Specific pathogen-free (SPF) white Leghorn chickens were purchased from the Laboratory Animal Center of Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences (Harbin, China). All chickens were housed in a sterile isolation chamber (IPQ-Type 3 negative pressure isolator; Shanxi Agricultural University, Taigu, China), and food and water were provided ad libitum. All animal experiments were performed according to the guidelines of Laboratory Animal Ethics Committee of Shanxi Agricultural University (Shanxi, China) in compliance with Laboratory animal-Guideline for ethical review of animal welfare (GB/T35892-2018, National Standards of the People's Republic of China).

Chickens were randomly assigned to experimental groups at day 28. As showed in Figure 1, normal gut microbiota group (N group) chickens were given normal drinking water, chickens were challenged by MG at day 48, samples were collected at day 51; disordered gut microbiota group (D group) chickens received a cocktail of antibiotics (ampicillin 1 g/L, neomycin sulfate 1 g/L, metronidazole 1 g/L, and vancomycin 0.5 g/L) in drinking water for 2 weeks to disturb gut microbiota as descried previously (4), chickens were challenged by MG at day 48, samples were collected at day 51; fecal bacteria transplantation group (F group) received a cocktail of antibiotic as described above for 2 weeks, then received gut microbiota transplantation from N group chickens at day 45 and challenged by MG at day 48, samples were collected at day 51. Details of TLR2 ligand, recombinant proteins, vitamin A and butyric acid were applied as showed in Figure legends.

Ampicillin, neomycin sulfate, metronidazole, vancomycin, vitamin A, and sodium butyrate were purchased from Solarbio life science (Beijing, China). TLR2 ligands Pam3CSK4 (P3C) was purchased from InvivoGen (Hongkong, China). Recombinant chicken GM-CSF protein (rGM-CSF) and recombinant chicken IL-17 protein (rIL-17) were purchased from Abcam (Cambridge, MA, UK).

MG strain (R_low) was kindly donated by Professor Xin Jiuqing of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. MG were grown in a modifed Hayfick medium, consisting of 0.1% nicotinamide adenine dinucleotide (NAD), 0.05% penicillin, 20% FBS, 0.05% thallium acetate and 10% freshly prepared yeast extract, as described in a previous study (9). Chickens were infected as described previously with some modification (2), a 0.3 mL aliquot solution containing 3 × 10⁹ CCU MG R_low were injected in the air sacs at indicated time points showed in Figure 1.

Fecal bacteria transplantation was performed as described previously (10). Fecal samples were weighed and homogenized (1:2 wt/vol) in sterile PBS, the fecal suspension vigorously mixed and the supernatant was collected. Chickens from F group were orally inoculated with 1 mL of fecal suspension at indicated time as showed in Figure 1.

To detect the extent of MG infection, absolute abundance of MG was measured by quantitative PCR (qPCR) using a recombinant plasmid consist of a cloned mgc2 gene of MG to establish a standard curve as previously described (11). The recombinant plasmid was diluted by a gradient of 10 times to establish a standard curve. The genomic DNA were extracted by using Bacterial Genomic DNA Extraction Kit (Solarbio life science, Beijing, China) according to manufacturer's recommended procedures, and mgc2 gene was amplified from genomic DNA by PCR and confirmed by Sanger sequence (Genewiz, Suzhou, China). The primers are: mgc2-F: 5′-TTGGGTTTAGGGATTGGGATT; mgc2-R: 5′ -CCAAGGGATTCAAC CATCTT. The reaction mixture was 1 × SYBR Green PCR Master Mix (Takara, Dalian, China), 0.2 mM of each forward and reverse primer, and 1 μL template DNA in a total reaction volume of 25 μL. The PCR reaction conditions are as follows: 1 cycle of 5 min at 95°C followed by 95°C for 10 s, 55°C for 30 s, 72°C for 60 s for 38 cycles and a final extension at 72°C for 5 min.

The procedure was carried out as described previously (2). Briefly, total RNA was extracted using Trizol reagent (Life Technologies, Grand Island, NY, USA) following the manufacturer's instructions. One microgram of total RNA were reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Transgen, Beijing, China). The gene expression levels were detected using real-time PCR with a SYBR premix Ex Taq kit (Transgen, Beijing, China) on a Roche 480 real-time PCR system thermocycler. Each sample was analyzed in triplicates. Target gene expression was normalized to β-actin. The primers are shown in Table 1.

DNA was extracted from the feces using Tiangen Stool DNA extraction kit (Tiangen, Beijing, China) according to the manufacturer's instructions. 16S rRNA gene V3 and V4 regions were amplified, and the primers are as follows: 341F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′ -GGACTACHVGGGTWTC TAAT-3′). 16S sequencing and data analysis were performed as previously described (19). Data has been uploaded to SRA under the accession number PRJNA601545.

SCFAs quantification was performed using a GC-MS method as previously described (19). The acetic acid, propionic acid, butyric acid standards (Solarbio, Beijing, China) was diluted by a gradient of 10 times of the working solution using ultrapure water. Fifty microliter working solution or 50 μL serum incubated with 400 μL NaCl and 50 μL of 3 mM hydrochloric acid sodium chloride solution, then the samples were exposed to ultrasonic treatment for 1 h at 4°C. The samples were added 1 mL of ice ether by vortex mixing for 15 min and centrifuged at 12,000 g for 15 min at 4°C. The supernatant was precipitated with an anhydrous sodium sulfate by vortex mixing for 5 min and was centrifuged at 5,000 g for 10 min at 4°C. The supernatant was used for SCFAs quantification using an Agilent 7890B/5977A GC-MS equipped with an Agilent HP-FFAP (25 m × 0.50 mm × 0.32 μm) column in a SIM mode.

Vitamin quantification was performed using a LC-MS method as stated previously (20). Hundred microliter serum of each group was transferred to a tube, added 1 mL of extract solvent (acetonitrile-methanol-water, 2:2:1), the mixture were vortexed for 45 s, homogenized at 45 Hz for 5 min, and sonicated for 5 min at 4°C. The procedure of homogenation and sonication were repeated twice, and incubation at −20°C for 1 h and centrifuged at 12,000 g at 4°C for 10 min. The supernatant was transferred to a new tube, then dried under nitrogen, added 100 μl of 50% acetonitrile/water. The mixture was centrifuged at 12,000 g for 15 min at 4°C. The supernatant was transferred to an autosampler bottle for UHPLC-MS/MS (ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry) analysis.

The data are expressed as mean ± SD and analyzed using GraphPad Prism 8.0 (GraphPad Software). Significant differences between the two groups were evaluated by two-tailed unpaired Student's t test or Mann-Whitney U test for samples that were not normally distributed. Statistical significance was determined by two-way ANOVA with Tukey tests for multiple-group comparisons. The level of significance was set at P < 0.05.

In order to explore the role of gut microbiota in MG infection, we first dysbiosed gut microbiota by feeding antibiotics cocktail for 2 weeks. The results showed that antibiotic treatment significantly changed the diversity and composition of gut microbiota (Figures 2A,B, P < 0.01). In addition, Fecal bacteria transplantation could restore gut microbiota diversity and composition in F group (Figures 2A,B, P < 0.01). Subsequent experiments were performed as follows.

MG isolation culture is time-consuming; therefore, we used a quantitative PCR detection method of MG (11). In D group, there was a significantly increased MG colonization in the lung at 3 days post MG inoculation compared to N group (Figure 2C, P < 0.01); there was a significantly decreased MG colonization in the lung at 3 days post MG inoculation in F group compared to D group (Figure 2C, P < 0.01). These results determined that gut microbiota dysbiosis aggravated MG colonization in lung.

We hypothesized that the decreased clearance of MG in D group might be related to the weakened immune response in lung. Therefore, a series of immune related genes were detected by qRT-PCR. Compared to N group and F group, D group exhibited significantly reduced pro-inflammatory genes and host defense peptide genes expression (Figure 3, P < 0.01), and significantly increased anti-inflammatory genes expression (Figure 3, P < 0.01). Among these different expression genes, TLR2, IL17 and GM-CSF have been confirmed to be associated with MG infection (9, 21), thus, the role of TLR2, IL17 and GM-CSF in gut microbiota dysbiosis aggravates MG colonization were explored. The results showed that intranasal administration of TLR2 ligand in D group promoted MG clearance (Figure 4A, P < 0.01). Consistent with the results of MG clearance, intranasal administration of TLR2 ligand increased IL-17 mRNA expression levels in D group (Figure 4B, P < 0.01). IL-17 plays a significant role in defense infection (4). To examine the role of IL-17 in regulating lung antibacterial immunity, chickens were administered rIL17 concomitant with MG inoculation. The rIL17 could promote MG clearance in D group (Figure 4C, P < 0.01). The rIL17A also could promote GM-CSF mRNA expression in D group (Figure 4D, P < 0.01). GM-CSF is critical signaling molecule in the innate response to respiratory infection as well as a member of the IL-17 signaling pathway. To examine the role of GM-CSF in regulating lung antibacterial immunity, chickens were administered rGM-CSF concomitant with MG inoculation and confirmed that the rGM-CSF could promote MG clearance in D group (Figure 4E, P < 0.01). Taken together, the above results showed that gut microbiota dysbiosis impairs pulmonary immune response against MG infection.

The profound influence of gut microbiota on the host is strongly associated with gut microbiota metabolites. Targeted metabolomics results showed that the contents of retinol, retinene and alpha-tocopherol were significantly decreased in the D group compared with the N and F groups (Figure 5, P < 0.01). Retinol and retinene both are bioactive molecules of vitamin A, and they can be transformed into each other in vivo and have same functions. The alpha-tocopherol is a fat-soluble vitamin that enhances vitamin A absorption. In addition, vitamin A deficiency is associated with Mycoplasma infection (22), therefore, vitamin A may be a key metabolite that contributes MG clearance. In D group, oral administration of vitamin A significantly reduced MG colonization in lung (Figure 6A, P < 0.01). Consistent with results of MG clearance, oral administration of vitamin A increased TLR2, IL-17 and GM-CSF mRNA expression levels in lung of D group (Figures 6B–D, all P < 0.01).

Targeted metabolomics results also showed that the contents of SCFAs (acetic acid, propionic acid, and butyric acid) were significantly decreased in the D group compared to the N and F groups (Figure 5, all P < 0.01). Since butyric acid decreased the most and was closely related to immune function (23), butyric acid was selected for further verification. In D group, oral administration of butyric acid significantly reduced MG colonization in lung (Figure 7A, P < 0.01). Consistent with results of MG clearance, oral administration of butyric acid increased hose defense peptide genes (AvBD3, AvBD9, AvBD10) mRNA expression levels in lung of D group (Figures 7B–D, all P < 0.01). Taken together, the above results showed that gut microbiota dysbiosis aggravated MG colonization in lung that may be due to disturbed SCFAs and vitamin A metabolism.