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[Individual and population variation in cercariae of bird schistosomes of the Trichobilharzia ocellata species group as revealed with the polymerase chain reaction].

The polymerase chain reaction with arbitrary (RAPD-PCR) or specific primers was used to study the population variation and to identify the species in cercariae of schistosomes of the Trichobilharzia ocellata species group (Trematoda, Schistosomatidae). In total, 28 cercariae were obtained from two spontaneously invaded mollusks Lymnaea stagnalis (LS) and L. ovata (LO), which were collected in different water bodies of Moscow. RAPD-PCR was carried out with two arbitrary primers, OPA9 and OPB11, which each detected different levels of individual and among-group variation and revealed considerable genetic differentiation of cercariae from different host mollusks. To check whether the cercariae of the two samples belong to one species, sequencing was performed with a region corresponding to intergenic transcribed spacer 2 (ITS2), which was earlier proposed for cercaria identification in three European species of bird schistosomes of the genus Trichobilharzia (T. franki, T. regenti, and T. szidati). The ITS2 sequences of two LO cercariae were identical, each consisted of 319 bp, and showed 100% homology to the T. franki ITS2 sequence. The ITS2 sequences of two LS cercariae were identical, each consisted of 323 bp, and showed 99.4% homology to the T. szidati counterpart. The causes of genetic variation in cercariae and prospects of using RAPD markers to study different stages of the life cycle in trematodes are discussed.

Animals↗

Chaetotaxy of cercariae of Opisthioglyphe ranae (Frölich, 1791) (Trematoda: Plagiorchidae).

A detailed observation of the sensory apparatus of Opisthioglyphe ranae (Frölich, 1791) cercariae was carried out. The species was determined on the basis of an experimental study of the life cycle and morphology of sporocysts, cercariae, metacercariae, and adults. In contrast to other species of the family Plagiorchidae, no papillae were present in the positions CId1, CId2, CII0, CII3 and CII4. Papillae with markedly ventral situation on the postacetabular zone were found very rarely. Some of the cercariae possessed ventral papillae variously distributed in the preacetabular zone. A comparative analysis of the chaetotaxy revealed that the cercaria studied by us was very similar to Cercaria 4 of Richard, 1971, with the exception of the number of acetabular papillae and some groups of ventral papillae. Compared to the cercaria of O. ranae studied by Dobrovolskiy (1965). AID papillae and a part of ventral papillae were distributed differently. The chaetotaxy was identical with that of experimentally obtained cercariae of O. ranae from L. stagnalis and of cercariae of the same species obtained from Bulgaria.

Animals↗

Degradation of extracellular matrix by larvae of Schistosoma mansoni. I. Degradation by cercariae as a model for initial parasite invasion of host.

We have examined the ability of cercariae of Schistosoma mansoni to degrade a model extracellular connective tissue matrix produced by rat vascular smooth muscle cells in culture. In this model, connective tissue macromolecules are present in the interactive framework that characterizes their structure in vivo. Cercariae were stimulated to degrade the matrix by skin lipid or linoleic acid. At the maximally stimulating concentration of linoleic acid (25 micrograms/cm2), 68% of the total matrix was degraded, including 57% of the glycoprotein, 79% of the elastin, and 8% of the collagen. Degradation of the matrix and transformation of cercariae to schistosomula began within minutes of exposure to maximally stimulating concentrations of linoleic acid. Degradation continued for 24 hours and was dependent on the number of cercariae. Some degradation occurred without exogenous stimulants but at a slower rate than with skin lipid or linoleic acid. Degradation of matrix was inhibited by alpha 1-proteinase inhibitor and soybean trypsin inhibitor. Ethylenediaminetetraacetic acid inhibited degradation by unstimulated but not linoleic acid-stimulated cercariae. Preacetabular gland secretions collected from cercariae also degraded the matrix with an activity 86% of that of live cercariae. Preacetabular gland proteolytic activity was also inhibited by alpha 1-proteinase inhibitor, soybean trypsin inhibitor, and ethylenediaminetetraacetic acid. The similar characteristics of matrix degradation by both live cercariae and cercarial preacetabular gland secretions support the idea that a proteinase secreted from cercarial preacetabular glands facilitates invasion of skin and connective tissue by these larvae. Degradation of elastin and glycoprotein constituents of extracellular matrix is probably essential for skin penetration.

Animals↗

Schistosoma mansoni: IgG reactivity to soluble adult worm antigen preparation (SWAP) in sera of immunized mice with irradiated attenuated and virulent cercariae.

The aim of this work was to identify the antigens, that elicit a greater or unique immune response in the immunized host against Schistosoma mansoni (Egyptian strain) infection. Moreover the difference in immune response to this antigen between mice immunized with radiation-attenuated cercariae or immunized with virulent cercariae were investigated. Immunobloting technique was used to monitor the fine specificity of host IgG to SDS-PAGE separated SWAP in the sera of different test groups immunized with either radiation-attenuated cercariae or low doses of virulent cercariae compared to non-immunized group. Data from this study showed that groups immunized with virulent and irradiated-attenuated cercariae showed 74.7% and 68.3% reduction in worm burden respectively. Differences of humoral immune response of sera of different tested groups against SWAP proteins were definite. Sera of non-immunized mice precipitated non-immunogenic proteins of 87 and 83 KD. SWAP proteins of 106 and 97 KD were identified only by the sera of vaccinated animals with irradiated attenuated cercariae whether challenged or not. Resistant sera from animals immunized by either irradiated-attenuated cercariae or virulent cercariae recognized low molecular weight antigens that ranged from 34 to 52 KD in addition to 57 KD. This indicated that the 106 and 97KD may be the target antigens for protection by radiation-attenuated larvae, while the marker of resistance induced by attenuated or virulent larvae may be 34-52 and 75 KD antigens.

Animals↗

Studies on lophocercous cercariae from Bithynia siamensis goniomphalus (Prosobranchia: Bithyniidae).

Cercariae from Bithynia siamensis goniomphalus were studied in an area endemic for opisthorchiasis. Snails emitted different types of cercariae when shedding was induced by illumination. In addition to two lophocercous cercariae, a monostome cercaria (probably Notocotylus sp), two different furcocercariae and a xiphidiocercaria were found. Two similar types of lophocercous cercariae were distinguishable and one could be identified as Opisthorchis viverrini. To date the cercaria of O. viverrini has been regarded as the only lophocercous cercaria in the endemic area of opisthorchiasis. Therefore, differences between these cercariae were further characterized by scanning electron microscopy to enable us to avoid misinterpretations of O. viverrini in Bithynia.

Animals↗

Ultrastructure of the daughter sporocyst and developing cercaria of Schistosoma japonicum in experimentally infected snails, Oncomelania hupensis hupensis.

Ultrastructure of daughter sporocysts and cercariae of Schistosoma japonicum were studied 2 and 4 months after infection of Oncomelania hupensis hupensis. The body walls of daughter sporocysts are similar at all infectious stages. They consist of an external syncytial tegument on a basement membrane, and an internal cellular subtegument surrounding a body cavity containing developing cercariae. The cercariae embryos develop 2 months after infection from germinal balls in the brood chamber of the daughter sporocyst. They are at first enveloped by a primitive epithelium rising from the daughter sporocyst. Four months after infection, the cercariae were almost fully developed and the primitive epithelium had degenerated. The body wall of the cercaria consists of a thin tegument covered by a surface coat of fibrous material and connected to the subtegumental cells by cytoplasmic processes. The matrix of the tegument contains numerous dense bodies, vacuoles, and spines. Two types of sensory structures - uniciliated and multiciliated - are found at the anterior tip of the cercaria. There are five pairs of penetration gland cells of two distinct types differentiated by the morphology of secretory granules. Flame cells are found in both daughter sporocysts and in cercariae. The cilia of the flame cells are characterized by the typical 9 and 2 cilium pattern.

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Finding and recognition of the bovine host by the cercariae of Schistosoma spindale.

The cercaria of Schistosoma spindale finds and identifies its bovine host with at least five behavioral phases. (1) Dispersal in and selection of midwater and water surface as the microhabitat are achieved by an intermittent swimming behavior with a weak geonegative but not photopositive orientation. (2) Attachments are stimulated by host-specific higher temperatures of the substrate but not by chemical host signals. (3) Remaining of the attached cercariae on the substrate is stimulated by host-specific higher temperatures of the substrate; chemical host signals have no effect. (4) The creeping of the cercariae is directed to the higher temperature in thermal gradients as weak as 0.07 degrees C/mm. Chemical gradients had no effect on the creeping direction. This behavior may enable the cercariae to migrate along hairs to the host's skin surface. (5) Penetrations are stimulated by the free fatty acid fraction of bovine skin-surface lipids. The characteristics of the stimulating fatty acids are the same as those identified for other schistosome species. Higher temperatures of the substrate alone do not stimulate penetrations. S. spindale cercariae do not use as many chemical host cues as stimuli for the identification of their host as do S. mansoni cercariae. S. spindale seems to be adapted to hairy hosts that are infected in shallow, muddy waters. The low host specificity of the cercarial host-finding behavior is compensated by an intimate parasite-snail intermediate host relationship, resulting in a high cercarial production of up to greater than 7,000 cercariae per snail per day.

Animals↗

The chetotaxy of the cercaria of Opisthioglyphe locellus Kossack 1910 (Trematoda, Plagiorchiidae).

The distribution of the tegumentary papillae (chetotaxy) was studied for the cercaria of Opisthioglyphe locellus. The cercariae, which emerge from the freshwater snail Planorbarius corneus L., were treated according to the method of Ginecinskaja and Dobrovol'skij (1963). The papillae were demonstrated after the nomenclature of Richard (1971). The results were identical for cercariae from naturally infected Planorbarius corneus from a pond near Ulm (Germany) and experimentally infected, laboratory-reared snails of the same species. The papillae were distributed symmetrically with few individual variations. The association of the trematode with the subfamily Opisthioglyphinae is corroborated by the chetotaxy of the cercaria. This method also allows the distinction of the cercaria from the most related species. The fact that the excretory system of the cercaria of Opisthioglyphe locellus shows common characteristics with other cercariae of the Opisthioglyphinae (Bock 1982), just like the chetotaxy, emphasizes the taxonomic importance of both criteria.

Animals↗

Experimental infection of Rana pipiens tadpoles with Echinostoma trivolvis cercariae.

Studies were done on laboratory-raised Rana pipiens tadpoles experimentally infected with Echinostoma trivolvis cercariae. Tadpoles exposed individually to 250 cercariae died within 24 h. They were edematous at death and their kidneys were heavily infected with metacercarial cysts. Of 20 tadpoles exposed to 100 cercariae each, 9 survived the infection, and their growth was compared for 4 weeks postinfection (p.i.) with that of 20 control tadpoles that had not been exposed to cercariae. There was a significant weekly decline in the total length and body weight of the infected versus control tadpoles. Surviving tadpoles retained their metacercarial infections in the kidneys following metamorphosis to frogs. Following exposure of tadpoles to cercariae, cercarial bodies were first seen in the kidneys by 0.5 h p.i. Metacercariae that were molding their inner and outer cyst walls were first seen at 2.3 h, and by 8.5 h the inner and outer cyst walls were clearly defined. Domestic chicks exposed to cysts aged 2.5 and 4.0 h did not become infected, whereas ovigerous adults of E. trivolvis were recovered from chicks fed 12-h-old cysts. Cercariae aged 6 to 8 h were more infective to tadpoles than were either 1- or 20-h-old cercariae. The E. trivolvis-R pipiens tadpole model is suitable for the study of host-parasite relationships of echinostome larvae in a cold-blooded vertebrate host.

Animals↗

Effect of ultraviolet radiation on survival, infectivity and maturation of Schistosoma mansoni cercariae.

S. mansoni cercariae exposed to ultraviolet radiation for 1, 3, 5, 10 and 20 s as well as non-irradiated cercariae remained actively motile 30 min post-irradiation. Thereafter the activity decreased with increasing dose level of radiation and age of cercariae. There was no significant difference between the rates of attachment of the batches of cercariae. The recovery rates (0-49% of cercariae to which mice were exposed) of adult worms were, however, significantly different from the number of cercariae calculated to have attached to the mice (93.5-100% of cercariae to which mice were exposed). Maturation and penetration rates were dependent on radiation exposure levels. Numbers of eggs deposited in the liver of mice as well as hatchability rate of eggs varied significantly with the levels of exposure to radiation.

Animals↗

Histochemical glycogen and neutral lipid in Echinostoma trivolvis cercariae and effects of exogenous glucose on cercarial longevity.

Histochemical glycogen and neutral lipid studies were conducted on Echinostoma trivolvis cercariae maintained in artificial spring water (ASW) at 24-25 degrees C for up to 24 h after emergence from host snails. Treatment of whole cercariae by the periodic acid Schiff (PAS) reagent with or without 1% malt diastase showed that cercariae depleted glycogen mainly from the tail by 6 to 24 h postemergence. The posterior tip of the tail remained PAS positive and diastase fast suggesting the presence of mucopolysaccharides there. Fresh cercariae or those stained up to 24 h postemergence with Oil Red O showed the presence of neutral lipid droplets in the excretory system. There was no discernible difference in the size, abundance, or distribution of these droplets in fresh or aged cercariae. Cercariae maintained in ASW plus 1% glucose for 12 or 23 h showed no evidence of resynthesizing glycogen. Nevertheless, cercariae survived longer in 1% glucose than in either 0.0, 0.1 or 0.5% glucose; but only at 23 h were any differences statistically greater (one way ANOVA, P < 0.05).

Animals↗

Histopathology of experimental Schistosoma incognitum infection in mice following exposure to normal and irradiated cercariae.

The tissue reactions in mice, experimentally infected with normal and irradiated cercariae of S. incognitum were studied. The lesions observed in the skin, liver, lungs and the intestine of mice infected with normal cercariae are briefly described, and compared with those observed with cercariae irradiated at 3000 r of gamma rays. In general, the reactions in mice exposed to normal cercariae were more intense than in those infected with irradiated cercariae. The severity of the reactions appeared largely due to the deposition of eggs in the tissues of the mice infected with normal cercariae. The experimental evidence suggested that most of the flukes from the irradiated cercariae are destroyed in the liver by tissue reaction.

Animals↗

Recognition and invasion of human skin by Schistosoma mansoni cercariae: the key-role of L-arginine.

The attachment of Schistosoma mansoni cercariae to mammalian skin is specifically stimulated by L-arginine. As L-arginine is an unsuitable signal for a specific identification of mammalian skin we examined the following 5 hypotheses to explain the advantage of the cercarial sensitivity to L-arginine. (1) A Schistosoma infection lowered the arginine concentration in the serum of mice, and this could enable the cercariae to avoid attachments to already infected mice. However, the infection did not reduce the arginine concentration in the skin and the cercarial attachment responses to it. (2) Creeping cercariae showed chemotactic orientation specifically along increasing L-arginine gradients. L-arginine could act as a pheromone which could guide cercariae towards common penetration sites. However, the cercarial acetabular gland contents were not attractive and they did not (in contrast to previous reports) contain much arginine. (3) Schistosomula (transformed cercariae) could use L-arginine to produce nitric oxide (NO) for blood vessel dilation during their migration in the host. However, in vitro the transformed cercariae did not convert L-arginine into citrulline and NO. (4) Schistosomula could bind L-arginine from the surrounding tissues and so escape the cellular immune attack (which needs L-arginine as the precursor of NO). However, transformed cercariae bound no more L-arginine than L-serine and L-lysine. (5) Schistosomula, migrating parallel to the surface in the mammalian epidermis, are dependent on information on their position between the inner and the surface layers of the skin. In the mouse skin, they adjusted their body axis with the ventral side toward the deeper (arginine-residue rich) epidermis layers. When migrating in agar, they showed chemo-orientation toward serum, and D-glucose and L-arginine were the stimulating compounds therein. The burrowing schistosomula adjusted their body axis (as in the epidermis) with the ventral side toward the higher concentration of L-arginine and not of glucose. We argue that the sensitivity for L-arginine has its primary function in orientation within mammalian skin and in location of blood vessels.

Animals↗

Age-dependent survival and infectivity of Schistosoma mansoni cercariae.

The age dependency of the mortality, spontaneous de-tailing and infectivity of cercariae of Schistosoma mansoni has been determined at 25 degrees C. Infectivity was assessed with respect to stratum corneum-like differentiated human keratinocyte cultures (validated by comparison with fresh human skin samples) and displayed a complex age-dependent pattern. From 1 to 9 h post-emergence cercariae showed a plateau of maximal infectivity (around 90% attachment). Thereafter, infectivity declined. Immediately after release, infectivity at around 60% was significantly lower than the plateau values and this could be an adaptation for spatial dispersal of cercariae. Age-dependent patterns of cercarial mortality and spontaneous de-tailing closely mirrored the infectivity pattern except in relation to the low initial infectivity value. These findings suggest that, at a population level, the age-dependent decline in cercarial infectivity towards human skin is essentially driven by cercarial mortality. The recently described phenomenon of delayed tail loss (DTL) in S. mansoni cercariae infecting human skin is confirmed in the present study. For cercariae aged up to 13.5 h post-emergence, 90% or more of invading cercariae took their tails with them into the keratinocyte culture. The infection dynamics described in this study suggest that diurnally shed S. mansoni cercariae, with peak emergence around mid-day, will have near maximal infectivity towards humans in contact with water through all remaining daylight hours in the tropics.

Age Factors↗

Schistosoma mansoni: the sex ratios of worms in animals infected with cercariae from three species of Biomphalaria.

The ratios of male to female worms of Schistosoma mansoni were determined in mice infected with cercariae from LE, SJ and AL strains shed by mollusc hosts of the parasite in Brazil. The sex ratios of worms in the animals were similar with cercariae from Biomphalaria glabrata and B. tenagophila varying from 1.1:1 to 1.6:1 with LE and AL strains and 1:1.1 with SJ. In the animals infected with cercariae from B. straminea the ratio of male to female worms was similar to those obtained using cercariae shed from the other two species of molluscs, 1.5:1 with LE strain. Inoculations by AL and SJ cercariae resulted in sex ratios of 3.1:1 and 6:1 respectively. The normal sex ratios of worms established in Brazil in animals inoculated with cercariae from B. glabrata and B. tenagophila is from 1:1 to 2:1. The higher number of male worms that developed from cercariae of the AL and SJ strains obtained from B. straminea indicate a lower compatibility of the snail concerning these strains of S. mansoni.

Animals↗

[The size polymorphism of the cercariae of a Venezuelan strain of Schistosoma mansoni].

By mean of the silver impregnation of a lot of 47 cercariae belonging to C5 venezuelan Schistosoma mansoni strain, a polymorphism of size was evidenced. The morph was conformed by the greatest cercariae (between 152.9 microns to 218.4 microns). This group was ever present in the population of cercariae in less percentage (29.8%). The length of the normal cercariae was comprised between 119 microns to 152 microns. This group was the dominant in the population: 70.2%. The comparison between the length of both lots of cercariae resulted with significative statistical differences (t = 5.5; P < 0.001). For the total studied cercariae the average of the length was X = 143.9 microns and for the wide X = 68.6 microns. It was found a positive significative correlation between the wide and the length of the cercariae r = 0.83 (P < 0.001).

Animals↗

Infection by Paragonimus miyazakii cercariae of their crab hosts, Geothelphusa dehaani, by percutaneous penetration.

Identification of the transmission routes of the trematode parasite Paragonimus miyazakii into different intermediate hosts would help to explain the natural distribution of the parasite. The behavior of P. miyazakii cercariae released from snails into water and in the presence of a living host or a whole crab leg was observed by stereoscopic or light microscopy at various times after exposure started. On encountering a crab leg or cheliped, the cercariae became entangled with the host via mucoid strands arising from the cercariae. Within 3 hr, most cercariae were attached to the host; cuticular penetration took between 5 and 6 hr, after which cercariae were found in the cavity of the leg. Crabs examined 102-149 days after exposure to the cercariae contained fully developed metacercariae. The metacercariae were fed to 2 rats, and the rats were killed 83 or 111 days later. Some of the metacercariae had reached maturity in the rats. That the cercariae were not ingested by the crabs but penetrated the crabs percutaneously (through hard as well as soft tissue) means that transmission can occur even in areas in which crabs and the host snails do not coexist, as they would if the usual route were oral (when the crabs ate infected snails).

Animals↗

Exposure of Dugesia tigrina (Turbellaria) to cercariae of Echinostoma trivolvis and Echinostoma caproni (Trematoda).

Laboratory-reared planarians, Dugesia tigrina, were exposed to cercariae of Echinostoma trivolvis or Echinostoma caproni. Of 100 D. tigrina exposed to 2,750 cercariae of E. trivolvis, 29 were infected with a total of 85 encysted metacercariae 24 hr postinfection (PI). None of 40 D. tigrina exposed to 1,100 cercariae of E. caproni was infected with metacercariae 24 hr PI. Cyst structure of E. trivolvis from the parenchyma of D. tigrina varied from normal to abnormal. Metacercariae removed from planarians could be excysted in an alkaline trypsin-bile salts medium. Cercariae of E. trivolvis were not attracted to dialysate material from D. tigrina. Cercariae of E. trivolvis that penetrated, but did not encyst in planarians, voided the contents of their paraesophageal glands. Cercariae of E. caproni lack paraesophageal glands. Secretions of those glands probably are involved in tissue penetration of D. tigrina by E. trivolvis cercariae.

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