DiagnÃstico microbiolÃgico, imunoenzimÃtico e molecular e perfil de genes associados à virulÃncia de Campylobacter

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoCampylobacter sp. à uma importante causa de enterite de origem alimentar com alta incidÃncia na populaÃÃo infantil de paÃses em desenvolvimento. No entanto, o diagnÃstico especÃfico de sua etiologia segue como um desafio, visto que mÃtodos moleculares e imunoenzimÃticos tÃm se mostrado mais sensÃveis. Postulamos que o conhecimento de sua virulÃncia e o diagnÃstico especÃfico possam ajudar na identificaÃÃo e potencial controle da campilobacteriose na infÃncia. Foi determinada a etiologia de diarreia por Campylobacter sp., em um estudo transversal sobre diarreia em crianÃas de 0-36 meses residentes da Ãrea urbana de Fortaleza, CearÃ, Brasil, que necessitaram de atendimento mÃdico de urgÃncia por causa de doenÃa diarreica. ApÃs a aprovaÃÃo Ãtica do estudo, um questionÃrio foi aplicado para qualificar as condiÃÃes clÃnicas apresentadas por cada crianÃa no momento da admissÃo. O DNA foi extraÃdo diretamente de amostras fecais coletadas de 226 crianÃas. Para a detecÃÃo do agente etiolÃgico, utilizamos diagnÃstico molecular (PCR e RT-PCR) e diagnÃstico imunoenzimÃtico (ELISA), alÃm da detecÃÃo de genes associados à virulÃncia de C. jejuni (PCR). Campylobacter sp. foi encontrado em 8,9% (20/225) das amostras, por diagnÃstico microbiolÃgico convencional. C. jejuni e C. coli foram detectados em 19,5% (44/226) e 1,3% (3/226) das amostras diarreicas, respectivamente, por PCR. Os diagnÃsticos por RT-PCR e ELISA alcanÃaram 26,7% (60/225) e 37,9% (58/153), respectivamente. Quando considerada a combinaÃÃo de diagnÃsticos (positividade no diagnÃstico microbiolÃgico ou no imunoenzimÃtico e ao menos em um dos testes moleculares) a prevalÃncia encontrada foi de 16,4% (37/226). A concordÃncia entre os testes para diagnÃstico utilizados foi de moderada a regular, de acordo com o Ãndice de Kappa. Genes associados à virulÃncia foram detectados nas seguintes proporÃÃes de amostras positivas para C. jejuni: flaA, 79,5% (35/44); racR, 97,7% (43/44) e dnaJ, 88,6% (39/44) â relacionados à adesÃo bacteriana e colonizaÃÃo; ciaB, 97,7% (43/44); pldA, 45,4% (20/44) e pVir 0% (0/44) â relacionados à invasÃo; e cdtABC em 95,4% (42/44) das amostras, operon relacionado à produÃÃo da toxina citoletal distensora (CDT). Sinais e sintomas especÃficos, tais como sangue nas fezes, vÃmito, febre e/ou dor abdominal, apesar de bastante frequentes, nÃo foram associados com a detecÃÃo de C. jejuni. O perfil de distribuiÃÃo dos genes de virulÃncia de C. jejuni nÃo apresentou correlaÃÃo com a apresentaÃÃo clÃnica da doenÃa, mesmo quando tal perfil foi categorizado de acordo com a funÃÃo das proteÃnas codificadas pelos genes, o que nos leva a crer que outros fatores, talvez relacionados à susceptibilidade do hospedeiro, possam ser mais importantes do que a variabilidade genÃtica do micro-organismo. ConcluÃmos que Campylobacter sp. foi detectado em percentual relevante da populaÃÃo estudada, principalmente quando os mÃtodos diagnÃsticos foram utilizados de forma combinada. Em geral, os genes de virulÃncia foram detectados em uma alta proporÃÃo das amostras positivas para C. jejuni, embora os genes relacionados à invasÃo tenham sido menos frequentemente encontrados. Corroboramos dados de outros grupos sobre a necessidade de revisÃo do diagnÃstico para Campylobacter sp. em prol da inclusÃo de metodologias mais sensÃveis e espÃcie-especÃficas, alÃm da busca por marcadores para inflamaÃÃo intestinal e fatores preditivos de cultura negativa.Campylobacter sp. is an important cause of food-borne gastroenteritis with high incidence in children living in developing countries. However, the specific diagnosis of its etiology remains as a challenge, since conventional diagnosis by culture is now challenged by molecular and immunoenzymatic methods, which have greater sensitivity. We postulate that the knowledge of its virulence and specific diagnosis may assist in identifying and potentially controling campylobacteriosis in childhood. We determined the etiology of Campylobacter sp. associated diarrhea, in a cross-sectional study of diarrhea in children aged 0-36 months from the urban area of Fortaleza, CearÃ, Brazil, who required emergency medical care because of diarrheal disease. After ethical approval of the study, a questionnaire was applied to describe the clinical conditions presented by each child at the time of admission. DNA was extracted directly from fecal samples collected from 226 children. For the determination of the etiologic agent we used molecular diagnostics (PCR and RT-PCR) and diagnostic immunoassay (ELISA), besides the detection of virulence associated genes of C. jejuni (PCR). Campylobacter sp. was found in 8.9% (20/225) of the samples by conventional microbiological diagnosis. C. jejuni and C. coli were detected in 19.5% (44/226) and 1.3% (3/226) of the diarrheic samples, respectively. The diagnostic RT-PCR and ELISA reached 26.7% (60/225) and 37.9% (58/153) of positivity, respectively. When considering the combination of diagnostic (positive in microbiological diagnosis or immunoassay and at least one of the molecular tests) the prevalence was 16.4% (37/226). The agreement between the tests used for diagnosis was moderate to regular, according to Kappa index. The presence of C. jejuniÂs virulence-associated genes that encode proteins related to the pathogenesis of micro-organism were detected in the following proportions of C. jejuni-positive DNA samples: flaA, 79.5% (35/44); racR, 97.7% (43/44) and dnaJ, 88.6% (39/44) â related to bacterial adhesion and colonization; ciaB, 97.7 % (43/44); pldA, 45.4% (20/44) and pVir 0% (0/44) â related to invasion, and cdtABC in 95.4% (42/44) of samples related to citoletal distending toxin (CDT). Specific signs and symptoms such as blood in the stool, vomiting, fever and/or abdominal pain, although quite frequent, were not associated with the detection of C. jejuni. The distribution profile of C. jejuniÂs virulence genes was not correlated with the clinical presentation of the disease, even when this profile was categorized according to the function of the proteins encoded by the genes, which leads us to believe that other factors, perhaps related to host susceptibility, may be more important than genetic variability of the microorganism. We conclude that Campylobacter sp. was detected in a significant percentage of the children 0-36 months with diarrhea, especially when the diagnostic methods were used in combination. In general, the virulence genes were detected in a high proportion of C. jejuni-positive samples, although the invasion-related genes have been found less frequently. Our data corroborates findings from other groups on the need to revise the diagnostic for Campylobacter sp. towards the inclusion of more sensitive and species-specific methods, as well as search for extra markers for intestinal inflammation and predictors of negative culture

Antigenicity of a whole parasite vaccine as promising candidate against canine leishmaniasis.

Human visceral leishmaniasis, one of the most important zoonoses, is caused by the protozoaLeishmania chagasi(syn. L. infantum ) and is present as a fatal disease common in South America and Europe where dogs and wild canids are the main reservoirs. A vaccine against visceral leishmaniasis would be an important tool in the control of this disease in dogs. Although the current strategies for vac-cination against leishmaniasis are based on the use of recombinant antigens, killed vaccines are still attractive in terms of stability of their biochemical composition and antigenicity, cost, and safety. Here we evaluate the immunogenicity of a whole parasite vaccine as a prom-ising candidate against canine leishmaniasis, demonstrated by cellular reactivity, changes in the cellular profile of the peripheral blood and by the differential production of immunoglobulins. Our results showed that immunization elicited mainly a strong cellular reactivity and increase in T-lymphocytes, particularly the subpopulation CD8 + that would be related to the control of tissue parasitism. In addi-tion, a higher production of anti- Leishmania total IgG, characterized by mixed isotypes profile (IgG1 and IgG2), was demonstrated

Antigenicity of a whole parasite vaccine as promising candidate against canine leishmaniasis.

Human visceral leishmaniasis, one of the most important zoonoses, is caused by the protozoaLeishmania chagasi(syn. L. infantum ) and is present as a fatal disease common in South America and Europe where dogs and wild canids are the main reservoirs. A vaccine against visceral leishmaniasis would be an important tool in the control of this disease in dogs. Although the current strategies for vac-cination against leishmaniasis are based on the use of recombinant antigens, killed vaccines are still attractive in terms of stability of their biochemical composition and antigenicity, cost, and safety. Here we evaluate the immunogenicity of a whole parasite vaccine as a prom-ising candidate against canine leishmaniasis, demonstrated by cellular reactivity, changes in the cellular profile of the peripheral blood and by the differential production of immunoglobulins. Our results showed that immunization elicited mainly a strong cellular reactivity and increase in T-lymphocytes, particularly the subpopulation CD8 + that would be related to the control of tissue parasitism. In addi-tion, a higher production of anti- Leishmania total IgG, characterized by mixed isotypes profile (IgG1 and IgG2), was demonstrated

Intestinal cell migration damage induced by enteropathogenic Escherichia coli strains

Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC

The Resin from Protium heptaphyllum Prevents High-Fat Diet-Induced Obesity in Mice: Scientific Evidence and Potential Mechanisms

Herbal compounds rich in triterpenes are well known to regulate glucose and lipid metabolism and to have beneficial effects on metabolic disorders. The present study investigated the antiobesity properties of resin from Protium heptaphyllum (RPH) and the possible mechanisms in mice fed a high-fat diet (HFD) for 15 weeks. Mice treated with RPH showed decreases in body weight, net energy intake, abdominal fat accumulation, plasma glucose, amylase, lipase, triglycerides, and total cholesterol relative to their respective controls, which were RPH unfed. Additionally, RPH treatment, while significantly elevating the plasma level of ghrelin hormone, decreased the levels of insulin, leptin, and resistin. Besides, HFD-induced increases in plasma levels of proinflammatory mediators TNF-α, IL-6, and MCP-1 were significantly lowered by RPH. Furthermore, in vitro studies revealed that RPH could significantly inhibit the lipid accumulation in 3T3-L1 adipocytes (measured by Oil-Red O staining) at concentrations up to 50 μg/mL. These findings suggest that the antiobese potential of RPH is largely due to its modulatory effects on various hormonal and enzymatic secretions related to fat and carbohydrate metabolism and to the regulation of obesity-associated inflammation

Immunogenicity in dogs of three recombinant antigens (TSA, LeiF and LmSTI1) potential vaccine for canine visceral leishmaniasis.

Control of canine visceral leishmaniasis (VL) remains a difficult and serious problem mostly because there is no reliable and effective vaccine available to prevent this disease. A mixture of three recombinant leishmanial antigens (TSA, LeIF and LmSTI1) encoded by three genes highly conserved in the Leishmania genus have been shown to induce excellent protection against infection in both murine and simian models of cutaneous leishmaniasis. A human clinical trial with these antigens is currently underway. Because of the high degree of conservation, these antigens might be useful vaccine candidates for VL as well. In the present study, using the dog model of the visceral disease, we evaluated the immunogenicity of these three antigens formulated with two different adjuvants, MPL-SE® and AdjuPrime®. The results were compared with a whole parasite vaccine formulated with BCG as the adjuvant. In order to investigate if sensitization with the recombinant antigens would result in recognition of the corresponding native parasite antigens upon infection, the animals were exposed for four weeks after the termination of the immunization protocol with the recombinant antigens to a low number of L. chagasi promastigotes, an etiological agent of VL. Immune response was evaluated by quantitative ELISA in the animal sera before and after exposure to the viable parasites. Both antigen specific IgG1 and IgG2 antibody levels were measured. Immunization of dogs with the recombinant antigens formulated in either MPL-SE® or AdjuPrime® resulted in high antibody levels particularly to LmSTI1. In addition, this immunization although to low levels, resulted in the development of antibody response to the whole parasite lysate. Importantly, experimental exposure with low numbers of culture forms of L. chagasi promastigotes caused a clear boost in the immune response to both the recombinant antigens and the corresponding native molecules. The boost response was predominantly of the IgG2 isotype in animals primed with the recombinant antigens plus MPL-SE®. In contrast, animals primed with the recombinant antigens formulated in AdjuPrime® as well as animals vaccinated with crude antigen preparation responded with mixed IgG1/IgG2 isotypes. These results point to the possible use of this antigen cocktail formulated with the adjuvant MPL-SE® in efficacy field trials against canine VL

Evaluation of 16S rRNA qPCR for detection of <i>Mycobacterium leprae</i> DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases

<p><i>Mycobacterium leprae</i> bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of <i>M. leprae</i>. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting <i>M. leprae</i> in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for <i>M. leprae</i> 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (<i>T</i>_<i>m</i> = 79.5 °C) and detection limit of qPCR was 20 fg of <i>M. leprae</i> DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 10³–8.02 × 10⁵, and in SB samples from MB patients were 1.87 × 10³–1.50 × 10⁶. Therefore, qPCR assays using SYBR Green targeting <i>M. leprae</i> 16S rRNA region can be employed in detecting <i>M. leprae</i> in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.</p