Schistosoma mansoni: stage-specific expression of muscle-specific genes

It was previously shown that an antigen preparation termed 9B obtained from Schistosoma mansoni cercarial extracts partially (34%) protects mice from challenge infection with cercariae (R. Tarrab-Hazdai et al., J. Immunol. 135, 2772, 1985). To characterize some of the proteins which comprise this preparation, rabbit antibodies to the 9B antigen preparation were used to screen cDNA libraries of cercariae and adult worms. We isolated and sequenced cDNA clones encoding three proteins: calcium-binding protein, paramyosin, and myosin. The calcium-binding protein was previously shown to be expressed in cercariae but not in sporocysts or adult worms (D. Ram et al., Mol. Biochem. Parasitol. 34, 167, 1989). Northern blots showed the presence of paramyosin and myosin mRNAs in sporocysts and adult worms but not in cercariae. Antibodies to paramyosin detected the protein in sporocysts and adult worms as well as in cercariae. These findings explain, in part, the protective activity of the 9B antigen preparation against challenge infection.     

The life cycle of Australapatemon magnacetabulum (Digenea: Strigeidae) from Northwestern Argentina

The life cycle of Australapatemon magnacetabulum Dubois, 1988 was resolved experimentally. Planorbid snails Biomphalaria tenagophila (d'Orbigny, 1835) collected in a small pond at the confluence of the San Lorenzo and Arias Rivers, near Salta City, Province of Salta, Argentina, were found to be shedding furcocercous cercariae possessing 4 pairs of penetration glands, 1 pair of unpigmented eyespots, 6 pairs of flame cells in the body, and 1 pair in the tail stem. Metacercariae were found encysted in naturally, and experimentally, exposed leeches Helobdella adiastola Ringuelet, 1972, Helobdella triserialis (Blanchard, 1849), Haementeria eichhorniae Ringuelet 1978, and Haementeria sp., and within their sporocysts in naturally infected planorbid intermediate hosts. Sexually mature adults were recovered from domestic chicks and a duck 8-28 days postexposure by metacercariae from leeches. The identification of the species was based upon the characteristic large ventral sucker and a genital cone, crossed by a hermaphroditic duct with internal folds, occupying approximately a 1/4 to 1/5 of the hindbody.     

Plagioporus sinitsini (Digenea : Opecoelidae): a one-host life cycle

Adult Plagioporus sinitsini occur within daughter sporocysts voided with the feces of prosobranch snails Elimia symmetrica in Basin Creek, North Carolina. These worms produced eggs containing active miracidia while still in the snail. Adults in snails and adults in rosyside dace, Clinostomus funduloides, collected from the same stream were indistinguishable morphometrically. Adults in snails develop from cotylocercous cercariae sequestered in daughter sporocysts that pass through the metacercaria stage. These observations, and previous study in Michigan, suggest that the life cycle of P. sinitsini has 3 potential pathways, i.e., a 3-host life cycle involving molluscan, arthropod, and piscine hosts, a 2-host life cycle involving only molluscan and piscine hosts, and a 1-host life cycle involving only the snail host. The truncated life cycles do not appear to be the result of paedomorphosis.     

Bge snail cell-line antigens: ineffectiveness as antischistosomal vaccine in mice.

Mice and rabbits were immunized with antigens derived from Bge cells, Biomphalaria glabrata hemolymph, or Schistosoma mansoni. Antisera from mice given molluscan antigens did not form immunoprecipitates with soluble antigen from adult worms, but their binding to surfaces of sporocysts, cercariae, and schistosomules suggests the presence of cross-reacting determinants. In vitro, cell-mediated immune responses to Bge antigens were not demonstrable in infected nor in immunized mice. Mice immunized with Bge cell-line antigens and challenged with S. mansoni cercariae showed no reduction in worm burden when compared with control mice.     

Schistosoma mansoni: topochemical features of cercariae, schistosomula, and adults

The teguments of developing and mature cercariae, recently transformed, and 1-wk-old schistosomula and adult worms were examined for the ultrastructural location of macromolecular carbohydrates and polyelectrolytes. The surface of mature cercariae within sporocysts and cercariae released from the snail is covered by a filamentous coat which reacts with cytochemical reagents for the demonstration of vicinal glycols, but neither the coat nor the surface of the tegument plasmalemma binds cationic colloidal iron at low pH.Upon penetrating mammalian skin, the cercaria sheds its surface coat; the tegument surface of newly transformed schistosomula, older schistosomula and adult worms stains en bloc with acidic colloidal iron, as does the tegument plasmalemma of mature cercariae if the overlying filamentous coat is first removed by physicochemical means. The cercarial coat thus serves to mask anionic groups at the surface of the tegument plasmalemma which become functionally exposed after penetration of the mammalian host. The distribution of colloidal iron binding sites coincides with those for the carbohydrate-complexing phytohemmagglutnin, concanavalin A, which suggests that these membrane-fixed anions are acid mucopolysaccharides, glycoproteins or glycolipids. Carbohydrate-containing material was also localized within membrane-bound vesicles of the tegument matrix and perikarya of developing cercariae and postcercarial schistosomes, suggesting that surface mucosubstances contributing to the tegument glycocalyx of these worms are elaborated, at least in part, by the tegument itself.     

Schistosoma mansoni: relationship between cercarial production levels and snail host susceptibility

Two populations of Biomphalaria glabrata snails differing slightly in their susceptibility to Schistosoma mansoni infection showed dramatic differences in cercarial output per snail. Exposed to five or more miracidia, snails from a group with a 90-100% susceptibility rate (Group A) produced nearly twice the number of cercariae as those from a group with a 70-80% susceptibility rate (Group B). Exposure of individual snails to known numbers of miracidia resulted in higher numbers of primary (mother) sporocysts in Group A snails than in Group B snails. However, monomiracidial exposure of snails from both groups resulted in equivalent numbers of cercariae produced per positive snail, indicating that, once established, all primary sporocysts possess a similar reproductive potential. Morphometric analysis of serially sectioned 9-day-old primary sporocysts supported this conclusion; the size of the primary sporocysts and the size and numbers of secondary (daughter) sporocysts within each primary sporocyst were comparable in snails from both groups. The data indicate cercarial production in this system is regulated prior to, and/or during, early development of the primary sporocyst.     

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.     

Observations on the life cycle of Carneophallus brevicaeca (Africa et Garcia 1935) comb. n. (Digenea: Microphallidae).

Metacercariae of Carneophallus brevicaeca (Africa et Garcia 1935) comb. n. from the muscles of naturally infected shrimps, Macrobrachium sp., were force-fed to laboratory-reared 4- to 5-day-old albino rats and day-old chicks. Ovigerous worms were recovered from the small intestines of the rats after 1 to 5 days, and gradually disappeared in 10 days. Only 1 of 10 chicks was positive 24 hr after exposure. These findings contribute to out knowledge of the mode of transmission, prevention, and control of this medically important trematode in some regions of the Philippines where raw Macrobrachium sp. is consumed.     

Schistosoma mansoni dermaseptin-like peptide: structural and functional characterization

Analysis of the Schistosoma mansoni peptidome for immunomodulatory molecules by solvent extraction and reverse-phase HPLC revealed a 27-amino-acid residue peptide from an extract of cercariae. Using matrix-assisted, laser desorption-ionization, time-of-flight mass spectrometry, the peptide yielded a protonated molecular ion [M + H]+ of m/z 2789. The unequivocal sequence was deduced by automated Edman degradation as: DLWNSIKDMAAAAGRAALNAVTGMVNQ. The peptide exhibited an 80.76% identity with dermaseptin 3.1 from the leaf frog Agalychnis annae, and was therefore named Schistosoma mansoni dermaseptin-like peptide (SmDLP). Immunocytochemical staining using a primary antidermaseptin B2 antibody located SmDLP in acetabular glands of cercariae, in and around schistosomula, and in adult worms and their eggs. Dot-blotting confirmed its presence in extracts (cercariae and worms) and excretion/secretion (E/S) products (transforming cercariae and eggs). This was corroborated by use of a MALDI-ToF spectra database of E/S products from cercariae. Functional characterization of the peptide indicated that SmDLP had typical amphipathic antimicrobial peptide properties, i.e., the ability to lyse human erythrocytes causing a decrease in the levels of nitric oxide produced by monocytic cells. This last function strongly suggests that SmDLP plays a vital role in the parasite's immunoevasion strategy. The possibility that schistosomes acquired this gene from amphibians has been discussed by constructing a phylogenetic tree.     

Host specificity of Pisidium coreanum (Bivalvia: Sphaeriidae) to larval infection with a human intestinal fluke Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea

The fingernail clam, Pisidium coreanum, has been traditionally consumed raw as a so-called drug therapy by patients with bone fractures in Korea. The present study was designed to determine the possible occurrence and, if present, the prevalence of Echinostoma cinetorchis in P. coreanum collected at a local site, and to determine the susceptibility of the clams in the laboratory to infection with miracidia and cercariae of E. cinetorchis. No cercariae or metacercariae of E. cinetorchis were observed in field-collected P. coreanum clams. In susceptibility experiments with laboratory-reared clams, individuals exposed to miracidia of E. cinetorchis did not release cercariae by 20 days after exposure; necropsy of exposed clams failed to show development of any sporocysts or rediae. To confirm the possibility of these clams serving as an experimental second intermediate host of E. cinetorchis, 20 of them were exposed to E. cinetorchis cercariae from experimentally infected Segmentina hemisphaerula that had been previously exposed to miracidia of E. cinetorchis; all exposed clams became infected. Metacercariae from clams at 14 days postinfection were fed to rats, and adult worms were recovered from the ileocecal regions. This is the first report of P. coreanum serving as second intermediate host of E. cinetorchis.     

Schistosoma mansoni: Long-term maintenance of clones by microsurgical transplantation of sporocysts

Schistosoma mansoni sporocysts originally derived from monomiracidially infected Biomphalaria glabrata snails were serially transplanted into the cephalopedal sinus of anesthetized snails by the microsurgical implantation of fragments of parasitized hepato-pancreas and ovotestis. Three to six passages each of five male and five female clones were maintained for as long as 2.0 years. Of the recipient snails which survived surgery, 87% released cercariae, usually beginning 5–7 weeks after surgery. The percentage of snails which released cercariae increased with successive passages. The mean survival time of surgically infected snails after cercarial emergence began was 9.2 ± 0.5 weeks, nearly the same as that of miracidially infected snails. Longevities of snails infected with male or female clones were similar. Recipient snail size and age did not influence cloning success. Beginning 5 weeks from the onset of cercarial emergence large numbers of cercariae (a mean of 3900/snail from male clones and 1300/snail from females) were obtained during each shedding period. These results clearly demonstrate that the microsurgical transplantation of sporocysts is a practical means of maintaining and expanding populations of genetically homogeneous schistosomes (clones).     

In vitro cultivation of Schistosoma japonicum-parasites and cells

Schistosomiasis is a serious parasitic zoonosis caused by blood-dwelling flukes of the genus Schistosoma. Understanding functions of genes and proteins of this parasite is important for uncovering this pathogen's complex biology, which will provide valuable information to design new strategies for schistosomiasis control. Effective applications of molecular tools reported to investigate schistosome gene function, such as inhibitor studies and transgenesis, rely on the developments of in vitro cultivation system of this parasite and cells. Besides the in vitro culture studies dealing with Schistosoma mansoni, there are also numerous excellent studies about the in vitro cultivation of Schistosoma japonicum, which were performed by Chinese researchers and published in Chinese journals. Nearly every stage of the life-cycle of S. japonicum, including miracidia, mother sporocysts, cercariae, schistosomula, and egg-laying adult worms, was employed for developing in vitro cultivation methods, being accompanied by the introduction of several media and supplements that helped to improve culture conditions. It was not only possible to generate mother sporocysts from miracidia in vitro, but also to obtain adult worms from cercariae through in vitro cultivation. The main obstacles to complete the life cycle of S. japonicum in the lab are the transition from mother sporocysts to cercariae, and the production of fertilized and completely developed eggs by adult worms generated in vitro. With regard to cells from S. japonicum, besides established isolation protocols and morphological observations, media optimizations were conducted by using different chemical reagents, biological supplements and physical treatment. Among these, mutagens like N-methyl-N-nitro-N-nitrosoguanidine and the addition of extracellular matrix were found to be able to induce mitogenic activities. Although enzyme activities or the level of silver-stained nucleolar region associated protein in cultured cells indicated still suboptimal conditions, the achievements made point to the possibility of reaching the aim of establishing cell lines for S. japonicum. Both the improvements of the in vitro culture of larval and adult worms of S. japonicum as well as the access of cells of this parasite provide excellent advances for research on this important parasite in the future.     

Up to what point are cercariogenesis and sporocystogenesis reversible in schistosomes?

A study of larval development of Schistosoma haematobium in the snail Planorbarius metidjensis infected by transplanted sporocysts showed that some embryos in daughter sporocysts have characters intermediate between cercariae and sporocysts. These embryos are interpreted to be nearly mature cercariae that have partially reverted to be secondary sporocysts. If this explanation is correct, it would mean that the embryos which develop within trematode daughter sporocysts can differentiate either into cercariae or into a new generation of sporocysts, but originate from a single type of germinal cell.     

The life cycle of Gyliauchen volubilis Nagaty, 1956 (Digenea: Gyliauchenidae) from the Red Sea

Although nothing is known about gyliauchenid life cycles, molecular phylogenetic studies have placed the Gyliauchenidae Fukui, 1929 close to the Lepocreadiidae Odhner, 1905. The gyliauchenid Gyliauchen volubilis Nagaty, 1956 was found in the intestine of its type-host, Siganus rivulatus, a siganid fish permanently resident in a lagoon within the mangrove swamps on the Egyptian coast of the Gulf of Aqaba. Larval forms of this trematode (mother sporocysts, rediae and cercariae) were found in the gonads and digestive gland of Clypeomorus clypeomorus (Gastropoda: Cerithiidae), a common snail in the same lagoon. So, this life cycle of G. volubilis was elucidated under natural conditions: eggs are directly ingested by the snail; mother sporocysts and rediae reach their maturity 3-6 and 11-13 weeks post-infection; rediae contain 23-29 developing cercariae; fully developed cercariae are gymnocephalus, without penetration glands, emerge from the snail during the night 16-18 weeks post-infection and rapidly encyst on aquatic vegetation (no second intermediate host); encysted metacercariae are not progenetic; 4-day-old metacercariae encysted on filamentous algae fed to S. rivulatus developed into fully mature worms 6-8 weeks post-infection. The cycle was completed in about 26 weeks and followed one of the three known patterns of lepocreadiid life cycles, and except for the gymnocephalus cercariae, the other larval stages are very similar to those of lepocreadiids. Generally, the life cycle of G. volubilis implicitly supports the phylogenetic relationship of Gyliauchenidae and Lepocreadiidae inferred from molecular phylogenetic studies.     

Susceptibility of 1- and 3-Day-Old Chicks to Infection with the Coccidium, Eimeria acervulina

SYNOPSIS. One-day-old chicks were less susceptible to experimental infection with E. acervidina than were 3-day-old chicks. Chicks fed intact oocysts when 3 days old produced 5.3, 6.7, and 42.7 times as many oocysts and had more extensive lesions than did those fed a similar number of oocysts when 1 day old. When oocyst suspensions that contained both liberated sporocysts and intact oocysts were administered, chicks infected when 3 days old produced only 1.8, 1.3, and 2.6 times as many oocysts as did those infected when 1 day old.
Examination of gizzard and intestinal contents of chicks killed 2–1½ hr after receiving massive numbers of intact oocysts showed that only a few sporocysts were liberated from oocysts in the gizzard of 1-day-old chicks, whereas more were liberated in the gizzard of 3-day-old chicks. Very few sporozoites were found in the duodenum of the 1-day-old chicks. but there was a linear increase in the percentages in samples from lower levels of the small intestine. In 3-day-old chicks, excystation in the duodenum was high and, instead of increasing, remained at about the same level in the jejunum.
The far smaller number of liberated sporocysts in the gizzards of 1-day-old chicks is attributed to less musculature and an incompletely developed grinding surface The delayed excystation of sporozoites in the intestine of 1-day-old chicks is thought to be due to suboptimal concentrations of trypsin and/or other pancreatic enzymes effecting excystation.
The lighter infections observed in 1-day-old chicks, as compared to those in chicks 3 days old, are attributed to (a) a smaller number of liberated sporocysts leaving the gizzard, (b) delayed excystation in the intestine, and (c) less opportunity for sporozoites to penetrate epithelial cells.     

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.     

Observations on the host-parasite relations between Echinostoma revolutum and lymnaeid snails.

Echinostoma revolutum from Taiwan was studied in lymneid snails at 29 +/- 0.5 degrees C. Given 3-5 miracidia, 95% of Lymnaea ollula and 40% of Lymnaea swinhoei became positive; the prepatent periods were 18 and 25 days, respectively. The following are based on the observations in Lymnaea ollula: The time required for miracidial penetration was about one hour. The sporocysts developed only in the ventricle of the snail but mother rediae developed in the heart and other organs. Mature daughter rediae were not found in the heart cavity. The sporocysts reached the ventricle within 3 days. Mother rediae were released after 6 days and daughter rediae after 8 days. Given 5 miracidia, 1-3 sporocysts reached the heart and 2-20 mother rediae were found per snail. The number of mature daughter rediae was usually 100-200 although more than a thousand may develop in a snail. The sporocysts and mother rediae attained maximal size 9 days postinfection and started degeneration 13 days postinfection. Daughter rediae were largest at the beginning of cercarial emergence and decreased in size thereafter. Simultaneous production of daughter rediae and cercariae by the mother redia was seen only once in this snail mature cercariae were obtained in 10 days postinfection. The cercariae emerged from a small area of mantle collar near the posterior corner of shell aperture. They were negatively phototactic and positively geotactic. An estimation showed that each snail shed about 350 cercariae a day. The cercariae reached the pericardial cavity of snail in one hour via the renal orifice and metacercariae were seen 4-5 hours after exposure. The infectivity of cercariae at various times after shedding, as expressed by cyst recovery rates, were: 51.6%, O-hr old; 76.1%, 2-hr; 68% 4-hr; 32%, 6-hr; 3%, 8 and 10-hr. Cyst recovery rates were not different within the dosage of 50-500 cercariae per snail. Most metacercariae recovered 1-2 days after cercarial exposure were viable; only 5 among 6,533 cysts were dead.     

Selection of genotypes of Schistosoma mansoni and their maintenance by sporocyst transplantation

Schistosoma mansoni was isolated by hatching eggs obtained from a naturally infected Rat in Grand Etang, Guadeloupe; fifty Biomphalaria glabrata were exposed to five miracidia each. The resulting cercariae were used to infect laboratory mice which were later sacrificed to provide worms for enzyme analyses and eggs for further infections. Seven enzymes in extracts of individual worms were examined by isoelectric focusing in polyacrylamide gels: AcP, G6PDH, PGM, GPI and HK showed no variation, whereas MDH and LDH proved to be polymorphic. Two MDH loci were recognised, MDH-2 was invariant whereas two alleles were assumed at the MDH-1 locus. It was not possible to make a genetic interpretation of the complex banding pattern of LDH, although 4 types (LDH-A, -B, -C, -D) were observed. Of the snail infections, one batch of snails was exposed to 5 miracidia per snail in the normal way whereas other snails were each exposed to a single miracidium. The latter were sacrificed to provide sporocysts to transplant into further groups of recipient snails. Cercariae from the recipient snails were used to infect mice and the adult worms were analysed and compared with the normally passaged material. In this way, three lines, defined by the possession of particular MDH and LDH types, were selected from the originally polymorphic population; two were identical. The combination of single miracidium infections and enzyme typing has illustrated the possibility of selecting parasite lines of known genotype; transplantation of sporocysts from snail to snail has demonstrated that such lines can be maintained exclusively in the intermediate host.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of inducible nitric oxide synthase in Schistosoma japonicum and S. mansoni

Schistosoma japonicum and S. mansoni were tested for reactivity with an anti-inducible nitric oxide (iNOS) antibody and the distribution of iNOS was studied by immunofluorescent tests in different stages of the parasites. Reactivity was associated with the tegument in both larval schistosomes (sporocysts and cercariae) and eggs. With adult worms, the majority of the immunofluorescence was predominantly subtegumental in S. japonicum and parenchymal in S. mansoni. Fluorescence was also observed in host tissues (snails and mouse liver). In Western blots, the enzyme of S. japonicum had an apparent molecular weight of about 210 kDa. The possible role of worm and host iNOS in the parasite-host interrelation remains to be clarified.