Populational structure of Schistosoma mansoni assessed by DNA microsatellites.

DNA microsatellites were used as molecular markers to analyse the population structure of the laboratory LE strain and of 10 field isolates of Schistosoma mansoni, the aetiologic agent of schistosomiasis. Out of 16,000 DNA sequences analysed in databases, 622 microsatellite loci were identified in 481 sequences (3.0%). The AT repetitions were the most frequent, followed by AAT and AC. Six loci showing perfect repetitions were selected and used in the polymerase chain reaction to evaluate polymorphisms in the number of repeats. Two groups of worms were studied. The first group consisted of 78 individuals, 39 of each sex, of the LE strain. The second group of worms consisted of 10 field isolates: seven from humans and three from snails. Four of the six loci were polymorphic, containing 11-17 alleles per locus. No linkage disequilibrium was observed among loci and none of the loci was sex linked. In both groups of worms, a significant deviation from Hardy-Weinberg equilibrium was observed. The observed heterozygosity was always lower than the expected one. The polymerase chain reaction primers were S. mansoni specific. The LE strain showed a lower total number of alleles or a lower average number of alleles/polymorphic locus than the field isolates, suggesting that 41 years of laboratory maintenance exerted selective pressure on the LE strain. The S. mansoni populations from the field were most genetically undifferentiated (R(ST)<0.027), suggesting a high gene flow among them. Our results showed the usefulness of microsatellites for population analysis of S. mansoni, offering a new alternative for a better understanding of schistosomiasis epidemiology.

Evolutionary relationships and biogeography of Biomphalaria (Gastropoda: Planorbidae) with implications regarding its role as host of the human bloodfluke, Schistosoma mansoni.

The wide geographic distribution of Schistosoma mansoni, a digenetic trematode and parasite of humans, is determined by the occurrence of its intermediate hosts, freshwater snails of the genus Biomphalaria (Preston 1910). We present phylogenetic analyses of 23 species of Biomphalaria, 16 Neotropical and seven African, including the most important schistosome hosts, using partial mitochondrial ribosomal 16S and complete nuclear ribosomal ITS1 and ITS2 nucleotide sequences. A dramatically better resolution was obtained by combining the data sets as opposed to analyzing each separately, indicating that there is additive congruent signal in each data set. Neotropical species are basal, and all African species are derived, suggesting an American origin for the genus. We confirm that a proto-Biomphalaria glabrata gave rise to all African species through a trans-Atlantic colonization of Africa. In addition, genetic distances among African species are smaller compared with those among Neotropical species, indicating a more recent origin. There are two species-rich clades, one African with B. glabrata as its base, and the other Neotropical. Within the African clade, a wide-ranging tropical savannah species, B. pfeifferi, and a Nilotic species complex, have both colonized Rift Valley lakes and produced endemic lacustrine forms. Within the Neotropical clade, two newly acquired natural hosts for S. mansoni (B. straminea and B. tenagophila) are not the closest relatives of each other, suggesting two separate acquisition events. Basal to these two species-rich clades are several Neotropical lineages with large genetic distances between them, indicating multiple lineages within the genus. Interesting patterns occur regarding schistosome susceptibility: (1) the most susceptible hosts belong to a single clade, comprising B. glabrata and the African species, (2) several susceptible Neotropical species are sister groups to apparently refractory species, and (3) some basal lineages are susceptible. These patterns suggest the existence of both inherent susceptibility and resistance, but also underscore the ability of S. mansoni to adapt to and acquire previously unsusceptible species as hosts. Biomphalaria schrammi appears to be distantly related to other Biomphalaria as well as to Helisoma, and may represent a separate or intermediate lineage.

Widespread heteroplasmy in schistosomes makes an mtVNTR marker "nearsighted".

Mitochondrial markers are often hailed as the preferred DNA elements for analyses of population subdivision. To this end we have employed a mitochondrial repeat element to examine the population structure in Schistosoma mansoni (human blood flukes). Schistosome isolates were collected from each of 21 different patients representing seven different areas of a Brazilian village. These parasite isolates demonstrate substantial genetic polymorphism, with an average of 10 genotypes infecting each patient, which is more readily detected because of high levels of heteroplasmy (i.e., 72.5% of the individual worms exhibit multiple versions of this repeat region with different numbers of repeats). Due to the high number of common haplotypes in the population, this repeat element from S. mansoni has a large proportion (47%) of its genetic variation described by differences among mitochondrial genomes within individual worms. However, when only rare haplotypes are considered, population structure can be detected. It seems that heteroplasmy in the schistosome population of Melquiades is both the source of plentiful genetic variation and a confounding factor in the analysis of that variation. Thus the schistosome population in Melquiades may actually be more strongly subdivided than we are able to detect using this mitochondrial marker.

Susceptibility of Biomphalaria tenagophila and Biomphalaria straminea to Schistosoma mansoni infection detected by low stringency polymerase chain reaction.

In order to determine Schistosoma mansoni infection rates in Biomphalaria tenagophila and B. straminea, low stringency polymerase chain reaction (LS-PCR) technique was used as a complementary method to light exposure technique. LS-PCR has already been standardized in our laboratory to detect the trematode DNA in B. glabrata. Higher S. mansoni infection rates were detected using conventional method and LS-PCR. The parasite DNA profile was detected in both species after 7-day exposure to miracidia, using LS-PCR. This technique enables early detection of schistosomiasis transmission focuses, in endemic areas, before the beginning of cercariae shedding.

Mitotic histone H3 phosphorylation by the NIMA kinase in Aspergillus nidulans.

Phosphorylation of histone H3 serine 10 correlates with chromosome condensation and is required for normal chromosome segregation in Tetrahymena. This phosphorylation is dependent upon activation of the NIMA kinase in Aspergillus nidulans. NIMA expression also induces Ser-10 phosphorylation inappropriately in S phase-arrested cells and in the absence of NIMX(cdc2) activity. At mitosis, NIMA becomes enriched on chromatin and subsequently localizes to the mitotic spindle and spindle pole bodies. The chromatin-like localization of NIMA early in mitosis is tightly correlated with histone H3 phosphorylation. Finally, NIMA can phosphorylate histone H3 Ser-10 in vitro, suggesting that NIMA is a mitotic histone H3 kinase, perhaps helping to explain how NIMA promotes chromatin condensation in A. nidulans and when expressed in other eukaryotes.

Centrosomal and cytoplasmic Cdc2/cyclin B1 activation precedes nuclear mitotic events.

The activation of cdc2/cyclin B is the trigger for entry into mitosis. The mechanism of cdc2/cyclin B activation is complex, but the final step is the dephosphorylation of the Thr14 and Tyr15 residues on the cdc2 subunit, catalyzed by a member of the Cdc25 family of phosphatases. Cdc2/cyclin B1 accumulates at the centrosome in late G2 phase and has been implicated in the conversion of the centrosome from an interphase to a mitotic microtubule organizing center. Here we demonstrate biochemically that cdc2/cyclin B1 accumulates at the centrosome in late G2 as the inactive, phosphotyrosine 15 form and that the centrosomal cdc2/cyclin B1 can be activated in vitro by recombinant cdc25B. We provide evidence that a portion of the cdc2/cyclin B1 translocated into the nucleus in prophase is the inactive tyrosine-15-phosphorylated form. At this time the centrosomal and cytoplasmic cdc2/cyclin B1 is already active. This provides evidence that the activation of cdc2/cyclin B1 is initiated in the cytoplasm and that full activation of the translocated pool occurs in the nucleus.

Checkpoint defects leading to premature mitosis also cause endoreplication of DNA in Aspergillus nidulans.

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMX(cdc2) in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsB(rad3) and uvsD(rad26) have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIME(cyclinB), but ectopic expression of active nondegradable NIME(cyclinB) does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMX(cdc2) and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.

[Lymnic snails from the microregion of Belo Horizonte, Minas Gerais, Brazil with an emphasis on parasite disease vectors]. / Moluscos límnicos da microrregião de Belo Horizonte, MG, com ênfase nos vetores de parasitoses.

A malacological survey to detect foci of transmission of schistosomiasis and other parasitic diseases was undertaken into water-courses from 13 municipalities of microregion of Belo Horizonte, MG, Brazil. From 1990 to 1996, 22,066 snails were collected. From those, 378 (1.7%) were found infected by trematodes: Biomphalaria glabrata (7,920), infected by Schistosoma mansoni (1.9%), Echinostomatidae (1.2%), Strigeidae (0.6%), Cercaria minense (0.1%) and Derogenidae (-0.1%); B. straminea (4,093) infected by Strigeidae (0.6%), Echinostomatidae (0.2%), Clinostomatidae (-0.1%) and two unidentified cercariae; B. tenagophila (1,338), infected by Strigeidae (0.1%) and Physa marmorata (1,776) by Echinostomatidae (1.6%). The snails Biomphalaria peregrina, B. occidentalis, B. schrammi, Drepanotrema depressissimum, D. lucidum, D. cimex, Physa cubensis, Lymnaea columella, Melania tuberculata, Idiopyrgus souleyetianus, Pomacea sp, Anodontites sp and Ancylidae were found noninfected. Snails from 9 municipalities were infected by S. mansoni and from 11 by other trematodes.

The molluscicidal activity of Crown of Christ (Euphorbia splendens var. hislopii) latex on snails acting as intermediate hosts of Schistosoma mansoni and Schistosoma haematobium.

The present study describes the action of the latex of Euphorbia splendens var. hislopii (E. milli) on species of the genus Bulinus and on Biomphalaria pfeifferi, intermediate hosts of schistosomiasis in Africa, and the Brazilian snails B. glabrata, B. tenagophila, and B. straminea, intermediate hosts of schistosomiasis in Brazil. The impact of the latex on the egg masses and embryos of B. glabrata was also evaluated. Using the standardized methodology of the World Health Organization for testing plant-derived molluscicides, we obtained a 90% lethal dose (LD90) ranging from 0.13 ppm for B. glabrata subjected to lyophilized latex to 4.0 ppm for B. pfeifferi tested with the natural latex. This material has proved to be one of the most potent and specific plant molluscicides discovered thus far, presenting advantages in terms of application so that it could be used in programs involving community participation in endemic areas in both Brazil and Africa.

Molecular characterisation of intermediate snail hosts and the search for resistance genes.

The relationship between schistosomes and their intermediate hosts is an extremely intricate one with strains and species of the parasite depending on particular species of snail, which in turn may vary in their susceptibility to the parasites. In order to gain a better understanding of the epidemiology of the disease we have been investigating the use of molecular markers for snail identification and for studying host-parasite relationships. In this paper we will draw on examples concerning schistosomiasis in West and East Africa to illustrate how a molecular analysis can be used as part of a "total evidence" approach to characterisation of Bulinus species and provide insights into parasite transmission. Particular emphasis is given to ribosomal RNA genes (rRNA), random amplified polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI). Snails resistant to infection occur naturally and there is a genetic basis for this resistance. In Biomphalaria glabrata resistance to Schistosoma mansoni is known to be a polygenic trait and we have initiated a preliminary search for snail genomic regions linked to, or involved in, resistance by using a RAPD based approach in conjunction with progeny pooling methods. We are currently characterising a variety of STSs, (sequence tagged sites) associated with resistance. These can be used for local linkage and interval mapping to define genomic regions associated with the resistance trait. The development of such markers into simple dot-blot or specific PCR-based assays may have a direct and practical application for the identification of resistant snails in natural populations.

Histopathologic features associated with susceptibility and resistance of Biomphalaria snails to infection with Schistosoma mansoni.

Resistance and susceptibility of Biomphalaria snails to Schistosoma mansoni sporocysts occur in different degrees. Histopathology reflects these differences. In a state of tolerance numerous sporocysts in different stages of differentiation are seen in the absence of host tissue reaction. However extensive diffuse and focal proliferation of amebocytes with sequestration and destruction of many parasitic structures appear in resistant snails. Some snails are totally resistant and when exposed to infecting miracidia may never eliminate cercarie. Sequential histopathological examination has revealed that in such cases the infected miracidia are destroyed a few minutes to 24 hr after penetration in the snail. However, B. glabrata that were exposed to S. mansoni miracidia and three months later failed to shed cercariae, exhibited focal and diffuse proliferation of amebocytes in many organs in the absence of pasitic structures. These lesions were similar to those observed in resistant snails that were still eliminating a few cercariae, with the difference that no recognizable sporocystic structures or remnants were present. Histological investigation carried out in similarly resistant B. tenagophila and B. straminea presented essentially normal histologic structures. Only occasionally a few focal proliferative (granulomatous) amebocytic reactions were seen in ovotestis and in the tubular portion of the kidney. Probably, there are two types of reactions to miracidium presented by totally resistant snails: one would implicate the immediate destruction of the miracidium leaving no traces in the tissues; the other involving late reactions that seem to completely destroy invading sporocysts and leave histological changes.

Comparative histopathology of Biomphalaria glabrata, B. tenagophila and B. straminea with variable degrees of resistance to Schistosoma mansoni miracidia.

A comparative histopathological study of three snails species--Biomphalaria glabrata, B. tenagophila and B. straminea--which had been infected with Schistosoma mansoni miracidia revealed similar qualitative features; consisting of areas of sporocyst proliferation and differentiation associated with reactive host reaction, at the time they were actively eliminating great number of cercariae. However, in specimens that were exposed to miracidia but failed to eliminate cercariae later on, different histopathological pictures were observed in different snail species. While B. glabrata exhibited frequent focal (granulomatous) proliferation of amebocytes in several organs, B. tenagophila and B. straminea only rarely showed such reactive changes, suggesting that the mechanism of resistance to miracidial infection probably follows different pathways in the snail species studied.

[The resistance of Biomphalaria glabrata to Schistosoma mansoni infection: variations in the prepatent period and in compatibility]. / Resistência de Biomphalaria glabrata à infecção pelo Schistosoma mansoni: variações no período pré-patente e na compatibilidade.

Biomphalaria glabrata from Belo Horizonte, Minas Gerais, Brazil, reared in laboratory, has a level of infection of 90% when exposed to 20 miracidia of the autochibonous LE strain. The prepatent period was of 5 to 7 weeks whereas 5 to 10% of exposed snails do not shed cercariae. The eggs of negative snails were collected and the progeny was again submitted to individual infection with 20 miracidia. The mean of infection from F14 to F20 was of 43.6%. Histological sections from F12, F14 and F15 snails showed tissue reactions in those specimens shedding less than 10 cercariae. A prepatent period of 17 to 32 weeks was observed in 35 (17.9%) of 195 infected snails. The index of cercariae of control was extremely compatible and for F12, F13 and F15 snails varied from very compatible class V to compatible class III, showing less compatibility in selected snails.

[Susceptibility to chemotherapeutic agents of Schistosoma mansoni isolates from patients treated with oxamniquine and praziquantel and not cured]. / Suscetibilidade aos agentes quimioterápicos de isolados de Schistosoma mansoni oriundos de pacientes tratados com oxamniquine e praziquantel e não curados.

Ten inhabitants of Itaquara, Bahia, Brazil treated with oxamniquine and subsequently praziquantel were not cured. Schistosoma mansoni isolates derived from these patients were studied. Snails were infected with miracidia derived from the feces of these patients and the cercariae produced used to infect albino mice. The animals were then treated with a single oral dose of oxamniquine (25, 50 and 100mg/kg) or praziquantel (100, 200 and 400 mg/kg). The response to chemotherapy was significantly different in some of the isolates although it was not possible to characterize any of them as resistant. In addition, DNA analysis of the isolates by means of "Random Amplified Polymorphic DNA" indicated a low degree of variability as compared with a laboratory strain, LE. Thus, it was not possible to characterize these organisms at a genetic level as a distinct strain.

Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells.

The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B. Here we present data that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of cdc25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic cdc25B activity, and that cdc25C regulates nuclear G2/M events. Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events.

Cholelithiasis in cirrhotic patients. (Analysis of cholelithiasis among patients with liver cirrhosis in São Paulo, Brazil).

During the past 20 years, several authors have reported increased prevalence of cholelithiasis in liver cirrhosis. This biliary disease has been implicated with the deterioration of liver function, liver disease of alcoholic origin or even the presence of hypersplenism in this patient population. This study analyzes the incidence and possible factors which are responsible for promoting cholelithiasis in cirrhosis. The study included 110 cirrhotic patients of a private center specialized in treating liver diseases. The incidence of cholelithiasis was 27.3% (25.3% in males and 33.3% in females). There was no correlation between liver function defined by Child's classification or through the laboratory examinations (AST, ALT, AP, GGT, PT, Alb, TB, DB, PA) and the presence of gallstones. No evidence was found that the etiology of cirrhosis or the presence/absence of hypersplenism affected the prevalence of cholelithiasis in this population. In conclusion, an increased prevalence of cholelithiasis was verified in this population of cirrhotics but the pathogenesis is still obscure.

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.

[Schistosoma mansoni: the levels of infectivity for intermediate host mollusks in Brazil]. / Schistosoma mansoni: níveis de infecciosidade para os moluscos hospedeiros intermediários do Brasil.

The levels of infectivity of Schistosoma mansoni for the three species of Biomphalaria, intermediate hosts of parasite in Brazil were studied after exposing of molluscs to miracidia in the laboratory and in the field. The LE and SJ strains of S. mansoni, maintained in laboratory were used in these experiments as well as the WVS and RFS strains obtained from faeces of schoolchildren from Belo Horizonte, MG. The results show the high level of infectivity of S. mansoni for B. glabrata with infection rates varying from 4.7 to 85.5%. The snail B. straminea was susceptible to LE, SJ and WVS strains, with infection rates of 11.0 to 24.6%, B. tenagophila was susceptible only to LE and SJ strains with infection rates of 2.5 to 6.5%. The mean number of cercariae of the WVS strain shed per day, by B. straminea and B. glabrata were 93 +/- 59 and 782 +/- 1,120, respectively.

Influence of temperature on development of Schistosoma mansoni female cercariae in Biomphalaria glabrata.

In these experiments the ratio of male to female S. mansoni larvae in B. glabrata from Belo Horizonte and Ribeirão das Neves Minas Gerais, Brazil, either reared in laboratory or collected in the field, varied from 1:1 to 1:1.3 or 1.4:1. Cercariae of LE strain of Schistosoma mansoni, shed by 39 snails maintained at 25 +/- 0.5 degrees C were used to infect mice on a weekly basis. Subsequent perfusion resulted in 76.6% male and 23.4% female worms. The cercariae produced by 32 infected snails maintained at 27 +/- 0.5 degrees C were inoculated into mice and produced 43.4% male and 56.6% female worms (p < 0.05). Cercariae eliminated by snails collected in Barreiro and Ressaca, Belo Horizonte, during hot months, produced 45.7 to 47.7% male and 52.3 to 54.3% female worms. A lower number of cercariae shed by snails collected in Gorduras, Belo Horizonte, at 20 +/- 3.0 degrees C, produced 51.6% male and 48.4% female worms. Thus, in this region the infection of vertebrate hosts with S. mansoni cercariae would be more severe in the summer due to the higher level of parasites and the number of eggs.

Development of Schistosoma mansoni in Biomphalaria tenagophila, Biomphalaria straminea and Biomphalaria glabrata.

A comparative study of the development of Schistosoma mansoni during the intra-molluscan phase was made by means of histological sections of Biomphalaria tenagophila, B. straminea and B. glabrata from Brazil. Two hundred snails of each species were individually exposed to 50 miracidia of the S. mansoni, AL line. No larvae were observed in the snails fixed 72 h after exposure. In specimens shedding cercariae, 31 days after exposure tissue reactions encapsulating the larvae were seen in B. tenagophila and B. straminea, in the head-foot, mantle collar and renal ducts. No tissue reactions occurred in the digestive glands of these two species. In B. glabrata the presence of numerous sporocysts and cercariae without tissue reactions was observed in the digestive gland, and other organs. The levels of infection of the snails and the average numbers of cercariae shed per day were 32.6% and 79 +/- 90 respectively for B. tenagophila, 11.3% and 112 +/- 100 for B. straminea and 75.3% and 432 +/- 436 for B. glabrata. The lower levels of infection and average numbers of cercariae shed by B. tenagophila and B. straminea are thus related to their more potent internal defense systems.