Identification of antigens in the Trichinella spiralis extracellular vesicles for serological detection of early stage infection in swine

Abstract

Background: Several studies have reported the roles of Trichinella spiralis extracellular vesicles in immune regulation and pathogen diagnosis. Currently, the T. spiralis muscle larvae excretory/secretory product (Ts-ML-ES) is the antigen recommended by the International Commission on Trichinellosis (ICT) for serological diagnosis of trichinellosis. However, it can only be used to detect middle and late stages of infections, and cross-reactions with other parasite detections occur. Therefore, there is a need to identify antigens for specific detection of early stage trichinellosis.

Methods: Extracellular vesicles of T. spiralis muscle larvae (Ts-ML-EVs) were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. Ts-ML-EVs protein profiles were analyzed by LC-MS/MS proteomics for identification of potential antigens (Ts-TTPA). Ts-TTPA were cloned into pMAL-c5X vector and expressed as recombinant proteins for evaluation of potential as detected antigens by western blot and ELISA.

Results: Isolated Ts-ML-EVs were round or elliptic (with diameters between 110.1 and 307.6 nm), showing a bilayer membrane structure. The specific surface markers on the Ts-ML-EVs were CD81, CD63, enolase and the 14-3-3 protein. A total of 53 proteins were identified by LC-MS/MS, including a variety of molecules that have been reported as potential detection and vaccine candidates. The cDNA of Ts-TTPA selected in this study has a total length of 1152 bp, encoding 384 amino acids with a molecular weight of 44.19 kDa. It contains a trypsin domain and can be recognized by anti-His antibody. It reacted with swine sera infected with 10,000 T. spiralis at 15, 25, 35 and 60 days post-infection (dpi). At 10 μg/ml, this antigen could detect T. spiralis antibodies from the swine sera at 13 dpi. There were no cross-reactions with the swine sera infected with other parasites including Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis.

Conclusions: This study identifies potential early stage detection antigens and more thoroughly characterizes a serine protease domain-containing protein. Extracellular vesicle proteins may be explored as effective antigens for the early stage detection of trichinellosis.

Keywords: Detection antigens of early stage; Extracellular vesicle; Proteomics; Trichinella spiralis.

Fig. 1

Characterization and identification of Ts-ML-EVs. A The morphology and size of T. spiralis extracellular vesicles visualized by scanning electron microscopy. White arrow indicates the extracellular vesicles (scale bar 100 μM). B NTA shows the size distribution of T. spiralis extracellular vesicle by intensity. The illustration is a combination of histograms and peak charts. C Identification of marker proteins 14-3-3, enolase in the excretory-secretory product (40 μg) and extracellular vesicles (40 μg) of T. spiralis. D CD81 and CD63 were identified on the extracellular vesicles of T. spiralis by flow cytometry

Fig. 2

Determination of components of Ts-ML-ES and Ts-ML-EVs. A SDS-PAGE of Ts-ML-ES and Ts-ML-EVs stained with Coomassie Bright Blue and silver. B Ts-ML-ES and Ts-ML-EVs were identified by serum at 25 dpi with 10,000 T. spiralis. Negative serum was used as a control

Fig. 3

Molecular weight and GO annotation of proteins screened by LC-MS/MS. A The distribution of all 53 protein numbers with different molecular weights was detected by LC-MS/MS. B The GO annotation shows the number of proteins involved in different functions detected by LC-MS/MS

Fig. 4

Bioinformatic analysis of Ts-TTPA. A Three-dimensional structural model of Ts-TTPA protein with secondary structure display. The alpha helix is covered in purple, the beta fold is covered in green, and the ring area is covered in gray. B Cladogram of analysis of Ts-TTPA. The maximum parsimony tree of tissue-type plasminogen activator protein in 12 species of Trichinella spiralis and Trichuris suis was drawn in MEGA. Protein sequence thumbnails of Ts-TTPA for each species are shown on the right. Different-colored squares represent different amino acids

Fig. 5

Expression and purification of rTs-TTPA. A The constructed vector was identified by double restriction enzyme digestion. From right to left are DNA markers, pMAL-c5X-Ts-TTPA plasmid digested with BamHI/EcoRI and pMAL-c5X-Ts-TTPA plasmid. The red arrow shows the Ts-TTPA gene. B The purified Ts-TTPA was stained with Coomassie Bright Blue. The red arrow shows the Ts-TTPA protein. C The purified rTs-TTPA was identified by monoclonal His-tag antibody

Fig. 6

Identification of rTs-TTPA reactivity. The rTs-TTPA was recognized by infected swine serum at different times (15 dpi, 25 dpi, 35 dpi, 60 dpi, 120 dpi), and negative serum was used as a control

Fig. 7

Establishment of rTs-TTPA-ELISA for early stage detection. A Eighty-six negative pig serum samples (1:100) were used to calculate the cut-off, with line representing mean + 3 SD (blue). B Kinetics of serum anti-Trichinella IgG in pigs experimentally infected with 10,000 muscle larvae (n = 3). C Cross-reactions assay of serum infected with Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis (n = 3)