Revisiting the Phlebotominae subfamily records in Bahia, Brazil.

Brazil is one of the countries that concentrates 90% of all tegumentary and visceral leishmaniases cases and Bahia is one of the highly affected states. In the present report, we consolidated secondary data from several complementary databases that allowed us to record the sand fly species identified including areas of Leishmania spp. transmission in the state of Bahia. We then overlayed the geographical distribution data onto maps of vegetational aspects found across the state. Overall, 21 602 records of phlebotomine sand flies occurrence between 1949 and 2016 were analysed, encompassing 85% of Bahia's municipalities. Seventy-six sand fly species under 17 genera were enlisted. Among described species, 27 were proven or putative Leishmania spp. vectors and three were considered exclusively endemic in the state. Lutzomyia longipalpis, Nyssomyia intermedia and Nyssomyia whitmani were found in 74, 29 and 27% of municipalities, respectively. Salvador, the state capital and major city presented records for 21 different sand fly species, including known vectors for leishmaniasis. In particular, a wide distribution of Evandromyia sallesi was detected for this city. This consolidated account on phebotomine fauna and distribution may be explored for improving the planning and deployment of vector-focused leishmaniasis control measures in affected areas of Bahia.

The human platelet alloantigen profile in blood donors from Amazonas, Brazil.

BACKGROUND: Human platelet antigens (HPAs) are alloantigens derived from polymorphisms in platelet-surface glycoproteins. The occurrence of alloantibodies against HPAs can lead to platelet destruction and subsequent thrombocytopenia. Brazilians have a high rate of racial admixture, and the knowledge of HPA polymorphisms in particular donors from north Brazil, who have a large Amerindian influence, is a relevant strategy to prevent alloimmunisation. OBJECTIVE: Our aim was investigate the HPA allele's frequencies in the Amazonas blood donors. METHODS: We performed HPA genotyping among 200 Amazonas blood donors by microarray for 11 HPA biallelic systems, including six of the most clinically significant systems (HPA-1 to -5 and -15) and five others (HPA-6 to -9 and -11) that have been also associated with alloimmunisation, amounting to 22 HPA alleles. RESULTS: The obtained allele frequencies were compared with data of 38 populations worldwide to determine the hierarchical relationship and estimated the probability of mismatch platelets. The allele frequencies were 0·862 for HPA-1a, 0·137 for HPA-1b, 0·852 for HPA-2a, 0·147 for HPA-2b, 0·665 for HPA-3a, 0·335 for HPA-3b, 0·995 for HPA-4a, 0·005 for HPA-4b, 0·892 for HPA-5a, 0·107 for HPA-5b, 0·997 for HPA-9a, 0·005 for HPA-9b, 0·502 for HPA-15a and 0·497 for HPA-15b. The incompatibility risks are higher for HPA-15 and HPA-3, followed by HPA-1, -2 and -5. CONCLUSION: We found differences among populations worldwide, and it is interesting to note the indigenous and European influences in this region, reinforcing the heterogeneity in the ancestry of Brazilians. The results will be helpful in providing information for platelet transfusion to avoid alloimmunisation.

IL-17 and Regulatory Cytokines (IL-10 and IL-27) in L. braziliensis Infection.

Cutaneous leishmaniasis (CL) is characterized by high production of pro-inflammatory cytokines and development of pathology. Individuals with subclinical L. braziliensis infection (SC) have a positive skin test to leishmania, but do not develop disease. We evaluated whether the downregulation of inflammatory response in SC is mediated by IL-10 and IL-27 and whether IL-17 is associated with control of infection. Participants include SC individuals, patients with CL and healthy subjects. Cytokines protein and mRNA were detected by ELISA and real-time PCR. IFN-γ and TNF-α levels were higher in CL than in SC group. The IL-10 levels and mRNA for IL-10 were similar in both SC and CL. mRNA for IL-27 was increased in cells from SC after stimulation with L. braziliensis antigen. There was a tendency for increased levels of IL-17 in SC compared to CL. The weak type 1 immune response observed in SC L. braziliensis infection is not because of the regulatory effects of IL-10 and IL-27. The control of Leishmania infection may be mediated by innate immune response with participation of IL-17. The results from this pilot study warrant further larger studies to investigate the potential contributions of IL-17 and IL-27 to the control of L. braziliensis infection.

Atypical manifestations of tegumentary leishmaniasis in a transmission area of Leishmania braziliensis in the state of Bahia, Brazil.

American tegumentary leishmaniasis (ATL) can occur in different forms, classically categorised as cutaneous leishmaniasis, mucosal leishmaniasis, diffuse cutaneous leishmaniasis and disseminated leishmaniasis. We analysed the presence of atypical manifestations (vegetative, verrucous, crusted and lupoid) among a cohort of patients presenting to the Health Post of Corte de Pedra, Bahia, Brazil. Among 1396 patients diagnosed with ATL in 2005-2006, 35 patients (2.5%) presented with atypical manifestations of the disease. Of these patients, 14 were pregnant women, 2 were co-infected with HIV and 19 had no co-morbidity or other apparent risk factors for the development of atypical ATL. The latter 19 patients were the focus of this study. They were predominantly adult males, frequently presenting with facial lesions [P<0.001; odds ratio (OR)=17.5, 95% CI 6.1-52.4] and had higher rates of treatment failure with antimonial therapy (P<0.001; OR=327, 95% CI 45-6668) compared with patients with classic ATL attending in the same period. Thirteen cases healed with amphotericin B, introduced after failure of three or more courses of antimony, suggesting that amphotericin B should be considered as the drug of choice for all patients diagnosed with atypical ATL.

Infective dermatitis has similar immunological features to human T lymphotropic virus-type 1-associated myelopathy/tropical spastic paraparesis.

Human T lymphotropic virus-type 1 (HTLV-1) is the causal agent of the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), adult T cell leukaemia/lymphoma and infective dermatitis associated with HTLV-1 (IDH). Over-production of proinflammatory cytokines and an increase in HTLV-1 proviral load are features of HAM/TSP, but the immunological basis of IDH has not been established. In addition to severe cutaneous manifestations, the importance of IDH relies on the observation that up to 30% of children with IDH develop HAM/TSP in childhood and adolescence. In this study we determined the immune response in patients with IDH measuring interleukin (IL)-4, IL-5, IL-10, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha levels as well as the HTLV-1 proviral load. Additionally, regulatory cytokines and anti-cytokines were added to cultures to evaluate the ability of these molecules to down-modulate TNF-alpha and IFN-gamma synthesis. HTLV-1 carriers and patients with HAM/TSP served as controls. TNF-alpha and IFN-gamma levels were higher in IDH than in HTLV-1 carriers. There was no difference in IFN-gamma and TNF-alpha concentrations in IDH and HAM/TSP patients. There was a tendency for higher IL-4 mRNA expression and immunoglobulin E (IgE) levels in IDH than in HTLV-1 carriers, but the difference did not reach statistical significance. The HTLV-1 proviral load was significantly higher in IDH patients than in HTLV-1 carriers. IDH is characterized by an exaggerated Th1 immune response and high HTLV-1 proviral load. The similarities between the immunological response in patients with IDH and HAM/TSP and the high proviral load observed in IDH provide support that IDH is a risk factor for development of HAM/TSP.

Expression of the virulence factor, BfpA, by enteropathogenic Escherichia coli is essential for apoptosis signalling but not for NF-kappaB activation in host cells.

Localized adherence (LA) of enteropathogenic Escherichia coli (EPEC) to epithelial cells results in attaching and effacing of the surface of these cells. LA depends on the gene bfpA, which codes for the BfpA protein. We found that EPEC-E. coli adherence factor (EAF)((+)), expressing BfpA, significantly reduced HeLa cell viability in comparison with EPEC-EAF((-)), as evaluated by the mitochondrial-dependent succinate dehydrogenase conversion of 3'-[4,5,-dimethylthiazol-2yl]2,5-diphenyltetrazolium bromide (MTT) to its formazan. Apoptosis accounts for a substantial loss of the cell viability, because the cells incubated with EPEC-EAF((+)) or with cloned BfpA (data not shown), but not with EPEC-EAF((-)), were positive for annexin-V binding, demonstrated chromatin condensation and nuclei fragmentation and exhibited a high level of caspase-3 activity. Because the blockade of bacterial cell-surface-associated BfpA by anti-BfpA immunoglobulin (Ig)Y antibody suppressed apoptotic death induced by EPEC-EAF((+)), BfpA may be the trigger for apoptosis. Both EPEC-EAF((+)) and EPEC-EAF((-)), as well as recombinant BfpA (data not shown), activated nuclear factor (NF)-kappaB in a similar manner as analysed by the electrophoretic mobility shift assay (EMSA). EMSA supershift analysis demonstrated the presence of p65/RelA in a DNA-binding complex. In contrast to DNA binding, NF-kappaB-dependent reporter gene transactivation was stimulated more strongly by EPEC B171/EAF((+)), suggesting a role for this virulence factor in the regulation of transcriptional activity of NF-kappaB. Because suppression of NF-kappaB activation by BAY11-7085, a NF-kappaB inhibitor, neither induced apoptosis by itself nor blocked apoptosis induction by EPEC-EAF((+)), it may be suggested that apoptosis is not regulated by the NF-kappaB pathway in HeLa cells.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Multiclonal Leishmania braziliensis population structure and its clinical implication in a region of endemicity for American tegumentary leishmaniasis.

In Corte de Pedra (CP), northeastern Brazil, Leishmania braziliensis causes three distinct forms of American tegumentary leishmaniasis (ATL). To test the hypothesis that strain polymorphism may be involved in this disease spectrum and accurately characterize the parasite population structure in CP, we compared one L. major, two non-CP L. braziliensis, one CP L. amazonensis, and 45 CP L. braziliensis isolates, obtained over a 10-year period from localized cutaneous, mucosal, and disseminated leishmaniasis patients, with randomly amplified polymorphic DNA (RAPD). Electrophoretic profiles were mostly unique across species. All typing protocols revealed polymorphism among the 45 CP L. braziliensis isolates, which displayed eight different RAPD patterns and greater than 80% overall fingerprint identity, attesting to the adequacy of the tools to assess strain variability in CP's geographically limited population of parasites. The dendrogram based on the sum of RAPD profiles of each isolate unveiled nine discrete typing units clustered into five clades. Global positioning showed extensive overlap of these clades in CP, precluding geographic sequestration as the mechanism of the observed structuralization. Finally, all forms of ATL presented a statistically significant difference in their frequencies among the clades, suggesting that L. braziliensis genotypes may be accompanied by specific disease manifestation after infection.

Egg yolk anti-BfpA antibodies as a tool for recognizing and identifying enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a major aetiological agent of childhood diarrhoea in developing countries. The structural repeating protein A subunit, BfpA, found in the bundle-forming pilus, is one of the virulent factors for EPEC pathogenesis. Recombinant BfpA in laying hens elicited sustained and vigorous antibody production. Immunoglobulin Y (IgY) anti-BfpA antibodies were recovered from egg yolk, purified and characterized. Immunoadsorption with whole extracts of the isogenic E. coli EPEC adherence factor (EAF) strain that lacks BfpA rendered the resulting IgY preparations capable of: (a) recognizing purified or recombinant BfpA proteins in a dose-dependent fashion; (b) blocking the colonization of HeLa cells by EPEC EAF+, in vitro; (c) specifically identifying E. coli bearing EAF+; and (d) inhibiting the growth of E. coli EAF+ but not the EAF strain. IgY anti-BfpA is potentially useful as a specific, low-cost immunobiological reagent to screen human faecal specimens for the presence of EPEC.

Expression of a pilin subunit BfpA of the bundle-forming pilus of enteropathogenic Escherichia coli in an aroA live salmonella vaccine strain.

Enteropathogenic Escherichia coli (EPEC) is a major cause of childhood diarrhea in developing countries and is a leading cause of severe diarrheal illness among Brazilian infants. As one approach to constructing a vaccine candidate against diarrhea caused by EPEC, we evaluated whether the pilin subunit (BfpA) of the bundle-forming pilus (BFP) could be expressed by a live Salmonella vaccine strain. Several copies of the coding region of BfpA (bfpA) were amplified by PCR from a preparation of the EAF plasmid of EPEC strain B171 and cloned into plasmid vectors. An intact copy of bfpA was subcloned into the heat inducible prokaryotic expression vector pCYTEXP1, and the resulting pBfpA was used to transform the aroA S. typhimurium strain SL3261, generating SL3261(pBfpA). The recombinant vaccine strain was able to express, but not to process, rBfpA as evidenced by a prominent 21 kDa protein that crossreacted with anti-BFP antiserum found only in extracts of heat-treated SL3261(pBfpA), but not in strains of untreated SL3261(pBfpA) or SL3261 not carrying the plasmid. Furthermore, rBfpA accumulation was not toxic to the Salmonella host, as evidenced by similar plating efficiencies between induced and uninduced strains of SL3261(pBfpA). Finally, SL3261(pBfpA) orally administered to BALB/c mice was capable of eliciting a sustained and vigorous humoral immune response to BfpA, achievable even with a single oral dose of approximately 10(9) organisms. Therefore, this pilin product may serve as a potential immunogen as part of a live combined vaccine strategy to prevent two of the major public health problems in Brazil--salmonellosis and EPEC childhood diahrrea.

Molecular cloning and characterization of ConBr, the lectin of Canavalia brasiliensis seeds.

ConBr, a lectin isolated from Canavalia brasiliensis seeds, shares with other legume plant lectins from the genus Canavalia (Diocleinae subtribe) primary carbohydrate recognition specificity for D-mannose and D-glucose. However, ConBr exerts different biological effects than concanavalin A, the lectin of Canavalia ensiformis seeds, regarding induction of rat paw edema, peritoneal macrophage spreading in mouse, and in vitro human lymphocyte stimulation. The primary structure of ConBr was established by cDNA cloning, amino acid sequencing, and mass spectrometry. The 237-amino-acid sequence of ConBr displays Ser/Thr heterogeneity at position 96, indicating the existence of two isoforms. The mature Canavalia brasiliensis lectin monomer consists of a mixture of predominantly full-length polypeptide (alpha-chain) and a small proportion of fragments 1-118 (beta-chain) and 119-237 (gamma-chain). Although ConBr isolectins and concanavalin A differ only in residues at positions 58, 70, and 96, ConBr monomers associate into dimers and tetramers in a different pH-dependent manner than those of concanavalin A. The occurrence of glycine at position 58 does not allow formation of the hydrogen bond that in the concanavalin A tetramer exists between Asp58 of subunit A and Ser62 of subunit C. The consequence is that the alpha carbons of the corresponding residues in ConBr are 1.5 A closer that in concanavalin A, and ConBr adopts a more open quaternary structure than concanavalin A. Our data support the hypothesis that substitution of amino acids located at the subunit interface of structurally related lectins of the same protein family can lead to different quaternary conformations that may account for their different biological activities.

Serum soluble markers in the evaluation of treatment in human visceral leishmaniasis.

Visceral leishmaniasis (VL) has a fatal course if not properly treated. Recovery from VL is linked to cellular immune response. Unresponsiveness to antimonial therapy reinforces the importance of determining parameters for treatment assessment. We analysed the pre- and post-treatment serum levels of soluble CD4 (sCD4), sCD8, sIL-2R, soluble intercellular adhesion molecule-1 (sICAM-1) and neopterin in groups of VL patients either responsive or not to standard antimonial therapy. Pretreatment serum levels of all markers except for sICAM-1 were significantly higher in VL patients than in healthy subjects from the same area (P < 0.05). sICAM-1 levels were similar in healthy controls and in VL patients refractory to antimonial therapy (P = 0.25), but significantly higher in patients responsive to treatment (P = 0.02). The comparison of pre- and post-treatment concentrations showed that all markers, except sCD4 and sICAM-1, presented a significant fall (P < 0.05) in patients responsive to antimonial therapy. However, only neopterin presented with levels compatible with those of healthy subjects at the end of treatment (P = 0.30). In refractory patients sICAM-1 presented with post-treatment levels significantly higher than the pretreatment determinations (P = 0.03), while sCD4 experienced a significant drop (P = 0.01). All markers displayed clearly distinct behaviour according to the patient's response to therapy. This makes all soluble molecules studied suitable for use as indicators of antimonial therapy response. Additionally the comparison of pretreatment levels of the markers between responders and refractory patients to antimonial therapy showed that serum concentrations of sIL-2R and sICAM-1 significantly differed among these two groups (P = 0.02 in each case), suggesting that they may be used in future as predictors of antimonial therapy response.

Serum levels of bothropic venom in patients without antivenom intervention.

Serum kinetics of bothropic venom were evaluated in eight snakebite patients, who due to a national shortage, received no specific antivenom therapy. The cases were clinically classified as mild, moderate, and severe. Patients were bled sequentially and serum levels of venom were assayed by ELISA. Venom level ranges differed among the groups, with peak levels of less than 13 ng/ml, 32 ng/ml, and 120 ng/ml for the mild, moderate, and severe groups, respectively. There was no clear pattern of kinetics in the groups. Regression analysis involving the variables severity and peak venom levels yielded a statistically significant correlation (rs = 0.80, P less than 0.05). These data indicate that different amounts of circulating venom correlate with clinical severity, even in highly complex venoms, and stress the importance of careful clinical classification in the proper management of bothropic incidents.

Immunological studies with the venom of the scorpion Tityus serrulatus.

1. We describe a "sandwich" enzyme-linked immunosorbent assay (ELISA) sensitive to quantities of scorpion (Tityus serrulatus) venom (TsV) in the range of 1-3 ng/ml sample. 2. Cross-reactivity with the venom from the rattlesnake Crotalus durissus terrificus and with venoms from several snakes of the Bothrops genus was detected only at concentrations higher than 1 microgram/ml sample. 3. A conventional ELISA is also described for the detection of antibodies against TsV. 4. Analysis by Western Blot (WB) demonstrated a 25-kDa protein band common to TsV and to the venoms of Bothrops moojeni, B. jararacussu and B. jararaca. 5. Venom from C. d. terrificus exhibited WB cross-reactive bands of 16 and 25 kDa with TsV.

Immunological studies with the venom of the scorpion Tityus serrulatus

We describe a "sandwich" enzyme-linkedimmunosorbent assay (ELISA) sensitive to quantities of scorpion (Tityus serrulatus) venom (TsV) in the range of 1-3 ng?ml sample. Cross-reactivity with the venom from the rattlesnake Crotalus durissus terrificus and with venoms from several snakes of the Bothrops genus was detected only at concentrations higher than 1 *g/ml sample. A conventional ELISA is also described for the detection of antibodies against TsV. Analysis by Western Blot (WB) demonstrated a 25-kDa protein band common to TsV and to the venoms of Bothrops moojeni, B. jararacussu and B. jararaca. Venom from C. d. terrificus exhibited WB cross-reactive bands of 16 and 25 kDa with TsV

Enzyme-linked immunosorbent assay for the detection of Bothrops jararaca venom.

This study reports an enzyme-linked immunosorbent assay for detecting Bothrops jararaca venom in fluids, employing the sandwich method with biotin/avidin amplification. The assay exhibits high accuracy in correlating optical densities with venom concentrations (r = 0.98), high reproducibility, low background and limited cross-reactivity with venom from other snake genera. Nevertheless, it was unable to distinguish among venoms from different bothropic species. Using this method we evaluated the serum kinetics of Bothrops jararaca venom in C57BL/6 mice. High concentrations were found in serum just 15 min after injection (151 +/- 41 ng/ml; mean +/- S.D.), followed by a progressive fall (102 +/- 46, 74 +/- 39 and 50 +/- 22 ng/ml after 1, 3 and 6 hr respectively), being undetectable by 24 hr. Such serum kinetics indicates a pattern of a rapid absorption of venom from the inoculation site, followed by a slow and progressive drop in its serum levels. This ELISA was a reliable tool in the determination of Bothrops jararaca venom levels in mouse serum, and may become useful in other fields of bothropic venom research.