Schistosoma manonsi: cercarial penetration of host epidermis at the ultrastructural level

Invasion of the outer layers of the epidermis of mouse ear skin by cercariae of Schistosoma mansoni within 7 min of their application to it has been studied with the optical and the scanning and transmission electron microscopes.Entrance of cercariae was under the edges of the dead flattened keratinized cells of the horny layer (squames), and penetration through this layer was by disarticulation of the stacks of squames at their interdigitations. Mucus from the postacetabular glands was recognized with the light and electron microscopes on the skin surface, especially at squame edges and between layers of squames and along the keratogenous zone. The findings suggested that disarticulation of the squames was not effected solely by the muscular probing and pushing of the parasite, but that it might be aided by swelling of the mucus secretion deposited in the area from the postacetabular glands. Loosening of the interlocked edges of the squames by enzymatic action is also a possibility, but was not evaluated for this report.The migration path along the keratongenous zone was marked by extensive damage to the transitional cells of the granular layer subjacent to the squames. Packets of secretion from the cercarial preacetabular glands were identified below the horny layer in the cytoplasm of these cells. It was considered that the host tissue damage in this area was the result not only of tearing of the tissues by passage of the spiny schistosomules, but also of enzymatic activity, the enzyme source being the granules in the packets of preacetabular gland secretion.     

Schistosoma mansoni: Cercarial degradation of a radioactively labeled collagen gel

The apparent penetration activity of Schistosoma mansoni cercariae was quantified by means of an in vitro assay with a radioactively labeled Type I collagen gel. Both live cercariae and cercarial preacetabular gland secretions degraded the collagen. The addition of skin lipid or linoleic acid to the gel surface enhanced the degradation by live cercariae.     

日本血吸虫皮肤型童虫透射电镜观察

本文报道应用透射电镜观察并比较0.5、3和12小时龄的日本血吸虫皮肤型童虫的超徽结构特征。结果表明,除了外质膜外,其他的超微结构,如体被、肌层、体被下细胞、胞质桥、头腺、钻腺和食道等结构在尾蚴感染后3小时均未见再有明显的变化。     

Schistosoma mansoni: biochemical evidence for morphogenetic change from cercaria to schistosomule

Conditions for obtaining in vitro the transformation of cercariae in larvae similar to schistosomules are described. They consist of either centrifuging cercarial suspension or packed cercariae at room temperature, or incubating at 30 C packed cercariae. The product obtained was a mixture of cercariae, tails, cercarial bodies, and schistosomule-like larvae as revealed by stereomicroscopy. Tails and cercariae were mechanically separated.Analysis of samples of schistosomule-like preparations obtained by centrifuging cercariae at room temperature revealed that many of the organisms were PAS negative and water sensitive, and showed no localized staining with alizarin and no tendency to form Cercarienhüllen Reaktion (envelopes) in immune human sera. Elimination of the contents of the preacetabular glands was followed during centrifugation or incubation by assaying the proteolytic and/or esterolytic activities in the pellets obtained at different times. The specific activity of the secretion collected increased as a function of time. Electrophoresis of extracts of organisms before and after centrifugation at room temperature showed decrease of PAS stained bands.     

Schistosoma mansoni: silver ion (Ag +) stimulates and reversibly inhibits lipid-induced cercarial penetration.

Certain long-chain polyunsaturated fatty acids (FA) found on mammalian skin trigger cercariae to penetrate and transform into schistosomules; however, the mechanism by which FAs stimulate cercariae is unknown. In order to determine whether argentophilic papillae concentrated at the apical region of the cercariae are the chemoreceptors that may mediate cercarial response to FAs, an assay assessed the proportion of cercariae that penetrated a 0.25% agar matrix in the presence (61%) and the absence (2.3%) of linolenic acid at 0.22 mM. Silver nitrate (Ag+) which selectively binds to cercarial papillae (Short and Cartrett, J. Parasitol. 59, 1041, 1973) is nontoxic (at 0.09 mM used in this study) as demonstrated by the ability of Ag+ treated cercariae to mature successfully into adult worms (8.8% maturation compared to 10.2% of untreated controls, n = 5) after subcutaneous injection. When Ag+ was added to cercarial suspensions, penetration into linolenic-impregnated agar was significantly inhibited (80.8%). Washing cercariae free of Ag+ reversed this inhibition. These data, as well as observations that both argentophilic papillae and cercarial response to FAs disappeared within 3 to 4 hr after mechanical conversion to schistosomules, implicate argentophilic papillae on cercariae as chemoreceptors for lipid stimulation.     

Schistosoma mansoni: Ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules

Against the background of cercarial fine structure, ultrastructural changes were compared in schistosomules of Schistosoma mansoni 30 min and 1 hr after their production in vivo by skin penetration and in vitro by shear pressure. The same developmental pattern was observed in schistosomules of both derivations. In vitro schistosomules, however, developed more slowly, resembled cercariae more closely, and varied less among organisms than did in vivo schistosomules. The greatest morphological changes were observed in the 1-hr in vivo schistosomules. These were as follows: (1) in tegument, formation of transient microvilli, a hepatalaminate outer membrane and accented surface invaginations, loss of glycocalyx, movement outward of cyton vesicles via bridges, accumulation of multilaminate bodies around bridge openings; (2) in the anterior organ (oral sucker), movement of head gland vesicles via the ducts into tegument followed by collapse of the gland fundus, disappearance of the circumfundal cells and two large support cells, and the appearance in these areas of membranes and parenchymal cells; (3) secretion of the acetabular gland contents, collapse of the glands and replacement by membranes and parenchymal cells; (4) peristaltic activity of the digestive tract as shown by alternate areas of lumen constriction and dilation; (5) loss of bladder and contraction of the small aboral collecting tubules; and (6) conversion of heterochromatic parenchymal cell nuclei to euchromatic. In contrast, the 1-hr in vitro shear schistosomules resembled 30-min in vivo schistosomules, retaining many cercarial features.     

Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion

Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula which migrate to the final location. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells – postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro-induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were somewhat inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati, employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far of the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled us to distinguish PA and CA glands for microdissection. Using 7.5 × 10⁶ μm³ sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least two of three replicates in either gland type (PA = 455, 40 exclusive; CA = 421, six exclusive; 60 proteins differed significantly in their abundance between the glands). Peptidases of five catalytic types accounted for ca. 8% and 6% of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. While PA glands generally had rich enzymatic equipment, CA glands were conspicuously abundant in venom allergen-like proteins.     

Schistosoma mansoni: partial characterization of enzyme(s) secreted from the preacetabular glands of cercariae.

Secretions from the preacetabular glands of cercariae of Schistosoma mansoni were collected over skin surface lipid on a warmed glass surface in a system providing a temperature gradient. Proteolytic activity of these secretions was linear with respect to the numbers of cercariae secreting. The enzyme was relatively storage stable in water or glycine-NaOH buffer. It was highly sensitive to the buffer system, glycine-NaOH buffer providing the highest proteolytic activity against Azocoll and gelatin substrates. In the glycine buffer, the pH optimum for the enzyme was 8.5–8.8; the temperature optimum was 51 C with the Q₁₀ for Azocoll hydrolysis in the range of 30–50 C approximately 2.2. Of the activity 0.4% was lost at 5 C, 1.7% at 35 C, and 100% at 60 C within 10–15 min. Two Sephadex column chromatography fractions showed proteolytic activity against Azocoll. The fraction with greatest activity was not associated with either of the two peaks of high absorbancy at 280 nm, but the lower proteolytic peak coincided with the lower absorbancy peak in the area of peptides below 10,000 MW. Calibration of the Sephadex column with proteins of known molecular weight showed the MW of the main proteolytic fraction of the secretion to be approximately 25,000–27,000.     

日本血吸虫尾蚴人工方法转变的童虫超微结构的观察

本文报道日本血吸虫尾蚴经注射器推压和血清孵育两种人工方法转变的童虫与载体皮肤型童虫的透射及扫描电镜的观察结果。描述了三种童虫在转变后3小时至12小时其糖膜、外质膜、体被内包含体及腺体的超微结构的变化。     

Schistosoma mansoni: localization of calcium-detecting reagents in electron-lucent areas of specific preacetabular gland granules

In an attempt to establish the exact location of calcium within the preacetabular glands of cercariae of Schistosoma mansoni, these larvae were exposed to reagents (potassium oxalate, potassium pyroantimonate, chloranilic acid, and silver nitrate) useful in the detection of calcium, and were subsequently observed with the aid of light and electron microscopes. Cercariae incubated in potassium oxalate and viewed in polarized light showed birefringence only in the preacetabular gland funduses. At the ultrastructural level, the preacetabular glands of potassium oxalate-treated cercariae had no electron-dense precipitate, but instead had translucent, irregularly shaped inclusions, similar to spaces left by volatilized calcium oxalate as described by others. Pyroantimonate treatment, on the other hand, localized the reaction in the electron-lucent areas of the light-spotted granules. The von Kossa silver nitrate procedure destroyed the secretory granules; therefore, an electron-dense precipitate was distributed throughout the gland. However, pretreatment with chloranilic acid before fixation preserved the granules, and subsequent exposure to the von Kossa silver nitrate gave a reaction identical to that obtained with the pyroantimonate alone. When viewed in polarized light, chloranilic acid-incubated cercariae showed birefringence in the fundus and duct areas.     

Schistosoma mansoni: cryopreservation of schistosomules.

Conditions were established for recovery of active schistosomules of Schistosoma mansoni after cryopreservation and storage in liquid nitrogen (?196 C). Schistosomules prepared from cercariae by a shear pressure technique were subjected to a two-step cooling process consisting of a slow cooling rate to an intermediate temperature, followed by rapid quenching of the sample in liquid nitrogen. Overall averages of 39 and 44% of the schistosomules, with a maximum of 88%, were recovered retaining normal activity with cooling rates of 0.4 C/min to ?32 C or 0.8 C/min to ?35 C, respectively. Methanol at 17.5% in Earle's lactalbumin hydrolysate was the freezing medium. As compared with 24 hr storage in liquid nitrogen, no loss in schistosomule motility was observed after 1 month. Following cryopreservation, attenuated schistosomules derived from ⁶⁰Co-irradiated cercariae (50 kR) exhibited structure and activity equivalent to that of unattenuated schistosomules. Infectivity for mice of unattenuated schistosomules derived from ⁶⁰Co-irradiated cercariae (50 Krad) exhibited structure and activity of unfrozen schistosomules ranged from 0.4 to 15.2%.     

Schistosoma mansoni: stimulus and transformation of cercariae into schistosomules

Schistosoma mansoni schistosomules prepared from cercariae by seven in vitro techniques had not all reached the same state of development at the end of the incubation period as scored by seven parameters: water tolerance; Cercarienhüllen Reaktion; presence of the glycocalyx; condition of the surface membrane; nuclear state; granule migration; and cryopreservability. At the end of the specific incubation period for each technique, the level of development was judged with respect to schistosomules which had developed in situ for 1 hr after penetration of the ear skin of mice. In descending order of their correspondence to in vivo schistosomules, those derived in vitro (by the procedures listed) ranked as follows: first, penetration of dried rat skin; second, centrifuging and vortexing, or incubation in serum-supplemented medium; and third, syringe passage, omnimixing, centrifuging, and incubating, or incubating alone. The only treatment common to all techniques was incubation in 37 C culture medium for 2 hr or more. This is suggested as the stimulus for the cercaria-to-schistosomule transformation.     

Penetration of human skin by the cercariae of Schistosoma mansoni: an investigation of the effect of multiple cercarial applications

It has previously been postulated that L-arginine emitted by penetrating Schistosoma mansoni cercariae serves as an intraspecific signal guiding other cercariae to the penetration site. It was suggested that penetrating in groups offers a selective advantage. If this hypothesis is correct and group penetration at one site on the host offers an advantage, it would follow that at such a site, successive groups of cercariae would be able to penetrate skin in either greater numbers or at a faster rate. This prediction was tested by the use of an in vitro model of cercarial penetration based on the Franz cell and using human skin. It was demonstrated that there was no increase in the percentage of cercariae able to penetrate the skin with subsequent exposures. Consequently, it seems unlikely that the release of L-arginine by cercariae during penetration could have evolved as a specific orientation system based on a selective advantage offered by group penetration.     

Schistosoma mansoni: vaccination of mice with cryopreserved irradiated schistosomules.

In our earlier experiments, NIH/Nmri (CV) mice developed protective immunity to a Schistosoma mansoni cercarial challenge when previously exposed percutaneously to highly ⁶⁰Co-irradiated homologous cercariae. Experiments reported here were conducted to assess the immunogenicity of unfrozen and frozen and thawed schistosomules derived from ⁶⁰Co-irradiated cercariae (irradiated schistosomules). Immunization of NIH/Nmri (CV) mice by ⁶⁰Co-irradiated unfrozen schistosomules reduced worm burdens from a subsequent percutaneous challenge with normal cercariae by 41 to 72%. Immunogenicity was not narrowly dependent on irradiation dose rates between 1 and 8 kR/min, or on the total dose of irradiation given the schistosomules between 25 and 50 kR. Comparable protective immunity developed after injection of irradiated schistosomules which had been frozen to ?196 C in liquid nitrogen and thawed. Cryopreservation appears to offer a solution to the problem of storage of attenuated, immunogenic S. mansoni schistosomules.     

Schistosoma mansoni: cercaricidal effect of 2-tetradecenoic acid, a penetration stimulant

Schistosoma mansoni cercariae are stimulated by 2-tetradecenoic acid (TDA) to penetrate agar substrates. TDA simultaneously causes tegumental transformation similar to that seen when cercariae transform to schistosomula, reduces the Cercarienhüllen reaction in immune human serum, and reduces larval tolerance to water. TDA damages cercariae that fail to penetrate or have no opportunity to do so. This damage apparently stems from increased tegumental permeability to water. Preincubation in TDA for 60 min reduces the percutaneous infectivity of cercariae to mice by from 95% at 0.2 ppm to 100% at 0.7 ppm TDA, but does not reduce the infectivity of subcutaneously injected cercariae. The interference with percutaneous infection seems to be entirely due to osmotic damage. TDA does not induce premature secretion of the acetabular glands or block host-recognition chemoreceptors. TDA may be a promising cercaricide for schistosomiasis control. It is highly specific for schistosome cercariae and is effective at low concentrations (0.2 to 0.7 ppm). Both cercariae and TDA tend to collect in the upper few millimeters of standing water. It is unlikely that cercariae can evolve resistance to a chemical that triggers the host penetration mechanisms.     

青海高原二种双腔吸虫尾蚴腺体组织化学的比较观察

本文报道应用组织化学反应方法观察了青海高原中华双腔吸虫尾蚴及暂称为“B”型双腔尾蚴(可能是枝双腔尾蚴)二种单细胞腺体的组化成分及其生理功能。大单细胞腺体12—13对,内含丰富的碳水化合物——蛋白质复合物象粘蛋白之类物质,及酸性粘多糖。小单细胞腺3对,含结合氨基的蛋白质及甲性糖蛋白。二种双腔吸虫在贝类宿主体内子胞蚴及尾蚴组化威分很相似但从尾蚴粘液腺的结构可以区分二虫种。中华双腔吸虫粘球内包绕在尾蚴体外的粘液含丰富的粘蛋白。双腔吸虫幼虫期组化成分与阔盘吸虫相应的幼虫期进行了比较。     

日本血吸虫尾蚴钻穿不同宿主皮肤时新转变童虫的死亡情况

日本血吸虫尾蚴在钻穿7种哺乳动物宿主皮肤而转变为童虫时,均发生了自然死亡,而且童虫的死亡率依宿主种类或同一种类的不同品系而异。它们皮肤内平均童虫死亡率分别为:小鼠3.7—7.2％、大鼠6.3％、金黄仑鼠7.0％、长爪沙鼠5.1％、豚鼠6.9％、兔4.9％、恒河猴12.8％。实验证明日本血吸虫尾蚴亦能钻入不易感的鸟类——鸽的皮肤,虽有1/4童虫立即在其皮肤内发生死亡,但部分童虫可移行至肺,然后在肺中消亡。