Takahiro Fujino

The expulsion of Echinostoma trivolvis : worm kinetics and intestinal cytopathology in conventional and congenitally athymic BALB/c mice

The infectivity and distribution of Echinostoma trivolvis were studied in male, conventional and congenitally athymic nude mice, each infected with 30 metacercarial cysts. In conventional mice, worm recoveries at 6 and 8 days post-exposure were 58.3 and 54.0%, respectively. Worm recovery declined to 44.0% by day 10, to 4.3% by day 13, and 0% by day 17. In athymic mice, worm recoveries at 6 and 8 days post-exposure were 61.7 and 36.3%, respectively. Worm recovery declined to 27.7% by day 10, to 0.7% by day 13, and 0% by day 17. The distribution of worms demonstrated a posteriad migration over time in both groups. Kinetic changes in the number of goblet and mucosal mast cells in the upper ileum of mice infected with E. trivolvis were examined. In conventional mice, the number of goblet cells increased rapidly to reach a peak at day 13 and then declined gradually. The number of goblet cells in athymic mice also increased to reach a peak at day 13, and then declined rapidly. However, the number of goblet cells in athymic mice was always less than that in conventional mice. The mast cell number in infected conventional mice increased rapidly to reach a peak at day 17 and then declined. There was no increase in the mast cell number of infected athymic mice throughout the experiment. Whereas common pathological changes occurred in the intestines of both mice groups infected with echinostomes some ultrastructural differences were observed in the gut epithelial cells of conventional versus athymic mice.

Expulsion ofEchinostoma trivolvis (Cort, 1914) Kanev, 1985 and retention ofE. caproni Richard, 1964 (Trematoda: Echinostomatidae) in C3H mice: pathological, ultrasturctural, and cytochemical effects on the host intestine

C3H mice were infected with 30 metacercarial cysts of either echinostome to study the pathological, ultrastructural, and cytochemical effects of the infection on the mouse small intestine. In mice infected withEchinostoma caproni, the intestine showed villous atrophy with fused or eroded villi. The microvilli of the enterocytes were sparse and distorted and showed reduced alkaline phosphatase activity. The crypts of Lieberkuhn were hyperplastic and showed a marked reduction in goblet and Paneth cells. As compared with uninfected controls, there was a marked reduction in glucose-6-phosphatase activity in the enterocytes of the infected gut. Collagen fibers and the number of fibroblasts were increased under the epithelium. In mice infected withE. trivolvis, the tips of the intestinal villi were bent and blunted. The microvilli of the enterocytes were less tightly packed than those of uninfected controls. The mitochondria in the enterocytes were irregularly shaped, contained intracristal bodies, and showed increased cytochrome oxidase activity as compared with those of uninfected controls. The crypts were hyperplastic but showed an increase in the numbers of goblet and Paneth cells. The fibroblasts and collagen fibers showed abnormal development. The ultrastructural and cytochemical differences seen in this study reflect the uniqueness of the host-parasite relationship of each of these echinostome species in the gut of the C3H mouse.

Rapid expulsion of the intestinal trematodes Echinostoma trivolvis and E. caproni from C3H mice by trapping with increased goblet cell mucins.

Echinostoma trivolvis (Cort, 1914) adults were rejected from C3H mice by 15 days post-exposure, corresponding to the increase in the number of goblet cells. Homologous and heterologous infections with the allopatric species E. caproni (Richard, 1964) were used to confirm the effect of increased secretion of goblet cell mucins in rejecting metacercariae of challenge infections of E. trivolvis or E. caproni on days 10, 16 and 20 p.i. after primary infections of E. trivolvis metacercariae. Five-day-old juveniles of E. trivolvis and E. caproni, which were recovered from C3H mice or hamsters, were also used for challenge infections on day 10 p.i. The metacercariae and juveniles, which were challenged homologously and heterologously on day 10 p.i., were almost all expelled. The metacercariae of E. trivolvis, which were challenged homologously on day 16, were completely rejected, but only a few challenged metacercariae of E. caproni in heterologous infection were recovered. Considerable numbers of E. caproni were recovered when challenge infections with the metacercariae were done on day 20 p.i., while only a small number of E. trivolvis was recovered. All controls without primary infections showed a recovery rate of over 50% of the worms. These results indicate that increased secretion of mucins by hyperplastic goblet cells associated with primary infections of E. trivolvis may be responsible for the expulsion of worms challenged homologously with E. trivolvis and heterologously with E. caproni from the mouse host.

Echinostoma caproni and E. trivolvis alter the binding of glycoconjugates in the intestinal mucosa of C3H mice as determined by lectin histochemistry

Mouse (C3H) mucosal glycoconjugates were examined in normal small intestines and intestines infected with Echinostoma caproni or E. trivolvis using six different fluorescein-conjugated lectins: Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin I (RCA-I), Glycine max soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and Arachis hypogaea peanut agglutinin (PNA). The expression of lectin-binding sites and the intensity of the binding of lectins in the mouse small intestines were changed by infection with the echinostomes. Specific differences in the reaction to glycoproteins were clearly observed between the mouse intestines infected with E. caproni and those infected with E. trivolvis. In E. caproni infection, binding of most of the lectins to the villi was remarkably reduced in accord with the villous atrophy and loss of goblet cells. In contrast, in E. trivolvis infection, the binding of WGA, RCA-I and DBA was reduced in the microvillar surfaces, but binding of UEA-I and SBA were unchanged compared to the control intestines. The lectin binding to goblet cells in E. trivolvis-infected mice mostly increased. These observations may reflect the marked increase in goblet cells and the less severe damage in the villi of E. trivolvis infection compared to E. caproni infection. Most of the glycoconjugates were slightly reduced in the hyperplastic crypts except for N-acetyl glucosamine. It is possible that glucose metabolism in the host intestines infected with E. trivolvis was activated, resulting in an increase in the rate of mucin synthesis as well as qualitative changes in mucus, thereby mediating the expulsion of the worms.

Immunosuppression by a neutral thiol protease from parasitic helminth larvae in mice

The composition and immunological suppression of a novel proteinaceous material, a neutral thiol protease (NTP), isolated from the metacercaria of the helminth Paragonimus westermani are reported. From cDNA cloning and sequencing, the protease was found to be composed of 215 amino acid residues and closely resembled the known cysteine proteases. Treatment of adult mice with the enzyme suppressed the delayed footpad reaction and haemagglutinin antibody production, and reduced expression of the major histocompatibility complex and interleukin 2 receptor on lymphocytes, and induced suppressor cells in the spleen. In addition, stable and long‐term skin graft survival was achieved by concomitant administration of the enzyme at a low dose.

Ultrastructural Studies on Spermatogenesis in a Parthenogenetic Type of Paragonimus westermani (Kerbert 1878) Proposed as P. pulmonalis (Baelz 1880)

Spermatogenesis in a parthenogenetic type of P. westermani (Kerbert 1878) called P. pulmonalis (Baelz 1880) throughout this study, was observed by light microscopy (LM), scanning (SEM), and transmission (TEM) electron microscopy. During spermatogenesis, most of the cells became degenerated or malformed as a result of aberrations during spermatogenesis. Vacuolated cells were often found in the testicular lumen. In some nuclei of spermatocytes, synaptonemal complexes were formed and this indicated that some pairing of homologous chromosomes did occur, but only rarely. Cytophores in some rosettes were broken down into small fragments and the cells separated from each other. Norman spermatozoa were very rarely found in the testis and never in the seminal receptacle, where egg and vitelline cells were present instead. Throughout spermatogenesis, Golgi complexes, mitochondria, ribosomes, and endoplasmic reticulum were not abundant, and this suggested that cell activities and protein synthesis were greatly reduced.

ARGENTOPHILIC AND SCANNING ELECTRON MICROSCOPIC OBSERVATIONS OF THE TEGUMENTARY PAPILLAE OF ECHINOSTOMA REVOLUTUM (TREMATODA) CERCARIAE

Argentophilic staining and scanning electron microscopy (SEM) were used to study the tegumentary papillae of Echinostoma revolutum cercariae. The most abundant papilla was uniciliate and arranged bilaterally on the body and tail of the cercaria. The distribution of these papillae was similar to that seen in other 37-collar spined echinostome cercariae. SEM revealed a total of 18 multiciliate papillae--16 on the anterior end and 2 at the middle of the body. These papillae contained up to 18 cilia per papilla and have not been reported previously from any cercaria.

The expulsion ofEchinostoma trivolvis (Trematoda) from ICR mice: extension/retraction mechanisms and ultrastructure of the collar spines

Ultrastructural observations of collar spines and surrounding tissues associated with extension/retraction mechanisms of the spines were made onEchinostoma trivolvis recovered from ICR mice or golden hamsters. The spine consisted mainly of a homogeneous, crystalline structure of moderate to heavy electron density. The spine was surrounded by an outer layer of tegument-like material. Interstitial material was located between this outer layer and the surrounding tegument. Muscle bundles associated with spine movements were anchored by hemidesmosomes to interstitial material. Contraction of these muscles caused an invagination of the tegument surrounding a spine, resulting in spinal protrusion. Relaxation of these muscles resulted in spinal retraction. An immunocytochemistry technique using colloidal gold confirmed the presence of actin in the collar spines. Most of the collar spines of excysted metacercariae and juveniles maintained in a defined medium supplemented with fresh hamster serum were extended, whereas worms maintained in mouse serum mainly showed retracted spines. Apparently, factors in mouse serum are involved in spinal retraction.

Rapid expulsion of the intestinal trematodes Echinostoma trivolvis and E. caproni from C3H/HeN mice after infection with Nippostrongylus brasiliensis

Abstract The number of goblet cells in the small intestines of C3H/HeN mice increased rapidly following their infection with about 500 third-stage larvae (L3) of the intestinal nematode Nippostrongylus brasiliensis. The number of goblet cells reached its peak on day 9 postinfection (p.i.). Worm burdens in the hosts’ small intestines were determined following a challenge infection with encysted metacercariae of the intestinal trematodes Echinostoma trivolvis or E. caproni on days 8 and 16 after primary infections with N. brasiliensis. All metacercariae of E. trivolvis or E. caproni used to challenge the hosts on day 8 p.i. were expelled. Considerable numbers of E. trivolvis (48.6%) and E. caproni (67.1%) remained in the intestines of hosts challenged with these echinostomes on day 16 p.i. All the controls used for E. trivolvis and E. caproni infections without primary infections with N. brasiliensis showed recovery rates greater than 70%. An enzyme-linked immunosorbent assay (ELISA) showed that the IgM titer rose remarkably and plateaued on day 11 p.i. No marked rise in the IgG or IgA titer occurred during the experiment. These results indicate that mucins increased by hyperplastic goblet cells associated with primary infections with N. brasiliensis are responsible for a rapid expulsion of the worms of the challenge infection with E. trivolvis or E. caproni from the mouse host.

Effects of diet on the lipid composition of the digestive gland-gonad complex of Biomphalaria Glabrata (Gastropoda) infected with larval Echinostoma Caproni (Trematoda)

This study examined the effects of a larval Echinostoma caproni infection on the neutral lipid composition of the digestive gland-gonad complex (DGG) of Biomphalaria glabrata snails fed hens egg yolk supplemented with lettuce (Y-L) or lettuce supplemented with Tetramin (L-T). Snails were experimentally infected with the miracidial stage of this echinostome, and their DGGs containing daughter rediae were analyzed for neutral lipids five weeks post-infection by qualitative and quantitative thin-layer chromatography. Light microscopy using Oil Red O (ORO) staining and transmission electron microscopy (TEM) were used to localize neutral lipids in the rediae. The DGGs of infected snails maintained on the Y-L diet showed a significant increase in free sterols and a significant decrease in triacylglycerols compared to uninfected snails maintained on the Y-L diet. The DGGs of infected snails maintained on the L-T diet showed no significant difference in free sterols or triacylglycerols compared to uninfected snails maintained on the L-T diet. ORO staining and TEM showed the presence of lipid droplets in rediae from snails on the Y-L diet. The significant decrease in triacylglycerols in the DGGs of infected snails maintained on the Y-L diet suggests that triacylglycerols were utilized by the rediae.