Crude saline extracts of Trichinella spiralis and T. pseudospiralis infective larvae were studied by Western blot analysis using a monoclonal antibody, named ES/TA2 and produced against T. spiralis larvae. This monoclonal antibody recognized seven major antigenic components in T. spiralis larvae with apparent Mr: 45, 48, 50, 68, 70, 92 and 105 kDa and five in T. pseudospiralis larvae: 38, 50, 70, 72 and 92 kDa. SDS-PAGE of both extracts did not reveal appreciable differences in the range of molecular weights recognized by ES/TA2. These facts show the existence of immunological differences among proteins with apparently identical molecular weights.

The two-dimensional patterns (isoelectrofocusing-IEF/polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate-SDS) of S3 fractions of muscle larvae of four Trichinella isolates were compared. The comparative study concerned six groups of polypeptides. It was observed that the Garkavi isolate of Trichinella pseudospiralis was clearly different from the other isolates, and it showed the simplest IEF/SDS polypeptide pattern. The C-76 isolate of T. nelsoni had only four of the six groups, distinguishing it from the GM-1 isolate of T. spiralis and the Boev isolate of T. nativa that showed all the indicated groups.

Infectivity of Trichinella pseudospiralis in Chinese hamsters was greater than that of Trichinella spiralis. Concurrent infection with these two species of parasite was accompanied by an increase in infectivity of T. spiralis. The infectivity of T. pseudospiralis, but not that of T. spiralis, was significantly influenced by host age. Regardless of host age, T. pseudospiralis was more infective for the Chinese hamster than was T. spiralis. Early during infection, enteritis (myeloperoxidase activity) in hamsters infected with T. pseudospiralis alone was significantly less than that seen in animals infected with T. spiralis alone or with both species concurrently. Myositis in hamsters infected with T. pseudospiralis alone was significantly less on days 14, 17 and 25 post-infection (p.i.) than that observed in concurrently infected or T. spiralis-infected animals. On days 11 and 14 p.i. inflammation in diaphragm muscle from concurrently infected hamsters was significantly less than that seen in animals infected with T. spiralis alone. Muscle histology supported these chemical data on myositis. Suppression of the host inflammatory response against early muscle-stage larvae of T. pseudospiralis underlies the greater infectivity of this parasite for the Chinese hamster.

The isometric mechanical properties of diaphragm muscle were studied in mice infected with either Trichinella spiralis or Trichinella pseudospiralis. Measurements of muscle stress were taken at 15, 20, 30 and 40 days post-infection. Infected diaphragm muscle showed a significant (P < 0·001) reduction in muscle stress during both twitch and tetanic contractions when compared with muscle from control animals. T. spiralis caused a significant reduction in resistance to muscle fatigue. The consequences of these changes in muscle function to host pathology are discussed, and related to previous work on the effects of Trichinella on host biochemistry and the immune response.

Heat shock proteins (HSPs) were documented for the first time in both somatic extracts and excretory/secretory (ES) products of the infective-stage larvae of Trichinella spiralis and T. pseudospiralis. Larvae recovered from muscles of infected mice were heat shocked at 37, 40, 43 and 45 °C in RPMI 1640 medium containing L-[35S]methionine. Somatic extracts and ES products of heat-shocked worms were then analysed by SDS-PAGE, autoradiography and laser densitometry. Prominent bands of HSPs were observed at 43 °C which is the optimal heat shock temperature. The major HSPs in somatic extracts of T. spiralis were 20, 47, 50, 70, 80 and 86 kDa. When the temperature was increased from 37 to 43 °C, the greatest increase in absorbance was observed in HSPs 70 and 86. In vitro translation of mRNA in a nuclease-treated rabbit reticulocyte lysate system showed an increase in the synthesis of the 80 kDa protein. This suggests that the production of HSP 80 is regulated at the transcriptional level. The major HSPs in the ES products were 11, 45, 53 and 64 kDa. In T. pseudospiralis, the major HSPs in the somatic extracts were 20, 26, 31, 50, 53, 70, 80 and 86 kDa, and in the ES products, 11, 35, 37, 41 and 64 kDa.

A cDNA library was constructed from muscle larvae of Trichinella pseudospiralis. A cDNA clone, designated as Tp8 contained a cDNA transcript of 1326 bp length with a single open reading frame, which encoded 303 amino acid residues (34187 Da, estimated molecular mass). The predicted amino acid sequence of the clone had an identity of approximately 60% to the Rcd1 (Required cell differentiation 1) -like proteins among a wide range of organisms. Real-time quantitative polymerase chain reaction results showed that the transcription level of Tp8 gene reached the highest value in adult worms, and that the transcription level in muscle larvae before stichosome formation was higher than in muscle larvae after stichosome formation. The recombinant Tp8 protein migrated at 37 kDa and reacted to antibody against T. pseudospiralis excretory-secretory (E-S) products and sera from mice infected with T. pseudospiralis. An antibody against the Tp8 recombinant protein could stain proteins migrating at approximately 34 kDa (which is the expected size from the sequence) on Western blotting of E-S products from muscle larvae. An immunocytochemical study showed that the Tp8 protein was present within the stichocyte of muscle larvae and adults worms.

A novel DNA-binding peptide of Mr∼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.

Thymidylate synthase, dihydrofolate reductase and dUTPase specific activities were found to remain at a high and constant level in crude extracts from adult worms of Trichinella spiralis, as well as from muscle larvae of both Trichinella spiralis (isolated 1–24 months after infection) and Trichinella pseudospiralis (isolated 5·5–13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. No thymidine kinase activity could be detected in muscle larvae of either species and thymidine phosphorylase could be found only in T. pseudospiralis larvae isolated without the use of pepsin. Muscle larvae of both species contained orotidylate phosphoribosyl transferase activity, pointing to a possibility of 5-fluorouracil activation. Uridine phosphorylase, another enzyme involved in 5-fluorouracil anabolism, was also present in T. pseudospiralis muscle larvae. Results of comparative studies on inhibition of purified T. spiralis and rat thymidylate synthases by substrate (4-thio-5-fluoro-dUMP, 2-thio-5-fluoro-dCMP and N4-hydroxy-dCMP) and cofactor (ZD 9331) analogues indicated only dUMP analogues to show feeble selectivity towards the parasite enzyme. A hypothesis is discussed, assuming high expression of thymidylate synthase in muscle larvae to be connected with their cells being arrested in the cell cycle.

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 ∼ 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.

This study characterized antigens of Trichinella spiralis and T. pseudospiralis muscle larvae recognized by mice infected with the worms. Two-dimensional (2-D) Western blot analysis revealed some profile of antigenic peptides including: (1) molecular weight (MW); (2) isoelectric points (pI), (3) reactivity to well-defined monoclonal antibodies (mAb) and (4) cross-reactivity between the 2 species. Antigenic peptides of T. spiralis consisted of about 100 spots. The MW ranged from 22 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. spiralis excretory–secretory (E–S) peptides migrating at 40, 45 and 50 kDa, and the mAb against TSL-4 stained non-E–S peptides. Antigenic peptides of T. pseudospiralis consisted of about 20 to 30 peptide spots. The MW ranged from 25 to 80 kDa, and pI ranged from 4 to 7. The mAb against TSL-1 stained most of the T. pseudospiralis E–S peptides migrating at 35 and 45 kDa, and the mAb against TSL-4 stained non-E–S peptides. Two-dimensional Western blots showed that the E–S products of T. spiralis and T. pseudospiralis were highly cross-reactive with each other. The non-E–S peptides were, however, not recognized by T. pseudospiralis-infected sera but were recognized by T. spiralis-infected sera. An immunoelectron microscopical study showed the similar result that stichocyte granules and cuticle surface (known to contain E–S antigen) had cross-reactive antigens between the two species. T. pseudospiralis-infected sera stained very weakly the cuticle inner layers and haemolymph (known to contain non-E–S antigen). This evidence implies that mice infected with T. pseudospiralis do not evoke antibodies against non-E–S antigen at the detectable level.

The ultrastructure of muscle larvae of Trichinella pseudospiralis was studied by electron microscopy. The overall structure of muscle larvae of T. pseudospiralis resembled that of T. spiralis except for the stichocyte granules. T. pseudospiralis had at least 3 kinds of stichocyte granules distinguishable from each other by their shape, size and inclusions. The granules had some resemblance to α granules or β granules of T. spiralis, but no resemblance to γ granules. In favour of these morphological differences and similarities among T. spiralis and T. pseudospiralis, excretory and secretory (E–S) products (originating from stichocyte granules) of the 2 species differed to some degree. In an analysis by 2-dimensional electrophoresis, some peptide spots migrating at 45 kDa were shared by the 2 species but the other spots were unique to each of the 2 species. Messenger RNA encoding the 43 kDa glycoprotein of stichocyte granules was detected in the muscle larvae of both species but mRNA encoding the 53 kDa glycoprotein was detected only in muscle larvae of T. spiralis.