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 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 cDNA library for Trichinella pseudospiralis was constructed to study the expression of speci®c antigens. Four positive clones were identi®ed 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 b-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 speci®c antigen for the dierential diagnosis of trichinellosis.
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