Mechanisms Mediating Antigenic Variation in Trypanosoma brucei

Antigenic variation in Trypanosoma brucei is a highly sophisticated survival strategy involving switching between the transcription of one of an estimated thousand variant surface glycoprotein (VSG) genes. Switching involves either transcriptional control, resulting in switching between different VSG expression sites; or DNA rearrangement events slotting previously inactive VSG genes into an active VSG expression site. In recent years, considerable progress has been made in techniques allowing us to genetically modify infective bloodstream form trypanosomes. This is allowing us to reengineer VSG expression sites, and look at the effect on the mechanisms subsequently used for antigenic variation. We can now begin a dissection of a highly complicated survival strategy mediated by many different mechanisms operating simultaneously.

Mitochondrial DNA variation of Triatoma infestans populations and its implication on the specific status of T. melanosoma

DNA sequence comparison of 412 base-pairs fragments of the mitochondrial cytochrome B gene was used to infer the genetic structure of nine geographical Triatoma infestans populations and their phylogenetic relationship with T. melanosoma and T. brasiliensis. T. infestans and T. melanosoma were compared by morphometry, allozyme and cytogenetic analyses, as well as subjected to reciprocal crosses, in order to clarify the taxonomic status of the latter. No differences were found to distinguish the two species and the crosses between them yielded progeny. T. infestans populations presented four haplotypes that could be separated in two clusters: one formed by the samples from Bolivia (Andes and Chaco) and the other formed by samples from Argentina and Brazil. Silvatic and domestic T. infestans populations from Bolivia (Andes) were genetically identical.

Variation in cuticular hydrocarbons among strains of the Anopheles gambiae sensu stricto by analysis of cuticular hydrocarbons using gas liquid chromatography of larvae

Cuticular hydrocarbons of larvae of individual strains of the Anopheles gambiae sensu stricto were investigated using gas liquid chromatography. Biomedical discriminant analysis involving multivariate statistics suggests that there was clear hydrocarbon difference between the Gambian(G3), the Nigerian (16CSS and, its malathion resistant substrain, REFMA) and the Tanzanian (KWA) strains. The high degree of segregation (95%) in hydrocarbons among the four strains investigated indicates that further analysis is needed to enable understanding of hydrocarbon variation in samples of An. gambiae especially from areas where these populations co-exist.

Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains

In this work, the susceptibility to benznidazole of two parental Trypanosoma cruzi strains, Colombian and Berenice-78, was compared to isolates obtained from dogs infected with these strains for several years. In order to evaluate the susceptibility to benznidazole two groups of mice were infected with one of five distinct populations isolated from dogs as well as the two parental strains of T. cruzi. The first group was treated with benznidazole during the acute phase and the second remained untreated controls. The animals were considered cured when parasitological and serological tests remained persistently negative. Mice infected with the Colombian strain and its isolates Colombian (A and B) did not cure after treatment. On the other hand, all animals infected with Berenice-78 were cured by benznidazole treatment. However, 100%, 50% and 70% of cure rates were observed in animals infected with the isolates Berenice-78 B, C and D, respectively. No significant differences were observed in serological profile of infected control groups, with all animals presenting high antibody levels. However, the ELISA test showed differences in serological patterns between mice inoculated with the different T. cruzi isolates and treated with benznidazole. This variability was dependent on the T. cruzi population used and seemed to be associated with the level of resistance to benznidazole.

Variation of the Oviposition Preferences of Aedes aegypti in Function of Substratum and Humidity

Two Aedes aegypti (L.) populations were studied in the laboratory regarding the preference for three types of breeding sites, i.e., flasks containing only water, flasks with a plant and flasks with a stick. Each of these breeding units was placed in one cage and the choice of the oviposition sites was determined for individual females and three females per experimental unit at two humidity levels. Preference for ovipositing on the water surface was observed and varied according to experimental unit and humidity. Mean hatching of eggs in water surface was 46.6%. Experiments with three females showed a more marked difference than when only one female was used. Inter and intrapopulation variability regarding oviposition sites was observed. The discrimination between the different oviposition substrates, hatching in water surface and its implication for mosquito control are discussed.

Time course of in vitro maturation of intra-erythrocytic malaria parasite: a comparison between Plasmodium falciparum and Plasmodium knowlesi

The schizont maturation assay for in vitro drug sensitivity tests has been a standard method employed in the global baseline assessment and monitoring of drug response in Plasmodium falciparum. This test is limited in its application to synchronous plasmodial infections because it evaluates the effect of drug on the maturation of parasite especially from ring to schizont stage and therefore synchronized P. falciparum cultures are required. On the other hand, P. knowlesi, a simian malaria parasite has a unique 24-h periodicity and maintains high natural synchronicity in monkeys. The present report presents the results of a comparative study on the course of in vitro maturation of sorbitol synchronized P. falciparum and naturally synchronous P. knowlesi. Ring stage parasites were incubated in RPMI medium supplemented with 10-15% pooled homologous serum in flat-bottomed 96-well micro plates using a candle jar at 37°C. The results suggest that the ideal time for harvesting the micro-assay plates for in vitro drug sensitivity test for sorbitol-synchronized P. falciparum and naturally synchronous P. knowlesi are from 26 to 30 h and from 22 to 25 h, respectively. The advantages of using P. knowlesi in chemotherapeutic studies are discussed.

Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil

Between 1985 and 2000, epidemiological surveys of the American tegumentary leishmaniasis (ATL) were carried out in several rural and urban communities in Espírito Santo, Brazil. A total of 100 stocks of Leishmania (comprising isolates from both human and canine hosts with ATL) were identified by two methods of molecular characterization, using specific monoclonal antibodies and multilocus enzyme electrophoresis. Parasite isolates from 19 municipalities were found to belong to the same zymodeme and serodeme type as of the Leishmania (Viannia) braziliensis reference strain. In contrast, our genotyping studies have shown intra-specific variation among these parasites (comparisons of the variability of the internal transcribed spacers between the small and large subunits of the rRNA genes of the 22 stocks studiedrevealed at least 11 genotypes). Two main clusters of L. (V.) braziliensis genotypes were observed, representing parasites collected from different endemic regions in the state, where transmission reflects distinct eco-epidemiological features. Infection with this pathogen was associated with the characteristic disease forms, but neither the clinical outcome nor the response to treatment could be related to the genetic polymorphism of the isolates, as defined by using the proposed methodology.

Louse infestation of the Chiribaya Culture, Southern Peru: variation in prevalence by age and sex

In order to improve the interpretive potential of archaeoparasitology, it is important to demonstrate that the epidemiology of ancient parasites is comparable to that of modern parasites. Once this is demonstrated, then we can be secure that the evidence of ancient parasitism truly reflects the pathoecology of parasitic disease. Presented here is an analysis of the paleoepidemiology of Pediculus humanus infestation from 146 mummies from the Chiribaya culture 1000-1250 AD of Southern Peru. The study demonstrates the modern parasitological axiom that 10% of the population harbors 70% of the parasites holds true for ancient louse infestation. This is the first demonstration of the paleoepidemiology of prehistoric lice infestation.

Re-infestation of houses by Triatoma dimidiata after intra-domicile insecticide application in the Yucatán peninsula, Mexico

In most countries, Chagas disease transmission control remains based on domestic insecticide application. We thus evaluated the efficacy of intra-domicile cyfluthrin spraying for the control of Triatoma dimidiata, the only Chagas disease vector in the Yucatán peninsula, Mexico, and monitored potential re-infestation every 15 days for up to 9 months. We found that there was a re-infestation of houses by adult bugs starting 4 months after insecticide application, possibly from sylvatic/peridomicile areas. This points out the need to take into account the potential dispersal of sylvatic/peridomestic adult bugs into the domiciles as well as continuity action for an effective vector control.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

Variation of snail's abundance in two water bodies harboring strains of Pseudosuccinea columella resistant and susceptible to Fasciola hepatica miracidial infection, in Pinar del Río Province, Cuba

The abundance of freshwater snails in two rural sites of Pinar del Río, Cuba, which harbor Pseudosuccinea columella susceptible and resistant to miracidia of Fasciola hepatica was followed for one year. Susceptible snails were found in the most anthropic site (IPA) whereas the resistant population inhabited the most preserved one (El Azufre). Only two snail species coexisted with P. columella at IPA site (Physa cubensis and Tarebia granifera) while five species were found at El Azufre, including an endemic from that province (Hemisinus cubanianus). Populations of both resistant and susceptible snails showed stable densities throughout the year, although the susceptible strain attained higher abundance. The highest densities were observed in April-May 2004 for the susceptible population whereas the resistant strain attained its highest abundance in January 2004. No record of Fossaria cubensis was made and the thiarid T. granifera occurred only at low densities. One of the sampled sites (IPA) meets all the conditions for the first report of P. columella naturally infected with larvae of F. hepatica.

Variation in the hatching response of Ochlerotatus albifasciatus egg batches (Diptera: Culicidae) in temperate Argentina

Egg hatching of winter-collected Ochlerotatus albifasciatus was studied for six months. Batches of eggs were divided into two groups, one of them was stored in the laboratory at 23°C and 12:12 photoperiod, and the other in the field under dead leaves. Every month, from July to December, eggs from the two groups were flooded under both laboratory and field conditions. Unhatched eggs were returned to the original condition and flooded two more times separated by ten-day intervals. Results show that egg diapause is expressed in different intensities, not only on eggs exposed to different conditions but also in those exposed to the same condition, even when they were laid by the same female. Successive inundations yielded incomplete hatches of eggs, and favored the hatching response in the next flooding. Low environmental temperatures before and during the flooding depressed hatching response. This shows that eggs need a warm period before flooding as well as warm temperatures during flooding, to hatch. As drought period was longer hatching response increased, but this was also accompanied by warmer environmental conditions. The experiment performed in laboratory did not show that increment. Field studies showed that a layer of dead leaves protected eggs from extreme temperatures.

Virulence attenuation and phenotypic variation of Paracoccidioides brasiliensis isolates obtained from armadillos and patients

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features.

Higher genetic variation estimated by microsatellites compared to isoenzyme markers in Aedes aegypti from Rio de Janeiro

Aedes aegypti populations from five districts in Rio de Janeiro were analyzed using five microsatellites and six isoenzyme markers, to assess the amount of variation and patterns of gene flow at local levels. Microsatellite loci were polymorphic enough to detect genetic differentiation of populations collected at small geographic scales (e.g. within a city). Ae. aegypti populations were highly differentiated as well in the city center as in the outskirt. Thus, dengue virus propagation by mosquitoes could be as efficient in the urban area as in the outskirt of Rio de Janeiro, the main entry point of dengue in Brazil.