The Use of Recombinant 31 kDa Antigens of Trichinella spiralis for Serodiagnosis of Experimental Trichinellosis.
ABSTRACT: BACKGROUND:We have previously reported that a 31 kDa protein was screened from the excretory-secretory (ES) proteins of Tichinella spiralis muscle larvae (ML) by immunoproteomics using early infection sera, and the gene encoding a 31 kDa protein from T. spiralis was cloned and expressed in an E. coli expression system. In this study, the recombinant 31 kDa antigens were used for detection of anti-Trichinella antibodies in serum of experimentally infected mice by ELISA. METHODS:Anti-Trichinella IgG antibodies in sera of mice infected with Trichinella were assayed by ELISA with recombinant 31 kDa antigens, and its sensitivity and specificity were compared with ELISA with ES antigen. RESULTS:The sensitivity and specificity of ELISA with recombinant antigens was 96.67% (29/30) and 96.87% (62/64), compared with 100% (30/30) and 98.44% (63/64) of ELISA with ES antigens was (P>0.05). In heavily, moderately and lightly infected mice (500, 300 and 100 larvae/mouse), anti-Trichinella antibodies were firstly detected by ELISA with recombinant antigens at 8, 12 and 14 dpi, respectively; then increased rapidly with a detection rate of 100% respectively at 28, 22 and 30 dpi. While the antibodies were firstly detected by ELISA with ES antigens at 10, 8 and 10 dpi, respectively, the antibody positive rate reached 100% at 14, 12 and 22 dpi, respectively. CONCLUSION:The recombinant 31 kDa antigens of T. spirali had a good sensitivity and specificity for detecting anti-Trichinella antibodies and might be the potential diagnostic antigen for trichinellosis.
Project description:Trichinellosis is a serious zoonositc parasitosis worldwide. Because its clinical manifestations aren't specific, the diagnosis of trichinellosis is not easy to be made. Trichinella spiralis muscle larva (ML) excretory-secretory (ES) antigens are the most widely applied diagnostic antigens for human trichinellosis, but the major drawback of the ES antigens for assaying anti-Trichinella antibodies is the false negative in the early Trichinella infection period. The aim of this study was to characterize the T. spiralis putative serine protease (TsSP) and to investigate its potential use for diagnosis of trichinellosis.The full-length TsSP sequence was cloned and expressed, and recombinant TsSP (rTsSP) was purified by Ni-NTA-Sefinose Column. On Western blotting analysis the rTsSP was recognized by T. spiralis-infected mouse serum, and the natural TsSP was identified in T. spiralis ML crude and ES antigens by using anti-rTsSP serum. Expression of TsSP was detected at various T. spiralis developmental stages (newborn larvae, muscle larvae, intestinal infective larvae and adult worms). Immunolocalization identified the TsSP principally in cuticles and stichosomes of the nematode. The sensitivity of rTsSP-ELISA and ES-ELISA was 98.11% (52/53) and 88.68% (47/53) respectively (P > 0.05) when the sera from trichinellosis patients were examined. However, while twenty-one serum samples of trichinellosis patients' sera at 19 days post-infection (dpi) were tested, the sensitivity (95.24%) of rTsSP-ELISA was distinctly higher than 71.43% of ES-ELISA (P < 0.05). The specificity (99.53%) of rTsSP-ELISA was remarkably higher than 91.98% of ES-ELISA (P < 0.01). Only one out of 20 serum samples of cysticercosis patients cross-reacted with the rTsSP. Specific anti-Trichinella IgG in infected mice was first detected by rTsSP-ELISA as soon as 7 dpi and antibody positive rate reached 100% on 10 dpi, whereas the ES-ELISA did not permit detection of 100% of infected mice before 16 dpi.The rTsSP is a potential early diagnostic antigen for human trichinellosis.
Project description:BACKGROUND:Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis. METHODS:The complete cDNA sequences of the TsEla gene were cloned and expressed, and recombinant (rTsEla) was purified. TsEla transcription and expression in different T. spiralis life-cycle stages was investigated by qPCR and western blotting, and its location in the nematodes was evaluated using an immunofluorescence assay (IFA). The antigenicity of rTsEla was investigated by western blotting analysis and ELISA. Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by rTsEla-ELISA and ES-ELISA. RESULTS:The results of the qPCR and western blotting showed that TsEla was expressed in various T. spiralis life stages. Natural TsEla was detected in the soluble proteins and ES proteins of different life stages. IFA revealed that TsEla was identified in the whole nematodes of various stages, especially in the cuticle, stichosome and genital primordium of the parasite. Serum anti-Trichinella IgM, IgG and IgE in infected mice was first detected by rTsEla-ELISA at 6, 10 and 12 days post-infection (dpi), and reached 100% at 8, 14 and 14 dpi, respectively. When rTsEla-ELISA and ES-ELISA were used to detect anti-Trichinella IgG in sera of trichinellosis patients, the sensitivity was 97.37% (37/38) and 89.74% (34/38) (P?>?0.05), and the specificity was 99.10% (220/222) and 98.20% (218/222), respectively (P?>?0.05). The rTsEla cross-reacted with only one serum sample out of 20 samples from paragonimiasis patients and 7 samples from clonorchiasis patients. CONCLUSIONS:rTsEla is valuable to early diagnosis of trichinellosis and could be an alternative diagnostic antigen to the ML ES antigens.
Project description: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.
Project description:<h4>Background</h4>Trichinellosis is a serious food-borne parasitic zoonosis, thus finding high quality antigens is the key to serodiagnosis of trichinosis. This article reports the characterization and sensitivity of four recombinant proteins expressed by four genes (<i>Wn10</i>, <i>Zh68</i>, <i>T668</i>, and <i>Wm5</i>) from different developmental stages of <i>Trichinella spiralis</i> for the diagnosis of trichinellosis in mice.<h4>Methods</h4>This study was conducted in Jilin University and National Institute of Parasitic Diseases of Chinese Center for Disease Control and Prevention in 2017-2018. The structures and functions of the proteins encoded by four genes were predicted by bioinformatics analysis. The four genes were cloned and expressed, and the recombinant proteins were purified. Anti-<i>Trichinella</i> IgM and IgG antibodies in the sera of mice infected with <i>T. spiralis</i> from 1-45 d post-infection (dpi) were evaluated by ELISA.<h4>Results</h4>The optimal antigen epitopes of four proteins (P1, P2, P3, and P4) encoded by the four genes from T- and B-cells were predicted, and four purified recombinant proteins (r-P1, r-P2, r-P3, and r-P4) were successfully produced. For IgM, the antibody levels detected by the four recombinant antigens were approximately equal to the cut-off value. Anti-<i>Trichinella</i> IgG antibodies were first detected by r-P1 at 8 dpi, followed by r-P2, r-P3, and r-P4 at 10 dpi, 14 dpi, and 16 dpi, respectively, and the antibody levels remained high until 45 dpi.<h4>Conclusion</h4>The recombinant antigens r-P1, r-P2, r-P3, and r-P4 could be antigens that react with antibodies, they showed high sensitivity in the detection of anti-<i>Trichinella</i> IgG antibodies in mice. Among these proteins, r-P1 may be a candidate antigen for the detection of anti-<i>Trichinella</i> IgG antibodies in the early infection phase and exhibited the best sensitivity among the antigens.
Project description:Trichinellosis, which is caused by nematodes of the genus Trichinella, is one of the most important zoonotic parasite diseases in the world. A rapid and sensitive immunochromatographic strip (ICS) based on Eu (III) nanoparticles (EuNPs) was developed for the detection of Trichinella spiralis (T. spiralis) infection in pigs. T. spiralis muscle larvae excretory secretory or preadult worm excretory secretory (ML-ES or PAW-ES) antigens were conjugated with EuNPs probes to capture T. spiralis-specific antibodies in pig sera, after which the complex bound to mouse anti-pig IgG deposited on the test line (T-line), producing a fluorescent signal. In the pigs infected with 100, 1000 and 10 000 ML, seroconversion was first detectable for the EuNPs-ML-ES ICS at 30, 25 and 21 days post-infection (dpi) and for the EuNPs-PAW-ES ICS at 25, 21 and 17 dpi. These results show that EuNPs-PAW-ES ICS detects anti-Trichinella IgG in pigs 4-5 days earlier that test using ML-ES antigens. Our ICS have no cross reaction with other parasite infection sera. Furthermore, the detection process could be completed in 10 min. This study indicated that our ICS can be used for the detection of the circulating antibodies in early T. spiralis infection and provide a novel method for on-site detection of T. spiralis infection in pigs.
Project description:Glutathione-S-transferase (GST) is a widespread multigene family of detoxification enzymes. The vaccination of mice with recombinant GST of 24 kDa from Trichinella spiralis elicited a low immune protection against challenge infection. The objective of this study was to characterize the T. spiralis putative GST gene (TspGST) encoding a 30.8 kDa protein and to evaluate its potential as a candidate antigen for anti-Trichinella vaccine.The full-length cDNA sequence of TspGST from T. spiralis muscle larvae (ML) was expressed in E. coli. The enzymatic activity and antigenicity of the rTspGST were identified by spectrophotometry, Western blot, and ELISA. The expression of TspGST at T. spiralis various stages was investigated by RT-PCR and indirect immunofluorescent test (IIFT). Serum level of total IgG, IgG1, and IgG2a antibodies against rTspGST were measured by ELISA. The immune protection produced by vaccination with rTspGST against T. spiralis was evaluated.The sequencing results showed that the cDNA of TspGST was 840 bp, and encoded a protein of 279 amino acids, which had a molecular size of 30.8 kDa and a pI of 5.21. Its amino acid sequence shares 37% similarity with TsGST. The rTspGST protein had enzymatic activity of GST. On Western blot and ELISA analysis, the native TspGST protein with 30.8 kDa in crude antigens derived from adult worms (AW), newborn larvae (NBL), infective intestinal larvae (IIL) and ML was recognized by anti-rTspGST sera, but the ML ES antigens could be not recognized by anti-rTspGST sera. Expression of TspGST was found in all of T. spiralis various stages (AW, NBL, ML, and IIL). An immunolocalization analysis identified TspGST in different stages (mainly in cuticles) of the nematode. The mice vaccinated with the rTspGST elicited Th2-predominant immune responses, showed a 34.38% reduction of adult worms and a 43.70% reduction of muscle larvae.Immunization with rTspGST produced a partial immune protection, and the rTspGST could be regarded as a potential candidate target for an anti-Trichinella vaccine.
Project description:Trichinella spiralis infection induces protective immunity against re-infection in animal models. Identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Trichinella infection and immunodiagnosis.The T. spiralis adult cDNA library was immunoscreened with sera from pigs experimentally infected with 20,000 infective T. spiralis larvae. Total 43 positive clones encoding for 28 proteins were identified; one of the immunodominant proteins was 20 kDa Ts-ES-1 secreted by Trichinella stichocytes and existing in the excretory/secretory (ES) products of T. spiralis adult and muscle larval worms. Ts-ES-1 contains 172 amino acids with a typical signal peptide in the first 20 amino acids. The expression of Ts-ES-1 was detected in both the adult and muscle larval stages at the mRNA and protein expression levels. Mice immunized with recombinant Ts-ES-1 (rTs-ES-1) formulated with ISA50v2 adjuvant exhibited a significant worm reduction in both the adult worm (27%) and muscle larvae burden (42.1%) after a challenge with T. spiralis compared to the adjuvant control group (p<0.01). The rTs-ES-1-induced protection was associated with a high level of specific anti-Ts-ES-1 IgG antibodies and a Th1/Th2 mixed immune response.The newly identified rTs-ES-1 is an immunodominant protein secreted by Trichinella stichocytes during natural infection and enables to the induction of partial protective immunity in vaccinated mice against Trichinella infection. Therefore, rTs-ES-1 is a potential candidate for vaccine development against trichinellosis.
Project description:BACKGROUND:Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop a vaccine in order to interrupt transmission from animals to humans. A 31 kDa protein from T. spiralis (Ts31) is an antigen targeted by protective antibodies, and Ts31 contains a domain of trypsin-like serine protease that might have the function of serine protease. The purpose of this study was to investigate the molecular characteristics of Ts31 and its induced immune protection. METHODS:Expression and localization of Ts31 in various T. spiralis phases were investigated using qPCR and immunofluorescent test (IFT). The specific binding between Ts31 and intestinal epithelium cells (IECs) was analyzed by Far-Western blotting, ELISA and IFT, and the cellular localization of binding sites was examined on confocal microscopy. The mice were subcutaneously vaccinated with recombinant Ts31 protein (rTs31), serum specific IgG was determined by ELISA, and immune protection induced by immunization with rTs31 was evaluated. Inhibition of anti-rTs31 IgG on IL1 invasion of IECs and ADCC-mediated killing of newborn larvae (NBL) was also determined. RESULTS:Ts31 was expressed at different life-cycle stages and located principally at the stichosome and cuticle of this parasite. rTs31 was capable to specially bond to IECs, and binding site was located in the cytoplasm of IECs. Immunization of mice with rTs31 elicited a significant humoral response and protection, as demonstrated by a 56.93% reduction of adult worms at 6 days post-infection (dpi) and a 53.50% reduction of muscle larvae at 42 dpi after larval challenge. Anti-rTs31 antibodies impeded T. spiralis penetration of enterocytes in a dose-dependent pattern, and participated in the destruction of NBL by an ADCC-mediated manner. CONCLUSIONS:Ts31 facilitated the T. spiralis penetration of intestinal epithelium, which could make it a vaccine candidate target molecule against Trichinella infection.
Project description: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.
Project description:Several outbreaks of trichinellosis associated with the consumption of raw pork have occurred in Laos since 2004. This cross-sectional study was conducted in four provinces of northern Laos to investigate the seroepidemiology of trichinellosis in the human population and determine the prevalence and species of Trichinella infection in the domestic pig population. Serum samples and questionnaire data were obtained from 1419 individuals. Serum samples were tested for Trichinella antibodies by ELISA using larval excretory-secretory (ES) antigens and a subset of 68 positive samples were tested by western blot. The seroprevalence of Trichinella antibodies was 19.1% (95% confidence interval (CI)?=?17.1-21.1%). The risk of having antibodies detected by ELISA using ES antigens increased with age, being of Lao-Tai ethnicity, living in Oudomxay province and being male. Tongue and diaphragm muscle samples were collected from 728 pigs and tested for Trichinella larvae by the artificial digestion method. Trichinella larvae were isolated from 15 pigs (2.1%) of which 13 were identified as T. spiralis by molecular typing; the species of the two remaining isolates could not be determined due to DNA degradation. Trichinella spp. are endemic in the domestic environment of northern Laos and targeted preventative health measures should be initiated to reduce the risk of further outbreaks occurring.