Animal procedures were performed in accordance with the National Standard Guideline for Ethical Review of Animal Welfare (GB/T 35892-2018) and approved by the Animal Care and Use Committee of Inner Mongolia Agricultural University. All efforts are made to take the least painful approach to animal handling and to avoid or reduce stress, pain and injury to the animal used in the experiment.

The Nematodirus oiratianus strain was isolated from lambs naturally grazing in Wushen Banner, Ordos (Latitude: 38.604136, Longitude: 108.817607), and confirmed as Nematodirus oiratianus through morphological examination of domestic nematodes and ITS-2 analysis (22). Following several generations of purification and culture, the strain was maintained in lambs. Fresh fecal samples were regularly collected using the sugar-saline floating method to obtain clean eggs, which were then repeatedly floated, cleaned by centrifugation, and cultured in distilled water at 27 °C for approximately 15 days to develop into infective larvae before being preserved for future use. Five Ordos fine wool sheep lambs, all of similar body condition and aged 3 months (weighing 15 kg ± 2 kg), were chosen as test subjects and underwent deworming with ivermectin, closantel, and sulfaquinoxaline prior to experimentation. Fecal samples were collected 7 days post-deworming and examined using the McMaster technique to confirm the absence of specific parasitic infections. The animals were housed in a controlled environment and provided with clean water and fecal-contamination-free forage (23).

After 15 days of acclimatization under uniform conditions, 20,000 Nematodirus oiratianus infective larvae were orally administered to each lamb. The third day post-infection constituted the infective L3 group, the seventh day the infective L4 group, the tenth day the L5 group, and the twenty-fifth day the infective adult (AD) group (24–26). A control group received an equivalent volume of distilled water. Each group was replicated three times. At 3, 7, 10, and 20 days post-infection, the anterior one-third segment of the duodenum was dissected following euthanasia via sodium pentobarbital injection. The contents were rinsed with pre-chilled PBS, and all collected samples were snap-frozen in liquid nitrogen and stored at −80 °C.

Based on the sequences of CLDN18, CCL19, STAT4, MUC5AC, and FGB obtained from the transcriptome, primers were designed using Primer 3, followed by fluorescence quantitative PCR. The reaction program was as follows: 95 °C for 30 s for denaturation (Stage 1), followed by denaturation at 95 °C for 15 s, annealing/extension at 60 °C for 30 s (Stage 2), and a gradual temperature increase from 65 °C to 95 °C at a rate of 0.5 °C per cycle with fluorescence signal collection at each increment (Stage 3). The results were analyzed using the ^ΔΔCT method. MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa, 9765), SweScript All-in-One RT SuperMix for qPCR (TaKaRa, G3337), 2 × Universal Blue SYBR Green qPCR Master Mix (TaKaRa, G3326), and RNAlater (Ambion, AM7020) were all purchased from TAKARA. (Primer information has been added in the appendix).

Serum: After 3, 7, 10, and 20 days post-infection, blood was collected from the jugular vein of lambs. The blood was left at 4 °C overnight and then centrifuged at 2,000 rpm for 20 min. The supernatant was collected and stored at −20 °C for further analysis.

The main indicators for the determination of immunoglobulins and cytokines in the serum include IgA, IgM, IL-2, IL-5, IL-6, IL-10, INF-γ, and TNF-β. Quantitative determination was performed using a sandwich ELISA method with double antibodies. The specific procedures for the determination of the above indicators were conducted according to the instructions provided with the reagent kits.

According to the HE staining procedure, the middle section of the fresh lamb duodenum at different stages of infection was immersed in 4% paraformaldehyde and fixed overnight at room temperature. The duodenal tissue was removed from the fixative and placed in a fume hood. Using a surgical blade, the targeted tissue was trimmed and flattened. The trimmed tissue and corresponding labels were placed in a dehydration box. The dehydration box was placed in a dehydrator, and dehydration was carried out using a gradient of alcohol. This included 75% ethanol for 4 h, 85% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol for 1 h, absolute ethanol I for 30 min, absolute ethanol II for 30 min, alcohol benzene for 5–10 min, xylene I for 5–10 min, xylene II for 5–10 min, melting paraffin I at 65 °C for 1 h, melting paraffin II at 65 °C for 1 h, and melting paraffin III at 65 °C for 1 h. Embedding: The tissue soaked in wax was embedded in an embedding machine. First, melted wax was placed into the embedding mold. Before the wax solidified, the tissue was removed from the dehydration box, placed into the embedding mold according to the embedding surface requirements, and labeled accordingly. The embedding mold was cooled on a −20 °C freezing stage. Once the wax solidified, the wax block was removed from the embedding mold and trimmed. Sectioning: The trimmed wax block was cooled on a −20 °C freezing stage and then placed in a paraffin microtome for sectioning at a thickness of 4 μm. The sections were floated on a water bath at 40 °C to flatten the tissue, then picked up onto glass slides. The slides were placed in a 60 °C oven to dry after water evaporation and paraffin melting. Once dried, they were stored at room temperature for future use. Hematoxylin and eosin (HE) staining: Sections were stained in hematoxylin for 3–5 min, rinsed in tap water, differentiated, rinsed again in tap water, counterstained in eosin, and then rinsed under running water. Eosin staining: Sections were sequentially dehydrated in 85 and 95% gradient ethanol for 5 min each, then stained in eosin solution for 5 min. Dehydration and sealing: Sections were successively dehydrated in absolute ethanol I for 5 min, absolute ethanol II for 5 min, absolute ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min. After transparency, they were sealed with neutral resin. Microscopic examination and image acquisition analysis.

Sample fixation: Fresh duodenum samples from lambs at different stages of N. oiratianus infection were gently rinsed with pre-cooled PBS to remove blood stains and intestinal contents from the intestinal surface. They were then quickly immersed in electron microscopy fixative and fixed at room temperature for 2 h before being transferred to 4 °C for storage. Post-fixation: The fixed samples were washed three times with 0.1 M phosphate buffer (pH 7.4) for 15 min each time. They were then fixed in 1% osmium tetroxide prepared in 0.1 M phosphate buffer (pH 7.4) at room temperature in the dark for 1–2 h. Afterward, the samples were washed three times with 0.1 M phosphate buffer (pH 7.4) for 15 min each time. Dehydration: The tissues were dehydrated sequentially in 30–50%, 70–80%, 90–95%, 100–100% ethanol for 15 min each, followed by immersion in isoamyl acetate for 15 min. Drying: The samples were dried in a critical point dryer. Sample conductive coating: The samples were tightly attached to a double-sided conductive carbon adhesive and placed on the sample stage of an ion sputtering instrument for gold sputtering for about 30 s. They were then observed and imaged under a scanning electron microscope.

According to the gene expression profiles of each group of samples, differential gene analysis was performed using DESeq2, with the criteria for selecting differentially expressed genes set as Q value ≤0.05 and |log2FC| ≥ 1. The results, as indicated in the Figure 1, show the following: Clustering analysis of the above DEGs (differentially expressed genes) revealed that when comparing free-living L1–L3 stages with eggs, upregulated genes were primarily associated with actin binding, symporter activity, and neuropeptide hormone activity, while downregulated genes were mainly related to carbohydrate binding. In contrast, when parasitic L4–L5 stages were compared with eggs, upregulated genes were predominantly involved in metal ion binding, lipid binding, and phospholipid metabolic process. For adult male and female worms versus eggs, upregulated genes were mainly linked to adherens junction, metal ion binding, lipid transporter activity, nutrient reservoir activity, lipid catabolic process, and chitin binding, while downregulated genes were primarily associated with arachidonic acid secretion. These findings indicate that during egg development, upregulated genes are predominantly related to the development of the muscular and nervous systems, whereas during the parasitic stage, they are mainly involved in biological processes such as nutrient metabolism and reproduction.

Based on the protein expression profiles of each group of samples, differentially expressed proteins were screened using Fold change >2 and p-value <0.05 as the criteria. The results, as indicated in the figure, show the following: Clustering analysis of the above DEPs (differentially expressed proteins) revealed that when comparing free-living L1–L3 stages with eggs, upregulated proteins were primarily associated with cuticle development, molting cycle, collagen and cuticulin-based cuticle, actin filament organization, myosin filament assembly, and locomotory behavior, while downregulated proteins were mainly related to negative regulation of translation, anaerobic respiration, lipid storage, regulation of mitotic cell cycle, and carbohydrate metabolic process. This reflects biological processes such as molting during larval development and energy consumption reduction in infective larvae. In contrast, when parasitic L4–L5 stages were compared with eggs, upregulated proteins were predominantly involved in muscle organ development, glycolytic process, nervous system process, and metabolic process. For adult male and female worms versus eggs, upregulated proteins were mainly linked to mitotic cytokinesis, innate immune response, carbohydrate metabolic process, lipid catabolic process, and chitin binding, while downregulated proteins were primarily associated with dauer larval development (Figure 1).

Through GO enrichment analysis, when compared with eggs, DEGs at each developmental stage were predominantly enriched in biological processes related to the integral component of membrane and metal ion binding, which may be associated with the blood-feeding biological characteristics of N. oiratianus. Additionally, in L3 stage, nucleus and DNA binding were significantly enriched, while in L4 stage, nucleus, RNA binding, and DNA binding were significantly enriched. Proteins can regulate various cellular functions through interactions with nucleic acids (DNA and RNA). The significant enrichment of DNA binding and RNA binding reflects the rapid transition of N. oiratianus from the free-living stage to the parasitic stage, where transcriptional regulation, alternative splicing, and modification of DEGs control the development of the nematode. In the female and male worm groups, the extracellular region was significantly enriched, indicating biological processes such as signal transduction with the host intestinal environment, cell adhesion, extracellular matrix formation, cell motility, and tissue structure maintenance (Figure 2).

Through KEGG enrichment analysis, compared with eggs, the Pathways of DEGs (differentially expressed genes) at each developmental stage were mainly enriched in Protein processing in endoplasmic reticulum and Longevity regulating pathway—multiple species/worm, and additionally, in L2 the DEGs were predominantly enriched in Spliceosome, in L4 they were mainly enriched in Neutrophil extracellular trap formation, mRNA surveillance pathway and MAPK signaling pathway, in L5 group the DEGs were primarily enriched in Lysosome and Autophagy—animal, while in the male worm group the DEGs were significantly enriched in Complement and coagulation cascades and Phagosome; these KEGG analyses of DEGs reflect the rapid transcription of genes from the free-living stage to the parasitic stage to adapt to different survival environments (Figure 3).

Through GO enrichment analysis, compared with eggs, DEPs (differentially expressed proteins) at each developmental stage were predominantly enriched in cellular process and metabolic process within biological processes, in cell and cell part within cellular components, and in binding and catalytic activity within molecular function GO terms. Through KEGG enrichment analysis, compared with the control group, DEPs in L1 were predominantly enriched in pathways such as Spliceosome, Focal adhesion, Toxoplasmosis, and Amino sugar and nucleotide sugar metabolism within cellular processes; DEPs in L2 were mainly enriched in pathways including RNA transport, Phagosome, and PPAR signaling pathway; DEPs in L3 were predominantly enriched in Protein processing in endoplasmic reticulum, Phagosome, and Glucagon signaling pathway; DEPs in L4, L5 groups and male worms were all mainly enriched in Metabolic pathways; DEPs in female worms were predominantly enriched in Metabolic pathways and Carbon metabolism. The GO and KEGG enrichment analyses of these DEPs reflect the protein expression differences of N. oiratianus from the free-living to parasitic stages, adapting to different survival environments and rapid development (Figures 4, 5).

Reverse Transcription quantitative PCR (RT-qPCR) was used to validate four stage-specific differentially expressed mRNAs across four infection stages. Results showed that MUC5AC, FGB, and CLDN18 were highly expressed only in the control group and L3 infection group, while CCL19 exhibited higher expression levels in the infection groups. The RT-qPCR results of differentially expressed mRNAs were consistent with those of RNA-Seq, further confirming the reliability of the RNA-Seq findings in this study (Figure 6).

After lambs are infected with N. oiratianus, the body initially produces specific IgM and IgA in the early stage of infection, with various cytokines all increasing (p < 0.05) compared to the uninfected group, thereby resisting N. oiratianus infection. However, as the infection duration prolongs, IFN-γ, IL-2, and TNF-β significantly decrease (p < 0.05), while IL-5 significantly increases (p < 0.05), indicating that Th2-type humoral immunity becomes the main anti-parasitic mechanism (Figure 7).

As shown in Figure 8, the intestinal villi in the control group exhibited a complete structure, with mucosal epithelial cells neatly arranged, goblet cells and striated borders clearly visible, normal lamina propria structure, and intact central lacteals. In the L3 infection group, intestinal villi remained relatively intact, but partial shedding of mucosal epithelial cells from villi was observed, along with edema in the lamina propria and infiltration of numerous mononuclear cells and a small number of eosinophils. In the L4 infection group, intestinal villi were still present, but severe shedding of mucosal epithelial cells occurred, exposing the lamina propria directly to the intestinal lumen, with dilation of central lacteals in the mucosa. In the L5 infection group, intestinal villi were preserved, but severe shedding of mucosal epithelial cells led to direct exposure of the lamina propria to the intestinal lumen, accompanied by edema in the lamina propria. In the adult worm infection group, intestinal villi were swollen due to mononuclear cell infiltration or edema in the lamina propria, with focal shedding of mucosal epithelium. As shown in Table 1, with the progression of infection, the height of intestinal villi significantly decreased (p < 0.05), crypt depth significantly increased (p < 0.05), and the villus height/crypt depth ratio (V/C ratio) also significantly decreased (p < 0.05), indicating a decline in the intestine’s ability to absorb nutrients and an increased probability of diarrhea.

Electron microscopy results showed that in uninfected lambs, intestinal villi were long and slender, with a smooth intestinal wall. After infection with N. oiratianus, severe shedding of epithelial cells occurred, and villi became dispersed. The lamina propria swelled due to cell infiltration. With the detachment of the worms, the length of intestinal villi shortened, cell gaps widened, and density decreased significantly (Figure 9). Although electron microscopy did not directly capture the parasitic state of N. oiratianus, the newly expelled worms exhibit a helical morphology, indirectly proving that they are not in a free state while parasitizing the intestinal wall. When the worms parasitize, they entwine around the intestinal villi, and the shedding and replacement of intestinal villi may be related to the expulsion (Figure 10).