Spirometra erinaceieuropaei casein kinase I (SeCKI) was analyzed using bioinformatical methods to predict its structure and function based on the deduced amino acid sequence from full length cDNA sequence of SeCKI gene with online sites and software programs. The longest open reading frame contains 448 amino acids, 50 kDa and theoretical pI of 4.73, with a complete tubulin domain, a SMART tubulin_C domain and a low complexity region. SeCKI has no signal sequence and no transmembrane domain, but is predicted to be located extracellularly. The secondary structure of SeCKI contains 12 α-helixes, 11 β-strands and 22 coils. SeCKI had 19 potential antigenic epitopes and 25 HLA-I restricted epitopes. Based on phylogenetic analysis of SeCKI sequence, S. erinaceieuropaei has the closest evolutionary status with Hymenolepis microstoma. Information from this study could provide important insights into the identification of diagnostic antigens and molecular targets of antisparganum drugs.

We have previously reported that the recombinant T. spiralis aminopeptidase (rTsAP) could induce a partial protective immunity against T. spiralis infection in mice. The aim of this study was to predict the structures and functions of TsAP protein by using the full length cDNA sequence of TsAP gene. TsAP sequence was 1515 bp length with a 1515 bp biggest ORF encoding 504-amino acid protein. The molecular weight and isoelectric point of TsAP were 54.7 kDa and 6.69, respectively. TsAP structure domains contained a Peptidase_M17_N and a Peptidase_M17 domain, which has the function of catalysis of the hydrolysis of N-terminal amino acid residues. TsAP had no signal peptide site and transmembrane domain, and located in cytoplasm. The secondary structure of TsAP contained 16 α-helix, 14 β-strand and 29 coils. The TsAP had 11 and 21 potential antigenic epitopes of T cell and B cell, respectively. Based on the phylogenetic analyses of TsAP, T. spiralis have the closest relationship with Plasmodium falciparum. TsAP was a kind of proteolytic enzyme with a variety of biological functions and its antigenic epitopes could provide important insights on the diagnostic antigens and target molecular of anti-Trichinella drugs

Previous studies showed that crude antigens from Trichinella spiralis adult worms (AW) can be recognized by mouse infection sera at 8 days post infection. The aim of this study was to identify the early diagnostic antigenic bands in soluble proteins from T. spiralis AW by Western blot using early infection sera. The affecting factors of adult recovery were firstly observed in this study, and the results showed that the maximum number of adults was collected from small intestine when the female BALB/c mice were orally infected with 4000 ML and sacrificed at 3 days post infection. The results of Western blot analysis showed that seven protein bands (31, 35.1, 39, 40.6, 41.9, 47 and 50.6 kDa) could be recognized by early infection sera as early as at 8-10 days post infection, and were strongly reacted with mouse infection sera at 11-12 days post infection. Our results suggested that the seven protein bands of T. spiralis AW soluble proteins might be the early expressed antigens during the intestinal stage of Trichinella infection and therefore have potential value for the early diagnosis of trichinellosis.

In previous studies, a Trichinella spiralis serine protease (TsSP) was identified in excretion/secretion (ES) products from intestinal infective L1 larvae (IIL1) using immunoproteomics. The complete cDNA sequence of TsSP gene was 1372 bp, which encoded 429 amino acids with 47.55 kDa. The TsSP was transcribed and expressed at all T. spiralis life cycle phases, as well as mainly located at the cuticle and stichosome of the parasitic nematode. Recombinant TsSP bind to intestinal epithelial cells (IEC) and promoted larva invasion, however, its exact function in invasion, development and reproduction are still unknown. The aim of this study was to confirm the biological function of TsSP during T. spiralis invasion and growth using RNA interference (RNAi) technology. The results showed that on 1 day after electroporation using 2.5 µM siRNA156, TsSP mRNA and protein expression of muscle larvae (ML) was suppressed by 48.35 and 59.98%, respectively. Meanwhile, silencing of TsSP gene by RNAi resulted in a 61.38% decrease of serine protease activity of ML ES proteins, and a significant reduction of the in vitro and in vivo invasive capacity of IIL1 to intrude into the IEC monolayer and intestinal mucosa. When mice were infected with siRNA 156-transfected larvae, adult worm and muscle larva burdens were decreased by 58.85 and 60.48%, respectively. Moreover, intestinal worm growth and female fecundity were evidently inhibited after TsSP gene was knockdown, it was demonstrated that intestinal adults became smaller and the in vitro newborn larval yield of females obviously declined compared with the control siRNA group. The results indicated that knockdown of TsSP gene by RNAi significantly reduced the TsSP expression and enzymatic activity, impaired larvae intrusion and growth, and lowered the female reproductive capacity, further verified that TsSP might participate in diverse processes of T. spiralis life cycle, it will be a new prospective candidate molecular target of anti-Trichinella vaccines.

Spirometra larvae are etiological agents of human sparganosis. However, the systematics of spirometrid cestodes has long been controversial. In order to determine the current knowledge on the evolution and genetic structure of Spirometra, an exhaustive population diversity analysis of spirometrid cestodes using the mitochondrial gene: cytochrome c oxidase subunit 1 (cox1) was performed. All publicly available cox1 sequences available in the GenBank and 127 new sequencing genes from China were used as the dataset. The haplotype identify, network, genetic differentiation and phylogenetic analysis were conducted successively. A total of 488 sequences from 20 host species, representing four spirometrid tapeworms (S. decipiens, S. ranarum, S. erinaceieuropaei and Sparganum proliferum) and several unclassified American and African isolates from 113 geographical locations in 17 countries, identified 45 haplotypes. The genetic analysis revealed that there are four clades of spirometrid cestodes: Clade 1 (Brazil + USA) and Clade 2 (Argentina + Venezuela) included isolates from America, Clade 3 contained African isolates and one Korean sample, and the remainders from Asia and Australia belonged to Clade 4; unclassified Spirometra from America and Africa should be considered the separate species within the genus; and the taxonomy of two Korea isolates (S. erinaceieuropaei KJ599680 and S. decipiens KJ599679) was still ambiguous and needs to be further identified. In addition, the demographical analyses supported population expansion for the total spirometrid population. In summary, four lineages were found in the spirometrid tapeworm, and further investigation with deeper sampling is needed to elucidate the population structure.

A T. spiralis serine protease 1.2 (TsSP1.2) was identified in the muscle larvae (ML) and intestinal larvae surface/excretory–secretory (ES) proteins by immunoproteomics. The aim of this study was to determine the TsSP1.2 function in the process of T. spiralis intrusion, growth and reproduction by using RNA interference (RNAi). RNAi was used to silence the expression of TsSP1.2 mRNA and protein in the nematode. On 2 days after the ML were electroporated with 2 µM of TsSP1.2-specific siRNA 534, TsSP1.2 mRNA and protein expression declined in 56.44 and 84.48%, respectively, compared with untreated ML. Although TsSP1.2 silencing did not impair worm viability, larval intrusion of intestinal epithelium cells (IEC) was suppressed by 57.18% (P < 0.01) and the suppression was siRNA-dose dependent (r = 0.976). Infection of mice with siRNA 534 transfected ML produced a 57.16% reduction of enteral adult burden and 71.46% reduction of muscle larva burden (P < 0.05). Moreover, silencing of TsSP1.2 gene in ML resulted in worm development impediment and reduction of female fertility. The results showed that silencing of TsSP1.2 by RNAi inhibited larval intrusion and development, and reduced female fecundity. TsSP1.2 plays a crucial role for worm invasion and development in T. spiralis life cycle, and is a potential vaccine/drug target against Trichinella infection.

Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop vaccine to prevent T. spiralis infection in food animals. T. spiralis aspartic protease-2 (TsASP2) has been demonstrated to play a crucial role in larval invasion of intestinal epithelium cells (IECs). The purpose of this study was to assess the interaction between TsASP2 and IECs and to investigate the immune protection elicited by vaccination with rTsASP2. The results showed that the enzymatic activity of native aspartic protease was detected in crude proteins of all T. spiralis development stages other than NBL stage, the highest activity was observed in the IIL stage. The results of Western blot showed that TsASP2 protein was expressed at ML, IIL and AW but not NBL, and the TsASP2 expression level at IIL stage was significantly higher than those of other three worm stages (P < 0.05). The specific binding between rTsASP2 and IECs was observed by immunofluorescence test (IFT) and confocal microscopy, and the binding site was localized at the IEC membrane and this binding ability was inhibited by aspartic protease specific inhibitor pepstain A. The results of ELISA showed that the binding ability was protein dose-dependent. Vaccination with rTsASP2 triggered a mixed Th1/Th2 humoral and mucosal immune responses, as demonstrated by the elevation levels of Th1/Th2 cytokines (IFN-γ and IL-4) secreted by the spleen and mesenteric lymph nodes (MLNs) of immunized mice. The mice vaccinated with rTsASP2 exhibited a 54.17% reduction in enteral adult worms and a 54.58% reduction in muscle larvae after T. spiralis challenge. The results demonstrated that TsASP2 might be a potential molecular target for anti-Trichinella vaccines.

Trichinellosis is an important zoonotic parasitic disease worldwide and is principally caused by ingesting animal meat containing Trichinella infective larvae. Aspartyl aminopeptidase is an intracytoplasmic metalloproteinase that specifically hydrolyzes the N-terminus of polypeptides free of acidic amino acids (aspartic acid and glutamate), and plays an important role in the metabolism, growth and development of organisms. In this study, a novel T. spiralis aspartyl aminopeptidase (TsAAP) was cloned and expressed, and its biological properties and roles in worm growth and development were investigated. The results revealed that TsAAP transcription and expression in diverse T. spiralis stages were detected by RT-PCR and Western blotting, and primarily localized at cuticle, stichosome and intrauterine embryos of this nematode by immunofluorescence test. rTsAAP has the enzymatic activity of native AAP to hydrolyze the substrate H-Glu-pNA. There was a specific binding between rTsAAP and murine erythrocyte, and the binding site was localized in erythrocyte membrane proteins. Silencing of TsAAP gene by specific dsRNA significantly reduced the TsAAP expression, enzymatic activity, intestinal worm burdens and female fecundity. The results demonstrated that TsAAP participates in the growth, development and fecundity of T. spiralis and it might be a potential target molecule for anti-Trichinella vaccines.

A putative serine protease of T. spiralis (TsSP) was expressed in Escherichia coli and its potential as a diagnostic antigen was primarily assessed in this study. Anti-Trichinella IgG in serum samples from T. spiralis different animal hosts (mice, rats, pigs and rabbits) were detected on Western blot analysis with rTsSP. Anti-Trichinella antibodies were detected in 100% (30/30) of experimentally infected mice by rTsSP-ELISA. Cross-reactions of rTsSPELISA were not found with sera from mice infected with other parasites (S. erinaceieuropaei, S. japonicum, C. sinensis, A. cantonensis and T. gondii) and sera from normal mice. There was no statistical difference in antibody detection rate among mice infected with the encapsulated Trichinella species (T. spiralis, T. nativa, T. britovi, and T. nelsoni) (P>0.05). The results of rTsSP-ELISA showed that serum specific antibody IgG in mice infected with 100 or 500 T. spiralis muscle larvae (ML) were detectable early at 7-8 dpi, but not detected by ML ES antigen-ELISA prior to 10-12 dpi. Specific anti-Trichinella IgG was detected in 100% (18/18) of infected pigs by rTsSP-ELISA and ES-ELISA, but no specific antibodies was not detected in 20 conventionally raised normal pigs by two antigens. The results showed the rTsSP had the potential for early serodiagnosis of animal Trichinella infection, however it requires to be assayed with early infection sera of swine infected with Trichinella and other parasites.

The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 105 labeled sporozoites and 0.5 μg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 105 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c+ levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c+ and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4+CD8- /CD8+CD4- in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.