Detection of toxigenic Clostridium perfringens and Clostridium botulinum from food sold in Lagos, Nigeria.

Food-borne diseases contribute to the huge burden of sickness and death globally and in the last decade, have become more frequently reported in Africa. In line with this, food safety is becoming a significant and growing public health problem in Nigeria. Diarrhoea is a common problem in Nigeria and has been reported but there has been little data on the possibility of clostridia as aetiological agents. Clostridium species are ubiquitous in the environment and in the gastrointestinal tract of man and animals and can serve as a marker for faecal contamination. We set out to determine the potential of these foods to transmit Clostridium species. A total of 220 food commodities from six local governments in Lagos State were sampled. Isolates obtained were identified based on cultural, morphological and biochemical characteristics. Toxinotyping was done using multiplex-PCR with primers specific for alpha, beta, epsilon and iota-toxin genes, enterotoxigenic cpe gene and neurotoxigenic BoNt gene. Fifty (22.7%) clostridial species were isolated of which 29 (58%) were identified as C. perfringens. Toxinotyping of the 29 strains showed that 28 (96.6%) were toxin producing C. perfringens type A while one (3.4%) was C. perfringens type D. Two (4%) C. botulinum species were isolated and identified by 16S rRNA sequencing, both harbouring BoNt/A gene. The contamination rates of food with Clostridium species show that food hygiene is a problem and Clostridium species may be a source of food borne disease in Lagos State, Nigeria.

Large-scale application of highly-diluted bacteria for Leptospirosis epidemic control.

BACKGROUND: Leptospirosis is a zoonotic disease of major importance in the tropics where the incidence peaks in rainy seasons. Natural disasters represent a big challenge to Leptospirosis prevention strategies especially in endemic regions. Vaccination is an effective option but of reduced effectiveness in emergency situations. Homeoprophylactic interventions might help to control epidemics by using highly-diluted pathogens to induce protection in a short time scale. We report the results of a very large-scale homeoprophylaxis (HP) intervention against Leptospirosis in a dangerous epidemic situation in three provinces of Cuba in 2007. METHODS: Forecast models were used to estimate possible trends of disease incidence. A homeoprophylactic formulation was prepared from dilutions of four circulating strains of Leptospirosis. This formulation was administered orally to 2.3 million persons at high risk in an epidemic in a region affected by natural disasters. The data from surveillance were used to measure the impact of the intervention by comparing with historical trends and non-intervention regions. RESULTS: After the homeoprophylactic intervention a significant decrease of the disease incidence was observed in the intervention regions. No such modifications were observed in non-intervention regions. In the intervention region the incidence of Leptospirosis fell below the historic median. This observation was independent of rainfall. CONCLUSIONS: The homeoprophylactic approach was associated with a large reduction of disease incidence and control of the epidemic. The results suggest the use of HP as a feasible tool for epidemic control, further research is warranted.

Induction of a protective response with an IgA monoclonal antibody against Mycobacterium tuberculosis 16kDa protein in a model of progressive pulmonary infection.

Mycobacterium tuberculosis is a facultative intracellular pathogen for which cell-mediated immunity is considered the major component of the immune response. For many decades, the prevailing scientific view has been the antibodies have little or no role in modifying the course of M. tuberculosis infection. In recent years, several studies have challenged this dogma, and there is a body of evidence that supports a role of antibodies against M. tuberculosis. In the present work, we evaluated the protective activity of two monoclonal antibodies (TBA61 and TBA84). Here, we chose the intratracheal model of pulmonary infection to evaluate bacterial load and morphometric and histological changes in the lungs of treated mice. Data obtained revealed the reduction of bacterial load and milder morphometric and histopathological changes in mice treated with TBA61 at 21 days post-infection with M. tuberculosis H37Rv compared to those treated with TBA84 and control mice. These results allow continuing exploring the potential use of monoclonal antibodies as prophylactic and therapeutic agents against intracellular pathogens such as M. tuberculosis.

The effect of the administration of human gamma globulins in a model of BCG infection in mice.

The effect of the administration of a commercial preparation of human gamma globulins has been evaluated in a mouse model of intranasal infection with BCG. First, we demonstrated the passage of specific antibodies to saliva and lung lavage following the intranasal or intraperitoneal administration to mice of human gamma globulins. This treatment of mice inhibited BCG colonization of the lungs (p < 0.01). A similar inhibitory effect was observed after infection of mice with gamma globulin opsonized BCG organisms (p < 0.01). These results are relevant for the development of new strategies for the control and treatment of tuberculosis.

Evaluation of enteric-coated tablets as a whole cell inactivated vaccine candidate against Vibrio cholerae.

A vaccine candidate against cholera was developed in the form of oral tablets to avoid difficulties during application exhibited by current whole cell inactivated cholera vaccines. In this study, enteric-coated tablets were used to improve the protection of the active compound from gastric acidity. Tablets containing heat-killed whole cells of Vibrio cholerae strain C7258 as the active pharmaceutical compound was enteric-coated with the polymer Kollicoat(®) MAE-100P, which protected them efficiently from acidity when a disintegration test was carried out. Enzyme-linked immunosorbent assay (ELISA) anti-lipopolysaccharide (LPS) inhibition test and Western blot assay revealed the presence of V. cholerae antigens as LPS, mannose-sensitive haemagglutinin (MSHA) and outer membrane protein U (Omp U) in enteric-coated tablets. Immunogenicity studies (ELISA and vibriocidal test) carried out by intraduodenal administration in rabbits showed that the coating process of tablets did not affect the immunogenicity of V. cholerae-inactivated cells. In addition, no differences were observed in the immune response elicited by enteric-coated or uncoated tablets, particularly because the animal model and immunization route used did not allow discriminating between acid resistances of both tablets formulations in vivo. Clinical studies with volunteers will be required to elucidate this aspect, but the results suggest the possibility of using enteric-coated tablets as a final pharmaceutical product for a cholera vaccine.

Caracterización de filtros comerciales para agua a base de carbón activado para el tratamiento de agua del río Tumbes - Perú / Characterization of water commercial filters based on activated carbon for water treatment of the Tumbes river - Peru / Caracterização de carvão ativado de filtros comerciais para tratamento da água do rio Tumbes -Perú

Resumen Se caracterizaron y se evaluaron carbones activados comerciales (A, B, C y D) utilizados en filtros para el tratamiento de agua en la descontaminación de metales pesados presentes en agua de río y en la eliminacion de microorganismos coliformes; los carbones comerciales resultaron tener estructuras microporosas y mesoporosas. Se determinaron areas superficiales entre 705 y 906 m2/g. Los carbones fueron amorfos y se detectó la presencia de agentes antibacterianos, tales como Ag, Cl, Cu y Si. Se determinó que para el As y Pb, cuyas concentraciones iniciales en el agua contaminada (agua del Río Tumbes-Perú) fueron 56,7 y 224,0 μg/L, respectivamente, el porcentaje de adsorción fue cercano al 100%. También se encontró que la relación entre el pH de carga cero de los carbones y pH del agua del río durante los experimentos juega un rol determinante en la adsorción de los elementos analizados. Por otro lado, la capacidad antibacteriana fue evaluada satisfactoriamente frente a las siguientes cepas de bacterias gram negativas fecales: Escherichia coli (ATCC® 25922™), Salmonella typhimurium (ATCC® 14028™) y Shigella flexneri (ATCC® 12022™). Esta capacidad se basa en la presencia superficial en los carbones de los agentes antibacterianos mencionados.

Abstract Comercial activated carbon samples (A, B, C, and D) used in filters for the treatment of water were characterized and evaluated in the decontamination of heavy metals present in river water and in the elimination of coliform microorganisms. The carbon samples had microporous and mesoporous structures. Surface areas of between 705 and 906 m2/g were found. The carbon samples were amorphous and the presence of antibacterial agents such as Ag, Cl, Cu, and Si was detected. It was determined that for As and Pb, whose initial concentrations in contaminated water (water of the Tumbes river-Peru) were 56.7 and 224.0 μg/L, respectively, the percentage of adsorption was close to 100%. The relationship between point of zero charge pH of the activated carbons and pH of the river water during the experiments plays a determinant role in the adsorption of the analyzed elements. The antibacterial capacity was evaluated satisfactorily against the following strains of fecal gram negative bacteria: Escherichia coli (ATCC® 25922™), Salmonella typhimurium (ATCC® 14028™), and Shigella flexneri (ATCC® 12022™). This ability is based on the surface presence in the carbons ofthe mentioned antibacterial agents.

Resumo Carvões ativados comerciais (A, B, C e D) usado em filtros para o tratamento de água foram caracterizados e avaliados na descontaminação de metais pesados da água do rio e para a remoção de microrganismos coliformes. Os carvões comerciais mostraram estruturas microporosas e mesoporosas. Foram determinadas as áreas superficiais entre 705 e 906 m2/g. As amostras de carvão foram amorfas e foi detectada a presença de agentes antibacterianos, tais como Ag, Cl, Cu e Si. Determinou-se que, no caso de As e Pb, cujas concentrações iniciais na água contaminada (água do rio Tumbes-Peru) foram de 56,7 e 224,0 μg/L respectivamente, a taxa de adsorção foi de quase 100%. Também foi econtrado que e a relação entre o pH de carga zero dos carvões e o pH da água do rio durante as experiências desempenha um papel decisivo na adsorção dos elementos analisados. Além disso, a capacidade antibacteriana foi avaliada com sucesso contra as seguintes variedades de bactérias gram negativas de origem fecal: Escherichia coli (ATCC® 25922™), Salmonella typhimurium (ATCC® 14028™) e Shigella flexneri (ATCC® 12022™). Esta capacidade esta baseada na presença de agentes bacterianos na superfície dos carvões.

Pharmacology and toxicology of an oral tablet whole cells inactivated cholera vaccine in Sprague Dawley rats.

Here we further investigate the pharmacological and toxicological properties of a cholera vaccine based on inactivated whole cells presented in either enteric coated (COA) or uncoated (U/C) tablet formulation from Vibrio cholerae C7258 strain. Tablets were dispersed in 2mL drinking water and administered orally to Sprague Dawley rats distributed in five groups (I COA7, II U/C7 immunized at 0, 7, 69days and III COA14, IV U/C14 immunized at 0, 14, 69days and V control group). Serum vibriocidal antibody response was measured after the administration of two doses with an interval of 7-14days. To further investigate the toxicological aspects a third dose was applied 10 weeks after the initial one. Animals were observed daily and water and food consumption was measured every other day. Periodic blood extractions were performed for hematology, biochemistry, and the titer of serum vibriocidal antibodies was determined. Anatomopathological analysis was performed at days 3 or 14 after the third dose. Results from clinical observations, as well as from water and food consumption and body weigh indicated no toxicity of the vaccine product. Meanwhile, no biological differences were found among different groups in hematological, hemo-chemistry, and anatomopathological studies. Moreover, enteric coated and uncoated tablets against human cholera were found to induce an immune response in rats.

Prophylactic effect of administration of human gamma globulins in a mouse model of tuberculosis.

The protective effect of human gamma globulins on Mycobacterium tuberculosis infection was evaluated in a mouse model of intratracheal infection. Animals receiving human gamma globulins intranasally, 2h before intratracheal challenge showed a significant decrease in lung bacilli load compared to non-treated animals in different time intervals of up to 2 months after challenge. The same effect was obtained when M. tuberculosis was pre-incubated with the gamma globulin before challenge. The protective effect of the gamma-globulin formulation was abolished after pre-incubation with M. tuberculosis. These results suggest a potential role of specific antibodies in the defence against mycobacterial infections.

Identification by genomic immunization of a pool of DNA vaccine candidates that confer protective immunity in mice against Neisseria meningitidis serogroup B.

We have shown previously that expression library immunization is viable alternative approach to induce protective immunity against Neisseria meningitidis serogroup B. In this study we report that few rounds of library screening allow identification of protective pools of defined antigens. A previously reported protective meningococcal library (L8, with 600 clones) was screened and two sub-libraries of 95 clones each were selected based on the induction of bactericidal and protective antibodies in BALB/c mice. After sequence analysis of each clone within these sub-libraries, we identified a pool of 20 individual antigens that induced protective immune responses in mice against N. meningitidis infection, and the observed protection was associated with the induction of bactericidal antibodies. Our studies demonstrate for the first time that ELI combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in a meningococcal vaccine.

Proteoliposome derived cochleate as novel adjuvant.

Cochleate structures (CS) consist in a highly stable lipid structures that have been reported to be a good antigen delivery system. The incorporation of pathogen associated molecular pattern (PAMP) from bacterial membranes into CS became in a promising approach to develop adjuvants, particularly mucosal adjuvants. Therefore, we prepare CS from proteoliposome (PL) obtained from Neisseria meningitidis B (PLCS) and evaluated it for its capability to stimulate the immune system as well as the adjuvant activity. The ability of PLCS to induce Thl polarization was also explored. The results and the easy capability for new antigen incorporation on CS support its use as adjuvant for immunization with a large variety of pathogen derived antigens and different routes of immunization.

Scale up of proteoliposome derived Cochleate production.

Cochleate are highly stable structures with promising immunological features. Cochleate structures are usually obtaining from commercial lipids. Proteoliposome derived Cochleate are derived from an outer membrane vesicles of Neisseria meningitidis B. Previously, we obtained Cochleates using dialysis procedures. In order to increase the production process, we used a crossflow system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The raw material and solutions used in the production process are already approved for human application. This work demonstrates that CFS is very efficient process to obtain Cochleate structures with a yield of more than 80% and the immunogenicity comparable to that obtained by dialysis membrane.

Proteliposome-derived Cochleate as an immunomodulator for nasal vaccine.

Proteoliposome (PL) has been recently used as a protective intramuscular (i.m.) anti-meningococcal BC vaccine. It induces a preferential Th 1 type of immune response. Nevertheless, mucosal protection is mainly mediated by IgA antibody response, which is not usually induced by i.m. vaccination route. IgA antibody production needs the stimulation of Th3 subpopulation, which is also related to the induction of small dose tolerance. We hypothesized that PL-derived Cochleate can induce a specific mucosal IgA and systemic IgG antibody responses. We could show that mice immunized with two or three intranasal doses of PL-derived Cochleate developed significantly increased levels of local anti PL IgA and systemic IgG antibody responses. Thus, our results suggest that PL-derived Cochleate can be used as a promising immunomodulator and delivery system for the development of mucosal, particularly nasal vaccines.

Novel adjuvant based on a proteoliposome-derived cochleate structure containing native lipopolysaccharide as a pathogen-associated molecular pattern.

Proteoliposomes (PL) from Neisseria meningitidis B have been widely used as a core antigen for antimeningococcal vaccination. PL contain major outer membrane proteins, LPS and phospholipids, and they induce a strong Th1 immune response, but they have low stability in solution. Attending to the need for new vaccine adjuvants, we developed a highly stable cochleate structure (CS) from PL using a technology that allows easy incorporation of new antigens. We explored the ability of PLCS to activate the immune system and its possible application as an adjuvant for parenteral and mucosal routes. Our results showed that PLCS were able to upregulate the expression of MHC class II and costimulatory molecules on human dendritic cells, as well as being able to stimulate the production of soluble mediators of a Th1 response, such as IL-12 and nitric oxide. High levels of anti-PL IgG were detected in serum after i.m. or mucosal (oral and nasal) administration, but also anti-PL secretory IgA was produced in saliva following nasal delivery. The immune response polarization to a Th1 pattern was confirmed by the induction of IgG2a antibodies, positive delayed type hypersensitivity reactions, and IFN-gamma production by splenocytes from immunized mice. The adjuvant potential was explored using PLCS containing ovalbumin (Ova). PLCS-Ova was able to elicit a substantial increase in anti-Ova IgG compared with Ova alone. In addition, a significant reduction in lesion size was observed in mice immunized with Leishmania major antigens in PLCS after challenge with virulent protozoa, suggesting at least partial modulation of the Th2 environment induced by this parasite. In conclusion, our results support the use of PLCS as a potent Th1 adjuvant for parenteral and mucosal vaccines.

Ultramicroelisa para la deteccion de anticuerpos IgM al Mycobacterium leprae utilizando muestras de sangre seca / Ultramicroelisa assay for the detection of IgM antibodies to Mycobacterium leprae using eluates of dried blood spots

En este trabajo se estebelecen las condiciones optimas para la deteccion de anticuerpos IgM al glicolipido fenolitico-I (GF-I) en muestras de sangre en papel de filtro utilizando el UltranicroELISA HANSEN y la tecnologia SUMA. Se estudiaron 30 doantes de sabgre y 58 pacientes leprosos. Para estas dos poblaciones se compararon los resultados de muestras de sangre seca colectadas en papel de filtro SS-2992 con los de suero, y se obtuvo una correlacion de 0.919 para doantes de sangre, 0.969 para pacientes y 0.954 para el total de las dos poblaciones....

Anti M. leprae IgM antibody determination by ultramicroimmunoenzimatic (UMELISA HANSEN) for the diagnosis and monitoring leprosy

Se analizo la relacion entre la carga antigenica y la respuesta de anticuerpos IgM, el comportamiento de dicha respuesta y la utilidad de su deteccion para el diagnostico y seguimento de la lepra. Se obtuvo un 82 por cento de coincidencia entre los resultados del UMELISA HANSEN y los de la baciloscopia. Este valor se vio afectado fundamentalmente por 16 pacientes con respuesta positiva a IgM y baciloscopia negativa. En estos pacientes de acuerdo a lo reportado, la respuesta IgM puede indicar la reaparicion de bacilos, precediendo a su deteccion en la piel. En uno de estos pacientes se demonstro la presencia de bacilos, dos meses despues de resultar positivo por el UNELISA HANSEN. Entre los pacientes coincidentes en IgM baciloscopia positivas con tratamiento quimioterapeutico...