The case came from more than 120 60-day-old Sanhuang chickens raised by a farmer in Guangling District, Yangzhou City, Jiangsu Province. On 19 April 2018, the flocks presented with symptoms including crouched head, drooping wings, and excretion of watery greenish yellow diarrhea. Necropsy of the dead chicken showed essentially the same symptoms, with ulcerated lesions in the liver with rounded depressions on the surface, a distinctive crater-like shape. The ceca were swollen and contained casein-like emboli.

Approximately 1 g of necrotic tissues from diseased chicken livers were aseptically removed and placed into a sterile 1.5 ml Eppendorf tubes. The tissues were smashed and inoculated into 10 ml medium that was preheated to 40°C, and then incubated anaerobically in an incubator at 40°C. The medium consisted of 9 ml of M199 medium (Gibco, California, USA), 1 ml of inactivated horse serum (Gibco), and 11 mg of rice flour (Sigma, St. Louis, MO, USA), and was inoculated with a ring of cecal bacteria grown on a Columbia blood agar plate. The cecal bacteria were obtained by inoculating Colombia blood agar plate with the cecal contents of healthy chickens and placing them overnight at 37°C. Cecal bacterial species were identified as Escherichia coli and Klebsiella pneumoniae by microbial mass spectrometry (MALDI Biotyper).

The cloning was carried out using the limiting dilution method of monoclonal antibodies. The cultured parasites reaching their growth peak were diluted to ~10³ cells/ml with preheated medium, and 1–2 μl of the medium was drawn onto cover slips to observe the parasites under a microscope. Single parasites were labeled and transferred to Eppendorf tubes containing 1 ml of medium and anaerobically cultured in an incubator at 40°C. After 3–4 days of cultivation, the culture broth containing propagated parasites was inoculated into fresh basal medium at a 1:10 ratio for the propagation of the parasites. The cloned parasite strain was named HM-JSYZ-D and cryopreserved.

The in vitro passage medium constituted of Medium 199 (Gibco, California, USA) and 10% inactivated horse serum (Gibco), 11 mg of sterilized rice flour (Sigma-Aldrich, St. Louis, MO, USA), with a total volume of 10 ml. Parasites were passaged every 3 days. Long term preservation use 10% dimethyl sulfoxide (Sigma-Aldrich, ShangHai, China) as cryoprotectant. In vitro, H. meleagridis with 10 passages was labeled as D10, and H. meleagridis with 168 passages was labeled as D168.

To assess whether H. meleagridis D168 strain has been attenuated. Thirty sanhaung chickens (purchased from the Institute of Poultry, Chinese Academy of Agricultural Sciences, Jiangsu, China) reared in steel cages with wire flooring under specific-pathogen-free conditions were randomly divided into three groups at 14 days of age: D168, D10, NC (10 chickens per group). These chickens were raised in an isolated environment after hatching and fed a drug-free feed formula with free access to water. Animal care and all experiments were performed according to the rules of the Animal Experiment Ethic Committee of Yangzhou University. On the day of grouping, the chickens in group D168 were inoculated cloacally at doses of 2 × 10⁵ JSYZ-D168 cells/chicken; the chickens in group D10 were inoculated cloacally at doses of 2 × 10⁵ JSYZ-D10 cells/chicken; the chickens in group NC served as negative control (unchallenged with virulent strain). On day 28 (14 days post challenge), all remaining chicks were euthanized and observed for cecal and hepatic lesions.

The D10 and D168 generations of parasites passaged in the same batch as the animal test were collected and designated as H. meleagridis/chicken/China/JSYZ-D/D10 and H. meleagridis/chicken/China/JSYZ-D/D168. Each sample was collected in triplicate, and the quality and quantity of H. meleagridis was assessed under a microscope using Trypan Blue Stain (TaKaRa, BeiJing, China), ensuring that each sample is no less than 1 × 10⁷ number of parasites. A purification method of continuous washing and centrifugation were used to collect H. meleagridis cells and remove most of the bacteria (15). Cultures were concentrated and transferred to 1.5 ml Eppendorf tubes, and histomonads were collected by centrifugation at 200 g for 5 min at 4°C. The bacteria enriched supernatant was discarded and the histomonads were resuspended in M199 medium at 4°C. This step was repeated for three times. The histomonads were finally centrifuged at 8,000 g for 2 min at 4°C, and the precipitate was collected after discarding the supernatant. The collection parasites were stored at −196°C before further use.

Parasite samples was sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer (8 M Urea, 1% Protease Inhibitor Cocktail). The remaining debris was removed by centrifugation at 12,000 g at 4°C for 10 min. The supernatant was collected and the protein concentration was determined with BCA (Beyotime, HangZhou, China) kit according to the manufacturer's instructions. Equal amounts of each sample protein were taken for enzymatic lysis, appropriate amounts of standard protein were added, the volume was adjusted to a consistent volume with the lysis solution, the final concentration of 20% TCA was slowly added, vortexed, and precipitated at 4°C for 2 h. Centrifuged at 4,500 g for 5 min, discard the supernatant, and wash the sample 2–3 times with pre chilled acetone. After air drying the sample was added to a final concentration of 200 mM TEAB (Sigma, HangZhou, China), the sample was disrupted by sonication. Trypsin (Promega, HangZhou, China) was added at a 1:50 ratio and enzymatically digested overnight at 4°C. Protein solution was reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer's protocol for TMT kit (Thermo, HangZhou, China). Briefly, one unit of TMT reagent were thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 h at room temperature and pooled, desalted, and dried by vacuum centrifugation.

Peptides were separated after dissolved them in solvent A (0.1% formic acid in 2% acetonitrile). The gradient was comprised of an increase from 7% to 25% solvent B (0.1% formic acid in 90% acetonitrile) over 24 min, 25% to 35% in 8 min and climbing to 80% in 4 min then holding at 80% for the last 4 min, all at a constant flow rate of 500 nl/min on an EASY-nLC 1000 UPLC system.

Peptides were injected into the NSI ion source for ionization after separation via the ultra-high-performance liquid phase system and then subjected to Q Exactive Plus mass spectrometry for analysis. The ion source voltage was set to 2.1 kV, and both peptide parent ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set to 400–1,500 m/z, and the scan resolution was set to 70,000. The secondary MS scan range was then fixed to a starting point of 100 m/z, and the secondary scan resolution was set to 17,500. Data acquisition mode used a data dependent scanning program whereby the top 20 peptide parent ions with the highest signal intensity were selected after the primary scan to sequentially enter the higher energy collision-induced dissociation (HCD) collision cell for fragmentation using 28% fragmentation energy, again followed sequentially by secondary mass spectrometry. To improve the effective utilization of the mass spectra, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 78,000 ions/s, the maximum injection time was set to 64 MS, and the dynamic exclusion time of the tandem MS scans was set to 30 s to avoid repetitive scanning of the parent ions.

The secondary mass spectrometry data were retrieved using MaxQuant (v1.5.2.8). Retrieval parameter setting: the BioProject number for the database is PRJNA935845 (25518 sequences, E. coli genomes ASM886v2 and ASM584v2, and Klebsiella pneumoniae genomes ASM24018v2 and ASM383019v1, genes from these genomes were deleted on the comparison), an reverse decoy database was added to calculate the false discovery rate (FDR) caused by random matching, and a common contaminated library was added to the database to eliminate the influence of contaminated proteins in the identification results. The enzyme digestion mode is set to Trypsin/P. The number of missing bits is set to two. The minimum length of the peptide was set to seven amino acid residues. The maximum number of peptide modifications was set to five. The mass error tolerance of primary parent ions of First search and Main search are set to 20 and 5 ppm, respectively, and the mass error tolerance of secondary fragment ions is 0.02 Da. Cysteine alkylation was set as fixed modification, variable modification were methionine oxidation, protein N-terminal acetylation, and deamidation. The quantitative method is set to TMT-6plex, and the FDR for protein identification and PSM identification is set to 1%.

The screening criteria of differential proteins were: D10/D168 ratio ≥1.2 and D10/D168 p-value <0.05 as up-regulated proteins; D10/D168 ratio ≤0.83 and D10/D168 p-value <0.05 as down regulated proteins. Sequence information for PRJNA935845 were mapped to the NR (NCBI non-redundant protein sequences), NT (NCBI non-redundant nucleotide sequence), SwissProt (a manually annotated and reviewed protein sequence database), KEGG (Kyoto Encyclopedia of Genes and Genomes, version 59), and KOG (Clusters of Eukaryotic Orthologous Groups) database by Blast software (version 2.2.23) with default parameters (under a threshold E-value ≤10⁻⁵) to get the isoform annotations (17). Gene Ontology (GO) annotations and functional classifications were obtained using Blast2GO program (version 2.5.0, E-value ≤10⁻⁵) based on NR annotations (20). InterProScan5 software (version 5.11–51.0) was used to get annotations from InterPro database (21).

Proteins were classified by Gene Ontology (GO) annotation into three categories: biological process, cellular compartment, and molecular function. For each category, a two-tailed Fisher's exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins. The GO with a corrected p-value < 0.05 is considered significant. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways by a two-tailed Fisher's exact test to test the enrichment of the differentially expressed protein against all identified proteins. The pathway with a corrected p-value < 0.05 was considered significant. Annotation information of KEGG was obtained by alignment using diamond (v0.9.26.127) software based on KOBAS (V3.0) (containing species sequence information) and KEGG database (sequence corresponding KO information). These pathways were classified into hierarchical categories according to the KEGG website. In the cluster analysis of functional enrichment of differentially expressed proteins, all the categories obtained after enrichment and their p-values were first collated, and then filtered for those categories which were at least enriched in one of the clusters with p-value <0.05. This filtered p-value matrix was transformed by the function x = –log₁₀ (p-value). Finally these x-values were z-transformed for each functional category. These z-scores were then clustered by one-way hierarchical clustering (Euclidean distance, average linkage clustering) in Genesis. Cluster membership were visualized by a heat map using the “heatmap.2” function from the “gplots” R-package.

Construction of protein–protein interaction (PPI) networks was also conducted by using the STRING (v11.0) database with R package “networkD3” tool. All identified protein name identifiers were blast to Trichomonas vaginalis (Taxonomy id:5722) and searched against the STRING database version 11.0 for protein–protein interactions. Only interactions between the proteins belonging to the searched data set were selected, thereby excluding external candidates. STRING defines a metric called “confidence score” to define interaction confidence; we fetched all interactions that had a confidence score ≥0.7 (high confidence).

To further validate the protein expression level gained through TMT quantification, additional quantification through parallel reaction monitoring (PRM) analysis was performed (22). Equal amounts of each sample protein were taken for enzymatic lysis, and the volume was adjusted to unity using lysis buffer. The final concentration of 20% TCA was slowly added, vortexed to mix, and precipitated at 4°C for 2 h. Centrifuge at 4,500 g for 5 min, discarded the supernatant and washed the precipitate 2–3 times with pre cooled acetone. After air drying the pellet was added to a final concentration of 200 mM TEAB, the pellet was disrupted by sonication, and trypsin was added at a 1:50 ratio (protease: protein, m/m), and enzymatically digested overnight. Dithiothreitol (DTT, Sigma, HangZhou, China) was added to a final concentration of 5 mm and reduced at 56°C for 30 min. After that, iodoacetamide (IAA, Sigma, HangZhou, China) was added to a final concentration of 11 mm and incubated for 15 min in the dark at room temperature. Peptides were separated after dissolved them in solvent A (0.1% formic acid in 2% acetonitrile). The gradient was comprised of an increase from 7% to 25% solvent B (0.1% formic acid in 90% acetonitrile) over 24 min, 25% to 35% in 8 min, and climbing to 80% in 4 min then holding at 80% for the last 4 min, all at a constant flow rate of 500 nl/min on an EASY-nLC 1000 UPLC system. Then the peptides were injected into the NSI ion source for ionization after separation by the ultra-high-performance liquid phase system and then subjected to Q Exactive^TM Plus mass spectrometry for analysis. The ion source voltage was set to 2.1 kV, and both peptide parent ions and their secondary fragments were detected and analyzed using the high-resolution 16/18 Orbitrap. The primary mass spectrometry scan range was set to 437–909 m/z, and the scan resolution was set to 70,000; the secondary mass spectrometry Orbitrap scan resolution was set to 17,500. Data acquisition mode a data-independent acquisition (DIA) program was used with the fragmentation energy of the HCD collision cell set to 27. Generated MS data were processed using skyline (v.3.6) to obtain the signal intensities for individual peptide sequences.

The symptoms after inoculation with the D10 strain in the chickens included depression and diarrhea, with four chickens experiencing symptoms and the morbidity reached 40%. No abnormal symptoms were found in D168 group. In the D10 group, crater-like circular ulcerative necrotic areas appeared on the liver surface of chickens, the cecal wall was thickened and intestinal emboli formed in the ceca (Figures 1A, B). No significant macroscopic lesions were observed in the livers and ceca of D168 group chickens (Figure 1C).

Based on TMT labeling and LC-MS/MS approaches, the difference between virulent strain and attenuated strain after continuous passage of H. meleagridis were analyzed. From MS analysis, 270,882 spectra were obtained, of which 24,857 effective spectra were available and the utilization rate was 9.2%. A total of 15,042 peptides were identified from spectral analysis, of which specific peptide numbered 13,624. A total of 3,849 proteins were identified, of which 3,494 were quantified (Figure 2A). When p-value < 0.05, the variation threshold of significant up-accumulation was set as the variation of differential expression ≥1.2, and the variation threshold of significant down-accumulation was set as the variation threshold ≤0.83. A total of 745 differentially expressed proteins were identified. Relative to D168, there were 192 up-regulated proteins and 553 down-regulated proteins in D10 (Figure 2B, Supplementary Table S1).

According to GO annotations, differentially expressed proteins were classified into three categories: Biological Processes, Cellular Components, and Molecular Functions. Biological processes analyses indicated proteins were involved in organic substance metabolic processes, primary metabolic processes, and cellular metabolic processes in both virulent and attenuated H. meleagridis. In cell composition analysis, proteins were primarily distributed in cytoplasm, organelles, cytosol, and membrane. In molecular function analysis, proteins were mainly exert the functions of protein binding, organic cyclic compound binding, heterocyclic compound binding (Figure 3).

Gene Ontology (GO) annotations were performed for up-regulated proteins in D10 relative to D168. Biological process analysis showed that proteins mainly participate in the organic substance metabolic process, regulation of biological process, and primary metabolic process. Cellular component analysis revealed that the proteins were mainly distributed in cytoplasm, organelles, and membranes. Molecular functional analysis revealed that the proteins mainly functioned as organic cyclic compound binding, heterocyclic compound binding, structural constituent of ribosome, and hydrolase activity (Figure 4).

Gene Ontology (GO) annotations were performed for down-regulated proteins in D10 relative to D168. Biological process analysis showed that proteins mainly participate in the organic substance metabolic process, primary metabolic process, and cellular metabolic process. Cellular component analysis revealed that the proteins were mainly distributed in cytoplasm, organelle, and cytosol. Molecular functional analysis revealed that the proteins mainly functioned in protein binding, organic cyclic compound binding, heterocyclic compound binding, and hydrolase activity (Figure 5).

These data suggested proteins were involved in a variety of biological processes, cellular components, and cellular molecular functions. The ribosome associated functions of the virulent strains were more vigorous and the attenuated strains were more adaptable to the in vitro culture environment.

To further clarify the functional relevance of differential proteins, they were divided into four parts according to the multiple of differences: Q1 < 0.769, 0.769 < Q2< 0.833, 1.2 < Q3< 1.3, Q4 > 1.3 (Figure 6), respectively, enriched the GO and KEGG pathways, and performed cluster analysis. From Figure 6, these analyses showed that for biological processes, proteins in Q1 were primarily involved in transcription by RNA polymerase III, DNA-templated transcription, termination, and mRNA processing. Proteins in Q2 were mainly involved in protein N-linked glycosylation via asparagine, peptidyl-asparagine modification, pseudouridine synthesis, and RNA-dependent DNA biosynthetic process. Proteins in Q3 were mainly involved in positive regulation of mRNA binding, cellular response to red or far red light, and negative regulation of cofactor metabolic process. Proteins in Q4 were mainly involved in negative regulation of gene expression, RNA processing, ribosomal small subunit assembly, and positive regulation of microtubule binding (Figure 7A).

For cellular components, proteins in Q1 were distributed in cell envelope. Proteins in Q2 were mainly distributed in nuclear envelope, organelle envelope, and endomembrane system. Proteins in Q3 were distributed in translation preinitiation complex. Proteins in Q4 were mainly distributed in preribosome, large subunit precursor, plasmodesma, and symplast (Figure 7B).

For molecular functions, proteins in Q1 were concentrated in mannosyl-oligosaccharide mannosidase activity, and DNA-binding transcription factor activity. Proteins in Q2 were mainly concentrated in isomerase activity, ATPase activity, and telomerase activity, etc. Proteins in Q3 were mainly concentrated in RNA cap binding, translation initiation factor activity, and protein serine/threonine phosphatase activity, etc. Proteins in Q4 were mainly concentrated in structural constituent of ribosome, protein phosphatase regulator activity, G protein-coupled receptor binding, and protein phosphatase activator activity (Figure 7C).

KEGG pathway analysis showed that proteins in Q1 were related to cytosolic DNA-sensing pathway, spliceosome, and ether lipid metabolism. Proteins in Q2 were mainly related to ribosome biogenesis in eukaryotes, MAPK signaling pathway, cAMP signaling pathway, and Toll-like receptor signaling pathway. Proteins in Q3 were mainly associated with carbohydrate digestion and absorption, inflammatory bowel disease, cellular senescence, and hepatocellular carcinoma. Proteins in Q4 were associated with ribosome, and TGF-beta signaling pathway (Figure 7D).

Proteins were selected for protein interaction network mapping based on pre-existing studies (13, 15, 16, 23), combined with protein function descriptions, including a total of 60 up-regulated proteins and 65 down-regulated proteins. They include virulence-related proteins, such as surface protein BspA-like, digestive cysteine proteinase 2, NADP-dependent malic enzyme; metabolism-related proteins, such as ferredoxin, enolase; cytoskeleton-related proteins, such as actin, alpha-actinin-1; ribosomal proteins, Ras-related proteins, and Ras-like proteins that were abundantly represented in this study (Supplementary Table S2). The results showed that most of the proteins in which the interactions occurred were ribosome associated proteins, which also included eukaryotic translation initiation factors and receptor expression enhancing proteins. The most abundant proteins that undergo interactions are 40S ribosomal protein S19S, 60S ribosomal protein L14, 60S ribosomal protein L17, and 60S ribosomal protein L10a (Figure 8).

In order to evaluate the reliability of TMT data, PRM was used to detect the relative levels of ten important functional proteins in the two strains. Seven cysteine related proteins (LCP2, PRPF8, Cts1, KYAT1, cysA, KANK1, metK), two cytoskeleton and movement related proteins (AP3D1, ACTN1), and one cell adhesion related protein (ME1) were selected for PRM analysis. As shown in Table 1, the proteins expression levels were quantified by PRM-MS analysis, which confirmed the protein expression levels obtained by TMT. The results of PRM assay were consistent with those of TMT analysis, indicating that the quantitative results were strong persuasive.