Abstract DNA vaccines have been evaluated as an option to prevent several diseases. In this study, the capacity of the xanthan biopolymer to improve the DNA vaccines immune response, administered intramuscularly, was evaluated. The experimental vaccines consisted of genes encoding fragments of the proteins LigA and LigB of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. The humoral immune response was evaluated by indirect ELISA. Cytokine expression levels were determined by RT-qPCR. Compared to the control group, the IgG antibody levels of animals immunized with pTARGET/ligAni and pTARGET/ligBrep plasmids associated with xanthan biopolymer were significantly higher than the control group. Additionally, there was a significant increase in IL-17 expression in animals vaccinated with pTARGET/ligBrep and xanthan.

Abstract Recombinant proteins are a suggested alternative for the diagnosis of toxocariasis. The current Escherichia coli recombinant protein overexpression system usually produces insoluble products. As an alternative, yeast such as Pichia pastoris have secretory mechanisms, which could diminish the cost and time for production. This study aimed to produce recombinant proteins in Pichia pastoris and verify their sensibility and specificity in an indirect ELISA assay. Two sequences (rTES-30 and rTES-120) of Toxocara canis excretory-secretory antigens were cloned in a pPICZαB vector and expressed in P. pastoris KM71H. Sera samples collected from human adults infected by Toxocara spp. were tested by indirect ELISA using rTES-30 and rTES-120 as antigens. Recombinant proteins were detected at 72 hours after induction, in the supernatant, as pure bands between 60~70 kDa with hyperglycosylation. Regarding diagnosis potential, recombinant antigens had high specificity (95.6%); however, sensitivity was 55.6% for rTES-30 and 68.9% for rTES-120. Further deglycosylation of the P. pastoris antigens did not seem to affect ELISA performance (p>0.05). The low sensitivity in the serodiagnosis diminished any advantage that P. pastoris expression could have. Therefore, we do not recommend P. pastoris recombinant TES production as an alternative for the diagnosis of toxocariasis.

BACKGROUND The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

Introduction: self-service restaurants in which food is served ready to be consumed are liable to have some products contaminated by pathogenic microorganisms causing food-transmitted diseases. Aim: evaluates the microbiological quality of lettuce salads in restaurants in Pelotas RS Brazil by counts of thermo-tolerant coliforms, E. coli, Staphylococcus spp. and detection of Salmonella spp. Antimicrobial resistance of Staphylococcus spp. isolates are also assessed. Methods: thirty-six samples of lettuce salads were collected from nine restaurants and thermotolerant coliforms, Escherichia coli and Staphylococcus spp. were quantified, coupled to a research on Salmonella spp., following methodology by the Bacteriological Analytical Manual. Staphylococcus spp. isolates underwent antimicrobial resistance test by the disc-diffusion method. Results and discussion: results showed that 61.1% of the salad samples contained more thermotolerant coliforms than allowed by Brazilian legislation and E. coli was confirmed in 5.6% of the samples. Positive and negative coagulase Staphylococcus occurred respectively in 5.6% and 77.8% of isolates, but no sample had Salmonella spp. Further, 56.7% of the thirty isolates of Staphylococcus spp. tested were resistant to penicillin; 46.7% to oxacillin; 26.7% to erythromycin and 23.3% were multi- resistant. Conclusion: inadequate quality of the salad was due to pathogenic microorganisms, while Staphylococcus spp. isolates had a high percentage of antimicrobial resistance.

Introducción: la procura por estabelecimientos que ofrecen alimentos prontos para consumo ha aumentado, sin embrago, los alimentos disponibles en estos locales pueden estar contaminados con microorganismos patogénicos, pudiendo causar enfermedades transmitidas por alimentos. Objetivos: evaluar la calidad microbiológica de las ensaladas de lechuga en los restaurantes de Pelotas RS Brasil, a través de los recuentos de coliformes termotolerantes, Escherichia coli, Staphylococcus spp. y la detección de Salmonella spp. Resistencia a los antimicrobianos de Staphylococcus spp. también se evalúan. Métodos: fueron colectadas 36 muestras de ensaladas de lechuga en nueve restaurantes y realizada la cuantificación de coliformes termotolerantes, Escherichia coli y Staphylococcus spp. e investigación de Salmonella spp., siguiendo la metodología del Bacteriological Analytical Manual. Los aislados de Staphylococcus spp. fueron sometidos al examen de resistencia a antimicrobianos por el método de difusión con discos. Resultados y discusión: de las 36 muestras de ensalada de lechuga, 61,1% presentaron cuantificación de coliformes termotolerantes superiores a lo permitido por la legislación brasileña, y hubo confirmación de E. coli en 5,6% de las muestras. La cuantificación de Staphylococcus coagulasa positiva representó 5,6% de los aislados y Staphylococcus coagulasa negativa representó 77,8%. Todas las muestras presentaron ausencia de Salmonella spp. De los 30 aislados de Staphylococcus spp. examinados, 56,7% fueron resistentes a penicilina, 46,7% a oxacilina, 26,7% a eritromicina y 23,3% fueron multirresistentes. Conclusión: la calidad microbiológica de las ensaladas de lechuga se mostró inadecuada debido a la presencia de microorganismos patogénicos, y los aislados de Staphylococcus spp. presentaron elevado porcentaje de resistencia antimicrobiana.

A separação imunomagnética (IMS) é uma técnica que tem sido associada a diferentes métodos de detecção de Salmonella em alimentos para aumentar a sensibilidade e a especificidade e diminuir o tempo de análise. Neste trabalho é comunicada a obtenção de microesferas magnéticas sensibilizadas com anticorpos anti-Salmonella e seu uso em associação com a metodologia de cultivo convencional para desenvolver um método de detecção de salmonelas em cortes de frango (IMS-plaqueamento). Inicialmente, microesferas cobertas com proteína A foram sensibilizadas com anticorpos policlonais contra lipopolissacarídeo e flagelo de salmonelas e usadas na padronização de um procedimento que captura Salmonella Enteritidis em cultivo puro e faz posterior detecção em ágar seletivo. A seguir, amostras de carne de frango experimentalmente contaminadas com S. Enteritidis foram analisadas imediatamente após a contaminação e após 24h de refrigeração utilizando três protocolos de enriquecimento. O limite de detecção foi cerca de 2x10 UFC/mL. O protocolo que incluiu enriquecimento não-seletivo por 6-8h, enriquecimento seletivo por 16-18h e pós-enriquecimento por 4h foi o que proporcionou melhor resultado na detecção de S. Enteritidis em carne de frango experimentalmente contaminada. Este protocolo foi comparado à metodologia convencional em estudo de detecção de salmonelas em cortes de frango naturalmente contaminados e obteve 100% de sensibilidade e 94% de especificidade. O método desenvolvido foi capaz de diminuir em pelo menos um dia o tempo de detecção de salmonelas em cortes de frango pela metodologia convencional.

The immunomagnetic separation (IMS) is a technique that has been used to increase sensitivity and specificity and to decrease the time required for detection of Salmonella in foods through different methodologies. In this work we report on the development of a method for detection of Salmonella in chicken cuts using in house antibody-sensitized microspheres associated to conventional plating in selective agar (IMS-plating). First, protein A-coated microspheres were sensitized with polyclonal antibodies against lipopolysacharide and flagella from salmonellae and used to standardize a procedure for capturing Salmonella Enteritidis from pure cultures and detection in selective agar. Subsequently, samples of chicken meat experimentally contaminated with S. Enteritidis were analyzed immediately after contamination and after 24h of refrigeration using three enrichment protocols. The detection limit of the IMS-plating procedure after standardization with pure culture was about 2x10 CFU/mL. The protocol using non-selective enrichment for 6-8h, selective enrichment for 16-18h and a post-enrichment for 4h gave the best results of S. Enteritidis detection by IMS-plating in experimentally contaminated meat. IMS-plating using this protocol was compared to the standard culture method for salmonellae detection in naturally contaminated chicken cuts and yielded 100% sensitivity and 94% specificity. The method developed using in house prepared magnetic microespheres for IMS and plating in selective agar was able to diminish by at least one day the time required for detection of Salmonella in chicken products by the conventional culture method.

O cumprimento da legislação que regulamenta a comercialização de alimentos e ingredientes contendo Organismos Geneticamente Modificados (OGMs) é totalmente dependente da sensibilidade e confiabilidade dos métodos de detecção e quantificação de OGMs. Na presente revisão, foram discutidos os métodos mais relevantes para tais fins, especialmente aqueles que se baseiam na detecção da proteína ou do DNA recombinante, destacando as suas principais propriedades, limitações e vantagens. A regulamentação e algumas sugestões de métodos alternativos para a detecção de OGMs também são abordadas.

The enforcement of legislation that regulates the presence of genetically modified organisms (GMOs) in food and food ingredients is totally dependent on the sensitivity and reliability of the GMO testing methods. In this review, the most relevant methods such as recombinant proteins or DNA-based methods were discussed, emphasizing their main properties, limitations and advantages. The regulamentation and some suggestions of alternative methods for the detection of GMOs were also discussed.

Biopolímeros são polissacarídeos microbianos. O biopolímero produzido por Beijerinckia sp. 7070 possui comportamento pseudoplástico e apresenta alta viscosidade em baixas velocidades de deformação, conferindo ao polímero excelentes características de suspensão. O objetivo desse trabalho foi caracterizar o biopolímero produzido por Beijerinckia sp. 7070 em diferentes tempos de cultivo, quanto à produção total, produção de polímeros de fibra longa e curta, produtividade, viscosidade e composição química. Os polímeros produzidos em meio YM líquido foram recuperados em diferentes tempos de cultivo, secos e pesados para determinação da produção e produtividade. O tipo de fibra produzido durante o cultivo foi avaliado microscopicamente. Viscosidades aparentes de solução aquosa 1% foram determinadas a 6, 12, 30 e 60rpm, a 25º C, em um viscosímetro Brookfield. A composição do biopolímero foi determinada por cromatografia em camada delgada comparativa. As maiores produções totais encontradas foram em 30 e 72h, a maior produtividade em 48h e a maior viscosidade em 72h. Os polímeros de fibra longa apresentaram uma tendência de tornarem-se mais longos com o tempo. A viscosidade do polímero de fibra longa foi maior que a do de fibra curta. Todos os biopolímeros apresentaram os mesmos componentes (glucose, galactose, fucose e ácido glucurônico) mas em concentrações diferentes.

Biopolymers are microbial polysaccharides. The biopolymer produced by Beijerinckia sp 7070 has pseudoplastic behaviour and shows high viscosity at low deformation rates, giving to polymer excellent suspension characteristics. The objective of this work was to characterize the biopolymer produced at different culture times by Beijerinckia sp 7070 in relation to total production, production of short and long fiber polymers, productivity, viscosity and chemical composition. The polymers produced in liquid YM medium were recovered at different culture times, dried and weighted for determination of the production and productivity. Fiber type produced during the culture times was evaluated microscopically. Apparent viscosities of the 1% (w/v) aqueous solutions were determined at 6, 12, 30 and 60rpm, at 25º C, in a Brookfield viscosimeter. Biopolymer composition was determined by comparative thin layer chromatography. The highest total biopolymer productions was found at 30 and 72h, the highest productivity was at 48h and the highest viscosity was at 72h. The long fiber polymer showed a tendency of becoming longer with time. The viscosity of long fiber polymer was higher than the short fiber one. All polymers showed the same components (glucose, galactose, fucose and glucuronic acid) but at different concentrations.

Biopolímeros microbianos são polissacarídeos produzidos por microrganismos. A Beijerinckia sp. 7070 produz o biopolímero denominado clairana®. Biopolímeros microbianos podem ser produzidos utilizando enzimas purificadas (via enzimática). O objetivo desse trabalho foi avaliar a possibilidade de produzir o biopolímero clairana® via enzimática, verificar se a síntese e as enzimas envolvidas na síntese são intra e/ou extracelulares, se as enzimas permanecem ativas no meio e estudar a produção do biopolímero via enzimática, através de inativação e lise celular, e pelo processo convencional com células viáveis. As amostras foram cultivadas com a bactéria em meio líquido e submetidas à morte celular por inativação e lise celular após 30h, através de antibiótico e ultrasom, respectivamente. Amostras foram retiradas após 32, 46 e 54h de incubação, junto com amostras produzidas com células viáveis. O mesmo procedimento foi realizado em 46h e amostras foram coletadas após 48, 52 e 54h de incubação. Os polímeros foram recuperados, secos, pesados e analisados. Os resultados sugerem que é possível produzir o biopolímero clairanaâ via enzimática e que, provavelmente enzimas intra e extracelulares estão envolvidas, permanecendo ativas no meio e começando a atuar antes de 30h de fermentação. O processo via enzimática através de lise foi mais eficiente pois libera os polissacarídeos produzidos intracelularmente para o meio externo.

Microbial biopolymers are polysaccharides produced by microorganisms. Beijerinckia sp. 7070 produces the biopolymer denominated as clairana®. Microbial biopolymers can be produced using purified enzymes (by enzymatic pathway). The aim of this work was to evaluate the possibility to produce the clairana® biopolymer using purified enzymes, to verify if the synthesis and the enzymes involved in the synthesis are intra and/or extracellular, if the enzymes remain active in the medium and to study biopolymer production by enzymatic pathway, through cellular inactivation and lyse, and through conventional process with viable cells. The samples were cultivated with bacteria in liquid medium, and subjected to cellular death through cellular inactivation and lyse after 30h, by antibiotic and ultrasom, respectively. Samples were removed 32, 46 e 54h after incubation period, together with samples produced with viable cells. The same procedure was performed after 46h and samples collected after 48, 52 e 54h of incubation period. The polymers were recovered, dried, weighed and analyzed. The results suggest that it is possible to produce the clairana® biopolymer by enzymatic pathway and that, probably, intra and extracellular enzymes are involved, remaining active in the medium and beginning to act before 30h of fermentation. The enzymatic pathway process through cellular lyse is more effective, because it releases the polysaccharides intracellularly produced to medium.