Cloning and differential expression of manganese superoxide dismutase (Mn-SOD) of Trichinella pseudospiralis.

The complete coding sequence of manganese superoxide dismutase (Mn-SOD) of Trichinella pseudospiralis (Tp) was obtained and characterized for the first time by degenerative reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of complementary DNA ends (RACE) reactions. The open reading frame of Tp Mn-SOD contained 663 nucleotides, encoding 220 amino acid residues. This included the conserved histidine and aspartate residues for metal binding, cysteine residues for disulfide bond formation, and arginine residue for directing the superoxide ion to the protein. The presence of mitochondrial transit peptides and maturation cleavage site suggest that the cloned Tp Mn-SOD gene is a mitochondrial enzyme. It is a single copy gene containing three introns. Northern blotting suggested that the expression level of Mn-SOD is lower than Cu/Zn SOD in infective stage larvae. Semi-quantitative RT-PCR demonstrated that a single dominant transcript of Tp Mn-SOD was highly expressed in the infective-stage larvae but not in adult worms. The information provides a better understanding of the highly compartmentalized superoxide dismutases of adenophorean nematodes.

Heat-inducible translationally controlled tumor protein of Trichinella pseudospiralis: cloning and regulation of gene expression.

To elucidate the mechanism of inducing translationally controlled tumor protein (TCTP) in stress adaptation of adenophorean nematodes, the complete coding sequence of TCTP of the infective-stage larvae of Trichinella pseudospiralis was characterized. Two cDNA clones with different 3' untranslated region were identified. Tp-TCTP contained an open reading frame of 534 bp encoding 177 residues. The gene with five introns was expressed as histidine-tagged fusion protein having a molecular mass of 17.5 kDa. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that TCTP RNA was not accumulated when the infective-stage larvae were heat-shocked for 1 h at 45 or 60 degrees C. Using enzyme-linked immunosorbent assay and antiserum against the fusion protein, the expression of TCTP was found to be up-regulated at the translational level. The data suggest that translational regulation of TCTP may play an important role in the early heat-stress adaptation of the trichinellid. Cluster analysis demonstrated that the TCTP sequence of T. pseudospiralis is closely related to that of T. spiralis, but is diverged from the secernentean species.

Cell-mediated response at the muscle phase of Trichinella pseudospiralis and Trichinella spiralis infections.

The cell-mediated response in BALB/c mice infected either by Trichinella pseudospiralis or Trichinella spiralis was compared at days 30-50 post-infection (muscle phase). The former species is non-encapsulated, whereas the latter is encapsulated in host muscles. The pattern of response against the two species was similar. Both species elicited T(H)0 or T(H)1/T(H)2 response, with the last one being dominant. Productions of interferon gamma (IFN-gamma), interleukin (IL)-4 and IL-5 were observed after antigenic restimulation of splenocytes from infected mice. No significant difference was observed between the levels of response to concanavalin A (Con-A) by the splenocytes from both infected and non-infected animals. There was a significant increase in serum IgG(1) and IgG(2a). Flow cytometric analysis revealed a marked proliferative response of splenocytes from infected mice to worm antigens, dominated by B (CD19) lymphoblasts. Only a few helper (CD4+) and cytotoxic (CD8+) T lymphoblasts were present. This was confirmed by an up-regulation of CD69, with a dominant expression on B lymphoblasts. In conclusion, the minimal or lack of intense cellular response against T. pseudospiralis in muscles is likely not due to depression of cell-mediated immunity.

Cloning and characterization of the Cu/Zn superoxide dismutase of Trichinella pseudospiralis.

Copper/zinc (Cu/Zn) superoxide dismutase (SOD) activity was identified for the first time in both crude somatic extracts (CE) and excretory/secretory (E/S) products of Trichinella pseudospiralis. It was the dominant SOD in infective-stage larvae. Native polyacrylamide gel electrophoresis of CE and E/S products yielded a prominent band, which was cyanide-sensitive and was partly inhibited by hydrogen peroxide in SOD assay. Cytosolic Cu/Zn SOD was cloned. The 471-bp full-length cDNA sequence contained an open reading frame of 157 amino acids. The gene contained three introns. Quantitative reverse transcription-polymerase chain reaction indicated that the expression of cytosolic Cu/Zn SOD was substantially higher in infective-stage larvae than in adult worms. Cluster analysis showed that the sequence of the Cu/Zn SOD of T. pseudospiralis, an adenophorean nematode, is related to those of Brugia pahangi, Acanthocheilonema viteae, Onchocerca volvulus, and Haemonchus contortus (all belonging to the sercenentean group).

Cloning and characterization of the mitochondrial heat-shock protein 60 gene of Trichinella spiralis.

The full-length cDNA of mitochondrial heat-shock protein (hsp) 60 of the infective-stage larva of Trichinella spiralis was cloned by degenerative PCR and rapid amplification of cDNA end reactions. The 1,945 bp full-length cDNA sequence contained an open reading frame of 576 amino acids. A mitochondrial signal peptide was located at the N-terminal and a GGM motif at the C-terminal. The gene contained ten exons and nine introns. RT-PCR analysis indicated that thermal, cold, acidic and oxidative treatment did not elicit significant changes in the expression of mitochondrial hsp 60 in the larvae. Cluster analysis showed that the sequence of the hsp 60 gene of T. spiralis is closely related to that of Drosophila melanogaster.

Characterization and cloning of metallo-proteinase in the excretory/secretory products of the infective-stage larva of Trichinella spiralis.

Inhibitor sensitivity assays using azocaesin and FTC-caesin as substrates showed that the excretory/secretory (E/S) products of the infective-stage larvae of Trichinella spiralis contained serine, metallo-, cysteine and aspartic proteinases. The activity of the metallo-proteinase was zinc ion dependent (within a range of ZnSO(4) concentrations). Gelatin-substrate gel electrophoresis revealed two bands of molecular mass 48 and 58 kDa which were sensitive to the metallo-proteinase inhibitor EDTA. The former peptide was probably a cleavage product of the latter. The authenticity of the 58 kDa metallo-proteinase as an E/S product was confirmed by immunoprecipitation. Using PCR and RACE reactions, a complete nucleotide sequence of the metallo-proteinase gene was obtained. It comprised 2,223 bp with an open reading frame encoding 604 amino acid residues. The 3' untranslated region consisted of 352 bp, including a polyadenylation signal AATAA. A consensus catalytic zinc-binding motif was present. The conserved domains suggest that the cloned metallo-proteinase belongs to the astacin family and occurs as a single copy gene with 11 introns and 10 exons. Cluster analysis showed that the sequence of the metallo-proteinase gene of T. spiralis resembles those of Caenorhabdites elegans and Strongyloides stercoralis.

Identification of some heat-induced genes of Trichinella spiralis.

Three heat-induced genes of the infective-stage larvae of Trichinella spiralis were successfully identified by the suppression subtractive hybridization (SSH) technique. As indicated by reverse Northern blotting, 19 of 25 clones were scored as differentially transcribed in the heat-shocked infective-stage larvae. The sequencing data showed the presence of 12 different genes. Three were homologous to histone H3, histone H2B and translationally controlled tumour protein (TCTP). A 0.6 kb cDNA of histone H3 was generated by the RACE method and sequenced. It contained an open reading frame of 136 amino acids that demonstrated 94% identity with genes from Drosophila hydei. Semi-quantitative RT-PCR indicated that after heat-shock treatment, the expression levels of histone H3, histone H2B and TCTP increased 4.8, 27 and 5.7-fold, respectively. Northern analysis confirmed the upregulation of histone H3, histone H2B and TCTP transcripts. The upregulation of these genes during stress conditions has not been reported in parasitic organisms. The stress proteins may play an active role to sustain the parasite after exposure to hostile host factors.

DNA-binding activity in the excretory-secretory products of Trichinella pseudospiralis (Nematoda: Trichinelloidea).

A novel DNA-binding peptide of Mr approximately 30 kDa was documented for the first time in the excretory-secretory (E-S) products of the infective-stage larvae of Trichinella pseudospiralis. Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E-S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of the Mr of proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E-S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E-S product.

Inflammatory response during the muscle phase of Trichinella spiralis and T. pseudospiralis infections.

An attempt was undertaken to determine whether a concurrent infection of Trichinella spiralis and T. pseudospiralis can reduce cellular infiltrations against the former species during the muscle phase of worm development. BALB/c, nude and CBA/N mice were orally infected with either species or a mixture of both. New-born larvae (NBL) of either or both species were also injected subcutaneously into the right/left leg of BALB/c mice. In T. spiralis oral infection, myositis was strongest in BALB/c, intermediate in CBA/N and weakest in nude mice. In T. pseudospiralis oral infection, slight cellular infiltrations were observed around the worms in BALB/c but not in nude or CBA/N mice. However, in mixed oral infections of two species, infiltrations around the sites of T. spiralis were not reduced. In mice injected with T. pseudospiralis NBL, infiltrations around the infective-stage larvae were mostly absent. However, in mice injected with T. spiralis NBL, prominent granulomatous reactions were observed near the sites of worms. The tissue reaction was substantially stronger than that in oral infections. In mice injected with NBL of both species (into different legs), a heavy infiltration was also observed at the site of T. spiralis. A marked increase in levels of IL-4 and IL-6 was detected in the popliteal lymphocytes of BALB/c mice injected with either live or dead NBL of T. spiralis at days 15 and 20 post-injection. This indicated that the worms mainly elicited a TH2 response during the muscle phase of development. An indirect fluorescent antibody test and laser confocal microscopic studies demonstrated the presence of CD4 and CD8 cells in the cytoplasmic region of the nurse cell complex of T. spiralis.

The detection and occurrence of circulating antigens of Trichinella spiralis during worm development.

Four enzyme-based immunoassays were compared in detecting circulating antigens (CA) of Trichinella spiralis, i.e. microfluorescence, dissociated enhanced lanthanide fluoroimmunoassay (DELFIA), enhanced chemiluminescence and enzyme-linked immunosorbent assay (ELISA). Parameters which could affect the sensitivity and specificity of the assays were evaluated. Different combinations of polyclonal antibody (PA) and five monoclonal antibodies (mAbs) against the excretory/secretory antigens of the nematode were tested to produce an optimal antigen-detecting system. DELFIA and a "sandwich" consisting of PA as capturing antibody and mAb 7C2C5 as detecting antibody, yielded the most stable and sensitive results; and 1 ng CA/ml could be detected. The assay did not cross-react with heterologous antigens of Angiostrongylus cantonensis, Ascaris suum, Cysticercus cellulosae, Fasciolopsis buski, Gnathostoma hispidum and Trichuris suis. Fluctuating levels of CA were observed in the serum of experimentally infected mice at various periods post-infection. As early as days 4 and 6, a significant amount of CA was detected. The level reached a peak at day 10, then declined and another peak was observed at day 18. The monitoring of the corresponding antibody response by ELISA showed that IgM was first detected at day 10, reaching a peak at day 16. A marked increase in IgG1 was noted from day 16 and its level was significantly higher than that of IgG2.

Single-stranded endonuclease activity in the excretory--secretory products of Trichinella spiralis and Trichinella pseudospiralis.

A novel acidic extracellular single-stranded endonuclease was demonstrated for the first time in the excretory-secretory (E-S) products of 2 species of Trichinella. Unlike the double-stranded endonuclease reported earlier, the single-stranded molecule is divalent cation independent and is detected in both T. spiralis and T. pseudospiralis E-S products. It hydrolysed single-stranded DNA and RNA at comparable rates. The single-stranded endonuclease was sensitive to inhibition by Zn2+ and to high concentrations of NaCl. Zymographic analysis indicated that it was encoded by at least 3 peptides of Mr approximately 50-60 kDa. The rate of hydrolysis of single-stranded targets by the E-S products was substantially higher than that of the double-stranded molecule. Due to the differences in peptide profile, divalent cation dependence, and species-specific expression, the single and double-stranded endonucleases are likely to be encoded by different proteins and may have different functions.

The distribution of excretory/secretory antigens during the muscle phase of Trichinella spiralis and T. pseudospiralis infections.

The in situ distribution of excretory/secretory (ES) antigens of the infective-stage larvae of Trichinella spiralis and T. pseudospiralis was compared at various periods of development by immunofluorescent laser confocal microscopy and the immunoperoxidase method. In the former infection, epitopes of the ES antigens were always confined exclusively within the nurse cell, i.e., in the cytoplasmic region, hypertrophic nuclei, stichocytes, and cuticular surface of worms. In the latter infection, as early as at day 15 postinfection, ES epitopes were located along the infected myofibers, in the adjacent muscles, hypertrophic nuclei, stichocytes, and cuticular surface of worms. By day 30 postinfection there was a marked increase in both the distribution and the intensity of ES antigens in infected as opposed to uninfected myofibers. A new method was also developed to reveal the number of hypertrophic nuclei, small cells, and larvae in intact nurse cells. As many as four worms could be accommodated within a single complex. The number of hypertrophic nuclei within each complex varied from 15 to 81.

A novel cDNA clone encoding a specific excretory/secretory antigen of larval Trichinella pseudospiralis.

A cDNA library for Trichinella pseudospiralis was constructed to study the expression of specific antigens. Four positive clones were identified using antibodies against the excretory/secretory (ES) products of the nematode as probe. Sequence analysis showed that they contained identical cDNA inserts of 606 bp, including a 5' non-translated region of 96 bp, a core translated segment of 408 bp and a poly(A)+ 3' terminus. It encoded a novel 136-amino-acid polypeptide. Southern blot analysis indicated that the cDNA did not cross-hybridize to the genomic digests of T. spiralis, mouse, or rat. A single copy only of its complementary sequence was found in the genome of T. pseudospiralis. Using the lambda ZAP expression system, the cDNA was induced to express a 23-kDa beta-galactosidase-fusion protein which did not cross-react with polyclonal and monoclonal antibodies against T. spiralis, heat shock proteins, or four heterologous species of nematodes. The antiserum against the fusion protein recognized a 15-kDa band from the ES products of T. pseudospiralis in immunoblotting. Immunocytolocalization demonstrated that the anti-fusion protein serum only recognized an epitope in the stichosome of T. pseudospiralis and not in T. spiralis. The protein can therefore serve as a specific antigen for the differential diagnosis of trichinellosis.

Characterization of endonuclease activity from excretory/secretory products of a parasitic nematode, Trichinella spiralis.

Double-stranded endonuclease activity was demonstrated for the first time in the excretory/secretory (ES) products of a parasitic nematode, Trichinella spiralis, which can reorganize host muscle cells. The endonuclease introduced double-stranded breaks to the native DNA. The ES double-stranded endonuclease(s) was sequence nonspecific, with a pH optimum below 6, and required divalent cations as a cofactor. Its activity was inhibited by the Zn2+ ion. It was detected mainly in the ES products of the infective-stage larvae of T. spiralis collected at 37 degrees C and was present in much smaller amounts in samples collected at 43 degrees C and in the products of T. pseudospiralis, a nonencapsulated species. The activity of endonuclease was blocked by antibodies against ES products. Zymographic analysis showed that the endonuclease activity was associated with at least three molecular forms, designated approximately 25, 30 and 58 kDa, respectively.

Evaluation of excretory/secretory products of larval Taenia solium as diagnostic antigens for porcine and human cysticercosis.

Excretory/secretory antigens (ES) of larval Taenia solium were obtained by maintaining the bladder worms in Medium 199 for 3 days. Analysis by SDS-PAGE showed that ES antigens consisted of at least 19 polypeptides, with M(r) ranging from 14-116 kDa. Analytical isoelectric focusing revealed eight bands with acidic pI. An immunocytolocalization study using the peroxidase method demonstrated the presence of ES epitopes on the tegument of the wall of the spiral canals of bladder worms. The specificity of ES antigens was evaluated by EITB, ELISA and FAST-ELISA using antisera against the common parasites of Chinese pigs and man. ES antigens cross-reacted with the antiserum against larval T. hydatigena of pigs. However, these antigens were generally more specific in diagnosing human cysticercosis. Three host-like molecules with molecular masses 43, 58 and 66 kDa were present in the ES products.

Specificity of isoelectric focusing-purified antigens in the diagnosis of human cysticercosis.

Specific antigens were isolated from the cystic fluid of larval Taenia solium by preparative isoelectric focusing (PIEF). A total of 20 fractions were produced by a rotating ampholine column with pI 3-10 ampholytes. The specificity of each fraction (F) was tested by double-antibody enzyme-linked immunosorbent assay (ELISA) using antisera from patients suffering from cysticercosis or one of six other parasitic diseases. F8-F15 cross-reacted strongly with sera from patients with hydatidosis. F9 and F10 also cross-reacted with the antisera against ascariasis and F15, with antisera against angiostrongylosis. However, F16 and F17 were highly specific as they yielded no cross-reaction with any of the heterologous antisera. PIEF is a good method for the production of specific antigens from larval T. solium because it is easy to perform and relatively inexpensive to run.

Purification of larval Taenia solium antigens by isoelectric focusing.

By preparative isoelectric focusing in a rotating ampholine column, crude cystic membrane (M) or fluid (F) antigens of larval Taenia solium were each separated into 20 fractions. M fractions were less specific and sensitive than F fractions in detecting cysticercosis antibodies in pig serum. Among the F fractions, F15 showed the best potential to serve as a screening antigen. It contained 18 polypeptides, with pI 5.3-8.2 and a specific epitope of 25 kDa which was detected by immunoblotting. Although F15 showed slight cross-reactions with heterologous antisera in double-antibody IgG enzyme-linked immunosorbent assays (ELISA), it yielded the highest absorbance values when tested against homologous antisera. The antigen was used to screen sera samples from 4870 pigs slaughtered in Hong Kong and five other Chinese cities for cysticercosis antibodies by double-antibody ELISA, Falcon Assay Screening Test (FAST)-ELISA and enhanced chemiluminescent immunoassay. The results varied significantly between assays. However, the samples collected from Shenzhen yielded the highest positive rates. Enhanced chemiluminescent immunoassay based on camera-luminometry was found suitable for use under field conditions.

In vitro effects of Trichinella spiralis on muscle cells.

Introduction of excretory/secretory (ES) products of both infective-stage and newborn larvae of Trichinella spiralis into cultures of primary rat myocytes elicited morphological and structural changes in the myotubes. They appeared more granular, thinner, and failed to form networks. The most prominent lesion was the formation of 'nodular' structures, each bearing an enlarged nucleus, along the myotubes. Each node contained numerous cavities enclosed by an intact sarcolemma. Co-culture of myocytes with newborn larvae also elicited nodular formation but each node contained a large central cavity encircled by smaller ones. An immunocytolocalization study using IFAT and laser confocal microscopy showed the presence of parasitic epitopes inside the nodes. However, ES products from adult worms did not affect the myotubes.

A brief update on the diagnosis of trichinellosis.

The latest immunological and molecular methods for the diagnosis of swine and human trichinellosis are briefly reviewed. The following topics are discussed in more detail: isolation of specific antigens by continuous elution-isoelectric focusing methods, production of recombinant antigens, nature of immunodominant antigens, potential use of heat shock proteins (HSPs) as diagnostic antigens, roles of specific IgE and circulating antigens (CA). The immunodominant antigens were found to be highly heat resistant. The specificity and sensitivity of colorimetric sandwich ELISA, microfluorescence (ELFA), enhanced chemiluminescence (ECIA) and dissociated enhanced lanthanide fluoroimmunoassay (DELFIA) in detecting CA were compared. The last method is the most sensitive, detecting as little as 1 ng of antigens/ml of serum. CA was detected as early as 7 days postinfection of mice. The serum from a patient suspected to have acute trichinellosis in Hong Kong was also tested positive for CA.

Production specific antigens from Trichinella spiralis using a continuous elution method and isoelectric focusing.

Immunodominant antigens of 45-53 kDa (one band per fraction) were obtained from excretory/secretory (E/S) and somatic products of infective-larvae of Trichinella spiralis using a continuous-elution method. They were further resolved by isoelectric focusing into different isoforms (45 kDa: pI4.47, 5.09, 5.47 and 5.86; 47 kDa: pI4.72 and 4.97; 53 kDa: pI4.86, 5.11, 5.44 and 5.78). In immunoblotting, the isoforms of pI 5.09, 5.86, 4.97, 5.44 and 5.78 did not cross-react with antisera against Trichuris suis, Metastrongylus apri, Gnathostoma hispidum and Stephanurus dentatus. Hence, they have the potential to serve as specific antigens for the serodiagnosis of trichinellosis.