Characterization of Two Trichinella spiralis Adult-Specific DNase II and Their Capacity to Induce Protective Immunity.

Deoxyribonuclease II (DNase II) is a widespread endonuclease, which can degrade the DNA. Trichinella spiralis adult-specific DNase II-1 (TsDNase II-1) and DNase II-7 (TsDNase II-7) were identified in excretory-secretory (ES) or surface proteins of adult worm (AW) and intestinal infective larvae (IIL) using immunoproteomics with early infection sera. The aim of this study was to characterize the two T. spiralis DNase II enzymes and to investigate their role as potential vaccine candidate target molecules. The cDNA sequences of the two DNase II enzymes from 3 days old AWs of T. spiralis were cloned and expressed. The sequencing results showed that the complete cDNA sequences of the two DNase II enzymes were 1221 and 1161 bp long, and the predicted open reading frames encoded 347 and 348 amino acids, respectively. On Western blot analysis, natural TsDNase II-1 and TsDNase II-7 in the crude extracts of IIL, AWs, and newborn larvae (NBL) and AW ES proteins were recognized by both anti-rTsDNase II-1 and anti-rTsDNase II-7 sera. Indirect immunofluorescence test and qPCR showed that the two DNase II enzymes were highly expressed at AW and NBL stages and were mainly located at the cuticle and stichosome of the nematode. Vaccination with the two recombinant DNase II enzymes triggered prominent humoral responses that exhibited significant immune protection against T. spiralis larval infection, as demonstrated by the notable reduction in intestinal AW and muscle larva burdens. Specific antibodies to the two molecules evidently inhibited the in vitro parasite invasion of enterocytes and participated in the killing of NBL by an antibody-dependent cell-mediated cytotoxicity (ADCC) mode. The enzymes DNase II-1 and DNase II-7 are the potential target molecules for anti-Trichinella vaccine for blocking both larval invasion and development.

The Immune Protection Induced by a Serine Protease Inhibitor From the Foodborne Parasite Trichinella spiralis.

Serine protease inhibitors (SPI) are a superfamily of the proteins able to suppress serine protease activity, and may exert the major biological function in complement activation, inflammation, and fibrinolysis. A SPI was identified from Trichinella spiralis adult worms (AW) by immunoproteomics with early infection sera. The aim of this study was to investigate the protective immune elicited by TsSPI. The complete TsSPI cDNA sequence was cloned into pQE-80 L and then expressed in Escherichia coli BL21. The rTsSPI was purified and its antigenicity was determined by Western blotting analysis. By using anti-rTsSPI serum the native TsSPI was identified in somatic and ES proteins from muscle larvae (ML). The results of qPCR and immunofluorescence assay (IFA) revealed that the expression of the TsSPI gene was observed throughout all developmental stages of T. spiralis (ML, intestinal infective larvale, 3- and 6-days AW, and newborn larvae, NBL), located principally in cuticles, stichosome, and embryos of this parasitic nematode. Vaccination of mice with rTsSPI triggered high level of anti-TsSPI IgG response, and showed a 62.2 and 57.25% worm burden reduction in the recovery of intestinal AW at 6 days post-infection (dpi) and ML at 35 dpi, respectively. The TsSPI might be a novel potential target for anti-Trichinella vaccine.

Morphological and Mitochondrial Genomic Characterization of Eyeworms (Thelazia callipaeda) from Clinical Cases in Central China.

Thelazia callipaeda, also called the oriental eyeworm, is the major etiological agent of human thelaziasis. Cases of thelaziasis have increased in recent years in China. Although this species is of medical importance, the genetics and phylogenetic systematics of T. callipaeda are poorly understood. In this study, we first reported three cases of thelaziasis in central China. All clinical isolates were identified as T. callipaeda according to morphological characteristics by light microscopy and scanning electron microscopy. Next, complete mitochondrial (mt) genomes for the three T. callipaeda isolates from different geographical locations were fully characterized using an Illumina sequencing platform. In addition, all available mt genomes of spirurid nematodes in GenBank were included to reconstruct the phylogeny and to explore the evolutionary histories of the isolates. The genome features of the T. callipaeda isolates contained 12 PCGs, 22 transfer RNA genes, two ribosomal RNA genes and a major non-coding region. The mtDNA nucleotide sequences of the T. callipaeda isolates from different hosts and different locations were similar. The nad6 gene showed high sequence variability among all isolates, which is worth considering for future population genetic studies of T. callipaeda. Phylogenetic analyses based on maximum parsimony and Bayesian inference methods revealed close relationships among Thelaziidae, Onchocercidae, Setariidae, Gongylonematidae, Physalopteridae, Dracunculidae, and Philometridae. The monophyly of the T. callipaeda isolates from different hosts and distinct geographical locations was confirmed. The entire mt genomes of T. callipaeda presented in this study will serve as a useful dataset for studying the population genetics and phylogenetic relationships of Thelazia species.

Proteomic Analysis of Trichinella spiralis Adult Worm Excretory-Secretory Proteins Recognized by Sera of Patients with Early Trichinellosis.

The most commonly used serodiagnostic antigens for trichinellosis are the excretory-secretory (ES) antigens from T. spiralis muscle larvae (ML), but the specific antibodies against the ML ES antigens are usually negative during early stage of Trichinella infection. The recent studies demonstrated that T. spiralis adult worm (AW) antigens were recognized by mouse or swine infection sera on Western blot as early as 7-15 days post-infection (dpi), the AW antigens might contain the early diagnostic markers for trichinellosis. The purpose of this study was to screen early diagnostic antigens in T. spiralis AW ES proteins recognized by sera of early patients with trichinellosis. T. spiralis AW were collected at 72 h post-infection (hpi), and their ES antigens were analyzed by SDS-PAGE and Western blot. Our results showed that 5 protein bands (55, 48-50, 45, 44, and 36 kDa) were recognized by sera of early patients with trichinellosis collected at 19 dpi, and were subjected to shotgun LC-MS/MS and bioinformatics analyses. A total of 185 proteins were identified from T. spiralis protein database, of which 116 (67.2%) proteins had molecular weights of 30∼60 kDa, and 125 (67.6%) proteins with pI 4-7. Bioinformatic analyses showed that the identified proteins have a wide diversity of biological functions (binding of nucleotides, proteins, ions, carbohydrates, and lipids; hydrolase, transferase, and oxidoreductase, etc.). Several enzymes (e.g., adult-specific DNase II, serine protease and serine protease inhibitor) could be the invasion-related proteins and early diagnostic markers for trichinellosis. Moreover, recombinant T. spiralis serine protease (rTsSP-ZH68) was expressed in E. coli and its antigenicity was analyzed by Western blot with the early infection sera. The rTsSP-ZH68 was recognized by sera of infected mice at 8-10 dpi and sera of early patients with trichinellosis at 19 dpi. T. spiralis AW proteins identified in this study, especially serine protease, are the promising early diagnostic antigens and vaccine candidates for trichinellosis.