Impact of Phellinus gilvus mycelia on growth, immunity and fecal microbiota in weaned piglets

Preparation of P. gilvus mycelia (SH)

P. gilvus strain (preserved at the Sericultural Research Institute, Zhejiang Academy of Agricultural Science, Hangzhou, China) was incubated in sterile liquid culture medium (ingredients are provided in Table S1) in a fermentation tank at 25 °C with stirring speed of 100 r/min. The entire culture process was sterile. After 2 weeks, the mycelia of P. gilvus growing in the fermentation tank was separated from the liquid culture medium with a filter and dried at 65 °C. The dried mycelia were then ground into fine powder and stored at −20 °C before use. The SH was tested to have 39.8% crude protein, 15.2% crude fiber, 11.1% crude fat, 4.3% ash, 7.5% water-soluble polysaccharide, and 2.5% polyphenol.

Experimental design

The experiment was conducted in Hangzhou Zhengxing Animal Husbandry co. LTD (Linan, Zhejiang, China), and all the piglets used in this experiment were from this company. The trial was approved by the farm’s veterinary consultant, and all procedures were compliant with Chinese regulations and were approved by the Medical Ethics Committee of Zhejiang Academy of Agricultural Sciences (No. 2017-050).

A total of 72 crossbred piglets [Duroc × (Landrace × Large White), 7.51 ± 0.88 kg BW, 36 barrows and 36 gilts] weaned at 28 d of age were used in a 28-day feeding trial. The piglets were assigned to three dietary treatment groups with four replicate pens per treatment and six piglets (three barrows and three gilts) per pen. Each pen was equipped with a self-feeder and a nipple waterer. Piglets had free access to water and diets.  A combination of daylight and artificial light was used, and room temperature was controlled at 24−26 °C. The treatments included basal diet without antibiotics or SH (C group), basal diet with 5 g/kg SH (SH group), and basal diet with antibiotics (ATB group). The antibiotic contents were 75 mg/kg aureomycin and 20 mg/kg kitasamycin. The composition and nutrient levels of the basal diet are shown in Table 1. On day 28, blood of eight pigs per treatment (two pigs per pen) was collected from the anterior vena cava using disposable syringes. Rectal fecal samples of ten pigs per treatment (two or three pigs per pen) were obtained. The sampling progress was performed by an experienced veterinary assistant, and no analgesia or anesthetic was given. None of the piglet died all through the experiment period. After the experiment was over, the piglets continued to be kept by Hangzhou Zhengxing Animal Husbandry co. LTD till they were marketed. Fecal samples were divided immediately into two aliquots; one aliquot was stored at −80 °C for microbial 16S rDNA sequencing analysis, and the other aliquot was stored on ice for plate count analysis of E. coli.

Growth performance measurement

The piglets were individually weighed on days 0, 14, and 28 of the experiment. Feed intake per pen was documented weekly, and the residual feed was measured at the end of the experiment. Average daily gain (ADG), average daily feed intake (ADFI), and feed intake to gain ratio (F/G) were calculated based on these parameters (n = 4, a pen was considered as an experimental unit).

Assay of immunological parameters in serum

Blood samples (n = 8 per group, two pigs per pen) were centrifuged at speed of 3,500 rpm for 10 min to obtain serum. The concentrations of immunoglobulin G (IgG), interleukin-1β (IL-1β), tumor necrosis factor (TNF)-α and myeloperoxidase (MPO) in serum were assessed using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The colorimetric reaction was read in an automated ELISA microplate reader (Versamax, Molecular Devices).

Viable plate counts of E. coli

Rectal fecal samples of ten pigs per treatment (two or three pigs per pen) were obtained and feces from two or three pigs in the same pen were mixed into one sample. The mixed fecal samples (n = 4 per group) were serially diluted (10⁻¹–10⁻⁷) in sterilized saline solution. An aliquot of 100 µL of the sample was plated onto MacConkey agar plates and spread evenly by using an L-shaped spreader. Three parallel plates were prepared for each sample dilution. The plates were then inverted and incubated at 37 °C for 24 h. The number of colonies in the plates with 30–300 available isolated colonies was used to calculate E. coli population in feces. The calculated equation is as follows:

Viable E. coli population in feces, colony-forming unit (CFU)/g = Number of E. coli colonies on the plate × dilution ratio of fecal sample/fecal weight.

Microbiota analysis of fecal samples via 16S rRNA gene sequencing

Microbial genome DNA of fecal samples (n = 10 per group, two or three pigs per pen) was isolated and purified using the QIAamp DNA Stool Mini Kit (Qiagen, GmbH Hilden, Germany) in accordance with the manufacturer’s instructions. The V3-V4 hypervariable regions of the 16S rRNA genes were PCR amplified from the microbial genomic DNA by using primers B341F–B785R (B341F: CCTACGGGNGGCWGCAG, B785R: GACTACHVGGGTATCTAATCC). PCR was performed in 25 µL of the reaction mixture with 12.5 µL of 2 × KAPA HiFi HotStart Ready Mix (KAPA Biosystems), 0.25 µL of 25 µM forward and reverse primers, and approximately 10 ng of template DNA. Thermal cycling involved initial denaturation at 95 °C for 3 min, followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 30 s and 72 °C for 5 min. The resulting amplicons were gel purified, quantified, pooled, and sequenced. Sequencing libraries were generated using a NEB Next® UltraTM DNA Library Prep Kit for Illumina (NEB, USA) in accordance with the manufacturer’s recommendations. The quality of the library was assessed with a Qubit@ 2.0 Fluorometer (Thermo Scientific) and an Agilent Bioanalyzer 2100 System (Agilent Technologies, Inc.). The library was sequenced on an Illumina Miseq platform, and 2 × 300 bp paired end reads were generated.

Raw sequence reads were filtered and assembled. Sequences with contaminated adapters, undetermined nucleotide, or low complexity were removed. The resulting sequences were clustered into operational taxonomic units (OTUs) by using USEARCH drive 7 at 97% sequence similarity. Chimeric OTUs were removed with UCHIME v4.2. Representative sequences for each OTU were picked and aligned using QIIME 1.8. The Ribosomal Database Project Classifier v2.2 was used to assign a taxonomic rank to each sequence in the representative set. The relative abundance of each OTU was examined at different taxonomic levels. Genetic diversity calculation and taxonomic community assessment were performed with QIIME 1.8 scripts. Sequencing and bioinformatics analyses were conducted by Hangzhou KAITAI Biotechnology Co., Ltd. (Hangzhou, China).

Statistical analysis

Data were expressed as means or means ± SEM. Significant differences in growth parameters, immunological parameters, viable plate counts of E. coli, and the relative abundance of the genus were analyzed by one-way ANOVA followed by LSD post hoc test by using SPSS 12.0 (Chicago, USA) and the significance was considered at P < 0.05. For phylotype analysis, the alpha diversity of the microbiome was calculated on the basis of the OTU level by using USEARCH 7.0. Principal coordinate analysis (PCoA) was performed using R and visualized with the R package ggplot2, and significant differences were evaluated by Adonis analysis (R package vegan) at P < 0.05. Comparison of OTU levels between groups were assessed using the edgeR test in R by using the Benjamini and Hochberg false discovery rate, and the significance was considered at P < 0.05 and FDR < 0.2.

Growth parameters

The ADG of the ATB group and SH group in the entire stage (1–28 days) increased significantly (P < 0.05) compared with that of the control group (Table 2). SH treatment increased the ADG greatly at the later stage (15–28 days). The ADFI and F/G throughout the entire experimental period were not significantly changed in ATB or SH group compared with those in control group (P > 0.05).

Immunological parameters

We quantified the IgG, the important antibody in adaptive immunity, TNF-α and IL-1β, cytokines secreted mainly by monocyte-macrophages, and MPO, one biomarker of active neutrophil in serum. Results (Fig. 1A) show that IgG levels did not varied among groups, suggesting that ATB and SH did not affected the adaptive immunity. However, the levels of TNF- α, IL-1β and MPO were both increased by treatment of ATB or SH (Figs. 1B–1D), and these cytokine levels in SH group were significant higher than in ATB group (P < 0.05). These results indicated the better innate immune response of pigs in SH group than that of the control group and ATB group.

Viable E. coli counts in feces

E. coli is a group opportunistic pathogens that may cause diarrhea in piglets. Figure 2 shows that both ATB and SH treatments decreased the viable E. coli counts in feces.

Fecal microbiota structure analysis

To further explore the influence of SH and ATB on the gut microbiota, we analyzed the structure of the fecal microbiota by 16S rRNA pyrosequencing. The diversity of the fecal microbes as displayed by the Richness, Chao1, Shannon, and Simpson indices (Figs. 3A–3D) was not significantly changed by SH or ATB treatment (P > 0.05). As displayed by PCoA on the OTU level (Fig. 3E), the microbiota of the SH group largely overlapped with the control. The ATB group was distinct from the control group in terms of position (P < 0.05), suggesting the significant ATB-induced change in the gut microbiota structure.

We also compared the relative abundance of each OTU in the SH and ATB groups with those in control group (Fig. 4). It shows that there were 271 OTUs significantly different in relative abundance between ATB group and control group; of these OTUs, 135 OTUs were significantly higher in relative abundance in ATB group than the control, and 136 OTUs were significantly lower in relative abundance in ATB group compared with the control. There were 68 OTUs significantly different in relative abundance between SH group and control group; of these OTUs, 30 OTUs were significantly higher in relative abundance in SH group than the control group, and 38 OTUs were significantly lower in relative abundance in SH group compared with the control.

Figure 5 displays the changes in the relative abundance of the top 10 phyla, families, and genera. SH treatment slightly influenced the top 10 phyla, families, and genera compared with the control group. By contrast, ATB remarkably changed the structures of families and genera compared with the control group. Figure 6 shows the genera that were significantly altered in relative abundances by SH or ATB treatment compared with the control. SH treatment increased the relative abundance of Alloprevotella (P < 0.05). ATB treatment increased the abundance of Prevotella, Megasphaera and Faecalibacterium significantly (P < 0.05).