Zinc Sulphate in the Treatment of Cutaneous Leishmaniasis: an in Vitro and Animal Study

This study was designed to evaluate the effectiveness of zinc sulphate both in vitro and in an animal model against both strains of old world cutaneous leishmaniasis. The in vitro sensitivities of promastigotes and axenic amastigotes of both Leishmania major and L. tropica to zinc sulphate was determined, the LD50 calculated and compared to the standard treatment for cutaneous leishmaniasis pentavalent antimony compounds. The results show that the two forms of both strains were sensitive to zinc sulphate and their respective LD50 were lower compared to the pentavalent antimony compound. Furthermore the sensitivities of the forms of both strains were tested using a simple slide method and compared to results of the standard method. To confirm this result, zinc sulphate was administered orally to mice infected with cutaneous leishmaniasis both therapeutically and prophylactically. Results showed that oral zinc sulphate was effective in both treatment and prophylaxis for cutaneous leishmaniasis. These results encourage the use of oral zinc sulphate in the treatment of cutaneous leishmaniasis clinically.

Schistosomiasis vaccine development: progress and prospects

The undisputed, worldwide success of chemotherapy notwithstanding, schistosomiasis continues to defy control efforts in as much rapid reinfection demands repeated treatment, sometimes as often as once a year. There is thus a need for a complementary tool with effect for the longer term, notably a vaccine. International efforts in this direction have been ongoing for several decades but, until the recombinant DNA techniques were introduced, antigen production remained an unsurmountable bottleneck. Although animal experiments have been highly productive and are still much needed, they probably do not reflect the human situation adequately and real progress can not be expected until more is known about human immune responses to schistosome infection. It is well-known that irradiated cercariae consistently produce high levels of protection in experimental animals but, for various reasons, this proof of principle cannot be directly exploited. Research has instead been focussed on the identification and testing of specific schistosome antigens. This work has been quite successful and is already at the stage where clinical trials are called for. Preliminary results from coordinated in vitro laboratory and field epidemiological studies regarding the protective potential of several antigens support the initiation of such trials. A series of meetings, organized earlier this year in Cairo, Egypt, reviewed recent progress, selecteded suitable vaccine candidates and made firm recommendations for future action including pledging support for large-scale production according to good manufacturing practice (GMP) and Phase I trials. Scientists at the American Centers for Disease Control and Prevention (CDC) have drawn up a detailed research plan. The major financial support will come from USAID, Cairo, which has established a scientific advisory group of Egyptian scientists and representatives from current and previous international donors such as WHO, NIAID, the European Union and the Edna McConnell Clark Foundation.

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 diferences. 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 moths 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 remmants 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.

Susceptibility of Nectomys rattus (Pelzen, 1883) to experimental infection with Schistosoma mansoni (Sambon, 1907): a potential reservoir in Brazil

The aim of the present research was to evaluate the potential of Nectomys rattus, the "water rat", to develop Schistosoma mansoni infection. Comparison with N. squamipes was carried out. Both species of rodents were submitted to transcutaneous infection using different infective cercariae loads: 50, 100 or 500. N. rattus showed high susceptibility to S. mansoni, with an infection rate of 71%. Rodents were able to excrete viable eggs of S. mansoni in the feaces during all infection period. For both species, the small intestine, followed by the liver and the large intestine, presented the highest concentration of eggs among the surveyed organs. Infection caused no animal death. Moreover, N. rattus accomplished the parasite's life cycle, by infecting the snails Biomphalaria glabrata and later Mus musculus. These evidences indicate that both N. rattus, as for N. squamipes are potential reservoirs for schistosomiasis in Brazil. Considering the fact that N. rattus and N. squamipes exist in the same natural ecosystems of S. mansoni, we suggest that these rodents must be regarded as influential factors in epidemiology surveys.

Ultrastructure and chaetotaxy of sensory eeceptors in the cercaria of a species of Allopodocotyle Pritchard, 1966 (Digenea: Opecoelidae)

Previous investigations of sensory systems in opecoelid cercariae have focused on chaetotaxy and ultrastructure of sensory receptors. They revealed chaetotaxic patterns within family, genus, and species as well as different receptors. Chaetotaxic and ultrastructural observations have rarely been combined. We investigated the ultrastructure of cercarial sensory receptors in conjunction with chaetotaxy and neuromorphology in a species of Allopodocotyle. Cercariae were treated with acetylthiocholine iodide and silver nitrate, and some were processed for light, scanning (SEM), and transmission (TEM) electron microscopy. Five nerve regions were distinguished. Chaetotaxy was consistent with that of other opecoelids. Five types of receptors were distinguished with SEM. Types differed in number of cilium-like structures (one or more), length of cilium-like structure (short, moderately long, or long), presence or absence of a tegumentary collar, and length of tegumentary collar (low, moderately low, or very high). Internal ultrastructure of some types revealed unsheathed cilium-like structures, basal body, and thickened nerve collars. Possible subtegumentary and sheathed receptors are introduced. Some receptor types were site-specific. For example, receptors with multiple cilium-like structures were concentrated on cephalic region whereas receptors with short cilium-like structure were widespread throughout most regions. Ultrastructure and site-specificity observations suggest that most receptors are mechanoreceptors.

Sequential histological changes in Biomphalaria glabrata during the course of Schistosoma mansoni infection

Biomphalaria glabrata, highly susceptible to Schistosoma mansoni, were seen to shed less and less cercariae along the time of infection. Histological examination kept a close correlation with this changing pattern of cercarial shedding, turning an initial picture of no-reaction (tolerance) gradually into one of hemocyte proliferation with formation of focal encapsulating lesions around disintegrating sporocysts and cercariae, a change that became disseminated toward the 142nd day post miracidial exposure. Findings were suggestive of a gradual installation of acquired immunity in snails infected with S. mansoni.

Effects of the desiccation on Biomphalaria tenagophila (Orbigny, 1835) (Mollusca) infected by Schistosoma mansoni Sambon, 1907

Specimens of Biomphalaria tenagophila exposed to miracidia of Schistosoma mansoni were submitted to different desiccation periods as follows: group I: 24 h after exposure, desiccated for 28 days; group II: after cercariae elimination, desiccated for 7 days; group III: 21 days after exposure, desiccated for 7 days; group IV: 14 days after exposure, desiccated for 14 days; group V: 7 days after exposure, desiccated for 21 days. From the obtained data it was verified that desiccation was not capable of interrupting the development of larvae of S. mansoni in mollusks. A delay in the development of S. mansoni larvae in groups I, III, IV and V was observed. A pause was verified in the development of S. mansoni larvae in groups II, III, IV and V. Some larvae, in groups I, III, IV and V, did not suffer as a result of desiccation and continued their development. Larvae in the cercariae stage were shown to be more sensitive to desiccation. It was possible to obtain clearing of mollusks infected by sporocysts II and cercariae using a period of 7 days of desiccation.

Cloning and characterization of SmZF1, a gene encoding a Schistosoma mansoni zinc finger protein

The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1.

r-Sm14 - pRSETA efficacy in experimental animals

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.

Freshwater snails and schistosomiasis mansoni in the State of Rio de Janeiro, Brazil: I - metropolitan mesoregion

In order to elaborate a planorbid chart of the State of Rio de Janeiro a survey of freshwater gastropods in the Metropolitan Mesoregion of this State was performed and revealed the occurrence of 20 species: Antillorbis nordestensis (Lucena, 1954); Biomphalaria glabrata (Say, 1818); Biomphalaria schrammi (Crosse, 1864); Biomphalaria straminea (Dunker, 1848); Biomphalaria tenagophila (Orbigny, 1835); Burnupia sp.; Drepanotrema anatinum (Orbigny, 1835); Drepanotrema cimex (Moricand, 1839); Drepanotrema lucidum (Pfeiffer, 1839); Ferrissia sp.; Gundlachia ticaga (Marcus & Marcus, 1962); Heleobia davisi Silva & Thomé, 1985; Lymnaea columella Say, 1817; Melanoides tuberculatus (Müller, 1774); Physa cubensis Pfeiffer, 1839; Physa marmorata Guilding, 1828; Pomacea sp.; Pomacea canaliculata (Lamarck, 1822); Pomacea lineata (Spix, 1827) and Pomacea sordida (Swainson, 1823). Among the planorbid species B. tenagophila was the most frequent, occurring in all municipalities surveyed. The present study extends the distribution of B. straminea in the State of Rio de Janeiro and reports new records for A. nordestensis, B. schrammi, G. ticaga, H. davisi and the genera Burnupia and Ferrissia. An account about the current transmission areas of schistosomiasis mansoni in this Mesoregion is presented as well.

Cercarial Chaetotaxy and Sex Differentiation of Schistosoma mansoni Deriving from Humans and Nectomys squamipes (Muridae: Sigmondontinae) in Brazil

A comparative study was made between sympatric isolates of Schistosoma mansoni: one from a wild rodent (R) Nectomys squamipes and another one from humans (H) isolated from a low endemic schistosomiasis transmission area in Brazil. Our purpose was to detect differences between them concerning chaetotaxy (number and pattern of distribution of the argentophilic papillae) of the cercariae by means of silver impregnation. No significant difference (x > 0.05) between isolates was noted. Nevertheless, a significant difference (x < 0.05) was observed in the cercarial index (ratio of the distance between the first and the second preacetabular papillae and the distance between the first and the second dorsal preacetabular papillae) of male and female cercariae in both isolates. Males presented a greater cercarial index than females. By means of multivariate analysis, male cercariae were distinguished from female cercariae through the following characteristics: average number of dorsal papillae on the right quadrant, average number of ventral middle papillae on the right quadrant (H isolate) and average number of dorsal middle papillae on the left quadrant (R isolate). The results suggest that R and H isolates belong to the same population that could complete its life cycle in rodent-snail-rodent fashion.

Altered Response of Strain of Schistosoma mansoni to Oxamniquine and Praziquantel

The susceptibility of a fourth generation Ouh strain (Paranapanema Valley, São Paulo, Brazil) of Schistosoma mansoni to oxamniquine (OXA) and praziquantel (PZQ) was studied. Ten groups of 13 female albino mice each were infected with 70 cercariae per animal. These mice were medicated orally on the 50th day after infection. Five groups were given OXA doses of 0, 100, 200, 300 and 400 mg/kg (single doses) and the rest were treated with PZQ doses of 0, 100, 200, and 250 mg/kg/5 days. Each group was sub-divided: 8 animals underwent perfusion after 15 days treatment, 5 mice followed up for oviposition and their feces were tested every 15 days for miracidia hatching. The efficacy of the OXA doses of 100 and 200 mg/kg was 66% and 91.4%, respectively and for the 100 mg/kg PZQ dose it was 90.1%. The follow-up groups with 100 and 200 mg/kg of OXA and PZQ, 100 and 150 mg/kg, showed that they re-established the oviposition after a period of 60 to 75 days of treatment. The ED50 was 69.6mg/kg OXA and 39.4 mg/kg PZQ. The results show the tolerance of the Ouh strain to a dose of 100 mg with both drugs and they appoint the need for a dose review during the follow up of the oviposition and in monitoring phenomena in the field.

Freshwater snails and Schistosomiasis mansoni in the State of Rio de Janeiro, Brazil: II - Centro Fluminense Mesoregion

During the course of a survey carried out from 2000 to 2001 in the Centro Fluminense Mesoregion of the State of Rio de Janeiro 22 molluscan species were found. Many of the records are new due to the dearth of previous studies. Concerning the snail hosts of Schistosoma mansoni, the most frequently encountered species was Biomphalaria tenagophila, as it occurred in all the surveyed municipalities. There are new records of Biomphalaria straminea and Biomphalaria peregrina which is regarded as a potential intermediate host. Drepanotrema lucidum and Antillorbis nordestensis were found to be shedding echinostome cercariae and strigid cercariae respectively. An account about the current schistosomiasis transmission sites in this Mesoregion is presented as well.

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37°C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.

Freshwater snails and Schistosomiasis mansoni in the state of Rio de Janeiro, Brazil: III - Baixadas Mesoregion

In this paper, the third of a series dealing with the survey of freshwater gastropods of the state of Rio de Janeiro, the results of collections carried out in the Mesoregion Baixadas from 2000 to 2002 are presented. Twenty-two species, belonging to seven families, were found. As to the snail intermediate hosts of Schistosoma mansoni, the most frequent species was Biomphalaria tenagophila besides some new findings of Biomphalaria straminea. No specimens were found harboring larval forms of S. mansoni although different kinds of cercariae had been observed.