Reproductive isolation between different forms of Lutzomyia longipalpis (Lutz & Neiva), (Diptera: Psychodidae), the vector of Leishmania donovani chagasi Cunha & Chagas and its significance to kala-azar distribution in South America

The males of the sandfly Lutzomyia longipalpis occur in two forms, one which bears a single pair of pale spots on tergite 4 and another in which an additional pair of spots characterizes tergite 3. In crosses between laboratory reared stocks of the two forms originating from allopatric and sympatric sites in Brazil nearly all males of one form fail to inseminate females of the other. In addition, insemination failure between some allopatric populaytions of Lu. longipalpis with similar tergal spot patterns is recorded, indicating the existence of additional forms in an apparent species complex. The possibility that Lu. longipalpis sensu latu represents more than a single taxon is discussed and the relevance of these findings to future epidemiological studies on kala-azar is considered.

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28§C and 37§C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page). Several proteins with molecular weights ranging from 34 kDa to 7 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could aldso be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discuted.

Methods for field detection of resistance to temephos in simuliids. Larval esterase level and topical application of the insecticide to adults

Two practical field methods for indirect detection of simuliid populations resistant to temephos are proposed. The first is based on high esterase activity in resistant larvae and involves adaptations of a filter paper test in which faintly stained spots indicate susceptible populations and strongly stained ones reveal populations resistant to temephos. The second is based on the resistance to the larvicide when adults are topically exposed, and involves the use of diagnostic doses obtained by the comparison between the LD50 for susceptible and resistant populations. The relevance of such methods is discussed in order to help resistance detection in Simulium pertinax Kollar control programmes.

Tegumental proteins of Schistosoma mansoni: complex biomolecules and potent antigens

The passive transfer of monoclonal antibodies, direct vaccination and in vitro assays have all shown that antigens associated with the tegumental membranes of Schistosoma mansoni are capable of mediating protective immune responses against the parasite in animal models. Furthermore, the principal antigens are highly antigenic during natural infection in man and stimulate strong humoral and cellular responses although, at present, their role in mediating protective immune responses in man remains equivocal. This presentation will review the current state of knowledge of the structure and expression of the major antigenic tegumental proteins of the schistosome and will attempt to relate the relevance of their structural features to possible function both in terms of protective immunity and parasite's ability to survive within the definitive host. A focus will be recent advances that have been made in the identification of means of anchoring of the antigenic proteins to the tegumental membrane. In addition, the implications of the structural complexity of the tegumental proteins in terms of their possible utility in vaccination and diagnosis will be considered.

Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro

We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels - hitherto observed in all experiments especially in those using HMC - CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continous fibroplastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background

A simple and economic slide micro-immunoenzymatic (Micro-SIA) test for epidemiological studies of toxoplasmosis

A slide micro-immunoenzymatic assay (micro-SIA) to detectantibodies to non-particulate Toxoplasma gondii antigens is described. This assay allows the diagnosis of toxoplasmosis infection in about 1 hr. Twenty-four determinations can be performed per slide. Five hundred ng of antigen and 5 or 10 µl drop of each reactive are necessary per well. The clear contrast of colours obtained for negative and positive sera after the test is finished, allows direct discrimination of the results. However, it is possible to quantify the results of the reaction using a minireader. Sera dilution cutoff value, determined as themost frequent titre for the general population, is 1:100. The toxoplasma micro-SIA correlates well with indirect immunofluorescence (IIF), its sensitivity is atleast three times as much as IIF. The test has an intra and inter assay variation coefficient of 5.46 per cent and of 6.24 per cent respectively. Sera obtained at random from argentinian people were analyzed and a 56 per cent of infection was found. The main features of the Toxoplasma micro-SIA are its simplicity, sensitivity, reproducibility, and the virtual absence of background making it very suitable for screening tests.

Schistosomiasis in a low prevalence area: incomplete urbanization increasing risk of infection in Paracambi, RJ, Brazil

The risk of schistosomiais infection and heavy infection in the locality of Sabugo was evaluated in relation to housing in areas with different urbanization development and to residential supply with snail-infested water. Critical sanitary conditions were found in areas of incomplete urbanization, where healthy water supply sources were scarce, and draining of sewage, without previous treatment, was made directly to the water-bodies used for domestic and leisure activities, despite being Biomphalaria tenagophila snail breeding-places. Stool examinations (Kato-Katz and Lutz methods) showed prevalence of 2.9%, mean intensity of 79 eggs per gram of stool and 47% of positive cases presenting intense infection. The use of snail-contaminated water for domestic purposes was considered a risk factor for infection. It is concluded that incomplete urbanization would facilitate transmission, probably enhancing the intensity of infection and that a low prevalence could hide a highly focal transmission. The relevance of these facts upon the efficiency of epidemiologic study methods and disease control planning are then discussed.

Defective production of interleukin 2 in patients with Chagas' disease: purified IL-2 augments in vitro response in patients with chagasic cardiomyopathy

The production of interleukin 2 (IL-2) by peripheral blood mononuclear cells, from patients with different clinical forms of Chagas disease and healthy controls, was evaluated after stimulation with Trypanosoma cruzi antigen, PPD and PHA. PHA induced higher production of IL-2 in infected patients than healthy controls. No diferences were found between infected groups. With PPD the trend was similar, the only difference was that asymptomatic infected patients (INF) showed higher levels of IL-2 production than patients with cardiomyopathy (CDM). With T. cruzi antigen, most patients showed little or no IL-2 production at 24 hr, a peak at 48 hr and an abrupt fall at 72 hr. A similar pattern of IL- 2 production was observed in INF and CDM. To evaluate the physiologic relevance of the deficit in IL-2 production, we studied the effect of non-mitogenic concentratios of IL-2 in the proliferative response to specific antigens. The addition of IL-2 only enhanced the proliferative response of CDM patients. These observations suggest that patients suffering Chagas' disease, particularly CDM, have a significant reduction in the capacity to produce IL-2. These findings could be of importance in the pathogenesis of Chagas' disease.

Mechanisms of cell accumulation induced by Mycobacterium bovis BCG

Mycobacteria, specially Mycobacterium tuberculosis are among the micro-organisms that are increasing dramatically the number of infections with death, all over the world. A great number of animal experimental models have been proposed to investigate the mechanisms involved in the host response against these intracellular parasites. Studies of airway infection in guinea-pigs and rabbits, as well as, in mice intravenously infected with BCG have made an important contribution to our understanding of the virulence, pathogenesis and the immunology of mycobacterial infections. Although, there are few models to study the mechanisms of the initial inflammatory process induced by the first contact with the Mycobacteria, and the relevance of the acute generation of inflammatory mediators, cytokines and leukocyte infiltration to the development of the mycobacterial infection. In this work we reviewed our results obtained with a model of M. bovis BCG-induced pleurisy in mice, describing the mechanisms involved in the leukocyte influx induced by BCG at 24 hr. Different mechanisms appear to be related with the influx of neutrophils, eosinophils and mononuclear cells and distinct inflammatory mediators, cytokines and adhesion molecules are involved in the BCG-induced cell accumulation.

External morphology of sensory structures of fourth instar larvae of neotropical species of phlebotomine sand flies (Diptera: Psychodidae) under scanning electron microscopy

In the present study, some morphological structures of antennae, maxillary palps and caudal setae of fourth instar larvae of laboratory-reared phlebotomine sand flies (Lutzomyia longipalpis, L. migonei, L. evandroi, L. lenti, L. sericea, L. whitmani and L. intermedia) of the State of Ceará, Brazil, were examined under scanning electron microscopy. The antennal structures exhibited considerable variation in the morphology and position. A prominent digitiform distal segment has been observed only on the antenna of species of the subgenus Nyssomyia. The taxonomic relevance of this and other antennal structure is discussed. The papiliform structures found in the maxillae and the porous structures of the caudal setae of all species examined may have chemosensory function. Further studies with transmission electron microscopy are needed to better understand the physiological function of these external structures.

Good bug, bad bug: in the case of enteric inflammatory disease does the epithelium decide?

Many studies demonstrate that intestinal inflammation is either initiated or exaggerated by a component of the normal microbiota, most likely commensal bacteria or products derived from these organisms. We review the nature of human inflammatory bowel disease, the evidence for the involvement of the normal bacterial flora in these disorders and the relevance of maintaining the integrity of the epithelial barrier. Moreover, we, and others, have shown abnormal mitochondria structure in tissue resections from patients with inflammatory bowel disease and tissues from rodents that demonstrated psychological stress-induced increases in epithelial permeability. Thus, we also consider the possibility that a defect in epithelial mitochondrial function would predispose an individual to respond to their commensal bacteria flora - no longer considering them as a beneficial passive inhabitant, but rather perceiving them as a threatening and pro-inflammatory stimulus. In support of this postulate, we discuss our recent findings from an in vitro model showing that the human colon-derived T84 cell line exposed to the metabolic stressor, dinitrophenol, and the non-pathogenic, non-invasive, Escherichia coli (strain HB101) display a loss of barrier function, increased signal transduction and increased production of the chemokine, interleukin 8.

Health at the time of Native-European contact in Southern Patagonia: First steps, results, and prospects

The objective of this paper is to present the first steps into the study of health in southern Patagonia during pre and post Native-European contact. Thus, our work has a double purpose. First, to discuss characteristics and relevance of human bone records of southern Patagonia, in order to study health in a population context. Second, to show some new lines of information, which include paleoparasitology, nutritional paleopathologies, and the study of lifestyles from human remains. In this context, we have started working on the first Spanish settlement "Nombre de Jesus", founded in 1584, and with historical documentation of "La Candelaria" Mission in Rio Grande (1896-1931).

Daily rhythm of aggregation in the haematophagous bug Triatoma infestans (Heteroptera: Reduviidae)

Triatomine bugs show a temporal modulation of many activities. Here, we analyse the daily modulation of the aggregation behaviour of Triatoma infestans larvae and its chronobiological basis. In the laboratory, groups of six bugs were released over an experimental arena during six consecutive days, where their aggregation behaviour was quantified every hour. When submitted to a 12/12 h photoperiod (L/D), the larvae of T. infestans exhibited a cyclic pattern of aggregation with a 24 h period, evincing the existence of a daily rhythm of aggregation in this species. Bugs exhibited the maximum aggregation tendency at the end of the scotophase (7:00 h), moment in which they naturally search for refuges. The minimum aggregation (i.e. maximal dispersion) was observed during the last part of the photophase and beginning of the scotophase (15:00 to 1:00 h). This cyclic pattern disappeared when constant conditions of illumination (L/L) or darkness (D/D) were imposed to the bugs, suggesting the absence of an endogenous circadian control of this behaviour. Insects submitted to L/L and D/D photoperiods presented lower global levels of aggregation than those submitted to L/D conditions. The lack of an endogenous control and the relevance of light cycles as a synchronization signal are discussed as the temporal modulation of this behaviour might play an important role in the nocturnal habits of this species.

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

Genomic analysis of a nontoxigenic, invasive Corynebacterium diphtheriae strain from Brazil

We report the complete genome sequence and analysis of an invasive Corynebacterium diphtheriae strain that caused endocarditis in Rio de Janeiro, Brazil. It was selected for sequencing on the basis of the current relevance of nontoxigenic strains for public health. The genomic information was explored in the context of diversity, plasticity and genetic relatedness with other contemporary strains.