Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.

Studies in a co-infection murine model of Plasmodium chabaudi chabaudi and Leishmania infantum: interferon-g and interleukin-4 mRNA expression

This work aimed to study the T helper type 1/2 (Th1/Th2) cytokine profile in a co-infection murine model of Plasmodium chabaudi chabaudi and Leishmania infantum. Expression of interferon-gamma (IFN-g) and interleukin-4 (IL-4) was analyzed, in spleen and liver of C57BL/6 mice, by reverse transcriptase-polymerase chain reaction. High levels of IFN-g expression did not prevent the progression of Leishmania in co-infected mice and Leishmania infection did not interfere with the Th1/Th2 switch necessary for Plasmodium control. The presence of IL-4 at day 28 in co-infected mice, essential for Plasmodium elimination, was probably a key factor on the exacerbation of the Leishmania infection.

Genetic structure of Triatoma venosa (Hemiptera: Reduviidae): molecular and morphometric evidence

Triatoma venosa presents a restricted geographical distribution in America and is considered as a secondary vector of Chagas disease in Colombia and Ecuador. A total of 120 adult insects were collected in domestic and peridomestic habitats in an endemic area of the department of Boyacá, Colombia, in order to determine their genetic structure through morphometric and molecular techniques. The head and wings of each specimen were used for the analyses of size, shape, and sexual dimorphism. A significant sexual dimorphism was found, although no differences in size among the studied groups were detected. Differences were found in the analyzed structures except for male heads. DNA was extracted from the legs in order to carry out the internal transcriber space-2 (ITS-2) amplification and the randon amplified polymorphic DNA (RAPD) analyses. Length polymorphisms were not detected in the ITS-2. Fst and Nm values were estimated (0.047 and 3.4, respectively). The high genetic flow found among the insects captured in the domicile and peridomiciliary environment does not permit a genetic differentiation, thus establishing the peridomicile as an important place for epidemiological surveillance.

Tethyan margins in space and time

Rifting processes, leading to sea-floor spreading, are characterized by a sequence of events: transtensive phase of extension with syn-rift volcanism; simple shear extension accompanied by lithospheric thinning and asthenospheric up-welling and thermal uplift of the rift shoulder and asymmetric volcanism. The simple shear model of extension leads to an asymmetric model of passive margin: a lower plate tilted block margin and an upper plate flexural, ramp-like margin- Both will be affected by thermal contraction and subsidence, starting soon after sea-floor spreading. Based on these actualistic models Tethyan margins are classified as one type or the other. Their evolution from the first transtensional phase of extension to the passive margin stage are analyzed. Four main rifting events are recognized in the Tethyan realm: an episode of lower Paleozoic events leading to the formation of the Paleotethys; a Late Paleozoic event leading to the opening of the Permotethys and East Mediterranean basin: an early Mesozoic event leading to the opening of the Pindos Neotethys and a Jurassic event related to the opening of the Alpine/Atlantic Neotethys. Type margins are given as example of each rifting event: -Northern Iran (Alborz) as a type area for the Late Ordovician to Silurian rifting of Paleotethys. -Northern India and Oman for the Late Carboniferous to early Permian rifting of Permotethys. -The East Mediterranean (Levant, Tunisia) as a Late Carboniferous rifting event. -The Neotethyan rifting phases are separated in two types: an eastern Pindos system found in Turkey and Greece is genetically linked to the Permotethys with a sea-floor spreading delayed until middle Triassic: a western Alpine system directly linked to the opening of the central Atlantic is characterized by a Late Triassic transtensive phase, an early to Middle Liassic break-away phase and. following sea-floor spreading, a thermal subsidence phase starting in Dogger. Problems related to the closure of the Paleozoic oceanic domains are reviewed. A Late Permian, early Triassic phase of `'docking'' between an European accretionary prism (Chios) and a Paleotethyan margin is supported by recent findings in the Mediterranean area. Back-arc rifting within the European active margin led to the formation of marginal seas during Permian and Triassic times and will contribute to the closure of the Paleozoic oceans.

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H); vector (Triatoma barberi) (RyC-V); and rodent reservoir (Peromyscus peromyscus) (RyC-R). The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes). Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40%) and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization

Incrimination of Anopheles (Nyssorhynchus) rangeli and An. (Nys.) oswaldoi as natural vectors of Plasmodium vivax in Southern Colombia

Malaria transmission in the Southern Colombian state of Putumayo continues despite the absence of traditional vector species, except for the presence of Anopheles darlingi near the southeastern border with the state of Amazonas. In order to facilitate malaria vector incrimination in Putumayo, 2445 morphologically identified Anopheles females were tested for natural infection of Plasmodium vivax by ELISA. Specimens tested included An. apicimacula (n = 2), An. benarrochi B (n = 1617), An. darlingi (n = 29), An. mattogrossensis (n = 7), An. neomaculipalpus (n = 7), An. oswaldoi (n = 362), An. peryassui (n = 1), An. punctimacula (n = 1), An. rangeli (n = 413), and An. triannulatus (n = 6). Despite being overwhelmingly the most anthropophilic species in the region and comprising 66.1% of the mosquitoes tested, An. benarrochi B was not shown to be a vector. Thirty-five An. rangeli and one An. oswaldoi were naturally infected with P. vivax VK210. Sequence data were generated for the nuclear second internal transcriber space region of 31 of these 36 vivax positive mosquitoes (86.1%) to confirm their morphological identification. An. oswaldoi is known to be a species complex in Latin America, but its internal taxonomy remains unresolved. Herein we show that the An. oswaldoi found in the state of Putumayo is genetically similar to specimens from the state of Amapá in Brazil and from the Ocama region in the state of Amazonas in Venezuela, and that this form harbors natural infections of P. vivax. That An. rangeli and this member of the An. oswaldoi complex are incriminated as malaria vectors in Putumayo, is a novel finding of significance for malaria control in Southern Colombia, and possibly in other areas of Latin America.

Evaluation of respiratory model employing conventional NIH mice to access the immunity induced by cellular and acellular pertussis vaccines

The increasing number of pertussis cases reported on the last twenty years and the existence of new acellular vaccines reinforce the need of research for experimental models to assure the quality of available pertussis vaccines. In this study, allotments of whole-cell and acellular pertussis vaccines were tested through the Intranasal Challenge Model (INM) using conventional NIH mice. The results have been compared to those achieved by the "Gold standard" Intracerebral Challenge Model (ICM). In contrast to ICM, INM results did not show intralaboratorial variations. Statistical analysis by Anova and Ancova tests revealed that the INM presented reproducibility and allowed identification and separation of different products, including three-component and four-component accellular pertussis vaccines. INM revealed differences between pertussis vaccines. INM provides lower distress to the mice allowing the reduction of mice number including the possibility of using conventional mice (less expensive) under non-aseptic environment. Thus, INM may be used as an alternative method of verifying the consistence of allotment production, including acellular pertussis vaccines.

Germline transformation of Aedes fluviatilis (Diptera:Culicidae) with the piggyBac transposable element

The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.

Morphological studies in a model for dengue-2 virus infection in mice

One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2). Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.

Histopathological study of Schistosoma mansoni infection in the murine model using the PC (Pará) and LILA (Maranhão) strains

Experimental models of Schistosoma mansoni infections in mammals have contributed greatly in understanding the pathology and pathogenesis of human infection. The absence of earlier reviews regarding specific strains of the Amazon region prompted research, which the main objective was to describe histopathological lesions in different phases of schistosomiasis in a murine model using PC (Pará) and LILA (Maranhão) S. mansoni strains. One hundred and eighty young female albino swiss mice (Mus musculus) were used and were randomly divided into five groups (PC-01, PC-02, LILA-01, LILA-02, and controls), according to the number of cercariae injected and the strain adopted. Animals were sacrificed in predetermined periods (35, 56, 112, 156, and 180 days) in an attempt to follow the evolution of the disease in the histological sections of their tissues at different phases of infection. Our findings were compatible with the data already described by others authors using different strains of S. mansoni, making it possible to identify some peculiarities, which are discussed in this work. In conclusion, the strains of parasite used did not modify the histopathological findings in the tissues of infected mice in any significant way when compared with the results of other studies using different strains.

Schistosome vaccine testing: lessons from the baboon model

The high level of protection elicited in rodents and primates by the radiation-attenuated schistosome vaccine gives hope that a human vaccine relying on equivalent mechanisms is feasible. In humans, a vaccine would be undoubtedly administered to previously or currently infected individuals. We have therefore used the olive baboon to investigate whether vaccine-induced immunity is compromised by a schistosome infection. We showed that neither a preceding infection, terminated by chemotherapy, nor an ongoing chronic infection affected the level of protection. Whilst IgM responses to vaccination or infection were short-lived, IgG responses rose with each successive exposure to the vaccine. Such a rise was obscured by responses to egg deposition in already-infected animals. In human trials it would be necessary to use indirect estimates of infection intensity to determine vaccine efficacy. Using worm burden as the definitive criterion, we demonstrated that the surrogate measures, fecal eggs, and circulating antigens, consistently overestimated protection. Regression analysis of the surrogate parameters on worm burden revealed that the principal reason for overestimation was the threshold sensitivity of the assays. If we extrapolate our findings to human schistosomiasis mansoni, it is clear that more sensitive indirect measures of infection intensity are required for future vaccine trials.

Biomphalaria tenagophila potencial vector of Schistosoma mansoni in the Paraná River basin (Argentina and Paraguay)

Susceptibility and compatibility experiments were carried out with 700 Biomphalaria tenagophila from the Paraná River basin exposed to infection with Schistosoma mansoni. Individual infection was performed with 10 miracidia of SJ2 strain from the Paraiba valley (Brazil) originally infective to B. tenagophila. These snails were laboratory-breed progeny of B. tenagophila collected from six localities of Argentina and one from Paraguay. From Argentina: Rincón de Vences (7%) and Posadas (11%) became infected with S. mansoni and the calculation of Frandsen's index (TCP/100) shows that they were Class II poorly compatible. Those snails from Goya (22%), Maloyas (5%), and Berón de Astrada (3%) were Class III compatible to the S. mansoni. None of the 100 snails exposed from Caá-Catí became infected (Class 0 incompatible). Tested samples from Paraguay (Encarnación) were infected (20%) and compatible (Class III). It was also studied the persistence of the infection in 244 snails of the first generation (F1) of those that were susceptible from three places. It was demonstrated an increment of the susceptibility in the F1 from Maloyas (chi2 = 27.22; p = 0.0001) and Posadas (chi2 = 4.24; p = 0.04). The results point out the possibility that schistosomiasis might be able to spread into the Paraná River basin where B. tenagophila exists.

Vector bionomics and malaria transmission in the Upper Orinoco River, Southern Venezuela

A longitudinal epidemiological and entomological study was carried out in Ocamo, Upper Orinoco River, between January 1994 and February 1995 to understand the dynamics of malaria transmission in this area. Malaria transmission occurs throughout the year with a peak in June at the beginning of the rainy season. The Annual Parasite Index was 1,279 per 1,000 populations at risk. Plasmodium falciparum infections accounted for 64% of all infections, P. vivax for 28%, and P. malariae for 4%. Mixed P. falciparum/P. vivax infections were diagnosed in 15 people representing 4% of total cases. Children under 10 years accounted for 58% of the cases; the risk for malaria in this age group was 77% higher than for those in the greater than 50 years age group. Anopheles darlingi was the predominant anopheline species landing on humans indoors with a biting peak between midnight and dawn. A significant positive correlation was found between malaria monthly incidence and mean number of An. darlingi caught. There was not a significant relationship between mean number of An. darlingi and rainfall or between incidence and rainfall. A total of 7295 anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite (CS) protein. Only An. darlingi (55) was positive for CS proteins of P. falciparum (0.42%), P. malariae (0.25%), and P. vivax-247 (0.1%). The overall estimated entomological inoculation rate was 129 positive bites/person/year. The present study was the first longitudinal entomological and epidemiological study conducted in this area and set up the basic ground for subsequent intervention with insecticide-treated nets.

A study of the scutellum in eight Chagas disease vector species from genus Triatoma (Hemiptera, Reduviidae) using optical and scanning electron microscopy

The aim of this study was to analyze the external morphology of the scutellum through optical microscopy and scanning electron microscopy (SEM) in male specimens of Triatoma costalimai, T. delpontei, T. eratyrusiformis, T. matogrossensis, T. infestans melanosoma, T. sherlocki, T. tibiamaculata, and T. vandae. A total of 30 photographs of the scutellum were made. Magnification varied from 50X to 750X. Regarding depth and forms of the central depression, the heart-shaped form was predominant, with some exceptions, so that this shape appears to be a common characteristic for species of genus Triatoma Laporte, 1832. In T. eratyrusiformis, a kind of sensillum with important taxonomic value was observed. The different sizes and shapes of the designs found on the posterior process of the scutellum were also of important taxonomic interest. The study of the scutellum based on SEM showed valuable characteristics, allowing the use of this structure to aid the diagnosis of triatomine species. Thus, more specimens in subsequent studies and analyses of morphometric parameters should contribute to agreement on phylogenetic aspects in this genus. A Key to eight species of Triatoma based on male scutellar morphology is presented.

Cloning and characterization of a V-ATPase subunit C from the American visceral leishmaniasis vector Lutzomyia longipalpis modulated during development and blood ingestion

Visceral leishmaniasis (VL) is a serious tropical disease that affects approximately 500 thousand people worldwide every year. In the Americas, VL is caused by the parasite Leishmania (Leishmania) infantum chagasi mainly transmitted by the bite of the sand fly vector Lutzomyia longipalpis. Despite recent advances in the study of interaction between Leishmania and sand flies, very little is known about sand fly protein expression profiles. Understanding how the expression of proteins may be affected by blood feeding and/or presence of parasite in the vector's midgut might allow us to devise new strategies for controlling the spread of leishmaniasis. In this work, we report the characterization of a vacuolar ATPase subunit C from L. longipalpis by screening of a midgut cDNA library with a 220 bp fragment identified by means of differential display reverse transcriptase-polymerase chain reaction analysis. The expression of the gene varies along insect development and is upregulated in males and bloodfed L. longipalpis, compared to unfed flies.