| Trichinellosis, caused by the ingestion of raw or inadequately cooked meat containing the infective larvae of the nematode genus Trichinella, is a food-borne parasitic zoonosis with worldwide distribution. The diagnosis of trichinellosis is rather difficult because the signs and symptoms are non-specific. Up to now, a definitive diagnosis of human trichinellosis can be made only by detecting larvae in a muscle biopsy or by highly specific immunodiagnositic tests. The sensitivity of muscle biopsy depends on the amount of muscle sample tested and the degree of infection. Muscle biopsy is not sensitive to the light infections and the early stage of infection. Even during the late stage of infection, the positive rate of muscle biopsy is only50%due to limitations of selecting muscular tissue. Besides, muscle biopsy is difficult to be accepted by patients. During the process of infection by Trichinella, the excretory-secretory (ES) proteins of muscle larvae (ML), mainly come from the stichocyte granules, are directly exposed to the host’s immune system, and can induce a strong immune response involving the generation of specific antibodies as they are easily targeted by the host’s immune system. The ES proteins of T. spiralis ML were recommended to be used in ELISA or Western blotting for detecting anti-Trichinella antibodies by International Commission on Trichinellosis (ICT), but their main disadvantages are the false negative results during the early stage of infection and cross-reaction with other parasites. In this study, the ES proteins from ML of T. spiralis were analysed by two dimensional gel electrophoresis (2-DE), Western blotting combined with mass spectrometry (MS), and the gene of antigen targeted by protective antibodies (ATPA, GenBank No. gi|404638) was selected to be cloned, expressed and anylased by immunological methods. Besides, the recombinant protein was applied to detect the specific anti-Trichinella antibodies in serum from the mice infected with T spiralis and patient’s sera with trichinellosis, and it will lay the foundation for searching the candidates of immunodiagnostic antigens for trichinellosis and the effective protective antigens for trichinellosis vaccine.Materials and methods1Trichinella spp., serum and experimental animalsT. spiralis isolate (T1) used in this study was maintained by serial passages in Kunming mice in our laboratory. Mouse serum samples were infected with Trichinella native (T2), T. britovi (T3), T. pseudospiralis (T4), T. nelsoni (T7), Toxoplasma gondii and Schistosoma japanicum, as well as patient’s sera with trichinellosis and other parasitosis. Experimental animals were female Kunming mice and BALB/c mice.2Identification of early diagnostic antigens from the ES proteins of T. spiralis muscle larvae using immunoproteomicsThe muscle larvae of T. spiralis were collected by artificial digestion and modified Baermann’s methods, and the ES proteins were obtained by culturing the ML in vitro. BALB/c mice were orally infected with300larvae/mouse and the tail vein blood was daily collected from each mouse before infection and during14-42days post-infection (dpi) on alternate days, respectively. Anti-Trichinella IgG antibodies in sera from infected mice at14-42dpi were assayed by ELISA and Western blotting methods using T. spiralis ML ES proteins as antigens. The sera which firstly detected the specific antibodies by the above-mentioned two methods were used in the following experiments. The ES proteins from T. spiralis ML were separated by2-DE, then were transferred onto polyvinylidene difluoride (PVDF) membranes and probed with early mouse sera infected with T. spiralis, and the immunoreactive protein spots were identified and characterized by MS.3Cloning, expression and identification of T. spiralis ATPA geneThe volumes of protein spots successfully identified by MS with30-40kDa from ES proteins of T. spiralis ML in2-DE and Western blotting and the ratios were compared. Out of these proteins, ATPA had the largest ratio and was selected for the further study. The gene of ATPA was cloned, expressed and purified. BALB/c mice were immunized with rATPA, and the immune sera were collected. The antigenicity and immunogenicity of rATPA were identified by Western blotting. RT-PCR was carried out to observe the transcription of ATPA gene in different development stages [adult worms (AD), new-born larvae (NBL), pre-encapsulated larvae (PEL) and ML] of Trichinella. The expression of ATPA protein in different development stage and location of rATPA were observed by IFT.4Application of rATPA for detection of Trichinella-specific antibodies in mouse sera infected with T. spiralis and patient sera with trichinellosisrATPA-ELISA and ES-ELISA were established using rATPA and ES proteins of T. spiralis ML, respectively. They were used to assay anti-Trichinella IgG antibodies in sera from mice infected with different species of Trichinella (T1, T2, T3, T4, and T7) and other parasites, as well as patient’s sera with trichinellosis and other parasitosis, and their sensitivity and specificity were evaluated.30female BALB/c mice were randomly divided into3groups (10mice/group):heavily infected group (500larvae/mouse), moderately infected group (300larvae/mouse), and lightly infected group (100larvae/mouse). The mice of infected group were orally inoculated with of T. spiralis muscle larvae. rATPA-ELISA and ES-ELISA were used to assay anti-Trichinella IgG antibodies in sera from mice infected with T. spiralis on alternate days during2-42dpi, and the value of rATPA-ELISA for diagnosis of trichinellosis during the the early stage of infection and light infections were evaluated. 5Statistical analysisAll statistical analyses of data were done with SPSS for Windows version17.0. Chi-square test and repeated measures of analysis of variance (ANOVA) were used, and the level of significance used was5%(P<0.05).Results1Identification of early diagnostic antigens from the ES proteins of T. spiralis muscle larvae using immunoproteomicsAnti-Trichinella IgG antibodies in sera from infected mice at14-42dpi were assayed by ELISA and Western blotting. The specific antibodies were firstly detected at18dpi by the above-mentioned two methods, and then these sera were used for the following experiments.200μg and800μg ES proteins of T. spiralis ML were separated by2-DE on10%and12%gels, the protein spots with40-60kDa and30-40kDa can be separated very well, and more than33and150spots were detected. After transferred to PVDF membranes, the spots were probed with18dpi mouse sera infected with T. spiralis, and there were31spots displaying reactivity to the infection sera at18dpi. These spots recognized by the infection sera at18dpi were digested by trypsin and then analysed by MALDI-TOF/TOF-MS, and these spots were identified to correlate with7different proteins of T. spiralis, including two serine proteases [serine proteases, SP-1.2(gi|168805931) and SP-1.3(gi|13641204, gi|168805933)], one deoxyribonuclease (DNase) Ⅱ (gi|339241449, gi|316974621), two kinds of trypsin (putative trypsin, gi|339241891and gi|339241897), one antigen targeted by protective antibodies (ATPA, gi|404638) and one conserved hypothetical protein (gi|316966524). The7kinds of proteins of T. spiralis identified were putatively annotated using the GO categories tool. The7proteins of T. spiralis had catalytic and hydrolase activity, and they were also associated with metabolic process.2Cloning, expression and identification of T. spiralis ATPA genePrediction of ATPA by soft wares of bioinformatics showed that the ATPA had no transmembrane domain, had a cleavable signal peptide (from1to17) and with possible cleavage site between17aa and18aa, and had good antigenicity. The ATPA gene obtained by RT-PCR was cloned to pMAL-c2X expression vector, and the recombinant plasmid pMAL-c2X-ATPA was constructed successfully. The recombinant plasmid pMAL-c2X-ATPA was induced by IPTG and the recombinant fusion protein74kDa (tag protein43kDa+ATPA31kDa) could be found by SDS-PAGE. The rATPA was expressed successfully, and existed in forms of both soluble protein and inclusion body in E. coli strain TB1. The desired protein was purified by Amylose pre-packed column after obtaining the supernatant. BALB/c mice were immunized with rATPA, and the immune sera were collected. The specific IgG antibody titer of immune sera were assayed by an indirect enzyme-linked immunosorbent assay (ELISA) using rATPA, and the IgG antibody titer of immune sera against rATPA was1:106. Western blotting showed that the rATPA could be recognized by the mouse sera infected with T. spiralis and immune sera against rATPA, but it was not immunostained with normal serum. And the native ATPA protein in soluble proteins and ES proteins from T. spiralis ML were recognized by immune sera against rATPA. RT-PCR results showed that ATPA mRNA was transcribed in different development stages (AD, NBL, PEL and ML) of Trichinella. The IFT results showed that the ATPA protein was expressed in the4different development stages, and the bright green fluorescence was mainly localized at the cuticle and stichosome of the worms.3Sensitivity and specificity of rATPA-ELISA for detection of Trichinella-specific antibodies in mouse sera infected with T. spiralisPositive rates of the specific enti-Trichinella IgG antibodies from mouse sera infected with Trichinella T1, T2, T3, T4, and T7were96.67%(29/30),90.00%(27/30),93.33%(14/15),40.91%(9/22), and93.75%(15/16) by rATPA-ELISA, while the positive rates of the above sera were100%(30/30),100%(30/30),100%(15/15),90.91%(20/22), and100%(16/16) by ES-ELISA. There was no significant difference in the positive rates of mouse sera infected with Trichinella T1, T2, T3, and T7between rATPA-ELISA and ES-ELISA (P>0.05); but as for the positive rates of mouse sera infected with Trichinella T4, rATPA-ELISA was lower than ES-ELISA (P<0.05).Positive rates of the specific anti-Trichinella IgG antibodies from mouse sera infected with T. spiralis by rATPA-ELISA and ES-ELISA were96.67%(29/30) and100%(30/30)(P>0.05). Positive rates of antibodies from mouse sera infected with plerocercoids of Spirometra mansoni (sparganum) by rATPA-ELISA and ES-ELISA were16.13%(5/31) and3.22%(1/31)(P>0.05). Positive rates of mouse sera infected with S. japanicum by rATPA-ELISA and ES-ELISA were68.75%(11/16) and6.25%(1/16)(P<0.05). However, the mouse sera collected from mice infected with T. gondii and before infection showed negative response by both rATPA-ELISA and ES-ELISA.4Serum IgG levels at different times in the mice experimentally infected with different dose of T. spiralis muscle larvaeIn lightly, moderately and heavily infected group, the specific anti-Trichinella IgG antibodies were firstly detected at14dpi,12dpi, and8dpi by rATPA-ELISA, while the antibodies were firstly detected at10dpi,8dpi, and10dpi by ES-ELISA, respectively. In heavily infected group, the positive rates of the specific anti-Trichinella IgG antibodies on12dpi and14dpi by rATPA-ELISA were20%and40%, while the positive rates of antibodies by ES-ELISA were80%and100%(P <0.05). In mouse sera of moderately infected group, the positive rates of the specific anti-Trichinella IgG antibodies on10dpi,12dpi, and14dpi by rATPA-ELISA were0,20%, and30%, while the positive rates of antibodies by ES-ELISA were70%,100%, and100%(P<0.05). In mouse sera of lightly infected group, the positive rates of the specific anti-Trichinella IgG antibodies on alternate days during12-24dpi by rATPA-ELISA were0,10%,10%,10%,30%,30%, and30%, while the positive rates of antibodies by ES-ELISA were10%,60%,80%,80%,80%,100%, and100%(P<0.05). The specific anti-Trichinella IgG antibodies were detected100%at30dpi,22dpi and28dpi a in lightly, moderately and heavily infected group by rATPA-ELISA, while the antibodies were detected100%at22dpi,12dpi and14dpi by ES-ELISA. There was no significant difference in the positive rates of mouse sera in lightly, moderately and heavily infected group during26dpi,16dpi and16dpi between rATPA-ELISA and ES-ELISA (P>0.05).There was no significant difference on the specific anti-Trichinella IgG antibodies level in heavily, moderately and lightly infected group by rATPA-ELISA (F=2.049,P>0.05); the difference of serum antibody levels at different times by rATPA-ELISA was statistically significant (F=219.924, P<0.05); there was interaction between time and the dose of inoculation (F=3.311,P<0.05). The results of paired comparison showed that the serum antibody level was raised during10-16dpi and24-28dpi (P<0.05). There was significant difference on the specific anti-Trichinella IgG antibodies level in heavily, moderately and lightly infected group by ES-ELISA (F=5.901, P<0.05); the difference of serum antibody levels at different times by ES-ELISA was statistically significant (F=478.276, P<0.05); there was interaction between time and the dose of inoculation (F=5.710, P<0.05). The results of paired comparison showed that the serum antibody level was raised during8-32dpi (P<0.05).5Sensitivity and specificity of rATPA-ELISA for detection of Trichinella-specific antibodies in patient’s sera with trichinellosisThe sensitivity of both rATPA-ELISA and ES-ELISA in detecting the serum samples of patients with trichinellosis was100%(22/22). Using rATPA-ELISA, only one case of patient’s sera with schistosomiasis was positive, while no positive reaction was detected in patients’sera with paragonimiosis, clonorchiosis, echinococcosis, cysticercosis and sparganosis, as well as healthy persons’ sera. The sensitivity and specificity of rATPA-ELISA for detecting specific anti-Trichinella antibodies in sera of patients with trichinellosis were100%and99.13%, respectively. Using ES-ELISA, Positive rates of the specific anti-Trichinella IgG antibodies from patients’ sera with schistosomiasis, paragonimiosis, clonorchiosis, echinococcosis, cysticercosis and sparganosis were20.00%(4/20)、5.00%(1/20、14.29%(1/7)、25.00%(5/20)、25.00%(5/20)'12.50%(1/8), but no positive reaction was detected in healthy persons’ sera. There was no significant difference in the detection of patients’ sera with schistosomiasis, paragonimiosis, clonorchiosis, and sparganosis (P>0.05) between rATPA-ELISA and ES-ELISA; but as for detection of patients’ sera with echinococcosis and cysticercosis, rATPA-ELISA was better than ES-ELISA (P <0.05).Conclusions1. Seven proteins of T. spiralis (two serine proteases, one DNase II, two kinds of trypsin, one antigen targeted by protective antibodies and one conserved hypothetical protein) were identified from muscle larval excretory-secretory proteins. All of the7proteins of T. spiralis had catalytic and hydrolase activity, and might have potential values of early diagnosis for trichinellosis.2. The recombinant plasmid pMAL-c2X-ATPA was constructed successfully, and the rATPA existed in forms of both soluble protein and inclusion body. The ATPA mRNA was transcribed in different development stages (AD, NBL, PEL and ML), the protein of ATPA was expressed in the4different development stages, and was mainly localized at the cuticle and stichosome of the worms.3. rATPA-ELISA which was used to detect the specific anti-Trichinella IgG antibodies in mouse sera infected with T. spiralis and patient sera with trichinellosis, had good sensitivity and specificity, and rATPA could be potential for serological diagnosis for trichinellosis. |
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