BackgroundTrichinellosis 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.Methodology/Principal findingsThe 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.ConclusionsThe rTsSP is a potential early diagnostic antigen for human trichinellosis.

Trichinellosis is a worldwide important food-borne zoonosis caused mainly by ingesting raw or undercooked pork infected with Trichinella spiralis larvae. The development of vaccine is needed for preventing swine from Trichinella infection to ensure pork safety. Previous studies showed that T. spiralis serine protease 1.2 (TsSP1.2) is a vaccine candidate against Trichinella infection. In this study, the complete TsSP1.2 cDNA sequences were cloned into pcDNA3.1, and the rTsSP1.2 DNA was transformed into attenuated Salmonella typhimurium strain ΔcyaSL1344. Oral vaccination of mice with Salmonella-delivered rTsSP1.2 DNA vaccine induced an obvious intestinal mucosal IgA response and a systemic Th1/Th2 immune response; the vaccinated mice showed a 33.45% reduction of intestinal adult worms and 71.84% reduction of muscle larvae after T. spiralis larval challenge. The protection might be due to the rTsSP1.2-induced production of specific anti-TsSP1.2 sIgA, IgG, IgG1/IgG2a, and secretion of IFN-γ, IL-4 and IL-10, which protected intestinal mucosa from the parasite invasion, inhibited worm development and reduced female fecundity. The results indicate that the attenuated Salmonella-delivered rTsSP1.2 DNA vaccine offers a prospective strategy for the prevention and control of animal Trichinella infection.Electronic supplementary materialThe online version of this article (10.1186/s13567-018-0582-2) contains supplementary material, which is available to authorized users.

BackgroundTrichinella spiralis is a major zoonotic tissue-dwelling nematode, which is a public health concern and a serious hazard to animal food safety. It is necessary to exploit an anti-Trichinella vaccine to interrupt the transmission of Trichinella infection among animals and from animals to humans. The purpose of the present study was to characterize the novel T. spiralis cathepsin B (TsCB) and to evaluate the immune protection elicited by immunization with recombinant TsCB (rTsCB).MethodsThe complete cDNA sequences of the TsCB gene were cloned, expressed and purified. The antigenicity of rTsCB was investigated by western blot analysis and ELISA. Transcription and expression of TsCB at various T. spiralis life-cycle stages were analyzed by RT-PCR and indirect immunofluorescent assay (IIFA). The mice were subcutaneously immunized with rTsCB, and serum level of TsCB-specific IgG (IgG1 and IgG2a) and IgE antibodies were assayed by ELISA. Immune protection elicited by vaccination with rTsCB was investigated.ResultsThe TsCB was transcribed and expressed in four T. spiralis life-cycle stages (adult worm, AW; newborn larvae, NBL; muscle larvae, ML; and intestinal infective L1 larvae), it was primarily located in the cuticle and stichosome of the parasitic nematode. Vaccination of mice with rTsCB produced a prominent antibody response (high level of specific IgG and IgE) and immune protection, as demonstrated by a 52.81% AW burden reduction of intestines at six days post-infection (dpi) and a 50.90% ML burden reduction of muscles at 35 dpi after oral larva challenge. The TsCB-specific antibody response elicited by immunization with rTsCB also impeded intestinal worm growth and decreased the female fecundity.ConclusionsTsCB might be considered as a novel potential molecular target to develop vaccines against T. spiralis infection.

The intestinal epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism of larval invasion of the gut epithelium is not fully elucidated. The aim of this study was to investigate whether the excretory/secretory proteins (ESPs) of T. spiralis intestinal infective larvae (IIL) degrade tight junction (TJ) proteins, to assess the main ESP proteases hydrolysing TJ proteins using various enzyme inhibitors and to define the key invasive factors in IIL invasion of the gut epithelium. The results of immunofluorescence, Western blot and Transwell assays showed that serine proteases and cysteine proteases in the ESPs played main roles in hydrolysing occludin, claudin-1 and E-cad and upregulating claudin-2 expression. Challenge infection results showed that IIL expulsion from the gut at 12 hpi was significantly higher in mice which were infected with muscle larvae (ML) treated with a single inhibitor (PMSF, E-64, 1,10-Phe or pepstatin) or various mixtures containing PMSF and E-64 than in mice in the PBS group or the groups treated with an inhibitor mixture not containing PMSF and E-64 (P < 0.0001). At 6 days post-infection, mice which were infected with ML treated with PMSF, E-64, 1,10-Phe or pepstatin exhibited 56.30, 64.91, 26.42 and 31.85% reductions in intestinal adult worms compared to mice in the PBS group (P < 0.0001). The results indicate that serine proteases and cysteine proteases play key roles in T. spiralis IIL invasion, growth and survival in the host and that they may be main candidate target molecules for vaccines against larval invasion and development.

In our previous studies, a novel T. spiralis peptidase (TsP) was identified among the excretory/secretory (ES) proteins of T. spiralis intestinal infective larvae (IIL) and T. spiralis at the adult worm (AW) stage using immunoproteomics, but the biological function of TsP in the life cycle of T. spiralis is not clear. The objective of this study was to investigate the biological properties and functions of TsP in larval intrusion and protective immunity induced by immunization with rTsP. The complete TsP cDNA sequence was cloned and expressed. The results of RT-PCR, indirect immunofluorescence assay (IIFA) and western blotting revealed that TsP is a surface and secretory protein expressed in T. spiralis at different stages (muscle larvae, IIL, AWs and newborn larvae) that is principally localized at the epicuticle of the nematode. rTsP facilitated the larval intrusion of intestinal epithelial cells (IECs) and intestinal mucosa, whereas anti-rTsP antibodies suppressed larval intrusion; these facilitative and suppressive roles were dose-dependently related to rTsP or anti-rTsP antibodies. Immunization of mice with rTsP triggered an obvious humoral immune response (high levels of IgG, IgG1/ IgG2a, and sIgA) and also elicited systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune responses, as demonstrated by an evident increase in the cytokines IFN-γ and IL-4. Immunization of mice with rTsP reduced the numbers of intestinal adult worms by 38.6% and muscle larvae by 41.93%. These results demonstrate that TsP plays a vital role in the intrusion, development and survival of T. spiralis in hosts and is a promising candidate target molecule for anti-Trichinella vaccines.