Development of an antibody against EtpA from enterotoxigenic Escherichia coli and evaluation of its use for bacterial isolation using magnetic beads.

The enterotoxigenic Escherichia coli (ETEC) strain is one of the most frequent causative agents of childhood diarrhea and travelers' diarrhea in low-and middle-income countries. Among the virulence factors secreted by ETEC, the exoprotein EtpA has been described as an important. In the present study, a new detection tool for enterotoxigenic E. coli bacteria using the EtpA protein was developed. Initially, antigenic sequences of the EtpA protein were selected via in silico prediction. A chimeric recombinant protein, corresponding to the selected regions, was expressed in an E. coli host, purified and used for the immunization of mice. The specific recognition of anti-EtpA IgG antibodies generated was evaluated using flow cytometry. The tests demonstrated that the antibodiesdeveloped were able to recognize the native EtpA protein. By coupling these antibodies to magnetic beads for the capture and detection of ETEC isolates, cytometric analyses showed an increase in sensitivity, specificity and the effectiveness of the method of separation and detection of these pathogens. This is the first report of the use of this methodology for ETEC separation. Future trials may indicate their potential use for isolating these and other pathogens in clinical samples, thus accelerating the diagnosis and treatment of diseases.

A hybrid protein containing MSP1a repeats and Omp7, Omp8 and Omp9 epitopes protect immunized BALB/c mice against anaplasmosis.

Anaplasma marginale (A. marginale) has a remarkable impact on livestock production, and an effective vaccine is not currently available due to the inexistence of a small animal model. Recently, BALB/c mice were successfully infected with A. marginale, resulting in an acute and persistent anaplasmosis infection. Here, we designed a hybrid protein containing repeats of polypeptide 1a from major surface protein-1 complex (MSP1a) repeats and common epitopes of outer membrane proteins (OMPs) OMP7, OMP8 and OMP9 expressed in Escherichia coli. Our proof-of-concept assessed vaccinal effectiveness against a challenge with live bacteria. The MSP1a/OMP7/8/9 immunized BALB/C mice exhibited a strong reduction in rickettsemia and had no signs of anaplasmosis or hepatic lesions. In contrast, the non-immunized mice exhibited signs of anaplasmosis and a body weight loss associated with increases in monocyte and neutrophil counts. Furthermore, the non-immunized mice displayed atrophies with chronic inflammatory infiltrates in the spleen and increased binucleation and hydropic degeneration in the hepatocytes. Our findings demonstrated that immunization with our hybrid protein induced a strong reduction in rickettsemia and conferred protection against anaplasmosis. Therefore, given the strong evidence of the protective effect against anaplasmosis, hybrid protein designs are potential candidates for the rational design of vaccinal subunits.

Phenotypic and Molecular Aspects of Staphylococcus spp. Isolated from Hospitalized Patients and Beef in the Brazilian Amazon.

The aim of this study was to characterize and compare Staphylococcus spp. isolated from hospitalized patients and beef marketed in the city of Porto Velho-RO, Brazil. The isolates were subjected to antibiogram tests, adherence capacity tests, detection of the mecA gene, and epidemiological investigation by the random amplified polymorphic DNA (RAPD) technique, using the primers M13 and H12. Among the 123 Staphylococcus spp. isolates, 50 were identified as S. aureus and 73 as coagulase-negative Staphylococcus; among the latter, 7 species were identified. It was observed that the coagulase-negative Staphylococcus isolates showed greater adhesion ability than S. aureus. The profile of antimicrobial susceptibility was different among isolates, all of which were susceptible to vancomycin and linezolid, and had high penicillin resistance rates, varying according to the bacterial class and the source. In this study, all strains were negative for mecA gene detection; however, 36% of S. aureus and 17% of coagulase-negative Staphylococcus were resistant to oxacillin. The genetic relationship of these bacteria, analyzed by RAPD, was able to discriminate the species of coagulase-negative Staphylococcus strains of S. aureus along its origin. It was concluded that the isolates of Staphylococcus spp. derived from beef and human infections differ genetically. Thus, it is suggested that isolates from beef, which were grouped within hospital isolates, were probably carried via contact with beef in hospital professionals or patients.

Proteomic analysis of Chromobacterium violaceum and its adaptability to stress.

BACKGROUND: Chromobacterium violaceum (C. violaceum) occurs abundantly in a variety of ecosystems, including ecosystems that place the bacterium under stress. This study assessed the adaptability of C. violaceum by submitting it to nutritional and pH stresses and then analyzing protein expression using bi-dimensional electrophoresis (2-DE) and Maldi mass spectrometry. RESULTS: Chromobacterium violaceum grew best in pH neutral, nutrient-rich medium (reference conditions); however, the total protein mass recovered from stressed bacteria cultures was always higher than the total protein mass recovered from our reference culture. The diversity of proteins expressed (repressed by the number of identifiable 2-DE spots) was seen to be highest in the reference cultures, suggesting that stress reduces the overall range of proteins expressed by C. violaceum. Database comparisons allowed 43 of the 55 spots subjected to Maldi mass spectrometry to be characterized as containing a single identifiable protein. Stress-related expression changes were noted for C. violaceum proteins related to the previously characterized bacterial proteins: DnaK, GroEL-2, Rhs, EF-Tu, EF-P; MCP, homogentisate 1,2-dioxygenase, Arginine deiminase and the ATP synthase ß-subunit protein as well as for the ribosomal protein subunits L1, L3, L5 and L6. The ability of C. violaceum to adapt its cellular mechanics to sub-optimal growth and protein production conditions was well illustrated by its regulation of ribosomal protein subunits. With the exception of the ribosomal subunit L3, which plays a role in protein folding and maybe therefore be more useful in stressful conditions, all the other ribosomal subunit proteins were seen to have reduced expression in stressed cultures. Curiously, C. violeaceum cultures were also observed to lose their violet color under stress, which suggests that the violacein pigment biosynthetic pathway is affected by stress. CONCLUSIONS: Analysis of the proteomic signatures of stressed C. violaceum indicates that nutrient-starvation and pH stress can cause changes in the expression of the C. violaceum receptors, transporters, and proteins involved with biosynthetic pathways, molecule recycling, energy production. Our findings complement the recent publication of the C. violeaceum genome sequence and could help with the future commercial exploitation of C. violeaceum.

Adherence and virulence genes of Escherichia coli from children diarrhoea in the Brazilian Amazon.

The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli . Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli . Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpA gene . EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg , aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea ( P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC.

Adherence and virulence genes of Escherichia coli from children diarrhoea in the Brazilian Amazon

The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli. Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli. Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpA gene. EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg, aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea (P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC.

Immunophenotyping in saliva as an alternative approach for evaluation of immunopathogenesis in chronic periodontitis.

BACKGROUND: To date, flow cytometric immunophenotyping has not been used to investigate immune patterns in saliva samples from individuals with inflammatory processes in the oral cavity, such as chronic periodontitis (CP). Saliva analysis could be a non-invasive method for evaluating oral health. The objective of this study is to determine the phenotype of leukocytes and total immunoglobulin A (IgA), IgG, and IgM titers in the saliva of individuals with CP. METHODS: Saliva samples were obtained from patients with CP (n = 12) and from a control group (n = 27) without oral diseases. Flow cytometry was performed to determine the frequency of T cells (CD4(+) and CD8(+)), B cells, and natural killer (NK) cells as well as the total leukocyte population. Immunoglobulin titers were determined by dot enzyme-linked immunosorbent assay. RESULTS: Cell immunophenotyping revealed that patients with CP had a higher frequency of total leukocytes (47.94% ± 5.1%; P < 0.001), B cells (43.93% ± 6.2%; P = 0.006), NK cells (0.16% ± 0.04%; P = 0.03), and CD4(+) T cells (38.99% ± 4.4%; P = 0.002) than individuals without oral pathologies (24.75% ± 2.2%, 20.60% ± 2.7%, 0.09% ± 0.03%, and 16.82% ± 3.5%, respectively). No significant differences in salivary total IgA, IgG, and IgM titers were found between the two cohorts studied. Nevertheless, higher total IgG levels were observed in patients with CP, which could indicate a possible correlation between clinical attachment level and salivary IgG (P = 0.07; r(2) = 0.08). CONCLUSION: These results show that cell phenotyping by flow cytometry could be an effective tool for determining leukocyte profiles in saliva samples from patients with CP and healthy individuals.

N-terminal Plasmodium vivax merozoite surface protein-1, a potential subunit for malaria vivax vaccine.

The human malaria is widely distributed in the Middle East, Asia, the western Pacific, and Central and South America. Plasmodium vivax started to have the attention of many researchers since it is causing diseases to millions of people and several reports of severe malaria cases have been noticed in the last few years. The lack of in vitro cultures for P. vivax represents a major delay in developing a functional malaria vaccine. One of the major candidates to antimalarial vaccine is the merozoite surface protein-1 (MSP1), which is expressed abundantly on the merozoite surface and capable of activating the host protective immunity. Studies have shown that MSP-1 possesses highly immunogenic fragments, capable of generating immune response and protection in natural infection in endemic regions. This paper shows humoral immune response to different proteins of PvMSP1 and the statement of N-terminal to be added to the list of potential candidates for malaria vivax vaccine.

High levels of IgG3 anti ICB2-5 in Plasmodium vivax-infected individuals who did not develop symptoms.

BACKGROUND: Plasmodium vivax has the potential to infect 2.85 billion individuals worldwide. Nevertheless, the limited number of studies investigating the immune status of individuals living in malaria-endemic areas, as well as the lack of reports investigating serological markers associated with clinical protection, has hampered development of vaccines for P. vivax. It was previously demonstrated that naturally total IgG against the N-terminus of P. vivax merozoite surface protein 1 (Pv-MSP1) was associated with reduced risk of malarial infection. METHODS: Immune response against Pv-MSP1 (N-terminus) of 313 residents of the Rio Pardo rural settlement (Amazonas State, Brazil) was evaluated in a cross-sectional and longitudinal follow up over two months (on site) wherein gold standard diagnosis by thick blood smear and rRNA gene-based nested real-time PCR were used to discriminate symptomless Plasmodium vivax-infected individuals who did not develop clinical symptoms during a 2-months from those uninfected ones or who have had acute malaria. The acquisition of antibodies against Pv-MSP1 was also evaluated as survival analysis by prospective study over a year collecting information of new malaria infections in surveillance database. RESULTS: The majority of P. vivax-infected individuals (52-67%) showed immune recognition of the N-terminus of Pv-MSP1. Interesting data on infected individuals who have not developed symptoms, total IgG levels against the N-terminus Pv-MSP1 were age-dependent and the IgG3 levels were significantly higher than levels of subjects had acute malaria or those uninfected ones. The total IgG anti ICB2-5 was detected to be an important factor of protection against new malaria vivax attacks in survival analysis in a prospective survey (p = 0.029). CONCLUSIONS: The study findings illustrate the importance of IgG3 associated to 2-months of symptomless in P. vivax infected individuals and open perspectives for the rationale of malaria vaccine designs capable to sustain high levels of IgG3 against polymorphic malaria antigens.

Gestational malaria associated to Plasmodium vivax and Plasmodium falciparum placental mixed-infection followed by foetal loss: a case report from an unstable transmission area in Brazil.

Gestational malaria is a multi-factorial syndrome leading to poor outcomes for both the mother and foetus. Although an unusual increasing in the number of hospitalizations caused by Plasmodium vivax has been reported in Brazil, mortality is rarely observed. This is a report of a gestational malaria case that occurred in the city of Manaus (Amazonas State, Brazil) and resulted in foetal loss. The patient presented placental mixed-infection by Plasmodium vivax and Plasmodium falciparum after diagnosis by nested-PCR, however microscopic analysis failed to detect P. falciparum in the peripheral blood. Furthermore, as the patient did not receive proper treatment for P. falciparum and hospitalization occurred soon after drug treatment, it seems that P. falciparum pathology was modulated by the concurrent presence of P. vivax. Collectively, this case confirms the tropism towards the placenta by both of these species of parasites, reinforces the notion that co-existence of distinct malaria parasites interferes on diseases' outcomes, and opens discussions regarding diagnostic methods, malaria treatment during pregnancy and prenatal care for women living in unstable transmission areas of malaria, such as the Brazilian Amazon.

On the cytoadhesion of Plasmodium vivax-infected erythrocytes.

BACKGROUND: Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. METHODS: Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. RESULTS: Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. CONCLUSIONS: These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.