chicks challenged by S. pullorum
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Since the essential oil of Artemisia argyit improved chick growth performance, we further analysed whether the growth-related hormones in chick serum were affected. We found that the levels of GH, Ghrelin, and IGF-1 secretion were significantly lower in the S. pullorum-infected group compared to the NC group. In contrast, the levels of Ghrelin and IGF-1 were dose-dependently elevated in chicks fed Artemisia argyit essential oil (30, 60, and 100 mg/kg) when compared to the SP group (P < 0.05). Notably, only the chicks supplemented with 100 mg/kg of Artemisia argyit essential oil showed increased levels of GH (P < 0.05, Figure 1B–D).

Next, we examined the effects of the 100 mg/kg supplementation of Artemisia argyit essential oil on chick physiology and gut microbiota in the following analyses.

Additionally, the supplementation of Artemisia argyit essential oil reduced the burden of S. pullorum in the liver, spleen, and ileum of chicks compared to the S. pullorum-infected group (P < 0.05, Figure 1E–G). Overall, these results suggested that Artemisia argyit essential oil may help alleviate S. pullorum-infection in chicks by reducing bacterial colonisation and preventing translocation of bacteria.

Artemisia argyit essential oil inhibits damage caused by S. pullorum and prevents the excessive release of inflammatory factors

Due to the negative effects of S. pullorum invading intestinal cells, we investigated the protective effect of Artemisia argyit essential oil on the intestines of chicks infected with S. pullorum. As illustrated in Figure 2A, the intestines of chicks infected with S. pullorum exhibited swelling of the ileum and significant accumulation of digestive products in the caecum. This condition was alleviated following treatment with Artemisia argyit essential oil. Specifically, the ileum crypt depth in the chicks decreased (P < 0.05), while both villus height and the V/C were significantly improved (P < 0.05) after treatment with Artemisia argyit essential oil (Figure 2B–D).

Figure 2
figure 2

Protective effect of Artemisia argyi essential oil on intestinal damage caused by S. pullorum infection. A Intestinal lesions and H&E staining in S. pullorum-infected chicks treated with or without Artemisia argyi essential oil. B–D The villus height (B), crypt length (C), and ratio of the villus to crypt length (D) of the ileum (n = 3). E–H Ileum MUC-2 (E), ZO-1 (F), Claudin-1 (G), and Occludin (H) mRNA levels were assessed by qPCR in chicks (n = 3). I–N Evaluation of chick ileum IL-1β (I), IL-6 (J), IL-10 (K), NF-κB (L), SOD (M), and MDA (N) levels with ELISA kits (n = 6). Data are expressed as the mean ± SD. #P < 0.05, ##P < 0.01 vs the uninfected (NC) group; ns P > 0.05, *P < 0.05, **P < 0.01 vs the S. pullorum infection (SP) group.

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Moreover, the expression of mucosa barrier-related genes in the ileum revealed that S. pullorum infection downregulated the expression of ZO-1, Claudin-1, MUC-2, and Occludin (P < 0.05), which was reversed by supplementation with 100 mg/kg of Artemisia argyit essential oil (P < 0.05, Figure 2E–H). This suggested that Artemisia argyit essential oil may help restore intestinal barrier integrity.

In terms of inflammatory cytokines in the ileum, the infiltration of pro-inflammatory factor IL-1β, IL-6, and NF-κB cytokines significantly increased in the ileum tissues of chicks infected with S. pullorum (P < 0.05). Conversely, the anti-inflammatory factor IL-10 was notably reduced due to the infection (P < 0.05). Notably, the antioxidant defense functions in chicks treated with S. pullorum were impaired, as indicated by a reduction in the antioxidant enzyme SOD and an increase in the peroxidation product MDA levels (P < 0.05).

However, treatment with Artemisia argyit essential oil mitigated the S. pullorum-induced inflammatory markers (P < 0.05, Figure 2I–L) and improved antioxidant function (P < 0.05, Figure 2M, N). Collectively, these results suggested that Artemisia argyit essential oil is effective in alleviating elevated intestinal inflammatory factors and restores intestinal barrier function in chicks infected with S. pullorum.

Artemisia argyit essential oil alters the structure of the gut microbial community in chick

To investigate the effects of Artemisia argyit essential oil on the gut microbiota of chicks, we conducted 16S rRNA sequencing on cecal content samples from chicks subjected to the different treatments. There were no significant differences in the alpha-diversity indicators of gut microbiota among the groups (P > 0.05, Additional file 2). To analyse the similarity or differences in gut microbial community structure across samples, we assessed the β-diversity of cecal microorganisms using PCoA analysis. As shown in Figure 3A, the PCoA analysis revealed a significant difference in the composition of gut microbiota among the three groups.

Figure 3
figure 3

Microbiome analysis of faecal samples from S. pullorum-infected chicks treated with or without Artemisia argyi essential oil. A PCoA analysis of the gut microbiota of healthy (NC), S. pullorum-infected (SP) and chicks treated with 100 mg/kg/day of Artemisia argyi essential oil and SP infection chicks (EOH), data were analysed using unweighted UniFrac (n = 6). B Comparative analysis of gut microbial OTUs of NC, SP, and EOH group chicks (n = 6). C The top 5 species at the phylum levels among different groups (n = 6). D The top 10 species at the genus levels among different groups (n = 6). E, F Differential caecal microbiota at genus levels among different groups (n = 6). NC: Uninfected chicks, SP: Chicks infected with S. pullorum (SP), EOH: Supplementation of 100 mg/kg/day Artemisia argyi essential oil (EOH) and S. pullorum-infected chicks.

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In this study, we identified 743 OTUs in the cecal contents of all groups, with 178 core OTUs shared among all groups. Additionally, there were 150, 145, and 158 OTUs unique to the NC, SP, and EOH groups, respectively (Figure 3B). Differences in microbial abundance at the phylum level among all groups were illustrated in Figure 3C. The top five dominant phyla identified were Bacteroidota, Firmicutes, Proteobacteria, Actinobacteriota, and Deinococcota.

Figure 3D highlights the major bacterial genera enriched in the cecal contents, with Bacteroides, Lactobacillus, Ruminococcus, Erysipelatoclostridium, and Sellimonas being the most prominent. Notably, at the genus level, Ruminococcus and Sellimonas were enriched in the EOH group, compared to the SP group. Conversely, Lactobacillus was found to be depleted in the SP group compared to the NC group (Figure 3E and F).

These findings demonstrated that S. pullorum infection disturbs the stability and function of the chick gut microbiota. However, significant changes in the composition of gut microbiota were observed following supplementation with Artemisia argyit essential oil. Importantly, the dominant gut microbiota in chicks was found to be significantly greater than in the SP group, suggesting that gut microbes may play a beneficial role in the inflammatory response to S. pullorum infection.

FMT of Artemisia argyit essential oil donor microbiota protects chicks from S. pullorum infection

The FMT model was established to investigate the potential of gut microbiota in mediating the protective effects of Artemisia argyit essential oil against S. pullorum infection in chicks (Figure 4A). Our findings indicated that the FMT group exhibited greater therapeutic benefits compared to the SP group. This was evident through increased levels of growth-related hormones (GH, Ghrelin, and IGF-1) (P < 0.05, Figure 4B–D), as well as a reduction in bacterial burden in tissue (P < 0.05, Figure 4E).

Figure 4
figure 4

FMT from Artemisia argyi essential oil treated chicks alleviates S. pullorum infection in chicks. A Illustration of FMT. FMT model in S. pullorum-infected chicks (NC: chicks without FMT treatment; SP: S. pullorum-infected chicks; FMT + SP: supplementation of FMT in S. pullorum-infected chicks (n = 6). B–D Effect of FMT on GH (B), Ghrelin (C), and IGF-1 (D) levels in serum of S. pullorum-infected chicks (n = 6). E Evaluation of S. pullorum colonization in chick visceral after FMT treatment (n = 6). F–I Intestinal lesions and histopathological (H&E staining) representative images of lesions in FMT treated S. pullorum-infected chicks (F), the villus height (G), crypt length (H), V/C (I) of the ileum (n = 3). J–M The mRNA levels of chick ileum mucosal genes Claudin-1 (J), MUC-2 (K), Occludin (L) and ZO-1 (M) were assessed by qPCR (n = 3). N–S Inflammatory parameters of ileum from different groups, including IL-1β (N), IL-6 (O), IL-10 (P), NF-κB (Q), MDA (R) and SOD (S) concentrations were measured (n = 6). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 vs the S. pullorum infection (SP) group.

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Additionally, chicks in the FMT group significant increases in body weight, ADG, and ADFI compared to the SP group from days 7 to 14. The F/G ratios for the FMT group were also considerably lower (P < 0.05) than those of the SP group (Table 3).

Table 3 Effect of FMT supplementation on growth performance of chicks challenged with S. pullorum
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Moreover, the FMT group exhibited less intestinal injury relative to the SP group, as shown by improved ileum villi height and V/C, along with a reduced crypt depth (P < 0.05, Figure 4F–I). Similar to the results observed with Artemisia argyit essential oil treatment, FMT corrected the decreases in the ileum TJ proteins Claudin-1, Occludin, ZO-1, as well as mucin MUC-2, which were caused by S. pullorum infection in chicks (P < 0.05, Figure 4J–M).

Consistently, FMT lowered the concentrations ileal cytokines, such as IL-1β, IL-6, NF-κB, and the peroxidation product MDA, while increasing levels of the anti-inflammatory factor IL-10 and the antioxidant enzyme SOD compared to the S. pullorum infection (P < 0.05, Figure 4N–S). Overall, FMT enhanced resistance to S. pullorum infection in chicks, suggesting that the effectiveness of Artemisia argyit essential oil against S. pullorum infection is mediated by the modulation of gut microbiota.

Oral administration of Artemisia argyit essential oil elevates the accumulation of probiotics in the gut microbiota

To investigate the interaction between Artemisia argyit essential oil, gut microbiota, and S. pullorum infection, we collected the caecal contents of chicks that were supplemented with Artemisia argyit essential oil for seven consecutive days. These samples underwent 16S rRNA sequencing to assess the effectiveness of the treatment in improving the gut microbiota of chicks.

Alpha-diversity indices—specifically Chao1, Shannon, Simpson, and Observed OTUs—were significantly lower in the EOH group compared to the NC group (Figure 5A–D, P < 0.05). Venn analysis indicated that the gut microbiota of chicks treated with Artemisia argyit essential oil showed marked improvements compared to the NC group. The EOH group exhibited a reduction in observed operational taxonomic units (OTUs) compared to the NC group (Figure 5E). Consistently, PCoA evaluation revealed a significant separation in gut microbial structure between the two groups (Figure 5F).

Figure 5
figure 5

Effects of Artemisia argyi essential oil on the microbial communities in the intestinal of recipient chicks. One-day-old chick were given 100 mg/kg/day of Artemisia argyi essential oil continuously for 7 days, and faecal microbiota was evaluated using 16S rRNA sequencing on day 7. A–D Chao1 index (A), Shannon index (B), Simpson index (C), and Observed_otus (D) showed that EOH group has reduced alpha-diversity, compared to the NC group (n = 6). E Vene analyses demonstrated different OTUs in the NC group and Artemisia argyi essential oil treatment group (EOH) (n = 6). F PCoA scores of chicks fecal based on unweighted unifrac scores (n = 6), indicated that the composition of the gut microbiota was different in the two groups. G–H Bacterial composition of the indicated groups at the phylum (G) and genus (H) levels (n = 6). I–K The relative abundance of Parabacteroides (I), Lactobacillus (J), and Butyricoccus (K) at genus levels (n = 6). L LEfSe was performed to show several bacterial taxa enriched in NC and EOH groups (log10LDA score > 4). NC: Uninfected chick group, EOH: Continuous feeding of 100 mg/kg/day of Artemisia argyi essential oil for 7 days. Data are expressed as the mean ± SD. **P < 0.01 vs the NC group.

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Subsequent calculations of the relative abundances of bacteria following species annotation (Figure 5G–H) showed that the abundances of Lactobacillus and Parabacteroides increased after treatment with Artemisia argyit essential oil, while the abundance of Butyricicoccus decreased (Figure 5I–K, P < 0.05). Additionally, a heatmap (Additional file 3A), and LEfSe (Figure 5L) were employed to analyse species differences. The results of these comparisons were consistent, with Lactobacillus being significantly after the treatment.

Furthermore, functional annotation analysis based on the KEGG database indicated that after treatment with Artemisia argyit essential oil, there was an increased enrichment of genes related to glycosyltransferase involved in cell wall biosynthesis pathways (Additional file 3B). These findings suggested that Artemisia argyit essential oil can remodel gut microbiota, potentially providing protection against S. pullorum infection.

Commensal L. reuteri alleviates S. pullorum infection-aggravated intestinal damage in chicks

According to the 16S rRNA sequencing analysis presented in Figure 5, the primary differential probiotics among gut microbes in chicks treated with Artemisia argyit essential oil were Lactobacillus and Parabacteroides. Notably, while Parabacteroides is considered a core member of the gut microbiota, it has been identified as a producer of LPS [42].

Furthermore, we investigated the intrinsic mechanisms by which L. reuteri may restore the dysfunction of the intestinal epithelial barrier caused by S. pullorum infection. We developed an L. reuteri protection model to assess its effects on the S. pullorum infection process in chicks (Figure 6A). As anticipated, the presence of L. reuteri reduced both the invasion of bacteria into internal organs (Figure 6B) and intestinal damage (Figure 6C). We observed a significant increase in the length of the ileal villi in chicks from the L. reuteri group (P < 0.05), as well as a substantially higher villus-to-crypt length ratio (P < 0.05) compared to the SP group (Figure 6D–F).

Figure 6
figure 6

Effects of supplementation with L. reuteri against S. pullorum-infected chicks. A Schematic diagram of L. reuteri fed to treat S. pullorum-infected chicks. Chicks were treated with L. reuteri for 14 days, and challenged with S. pullorum on days 8–10. B Protective effect of L. reuteri feeding against bacterial colonization of the visceral of S. pullorum-infected chicks (n = 6). C Effects of L. reuteri supplementation on the morphological structure of the ileum intestine of S. pullorum-infected chicks. D–F The villus height (D), crypt length (E), and ratio of the villus to crypt length (F) of the ileum (n = 3). G–J Gene expression levels of Claudin-1 (G), MUC-2 (H), Occludin (I), and ZO-1 (J) in chick ileum were determined by qPCR, and normalised by β-actin expression (n = 3). K–P Determination of inflammatory factor IL-1β (K), IL-6 (L), IL-10 (M), NF-κB (N) and SOD (O), MDA (P) levels in chick ileum by ELISA (n = 6). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 vs the S. pullorum infection (SP) group.

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Additionally, we measured indicators related to barrier integrity and inflammation in the ileum. As depicted in Figure 6G–J, the expression levels of ZO-1, MUC-2, Claudin-1, and Occludin mRNA were significantly higher in the ileum of the L. reuteri group (P < 0.05). Correspondingly, the Elisa results indicated that the L. reuteri intervention group had significantly lower levels of IL-1β, IL-6, NF-κB, and the overoxidation product MDA compared to the SP group. In contrast, the levels of IL-10 and the antioxidant enzyme SOD was markedly elevated in the L. reuteri intervention group (P < 0.05, Figure 6K–P).

Collectively, these findings suggested that the ingestion of L. reuteri may provide beneficial physiological effects to mitigate S. pullorum infection in chicks.

Artemisia argyit essential oil activates the Wnt/β-catenin pathway to repair S. pullorum-induced intestinal epithelia damage

Since higher resistance to S. pullorum and lower bacterial burdens were observed in the groups treated with Artemisia argyit essential oil and L. reuteri, we further examined the protective mechanisms of these treatments against S. pullorum infection. The alteration in the intestinal barrier and the structure of the intestinal mucosa are closely linked to the differentiation of enterocytes. As expected, several intestinal cell differentiation genes—including olfactomedin 4 (OLFM4), proliferating cell nuclear antigen (PCNA), arginosuccinate synthetase 1 (Ass1), and β-galactosidase (Gleb), and adenosine deaminase (Ada)—were upregulated in the L. reuteri treatment group compared to the SP group (P < 0.05, Figure 7A). This upregulation indicates that L. reuteri positively influences the development of intestinal epithelial cells. The observed increase in epithelial proliferation may be attributed to the activation of the Wnt/β-catenin signalling pathway and the inhibition of the Notch pathway. As shown in Figure 7B, the L. reuteri group significantly increased mRNA expression of Wnt3a, β-catenin, Axin2, and Lrp5, indicating activation of the Wnt/β-catenin signalling pathway (P < 0.05). Additionally, the L. reuteri group exhibited a decreased expression of Notch signalling pathway genes (Dll1, Notch1, and Hes1) (P < 0.05, Figure 7C). Finally, we also investigated several promising gut-protecting genes. The results showed that L. reuteri treatment substantially upregulated the expression of HSP70, sodium-dependent glucose transporter 1 (SGLT-1), fatty acid-binding protein 6 (FABP-6), and glucagon-like peptide 2 (GLP-2), compared to the SP group (P < 0.05, Figure 7D).

Figure 7
figure 7

Supplementation with Artemisia argyi essential oil or L. reuteri activates the Wnt/β-catenin signalling pathway in S. pullorum-infected chicks. A and E Supplementation with L. reuteri (A) or Artemisia argyi essential oil (E) upregulated mRNA expressions of genes associated with intestinal epithelial differentiation during S. pullorum infection (n = 3). B and F The comparison of Wnt/β-catenin signalling genes expression in the ileum of L. reuteri (B) or Artemisia argyi essential oil (F) treated S. pullorum-infected chicks (n = 3). C and G The expression levels of Notch signalling genes in the ileum of S. pullorum-infected chicks treated with L. reuteri (C) or Artemisia argyi essential oil (G) were measured by qPCR (n = 3). D and H L. reuteri (D) or Artemisia argyi essential oil (H) treatment increased the expression of intestinal protective genes in the ileum of S. pullorum-infected chicks (n = 3). Data are expressed as the mean ± SD. ns P > 0.05, *P < 0.05, **P < 0.01 vs the S. pullorum infection (SP) group.

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Considering that Artemisia argyit essential oil may mediate the enrichment of gut microbes, particularly Lactobacillus, we investigated whether this oil activated the Wnt/β-catenin pathway. Notably, Artemisia argyit essential oil enhanced the mRNA expression of enterocyte differentiation markers such as OLFM4. Additionally, it significantly increased the expression levels of PCNA, Ass1, Gleb, and Ada (P < 0.05, Figure 7E). These findings suggested that Artemisia argyit essential oil promotes the proliferation and differentiation of intestinal epithelial cells.

Compared to the S. pullorum infection group, the treatment with Artemisia argyit essential oil markedly upregulated the expression of Wnt3a, β-catenin, Axin2, and Lrp5. This was consistent with the enhanced expression observed with L. reuteri treatments, indicating that the Wnt/β-catenin signalling pathway was activated by Artemisia argyit essential oil (P < 0.05, Figure 7F). In contrast, Paneth cells play a crucial role in the proliferation of intestinal stem cells (ISCs) through the secretion of the Notch ligand Dll [43]. However, Artemisia argyit essential oil showed a decreasing trend in the expression of Notch signalling pathway genes (Notch1 and Hes1), suggesting that the Notch signalling pathway was effectively inhibited by this essential oil (P < 0.05, Figure 7G).

Furthermore, Artemisia argyit essential oil promoted the expression of intestinal protective genes, including HSP70, Glp-2, SGLT-1, and FABP-6 (P < 0.05, Figure 7H). These results indicated that L. reuteri, produced in the presence of Artemisia argyit essential oil, mediates the activation of the Wnt/β-catenin pathway and mitigates the intestinal damage induced by S. pullorum in chicks.

Discussion

Pullorum disease, caused by S. pullorum, poses a significant global health challenge for livestock, leading to reduced production performance, intestinal colonisation, and invasion of internal organs in chicks [44, 45]. Notably, chicks are highly susceptible to S. pullorum infection during their first 14 days of life due to their developing gut microbiota [46]. Traditionally, antibiotics have been used to manage Salmonella infections; however, the rapid emergence of multidrug-resistant (MDR) Salmonella strains presents a serious threat to the poultry industry and public health [47]. Consequently, reducing antibiotic has become a crucial necessity. Since 2020, China has implemented strict policies to limit and reduce antibiotic usage in agriculture [48].

In light of this, essential oils are being considered as suitable candidates due to their safety and potential health benefits [49]. This study specifically investigates the effects of Artemisia argyit essential oil on preventing and treating S. pullorum infections by examining its impact on gut microbiota. Our research provides evidence that treatment with Artemisia argyit essential oil alleviates intestinal mucosal barrier damage and intestinal inflammation caused by S. pullorum infection. This is achieved through the remodelling of gut microbiota, particularly through the promotion of L. reuteri. These findings offer a new perspective on regulating intestinal health for the treatment of S. pullorum infections.

This further study investigated the effectiveness of Artemisia argyit essential oil in reducing the inflammatory response and restoring the integrity of the intestinal mucosal barrier in chicks infected with S. pullorum. Changes in chick weight may correlate with intestinal barrier integrity. Research shows that Salmonella can invade the spleen and liver by disrupting gut integrity, leading to inflammation in these organs [50].

The present study found that Artemisia argyit essential oil significantly improved growth performance, as evidenced by increased BW and ADG. Key indicators of intestinal health, mucosal barrier function, permeability, and healing include intestinal morphology (such as villus height, crypt depth, and V/C ratio), intestinal epithelial tight junction proteins (including claudin, occludin, ZO-1, etc.), bacterial colonisation, and the secretion of intestinal inflammatory factors [51]. Our investigation of these parameters revealed that S. pullorum infection led to enhanced colonisation, intestinal damage, and an increased inflammatory response, consistent with previous studies [52].

Furthermore, high-throughput 16S rRNA sequencing demonstrated a significant improvement in the balance and diversity of gut microbiota in the intestines of chicks fed Artemisia argyi essential oil. It has been shown that a reduction in the number of intestinal Firmicutes (such as Bacillus and Lactobacillus) is closely associated with intestinal damage and poor growth in S. pullorum-infected chicks [53]. Interestingly, FMT of gut microbes from Artemisia argyit essential oil donors helped rehabilitate intestinal damage in the infected chicks.

Collectively, these findings suggest that Artemisia argyit essential oil can alleviate S. pullorum infection and intestinal inflammation in chicks by modifying the composition of the gut microbiota.

The intestinal tract of chicks is home to thousands of organisms, forming a complex microbiome. Dysbiosis in gut microbiota can have detrimental effects on the metabolism, immunity, and overall health of the host [54]. To investigate the impact of Artemisia argyit essential oil on gut microbiota, 16S rRNA gene sequencing was performed. Compared to the NC group, significant changes in the levels of Lactobacillus and Parabacteroides were found in the EOH group.

Lactobacillus is noteworthy for its role in defending against pathogenic microorganisms, regulating inflammation, managing gut microbiota, and preventing bacterial infections, along with its recognised safety profile [55]. Studies have indicating that feeding Lactobacillus can improve gut microbiota in chicks [56]. Specifically, Lactobacillus species such as L. reuteri, L. casei, and L. plantarum have all shown protective effects in chicks infected with S. pullorum-infected chicks [57,58,59], which is consistence with our findings. Probiotics from the Lactobacillus genus, particularly L. reuteri, are well-regarded for their excellent probiotic properties. Therefore, this study suggests that Artemisia argyi essential oil may alleviate S. pullorum infection in chicks by enhancing the presence of L. reuteri.

To understand how Artemisia argyi essential oil affects S. pullorum infections by altering gut microbiota with L. reuteri, we analysed the gene expression related to the proliferation and differentiation of chick intestinal epithelial cells. The results showed that Artemisia argyi essential oil significantly increased PCNA mRNA levels in the chick ileum, which serves as a concrete marker of cell proliferation and correlates positively with the number of crypt cells [41]. When pathogenic bacteria threaten the intestine, the intestinal mucosa functions as a barrier [60]. Our findings align with observations of changes in ileum villus height and crypt depth in chicks, indicating that supplementation with Artemisia argyi essential oil enhances the proliferation and maturation of the ileum epithelium during S. pullorum infection.

Moreover, the Wnt/β-catenin signalling is crucial for intestinal development and maintaining intestinal microenvironmental homeostasis, with its dysregulation linked to intestinal injury [61]. The activation of Wnt/β-catenin signalling is associated with the downregulation of Notch genes (Notch1, Dll1, and Hes1), which promotes the differentiation of chick intestinal epithelial cells, in line with previous research [62]. Additionally, it has been reported that Artemisia argyi essential oil enhances the expression of endogenous antimicrobial peptides, which contribute to various biological activities such as immunomodulation and protecting the intestinal barrier [63].

While our study indicates that Artemisia argyit essential oil may alleviate S. pullorum infection progression by increasing Lactobacillus abundance, we recognise several limitations. Besides Lactobacillus, which was the focus of this investigation, there are other beneficial intestinal microorganisms likely have unique mechanisms of resistance to S. pullorum yet to be explored.

Our results indicate that supplementation with Artemisia argyi essential oil alleviated intestinal mucosal damage and substantial intestinal inflammation caused by S. pullorum infection by regulating the gut microbiota (Figure 8). Notably, the relative abundance of L. reuteri in the gut microbiota increased after supplementation. Moreover, with L. reuteri supplementation demonstrated the ability to activate the Wnt/β-catenin pathway and improve intestinal barrier function in chicks infected with S. pullorum. This suggests that targeting this pathway could be crucial in preventing intestinal infections caused by S. pullorum. Overall, these findings provide a mechanistic explanation for the effectiveness of Artemisia argyi essential oil against S. pullorum infection, highlighting potential therapeutic strategies for managing pullorum disease.

Figure 8
figure 8

Potential mechanisms underlying the prevention effect of Artemisia argyi essential oil against S. pullorum infections.

Full size image

Availability of data and materials

The data analysed during the current study are available from the corresponding author on reasonable request. Part of the material in graphic was modified from Servier Medical Art, licensed under a Creative Common Attribution 4.0 Unported License.

Abbreviations

ADG:

average daily gain

ASV:

amplicon sequence variants

ADFI:

average daily feed intake

ASS1:

arginosuccinate synthetase 1

Ada:

adenosine deaminase

Axin2:

axis inhibition protein 2

CMC-Na:

Carboxymethyl cellulose

CFU:

Colony forming units

Dll1:

delta-like 1

DSS:

dextran sulphate sodium

ELISA:

enzyme linked immunosorbent assay

FABP-6:

fatty acid-binding protein 6

F/G:

feed/gain ratio

FMT:

faecal microbiota transplantation

GH:

growth hormone

Gleb:

β-galactosidase

GLP-2:

glucagon-like peptide 2

H&E:

haematoxylin and eosin

HSP70:

heat shock protein 70

Hes1:

hairy and enhancer of split 1

IGF-1:

insulin-like growth factor-1

IL-6:

interleukin-6

IL-10:

interleukin-10

IL-1β:

interleukin-1β

KEGG:

Kyoto encyclopaedia of genes and genomes

LDH:

lactate dehydrogenase

L. reuteri :

Lactobacillus reuteri

Lrp5:

lipoproteins receptor related protein 5

LB:

Luria–Bertani

LDA:

linear discriminant analysis

LEfSe:

linear discriminant analysis effect size

MDA:

malondialdehyde

MUC-2:

mucin-2

MRS:

De Man, Rogosa and Sharpe

MDR:

multi drug resistance

NF-κB:

nuclear factor kappa-B

OTU:

operational taxonomic unit

OLFM4:

olfactomedin 4

PCoA:

principal coordinate analysis

PCNA:

proliferating cell nuclear antigen

PICRUSt:

phylogenetic Investigation of Communities by Reconstruction of Unobserved State

PBS:

phosphate buffered saline

S. pullorum :

Salmonella pullorum

SOD:

superoxide dismutase

SGLT-1:

sodium-dependent glucose transporter 1

TCM:

Traditional Chinese M edicine

V/C:

villus height to crypt depth ratio

XLD:

xylose lysine deoxycholate

ZO-1:

zonula occludens-1

16S RNA:

16S ribosomal RNA

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Acknowledgements

The authors are grateful to Prof. Jingmin Gu and Prof. Yunhe Fu of Jilin University for providing S. pullorum and L. reuteri for this study.

Funding

This research was supported by the National Key Research and Development Program of China (2023YFD1800903), the National Natural Science Foundation of China (grant U22A20523), the Scientific Research Project of the Education Department of Jilin Province (No. JJKH20250155KJ), the Fundamental Research Funds for the Central Universities under Grant 2023-JCXK-01, and the China Postdoctoral Science Foundation (2024T170330, 2024M751096 and GZB20240268). All authors report no potential conflicts.

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Author notes

  1. Linlin Ding and Kaige Qi have contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun, 130062, China

    Linlin Ding, Kaige Qi, Yutong Zhou, Minda Liu, Na Hu, Jianfeng Wang, Jiazhang Qiu, Xuming Deng & Lei Xu

  2. Research Center of Traditional Chinese Medicine, The Affiliated Hospital to Changchun University of Chinese Medicine, Changchun, 130021, China

    Qingjie Li

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  1. Linlin Ding
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Contributions

LD and KQ contributed equally to this work. LD and LX conceived the project, contributed to the experimental design, and wrote the manuscript; KQ and NH performed the animal experiments; ML and YZ performed data analysis and managed and archived the data; JW and JQ conducted data analysis; XD and QL supervised the research and coordinated the project. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Xuming Deng or Lei Xu.

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Ethics approval and consent to participate

The animal experiments were approved by Jilin University Animal Care and Use Committee (Changchun, China) (approval number: SY202405028).

Competing interests

The authors declare that they no competing interests.

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Supplementary Information

Additional file 1. The primers for qPCR assays.

Additional file 2.

Effects of supplementation Artemisia argyi essential oil on the Alpha-diversity indices of caecal microbiota communities of the S. pullorum-infected chicks. A Chao1 index, B Simpson index, C Shannon index, D Observed_otus index, and E Goods_coverage index (n = 6). NC: Uninfected chicks, SP: S. pullorum-infected chicks, EOH: 100 mg/kg/day of Artemisia argyi essential oil and S. pullorum-infected chicks. Data are expressed as the mean ± SD. ns, P > 0.05.

Additional file 3.

PICRUSt2 metagenome and Heatmap inference analysis based on 16S rRNA dataset of intestinal microorganisms after Artemisia argyi essential oil treatment. A Heatmap analysis of microbial taxonomic composition at the genus level. B Prediction of significant KEGG pathways that were different in the Artemisia argyi essential oil supplementation group compared to the NC group.

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Ding, L., Qi, K., Zhou, Y. et al. Ingestion of Artemisia argyit essential oil combats Salmonella pullorum infections by altering gut microbiota composition in chicks. Vet Res 56, 98 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-025-01527-7

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Keywords

Veterinary Research

ISSN: 1297-9716