Immunoproteomic analysis of Trichinella spiralis and Trichinella britovi excretory-secretory muscle larvae proteins recognized by sera from humans infected with Trichinella

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.

Insight into Trichinella britovi infection in pigs: effect of various moderate infectious doses on larvae density and spatial larvae distribution in carcasses and comparison of the detection of anti-T. britovi IgG of three different commercial ELISA tests and immunoblot assay

“…It should be noted, however, that all global studies on the use of Western blot for pig trichinellosis detection were performed using T. spiralis ML ES antigens as well as serum samples collected from pigs infected with T. spiralis [30,31]; the results of this research have been discussed elsewhere [32]. It was also proved that all Trichinella species share some ES proteins and, as a consequence, an ES antigen obtained from one Trichinella species is able to detect infections induced by other Trichinella species and genotypes [29]; however, recently Grzelak et al [33] showed that sera from Trichinella-infected persons displayed some differences in terms of their speci c band patterns when T. spiralis and T. britovi ML ES antigens were used. It was also found by using one-dimensional electrophoresis that the native T. spiralis ES antigen shows more protein bands than the T. britovi ML ES antigen [33].…”

“…It was also proved that all Trichinella species share some ES proteins and, as a consequence, an ES antigen obtained from one Trichinella species is able to detect infections induced by other Trichinella species and genotypes [29]; however, recently Grzelak et al [33] showed that sera from Trichinella-infected persons displayed some differences in terms of their speci c band patterns when T. spiralis and T. britovi ML ES antigens were used. It was also found by using one-dimensional electrophoresis that the native T. spiralis ES antigen shows more protein bands than the T. britovi ML ES antigen [33]. However, on the basis of our results and studies conducted by Gomez Morales et al [30], it should be stated that the use of T. britovi ML ES antigen and serum samples of pigs infected with T. britovi did not signi cantly affect on Western blot band pattern as compared to the pattern generated by serum of pigs infected with T. spiralis tested by using T. spiralis ES-WB.…”

“…The T. britovi ML ES antigen was produced as described previously [33,36] and kept frozen at −70 °C. Brie y, T. britovi ML were washed with Roswell Park Memorial Institute medium (RPMI)-1640 and resuspended at 5000 ML/ml in RPMI-1640 supplemented with 20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 200 mM L-glutamine, 100 mM Na-pyruvate and 100 units each of penicillin and streptomycin.…”

Identification and profiling of Trichinella spiralis circulating antigens and proteins in sera of mice with trichinellosis

“…In addition, previous immunoproteomic studies have identified hypothetical protein Tsp_10331 (XP_003372574.1), conserved hypothetical protein (XP_003374582.1), putative LNS2 protein (OUC42377.1), hypothetical protein D917_07875 (OUC46244.1), peptidase S1C family (XP_003375617.1), phosphatidylinositol phosphatase PTPRQ (KRY42983.1) and putative zinc finger protein C06E1.8 (KRY37548.1) as T . spiralis antigens [ 20 , 21 ].…”

Prospects of Using High-Throughput Proteomics to Underpin the Discovery of Animal Host–Nematode Interactions

“…Trichinella species. Immunoproteomic studies of Trichinella spiralis , Trichinella pseudospiralis and Trichinella britovi have focused on ES proteins [ 51 , 52 , 53 , 54 , 55 ], with the intention of improving the immuno- or sero-diagnosis of trichinellosis. Numerous immunogenic proteins, including serine protease, enolase and peptidase inhibitors, were recognised by antibodies in the sera from patients or from experimental animals.…”