Luiz Augusto Magalhães

Origin and diversification of the human parasite Schistosoma mansoni

Schistosoma mansoni is the most widespread of the human‐infecting schistosomes, present in 54 countries, predominantly in Africa, but also in Madagascar, the Arabian Peninsula, and the Neotropics. Adult‐stage parasites that infect humans are also occasionally recovered from baboons, rodents, and other mammals. Larval stages of the parasite are dependent upon certain species of freshwater snails in the genus Biomphalaria, which largely determine the parasites geographical range. How S. mansoni genetic diversity is distributed geographically and among isolates using different hosts has never been examined with DNA sequence data. Here we describe the global phylogeography of S. mansoni using more than 2500 bp of mitochondrial DNA (mtDNA) from 143 parasites collected in 53 geographically widespread localities. Considerable within‐species mtDNA diversity was found, with 85 unique haplotypes grouping into five distinct lineages. Geographical separation, and not host use, appears to be the most important factor in the diversification of the parasite. East African specimens showed a remarkable amount of variation, comprising three clades and basal members of a fourth, strongly suggesting an East African origin for the parasite 0.30–0.43 million years ago, a time frame that follows the arrival of its snail host. Less but still substantial variation was found in the rest of Africa. A recent colonization of the New World is supported by finding only seven closely related New World haplotypes which have West African affinities. All Brazilian isolates have nearly identical mtDNA haplotypes, suggesting a founder effect from the establishment and spread of the parasite in this large country.

Phylogeography of Biomphalaria glabrata and B. pfeifferi, important intermediate hosts of Schistosoma mansoni in the New and Old World tropics

The historical phylogeography of the two most important intermediate host species of the human blood fluke Schistosoma mansoni, B. glabrata in the New World, and B. pfeifferi in the Old World, was investigated using partial 16S and ND1 sequences from the mitochondrial genome. Nuclear sequences of an actin intron and internal transcribed spacer (ITS)‐1 were also obtained, but they were uninformative for the relationships among populations. Phylogenetic analyses based on mtDNA revealed six well‐differentiated clades within B. glabrata: the Greater Antilles, Venezuela and the Lesser Antilles, and four geographically overlapping Brazilian clades. Application of a Biomphalaria‐specific mutation rate gives an estimate of the early Pleistocene for their divergence. The Brazilian clades were inferred to be the result of fragmentation, due possibly to climate oscillations, with subsequent range expansion producing the overlapping ranges. Within the Venezuela and Lesser Antilles clade, lineages from each of these areas were estimated to have separated approximately 740 000 years ago. Compared to B. glabrata, mitochondrial sequences of B. pfeifferi are about 4× lower in diversity, reflecting a much younger age for the species, with the most recent common ancestor of all haplotypes estimated to have existed 880 000 years ago. The oldest B. pfeifferi haplotypes occurred in southern Africa, suggesting it may have been a refugium during dry periods. A recent range expansion was inferred for eastern Africa less than 100 000 years ago. Several putative species and subspecies, B. arabica, B. gaudi, B. rhodesiensis and B. stanleyi, are shown to be undifferentiated from other B. pfeifferi populations.

Schistosoma mansoni : Evaluation of an RNAi-based treatment targeting HGPRTase gene

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential gene of the parasite Schistosoma mansoni and it is well conserved in its hosts (mouse and human) at the protein but not at the RNA level. This feature prompted us to assess RNA interference (RNAi) to combat schistosomiasis. Small interfering RNAs (siRNAs) were produced against HGPRTase, injected in infected mice and the number of worms was counted six days after injection. The total number of parasites was reduced by approximately 27% after treatment. RT-PCR analyzes showed a significant reduction in parasite target mRNA but not in hosts homologue. The use of low doses of molecules did not oversaturate si- or miRNA pathways as mice survival rates were not affected by siRNAs. This is the first successful in vivo demonstration of a RNAi-based treatment against schistosomiasis. We believe that improvements in molecule delivery and an increase on siRNA dose could rapidly eliminate parasite.

Evolução dos esporocistos de Schistosoma mansoni Sambon, 1907 em Biomphalaria glabrata (Say, 1818) e Biomphalaria tenagophila (D'Orbigny, 1835)

Estudou-se a evolucao dos esporocistos de Shistosoma mansoni das linhagens BH e SJ respectivamente em Biomphalaria glabrata e Biomphalaria tenagophila. Utilizando-se cortes histologicos foram avaliados o aspecto e numero de esporocistos primarios desde a primeira ate a oitava semana de infeccao, a contar do dia em que cada molusco foi exposto a 100 miracidios. No decorrer da primeira semana constataram-se diferencas significativas entre as linhagens estudadas quanto ao numero e aspecto dos esporocistos primarios, A distribuicao por orgaos e a evolucao dos esporocistos foi observada ate a fase de formacao das cercarias infectantes.

Saponins from Swartzia langsdorffii: Biological Activities

The presence of saponins and the molluscicidal activity of the roots, leaves, seeds and fruits of Swartzia langsdorffii Raddi (Leguminosae) against Biomphalaria glabrata adults and eggs were investigated. The roots, seeds and fruits were macerated in 95% ethanol. These extracts exerted a significant molluscicidal activity against B. glabrata, up to a dilution of 100 mg/l. Four mixtures (A2, B2, C and D) of triterpenoid oleanane type saponins were chromatographically isolated from the seed and fruit extracts. Two known saponins (1 and 2) were identified as beta-D-glucopyranosyl-[alpha-L-rhamnopyranosyl-(1->3)- beta-D-glucuronopyranosyl-(1->3)]-3beta-hydroxyolean-12-ene-28 -oate, and beta-D-glucopyranosyl-(1->3)-beta-D-glucuronopyranosyl-(1 ->3)]-3beta-hydroxyolean-12-ene-28-oate, respectively. These two saponins were present in all the mixtures, together with other triterpenoid oleane type saponins, which were shown to be less polar, by reversed-phase HPLC. The saponin identifications were based on spectral evidence, including H- H two-dimensional correlation spectroscopy, nuclear Overhauser and exchange spectroscopy, heteronuclear multiple quantum coherence, and heteronuclear multiple-bond connectivity experiments. The toxicity of S. langsdorffii saponins to non-target organisms was prescreened by the brine shrimp lethality test.

Seleção de linhagens de Biomphalaria tenagophila e Biompnalaria glabrata visando maior suscetibilidade ao Schistosoma mansoni

Utilizando um esquema de selecao individual em progenies autofecundadas, foram obtidas, apos quatro geracoes, populacoes de Biomphalaria tenagophila e de Biomphalaria glabrata, altamente suscetiveis as linhagens do Schistosoma mansoni do Vale do Rio Paraiba do Sul, SP e de Belo Horizonte, MG (Brasil), respectivamente. Os rapidos ganhos geneticos obtidos confirmam ser a suscetibilidade de moluscos a infeccao esquistossomotica, uma caracteristica de alta herdabilidade, sendo aparentemente condicionada por um pequeno numero de genes.

Genetic markers between Biomphalaria glabrata snails susceptible and resistant to Schistosoma mansoni infection

The analysis of the genetic variability related to susceptibility to Schistosoma mansoni infection in the vector of the genus Biomphalaria is important in terms of a better understanding of the epidemiology of schistosomiasis itself, the possible pathological implications of this interaction in vertebrate hosts, and the formulation of new strategies and approaches for disease control. In the present study, the genetic variability of B. glabrata strains found to be resistant or susceptible to S. mansoni infection was investigated using DNA amplification by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The amplification products were analyzed on 8% polyacrylamide gel and stained with silver. We selected 10 primers, since they have previously been useful to detect polymorphism among B. glabrata and/or B. tenagophila. The results showed polymorphisms with 5 primers. Polymorphic bands observed only in the susceptible strain. The RAPD-PCR methodology represents an adequate approach for the analysis of genetic polymorphisms. The understanding of the genetic polymorphisms associated to resistance may contribute to the future identification of genomic sequences related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni and to the development of new strategies for the control of schistosomiasis.

Morphologic studies of Schistosoma mansoni strains from Belo Horizonte, MG and S. José dos Campos, SP (Brazil).

Two strains of Schistosoma mansoni (Belo Horizonte, MG and S. Jose dos Campos, SP, Brazil) are morphologically studied. Some numerical characteristics are found to be different between the strains. These differences are as follows: lenght, distance from the oral sucker until the ventral sucker, distance between the anterior pole of the worm and the distal part of the gonads and finally the number of testes.Foram estudados morfologicamente exemplares de S. mansoni adultos das linhagens de Belo Horizonte (MG), e de Sao Jose dos Campos (SP), Brasil. Concluiu-se haver diferencas significativas referentes as medidas de comprimento do verme, distância entre as ventosas oral e acetabular, distância entre a parte anterior dos vermes e a extremidade distal das gonadas e quanto ao numero de testiculos.

Liposomal-praziquantel: efficacy against Schistosoma mansoni in a preclinical assay.

Currently, schistosomiasis mansoni is treated clinically with praziquantel (PZQ). Nevertheless, cases of tolerance and resistance to this drug have been reported, creating the need to develop new drugs or to improve existing drugs. Considering the small number of new drugs against Schistosoma mansoni, the design of nanotechnology-based drug delivery systems is an important strategy in combating this disease. The aim of this study was to evaluate the activity of PZQ containing liposome (lip.PZQ) on S. mansoni, BH strain. Mice were treated orally with different concentrations of PZQ and lip.PZQ 30 and 45 days following infection. The number of worms, recovered by perfusion of the hepatic portal system, and the number of eggs found in the intestine and liver were analysed. Parasite egg counts were also performed. The most active formulation for all parameters was 300mg/kg of lip.PZQ, since as it decreased the total number of worms by 68.8%, the number of eggs in the intestine by 79%, and the number of hepatic granulomas by 98.4% compared to untreated controls. In addition, this concentration decreased egg counts by 55.5%. The improved efficacy of the treatment with lip.PZQ, especially when administered 45 days following infection, compared with the positive-control group (untreated) and the groups that received free PZQ, can be explained by greater bioavailability in the host organism; the preferred target of lip.PZQ is the liver, and lip.PZQ is better absorbed by the tegument of S. mansoni, which has an affinity for phospholipids.

Avaliação da atividade tóxica em Artemia salina e Biomphalaria glabrata de extratos de quatro espécies do gênero Eleocharis (Cyperaceae)

The genus Eleocharis R. Br. comprises about 200 species, occurring in wet environments like swamps, lakes and river margins. In the search for new molluscicides, extracts from Eleocharis acutangula (Roxb.) Schult., Eleocharis interstincta (Vahl) Roem. & Schult., Eleocharis maculosa (Vahl) Roem. & Schult. and Eleocharis sellowiana Kunth were tested for molluscicidal activity (spawns and adult snails) and toxicity (Brine Shrimp Lethality - BSL - bioassay). The hexane extract of Eleocharis acutangula (fresh subterraneous parts) was active in the BSL bioassay (LC50 = 476 mg/mL), while the other extracts showed LC50 >> 103 mg/mL, suggesting they have low toxicity. The aqueous ethanol extract of Eleocharis sellowiana (fresh subterraneous parts) was active against Biomphalaria glabrata spawns (LC50 = 24.27 mg/mL) but it was not lethal to adult snails. No other plant extract tested in this study showed molluscicidal activity.