Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes.

As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum.     

Schistosoma haematobium: the pathology of experimental infection

The pathologic changes in experimental animals infected with Schistosoma haematobium are reviewed and compared to the pathology in infected humans. The clinically important lesions in persons infected with S. haematobium are generally confined to the urogenital system. In experimental animals, functionally important lesions of the urogenital system are the exception but do occur in a significant proportion of infected primates. The acute lesions of the urinary tract in primates are similar to those in infected persons. Chronic lesions characterized by the extensive submucosal accumulation of calcified eggs are common in infected humans but uncommon in S. haematobium-infected animals.     

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.     

Isoenzyme analysis of Schistosoma haematobium,S. intercalatum and their hybrids and occurrences of natural hybridization in Cameroon

Isoelectric focusing of glucose-6-phosphate dehydrogenase (G6PD) produced clearly identifiable profiles for S. haematobium and S. intercalatum and their hybrids. To provide a more detailed analysis of the interactions of S. haematobium and S. intercalatum in South West Cameroon over the last 12 years, G6PD analyses were carried out on individual schistosomes collected in Kumba in 1990, Loum in 1990, 1999 and 2000 and Barombi Mbo and Barombi Kotto in 1999. Studies were also carried out on the two parental species S. haematobium Barombi Mbo, S. intercalatum Edea and subsequent generations of hybrids resulting from laboratory crosses of the two parental species. The isoenzyme analysis demonstrated that the 1990 isolate from Kumba, was a recombinant of S. intercalatum x S. haematobium, and that 30% of individual schistosomes collected in 1990 in Loum were also recombinants. The remainder gave data indicative of S. haematobium. In 1999, 12.5% of individuals from Loum showed recombination and 10% in 2000. Results from the most recent parasitological survey in October 2000 showed the persistence of the recombinant population in addition to that of S. haematobium. There was also evidence of recombination having taken place in Barombi Kotto but not Barombi Mbo. This study demonstrates how the situation has changed over the last 12 years, and emphasizes the importance of assessing morphological, biological and molecular data together to gain a true picture of the rapidly evolving situation.     

Schistosoma haematobium and S. japonicum: analysis of the ribosomal RNA genes and determination of the "gap" boundaries and sequences

We have determined the intragenic organization of the rRNA genes of Schistosoma haematobium and S. japonicum and found them to be similar to that of S. mansoni and other eukaryotes. An entire ribosomal repeat approximately 10 kbp in size from each species was isolated as a SalI fragment from a genomic library constructed in bacteriophage lambda. The segments encoding both the small and large rRNAs have been identified using three cloned EcoRI fragments of S. mansoni as probes. There were three EcoRI fragments (4.2, 3.0, 1.6 kbp) from S. haematobium and four EcoRI fragments (4.6, 2.3, 1.7, 1.0 kbp) from S. japonicum. As in a wide variety of organisms within the protostome phyla, the 28S rRNA in schistosomes contains a "gap" which separates it into two fragments. The length of the gap sequence in S. haematobium is 54 bases and it is identical to that in S. mansoni in both length and sequence. However, in S. japonicum the sequence is between 64-67 bases long. In each case, irrespective of the species, the gap is located at the same position within the 28S rRNA. Secondary structures of the gap sequence derived by computer analysis predict a conformation with the minimum free energy that has an UAAU tract in a hairpin loop for S. haematobium and an UAUU tract for S. japonicum.     

Identification of a putative eggshell precursor protein of the female Schistosoma japonicum

In adult worms of Schistosoma japonicum, a prominent radiolabelled female-specific protein (34 kDa) was demonstrated on fluorography of SDS gels with the pulse incorporation of 14C-tyrosine in vitro, though it was difficult to detect major female-specific proteins by direct staining methods. This female-specific protein was demonstrated to localize exclusively in the vitelline cells by indirect immunofluorescence using the rabbit anti-34 kDa female protein antiserum. It was shown that 14C-tyrosine was selectively incorporated into the vitelline cells by the pulse labelled autoradiographs. Two days after the exposure of worms to radio-tyrosine, the shells of eggs in the uterus were demonstrated to have become radioactive, indicating that 14C-tyrosine-labelled protein was used as a material for the eggshell. In the fluorograph of proteins extracted from newly laid eggs in vitro, the prominent band was not found at the 34 kDa region, but a lot of radioactivity appeared at higher than 100 kDa. The results suggested that a 34 kDa female protein was a precursor of the eggshell and became a much larger protein molecule as a result of cross-linking during eggshell hardening.     

Schistosoma haematobium: oxidoreductase histochemistry and ultrastructure of niridazole-treated females

Administration of niridazole to Saccostomus campestris produced changes in enzyme activity in Schislosoma haematobium females as indicated histochemically by a decrease in the activity of cytochrome oxidase (EC 1.9.3.1), malate (NAD) dehydrogenase (EC 1.1.1.37), malate (NADP) dehydrogenase (EC 1.1.1.40), succinate dehydrogenase (EC 1.3.99.11), isocitrate (NAD) dehydrogenase (EC 1.1.1.41), isocitrate (NADP) dehydrogenase (EC 1.1.1.42), lactate dehydrogenase (EC 1.1.1.27), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), NADH: tetrazolium oxidoreductase, NADPH: tetrazolium oxidoreductase, and a disappearance of both the activity of phenolase (EC 1.10.3.1) and the reactivity of vitelline phenols. These changes were associated with the following alterations in the ultrastructure of the parasites: a decrease in number of immature vitelline cells of gonial type, a disruption of the tegument surface, a swelling of mitochondria in vitelline cells, a disappearance of the regular structure of the endoplasmic reticulum and a vaeuolization of the cytoplasm in vitelline cells, an appearance of areas of focal cytoplasmic degradation in vitelline cells, and a disruption of shell globules. The degree of changes in enzyme activity and ultrastructure increased both with increase in the dose of niridazole administered to the hosts, and with length of time after treatment.Preincubation of control sectioned material in a buffered niridazole-sucrose solution produced total inhibition of succinate dehydrogenase activity, whereas the activity of other enzymes examined remained unchanged.     

Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded.     

Praziquantel: a new schistosomicide against Schistosoma haematobium.

The effectiveness of the new schistosomicide praziquantel was assessed in African schoolchildren infected with Schistosoma haematobium. They were stratified according to the severity of their infection and were then randomly allocated to treatment with two single-dose regimens (30 and 40 mg/kg) and a split regimen of two doses of 20 mg/kg given four hours apart. All three regimens were highly effective and produced few side effects. Children who initially had very high pretreatment egg loads showed a poorer therapeutic response at all dose levels, and further investigations are necessary to find the optimum dose. Because of its effectiveness in a single dose and lack of toxicity, praziquantel may prove to be the ideal schistosomicide.     

Epidemiology of mixed Schistosoma mansoni and Schistosoma haematobium infections in northern Senegal

Due to the large overlap of Schistosoma mansoni- and Schistosoma haematobium-endemic regions in Africa, many people are at risk of co-infection, with potential adverse effects on schistosomiasis morbidity and control. Nonetheless, studies on the distribution and determinants of mixed Schistosoma infections have to date been rare. We conducted a cross-sectional survey in two communities in northern Senegal (n=857) to obtain further insight into the epidemiology of mixed infections and ectopic egg elimination. Overall prevalences of S. mansoni and S. haematobium infection were 61% and 50%, respectively, in these communities. Among infected subjects, 53% had mixed infections and 8% demonstrated ectopic egg elimination. Risk factors for mixed infection - i.e. gender, community of residence and age - were not different from what is generally seen in Schistosoma-endemic areas. Similar to overall S. mansoni and S. haematobium infections, age-related patterns of mixed infections showed the characteristic convex-shaped curve for schistosomiasis, with a rapid increase in children, a peak in adolescents and a decline in adults. Looking at the data in more detail however, the decline in overall S. haematobium infection prevalences and intensities appeared to be steeper than for S. mansoni, resulting in a decrease in mixed infections and a relative increase in single S. mansoni infections with age. Moreover, individuals with mixed infections had higher infection intensities of both S. mansoni and S. haematobium than those with single infections, especially those with ectopic egg elimination (P<0.05). High infection intensities in mixed infections, as well as age-related differences in infection patterns between S. mansoni and S. haematobium, may influence disease epidemiology and control considerably, and merit further studies into the underlying mechanisms of Schistosoma infections in co-endemic areas.     

Schistosoma mansoni: eggshell formation is regulated by pH and calcium

The protein precursors of the schistosome eggshell are synthesized and packaged into secretory vesicles in the vitelline cells. These vesicles appear to contain an emulsion of eggshell precursor material. Evidence is presented to show that these secretory vesicles are acidic as in other systems and that this acidity stabilizes the emulsion and prevents the eggshell cross-linking reactions from occurring. Alkalinizing treatments trigger eggshell formation within the secretory vesicles as shown by (1) the induction of autofluorescence and (2) by electron microscopy which shows that the eggshell precursors have aggregated within the secretory vesicles into spherical particles bearing microspines. These aggregates formed in the secretory vesicles were isolated and shown to have the same protease resistance and amino acid composition as authentic eggshell. The calcium ionophore A23187 induces scattered autofluorescence in intact female worms which electron micrographs show to be due to exocytosis of eggshell material. Based on these observations we propose a model for the formation of schistosome eggshell and suggest that it may apply to all trematodes in which the eggshell precursors are present as stable emulsions in the secretory vesicles of the vitelline cells.     

Cloning and expression analysis of Phytoplasma protein translocation genes

Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.     

Hybridisation between the digeneans Schistosoma haematobium and S. mattheei : viability of hybrids and their development in sheep

The F1 and F2 hybrids of Schistosoma haematobium male × S. mattheei female were studied with regard to infectivity to intermediate and definitive hosts, isoenzymes (phosphoglucomutase) of individual male worms, randomly amplified polymorphic DNAs of individual adult worms and scanning electron micrographs of the tubercles of male worms. The infection rate of the F1 hybrid miracidia in Bulinus globosus (41.7%) was greater than that achieved in B. wrighti (16.3%); the infection rate of the F2 in B. wrighti was 15.4%. In the definitive hosts: in sheep only male F1 hybrids (i.e. no females and no F2 worms)were recovered; but in hamsters both paired F1 worms and unpaired F1 males were recovered, as were one pair of worms and unpaired males of the F2 generation. The S. mattheei and S. haematobium male worms showed very distinctive PGM patterns, and the F1 hybrids showed additive patterns and a polymorphism with two distinct types of band patterns which are the result of polymorphism in the S. haematobium. The RAPD profiles of the F1 hybrids were also composite of the two parental species. Scanning electron micrographs of the tubercles of male S. haematobium showed them to be heavily spined, whereas those of S. mattheei males were devoid of spines. The F1 hybrids did show variation ranging from non-spined, some with partial spination, to those with heavily spined tubercles. Male worms of the F2 generation possessed tubercles either with or without spines. The potential significance of hybridisation in areas of sympatry between S. haematobium and S. mattheei is discussed.     

Schistosoma haematobium in Morocco: moving from control to elimination

In this article, Hammou Laamrani and colleagues summarize the Moroccan schistosomiasis control programme and discuss the challenges ahead for schistosomiasis elimination. In 1994, a programme was initiated by the Moroccan Ministry of Health to eliminate schistosomiasis from Morocco by the year 2004. In 1997, this objective had been achieved in three out of 20 affected provinces. This article discusses the background and strategies of this programme, as well as the achievements, the problems encountered and the challenges ahead, along with suggestions as to how to reach the goal of elimination of urinary schistosomiasis in Morocco, and possibly elsewhere in Africa.     

Population genetics of Schistosoma haematobium: development of novel microsatellite markers and their application to schistosomiasis control in Mali

The recent implementation of mass drug administration (MDA) for control of uro-genital schistosomiasis has identified an urgent need for molecular markers to both directly monitor the impact of MDA, for example to distinguish re-infections from uncleared infections, as well as understand aspects of parasite reproduction and gene flow which might predict evolutionary change, such as the development and spread of drug resistance. We report the development of a novel microsatellite tool-kit allowing, for the first time, robust genetic analysis of individual S. haematobium larvae collected directly from infected human hosts. We genotyped the parasite populations of 47 children from 2 schools in the Ségou region of Mali, the first microsatellite study of this highly neglected parasite. There was only limited evidence of population subdivision between individual children or between the two schools, suggesting that few barriers to gene flow exist in this population. Complex relationships between parasite reproductive success, infection intensity and host age and gender were identified. Older children and boys harboured more diverse infections, as measured by the number of unique adult genotypes present. Individual parasite genotypes had variable reproductive success both across hosts, a pre-requisite for evolutionary selection, and, phenotypically, in hosts of different ages and genders. These data serve as a baseline against which to measure the effect of treatment on parasite population genetics in this region of Mali, and the tools developed are suitable to further investigate this important pathogen, and its close relatives, throughout their range.