Abstract
The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.
Highlights
Trichinellosis is a zoonotic parasitic disease caused by nematodes of the genus Trichinella [1]
The purpose of the present study was to compare the immunogenic patterns of the muscle larvae excretory-secretory proteins (ML E-S) proteins of T. spiralis and T. britovi and select immunoreactive proteins recognized by human Trichinella-infected sera by a combination of two-dimensional electrophoresis (2-DE) with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/ MS analysis
The main disadvantage of using enzyme-linked immunosorbent assay (ELISA) based on muscle larvae (ML) E-S antigens is that a high rate of false negative results is typically observed during the early stage of infection, indicating that ML E-S antigens are stage-specific and are not recognized by antibodies during intestinal phase of infection
Summary
Trichinellosis is a zoonotic parasitic disease caused by nematodes of the genus Trichinella [1]. The complete life cycle of Trichinella occurs in a single host, including all stages of the adult worms (Ad), newborn larvae (NBL) and muscle larvae (ML). Trichinella express many immunodominant antigens that have the ability to elicit a significant immune response. In T. spiralis and T. britovi muscle larvae (ML), the excretory-secretory (E-S) proteins are produced mainly by the excretory granules of the stichosome and the cuticles (membrane proteins); these are directly exposed to the host immune system and are the main target antigens which induce immune responses [3]. Most human infections and deaths caused by trichinellosis globally have been attributed to T. spiralis. A number of other encapsulating and non-encapsulating species can cause human infections, including T. britovi, T. nativa, T. pseudospiralis and T. murelli [4,5,6,7,8]
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