Impacts of diarrhea on the immune system, intestinal environment, and expression of PGRPs in New Zealand rabbits

Ethics statements

All animal experiments used in this study were approved by the Institutional Animal Care and Use Committee of Yangzhou University (Jiangsu, China) and were strictly implemented according to the regulations for experimental animals. An ordinary housing facility was used and was in keeping with the national standard Laboratory Animal Requirements of Environment and Housing Facilities (GB 14925-2001). The care of laboratory animals and the animal experimental operations conformed to the Jiangsu Administration Rule of Laboratory Animal. The rabbits were anesthetized by an intraperitoneal injection of sodium pentobarbital.

Animals and sampling

New Zealand rabbits weaned at 35 days of age were used as experimental animals to study the impacts of diarrhea. Rabbits with spontaneous diarrhea were collected 10 days after weaning. Moreover, eight weaned rabbits with diarrhea and eight healthy weaned rabbits were obtained and sacrificed. The cecal contents of each rabbit were collected in three 2-mL cryotubes and stored in liquid nitrogen for later determination of VFA and ammonia nitrogen (NH₃-N) levels, as well as 16S high-throughput sequencing. The duodenum and cecum samples were collected at the same position in each rabbit and stored in liquid nitrogen for later RNA extraction. A fragment of the duodenum was removed at the same position, rinsed with 0.9% saline, and fixed in paraformaldehyde for the preparation of intestinal sections.

Assessment of intestinal environment

Cecal contents were collected, and the pH was measured immediately with a pH meter (DELTA 320, Shanghai, China). Duodenal contents (2 mL) were mixed with 2 mL distilled water and recovered by centrifugation at 10,000 rpm for 10 min. Subsequently, 1 mL supernatant was collected and mixed with 0.2 mL 20% metaphosphoric acid solution containing 60 mM crotonic acid. VFA levels were determined using a GC-9A Gas Chromatograph (Shimadzu, Japan) with nitrogen as the carrier gas, a flow-rate of 30 mL/min. A CP-WAX capillary column with a length of 30 m, an inner diameter of 0.53 mm, and a membrane thickness of 1 µm was used, and the sample input was 0.6 µL.

Diluted cecal contents (4 mL) were centrifuged for 10 min, and 50 µL supernatant was collected in a 10-mL tube. Phenol and sodium hypochlorite (3 mL each) were added to the supernatant, and the mixture was incubated in a 60 °C water bath for 10 min, followed by immediate cooling. The optical density (OD) at 546 nm was measured, and the concentration of NH₃-N in the cecum was analyzed based on a standard curve of ammonium chloride.

Determination of serum indicators

Blood was drawn from rabbits with diarrhea and healthy rabbits, allowed to settle for 30 min, and centrifuged at 3,500 rpm for 15 min to isolate serum. Serum indicators were detected according to the instructions of the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The factors used to evaluate the serum biochemical profile included serum glucose (GLU), total protein (TP), urea nitrogen (UN), and serum alkaline phosphatase (AKP). In particular, serum immune indicators, including interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), immunoglobulin G (IgG), and immunoglobulin M (IgM), reflected the immune response to diarrhea.

The level of GLU was measured using the glucose oxidase method. TP content in the serum was determined by the biuret method. The level of UN in the serum was determined using the urease method. AKP was measured by colorimetry. The serum levels of IL-2, IL-6, TNF-α, IgG, and IgM were determined by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).

Measurement of intestinal villus length and crypt depth

The intestine was examined by routine HE staining of tissue paraffin sections. Five consecutive longitudinal sections were obtained from each segment of the intestinal tract, and six typical microscopic fields were assessed for each tissue section. The lengths of the longest villus and the corresponding crypt in each field were measured. The intestinal wall, villi, and crypt are shown in Fig. 1. The mean values for villus length and crypt depth were calculated.

Real-time PCR

Real-time PCR was carried out on Applied Biosystems 7500 Real-Time RT-PCR System with the following program: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. All cDNA samples were tested three times, and the results were normalized to the levels of GAPDH expression using the SYBR Premix Ex Taq II system (Takara, Japan). Based on the mRNA sequences of rabbit PGRPs published in GenBank, real-time PCR primers for PGLYRP-1, PGLYRP-2, and PGLYRP-3 were designed and are shown in Table 1. The following equation was used to obtain the fold change: $RQ=\frac{{\left(1+E_a\right)}^{C{T}_{ca}-C{T}_{ta}}}{{\left(1+E_k\right)}^{C{T}_{ck}-C{T}_{tk}}}$, where E_a is the amplification efficiency of the target gene, E_k is the amplification efficiency of the internal control gene, CT_ca is the C_t value of the target gene in the control group, CT_ta is the C_t value of the target gene in the test group, CT_ck is the C_t value of the internal control gene in the control group, and CT_tk is the C_t value of the internal control gene in the test group.

Statistical analysis

Each experiment was repeated at least three times, and the data were analyzed using t-tests for independent samples and one-way ANOVA (SPSS version 21). The results are presented as the mean ± SD at two levels of significance, P < 0.05 and P < 0.01.

16S rDNA amplicon pyrosequencing

Homogenized cecal contents weighing 3 g (wet weight) from each healthy rabbit and rabbit with diarrhea were used (three rabbits from each group) for DNA extraction. Genomic DNA was extracted using hexadecyl trimethyl ammonium bromide (CTAB). After testing for DNA purity and concentration, an appropriate amount of sample was diluted with sterile water to a final concentration of 1 ng/µL.

The 16S V4 amplicon was amplified by PCR using specific barcode-containing primers (515F 5′-GTGCCAGCMGCCGCGGTAA-3′, 806R 5′-GGACTACHVGGGTWTCTAAT-3′). The PCR products were mixed in equal quantities based on their concentrations, and the mixture was subjected to electrophoresis on a 2% agarose gel (1 × TAE) for the purification of PCR products. The major bands at 400–450 bp were collected using GeneJET (Thermo Scientific). Thereafter, the library was constructed using the TruSeq DNA PCR-Free Library Preparation Kit (Illumina, San Diego, CA, USA), and the quality of the library was assessed using a Qubit® 2.0 Fluorometer and Agilent Bioanalyzer 2100 system. After quality testing, libraries were sequenced by Illumina HiSeq.

Sequence analysis

We merged paired-end reads from the original DNA fragments and applied quality filters using FLASH (Magoc & Salzberg, 2011). The resulting reads were analyzed by assignment to each sample based on their unique barcodes. We processed the sequences using the QIIME (Quantitative Insights Into Microbial Ecology) software package. The alpha- and beta- diversity were parsed by in-house Perl scripts (Caporaso et al., 2010) to assess diversity within and among samples, respectively. The pick_de_novo_otus.py workflow was used to select operational taxonomic units (OTUs) after applying QIIME quality filters. Sequences with >97% similarity were defined as the same OTU. Taxonomic classifications for representative sequences were determined used the Ribosomal Database Project (RDP) classifier (Wang et al., 2007b). We used principal components analysis (PCA) to examine differences between samples along two axes and visualize them (Avershina, Frisli & Rudi, 2013). To further investigate differences in microbial diversity among the samples, statistical tests were conducted using MetaStat. The 16S rDNA sequences were deposited with the NCBI (accession number: SRP114741).

Comparison of intestinal morphology and environment between rabbits with diarrhea and healthy rabbits

Anatomical examination showed serious flatulence in the ceca and recta of young rabbits with diarrhea, accompanied by thinning of the cecal and rectal walls and watery contents. Figure 2 shows the duodenal morphology and cecal environment of the rabbits. Histological analysis revealed that the density of duodenal villi in rabbits with diarrhea was lower than that in the healthy rabbits, and the morphology of the diarrheal duodenum was not intact (Fig. 3). Statistical analysis indicated that, compared with the healthy rabbits, rabbits with diarrhea had markedly shorter villous lengths and smaller villus/crypt ratios (P < 0.01), as well as significantly thinner duodenal walls (P < 0.05). However, crypt depth was not significantly different between the two groups (Table 2). These results indicate that diarrhea leads to severe damage to the duodenal structure in rabbits.

The characteristics of the cecal environment are shown in Table 3. The concentration of acetic acid was significantly reduced after diarrhea (P < 0.05), but the concentrations of propionic acid and butyric acid as well as the ratio of acetic acid/(propionic acid + butyric acid) were comparable to those in the healthy group (P > 0.05). Moreover, diarrhea led to markedly increased pH (P < 0.05) and NH₃-N content (P < 0.01). Under normal conditions, propagating probiotics produce lactic acid and acetic acid, reducing the pH in the digestive tract and modulating the microenvironment to inhibit the growth of harmful bacteria.

Comparison of serum indicators between healthy rabbits and those with diarrhea

Changes in serum indicators after diarrhea were examined. As shown in Table 4, serum TP in rabbits with diarrhea was significantly lower than that in the healthy group (P < 0.05), whereas AKP, UN, TNF-α, and IL-6 were significantly higher in the diarrhea group than the healthy group (P < 0.05). Levels of GLU, IgG, IgM, and IL-2 were not significantly different between the two groups (P > 0.05). These results suggest that protein breakdown and consumption increases in diarrhea, thus impairing body development.

Expression profile of PGRPs in healthy rabbits and those with diarrhea

Compared with the healthy group, the expression of antibacterial PGRPs increased in the ceca and duodena of rabbits with diarrhea (Fig. 4). PGLYRP-1 and PGLYRP-2 were both significantly upregulated in the ceca and duodena of rabbits with diarrhea compared with levels in healthy rabbits (P < 0.05), while PGLYRP-3 was significantly upregulated in the ceca of rabbits with diarrhea (P < 0.05) but not significantly altered in the duodena (P > 0.05). These data suggest that young rabbits might combat harmful, diarrhea-causing intestinal bacteria by upregulating intestinal PGRPs.

Analysis of floral differences between healthy rabbits and those with diarrhea

High-throughput sequencing data indicated that 57,286 ± 6,637 (mean ± SD) tags were detected in each sample, with no significant difference between the healthy (57,127 ± 7,484, mean ± SD) and diarrhea groups (57,445 ± 7,352, mean ± SD) (P > 0.05). To assess microbial diversity, clustering of effective tags was performed on all samples by sorting tags with ≥97% identity into an OTU. There were 916 ± 28 and 880 ± 46 OTUs in the healthy group and the diarrhea group, respectively, implying a higher diversity of cecal flora in healthy New Zealand rabbits. However, floral diversity was not significantly different between the diarrhea and healthy groups (P > 0.05) (Table 5).

The rarefaction curve plateaued when the sequence number exceeded 6,000, and the OTU number did not change significantly with an increasing number of sequences thereafter. A minimum of 50,657 sequences were obtained from the tested samples, indicating that the sequence number was sufficient for analysis (Fig. 5A). PCA based on OTU levels (Fig. 5B) showed two prominent clusters from healthy and diarrhea samples, suggesting that healthy and diarrhea samples varied significantly.

Differences in the cecal floras of the diarrhea and healthy groups were analyzed at the phylum, class, order, family, genus, and species levels (Table 6). At the family level, the prevalences of three families of bacteria were significantly different between the healthy and diarrhea groups (P < 0.05), including Planococcaceae, which was 3.20-fold higher in the healthy group. Compared with healthy rabbits, five genera were significantly reduced in rabbits with diarrhea (P < 0.05), including Clostridium (2.38-fold lower), Roseburia (2.62-fold lower), and Alistipes (3.52-fold lower). At the species level, two species varied significantly between the healthy and diarrhea groups (P < 0.05), including Alistipes indistinctus (3.53-fold higher in healthy group) and Coprococcus catus (2.89-fold higher in the diarrhea group).