Li Na Liu

Cloning, expression and characterization of a Trichinella spiralis serine protease gene encoding a 35.5 kDa protein

Serine proteases are found in the excretory-secretory (ES) products from Trichinella spiralis muscle larvae, have collagenolytic and elastolytic activities, and may be related to the larval invasion of intestinal epithelial cells. In this study, the serine protease gene (TspSP-1.2, GenBank accession No. EU302800) encoding a 35.5 kDa protein from T. spiralis was cloned, and recombinant TspSP-1.2 protein was produced in an Escherichia coli expression system. An anti-TspSP-1.2 serum recognized the native protein migrating at 35.5 kDa by the Western blotting of the crude or ES antigens from muscle larvae at 42 days post infection. An immunolocalization analysis identified TspSP-1.2 in the cuticle and internal organs of the parasite. Transcription and expression of the TspSP-1.2 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and muscle larvae). An in vitro invasion assay showed that, when anti-TspSP-1.2 serum, serum of infected mice and normal mouse serum were added to the medium, the invasion rate of the infective larvae in an HCT-8 cell monolayer was 33.0%, 89.4%, and 96.2%, respectively (P<0.05), indicating that the anti-TspSP-1.2 serum partially prevented the larval invasion of intestinal epithelial cells. After a challenge infection with T. spiralis infective larvae, mice immunized with the recombinant TspSP-1.2 protein displayed a 34.92% reduction in adult worm burden and 52.24% reduction in muscle larval burden. The results showed that the recombinant TspSP-1.2 protein induced a partial protective immunity in mice and could be considered as a potential vaccine candidate against T. spiralis infection.

Survey of Trichinella infections in domestic pigs from northern and eastern Henan, China

The aim of this work was to investigate the current situation of Trichinella infections in swine in the cities of Anyang and Shanqiu in the Henan province historically designated as trichinellosis-free. A total of 475 diaphragm muscle samples were collected from 2010 to 2011 and examined by trichinelloscopy and artificial digestion. No Trichinella larvae were detected by trichinelloscopy; however, using the digestion method, 3.79% (18/475) of domestic pigs were deemed positive for Trichinella. Among the 475 pigs examined, 112 from an industrialized pig farm were negative. However. Trichinella larvae were detected in 10% (9/90) of pigs from small pig farms, which was significantly higher than the 3.3% (9/273) of pigs found positive from backyard farms (P<0.05). The larval burdens in infected animals ranged from 0.1 to 1.58 larvae per gram. The larvae were identified by multiplex PCR as Trichinella spiralis. Our study confirms the existence of porcine trichinellosis in northern and eastern parts of Henan. The results will be useful for evaluating the risk of infection for humans. Given this new found data, public health officials should consider implementing strategies to eliminate human transmission.

Characterization of a Trichinella spiralis 31 kDa protein and its potential application for the serodiagnosis of trichinellosis

The Trichinella spiralis 31 kDa protein (Ts31) was screened from the excretory-secretory (ES) proteins of muscle larvae (ML) by immunoproteomics using serum from mice infected with T. spiralis at 18 days post infection (dpi). The aim of this study was to characterize the Ts31 protein and to evaluate the potential of the recombinant Ts31 protein (rTs31) for serodiagnosis of human trichinellosis. Ts31 gene was cloned and rTs31 was produced in an E. coli expression system. An anti-rTs31serum recognized the native protein migrating in a 25-55 kDa range by Western blotting of ML crude or ES antigens. Expression of Ts31 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and ML). An immunolocalization analysis identified Ts31 in the cuticle and stichocytes of the parasite. The sensitivity of rTs31-ELISA and ES antigen ELISA for detecting anti-Trichinella IgG antibodies in sera of patients with trichinellosis was 97.83% (45/46) and 86.78% (39/46), respectively (P>0.05); The specificity of rTs31-ELISA was 99.13% (114/115), which was significantly higher than 85.22% (98/115) of ES antigen ELISA (P<0.01). The rTs31 protein of T. spiralis could be considered as a potential diagnostic antigen for trichinellosis.

Detection of circulating antigen in serum of mice infected with Trichinella spiralis by an IgY-IgM mAb sandwich ELISA.

In this study, a sandwich ELISA based on IgY (egg yolk immunoglobulin) and IgM monoclonal antibody (mAb) against excretory-secretory (ES) antigens of Trichinella spiralis muscle larvae was developed for detection of circulating antigens (CAg) in serum from mice infected with T. spiralis. The IgY-IgM sandwich ELISA involved the use of chicken antibody IgY as a capture antibody and mouse IgM mAb 5A7G4 as a detecting antibody. This assay was able to detect as little as 1 ng/ml of ES antigens added to normal mouse serum. Two groups of BALB/c mice infected with T. spiralis larvae were used: heavily infected mice (20 mice infected with 300 larvae) and lightly infected mice (20 mice infected with 100 larvae) and 10 normal mice as control. The CAg was detectable as early as 4 days post infection (dpi) in the sera from both groups of infected mice, then increased rapidly, reached a peak with detection rate of 100% in heavily infected mice at 10 dpi and 80% in lightly infected mice at 22 dpi, respectively. The anti-Trichinella IgM antibodies was first detected in 40% of heavily infected mice and 20% of lightly infected mice at 8 dpi, and reached a peak positive rate of 100% in heavily infected mice at 16 dpi and in lightly infected mice at 26 dpi, respectively. The novel assay appears to be sensitive for detection of antigens of T. spiralis and valuable to the early diagnosis of trichinellosis.

Oral vaccination of mice with Trichinella spiralis nudix hydrolase DNA vaccine delivered by attenuated Salmonella elicited protective immunity

We have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity against challenge infection in mice. In this study, the full-length cDNA sequence of TsNd gene was cloned into the eukaryotic expression plasmid pcDNA3.1, and the recombinant TsNd DNA was transformed into attenuated Salmonella typhimurium strain ⊿cyaSL1344. Oral immunization of mice with TsNd/S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, 10) responses in splenocytes of immunized mice upon stimulation with the rTsNd. The oral immunization of mice with TsNd/S. typhimurium displayed a statistically significant 73.32% reduction in adult worm burden and a 49.5% reduction in muscle larvae after challenge with T. spiralis muscle larvae, compared with PBS control group. Our results demonstrated that TsNd DNA delivered by attenuated live S. typhimurium elicited a local IgA response and a mixed Th1/Th2 immune response, and produced a partial protection against T. spiralis infection in mice.

Trichinella spiralis: Low vaccine potential of glutathione S-transferase against infections in mice

We have previously reported that Trichinella spiralis glutathione-S-transferase (TsGST) gene is an up-regulated gene in intestinal infective larvae (IIL) compared to muscle larvae (ML). In this study, the TsGST gene was cloned, and recombinant TsGST (rTsGST) was produced. Anti-rTsGST serum recognized the native TsGST by Western blotting in crude antigens of ML, adult worm (AW) and newborn larvae (NBL) of T. spiralis, but not in ML excretory-secretory (ES) antigens. Expression of TsGST was observed in all different developmental stages (IIL, AW, NBL and ML). An immunolocalization analysis identified TsGST in the cuticle, stichosome and genital primordium of the parasite. The rTsGST had GST enzymatic activity. After a challenge infection with T. spiralis larvae, mice immunized with rTsGST displayed a 35.71% reduction in adult worms and a 38.55% reduction in muscle larvae. The vaccination of mice with rTsGST induced the Th1/Th2-mixed type of immune response with Th2 predominant (high levels of IgG1) and partial protective immunity against T. spiralis infection.

Characterization of Spirometra erinaceieuropaei Plerocercoid Cysteine Protease and Potential Application for Serodiagnosis of Sparganosis

Background Sparganosis is a neglected but important food-borne parasitic zoonosis. Clinical diagnosis of sparganosis is difficult because there are no specific manifestations. ELISA using plerocercoid crude or excretory–secretory (ES) antigens has high sensitivity but has cross-reactions with other helminthiases. The aim of this study was to characterize Spirometra erinaceieuropaei cysteine protease (SeCP) and to evaluate its potential application for serodiagnosis of sparganosis. Methodology/Principal Findings The full length SeCP gene was cloned, and recombinant SeCP (rSeCP) was expressed and purified. Western blotting showed that rSeCP was recognized by the serum of sparganum-infected mice, and anti-rSeCP serum recognized the native SeCP protein of plerocercoid crude or ES antigens. Expression of SeCP was observed at plerocercoid stages but not at the adult and egg stages. Immunolocalization identified SeCP in plerocercoid tegument and parenchymal tissue. The rSeCP had CP activity, and the optimum pH and temperature were 5.5 and 37°C, respectively. Enzymatic activity was significantly inhibited by E-64. rSeCP functions to degrade different proteins and the function was inhibited by anti-rSeCP serum and E-64. Immunization of mice with rSeCP induced Th2-predominant immune responses and anti-rSeCP antibodies had the potential capabilities to kill plerocercoids in an ADCC assay. The sensitivity of rSeCP-ELISA and ES antigen ELISA was 100% when performed on sera of patients with sparganosis. The specificity of rSeCP-ELISA and ES antigen ELISA was 98.22% (166/169) and 87.57% (148/169), respectively (P<0.05). Conclusions The rSeCP had the CP enzymatic activity and SeCP seems to be important for the survival of plerocercoids in host. The rSeCP is a potential diagnostic antigen for sparganosis.

Molecular identification and phylogenetic analysis of Trichinella isolates from different provinces in mainland China

Polymerase chain reaction (PCR) and sequencing are useful for species identification of Trichinella spp., especially when their morphological characteristics useful for identifying taxa are lacking. In the present study, nine Trichinella isolates from different provinces in mainland China were identified by the PCR-based method using the 5S ribosomal DNA intergene spacer region (5S ISR) and the mitochondrial large subunit ribosomal DNA genes as molecular markers. The results indicated that eight isolates originating from domestic pigs and one isolate originating from civet cat (Paguma larvata) showed identical DNA banding pattern to Trichinella spiralis. Sequence analysis of the 5S ISR gene further confirmed that the nine Trichinella isolates were T. spiralis and revealed the intraspecies genetic variation within T. spiralis.

Genetic Structure Analysis of Spirometra erinaceieuropaei Isolates from Central and Southern China

Background Sparganosis caused by invasion of the plerocercoid larvae (spargana) of Spirometra erinaceieuropaei have increased in recent years in China. However, the population genetic structure regarding this parasite is still unclear. In this study, we used the sequences of two mitochondrial genes cytochrome b (cytb) and cytochrome c oxidase subunit I (cox1) to analyze genetic variation and phylogeographic structure of the S. erinaceieuropaei populations. Methodology/Principal Findings A total of 88 S. erinaceieuropaei isolates were collected from naturally infected frogs in 14 geographical locations of China. The complete cytb and cox1 genes of each sample was amplified and sequenced. Total 61 haplotypes were found in these 88 concatenated sequences. Each sampled population and the total population have high haplotype diversity (Hd), accompanied by very low nucleotide diversity (Pi). Phylogenetic analyses of haplotypes revealed two distinct clades (HeN+HuN+GZ-AS clade and GX+HN+GZ-GY clade) corresponding two sub-networks yielded by the median-joining network. Pairwise F ST values supported great genetic differentiation between S. erinaceieuropaei populations. Both negative Fu’s F S value of neutrality tests and unimodal curve of mismatch distribution analyses supported demographic population expansion in the HeN+HuN+GZ-AS clade. The BEAST analysis showed that the divergence time between the two clades took place in the early Pleistocene (1.16 Myr), and by Bayesian skyline plot (BSP) an expansion occurred after about 0.3 Myr ago. Conclusions S. erinaceieuropaei from central and southern China has significant phylogeographic structure, and climatic oscillations during glacial periods in the Quaternary may affect the demography and diversification of this species.

Analysis of Structures, Functions, and Epitopes of Cysteine Protease from Spirometra erinaceieuropaei Spargana

Spirometra erinaceieuropaei cysteine protease (SeCP) in sparganum ES proteins recognized by early infection sera was identified by MALDI-TOF/TOF-MS. The aim of this study was to predict the structures and functions of SeCP protein by using the full length cDNA sequence of SeCP gene with online sites and software programs. The SeCP gene sequence was of 1 053 bp length with a 1011 bp biggest ORF encoding 336-amino acid protein with a complete cathepsin propeptide inhibitor domain and a peptidase C1A conserved domain. The predicted molecular weight and isoelectric point of SeCP were 37.87 kDa and 6.47, respectively. The SeCP has a signal peptide site and no transmembrane domain, located outside the membrane. The secondary structure of SeCP contained 8 α-helixes, 7 β-strands, and 20 coils. The SeCP had 15 potential antigenic epitopes and 19 HLA-I restricted epitopes. Based on the phylogenetic analysis of SeCP, S. erinaceieuropaei has the closest evolutionary status with S. mansonoides. SeCP 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 antisparganum drugs.