Trichinella britovi muscle larvae and adult worms: stage-specific and common antigens detected by two-dimensional gel electrophoresis-based immunoblotting

Abstract

BackgroundTrichinella britovi is the second most common species of Trichinella that may affect human health. As an early diagnosis of trichinellosis is crucial for effective treatment, it is important to identify sensitive, specific and common antigens of adult T. britovi worms and muscle larvae. The present study was undertaken to uncover the stage-specific and common proteins of T. britovi that may be used in specific diagnostics.MethodsSomatic extracts obtained from two developmental stages, muscle larvae (ML) and adult worms (Ad), were separated using two-dimensional gel electrophoresis (2-DE) coupled with immunoblot analysis. The positively-visualized protein spots specific for each stage were identified through liquid chromatography-tandem mass spectrometry (LC-LC/MS).ResultsA total of 272 spots were detected in the proteome of T. britovi adult worms (Ad) and 261 in the muscle larvae (ML). The somatic extracts from Ad and ML were specifically recognized by T. britovi-infected swine sera at 10 days post infection (dpi) and 60 dpi, with a total of 70 prominent protein spots. According to immunoblotting patterns and LC-MS/MS results, the immunogenic spots recognized by different pig T. britovi-infected sera were divided into three groups for the two developmental stages: adult stage-specific proteins, muscle larvae stage-specific proteins, and proteins common to both stages. Forty-five Ad proteins (29 Ad-specific and 16 common) and thirteen ML proteins (nine ML-specific and four common) cross-reacted with sera at 10 dpi. Many of the proteins identified in Ad (myosin-4, myosin light chain kinase, paramyosin, intermediate filament protein B, actin-depolymerizing factor 1 and calreticulin) are involved in structural and motor activity. Among the most abundant proteins identified in ML were 14-3-3 protein zeta, actin-5C, ATP synthase subunit d, deoxyribonuclease-2-alpha, poly-cysteine and histide-tailed protein, enolase, V-type proton ATPase catalytic and serine protease 30. Heat-shock protein, intermediate filament protein ifa-1 and intermediate filament protein B were identified in both proteomes.ConclusionsTo our knowledge, this study represents the first immunoproteomic identification of the antigenic proteins of adult worms and muscle larvae of T. britovi. Our results provide a valuable basis for the development of diagnostic methods. The identification of common components for the two developmental stages of T. britovi may be useful in the preparation of parasitic antigens in recombinant forms for diagnostic use.

Proteomic Analysis Of Trichuris Trichiura Egg Extract Reveals Potential Immunomodulators And Diagnostic Targets

“…The most abundant transcripts found in this extensive study, included proteins we have also identi ed in the EE proteome such as two WAP domain containing SLP-like proteins, protease inhibitors such as Cystatin-domain containing protein and nematode cuticle collagen N-terminal domain containing proteins and Chitin binding domain containing proteins such as CBM14 domain containing proteins. Furthermore, with more or less representation, but of particular interest within the context of the present work, we have found trichuris egg proteins with known immunomodulatory properties such as Macrophage migration inhibitory factor homolog (MIF), previously identi ed in T. trichiura adult [14], and 14-3-3 protein which has also been identi ed in several developmental stages of other nematodes, Trichinella britovi [31] and Trichinella spiralis [32] and trematodes, Schistosoma japonicum [33]. Both proteins are considered as enhancers of humoral and cellular immune responses [34].…”

“…Actin, Tropomyosin, Paramyosin, Intermediate lament protein IFA 1 and Epididymal secretory protein E1 were found with a high number of distinct peptides. These proteins are essential to enhance the motility of the nematodes and have also been recorded in many helminthic proteomes: somatic extract of adults of T. spiralis [43], T. britovi [31], Syphacia muris [42] and Echinostoma caproni [44]; and in egg secretions of Schistosoma mansoni [17]. Speci cally, Intermediate lament protein IFA1 has been studied in Caenorhabditis elegans demonstrating that in nematodes they allow epidermal elongation in the larval stages to grow into adults [45].…”

“…This type of immunoproteomic approach has been was applied in previous studies to determine both the antigenic proteins of different helminths developmental stages (larvae and adults), and evaluate the serological response to the soluble protein extracts of Ascaris lumbricoides [53], T. britovi [31], Schistosoma japonicum [54] and Taenia solium [55].…”

“…HSP-70 and heat shock proteins in general, have caught the attention of researchers for acting typically as immunodominant antigens eliciting strong humoral responses as major targets of host immune responses, suggesting them out as possible candidates for antiparasitic, allergic and autoimmune diseases treatments [61,62]. The HSP70 is amongst the most highly abundant protein identi ed in egg secretions of S. mansoni and H. polygyrus [17,60], and also heavily represented in E. caproni, F. hepatica, H. polygyrus, Schistosoma bovis, T. trichiura, T. britovi and Zygocotyle lunata adult worms extract [14,31,41,44,63,64]. Others have reported on their immunogenicity linked to stimulation of IgG and IgM responses [39,65,66] and they have been suggested as possible vaccine targets [67].…”

“…Furthermore, Enolase plays an important role in brinolysis and degradation of the intracellular matrix through the activation of plasminogen, which may induce plasmin-mediated proteolysis and facilitate the invasion, migration and xation in the host [15,17,29,42]. In T. spiralis [39] and T. britovi [31] this enzyme has been con rmed as immunodominant suggesting that it may assist in tissue migration of the larvae. Enolase and heat shock proteins have also been classi ed as exosome markers [37,70].…”