Effects on Trichinella spiralis of host responses to purified antigens.

Purification of two antigens (48-kilodalton polypeptide and a group with major subunits of 50 and 55 kilodaltons) from the infective larvae of the parasitic nematode Trichinella spiralis was recently reported. Immunization of mice with either of these antigens induces strong resistance to a subsequent challenge infection. In the study reported here the mechanism of this resistance was investigated by monitoring the parasite's life cycle in mice immunized with the antigens. Immunized mice were able to expel intestinal adult worms and to inhibit the fecundity of adult female worms at an accelerated rate compared to control mice. Accelerated expulsion and inhibition of fecundity may account entirely for the level of resistance induced by immunization. Although the effects of the immune response apparently are exerted on adult worms, the target antigens are expressed only by developing larvae. This suggests that immune effector mechanisms act on intestinal larvae in such a way that they develop into defective adults.

Partial characterization of two antigens secreted by L1 larvae of Trichinella spiralis.

Two protein antigens were isolated from excretory-secretory products of Trichinella spiralis by biochemical methods and characterized with respect to their chemical and immunological properties. One antigen, of apparent Mr 43,000, is an abundant secreted protein of infective L1 larvae, while the other, of 45-50 kDa, is present in smaller amounts. Yields, extinction coefficients, isoelectric points, amino acid compositions, and partial N-terminal amino acid sequences for each are reported. Partial amino acid sequences of peptides derived from the 43-kDa protein by cyanogen bromide cleavage have been determined. Treating a reduced-pyridylethylated derivative of the 43-kDa protein with glycopeptidase F (N-glycanase) resulted in formation of a transient product of 37 kDa followed by a stable polypeptide of 32 kDa (by SDS-PAGE), suggesting the presence of two N-linked carbohydrate groups. A similar result was obtained with the 45-50-kDa protein, which gave a transient doublet of 38 and 40 kDa and a final, stable product of 33 kDa, with a minor component of 35 kDa. Two glycosylation sites of the 43-kDa protein and one site of the 45-50-kDa protein can be identified in the amino acid sequences. Polyclonal antibodies prepared against the two proteins cross-reacted extensively, but failed to react with the doubly deglycosylated polypeptides in Western blots. The dominant epitopes present in the reduced-pyridylethylated polypeptides are, therefore, N-linked carbohydrate, although the presence of peptide epitopes in the native proteins cannot be excluded.

Failure to detect Trichinella spiralis p43 in isolated host nuclei and in irradiated larvae of infected muscle cells which express the infected cell phenotype.

Infection by Trichinella spiralis induces host muscle cells to become repositioned within the cell cycle and to lose differentiated skeletal muscle characteristics. Antibodies to a 43-kDa excretory-secretory (ES) protein (p43) also bind to infected host cell nuclei. Neither the identity of these nuclear antigens nor their role in inducing the infected cell phenotype is known. To address these issues, infected cell nuclei were isolated and nuclear antigens analyzed with several antibody preparations to p43. Four antibody preparations to p43 recognized 43-, 45-, 50-, 67- and 71-kDa proteins in ES extracts. The prominent proteins recognized by these antibodies in host nuclear antigen extracts were 71, 79, 86 and 97 kDa. Less prominent proteins of approximately 43 and 45 kDa were detected in nuclear extracts. However, antibodies which specifically recognized p43 failed to bind detectably with in situ and isolated host nuclei and nuclear extracts. Expression of p43 was analyzed in host cells infected by newborn larvae irradiated with 60Co. This treatment prevented expression of detectable levels of p43 in resulting muscle larvae, while infected muscle cells displayed typical infected cell characteristics. However, anti-p43 antibodies which recognized multiple ES and nuclear proteins did stain nuclei of irradiated larva-infected cells, albeit at reduced levels. The results raise doubts that p43 is required for induction of the infected cell phenotype. Nevertheless, nuclear antigens recognized by anti-p53 antibodies remain as candidates for influencing this phenotype.

Developmental expression of a 43-kDa secreted glycoprotein from Trichinella spiralis.

Trichinella spiralis is an intracellular parasitic nematode that infects skeletal muscle cells. Infection results in loss of tissue specific characteristics and conversion of the muscle cell to a Nurse cell. The characteristic changes leading to the formation of the Nurse cell appear complete by day 12 after intramuscular infection. Proteins synthesized in the stichocytes (secretory cells) of T. spiralis and secreted in the host cell are believed to be involved in the process of Nurse cell formation. One secreted glycoprotein of 43 kDa has been considered as a candidate factor involved in Nurse cell formation. We determined the timing of synthesis and secretion of the 43-kDa glycoprotein and its temporal correlation to the changes of the infected host cell, to gain an understanding of the role of the 43-kDa glycoprotein in T. spiralis infection. We show that the 43-kDa glycoprotein is first expressed on day 11 following intramuscular infection, several days after the changes in the infected muscle cell have been initiated. Protein(s) immunologically related to the 43-kDa glycoprotein but not the 43-kDa glycoprotein itself are detected in the nuclei of mature Nurse cells. During the intramuscular stage the 43-kDa glycoprotein appears to be stored in the alpha-stichocytes of T. spiralis and appears to be secreted immediately following invasion of the intestinal columnar epithelial cells by the L1 larva. The role of the 43-kDa glycoprotein remains unknown, however, these findings argue against involvement of the 43-kDa glycoprotein in Nurse cell formation.

Trichinella pseudospiralis secretes a protein related to the Trichinella spiralis 43-kDa glycoprotein.

A 43-kDa secreted glycoprotein from the intracellular parasitic nematode Trichinella spiralis has been considered as a factor involved in the formation of the Nurse cell in infected muscle. The closely related intracellular parasitic nematode Trichinella pseudospiralis that also infects muscle cells, does not form Nurse cells and was thought not to secrete the 43-kDa glycoprotein. This implied a unique role for the 43-kDa glycoprotein in T. spiralis infection and supported the hypothesis of involvement of the 43-kDa glycoprotein in Nurse cell formation. Following cloning of a full length cDNA encoding the 43-kDa protein, antibodies were raised against several domains of the 43-kDa glycoprotein. Here we show that a protein related to the 43-kDa glycoprotein exists in T. pseudospiralis. Immunohistochemical studies reveal important similarities in the distribution of the 43-kDa glycoprotein and the related protein from T. pseudospiralis in muscle infections with either of the two parasites. The 43-kDa glycoprotein may therefore play a common role in the life cycles of these two parasites and probably is not involved in Nurse cell formation.

Cryptosporidiosis of the human small intestine: a light and electron microscopic study.

Intestinal infection by the coccidian parasite Cryptosporidium is a well-recognized condition in immunocompromised hosts and in some normal persons. The authors studied a patient with acquired immunodeficiency syndrome and cryptosporidiosis of the small intestine. The parasite inhabits the microvillous brush border of the intestinal epithelium and must be carefully sought on light microscopic examination of intestinal biopsy specimens. Characteristic life cycle stages are observed on electron microscopy. The absence of significant light microscopic alterations of the villous architecture in this patient's biopsy specimen and in other cases suggests that other factors, such as toxin elaboration by cryptosporidia or other organisms, may be involved in the pathogenesis of diarrhea. Abnormal aggregation of lysosomes at the apices of intestinal epithelial cells may reflect ineffective host phagocytic mechanisms.

Enzyme immunoassay for swine trichinellosis using antigens purified by immunoaffinity chromatography.

Various preparations of crude and a purified preparation of Trichinella spiralis antigens were compared in a rapid, micro-enzyme immunoassay (EIA) for detecting trichinellosis in swine. The crude antigen preparations (XM-300 or S3 fraction) were lipid-free, cell-free fractions of muscle larvae, and the purified antigen was prepared by immunoaffinity chromatography of the soluble fraction of stichocyte secretory granules from rat muscle larvae. The antigens were tested against normal and immune swine sera for sensitivity and specificity, and for their ability to detect seroconversions early in the immune response. At optimum concentrations, absorbance values from immune and nonimmune sera produced sample to noise (S/N) ratios three-fold higher for the column antigen than for XM-300. Tests of sequential sera from experimentally-infected pigs showed that the column antigen produced lower absorbances with pre-infection sera and, from 18 days post-infection, higher absorbances with positive sera. From 21-28 days post-infection, absorbances and S/N ratios with column antigen were nearly twice those with XM-300. Column antigen detected antibodies more often than XM-300 antigen in sera collected prior to the appearance of larvae. Crude antigen did not distinguish all true negatives from weakly positives in a study involving 100 sera from muscle digestion-negative pigs and 75 sera from experimentally infected pigs, whereas the column antigen distinguished all negatives from positives. In a larger scale test of the column antigen, 1,130 pigs from Puerto Rico were tested in the micro-EIA test. Puerto Rico has no endogenous trichinellosis, and all 1,130 pigs were shown to be muscle digestion negative. These same pigs were all negative using the column antigen. These results show that the column antigen out-performs the crude antigens in sensitivity, specificity, and early detection. The column antigen is therefore a major improvement in the EIA for swine trichinellosis.

Immunization of swine against Trichinella spiralis.

Swine were immunized with partially purified stichosome antigens derived from Trichinella spiralis muscle larvae. In 3 trials, 500 to 600 micrograms of the solubilized particle component (S3) induced moderate levels of resistance to challenge inoculation; the percent reduction in larvae per gram of muscle ranged from 43 to 55. The effect of immunization with S3 on pig intestinal expulsion of adult worms was determined in one experiment and the results indicated that S3 immunization was only weakly effective in enhancing gut expulsion. These results suggest that immunization with S3 induces a protective immune response in the pig that may be directed either against the migrating newborn larvae or against adult worm fecundity.

Trichinella spiralis and the concept of niche.

Trichinella spiralis is an intracellular parasite as both a larva and an adult. The first-stage larva lives in a modified portion of a skeletal muscle cell, the nurse cell, and can reside there for the life span of the host. Adult worms occupy a nonmembrane-bound portion of columnar epithelium, living there as intramulticellular parasites. The newborn larva is the only nonintracellular stage, living free in the circulation. Trichinella spiralis induces modifications in each of its intracellular niches. Parasite signals secreted into the milieu of the developing nurse cell results in the reprogramming of host genomic expression, reflected in loss of muscle-specific proteins, over-expression of collagen, and the development of a circulatory rete. Formation of the nurse cell is complex, presumably involving many steps; yet there is not a large series of related intermediate forms in nature. Trichinella pseudospiralis induces an incomplete nurse cell. Adult parasites cause the death of the infected epithelium. The precise nature of most of the signals from parasite to host and from host to parasite has not been determined. As a direct consequence of exposure to some of them, the host develops long-lasting immunity to reinfection. This may confer advantages both for the parasite, as well as the host, because strong immune responses should reduce intraspecific competition.

Blood vessels in Trichinella spiralis infections: a study using vascular casts.

Larvae of Trichinella spiralis initiate the transformation of myocytes into nurse cells that become surrounded by elaborate networks of blood vessels. To examine the structure of these networks (i.e., retes), transcardic perfusion was used to clear the vascular tree of Trichinella-infected mice and to inject a plastic that polymerized in situ. Vascular complexes were found only around infected myocytes and were characterized by large circumferential vessels that give rise to the smaller channels of the retes. The secondary vessels vary widely in caliber and are distributed in a random fashion. Three types of network were found: simple, complex, and hypercomplex, and they were distributed normally, with the complex retes the most common. Comparison of the structure of the baskets with that of vessels in surrounding uninfected muscle strongly suggests that the vascular retes are the result of de novo angiogenesis induced during the infection. The parasite may elicit angiogenesis directly through secretion of unique products or may elicit a change in the nurse cell that, in turn, results in growth of new blood vessels.

The stichosome and its secretion granules in the mature muscle larva of Trichinella spiralis.

The stichosome of the mature muscle larva of Trichinella spiralis consists of a single row of 45 to 55 stichocytes. Each stichocyte is about 25 mum in diameter and possesses a single nucleus. A duct leads from each stichocyte to the lumen of the esophagus. The stichocyte cytoplasm contains mitochondria, structures resembling Golgi-complexes, rough endoplasmic reticulum, and usually 1 of 2 types of secretory granules. The alpha-granule measures about 800 mn in diameter, contains a prominent inclusion, and has a granular matrix. The beta-granule is about 600 mn in diameter and is homogeneous in appearance. Both granule types are surrounded by a single membrane. Ten to thirteen stichocytes containing alpha-granules are confined to the posterior portion of the stichosome. After isopycnic centrifugation in sucrose gradient of large granule fractions obtained from cell-free homogenates, the alpha- and beta-granules show characteristic distribution patterns as revealed by the morphology of the fractions. The median equilibrium density of the alpha-granules is 1.245, while that of the beta-granules is 1.230. There is a correlation between the distribution of the granules and of antigens reacting with hyperimmune antitrichinella seruma. At least 4 unique antigens can be attributed to each of the granule types. Fractions enriched in mitochondria do not contain specific antigens. Antigens from both types of secretory granules cross react totally with those present in the excretion-secretion products of living muscle larvae. Cytoimmunochemical data show that antigens are distributed in a patchy fashion throughout the stichocyte cytoplasm. This finding is consistent with the distribution of the secretory granules in the intact stichocyte.

Trichinella spiralis: partial characterization of antigens isolated by immuno-affinity chromatography from the large-particle fraction of the muscle larvae.

A large-particle fraction, derived from muscle larvae of Trichinella spiralis by a combination of homogenization and differential centrifugation, was treated with 0.1% Triton X-100 and then centrifuged at high speed in order to obtain the soluble portion of this fraction (i.e., S3 fraction). The S3 fraction was then subjected to immuno-affinity chromatography. The antigens were eluted from the column using glycine-HCl buffer (pH 2.5). All 20 antigens were recovered from the S3 fraction in quantities that permitted physical, chemical, and biological determinations to be made on them. Antigens analyzed on SDS-PAGE ranged in molecular weight from 105,000 to 11,000 daltons Their isoelectric points were estimated with slab gel isoelectric focusing and ranged in pI from 4.0 to 9.0. The column-purified antigens were injected into mice together with an equal volume of Freund's complete adjuvant, and 2 wk later all mice were each challenged with 200 muscle larvae given orally. Immunized mice harbored 84% fewer muscle larvae at day 30 postinfection than controls, and were as immune as those mice receiving whole S3 (86% reduction). These results showed that protection-inducing antigens can be separated from the nonprotective portion of the muscle larva by immuno-affinity chromatography without loss of protection-inducing activity.

Changes in nurse cell nuclei during synchronous infection with Trichinella spiralis.

The rate of enlargement of nuclei was determined on 4-microns-thick sections of synchronously infected mouse thigh muscle. Normal muscle nuclei had a geometric mean volume of 84 microns and a range of 42-170 microns 3. At days 5, 6, 7, 8, and 10 and 6 mo after infection, mean nuclear volume was 177 (100-315) microns 3, 254 (140-462) microns 3, 278 (172-447) microns 3, 681 (407-1,138) microns 3, 512 (326-804) microns 3, and 509 (298-870) microns 3, respectively. Size of nuclei for any given day followed a log normal distribution. On days 7 and 8 after infection, 31% of enlarged nuclei had 2 nucleoli, whereas only 15% had 2 nucleoli on day 10. One percent of enlarged nuclei in 6-mo-old nurse cells had double nucleoli. The number of enlarged nuclei in 6-mo-old nurse cells was determined from serial sections of infected tongue muscle. Each nurse cell contained an average of 40 enlarged nuclei. Sixty-four percent of nurse cells examined (n = 55) had between 30 and 60 enlarged nuclei. However, there was great variation in the range (7-142). These results are discussed in relation to the development of the nurse cell.

Immunocytolocalization of two protection-inducing antigens of Trichinella spiralis during its enteral phase in immune and non-immune mice.

Monoclonal antibodies (mAb) recognizing epitopes on the 48K (beta stichocyte specific) and the 50/55K antigen (alpha stichocyte specific) were used as first ligands for immunocytolocalization on de-paraffinized sections of infected gut tissue of non-immune and immune CFW strain mice. The enteral phase was studied at 6, 14, 23, 30 hr and 7 days after initiation of infection via the oral route, times corresponding in worm development to the first (L1), second (L2), and third (L3) stage larva and adult. No change in the intensity of the immune reaction with either mAb was noted in parasites developing within immune or non-immune mice for any of the time-points studied. The 48K and the 50/55K antigens were present within the stichocytes at 6 hr. Enterocytes adjacent to some worms also stained positive for both epitopes at this time. Throughout worm development, the amount of each antigen within the worm diminished, until almost none was left at 30 hr. At day 7, the 48K antigen was present within a few stichocyte cells, the canalicular tree, and within the lumen of the midgut. The 50/55K antigen at this time point was localized within only a few stichocyte granules and on the lining of the worm's gut. Embryo stages did not possess either the 48K or 50/55K epitopes. A marked increase in cells bearing IgG in the lamina propria was noted in immune mice when compared with their non-immune counterparts.

Eosinophilia in murine trichinellosis.

In both primary and secondary infections using 3 different strains of mice (DBA/1, B10 X D2, and C3H/HeJ), 2 peaks of peripheral blood eosinophilia were noted. Depending upon the strain, the first of the biphasic peaks in primary infections occurred between days 5 and 9, and the second peak occurred between days 16 and 22. The relative heights of the peak varied with the strain. In secondary infections, the initial peak was lower and the larger second peak occurred earlier. Methyridine treatment to eliminate adults prior to release of newborn larvae altered neither the course of eosinophilia in primary infection nor the biphasic distribution of eosinophilia. These studies demonstrate that the adult worm is responsible for inducing eosinophilia in murine infections, that 2 peaks of eosinophilia exist in primary and secondary infections, and that eosinophilia in secondary infections is an anamnestic response.

Trichinella spiralis: vascular endothelial growth factor is up-regulated within the nurse cell during the early phase of its formation.

The L1 larval stage of Trichinella spiralis induces modification in a portion of striated skeletal muscle cell resulting in the formation of the nurse cell. This specialized host cell is completely encased in a capsule composed mainly of collagen type IV and type VI, which, in turn, is surrounded by a unique rete of vessels whose formation begins on around day 12 after intracellular infection. We investigated the possibility that vascular endothelial growth factor (VEGF) may be up-regulated during nurse cell formation by employing immunohistochemistry and in situ hybridization on synchronously infected mouse muscle tissue. Both VEGF mRNA and VEGF peptide were detected in the developing nurse cell cytoplasm from day 7 up to 16 mo after infection. In addition, VEGF was also detected in cells in the area immediately surrounding the nurse cell on days 15 and 17. On the basis of these results, we propose that hypoxia is induced by T. spiralis within the developing nurse cell some time prior to the up-regulation of VEGF, perhaps as early as day 7. We further propose, on the basis of the continued presence of VEGF in nurse cell cytoplasm, that a constant state of hypoxia cell is maintained.

A method for isolation and partial purification of Trichinella spiralis nurse cells.

Invasion of vertebrate muscle cells by larvae of Trichinella spiralis is accompanied by redifferentiation of the host myofiber into a novel structure called the nurse cell. The nurse cell protects and nurtures the enclosed parasite during its long stay in host muscle. It is anatomically independent of the surrounding uninfected muscle cells and can be isolated from host tissue by mechanical or enzymatic means. Current methods employed for this purpose have yielded only small numbers of nurse cells. An apparatus designed to isolate large numbers of nurse cells and a method for removal of all free larvae and most host muscle debris is described. Homogenization and trypsin digestion of muscle tissue was followed by passage of muscle/parasite suspensions maintained at 37 C through a jacketed glass column fitted with a 40-mesh stainless steel screen at the top and a Nitex screen with 150-microns-diameter pores at the bottom. Nurse cells were retained by the Nitex screen. Density gradient centrifugation using Percoll removed all free larvae and most contaminating muscle debris from nurse cell suspensions. The large quantities of nurse cells made available by this method will allow evaluation of the molecular biology, nutrition, biochemistry, and metabolism of the enclosed parasite and of the Trichinella-modified host muscle cell.